Centro De

Ciências Exatas

E De Tecnologia

Programa De Pós-Graduação

E M Ciência

Engenharia De Materiais

Liliane Samara

Ferreira Leite

São Carlos-Sp

Universidade Federal

D E São

Carlos Centro De

Liliane Samara Ferreira

Leite Tese

Prof. Dr Henrique Capparelli Luiz

Capparelli Mattoso

Prof. Dr Julien Bras

Dr Francys Clay

Vieira Moreira

Henrique Leite

Dr Julien Bras Mattoso

Dr Francys Kley

Dra Maria

Carmo Gonçalves

Dr Mattoso

Profa Dra

Helena Sílvia

UFSCar Bettini Prado

Rosa Morsyleide De Freitas

Prof. Dr Eric Pollet

Prof. Dr Mohamed Naceur Belgacem

Dr Francys

Dr Dra Henriette Azeredo

Osvaldo Novais De Oliveira

Dr Emily Cranston

Amanda Carla

Ana Carolina

Caio Moreira

Vitor, Stanley, Rafaella Alex Naja

Caio Gomide

Merci Donc À Eva

Lorelei Claire

Manon Ardilla

Estelle Malek

Rim, Justine, Hippolyte, Bastian Amina Hugo

Gabriel, Mathieu Max A Todos

D E Proteína

Nanocristais De

Celulose Por

Casiting Continue

C Preparação

L S F ; Leite

F K V L Moreira

UNIVERSIDADE FEDERAL DE SÃO CARLOS

Keywords: Gelatin, Cellulose Nanocrystals, Packaging, Physical Properties, Continuous Casting. vi vii Gélatine, Nanocristaux de Cellulose, Emballage, Propriétés Physiques, Casting Continue

This Ph.D. thesis covers the production and characterization of gelatincellulose nanocrystals (CNCs) bionanocomposite films intended for multifunctional food packaging. The up-scalability of the gelatin/CNCs films was attained by continuous casting and the rheological parameters of the film-forming solutions and drying temperature required to produce 12 m film/h were established. The continuous casting processing was proven to be advantageous when compared to the bench solution casting method. Tensile test, thermogravimetric analysis, and water vapor permeability analysis showed that the continuously cast gelatin/CNCs films had exceptionally better performance than the films obtained by bench casting. The physical and structural properties of the gelatin/CNCs bionanocomposites as tailored by pH-dependent electrostatic interactions were investigated. The suitability of CNCs was successfully extended by functionalization with rosin (r-CNCs), thus giving rise to antimicrobial reinforcing agents. Tensile test and antimicrobial analysis showed that r-CNCs served as bacterial nanofillers in gelatin-based films, which otherwise would suffer from limited physical and biological properties. Plantderived tannic acid was successfully used as an antioxidant and bactericidal additive, as another perspective of extending the potential of the gelatin/CNCs films as a packaging material. Non-oxidized tannic acid favored non-covalent interactions and intermolecular connections between gelatin, CNCs, and tannic acid. These physico chemical interactions showed to have a greater influence on the antioxidant and physical properties of gelatin/CNCs/Tannic acid films. This thesis provides a comprehensive understanding of how CNCs can be explored to develop biodegradable films based on gelatin with enhanced properties or extra functionalities. A continuous solution casting was applied for scaling up the production of gelatin/CNCs-based films making them highly desirable for packaging applications.

EXTENDED FRENCH ABSTRACT

SYMBOLS AND ABBREVIATIONS

Chemical and materials

wt% CNCs

Gelatin film with 0 wt% cellulose nanocrystals 0.5 wt% CNCs Gelatin film with 0.5wt% cellulose nanocrystals 0.

GENERAL INTRODUCTION

The growing plastic production has led to a global waste disposal crisis.

Nowadays, researches are focused on solving this problem by obtaining biodegradable packaging. Biopolymers, including proteins and polysaccharides, are the main natural resources employed to develop biodegradable films [START_REF] Esteghlal | Physical and mechanical properties of gelatin-CMC composite films under the influence of electrostatic interactions[END_REF].

Proteins such as gelatin can be suitable for food packaging due to their renewability, biodegradability, low costs, film-forming ability, and edible nature [START_REF] Esteghlal | Physical and mechanical properties of gelatin-CMC composite films under the influence of electrostatic interactions[END_REF][START_REF] Al-Tayyar | Antimicrobial food packaging based on sustainable Bio-based materials for reducing foodborne Pathogens: A review[END_REF][START_REF] Moustafa | Eco-friendly polymer composites for green packaging: Future vision and challenges[END_REF]. Gelatin is a water-soluble protein produced from partial hydrolysis of collagen, one of the most used biopolymers in food and pharmaceutical fields [START_REF] Liu | Chemical mapping analysis of compatibility in gelatin and hydroxypropyl methylcellulose blend films[END_REF].

Gelatin films have suitable properties, such as transparency, biodegradability, and low oxygen permeability [START_REF] Wang | The effects of ultrasonic/microwave assisted treatment on the water vapor barrier properties of soybean protein isolate-based oleic acid/stearic acid blend edible films[END_REF]; but they show poor barrier properties against moisture and only moderate mechanical strength in high relative humidity conditions [START_REF] Gómez-Guillén | Fish gelatin: a renewable material for developing active biodegradable films[END_REF]. This drawback could be overcome by incorporating reinforcing nanoparticles, such as cellulose nanocrystals (CNCs), in gelatin films [START_REF] Moustafa | Eco-friendly polymer composites for green packaging: Future vision and challenges[END_REF][START_REF] Hosseini | A state-of-the-art review on the elaboration of fish gelatin as bioactive packaging: Special emphasis on nanotechnology-based approaches[END_REF][START_REF] Azeredo | Nanocomposites for food packaging applications[END_REF][START_REF] Squinca | Nanocellulose production in future biorefineries: an integrated approach using tailor-made enzymes Nanocellulose production in future biorefineries : an integrated approach using tailor-made enzymes[END_REF][START_REF] Thomas | a versatile green platform: from biosources to materials and their applications[END_REF].

Cellulose nanocrystals are rigid rod-like crystalline particles extracted from biosourced cellulosic materials. Since the last two decades, interest in cellulose nanocrystals has been growing as shown in Figure 1 by the exponential number of publications per year, owing to their biodegradability, renewability, abundance, low density, and excellent mechanical properties [START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF]. CNCs are one of the most studied polysaccharide-based nanomaterials in polymer nanocomposites [START_REF] Dufresne | Polysaccharide nanocrystal reinforced nanocomposites[END_REF] and can not only enhance the physical properties of protein films [START_REF] Zhuang | Eco-friendly biorefractory films of gelatin and TEMPO-oxidized cellulose ester for food packaging application[END_REF][START_REF] Sukyai | Effect of cellulose nanocrystals from sugarcane bagasse on whey protein isolate-based films[END_REF][START_REF] George | High performance edible nanocomposite films containing bacterial cellulose nanocrystals[END_REF][START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] but can also be chemically modified with various compounds to provide the ensuing nanocomposite with other functionalities [START_REF] Gars | Polymerization of glycidyl methacrylate from the surface of cellulose nanocrystals for the elaboration of PLA-based nanocomposites[END_REF][START_REF] Tang | Functionalization of cellulose nanocrystals for advanced applications[END_REF][START_REF] Espino-Pérez | Cellulose nanocrystal surface functionalization for Liliane Leite -2020 the controlled sorption of water and organic vapours[END_REF][START_REF] Kwak | Chemical and physical reinforcement of hydrophilic gelatin film with di-aldehyde nanocellulose[END_REF], such as antibacterial activity with grafting of natural antimicrobial agents.

The last decades have been marked by intensive research on natural polymer processing as an effort to fabricate biodegradable plastics at a large scale [START_REF] Reddy | Biobased plastics and bionanocomposites: Current status and future opportunities[END_REF].

Most studies have been focused on melt processing techniques due to technologies already established for petroleum-based polymers. However, melt processing of natural polymers, such as proteins, has been found to be challenging due to the thermomechanical instability of these macromolecules. In this project, continuous casting is proposed as a suitable approach to scale up the production of gelatin-cellulose nanocrystals bionanocomposites and produce rolls of bioplastics.

This Ph.D. proposes the production and characterization of multifunctional gelatin-cellulose nanocrystals films intended for packaging. Several objectives can be announced for this project:

i.

Investigate the parameters necessary to produce gelatin/CNCs films by pilot solution casting method (continuous casting method).

ii.

Characterize the morphological, thermal, mechanical, and barrier properties of gelatin/CNCs films obtained by continuous casting.

iii.

Functionalize the surface of CNCs and produce active gelatin films and functionalized CNCs.

In order to achieve these objectives, an active collaboration was developed during the four years with experts for nanocellulose production and functionalization at the Laboratory of Pulp and Paper Science and Graphic Arts (France) and experts for the production of bio-based materials films packaging at the Embrapa Instrumentação (Brazil). Therefore, this joined Ph.D. took place in a very international and multidisciplinary context.

The results obtained from this Ph.D. are organized in four chapters based on scientific papers.

In Chapter I, a literature review describes the context and objectives of this Ph.D.

A focus on gelatin and cellulose nanocrystals and their potential uses as sustainable packaging is exposed. Besides, a scaled-up processing method for natural biodegradable films is provided.

In chapter II focuses on the parameters such as the rheological behavior of the film-forming solution and drying temperature required to produce gelatin/CNCs films by continuous casting. In addition, the physical properties of gelatin/CNCs obtained by continuous casting will be analyzed and two different types of CNCs suspensions were compared.

Chapter III describes the production of gelatin/CNCs with tunable physical properties by controlling the physical interactions between CNCs and gelatin but also checking the influence of the CNCs content. In addition, the effect of drying temperature on gelatin renaturation triple helices and the physical properties of gelatin/CNCs films will be investigated.

Finally, chapter IV describes the functionalization and characterization of the surface of CNCs with rosin, a natural antimicrobial molecule, and with tannic acid, a natural antioxidant. The physical and antimicrobial properties of gelatin-based films with functionalized CNCs are investigated. Besides, the effects of various types of tannic acid, and CNCs on the physical, morphological, antioxidant, and antimicrobial properties of the gelatin-based films will be also analyzed.

I Literature Review

Introduction to chapter I

This chapter aims to introduce the concepts related to this Ph.D. thesis both to "non-expert" and "expert" readers by providing them with general knowledge and relevant data through tables and schematic representations on every topic, extracted from around 200 references. The link between the Ph.D. thesis and literature review is emphasized in italic dark gray letters.

The first part of the literature review offers an overview of the importance of food packaging to the economic sector, as well as the environmental issues generated by the disordered use of plastic packaging. Besides, this part explains the main properties of food packaging materials and provides the reader with insight into the reference data of commercial plastic packaging to easily go over the next chapters.

The second part focuses on natural biodegradable materials -gelatin and cellulose nanocrystals, and their potential use as sustainable packaging. Gelatin and cellulose nanocrystals origins, chemical structure, toxicology, and application in food packaging are described.

Finally, the last part covers the processing of natural biodegradable films and introduces continuous casting as an alternative method -not yet explored for processing protein films at a pilot scale.

11

Liliane Leite -2020

Food packaging: current challenges and sustainability

The use of packaging, throughout human history, has brought several benefits for the conservation, convenient usage, storage, and transportation of foods; contributing significantly to the development of trade markets and the growth of cities [START_REF] Robertson | History of food packaging[END_REF]. From the view of many consumers, however, packaging is often regarded as a necessary evil or an unnecessary cost, a serious waste of resources, and an environmental menace. This happens because consumers do not exactly know all the packaging functions for a product. Usually, when consumers are in contact with packaging its job is almost done [START_REF] Smith | Food packaging -Principles and practice[END_REF].

Packaging has been defined as a socio-scientific discipline that operates in society to ensure the delivery of goods to the ultimate consumer in the best conditions intended for their use [START_REF] Lackhart | A paradigm for packaging[END_REF]. Other definitions of packaging include products, items, or packages in a bag, bottle, box, can, cup, tray, pouch, or another container form with the role of containing and protecting the product, as well as providing convenience and communicating to the consumer. The device or container is considered as a package if it performs one or more of these functions [START_REF]ANON. Glossary of packaging terms[END_REF].

The packaging sector represents about 2% of the gross national product (GNP) in developed countries, and about half of all packaging is used to package food [START_REF] Smith | Food packaging -Principles and practice[END_REF]. According to [START_REF] Guillard | The Next Generation of Sustainable Food Packaging to Preserve Our Environment in a Circular Economy Context[END_REF] [START_REF] Guillard | The Next Generation of Sustainable Food Packaging to Preserve Our Environment in a Circular Economy Context[END_REF], packaging has been identified

as an essential element to address the key challenge of sustainable food consumption. The primary fundamental role of food packaging is to maintain the quality and safety of food products during storage and transportation, controlling gas and vapor exchanges with the external atmosphere, thus extending the food product shelf-life and avoiding unfavorable factors or conditions, such as chemical contaminants, spoilage microorganisms, oxygen, moisture, light, physical damage, etc. [START_REF] Youssef | Bionanocomposites materials for food packaging applications: Concepts and future outlook[END_REF]. To perform such functions, the packaging provides a physical barrier between the outside environment and the food, creating the physicochemical conditions that are essential for obtaining a satisfactory shelf life and maintaining food quality and safety [START_REF] Rhim | Bio-nanocomposites for food packaging applications[END_REF]. It can also be mentioned, as a secondary role, that packaging is used to attract customers while products are on the shelf. Companies spend millions of dollars every year using packaging as a form of marketing. Figure 1.1 -shows the primary functions required for food packaging materials.

Figure 1.1 -Primary functions of food packaging.

Impact of food losses and waste on sustainability

Food packaging plays a significant role in terms of global food waste reduction thanks to reducing damage in transport and handling, as well as shelf life extension, especially by using a well-designed packaging system adapted to the food in terms of presentation [START_REF] Beitzen-Heineke | The prospects of zero-packaging grocery stores to improve the social and environmental impacts of the food supply chain[END_REF]. According to the Swedish Institute for Food and Biotechnology, report for Food and Agriculture Organization (FAO) of the United Nations [START_REF] Hlpe | Food Losses and Waste in the Context of Sustainable Food Systems[END_REF], almost 30 percent of all food produced for human consumption, approximately 1.3 billion tons per year, are either lost or wasted globally. In a context of uncertainties about the capacity of ecosystems and natural resources to sustain an increasing demand for foods, food losses and waste are at the same time a waste of resources and an environmental issue and represent a symbol of inefficiency, unfairness, and unsustainability of the food Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] chain. Therefore, reduce them is a priority to improve the sustainability of food systems.

Sustainable food systems are defined as "food systems that ensure food security and nutrition for all in such a way that the economic, social, and environmental bases to generate food security and nutrition for the future generations are not compromise"' [START_REF] Hlpe | Food Losses and Waste in the Context of Sustainable Food Systems[END_REF]. The subject was highlighted in the United Nations Conference on Sustainable Development (Rio+20) in 2012 [START_REF] Rio+20 | Rio+20 conferência das Nações Unidas sobre desenvolvimento sustentável[END_REF], which connected the food loss and waste reduction to the issue of more sustainable food systems. In 2016, the European Commission set a target to reduce by 50% the food waste in Europe by 2030 [START_REF]EU actions against food waste[END_REF], mobilizing a vast range of disciplines, from biology of food products and conservation technologies to organization and economics of food chains and markets, as well as consumer behavior. Figure 1.2 presents the food loss and waste that occur along the entire food chain.

Figure 1.2 -Food loss and waste that occur along the entire food chain.

Plastic food packaging

The most common materials used for food packaging are plastic, glass, paper and paperboard, and metals. Plastic is currently the most well-known food packaging material [START_REF] Robertson | History of food packaging[END_REF], which is a specific type of polymer, synthetic or natural, classified either as thermoset polymers (solidify into a permanent design and shape) or thermoplastic polymers (flows upon application of heat and/or pressure). Polymers are macromolecules composed of many repeating units, called meros (single units) connected by covalent bonds in a variety of ways [START_REF] Koltzenburg | [END_REF].

In our plastic-based economy, plastic packaging materials are most time referred to as those made from petrochemicals.

In 2018, the worldwide annual plastics manufacturing reached 359 million metric tons, with 62 million metric tons produced in Europe only [START_REF] Gardside | Global plastic production from 1950 to 2018[END_REF]. More than 40% of all plastics are used in the packaging sector, and almost half of them are used for food packaging (Figure 1.3). In Europe, 23 million tons of plastic packaging are produced each year (92 million tons expected in 2050) [START_REF] Hlpe | Food Losses and Waste in the Context of Sustainable Food Systems[END_REF] in a wide range of forms, such as films, sheets, bottles, cups, tubs, and trays, etc. The different types of plastic packaging can provide choices with respect to transparency, color, print, footprint, and heat sealing and can offer specific features regarding durability, shelf life, lightweight, temperature range -they can be used for hot and cold filling and microwave reheating, and barrier properties against water, carbon dioxide, oxygen and nitrogen [START_REF] Tajeddin | Polymers and food packaging[END_REF]. Polyethylene (PE) have been used in packaging since the late 1940s and is the most widely used plastic today, followed by poly(ethylene terephthalate) (PET), polypropylene (PP), polystyrene (PS), and poly(vinyl chloride) (PVC) [START_REF] Kirwan | Plastics in Food Packaging[END_REF].

Figure 1.3 -Plastic demand by segment in 2018 (adapted from [START_REF] Gardside | Global plastic production from 1950 to 2018[END_REF]).

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] Plastic packaging volumes are expected to continuously increasing, quadrupling by 2050, to 318 million tons annually [START_REF] Payne | Key Debates in New Political Economy[END_REF]. However, plastic wasteespecially from food packaging designed for single or short-term use, i.e.

packaging or other consumer products that are thrown away after one brief use, results nowadays in an over-accumulation of non-degradable materials on land and seas, becoming a global threat to our environment [START_REF] Ferraro | Governing plastic pollution in the oceans: Institutional challenges and areas for action[END_REF]. It is estimated that 72% of plastic packaging is not recovered at all: 40% ends up in landfills, corresponding to 9 million tons of waste that are accumulates in the soil, and 32% leak out of collecting system and finally end in the soil and oceans [START_REF] Payne | Key Debates in New Political Economy[END_REF]. The presence of hundreds of millions of tons of plastics and microplastics (micro-and nano-sized particles) in the ocean has caused negative impacts on the economic activities in the marine ecosystems [START_REF] Ferraro | Governing plastic pollution in the oceans: Institutional challenges and areas for action[END_REF]. The United Nations Environment Programme (UNEP) estimates that damage to marine environments is at least UDS 8 billion per year globally [START_REF]Addressing Land-Based Pollution[END_REF]. Additionally, in 2006, only 14% of plastic packaging were collected for recycling and only 5% of the original material value were retained for subsequent use, while 14% were sent to incineration and/or energy recovery process [START_REF] Payne | Key Debates in New Political Economy[END_REF]. However, the recycling rate has been increasing over the years, encouraged by a favorable regulation and Circular Economy (CE) models.

CE models are described as economic systems "where the value of products, materials, and resources is maintained in the economy as long as possible, and the generation of waste is minimal"' [START_REF] Robaina | Circular economy in plastic waste -Efficiency analysis of European countries[END_REF]. In this context, 42% of plastic packaging waste was recycled in the European Union (EU) in 2017, but this trend was observed at varying levels in all EU Member States, for example, only 27% of plastic waste were recycled in France [START_REF]How much plastic packaging waste do we recycle? Disponível[END_REF]. The European Commission identifies plastic waste as a key priority in its Action Plan for the CE and in 2030, 65% of municipal waste and 70% of packaging waste should be recycled [START_REF] Robaina | Circular economy in plastic waste -Efficiency analysis of European countries[END_REF].

In the case of Brazil, according to a report published in 2019 by the World Wildlife Fund (WWF), the country ranks as the fourth global plastic waste producer, with 11.3 billion tons. Out of this total, WWF states that 91% are collected, but only 1.28% is effectively reinserted in the production chain, which is below the global average of 9% [START_REF] Wit | Solucionar a poluição plástica: Transparência e responsabilizaçãoWWF: Fundo Mundial para a Natureza[END_REF].

Plastic is essential in modern economies, and also, the plastic used for packaging is almost exclusively single-use, especially in business-to-consumer applications. The current way by which plastics are produced, used, and discarded fails to capture the economic benefits and is dramatically harming the environment [START_REF] Thompson | Plastics, the environment and human health: current consensus and future trends[END_REF]. However, recycling is not a unique solution for the plastic economy issue. The challenges associated with the entire plastic chain can be turned into opportunities for innovation, sustainability, competitiveness, and job creation. Bio-based and biodegradable plastics can play a key role in achieving a sustainable model, as they can contribute to reduce greenhouse gas emissions and the demand for fossil resources.

Main properties of food packaging materials

Effective food packaging is designed by identifying the product requirements. The choice should take into account the product natural, use, distribution and storage, expiration date and packaging disposal, as well as environmental and waste management issues. Furthermore, ensuring food safety concerning biological risks and needs relating to flavor, color, and texture of the food is essential. In this context, the main properties evaluated in food packaging are listed below:

Permeability properties

The permeability properties, also called barrier properties, indicate the resistance of food packaging materials to sorption and diffusion of substances (mass transfers of gases, water vapor, aroma compounds, and other low molecular weight compounds). Barrier properties play a major role in the control of food degradation reactions by defining around the product an atmosphere whose composition is favorable to the slowing down of the reactions.

Unfortunately, a single packaging material rarely fully meets all the food requirements. According to [START_REF] Guillard | The Next Generation of Sustainable Food Packaging to Preserve Our Environment in a Circular Economy Context[END_REF] [START_REF] Guillard | The Next Generation of Sustainable Food Packaging to Preserve Our Environment in a Circular Economy Context[END_REF], the barrier properties are

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] either too low (case of O2 -sensitive food products for which high O2 barrier packaging is queried) or too high. They cite, for example, the case of respiring products such as fresh fruits and vegetables, where the plastic films are perforated to compensate for their high O2 barrier properties.

The protection of food from vapor and gas exchange with the environment depends on several factors, including food contact and environmental conditions (temperature, pH, relative humidity), film structure and permeability, thickness, area, difference in pressure or concentration gradient across the film [START_REF] Siracusa | Food packaging permeability behaviour: A report[END_REF]. There are two processes by which vapor and gases (permeants) may pass through materials -pore effect and solubility-diffusion effect. In the pore effect, the permeant flow through the microscopic pores, pinholes, and cracks in the material structure. The porosity falls very sharply as the thickness of the materials increases, reaching practically zero with many of the thicker types of commercially available materials. Yet in the solubility-diffusion effect, described as true permeability, the permeant dissolves in the film surface, diffuses through the film matrix, driven by the concentration gradient, and evaporates at the other film surface. The first step depends on the solubility of the specific gas in the polymer and influences the diffusivity of gases across the material, and the second step, relates to the diffusion itself, which depends on the size, and shape, and polarity of the permeant, and the crystallinity, cross-linking degree of and polymer chain segmental motion of the polymer matrix. This process is shown diagrammatically in Figure 1.4.

The true permeability is normally associated with the quantitative evaluation of the barrier properties of materials. In a material without pore defects, the primary mechanism for vapor and gases flow through a film is an active diffusion. A material that is a good barrier to gas/water vapor diffusion has a low permeability. The permeability is inversely proportional to the material thickness [START_REF] Figura | Food physics: physical propertiesmeasurement and application[END_REF]. The gas permeation mechanism is described by the Henry's and Fick's law by which the equations that relate the permeation rate with the area and thickness of the film are obtained [START_REF] Figura | Food physics: physical propertiesmeasurement and application[END_REF]. The calculation of transport rate density (flux) is proportional to the permeant potential gradient, as shown in Equation 1.

-. = Eq. 1

: = ! " #$%!
Where A is the area, m is the amount of set, t is the time, φ is the potential,

x is the length, K is the coefficient and TR is the transmission rate through the film and the SI units are kg. m -2 . s -1 .

The permeability coefficient (P) is calculated using the following Equation 2.

& = '( ) ∆+ Eq. 2
Where l is the film thickness and ∆p is the partial pressure gradient across the film. The SI units for P are kg. s -1 . m -2 . Pa.

The most important barrier properties of polymer films used in food packaging are water vapor permeability and oxygen transmission rate. The water vapor barrier is quantified by the Water Vapor Transmission Rate (WVTR), which indicates the amount of water vapor that permeates a unit area of packaging film and in a time and it is expressed in cm 3 /m 2 .s or g/m 2 .day. The water vapor barrier properties for food packaged products are related to its equilibrium moisture content and are of great importance for maintaining or extending their shelf life.

It is important to avoid dehydration in fresh food, while for bakery or delicatessen it is important to avoid water absorption.

The Oxygen Transmission Rate (OTR) indicates the amount of oxygen that permeates a packaging film per unit of area and time, expressed in cm 3 /m 2 .day.

The oxygen barrier plays an important role in the oxidation of packed food (fruits, salad, ready-to-eat meals, etc.). Polymer films with low oxygen permeability rate delay the oxidation and extend the food shelf life.

Mechanical properties

The feasibility of commercial utilization of packaging films also depends on the mechanical properties. Tensile properties are composed of the reaction of the materials to resist when tensile forces are applied. Tensile properties are determined by tensile testing and provide information about the modulus of elasticity (EM), also called Young's modulus, that measure the tensile stiffness of solid material and is determined by the pulling force/area to film stretching degree ratio (Equation 3); tensile strength (TS) -maximum pulling force per film crosssectional area that a material can support without fracture when being stretched (Equation 4); and elongation at break (EAB) -stretching ability before breaking (Equation 5) [START_REF] Rahman | Tensile properties of natural and synthetic fiber-reinforced polymer composites[END_REF]. These properties are used to predict the performance of packaging films upon handling and storage. Eq. 5

Where F is the maximum force supported, L0 is the original length, L is the axial deformation at rupture and A0 is the initial area of cross-section of the film.

Optical properties

The optical properties, such as color and transparency, are essential for packaging materials. These parameters are important and can provide visual evidence of the food content, which directly influences the consumer purchasing decision [START_REF] Shiku | Effect of pH on the preparation of edible films based on fish myofibrillar proteins[END_REF]. If films are colorless, then they are generally accepted by consumers. Besides, packaging with a high light barrier (low UV light penetration) has received special attention due to the protective effect against the oxidative rancidity catalyzed by UV light, especially for oily food [START_REF] Kwon | A Short Review of Light Barrier Materials for Food and Beverage Packaging[END_REF].

Heat sealing properties

Heat sealing is a process of welding thermoplastic polymer surfaces together in order to produce seals or joints of sufficient strength to withstand stresses in the distribution and consumer environment. A seal can be nonpeelable (tearable) or peelable (so-called easy open) [START_REF] Smith | Food packaging -Principles and practice[END_REF]. The heat sealability of a packaging film is essential for maintaining the desired gas composition within the pack, avoid contamination, and have a direct impact on the food shelf life.

Additionally, the seal must be strong enough to withstand the rigors of shipping and handling. Heat sealable films are those films that can be bonded together by the normal application of heat, such as by conductance from a heavy heatresistant metal bar. A range of polymers can be heat sealed, such as PE, PP, PS, PLA, PVC, etc. [START_REF] Smith | Food packaging -Principles and practice[END_REF].

Bio-based and biodegradable materials: definitions

Bio-based and biodegradable materials have gained attention on the food market over the last decades as alternatives for the classical petrochemicalbased plastic packaging materials, especially for short-time and disposable packaging applications [START_REF] Han | Food Packaging: A Comprehensive Review and Future Trends[END_REF]. This is a consequence of the environmental concerns, as well as the advances in material science and processing technologies.

According to a statement of the European Bioplastics Organization (EUBP) [START_REF]European bioplastics[END_REF] Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] The International Union of Pure and Applied Chemistry (IUPAC) states that biodegradable polymers refer to "macromolecules or polymeric substances susceptible to degradation by biological activity by lowering the molar mass of macromolecules that form the substance" [START_REF] Vert | Terminology for biorelated polymers and applications (IUPAC Recommendations[END_REF]. In other words, they are materials that undergo a biochemical process, in which microorganisms (yeast, bacteria, and fungi) metabolize the material into water, carbon dioxide, methane, and others [START_REF] Kale | Compostability of Bioplastic Packaging Materials: An Overview[END_REF]. This phenomenon can occur both under aerobic and anaerobic conditions and several factors such as moisture, pH, and oxygen availability, determine the rate and the final products of the biodegradation process [START_REF] Licciardello | Packaging and food sustainability[END_REF].

Biodegradation leads to fragmentation or disintegration of the polymers with no toxic or environmentally harmful residues. The material biodegradability depends on its chemical structure and molecular weight, and not on its feedstock type [START_REF] Vroman | Biodegradable Polymers[END_REF]. This is the case of poly(ε-caprolactone) (PCL) and poly(butylene adipate terephthalate) (PBAT). These polymers are biodegradable and derived from petroleum. It should be emphasized that biodegradability is different from compostability. The term compostability refers to the aerobic process, including the level of biodegradation, timeframe, surrounding conditions, and the safety of final products (ISO 17088, EN 13432, EN 14995, or ASTM D6868) [START_REF] Licciardello | Packaging and food sustainability[END_REF].

To illustrate the distinctions between the various types of plastics (bioplastic and conventional plastics), the EUBP has provided a simple two-axis model that encompasses the plastic types, classified according to the raw material source (bio-based or petrochemicals-based) and functionality (biodegradable or non-biodegradable) (Figure 1.7).

Figure 1.7 -Overview of all plastics types, classified according to raw material source and functionality (adapted from EUBP [START_REF]European bioplastics[END_REF]).

The global bioplastic production is expected to increase from 2.11 million tons in 2019 to approximately 2.43 million tons in 2024 [START_REF]European bioplastics[END_REF]. Compared to the annual production capacity of 359 million tons of conventional plastics [START_REF] Gardside | Global plastic production from 1950 to 2018[END_REF], the bioplastics represent a very small share of the market. Nevertheless, the demand for sophisticated bioplastics, applications, and products is increasing, the bioplastics market is, therefore, expected to grow continuously.

Currently, 25% of the global bioplastic production capacity is located in Europe, but Asia continues to be the major production hub with 45% of the total bioplastics production. According to EUBP [START_REF]European bioplastics[END_REF], biodegradable plastics as a whole, including PLA, PHA, starch blends, and others, account for over 55.5%

(over one million tons) of the global bioplastic production capacity. The production of biodegradable plastics is expected to increase to 1.33 million by 2024.

It is known that not all applications should be made from bioplastic, but several key products and applications can amplify the benefits and contributions of biodegradable plastics to prevent and reduce waste. Bioplastics are used in an

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] increasing number of markets and the portfolio of applications continues to diversify. Segments, such as automotive, transport, and building and construction, significantly increased their share (Figure 1.8). However, packaging remains as the largest field of application for bioplastics with more than 53% (1.14 million tonnes) of the total bioplastics market in 2019, and almost half of them were made from biodegradable bioplastics [START_REF]European bioplastics[END_REF]. Biodegradable plastics can be used as flexible or rigid packaging in the food industry. Flexible packaging such as films is applied in a variety of foods, and are particularly suitable for fresh products, such as fruit and vegetables [START_REF] Falguera | Edible films and coatings: Structures, active functions and trends in their use[END_REF].

For instance, searching on the ACS SciFinder tool concerning the last 20 years, with the following keywords: biodegradable and food packaging, 2335 articles, and 1328 patents were found (done September in 2020) proving the dynamic research in the field (Figure 1.9).

Figure 1.9 -Publications extracted from ACS SciFinder in September 2020 with keywords: biodegradable and food packaging.

Bio-based plastics: competition with food production

The land used to grow the renewable feedstock for bioplastics production, as reported by the EUBP [START_REF]European bioplastics[END_REF], corresponds to approximately 0.79 million hectares in 2019, which represents 0.02% of the global agricultural area (4.8 billion hectares). Nowadays the area for biofuel production is equivalent to 1% of the global agricultural area. Despite the expected market growth for the next five years, the share of land use for bioplastics will remain around 0.02% (1.00 million hectares by 2024). This clearly shows that there is no competition between renewable raw materials for food, feed, and the production of bioplastics.

Besides, bioplastic can use the marginal land (areas not suitable for food production), and, mainly, residual and co-products from agriculture and forestry.

Liliane Leite -2020

Natural polymers

In The two next sub-chapters are dedicated to gelatin and cellulose nanocrystals that are both naturally occurring bio-based materials of importance for this Ph.D.

work. A more detailed review of their physicochemical properties and food packaging applications is thus presented.

Protein

Proteins are renewable natural macromolecules synthesized by animals (casein, collagen, silk fibroin, etc.) plants (soy protein, zein, wheat proteins, etc.), and bacteria (lactate dehydrogenase, chymotrypsin, fumarase, etc.). They are composed of up to twenty different amino acid residues, which can be characterized as polar, non-polar, aromatic, anionic, and cationic (Figure1.11)

Liliane Leite -2020 [START_REF] Silva | Protein-based materials: from sources to innovative sustainable materials for biomedical applications[END_REF]. Due to the amphoteric nature of amino acids, protein can act as acids (the carboxyl group is able to lose a proton) or alkali (the amine group is able to accept a proton), or both at the same time, leading to the well-known zwitterionic form of amino acids [START_REF] Silva | Protein-based materials: from sources to innovative sustainable materials for biomedical applications[END_REF]. The diversity of amino acid composition and sequence leads to the different structures, conformations, and functionality of proteins. The structure of proteins is organized in four levels and are distinguished from one another by the degree of complexity in the polypeptide chain conformation [START_REF] Silva | Protein-based materials: from sources to innovative sustainable materials for biomedical applications[END_REF], as shown in The primary structure is related to the linear amino acid sequence in the protein chain. This sequence is formed by linkages between the -carboxyl group

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] of one amino acid and the  -amino group of another amino acid by a peptide bond (also called an amide bond). The order of amino acids in a polypeptide chain is unique and specific to a particular protein. The secondary structure of proteins refers to the spatial conformation of the primary structure. The two most important secondary structures of proteins are α-helices and a β-sheets. The α-helix structure is formed by hydrogen bonding between the amino groups and the carbonyl oxygen of the fourth amino acid on the amino terminal side of the peptide bond. The β-sheets structure appears to be folded or pleated, and it is held together by hydrogen bonding between polypeptide units of the folded chain that lie adjacent to one another [START_REF] Silva | Protein-based materials: from sources to innovative sustainable materials for biomedical applications[END_REF].

The tertiary structure refers to the three-dimensional structure of the polypeptide chain of a protein, which is generally stabilized by nonlocal interactions, salt bridges, hydrogen bonding, and disulfide bonds between thiol groups (-SH) from cysteine units. These tertiary structures can further associate with non-covalent bonding into more complex assemblies called quaternary structures (specific association of multiple polypeptide chains).

Proteins are currently being investigated to prepare edible and non-edible films due to their good functional properties as a food packaging material and biodegradability [START_REF] Cuq | Proteins as Agricultural Polymers for Packaging Production[END_REF]. Proteins confer a wide range of functional properties, especially a high potential to form intermolecular bonds in different positions, due to their unique structure that can be modulated by pH [START_REF] Zhang | Proteins as Sources of Materials[END_REF]. Protein-based films display excellent barrier properties against carbon dioxide and oxygen, as well as moderate mechanical properties when compared with polysaccharide films [START_REF] Calva-Estrada | Protein-Based Films: Advances in the Development of Biomaterials Applicable to Food Packaging[END_REF].

Gelatin

Gelatin is a water-soluble protein derived from collagen [START_REF] Cuq | Proteins as Agricultural Polymers for Packaging Production[END_REF]. Collagen is one of the most abundant animal proteins [START_REF] Shankar | Gelatin-based nanocomposite films[END_REF]. This protein is present in the skin, tendons, bone, cartilage, and most of the internal organs of animals, providing structural and mechanical support to the tissues. This fibrous protein is formed by individual chains held in a triple helix suprastructure by non-covalent interactions. Collagen differs from gelatin due to the far more tertiary structures, leading to a lower aqueous solubility [START_REF] Gómez-Guillén | Fish gelatin: a renewable material for developing active biodegradable films[END_REF].

Converting insoluble native collagen into gelatin requires pre-treatments involving acidic or alkaline conditions, to breakdown the non-covalent bonds and disorganize the protein structure, allowing swelling and cleavage of intra and intermolecular bonds to solubilize the collagen. Subsequent heat treatment up to 45 °C cleaves the hydrogen and covalent bonds to destabilize the triple helix, resulting in helix-to-coil transition and conversion into soluble gelatin [START_REF] Harrington | The Structure Of Collagen And Gelatin[END_REF].

Gelatin can form homogeneous gels for concentrations in the range of about 1-50%. When a gelatin solution is cooled below the sol-gel transition temperature, the coil molecules form triple-chain helices through the renaturation of collagen-like spirals, and the solution transforms into a three-dimensional gel network (Figure 1.12). Since the transition is physical such as hydrogen and Van der Waals bonds, the gelatin process is thermoreversible [START_REF] Nunes | Preparação E Caracterização De Bioblendas Poliméricas a Partir De Gelatina[END_REF].

Figure 1.12 -Conversion of insoluble native collagen into soluble gelatin (adapted from [START_REF] Campiglio | Cross-linking strategies for electrospun gelatin scaffolds[END_REF]).

Gelatin is a combination of hydrolysate macromolecule chains with different molecular weights including -chains (80-125 kDa), -chains (160-250 kDa), and other low molecular substances [START_REF] Huang | Fish gelatin modifications: A comprehensive review[END_REF]. Gelatin chains are made up of 18 specific amino acids. Table 1.2 shows the amino acid composition of different sources of gelatin. Glycine (Gly), proline (Pro), and hydroxyproline (Hyp) are the most dominant amino acids and represent almost two-thirds of the total amino acid constituents [START_REF] Huang | Fish gelatin modifications: A comprehensive review[END_REF].

Gelatin-based films for food packaging

Gelatin has attracted much attention as an alternative plastic material due to its abundance, low cost, biodegradability, excellent gelling properties, ability to form films and non-toxicity that poses no hazard for food contact applications.

However, gelatin films are mechanically weak and brittle [START_REF] Ramos | Gelatin-based films and coatings for food packaging applications[END_REF]. To overcome this problem, plasticizers are added to gelatin films. Plasticizers are low volatility molecules that are added to polymers to increase their extensibility, flexibility, and toughness, lowering the glass transition temperature and reducing the polymerpolymer interactions and molecular cohesion [START_REF] Vieira | Natural-based plasticizers and biopolymer films: A review[END_REF]. Polyols such as glycerol and sorbitol are commonly applied plasticizers for gelatin-based films. These plasticizers are added to the film-forming solution at approximately 10-60% on a dry basis [START_REF] Suderman | The effect of plasticizers on the functional properties of biodegradable gelatin-based film: A review[END_REF]. Nevertheless, these plasticizers have hydrophilic groups (-OH) that are known to increase water vapor permeability and decrease the film strength, and this effect is directly related to plasticizer concentration [START_REF] Suderman | The effect of plasticizers on the functional properties of biodegradable gelatin-based film: A review[END_REF].

Gelatin-based films can be made from a variety of sources with the most commonly used forms in food packaging derived from porcine [START_REF]Fabrication of cationized gelatin nanofibers by electrospinning for tissue regeneration[END_REF], fish [START_REF] Hanani | Effect of pomegranate (Punica granatum L.) peel powder on the antioxidant and antimicrobial properties of fish gelatin films as active packaging[END_REF], and bovine [START_REF] Quero | Stress transfer and matrix-cohesive fracture mechanism in microfibrillated cellulose-gelatin nanocomposite films[END_REF] mechanical properties and water vapor barrier at high relative humidity [START_REF] Suderman | The effect of plasticizers on the functional properties of biodegradable gelatin-based film: A review[END_REF].

Therefore, new approaches are focused on enhancing the functional properties of gelatin films for augmenting their function as food packaging materials.

The mechanical and water resistance of gelatin films can be enhanced by producing blends or biocomposites with other polysaccharides, proteins, and synthetic plastic. Various types of biopolymers have been mixed with gelatin including soy protein isolate [START_REF] Cao | Preparation and physical properties of soy protein isolate and gelatin composite films[END_REF], starch [START_REF] Fakhouri | Edible films and coatings based on starch/gelatin: Film properties and effect of coatings on quality of refrigerated Red Crimson grapes[END_REF], pectin [START_REF] Muhoza | Gelatin and high methyl pectin coacervates crosslinked with tannic acid: The characterization, rheological properties, and application for peppermint oil microencapsulation[END_REF], chitosan [START_REF] Hosseini | Preparation and functional properties of fish gelatinchitosan blend edible films[END_REF], and carboxymethyl cellulose (CMC) [START_REF] Esteghlal | Physical and mechanical properties of gelatin-CMC composite films under the influence of electrostatic interactions[END_REF].

Crosslinking reactions can also improve the physical properties of gelatin films. The crosslinking of proteins can be induced by heating, irradiation (plasma, -irradiation, high energy electron beam, etc.), enzymatic (transglutaminase), and chemical crosslinkers [START_REF] Calva-Estrada | Protein-Based Films: Advances in the Development of Biomaterials Applicable to Food Packaging[END_REF]. The latter is the most used and involves the formation of covalent bonds between the gelatin chains and the crosslinking agent.

Crosslinking agents, in general, increase the tensile strength and water resistance, while decreasing the elongation of the films. Several crosslinking agents including, carbodiimide [START_REF] Kuijpers | Cross-linking and characterisation of gelatin matrices for biomedical applications[END_REF], tannic acid [START_REF] Zhang | Chemical modification of gelatin by a natural phenolic cross-linker, tannic acid[END_REF], acid citric [START_REF] Santos | Crosslinking agents effect on gelatins from carp and tilapia skins and in their biopolymeric films[END_REF], genipin [START_REF] Iahnke | Gelatin capsule residue-based films crosslinked with the natural agent genipin[END_REF] were recently used to crosslink gelatin-based films, as they are significantly less cytotoxic than formaldehyde and glutaraldehyde [START_REF] Sung | Evaluation of gelatin hydrogel crosslinked with various crosslinking agents as bioadhesives:In vitro study[END_REF].

Recently, nanotechnology has been used not only to enhance the physical properties of gelatin-based films but also to provide extra functional properties, such as antioxidant and antimicrobial functions. Polymer nanocomposite is a multiphase solid material in which at least one type of the phases domain is a continuous phase and in which at least one of the phases has at least one dimension smaller than 100 nm [START_REF] Oksman | Manufacturing process of cellulose whiskers/polylactic acid nanocomposites[END_REF]. The development of bionanocomposite films with antimicrobial properties especially broadens the application range of gelatin in food packaging. Several metallic or inorganic nanoparticles such as silver [START_REF] Menezes | Effect of tannic acid as crosslinking agent on fish skin gelatin-silver nanocomposite film[END_REF], tin dioxide [START_REF] Voon | Effect of Addition of Halloysite Nanoclay and SiO2 Nanoparticles on Barrier and Mechanical Properties of Bovine Gelatin Films[END_REF], zin oxide [START_REF] Marvizadeh | Preparation and characterization of bionanocomposite film based on tapioca starch/bovine gelatin/nanorod zinc oxide[END_REF], and clay [START_REF] Jang | Original article: Use of nano-clay (Cloisite Na+) improves tensile strength and vapour permeability in agar rich red algae (Gelidium corneum)-gelatin composite films[END_REF] have been used to improve the functional properties of gelatin. Additionally, other organic nanoparticles, such as starch [START_REF] Tao | Preparation and physicochemistry properties of smart edible films based on gelatin-starch nanoparticles[END_REF], chitin [START_REF] Sahraee | Effect of corn oil on physical, thermal, and antifungal properties of gelatin-based nanocomposite films containing nano chitin[END_REF], and cellulose nanocrystals [START_REF] George | High performance edible nanocomposite films containing bacterial cellulose nanocrystals[END_REF] have been incorporated with the gelatin matrix to prepare fully biodegradable films.

Cellulose

Cellulose is the most abundant polymer in nature, with approximately 250 megatons produced annually through photosynthesis by green plants from carbon dioxide, water, and sunlight [START_REF] Urruzola | Nanopaper from almond (Prunus dulcis) shell[END_REF]. Cellulose is widely biosynthesized by several living organisms ranging from higher to lower plants, several marine animals (for example, tunicates), algae, fungi, bacteria, invertebrates, and even amoeba (protozoa) [START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF]. Cellulose is a renewable, biodegradable, biocompatible, and non-toxic material [START_REF] Thomas | a versatile green platform: from biosources to materials and their applications[END_REF].

Cellulose was discovered in 1838 by the French chemist Anselme Payen, who isolated it from a plant matter and determined its chemical formula [START_REF] Payen | Comptes rendus de l academie des sciences serie lii -science de la vie[END_REF], and since then, multiple physical and chemical aspects of cellulose have been Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] One of the most distinguishing and useful characteristics of cellulose is the high degree of hydroxylation along the polymer chain. Each AGU has three hydroxyls groups, which form strong intra and inter hydrogen bonds in the same and neighboring cellulose chains. Intra-and inter-chain hydrogen bonding play a major role in directing the crystalline packing and also governing the physical properties of cellulose, namely, its high mechanical strength and flexibility [START_REF] Thomas | a versatile green platform: from biosources to materials and their applications[END_REF].

In nature, cellulose does not occur as an isolated individual molecule. The Van der Waals and intermolecular hydrogen bonds promote parallel stacking of multiple cellulose chains forming elementary fibrils that further aggregate into large microfibrils (5-50 nm in diameter and several microns in length) [START_REF] Moon | Cellulose nanomaterials review: structure, properties and nanocomposites[END_REF]. The fibrils contain regions where the cellulose chains are arranged in a hilly ordered structure (crystalline), and regions that they are disordered (amorphous). The organization of the amorphous and crystalline domains in the cellulose fibers is dependent on the cellulose source. According to Mariano et al. ( 2014) [START_REF] Mariano | Cellulose nanocrystals and related nanocomposites: Review of some properties and challenges[END_REF], for some plant species, the amorphous regions represent up to 50% of the structure, while in bacterial cellulose and celluloses extracted from some algae the crystalline domains correspond to almost 100% of the fibril.

Cellulose nanocrystals

Cellulose nanocrystals (CNCs), also referred to as cellulose whiskers, The discovery of CNCs is attributed to Rånby et al. in 1950 [126], who reported for the first time that colloidal cellulose particles with crystalline character can be obtained by controlled sulfuric acid degradation of cellulosic fibers. A typical process for the production of CNCs from fibers begins with pre-treatments (bleaching, alkaline treatment, etc.) to remove non-cellulosic components (hemicellulose, lignin, waxes, etc.), followed by isolation of CNCs, washing, centrifugation, dialysis, and ultrasonication to form a suspension which may be further subjected to lyophilization (freeze-drying) or spray-drying [START_REF] Thomas | a versatile green platform: from biosources to materials and their applications[END_REF]. The methods used to release CNCs from the cellulose source include mechanical, chemical, and enzymatic processes or a combination of them. However, the main process for the isolation of CNCs is the acid hydrolysis [START_REF] Taipina | Morphological evolution of curauá fibers under acid hydrolysis[END_REF][START_REF] Taipina | Surface modification of cotton nanocrystals with a silane agent[END_REF][START_REF] Yang | The transparency and mechanical properties of cellulose acetate nanocomposites using cellulose nanowhiskers as fillersCellulose[END_REF]. According to De Souza Lima et al. ( 2004) [START_REF] Souza Lima | Rodlike Cellulose Microcrystals: Structure, Properties, and Applications[END_REF], the acid hydrolysis mechanism involves the diffusion of hydronium ions into the amorphous domains of native cellulose,

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] promoting the hydrolytic cleavage of glycosidic bonds and releasing highly order cellulose in the form of rod-shaped particles. The CNCs dimensions depend on the cellulose source and hydrolysis conditions, ranging from 100 to 500 nm in length (L) and from 5 to 10 nm in width (d), resulting in high aspect ratios (L/d) [START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF][START_REF] Dufresne | Nanocellulose: a new ageless bionanomaterial[END_REF]. Figure 1.16 shows transmission electron microscopy images of CNCs obtained by acid hydrolysis from different sources. Dufresne (2018) [START_REF] Dufresne | Cellulose nanomaterials as green nanoreinforcements for polymer nanocomposites[END_REF].reported that the stability of CNCs suspensions depends on the dimensions of their dispersed particles, size polydispersity, and surface charge. Strong mineral acids like sulfuric, hydrochloric [START_REF] Araki | Flow properties of microcrystalline cellulose suspension prepared by acid treatment of native cellulose[END_REF], but also phosphoric [START_REF] Espinosa | Isolation of thermally stable cellulose nanocrystals by phosphoric acid hydrolysis[END_REF] and hydrobromic [START_REF] Sadeghifar | Production of cellulose nanocrystals using hydrobromic acid and click reactions on their surface[END_REF] have been applied for obtaining CNCs.

However, sulfuric acid (~64 wt%) is classically used because it promotes grafting of anionic sulfate groups (OSO3 -) onto the CNCs nanoparticles, resulting in stable aqueous dispersions [START_REF] Reid | Benchmarking cellulose nanocrystals: from the laboratory to industrial production[END_REF]. CNCs encompass a suitable combination of properties such as high aspect ratio (~10-70), low density (1.5-1.6 g/cm 3 ), low thermal expansion coefficient, optical transparency and anisotropy, negative diamagnetic anisotropy, and shear-thinning rheology [START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF][START_REF] Huber | A critical review of all-cellulose composites[END_REF][START_REF] Lagerwall | Cellulose nanocrystal-based materials: from liquid crystal self-assembly and glass formation to multifunctional thin films[END_REF]. In addition, the welldefined CNCs dimensions, their high crystallinity (70-90%), and their bond packing organization induce excellent mechanical properties such as high axial stiffness (~150 GPa), high Young's modulus (20-50 GPa) [START_REF] Pandey | Fabrication and applications of cellulose nanoparticle-based polymer composites[END_REF], and high tensile strength (~7 GPa) [START_REF] Huber | A critical review of all-cellulose composites[END_REF]. making they are excellent candidates for the design and development of high-performance nanomaterials in several applications. Today, CNC-based materials are used in a wide range of applications such as security paper [START_REF] Zhang | Nanocrystalline cellulose for covert optical encryption[END_REF], energy & electronics [START_REF] Hoeng | Use of nanocellulose in printed electronics: A review[END_REF], environment [START_REF] Carpenter | Cellulose nanomaterials in water treatment technologies[END_REF],

biomedical [START_REF] Zhou | Electrospun bio-nanocomposite scaffolds for bone tissue engineering by cellulose nanocrystals reinforcing maleic anhydride grafted PLA[END_REF], and composites and nanocomposites [START_REF] Rafieian | thermal and barrier properties of nanobiocomposite based on gluten and carboxylated cellulose nanocrystals[END_REF]. The use of CNCs in nanocomposites has been detailed in several papers that focused on demonstrating the reinforcing potential of high aspect ratio CNCs for different polymer matrixes [START_REF] Dufresne | Cellulose nanomaterials as green nanoreinforcements for polymer nanocomposites[END_REF].

Cellulose nanocrystals in food packaging

CNCs can provide many benefits for packaging films in terms of performance, including biodegradability, mechanical properties, and gas, and vapor barrier. According to Criado et al. ( 2019) [START_REF] Criado | Cellulose Nanocrystals in Food Packaging[END_REF], the improvement of polymer film mechanical properties due to CNCs addition is attributed to the good dispersion of the CNCs in the polymer matrix, effective filler-matrix interaction, and the creation of a CNC percolation network. This behavior, explained by the

Halpin & Kardos percolation model (1972) [START_REF] Halpin | Moduli of crystalline polymers employing composite theory[END_REF], led to the conclusion that infinite networks of nanoparticles stabilized by hydrogen bonding were responsible for improving the mechanical properties of the polymer matrix. The percolation threshold for rod-like nanoparticles can be linked to the CNCs aspect ratio following Equation 6 [START_REF] Samir | Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field[END_REF]. The effect of adding CNCs has been explored in different types of biodegradable polymers, such as poly(butylene adipate terephthalate) (PBAT) [START_REF] Ferreira | Cellulose nanocrystal-based poly(butylene adipateco-terephthalate) nanocomposites covered with antimicrobial silver thin films[END_REF], PCL [START_REF] Goffin | Poly(ε-caprolactone) based nanocomposites reinforced by surface-grafted cellulose nanowhiskers via extrusion processing: Morphology, rheology, and thermo-mechanical properties[END_REF], alginate [START_REF] Lin | TEMPO-Oxidized Nanocellulose Participating as Crosslinking Aid for Alginate-Based Sponges[END_REF], polyhydroxybutyrate (PHB) [START_REF] Wei | Grafting of Bacterial Polyhydroxybutyrate (PHB) onto Cellulose via In Situ Reactive Extrusion with Dicumyl Peroxide[END_REF], gum arabic, chitosan [START_REF] Vuillemin | Gum Arabic and chitosan self-assembly: Thermodynamic and mechanism aspects[END_REF] and whey protein [START_REF] Sukyai | Effect of cellulose nanocrystals from sugarcane bagasse on whey protein isolate-based films[END_REF].

? = @.A 3/C
Eq. 6

Parameters such as cohesive energy density, free volume, and crystallinity play a role in the oxygen barrier properties of polymers [START_REF] Criado | Cellulose Nanocrystals in Food Packaging[END_REF]. As reported by [START_REF] Kofinas | Gas permeability of polyethylene/poly(ethylene-propylene) semicrystalline diblock copolymers[END_REF] [START_REF] Kofinas | Gas permeability of polyethylene/poly(ethylene-propylene) semicrystalline diblock copolymers[END_REF], gas molecules are unable to permeate the crystalline regions of polymers. Thus, gas permeation in semicrystalline polymers is confined in the amorphous regions [START_REF] Siracusa | Food packaging permeability behaviour: A report[END_REF]. Garcia et al. ( 2004) [START_REF] García | Hybrid organic inorganic nylon-6/SiO2 nanocomposites: Transport properties[END_REF] described that the presence of crystalline nanoparticles, such as CNCs, can result in a more compact material and create a large tortuous path inside the matrix, which interrupts the gas path or a gas "jump" response in diffusion. Therefore, a decreased gas permeation is expected after the CNCs are appropriately dispersed in the polymer matrix [START_REF] Dufresne | From nature to high performance tailored materials[END_REF]. This effect has been described in detail in several papers [START_REF] Luzi | Production and characterization of PLA_PBS biodegradable blends reinforced with cellulose nanocrystals extracted from hemp fibres[END_REF][START_REF] Li | Tunable green oxygen barrier through layer-by-layer selfassembly of chitosan and cellulose nanocrystals[END_REF][START_REF] Herrera | Gas permeability and selectivity of cellulose nanocrystals films (layers) deposited by spin coating[END_REF].

A decrease in water vapor permeability in CNC-based nanocomposites has also been observed. Although the fact that CNCs are hydrophilic due to the OH groups, CNCs have shown to enhance the moisture barrier properties and decrease the WVP of several biopolymers [START_REF] Silva | Mango kernel starch films as affected by starch nanocrystals and cellulose nanocrystals[END_REF][START_REF] Pereira | Wheat straw hemicelluloses added with cellulose nanocrystals and citric acid. Effect on film physical properties[END_REF][START_REF] Kumar | Effect of nano-cellulosic fiber on mechanical and barrier properties of polylactic acid (PLA) green nanocomposite film[END_REF]. This behavior is attributed to the formation of hydrogen networks that allows better cohesiveness in the nanocomposite structure. However, it is important to highlight that the addition of excessive amounts of CNCs results in aggregated and accelerate preferential paths for water diffusion, thus reducing the material barrier performance [START_REF] Paralikar | Poly(vinyl alcohol)/cellulose nanocrystal barrier membranes[END_REF].

Table 1.4 shows the effect of CNCs on the physical properties of gelatin films (done September in 2020). Regarding the objective of this Ph.D. to produce gelatin packaging with CNCs, no more than 6 papers can be cited on this date. Of this total, only one paper studied the effect of functionalized CNCs on the properties of gelatin film, being an area with many possibilities for innovation.

Functionalization of cellulose nanocrystals

The surface of CNCs can be easily modified due to the reactivity of the hydroxyl group, which makes CNCs a unique platform amenable to various surface functionalization, extending the specific applications of CNCs, particularly to alleviate difficulties in dispersing them in apolar solvent or polymers [START_REF] Kargarzadeh | Advances in cellulose nanomaterials[END_REF].

Though, it is important to keep the crystalline structure intact and avoid the degradation of CNCs during the functionalization, since their mechanical properties are important for most applications.

The functionalization of CNCs can generally be categorized into 2 distinct groups (i) non-covalent and (ii) covalent surface functionalization or direct chemical modification. Non-covalent functionalization is typically achieved via adsorption of molecules or macromolecules such as surfactant to the CNCs surface. These additives interact with the CNCs surface by electrostatic attractions, Van der Waals forces, or hydrogen bonding, thus improving the CNCs stability and dispersion [START_REF] Habibi | Key advances in the chemical modification of nanocelluloses[END_REF]. Dispersants as stearic acid and cetyltrimethylammonium-bromide (CTAB) are the most common molecules applied for non-covalent functionalization of CNCs [START_REF] Taipina | Nanocristais de celulose: obtenção, caracterização e modificação de superfície[END_REF]. In the covalent functionalization, also referred to as grafting, many molecules or polymers are covalently attached to the CNCs surface. This has been achieved by reacting the hydroxyl groups using various methods such as silylation [START_REF] Gousse | Stable suspensions of partially silylated cellulose whiskers dispersed in organic solvents[END_REF][START_REF] Siqueira | Processing of cellulose nanowhiskers/cellulose acetate butyrate nanocomposites using sol-gel process to facilitate dispersion[END_REF], coupling with isocyanate [START_REF] Siqueira | New Process of Chemical Grafting of Cellulose Nanoparticles with a Long Chain Isocyanate[END_REF], macromolecular grafting [START_REF] Jean | Structural details of cellulose nanocrystals/polyelectrolytes multilayers probed by neutron reflectivity and AFM[END_REF][START_REF] Sobkowicz | Decorating in green: surface esterification of carbon and cellulosic nanoparticles[END_REF], acetylation [START_REF] Sassi | Ultrastructural aspects of the acetylation of cellulose[END_REF],

acetylation with acetic anhydride [START_REF] Missoum | Nanofibrillated cellulose surface grafting in ionic liquid[END_REF], esterification [START_REF] Sobkowicz | Decorating in green: surface esterification of carbon and cellulosic nanoparticles[END_REF], covalent grafting by polymer ring-opening polymerization [START_REF] Hatton | Xyloglucan-Functional Latex Particles via RAFT-Mediated Emulsion Polymerization for the Biomimetic Modification of Cellulose[END_REF], etc. Alternatively, many groups seek to change the chemistry of the hydroxyl groups by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidation that converts alcohol groups to carboxylic acid moieties for better CNCs dispersibility [START_REF] Kedzior | Recent advances and an industrial perspective of cellulose nanocrystal functionalization through polymer grafting[END_REF]. The hydrolysis method using sulfuric acid can also be used to form sulfate esters, as mentioned before [START_REF] Taipina | Nanocristais de celulose: obtenção, caracterização e modificação de superfície[END_REF]. Figure

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] 1.17 gives a wide overview of the common reactions used to modify CNCs surface.

Figure 1.17 -Common surface functionalization of CNCs: (clockwise from topright) sulfuric acid hydrolysis, carboxylic acid halides, acid anhydrides, epoxides, isocyanates, TEMPO oxidation, halogenates acetic acids, and chlorosilanes [START_REF] Moon | Cellulose nanomaterials review: structure, properties and nanocomposites[END_REF].

One recent strong trend is to focus on the functionalization of CNCs to develop novel active packaging with diverse advanced functionalities such as antimicrobial and antioxidant. Active packaging is an innovative concept that offers positive impacts on consumer health by integrating functional ingredients in the packaging material structure to actively enhance the shelf life of food products while ensuring their quality, safety, and integrity. The most common types of active packaging are those that release active compounds such as antimicrobial agents onto the food surface [START_REF] Yildirim | Active Packaging Applications for Food[END_REF].

CNCs themselves have no antibacterial or antimicrobial activity [START_REF] Criado | Cellulose Nanocrystals in Food Packaging[END_REF].

Nevertheless, the CNCs have allowed for adsorption or grafting of active compounds. Also, CNCs have been studied based on the controlling release of bioactive compounds generated by the CNCs in polymer matrices [START_REF] Bras | Nanocellulose in functional packaging[END_REF]. Different active packaging systems based on functionalized CNCs can be produced by different routes, as summarized in recent reviews [START_REF] Yildirim | Active Packaging Applications for Food[END_REF][START_REF] Sharma | Antimicrobial bionanocomposites and their potential applications in food packaging[END_REF][START_REF] Vilarinho | Nanocellulose in green food packaging[END_REF]. Of particular interest are treatments with food-grade compounds such as sorbic acid [START_REF] Dobre | Modelling of sorbic acid diffusion through bacterial cellulose-based antimicrobial films[END_REF] or bio-based cationic polymers like chitosan [START_REF] Khan | Mechanical and barrier properties of nanocrystalline cellulose reinforced chitosan based nanocomposite films[END_REF]. Also, there has been much

interest in applying nanomaterials such as colloidal silver particles in combination with nanocelluloses for antimicrobial active packaging. The silver nanoparticle/CNCs-based materials showed enhanced antibacterial properties against E. coli and Staphylococcus aureus due to the metal ion reducing and chelating capacity of the modified CNCs [START_REF] Li | Nanocellulose-Based Antibacterial Materials[END_REF].

Cellulose nanocrystals safety and toxicology assessment

Cellulose is considered as a non-toxic material. However, the particularities of CNCs, especially regarding the nanometric size and the high aspect ratio, are expected to increase their toxicity as compared to that of bulk cellulose. In-depth research has been carried out to assess the in vitro or in vivo toxicity of CNCs. According to Ventura et al. (2020) [START_REF] Ventura | On the toxicity of cellulose nanocrystals and nanofibrils in animal and cellular models[END_REF], in general, the CNCs uptake into cells is generally low, with no induction of oxidative stress and no significant cytotoxic and genotoxic effects. Several studies have reported that CNCs are not significantly toxic to human cells or the environment [START_REF] Roman | Toxicity of Cellulose Nanocrystals: A Review[END_REF]. The impact of chemical modification on the toxicity of several nanocellulose materials has also been investigated [START_REF] Harper | Impacts of chemical modification on the toxicity of diverse nanocellulose materials to developing zebrafish[END_REF]. Also, CNCs are currently exempted from registration in the REACH list (list of restrictions of certain hazardous substances, mixtures, and articles for their marketing and use in the European market).

Processing of natural polymer-based film packaging

The film-forming process plays an important role in the physical properties of polymer films. Two processes have been investigated to make packaging materials based on proteins: "dry process (extrusion)" based on the thermoplastic behavior of protein at low moisture levels, and the "wet (or solution casting)

process" based on the solubilization of the protein in a solvent medium [START_REF] Cuq | Proteins as Agricultural Polymers for Packaging Production[END_REF]. Films can be produced by extrusion followed by thermoforming that is ordinarily higher than 80 °C [START_REF] Gómez-Guillén | Fish gelatin: a renewable material for developing active biodegradable films[END_REF]. This process would enhance the commercial potential of protein films. However, the extrusion/thermoforming process affects film properties.

Krishna et al (2012) [START_REF] Krishna | Development of fish gelatin edible films using extrusion and compression molding[END_REF]9, compared the properties of fish gelatin films produced either by extrusion/heat-processing or by casting and found that extruded films higher WVP values than the corresponding cast films. They explained these differences in terms of the moisture evaporation rate, which was lower in the cast films and, as a result, favored the formation of a more ordered and compact film network.

Recently, Chuaynukul et al. ( 2018) [START_REF] Chuaynukul | Comparative Characterization of Bovine and Fish Gelatin Films Fabricated by Compression Molding and Solution Casting Methods[END_REF] compared the proprieties of gelatin films obtained by compression molding and solution casting. The authors found that the compression-molded gelatin films exhibited lower thermal stability and WVP than the cast films. This behavior was attributed to the higher thermal degradation of gelatin during the film thermoforming step that requires high temperatures.

The wet process is the most often use film-forming method for protein film production. In this Ph.D. work, the gelatin films were produced using this bench and continuous casting. Details on this method are described in the two next subchapters.

Bench casting

Bench casting is the wet process most widely used to produce gelatin films. Film production by bench casting consists of dissolving the polymer and blending it with plasticizers and/or additives to obtain a film-forming solution, which is poured on plates and then dried by driving off the solvent [START_REF] Salgado | Edible films and coatings containing bioactives[END_REF]. Film formation by solvent -water evaporation is due to increased polymer concentration in the medium, inducing the macromolecules to rearrange into a cohesive matrix stabilized by new interactions and bonds [START_REF] Koshy | Environment friendly green composites based on soy protein isolate -A review[END_REF]. The final film thickness is controlled by the amount of solution poured on plates. The air-drying procedure is a very important step on the physical properties of the films and it is generally carried out at room temperature or in an air circulating oven at temperatures not exceeding 30 to 40 °C and for 12 to 48 h. Indeed, the long drying times are the most remarkable disadvantage of this technique, making it not feasible for commercial production [START_REF] Moraes | Scale-up of the production of cassava starch based films using tape-casting[END_REF]. Information on parameters such as wet thickness, relative humidity, and air flow speed is critical [START_REF] Otoni | Recent Advances on Edible Films Based on Fruits and Vegetables-A Review[END_REF].

According to Suhag et al. (2020) [START_REF] Suhag | Film formation and deposition methods of edible coating on food products: A review[END_REF], the greatest challenge is to convert film production from laboratory to production scale because many variables, including heating, speed, and temperature, could cause quality differences and prevent the constant development for commercial scales.

Continuous solution casting

Recently, a continuous solution casting method has been proposed for the production of biopolymer films on a pilot scale. Continuous film casting is carried out on a coating line such as a tape-casting machine. In this process, solutions are continuously spread onto a moving substrate such as polyester or coated paper, with a blade whose height can be adjusted to control film thickness (Figure 1.18). The coated substrate then passes through a pre-drying infrared heater and drying chambers and at the end, the dry film is cooled to room temperature and wound on a roller while still adhered to the substrate [START_REF] Otoni | Recent Advances on Edible Films Based on Fruits and Vegetables-A Review[END_REF]. The distance between the two drying chambers is approximately 2 meters. The continuous solution casting approach may be considered a scale-up of the previously described bench casting procedure. The main advantage of continuous casting consists in the fact that this method requires short processing times, thanks to the use of infrared radiation in the pre-drying stage, much higher temperatures, and intense air circulation. Other advantages are the high wet thickness control, no processing additives required, allows the use of aqueous solutions/dispersion, and the less need for space and labor.

Edible films based on fruits have been produced by using continuous casting in a few minutes [START_REF] Du | Antibacterial activity against E. coli O157:H7, physical properties, and storage stability of novel carvacrol-containing edible tomato films[END_REF]. Du et al (2008) [START_REF] Du | Storage stability and antibacterial activity against Escherichia coli O157:H7 of carvacrol in edible apple films made by two different casting methods[END_REF], compared the properties of apple films with carvacrol produced either by continuous casting or bench casting.

The authors concluded that the higher temperatures used in the continuous casting increased carvacrol evaporation and made the films thinner and denser,

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] probably reducing interstitial spaces for molecular diffusion, which explain the lower oxygen permeability and WVP of continuously cast films. No significant difference in tensile properties when compared to cast films was observed.

Recently, Munhoz et al. ( 2018) [START_REF] Munhoz | Sustainable Production and in vitro Biodegradability of Edible Films from Yellow Passion Fruit Coproducts via Continuous Casting[END_REF] produced yellow passion fruit (passiflora

CONCLUSIONS

Proteins are a suitable alternative for food packaging applications, replacing conventional plastics, especially those made from petrochemicals. However, using only proteins is not enough for producing films with satisfactory mechanical and barrier properties for food packaging. The excellent mechanical and barrier properties of CNCs and the fact that they can be considered as green nanomaterials because they are renewable and biodegradable, open excellent prospects for their increased use in the design of novel high performance food packaging materials. The assessment of the safety and toxicology of CNCs has been monitored and, to date, studies have shown that they had no harmful effects on human health and the environment. Besides, the functionalization of CNCs can also create other desired functions in food packaging materials, such as antimicrobial and antioxidant properties, contributing to extending food products shelf-life Therefore, proteins and CNCs offer excellent opportunities for a new generation of bionanocomposite packaging materials. However, the processing of protein films still limits their widespread use in the food packaging industry.

In line with this context, it seems very innovative (i) to use the continuous casting as an appropriate approach to increase the production of gelatin-cellulose nanocrystalline bionanocomposites films and (ii) to functionalize the surface of CNCs with natural active molecules and to use such materials in combination with gelatin for the design of new active packaging. The strategies adopted in this Ph.D. work to achieve these objectives will be detailed in the following chapters.

II. Gelatin-based films produced by continuous casting

Introduction to chapter II

As described in Chapter I, the greatest challenge is to convert the film production of natural bio-based materials from laboratory to production. Besides, the quality of the dispersion of CNCs particles in the film-forming solution also plays an important role in the physical properties of the film produced by spreading. One of the main objectives of this Ph.D. is to develop a suitable approach to scale up the production of gelatin-cellulose nanocrystals (CNCs).

In the first part of this chapter (Chapter II.1) will investigate the parameters such as the rheological behavior of the film-forming solution and drying temperature required to produce gelatin/CNCs films by continuous casting. In addition, the physical properties of gelatin/CNCs obtained by continuous casting will be analyzed. The results show that continuous casting is a suitable technique to hasten the production of bionanocomposites based on gelatin/CNCs from aqueous formulations.

The second part (Chapter II.2) will focus on the effects of the dispersion of CNCs on the properties of the gelatin-based films. We particularly examined the dispersibility of the heterogeneous CNCs and homogeneous CNCs suspensions in gelatin film-forming solutions. Based on the work realized in Chapter II.1, continuously cast gelatin/CNCs films with better CNCs dispersion and their counterparts obtained by bench casting will be analyzed. The results show that continuous casting not only allowed scaling-up the production of these bionanocomposites films but also led to films with relatively superior mechanical, barrier, and thermal performance.

introduction

Gelatin can be used in food packaging and biomedical applications, including coatings, wound dressing, adhesives, adsorbent materials, and drug delivery as hard or soft capsules due to its film-forming properties, low cost, biodegradability, and biocompatibility [START_REF] Shit | Edible polymers: challenges and opportunities[END_REF]. Several strategies have been reported in the literature to enhance the physical properties of gelatin films for different applications. One of these strategies is the use of nano-sized structures as reinforcing fillers in gelatin films [START_REF] Bhowmik | Reinforcement of gelatin-based nanofilled polymer biocomposite by crystalline cellulose from cotton for advanced wound dressing applications[END_REF][START_REF] González | Nanocrystal-reinforced soy protein films and their application as active packaging[END_REF][START_REF] Castro | Whey protein as a key component in food Liliane Leite -2020 systems: Physicochemical properties, production technologies and applications[END_REF], which usually yields nanocomposites with superior properties, compared to the corresponding microcomposites without impairing density, transparency, and film processability.

Cellulose nanocrystals (CNCs) are one of the most studied polysaccharide-based nanomaterials in polymer nanocomposites [START_REF] Santos | Fish gelatin films as affected by cellulose whiskers and sonication[END_REF]203].

Reports have demonstrated that CNCs can be applied as reinforcing fillers for gelatin. George & Siddaramaiah (2012) [START_REF] George | High performance edible nanocomposite films containing bacterial cellulose nanocrystals[END_REF] observed that the addition of 2 wt% CNCs increased the modulus and tensile strength of bench-cast gelatin films from 2190 MPa to 2273 MPa, and 84 MPa to 95 MPa, respectively. Santos et al. ( 2014) [START_REF] Santos | Fish gelatin films as affected by cellulose whiskers and sonication[END_REF] found that gelatin nanocomposites produced by bench casting exhibited high stiffness and good ductility up to 5 wt% CNCs. After this point, the mechanical properties tended to level off or even to decrease, which was attributed to CNCs agglomeration effects. However, all these reports were based on gelatin/CNCs films obtained by bench casting, which is an important technique for small-scale studies, but fails in achieving high productivity because the drying times are relatively long (e.g. 12-24 h) [START_REF] Ortiz | Scale-up of the production of soy (Glycine max L.) protein films using tape casting: Formulation of film-forming suspension and drying conditions[END_REF]. In this sense, researches are still necessary to develop scaled-up routes for producing gelatin-CNCs bionanocomposites films. To the best of our knowledge, such a route has not been efficiently developed yet, neither the examination of the bionanocomposite film properties obtained in such a pilot-scale scenario.

In the present work a continuous solution casting approach was applied for scaling up the production of gelatin-CNCs bionanocomposite films. A pre-pilot labcoater machine was used to speed-up the casting drying step and to provide green and safety aspects to the films for food packaging applications. We particularly examined the effect of the CNCs concentration on the physical properties of the bionanocomposite films. The continuously cast films were also compared for their mechanical properties to their counterparts obtained by bench casting.

Materials and Methods

Materials

Bovine gelatin (Bloom Strength-180) and eucalyptus kraft pulp were kindly supplied by Gelco Gelatinas do Brazil Ltda (Pedreira, SP) and Suzano Papel e

Celulose Ltda (Limeira, SP), respectively. Sulfuric acid (98.08 wt%) was purchased from Sigma-Aldrich Ltd. (st. Louis, USA). Glycerol was purchased from Dinâmica Química (Indaiatuba, SP). All aqueous solutions were prepared using ultrapure water (18 MΩ.cm) obtained from a Milli-Q system (Millipore).

Preparation of CNCs

The CNCs were prepared by acid hydrolysis of 10 g of eucalyptus kraft pulp using 200 mL of 60% w/w sulfuric acid solution at 45 °C for 60 min with constant stirring. After hydrolysis, the suspension was diluted in 500 mL of cold water to quench the reaction and centrifuged at 10000 rpm for 10 min. The resultant pellet was rinsed, re-centrifuged, and dialyzed in water with a cellulose membrane (6-8 kDa) until neutral pH. Afterward, the suspension was sonicated for 5 min and stored in a refrigerator after adding chloroform drops to prevent microbial growth. The final solid content was 3.5% (w/w).

Preparation of gelatin/CNCs film-forming solutions (FFS)

FFS based on gelatin and CNCs were prepared according to the following steps: first, gelatin powder was hydrated in ultrapure water at concentrations of 10, 20, and 30 wt% (for continuous casting) and 10 wt% (for bench casting) at 24 °C for 5 min, and then heated at 60 °C under mechanical stirring for 15 min. Then, glycerol (20 wt% on a dry gelatin basis) was added to the gelatin solution, which was then homogenized with an Ultra Turrax T-25 (Ika, Staufen, Germany) at 60 12000 rpm for 10 min. The CNCs suspension was gradually added to the gelatin/glycerol solution over the first 2 min. Four film-forming solutions were prepared with the following CNCs concentrations (on a dry gelatin basis): 0 wt%, 0.5 wt%, 1.0 wt%, and 2.5 wt%. The solutions were allowed to rest for 60 min to eliminate bubbles before casting.

Continuous casting

Gelatin-based FFS were turned into monolayer films on a KTF-S labcoater casting machine (Werner Mathis AG, Switzerland), as schematically depicted in

Bench casting

Gelatin-CNCs FFS (60 mL) were cast on glass plates (40 cm × 25 cm) covered with a polyester film (Mylar®, DuPont, Brazil) to facilitate peeling off the films after drying at room temperature for 24 h. Film samples were conditioned in a desiccator at 50% RH and 25 °C for at least 48 h prior to testing.

Transmission electron microscopy (TEM)

Transmission electron microscopy was carried out on a Tecnai TM G2 F20 microscope (FEI Company, Netherlands) using the SEM (Transmission Electron Microscopy) mode. The images were acquired with a dark field (DF) detector. A droplet of diluted CNCs suspension was deposited on a carbon microgrid with formvar (400 mesh) and allowed to dry. The grid was stained with 1.5 wt% uranyl acetate solution and dried at room temperature before analysis. Average CNCs sizes were obtained from a minimum of 100 particles by using the ImageJ software.

Determination of Zeta potential (ζ-potentials)

ζ-potentials of aqueous CNCs and gelatin suspensions with concentration of 0.25 wt% measured at 25 °C using a Zetasizer Nano-ZS (Malvern Instruments Inc., UK). The measurements were performed in triplicate.

Rheological study

The apparent viscosity and flow behavior of the different FFS were evaluated on a Physica MCR101 rheometer (Anton Paar, Áustria) using concentric cylinder geometries at 25 and 30 °C. The shear rate was increased from 0 to 150 s -1 in 10 min. Oscillatory shear deformation tests were also carried out to determine the storage modulus (G') and loss modulus (G") of the FFS at a constant strain of 1% and frequency of 10 rad s -1 in the temperature range of 80-20 °C by cooling rate of 0.09 °C s -1 . Strain values were chosen to be within the linear viscoelastic region based on preliminary tests. The flow behavior was described by fitting the experimental data with the Power Law model Equation 1.

 = k n (1)
Where  is the shear stress (Pa),  is the shear rate (s -1 ), k is the consistency coefficient (Pa s n ) and n is the flow behavior index.

Frequency sweep tests were performed in the 0.1-100 Hz range at strain 1% in the linear viscoelastic range. Storage modulus (G′), loss modulus (G″) were recorded as a function of frequency.

Scanning electron microscopy (SEM)

The cross-sectional surface of the film samples was investigated using a scanning electron microscope (SEM), model JMS 6510, (JEOL, Japan). Samples were first cryo-fractured in liquid N2 and then fixed onto 90° specimen mounts.

Once coated with a ca. 5-nm-thick gold layer in an argon atmosphere, the samples were analyzed using an accelerating voltage of 2 kV and a working distance of ca. 5 mm. All SEM images were taken using the secondary electron mode.

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR)

FTIR spectra were recorded on a FT-NIR VERTEX spectrometer (Bruker, Germany) operating in the attenuated total reflectance (ATR) mode. The ATR-FTIR spectra were registered from 400 to 4000 cm -1 using accumulation of 32 scans and resolution of 1 cm -1 .

Color measurement

Color parameters (L*, a*, b*) were determined using a colorimeter model Miniscan XE (HunterLab, USA). Gelatin/CNCs films were placed on the surface of a white standard plate and color parameters were measured using the CIELab color scale: L* = 0 (black) to L* = 100 (white), -a* (greenness) to +a* (redness), .

. and -b* (blueness) to +b* (yellowness). The measurements were done in triplicate. Color difference (ΔE*) was calculated using Equation 2 [START_REF] Gennadios | Mechanical and barrier properties of egg albumen films[END_REF].

D/ * = F(DG * ) H + (DJ * ) H + (DK * ) H (2)
where ΔL, Δa, and Δb are the differences between the color parameter of the samples and the color parameter of the white standard.

Light transmission and transparency

The barrier properties of film samples against ultraviolet (UV) and visible light were measured at selected wavelengths between 190 and 700 nm using a UV-1650 spectrophotometer (Shimadzu, Japan). Three replicates were performed for each film. Transparency values were calculated using Equation 3[206]:

J LJ M NO = P.. ( 3
)
where A600 is the absorbance at 600 nm and x is the film thickness (mm). Film thickness was measured using a digital micrometer (Mitutoyo Corp., Kanogawa, Japan) from Ten random measurements per film.

Thermogravimetric analysis (TGA)

Thermogravimetric analysis was carried out in a Q500 equipment (TA Instruments, USA). About 10 mg of each film sample was taken in a standard Pt crucible and heated from 25 °C to 700 °C with heating rate of 10 °C min -1 , under synthetic air atmosphere (60 mL/min). Samples before the TGA tests were conditioned in a ventilated climatic chamber at 23 ± 1 °C and 50 ± 5% RH for 48 h.

Tensile tests

The mechanical properties tensile strength (TS), elongation at break (EAB), and elastic modulus (EM) were measured according to ASTM standard method D882-09 (2009). Film specimens were prepared and equilibrated at 23 °C and 50% RH for 48 h to be tested on a universal testing machine EMIC DL3000 (EMIC Equipamentos e Sistemas de Ensaio LTDA, Brazil) equipped with a 100 N load cell. Specimens were stretched using a crosshead speed of 10 mm min -1 and initial distance between clamps of 100 mm. Tensile tests were performed with five replicates for each film.

Contact angle measurements

Contact angle measurements were conducted with CAM 101 Optical Contact Angle Meter (KSV Instruments, Finland) equipped with a CCD KSV-5000 digital camera. For each measurement, 5 µL of ultrapure water was dropped on the film surface and 100 images were automatically recorded within an experimental time of 60 s using the KSV CAM2008 software. Contact angle determinations were performed with 5 replicates as per ASTM D5725-99 (2008).

Statistical analysis

Data were submitted to analysis of variance (ANOVA). Mean values were compared using the Tukey's test at a confidence level of 95% (p<0.05) using the software Origin (OriginLab, Northampton, USA). were 360 ± 104 nm and 16 ± 3 nm, respectively. The aspect ratio (L/D) was around 22.5. These values are in agreement with a previous report [START_REF] Tonoli | Cellulose micro/nanofibres from Eucalyptus kraft pulp: preparation and properties[END_REF].

Results and discussion

Characterization of CNCs

Liliane Leite -2020 At pH values lower than the Ip, the gelatin chains were positively charged, whereas, for pH values higher than the Ip, the gelatin chains exhibited predominantly negative charges (Figure 2.3). The presence of significant surface negative charges on CNCs was confirmed with the measured ζ-potential value of around -50 mV, which is consistent with previous reports [START_REF] Tonoli | Cellulose micro/nanofibres from Eucalyptus kraft pulp: preparation and properties[END_REF][START_REF] Corrêa | Cellulose nanofibers from curaua fibers[END_REF]. This is due to the acid hydrolysis 66 carried out with sulfuric acid, which results in sulfate groups on the CNCs surface.

This result indicates electrostatic repulsive forces between nanocrystals and good stability for the CNCs colloidal suspension [START_REF] Qiao | Structure and rheological properties of cellulose nanocrystals suspension[END_REF] over an extended time, which is critical for the preparation of bionanocomposites by casting. To avoid the formation of coacervates, which may occur through electrostatic cross-linking between oppositely charged biopolymers [START_REF] Li | Investigation on complex coacervation between fish skin gelatin from cold-water fish and gum arabic: Phase behavior, thermodynamic, and structural properties[END_REF], the FFS used in this study were prepared at pH 6, in which the average ζ-potentials of the CNCs and gelatin were negative.

The The apparent viscosity (η) and consistency index (K) increased, and the flow index (n) was lower than 1 and decreased, with the increasing gelatin concentration (Table 2.1). This means that the gelatin solutions were pseudoplastic (shear-thinning) behavior and became progressively more viscous at high gelatin contents. This behavior is suitable for producing films by continuous casting, which required solutions that could be sheared at the low shear rates at the doctor blade (γ = 3 s -1 ) of the casting process ( . . also exhibit high viscosity at low shear rates to prevent undesired flow onto the moving substrate [START_REF] Ortiz | Scale-up of the production of soy (Glycine max L.) protein films using tape casting: Formulation of film-forming suspension and drying conditions[END_REF].

Usually, gelatin films are obtained by bench casting using solutions containing  10 wt% gelatin [START_REF] Mondragon | Bionanocomposites based on gelatin matrix and nanocellulose[END_REF][START_REF] Bhowmik | Reinforcement of gelatin-based nanofilled polymer biocomposite by crystalline cellulose from cotton for advanced wound dressing applications[END_REF][START_REF] Ning | Enhanced electromechanical performance of bio-based gelatin/glycerin dielectric elastomer by cellulose nanocrystals[END_REF][START_REF] Fadel | Improving tensile strength and moisture barrier properties of gelatin using microfibrillated cellulose[END_REF][START_REF] Paschoalick | Characterization of some functional properties of edible films based on muscle proteins of Nile Tilapia[END_REF][START_REF] Chiou | Cold water fish gelatin films: Effects of cross-linking on thermal, mechanical, barrier, and biodegradation properties[END_REF]. Nevertheless, this concentration was not adequate for our continuous casting technique because the solution flowed off from the substrate before the drying step due to its low viscosity. On the other hand, it was not possible to obtain homogeneous films from solutions with gelatin concentrations higher than 20 wt% due to their very high viscosity and fast gelation under cooling, which resulted in incomplete spreading on the moving substrate and discontinuous films. Therefore, the FFS containing 20 wt% gelatin, which exhibited adequate flow properties and could be spread properly on the substrate, was selected to evaluate the influence of the CNCs on the bionanocomposite film properties. The wet layer thickness was set at 1.00 mm.

In this way, the continuous casting operated at a shear rate of approximately 3 s -1 . In addition, the apparent viscosity decreased with the increasing temperature at around 40 °C [START_REF] Liu | Rheological properties of soy protein isolate solution for fibers and films[END_REF]. This behavior is typical of physical gels which present sol-gel (Tsol-gel) and gel-sol (Tgel-sol) transitions, whenever the solutions are cooled or heated, respectively [START_REF] Ahmed | Rheological Properties of Gelatin and Advances in Measurement[END_REF]. The gelation of gelatin is a physical process that is mostly dependent on intermolecular interactions among protein chains through electrostatic, hydrogen bonding as well as hydrophobic interactions, which largely lead to the formation of a three-dimensional network [START_REF] Wang | Mechanical reinforcement of gelatin hydrogel with nanofiber cellulose as a function of percolation concentration[END_REF].

Production capacity of gelatin/CNCs films by continuous casting

The gelatin/CNCs FFS were used at 30 °C to prevent their gelation during the continuous casting process. The major advantage of the continuous casting approach used herein is the shorter drying time due to the use of much higher temperatures concerning bench casting, and intense air circulation.

Temperatures below

Morphological characterization

The morphology of the gelatin/CNCs bionanocomposites was investigated by SEM. within the polymer matrix [START_REF] Leite | Morphological investigation of cellulose Liliane Leite -2020 acetate/cellulose nanocrystal composites obtained by melt extrusion[END_REF] or CNCs-Gelatin aggregates or coacervate phases [START_REF] Schmitt | Protein-polysaccharide complexes and coacervates[END_REF]. In this sense, the SEM micrographs corroborate with the ζ-potentials results (Table 2.1). Moreover, all films were transparent with no significant differences (p>0.05).

Transparency is a microstructural parameter and high values of transparency suggest the absence of micrometric agglomerates that could scatter light [START_REF] Uranga | Cross-linking of fish gelatins to develop sustainable films with enhanced properties[END_REF]. In this case, the transparency data suggest a good macroscopic dispersion of the CNCs within the gelatin matrix of the bionanocomposites made by continuous casting. Photo-oxidation is one of the main mechanisms of food deterioration.

Packaging with a light barrier has received special attention due to the protective effect against the oxidation of lipids and some other nutrients catalyzed by UV light [START_REF] Kwon | A Short Review of Light Barrier Materials for Food and Beverage Packaging[END_REF]. Figure 2.9 shows the UV-Vis transmittance spectra of the gelatin/CNCs films. The bionanocomposites showed good light barrier properties, mainly in the UVC region, where they were able to block more than 99% of the radiation. This can be attributed to the chromophore groups presented in gelatin amino acid blocks, such as tyrosine and phenylalanine, which have unsaturated bonds capable of absorbing UV radiation [START_REF] Chuaynukul | Comparative Characterization of Bovine and Fish Gelatin Films Fabricated by Compression Molding and Solution Casting Methods[END_REF]. Furthermore, the addition of 2.5 wt% CNCs slightly reduced by 10% and 6% the UVA transmittance at 320 and 400 nm, respectively (p<0.05). This is due to the hydroxyl groups of cellulose, which can act as auxochrome groups. These groups when conjugated to a chromophore are capable of modifying the ability of the chromophore to absorb light [START_REF] Uranga | Cross-linking of fish gelatins to develop sustainable films with enhanced properties[END_REF]. In summary, all the gelatin/CNCs bionanocomposites analyzed exhibited good barrier properties against the UV light, suggesting that this type of film could be used to protect light-sensitive products, including foods and pharmaceuticals.

Conclusions

We demonstrated that continuous casting is a suitable technique to hasten the production of bionanocomposites based on gelatin and cellulose nanocrystals from aqueous formulations. The continuous casting involved relatively low drying temperatures and allowed the production of 12 m/h of bionanocomposite film. All gelatin/CNCs bionanocomposites were highly transparent and easy to handle.

More importantly, the continuously cast gelatin/CNCs films reached higher mechanical resistance at a low CNCs content (0.5 wt%) than similar nanocellulose-based nanocomposites produced by bench casting. This means that the continuous casting not only allowed scaling-up the production of these

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] bionanocomposites but also led to films with relatively superior mechanical performance, in addition to good thermal and UV barrier properties. It is noteworthy that improvements in such properties are of great interest to produce flexible films for food packaging, membranes for wound healing, and so on. We particularly expect that this continuous casting approach will serve as an alternative pilot-scale route to produce nanocellulose-based nanocomposites when the nanocellulose dispersion is not easily achieved through melt processing routes.

Introduction

The and hydrogen bond interactions). However, there is no guarantee that all agglomerated particles will separate during mixing, and some of them remain intact. This is because the hydrodynamic force (by pulling mechanism) required to fully separate two parallel CNCs particles is 115 nN, much greater than the break-up force for the C-C covalent bond (2.5-13 nN) [START_REF] Khoshkava | Effect of drying conditions on cellulose nanocrystal (CNC) agglomerate porosity and dispersibility in polymer nanocomposites[END_REF]. Thus, if this amount of force is provided, the polymer chain scission and degradation can take place before particle separation.

Therefore, the separation of CNCs nanoparticles into individual nanoparticles is quite challenging. The CNCs microstructure depends highly on the employed hydrolysis conditions and drying techniques. In this study, a tipsonication was performed to disperse the heterogeneous CNCs and homogeneous CNCs in film-forming solutions. The aggregates of both CNCs and their dispersibility in gelatin film-forming solutions were analyzed. The morphological, mechanical, thermal, and barrier properties of gelatin-based films obtained by bench casting and continuous casting were also investigated.

Materials and Methods

Materials

Bovine gelatin (Bloom Strength-190) and eucalyptus kraft pulp were kindly supplied by Gelco Gelatinas do Brazil Ltda (Pedreira, SP) and Suzano Papel e Celulose Ltda (Limeira, SP), respectively. Homogeneous CNCs isolated from wood pulp by sulfuric acid hydrolysis and delivered as a dried powder, were purchased from Celluforce Inc. (Windsor, Québec, Canada). Sulfuric acid and glycerol were purchased from Across Organics (USA). All aqueous solutions were prepared using ultrapure water (18 MΩ.cm) obtained from a Milli-Q system (Millipore).

Hydrolyses of CNCs from eucalyptus kraft pulp

The CNCs were prepared by acid hydrolysis of 10 g of eucalyptus kraft pulp using 200 mL of 60% w/w sulfuric acid solution at 45 °C for 60 min with constant stirring. After hydrolysis, the suspension was diluted in 500 mL of cold water to quench the reaction and centrifuged at 10000 rpm for 10 min. The resultant pellet was rinsed, re-centrifuged, and dialyzed in water with a cellulose membrane (6-8 kDa) until neutral pH. Afterward, the suspension was sonicated for 5 min and stored in a refrigerator after adding chloroform drops to prevent microbial growth. The final solid content was 3.5% (w/w). These CNCs were call in this work as heterogeneous CNCs.

Preparation film-forming solutions (FFS)

FFS based on gelatin and CNCs were prepared according to the following steps: first, gelatin powder was hydrated in ultrapure water at concentrations of 20 wt% (for continuous casting) and 10 wt% (for bench casting) at 24 °C for 5 min, and then heated at 60 °C under mechanical stirring for 15 min. Glycerol (20 wt% on a dry gelatin basis) was added to the gelatin solution under stirring. The solution obtained was mixed with the heterogeneous CNCs at various contents (0 wt%, 0.5 wt%, 1.0 wt%, and 2.5 wt%, on a dry gelatin mass basis) and stirred for 5 min. The maximum concentration of heterogeneous CNCs added was 2.5%.

Above this concentration, it was observed with the naked eye aggregates in the FFS. FFS with homogeneous CNCs at various contents (0 wt%, 0.5 wt%, 4.0 wt%, and 6.0 wt% on a dry gelatin mass basis) were prepared in the same manner described above for comparison. These concentrations were chosen based on the percolation threshold calculation for homogeneous CNCs, as discussed in more detail in the results and discussion section.

Polarized light optical microscopy (POM)

Optical images of gelatin/CNCs FFS were taken between crossed polarizers to evaluate the dispersion of the heterogeneous CNCs within the gelatin matrix. The dispersion of 1.0 wt% of aqueous heterogeneous and homogeneous CNCs suspensions was also analyzed. The samples were imaged by polarized optical microscopy taken at 10-fold magnification using a microscope Zeiss Axio Vert.A1 (Carl Zeiss Microscopy).

Transmission electron microscopy (TEM)

Transmission electron microscopy was carried out on a Tecnai TM G2 F20 microscope (FEI Company, Netherlands) using the TEM (Transmission Electron Microscopy) mode. The images were acquired with a dark field (DF) detector. A droplet of diluted heterogeneous and homogeneous CNCs suspension was deposited on a carbon microgrid with formvar (400 mesh) and allowed to dry. The grid was stained with 1.5 wt% uranyl acetate solution and dried at room temperature before analysis. Average CNCs sizes were obtained from a minimum of 100 particles by using the ImageJ software.

X-ray diffraction (XRD)

The crystallinity of heterogeneous and homogenous was estimated by The crystallinity index (CI) was calculated according to the method of Segal et al. [START_REF] Segal | An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer[END_REF]:

Q (%) = S1 - U V U W X Y100 (1)
where I1 is the intensity at the minimum (2θ = 18°) and I2 in the intensity associated with the crystalline region of cellulose (2θ = 22.7°).

XRD patterns of the gelatin/CNCs films were acquired from 2 ° to 60 ° (2θ).

At least duplicate measurements were performed for each sample. The amount of triple helix configuration (Xc in %) in the gelatin/CNCs films was calculated using Equation. 2.

[N = [0 ? (0 ?] 0 )] ⁄ Y100

where Ac is the area of the diffraction peak at ~2θ = 8°, corresponding to the triple helix configuration of gelatin chains, and Aa is the area under the hump located at ~2θ = 21°, corresponding to the fraction of amorphous domains in the gelatin films [START_REF] Díaz-Calderón | Influence of extraction variables on the structure and physical properties of salmon gelatin[END_REF].

Bench casting

Gelatin-based FFS with homogeneous CNCs (60 mL) were cast on glass plates (40 cm × 25 cm) covered with a polyester film (Mylar®, DuPont, Brazil) to facilitate peeling off the films after drying at room temperature for 24 h. Film samples were conditioned in a desiccator at 50% RH and 25 °C for at least 48 h prior to testing.

Continuous casting

Gelatin-based FFS with homogeneous CNCs were turned into monolayer films on a KTF-S labcoater casting machine (Werner Mathis AG, Switzerland). In this technique, the polymer solution is continuously spread onto a moving substrate (Mylar®, DuPont, Brazil) with a doctor blade device, which allows controlling the wet layer thickness. The wet solution layer then passes through two drying chambers with controlled temperatures. The wet solution layer then passes through two drying chambers with controlled temperatures The dried film is wound into rolls. The distance between the substrate and doctor blade was set at 1.0 mm. The conveyor speed and the drying ovens were 12 m/h and 80 °C, respectively. All samples were conditioned in a desiccator at 50% RH and 25 ± 2 °C for at least 48 h prior to testing.

Tensile test

The mechanical properties were measured according to ASTM standard method D882-09 (2009). Film specimens were prepared and equilibrated at 23 °C and 50% RH for 48 h to be tested on a universal testing machine EMIC DL3000 (EMIC Equipamentos e Sistemas de Ensaio LTDA, Brazil) equipped with a 100 N load cell. Specimens were stretched using a crosshead speed of 10 mm min -1 and initial distance between clamps of 100 mm. Tensile tests were performed with five replicates for each film.

Scanning electron microscopy (SEM)

The cross-sectional surface of the gelatin/CNCs film samples was investigated using a scanning electron microscope (SEM), model JMS 6510, (JEOL, Japan). Samples were first cryo-fractured in liquid N2 and then fixed onto 90° specimen mounts. Once coated with a ca. 5-nm-thick gold layer in an argon atmosphere, the samples were analyzed using an accelerating voltage of 2 kV and a working distance of ca. 5 mm. All SEM images were taken using the secondary electron mode.

Field emission gun scanning electron microscopy (FESEM)

The morphology of the cryogenically fractured gelatin/CNCs films was analyzed by micrographs in a field emission gun scanning electron microscopy, model JSM 6701F (JEOL®), using an accelerating voltage of 2 kV. The FEG-SEM samples were mounted on copper stubs and then coated with carbon.

Water Vapor Permeability (WVP)

The WVP of the gelatin/CNCs films was determined following ASTM E-96-01. The preconditioned film was sealed onto the opening of an aluminum permeation cup (28 mm internal diameter) containing dried calcium chloride. The cup was kept in a controlled chamber at 23 ± 1 °C and 50 ± 5% RH. The cup was weighed at least four times for at least 7 days under this controlled environment.

For each film, three replicates were performed. WVP [(g mm)/ m 2 h kPa) was determined using Eq. 2:

WVP = WVTR x L/∆p (2)
in which WVTR is the water vapor transmission rate (g/m 2 h) through the film area; L is the film thickness (mm), and ∆p is the partial pressure gradient (kPa) across the film.

Contact angle measurements

Contact angle measurements were conducted with CAM 101 Optical Contact Angle Meter (KSV Instruments, Finland) equipped with a CCD KSV-5000 digital camera. For each measurement, 5 µL of ultrapure water was dropped on the film surface and 100 images were automatically recorded within an experimental time of 60 s using the KSV CAM2008 software. Contact angle determinations were performed with 5 replicates as per ASTM D5725-99 (2008).

Thermogravimetric analysis (TGA)

Thermogravimetric analysis was carried out in a Q500 equipment (TA Instruments, USA). About 10 mg of each film sample was taken in a standard aluminum cup and heated from 25 °C to 600 °C with heating rate of 10 °C min -1 , under an inert atmosphere of nitrogen (60 mL/min). Samples before the TGA tests were conditioned at 23 ± 1 °C and 50 ± 5% RH for 48 h.

Statistical analysis

Data were submitted to analysis of variance (ANOVA). Mean values were compared using the Tukey's test at a confidence level of 95% (p<0.05) using the software Origin (OriginLab, Northampton, USA).

Results and discussion

CNCs isolated from eucalyptus pulp into gelatin FFS

Polarized light images of gelatin and gelatin/CNCs FFS at different heterogeneous CNCs concentrations are shown in Figure 2.13a. As expected, no macroaggregated was observed in the pure gelatin FFS (0 wt% CNCs).

However, gelatin/CNCs FFS showed a broad range of aggregates (ca. 5-60 m)

that increased with increasing heterogeneous CNCs content, suggesting a poor dispersion. Photographs of pure gelatin and gelatin/CNCs films are shown in The maximum amount of CNCs incorporated in the FFS was 2.5 wt%.

Above this concentration, the gelatin/CNC films obtained showed macroscopic aggregates with the naked eyes in the FFS. This is may be attributed to the presence of some partially hydrolyzed fibers (arrow in 2.4. All CNCs samples presented needle-like morphology. Heterogenous CNCs showed the widest distribution with particles that range from 250-460 nm in length and 13-19 nm in diameter, which resulted in a lower aspect ratio. Homogeneous CNCs showed a relatively narrow distribution of length and diameter, and a higher aspect ratio. The crystallinity index was 76% for heterogenous CNCs and 82% commercial CNCs (Table 2.4).

The low crystallinity index found for heterogeneous CNCs can be attributed to the presence of amorphous components such as hemicellulose in the partially hydrolyzed fibers present in heterogeneous CNCs, as showed in Figure 2.14. while the elongation at break decreased to about 3%. This may be attributed to the combination of infrared radiation (IR) and higher drying temperatures applied in the continuous casting processing. The IR is a very efficient non-contact irradiation drying process that allows fast drying when containing molecules that can absorb IR as water, glycerol, and gelatin [START_REF] Lecorre | All starch nanocomposite coating for barrier material[END_REF]. The first hypothesis is the evaporation of the plasticizer. Glycerol has a boiling point of around 290 °C.

Although IR induces high temperatures in the laminated film layer, the glycerol boiling point could not be achieved without the deterioration of the gelatin matrix.

Which was not the case. Therefore, it is unlikely that its evaporation could have caused the improvement in mechanical properties and this hypothesis can be eliminated.

Another hypothesis is molecular changes in the coating. According to Ortiz et al. (2017) [START_REF] Ortiz | Scale-up of the production of soy (Glycine max L.) protein films using tape casting: Formulation of film-forming suspension and drying conditions[END_REF], the IR applied during the casting manufacture of protein films may affect protein conformation and the protein chains can interact with each other, conditioning the physicochemical properties of the resulting materials. In addition, previous studies have shown that disulfide bridges are favored at high temperatures, presenting a major role in the stabilization of protein, which in turn influences the mechanical properties [START_REF] Ortiz | Scale-up of the production of soy (Glycine max L.) protein films using tape casting: Formulation of film-forming suspension and drying conditions[END_REF]. [START_REF] Salgado | Biodegradable sunflower protein films naturally activated with antioxidant compounds[END_REF]. The results of the mechanical properties of the pure gelatin film suggest that the film processing method 92 affected the level of protein interactions and molecular structure in the film matrix and will be better explored in the next section.

Gelatin/CNCs produced by bench casting showed a significant increase (p< 0.05) in the tensile strength and Young's modulus and decrease in the elongation at break with increasing CNCs content and reached a plateau between 4 wt% and 6 wt% CNCs, suggesting that percolation was achieved. To calculate volume fraction, Favier et al. (1997) [START_REF] Favier | Mechanical Percolation in Cellulose Whisker[END_REF] developed the following relation by using a statistical percolation theory for cylindrically shaped particles: v= 0.7/(A),

where A is the aspect ratio of CNCs. Therefore, considering the aspect ratio of CNCs as 28.5 (Table 2.4), the calculated volume fraction was 2.5% (v/v), which corresponds to 4 wt%, corroborating the tensile results.

Gelatin/CNCs films obtained by continuous casting showed a significant increase (p< 0.05) in Young's modulus with increasing CNCs content. Gelatin film with 0.5 wt% CNCs showed the highest tensile strength, compared with pure gelatin obtained by the same processing. No significant differences in the elongation at break have been detected. However, all the gelatin/CNCs produced by continuous casting became less ductile than their counterpart films obtained by bench casting, as shown in Figure 2.16d, indicating that films had a strong intermolecular interaction.

Structure analyses of gelatin-based films

The drying temperature used in the bench casting and also the IR used in the continuous process is a significant factor that affects the structure and physical properties of protein films. The XRD patterns of gelatin-based films are shown in Figure 2.17 and the triple helix content of them is represented in Table 2.5. The formation of gelatin film is accompanied by the partial renaturation of triple helix of the collagen structure [START_REF] Lin | The modification of gelatin films: Based on various cross-linking mechanism of glutaraldehyde at acidic and alkaline conditions[END_REF], which is known to be crystalline. The XRD diffractograms showed a wide background with a band at 2θ ~ 21°, related to the large amorphous fraction of gelatin [START_REF] Bigi | Relationship between triple-helix content and mechanical properties of gelatin films[END_REF]. The band at 2θ ~8° has been previously attributed to the presence of triple helix structure present in collagen and renaturated gelatin [START_REF] Quero | Stress transfer and matrix-cohesive fracture mechanism in microfibrillated cellulose-gelatin nanocomposite films[END_REF], and the area of this peak has been found to be directly related to the triple helix content of gelatin-based composites [START_REF] Bigi | Relationship between triple-helix content and mechanical properties of gelatin films[END_REF][START_REF] Badii | The Effect of Drying Temperature on Physical Properties of Thin Gelatin Films[END_REF].

Water vapor barrier analysis of gelatin-based films

The water vapor barrier of gelatin-based films obtained by bench and continuous casting is presented in Figure 2.19. The addition of CNCs significantly reduced (p <0.05) the WVP of the gelatin matrix for all the samples analyzed, regardless of the processing method. This phenomenon is the result of interactions between CNCs and gelatin, as well as an increased tortuosity of the pathway for vapor diffusion through the film matrix [START_REF] Slavutsky | Water barrier properties of starch films reinforced with cellulose nanocrystals obtained from sugarcane bagasse[END_REF]. Besides, gelatin-based films obtained by continuous casting showed lower WVP value than films produced by bench casting. This can be attributed to the use of IR and higher WVP values twice as low as those dried at 40 °C, suggesting that films dried at a higher temperature could have more intense structural collapse, which would probably bring films with lower volume and, therefore, higher density. Higher density is generally associated with lower WVP films. Gelatin-based films obtained by bench casting showed water contact angle values higher than 90 ° over time, which indicates that the surface analyzed was hydrophobic, except for the gelatin film with 6.0 wt% CNCs, which after approximately 45 s became hydrophilic. Gelatin has been widely known for its high hydrophilicity [START_REF] Chen | Superhydrophobic coatings on gelatin-based films: Fabrication, characterization and cytotoxicity studies[END_REF]. However, the hydrophobic surface for gelatin-based films has been reported [203, [START_REF] Chen | Superhydrophobic coatings on gelatin-based films: Fabrication, characterization and cytotoxicity studies[END_REF][START_REF] Syahida | Effects of palm wax on the physical, mechanical and water barrier properties of fish gelatin films for food packaging application[END_REF]] and the explanation given by the authors is that during film drying, the randomly coiled gelatin molecules are capable of spontaneously rearranging their hydrophobic parts towards the film-air [START_REF] Białopiotrowicz | The changes of the surface free energy of the adsorptive gelatin films[END_REF].

By contrast, the fast drying time used in the continuous casting did not allow this rearrangement and hydrophilic surfaces over time were observed for all the films analyzed. Besides, the increasing concentration of CNCs decreased the water contact angle value of gelatin/CNCs films obtained by continuous casting. This result demonstrated that the film was more hydrophilic due to the OH-group in the CNCs structure since the polarity around the structure could create H-bonding to react with water.

Thermostability of gelatin-based films

The decomposition temperatures of CNCs power and gelatin-based films obtained by bench and continuous casting are reported in Table 2.6. The thermal stability is one of the main issues in the processing of natural bio-based materials.

The CNCs showed onset thermal decomposition temperature at 280 °C, which was in agreement with previously reported [START_REF] Espinosa | Isolation of thermally stable cellulose nanocrystals by phosphoric acid hydrolysis[END_REF]. The thermostability of gelatinbased films produced by bench casting decreased with the addition of CNCs. By contrast, the results showed that films produced by continuous casting became more thermally stable. Pure gelatin registered an increase in onset thermal decomposition temperature by 9 °C compared to the same film obtained by bench casting. The highest thermostability was found for gelatin film with 4 wt% CNCs, which showed an increase by 24 °C compared to their counterpart films obtained by bench casting. These findings corroborate the hypothesis that the IR and the higher drying temperature applied in the continuous casting contributed to better cohesiveness of the matrix, stabilized by strong inter-and intramolecular bonds, which requires a higher temperature for breakouts. [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF].

Conclusion

The results demonstrated that the good dispersion of CNCs into the matrix can improve the mechanical, barrier, and thermal properties of the gelatin films.

The results obtained in this part give important insights concerning the film properties, showing that to achieve the percolation threshold the CNCs 100 dispersion is as important as their content. All the gelatin/CNCs films obtained by bench casting and continuous casting were highly homogeneous and easy to handle. The infrared radiation and high drying temperatures applied in continuous casting processing promoted strong intermolecular interaction and intense structural collapse, which led to the formation of more cohesive films with a better barrier, mechanical and thermal properties. Continuous casting processing has been proven to be advantageous when compared to the bench casting, as it allows not only scaling-up the production of gelatin-based films but also allows better performance of these films. This is a straightforward alternative to bench casting. under electrostatic attractive forces and by 39% at isoelectric point. The addition of 5 wt% CNCs at pH 3 resulted in the formation of complex coacervates, which contributed to decrease the mechanical properties and increase the WVP of gelatin/CNCs films. Increasing pH above the gelatin pI remarkedly increased the gelatin renaturation as triple helices, which was found to be key for increasing by 152% and 56% in Young's modulus and tensile strength of gelatin/CNCs films with 0.5 wt% CNCs, respectively. In contrast, the film-forming drying temperatures of these films had an inverse effect on the triple helix content, and, consequently, on their physical properties. These findings can be explored to modulate the mechanical strength, permeability, and thermal stability of nanocellulose-added gelatin films for a variety of applications, including flexible food packaging.

Introduction

Proteins are natural polyampholytes that have attracted great interest due to their unique properties [START_REF] Al-Tayyar | Antimicrobial food packaging based on sustainable Bio-based materials for reducing foodborne Pathogens: A review[END_REF], in particular, their ability to form self-assembled structures, such as polyelectrolyte complexes obtained by interactions between proteins and polysaccharides carried opposite charges in aqueous medium [START_REF] Derkach | Polyelectrolyte Polysaccharide-Gelatin Complexes: Rheology and Structure[END_REF].

These structures are promising for the creation of new materials applicable in pharmacy, medicine [START_REF] Vuillemin | Gum Arabic and chitosan self-assembly: Thermodynamic and mechanism aspects[END_REF][START_REF] Zhao | Polyelectrolyte-based platforms for the delivery of peptides and proteins[END_REF], and food packaging [START_REF] Silva | Physical chemical study of zein and arabinogalactans or glucuronomannans polyelectrolyte complexes and their filmforming properties[END_REF][START_REF] Hosseini | Fabrication of bio-nanocomposite films based on fish Liliane Leite -2020 gelatin reinforced with chitosan nanoparticles[END_REF].

Gelatin is a protein obtained by the partial hydrolysis of collagen at controlled temperature and pH conditions [START_REF] Joshi | pH and ionic strength induced complex coacervation of Pectin and Gelatin A[END_REF]. The mechanical behavior of gelatin gels and films is related to the regeneration of gelatin as crystalline triple helixes. Gioffrè et al. ( 2012) [START_REF] Gioffrè | Role of pH on stability and mechanical properties of gelatin films[END_REF] investigated the pH effect of film-forming solutions on the mechanical properties of pigskin type A gelatin films and observed that pH values higher than 9 and lower than 5 considerably reduced the triple helix content and, consequently, the mechanical properties of the films.

These results indicate that pH can be used to modulate the mechanical behavior of gelatin-based materials.

Gelatin films have also been found to be moisture-sensitive and exhibit a reduced water vapor barrier and mechanical resistance under high relative humidity, due to the hydrophilicity of the polypeptide chains. A suitable approach to overcoming these limitations of gelatin films is the use of cellulose nanocrystals (CNCs) as nano-sized fillers [START_REF] Hosseini | A state-of-the-art review on the elaboration of fish gelatin as bioactive packaging: Special emphasis on nanotechnology-based approaches[END_REF]. The use of CNCs has been proven to enhance the mechanical and barrier properties of gelatin films. Several works have described a joint synergistic effect between gelatin and CNCs toward the formation of percolated networks stabilized by hydrogen bonding [START_REF] George | High performance edible nanocomposite films containing bacterial cellulose nanocrystals[END_REF], [START_REF] Santos | Fish gelatin films as affected by cellulose whiskers and sonication[END_REF].

To the best of our knowledge, no research has been published on the effect of reinforcing CNCs on the properties of gelatin films in function of the electrostatic interactions. The knowledge of these intermolecular interactions is crucial for understanding the structure and physical properties of gelatin/CNCs films, as well as for the development of packaging films with tunable mechanical properties. Furthermore, there is no report that examines the structure and mechanical properties of gelatin/CNCs films under the influence of drying temperature. This work aimed to investigate the effect of CNCs, pH, and drying temperature on the triple helix content, mechanical, thermal, and barrier properties of gelatin films obtained by bench casting.

Materials and methods

Materials

Bovine gelatin (Bloom Strength-190) was kindly supplied by Gelco Gelatinas do Brazil Ltda (Pedreira, SP). CNCs isolated from wood pulp by sulfuric acid hydrolysis and delivered as a dried powder were purchased from Celluforce Inc. (Windsor, Québec, Canada). Glycerol and ethanol were purchased from Across Organics (USA). All chemicals were analytical grade and were used as purchased.

Preparation of gelatin/CNCs films

The bionanocomposite films were prepared by solvent casting. The gelatin powder was hydrated in distilled water (10 g/100 g) at 24 °C for 5 min and heated

Determination of Zeta potentials (ζ-potentials)

ζ-potentials of aqueous CNCs dispersion, pure gelatin solution, and gelatin/CNCs FFS (0.5 wt% and 5.0 wt% CNCs on a dry gelatin basis) diluted to 0.25 wt% were analyzed at 25 °C using a Zetasizer Nano-ZS (Malvern Instruments Inc., UK). Each sample was mixed with a sonicator (Branson 450 Sonifier, USA) for 5 min with a microtip before analysis. The measurements were performed in triplicate.

Rheological study

The apparent viscosity and flow behavior of the FFS (pure gelatin and gelatin/CNCs with 0.5, and 5 wt% CNCs at pH 3, pH 6, and pH 8) were evaluated on a Physica MCR101 rheometer (Anton Paar, Austria) using the concentric cylinder geometry at 40 °C. The shear rate was varied from 0 to 150 s -1 in 10 min.

Oscillatory shear deformation tests were also carried out to determine the storage modulus (G') and loss modulus (G") of the FFS at a constant strain of 1% and frequency of 1 Hz in the temperature range of 60-20 °C, using heating/cooling rate of 0.02 °C s -1 . Strain values were chosen to be within the linear viscoelastic region based on preliminary measurements. The flow behavior was described by fitting the experimental data to the Power Law model (Equation 1).

 = k n

Eq. 1

where  is the shear stress (Pa),  is the shear rate (s -1 ), k is the consistency coefficient (Pa s n ) and n is the flow behavior index.

Polarized light optical microscopy

Optical images of gelatin/CNCs films were taken between crossed polarizers to evaluate the dispersion of the CNCs within the gelatin matrix. The samples were imaged by polarized optical microscopy taken at 10-fold magnification using a microscope Zeiss Axio Vert.A1 (Carl Zeiss Microscopy).

Scanning electron microscopy (SEM)

The film surface and the cross-sectional surface were investigated using a JEOL Scanning Electron Microscope (SEM, model Quanta200, FEI, the .

.

Liliane Leite -2020

Netherlands). The samples were first cryo-fractured in liquid N2 and then fixed onto 90° specimen mounts to be coated with a ca. 5-nm-thick gold layer in an argon atmosphere. The SEM images were taken at accelerating voltages below 10 kV using the secondary electron mode. At least 10 images per sample were obtained and the most representative was used in the discussion.

X-ray diffraction (XRD) analysis

X-ray diffraction patterns were acquired from 2 ° to 60 ° (2θ) using a Panalytical diffractometer (X'Pert Pro MPD-Ray) with Ni-filtered Cu Ka radiation (k = 1.54 Å) generated at a voltage of 45 kV and current of 40 mA. At least duplicate measurements were performed for each sample. The amount of triple helix configuration (Xc in %) in the gelatin/CNCs films was calculated using Equation 2.

[N = [0 ? (0 ?] 0 )] ⁄ Y100 Eq. 2
where Ac is the area of the diffraction peak at ~2θ = 8°, corresponding to the triple helix configuration of gelatin chains, and Aa is the area under the hump located at ~2θ = 21°, corresponding to the fraction of amorphous domains in the gelatin films [START_REF] Díaz-Calderón | Influence of extraction variables on the structure and physical properties of salmon gelatin[END_REF].

Tensile tests

The tensile properties of the films were determined by the ASTM D882-09 standard method (2009). The film specimens (17 cm x 1 cm) were prepared and equilibrated in a ventilated climatic chamber at 23 ± 1 °C and 50 ± 5% RH for 48

Water vapor permeability (WVP)

WVP was determined using the ASTM E-96-01. The preconditioned film was sealed onto the opening of an aluminum permeation cup (28 mm internal diameter) containing dried calcium chloride. The cup was kept in a ventilated climatic chamber at 23 ± 1 °C and 50 ± 5% RH. The cup was weighed at least four times for at least 7 days under this controlled environment. For each film, three replicates were performed. The WVP g mm m -2 h -1 kPa -1 was determined using Equation 3:

WVP = (∆c ∆d) ⁄ Y (G 0∆&) ⁄ Eq. 3
where ∆m/∆t is the mass gain over time (g h -1 ), A is the exposed film area (m 2 ), L is the film thickness (mm), and ∆P is the difference of partial pressure.

Water solubility (WS) determination

The gelatin films were cut into rectangular (1 cm × 1 cm) specimens and pre-dried at 105 °C for 24 h. The dried specimens were accurately weighed (initial dry weight, w0), placed in distilled water (30 mL), and stirred at 25 ± 1 °C for 24 h. Filter paper number 1 was prepared by drying at 105 °C for 24 h. Then, the film was poured onto the filter paper and dried at 105 °C for 24 h and weighed (final dry weight, w1). WS was measured in triplicate following calculated using Equation 4.

WS (%) = [( @ -) @ ⁄ ] x 100

Eq. 4

Thermogravimetric analysis (TGA)

Thermogravimetric analysis was carried out in a Q500 equipment (TA Instruments, USA). About 10 mg of each film sample was taken in a standard Pt crucible and heated from 25 °C to 600 °C with heating rate of 10 °C min -1 , under an inert atmosphere of nitrogen (60 mL/min). Samples before the TGA tests were conditioned in a ventilated climatic chamber at 23 ± 1 °C and 50 ± 5% RH for 48 h.

Statistical analysis

Data were subject to analysis of variance (ANOVA). The mean values were compared using the Tukey's test at a confidence level of 95 % (p<0.05) using the Origin software (OriginLab, Northampton, USA). This is in agreement with previously reported data for type B gelatins (pH 4.5-6.0) [START_REF] Ahmed | Rheological Properties of Gelatin and Advances in Measurement[END_REF]. Gelatin has the ability to present different charges depending on the pH [START_REF] Gioffrè | Role of pH on stability and mechanical properties of gelatin films[END_REF]. At pH < pI, the gelatin solution showed a net positive charge, which was attributed to the protonation of the amino moieties (-NH3 + ), while at pH > pI the solution showed a net negative charge, due to the ionization of the carboxyl moieties (-COO -). For the CNCs suspension, the ζ-potential value was found to be negative regardless of the pH. The ζ-potential value of CNCs suspension was around -50 mV, indicating a stable suspension in water.

According to Lin, Gu, & Cui (2019) [START_REF] Lin | Moringa oil/chitosan nanoparticles embedded gelatin nanofibers for food packaging against Listeria monocytogenes and Staphylococcus aureus on cheese[END_REF], ζ-potentials (< -30mV and > + 30 mV) could maintain a stable colloidal system due to the repelling force between particles. This value can be attributed to the sulfate groups on the CNCs surface, agreeing with a previous study [START_REF] Silva | Mango kernel starch films as affected by starch nanocrystals and cellulose nanocrystals[END_REF]. Dynamic rheological measurements can be used to monitor structures in weak gels or viscoelastic fluids [START_REF] Behrouzain | Steady shear rheological properties of emerging hydrocolloids[END_REF]. These measurements provide key information for gelatin processability in continuous solvent casting or 3D printing. All the samples presented a similar rheological behavior, regardless of the pH or CNCs content. The viscoelastic modulus (G', G") were higher at lower temperatures (gel-like domain) than at higher temperatures (sol-like domain), with a well visible inflection between both domains (G'= G"). This inflection is typical of physical gels and on the heating and cooling curves is defined as the gel-sol transition (Figure 3.4a,c) and sol-gel transition (Figure 3.4b,d), respectively. Similar profiles have been observed in gelatin-based systems by several authors [START_REF] Jorge | Viscoelastic and rheological properties of nanocomposite-forming solutions based on gelatin and montmorillonite[END_REF][START_REF] Alexandre | Gelatin-based films reinforced with montmorillonite and activated with nanoemulsion of ginger essential oil for food packaging applications[END_REF]. The temperatures of sol-gel (cooling curves) and gelsol (heating curves) of the gelatin/CNCs FFS were slightly affected by the electrostatic interactions, which was more pronounced for the gel-sol transitions (Table 1). Furthermore, the addition of 5.0 wt% CNCs to the gelatin solution at Besides, the gelatin/CNCs FFS with 5% CNCs at pH 8 also did not exhibit the sol-gel transition and presented a viscoelastic solid-like behavior, indicating the existence of physical crosslinks by hydrogen bonding as mentioned before. *Triple helix content of gelatin films was calculated using Equation 1 from X-ray diffraction spectra.

Mechanical properties

The Young's modulus, elongation at break, and tensile strength of gelatin/CNCs films are presented in Figure 3. The Young's modulus and tensile strength of the gelatin/CNCs films reached a maximum value with the addition of 0.5 wt% CNCs and then decreased after the addition of 5.0 wt% CNCs, at both acidic and alkaline conditions. This behavior can be attributed to high concentrations of aggregates formed by the coacervates at pH 3 [START_REF] Celebi | Effects of processing on the properties of chitosan/cellulose nanocrystal films[END_REF], which provide a heterogeneous distribution of CNCs within the gelatin matrix, as revealed by SEM (Figure 3.5b). These aggregates can lead to a decrease in the strength of the nanocomposites since they act as defects or stress concentrators [START_REF] Cao | The relationship between cellulose nanocrystal dispersion and strength[END_REF]. At pH 8, the electrostatic repulsion in alkaline gelatin/CNCs film can increase the free volume and flexibility of the gelatin matrix [START_REF] Lin | The modification of gelatin films: Based on various cross-linking mechanism of glutaraldehyde at acidic and alkaline conditions[END_REF]. The moderate but gradual increases in Young's modulus and tensile strength at pH 6 may relate to the lower triple helix content compared to pH 8. These lowered effects also led the gelatin/CNCs bionanocomposites obtained from the gelatin pI to show a more ductile mechanical behavior, as illustrated in Figure 3.7d. Overall, these results denote that pH and CNCs can be used to modulate the mechanical properties of gelatin films.

Water sensitive properties

The WVP of the gelatin films with different pH and CNCs content are presented in Table 3.3. No significant differences (p > 0.05) among the WVP for gelatin films with 0% CNCs were observed concerning the pH variations. These results agree with previous findings on fish myofibrillar protein films [START_REF] Shiku | Effect of pH on the preparation of edible films based on fish myofibrillar proteins[END_REF], but differ from those previously reported for soy protein isolate films, which showed lower WVP at pH values above the isoelectric point [START_REF] Gennadios | Effect of pH on properties of wheat gluten and soy protein isolate films[END_REF]. However, the WVP of the gelatin/CNCs films obtained in this work at 50% RH were lower than those reported for other films based on gelatin at 0% RH [START_REF] Chiou | Cold water fish gelatin films: Effects of cross-linking on thermal, mechanical, barrier, and biodegradation properties[END_REF], the results found were close to 1.8 g mm/m 2 h kPa (25 °C at 0% RH) for bovine and porcine gelatin films.

123

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] The addition of 0. The water solubility results are summarized in Table 3.3. No film fully dissolved or broke apart after 24 h at 25 °C, suggesting that they were formed by a stable network. The solubility of the gelatin films decreased by increasing the pH since higher pH favors the increase of protein-protein interactions and the exposition of hydrophobic groups that increase surface hydrophobicity gelatin and decrease the water solubility [START_REF] Díaz | Whey protein film properties as affected by ultraviolet treatment under alkaline conditions[END_REF], the addition of CNCs at pH 6 and pH 8

did not significantly (p < 0.05) change the film solubility. However, a drastic decrease in water solubility was observed at 0.5 wt% CNCs and pH 3. This improvement in the water resistance can be attributed to the formation of the gelatin-CNCs complex coacervates, as shown in the SEM image (Figure 3.5b).

These results are in agreement with Esteghlal et al. ( 2018) [START_REF] Esteghlal | Physical and mechanical properties of gelatin-CMC composite films under the influence of electrostatic interactions[END_REF], who suggested that the decrease in solubility is due to charge reduction after complexation and formation of cross-linked gelatin-CMC films. [START_REF] Yin | A preliminary study on chitosan/gelatin polyelectrolyte complex[END_REF] [263] also found similar behavior for chitosan/gelatin polyelectrolyte complexes.

Thermostability

The gelatin/CNCs films were further characterized by TGA and the decomposition temperatures are reported in Table 3.4. All samples followed a similar decomposition profile. The first mass loss was due to moisture and volatiles evaporation [203]. Pure gelatin films at pH 8 showed the lowest moisture content (3%) and the addition of 5.0 wt% CNCs increased the weight loss at this stage, except the gelatin/CNCs film at pH 6, which may be due to less availability of free hydroxyl groups of the gelatin matrix to hydrogen bonding with CNCs. This result is in line with the increase in Young's Modulus (Figure 3.7) and reduction in the WVP of the gelatin/CNCs films with 5.0 wt% at pH 6 (Table 3.

3).

A significant mass loss occurred at temperature (Tonset) above 200 °C, mainly associated with the decomposition of gelatin and glycerol molecules. The thermal stability of gelatin films increased near the gelatin isoelectric point, which is the result of a more compact form of gelatin [START_REF] Alberto | Hydrodynamic Properties of Gelatin -Studies from Intrinsic Viscosity Measurements[END_REF]. This result is in agreement with Gioffrè et al. (2012) [START_REF] Gioffrè | Role of pH on stability and mechanical properties of gelatin films[END_REF], who reported a reduction in the thermal stability of gelatin films prepared at relatively high and low pH. The higher thermal stability was found for the gelatin/CNCs films with 0.5% CNCs at pH 3 and pH 8, which showed increases of 14.4 and 15.3 °C in the Tonset, respectively, compared to pure gelatin films at the same pH. This improvement might be attributed to the formation of more rigid structures, like the gelatin-CNCs coacervate complexes formed at pH 3. This may also be related to the high concentration of triple helix structures present in gelatin/CNCs films at pH 8, as shown by XRD (Figure 3.6).

In addition, the formation of hydrogen bonds between CNCs and gelatin can also influence the protein denaturation process and, as a consequence, lead to an increased thermal stability, as justified by George & Siddaramaiah, (2012) [START_REF] George | High performance edible nanocomposite films containing bacterial cellulose nanocrystals[END_REF].

Previous studies found an increase of 9 °C for gelatin films with 10 wt% CNCs [START_REF] Mondragon | Bionanocomposites based on gelatin matrix and nanocellulose[END_REF], 10.6 °C for gelatin films with 2.5 wt% [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF], and a decrease of around 6 °C for gelatin films with 1.5 wt% CNCs, when compared to pure gelatin films [START_REF] Alves | Effect of cellulose nanocrystals and gelatin in corn starch plasticized films[END_REF]. The gelatin films with 0.5 wt% CNCs obtained from pH 8, which had the highest triple helix content, were chosen to evaluate the effect of the drying temperature on the mechanical, thermal, and barrier properties of gelatin/CNCs films. The obtained results for film samples dried at 25 °C ("cold-dried"), 35 °C (gelation temperature), and 80 °C ("hot-dried") are listed in Table 3.5. The gelatin films dried at 25 °C showed the highest Young's modulus and tensile strength.

This was likely due to the fact that cold-dried films had a longer drying time, i.e.

sufficient time for the reorganization of gelatin structures as triple helices, than the hot-dried films. [START_REF] Chiou | Effects of drying temperature on barrier and mechanical properties of cold-water fish gelatin films[END_REF] [START_REF] Chiou | Effects of drying temperature on barrier and mechanical properties of cold-water fish gelatin films[END_REF] observed that gelatin films dried below their gelation temperature contained triple-helix structures, whereas films dried above their gelation temperature displayed lower crystallinity degree or amorphous structure. It has been previously reported that films with helical structures exhibit higher tensile strength and Young's modulus than amorphous films [START_REF] Badii | The Effect of Drying Temperature on Physical Properties of Thin Gelatin Films[END_REF].

Furthermore, the thermal stability of gelatin films increased when the drying temperature was decreased. These results might be explained by the larger amount of energy needed to break the hydrogen bonds in the triple helix structures formed in the cold-dried gelatin films (Figure 3.8). The WVP results

showed no statistical difference concerning the drying temperature (p > 0.05).

Bor Sen Chiou et al. (2008) [START_REF] Chiou | Cold water fish gelatin films: Effects of cross-linking on thermal, mechanical, barrier, and biodegradation properties[END_REF] showed that mammalian gelatin films had higher levels of renaturation than fish gelatin films. However, the authors observed that this greater amount of helical structures did not lead to better water barrier properties, proving that WVP is not proportional to the renaturation level in gelatin films. The mechanical, thermal, and barrier results suggest that the drying temperature used in the casting process and the addition of CNCs are significant factors that affect the structure and physical properties of gelatin/CNCs films.

Abstract

We report on gelatin films incorporating rosin-grafted cellulose nanocrystals (r-CNCs), which fulfill the most relevant requirements for an antimicrobial food packaging material. The gelatin/r-CNCs bionanocomposites (0.5 -6 wt% r-CNCs) were obtained by water-based casting and displayed UVA-and UVBbarrier properties (over 95% for UVB, and 59.0% for UVA) which are superior to the most used plastics while keeping the optical transparency. The films exhibited a moderate water vapor permeability (0.09 g mm/m 2 h kPa), lower than bio-based materials, and high tensile strength (40 MPa) and Young's modulus (1.9 GPa).

These favorable properties should be attributed to Van der Waals interactions with the gelatin matrix in addition to hydrogen bonding. Grafting of rosin on CNCs resulted in a nanocellulose that inhibits Staphylococcus aureus and Escherichia coli growth, including in films as demonstrated in proof-of-principle experiments with the prevention of microbial spoilage of cheese samples.

Materials and Methods

Materials

Bovine gelatin powder (Bloom Strength-190) was kindly supplied by Gelco Gelatinas do Brazil Ltda (Pedreira, SP). CNCs, isolated from wood pulp by sulfuric acid hydrolysis and delivered as a dried powder, were purchased from Celluforce Inc. (Windsor, Québec, Canada). Rosin was purchased from Aldrich Chemical (USA). Glycerol and ethanol were purchased from Across Organics (USA). All chemicals were of analytical grade and were used as purchased.

Staphylococcus aureus (S. aureus, Gram-positive, ATCC 25923) and

Escherichia coli (E. coli, Gram-negative, ATCC 25922) were supplied by Cefar Diagnostica (Brazil). Distilled water was used in all experiments.

Synthesis of rosin-grafted CNCs (r-CNCs)

The CNCs were functionalized with rosin by a SolReact process adapted from Castro et al. [START_REF] Castro | Surface grafting of cellulose nanocrystals with natural antimicrobial rosin mixture using a green process[END_REF]. The CNCs were dispersed in water at 1.0 wt% and ultrasonicated for 2 min using a Branson sonicator, and the pH was adjusted to 4.0 with HCl (0.1 mol/L). The CNCs dispersion was placed in a closed distillation system in an oil bath at 130 °C. After 10 min, an excess of rosin (10 equiv.

according to the CNCs dry weight) was added slowly to ensure its melting and adsorption on the CNCs surface. The system was kept under stirring for 9 h at 130 °C. After the reaction, the rosin-grafted CNCs (r-CNCs) were purified from unreacted rosin by six dispersion-centrifugation cycles (10000 rpm at 4 °C for 10 min) with a large excess of ethanol, until no more rosin was detected by FTIR in the supernatant. Afterward, the r-CNCs were recovered by centrifugation and washed thoroughly with water, and then sonicated for 5 min and stored in a refrigerator. The final solid content was 3.5% (w/w).

Preparation of gelatin/r-CNCs bionanocomposite films

The bionanocomposite films were prepared by solvent casting. The gelatin powder was hydrated in distilled water (10 g/100 g) at 24 °C for 5 min and then heated at 60 °C under stirring for 15 min. Glycerol (20 wt% on a dry gelatin basis) was added to the gelatin solution under stirring. The solution obtained was mixed with the r-CNCs suspension at various contents (0 wt%, 0.5 wt%, 4.0 wt% and 6.0 wt%, on a dry gelatin basis) and stirred for 5 min. The suspensions (45 mL)

were then cast on glass plates (40 cm × 25 cm) covered with a polyester film (Mylar®, DuPont, Brazil) to facilitate film peeling off after drying at room temperature for 24 h. Film samples were conditioned at 50% RH and 25 °C for at least 24 h prior to testing. Non-grafted gelatin/CNCs films were prepared at the same concentrations described above for comparison.

X-ray photoelectron spectroscopy (XPS)

The functionalization of CNCs with rosin was characterized by XPS (XR3E2, Vacuum Generator, UK) equipped with monochromated Mg Kα X-ray source (1253.6 eV) operated at 15 kV and 20 mA.

Fourier Transform Infrared Spectroscopy (FTIR)

CNCs and r-CNCs were characterized by FTIR (FT-NIR VERTEX spectrometer, Bruker, Germany) in the attenuated total reflection (ATR) mode.

Spectra were recorded between 4000 and 400 cm -1 with a resolution of 1 cm -1 and 32 spectral accumulations. The FTIR measurements of gelatin and PVC films were carried out with five repetitions for each film sample.

Transmission Electron Microscopy (TEM)

The morphology of CNCs and r-CNCs was investigated using TEM (TecnaiTM G2 F20 microscope, FEI Company, USA) with an accelerating voltage of 80 kV. The samples were prepared by depositing a 0.1 wt% suspension droplet on a carbon microgrid with formvar (400 mesh) stained with 1.5 wt% uranyl acetate solution. A minimum of 10 images was recorded and the most representative one was used for discussion. The CNCs and r-CNCs sizes were determined from the analysis of a minimum of 100 particles using the ImageJ software.

X-ray diffraction (XRD)

The crystallinity of CNCs and r-CNCs was estimated by XRD (Panalytical diffractometer, X'Pert Pro MPD-Ray, The Netherlands) with Ni-filtered Cu Kα radiation (k = 1.54 Å, 45 kV, 40 mA) in the 2θ range from 5° to 60°. The crystallinity index (CI) was calculated according to the method of Segal et al. [START_REF] Segal | An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer[END_REF]:

Q (%) = S1 - U V U W X Y100 (1)
where I1 is the intensity at the minimum (2θ = 18°) and I2 in the intensity associated with the crystalline region of cellulose (2θ = 22.7°).

Scanning Electron Microscope (SEM)

The film cross-sectional surface was studied using a JEOL (SEM, model Quanta200, FEI, the Netherlands). The samples were first cryo-fractured in liquid N2 and then fixed onto 90° specimen mounts to be coated with a ca. 5-nm-thick gold layer in an argon atmosphere. The SEM images were taken at accelerating voltages below 5 kV using the secondary electron mode.

Water Vapor Permeability

The WVP of the films was determined following ASTM E-96-01. The preconditioned film was sealed onto the opening of an aluminum permeation cup (28 mm internal diameter) containing dried calcium chloride. The cup was kept in a controlled chamber at 23 ± 1 °C and 50 ± 5% RH. The cup was weighed at least four times for at least 7 days under this controlled environment. For each film, three replicates were performed. WVP [(g mm)/ m 2 h kPa) was determined using Eq. 2:

WVP = WVTR x L/∆p (2)
in which WVTR is the water vapor transmission rate (g/m 2 h) through the film area; L is the film thickness (mm), and ∆p is the partial pressure gradient (kPa) across the film.

Oxygen Transmission Rate (OTR)

The OTR of the films was determined in duplicate at 0%, 50%, and 80%

RH according to the ASTM D3985 method (2002a) using an oxygen transmission rate analyzer (Systech Illinois 8500, USA).

Tensile tests

The mechanical properties of the films were determined as per the ASTM D882-09 standard method (2009). Film specimens were prepared and equilibrated at 23 ± 1 °C and 50 ± 5% RH for 48 h. The tests were carried out on an Instron Universal Testing Machine (model 5569, Instron Corp., USA) with a 100 N load cell. The specimens were stretched using crosshead speed of 10 mm/min with clamps initially separated by 100 mm. Tensile strength, Young's modulus, and elongation at break were calculated from the stress-strain curves.

The tests were performed with five replicates for each film. Film thickness was measured with a digital micrometer (Mitutoyo Corp., Kanogawa, Japan) to the nearest 0.001 mm. At least 10 specimens of each composition were analyzed.

Antimicrobial activity tests

The bacterial strains to evaluate the effectiveness of CNCs-and r-CNCsloaded gelatin solutions were Staphylococcus aureus and Escherichia coli, which are foodborne pathogens [START_REF] Paudyal | Microbiology prevalence of foodborne pathogens in food from selected African countries -a meta-analysis[END_REF][START_REF] Ferrario | Microbiology next generation sequencing-based multigene panel for high throughput detection of food-borne pathogens[END_REF]. The inoculum cultures were prepared inoculating a selected single colony in 15 mL of Muller-Hinton Broth medium (MHB), followed by overnight incubation at 35 ˚C. The optical densities at 625 nm were measured by UV-Vis absorption spectroscopy and compared with the turbidity of a 0.5 McFarland standard at the same wavelength, equivalent to 1.5  10 8 colony forming units per mL (CFU/mL). The overnight cultures were diluted in MHB until reaching 10 6 CFU/mL. The antimicrobial activity of the CNCs and r-CNCs suspensions was assessed by determining the minimum inhibitory concentration (MIC) in a 96 flat-bottomed-well tissue culture microplate. Briefly, 100 μL of sterile Mueller Hinton were added to all wells. In the first column, the wells were filled with 50 μL of CNCs (50 mg.ml -1 ) and r-CNCs (22 mg.ml -1 ) suspensions. Then, 50 μL of each antimicrobial substance were serially transferred from the well to the corresponding wells. Then, 50 μL of the culture suspension were added to all wells. Positive and negative controls (streptomycin and culture medium -data not shown) were included in each assay plate.

Afterward, the inoculated plates were incubated in a wet chamber for 24 h at 35 [START_REF] Ly | Surface functionalization of cellulose by grafting oligoether chains[END_REF]. The esterification of CNCs surface with rosin changed the relative proportion of C4 and C1 peaks due to the higher amount of aliphatic carbon from rosin. Furthermore, the O/C ratio decreased from 0.61 to 0.33 (Table 4.1), also confirming the successful grafting of rosin onto the CNCs. Niu et al.

[277] found similar behavior for rosin-modified cellulose nanofiber. observed in the spectrum of r-CNCs, assigned to ester carbonyl groups from the reaction between CNCs OH groups and -COOH groups of rosin. Likewise, the increased intensity of the 1647 cm -1 band can be related to C=C stretching from rosin cyclic alkene rings. There was no significant reduction in the intensity of the O-H band at 3340 cm -1 , indicating that esterification occurred mainly with hydroxyl groups accessible at the CNCs surface, as reported by Castro et al. [START_REF] Castro | Surface grafting of cellulose nanocrystals with natural antimicrobial rosin mixture using a green process[END_REF]. The absence of a band at ~1700 cm -1 from rosin carbonyl groups indicates that the residual rosin was completely removed after the purification step. showed a reduction of 20.8 and 29.1 in the UVB and UVA transmittance, compared to the gelatin/CNCs films, respectively. Narayanan et al. [START_REF] Narayanan | UV protective poly(lactic acid)/rosin films for sustainable packaging[END_REF] observed a reduction of 1% and 6% for UVB and UVA, respectively, in PLA bionanocomposites with 20 wt% rosin. The authors attributed this reduction to the absorbance by rosin which prevents light transmission through the films.

Contact angle of gelatin films reinforced with CNCs and r-CNCs

However, gelatin films with 6.0 wt% r-CNCs showed a significant reduction in the Table 4.2 presents a comparison of light transmission of the gelatin/r-CNCs bionanocomposites films with some synthetic packaging films.

The gelatin/r-CNCs films had low transmittance over the entire spectral range, indicating that they absorb light much more efficiently than several synthetic polymer films. These results suggest that the gelatin/r-CNCs bionanocomposites are suitable for packaging to protect light-sensitive food products. a adapted from [START_REF] Ramos | Edible Films and Coatings from Whey Proteins: A Review on Formulation, and on Mechanical and Bioactive Properties[END_REF].

Barrier properties of gelatin/CNCs and gelatin/r-CNCs films

Water vapor permeability (WVP) and oxygen transmission rates (OTR) are barrier properties that determine the ability of bio-based films to protect food products from moisture transfer, oxygen uptake, lipid oxidation, and loss of volatile aromas and flavors. Gelatin films generally display good barrier against oxygen at low and intermediate relative humidity [START_REF] Rhim | Bio-nanocomposites for food packaging applications[END_REF]. Our results show that the OTR of gelatin films with 0.5 and 6.0 wt% CNCs and r-CNCs at 0% and 80% RH According to Ooi, Ahmad, & Amin [START_REF] Ooi | Cellulose nanocrystals extracted from rice husks as a reinforcing material in gelatin hydrogels for use in controlled drug delivery systems[END_REF], nanoparticles can reduce WVP by increasing the biopolymer crystallinity or by reducing the free hydrophilic groups (OH, NH) in the gelatin matrix, thereby creating a tortuous pass for water vapor diffusion through the film. Santos et al. [START_REF] Santos | Fish gelatin films as affected by cellulose whiskers and sonication[END_REF] reported decreased WVP of protein films with increasing CNCs contents. They found that fish gelatin films with 15 wt% CNCs had WVP values of approximately 2 g mm/m 2 h kPa (25 ˚C at 85% RH). George & Siddaramaiah [START_REF] George | High performance edible nanocomposite films containing bacterial cellulose nanocrystals[END_REF] reported that 4.0 wt% bacterial cellulose nanocrystals reduced WVP of gelatin, and attributed this outcome to the low hygroscopicity of highly crystalline CNCs. They found WVP values around 0.175 g mm/m 2 h kPa (25 ˚C at 50% RH), which were higher than those of our gelatin films reinforced with CNCs or r-CNCs. The mechanical properties, Young's modulus, tensile strength, and elongation at break of bionanocomposite films were investigated using tensile tests. The Young's modulus and tensile strength of pure gelatin film were 743 and 17 MPa, respectively, as shown in Figure 4.12. For gelatin/CNCs bionanocomposite films, the tensile strength and Young's modulus increased significantly with increasing CNCs content. These increases can be attributed to the CNCs reinforcing effect through effective stress transfer from the gelatin matrix to CNCs [START_REF] Dufresne | Polysaccharide nanocrystal reinforced nanocomposites[END_REF]. For the same filler content of 4.0 wt%, the gelatin film with r-CNCs exhibited tensile strength ~30% higher than that of the film with CNCs.

This improvement is likely due to the intermolecular interactions such as hydrogen bonds and Van der Waals forces between the rosin graphitized onto CNCs and gelatin matrix. Besides, the slightly increased crystallinity of r-CNCs may have contributed to the higher stiffness of the bionanocomposites. However, for gelatin/r-CNCs films with 6.0 wt% r-CNCs, a reduction in Young's modulus and tensile strength was observed. In addition, there was an increase in elongation at break, which can be attributed to the formation of aggregates, as revealed by SEM. Furthermore, the gelatin film with 6.0 wt% r-CNCs showed an elongation at break ~145% higher than that of gelatin film, indicating that the gelatin/r-CNCs films were much more ductile and flexible. The interaction formed between the gelatin and r-CNCs weakened the protein-protein interactions, which were effective in stabilizing the gelatin network, as described by Zhuang et al [START_REF] Zhuang | Eco-friendly biorefractory films of gelatin and TEMPO-oxidized cellulose ester for food packaging application[END_REF].

Therefore, it is clear that r-CNCs play an effective role in enhancing the mechanical properties of gelatin, with promising features for flexible food packaging.

Antimicrobial properties of r-CNCs and gelatin/r-CNCs films

The minimum inhibitory concentration (MIC) value was determined as the lowest concentration of r-CNCs suspension that inhibited the growth of tested microorganisms. The results in Figure 4.13 show the highest bactericidal effect against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli with the r-CNCs suspension. As expected, there was no inhibitory effect for the CNCs suspension. A low concentration of r-CNCs (5.5 mg/mL) inhibited the growth of S. aureus, whereas for E. coli inhibition required 22 mg/mL. This may be due to the external lipopolysaccharide layer of the cell membrane of Gramnegative bacteria, which restricts the diffusion of hydrophobic compounds [START_REF] Santos | Recent developments in antimicrobial polymers: A review[END_REF].

The proposed mechanism for r-CNCs antimicrobial activity is based on its interaction with the phospholipid cell membrane, which causes increased effective antibacterial property, especially against E. Coli, since these strains did not grow in the inhibition area covered with gelatin/r-CNCs films. Neither of the tested films yielded a halo or a surrounding clearing zone, which shows that r-CNCs do not diffuse through the adjacent agar media and their antimicrobial effect is only by contact. The lack of diffusion is related to the hydrophobic nature of rosin molecules grafted onto the r-CNCs. Indeed, migration is linked to factors such as molecule size, polarity, shape, and quantity of water in the agar, in addition to the chemical structure and crosslinking in the films [START_REF] Cagri | Antimicrobial, mechanical, and moisture barrier properties of low pH whey protein-based edible films containing p-aminobenzoic or sorbic acids[END_REF]. was no microbial growth in the sample packed in 6 wt% r-CNCs-loaded bionanocomposite films. As a proof of concept, we illustrated that the gelatin/r-CNCs bionanocomposite films can extend the shelf-life of mozzarella cheese, also providing a direct indication of the antibacterial activity of r-CNCs even after forming bionanocomposites with gelatin. However, it is possible that r-CNCs migrated towards the cheese surface in contact with the film, but this has to be confirmed with further quantitative studies.

Conclusion

CNCs were successfully functionalized with rosin and used as a bactericidal nanofiller in gelatin for achieving multifunctional packaging. In particular, the r-CNCs consistently improved the optical, and water vapor barrier properties of gelatin films as compared to conventional CNCs. Besides, the mechanical strength of the gelatin matrix was increased and could be tuned by varying the r-CNCs content. This study demonstrates how grafting reactions can extend the functionalities of nanocelluloses for use in flexible packaging materials, which otherwise would suffer from limited physical and biological properties, as well as microbial counting assays to assess the performance of the gelatin/r-CNCs bionanocomposites as antimicrobial packaging materials for cheese and other food items stored at different conditions. These results suggest that the incorporation of low amounts of r-CNCs as active agents in gelatin films may be highly suitable for producing antimicrobial packaging materials.

Abstract

In this work, we explored gelatin (G) films incorporated with tannic acid (TA) and cellulose nanocrystals (CNCs), which fulfill the most relevant requirements for a natural antioxidant and antimicrobial food packaging material. The effects of nonoxidized TA, oxidized TA (OTA), and CNCs on the physical, morphological, antioxidant, and antimicrobial properties of the films were investigated. QCM-D and rheological measurements confirmed the interactions of the gelatin with TA and the formation of hydrogen bonds between CNCs and gelatin. The decreased crosslinking effect in the G/TA film obtained from non-oxidized TA increased the antioxidant capacity of the film by 52% due to the higher TA availability as an antioxidant. This also allowed the gelatin groups to form hydrogen bonds with CNCs to a larger extent, which resulted in a substantial increase in the viscosity (410%) and mechanical stability (G' 1697%) of the films. The addition of 6 wt% CNCs resulted in films with a denser structure, with reduced interlayer space and low porosity, as revealed by SEM. As a result, the bionanocomposite displayed lower WVP (67%) and greater tensile strength (79%) compared to the G-TA films.

The G-TA-CNCs films also demonstrated an inhibitory effect against Staphylococcus aureus and Escherichia coli growth.

Introduction

A new generation of multifunctional bionanocomposites based on ecofriendly polymers has emerged as a new trend in food packaging. In this context, researches have been focused on incorporating antimicrobial and antioxidant agents in packaging films to extend food shelf life [START_REF] Sivakanthan | Antioxidant and antimicrobial applications of biopolymers: A review[END_REF], especially concerning lipid oxidation and microbial spoilage, which are the major causes of deterioration in a great variety of foods [START_REF] Gómez-Estaca | Advances in antioxidant active food packaging[END_REF][START_REF] Wang | Decrease of microbial community diversity, biogenic amines formation, and lipid oxidation by phloretin in Atlantic salmon fillets[END_REF].

Traditionally, synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butylated hydroquinone (TBHQ) are used in food products. However, the potential toxicity of these compounds [START_REF] Vilela | A concise guide to active agents for active food packaging[END_REF] has made natural antioxidants like plant essential oils [START_REF] Chollakup | Antioxidant and antibacterial activities of cassava starch and whey protein blend films containing rambutan peel extract and cinnamon oil for active packaging[END_REF] and extracts [START_REF] Zhang | Plant extracts such as pine nut shell, peanut shell and jujube leaf improved the antioxidant ability and gas permeability of chitosan films[END_REF] to become increasingly valuable among consumers. Tannic acid (TA) is an inexpensive plant-derived polyphenol that has received considerable attention due to its multifunctionality, including antioxidant [START_REF] Gülçin | Radical scavenging and antioxidant activity of tannic acid[END_REF], antimutagenic [START_REF] Chen | Mutagenicity and antimutagenicity studies of tannic acid and its related compounds[END_REF], anticarcinogenic and antimicrobial properties [START_REF] Guo | Development of tannin-inspired antimicrobial bioadhesives[END_REF]. TA is also classified as a Generally Recognized as Safe (GRAS) ingredient by the Food and Drug Administration (FDA), meaning that it can be directly used in food products [START_REF] Aewsiri | Antioxidative activity and emulsifying properties of cuttlefish skin gelatin-tannic acid complex as influenced by types of interaction[END_REF].

Tannic acid has been used to develop multifunctional films and coatings, and as a natural crosslinker for gelatin [START_REF] Park | Developing regulatory property of gelatin-tannic acid multilayer films for coating-based nitric oxide gas delivery system[END_REF]. Crosslinking of proteins with polyphenols has been found to enhance film performance, depending on the type of polyphenol-protein interaction (covalent or non-covalent). For instance, oxidation of polyphenols produces quinone compounds, which react with amino acid residues of gelatin chains via covalent bonds (C-N or C-S), generating crosslinked films [START_REF] Choi | Effect of oxidized phenolic compounds on cross-linking and properties of biodegradable active packaging film composed of turmeric and gelatin[END_REF]. By contrast, non-covalent bonds, such as hydrogen bonding and hydrophobic interactions, can be formed between gelatin and polyphenols at acidic or neutral pH, and are reversible in their nature [START_REF] Zhang | Chemical modification of gelatin by a natural phenolic cross-linker, tannic acid[END_REF] All chemicals were analytical grade and were used as purchased.

Staphylococcus aureus (S. aureus, Gram-positive, ATCC 25923) and

Escherichia coli (E. coli, Gram-negative, ATCC 25922) were supplied by Cefar Diagnostica (Brazil). Distilled water was used in all experiments.

Film-forming solution (FFS) preparation

A 10 wt% gelatin solution was prepared by first hydrating the gelatin powder in distilled water at 24 °C for 5 min, followed by heating at 60 °C under mechanical stirring for 15 min. Glycerol (20 wt% on a dry gelatin mass basis) was added to the gelatin solution under stirring at 60 °C for 5 min. Afterward, 10 mL of a TA solution (1.8 wt% in water) were slowly added to the gelatin solution and stirred for 1 h at 60 °C. The obtained solution was mixed and stirred for 1 h at 60 °C with 10 mL of CNCs dispersion at designed CNCs contents (4.0 wt% and 6.0 wt% on a dry gelatin mass basis) previously sonicated for 5 min. Pure gelatin (without TA and CNCs) and gelatin-TA solution (without CNCs) solutions were prepared likewise for comparison purposes. The samples were codified as Gel, G-TA, G-TA-CNC4, and G-TA-CNC6. The latter formulation (G-TA-CNC6) was also produced using a different mixing sequence, namely, the TA solution was first stirred with the CNCs suspension for 1 h at 25 °C to oxidize the TA molecules into quinone, followed by the addition to the plasticized gelatin solution under stirring for 1 h at 60 °C. This sample was codified as G-OTA-CNC6. All the solutions were allowed to rest for 60 min to eliminate bubbles before casting.

Film casting

The FFS samples (45 mL) were cast on leveled glass plates (40 cm × 25 cm) covered with a polyester film (Mylar®, DuPont, Brazil), to facilitate film solutions/suspensions at 25 o C. Briefly, the CNC-coated silica sensors were equilibrated overnight in water (flowed at 10 μL/min) in the QCM-D instrument to achieve a stable baseline. The samples were then injected at a flow rate of 100 μL/min, allowed to adsorb for 50 min, followed by 30 min of rinsing with water.

The order of injections varied for each run while keeping constant the time of each injection (TA solution, gelatin solution, glycerol solution, or CNC dispersion).

For the QCM-D measurements, 13 overtones were monitored and representative curves from 3 replicates of the third overtone were used.

Rheological properties

The rheological measurements were carried out on a Physica MCR101 rheometer (Anton Paar, Austria) using the concentric cylinder geometry. The apparent viscosity of FFS was measured within the shear rate range 0-500 s -1 at 35 °C The flow behavior was described by fitting the experimental data to the Power Law model (Eq. 2).

 = k

(2

)
Where  is the shear stress (Pa),  is the shear rate (s -1 ), k is the consistency coefficient (Pa s n ) and n is the flow behavior index.

Oscillatory shear testing was carried out to determine the storage modulus (G'), loss modulus (G") and tan δ (G'/G'') of the FFS at a constant strain of 1% and frequency of 1 Hz with heating from 10 to 50 °C and then cooling down to 10 °C using constant rate of 0.02 °C/s. Frequency sweep tests were performed in the 0.1-100 Hz range at strain 1 %. The films were cut into 11-mm discs and the frequency sweep tests were conducted from 0.1 to 100 Hz at a fixed strain of 0.1%, using a 25-mm plate-plate geometry Amplitude sweep experiments were previously carried out to determine the linear viscoelastic region of each sample.

Determination of crosslinking degree

. .

The tannic acid-gelatin crosslinking degree in FFS and films was determined by UV/VIS spectrophotometry at l = 275 nm (Model PC, Shimadzu, Kyoto, Japan), as described by [START_REF] Picchio | Casein films crosslinked by tannic acid for food packaging applications[END_REF]. TA was added to the FFS at 3 x 10 -5 mol/L.

The TA extraction from the gelatin films was done through several washing cycles with 50 mL of water until the phenolic compound was not observed in the UV spectrum. The amount of free TA was then calculated for each washing cycle using an absorbance vs concentration calibration curve. The crosslinking degree was estimated as the difference between the initial TA amount added to the FFS and the amount of free TA extracted from the samples.

Scanning electron microscopy (SEM)

The cross-sectional surface of films before and after swelling in water for 24 h was investigated using a JEOL Scanning Electron Microscope (SEM, model Quanta200, FEI, The Netherlands). The film samples were first cryo-fractured in liquid N2. Swollen films were freeze-dried for 24h. The films were, fixed onto 90° specimen mounts and then coated with a ca. 5-nm-thick gold layer in argon atmosphere. The SEM images were obtained from the secondary electron mode at accelerating voltages below 5 kV.

Determination of antioxidant activity

Antioxidant capacity was determined in triplicate using the DPPH (2,2diphenyl-1-1picrylhydrazyl) free radical scavenging method. The samples were cut into small pieces (45 mg) and immersed into 10 ml of DPPH * solution (0.003 % in ethanol 95%). The mixtures were then stirred at 23 °C and 100 rpm in the dark. The absorbance of the resulting solution was measured at 517 nm by UV/Vis spectrophotometry (Model PC, Shimadzu, Kyoto, Japan) at different times. The DPPH radical scavenging activity (RSA) was determined according to Eq. 3.

,0(%) = ⌊ h ijklmn 4 omjpq r omjpq Y100⌋

where Asample is the absorbance of DPPH * solution containing the film sample and Ablank is the absorbance of DPPH * solution. For each film, three replicates were performed.

UV-Vis spectroscopy and transparency determination

UV-Vis transmittance spectra were acquired on a UV-1650 spectrophotometer (Model PC, Shimadzu, Kyoto, Japan) at λ = 190 -800 nm.

Three replicates were performed for each film. Film transparency was calculated using Eq. 4 [START_REF] Han | Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity[END_REF]:

J LJ M NO = P.. ( 4
)
where A600 is the absorbance at 600 nm and x is the film thickness (mm).

Film thickness was measured with a digital micrometer (Mitutoyo Corp., Kanogawa, Japan) to the nearest 0.001 mm.

Contact angle measurements

Contact angle measurements were conducted with CAM 101 Optical Contact Angle Meter (KSV Instruments, Finland) equipped with a CCD KSV-5000 digital camera. For each measurement, 5 µL of ultrapure water was dropped on the film surface and 100 images were automatically recorded within an experimental time of 60 s using the KSV CAM2008 software. Contact angle determinations were performed with 5 replicates as per ASTM D5725-99 (2008).

Tensile tests

The mechanical properties were measured according to ASTM D882-09

Water vapor permeability and moisture content determination

Water vapor permeability (WVP) was determined as per ASTM E-96-01.

Briefly, the preconditioned film sample was used to seal an aluminum permeation cup (28 mm in internal diameter) containing dried calcium chloride. The cup was kept in a controlled chamber at 23 ± 1 °C and 50 ± 5% RH and weighed every 4 h until 7 days. For each film, three replicates were performed. The WVP [(g mm)/ m 2 h kPa)] was determined using Eq. 5:

tu& = vw'( 3 <∆x ( 5
)
where WVTR is the water vapor transmission rate (g/h) obtained as the slope of the mass vs time curve, S is the effective film area; L is the film thickness (mm), and ∆P is the difference of partial pressure (kPa) across the film.

The moisture absorption of the films was analyzed using the method described by Zhuang et al. (2017) [START_REF] Zhuang | Eco-friendly biorefractory films of gelatin and TEMPO-oxidized cellulose ester for food packaging application[END_REF] with slight modifications. Rectangular (20 mm x 10 mm) film specimens were conditioned at 24°C and 50 ± 5% RH for 2 days until achieving constant weight (mi). The samples were transferred to desiccators at 90% ± 5% RH (supersaturated CuSO4.5H2O solution) at 24 °C for 2 days and then they were gently blotted and weighed (mf). The moisture absorption was calculated using Eq. 6. Three measurements were done for each film.

6 ydz M JK Ldy (%) = { | Y100 (6)

Antimicrobial tests

The bacterial strains to evaluate the effectiveness of CNCs-and TAloaded solutions were Staphylococcus aureus and Escherichia coli, which are foodborne pathogens [START_REF] Paudyal | Microbiology prevalence of foodborne pathogens in food from selected African countries -a meta-analysis[END_REF][START_REF] Ferrario | Microbiology next generation sequencing-based multigene panel for high throughput detection of food-borne pathogens[END_REF]. The inoculum cultures were prepared

Statistical analysis

The results were expressed as mean value ± standard deviation and were subjected to analysis of variance (ANOVA). The mean values were compared using the Tukey's test at a confidence level of 95% (p < 0.05).

Results and discussion

Cellulose nanocrystals characterization

TEM revealed that the CNCs used in this work presented needle-like morphology with average length (L) and diameter (D) of 108 ± 33 nm and 3.8 ± 0.9 nm, respectively, resulting in an aspect ratio (L/D) of around 28.4 (Figure 4.

Gelatin, tannic acid, and CNCs interactions

The QCM-D experiments allowed us to predict the interactions between the film components Figure 4.18. As reported by [START_REF] Rodahl | Quartz crystal microbalance setup for frequency and Qfactor measurements in gaseous and liquid environments[END_REF] [START_REF] Rodahl | Quartz crystal microbalance setup for frequency and Qfactor measurements in gaseous and liquid environments[END_REF], the change in resonance frequency is related to the mass added to the crystal, and thus adsorption or desorption of material induces a frequency shift. From the frequency and dissipation data, it can be suggested that TA adsorbed weakly to the CNCs surface (Figure 4.18a). This minimal adsorption is depicted as a minor decrease in frequency (-8 Hz) along with a minor increase in the dissipation when TA is injected over the CNCs-coated sensor. However, this interaction was found to be reversible since the water rinse brought the frequency back to the baseline, indicating that TA was washed off the sensor surface. This is in contrast to past studies [START_REF] Hu | One-Pot Water-Based Hydrophobic Surface Modification of Cellulose Nanocrystals Using Plant Polyphenols[END_REF][START_REF] Palange | The effect of the dispersion of microfibrillated cellulose on the mechanical properties of melt-compounded polypropylene-polyethylene copolymer[END_REF] that have shown that TA can modify the nanocellulose surface by forming a "primer" layer. However, the interaction/reaction between components was promoted through conditions that allowed for oxidative processes to occur, and by increasing the pH, which is different from the neutral pH and closed flow cell conditions used herein.

Unlike the gelatin-TA interaction, the gelatin-CNCs interaction was seen to be strong (Figure 4.18a-c). with a normalized decrease in frequency of about -500 Hz. This was seen most directly when the gelatin FFS flowed over the neat CNCs surface (Figure 4.18b). This was also apparent if the gelatin adsorption step came after the TA or glycerol injection (since these two components do not bind irreversibly to CNCs). The large frequency change after gelatin injection and the lack of frequency change after the water rinse supports the interactions between cellulose and gelatin reported previously [START_REF] George | High performance edible nanocomposite films containing bacterial cellulose nanocrystals[END_REF]. The largest frequency shifts were seen for TA adsorbing to gelatin (-1900 Hz) (Figure 4.18b,c). The frequency shift was larger when gelatin was adsorbed to the CNCs surface prior to TA, in comparison to when TA was added first to CNCs followed by gelatin.

The adsorption of TA to gelatin may be explained by the physical interaction and/or cross-linking reaction that occurs between the amine groups of gelatin chains and the catechol groups of TA [START_REF] Muhoza | Gelatin and high methyl pectin coacervates crosslinked with tannic acid: The characterization, rheological properties, and application for peppermint oil microencapsulation[END_REF]. This large frequency shift was accompanied by a spike in dissipation upon injection of TA, indicating that the TA-gelatin interaction results in a "soft" binding and, consequently, some TA molecules are washed off during the rinsing step (Figure 4.18b,c).

To further investigate the nature of the cross-linking between TA and gelatin, a longer water rinsing step followed by CNCs injection was tested (Figure 4.18d). It was observed that CNCs do not adsorb onto the outer cross-linked gelatin-TA layer. The CNCs injection step was likely to continue the rinsing step, the same trend of slightly increasing frequency occurred during water rinsing and CNCs flow. The QCM-D data showed that glycerol does not adsorb to any of the components (i.e., no decrease in frequency was observed upon glycerol injection), regardless of the previous layer adsorbed to the sensor. Thus, glycerol is most likely acting as a chemically inert plasticizer in the film.

Crosslinking degree in the gelatin-based films with TA or OTA

The amount of free TA released from the gelatin-based films was calculated for each washing cycle using a concentration curve that correlates concentration with absorbance (Figure 4.23a). Finally, the crosslinking degree was estimated by the difference between the initial amount of TA added to the FFS and the amount extracted in the washing water of each film. The amount of TA released from all the film samples is shown in Figure 4.23b. The amount of crosslinked TA in the G-TA film was around 45% and decreased to 36% when the CNCs were added at 6 wt% to the gelatin-TA FFS, indicating that the G-TA-CNC6 film had a higher free TA content (not covalently bonded to gelatin). This was probably because hydrogen bonding and electrostatic interactions between sulfate groups of CNCs and the cationic amine groups of gelatin decreased the gelatin-TA interactions, as previously observed for CNCs and chitosan [START_REF] Rubentheren | Physical and chemical reinforcement of chitosan film using nanocrystalline cellulose and tannic acid[END_REF][START_REF] Pereda | Polyelectrolyte films based on chitosan/olive oil and reinforced with cellulose nanocrystals[END_REF].

The crosslinking degree in the G-TA-CNC6 films was lower than the previous results. Picchio et al. ( 2018) [START_REF] Picchio | Casein films crosslinked by tannic acid for food packaging applications[END_REF] found that 97% of phenolic compounds were crosslinked to casein at pH 9. In this case, the alkaline conditions favored the TA oxidation and crosslinking with proteins. The G-OTA-CNC6 film showed 53% TA crosslinked, due to the oxidation of TA molecules to quinones, as previously mentioned, which favors the crosslinking reaction between quinones and amino acid groups [START_REF] Muhoza | Gelatin and high methyl pectin coacervates crosslinked with tannic acid: The characterization, rheological properties, and application for peppermint oil microencapsulation[END_REF][START_REF] Santos | Fish gelatin films as affected by cellulose whiskers and sonication[END_REF]. films, compared to the physical Gel film. This result confirms that gelatin was effectively crosslinked by TA. Furthermore, both G' and G" exhibited a plateau over all the frequency range analyzed, which is indicative of a stable crosslinked network. Similar results were observed for gelatin films crosslinked with oxidized

Viscoelastic behavior of the gelatin-based films

CNCs [START_REF] Dash | Improving the mechanical and thermal properties of gelatin hydrogels cross-linked by cellulose nanowhiskers[END_REF]. The highest G' was found for the G-TA-CNC6 films, which increased by a factor of 15 to G-OTA-CNC6 films. This is most likely due to the decrease in the crosslinking degree between the gelatin chains and TA molecules in the G-TA-CNC6 films, which favored the non-covalent interactions between the CNCs, gelatin matrix, and TA. corroborating with the results from the rheological analysis (Table 4. 4 and Figure 4.19). This also indicates that a higher degree of noncovalent intermolecular connectivities led to a significantly higher mechanical stability to the gelatin films than the covalently crosslinked films with OTA (G-OTA-CNC6).

Morphological characterizations of gelatin-based films with TA or OTA

The microstructure of the gelatin/CNCs film with TA or OTA was examined by SEM. To further assess the effect of the TA crosslinking and CNCs percolation network on the stability of the gelatin matrix, the samples were also characterized after swelling in water for 24 ºC at room temperature. Figure 4.25 shows the SEM images of the fractured cross-sectional surface of the films before and after swelling for 24h in water. The Gel film (Figure 4.25a) presented a homogenous and smooth surface with good structural integrity, before and after swelling. By contrast, the G-TA film before swelling (Figure 4.25b) showed an irregular and compact surface, with particulate-like structure without any phase separation.

[303] also found a similar morphology for TA-crosslinked chitosan films.

Nevertheless, the swollen G-TA film presented an open network structure with interconnected pores of regular sizes. Similar findings were reported by Bertolo et al. (2020) [START_REF] Bertolo | Rheological and antioxidant properties of chitosan/gelatin-based materials functionalized by pomegranate peel extract[END_REF], who attributed this morphology to the chemical protein/phenolic crosslinking, which offers more intermolecular association and junction points to the film structure.

The G-TA-CNC6 film (Figure 4.25c) had a more regular and compact surface before swelling, and the related swollen film had a denser structure than that of G-TA, with decreased interlayer space and low porosity. This is due to the greater number of covalent and non-covalent bonding, which is supported by the 2018) [START_REF] Choi | Effect of oxidized phenolic compounds on cross-linking and properties of biodegradable active packaging film composed of turmeric and gelatin[END_REF] reported that an active compound is usually released from a swollen polymeric matrix by hydration, followed by diffusion of the active compound through the expanded polymeric matrix. The gelatin-TA crosslinking through covalent bonds leads to a lower TA release from the crosslinked films, consequently, a lower hydrogen donor ability, which results in a reduction of antioxidant activity when compared with free TA (89%). This was in accordance with Aewsiri, Benjakul, Visessanguan, Wierenga, & Gruppen, (2010) [START_REF] Aewsiri | Antioxidative activity and emulsifying properties of cuttlefish skin gelatin-tannic acid complex as influenced by types of interaction[END_REF] who found similar results for gelatin-TA complexes. Accordingly, the G-TA-CNC4 and G-TA-CNC6 films showed higher antioxidant activity in comparison with the G-OTA-CNC6 (56%) and G-TA (56%) films, which presented the highest crosslinking degrees [START_REF] Sun | Use of sulfated cellulose nanocrystals towards stability enhancement of gelatin-encapsulated tea polyphenols[END_REF].

Furthermore, the addition of CNCs sustained the enhanced antioxidant capacity of the gelatin films over three months (Figure 4.27b). This trend was similar to that observed by [START_REF] Sun | Use of sulfated cellulose nanocrystals towards stability enhancement of gelatin-encapsulated tea polyphenols[END_REF], which demonstrated that the antioxidant capacity of gelatin-tea polyphenols (G-TPP) dispersions were dependent on storage time and temperature, and also that the addition of sulfated cellulose nanocrystals (SCNC) resulted in improvement in their antioxidative stabilities.

The decrease in the antioxidative stabilities was only 8.9% for SCNC-G-TPP against 17.2% for G-TPP suggesting that there is a strong effect of matrix interactions, and the presence of SCNC increases the physical strength of gelatin-based capsules by hydrogen bonding. Therefore, as the gelatin-based films with non-oxidized TA showed the best antioxidant properties, they were chosen to evaluate the influence of CNCs and TA on the physical properties of the gelatin nanocomposite films, which will be presented below.

Optical properties of gelatin-based films

Photographs of the gelatin-based films are presented in Figure 4.28. All the gelatin films showed good macroscopic homogeneity and continuity, with no cracks or visible aggregates on the surface. Table 4.5 shows the light transmission at different wavelengths and the transparency value of the samples.

The Gel film exhibited a high barrier against UVC (200-280 nm), due to the chromophore groups present in the amino acid residues of gelatin chains, such as tyrosine and phenylalanine [START_REF] Chuaynukul | Comparative Characterization of Bovine and Fish Gelatin Films Fabricated by Compression Molding and Solution Casting Methods[END_REF]. After the addition of TA, the UVC transmission became negligible, due to the presence of the TA polyphenol groups, which are well-known as sensitive chromophores concerning the absorption of light at wavelengths below 300 nm. [START_REF] Halim | Chitosan, gelatin and methylcellulose films incorporated with tannic acid for food packaging[END_REF] [109] also found similar results

for G-TA films.

The addition of CNCs reduced the UV light transmission through the films.

This is attributed to the hydroxyl groups of cellulose, which act as auxochrome groups [START_REF] Uranga | Cross-linking of fish gelatins to develop sustainable films with enhanced properties[END_REF]. As presented in Table 4.5, the gelatin films had improved light barrier from 13% to 19%, as similarly observed in a previous report [START_REF] Halim | Chitosan, gelatin and methylcellulose films incorporated with tannic acid for food packaging[END_REF]. This was due to the plasticizing effect of TA on the gelatin matrix. Nevertheless, the addition of CNCs to the gelatin matrix caused a significant increase (p < 0.05) by 79% in TS compared to the G-TA films. This effect is mainly due to the formation of a rigid percolated network with a large amount of hydrogen bond interactions between the hydroxyl groups of CNCs and TA with polar groups of gelatin. In a previous study, the contact percolating network of 7 wt% CNCs had a considerable impact on the mechanical properties of collagen-CNCs nanocomposites [START_REF] Rodrigues | Control of properties of nanocomposites biobased collagen and cellulose nanocrystals[END_REF]. These interactions can also lead to the enhancement of mechanical properties even if the volume fraction of CNCs is low, as demonstrated in Chapter II.

Mechanical properties of gelatin-based films

The resistance of the films to be perforated was determined by puncture tests and the results for the gelatin-based films are shown in Figure 4.30b.

Comparing the values of the puncture force of the G-TA and G-TA-CNC6 films,

Water barrier properties and water vapor absorption of gelatin-based films

The water vapor barrier is an important property for packaging films since moisture exchanges are a major cause of quality loss in foods, especially those with low water activity. As presented in Figure 4.31 the films containing CNCs had a lower water vapor permeability (WVP) value than the other samples. The gelatin films showed a WVP of 0.18 g mm/(kPa h m 2 ). WVP values in the range of 0.17-2.0 g mm/(kPa h m 2 ) have been reported for gelatin films [START_REF] Suderman | The effect of plasticizers on the functional properties of biodegradable gelatin-based film: A review[END_REF][START_REF] Menezes | Effect of tannic acid as crosslinking agent on fish skin gelatin-silver nanocomposite film[END_REF][START_REF] Santos | Fish gelatin films as affected by cellulose whiskers and sonication[END_REF].

The addition of TA, as a crosslinking agent, significantly decreased (p < 0.05) by 28% in WVP of these films, as presented in Figure 4.31. A previous study [START_REF] Carvalho | Effect of chemical treatment on the mechanical properties, water vapour permeability and sorption isotherms of gelatin-based films[END_REF] had also found that incorporating glyoxal and formaldehyde as crosslinkers to bovine gelatin films decreased the WVP of the films by 12% and 22%, respectively, in comparison with pure gelatin films. An advantage of this thesis work is that TA is a nontoxic crosslinker, in contrast to the high toxicity of compounds such as formaldehyde, which may not be tolerable in many applications, including food contact materials. Furthermore, the addition of 4.0 wt% and 6.0 wt% CNCs in the G-TA film significantly reduced (p <0.05) the WVP of the gelatin matrix by 83% and 67% respectively. This phenomenon is the result of interactions between CNCs and gelatin, as well as an increased tortuosity of the pathway for vapor diffusion through the film matrix [START_REF] Slavutsky | Water barrier properties of starch films reinforced with cellulose nanocrystals obtained from sugarcane bagasse[END_REF].

The swelling properties of gelatin films were also evaluated from their moisture uptake value at 90% RH. All the films attained equilibrium in 2 days. As shown in Figure 4.31, the moisture content did not change significantly after crosslinking the gelatin films with TA. However, the addition of CNCs to the gelatin matrix decreased the moisture content, which is attributed to the formation of a rigid network, as observed by rheological experiments, which consequently has less ability to uptake water. The moisture uptake value decreased from 136% (for Gel) to 129% (for G-TA-CNC6). These results and WVP are in line with the compact structures observed by SEM (Figure 4.25c).

4.33c,d, g, h, and insets). The control films made with pure gelatin or gelatin/CNCs displayed no inhibitory effect, being totally covered by bacteria as well as the area under the film (Figure 4.33a,b, e, and f). According to the Standard SNV 195920-1992 [START_REF] Villanueva | Chitin nanowhiskers as alternative antimicrobial controlled release carriers[END_REF], the antibacterial property of a material may be classified as "good" when resulting in an inhibition zone around and under the sample, "sufficient" when the growth inhibition area is only under the sample, and "not sufficient" when the sample, as well as the area under the sample, is totally covered by bacteria. It is important to point out the higher film stability with CNCs (Figure 4.33b, f, d, and h) compared with ones without CNCs (Figure 4.33a, e, c, and g). The last one presented a drained path after the gelatin melting or diffusion, probably due to the similarities between the gelatin and the agar.

This result also reinforces the antimicrobial activity of TA, showing the absence of bacteria across this pathway. Therefore, the antibacterial property of these films can be classified as "sufficient" for antimicrobial packaging applications. However, future quantitative studies may provide more accurate information about the susceptibility of these organisms to the antibacterial action of the film G-TA-CNC6. Os filmes a base de gelatina podem ser feitos de uma variedade de matériasprimas, sendo as mais utilizadas em embalagens de alimentos as derivadas de tecidos suínos [START_REF]Fabrication of cationized gelatin nanofibers by electrospinning for tissue regeneration[END_REF], peixes [START_REF] Hanani | Effect of pomegranate (Punica granatum L.) peel powder on the antioxidant and antimicrobial properties of fish gelatin films as active packaging[END_REF] e bovinos [START_REF] Quero | Stress transfer and matrix-cohesive fracture mechanism in microfibrillated cellulose-gelatin nanocomposite films[END_REF]. As propriedades físico-químicas dos filmes de gelatina dependem da fonte, das condições de produção do filme (concentração, solvente, pH, temperatura, tempo de secagem do filme, etc.) e do tipo e concentração dos aditivos empregados (plastificante, carga de reforço, agentes reticulantes, etc.).

Em geral, os filmes de gelatina são incolores e exibem alta transparência e excelentes propriedades de barreira na região do ultravioleta. Outra grande vantagem da gelatina como material de embalagem é sua excelente barreira contra dióxido de carbono e oxigênio, devido à sua natureza hidrofílica [START_REF] Avena-Bustillos | Gelation, oxygen permeability, and mechanical properties of mammalian and fish gelatin films[END_REF]. No entanto, a natureza higroscópica da gelatina também representa a sua principal desvantagem como filmes. Esses filmes são sensíveis à umidade e apresentam baixas propriedades mecânicas e barreira ao vapor de água em altas umidades relativas [START_REF] Suderman | The effect of plasticizers on the functional properties of biodegradable gelatin-based film: A review[END_REF]. Entretanto, essas desvantagens podem ser superadas pela incorporação de nanopartículas de reforço à matriz de gelatina, como por exemplo, os nanocristais de celulose (CNCs) [START_REF] Moustafa | Eco-friendly polymer composites for green packaging: Future vision and challenges[END_REF][START_REF] Hosseini | A state-of-the-art review on the elaboration of fish gelatin as bioactive packaging: Special emphasis on nanotechnology-based approaches[END_REF][START_REF] Azeredo | Nanocomposites for food packaging applications[END_REF][START_REF] Squinca | Nanocellulose production in future biorefineries: an integrated approach using tailor-made enzymes Nanocellulose production in future biorefineries : an integrated approach using tailor-made enzymes[END_REF][START_REF] Thomas | a versatile green platform: from biosources to materials and their applications[END_REF].

CNCs são domínios cristalinos extraídos de várias fontes naturais, incluindo madeira, plantas, microorganismos e animais que contêm celulose. CNCs apresentam uma combinação adequada de propriedades, como biodegradabilidade, alta razão de aspecto (~ 10-70), baixa densidade (1,5 -1,6

g/cm 3 ), baixo coeficiente de expansão térmica e transparência [START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF][START_REF] Huber | A critical review of all-cellulose composites[END_REF][START_REF] Lagerwall | Cellulose nanocrystal-based materials: from liquid crystal self-assembly and glass formation to multifunctional thin films[END_REF].

Além disso, as dimensões bem definidas dos CNCs e sua alta cristalinidade (70-90%) induzem excelentes propriedades mecânicas, como alta rigidez axial (~ 150 GPa), alto módulo de elástico (20-50 GPa) [START_REF] Gars | Polymerization of glycidyl methacrylate from the surface of cellulose nanocrystals for the elaboration of PLA-based nanocomposites[END_REF] e alta resistência à tração (~ 7 GPa) [START_REF] Sukyai | Effect of cellulose nanocrystals from sugarcane bagasse on whey protein isolate-based films[END_REF].

Além disso, a superfície dos CNCs pode ser facilmente modificada devido à reatividade dos seus grupos hidroxilas, o que torna os CNCs uma plataforma versátil para diversos tipos de funcionalização de superfícies. Essa característica permite expandir as aplicações específicas dos CNCs, tornando-os excelentes candidatos para desenvolvimento de nanocompósitos de alto desempenho em diversas aplicações [START_REF] Habibi | Key advances in the chemical modification of nanocelluloses[END_REF].

O que concerne ao processamento de filmes poliméricos para embalagens, a maioria dos estudos tem se concentrado em técnicas de processamento por extrusão, injeção ou moldagem por compressão a quente, devido às tecnologias já estabelecidas para polímeros à base de petróleo [START_REF] Reddy | Biobased plastics and bionanocomposites: Current status and future opportunities[END_REF]. No entanto, o processamento utilizando essas técnicas para polímeros naturais como proteínas, tem se mostrado um desafio devido principalmente à instabilidade térmica dessas macromoléculas.

Recentemente, um método de casting continuo foi proposto para a produção de filmes a base de biopolímeros em escala piloto. A produção de filmes por casting contínuo é realizada em uma linha de coating. Neste processo, as soluções filmogênicas são continuamente espalhadas sobre um substrato móvel, como poliéster ou papel revestido, com uma lâmina cuja altura pode ser ajustada para controlar a espessura do filme. Após, o substrato revestido com a solução laminada passa por um estágio de pré-secagem com aquecedores infravermelho e em seguida por câmaras de secagem. No final, o filme seco é enrolado em um rolo enquanto ainda está aderido ao substrato [START_REF] Otoni | Recent Advances on Edible Films Based on Fruits and Vegetables-A Review[END_REF].

A abordagem de casting contínua pode ser considerada uma ampliação do processamento por casting em bancada. A principal vantagem do casting continuo consiste no fato deste método requerer tempos de secagem curtos, graças ao uso da radiação infravermelha na fase de pré-secagem, temperaturas muito mais elevadas e intensa circulação de ar. Outras vantagens são o alto controle de espessura do filme, não requer o uso de aditivo de processamento, permite o uso de soluções/dispersões aquosas e possui menor necessidade de espaço e mão de obra. Essas características tornam o casting contínuo um processo atraente para a produção em larga escala de filmes a base de polímeros naturais.

Em linha com este contexto, vários objetivos podem ser anunciados para este doutorado: Les films à base de gélatine peuvent être composés d'une variété de matières premières, dont les tissus d'origine animale sont les plus utilisés [START_REF]Fabrication of cationized gelatin nanofibers by electrospinning for tissue regeneration[END_REF], [START_REF] Hanani | Effect of pomegranate (Punica granatum L.) peel powder on the antioxidant and antimicrobial properties of fish gelatin films as active packaging[END_REF], [START_REF] Quero | Stress transfer and matrix-cohesive fracture mechanism in microfibrillated cellulose-gelatin nanocomposite films[END_REF].

Les propriétés physico-chimiques des films à base de gélatine dépendent de la source de gélatine, de la production du film (comprenant la concentration de gélatine, le solvant, le pH, la température, le séchage) et du type/concentration des additifs (plastifiant, charges renforçantes, agents de réticulation).

En général, les films à base de gélatine sont incolores, transparent, avec Concernant la mise en forme des films polymères pour les emballages, la plupart des études sont concentrés sur les techniques par extrusion, injection ou moulage par compression à chaud, en raison de la technologie bien défini pour les polymères à base du pétrole [START_REF] Reddy | Biobased plastics and bionanocomposites: Current status and future opportunities[END_REF]. Cependant, l'utilisation de ces techniques appliquées aux polymères naturels, comme les protéines, sont considérés un vrai défi à cause de l'instabilité thermique de ces macromolécules.

Récemment, une méthode de casting continue a été proposée pour la production des films à base de biopolymères à l'échelle pilote. La production des films par casting continue est réalisée dans une ligne de casting. Dans ce processus, des solutions filmogènes sont disposées en continu sur un substrat mobile, par exemple polyester ou papier couché, avec une lame dont la hauteur est ajustée en fonction de l'épaisseur désirée. Ensuite, le substrat recouvert par la solution passe par une étape de pré-séchage par chauffage infrarouge puis par des chambres de séchage. À la fin, le film sec est roulé alors qu'il est encore adhéré au substrat [START_REF] Otoni | Recent Advances on Edible Films Based on Fruits and Vegetables-A Review[END_REF].

L'approche de la méthode de casting continue peut être considérée comme un

CHAPTER III:

  Evalution of physical properties of gelatin-based films under the influence of electrostatic interactions .................................................. Introduction to chapter III ................................................................................ 1 Evaluation of physical properties of gelatin-based films under the influence of electrostatic interactions ................................................................................. 1 Introduction .................................................................................................. 1.2 Materials and methods ............................................................................. 1.2.1 Materials ................................................................................................ 1.2.2 Preparation of gelatin/CNCs films .......................................................... 1.2.3 Determination of Zeta potentials (ζ-potentials) ...................................... 1.2.4 Rheological study .................................................................................. 1.2.5 Polarized light optical microscopy .......................................................... 1.2.6 Scanning electron microscopy (SEM) .................................................... 1.2.7 X-ray diffraction (XRD) analysis ............................................................. 1.2.8 Tensile tests .......................................................................................... 1.2.9 Water vapor permeability (WVP) ........................................................... 1.2.10 Water solubility (WS) determination..................................................... 1.2.11 Thermogravimetric analysis (TGA) ...................................................... 1.2.12 Statistical analysis ............................................................................... 1.3 Results and discussion ........................................................................... 1.3.1 Physical and rheological properties of gelatin/CNCs film-forming solutions….. .................................................................................................... 1.3.2 Characterization of gelatin/CNCs films .................................................. 1.3.2.1 Morphological and structural characterization .................................... 1.3.2.2 Mechanical properties ......................................................................... 1.3.2.3 Water sensitive properties .................................................................. xix 1.3.2.4 Thermostability ................................................................................... 1.4 Conclusion ............................................................................................... CHAPTER IV: Functionalization of CNCs and active gelatin-based films Introduction to chapter IV ............................................................................... 1 Functionalization of CNCs with natural antimicrobial rosin mixture and evaluation of their gelatin-based films ............................................................ 1.1 introduction ............................................................................................... 1.2 Materials and Methods ............................................................................. 1.2.1 Materials ................................................................................................ 1.2.2 Synthesis of rosin-grafted CNCs (r-CNCs) ............................................ 1.2.3 Preparation of gelatin/r-CNCs bionanocomposite films ......................... 1.2.4 X-ray photoelectron spectroscopy (XPS) .............................................. 1.2.5 Fourier Transform Infrared Spectroscopy (FTIR) .................................. 1.2.6 Transmission Electron Microscopy (TEM) ............................................. 1.2.7 X-ray diffraction (XRD) .......................................................................... 1.2.8 Scanning Electron Microscope (SEM) ................................................... 1.2.9 Water Vapor Permeability ..................................................................... 1.2.10 Oxygen Transmission Rate (OTR) ...................................................... 1.2.11 Tensile tests ........................................................................................ 1.2.12 Antimicrobial activity tests ................................................................... 1.2.13 Statistical analysis ............................................................................... 1.3 Results and discussion............................................................................. 1.3.1 Functionalization of CNCs with rosin ..................................................... 1.3.2 Morphology of gelatin films reinforced with CNCs and r-CNCs ............. 1.3.3 Contact angle of gelatin films reinforced with CNCs and r-CNCs .......... 1.3.4 Optical properties of gelatin films reinforced with CNCs and r-CNCs .... 1.3.5 Barrier properties of gelatin/CNCs and gelatin/r-CNCs films ................. 1.3.6 Antimicrobial properties of r-CNCs and gelatin/r-CNCs films ................ 1.3.7 Use of gelatin films reinforced with r-CNCs for actual packaging .......... 1.4 Conclusion ............................................................................................... xx 2 Effect of tannic acid and cellulose nanocrystals on antioxidant, antimicrobial, and physical properties of gelatin-based films ................................................ 2.1 Introduction ............................................................................................... 2.2 Materials and Methods .............................................................................. 2.2.1 Materials ................................................................................................ 2.2.2 Film-forming solution (FFS) preparation ................................................ 2.2.3 Film casting ........................................................................................... 2.2.4 Transmission electron microscopy (TEM) .............................................. 2.2.5 X-ray diffraction (XRD) ........................................................................... 2.2.6 Quartz crystal microbalance with dissipation (QCM-D) .......................... 2.2.7 Rheological properties ........................................................................... 2.2.8 Determination of crosslinking degree .................................................... 2.2.9 Scanning electron microscopy (SEM) .................................................... 2.2.10 Determination of antioxidant activity .................................................... 2.2.11 UV-Vis spectroscopy and transparency determination ........................ 2.2.12 Contact angle measurements .............................................................. 2.2.13 Tensile tests ........................................................................................ 2.2.14 Puncture test ....................................................................................... 2.2.15 Water vapor permeability and moisture content determination ............ 2.2.16 Antimicrobial tests................................................................................ 2.2.17 Statistical analysis ............................................................................... 2.3. Results and discussion ............................................................................ 2.3.1 Cellulose nanocrystals characterization ................................................. 2.3.2 Gelatin, tannic acid, and CNCs interactions .......................................... 2.3.3 Crosslinking degree in the gelatin-based films with TA or OTA ............. 2.3.4 Viscoelastic behavior of the gelatin-based films .................................... 2.3.5 Morphological characterizations of gelatin-based films with TA or OTA 2.3.6 Antioxidant activity of gelatin-based films with TA or OTA ..................... 2.3.7 Optical properties of gelatin-based films ................................................ 2.3.8 Contact angle of gelatin-based films ...................................................... 2.3.9 Mechanical properties of gelatin-based films ......................................... 2.3.10 Water barrier properties and water vapor absorption of gelatin-based films.. ..............................................................................................................xxi 2.3.11 Antimicrobial activities of tannic acid and gelatin-based films with TA 2.4 Conclusion ............................................................................................... CHAPTER V: General conclusions and perspectives ............................... General conclusions and perspectives ........................................................... EXTENDED PORTUGUES ABSTRACT -Resumo Português ................... EXTENDED FRENCH ABSTRACT -Résumé Français .............................. REFERENCES ...............................................................................................

2 - 71 Table 2 . 3 - 90 Table 2 . 5 - 95 Table 2 . 6 - 114 Table 3 . 2 - 120 Table 3 . 3 - 142 Table 4 . 2 - 175 Table 4 . 5 -Figure 1 -Figure 1 . 1 - 12 Figure 1 . 2 - 13 Figure 1 . 3 - 14 Figure 1 . 4 - 18 Figure 1 . 5 - 20 Figure 1 . 6 - 20 Figure 1 . 7 - 24 Figure 1 . 8 - 25 Figure 1 . 9 - 26 Figure 1 . 10 - 28 Figure 1 . 11 - 30 Figure 1 . 12 -Figure 1 . 13 - 38 Figure 1 . 14 - 40 Figure 1 . 15 - 41 Figure 1 . 16 - 42 Figure 1 . 17 - 47 Figure 1 . 18 -Figure 2 . 1 - 60 Figure 2 . 2 - 65 Figure 2 . 3 - 65 Figure 2 . 4 - 67 Figure 2 . 5 -Figure 2 . 6 - 72 Figure 2 . 7 - 73 Figure 2 . 8 - 74 Figure 2 . 9 - 76 Figure 2 . 77 Figure 2 . 78 Figure 2 . 12 - 81 Figure 2 . 88 Figure 2 . 90 Figure 2 . 93 Figure 2 . 16 - 96 Figure 2 . 17 -Figure 2 . 18 - 94 Figure 2 . 19 -Figure 3 . 1 - 112 Figure 3 113 Figure 3 . 3 - 115 Figure 3 . 4 - 117 Figure 3 . 5 - 118 Figure 3 . 6 - 120 Figure 3 . 7 - 122 Figure 3 . 8 -Figure 4 . 1 -Figure 4 . 2 - 141 Figure 4 . 3 - 142 Figure 4 . 4 - 143 Figure 4 . 5 - 144 Figure 4 . 6 - 144 Figure 4 . 7 - 146 Figure 4 . 8 - 147 Figure 4 . 9 - 148 Figure 4 .

 27123902595261143212033142421754511112121313141418152016201724182519261102811130112113381144011541116421174711821602265236524672526722773287429762772782128128829029321696217218942193111231133311534117351183612037122384142141431424414345144461444714648147491484 Figure 1 -Number of papers and patents releases each year dealing with CNCs until 2020 (extracted from ACS SciFinder in September 2020, descriptors are cellulose nanocrystals, cellulose nanowhiskers, nanocrystalline cellulose, and cellulose whiskers). ............................................................................................ 4

Figure 4 . 11 - 150 Figure 4 . 152 Figure 4 . 13 - 153 Figure 4 . 14 - 154 Figure 4 . 15 -Figure 4 . 16 - 159 Figure 4 . 169 Figure 4 . 171 Figure 4 . 19 - 172 Figure 4 . 173 Figure 4 . 174 Figure 4 . 176 Figure 4 . 177 Figure 4 . 178 Figure 4 . 25 - 180 Figure 4 . 181 Figure 4 . 183 Figure 4 . 28 - 184 Figure 4 . 29 -Figure 4 . 187 Figure 4 . 31 - 189 Figure 4 . 32 - 190 Figure 4 . 33 - 191 PageFigura 1 . 1 - 204 Figura 1 . 2 -

 411150415241315341415441541615941694171419172417341744176417741784251804181418342818442941874311894321904331911120412 Figure 4.11 -Water vapor permeability and water vapor transmission rate (WVTR) of gelatin bionanocomposites as functions of CNCs and r-CNCs contents. ........................................................................................................ 150 Figure 4.12 -(a) Young's modulus, (b) tensile strength (c) elongation at break, and (d) stress-strain curves as a function of CNCs and r-CNCs content in bionanocomposite gelatin films obtained by casting. ..................................... 152 Figure 4.13 -Minimum inhibitory concentration (MIC) of CNCs and r-CNCs suspension tested on Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. ............................................................................................. 153 Figure 4.14 -Agar overlay assay of gelatin film discs against S. aureus and E.coli. ....................................................................................................................... 154Figure 4.15 -Schematic comparison of accelerated storage for mozzarella cheese slices packed in a PVC cling film, pure gelatin film, and gelatin/r-CNCs nanocomposite (6 wt%) film over 30 days at 25 ºC. The control refers to freestanding, unpacked cheese slices (The Brazilian version of mozzarella cheese can be sliced, in contrast to the Italian mozzarella). Microbial spoilage is indicated by arrows in the images. ................................................................................ 155

Figure 1 . 1 - 216 Figure 1 . 2 -

 1121612 Figure 1.1 -Viscosité en fonction du taux de cisaillement pour (a) SF avec différentes concentrations de gélatine et (b) viscosité et contrainte de cisaillement en fonction du taux de cisaillement pour SF 20%..............................................216 Figure 1.2 -Résumé schématique de tous les résultats les plus pertinents et étapes développées dans ce travail………………………………………………220

Figure 1 -

 1 Figure 1 -Number of papers and patents releases each year dealing with CNCs until 2019 (extracted from ACS SciFinder in September 2020, descriptors are cellulose nanocrystals, cellulose nanowhiskers, nanocrystalline cellulose, and cellulose whiskers).

Figure 1 . 4 -

 14 Figure 1.4 -General mechanism of gas or vapor permeation through a polymer packaging film.

  Figures 1.5 and 1.6 shows the packaging barrier requirements for different foods and the barrier properties of different polymers, respectively.

Figure 1 . 5 -Figure 1 . 6 -

 1516 Figure 1.5 -Requirements on packaging for different foods (adapted from [48])

Figure 1 . 8 -

 18 Figure 1.8 -Global production capacities of bioplastics by market segment in 2019 (source: https://www.european-bioplastics.org/market/ [56])

Figure 1 . 10 -

 110 Figure 1.10 -Advantages and disadvantages of natural polymers and applications (source: [62] [63][37] [64][65]).

Figure 1

 1 Figure 1.11.

Figure 1 . 11 -

 111 Figure 1.11 -Amino acid residues, peptide bonds, and levels of protein organization (adapted from [67]).

  extensively studied. According to the work of[START_REF] Haworth | The existence of the cellobiose residue in cellulose[END_REF] [START_REF] Haworth | The existence of the cellobiose residue in cellulose[END_REF], cellulose is a linear homopolysaccharide composed of D-glucopyranose units linked by β(1→4) glycosidic bonds, resulting in anhydroglucose units (C6H10O5).The repeating unit of cellulose is cellobiose that comprises two anhydroglucose units (AGU). AGU is commonly used to define the degree of polymerization (DP = n/2) of cellulose. According to[START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF] [START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF], the degree polymerization is up to 20,000, but shorter cellulose chains can occur and are mainly localized in the primary cell walls. Figure1.13 shows the basic chemical structure of cellulose with three types of AGU: (i) the internal UGA, (ii) a reducing end (i.e., a hemiacetal unit), and (ii) the non-reducing end with a pendant hydroxyl group.

Figure 1 . 13 -

 113 Figure 1.13 -Representation of a cellulose chain showing the anhydroglucose unit, the glycosidic link, and both reducing and non-reducing ends of the polymer.

  nanowhiskers, or nanocrystalline cellulose, are crystalline domains extracted from several natural sources, including wood, plants, cellulose-containing animals, and microorganisms. The main characteristics of CNCs are related to their low density, excellent mechanical properties, flexibility regarding chemical modifications, and the possible myriad applications, which explains the exponential growth interest from researchers and companies for this material over the last 20 years (Figure 1.14).

Figure 1 . 14 -

 114 Figure 1.14 -Number of papers and patents releases each year dealing with CNCs until 2019 (extracted from ACS SciFinder in September 2020, descriptors are cellulose nanocrystals, cellulose nanowhiskers, nanocrystalline cellulose, and cellulose whiskers).

Figure 1 .

 1 [START_REF] Zhuang | Eco-friendly biorefractory films of gelatin and TEMPO-oxidized cellulose ester for food packaging application[END_REF] illustrates the preparation of CNCs by sulfuric acid hydrolysis.

Figure 1 . 15 -

 115 Figure 1.15 -Schematic acid hydrolysis production of CNCs resulting in sulfate half ester surface groups (adapted from [131]).

Figure 1 . 16 -

 116 Figure 1.16 -Transmission electron microscopy images of CNCs suspensions obtained from (a) tunicate, (b) ramie, (c) cotton, (d) beet sugar, (e) microcrystalline cellulose, and (f) bacterial cellulose [8].

  In the last decades, the interest in the use of CNCs encouraged the development of large-scale CNCs production facilities around the world. For instance, North America dominates the market with a share of almost 45 %, which is expected to grow in the coming years. The key players and their industrial capacity (tonnes per year, dry basis) are Celluforce (Canada) 300 tons, American Process Inc. (USA) 200 tons, Melodea (Sweden)[START_REF] Koltzenburg | [END_REF] tons, Alberta InnovatesLiliane[START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] (Canada) 7 tons, US Forest Products Lab (USA), and Blue Goose Biorefineries (Canada), FPInnovations (Canada), University of Maine (USA) with 4 tons each.

  edulis) packaging films by continuous casting at 120 °C. During the continuous production, the authors achieved productivity of 0.03 m 2 of film per minute with mechanical strength comparable to traditional PVC. These characteristics make continuous casting an attractive process for large-scale bio-based film production.

Figure 1 . 18 -

 118 Figure 1.18 -Schematic representation of gelatin-CNCs bionanocomposite production by continuous casting [18].

Figure 2 . 1 .

 21 Figure 2.1. In this technique, the polymer solution is continuously spread onto a moving substrate (Mylar®, DuPont, Brazil) with a doctor blade device, which allows controlling the wet layer thickness. The wet solution layer then passes through two drying chambers with controlled temperatures. The distance between the two drying chambers is approximately 2 meters. The dried film is wound into rolls. The distance between the substrate and the doctor blade was set at 1.0 mm. The conveyor speed and the drying ovens were 12 m/h and 80 °C, respectively. All samples were conditioned in a desiccator at 50% RH and 25 ± 2 °C for at least 48 h prior to testing.

Figure 2 . 1 -

 21 Figure 2.1 -Schematic representation of gelatin-CNCs bionanocomposite production by continuous casting.

Figure 2 .

 2 Figure 2.2 shows a typical TEM micrograph of the CNCs isolated from the eucalyptus fiber pulp by acid hydrolysis. The micrograph reveals the effective hydrolysis of the cellulosic fibers, giving rise to nanoparticles with needle-like morphology, as expected. The average length (L) and diameter (D) of the CNCs

Figure 2 . 2 -

 22 Figure 2.2 -TEM micrograph of CNCs isolated from eucalyptus pulp.

Figure 2 . 3 -

 23 Figure 2.3 -Structure of ionized and zwitterion forms of glycine.

Figure 2 . 4 -

 24 Figure 2.4 -Power Law model fitting for the film-forming solutions as a function of gelatin concentration at 30 °C.

  Figure 2.1) but ln = 0,9379 ln -3

Figure 2 .

 2 Figure 2.5a shows the flow curves of the gelatin-based FFS containing different CNCs concentrations. The apparent viscosity of all FFS decreased faster at low shear rates (< 100 s -1 ), indicating their pseudoplastic behavior, regardless of the CNCs concentration.Figure 2.5b shows the influence of temperature on the rheological behavior of the gelatin/CNCs FFS. The increase of the CNCs concentration led to more viscous FFS compared to the pure gelatin

Figure 2 .

 2 Figure 2.5c shows the viscoelastic moduli (storage modulus G' and loss modulus G") of the FFS with different CNCs concentrations obtained from temperature sweep tests. All samples presented a similar viscoelastic behavior:high G' and G" and low G' and G" moduli at temperatures below and above the Tsol-gel, respectively. The gelatin/CNCs FFS exhibited higher viscoelastic moduli compared to those of the pure gelatin solution at a fixed temperature. For the highest CNCs concentration, i.e., 2.5 wt% CNCs, the FFS still presented a gellike behavior (G' > G"). Furthermore, the sol-gel transition temperature increased from 24.5 °C for the pure gelatin solution to 28.3 °C for the FFS with 0.5 wt% CNCs. These results may be due to the high stiffness of the CNCs and CNCgelatin and CNCs-CNCs interactions, resulting in a more elastic behavior, as observed in Figure2.5c[START_REF] Mukherjee | Thermal transitions of gelatin evaluated using DSC sample pans of various seal integrities[END_REF][START_REF] Ching | Rheological properties of cellulose nanocrystalembedded polymer composites: a review[END_REF].

Figure 2 . 5 -

 25 Figure 2.5 -Rheological characterizations of gelatin-CNCs FFS containing different CNCs concentrations (a) apparent viscosity vs. shear rate (25 °C), (b) apparent viscosity vs. temperature curves, and (c) storage modulus (G') and loss modulus (G") vs. temperature curves.

Figure 2 .

 2 [START_REF] Gómez-Guillén | Fish gelatin: a renewable material for developing active biodegradable films[END_REF] shows the micrographs of the cryo-fractured cross-sectional surface of the films. As observed in Figure2.6a, the pure gelatin film displayed a continuous and homogeneous matrix with a smooth fractured surface. The addition of CNCs gradually promoted the formation of a rougher fractured surface, indicating a microstructural change in the gelatin matrix. There was no clear evidence of CNCs agglomerates in all the bionanocomposite micrographs analyzed, suggesting that the CNCs were homogeneously distributed within the gelatin matrix. The presence of CNCs is typically indicated by the appearance of shiny dots, which have been related to small CNCs agglomerates embedded

Figure 2 . 6 -

 26 Figure 2.6 -SEM images of cross-sectional surfaces of films (a) pure gelatin and with (b) 0.5 wt% CNCs, (c) 1.0 wt% CNCs and (d) 2.5 wt% CNCs.

Figure 2 . 7 - 1 . 3 . 6

 27136 Figure 2.7 -ATR-FTIR spectra of (a) CNCs powder, (b) gelatin powder and gelatin/CNCs bionanocomposite films with (c) 0 wt% CNCs and (d) 2.5 wt% CNCs.

Figure 2 . 8 -

 28 Figure 2.8 -Photography of gelatin/CNCs bionanocomposite films obtained by continuous casting.

Figure 2 . 9 -

 29 Figure 2.9 -UV-Vis spectra of pure gelatin film and its bionanocomposites with CNCs.

Figure 2 . 1 . 3 . 7

 2137 Figure 2.10 -(a) Thermogravimetric and (b) DTG curves of the gelatin/CNCs bionanocomposites obtained by continuous casting.

Figure 2 .

 2 Figure 2. 11 -(a) Tensile strength, (b) Young's modulus (c) strain at break, and (d) stress-strain curves as a function of CNCs content in gelatin films obtained by bench casting and continuous casting.

  quality of the dispersion of CNCs particles in the film-forming solution plays an important role in the properties of the film produced by spreading. CNCs have a high specific surface area and surface energy. Consequently, these nanoparticles have a strong tendency for agglomeration (Figure2.12a,b). CNCs agglomeration can lead to a decrease in the strength of the nanocomposites since they act as defects or stress concentrators[START_REF] Cao | The relationship between cellulose nanocrystal dispersion and strength[END_REF]. Previous studies have demonstrated that a good dispersion and distribution of CNCs into the matrix (Figure2.12d) can improve the thermal and mechanical properties of the films[START_REF] Oksman | Review of the recent developments in cellulose nanocomposite processing[END_REF]. The challenge consists therefore in avoiding agglomeration and promoting the homogeneous dispersion and distribution of the CNCs, which favor CNCsmatrix interactions, and at the same time promoting CNCs-CNCs interactions to allow the beneficial formation of a percolating network of nanoparticles[START_REF] Mariano | Cellulose nanocrystals and related nanocomposites: Review of some properties and challenges[END_REF]

Figure 2 . 12 -

 212 Figure 2.12 -Schematic of poor and well dispersion and distribution of particles

XRD(

  Panalytical diffractometer, X'Pert Pro MPD-Ray, The Netherlands) with Nifiltered Cu Kα radiation (k = 1.54 Å, 45 kV, 40 mA) in the 2θ range from 5° to 60°.

Figure 2 .

 2 Figure 2.13b. All the gelatin-based films showed good continuity with no visible cracks. Nevertheless, the addition of heterogenous CNCs into the gelatin matrix induced the formation of visible roughness on the dry surface in contact with the air. This can be attributed to the presence of CNCs aggregation. Since the increase in roughness is an indication of poor dispersion and interfacial interaction between the reinforcement phase (CNCs) and the matrix phase (gelatin) [231].

Figure 2 .

 2 Figure 2.13 -(a) Polarized light images of gelatin FFS with heterogeneous CNCs and (b) photographs of gelatin films with heterogeneous CNCs obtained by continuous casting (Chapter II.1).

Figure 2 .

 2 [START_REF] Dufresne | Polysaccharide nanocrystal reinforced nanocomposites[END_REF]. The presence of these microparticles is indicative of the lower yields attained during the sulfuric hydrolysis, which can result in a heterogeneous CNCs size distribution Liliane[START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF]

Figure 2 .

 2 Figure 2.14 -(a) TEM micrograph of heterogeneous CNCs with partially hydrolyzed fiber (arrow) and (b) an magnified view of the image (a) showing the agglomeration of CNCs particles (arrow).

2. 3 . 2

 32 Heterogeneous CNCs vs. homogeneous CNCs Polarized light images of heterogeneous and homogeneous CNCs are shown in Figure 2.15a. The aqueous suspension of heterogeneous CNCs showed more microscopic aggregates after sonication than homogeneous CNCs suspension, which suggests a better dispersion of the last nanoparticles. TEM images of both CNCs nanoparticles are shown in Figure 2.15b and their average dimensions values are presented in Table

Figure 2 .

 2 Figure 2.15 -(a) Polarized light images of heterogeneous and homogeneous CNCs aqueous suspensions at 1.0 wt% and (b) TEM micrograph of heterogeneous and homogeneous CNCs.

93 Liliane

 93

Figure 2 .

 2 Figure 2.16 -(a) Young's modulus, (b) tensile strength, (c) strain at break, and (d) stress-strain curves as a function of CNCs content in gelatin films obtained by bench casting and continuous casting.

Figure 2 .

 2 Figure 2.18 -SEM micrograph of the cross-sectional surface of (a) pure gelatin and (b) gelatin film with 6.0 wt% CNCs obtained by bench casting and (d) pure gelatin and (e) gelatin films with 6.0 wt% CNCs obtained by continuous casting. The magnified view of the image of (b) and (c) are shown in (c) and (f) image, respectively.

  drying temperatures in continuous casting, which contributed to better cohesiveness of the matrix resulting in more compacted films, as shown by SEM image (Figure2.18b,e).[START_REF] Chiou | Effects of drying temperature on barrier and mechanical properties of cold-water fish gelatin films[END_REF] [START_REF] Chiou | Effects of drying temperature on barrier and mechanical properties of cold-water fish gelatin films[END_REF] reported the same behavior for fish gelatin and observed that cold-cast gelatin films had WVP values approximately two to three times higher than those of hot-cast gelatin films.Fernández-Pan et al. (2010)[START_REF] Fernández-Pan | Effect of drying conditions on the mechanical and barrier properties of films based on Chitosan[END_REF] found that chitosan films dried at 80 °C showed 97 Liliane[START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF]

Figure 2 . 19 -

 219 Figure 2.19 -Water vapor permeability of gelatin-based films obtained by bench and continuous casting

Figure 2 . 20 -

 220 Figure 2.20 -Contact angle of gelatin-based films obtained by bench and continuous casting.

101 Liliane

 101 focused on the use of gelatin and cellulose nanocrystals (CNCs) as a bio-based and biodegradable material for food packaging applications. One of the main objectives of this Ph.D. is the production of gelatin-based films, which fulfill the most relevant requirements for packaging material. The knowledge of the intermolecular interactions is crucial for understanding the structure and physical properties of gelatin-based films. Therefore, this chapter investigates the electrostatic interactions between gelatin and CNCs as a function of pH using zeta potential and rheological measurements. The physical properties such as mechanical, thermal, and water vapor permeability of gelatinbased films at different pH and CNCs amount is evaluated. properties of gelatin/CNCs films covering different CNCs contents. The interactions between CNCs and gelatin were studied by assessing the ζ-potential of gelatin/CNCs suspensions under acidic (pH 3) and alkaline (pH 8) conditions and at gelatin isoelectric point (pI, pH 6). pH 3 promoted the electrostatic attraction, while pH 8 favored the electrostatic repulsion in the gelatin-CNCs pair, increasing the suspension viscosity in both cases. The addition of 0.5 wt% CNCs decreased the water vapor permeability (WVP) of the gelatin/CNCs films by 68%

at 60 °C

 60 under stirring for 15 min. The gelatin solution pH was adjusted to 3, 6, and 8 with 3M HCl or NaOH. Glycerol (2 g/100 g of protein) was added and the gelatin solution under stirring for 5 min. The obtained solution was mixed with the CNCs suspension at desired contents (0 wt%, 0.5 wt%, and 5.0 wt%, on a dry gelatin mass basis) and stirred for an additional 5 min. The final film-forming solution (FFS) (45 mL) were cast on glass plates (40 cm × 25 cm), covered with a polyester film (Mylar®, DuPont, Brazil) to facilitate film peeling off after drying in an oven at 25, 35, and 80 °C for 24 h. Film samples were conditioned at 50% relative humidity (RH) in a ventilated climatic chamber and 25 °C for at least 24 h prior to any testing.

h.

  The tests were carried out in an Instron Universal Testing Machine model 5569 (Instron Corp., USA) equipped with a 100 N load cell. The specimens were stretched using crosshead speed of 10 mm/min with clamps initially separated by 100 mm. Tensile strength, Young's modulus, and elongation at break were calculated from the stress-strain curves. Thicknesses were measured using a digital micrometer (Adamel Lhomargy) to the nearest 0.001 mm with ten random measurements per film. The tensile tests were performed with five replicates per film sample.

1. 3 and discussion 1 . 3 . 1

 3131 Results Physical and rheological properties of gelatin/CNCs film-forming solutions ζ-potentials provide evidence of the net charges on particle surfaces as a function of pH. Figure 3.1 illustrates the evolution of the ζ-potential of gelatin solution and CNCs suspension upon pH variation. The isoelectric point (pI) of gelatin solution occurred at around pH 6, at which a null ζ-potential was observed.

Figure 3 . 1 -

 31 Figure 3.1 --Potential values of pure gelatin solution and CNCs suspension (0.25 wt%) at and 25 °C as a function of pH.

Figure 3 . 3 . 3 .

 333 Figure 3.2 -ζ-potential curves of gelatin/CNCs FFS at different pH as a function of CNCs concentration.

Figure 3 . 3 -

 33 Figure 3.3 -Apparent viscosity (left) and shear stress (right) curves as a function of the shear rate of gelatin/CNCs films at different pH values. (___) increase in shear rate and (------) decrease in shear rate.

Figure 3 .

 3 Figure 3.4 shows the storage (G') and loss (G") modulus of the gelatin/CNCs FFS with 0 and 5 wt% CNCs at different pH obtained from heating and cooling scans.

pH 3 3 (

 33 resulted in an elastic gel-like behavior (G' > G") even above the gel-sol temperature. According to[START_REF] Qiao | Structure and rheological properties of cellulose nanocrystals suspension[END_REF] [START_REF] Qiao | Structure and rheological properties of cellulose nanocrystals suspension[END_REF], proteins can form complex coacervates through interactions of oppositely charged particles, as previously indicated by the ζ-potential values of the gelatin and CNCs solutions at pH Figure3.1). Complex coacervation can lead to precipitation of polymer complexes through the formation of a dense media that can be treated as being composed of interconnected gel-like network structures, consistent with the assumption that the CNCs are electrostatically "cross-linked" along the macromolecules. Consequently, the gel-sol and sol-gel transitions of the gelatin/CNCs FFS due to electrostatic attractive forces were not observed.

Figure 3 . 4 - 1 . 3 . 2 1 . 3 . 2 . 1

 341321321 Figure 3.4 -Viscoelastic modulus of gelatin/CNCs FFS at different pH and CNCs contents. Heating (a and c) and cooling (b and d) at 1 °C min -1 .

Figure 3 . 5 -

 35 Figure 3.5 -(a) Photographs of gelatin/CNCs films with different CNCs contents, (b) SEM micrographs of air-drying and cross-sectional surfaces of gelatin/CNCs films with 5.0 wt% CNCs at pH 3, 6 and 8. White arrows indicate CNCs-gelatin coacervates at pH 3.

Figure 3 . 6 -

 36 Figure 3.6 -XRD patterns of (a) pure gelatin films obtained from FFS films at pH 3, 6, and 8 and (b) gelatin/CNCs films obtained from pH 8 and different CNCs amount. The magnified view of the 2θ range 3 -12 ° is shown as an insert.

7 . 5 wt%

 75 The gelatin films containing 0.CNCs at pH 3 and 8 showed significantly increased Young's modulus and tensile strength and decrease elongation at break compared to the pure gelatin films (p < 0.05). This effect was more pronounced at pH 8, whose Young's modulus and tensile strength increased by 152% and 56%, respectively, compared to those of the pure gelatin film (p < 0.05). The mechanical properties of gelatin films are related to the regeneration of triple helixes, which represent crystalline domains within the film matrix and may act as reinforcing fillers in these films[START_REF] Bigi | Relationship between triple-helix content and mechanical properties of gelatin films[END_REF]. Similar behavior was reported by[START_REF] Quero | Stress transfer and matrix-cohesive fracture mechanism in microfibrillated cellulose-gelatin nanocomposite films[END_REF] [START_REF] Quero | Stress transfer and matrix-cohesive fracture mechanism in microfibrillated cellulose-gelatin nanocomposite films[END_REF] that attributed the improved mechanical performance of microfibrillated cellulose (MFC)-salmon gelatin films at a MFC fraction of 15% w/w to the high content of triple-helix configurations present in these nanocomposites compared to the MFC-bovine gelatin nanocomposites.[START_REF] Gioffrè | Role of pH on stability and mechanical properties of gelatin films[END_REF] [START_REF] Gioffrè | Role of pH on stability and mechanical properties of gelatin films[END_REF] investigated the pH effect on the mechanical properties of pigskin type A gelatin films and observed that pH values higher than 9 and lower than 5 considerably reduced the triple helix content and consequently Young's modulus and elongation at break of the films.

Figure 3 . 7 -

 37 Figure 3.7 -(a) Young's modulus, (b) elongation at break, (c) tensile strength, and (d) stress-strain curves of gelatin/CNCs films as a function of pH and CNCs content.

127 Liliane

 127

Figure 3 . 8 -1. 4 Conclusion 129 LilianeIV 133 Liliane Leite - 2020 1

 3841291332020 Figure 3.8 -XRD spectra of the gelatin/CNCs films with 0.5% at pH 8 obtained at different temperatures.

Figure 4 . 1 -

 41 Figure 4.1 -The most representative structures found in Rosin.

Figure 4 . 2 -

 42 Figure 4.2 -Schematic representation of the rosin-functionalized CNCs.

Table 4 . 1 -Figure 4 . 3 -

 4143 Figure 4.3 -C1s XPS spectra of (a) CNCs and (b) r-CNCs.

Figure 4 . 5 -

 45 Figure 4.5 -TEM micrograph of CNCs and (e) r-CNCs. (f) schematic representation of rosin-functionalized CNCs.

Figure 4 . 6 -

 46 Figure 4.6 -XRD spectra of the CNCs and r-CNCs.

Figure 4 . 7 -

 47 Figure 4.7 -SEM micrographs of the cross-sectional surface of (a) pure gelatin films and gelatin bionanocomposites with 4.0 wt% (b) CNCs and (c) r-CNCs, and with 6.0 wt% (d) CNCs and (e) r-CNCs content.

Figure 4 . 8 -

 48 Figure 4.8 -displays the dynamic changes in the water contact angle of pure gelatin, gelatin/CNCs, and gelatin/r-CNCs films. Gelatin films presented water contact angle values higher than 90° over time, which indicates that the surface studied was hydrophobic. This behavior is attributed to the hydrophobic parts of the gelatin that are able to spontaneously rearrange towards the air-film [203,241-243]. Nevertheless, the increasing concentration of CNCs decreased

  visible light range, which may indicate the light scattering by r-CNCs aggregates, as shown by the SEM images (Figure 4.7e).

Figure 4 . 9 -

 49 Figure 4.9 -Photography of (a) gelatin/CNCs and (b) gelatin/r-CNCs bionanocomposite films.

Figure 4 . 149 Liliane

 4149 Figure 4.10 -UV-Vis spectra of pure gelatin, gelatin/CNCs, and gelatin/r-CNCs films.

were < 0 .

 0 01 cm³/m² day, indicating that the addition of CNCs or r-CNCs did not change significantly the already high oxygen barrier of gelatin. The values of WVP and WVTR of the gelatin bionanocomposites with CNCs and r-CNCs at 25 ˚C and 50% RH are presented in Figure 4.11. Pure gelatin film exhibited a WVP of 0.20 ± 0.03 g mm/m 2 h kPa. The incorporation of CNCs or r-CNCs reduced the WVP of the gelatin films. The addition of 0.5 wt% r-CNCs significantly decreased WVP of the gelatin matrix by 55% (p < 0.05).

Figure 4 . 11 -

 411 Figure 4.11 -Water vapor permeability and water vapor transmission rate (WVTR) of gelatin bionanocomposites as functions of CNCs and r-CNCs contents.

Figure 4 .

 4 Figure 4.12 -(a) Young's modulus, (b) tensile strength (c) elongation at break, and (d) stress-strain curves as a function of CNCs and r-CNCs content in bionanocomposite gelatin films obtained by casting.

Figure 4 . 14 - 1 . 3 . 7

 414137 Figure 4.14 -Agar overlay assay of gelatin film discs against S. aureus and E.coli.

Figure 4 .

 4 Figure 4.15 shows evident microbial spoilage in the control and gelatin-packed cheese samples, and especially in the sample packed in PVC. In contrast, there

Figure 4 . 15 -

 415 Figure 4.15 -Schematic comparison of accelerated storage for mozzarella cheese slices packed in a PVC cling film, pure gelatin film, and gelatin/r-CNCs bionanocomposite (6 wt%) film over 30 days at 25 ºC. The control refers to freestanding, unpacked cheese slices (The Brazilian version of mozzarella cheese can be sliced, in contrast to the Italian mozzarella). Microbial spoilage is indicated by arrows in the images.

Figure

4. 16 .

 16 purchased as a spray-dried powder from CelluForce Inc. (Windsor, Québec, Canada) and used as received. Tannic acid (TA) was purchased from Roth (France). Glycerol and ethanol were purchased by Across Organics (USA). 1,1diphenyl-2-picrylhydrazyl (DPPH) was purchased by Sigma Chemical Co. (USA).

( 2 . 2 .

 22 2009). Film specimens were prepared and equilibrated at 23 ± 1 °C and 50 ± 5% RH for 48 h. The tests were carried out on an Instron Universal Testing Machine (model 5569, Instron Corp., USA) equipped with a 100 N load cell. The specimens were stretched at a crosshead speed of 10 mm/min with clamps initially separated by 100 mm. Tensile strength and elongation at break were calculated from the stress-strain curves. At least five specimens of each film were tested. 14 Puncture test The puncture test was performed according to EN 14777 (2005). The films were cut into a tape of 20 mm by at least 250 mm and perforated to the breaking point using a Multitest-xt Mecmesin machine (Mecmesin, UK) with a round-ended stainless-steel plunger 0.88 mm in diameter at a crosshead speed of 10 mm/min and a 12 N load cell. All determinations were done at least five replicates.

5 

 5 inoculating a selected single colony in 15 mL of Muller-Hinton Broth medium (MHB), followed by overnight incubation at 35 ˚C. The optical densities at 625 nm were measured by UV-Vis absorption spectroscopy and compared with the turbidity of a 0.5 McFarland standard at the same wavelength, equivalent to 1.10 8 colony forming units per mL (CFU/mL). The overnight cultures were diluted in MHB until reaching 10 6 CFU/mL. The antimicrobial activity of the CNCs and TA suspensions was assessed by determining the minimum inhibitory concentration (MIC) in a 96 flat-bottomed-well tissue culture microplate. Briefly, 100 μL of sterile Mueller Hinton were added to all wells. In the first column, the wells were filled with 50 μL of CNCs (50 mg/ml) and TA (50 mg/ml) suspensions.Then, 50 μL of each antimicrobial substance were serially transferred from the well to the corresponding wells. Then, 50 μL of the culture suspension were added to all wells. Positive and negative controls (streptomycin and culture medium -data not shown) were included in each assay plate. Afterward, the inoculated plates were incubated in a wet chamber for 24 h at 35 °C and then chlorinated with 50 μL of 2,3,5 -Triphenyltetrazolium chloride (0.1%). The lowest sample concentration with an inhibition effect on microbial growth was considered as the MIC for each tested microorganism. The antibacterial activity of the film samples Gel, G-TA, G-CNC6, and G-TA-CNC6 was assessed by the disk diffusion method using the pathogenic microorganisms Staphylococcus aureus and Escherichia coli, as model Grampositive and Gram-negative bacteria, respectively. Briefly, the bacteria were cultured in Muller-Hinton Broth medium (MHB) and incubated overnight at 35 ˚C. The ensuing inoculum of each bacterium was diluted in MHB to 1× 10 6 CFU/mL (turbidity = 0.5, McFarland barium sulfate standard), and further spread (100 µL) over solid Muller-Hinton Agar (MHA) Petri dishes. Dried and sterile film sample disks (10 mm) were placed onto the inoculated Petri dishes and incubated overnight at 35 ˚C for optimum bacterial growth. The antibacterial activity was investigated by the presence of inhibition zones (colony-free area) surrounding the film disks.

  17a). These values were in accordance with Reid, Villalobos, & Cranston, (2017).The L/D value can be used to estimate the critical CNCs volume fraction required to achieve the CNCs percolation network (percolation threshold) using the equation v= 0.7/(L/D)[START_REF] Favier | Mechanical Percolation in Cellulose Whisker[END_REF] [START_REF] Favier | Mechanical Percolation in Cellulose Whisker[END_REF]. According to the experimental L/D, the calculated volume fraction was 2.5 % (v/v), which corresponds to 4 wt%.The XRD pattern of CNCs exhibited typical diffraction peaks at around 16.5º, 22.5º, and 34.5˚of 2θ, assigned to the planes (110),[START_REF] Bhowmik | Reinforcement of gelatin-based nanofilled polymer biocomposite by crystalline cellulose from cotton for advanced wound dressing applications[END_REF], and (004), respectively, representing the cellulose I polymorph (Figure4. 17b)[START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF]. The crystallinity index was 82%. This result was in agreement with Hu et al.

Figure 4 .

 4 Figure 4. 17 -(a) TEM micrograph and (b) XRD spectra of CNCs.

Figure 4 .

 4 Figure 4.18 -(a-d) Normalized third overtone frequency (black) and dissipation (gray) data versus time from QCM-D analysis of CNCs-coated silica sensors exposed to three different injection orders of tannic acid (TA), gelatin (GEL), and glycerol (GLYC) with water (W) rinsing steps in between each injection step

Figure 4 . 19 -

 419 Figure 4.19 -Storage modulus and loss modulus vs. temperature curves of (a), (b) G-TA-CNC6, and (c), (d) G-OTA-CNC6 FFS under heating and cooling scans.

Table 4 . 3 -

 43 Transitions temperatures for the gelatin-based FFS with TA and CNCs. polyphenols and protein, as demonstrated for chitosan films crosslinked with tannic acid[START_REF] Rivero | Crosslinking capacity of tannic acid in plasticized chitosan films[END_REF].

Figure 4 .

 4 Figure 4.21 -(a) Free TA released from FFS and (b) UV-Vis spectra of FFS and photography of the (c) G-OTA-CNC6 and G-TA-CNC6 FFS, and (d) G-OTA-CNC6 and G-TA-CNC6 films.

Figure 4 .

 4 Figure 4.22 -(a) Apparent viscosity and (b) shear stress as a function of shear rate of the gelatin-based FFS at 35 °C.

Figure 4 .

 4 Figure 4.23 -(a) Calibration curve of TA in water and (b) free TA extracted from gelatin-based films.

Figure 4 .

 4 Figure 4.24 shows the storage modulus (G') and loss modulus (G") of the Gel, G-TA G-TA-CNC6, and G-OTA-CNC6 films at room temperature. It can be observed that the G" component was considerably smaller than G', exhibiting the formation of an elastic network. Besides, there was a significant increase in the G' of chemically crosslinked TA in the G-TA, G-TA-CNC6, and G-OTA-CNC6

Figure 4 .

 4 Figure 4.24 -(a) Storage and (b) loss modulus of the gelatin-based films with TA or OTA.

Figure 4 .

 4 Figure 4.25 -SEM images of cross-section surface of (a) Gel, (b) G-TA (c) G-TA-CNC6, and (e) G-OTA-CNC6 films before and after swelling in water for 24. The low-magnified view of the film is shown as an insert.

Figure 4 .

 4 Figure 4.27a shows DPPH radical scavenging activities of free TA solution and TA-loaded gelatin films. The antioxidant effect on the DPPH radicalscavenging was substantially increased with the addition of CNCs, and the maximum activity was observed for the G-TA-CNC6 film (85%).[START_REF] Choi | Effect of oxidized phenolic compounds on cross-linking and properties of biodegradable active packaging film composed of turmeric and gelatin[END_REF]

Figure 4 .

 4 Figure 4.27 -(a) DPPH radical scavenging activity of free TA solution and gelatinbased films with CNCs and TA and (b) stability of gelatin-based films.

Figure 4 . 29 -

 429 Figure 4.29 -Water contact angle measurements of gelatin-based films.

Figure 4 .

 4 Figure 4.30a presents the tensile strength (TS) and elongation at break (EAB) of gelatin-based films. After reaction with TA, the TS value decreased significantly (p < 0.05) from 17 MPa to 10 MPa, and the EAB value increases

Figure 4 . 33 -

 433 Figure 4.33 -Agar overlay assay of gelatin-based film discs against S. aureus and E. coli. Inset of the growth inhibition area observed under the sample.

  i.Investigar os parâmetros reológicos necessários para produzir filmes de gelatina/CNCs em escala piloto usando o método de casting continuo.ii. Caracterizar as propriedades morfológicas, térmicas, mecânicas e de barreiras dos filmes de gelatina/CNCs obtidos por casting contínuo iii. Funcionalizar a superfície dos CNCs com ativos naturais e produzir filmes ativos de gelatina e CNCs funcionalizados. Para alcançar esses objetivos, foi desenvolvida uma parceria colaborativa durante os quatro anos com especialistas em produção e funcionalização de nanocelulose do Laboratório de Ciência da Celulose e Papel e Artes Gráficas (França) e, especialistas em produção de filmes de origem natural da Embrapa Instrumentação (Brasil). Portanto, este doutorado ocorreu em um contexto dinâmico e interdisciplinar. Os resultados obtidos neste doutorado foram organizados em quatro capítulos. O Capítulo I apresentam uma revisão da literatura que descreve o contexto e os 204 objetivos deste doutorado, focando no potencial uso de gelatina e CNCs como embalagens sustentáveis. Além disso, é descrito um método de processamento em escala piloto para filmes biodegradáveis de fontes naturais. Conforme mencionado no início deste resumo. O capítulo II descreve os parâmetros reológicos das soluções formadoras de filmes (SFF) necessário para produção de filmes de gelatina/CNCs por casting contínuo. O estudo das propriedades reológicas da SFF é de grande importância para a avaliação da capacidade de laminação e determinação da formulação mais adequada para produção de filmes por casting contínuo. Todas as soluções analisadas apresentaram comportamento pseudoplástico em função da taxa de cisalhamento aplicada (Figura 1.1). O comportamento pseudoplástico é considerado o mais adequado para produção de filmes por casting contínuo, uma vez que as SFF devem apresentar baixa viscosidade sobre cisalhamento, para garantir condições de fluxo apropriadas sob a lâmina, e viscosidades mais elevadas em baixas tensões de cisalhamento geradas imediatamente após a faca de coating, para minimizar fluxo indesejado e efeitos de sedimentação.

Figura 1 . 1 -

 11 Figura 1.1 -Viscosidade em função da taxa de cisalhamento para (a) SFF com diferentes concentrações de gelatina e (b) viscosidade e tensão de cisalhamento em função taxa de cisalhamento para SFF 20%.

Figura 1 . 2 - 209 Liliane Leite - 2020 213 Liliane

 122092020213 Figura 1.2 -Resumo esquemático dos resultados obtidos nesse trabalho de doutorado.

  d'excellentes propriétés barrières contre la lumière UV. Par ailleurs, la gélatine présente d'excellentes propriétés barrières au dioxyde de carbone et à l'oxygène grâce à son caractère hydrophile[START_REF] Avena-Bustillos | Gelation, oxygen permeability, and mechanical properties of mammalian and fish gelatin films[END_REF]. Cependant, son caractère hygroscopique est considéré comme le principal inconvénient lorsqu'elle est utilisée pour les emballages, puisque les films sont sensibles à l'humidité et présentent des propriétés mécaniques et barrières faibles dans des conditions d'haute humidité relative[START_REF] Suderman | The effect of plasticizers on the functional properties of biodegradable gelatin-based film: A review[END_REF]. Cependant, ces inconvénients peuvent être maîtrisés par l'incorporation de nanoparticules renforçantes, comme par exemple, les nanocristaux de cellulose (CNCs), dans la matrice de gélatine[START_REF] Moustafa | Eco-friendly polymer composites for green packaging: Future vision and challenges[END_REF][START_REF] Hosseini | A state-of-the-art review on the elaboration of fish gelatin as bioactive packaging: Special emphasis on nanotechnology-based approaches[END_REF][START_REF] Azeredo | Nanocomposites for food packaging applications[END_REF][START_REF] Squinca | Nanocellulose production in future biorefineries: an integrated approach using tailor-made enzymes Nanocellulose production in future biorefineries : an integrated approach using tailor-made enzymes[END_REF][START_REF] Thomas | a versatile green platform: from biosources to materials and their applications[END_REF].Les CNCs sont des domaines cristallins extraits à partir de différentes sources naturelles, dont le bois, les plantes, les microorganismes et les animaux contenant de la cellulose. Les CNCs sont biodégradables, ils présentent un rapport longueur/diamètre élevé (environ 10-70), une faible densité (1,5-1,6 g/cm 3 ), un faible coefficient de dilatation thermique et ils forment des films transparents[START_REF] Habibi | Cellulose Nanocrystals: Chemistry, Self-Assembly, and Applications[END_REF][START_REF] Huber | A critical review of all-cellulose composites[END_REF][START_REF] Lagerwall | Cellulose nanocrystal-based materials: from liquid crystal self-assembly and glass formation to multifunctional thin films[END_REF]. De plus, les dimensions bien définis des CNCs et leur taux de cristallinité élevé (70-90 %) conduisent à d'excellentes propriétés mécaniques, comme une grande rigidité axiale (environ 150 GPa), un module de Young élevé (20-50 GPa)[START_REF] Gars | Polymerization of glycidyl methacrylate from the surface of cellulose nanocrystals for the elaboration of PLA-based nanocomposites[END_REF] et une résistance à la traction d'environ 7 GPa[START_REF] Sukyai | Effect of cellulose nanocrystals from sugarcane bagasse on whey protein isolate-based films[END_REF].Par ailleurs, la surface des CNCs peut être modifiée grâce à la réactivité du groupe hydroxyle, ce qui rend les CNCs versatiles pour diverses possibilités de fonctionnalisation des surfaces. Cette caractéristique permet une augmentation des applications spécifiques des CNCs pour le développement des nanomatériaux haute performance avec applications diverses[START_REF] Habibi | Key advances in the chemical modification of nanocelluloses[END_REF].

  élargissement du processus par casting traditionnel (normalement réalisé sur une paillasse de laboratoire). L'avantage principal du casting continue se compose du fait que cette méthode demande un temps de séchage plus court, grâce à l'utilisation du chauffage infrarouge dans la phase de pré-séchage, des températures plus élevées et une circulation d'air intense. D'autres avantages du casting continu sont le contrôle de l'épaisseur du film, l'absence d'additifs de traitement, l'utilisation de solutions/dispersions aqueuses et, la nécessité de moins d'espace et de personnel. Dans ce contexte, les objectifs de ce travail de doctorat sont : i. Chercher les paramètres rhéologiques nécessaires pour la production des films à base de gélatine/CNCs à l'échelle pilote en utilisant la méthode de casting continu. ii. Caractériser les propriétés morphologiques, thermiques, mécaniques et barrières des films à base de gélatine/CNCs obtenus par la méthode de casting continue. iii. Fonctionnaliser la surface des CNCs par des actifs naturels et obtenir des films actifs à base de gélatine et CNCs fonctionnalisés. Pour atteindre ces objectifs, une collaboration avec des spécialistes de la production et de la fonctionnalisation de nanocelluloses du Laboratoire de Génie des Procédés Papetiers (France) et des spécialistes de la production de films à base de sources naturelles à Embrapa Instrumentation (Brésil) a été développé au cours de ces quatre années de thèse. Ce doctorat s'est déroulé, par conséquent, dans un contexte dynamique et interdisciplinaire. Les résultats obtenus pendant la préparation de cette thèse ont été organisés en quatre chapitres. Le Chapitre I présente une revue de la littérature et décrit le contexte et les objectifs de cette thèse, avec un intérêt particulier pour l'utilisation potentielle de la gélatine et des nanocristaux de cellulose (CNC) dans le domaine des emballages écologiques. De plus, une méthodologie de mise en forme des films biodégradables et biosourcés à une échelle pilote est décrite. Le Chapitre II décrit les propriétés rhéologiques des solutions filmogènes pour préparer les films (SF) à base de gélatine/CNCs en utilisant la méthode de casting continue. Cette étude est essentielle pour évaluer la capacité de lamination et déterminer la composition la plus appropriée pour la production des films en continu. Toutes les solutions analysées présentent un comportement pseudoplastique avec le taux de cisaillement appliqué (Figure 1.1). Ce comportement est considéré comme le plus convenable pour la production des films par la méthode de casting continu, une fois que les doivent posséder une faible viscosité lorsqu'un cisaillement est appliqué. Cette caractéristique permet d'assurer un écoulement approprié en dessous de la lame. Une haute viscosité dans les basses fréquences de cisaillement qui sont générées après le couteau d'écoulement est nécessaire pour minimiser l'écoulement non désiré et des effets de sédimentation.

Figure 1 . 1 -Figure 1 . 2 -

 1112 Figure 1.1 -Viscosité en fonction du taux de cisaillement pour (a) SF avec différentes concentrations de gélatine et (b) viscosité et contrainte de cisaillement en fonction du taux de cisaillement pour SF 20 %.

Table 1 .

 1

	1 -Common biodegradable materials used in food packaging
	applications ………………………………………………………………………..…27

Table 1 .

 1 2 -Amino acid composition (expressed as no. residues/1000 residues) from various sources of gelatin……………………….…………………………....[START_REF] Rio+20 | Rio+20 conferência das Nações Unidas sobre desenvolvimento sustentável[END_REF]

Table 1 .

 1 3 -Physical-chemical properties of gelatin-based films………………..35

Table 1 .

 1

	4 -Physical-chemical properties of gelatin-nanocellulose
	bionanocomposites…………………………………………………………………..45
	Page
	CHAPTER II

Table 2 .

 2 1ζ-potential of gelatin and CNCs suspensions and viscosity and rheological parameters of gelatin FFS calculated by the Power Law model. ... 68

Table 2 .

 2

Table 1 .

 1

	2 -Amino acid composition (expressed as no. residues/1000 residues)
	from various sources of gelatin.
	Amino acid	Bovine skin [76] Pork skin [77] Tilapia [78]
	Alanine (Ala)	81	112	123
	Arginine (Arg)	52	49	47
	Aspartic acid (Asp)	32	46	48
	Cystine (Cys)	5	0
	Glutamic acid (Glu)	54	72	69
	Glycine (Gly)	365	330	347
	Histidine (His)	-	4	6
	Hydroxylysine (Hyl)	-	6	8
	Hydroxyproline (Hyp)	102	91	79
	Isoleucine (Ile)	10	10	8
	Leucine (Leu)	19	24	23
	Lysine (Lys)	49	27	25
	Methionine (Met)	2	4	9
	Phenylalanine (Phe)	16	14	13
	Proline (Pro)	101	132	119
	Serine (Ser)	29	35	35
	Threonine (The)	8	18	24
	Tyrosine (Tyr)	11	3	2
	Valine (Val)	20	26	15

Table 1 .

 1 tissues. The physicochemical properties of gelatin films depend on the gelatin source, film manufacturing conditions (concentration, solvent, pH, temperature, drying time), and type and concentration of additives (plasticizer,

	Matrix film	Additive	TS (MPa)	EAB (%)	WVP (g mm/h m 2 kPa)	Solubility (%)	Ref.
	Gelatin		74.2	5.26			[91]
	Gelatin/starch + 10%			11.13	1.57	16.52	[92]
	Gly			4.47	1.43	10.86
	Gelatin/starch + 10 Sor
	Gelatin/agar + 10%		13.62 38.89 0.873	20.85	[93]
	Gly
	Gelatin + 20% Glycerol 20% SPI	49.45 3.5
	Gelatin + 25% Glycerol 10% MMT	63.6	9.7	0.002		[94]
	Gelatin + 30% Glycerol 7% MMT	40.2		0.43		[95]
	Gelatin + 25% Glycerol		66.11 1.01	2.81		[96]
	Gelatin + 10% Glycerol	BTCAD-NHS -N 30% Starch-	48.54 31.3%			[97]
	Gelatin + 20% Glycerol 40% CA	35	16	1.16		[98]
	Gelatin + 60% Glycerol 0.15% Genipi	4.4	244% 0.88	36.3	[99]
	Gelatin + 20% Glycerol	6% CA 5% Gallic acid	26.35 28.34	4.92 3.62	0.048 0.064		[100]
	Gelatin + 25% Glycerol 1.8% GTA	16.13 53.34			[101]
		Active packaging
		1% AgNP	3.18		0.55	4.3	[102]
	Gelatin + 20% Glycerol	AgNP+5%TA	8.12		0.60	1.8

reinforcement, crosslinkers). Having said this, data from the scientific literature are not readily comparable since methodologies may differ considerably from one work to another. The physical properties of gelatin-based films are shown in Table 1.3. Table 1.3 -Physical-chemical properties of gelatin-based films. Abbreviations: WVP: water vapor permeability; TS: tensile strength; EAB: elongation at break; Sor: sorbitol; CA: citric acid; GTA: glutaraldehyde; BTCAD: 1,2,3,4-butanetetracarboxylic acid; NHS, N-hydroxysuccinimide; SPI soy protein isolate; TA: tannic acid; MC: methylcellulose; PP: polypropylene; HDPE: high-density polyethylene; LDPE: low-density polyethylene; PVC: poly(vinylalcohol); PET: pole(ethylene terephthalate).

In general, gelatin films are nearly colorless and display high transparency and excellent UV barrier properties. Gelatin-based films exhibit very low UV light transmission (200-280 nm) (0.01%-30.3% transmission), compared to commercial films such as PVC and LDPE (12.2-87.4% transmission). This is due to the light absorption ability of the disulfide bonds, aromatic amino acids, and carbonyl groups of peptide bonds

[START_REF] Ramos | Gelatin-based films and coatings for food packaging applications[END_REF]

. Another major advantage of gelatin as a packaging material is its excellent barrier against carbon dioxide and oxygen due to its hydrophilic nature. Avena-Bustillos et al.

[START_REF] Avena-Bustillos | Gelation, oxygen permeability, and mechanical properties of mammalian and fish gelatin films[END_REF]

reported oxygen permeability from 0.2 to 0.7 [(cm 3 mm)/(m 2 d bar)] at 23 ˚C and 55 % RH for gelatin films from different sources. The hygroscopic nature of gelatin also represents the main drawback of gelatin films. These films are moisture sensitive and exhibit low Liliane

[START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF]

Table 1 .

 1 4 -Physical-chemical properties of gelatin-nanocellulose bionanocomposites

	Ref.		[17]		[164]		[165]	[166]		[167]		[22]
	Tonset	(°C)		-		-		-	-		-		-
	RH	(%)		-		50%		85%	100%		50%		-
		h
	WVP	(g mm/ m 2	kPa)	0.0002-	0.0017	0.0003-	0.0002		2.5-1,6	1.21-1.89	1.21-1.89	2.30-1.37		-
	EAB	(%)		33.7-20.8		27.5-38.3		19-15	4.2-5.8	4.2-4.4		18-43		110-38
	TS	(MPa)		83.7-108.6		32-33		16.3-17	110-80	100-110		8.2-13.6		12-38
	Drying	Temp.	(°)	37 °C		25 °C		24 °C	24 °C		25 °C		25 °C
	Additive		-		-		-	-		-		-
	CNCs	(wt%)		0-5%	BCNCs	0-1.5%	CNCs		0-15%	CNCs	0-10% CNCs	0-10% CNF		0-1%	CNCs	0-20% di-	aldehyde	CNCs
	Plasticizer	(wt%)		-		20%	Glycerol		25%	Glycerol	-		4%	Glycerol	1-5%	Glycerol
	Bloom		-		-		139	225-325		-		-
	Matrix	(wt%)		10%	gelatin	10%	bovine	gelatin	9.6% fish	gelatin	5-10%	pigskin	gelatin	10%	gelatin	10%	gelatin

Abbreviation: WVP: water vapor permeability, TS: tensile strength, EAB: elongation at break; BCNCs: bacterial cellulose nanocrystal

10 wt% Gelatin FFS 20 wt% Gelatin FFS 30 wt% Gelatin

  rheological behavior of the gelatin solutions with different polymer concentrations was studied to find the most appropriate formulation to produce the bionanocomposite films by continuous casting. Experimental shear stress vs.

	Power Law model fitting
	Sample 1	Sample 2
	FFS
	Liliane Leite -2020

shear rate curves were fitted by the classical Power Law model (R 2 ≥ 0.996) (Figure

2

.4), which has been widely used for non-Newtonian fluids over the most important shear rate range

[START_REF] Ahmed | Rheological Properties of Gelatin and Advances in Measurement[END_REF]

.

Table 2 .

 2

	pH	ζ-potential (mV) Gelatin CNCs	FFS (wt%)	Viscosity (Pa s) at 3 s -1	k (Pa s n )	n
	3	19.0 ± 1.1 a -50.3 ± 2.2 a	10	0.04 ± 0.01 a	0.03 ± 0.01 a	0.89 ± 0.06 a
	6	-0.5 ± 0.1 b -50.4 ± 2.2 a	20	3.95 ± 0.35 b	1.54 ± 1.02 b	0.79 ± 0.07 a
	8	-5.3 ± 0.1 c -48.9 ± 0.4 a	30	169.33 ± 54.70 c 11.67 ± 2.18 c 0.48 ± 0.03 b
	Values with a different superscript in the same column are significantly different (p < 0.05)
	according to Turkey's test.

1ζ-potential of gelatin and CNCs suspensions and viscosity and rheological parameters of gelatin FFS calculated by the Power Law model.

  [START_REF] Ahmed | Rheological Properties of Gelatin and Advances in Measurement[END_REF] °C did not allow the efficient drying of the gelatin/CNCs FFS. On the other hand, temperatures above 80 °C led to gelatin/CNCs film More importantly, the continuous casting approach used in this work had significantly higher productivity compared to nanocellulose-based nanocomposites and other films produced by bench casting reported in the literature (Table2.2).

	Table 2.2 -Productivity of biopolymers-based films produced by
	casting/evaporation.
	Polymer	Reinforcement	Drying conditions	Productivity	Ref.
	Cassava	Fibers	60 °C	0.001 m 2 /min of film	[193]
	k-carrageenan	CNCs	40 °C	Glass petri dish/24h	[221]
	Soy protein	SNC	50 °C	Petri dishes/24h	[201]
	Soy protein	-	60 °C	0.002 m 2 /min of film	[204]
	Soy protein	CNF	37 °C	3.3 x 10 -6 m 2 /min of film	[222]
	Gelatin	CNCs	24 °C	Glass plates/24h	[165]
	Gelatin	CNCs, CNF	24 °C	Petri plates/48h	[166]
	Gelatin	Montmorillonite	30 °C	5.4 x 10 -5 m 2 /min of film	[95]
	Gelatin	Acid citric	24 °C	Petri dishes/48h	[98]
	Gelatin	-	24 °C	3.5 x 10 -6 m 2 /min of film	[96]
	Gelatin	Pomegranate peel	24 °C	Petri dishes/24h	[89]
	Gelatin	Melanin	23 °C	2.5 x 10 -5 m 2 /min of film	[223]
	Gelatin	CNCs	80 °C	0.03 m 2 /min of film	This work

degradation due to extreme fast drying (data not shown). The continuous casting produced 12 m/h of gelatin/CNCs films. The wet layer thickness of 1 mm was used to obtain dry films with a thickness of about 0.065 mm.

Table 2 .

 2 3 -Color parameters, transparency, and thermal properties of gelatin/CNCsMean values followed by the same letter in the same column are not significantly (p > 0.05) different according to the Tukey's test.

	bionanocomposite films.
	CNCs (wt%)	∆L*	∆a*	∆b*	∆E*	Transparency (mm -1 )	Tonset (°C)
	0	2.9 ± 0.4 0.8 ± 0.1 1.7 ± 0.3	3.5 ± 0.1 a	0.7 ± 0.1 a	205.9
	0.5	2.4 ± 0.4 0.8 ± 0.1 1.6 ± 0.1 3.0 ± 0.4 a 0.7 ± 0.1 a	207.3
	1.0	2.9 ± 0.1 0.8 ± 0.1 1.6 ± 0.2 3.4 ± 0.1 a 0.6 ± 0.1 a	214.9
	2.5	2.6 ± 0.7 0.7 ± 0.1 1.9 ± 0.2 3.2 ± 0.4 a 0.7 ± 0.2 a	216.5

Table 2 . 4

 24

	Nanoparticles	Length	Diameter	Aspect ratio	Crystallinity
		(nm)	(nm)	(L/D)	(%)
	Heterogeneous CNCs	360 ± 104	16 ± 3	22.5	76
	Homogenous CNCs	108 ± 33	3.8 ± 0.9 28.4	82

-Average length, diameter, aspect ratios measured by TEM, and percent crystallinity determined by XRD, of heterogeneous and homogeneous CNCs.

Therefore, as homogeneous CNCs showed the best dispersion in water, higher aspect ratio, and higher percent crystallinity compared to heterogenous CNCs, they were chosen to assess the influence of CNCs on the properties of the

3 Physical properties of gelatin-based films obtained by bench casting vs continuous casting 2.3.3.1 Mechanical properties of gelatin-based films

	The mechanical properties, Young's modulus, tensile strength, and
	elongation at break of gelatin-based films were investigated using tensile tests
	and the results are shown in Figure 2.16. The Young's modulus and tensile
	strength of pure gelatin film obtained by bench casting were 743 and 17 MPa,

respectively. The same film produced by continuous casting showed a significant increase (p < 0.05) of 106% for Young's modulus and 111% of tensile strength,

Table 2 .

 2 5 -Triple helix content of gelatin-based films obtained by bench and

	continuous casting.
	Gelatin-based films	Triple helix content (%)*
	Bench casting
	0 wt% CNCs	6.06
	6.0 wt% CNCs	4.23
	Continuous casting
	0%	4.62
	0.5 wt% CNCs	4.41
	4.0wt% CNCs	4.46
	6.0 wt% CNCs	7.41
	2.3.3.

3 Morphological analysis of gelatin-based films

In an attempt to evaluate CNCs dispersion and distribution in gelatinbased films obtained by different methods, scanning electron microscope analyses were carried out. The micrographs of cryogenically fractured samples are shown in Figure

2

.18. A roughness fractured surface was observed for pure gelatin obtained by both methods (Figure

2

.18a, d). Gelatin films with 6.0 wt% obtained by bench casting showed a significant decrease in the fracture roughness (Figure

2

.18b,c). On the other hand, gelatin/CNCs film obtained by continuous casting (Figure

2

.18e,f) showed a homogenous and smooth fractured surface, suggesting a compact surface and good interfacial adhesion of CNCs to the matrix. Besides, no small CNCs aggregates were observed for gelatin/CNCs films obtained by both methods, indicating that even using high CNCs content, these nanoparticles show a good dispersion into the gelatin matrix

[START_REF] Leite | Morphological investigation of cellulose acetate/cellulose nanocrystal composites obtained by melt extrusion[END_REF]

.

Table 2 .

 2

	6 -Onset of thermal decomposition, 50% weight loss, and maximum
	thermal decomposition temperatures for CNCs powder and gelatin-based
	obtained by bench casting and continuous casting.
	Sample	Tonset (°C)	T50 (°C)	Tmax (°C) Residue
	CNCs	281	296	293	21
		Films obtained by bench casting
	0%	256	317	327	17
	0.5 wt% CNCs	244	311	313	17
	4.0wt% CNCs	247	310	312	18
	6.0 wt% CNCs	250	314	313	19
		Films obtained by continuous casting
	0%	265	331	325	20
	0.5 wt% CNCs	271	333	325	21
	4.0wt% CNCs	271	332	326	20
	6.0 wt% CNCs	268	327	331	19

Table 3 .

 3

	1 -Apparent viscosity (ηap), parameters of Ostwald de Waele model
	(consistency index (K), and flow index (n)) and transition temperatures of
	gelatin/CNCs FFS at pH 3, 6 and 8.
			Gelatin/CNCs film-forming solution
		Electrostatic attractive forces pH 3	Isoelectric point pH 6	Electrostatic repulsive forces pH 8
	CNCs (wt%)	0	0.5	5.0	0	0.5	5.0	0	0.5	5.0
			Parameters of Ostwald de Waele model
	k (mPa s n )	0.01	0.03	1.03	0.01	0.03	0.01	0.01	0.02	2.25
	n	0.92	0.83	0.32	0.97	0.97	0.90	0.90	0.96	0.24
	r 2	0.993 0.998 0.997 0.999 0.999 0.992 0.999 0.999 0.990
	ηap (mPa) at 1 s -1	10.1	25.0 887.3 10.9	17.5	12.7	7.79	14.0 1788
				Transition temperature (°C)
	Gel-sol	32.1	32.0	Nd	34.6	35.1	35.0	35.6	35.1	37.8
	Sol-gel	19.4	22.7	Nd	22.7	22.8	22.6	22.4	22.6	Nd

Nd: not detected.

  5 wt% CNCs to gelatin at pH 3 and pH 6, significantly decreased (p < 0.05) the WVP of by 68% and 39%, respectively the resulting films. According

	to Qiao et al. (2013) [261], the addition of counterions screens the electrostatic
	repulsions between charges in the polymer, which, in turn, allows the chains to
	collapse and assume a more compact conformation. In a previous study, Santos
	et al. (2014) [165] reported that the WVP of protein films decreased by increasing
	the CNCs loading. They found that fish gelatin films with 15 wt% CNCs exhibit
	WVP values of approximately 2 g mm/m 2 h kPa (25 °C at 85% RH). George &
	Siddaramaiah (2012) [17] reported that 4 wt% of bacterial cellulose nanocrystals
	reduced the WVP of gelatin and attributed this to the low hygroscopicity of the
	highly crystalline cellulose nanocrystals. They found WVP values around 0.175 g
	mm/m

2

h kPa (25 °C at 50% RH). By contrast, as expected, the addition of CNCs to the gelatin matrix at pH 8 increased the WVP since the electrostatic repulsion in alkaline gelatin/CNCs films can increase the free volume, which resulted in greater gas diffusion. Overall, the WVP values of the gelatin films in the present study were lower than those of previous gelatin films reinforced with CNCs reported above.

Table 3 .

 3 3 -Water vapor permeability at 50% RH and 25 °C and water solubility of the gelatin films with different pH and CNCs content.

	CNCs		WVP (g mm/ m 2 h KPa)
	content (%)	Electrostatic	Isoelectric	Electrostatic
		attractive	point	Repulsive
		force		force
		pH 3	pH 6	pH 8
	0	0.204 ± 0.085 aA	0.180 ± 0.027 aA	0.131 ± 0.020 aA
	0.5	0.065 ± 0.013 bA	0.111 ± 0.022 bB	0.158 ± 0.022 aB
	5.0	0.189 ± 0.009 aA	0.109 ± 0.019 bB	0.262 ± 0.034 bC
			Water solubility (%)
	0	65.3 ± 2.55 aA	31.7 ± 4.81 aB	15.7 ± 3.30 aC
	0.5	14.6 ± 1.24 bA	21.5 ± 6.55 aA	16.9 ± 4.97 aA
	5.0	42.1 ± 2.52 cA	32.4 ± 1.30 aB	17.1 ± 9.46 aB

Mean ± standard deviation. Lowercase letters (a, b, c) -Mean values in the same column bearing the same letter are not statistically different according to the Tukey's test (p > 0.05). Capital letters (A, B, C) -Mean values in the same row bearing the same letter are not statistically different according to the Tukey's test (p > 0.05).

Table 3 .

 3 4 -TGA results of gelatin/CNCs films at different pH.

		1° Stage	2° Stage
	Films	T (°C)	Weight (%)	Tonset (°C)	Weight (%)
		Electrostatic attractive forces -pH3
	0% CNCs	105	6.1	253.9	23.7
	0.5% CNCs	105	7.1	268.3	19.7
	5.0% CNCs	105	12.9	259.5	24.0
		Isoelectric point -pH6
	0% CNCs	105	9.8	258.6	25.3
	0.5% CNCs	105	8.5	248.3	23.5
	5.0% CNCs	105	7.1	252.6	24.1
		Electrostatic repulsive forces -pH8
	0% CNCs	105	3.0	251.2	17.4
	0.5% CNCs	105	8.7	266.5	18.7
	5.0% CNCs	105	7.1	253.4	23.7

Table 3 .

 3 5 -Tensile, thermal properties, water vapor permeability, and triple-helix content of gelatin films with 0.5% CNCs at pH 8 as a function of different drying temperatures.

	Drying temperature (°C)	Young's modulus (GPa)	Tensile strength (MPa)	Elongation at break (%)	Tonset (°C)	WVP (g mm/ m 2 h kPa)	Triple helix content (%)
	25	2.1 ± 0,2 a 51.5 ± 8,95 a 3.31 ± 0.53 a 266.5 0.16 ± 0.02 a 13.5
	35	1.0 ± 0,2 b 20.9 ± 5,01 b 3.57 ± 0.71 a 252.6 0.23 ± 0.08 a 8.7
	80	1.2 ± 0,1 b 19.1 ± 0,45 b 2.98 ± 0.29 a 251.4 0.24 ± 0.16 a 0.3

Mean ± standard deviation. Mean values in the same column bearing the same letter are not statistically different according to the Tukey's test (p > 0.05).

Table 4 .

 4

	2 -Optical properties of gelatin/r-CNCs bionanocomposite and synthetic
	films currently applied in food packaging.
	Film	200 nm	Light transmission (%) 280 nm 400 nm 600 nm
	0.5 wt% gelatin/r-CNCs 0.0	4.5	77.3	87.4
	6.0 wt% gelatin/r-CNCs 0.0	5.4	60.5	66.7
	Synthetic films currently applied in food packaging a
	LDPE a	13.1	67.5	83.4	86.9
	OPP a	4.6	80.0	87.9	89.1
	PVC	20	83.9	87.9	88.7

LDPE: low-density polyethylene; OPP: oriented polypropylene; PVC: polyvinyl chloride.

2 Effect of tannic acid and cellulose nanocrystals on antioxidant, antimicrobial, and physical properties of gelatin-based films

	This section is adapted from Liliane S. F. Leite, Cynthia Pham, Stanley Bilatto,
	Emily D. Cranston, Henriette M.C. Azeredo, Francys K. V. Moreira, Luiz H. C.
	Mattoso, Julien Bras. Active properties of gelatin films as affected by tannic acid
	and cellulose nanocrystals. Submitted in October 2020.

Table 1 -

 1 use of oxidized TA hindered the formation of hydrogen bonds between gelatin, tannic acid, and CNCs, which showed to have a greater influence on the physical properties of gelatin/CNCs-TA films. The main results of this Ph.D. work are summarized in Table1. main results of this Ph.D. work.

	Chapter	Scientific challenges	Key results
	II.1	Scale up the production of gelatin-CNCs films	A roll at 12 m/h of gelatin/CNCs films was produced by continuous casting
		Avoid the formation of	High dispersion of CNCs was achieved using homogenous
		CNCs aggregates	CNCs
	II.2	Gelatin films show moderate mechanical strength and poor barrier properties against moisture	Gelatin film 4.0 wt% CNCs obtained by continuous casting exhibited high thermal and mechanical properties and its WVP decreased by 50% compared with its counterpart obtained by bench casting.
			pH modulated the electrostatic interactions in the CNCs-
			gelatin pair and physical gelatin/CNCs films
	III.1	Produces gelatin/CNCs with tunable physical properties by controlling the pH and the CNCs content	High CNCs contents at pH3 and pH8 decreased the mechanical properties and increased WVP of films Lower WVP of films was attained at pH 3, however, a drastic reduction in their ductility was observed Gelatin/CNCs at pH 6 showed a significant increase in
			mechanical properties and a decrease in WVP at high
			CNCs content.
			Functionalized CNCs with rosin showed antimicrobial
			activity against S. aureus and E. coli bacteria
	IV.1	Active films packaging with antimicrobial properties using a natural agent	Functionalized CNCs consistently improved the optical, and WVP properties of gelatin films as compared to conventional CNCs The antimicrobial activity of rosin in the gelatin films with high
			r-CNCs contents was demonstrated in a proof of concept test
			using mozzarella cheese
	IV.2	Active films packaging with antioxidant and antimicrobial properties using a natural agent	The addition of non-oxidized tannic acid favored non-covalent interactions and intermolecular connections, improving the mechanical stability and increased the antioxidant capacity of the film by 52% compared with gelatin film with oxidized tannic acid.

animais, fornecendo suporte estrutural e mecânico aos tecidos. As cadeias de gelatina são compostas por 18 aminoácidos. Glicina (Gly), prolina (Pro) e hidroxiprolina (Hyp) são os aminoácidos mais dominantes e representam quase dois terços do total de aminoácidos constituintes. A gelatina tem atraído muita atenção como um material plástico alternativo devido à sua abundância, baixo custo, biodegradabilidade, excelentes propriedades de gelificação, capacidade de formar filmes e não toxicidade para aplicações em contato com alimentos.

Liliane[START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF]

Liliane[START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] augmentation de 52 % de la capacité antioxydante de ces films. Cela a également permis la disponibilité de la gélatine pour interagir avec les CNCs et, par conséquent, augmenter de manière significative la viscosité (410 %) et la stabilité mécanique (G' 1697 %) des films.De plus, l'addition de CNC (6 %) aux films à base de gélatine-TA a conduit à la formation de matériaux plus denses avec une porosité réduite, observée par les images de microscopie électronique à balayage (MEB). Les films présentent une PVE réduite (67 %) et une résistance à la traction plus élevée (79 %). Les films à base de gélatine-TA-CNCs ont aussi démontré un effet inhibiteur à la croissance de Staphylococcus aureus et Escherichia coli.La figure 1.2 présente un résumé schématique de toutes les étapes développées dans ce travail, ainsi que les résultats les plus pertinents de chacune d'elles.

Liliane[START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] Les résultats de ce travail ont montré que l'utilisation de la méthode de casting continue pour l'obtention des films à base de gélatine avec propriétés optiques, mécaniques et thermiques d'intérêt est prometteuse pour le développement des emballages. Cependant, des études supplémentaires sont encore nécessaires pour l'utilisation de ces matériaux. Concernant les emballages actifs, des études quantitatives de l'activité antibactérienne sont essentielles pour prouver son efficacité. De plus, des tests de durée de vie en utilisant des aliments différents peuvent étendre l'application de ces films. Des films actifs pourront ensuite être produits et caractérisés par casting continu, en utilisant les paramètres pour les films à base de gélatine/CNCs décrit dans ce travail. Enfin, malgré que tous les composants utilisés dans l'obtention des films étudiés au cours de ce projet soient reconnus comme des produits biodégradables, des tests de biodégradation et compostage sont nécessaires pour prouver leur efficacité.

Program in Materials Science and Engineering (PPG-CEM) À FAPESP -Fundação -3 e processo n° 2018/00278-2. FAPESP -São Paulo Research Foundationgrant 2016/03080-3 and grant 2018-00278-2.

O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Código de Financiamento 001 .

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Financial Code 001.

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] Chapter II Gelatin Therefore, since these aggregates can influence the performance of the films, for a better evaluation of the continuous casting method on the physical properties of gelatin-based films, it is also necessary to investigate the quality of the CNCs dispersion. In this second part of chapter II, the dispersion of heterogeneous and homogeneous CNCs suspension and their morphology were analyzed.

Homogeneous CNCs showed a relatively narrow distribution of length and diameter, and a higher aspect ratio and crystallinity. Therefore, they are chosen to assess the influence of CNCs on the properties of the gelatin-based films obtained by different methods. Gelatin films with 4.0 wt% homogeneous CNCs produced by continuous casting exhibited a decrease of 50% in water vapor permeability and high tensile strength (36 MPa) and Young's modulus (1.8 GPa) compared with its counterpart obtained by bench casting. In addition, gelatin/CNCs films produced by continuous casting presented hydrophilic surfaces and became more thermally stable compared with gelatin/CNCs films obtained by bench casting.

The pure gelatin film dried at 35 °C (bench casting processing) showed a higher triple helix content than pure gelatin film dried at 80 °C (continuous casting processing). This is due to the cold-dried films have a longer drying period, i.e.

sufficient time for the reorganization of gelatin structures, than hot-dried films.

Surprisingly, the addition of 6.0 wt% CNCs increased the triple helix content by 60% in the gelatin/CNCs film, compared with the pure gelatin film obtained by obtained continuous casting. The CNCs contribute to the molecular reorganization of the gelatin and play an important role in the stabilization of the triple helix structure by intramolecular hydrogen bonds [START_REF] Yakimets | Mechanical properties with respect to water content of gelatin films in glassy state[END_REF]. Though, this effect was not observed for the gelatin/CNCs film with the same amount of CNCs obtained by bench casting. This suggesting that there is a synergism between the high concentration of CNCs, IR, and high drying temperature in the formation and stabilization of the triple helix structure. However, the mechanic is still not entirely clear. Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] The XRD patterns of the gelatin-based films obtained at different pH and CNCs contents are shown in Figure 3.6. The formation of gelatin film is accompanied by the renaturation of triple helical structures of protein molecules [START_REF] Lin | The modification of gelatin films: Based on various cross-linking mechanism of glutaraldehyde at acidic and alkaline conditions[END_REF]. The XRD diffractograms showed a wide background with a band at 2θ ~21° [START_REF] Bigi | Relationship between triple-helix content and mechanical properties of gelatin films[END_REF] and band at 2θ ~8°, which has been previously attributed to the presence of triple helix structure present in collagen and renaturated gelatin [START_REF] Quero | Stress transfer and matrix-cohesive fracture mechanism in microfibrillated cellulose-gelatin nanocomposite films[END_REF].

The area of this peak has been found to be directly related to the triple helix content of gelatin-based composites [START_REF] Bigi | Relationship between triple-helix content and mechanical properties of gelatin films[END_REF][START_REF] Badii | The Effect of Drying Temperature on Physical Properties of Thin Gelatin Films[END_REF][START_REF] Yakimets | Mechanical properties with respect to water content of gelatin films in glassy state[END_REF]. As seen in the insert in Figure 3.6a,b the band area at 2θ ~8° of gelatin/CNCs, changed with the pH and CNCs content. The triple helix content of gelatin/CNCs films is presented in Table 3.2.

The triple helix content of pure gelatin film decreased with decreasing pH.

According to Ahsan & Rao (2017) [START_REF] Ahsan | Structural studies on aqueous gelatin solutions: Implications in designing a thermo-responsive nanoparticulate formulation[END_REF] reported the same behavior for type B gelatin, which was attributed to most likely due to the structural changes in gelatin to mild denaturation, which was complete at around pH 2.5.

The highest content of triple helix (13.5%) was observed in the gelatin films with 0.5 wt% CNCs at pH 8. This behavior may result from deprotonation of the gelatin molecules at alkaline pH, favoring the formation of hydrogen bonding [START_REF] Ahmed | Rheological Properties of Gelatin and Advances in Measurement[END_REF],

which are essential to stabilize the gelatin triple helices. In addition, the formation of hydrogen bonds between the CNCs and proteins (both inside and outside helical fragments), also favors the regeneration and stabilization of triple helix structures [START_REF] Bigi | Relationship between triple-helix content and mechanical properties of gelatin films[END_REF][START_REF] Gioffrè | Role of pH on stability and mechanical properties of gelatin films[END_REF]. However, the addition of 5.0 wt% CNCs amount markedly contributes to the reorganization of the gelatin molecular order and diminished the triple helix content, suggesting an increase in random coil conformation of gelatin chains. Similar behavior was observed [START_REF] Yakimets | Mechanical properties with respect to water content of gelatin films in glassy state[END_REF] [237] who found changes in the structural and molecular level of gelatin films induced by water content. These results also suggest that the process of folding the random coil to the triple helix is influenced by not only the pH but also by the interactions with CNCs and by the amount of nanoparticles added.

introduction

Sustainability related to food plastic packaging has worldwide relevance owing to the need for safe disposal of post-consumer plastic wastes [START_REF] Al-Tayyar | Antimicrobial food packaging based on sustainable Bio-based materials for reducing foodborne Pathogens: A review[END_REF]. These problems can be mitigated if natural biodegradable polymers are employed as a packaging material, for which biopolymers have to exhibit suitable physical properties and increase the quality and safety of food [START_REF] Shankar | Preparation, characterization, and antimicrobial activity of gelatin/ZnO nanocomposite films[END_REF][START_REF] Garavand | Improving the integrity of natural biopolymer films used in food packaging by crosslinking approach: A review[END_REF]. Desirable properties of a packaging material include adequate mechanical strength, thermal stability, recyclability, biodegradability, and barrier against water vapor [START_REF] Hassan | Recent advances on polysaccharides, lipids and protein Liliane Leite -2020 based edible films and coatings: A review[END_REF]. Recently, several researches focused on incorporating antimicrobial agents in packaging films to delay microbial growth [START_REF] Sivakanthan | Antioxidant and antimicrobial applications of biopolymers: A review[END_REF], which is the major cause of deterioration of foods [START_REF] Gómez-Estaca | Advances in antioxidant active food packaging[END_REF], causing off-flavor development, textural changes, loss of nutritive value, shelf-life reduction, increased risk of foodborne illnesses [START_REF] Han | Food Packaging: A Comprehensive Review and Future Trends[END_REF], rendering the product unacceptable for human consumption [START_REF] Wang | Decrease of microbial community diversity, biogenic amines formation, and lipid oxidation by phloretin in Atlantic salmon fillets[END_REF].

Rosin is an abundant natural product of pine resins, which is produced in more than 1 million tons annually [START_REF] Zheng | Well-defined renewable polymers derived from gum rosin[END_REF][START_REF] Kugler | Advances in rosin-based chemicals: The latest recipes, applications and future trends[END_REF], and contains a mixture of acids (ca.

90%) with hydrogenated phenanthrene ring structures (Figure 4.1) [START_REF] Liu | Rosin-based acid anhydrides as alternatives to petrochemical curing agents[END_REF]. These acids are used in numerous applications as renewable feedstocks for polymer synthesis due to their biodegradability and nontoxicity [START_REF] Zheng | Well-defined renewable polymers derived from gum rosin[END_REF][START_REF] Kugler | Advances in rosin-based chemicals: The latest recipes, applications and future trends[END_REF][START_REF] Narayanan | UV protective poly(lactic acid)/rosin films for sustainable packaging[END_REF]. Cellulose nanocrystals grafted with natural rosin mixture (r-CNCs) showed the antimicrobial activities for Gram-negative bacteria and Gram-positive bacteria [START_REF] Castro | Surface grafting of cellulose nanocrystals with natural antimicrobial rosin mixture using a green process[END_REF]. Recently, rosin-based bionanocomposites have been studied for the production of active films [START_REF] Li | Bifunctional Reinforcement of Green Biopolymer Packaging Nanocomposites with Natural Cellulose Nanocrystal-Rosin Hybrids[END_REF][START_REF] Niu | Rosin modified cellulose nanofiber as a reinforcing and coantimicrobial agents in polylactic acid/chitosan composite film for food packaging[END_REF]. However, these rosin-based bionanocomposites in previous works require the use of chloroform or dichloromethane to disperse the polymer, limiting their application in the food industry.

°C and then chlorinated with 50 μL of 2,3,5 -Triphenyltetrazolium chloride (0.1%).

The lowest sample concentration with an inhibition effect on microbial growth was considered as the MIC for each tested microorganism.

In the antimicrobial film test, all films were cut into discs of 10 mm diameter and both sides were sterilized by UV treatment in a laminar flow hood for 30 min.

The discs were placed on Muller-Hinton Agar (MHA) plates that had been previously seeded with 100 µL of inoculums containing S. aureus or E. coli at a concentration of 1  10 6 CFU/mL. The plates were then incubated at 35 ˚C for 24 h. Two independent experiments were performed in triplicate.

The antibacterial activity of the gelatin/r-CNCs films on mozzarella cheese was also investigated. Fresh mozzarella samples (20 mm x 20 mm x 10 mm; four replicates) were wrapped with gelatin and gelatin/r-CNCs films. All samples were packaged, sealed by heating and stored at room temperature for 1 month.

Cheese samples without film and wrapped in PVC cling film were used as control.

Statistical analysis

All data were subjected to analysis of variance (ANOVA). Mean values were compared using the Tukey's test at a confidence level of 95% (p<0.05).

Results and discussion

Functionalization of CNCs with rosin

The functionalization of the surface of CNCs occurred by the esterification reaction between the -OH groups on the CNCs and the -COOH groups on the Rosin. A schematic representation of the rosin-functionalized CNCs is shown in Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] the water contact angle value of gelatin/CNCs films. This result demonstrated that the film was more hydrophilic due to the OH-group in the CNCs structure.

The polarity around the structure could create H-bonding to react with water. This

Optical properties of gelatin films reinforced with CNCs and r-CNCs

All gelatin/CNCs and gelatin/r-CNCs bionanocomposites were flexible, and exhibited good macroscopic homogeneity and high transparency, as displayed in Figure 4.9 and the UV-Vis transmittance spectra in Figure 4.10.

Gelatin films exhibited a high barrier against UV radiation, nearly 100% for UVC, over 93.3% for UVB, and 54.0% for UVA, due to chromophore groups such as tyrosine and phenylalanine [START_REF] Chuaynukul | Comparative Characterization of Bovine and Fish Gelatin Films Fabricated by Compression Molding and Solution Casting Methods[END_REF]. The addition of r-CNCs led to a significant reduction in the transmittance over all the UV regions compared to the pure gelatin and gelatin/CNCs film samples. The gelatin film with 0.5 wt% r-CNCs

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] permeability and leakage of cytoplasm, or its reaction with enzymes located at the cell wall [START_REF] Ahmad | Physico-mechanical and antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils[END_REF]. This is consistent with findings that rosin-derived cationic compounds have antimicrobial activity against many bacteria due to the hydrophobicity and structure of resin acids [START_REF] Wang | Robust antimicrobial compounds and polymers derived from natural resin acids[END_REF]. between protein and tannic acid (adapted from [START_REF] Corrêa | Cellulose nanofibers from curaua fibers[END_REF]).

Tannins, particularly TA in its oxidized form, have been reported to improve the mechanical and rheological properties of gelatin films and gels [START_REF] Zhang | Chemical modification of gelatin by a natural phenolic cross-linker, tannic acid[END_REF] [108].

To the best of our knowledge, however, the use of non-oxidized TA, which shows remarkable in vitro antioxidant activity [START_REF] Aewsiri | Antioxidative activity and emulsifying properties of cuttlefish skin gelatin-tannic acid complex as influenced by types of interaction[END_REF], to provide functional properties to gelatin films has not been previously reported. Moreover, the concomitant modification of gelatin films with TA and CNCs is a relevant topic for food packaging technologies that remains still unexplored.

In this study, gelatin/CNCs bionanocomposite films added with nonoxidized TA were produced to explore the individual and combined effects of both agents on the antioxidant, antimicrobial and physical properties of the films aiming at multifunctional food packaging applications. The films were also compared with their counterparts obtained with oxidized TA.

Materials and Methods

Materials

Bovine gelatin powder (Bloom Strength-190) was kindly supplied by Gelco Gelatinas do Brazil Ltda (Pedreira, SP). Cellulose nanocrystals (CNCs) were

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] peeling off after drying at 35 °C for 24 h. The film samples were conditioned at 50% RH and 24 °C for at least 24 h prior to testing.

Transmission electron microscopy (TEM)

The CNCs morphology was investigated by transmission electron microscopy (TecnaiTM G2 F20 microscope, FEI Company, USA) using the STEM (Scanning Transmission Electron Microscopy) mode with an accelerating voltage of 80 kV. The samples were prepared by depositing a 0.1 wt% CNCs dispersion drop on a carbon microgrid with formvar (400 mesh) stained with 1.5 wt% uranyl acetate solution. The CNCs sizes were determined from a minimum of 100 particles using the ImageJ software.

X-ray diffraction (XRD)

The crystallinity of the CNCs was analyzed by X-ray diffraction (Panalytical diffractometer, X'Pert Pro MPD-Ray, The Netherlands) with Ni-filtered Cu Ka radiation (k = 1.54 Å, 45 kV, 40 mA) by using 2θ range from 5° to 60°. The crystallinity index (CI) was calculated according to the Segal method [START_REF] Segal | An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer[END_REF] as shown in Eq. 1:

where I1 is the intensity at the minimum (2θ = 18°) and I2 in the intensity associated with the crystalline region of cellulose (2θ = 22.7°). A pre-spin step with water at 4000 rpm for 30 s was done followed by depositing the 2 wt% CNCs dispersion and spinning at 4000 rpm for 30 s. The CNCs films on spin-coated sensors were annealed at 80 o C overnight in an oven [START_REF] Hu | Tuning cellulose nanocrystal gelation with polysaccharides and surfactants[END_REF]. The nominal CNCs film thickness is estimated to be 30-40 nm [START_REF] Hu | Tuning cellulose nanocrystal gelation with polysaccharides and surfactants[END_REF][START_REF] Benselfelt | Adsorption of Xyloglucan onto Cellulose Surfaces of Different Morphologies: An Entropy-Driven Process[END_REF]. Chung (2016) [START_REF] Anvari | Dynamic rheological and structural characterization of fish gelatin -Gum arabic coacervate gels cross-linked by tannic acid[END_REF] who found that fish gelatin/gum arabic systems with nonoxidized TA presented a higher degree of intermolecular connectivities in comparison with those containing OTA. observed similar behavior for gelatin-encapsulated tea polyphenols dispersion added with sulfated CNCs, which exhibited a positive effect on viscosity, increasing from 67 mPa s to 122 mPa s. This is in agreement with the formation of a percolation CNCs network, as previously discussed, which can result in high resistance to flow. The lower viscosity was found to G-TA FFS, which could be attributed to the plasticizing effect of TA at high concentrations [START_REF] Ortiz-Zarama | Rheological characterization of solutions of gelatin with bentonite and tannic acid[END_REF].

Quartz crystal microbalance with dissipation (QCM-D)

Preparation of solutions and CNCs dispersions for QCM-D experiments

QCM-D experiments

Besides, all the FFS exhibited a shear-thinning behavior (n < 1), and the non-Newtonian index (n) decreased and the consistency coefficient (k) increased upon the addition of CNCs. This means that the viscosity is more dependent on the shear rate [START_REF] Liu | Rheological properties of soy protein isolate solution for fibers and films[END_REF] and gelatin-based films became more viscous. For all the samples, r 2 was greater than 0.99, confirming that the Power Law model was very suitable to describe the rheological property of the gelatin/CNCs FFS containing TA and OTA. higher viscoelastic modulus observed for the G-TA-CNC6 film (Figure 4.24).

However, the addition of OTA, consequently, increased the crosslinking degree and decreased the hydrogen bonding the G-OTA-CNC6 film (Figure 4.25d), which affected the swollen films, which presented an open network with irregular shape and large pores and discontinuous zones, compared to the G-TA and G-TA-CNC6 films.

Antioxidant activity of gelatin-based films with TA or OTA

The free radical chain reaction is extensively accepted as a common mechanism of lipid peroxidation. Assays based upon the use of DPPH * radicals are the most popular spectrophotometric method for the determination of the antioxidant capacity of different samples, such as pure compounds, plant extracts, fruit, vegetables, lignocellulosic agro-waste, etc. [START_REF] Collazo-Bigliardi | Improving properties of thermoplastic starch films by incorporating active extracts and cellulose fibres isolated from rice or coffee husk[END_REF]. In the DPPH assay, the purple-colored DPPH * is a stable free radical, which is reduced to the yellow non-radical form DPPH-H (diphenyl-picrylhydrazine) by reacting with a hydrogen-donating antioxidant [START_REF] Hanani | Effect of pomegranate (Punica granatum L.) peel powder on the antioxidant and antimicrobial properties of fish gelatin films as active packaging[END_REF] (Figure 4.26a). Antioxidant as TA is a substance that inhibits or delays the free radical chain oxidation by donating hydrogen from hydroxyl groups to form a stable end product, which does not initiate or propagate further oxidation of lipid (Figure 4.26b) [START_REF] Gülçin | Radical scavenging and antioxidant activity of tannic acid[END_REF]. Mean values followed by the same letter in the same column are not significantly (p > 0.05) different according to the Tukey's test. Nevertheless, the addition of TA and 4 wt% CNCs decreased the water contact angle value of G-TA-CNC4 film to 70.6 ± 2.2 °. This result demonstrated that the film was more hydrophilic due to the OH-group in the TA and CNCs structure. The polarity around the structure could create H-bonding to react with water. However, adding 6 wt% of CNCs to gelatin-TA matrix increased significantly the water contact angle value to 90.0 ± 5.9 o , which can be attributed to the higher degree of hydrogen bonding between the CNCs, TA, and gelatin, which is in agreement with rheological results. These results suggest the less water-susceptible surface of the G-TA-CNC6 film. Mahajer et al. (2017) [START_REF] Mohajer | Physico-chemical and microstructural properties of fish gelatin/agar bio-based blend films[END_REF] observed the same effect on the surface of gelatin films after the addition of agar.

Contact angle of gelatin-based films

The authors attributed the decrease in surface hydrophilicity of the gelatin films due to the formation of intermolecular hydrogen bonds between hydroxyl groups of agar and amino groups in the gelatin chains, reducing their ability to form hydrogen bonds with water molecules.

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] no significant difference (p > 0.05) was noticed concerning the addition of CNCs.

However, the puncture deformation of the G-TA-CNC films was significantly reduced (p < 0.05) by 38% compared with that of the G-TA films, which may be attributed to the CNCs reinforcing effect. These results are in agreement with Nazimi et al. (2017) [START_REF] Nazmi | Preparation and characterization of chicken skin gelatin/CMC composite film as compared to bovine gelatin film[END_REF] who found 6.31 N of puncture force for bovine gelatin blended with carboxymethyl cellulose films. those of synthetic polymeric film (adapted from [START_REF] Kwak | Chemical and physical reinforcement of hydrophilic gelatin film with di-aldehyde nanocellulose[END_REF]).

Liliane Leite -2020 The increase in the elongation of gelatin-based films may expand its applicability to different types of food packaging.

 Expand the use of continuous casting to vegetal proteins

Although gelatin is considered a by-product of the meat industry, a culture of continuous improvement must be adopted to improve sustainability. The use of protein such as soybean can be an alternative to gelatin.

 Scale-up the production of active gelatin films using the parameters set in section 4.1 and 4.2.



Investigate the infrared radiation effects on the physical properties of active gelatin films. Active films packaging with antioxidant and antimicrobial properties using a natural agent

The addition of 6% CNCs in Gelatin-TA films resulted in a denser structure, low porosity, and lower WVP (67%), and greater tensile strength (79%) compared to the gelatin-TA films.

Gelatin-TA-CNCs films also demonstrated an inhibitory effect against S. aureus and E. coli growth.

Liliane [START_REF] Leite | Scaled-up production of gelatin-cellulose nanocrystal bionanocomposite films by continuous casting[END_REF] Extended French Abstract Résumé Français