Gabriela Vollet Marson
email: gabrielavollet@gmail.com

Rafaela Polessi Saturno

Aline Talita

Larissa Comunian

Mariana Consoli

Miriam Teixeira Da Costa Machado

Dupas Hubinger

Aline Comunian

Larissa Consoli

Mariana Teixeira Da Costa Machado

Miriam Dupas Hubinger

Maillard conjugates from spent brewer's yeast by-product as an innovative encapsulating material

Keywords: ascorbic acid, spray drying, yeast-based by-products, Saccharomyces pastorianus, hydrolyzed yeast protein, yeast cell debris, food-grade carrier agents 0 glass transition temperature, ∆E: Total Color Difference, BI: Browning Index, BSA: bovine serum albumin, WPI: whey protein isolate

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Great amounts of by-products and residues are produced by the food industry as a result of raw materials processing. In 2010, about 90 million tons of food waste were produced due only to food manufacturing [START_REF] Ravindran | Exploitation of food industry waste for high-value products[END_REF]. These materials usually have high moisture content, biological instability and organic load, requiring an appropriate handling and waste management system that considers environmental and economical aspects [START_REF] Nayak | An overview of the recent trends on the waste valorization techniques for food wastes[END_REF]. Some of those food by-products are underutilized even though they present great potential to become added-value innovative ingredients for the food and pharmaceutical industries once adequately processed [START_REF] Nayak | An overview of the recent trends on the waste valorization techniques for food wastes[END_REF][START_REF] Ravindran | Exploitation of food industry waste for high-value products[END_REF].

Yeast-based by-products may be an attractive option due to their great availability, nutritional composition, biocompatibility and environmental perspective [START_REF] Pérez-Torrado | Yeast biomass, an optimised product with myriad applications in the food industry[END_REF][START_REF] Paramera | Chapter 23 -yeast cells and yeast-based materials for microencapsulation[END_REF][START_REF] Ciamponi | Yeast cells as microcapsules. analytical tools and process variables in the encapsulation of hydrophobes in S. cerevisiae[END_REF]. The spent brewer yeast (SBY) consists of the spent yeast cells collected after beer fermentation and is the second most relevant by-product of the brewing industry [START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF][START_REF] Ferreira | Brewer's Saccharomyces yeast biomass: characteristics and potential applications[END_REF], which is responsible for the generation and management of about 2.3 g of this waste material per litre of beer produced [START_REF] Kumar | Valorization of Food Processing By-Products[END_REF][START_REF] Pinto | Valuation of brewers spent yeast polysaccharides: A structural characterization approach[END_REF]. SBY is rich in carbohydrates (about 40% in dry basis) and proteins (about 50% in dry basis) as well as in vitamins from the B complex and minerals [START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF][START_REF] Mussatto | Biotechnological potential of brewing industry by-products[END_REF]. Despite presenting a notably high nutritional value and a steady availability throughout the year [START_REF] Podpora | Spent brewer's yeast extracts as a new component of functional food[END_REF][START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF][START_REF] Mussatto | Biotechnological potential of brewing industry by-products[END_REF], it is still underutilized as animal feed, with low commercial value [START_REF] Vieira | Valorization of brewers' spent grain and spent yeast through protein hydrolysates with antioxidant properties[END_REF][START_REF] Shurson | Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods[END_REF].

Technological and processing strategies as well as value-added applications for yeast by-products are being investigated, but the exploration of SBY is still under development (Marson et al., 2019a,b;[START_REF] Love | The yeast stands alone: the future of protein biologic production[END_REF][START_REF] Vieira | Antioxidant and antihypertensive hydrolysates obtained from by-products of cannery sardine and brewing industries[END_REF][START_REF] Amorim | Valorization of spent brewer's yeast: Optimization of hydrolysis process towards the generation of stable ACE-inhibitory peptides[END_REF].

The first step in processing SBY material is to disrupt yeast cell walls so that cell debris and intracellular compounds may be accessed [START_REF] Liu | Disruption and protein release by ultrasonication of yeast cells[END_REF]. Enzymatic hydrolysis can promote the rupture of the yeast cells, enhance the cell wall porosity, enlarge cell wall pore size and extract proteins, producing smaller fractions of peptides [START_REF] Ed. Chae | Utilization of brewer's yeast cells for the production of food-grade yeast extract. part 1: effects of different enzymatic treatments on solid and protein recovery and flavor characteristics[END_REF][START_REF] Wei | Enzymatic hydrolysis of flaxseed (Linum usitatissimum L.) protein and sensory characterization of Maillard reaction products[END_REF]Marson et al., 2019a,b). After centrifugation, the Saccharomyces sp. cell wall debris are recovered. The cell wall of Saccharomyces sp. is approximately 100-200 nm thick and comprises 15-25% of the dry mass of the cell. It is made of a β-1,3-glucan network crosslinked to β-1,6-glucans, a mannoprotein layer, and a small amount of chitin [START_REF] Zechner-Krpan | Characterization of β-glucans isolated from brewer's yeast and dried by different methods[END_REF][START_REF] Paramera | Chapter 23 -yeast cells and yeast-based materials for microencapsulation[END_REF][START_REF] Shurson | Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods[END_REF][START_REF] Nelson | Microencapsulation in yeast cells and applications in drug delivery[END_REF]. Glucans represents 50-60% of the dry weight of yeast cell walls) [START_REF] Shurson | Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods[END_REF][START_REF] Nakhaee Moghadam | Saccharomyces cervisiae as an efficient carrier for delivery of bioactives: a Review[END_REF][START_REF] Zechner-Krpan | Characterization of β-glucans isolated from brewer's yeast and dried by different methods[END_REF]

and along

Eugène Bataillon, 34095 Montpellier, France with chitin, are responsible for the mechanical rigidity of the cell wall [START_REF] Paramera | Chapter 23 -yeast cells and yeast-based materials for microencapsulation[END_REF]. The porosity and molecular flux passing through yeast cell walls are controlled by the plasma membrane and mannoproteins [START_REF] Pham-Hoang | Molecule structural factors influencing the loading of flavoring compounds in a natural-preformed capsule: Yeast cells[END_REF][START_REF] Paramera | Chapter 23 -yeast cells and yeast-based materials for microencapsulation[END_REF]. β-glucans are functional ingredients successfully employed for microencapsulation and reported to play a role in the retention capability of yeast as a coating material [START_REF] Ahmad | Microencapsulation of saffron anthocyanins using β glucan and β cyclodextrin: Microcapsule characterization, release behaviour & antioxidant potential during in-vitro digestion[END_REF][START_REF] Sobieralska | Beta-glucan as wall material in encapsulation of elderberry (Sambucus nigra) extract[END_REF][START_REF] Sultana | Stability and release behavior of encapsulated flavor from spray-dried Saccharomyces cerevisiae and maltodextrin powder[END_REF]. The hydrophobicity and composition of the yeast cell walls can be changed by factors such as growth media and strain, influencing the encapsulation properties of yeast cells [START_REF] Pham-Hoang | Molecule structural factors influencing the loading of flavoring compounds in a natural-preformed capsule: Yeast cells[END_REF]. Because of its composition and unique characteristics, the yeast cell wall is currently being used as a novel coating for the encapsulation of different materials in the food industry (Mokhtari et al., 2017a).

Microencapsulation is a technique in which a core compound of interest is surrounded by another material, a carrier, resulting in the formation of small particles. Spray drying is one of the most used techniques in the food industry for this purpose [START_REF] O'sullivan | Atomisation technologies used in spray drying in the dairy industry: A review[END_REF], turning liquids into powders of higher stability and lower transportation costs [START_REF] Assadpour | Advances in spray-drying encapsulation of food bioactive ingredients: From microcapsules to nanocapsules[END_REF]. The encapsulation impacts the stability of the core compound because the carrier protects them against environmental conditions (light, oxygen, moisture and heating), undesired biological reactions, masks off-flavors and influences their release in products and in complex biological systems [START_REF] Pham-Hoang | Molecule structural factors influencing the loading of flavoring compounds in a natural-preformed capsule: Yeast cells[END_REF][START_REF] Reineccius | Multiple-core encapsulation: The spray drying of food ingredients[END_REF][START_REF] Reineccius | The spray drying of food flavors[END_REF][START_REF] Timilsena | Complex coacervation: Principles, mechanisms and applications in microencapsulation[END_REF][START_REF] Saifullah | Micro and nano encapsulation, retention and controlled release of flavor and aroma compounds: A critical review[END_REF][START_REF] Anandharamakrishnan | Selection of wall material for encapsulation by spray drying[END_REF]. The extent of those effects is highly dependent on processing parameters and carrier composition [START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF]. A suitable carrier is selected based on process parameters and technique, cost, functionality, nutritional characteristics, stability during storage, and other applicable restraints [START_REF] Assadpour | Advances in spray-drying encapsulation of food bioactive ingredients: From microcapsules to nanocapsules[END_REF]Mokhtari et al., 2017a;[START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF].

The most commonly employed biopolymers used as carrier agents for encapsulation are proteins and polysaccharides, namely Arabic gum, maltodextrin, gelatin, soy and whey proteins. In spray-drying microencapsulation, maltodextrin is widely used because it is cost-effective, presents high water solubility, low viscosity and moderate sweet taste, allowing to attain high solid ratios, advantageous for core retention [START_REF] Sultana | Stability and release behavior of encapsulated flavor from spray-dried Saccharomyces cerevisiae and maltodextrin powder[END_REF]. Maltodextrin rapidly forms a dense film around the core, providing an excellent protection for the core material, specially when combined with proteins, which increase their emulsification capacity [START_REF] Anandharamakrishnan | Selection of wall material for encapsulation by spray drying[END_REF].

Great interest surrounds the investigation of new food-grade wall materials. Agro-industrial by-products may be a compelling alternative, proposing water-soluble, protein and polysaccharides-rich materials for biomolecules encapsulation associated with a sustainable approach and waste management solution. Alternative sources of protein are also increasingly in demand as substitutes for animal proteins in human nutrition, and SBY is considered as an option [START_REF] Mussatto | Biotechnological potential of brewing industry by-products[END_REF][START_REF] Love | The yeast stands alone: the future of protein biologic production[END_REF].

Yeast materials are reported successfully in the encapsulation of several hydrophilic and hydrophobic food compounds [START_REF] Pham-Hoang | Molecule structural factors influencing the loading of flavoring compounds in a natural-preformed capsule: Yeast cells[END_REF][START_REF] Sultana | Microencapsulation of flavors by spray drying using Saccharomyces cerevisiae[END_REF][START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF][START_REF] Paramera | Chapter 23 -yeast cells and yeast-based materials for microencapsulation[END_REF][START_REF] Nelson | Microencapsulation in yeast cells and applications in drug delivery[END_REF][START_REF] Shi | Characterization of yeast cells as a microencapsulation wall material by fourier-transform infrared spectroscopy[END_REF]. The main cell components responsible for the yeasts' excellent encapsulating agent properties are cell wall constituents and inner plasma membrane [START_REF] Paramera | Chapter 23 -yeast cells and yeast-based materials for microencapsulation[END_REF]. Yeasts can be used as microcapsules (the core material penetrates the cells via passive diffusion) in viable or not, entire or plasmolysed yeast [START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF][START_REF] Pham-Hoang | Molecule structural factors influencing the loading of flavoring compounds in a natural-preformed capsule: Yeast cells[END_REF][START_REF] Shi | Characterization of yeast cells as a microencapsulation wall material by fourier-transform infrared spectroscopy[END_REF]. Yeast cell debris are also suitable carriers for food microencapsulation (Mokhtari et al., 2017a;[START_REF] Paramera | Chapter 23 -yeast cells and yeast-based materials for microencapsulation[END_REF]. Broken yeast cell wall material from S. cerevisiae was used in the encapsulation of probiotic bacteria which are bigger in size (Mokhtari et al., 2017a). The yeast coating impacted positively the resistance of the encapsulated probiotic bacteria in triple layered microcapsules with alginate, retarding gastric fluid permeation (Mokhtari et al., 2017a). The yeast cell wall is reported to promote a successful protection against the oxidation of compounds and some authors stated that the yeast cell wall may have a better antioxidative ability than isolated β-glucan [START_REF] Sultana | Stability and release behavior of encapsulated flavor from spray-dried Saccharomyces cerevisiae and maltodextrin powder[END_REF][START_REF] Jaehrig | In vitro potential antioxidant activity of (1→3),(1→6)β--glucan and protein fractions from Saccharomyces cerevisiae cell walls[END_REF]. Yeast cell mannans from S. cerevisiae are also reported to have antioxidant and antimutagenic properties [START_REF] Križková | Antioxidative and antimutagenic activity of yeast cell wall mannans in vitro[END_REF][START_REF] Liu | Extraction, characterization and antioxidant activities of mannan from yeast cell wall[END_REF]. Indeed, there seem to be a tendency to procuce capsules with dual health-promoting properties, from both the core and coating materials [START_REF] Drozińska | Microencapsulation of sea buckthorn oil with β-glucan from barley as coating material[END_REF].

The Maillard reaction is defined as the reaction between amino acids and reducing sugars, eventually forming melanoidins, brown-colored polymers [START_REF] Mirafzali | Chapter 13 -application of liposomes in the food industry[END_REF]. The Maillard reaction can also result in an increased solubility and decreased allergic effects, caused mainly by the glycation of carbohydrates to proteins [START_REF] Consoli | Sodium caseinate-corn starch hydrolysates conjugates obtained through the Maillard reaction as stabilizing agents in resveratrolloaded emulsions[END_REF][START_REF] Yang | Optimization of maillard reaction with ribose for enhancing anti-allergy effect of fish protein hydrolysates using response surface methodology[END_REF]. Moreover, the enhancement of antioxidant properties may occur, and have been related to the development of advanced Maillard Reaction Products (MRPs) [START_REF] O'regan | Preparation, characterisation and selected functional properties of sodium caseinatemaltodextrin conjugates[END_REF][START_REF] Consoli | Sodium caseinate-corn starch hydrolysates conjugates obtained through the Maillard reaction as stabilizing agents in resveratrolloaded emulsions[END_REF][START_REF] Li | Functional properties of Maillard reaction products of rice protein hydrolysates with mono-, oligo-and polysaccharides[END_REF]. In this context, the Maillard reaction may be seen as an option to obtain ingredients of improved functional and sensorial properties. Protein hydrolysates have been studied as substrate for the Maillard reaction to obtain ingredients of improved functional and sensorial properties [START_REF] Hou | Optimization of enzymatic hydrolysis of Alaska pollock frame for preparing protein hydrolysates with low-bitterness[END_REF][START_REF] Wei | Enzymatic hydrolysis of flaxseed (Linum usitatissimum L.) protein and sensory characterization of Maillard reaction products[END_REF][START_REF] Liu | Effect of thermal treatment on the flavor generation from Maillard reaction of xylose and chicken peptide[END_REF]. Regarding the SBY material, this reaction was previously reported as a processing step for brewer yeast extract production as a flavor enhancing step [START_REF] In | Downstream process for the production of yeast extract using brewer's yeast cells[END_REF], but was not employed with the interest of improving functional properties as the solubility of yeast compounds and development of color.

In previous works, standard, plasmolysed or milled compressed baker's yeast (Saccharomyces cerevisiae) [START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF]Mokhtari et al., 2017a,b) or spent yeast by-product from β-glucan production (Sac-charomyces cerevisiae) [START_REF] Sultana | Microencapsulation of flavors by spray drying using Saccharomyces cerevisiae[END_REF][START_REF] Sultana | Stability and release behavior of encapsulated flavor from spray-dried Saccharomyces cerevisiae and maltodextrin powder[END_REF] were used for microencapsulation purposes. In Mokhtari et al. (2017a,b) works, baker's yeast cells were used to encapsulate probiotics through coating of alginate capsules. [START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF][START_REF] Sultana | Microencapsulation of flavors by spray drying using Saccharomyces cerevisiae[END_REF][START_REF] Sultana | Stability and release behavior of encapsulated flavor from spray-dried Saccharomyces cerevisiae and maltodextrin powder[END_REF]; [START_REF] Shi | Characterization of yeast cells as a microencapsulation wall material by fourier-transform infrared spectroscopy[END_REF] encapsulated, respectively, curcumin, flavors and chlorogenic acid in yeast cells as preformed natural capsules, by passive cell transport, and then dried the obtained material. Spray drying using hydrolyzed SBY cell walls was not yet reported.

The SBY proposed in this work is still a different material because it is not an yeast pure material -which is usually used in microencapsulation studies -it contains compounds from the beer production process and are exhausted cells after fermentation, which changes their characteristics and cell wall composition (Marson et al., 2019b;[START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF]. Moreover, in those works the yeast cells were not enzymatically hydrolyzed -what causes important changes on the physico-chemical characteristics of the material -and were β-glucan depleted (because of their previous application). Because of all those particularities and because of the recently reported importance of β-glucan in the microencapsulation capacity of yeast cells [START_REF] Ahmad | Microencapsulation of saffron anthocyanins using β glucan and β cyclodextrin: Microcapsule characterization, release behaviour & antioxidant potential during in-vitro digestion[END_REF]; [START_REF] Pham-Hoang | Molecule structural factors influencing the loading of flavoring compounds in a natural-preformed capsule: Yeast cells[END_REF], the evaluation of this material is of great interest. In this work, we propose the use of Maillard-reacted SBY cell wall debris as a coating material for ascorbic acid, a model core material. Ascorbic acid is a widely used ingredient in the food industry added as a vitamin supplement and antioxidant, but its high reactivity and low stability to oxygen and metallic ions limits their direct application as an ingredient. Acids can also promote undesirable changes in foods such as colour degradation, undesirable odours, changes in pH and flavor when incorporated directly [START_REF] Trindade | The stability of ascorbic acid microencapsulated in granules of rice starch and in gum arabic[END_REF].

Material and Methods

Material

Microencapsulation materials

The wall material proposed was the enzymatically hydrolyzed yeast cells of SBY (Section 2.2). The raw material (S. pastorianus) from Lager Pilsen beer production (Diamond Lager, Lallemand, Canada), collected after 11 days of beer maturation without any repitching at Haus Bier Brewery (São José dos Campos, Brazil), was homogenized and kept at 2 °C until processing. Enzymes used were Alcalase™ 2.4 L and Protamex™, supplied by Novozymes (Denmark), and Brauzyn ® 100 L, supplied by Prozyn (Brazil).

Maltodextrin with a dextrose equivalent of 20% (MOR-REX ® 1920) was supplied by Ingredion (Brazil) and L-ascorbic acid (99%), which was used as the core material, was purchased from Sigma-Aldrich (Steinheim, Germany).

Reagents

Bovine serum albumine (BSA) (electrophoretic grade) was purchased from Sigma-Aldrich (Steinheim, Germany). All other reagents were of analytical grade.

Production of spent brewer's yeast hydrolyzed cells

The schematic representation of all experimental steps taken in this work to study the hydrolyzed yeast cells as carrier materials for ascorbic acid microencapsulation are presented in Figure 1. The hydrolyzed yeast cell debris (HP) were produced using the method of Marson et al. (2019a). Briefly, the yeast slurry was heat-treated, at 70.0 ± 0.2 °C, under 1000 rpm of agitation for an hour, followed by an ice bath. Then hydrolysis was done at pH 7.0, 50 °C, under agitation, for 2 hours. A mixture of enzymes was added (Alcalase™, Protamex™, Brauzyn ® ) in an enzyme:substrate ratio of 666.67 U g protein -1 each. Enzymes were then inactivated by heating (95 °C for 15 min). Non-treated raw material and hydrolysate were centrifuged at 15,300 × g for 30 min at 4 °C (Allegra 25R, Beckman Coulter, United States), to harvest the yeast cells, which were kept at -20 °C in polypropylene bottles until further analysis.

Characterization of spent brewer's yeast material and fractions

Physico-chemical and proximal composition analysis were performed in the non-treated and hydrolyzed SBY cell debris. Total solids, pH value, total titratable acidity, crude protein content, total sugars, fibers, ash and ribonucleic acids (RNA) content were determined as described below in section 2. , 2006) considering citric acid as the major acid present in the samples. Total sugars content was measured according to the Phenol-Sulphuric Acid Assay [START_REF] Fournier | Colorimetric quantification of carbohydrates[END_REF] and reducing sugars by the method of Somogyi-Nelson [START_REF] Fournier | Colorimetric quantification of carbohydrates[END_REF] using glucose as standard. Crude protein content was determined by the Dumas method [START_REF] Wrolstad | Measurements of protein content[END_REF] in an element analyser CHNS-O (Flash 2000, ThermoScientific, USA). A nitrogen conversion factor of 5.5 was considered because of the high content of non-proteic nitrogen in yeast, as previously reported by other papers which measured crude protein in yeast products [START_REF] Reed | Food and feed yeast[END_REF][START_REF] Caballero-Córdoba | Nutritional and toxicological evaluation of yeast (Saccharomyces cerevisiae) biomass and a yeast protein concentrate[END_REF][START_REF] De La Hoz | Iron-binding properties of sugar cane yeast peptides[END_REF]. RNA was extracted according to the method of [START_REF] Webb | Studies on the determination of total nucleic acids by ultraviolet absorption methods[END_REF]; [START_REF] Webb | A sensitive method for the determination of deoxyribonucleic acid in tissues and microorganisms[END_REF] and determined based on the calculation presented by [START_REF] Sceni | Thermal and surface behavior of yeast protein fractions from Saccharomyces cerevisiae[END_REF].

Particle size

Estimation of particle size and particle size distribution in the yeast materials was determined by the angular variation in the intensity of scattered light, as a laser beam passes through the sample (Mastersizer 2000, Malvern Instruments Ltd., UK), using water as dispersant. Non-treated yeast cells (YP) and hydrolyzed yeast cell debris (HP) were diluted in distilled water to reach a concentration of 3% (m/v, wet basis). Whole yeast (Y) was not diluted. Mean diameter of particles was expressed as the volume-weighted [START_REF] Fan | Size and properties of particles[END_REF], as presented in Equation 1, where d i represents the particles diameter and n is the amount of particles.

mean diameter, D [4.3] (Fan
D [4.3] = n i=1 n • d 4 i n i=1 n • d 3 i (1)
The particle size distribution was measured as the span, which considers the equivalent volume diameters at 90% (D (0.9) ), 10% (D (0.1) ), and 50% (D (0.5) ) cumulative volume [START_REF] Jinapong | Production of instant soymilk powders by ultrafiltration, spray drying and fluidized bed agglomeration[END_REF], as presented in Equation 2.

Span = D (0.9) -D (0.1) D (0.5) (2)

Surface tension

Non-treated (YP) and hydrolyzed yeast cell debris (HP) were diluted prior to analysis to a concentration of 3% (m/v, wet basis). The solution was left 1h at 2 °C, the supernatant was collected and the pH adjusted using NaOH or HCl. Multiple measurements via camera were made of a sample's drop of 6 µL in contact with air for 2000 s in a tensiometer (Teclis, Tracker, France), on pendant drop mode. For the hydrolyzed yeast cell debris (HP) measurements were made for pH values of 5, 6, 7 and 8 and for the other yeast materials, at pH 7. Six replicates were determined for each sample. Results were expressed as mN m -1 .

Zeta potential

The ζ-potential of yeast materials was determined using a Zetasizer Nano Series (Malvern Instruments, UK) at 25 °C. HP samples were suspended in water and diluted as described previously (section 2.3.3) and the non-treated yeast (Y) was diluted to 0.1% (m/v, wet basis). For HP, measurements were made for pH values of 5, 6, 7 and 8 and for the other yeast materials, at pH 7. The average pI of HP proteins was determined as the pH where the surface charge density was equal to zero. The influence of the addition of maltodextrin in the production of hydrolyzed yeast Maillard conjugates was evaluated. The control (without maltodextrin) suspension was prepared mixing the hydrolyzed yeast cells (HP) directly in distilled water at the concentration of 4% (m/v, wet basis), resulting in yeast cells Maillard conjugates (YMC). The experimental suspension was produced the same way, with the addition of maltodextrin in the proportion of 1:5 (protein content in HP: maltodextrin, m/m), resulting in another yeast cells Maillard conjugates with added maltodextrin (YMC20) (Figure 1).

Maillard reaction

The Maillard reaction in the HP was prepared in wet medium, according to the methodology of [START_REF] Augustin | Maillard reaction products as encapsulants for fish oil powders[END_REF], with the modifications proposed by [START_REF] Consoli | Sodium caseinate-corn starch hydrolysates conjugates obtained through the Maillard reaction as stabilizing agents in resveratrolloaded emulsions[END_REF]. Briefly, the suspensions were kept under agitation (400 rpm) at 75 °C for 12 h and after, cooled down in an ice bath. Non-heated control suspensions were prepared as well.

Evaluation of the extent of the Maillard Reaction

The extent of Maillard reaction was evaluated by the pH measurement before and after the reaction, supernatant mass recovery, soluble solids content, peptide content in the supernatant fraction and evolution of color.

The pH values of the suspensions before and after the Maillard reaction were determined in triplicate using a two-point calibrated pH meter (K39-1014B, Kasvi, Brazil). Then, it was adjusted at the isoelectric pH (pI) (section 2.3.4) prior to centrifugation at 15,300 × g for 15 min at 4 °C (Allegra 25R, Beckman

Coulter, United States). The mass of recovered supernatant was recorded. Soluble solid content (°BRIX) was determined at 25 °C in a refractometer (N-1 alpha, ATAGO, Japan). (AOAC, 2006).

The peptide concentration of the supernatants was measured by Far-UV (2800, Unico, United States)

following the method of [START_REF] Aitken | Protein determination by UV absorption[END_REF] with some modifications. Briefly, samples were carefully diluted 55 fold using a 5 mM phosphate buffer (pH 7) with 50 mM of sodium sulfate and the absorbance at 205 nm was registered. A standard curve of BSA diluted under the same conditions was prepared (concentration range of 2 -25 mg BSA L -1 ).

Color of suspensions before and after the Maillard reaction was measured in an UV-Vis spectrophotome- The magnitude of color changes between control and treated samples were evaluated through the ∆E, using the first formula of the Euclidean distance between two points in the CIE -L*a*b* space [START_REF] Mokrzycki | Colour difference Delta E -A survey[END_REF], as presented in Equation 3.

∆E = [(L * control -L * sample ) 2 + (a * control -a * sample ) 2 + (b * control -b * sample ) 2 ] 1 2 (3)
The BI was calculated using Equations 4 and 5. It measures the shift towards a more brown color, used to describe non-enzymatic color development, such as that caused by the MRPs [START_REF] In | Downstream process for the production of yeast extract using brewer's yeast cells[END_REF][START_REF] Yu | Antioxidant activity and sensory characteristics of Maillard reaction products derived from different peptide fractions of soybean meal hydrolysate[END_REF][START_REF] Song | Antioxidative, antibacterial, and food functional properties of the half-fin anchovy hydrolysates-glucose conjugates formed via maillard reaction[END_REF].

x = a * + (1.75 • L * ) (5.65 • L * ) + a * -(3.01 • b * ) (4) BI =
x -0.31 0.17 were prepared in 500 mL jacketed glass beckers and mixed at 10230 rpm for 8 min in a rotor stator (L5M-A, Silverson, United States). The Maillard reaction took place as previously described (section 2.4.2). After the reaction, the pH of the solution was adjusted to 6.0 and AA was added to one of the suspensions and submitted to the agitation on the rotor stator using the same conditions. Solid content was 29% with a ratio of wall material to ascorbic acid of 5:1 (m/m). Both formulations were dried in a mini Spray Dryer (B-290, Büchi, Switzerland), with a double-fluid-type atomizer nozzle with a diameter of 0.7 mm operated by compressed air. Constantly stirred feeding solutions were pumped into the drying chamber by a peristaltic pump. Inlet temperature was determined at 150 °C based on previous research of the drying of yeast materials [START_REF] Zechner-Krpan | Characterization of β-glucans isolated from brewer's yeast and dried by different methods[END_REF][START_REF] Luna-Solano | Optimization of brewer's yeast spray drying process[END_REF][START_REF] Sultana | Microencapsulation of flavors by spray drying using Saccharomyces cerevisiae[END_REF][START_REF] Sultana | Stability and release behavior of encapsulated flavor from spray-dried Saccharomyces cerevisiae and maltodextrin powder[END_REF] and spray-drying of ascorbic acid using other wall materials [START_REF] Trindade | The stability of ascorbic acid microencapsulated in granules of rice starch and in gum arabic[END_REF]. Aspiration rate was about 37 m 3 h -1 , gas flow of 742 L h -1 and feeding rate of the solutions was approximately 6.5 mL min -1 .

Particles were characterized regarding their encapsulation yield (section 2.5.2), moisture content, water activity, hygroscopicity (section 2.5.3), glass transition temperature (section 2.5.4), morphology by optical microscopy (section 2.5.5) as well as particles size distribution (section 2.3.2, with compressed air as dispersant).

Encapsulation yield

Encapsulation yield (EY) was calculated according to Equation 6, considering the initial and final concentration of ascorbic acid (AA) present in the particles.

EY (%) = x encapsulated x total × 100 (6)
Where x encapsulated is the concentration of AA quantified in the particles (mg g -1 ); x total is the concentration of AA quantified in feed before drying (mg g -1 ), on dry basis.

The content of AA in the particles was determined using the methodology of Estevinho et al. ( 2016) with modifications. Briefly, particles were solubilized in deionized water (58.3 mg particles L -1 ) and the absorbance was measured at 260.6 nm in quartz cuvettes after the samples stood for 10 min. A standard curve of AA was prepared (concentration range of 0.5 -22 mg AA L -1 ) and results were expressed as mg AA g particles -1 .

Particles produced without AA were considered as the controls.

Physico-chemical properties of the particles

The moisture content of the particles was determined using a moisture-determining balance (model Moc63u, Shimadzu, Japan). The samples were heated until 105.0 °C and remained at that temperature until the moisture content, which was measured every 30 seconds, had a variation coefficient smaller then 0.01%. Water activity (a w ) was measured using Aqualab digital meter (model 3TE series, Decagon, USA), and hygroscopicity was determined according to the method of [START_REF] Cai | Production and properties of spray-dried amaranthus betacyanin pigments[END_REF], with modifications.

One gram of particles was stored in an hermetic container with saturated NaCl solution (relative humidity of 75%) at 25 °C. Samples were weighed on the first and seventh days of conditioning. Hygroscopicity was expressed as g of absorbed H 2 O per 100 g of sample.

Glass transition temperature

Glass transition temperature (T g ) and heat flow of the dried hydrolyzed yeast cells (HP) and of the spray-dried particles (control and with AA) were determined by differential scanning calorimetry (DSC) (TA-MDSC-2920, TA Instruments, USA) equipped with a mechanical refrigeration system (RCS-refrigerated cooling accessory). Approximately 3 mg of powder were placed into DSC aluminum pans (20 µL). Samples were heated at 5 °C min -1 from 0 to 150 °C and an empty pan was used as reference. Equipment calibration was performed with indium (melting temperature of 156.6 °C). Helium at 25 mL min -1 was used as the purge gas. All data was treated using the software Universal Analysis 2.6 (TA Instruments, USA).

Morphology of particles by optical and scanning electron microscopy

The particle morphology was analyzed using a Carls Zeiss optical microscope (Axio Scope A1 model, Gottingen, Germany), with an increase of 100 ×, and a scanning electron microscope with X-ray dispersed energy detector, model Leo 440i, EDS 6070 (LEO Electron Microscopy, Cambridge, UK). Metallic coating was applied to the samples using a sputter coater, model k450 (Emitech, Kent, UK), with a gold thinkness estimated at 200 Å. Tensions of 5, 10 and 15 kV were used, an electric current of 50 mA and amplifications from 150 to 6000 times.

Fourier-Transform Infrared Spectroscopy (FT-IR)

Produced particles as well as vitamin C, maltodextrin and dried yeast cell wall debris were analyzed by Fourier Transform Infrared Spectroscopy (FT-IR) using a spectrophotometer model IRPrestige-21 (Shimadzu, Kyoto, Japan). Data was analyzed using the software IRSolution, version 1.6. Powder samples were mixed to potassium bromate into a 13 mm pellet in the proportion of 1:100 (sample:KBr). The spectra were obtained in the range from 4000 to 400 cm -1 .

Statistical analysis

All experiments were studied in duplicate and all analyses were performed at least in triplicate. et al., 2015;[START_REF] Borchani | Enzymatic process for the fractionation of baker's yeast cell wall (Saccharomyces cerevisiae)[END_REF]. Nucleic acid content was smaller than the previously presented levels (from 6.9% to 10%), but as it is strain-dependent, high variations are expected [START_REF] Castrillo | Chapter 22 -Mycoprotein and Related Microbial Protein Products[END_REF][START_REF] Sceni | Thermal and surface behavior of yeast protein fractions from Saccharomyces cerevisiae[END_REF][START_REF] Caballero-Córdoba | Nutritional and toxicological evaluation of yeast (Saccharomyces cerevisiae) biomass and a yeast protein concentrate[END_REF]. Ash contents are also within the reported range

(5.9% for SBY and 8.5% to S. cerevisiae yeast biomass) [START_REF] Caballero-Córdoba | Nutritional and toxicological evaluation of yeast (Saccharomyces cerevisiae) biomass and a yeast protein concentrate[END_REF][START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF]. Fibers and lipids usually represent, respectively, less than 12% and 4% (dry basis) of global composition in S. cerevisiae yeast biomass [START_REF] Pacheco | Composition and nutritive value of yeast biomass and yeast protein concentrates[END_REF].

It is important to emphasize that SBY chemical composition as well as its susceptibility to cell wall breakage is reported to vary greatly among yeast strains and suppliers, brewing conditions, the number of times it is reused and also depends on the stage of fermentation/maturation at which it is removed [START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF].

The precipitate fraction of the non-treated yeast presents a 2.2 times higher total solids content than the whole yeast slurry. Indeed, following centrifugation, yeast cell wall components such as glucans and mannans, which are mostly insoluble, are concentrated on the precipitate [START_REF] Shurson | Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods[END_REF]. Higher amounts of crude protein, RNA and fibers were found, by 22%, 58% and 71%, respectively, whereas the total sugar content and ash content were lower (by 29% and 36%) in comparison to the whole residual yeast.

The effect of the hydrolysis in the chemical composition of yeast cells before and after hydrolysis are also presented in Table 1. A 13% decrease in crude protein content can be observed. The enzymatic hydrolysis promotes the breakdown of the peptide bonds of the proteins, resulting in lower molecular weight peptides [START_REF] Kunst | Ascorbic acid degradation of papaya during drying: Effect of process conditions and glass transition phenomenon[END_REF]. These molecules become more soluble and are more susceptible to migrate to the supernatant fraction [START_REF] Borchani | Enzymatic process for the fractionation of baker's yeast cell wall (Saccharomyces cerevisiae)[END_REF]. This solubilization, therefore, is traduced into a decrease in the protein content in the precipitate.

The RNA content of hydrolyzed precipitate (HP) was 33% lower than the non-treated precipitate (YP).

Proteins are the components to which, in acid medium, about 80% of the RNA is complexed [START_REF] Oliveira | Improvement in RNA extraction from S. cerevisiae by optimization in the autolysis and NH3 hydrolysis[END_REF]. Both the heat treatment and the hydrolysis alter the structure of the proteins, releasing their amino acids and, consequently, the nucleic acids, which become more soluble and migrate to the supernatant fraction. Moreover, [START_REF] Sgarbieri | Produção piloto de derivados de levedura (Saccharomyces sp.) para uso como ingrediente na formulação de alimentos[END_REF] also reported a decrease in RNA content and protein in the precipitate fractions of yeast after rupture of the cell wall. A RNA content of 5.7% was reported for the precipitate fraction of yeast whole cells but after the autolysis procedure, the content decreased to 1.8%

for the yeast cell debris.

Total sugar content increased 20% in the precipitate fraction after hydrolysis. As proteins were cleaved and the yeast cell wall was ruptured, carbohydrates could also be released, but because they were not soluble or were complexed to other molecules, they stayed in the precipitate fraction. The content of reducing sugars in the HP was higher (9.0%) than the one reported value in SBY slurry (1.3%) [START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF]. They appeared to be released during hydrolysis and were present in the precipitate fraction.

The contents of ash and fibers seemed to be slightly higher in the precipitate fraction after the hydrolysis step. Yeast cell wall components such as glucans and chitin may be determined as fibers, further confirming that the precipitate fraction was enriched in cell wall components [START_REF] Shurson | Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods[END_REF].

In sum, HP was considered a potential substrate for the Maillard reaction due to the high content of proteins and total sugars (Table 1). The effect of the addition of maltodextrin was investigated in order to supplement the reducing sugars content and ensure a good rate of reaction. The effect of the addition of amino acids to improve the Maillard reaction was previously reported for SBY, but it did not contribute to the extent of the reaction in comparison to the effect of added sugars because yeast hydrolysates usually have already enough amino acids and amino components [START_REF] In | Downstream process for the production of yeast extract using brewer's yeast cells[END_REF].

Physico-chemical properties

The values obtained for total titratable acidity are shown in Table 1. Yeast slurry value was smaller than that previously reported for SBY (32.7%) but this parameter is strongly affected by beer composition and process conditions [START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF]. After the hydrolysis, total titratable acidity was reduced by 47%. A hydrolysis treatment releases fatty acids and amino acids in the medium, and these components are believed to influence this parameter [START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF]. Indeed, after centrifugation, these components probably migrated to the supernatant fraction [START_REF] Kunst | Ascorbic acid degradation of papaya during drying: Effect of process conditions and glass transition phenomenon[END_REF], resulting in a decrease of total titratable acidity in the HP.

The mean values of particle diameter and span are presented in Table 1. The mean diameter of the particles in the precipitate fraction of non-treated yeast (YP) was 2 times bigger than that of the noncentrifuged yeast (Y). After hydrolysis, the mean particle size was smaller by 1.5 fold. Although native yeast cells are reported to have a diameter of approximately 2 to 5 µm [START_REF] Paramera | Chapter 23 -yeast cells and yeast-based materials for microencapsulation[END_REF], non-treated S. cerevisiae cell wall was reported to contain particles ranging from 1 to 500 µm [START_REF] Borchani | Physical, functional and structural characterization of the cell wall fractions from baker's yeast Saccharomyces cerevisiae[END_REF], 10 times higher than the hydrolyzed yeast cell debris that produced in this study. Despite of size, compounds larger than yeast cell wall (4-40 µm) were encapsulated recently (Mokhtari et al., 2017b). High span values indicate a high polidispersion of the particles' size (from 10.6 to 20.9). In other words, the span suggests a non-uniform size distribution which can be explained due to the high degree of complexity of the composition of the material, as presented in section 3.1.1. All those chemical components differ in size and structure and are probably interacting with each other, resulting in a wider size distribution.

The surface tension of the non-treated yeast precipitate (YP) was kept constant after the hydrolysis (HP) at pH 7 (Table 1). Surface tension is known to depend on protein conformation as well as on pH value of surface-active species. Recently, a method to obtain the hydrophobicity of proteins via surface tension measurements was proposed, taking into account the long known inverse correlation between surface tension and relative hydrophobicity of proteins [START_REF] Amrhein | Non-invasive high throughput approach for protein hydrophobicity determination based on surface tension[END_REF]. According to this relationship, hydrophobic amino acids reduce the surface tension while hydrophilic ones increase it [START_REF] Amrhein | Non-invasive high throughput approach for protein hydrophobicity determination based on surface tension[END_REF]. Proteins with a hydrophobic character play a key role in the biological and technological properties of proteins (solubility, and aggregation tendency) which are of interest in the pharmaceutical and food industries and needed for application studies [START_REF] Phongthai | Fractionation and antioxidant properties of rice bran protein hydrolysates stimulated by in vitro gastrointestinal digestion[END_REF][START_REF] Amrhein | Non-invasive high throughput approach for protein hydrophobicity determination based on surface tension[END_REF][START_REF] Xia | Fractionation and characterization of antioxidant peptides derived from barley glutelin by enzymatic hydrolysis[END_REF]. Because of this, we determined surface tension of yeast hydrolysate suspension at different pHs (Figure 2). Among pH values evaluated, surface tension was maximum at pH 5. Maximum hidrophobicity of the proteins in solution was achieved around pH 7, when the surface tension was smaller.

In food ingredients microencapsulation, various wall materials with ionic properties are used, such as phospholipids, proteins and polysaccharides. These chemical compounds have electric charges at their surface which play a role in the stability and physico-chemical properties of encapsulated products [START_REF] Arpagaus | Nano spray drying for encapsulation of pharmaceuticals[END_REF]. In suspensions, the ζ potential evinces the surface charge density of a dispersed phase [START_REF] Ye | Microencapsulation of active ingredients in functional foods: From research stage to commercial food products[END_REF][START_REF] Wen-Qiong | Cheese whey protein recovery by ultrafiltration through transglutaminase (TG) catalysis whey protein cross-linking[END_REF] and is an indicator of the stability or ability of aggregation of the dispersed particles [START_REF] Ye | Microencapsulation of active ingredients in functional foods: From research stage to commercial food products[END_REF][START_REF] Arpagaus | Nano spray drying for encapsulation of pharmaceuticals[END_REF]. Strongly charged surfaces (ζ > ±30 mV) are usually considered as more stable and may result in minimum aggregation due to intense repulsive forces [START_REF] Arpagaus | Nano spray drying for encapsulation of pharmaceuticals[END_REF][START_REF] Wen-Qiong | Cheese whey protein recovery by ultrafiltration through transglutaminase (TG) catalysis whey protein cross-linking[END_REF]. For protein-rich solutions, around the pI, the surface charge is close to zero and aggregation occurs [START_REF] Ye | Microencapsulation of active ingredients in functional foods: From research stage to commercial food products[END_REF]. The approximate pI of HP was 3.6 (determined as described in section 2.3.4). Debittered SBY biomass (Saccharomyces sp.) pI was previously reported as 4.5 [START_REF] Pacheco | Composition and nutritive value of yeast biomass and yeast protein concentrates[END_REF] but it can change with the proteins present in the material and thus with SBY provenance.

We evaluated the surface charge density and surface tension in the HP samples in pH values above the pI (Figure 2).

The zeta potential values (mV) for all yeast samples are presented in Table 1. Comparing the surface charge density of the non-treated yeast (YP) with that of HP at pH 7, a decrease in the surface charge density value was observed. The hydrolysis and heating pre-treatment process contribute to the degradation of the phospholipid membrane and yeast cell wall, potentially modifying the physico-chemical characteristics of the material, such as surface charge density [START_REF] Pradelles | Influence of the drying processes of yeasts on their volatile phenol sorption capacity in model wine[END_REF].

At highly positive or negative ζ potentials, the suspension is more likely to be stable [START_REF] Ye | Microencapsulation of active ingredients in functional foods: From research stage to commercial food products[END_REF].

Yeast hydrolysate (HP) presented negative ζ potentials for pH values ranging from 5 to 8 (Figure 2). In Figure 2 it can be observed that as the pH increased, there was an increase in the ζ potential of the solutions, which occurs due to the elevation of the electrostatic repulsion between the proteins as the pH is farther away from the pI [START_REF] Arpagaus | Nano spray drying for encapsulation of pharmaceuticals[END_REF][START_REF] Magdassi | Chapter 2 -Enhanced Hydrophobicity: Formation and Properties of Surface-Active Proteins[END_REF]. In the studied range, highest surface charge density was attained around pH 8, but at this pH, the surface tension went up, which could negatively influence the stability of encapsulated products in which there is a surface-active stabilization mechanism.

Moreover, the chosen active for the microencapsulation application study using HP was AA, which is a compound sensible to pH and stable in the pH range of 4 to 6. Above this range, the ascorbic acid is irreversibly changed to 2,3-diketogulonic acid, which is an inactive form [START_REF] Eitenmiller | Soluble vitamins (vitamin B12 and vitamin C) microencapsulated with different biopolymers by a spray drying process[END_REF].

Maillard reaction

Solubilisation of proteins and extent of reaction

The Maillard reaction was used to increase the solubilization of yeast cell debris in water. One indication that the reaction effectively caused an increase in solubility is the mass transfer from the precipitated material to the supernatant after centrifugation. As the reaction proceeds, the basic amino group is consumed and the media becomes more acidic [START_REF] Mirafzali | Chapter 13 -application of liposomes in the food industry[END_REF]. The evolution of soluble peptide content, pH and BI in the samples before and after the Maillard reaction are shown in Figure 3.

It was observed that supernatant mass percentage after centrifugation was not statistically different both for the HP-only dispersions and those containing HP and M20. Supernatant recovery was, in average 96% (m/m) for all experiments. Even before the Maillard reaction took place, the supernatant fraction already represented 95% of the recovered mass after centrifugation and maybe because of the low solid concentration of the suspensions, significant differences in solids migration could not be detected. However, in HP dispersions there was a increase of 20% in soluble solids content, probably due to solubilization of yeast compounds. After the Maillard reaction, soluble solids content in the dispersions with and without maltodextrin were 0.6 and 2.5 °BRIX, respectively.

According to Figure 3a, it was observed that the Maillard reaction raised the concentration of peptides in solution in the suspensions with maltodextrin by 14.3%, in comparison with the control. The Maillard reaction is indeed reported to cause the solubilization of components [START_REF] Oliver | Creating proteins with novel functionality via the Maillard Reaction: A Review[END_REF][START_REF] Consoli | Sodium caseinate-corn starch hydrolysates conjugates obtained through the Maillard reaction as stabilizing agents in resveratrolloaded emulsions[END_REF].

The extent of the Maillard reaction of the yeast hydrolysate solution was also checked by monitoring the pH, Browning Index and ∆E against a control sample. The extent of the change in color was already reported as a measure of the rate of the Maillard reaction. The rate of brown pigment formation in SBY was found proportional to the square of amino content, reducing sugar concentration and time of reaction [START_REF] In | Downstream process for the production of yeast extract using brewer's yeast cells[END_REF]. The values of pH and Browning Index (BI) of the samples before and after Maillard reaction, containing or not maltodextrin, are shown in Figures 3b and3c. A reduction of the pH values of 4.5 and 5.4%

and an increase in BI values of 227.6 and 230.3% in the suspensions without and with added maltodextrin were observed, respectively, corroborating the development of the Maillard reaction. In addition, according to the ∆E, the color variation of the sample containing maltodextrin was slightly higher (5.5 ± 0.1) than the sample that did not contain it (5.3 ± 0.1). It may be explained due to a higher amount of sugars in the suspension containing maltodextrin, allowing the reaction to occur more intensely [START_REF] Oliver | Creating proteins with novel functionality via the Maillard Reaction: A Review[END_REF][START_REF] Ames | Chapter 4 -The Maillard Reaction[END_REF].

Microencapsulation by spray drying

Considering the protein and polysaccharide-rich composition of the hydrolyzed yeast cell debris, this material was used along with maltodextrin after the Maillard reaction as a carrier material for encapsulation of ascorbic acid (AA). Vitamin C is the most unstable of vitamins, very susceptible to degradation when in contact with light, heating and oxygen [START_REF] Eitenmiller | Soluble vitamins (vitamin B12 and vitamin C) microencapsulated with different biopolymers by a spray drying process[END_REF].

Encapsulation yield

In the microencapsulation of materials by spray drying, a high product yield and a maximum encapsulation efficiency are intended [START_REF] Assadpour | Advances in spray-drying encapsulation of food bioactive ingredients: From microcapsules to nanocapsules[END_REF]. The EY for the ascorbic acid particles was 101.90 ± 5.5%, showing that the use of yeast as a carrier agent ensured very little loss of the active materials during the process. In spray dried particles, the encapsulated compound is dispersed in a porous and hollow matrix, potentially resulting in lower values of retention, which was not observed in AA particles with yeast hydrolysate debris as carrier. [START_REF] Lee | L-ascorbic acid microencapsulated with polyacylglycerol monostearate for milk fortification[END_REF] encapsulated ascorbic acid using polyacylglycerol monostearate by spray drying, obtaining EY values in the range of 80.7% to 94.2%. The microencapsulation of ascorbic acid in gelatin, rice starch and Arabic gum by spray drying resulted in EYs higher than 98% [START_REF] Trindade | The stability of ascorbic acid microencapsulated in granules of rice starch and in gum arabic[END_REF]. [START_REF] Carvalho | Characterization of ascorbic acid microencapsulated by the spray chilling technique using palm oil and fully hydrogenated palm oil[END_REF] studied the characterization of ascorbic acid encapsulated by spray chilling using palm oil and fully hydrogenated palm oil as carriers and obtained values of encapsulation efficiency in the range of 82.6-96.1%. In other research, Hoyos-Leyva et al. ( 2018) studied the physical and chemical stability of L-ascorbic acid microencapsulated into taro starch spherical aggregates by spray drying and obtained total retention of 99.0 ± 0.40%. The encapsulation of vitamin C by spray drying using YMC20 as carrier material promoted similar retention of AA when compared to the results obtained by these authors.

However, our approach presents advantages since we used a material from a agro-industrial underutilized residue (food-grade material) with high protein content (nutritive). In addition, the reuse of this material presents a sustainable approach and waste management solution. In other words, YMC20 may be considered a better carrier in relation to polyacylglycerol monostearate, palm oil and fully hydrogenated palm oil and taro starch spherical aggregates, as a new sustainable alternative for encapsulation of bioactive compounds by spray drying.

Moisture, water activity and hygroscopicity of particles

The values of moisture, a w and hygroscopicity of the particles obtained by spray drying are presented in Table 2. Particles containing AA presented lower values of moisture and water activity. However, higher values of hygroscopicity when compared with the control particles (without AA) were found. All these values obtained for the AA particles using SBY-maltodextrin-based Maillard conjugates as carrier material resulted in low values of moisture, a w and hygroscopicity, which is expected for proper handling, storage and application of the powder. In addition, a w values lower than 0.6 were obtained, suggesting microbiological stability of the powder, limiting the microorganisms development below this value. Moreover, particles with a w values below 0.2 have lower susceptibility to enzymatic and non-enzymatic reactions during storage [START_REF] Belitz | Food Chemistry[END_REF][START_REF] Comunian | Microencapsulation of ascorbic acid by complex coacervation: Protection and controlled release[END_REF][START_REF] Croguennec | Non-enzymatic browning[END_REF][START_REF] Damodaran | Química de Alimentos de Fennema[END_REF].

Hygroscopicity of the encapsulated AA powder was slightly higher than the control. As the amount of carrier agent is smaller, the higroscopicity of the powders tends to get higher [START_REF] Breternitz | Volatile composition and physicochemical characteristics of mussel (Perna perna) protein hydrolysate microencapsulated with maltodextrin and n-OSA modified starch[END_REF].

This parameter is dependent inversely on the components molecular weight as well as the physico-chemical structure of the material (chains arrangements). The complex composition of YMC20 may form a fairly open structure that provides available interaction with water [START_REF] Botrel | Influence of wall matrix systems on the properties of spray-dried microparticles containing fish oil[END_REF]. [START_REF] Choi | Functional enhancement of ultrafine Angelica gigas powder by spray-drying microencapsulation[END_REF] studied the enhancement of ultrafine Angelica gigas powder by spray drying encapsu-lation using maltodextrin as carrier material and obtained moisture values in the range of 4.0%5.5%. On the other hand, [START_REF] Otálora | Encapsulation of cactus (Opuntia megacantha) betaxanthins by ionic gelation and spray drying: A comparative study[END_REF] obtained a moisture content of 1.2 ± 0.1% for particles obtained by spray drying when cactus (Opuntia megacantha) betaxanthins was encapsulated using a mixture of maltodextrincactus cladode mucilage as carrier. In another recent research, [START_REF] Machado | Encapsulation of anthocyanin-rich extract from blackberry residues by spray-drying, freeze-drying and supercritical antisolvent[END_REF] evaluated the encapsulation of anthocyanin-rich extract from blackberry residues by spray drying using polyvinylpyrrolidone as carrier. These authors obtained moisture values of 5.3 ± 0.1%. These results indicate that particles obtained with HP result in moisture values lower than particles with maltodextrin or olyvinylpyrrolidone as carriers, but higher than when maltodextrin is used as carrier in combination with another material. However, all these results are appropriate for application since they are considered low for a powdered product.

The microencapsulation of jabuticaba, jussara and blueberry phenolic extracts blends by spray drying using maltodextrin, gum Arabic and whey protein concentrate as wall materials resulted in water activity and hygroscopicity values in the range of 0.34%-0.45% and 9.1%-15.5%, respectively [START_REF] Rocha | Microencapsulation by atomization of the mixture of phenolic extracts[END_REF].

Otálora et al. ( 2018) obtained a a w value of 0.39 ± 0.01 for cactus betaxanthins particles with maltodextrincactus cladode mucilage as carrier. It seems that particles with HD as carrier resulted in samples with lower a w and hygroscopicity values similar to particles with different wall materials obtained by spray drying.

Matos-Jr et al. ( 2017) evaluated the effect of feed preparation on the properties and stability of ascorbic acid microparticles produced by spray chilling using interesterified fat as carrier and obtained values of a w in the range of 0.63 -0.97. These high values were already expected since interesterified fat was used as carrier. [START_REF] Rezende | Microencapsulation of extracts of bioactive compounds obtained from acerola (Malpighia emarginata dc) pulp and residue by spray and freeze drying: Chemical, morphological and chemometric characterization[END_REF] studied the encapsulation of extract obtained from acerola by spray and freeze drying using Arabic gum and maltodextrin as encapsulating agents and obtained hygroscopicity values in the range of 9-12.5%. It seems that particles with YMC20 as carrier resulted in samples with lower a w values and hygroscopicity similar to particles with different wall materials obtained by spray drying.

DSC and FT-IR study of particles

Spray-dried samples were studied with Differential Scanning Calorimetry (DSC) and Fourier-Transform Infrared Spectroscopy (FT-IR) in order to evaluate their changes in structure after spray-drying. DSC thermograms indicate system changes in terms of exothermic, endothermic and heat capacity processes as a function of temperature. The glass transition temperature (T g ), which is an important parameter for particulate systems such as samples obtained by spray drying, can also be determined. Particles are either in a glassy or a rubbery state. Molecular mobility is reduced in the glassy state, improving the material stability and properly protecting the encapsulated compounds [START_REF] Kasapis | Glass transition phenomena in dehydrated model systems and foods: A review[END_REF]Kurozawa et al., 2014). Above the T g , the particles' structure are in a physico-chemical state called "gummy state". This condition is characterized by a higher mobility where physico-chemical modifications are more likely to happen, possibly resulting in agglomeration, crystallization, loss of volatiles, darkening or oxidation reactions and consequently destabilization of the particulate system. Therefore, a T g as far as possible from room temperature is desirable to ensure stability and quality of the particles obtained after drying and during storage [START_REF] Bhandari | Implication of glass transition for the drying and stability of dried foods[END_REF].

DSC thermograms in Figure 5a show slightly different profiles for control particles (YMC20) (i), encapsulated ascorbic acid in YMC20 (ii) and hydrolyzed yeast cell debris (HP) (iii) but in all three, an exothermic peak that starts around 60-70 °C can be observed. This peak may be attributed to the aggregation of yeast proteins or rearranging of the structure. Thermally induced-transitions are represented by changes in peaks shape, temperature and area, providing information about the interactions of a system [START_REF] Kaletunç | Calorimetric methods as applied to food: An overview[END_REF].

Differences in composition may help to explain the observed changes. Only in YMC2 control particles, an endothermic peak around 45 °C can be seen. Gelatinization of β--glucan is an endothermic process and was reported previously with an onset temperature of 52 °C [START_REF] Khan | Structural, thermal, functional, antioxidant & antimicrobial properties of β--glucan extracted from bakers yeast (Saccharomyces cerevisiae) -Effect of γ-irradiation[END_REF]. In protein-rich materials, endothermic peaks are also usually related to the denaturation of proteins or melting of solids [START_REF] Kaletunç | Calorimetric methods as applied to food: An overview[END_REF]. To the authors best knowledge, until now, DSC thermograms of disrupted BSY were not reported.

Other authors reported structure changes in whole yeast cells regarding the phase transition temperature of yeasts phospholipid bi-layer (endothermic) and destruction of yeast cell (exothermic) at higher temperatures (160 and 265 °C) [START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF].

The glass transition temperature of the yeast cell debris, control and AA particles ranged from 70 to 72

°C. This result agrees with the values obtained by [START_REF] Schebor | Glass transition temperatures and fermentative activity of heat-treated commercial active dry yeasts[END_REF], which determined the glass transition FT-IR spectra of wall materials, vitamin C and particles are presented in Figure 5b. Wall materials and particles showed peaks in the range of 3410-3315 and at 2926-2924 cm -1 , which are related to O-H stretch-ing, residual water and C-H stretching [START_REF] Akbarbaglu | Influence of spray drying encapsulation on the retention of antioxidant properties and microstructure of flaxseed protein hydrolysates[END_REF]. The spectra obtained for maltodextrin is similar to reported spectra when this material was used in the study of spray drying encapsulation on the retention of antioxidant properties and microstructure of flaxseed protein hydrolysates [START_REF] Akbarbaglu | Influence of spray drying encapsulation on the retention of antioxidant properties and microstructure of flaxseed protein hydrolysates[END_REF]. According to these authors, the peaks at 1658, 1413-1238, 1155 and 1022 cm -1 correspond to O-H, CH 2 , C-O and C-O-H and =CH/=CH 2 bonds, respectively. Regarding to yeast cell wall debris, besides the peaks in the range of 3377-3323 cm -1 , peaks at 2924, 1629, 1546 and 1047 cm -1 were observed. Spectra in the range of 3377-3323 cm -1 correspond, besides O-H stretching and residual water, to NH stretching vibrations of proteins and peptides and to amide II overtone [START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF]. Moreover, the changes in the peaks at 1629 and 1546 cm -1 (present in yeast cell wall debris) to 1597-1409 and to 1656-1411 cm -1 (present in YCM20+AA and YCM20, respectively), could be related to partial protein degradation associated to yeast cell wall [START_REF] Paramera | Microencapsulation of curcumin in cells of Saccharomyces cerevisiae[END_REF]. Peaks from 1151 to 1026 cm -1 are related to absorptions of mannans and glucans. The spectra of particles (YMC20+AA and YMC20) presented characteristics similar to wall materials, with some peaks shifted, which are related to presence of vitamin C and formation of compounds during the Maillard reaction. The FT-IR spectra of AA encapsulated particles is very similar to that of control particles (just the carrier material), which confirms the presence of YMC20 in the surface of the particles.

Microstructure and particle size

The micrographs of the particles obtained by optical microscopy are shown in Figures 4a and4b. The micrographs did not indicate relevant morphological differences in the samples with and without AA. In other words, the incorporation of vitamin C into the formulation and the use of HP with maltodextrin as carriers did not seem to influence particle's morphology. As confirmed by SEM images of the control and vitamin C particles (Figures 6c and6d, respectively), the encapsulated vitamin C particles presented a rounded shape, as expected using this method of encapsulation [START_REF] Bastan | Spray drying of hydroxyapatite powders: The effect of spray drying parameters and heat treatment on the particle size and morphology[END_REF]. A smooth and shrinkaged surface was observed, with hollows and without pores. The observed shrinking characteristic is explained by the sudden evaporation of water that takes place during the atomization process. SEM images of the carrier materials show a very random morphology of maltodextrin (Figure 6a). Yeast cell debris hydrolyzed material (HP) (Figure 6b) shows clearly disrupted yeast cells, very different from rounded-shaped yeast cells non-hydrolyzed as previously reported [START_REF] Sultana | Microencapsulation of flavors by spray drying using Saccharomyces cerevisiae[END_REF].

The particle size distribution for the formulations is shown in Figure 4c. A bimodal behavior for the particles with AA and control was observed with only a peak with higher intensity. This behavior was already expected for this type of material since it is not possible to control the particle size during atomization. The particles were not homogeneous, which makes the small particles occupy the space among larger ones, reducing the volume and favoring their food application. A wide range of size distribution (24 to 280 µm)

was also obtained for alginate-calcium microcapsules containing probiotics and a coating of yeast cell wall material (Mokhtari et al., 2017a) although this higher span could be attributed to the addition of more layers and not specifically to the addition of the yeast coating.

Average particle size D [4.3] obtained for the particles containing ascorbic acid and the control (only wall material without AA) were 14.7 ± 0.6 and 11.6 ± 0.7 µm, respectively. The addition of AA resulted in 21% larger particles. Despite particles' size being different, the particles loaded with vitamin C resulted in appropriate average sizes for application in food products. Although the absolute size detection threshold and acceptability are matrix specific and depends on particles surface morphology, diameters below 100 µm are often reported as acceptable for most applications in foods because at that size there is little influence on perceived texture [START_REF] Annan | Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions[END_REF][START_REF] Yan | Chapter 12 -coacervation processes[END_REF][START_REF] Lee | Chapter 28 -stability characterization and sensory testing in food products containing microencapsulants[END_REF].

The encapsulation of peppermint oil in whole non-viable S. cerevisiae and S. boulardi cells, approximately 30 µm diameter particles were obtained [START_REF] Nelson | Microencapsulation in yeast cells and applications in drug delivery[END_REF]. Those spray-dried particles formed aggregates, resulting in 6 times bigger particles than non-treated yeast cells (approximately 5 µm) [START_REF] Nelson | Microencapsulation in yeast cells and applications in drug delivery[END_REF].

Particles with mean diameters around 7 and 35 µm were obtained in the encapsulation of flavors using yeast cells by-product from β-glucan production using mini and pilot-scale spray dryers, respectively [START_REF] Sultana | Microencapsulation of flavors by spray drying using Saccharomyces cerevisiae[END_REF]. The particles obtained in this paper using yeast cell debris were much smaller even though some agglomerates bigger than 100 µm were detected (Figure 4c). The encapsulation of AA using Arabic gum, gelatin and rice starch by spray drying resulted in particles of a mean diameters of 8 µm and 18-21 µm, respectively. Multimodal distributions were observed, with particles ranging from 0.3 to 90 µm for Arabic gum and to 224 µm for gelatin and rice starch [START_REF] Trindade | The stability of ascorbic acid microencapsulated in granules of rice starch and in gum arabic[END_REF]. [START_REF] Hoyos-Leyva | Physical and chemical stability of L-ascorbic acid microencapsulated into taro starch spherical aggregates by spray drying[END_REF] studied the microencapsulation of L-ascorbic acid into starch spherical aggregates by spray drying and obtained particle size of 16.4 µm and particle size distribution similar to particles with HP, maltodextrin and AA,

showing that the use of the precipitate fraction of spent brewer's yeast hydrolysate as carrier promoted the formation of particles with the similar size and distribution when compared to standard encapsulating agents.

Conclusions

Spent yeasts from brewing are an inexpensive raw material and a good source of proteins and carbohydrates that can be used in the development of food ingredients while promoting the valorization of relevant agricultural by-products. Our work demonstrated the potential of SBY hydrolyzed cell debris to generate MRPs and encapsulate compounds of interest. SBY-based Maillard conjugates were able to encapsulate ascorbic acid, resulting in particles of high values of EY, low a w , hlmoisture and hygroscopicity. The effects of yeast cell wall material in the particle matrix was clearly depicted through SEM and FT-IR spectroscopy.

In average, particles were smaller than 15 µm and the glass transition temperature was relatively high (72 °C), indicating product stability. Future studies are committed to evaluate the viability of encapsulated compounds using yeast cell debris as wall material, the stability of the particles along storage as well as appraise particles' nutritional and potential health-promoting characteristics. Determined by difference from total composition (crude proteins, total sugars, ribonucleic acids (RNA), fiber and ash). a,b Difference letters in the same row indicate significant differences among non-treated yeast cells (YP) and hydrolyzed yeast cells obtained after heating pre-treatment followed by hydrolysis (HP) with a confidence level of 5%. All proximal composition results were expressed in dry basis.

Figure captions

Table 2: Moisture, water activity (aw) and hygroscopicity of control (pure wall material) and ascorbic acid (AA) particles produced by spray drying with SBY-maltodextrin-based Maillard conjugates (YMC20) as a wall material.

Characterization

2. 4 .

 4 Preparation of SBY-based Maillard conjugates 2.4.1. Preparation of the yeast suspensions

  ter in reflectance mode using D65 illuminant (model UltraScan, Hunterlab, United States). Results were expressed as the Total Color Difference (∆E) and Browning Index (BI), which were calculated considering the parameters of the CIE -L*a*b* scale where L* represents the luminosity (L = 0 corresponds to the darkest black and L = 100 to the brightest white), a* represents the green (-)/red (+) colors and b* the blue (-)/yellow (+) colors.

  Application of SBY-based Maillard conjugates as a wall material 2.5.1. Encapsulation of ascorbic acid Two suspensions of 4% (m/v) HP and maltodextrin (1:5, protein content in HP:maltodextrin, m/m)

  temperature of dried samples of S. cerevisiae, obtaining values in the range of 64 -87 °C, according to the moisture content of the samples. No statistical difference (p > 0.05) between values of T g for particles with and without AA was found. The T g obtained for the microparticles with vitamin C (72.3 ± 0.3 °C) was high compared to the ambient temperature, which, in combination with the other parameters, assures the stability of the system and of the encapsulated material.[START_REF] Zhong | Redness generation via maillard reactions of whey protein isolate (WPI) and ascorbic acid (vitamin c) in spray-dried powders[END_REF] studied the redness generation via Maillard reaction of whey protein isolate (WPI) and AA in spray dried powders and observed T g values around 80 °C due to the presence of WPI. Particles obtained with YMC20 as carrier showed T g values lower than particles with WPI. Both are considered good for samples obtained by spray drying for future food applications.

Figure 1 :

 1 Figure 1: Full schematic representation of experimental plan for the production of hydrolyzed yeast cells (HP) from spent brewer's yeast (Y) and encapsulation of ascorbic acid (AA) using SBY-based Maillard conjugates and maltodextrin (YMC20) as a wall material.

Figure 2 :

 2 Figure 2: Surface tension (mN m -1 ) and surface charge density (mV) of the hydrolyzed yeast cell (HP) suspensions at pH 5, 6, 7 and 8.

Figure 3 :

 3 Figure 3: Effect of maltodextrin (M20) addition in the hydrolyzed yeast cells (HP) suspension after the Maillard Reaction on peptide concentration (a), pH (b) and Browning Index (c).

Figure 4 :

 4 Figure 4: DSC thermograms (a) of (i) control particles (YMC20), (ii) encapsulated ascorbic acid in YMC20 and (iii) hydrolyzed yeast cell (HP); and FT-IR spectra (b) of particles and separated materials (vitamin C, hydrolyzed yeast cell debris (HP) and maltodextrin).

Figure 5 :

 5 Figure 5: Optical microscopy (increase of 100x) of the particles produced: control (only wall material -YMC20) a) and ascorbic acid encapsulated in YMC20 (b); and the particle size distribution of those formulations (c).

Figure 6 :

 6 Figure 6: Scanning electron microscopy of (a) maltodextrin and (b) yeast cell wall debris (HP), control particles (only wall material -YMC20) (c) and ascorbic acid encapsulated in YMC20 (d)

Figure 1 SBY

 1 Figure 1

  Figure 4

  Figure 6

  the same line indicate significant differences between treatments (p < 0.05).

Table 1 .

 1 Resultswere expressed as average values ± standard deviation and were submitted to analysis of variance (ANOVA) and comparison of means by Tukey HSD test. ANOVA assumptions were checked through analysis of the residues, tests for data distribution (Ryan Joiner and Shapiro Wilk) and homogeneity of variances (Multiple Comparisons' and Levene). Differences were considered significant at a level of 5%. Correlations between data were determined by Pearson test followed by paired Student test. SBY yeast slurry contains 12.3% of dry matter, which mainly consists of proteins (41%) and total sugars (44%), with lower amounts of ash (7%), fibers (7%), RNA (2%) and lipids (0.2%). Results were in agreement with centesimal data for Saccharomyces sp. whole cells and SBY, which usually present a total sugar content of 21.5%-42.3%[START_REF] Caballero-Córdoba | Nutritional and toxicological evaluation of yeast (Saccharomyces cerevisiae) biomass and a yeast protein concentrate[END_REF][START_REF] Sceni | Thermal and surface behavior of yeast protein fractions from Saccharomyces cerevisiae[END_REF][START_REF] Borchani | Enzymatic process for the fractionation of baker's yeast cell wall (Saccharomyces cerevisiae)[END_REF] and crude protein content varying from 27.6% to 50.5%[START_REF] Caballero-Córdoba | Nutritional and toxicological evaluation of yeast (Saccharomyces cerevisiae) biomass and a yeast protein concentrate[END_REF][START_REF] Mathias | Characterization and determination of brewer's solid wastes composition[END_REF]

	3. Results and Discussion
	3.1. Characterization of spent brewer's yeast materials
	3.1.1. Proximal composition
	The proximal analysis of non-centrifuged whole SBY (Y) and its precipitate fraction (YP) are presented
	in

Table 1 :

 1 Proximal composition and physico-chemical data of non-treated spent brewer's yeast (SBY), non-treated yeast cells (YP) and hydrolyzed yeast cells (HP) Particle size in suspension -Diameter (µm) 42.8 ± 2.4 86.1 ± 3.8 a 57.0 ± 2.7 b Particle size in suspension -Span (d.u.) 13.2 ± 0.4 10.6 ± 0.9 a 20.9 ± 1.1 b Surface tension at pH 7 (mN m -1 ) nd 40.1 ± 0.3 a 40.4 ± 0.1 a Surface charge density at pH 7 (mV) -8.7 ± 0.3 -19.2 ± 1.3 a -13.3 ± 0.6 b Nd: not determined; d.u.: dimensionless units;

	Analysis		SBY	YP	HP
	Total solids (%)		12.3 ± 0.1 26.9 ± 0.2 a	26.3 ± 0.3 b
	Crude protein (%)		40.8 ± 0.1 49.6 ± 0.9 a	43.0 ± 0.3 b
	RNA (%)		1.9 ± 0.1	3.0 ± 0.1 a	2.0 ± 0.1 b
	Total sugars (%)		43.5 ± 2.4 30.9 ± 6.8 a	37.2 ± 1.0 a
	Reducing sugars (%)		nd	nd	9.0 ± 0.2
	Ash (%)		7.0 ± 0.1	4.5 ± 0.1 a	5.1 ± 0.1 b
	Fiber (%)		6.6 ± 0.1	11.3 ± 0.3 a	18.4 ± 0.6 b
	Lipids 1 (%)		0.21	0.7	0.0
	Total titratable acidity (g 100g citric acid	-1 )	8.2 ± 0.1	3.8 ± 0.1 a	2.0 ± 0.1 b

Acknowledgements

This work was supported by FAPESP (São Paulo Research Foundation) [grant numbers #2018/04067-6, #2016/18465-8, #2015/24351-2, #2015/11984-7, #2012/50411-4, #2011/51707-1, #2011/06083-0, #2009/ 50593-2, #2009/50591-0, #2007/58017-5, #2007/54520-4, #2006/03263-9, and #96/08366-5]; EMU [grant numbers #2009/54137-1]; FAEPEX (UNICAMP) [#2422/16; 519.292]; and CNPq [# 306461/2017-0, #148665/2017-9]. The authors thank Prozyn and Novozymes for the kind supply of enzymes; Pedro Moura (Haus Beer Brewery) for donating SBYs. Funding sources had no involvement in any steps of this research.

Declaration of interests

None.