Camila Gonçalves Teixeira

D E Campos

Das Vertentes

Minas Gerais

Pr M Djamel Drider

M Antônio

Pr Fernandes De Carvalho

M Luís

Pr Augusto Nero

Mme Claire

Pr M Michel Federighi

Cinzia Mme

Pr Randazzo

Mme Elaine

Pr Cristina Pereira De Martinis

Rabia, Désiré Adrian Yanath

Camila Gonçalves Teixeira

Evandro Martins

Andressa Fusieger

Rosângela De Freitas

Tatiana Santos Lima

Júlia Oliveira Lopes

Luis Gustavo

Lima Nascimento

Antônio Fernandes

De " Carvalho

Technological

Antônio Fernandes De Carvalho

Djamel Drider " Brazilian

Rafaela Da

Silva Rodriguesa

Ricardo Seiti Yamatogi

Anca Lucau-Danila

Djamel Drider

Augusto Luís

Antônio Nero

Fernandes

Yanath Belguesmia

Silva Rodrigues

Antônio Nero

Djamel Drider Fernandes De Carvalho

D.Sc Gonçalves

Djamel Drider Co-

Luis Augusto Nero

Anca Lucau

PROPRIÉTÉS ANTIMICROBIENNES DES SOUCHES DE Weissella cibaria ISOLÉES

To God, for walking with me and for carrying me on during the most difficult moments of my walk in my work.

To my family, especially my mothers, Francisca and Aparecida, and my fathers, Gerônimo and Genilson, for the examples of wisdom and the incentives that have always motivated me.

To my brothers, Guilherme and Henrique, and sisters Lívia and Lucimar for the moments of distraction, love, and affection

bacteriocin identified as Bacteriocin_IIc. Also, we compared the nucleotides and the amino acids of this putative bacteriocin in the NCBI platform and there was no similarity with no other so far described bacteriocin from the genus Weissella. Finally, we analyze the safety of using these three strains for human and/or animal consumption and their capacity to be used as probiotics. All three strains have no acquired antimicrobial resistance genes and no mobile elements in the genome. W. cibaria strains ware resistant to one antibiotic with intrinsic resistances confirmed by genomic analysis, no hemolytic activity and no one of the known virulence factor ware detected. They possess the defense system CRISPR-Cas.

Besides they didn't present any inflammation or cytotoxicity capacity. W. cibaria W21 presents higher potential, once it demonstrates excellent results of adhesion to intestinal cells and exclusion of S. aureus MRSA SA1. However none of the three strains survived completely through the simulated gastro intestinal tract, but some protective ways can be further study. In conclusion, this work allowed us to understand the diversity and the distribution of Weissella species found in the different regions producing artisanal cheeses in Brazil, besides helping to understand the role of these strains in artisanal cheeses. It also showed that some of them have a favorable technological and safety potential for future uses in the food industry or as a probiotic culture. O gênero Weissella é composto por bactérias pertencentes ao grupo conhecido como bactérias láticas (BAL), portanto é capaz de produzir ácido lático através da fermentação de carboidratos. Bactérias desse gênero são autóctones tanto para ambientes lácteos quanto não lácteos e muitas linhagens têm a capacidade de produzir diversos compostos de interesse para a indústria alimentícia, como exopolissacarídeos, bacteriocinas e peróxido de hidrogênio, por exemplo. Este trabalho começou com uma profunda pesquisa culminando em duas revisões de literatura compreendendo o uso de cepas de Weissella para a indústria alimentícia e farmacêutica e o potencial probiótico deste gênero. Um grande número de estudos científicos relataram e apoiam o uso de cepas de Weissella neste campo devido às suas propriedades promotoras da saúde. Do ponto de vista da tecnologia de alimentos, algumas cepas apresentam potencial na produção de exopolissacarídeos, oligossacarídeos não digeríveis, que está além de seu potencial probiótico. É importante notar que a maioria das cepas de Weissella com propriedades interessantes para uso para consumo humano são seguras, devido à ausência ou baixa ocorrência de genes de virulência ou de resistência a antibióticos. Em seguida, realizou-se o estudo da biodiversidade de cepas de Weissella isoladas em regiões produtoras de queijos artesanais no Brasil. Para isso, foram realizadas as análises de PFGE e Rep-PCR, que permitiram o agrupamento das cepas estudadas. Com essas análises, observou-se que cada região de estudo representa um nicho ecológico específico e, além disso, observou-se também que a técnica de PFGE possibilita obter um melhor resultado de heterogeneidade entre as linhagens de uma mesma espécie. Após a seleção dos representantes de cada grupo foi analisado o potencial antimicrobiano dessas cepas por meio de testes antagônicos e produção de bacteriocinas contra os patógenos alimentares mais comuns. A maioria dos isolados testados apresentou boa inibição dos patógenos, com percentual de inibição superior a 60%. No entanto, não houve inibição significativa de patógenos quando o teste de produção de bacteriocina foi realizado. Também foi avaliado o potencial tecnológico dessas culturas. Treze cepas foram selecionadas e testadas para determinar a capacidade de coagulação, produção de diacetil, produção de exopolissacarídeos, capacidade de acidificação, atividade proteolítica extracelular e características do coágulo formado.

Résumé

Algumas cepas apresentaram resultados positivos para a maioria dos testes realizados, exceto para a produção de exopolissacarídeos, com destaque para a cepa número 16 isolada de queijos do sul do Pará, apresentando boa capacidade de acidificação e diminuição do pH do leite, e resultados positivos para os testes de produção de diacetil, atividade proteolítica e formação de coágulos homogêneos. Logo depois selecionamos três cepas com boa atividade inibitória dos patógenos, a primeira com a qual trabalhamos foi Weissella cibaria W25, sequenciamos e montamos a sequência preliminar do genoma, que consiste em 41 contigs totalizando ~2,4Mbp, com um teor de G+C de 45,04%. Em seguida, realizamos uma análise genômica comparativa abrangente com W. cibaria 110, conhecida por produzir a bacteriocina weissellicina 110, e quatro outras cepas de W. cibaria não produtoras de bacteriocina. W. cibaria W25 tem grande potencial para ser utilizada para consumo humano, uma vez que não foi prevista como um patógeno para humanos. Além disso, as análises mostraram que esta cepa possui genes únicos de agrupamento de proteínas que podem ser relacionados a genes de bacteriocinas indicados pelo AntiSmash, confirmando a possibilidade de produzir duas bacteriocinas diferentes. Com base nesses resultados, analisamos o genoma das três linhagens de W. cibaria (W21, W25 e W42), e o genoma de W21 e W42 também foram sequenciados. Seguidamente, investigamos a presença de genes para metabólitos secundários no genoma das cepas e descobrimos que eles possuem uma região comum no genoma com uma putativa bacteriocina identificada como Bacteriocin_IIc. Posteriormente, testamos a atividade antimicrobiana in vitro do sobrenadante dessas cepas. Os resultados mostraram que a cepa W21, que estava faltando um gene relacionado ao transporte na análise do genoma, também não apresentou diferença no teste antimicrobiano in vitro, comparando os resultados após e antes da neutralização do sobrenadante e tratamento com proteinase K. Além disso, comparamos os nucleotídeos e os aminoácidos desta putativa bacteriocina na plataforma NCBI e não houve similaridade com nenhuma outra bacteriocina do gênero Weissella até agora descrita. Por fim, analisamos a segurança do uso dessas três cepas para consumo humano e/ou animal e sua capacidade de uso como probióticos. A sequência completa do genoma foi avaliada para genes relacionados à segurança. Além disso, a suscetibilidade aos antibióticos, citotoxicidade, capacidade de inflamação e atividade hemolítica foram testados. Para os testes probióticos, analisamos a sobrevivência através da simulação do trato gástrico, adesão à cultura de células intestinais e exclusão de S. aureus MRSA SA1 e E. coli 184 também em cultura de células intestinais. Todas as três estirpes não possuíram genes de resistência antimicrobiana adquiridos e nenhum elemento móvel no genoma. As cepas de W. cibaria foram resistentes a um antibiótico com resistências intrínsecas confirmadas por análise genômica, nenhuma atividade hemolítica e nenhum fator de virulência conhecido foi detectado. As estirpes possuem o sistema de defesa CRISPR-Cas. Além de não apresentarem nenhuma capacidade de inflamação ou citotoxicidade e as três estirpes de Weissella não foram preditas como patógenos humanos. W. cibaria W21 apresentou maior potencial probiótico, pois demonstrou excelentes resultados de adesão às células intestinais e exclusão de S. aureus MRSA SA1. No entanto, nenhuma das três cepas sobreviveu completamente à simulação a passagem pelo trato gástrico, mas algumas formas de proteção para esse fim podem ser mais estudadas. Em conclusão, este trabalho permitiu compreender a diversidade de espécies de Weissella encontradas nas diferentes regiões produtoras de queijos artesanais no Brasil e como estão distribuídas nesses ambientes, além de ajudar a compreender o papel dessas linhagens nos queijos artesanais. Também mostrou que alguns deles têm um potencial tecnológico e de segurança favorável para usos futuros na indústria de alimentos ou como cultura probiótica.

GENERAL INTRODUCTION

Context

The study of microbial diversity in dairy and non-dairy environments has great importance for the understanding of the presence of different microorganisms in these environments, as well as the impact of these microorganisms on the final product when we refer to artisanal dairy products. Each environment has unique characteristics that favor and allow the development of different bacterial species.

Handmade cheeses and raw milk are considered potential sources of new strains of lactic acid bacteria. The way of making these cheeses causes the fermentation to be conducted by bacteria contaminating the grazing, animal skin, utensils, surfaces, and other environments that may come into contact with the cheese during manufacture. The study of the bacterial community present in artisanal cheeses has revealed the presence of species that had not yet been related to cheeses and a great diversity of lactic bacteria with differentiated technological characteristics. In addition, non-dairy environments such as grass, different types of silage, and even animal skin have also been an important source of new strains that have adapted and therefore, can provide interesting features to be explored.

The diversity of Weissella isolated from dairy and non-dairy environments is of great interest for the understanding of this microorganism in the final products and can be studied by molecular techniques such as PFGE and Rep-PCR that can phylogenetically relate the strains. In addition, phenotypic characterization is necessary to understand the characteristics and potential of each of them.

Objectives

This study aims to understand the microbial diversity in different environments (dairy and non-dairy) seeking the application of the strains with the potential to be used as a bacteriocin producer for the food industry or the pharmaceutical industry as a probiotic.

Manuscript organization CHAPTER I: Literature review

This chapter is divided into two parts, the first part presents overall information about the Weissella genus and its potential application for the food and pharmaceutical industry.

The physiological and metabolic characteristics, ecology, and application in the food industry are part A. In the second part (pat B), the Weissella strains are approached for their species identification and characterization, beneficial properties, health benefits and exopolysaccharides production, and possibility for being used as probiotic culture. The first part of this review is published as "The Weissella Genus in the Food Industry: A Review" in the Research, Society, and Development. The second part of this review was published as "Weissella: An Emerging Bacterium with Promising Health Benefits" in the Probiotics and Antimicrobial Proteins.

CHAPTER IV: Weissella cibaria strains isolated from Campo das Vertentes (MG), a potential bacteriocin producers

This part aims to identify the antimicrobial compounds produced by Weissella strains isolated from Campos das Vertentes, Minas Gerais, Brazil, scheduling their future application as pathogens inhibitors in the food industry and preservation. The content of this chapter will be submitted as "Genome characterization of Weissella strains isolated from Campos das Vertentes, Minas Gerais, Brazil focused revealed new bacteriocins with a large spectrum of activity".

CHAPTER V: Weissella cibaria strains isolated from Campo das Vertentes (MG), biosecurity and probiotics analyses

In this last chapter, we evaluated the safety capacity of three strains of Weissella cibaria, isolated from different producing artisanal cheese regions of Campos das Vertentes, in Minas Gerais, Brazil to be used as probiotics for human consumption or animal feed. This chapter will be valorized as "Biosecurity of Weissella strains isolated from Campos das Vertentes, Minas Gerais, Brazil focused on its potential as probiotic"

General conclusions and perspectives

The genus Weissella has many interesting properties and can be explored to aggregate more to the food and pharmaceutical industries. The main conclusions derived from the study are lined out with the future research needed to increment the knowledge about the strains regarding their use as probiotics or as an ingredient in the food industry. grupo das bactérias lácticas (BAL), principalmente pela produção de ácido láctico a partir da fermentação de carboidratos. As espécies de Weissella estão distribuídas em diferentes habitats, como solos, ordenhadeiras, cana-de-açúcar e algumas cepas com características tecnológicas interessantes podem ser isoladas de alimentos fermentados, como queijos de leite cru, vegetais fermentados e leite fermentado. Do ponto de vista da tecnologia de alimentos, algumas cepas apresentam potencial na produção de exopolissacarídeos, oligossacarídeos não digeríveis, que está além de seu potencial probiótico. Portanto, as bactérias pertencentes ao gênero Weissella podem ter grande importância tecnológica, estando também envolvidas no controle de doenças de origem alimentar pela produção de bacteriocinas e peróxido de hidrogênio. Este gênero possui grande potencial para uso na indústria alimentícia.

Palavras-chave: Weissella; Indústria alimentícia; Metabolismo; Ecologia; Potencial tecnológico; Bacteriocinas.

Resumen

El género Weissella está compuesto por bacterias clasificadas como gram-positivas, catalasa negativas, no formadoras de esporas, de morfología cocoide o bacilos cortos.

Pertenecen al grupo de las bacterias del ácido láctico (BAL), principalmente por la producción de ácido láctico a partir de la fermentación de carbohidratos. Las especies de Weissella se encuentran distribuidas en distintos hábitats, como suelos, ordeñadoras, caña de azúcar y algunas cepas con interesantes características tecnológicas pueden aislarse de alimentos fermentados, como quesos elaborados a partir de leche cruda, vegetales fermentados y leche fermentada. Desde el punto de vista de la tecnología de los alimentos, algunas cepas tienen potencial en la producción de exopolisacáridos, oligosacáridos no digeribles, que está más allá de su potencial probiótico. Por tanto, las bacterias pertenecientes al género Weissella podrían tener una gran importancia tecnológica, estando también implicadas en el control de enfermedades transmitidas por alimentos mediante la producción de bacteriocinas y peróxido de hidrógeno. Este género tiene un gran potencial para su uso en la industria alimentaria.

Palabras clave:

Weissella; Industria de alimentos; Metabolismo; Ecología; Potencial tecnológico; Bacteriocinas.

Introduction

At first, microbiology techniques for the identification of genera and species were based only on phenotypic tests such as morphological and biochemical tests for typing, subtyping, and identification of microbial genera, species, and subspecies. However, despite presenting atypical characteristics, many microorganisms were mistakenly grouped due to the low discriminatory power of these techniques when it comes to microorganisms with little genetic variability. Nevertheless, since the '80s, the techniques of manipulation of the genetic material, allow the evaluation of the bacterial genome. Because of the advancement in the studies of molecular biology, it is now possible to construct more and They showed that the strains analyzed were phylogenetically closer to the Leuconostoc paramesenteroides than to the other Leuconostoc species (Figure 1). They proposed that L. paramesenteroides, the new species isolated from the fermented sausages, and some heterofermentative Lactobacillus, should belong to the new genus, named Weissella. The name Weissella hellenica was proposed to name the new species. Bacteria belonging to the Weissella genus are hardly differentiated from Leuconostoc strains and heterofermentative Lactobacillus by phenotypic characteristics; thus, the description was only possible through molecular taxonomic analyzes. Collins et al. (1993) proposed the reclassification of L. paramesenteroides, Lb. confusus, Lb. halotolerans, Lb. kandleri, Lb. minor, and Lb. viridescens to W. paramesenteroides, W. confusa, W. halotolerans, W. kandleri, W. minor, and W. viridescens, respectively. Subsequently, other studies have identified new species, and currently 25 species are validated: W. viridescens, [START_REF] Niven | Species That Produces a Green Discoloration of Cured Meat Pigments[END_REF], W. paramesenteroides (Garvie, 1967), W. confusa (Holzapfel e Kandler, 1969apud Collins et al., 1993), W. kandleri [START_REF] Holzapfel | Lactobacillus kandleri sp. nov., a New Species of the Subgenus Betabacterium, with Glycine in the Peptidoglycan[END_REF], W. halotolerans (Kandler, Schillinger & Weiss, 1983), W. minor (Kandler et al., 1983), W. hellenica (Collins et al., 1993) (Tohno et al., 2013), W. diestrammenae [START_REF] Oh | Weissella diestrammenae sp. nov., isolated from the gut of a camel cricket (Diestrammena coreana)[END_REF], W. uvarum (Nisiotou, Dourou, Filippousi, Banilas, & Tassou, 2014), W. cryptocerci [START_REF] Heo | Weissella cryptocerci sp . nov ., isolated from gut of the insect Cryptocercus kyebangensis[END_REF], W. bombi [START_REF] Praet | Novel lactic acid bacteria isolated from the bumble bee gut : Convivina intestini gen. nov., sp. nov., Lactobacillus bombicola sp. nov., and Weissella bombi sp. nov. Antonie van Leeuwenhoek[END_REF], W. jogaejeotgali (Lee et al. 2015), W. kimchi (Choi et al., 2002), W. muntiaci (Lin et al., 2020) and W. sagaensis (Li, Tian & Gu, 2020).

The genus Weissella belongs to the phylum Firmicutes, class Bacilli, order Lactobacillales, and family Leuconostocaceae. The bacteria belonging to this genus may have different morphologies, being short rods with rounded to tapered ends or coccoid in shape, which is the morphology of microorganisms belonging to the genus Leuconostocs, Oenococcus, and Streptococci (Collins et al., 1993). In some species, a tendency to pleomorphism occurs depending on the stress condition to which the bacterium is submitted.

Concerning the organization, they can also be found in pairs, alone, or in small chains [START_REF] Gandra | Técnicas moleculares aplicadas à microbiologia de alimentos[END_REF], Collins et al., 1993). Weissella is a relatively new genus, which is not yet used as a starter or adjunct culture by the food industry, as there are still few studies on its use as such. The purpose of this review is to bring the most relevant research in this genus to the food industry, presenting the potential application of the strains of the genus Weissella in food.

Methodology

This paper is a bibliographic research (Pereira, Shitsuka, Parreira & Shitsuka, 2018).

Therefore, the objective is the verification of scientific data and the discussion about them on the proposed theme. This article was done descriptively and qualitatively to present how Weissela sp. can be applied in the food industry.

To obtain the theoretical foundation it was used database websites: such as Google academic, Scielo, PubMed, and Science Direct, and for the research was used keywords such as Weissela, bacteriocin, exopolysaccharides, and lactic acid bacteria. The period used was from the year 1993, when the species was recognized, until the year 2021 as a form to compare the evolution of research and application of the Weisella genus and their compounds. Also for this research, there was no limitation to language as a way to gather as much information as possible. By the end, was select 90 sources of information.

Physiological and metabolic characteristics

Bacteria from the genus Weissella are chemoorganotrophic, facultatively anaerobic, Gram-positive, non-spore-forming, catalase-negative (Collins et al., 1993) and have no motility except to W. beninenses that has peritrichous flagella (Padonou, et al. 2010). All the microorganisms of this genus are compulsory hetero-fermenters producing lactic acid, carbon dioxide, ethanol, and/or acetic acid from carbohydrate fermentation. They use the phosphoketolase and the hexose monophosphate pathways to perform carbohydrate fermentation (Garvie, 1967[START_REF] Holzapfel | Lactobacillus kandleri sp. nov., a New Species of the Subgenus Betabacterium, with Glycine in the Peptidoglycan[END_REF].

Weissella sp. has very complex nutritional needs requiring peptides, amino acids (arginine, aspartic acid, cystine, glutamic acid, histidine, isoleucine, phenylalanine, serine, threonine, tryptophan, and valine), fatty acids, nucleic acids, fermentable carbohydrates (glucose, fructose, mannose, maltose, sucrose, trehalose) and vitamins (riboflavin, pyridoxal, folic acid, biotin, nicotine, thiamine, pantothenic acid) for their development [START_REF] Gandra | Técnicas moleculares aplicadas à microbiologia de alimentos[END_REF][START_REF] Björkroth | The genus Weissella, in Lactic Acid Bacteria, Biodiversity and Taxonomy[END_REF], Kandler et al., 1983). However, culture media used as Man Rogosa and Sharp (MRS) and M17 for lactic acid bacteria are easily employed for the multiplication of these microorganisms. Moreover, all the nutritional requirements can be found in many raw materials used in the food industry such as milk, meat, and vegetables, providing the use of such cultures for fermentation thereof.

As part of the LAB group, the species of the genus Weissella are chemoorganotrophic microorganisms with strictly fermentative metabolism being unable to synthesize porphyrinic groups (e.g. heme). All species can multiply at 15 °C while some multiply at temperatures between 42 °C and 45 °C, having an optimum temperature of multiplication between 20 °C and 30 °C (Garvie, 1967). These bacteria can be applied in fermented dairy products like fermented milk which has the temperature of incubation of the microorganisms around 40 °C and fermented meat sausages with an incubation temperature of 25 °C.

Different species produce dextran, hydrolyzed esculin, and produces ammonia from arginine. Dextran is an exopolysaccharide important for the manufacture of products such as yogurt, in which it is desired to have viscosity [START_REF] Mende | Influence of exopolysaccharides on the structure, texture, stability and sensory properties of yoghurt and related products[END_REF]. In addition, these bacteria can ferment several sugars such as cellobiose, fructose, galactose, maltose, raffinose, ribose, trehalose, and xylose (Fusco et al., 2015). they can be used as starter cultures for the manufacture of products with different sensory characteristics.

W. cibaria MG1 possesses all the necessary genes for the use of the phosphoketolase pathway and metabolizes the galactose via the Leloir pathway in figure 2 is possible to understand how it is the metabolism. It can also use maltose, fructose, ribose, xylose, sucrose and gluconate as carbon sources. In addition, a β-galactosidase has been known to indicate that lactose can also be metabolized (Lynch et al., 2015). which suggests that this strain can be used in the fermentation of dairy products that have lactose as their main sugar. Most EPS produced by strains of the genus Weissella are homopolysaccharides (HoPS) such as dextran, fructan, galactan, levan, and inulin for example (Björkroth, et al., 2002, Lee, et al., 2002). Strains of the genus Weissella use sucrose as the obligatory substrate for the extracellular synthesis of HoPS. The enzymes used to hydrolyze sucrose are glycosyltransferase, glucan-sucrase (GS), or fructan-sucrase (FS), these being highly specific [START_REF] Zeidan | Polysaccharide production by lactic acid bacteria: from genes to industrial applications[END_REF]. The GS and FS enzymes cleave the glycosidic linkage of sucrose and couple a glucose (GS)/fructose (FS) unit to a glucan (GS)/fructan (FS) chain, water, sucrose, or other acceptors [START_REF] Meng | Structurefunction relationships of family GH70 glucansucrase and 4, 6-α-glucanotransferase enzymes, and their evolutionary relationships with family GH13 enzymes[END_REF].

The Weissella strains are dependent on the proteolytic system to obtain essential amino acids, which are precursors of peptides, proteins, and aromatic compounds. Lynch et al. (2015) studied the metabolic traits of W. cibaria and noted that the secretome of the genus Weissella includes both extracellular proteins, and cell wall proteins, that are secreted. It has been found that many of the secretome proteins were large, containing multiple domains, and greater than 1000 amino acids. They also showed that W. cibaria and W. confusa have a similar number of secretory proteins as Lb. rhamnosus GG, which is a known probiotic strain.

The strain W. cibaria MG1 has the production capacity of acetate, via the pyruvate oxidase pathway in the presence of oxygen, and the production of lactic acid, diacetyl, or acetoin via the diacetyl/acetoin pathway in anaerobiosis (Lynch et al., 2015). Some strains of W. confusa, W. paramesenteroides, and W. cibaria can metabolize D-xylose, glucose, Dfructose, D-mannose, sucrose, D-maltose and cellobiose and also showed β-glucosidase and β-galactosidase activity (López-Hernández, Rodríguez-Alegría, López-Munguía, & Wacher, 2017).

Ecology

Although Weissella sp. has a very complex nutritional requirement, it is found as autochthonous bacteria in different ecosystems. Due to this fact, they may contaminate food since it can spread easily in the processing environment. Different strains were isolated from soil (Padonou, et al. 2010), sediments of swamps (De Bruyne, et al., 2010), and lake water (Vela et al., 2011), being identified, mainly, in fermented foods such as cheese made with raw milk (Snauwaert et al., 2013, Tohno et al., 2013), fermented milk [START_REF] Oh | Weissella diestrammenae sp. nov., isolated from the gut of a camel cricket (Diestrammena coreana)[END_REF], Nisiotou, et al., 2014, 85], vegetables (Wouters, Grosu-Tudor, Zamfir, & De Vuyst, 2013). and sausages (Huys, Leisner, & Björkroth, 2012, Mende, Rohm, & Jaros, 2016) in Table 1 those species and their habitat are better explained. Lynch and colleagues investigated the genome of the W. cibaria species and according to the study of pan-proteome and core-proteome at the species level. Panproteome is all the proteins that are present in a given condition for all the species of a given life branch and the core-proteome are the proteins that are conserved in all the species of a given life branch and produced for a given condition [START_REF] Trapp | Proteogenomic insights into the core-proteome of female reproductive tissues from crustacean amphipods[END_REF]. Lynch and colleagues noted that the pan-proteome was much smaller and the core-proteome much larger in a level of genus. The fact that the core-proteome is much larger (corresponding to 69% of the pan-proteome of the species) may explain the ability of W. cibaria to survive in several ecological niches where they were found since they have a higher number of proteins (729 proteins) that help in their adaptation (Lynch et al., 2015).

Some strains of Weissella such as W. cibaria MG1 (Lynch et al., 2015) and W. cetti (Ortega, et al., 2018) ). These products may not have the same characteristics if they were manufactured without the presence of these strains.

Some applications of bacteria from genus Weissella in food

Weissella sp. has great potential in its application in food and for this reason, it has been studied. Some strains show antagonistic activity against pathogens due to the production of several compounds like bacteriocins, organic acids, and hydrogen peroxide, among others (Fusco et The first new bacteriocin produced by Weissella strains to be discovered was Weissellicin 110 in the year 2007. This compound is produced by the strain W. cibaria 110 isolated from the Thai fermented fish product plaa-som. This bacteriocin has antimicrobial activity against some Gram-positive microorganisms and it is resistant to high temperatures and catalase, but loses its activity when exposed to proteinase K and trypsin (Srionnual et al., 2007).

In the year 2014, Chen and colleagues (Chen, et Bacteriocins are also of great importance in the food industry, for example in the production of biodegradable food packaging with antimicrobial properties (Teixeira, et al., 2021). By incorporating the Bacteriocin 7293 produced by the strain W. hellenica BCC 7293, it was possible to control pathogenic bacteria in fillets of pangasius fish. The film produced inhibited the multiplication of both Gram-positive bacteria such as L. monocytogenes and S.

aureus as well as Gram-negative bacteria such as Pseudomonas aeruginosa, Aeromonas hydrophila, E. coli and Salmonella Typhimurium [START_REF] Woraprayote | Antimicrobial biodegradable food packaging impregnated with Bacteriocin 7293 for control of pathogenic bacteria in pangasius fish fillets[END_REF].

Weissella can also be useful for the food industry due to its capacity of some strains to produce EPS in a sucrose culture medium [START_REF] Woraprayote | Antimicrobial biodegradable food packaging impregnated with Bacteriocin 7293 for control of pathogenic bacteria in pangasius fish fillets[END_REF]. EPS are structures of high molecular mass, composed of carbohydrates, which when added to the food, behave mainly as a thickening and emulsifying agent. The polysaccharides that add thickening and gelling properties are irreplaceable in the food industry formulations. In addition to its ability as a probiotic, EPS imposes highly desirable rheological changes in the food matrix, such as viscosity increase, improved texture, and reduced syneresis (Lakra, Domdi, Tilwani & Arul,2020). They can be used to replace corn starch in manufacturing puddings, for example, where these characteristics are desired. Some studies have tested strategies to improve the EPS characteristics to enlarge its application in the food industry [START_REF] Kavitake | Effect of cirradiation on physico-chemical and antioxidant properties of galactan exopolysaccharide from Weissella confusa KR780676[END_REF]. Most of the strains of Weissella can produce the EPS dextran and can also produce other EPS as shown in Table 3. The EPS dextran has great efficacy as soluble dietary fiber since it can be fermented by the probiotic intestinal microbiota due to its low digestibility when compared to commercial prebiotic inulin (Teixeira, et al., 2021). The basis for EPS production by

Weissella strains is diverse, some studies use wheat flour and rye as the basis for fermentation. W. confusa presented higher rye meal production due to the higher optimum pH period for the synthesis of dextran during fermentation [START_REF] Cotter | Bacteriocins-a viable alternative to antibiotics[END_REF][START_REF] Kavitake | Effect of cirradiation on physico-chemical and antioxidant properties of galactan exopolysaccharide from Weissella confusa KR780676[END_REF].

The strains that can produce more than one exopolysaccharide are of great interest when it comes to the food industry because of their synergistic effect on texture and also nutritional improvement [START_REF] Malang | Characterization of exopolysaccharide and ropy capsular polysaccharide formation by Weissella[END_REF]. Some EPS that are being produced, such as galactan, has good emulsifying and stabilizing capacities which provide its future use in cosmetic and food emulsions (Ortega, et al., 2018[START_REF] De Angelis | Arginine catabolism by sourdough lactic acid bacteria: purification and characterization of the arginine deiminase pathway enzymes from Lactobacillus sanfranciscensis CB1[END_REF], Kavitake, Balvan, Devi & Shetty, 2020). The high molecular weight dextran produced by W. confusa QS813 can form hydrogens bonds and steric interactions with proteins. When this EPS is used in the frozen dough it's maintained the structural integrity of gluten during the freezethaw-cycles being appropriated to use as a cryoprotectant in wheat gluten-based frozen food [START_REF] Tang | Food Hydrocolloids Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813[END_REF].

Because of the production of exopolysaccharide, the application of W. cibaria as an assistant in the manufacture of cheddar cheese was performed resulting in a product with higher retention of humidity and without alteration of the proteolysis degree (Lynch, et al., 2014). In addition, W. cibaria MG1 produces exopolysaccharides (dextran) and oligosaccharides (glucooligosaccharides) and because of that was studied with the intention of producing a new fermented drink from wort sucrose-supplemented barley-malt-derived (Zannini, et al., 2013). The oligosaccharides are being applied in some foods as prebiotics when it is impossible to use probiotics, an example of this is infant formulas. In developing countries, infant formulas are heated before consumption, because of their dubious quality of water. In this way, the addition of probiotics is impossible, and prebiotics is added instead.

The oligosaccharides produced by Weissella can be a source of prebiotics for the food industry.

Rosca et al. (2018), verified that W. confusa produced a dextran with high structural stability and purity that have pharmaceutical importance due to its antifungal characteristics against the pathogenic yeast Candida albicans. Some strains such as the W. cibaria strain isolated from goat's milk also have been shown to have great potential as probiotics, such as resistance to 1% bile salt and tolerance to pH 3.0 (Elavarasi, Pugazhendhi, Poornima Priyadharsani, Valsala, & Thamaraiselvi, 2014). In addition, W. cibaria, jointly with Lb.

plantarum, showed in vitro tests of high antioxidant capacity, survived simulated gastric and intestinal transit, and tolerated bile acids and salts (Yu et al., 2019). Furthermore, the two strains were administered to male Wistar albino rats and showed an improvement in liver and kidney functions, damaged by heavy metals compared to rats that received only the heavy metals in their diet (Ojekunle, Banwo, & Sanni, 2017).

In addition, the Weissella species-pair W. cibaria/confusa, considered potentially On the other hand, the EPS glucan-sucrase produced by Weissella sp. TN610 can solidify semi-skimmed milk supplemented with sucrose which shows its potential in the application as a safe additive food to improve the texture of dairy products [START_REF] Bejar | Characterization of glucansucrase and dextran from Weissella sp . TN610 with potential as safe food additives[END_REF]. Besides that, a novel quinoa-based yogurt fermented with dextran producer W. The EPS dextran, levan, and ropy capsular polysaccharide, produced by W. confusa were evaluated in bread when it comes to the delaying of the deterioration by fungus, as well as improving the texture was observed (Tinzl-Malang, Rast, Grattepanche, Sych, & Lacroix, 2015). In addition to the production of EPS, some strains of W. cibaria and W. confusa as well as producing lactic acid also can produce folate (vitamin B9), which allows the nutritional improvement of fermented products that use these strains in the fermentation process (Deatraksa et al., 2018).

Conclusion

Weissella sp. may have a wide range of potential applications in food products since they can produce a high variety of compounds from the production of EPS, bacteriocins, and even vitamins such as B9. However, a few studies have been conducted and the industrial application of Weissella strains is still not a reality. There are still some obstacles that prevent the use of Weissella as a starter culture in the food industry, such as the lack of knowledge about the pathogenicity of some strains of the genus and its antagonistic capacity against other microorganisms of industrial interest due to its production of bacteriocins. Because some strains have a potential for opportunistic infection in humans, the food industry should always be vigilant for the safety testing of any strain before its technological application.

Thereby, for the future use of this potential species in the food industry, is necessary more researches about how Weissella sp. and their compounds can be applied, and with this review article, it's possible to understand that it's necessary to research the safety use of this strains in food.

Introduction

Advances in molecular biology can be credited with the discovery of the Weissella genus. Collins et al. [1] reported that strains with very similar phenotypic characteristics that had previously been included in the Leuconostoc genus exhibited different DNA profiles from other bacteria in the group. The authors used 16S rRNA sequences to study Weissella.

They found that certain strains demonstrated specific features that were phylogenetically closer to Leuconostoc paramesenteroides than others. Collins et al. [1] Leuconostoc, Oenococcus, and streptococci [1]. Nevertheless, some Weissella species can present pleomorphisms under stress conditions. Cells can be present alone, in pairs, or even in small chains [1,3]. Currently, 25 Weissella species have been determined: W.

viridescens, [4], W. paramesenteroides [5], W. confusa [1], W. kandleri [6], W. halotolerans [7], W. minor [7], W. hellenica [1], W. thailandensis [8], W. soli [9], W. cibaria [10], W.

koreensis [11], W. ghanensis [12], W. beninenses [13], W. fabaria [14], W. ceti [15], W.

fabalis [16], W. oryzae [17], W. diestrammenae [18], W. uvarum [19], W. cryptocerci [20], W.

bombi [21], W. jogaejeotgali [22], W. kimchi [23], W. muntiaci [24], and W. sagaensis [25].

A clear, well-documented summary of Weissella taxonomy and ecology can be found in Fusco et al. [2]. Although Weissella remains a relatively new genus compared with other LAB, it has been the focus of research in the past 5 years and has attracted strong interest from the food industry. Specific Weissella strains have been studied for their ability to produce (i) antimicrobial compounds like bacteriocins and hydrogen peroxide and (ii) exopolysaccharides (EPSs) and diacetyl. The latter is of major importance to dairy applications [26][27][28][29]. Different strains have been isolated from artisanally produced foods, which demonstrates the contributing role Weissella plays in the foods' characteristic features. Other studies have highlighted Weissella strains' ability to survive in the gastrointestinal tract (GIT) and even grow in the gut. Both of these attributes suggest the use of Weissella species as beneficial applied probiotics [30][31][32].

The purpose of this review is to provide an overview of the Weissella genus and present the latest research and findings from the past 5 years. The paper presents the most recent techniques and approaches used in Weissella identification and characterization. It also covers the bacteria genus' potential health benefits and its main technological features which are of interest to the food industry.

Weissella Species Identification and Characterization

The past 5 years have seen the development of new methods for identifying Weissella species. They now make it possible to distinguish Weissella species from other closely related LAB. The polyphasic identification method and sequencing and analysis of the 16S and 23S rRNA genes are two new molecular-based identification methods that allow for rapid differentiation between Lactiplantibacillus plantarum, Pediococcus pentosaceus, and Weissella confusa [33]. Complete genome sequence analysis using bioinformatic analysis can be used to describe metabolic routes and to predict potential uses for the identified strains [34][35][36][START_REF] Kang | Effect of Weissella cibaria on Fusobacterium nucleatum -induced Interleukin-6 and Interleukin-8 Production in KB Cells[END_REF][38]. These methods are the foundation of modern microbial taxonomy, as are the matrix-assisted laser desorption ionization-time of flight method (MALDI-TOF)

and database mass spectrometry [39,[START_REF] Nacef | MALDI-TOF mass spectrometry for the identi fi cation of lactic acid bacteria isolated from a French cheese : the Maroilles[END_REF]. One recent study has shown that the colony morphology of W. confusa can change according to the adopted culture media and incubation conditions. W. confusa's morphology can vary from rod-shaped coccobacilliwith characteristic, small, cream-colored colonies gathered in single, short or long chainsto a large, irregularly shaped, transparent morphology that lacks characteristic chain cell organization [41]. Weissella strains can also thrive in high-sugar niches and produce large amounts of dextran [41]. Weissella strains' behavior in different substrates is key to differentiating them from other LAB.

Genomic and metatranscriptomic methods have been used to assess the metabolic and fermentative traits of W. koreensis during the kimchi fermentation process. This has led to the characterization of metabolic pathways for certain carbohydrates fermented by W.

koreensis. These include D-glucose, D-mannose, D-lactose, L-malate, D-xylose, Larabinose, D-ribose, N-acetyl-glucosamine, and gluconate [42]. The determination of carbohydrate metabolic pathways makes it possible to predict how specific Weissella substrates will ferment, as well as pinpoint those which promote higher cell multiplication rates. It also allows for the estimation of type and quantity of products generated by the fermentation process. With this information, it is possible to predict the ecosystems where

Weissella will survive and thus potential food and pharmaceutical applications for specific species.

Rizzello et al. [43] delved into Weissella genus metabolism by focusing on W. cibaria and W. confusa strains. The resulting data showed that W. cibaria and W. confusa. were able to both produce phytase and use phytic acid, thus counteracting an anti-nutritional factor in legumes. The two strains were also able to reduce the raffinose oligosaccharide (RFO) concentrations of α-galactosides raffinose, verbascose, and stachyoserafinose. RFO concentrations represent another anti-nutritional factor in vegetables, especially fava beans.

When W. cibaria and W. confusa strains undergo the formation process, RFO hydrolysis enables galactose production. [43]. Legumes such as fava beans, peas, and lentils are good sources of vegetable proteins. Their anti-nutritional factors include saponins, condensed tannins, and protease inhibitors. α-Galactosides and phytic acid [44] have been shown to inhibit them. Fermentation and its subsequent reduction of these anti-nutritional factors would allow these foods to be used as new protein sources for both humans and animals.

These new protein sources could in turn be used to develop new products in the food industry.

Many Weissella strains have been isolated from fermented products such as kimchi, pozol, jeotgal, and ogi [33,39,42,45]. These strains have been characterized in terms of the carbon source they use and the compounds they can produce. While studying carbon source metabolism, Lopez-Hernandez et al. [45] observed that strains W. cibaria, W.

confusa, and W. paramesenteroides were able to metabolize D-xylose, glucose, D-fructose, D-mannose, sucrose, and D-maltose. Some strains were also able to use ribose and esculin ferric citrate. Others were able to use galactose, metabolize cellobiose, and demonstrate βglucosidase and β-galactosidase activity. The latter is a desirable characteristic in probiotic microorganisms, as β-glucosidase breaks down certain compounds and makes them easier for the body to absorb. Anthocyanins, which have antioxidant and anti-inflammatory properties, are among these compounds [46].

New identification methods have allowed for rapid and accurate identification of Weissella strains and reduced the risk of misidentification. Characterizing Weissella strains based on their metabolic pathways opens up the possibilities for their use in the medical and food production sectors.

Beneficial Properties of Weissella

Once the metabolic pathways of Weissella have been characterized, isolated strains can be studied for use in the pharmaceutical and food industries. Weissella isolates have beneficial, probiotic features and are able to produce EPS. These make them important bacteria in both pharmaceutical and food production sectors. Abriouel et al. [47] described the technological, functional, and pathogenic potential of Weissella genus applications in the food industry, but their review only covered research carried out before 2015. Below, our review presents and discusses the primary benefits of Weissella based on these areas of study.

Health Benefits of Weissella

Few studies in the past 5 years have examined the effects of Weissella strains on human health [48][49][50]. Some research has evaluated their effects on mice [51][52][START_REF] Kim | Inula britannica fermented with probiotic Weissella cibaria D30 exhibited anti-inflammatory effect and increased viability in RAW 264.7 cells[END_REF][START_REF] Lim | Weissella cibaria WIKIM28 ameliorates atopic dermatitislike skin lesions by inducing tolerogenic dendritic cells and regulatory T cells in BALB/c mice[END_REF][START_REF] Park | Immune enhancing effects of Weissella cibaria JW15 on BALB/c mice immunosuppressed by cyclophosphamide[END_REF], gerbils [START_REF] Fonseca | Probiotic effect of Bifidobacterium longum 51A and Weissella paramesenteroides WpK4 on gerbils infected with Giardia lamblia[END_REF], and beagles [START_REF] Do | Effects of Weissella cibaria CMU on halitosis and calculus, plaque, and gingivitis indices in beagles[END_REF]. Most studies have demonstrated how Weissella's probiotic characteristics and antimicrobial properties may offer health benefits. Only two strains of W.

cibaria have been studied for their probiotic potential for humans. W. cibaria JW15 was studied because of how it affects cytokine and immunoglobulin natural killer cell activity, and W. cibaria CMU has been the subject of research because of its oral epithelial cell adhesion and oral colonization; W. cibaria CMU's antibacterial activity against Fusobacterium nucleatum and Streptococcus mutans [48][49][50] represents another important species feature. A recent study found that immune functions were enhanced by an increase in natural killer cell activity for subjects who consumed probiotic W. cibaria capsules [50].

Moreover, oral ingestion of W. cibaria CMU can help to reduce halitosis and microbiota numbers in the gingival sulcus, thus improving oral health [48,49].

Fonseca et al. [START_REF] Fonseca | Probiotic effect of Bifidobacterium longum 51A and Weissella paramesenteroides WpK4 on gerbils infected with Giardia lamblia[END_REF] tested strains of Bifidobacterium longum and W.

paramesenteroides to assess their probiotic potential and their impact on parasite load in gerbils. They observed the positive effect these two strains had in reducing the parasitic load of animals infected with Giardia. Strains of W. cibaria have been studied as a way to reduce cancer treatment drug side effects in mice [START_REF] Park | Immune enhancing effects of Weissella cibaria JW15 on BALB/c mice immunosuppressed by cyclophosphamide[END_REF]. W. cibaria strains were shown to assist in the recovery of lymphocytes, hemoglobins, and platelet levels that drop when cyclosporamide (an anti-cancer agent) is administered. Cyclosporamide is widely used in the treatment of acute and chronic leukemia, lymphoma, and several autoimmune diseases [START_REF] Park | Immune enhancing effects of Weissella cibaria JW15 on BALB/c mice immunosuppressed by cyclophosphamide[END_REF]. W. cibaria strains also proved to be efficient in treating atopic dermatitis when administered orally to mice. W. cibaria improved the clinical symptoms of lesions, such as erythema/hemorrhage, edema/excoriation, erosion, scarring/dryness, and lichenification [START_REF] Lim | Weissella cibaria WIKIM28 ameliorates atopic dermatitislike skin lesions by inducing tolerogenic dendritic cells and regulatory T cells in BALB/c mice[END_REF]. In yet another study, W. cibaria was shown to reduce the destruction of periodontal tissue in mice and also protected against alveolar bone destruction in mice. W. cibaria coaggregates with periodontal bacteria to produce hydrogen peroxide and bacteriocins that inhibit the production of proinflammatory cytokines [58]. Although clinical studies using mice are an effective research technique, these studies cannot reliably predict the outcome of human studies [START_REF] Perlman | Mouse models of human disease: an evolutionary perspective[END_REF]. Further study is needed to determine the health benefits of Weissella strains for humans.

Although there have been few human or animal studies using Weissella strains in vitro research indicates a potential for future use as antimicrobial agents (Table 1) and probiotics (Table 2). Further study on animals and/ or humans is necessary to prove their efficiency as a functional species. The W. confusa DD_A7 strain has been shown to be a natural alternative prophylactic agent against multidrug-resistant (MDR) ESBL (extendedspectrum β-lactamase) positive E. coli bacteria [27,29]. W. confusa has also been shown to be effective in destroying Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 12600), Salmonella Typhimurium (ATCC 43174), Bacillus cereus (ATCC 13061), and E. coli O157: H7 (ATCC 43889) pathogens. Several studies have shown that the supernatant from W. confusa culture weakened the membrane of the pathogen E. coli O157: H7 (ATCC 43889) and damaged the integrity of its DNA [27,29]. Another study that paired W. viridescens with high-pressure processing showed strong results when it came to protecting ready-to-eat salads from L. monocytogenes. The research showed that high pressure was applied to the weakened pathogen membranes which then facilitated the antimicrobial activity produced by the W. viridescens strain [START_REF] Stratakos | Effect of high pressure processing in combination with Weissella viridescens as a protective culture against Listeria monocytogenes in ready to-eat salads of different pH[END_REF].

Species Probiotic property References

Weissella spp Cholesterol-reducing potential Anandharaj et al. [30] W. cibaria Enhance immune functions Lee et al. [50] Reduce the side effects of drugs Park and Lee [START_REF] Park | Immune enhancing effects of Weissella cibaria JW15 on BALB/c mice immunosuppressed by cyclophosphamide[END_REF] Improve atopic dermatitis Lim et al. [START_REF] Lim | Weissella cibaria WIKIM28 ameliorates atopic dermatitislike skin lesions by inducing tolerogenic dendritic cells and regulatory T cells in BALB/c mice[END_REF] Protective effects against alveolar bone destruction

Kim et al. [58]

Short-chain fatty acids production and adhesion to HT-29 cells.

Silva et al. [64]

W. confusa inhibition of -α-amylase activity Xia et al. [32] Thermostability, cholesterol removal, β-galactosidase production, and proteolytic activity Sharma et al. [START_REF] Sharma | Probiotic characterization and antioxidant properties of Weissella confusa KR780676, isolated from an Indian fermented food[END_REF] W. confusa and W. cibaria reduction in cholesterol, DPPH free radical and inhibition of linoleic acid peroxidation.

Xia et al. [32]

Lakra et al. [START_REF] Lakra | Some probiotic potential of Weissella confusa MD1 and Weissella cibaria MD2 isolated from fermented batter[END_REF] W. paramesenteroides Formation of short-chain fatty acids and antimicrobial compounds.

Pabari et al. [START_REF] Pabari | Evaluation of probiotic properties and prebiotic utilization potential of Weissella paramesenteroides isolated from fruits[END_REF] Recent in vitro studies have shown that W. cibaria, W. koreensis, W. confusa, and W. paramesenteroides strains are strong candidates for probiotic applications [30,32,[START_REF] Lakra | Some probiotic potential of Weissella confusa MD1 and Weissella cibaria MD2 isolated from fermented batter[END_REF][START_REF] Pabari | Evaluation of probiotic properties and prebiotic utilization potential of Weissella paramesenteroides isolated from fruits[END_REF][START_REF] Sharma | Probiotic characterization and antioxidant properties of Weissella confusa KR780676, isolated from an Indian fermented food[END_REF][START_REF] Silva | Probiotic properties of Weissella cibaria and Leuconostoc citreum isolated from tejuinoa typical Mexican beverage[END_REF]. The survival of these strains in the GIT and their tolerance levels when in contact with bile and acids are important factors for intestinal colonization and pathogen protection. W.

paramesenteroides has also been shown to form a biofilm that facilitates mucin adherence.

It uses fructooligosaccharides (FOS) and galactooligosaccharides (GOS) as prebiotic fibers

to form compounds that can survive the gut microbiome. W. paramesenteroides has also demonstrated antimicrobial activity against E. coli and S. aureus [START_REF] Pabari | Evaluation of probiotic properties and prebiotic utilization potential of Weissella paramesenteroides isolated from fruits[END_REF]. In another study, Anandharaj et al. [30] demonstrated that Weissella species may help reduce cholesterol due to its ability to use cholesterol from de Man, Rogosa, and Sharpe (MRS) agar with added Oxgall and water-soluble cholesterol. Cholesterol reduction is common in probiotic microorganisms isolated from the intestine [START_REF] Seddik | Antipathogenic and probiotic attributes of Lactobacillus salivarius and Lactobacillus plantarum strains isolated from feces of Algerian infants and adults[END_REF][START_REF] Cavalcante | The probiotic Lactobacillus fermentum 296 attenuates cardiometabolic disorders in high fat diet-treated rats[END_REF][START_REF] Liang | Study on intestinal survival and cholesterol metabolism of probiotics[END_REF]. Interestingly, Kim et al.

[58] found that using the W. cibaria D30 probiotic strain in fermented Inula britannica created a product with improved anti-inflammatory capacity. W. confusa and W. cibaria-isolated from Plaa Som Fug-have also been shown to produce folate. Folate, or Vitamin B9, plays an important role in DNA replication, repair, and methylation. It can also act as an antioxidant [START_REF] Deatraksa | Isolation of folate-producing Weissella spp. from Thai fermented fish (Plaa Som Fug)[END_REF]. Folate is important for immune and nervous system functions because it plays an integral part in the synthesis of neurotransmitters in the central nervous system [START_REF] Hsieh | Serum folate levels in bipolar disorder: a systematic review and meta-analysis[END_REF]. This is especially the case during the early stages of uterine growth and development.

Extensive research has been carried out on the Weissella genus's antimicrobial activity and its potential use of strains as probiotics. However, other beneficial compounds produced by Weissella, including vitamins, have only been touched on in research thus far.

EPS Production by Weissella

EPSs are extracellular macromolecules that have potential applications in the food, medical, and pharmaceutical industries. It has been shown that Weissella strains can produce these molecules, and EPS production has been the focus of several Weissella studies (Table 3). EPS production has been primarily documented for W. confusa and W.

cibaria strains, with dextran as the dominant EPS produced by the strains. Structurally, dextran molecules are highly linear. They are formed by α-1,6linked glucose residues with branches linked to α-1,2, α-1,3, or α-1,4. The α-1,2 bonds are representative of prebiotic properties because they are resistant to adverse GIT conditions and can help to modulate the intestinal microbiota [41,[START_REF] Bounaix | Biodiversity of exopolysaccharides produced from sucrose by sourdough lactic acid bacteria[END_REF][START_REF] Hu | Effect of temperature on productionof oligosaccharides and dextran by Weissella cibaria 10 M[END_REF][START_REF] Kanimozhi | Optimization of dextran production by Weissella cibaria NITCSK4 using response surface methodology-genetic algorithm based technology[END_REF]. EPS produced by LAB are generally recognized as safe (GRAS), which means it can be used in foods, prebiotics, and medical applications.

oligosaccharide production was higher at temperatures at or below 20 °C. The greatest dextran production occurred at 15 °C.

The physicochemical properties of bacteria-produced EPS are an important factor for functionality and use. Devi et al. [START_REF] Devi | Physico-chemical characterization of galactan exopolysaccharide produced by Weissella confusa KR780676[END_REF] carried out physicochemical characterizations of galactan EPS produced by a W. confusa strain. They showed that the EPS displayed a high oil absorption capacity, strong emulsifying activity, and an emulsion kinetic stability of up to 15 days. These results supported the theory that galactan EPS may be a good candidate as an emulsifier in forming long-term colloidal systems in food, pharmaceutical, and cosmetic products. Other studies have shown that several Weissella strains use a dextransucrase enzyme to produce dextran. This enzyme has been the focus of isolation and purification studies for the production of EPS [START_REF] Baruah | Purification and characterization of dextransucrase from Weissella cibaria RBA12 and its application in in vitro synthesis of prebiotic oligosaccharides in mango and pineapple juices[END_REF][START_REF] Shi | Lactose-and cellobiose derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase[END_REF] and caffeic acids [START_REF] Nolte | Glycosylation of caffeic acid and structural analogues catalyzed by novel glucansucrases from Leuconostoc and Weissella species[END_REF].

Dextransucrase produced by a W. confusa strain has been shown to synthesize a rare oligosaccharide called iso-melezitose. This oligosaccharide has a potent nutraceutical effect due to its ability to promote bifidobacteria growth in the colon [START_REF] Shi | Lactose-and cellobiose derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase[END_REF].

The EPS produced by LAB has antifungal [START_REF] Rosca | Biosynthesis of dextran by Weissella confusa and its in vitro functional characteristics[END_REF], antiinflammatory, and immunomodulatory properties [51] that may be of great interest to the medical sector. EPS can also act as antioxidants [26,28,51,[START_REF] Zhu | Purification, characterization and antioxidant activity of the exopolysaccharide from Weissella cibaria SJ14 isolated from Sichuan paocai[END_REF] and help probiotics survive passage through the GIT [START_REF] Kavitake | Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food[END_REF]. The linear EPS galactan produced by the W. confusa strain shows strong potential for encapsulation technology that is used to deliver bioactive compounds, probiotics, and drugs. The EPS produced by a W. confusa strain was shown to inhibit the multiplication of Candida albicans and was able to destroy up to 70% of the biofilm formed by this pathogen [START_REF] Rosca | Biosynthesis of dextran by Weissella confusa and its in vitro functional characteristics[END_REF]. This suggests that it could be a promising candidate for use in antifungal treatments. Another recent study showed that W. confusa can produce mannan, an EPS made up of only monomeric units of mannose. This EPS displayed antibiofilm activity against pathogenic bacteria such as S. aureus, L. monocytogenes, S. enterica, and S. typhi. It also demonstrated potential as an antioxidant compound [START_REF] Lakra | Some probiotic potential of Weissella confusa MD1 and Weissella cibaria MD2 isolated from fermented batter[END_REF].

EPS may also have prebiotic properties. This is the case for the EPS produced by the W. cibaria strain isolated from pomelos (Citrus maximum) grown in India [START_REF] Baruah | Functional food applications of dextran from Weissella cibaria RBA12 from pummelo (Citrus maxima)[END_REF]. To be categorized as a prebiotic compound, the EPS carbohydrates must demonstrate higher metabolization levels from probiotic bacteria than from enteric bacteria present in the intestine. Dextran produced by W. cibaria was shown to stimulate the growth of L. plantarum, L. acidophilus, B. animalis, B. bifidum, and B. infantis more than E. coli and E. aerogenes [START_REF] Baruah | Functional food applications of dextran from Weissella cibaria RBA12 from pummelo (Citrus maxima)[END_REF]. This activity confirmed its classification as a prebiotic compound.

In the food industry, EPS can be used to improve the rheological characteristics and texture of fermented products. EPS can also play a role as an emulsifier and stabilizer [28,[START_REF] Ye | Purification and characterization of exopolysaccharide produced by Weissella cibaria YB-1 from pickle Chinese cabbage[END_REF][START_REF] Kavitake | Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food[END_REF]. EPS-producing LABs can be used as starter or secondary cultures in the production of fermented products such as yogurt [START_REF] Zannini | Production, properties, and industrial food application of lactic acid bacteria-derived exopolysaccharides[END_REF]. The use of these cultures leads to the production of EPS in situ, where they act as natural thickeners and can eliminate the need for artificial additives [START_REF] Zhu | Purification, characterization and antioxidant activity of the exopolysaccharide from Weissella cibaria SJ14 isolated from Sichuan paocai[END_REF]. Weissella strains have been used to ferment quinoa-based yogurt [START_REF] Zannini | Development of novel quinoa-based yoghurt fermented with dextran producer Weissella cibaria MG1[END_REF] and fruit juices [START_REF] Rolim | Sensory analysis of juice blend containing isomaltooligosaccharides produced by fermentation with Weissella cibaria[END_REF][START_REF] Tang | Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813[END_REF]. The EPS produced can improve the sensory characteristics of such products. One example: Orange juice concentrate was used to produce oligosaccharides.

Then, after a fermentation process involving the sucrose present in the juice itself, a drink was obtained with reduced sugar content and specific acidity and sweetness levels [START_REF] Rolim | Sensory analysis of juice blend containing isomaltooligosaccharides produced by fermentation with Weissella cibaria[END_REF][START_REF] Tang | Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813[END_REF].

EPS are also of interest to the frozen dough industry, as it can modify the texture and improve the end product characteristics of frozen and thawed doughs. Studies have shown that dextrans produced by different microorganisms have different functionalities. For instance, dextran produced by the W. cibaria strain demonstrated a greater ability to reinforce gelling properties compared with dextran produced by L. pseudomesenteroides when the strains were mixed with fava bean (Vicia faba L.) protein isolate [START_REF] Xu | Interactions between fava bean protein and dextrans produced by Leuconostoc pseudomesenteroides DSM 20193 and Weissella cibaria Sj 1b[END_REF][START_REF] Xu | In situ synthesis of exopolysaccharides by Leuconostoc spp. and Weissella spp. and their rheological impacts in fava bean flour[END_REF].

Weissella
EPS has also been shown to improve the characteristics of wheat gluten during dough freezing and thawing. In general, the freezing-thawing process leads to an increase in water loss, a decrease in gluten viscoelasticity, and changes in water mobility and distribution.

During freezing and thawing, the continuous structure of gluten is destroyed by mechanical damage caused when ice recrystallizes. An addition of W. confuse-produced EPS to doughs helped the gluten to maintain its qualities and structural integrity during the freezing-thawing process. This makes Weissella EPS promising cryoprotectant candidates for the frozen dough industry [START_REF] Tang | Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813[END_REF].

Weissella as an Emerging Pathogen

Weissella isolates with potential health benefits must also be assessed for any adverse effects. Testing is standard procedure for isolates to be used as food supplements and starter cultures to determine if they harbor virulent or antibiotic-resistant genes, even when the negative effects are not active or apparent. [START_REF] Mathur | Antibiotic resistance in food lactic acid bacteria -a review[END_REF]. If or when any noxious genetic properties are present in the isolate genome, they represent a health risk because they may be transferred to other bacteria present in the food matrix or gut [START_REF] Toomey | Characterisation and transferability of antibiotic resistance genes from lactic acid bacteria isolated from Irish pork and beef abattoirs[END_REF][START_REF] Džidić | Antibiotic resistance mechanisms in bacteria: biochemical and genetic aspects[END_REF][START_REF] Ammor | Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria[END_REF].

Although many LAB cultures are found in foods and are considered safe, the biosafety of new cultures must be established before they can be used in food production [START_REF] Perin | Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk[END_REF]. Most of the studies carried out to determine Weissella strains' probiotic potential also evaluate the strains' health risks. These assessments include tests for hemolytic capacity and resistance to antibiotics, PCR detection of virulence genes, and biogenic amine production [START_REF] Fhoula | Functional probiotic assessment and in vivo cholesterol-lowering efficacy of Weissella sp. associated with arid lands living-hosts[END_REF][START_REF] Mortezaei | In vitro assessment of potential probiotic characteristics of indigenous Lactococcus lactis and Weissella oryzae isolates from rainbow trout (Oncorhynchus mykiss Walbaum)[END_REF][START_REF] Quattrini | A polyphasic approach to characterize Weissella cibaria and Weissella confusa strains[END_REF]. Kang et al. [START_REF] Kang | Safety evaluation of oral care probiotics Weissella cibaria CMU and CMS1 by phenotypic land genotypic analysis[END_REF] assessed the safety of W. cibaria CMU and CMS1 when used for oral care probiotics. Their results confirmed the strain is resistant to vancomycin and kanamycin (antibiotics) and that the resistance is intrinsic to the genus. They also demonstrated that the antibiotic resistance of W. cibaria CMU was not transferred to recipient strains. This was confirmed by the fact that no homology for a conjugative transposon integron-specific gene was present, i.e., there was no trace of antibiotic resistance genes in either chromosomal or plasmid DNA samples. They found that the strain had negative results for β-hemolysis, mucin degradation, and platelet aggregation tests. The strain also tested negative for various toxic secondary metabolites [START_REF] Kang | Safety evaluation of oral care probiotics Weissella cibaria CMU and CMS1 by phenotypic land genotypic analysis[END_REF].

Until now, Cupi and Elvig-Jorgensen [106] carried out a series of toxicity tests which included genotoxicity, skin irritation, eye irritation, and sub-chronic oral toxicity in rats. They found that the W. confusa strain did not show any toxic effects and therefore can be used as a safe, direct-feed microbial product for animals. Kang et al. [START_REF] Kang | Safety evaluation of oral care probiotics Weissella cibaria CMU and CMS1 by phenotypic land genotypic analysis[END_REF] showed that W. cibaria strains could be granted GRAS status in the future because: (i) they do not have antibiotic-resistant genes, (ii) they lack antibiotic resistance transferability, (iii) their genomic sequences do not include virulent genes related to pathogenic bacteria, and (iv) they tested negative for most virulent factors (β-hemolysis, mucin degradation, etc.) and toxic metabolic production (ammonia production, β-glucuronidase activity, etc.).

Although [START_REF] Quattrini | A polyphasic approach to characterize Weissella cibaria and Weissella confusa strains[END_REF]. To be granted the QPS status, a microorganism must meet the following criteria: (i) its taxonomic identity must be well defined, (ii) the available body of knowledge must be enough to establish its safety, (iii) the lack of pathogenic properties must be established and substantiated, and (iv) its intended use must be clearly described [107].

Conclusion

Over the past 5 years, the Weissella genus has been the focus of many studies due to its strong potential as a probiotic that could be used in both the food and pharmaceutical industries. Weissella strains' probiotic potential is attributed to their remarkable ability to survive passage through the GIT, produce antimicrobial substances for a variety of pathogens, and promote the formation of compounds that stimulate the gut microbiome. The ability of some Weissella strains to produce large amounts of EPS represents another major attribute, as EPS has prebiotic properties. Weissella EPS production also demonstrates the strains' potential as a natural thickener for foods, among other possibilities.

Moreover, recent studies have demonstrated that Weissella strains do not have antibiotic-resistant genes and they generally test negative for virulent factors. Thus, there is a low probability that the genus could cause foodborne diseases or carry on virulent genes that may be transferred to other bacteria pathogens.

Introduction

Lactic acid bacteria (LAB) is a microbial group naturally present in a wide variety of foods. LAB play an important role in the production of fermented foods due to their ability to produce aroma, flavor, and texture. BioNumerics software, version 6.6 (Applied Maths, Kortrijk, Belgium). The normalized band profiles were compared using the Dice similarity coefficient (1%). Cluster analysis for the compiled matrix was established using the unweighted pair group method and an arithmetic average (UPGMA) clustering algorithm.

Rep-PCR characterization of Weissella spp.

Aliquots of 1 mL of Weissella spp. cultures were centrifuged at 10,000×g for 10 min at 4 °C, and the obtained pellets were subjected to DNA extraction using a DNA Purification Wizard® Genomic kit (Promega). DNA concentration was standardized at 100 ng/μL (NanoDrop™ Lite Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA).

Rep-PCR analysis was performed according to the protocol described by Dal Bello The PCR products were electrophoresed on 2% (w/v) agarose gel for 2 h at a constant voltage of 75 V in 0.5x TBE buffer. The gels were stained using GelRed (Biotium Inc.) and developed using an LPIX transilluminator (Loccus Biotechnology, São Paulo, SP, Brazil).

The band profile was analyzed using BioNumerics software 6.6 (Applied Maths). The similarities between the profiles were calculated using the Dice similarity coefficient (1%).

Dendrograms were constructed using the UPGMA method.

Antagonism analysis and bacteriocin production

Based on the fingerprinting profiles, a representative number of isolates (n = 26) were selected for antimicrobial activity evaluation. Weissella spp. isolates were cultured in MRS broth (Oxoid) at 30 °C for 24 h and a cell-free supernatant (CFS) was obtained by centrifugation (10,000×g for 10 min at 4 °C) and filtration (Ø = 0.45 μm). The strains' CFS levels were tested using microdilution assays (Arena et OD from positive controls (ODc) was compared with OD from CFS-treated cultures (ODt) and inhibition values were calculated based on the following equation:

Inhibition = 100 -(ODt × 100/ODc)
Based on the antagonism test results, isolates that presented antimicrobial activity higher than 60% were selected to verify their bacteriocinogenic activity. CFS of the selected isolates was obtained as previously described. Furthermore, it was treated as follows: pH was adjusted to 6.5 with 1 mol/L NaOH to neutralize the medium and a heat treatment was applied for 10 min, at 80 °C, to eliminate peroxides (Cavicchioli, Camargo, Todorov, & Nero, 2017). The treated CFS were tested using the same target strains as described above. The same positive and negative controls were used, and the same result interpretation methods were used. This assay was conducted in triplicate and over two independent repetitions.

Technological potential

Selected isolates (n = 13) were also subjected to phenotypical analysis in order to characterize the following technological features: diacetyl production, coagulation, proteolysis, acidifying activity, and exopolysaccharide (EPS) production. Before each assay, the isolates were cultured in MRS (Oxoid) at 30 °C for 14 h and centrifuged at 14,000×g for 2 min at 4 °C. After the supernatant was discarded, the cell pellets were suspended in NaCl 0.85% (w/v) until a turbidity similar to McFarland tube 1 (approximately 3 × 10 8 CFU/mL).

The obtained cell suspension of the cultures was used in the assays described below, which were conducted in triplicate and over two independent repetitions. MilliQ water was used as a negative control. To assess diacetyl production, 0.1 mL aliquots of the cell suspension of the cultures were transferred to 10 mL of sterile reconstituted skim milk (10% w/v, Nestlé, São Paulo, SP, Brazil), and incubated at 30 °C for 24 h. Then, 1 mL aliquots of the obtained cultures were added to 0.5 mL of α-naphthol (1% w/v) and KOH (16% w/v) and incubated at

Results

Biodiversity by molecular analysis

Following SmaI macro-restriction, the Weissella spp. isolates presented 51 pulsotypes. These were grouped in three major clusters with similarity indexes among the grouped isolates varying from 60 to 80% (Fig. 2). Clusters I and II included isolates from different species and from all four geographical regions, while cluster III was composed only of isolates from Campos das Vertentes and Southern Pará. Also, cluster III presented the highest index similarity among isolates (around 80%), and included isolates from different origins that shared identical pulsotypes: isolates 28, 29, 46, and 53, obtained from silage, pasture, and cow milk (W. cibaria), respectively, and isolates 26, 27 and 44, obtained from soil and pasture samples (W.

cibaria).

Based on the rep-PCR tests, the isolates presented genetic profiles with bands ranging from 500 to 5000 bp. This allowed the characterization of 45 different profiles (Fig. 3). Unlike PFGE, the rep-PCR profiles were visually more diverse, which allowed the isolates to be clustered into two major groups, with similarity indexes among the included isolates ranging from 35 to 45%. Isolates that presented identical profiles according to rep-PCR analysis did not show the same SmaI restriction profiles (Figs. 2 and3). However, as confusa).

Inhibitory activity

Most of the tested isolates demonstrated antagonism against the target strains, with a minimum level of 60% of inhibitory activity (Table 1). The pH range of the inoculated MRS after the isolates was added and incubated for 24 h is shown in Table

Technological features

Among the 13 tested Weissella isolates, six exhibited diacetyl production (Table 2): isolate 25 (W. cibaria) results exhibited the most intense red color (data not shown). Four of these six isolates were obtained from cheese (Southern Pará) and two from pasture samples (Campo das Vertentes). W. confusa isolates demonstrated no diacetyl production.

In our research, four of the Weissella spp. isolates tested showed uniform and consistent clotting after milk coagulation ( Percentage of inhibition is expresses as (%) = 100 -(mean OD600 nm / mean OD600 nm target strain) * 100 , where clustering tendencies between isolates species and regions have been observed (Figs. 2 and3). However, similarity and distribution levels within the groups were not the same for the two techniques: PFGE was able to establish clearer associations between similar isolates, thus demonstrating better discriminatory properties and genetic characterizations of these isolates. As PFGE and rep-PCR require very different approaches, the first based on the digestion of entire DNA and the second based on the amplification of specific DNA regions, the profiles recorded were expected to be different [START_REF] Tenover | Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing[END_REF]. Using PFGE, identical pulsotypes were found within the same geographical regions (Fig. 2). In addition to belonging to the same regional classification (Campo das Vertentes), these isolates were taken from very closely related and/or similar samples, such as isolates 20 and 21 from pasture and isolate 44, 26 and 27, from soil and pasture from the same region (Fig. 1). Decreases in pH indicate that acids were produced during the incubation of isolates in MRS. Because MRS includes dextrose and ammonium citrate in its formulation, it can be assumed that the acids produced by the strains are lactic acid and acetic acid, via glycolytic and phosphoketolase pathways, as well as acetic acid via a diacetyl/acetoin pathway (Lynch et al., 2015). These organic acids have antimicrobial capacities when the pathogenic bacteria's cytoplasmic medium experiences a decrease in pH. According to [START_REF] Russell | Another explanation for the toxicity of fermentation acids at low pH: Anion accumulation versus uncoupling[END_REF], the non-dissociated form of organic acids is liposoluble and able to passively pass through cell cytoplasmic membranes. It changes the pH within cells by modifying the proton gradient and the electric charge when it interferes with the amino acid transport system and inactivates enzymes.

Weissella sp. is autochthonous to certain Brazilian raw milk artisanal cheeses and their production environments. The fact that they present antimicrobial activity against important foodborne pathogens indicates the microbiota may interfere with pathogens present in raw milk and inhibit them. According to [START_REF] Jay | Microorganisms in fresh ground meats: The relative safety of products with low versus high numbers[END_REF], the effects of autochthonous microorganisms on pathogens are well established. This effect is greater in animal-derived products containing a rich autochthonous microbiota.

The six isolates capable of producing diacetyl included two W. cibaria and four W.

paramesenteroides (Table 2 Of the four isolates that were able to coagulate milk, two were W. cibaria (Table 2).

Some studies have shown the ability of W. cibaria strains to ferment lactose and galactose Presumed proteolytic activity was observed for almost all the isolates selected in our study (Table 2). Lynch et al. (2014) have also evaluated the proteolysis activity of strains of W. cibaria MG1 using tests in milk agar and report that this enzymatic activity is strainspecific. LAB proteolytic and lipolytic activities in cheese lead to aroma and flavor generation and contribute to the release of bioactive peptides in milk that are beneficial to consumer health [START_REF] Meisel | Bioactive peptides encrypted in milk proteins: Proteolytic activation and thropho-functional properties[END_REF][START_REF] Wouters | Microbes from raw milk for fermented dairy products[END_REF].

According to [START_REF] Cogan | Characterization of the lactic acid bacteria in artisanal dairy products[END_REF], isolates with acidification capacities must reduce milk pH to 1.3 units after 6 h of incubation. The isolates examined in this study can be characterized as poor acid producers, except for isolate 16 (W. paramesenteroides), which was the only one able to reduce the pH to 5.41 after 6 h of incubation at 30 °C. It also produced a large amount of acid after 24 h of incubation (Table 2). Isolate 25 (W. cibaria) also produced a large amount of acid at the end of 24 h, but it required 12 h for it to reach a pH below 5.75 (Table 2).

Isolate 16 (W. paramesenteroides) was isolated from cheese samples (Southern Pará) which could justify its strong acidification properties given the optimal adaptation it has undergone. In addition, we can conclude that this isolate may play an important role in cheese. Not only did it show positive results for acidification and pH reduction in milk, but it also showed promising results for diacetyl production (which generates aroma), proteolytic activity (texture), and homogeneous clot formation.

Conclusion

This study shows the genetic diversity of Weissella strains isolated from different sources and different Brazilian regions that produce artisanal cheeses. It demonstrates the relevance of characterizing the molecular diversity of autochthonous Weissella in various environments. The technological characterizations helped us understand the role Weissella strains play in artisanal cheese microbiota and demonstrated their potential features. Further studies will be conducted to elucidate the contributing of Weissella species on cheese quality and sensory aspects, allowing propose their use as starter cultures in the dairy industry.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.lwt.2021.111474.

CHAPTER III:

Weissella cibaria W25: Comparative genomic analyses

Introduction

The study of microbial diversity in dairy and non-dairy environments plays a pivotal role in understanding the presence of these microorganisms in such ecosystems and their impact on the final product, especially when we refer to traditional and artisanal products.

Each environment has unique and specific characteristics that favor and allow the development of different bacterial species [1].

Handmade cheeses and raw milk are considered potential sources of new strains of LAB [2]. The way these cheeses are made can determine the fermentation to be con-ducted by bacteria present in the grazing, animal skin, utensils, surfaces, and other places that may come into contact with the cheese during production [3]. The study of the bacterial community present in artisanal cheeses revealed the presence of species that had not yet been related to cheeses and a high diversity of lactic bacteria with differentiated technological characteristics [4]. In addition, non-dairy environments such as grass, different types of silage, and even animal skin have also been important sources of novel strains that have adapted and therefore, can provide interesting features to be explored [5].

The diversity of Weissella isolated from dairy and non-dairy environments is of great interest for the enrichment of knowledge of this microorganism in the final products. The genus Weissella is composed of bacteria classified as Gram-positive, catalase-negative, non-spore-forming, coccoid morphology, or short bacilli. They belong to the group LAB, mainly due to their production of lactic acid from the fermentation of carbohydrates [6]. The main purpose of this study is to announce and analyze the sequencing and annotation of the Weissella cibaria W25 genome and carry out a comprehensive comparative genomic analysis with W. cibaria 110, known to produce the weissellicin 110 bacteriocin and four other non-bacteriocin-producing W. cibaria strains.

Materials and Methods

Bacterial strain

The strain W25 was previously isolated from pasture sampled from a dairy farm located in the Campos da Vertentes region, in the Southeast of the Minas Gerais state, Brazil. This strain was named "isolate id 25" by Teixeira, et al. [7], identified as Weissella cibaria after sequencing of the gene 16S rRNA and characterized as possessing a technological potential due to the ability to coagulate milk, producing diacetyl and non- No plasmid gene was detected by plasmidFinder in W. cibaria W25 and this input organism was predicted as a non-human pathogen by PathogenFinder. These results are indicative of the safe use of this strain for future human consumption as a probiotic or as a bioprotective culture in food, for example.

According to AntiSMASH W. cibaria W25 possess two putative bacteriocins gene clusters, one lasso peptide (MicJ25), and one RiPP-like bacteriocin_IIc. Of, note, the Bagel4 software did not allow the identification of any bacteriocin gene. Previously, we showed that W. cibaria W25 has a narrow spectrum of inhibition against the most common foodborne pathogens [7] reinforcing that this strain is a suitable probiotic candidate. Li et al. (2017) [25] showed that W. cibaria 110 present similar results showing a large spectrum of inhibition against other LAB but the bacteriocin weissellicin 110, produced by W. cibaria 110, unlike most class II bacteriocins, has no inhibitory activity against Listeria monocytogenes.

Phylogenetic analyses and comparative genomic

Weissella phylogeny was constructed using available 16S rRNA sequences of sequenced Weissella species, including complete and draft genomes (Figure 1) and using the whole genome sequences (Figure 2). Figure 1 and 2 shows the formation of two major clusters, using only the 16S rRNA sequence we noticed that cluster 1 com-prised 4 strains (W. confusa, W. cibaria, and W. muntiaci) and cluster 2 comprises the other strains. On the other hand when we used the whole genome sequence, cluster 1 comprised W. ceti and W. diestrammenae, and cluster 2 all the other strains. Also figures 1 and 2 show that W. cibaria with the one from KmerFinder software confirmed the genus Weissella and the species cibaria for the strain W25 as previously announced by Teixeira et. al (2021). average branch support of 55.9 %. The tree was rooted at the midpoint [29].

The digital DDH genomic similarity revealed that between the strains tested in this study, W. cibaria W25 possesses more similarity with W. cibaria JCM 12495 (87.10%), a non-bacteriocin producer, and less similarity with W. cibaria 110 (63.80%), a strain known as a bacteriocin producer.

According to the Veen diagram from OrthoVenn2, W. cibaria W25 shares the same quantity of protein cluster genes with W. cibaria 110 and W. cibaria JCM 12495 (Figure 3) when comparing just the three of them. When we compared with all the strains used in this study, W. cibaria W25 shares more unique protein cluster genes [18] with W. cibaria 110 (Figure 4, B) than with the others. Besides that, W. cibaria W25 shares 25 with all the nonbacteriocin-producing strains (Figure 4,C). There were 1,852 protein cluster genes conserved in all W. cibaria strains, and five of them are unique to W. cibaria W25 (Figure 4, A) with 10 paralogs. Among the five protein cluster coding genes OrthoVenn2 identified three of them, one related to the lipopolysaccharide biosynthetic process, one to the O antigen biosynthetic process, and one to oxidoreductase activity. We constructed a comparative genetic map using CGview server to help demonstrate the similarity between the strains (Figure 5). We used the W. cibaria 110 as the reference for comparison because it is a well-known bacteriocin-producing strain. Moreover, gene confirmation for weissellicin 110 was performed by the BLASTn program at the National Center for Biotechnology Information (NCBI) using the deposited sequence under accession number LC010242 and the predicted genes of the genome from W. cibaria 110. We obtained results to query coverage and percent identity of 100%. Among the regions that showed to be similar between W. cibaria W25 and W. cibaria 110, we observed that the strain under study presented a similar and more complete region, when compared to the region containing weissellicin 110, than the non-bacteriocin-producing strains (Figure 5,B). This indicates that perhaps in this region of W. cibaria W25, there are genes for a bacteriocin, however a distinct one.

Conclusions

According to the bioinformatics results obtained in this study, W. cibaria W25 has great potential to be used for human consumption once it was predicted as a non-human pathogen. Also, despite W. cibaria W25 showing more genomic similarity with W. cibaria JCM 12495 (according to DDH similarly) OrthoVenn2 showed that it has its unique protein cluster genes which can be related with the bacteriocins genes indicated by AntiSMASH confirming the possibility of producing two different bacteriocins.

Introduction

For many years antibiotics have been used as the basis of treatment against bacterial infections and their misuse conducted to the development of antibiotic-resistant species [START_REF] Blair | Molecular mechanisms of antibiotic resistance[END_REF]. The World Health Organization (WHO) encourages new strategies and interventions to avoid the development of new multi-drug-resistant pathogens [START_REF] Neill | An audience with[END_REF].

LAB and its diverse metabolites such as organic acids, hydrogen peroxide (H2O2), and Bacteriocins can be active against phylogenetically related or phylogenetically distant target strains, but will not be against producing strains themselves because of their developed specific immunity proteins [START_REF] Drider | The Continuing Story of Class IIa Bacteriocins[END_REF]. As bacteriocins are commonly produced by strains from the gut microbiota and natural food sources, in which some are "Generally Recognized as Safe" (GRAS status), interest in LAB-bacteriocins is ever-growing for the Despite some LAB genera being known for their antimicrobial activity against food spoilage and pathogenic microorganisms, not all of them are effective. Some of them produce a low amount of antimicrobial compounds or have a limited spectrum of activity (Tenea et al., 2020). Therefore, the discovery of novel antimicrobial compounds endowed with broad inhibitory spectra is of interest for their potential applications as alternatives to overage antibiotics or as bio-preservative agents for the food industry. Of note, applications of LAB-bacteriocins in the food industry as bio-preservative agents are feasible and constitute an eco-friendly approach that could replace the use of chemical preservatives.

The biopreservative approach is timely as it meets the consumers' expectations for natural and bio-protected foods (Venegas- Related to that, we isolated strains of W. cibaria from different artisanal cheese-producing regions in Brazil and established their technological and bacteriocinogenic traits (Teixeira et al., 2021). Most of the studied isolates demonstrated broad inhibition against several pathogens, both gram-positive and gram-negative (Teixeira et al., 2021).

This study aims to identify the antimicrobial compounds produced by Weissella strains isolated from Campos das Vertentes, Minas Gerais, Brazil, to schedule their future application as pathogens inhibitors in the food industry and preservation.

Material and Methods

Microorganisms and Culture Conditions

Three different strains of Weissella cibaria, previously isolated from dairy farms located in the Campos da Vertentes region, in the southeast of the Minas Gerais state, Brazil were used in this work. The strains W21 (isolate id 21) and W25 (isolate id 25) were isolated from pasture samples, whereas strain W42 (isolate id 42) was isolated from soil (Teixeira et al., 2021). These 3 strains were identified as Weissella cibaria after sequencing of the gene 16S rRNA and presented antimicrobial activity against a panel of Gram-positive and Gram-negative foodborne pathogens (Teixeira et al., 2021). The isolates obtained were stored at -80°C in the Man Rogosa and Sharpe (MRS) broth (Oxoid Ltd., Basingstoke, England) and supplemented with 30% (v/v) glycerol (Synth, São Paulo, SP, Brazil). Before analysis, stock cultures (1% v/v) were transferred to MRS broth (Oxoid) and incubated at 30 °C overnight.

Genome Sequencing, Assembly, and Annotation

The genome sequence of the W25 strain was previously deposited in GenBank under accession number JAFNKE000000000 [START_REF] Teixeira | Genomic Analyses of Weissella cibaria W25, a Potential Bacteriocin-Producing Strain Isolated from Pasture in Campos das Vertentes, Minas Gerais, Brazil[END_REF]. The whole genomes of W. cibaria W21 and W42 were sequenced, trimmed, and assembled as recently performed for the W25 strain [START_REF] Teixeira | Genomic Analyses of Weissella cibaria W25, a Potential Bacteriocin-Producing Strain Isolated from Pasture in Campos das Vertentes, Minas Gerais, Brazil[END_REF]. Gene prediction and annotation were performed using the Rapid Prokaryotic Genome Annotation (PROKKA) v.1.14.5 (Seemann, 2014), executed with default parameters, and also performed by RAST automated web server (Aziz et al., 2008). The assembled genome was conducted by using MIRA Assembler v.4.9.6 (Chevreux et al., 1999), mode "genome, accurate". The assembling quality was determined with

We tested the antimicrobial activity against Escherichia coli ATCC 8739, Kocuria rhizophila CIP 5345, Listeria innocua CIP 80.11, and Salmonella enterica using the microdilution assays according to Teixeira et al. (Teixeira et al., 2021) and the spot on the lawn assay according to Moraes et al. [START_REF] Moraes | Protocols for the isolation and detection of lactic acid bacteria with bacteriocinogenic potential[END_REF]. Weissella spp. strains were cultured in MRS broth (Oxoid) at 30°C for 24 h and a cell-free supernatant (CFS) was obtained by centrifugation (10,000 × g for 10 min at 4 ºC) and filtration (Ø = 0.45 μm). Target strains were cultured in a brain heart infusion (BHI, Oxoid) at 37 °C for 24 h. For the microdilution assay aliquots of the supernatant (100 μL, at a concentration of about 10 4 CFU/mL) were transferred to 96-well microtiter plates. Then, 50 μL aliquots of the selected Weissella spp.

strains' CFS were added to each well. The microplates were incubated at 37 ºC for 24 h in a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cultures' optical densities (OD) were measured every 30 min (λ = 600 nm). For the spot-on-the-lawn assay, the supernatant was ten-fold concentrated and spotted on BHI agar plate. After absorption at room temperature (10-15 min), the agar was covered with an overlayer of semi-solid BHI (8 ml) inoculated with 10 5 CFU/mL of the target and incubated at 37 °C for 24 h. The absence of an inhibition zone in the proximity of the well indicates no antimicrobial activity of the supernatant.

After confirming the antimicrobial activity against E. coli, K. rhizophila, L. innocua, S.

enterica, and S. aureus, the supernatant of the Weissella strains were treated with proteinase K in order to confirm the proteinaceous nature of the inhibitory substances. On the other hand, the pH of the culture supernatant was adjusted/neutralized to discard the effect of any inhibition linked to acid production. Of note, after these treatments, the antibacterial activity was confirmed against the aforementioned target organisms with both assays as described above.

Results and Discussion

Genome Sequencing, Assembly, and Annotation

The information obtained after genome sequencing, assembly and annotation is presented in table 1. A slight difference between the three genomes regarding the number of contigs and the size of the genomes (Table 1). According to Björkroth et al. (Björkroth et al., 2002), the G+C content in W. cibaria species should be 44 to 45 mol% DNA base ratio, which was what we observed. We can also see that the quantities of rRNA and tRNA genes are comparable between the same species but slightly different due to the fact to be different strains from the same species.

Bacterial Species Confirmation

The identification of the genus and species using KmerFinder (Hasman et The results from the genetic relatedness of W. cibaria W21, W25, and W42, constructed using their whole-genomes and those from other Weissella, are expressed in the phylogenetic tree in figure 1. with average branch support of 56.6 %. The tree was rooted at the midpoint (Farris, 1972).

Figure 1 besides confirming the genus and species of the three sequenced strains also confirmed the similarity of the results from table 1 grouping the three strains in the same cluster with a high percentage of similarity.

Antimicrobial potential

In the previous report [15], we mentioned the presence of two putative bacteriocins genes cluster [START_REF] Teixeira | Genomic Analyses of Weissella cibaria W25, a Potential Bacteriocin-Producing Strain Isolated from Pasture in Campos das Vertentes, Minas Gerais, Brazil[END_REF], one lasso peptide (MicJ25), and one RiPP-like bacteriocin_IIc. According to the analysis for W. cibaria W25, AntiSMASH (Blin et al., 2021) detected three regions in its genome, C6, C18, and C75.

The C6 region contains 2 core biosynthetic genes, one additional biosynthetic gene, one transport-related gene, and nine putative genes. The two core biosynthetic genes are a possible bacteriocin classified as Bacteriocin_IIc (Figure 2) and a possible protein related to the ABC transport. The additional biosynthetic gene was identified as a phenylalaninespecific permease and the transport-related gene was identified as a possible ABC transporter ATP-binding protein.

In the C18 region, AntiSMASH (Blin et al., 2021) Finally, in the C75 there was one core biosynthetic gene named biosynthetic (rulebased clusters) lasso peptide: micJ25 and three putative genes.

In the genome of W21, we detected two regions C1 and C9 with two and one core biosynthetic genes, respectively. In the C1 region, we found, as in the W25, a possible bacteriocin gene named Bateriocin_IIc. The difference between this region compared to the C6 in the genome of the W25 strain is that there are all the same genes except for the transport-related gene, as we found in W25. Region C9 of W21 has the same genes as region C18 in the W25 genome strain.

In the genome of strain W42, AntiSMASH (Blin et al., 2021) detected two different regions and one possible bacteriocin. Region C3 has the same genes as the C6 region in the W25 genome strain. Also, the C2 region has the same genes as the region C18 in W25.

The bacteriocin_IIc detected by AntiSMASH in the region C6 of W. cibaria W25 is represented in figure 2. The gene for the bacteriocin_IIc was identified in all three Weissella strains, and the region containing the gene has also other genes such as the ABC transport protein-related gene and other Orfs non-identified by AntiSMASH. However, the strain W21 is missing one transport-related gene. When the sequence of amino acids of this putative bacteriocin was blasted in the NCBI platform it shows a 98.48% of identity with Blp family class II bacteriocin from W. cibaria.

The putative sequences of the promoter region for the bacteriocin_IIc gene are depicted in Figure 2. The results for the terminator region indicate that it is a Rhoindependent terminator which is based on a palindrome sequence downstream from the target gene. The distance between the Orf05 and Orf06 is very short, (3 nucleotides) and therefore we could not identify any putative promoter sequences for gene Orf06 and terminator sequences for gene Orf05 which means that these Orfs could be co-transcribed.

There are already some well-known bacteriocins from the The Weissellicin 110 is the most characterized bacteriocin from the specie W. cibaria, despite of that no other bacteriocin was well-study from the strain W. cibaria and the identified sequence by AntiSMASH didn't reveal any similarity with the studied bacteriocins from Weissella genus.

The in vitro evaluation of antimicrobial activity against E. coli, K. rhizophila, L.

innocua, and S. enterica is presented in Figure 3 for the microdilution assay. The results of the spot-on-the-lawn assay show that all the supernatant previous to the treatment presented a clear zone around the spot, but in the test with the treated supernatant, no clear zone was detected. The results showed that when we used the supernatant without treatment there was no growth of any target, but when the supernatant is neutralized or neutralized and treated with proteinase K there was a huge decrease in the antimicrobial activity. However, as we can see in some growth curves from figure 3 (A, C, D, F, andI) after neutralization of the supernatant there was a remaining antimicrobial activity and this one was lost after the treatment with proteinase K. This is an indication that the major compound responsible for antimicrobial activity can be some organic acids produced by the tested Weissella strains or a combination between produced compounds. Several compounds produced by Weissella strains has antimicrobial activity such as organic acids, hydrogen peroxide, bacteriocins, and other (Cheng et al., 2016;[START_REF] Fhoula | Antimicrobial activity and safety features assessment of Weissella spp . from environmental sources[END_REF][START_REF] Yeu | Antimicrobial and Antibiofilm Activities of Weissella cibaria against Pathogens of Upper Respiratory Tract Infections[END_REF]Yu et al., 2019). Also, the synergism between two or more compounds, such as lactic acid and acid citric with bacteriocin, can increase the antimicrobial activity of the supernatant [START_REF] Soltani | Bacteriocin-Based Synergetic Consortia: a Promising Strategy to Enhance Antimicrobial Activity and Broaden the Spectrum of Inhibition[END_REF].

CHAPTER V: Weissella cibaria strains isolated from

Campo das Vertentes (MG), biosecurity and probiotics analyses

Introduction

Brazil is a country, in which several regions produce their own artisanal cheeses, offering, therefore, access to rich bacterial microbiota and beneficial microorganisms such as lactic acid bacteria (LAB), that could be employed in the food and pharmaceutical sectors [1]. Related to that, handmade cheeses and raw milk are endless sources of novel LAB strains [2], and each handmade cheese has its specific microbiota. Environmental microorganisms can also reach the product during the manufacturing process, influence the fermentation process and consequently impact the organoleptic and aromatic qualities of the final product [3]. LAB assigned to the Weissella genus are Gram-positive bacteria, catalase-negative, asporogenous, coccoid morphology, or short bacilli with heterofermentative metabolism and CO2 production from carbohydrate uptake, making them suitable for use in fermentation processes. Weissella, as well as Fructilactobacillus, Convivina, Leuconostoc, and Oenococcus, are known as Fructophilic LAB because they usually metabolize fructose instead glucose under certain conditions. Weissella spp. inhabit a wide range of ecological niches including plants, vegetables, soil, water, and fermented foods, where they assume a probiotic role [4]. Weissella species can also be found in habitats associated with the human or animal body, e.g., the gastrointestinal tract, vaginal microbiota, or human breast milk [5,6].

Weissella strains endowed with antagonistic activities against different foodborne pathogens have been isolated from different Brazilian regions producing artisanal cheeses [7]. Of note, the Weissella genus has emerged in 1993, after reconsidering taxonomically atypical Leuconostoc [4]. Phylogenetically, the genus Weissella belongs to the Leuconostocaceae family, order Lactobacillales, class Bacilli, phylum Bacillota and domain Bacteria. Currently, the genus Weissella contains 35 species (www.bacterio.net). Weissella species are facultatively anaerobic, obligately fermentative, and non-motile bacteria, except W. beninsensis which is reported as being motile [8]. The accurate identification of Weissella strains is to be performed by sensitive methods like 16SrRNA gene sequencing or Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), rather than traditional phenotypic methods, which lead to their misidentification [5]. Weissella is enormously reported for diversity and high production of exopolysaccharides with potential for industrial applications and bio-functional properties [9,10].

The ability of Weissella and overall LAB to produce antimicrobial compounds retain very strong interest because of potential applications in the food sector. Indeed, the last decade has seen a new challenge for the food industry, which consists to identify natural preservative compounds that will augment food safety and security after their incorporation into foods [11]. Foods free of chemicals are new demands of consumers and not necessarily of consumers of high living standards. Thus, alternatives based on the incorporation of beneficial microorganisms or their metabolites in the food matrix might be the next solution.

The use of a novel strain as an ingredient either for human or animal feed requires one to meet strict criteria related to safety by discarding any adverse effect like hemolysis, carriage of genes coding for antibiotic resistance, or pro-inflammation reaction [12,13].

To discover novel lactic acid bacteria, we aim in this work to characterize W. cibaria W21, W25, and W42, isolated in different Brazilian farms, in Campos das Vertentes (at Minas Gerais state), known for producing artisanal cheeses with raw milk, and address their probiotic profiles.

Material and Methods

Safety of Weissella cibaria W21, W25, and W42 strains

Bioinformatics analyses of Weissella's genomes

To locate genes coding for the well-known virulence factors in the genomes of W. cibaria W21, W25, and W42 strains, we used web tools recommended by the European Food Safety Authority. The search for MGE-like plasmids was done by employing plasmidFinder [14], while the PathogenFinder web tool [15] enabled their profile as potential human pathogens. Genes coding for the virulence factors were investigated by using the "Virulence Factors Database" (VFDB) (http://www.mgc.ac.cn/VFs/main.htm) [16], while the search for genes coding for antibiotics resistance was realized by using the RGI (Resistance Gene Identifier) tool and the "Comprehensive Antibiotic Resistance Database" (CARD) (https://card.mcmaster.ca/) [17]. To this end, we import the contigs selecting the parameters "perfect and strict hits only" and "high-quality coverage". Finally, the ResFinder 4.1 [18] server (https://cge.cbs.dtu.dk/services/ResFinder/) was used to identify the acquired antimicrobial resistance genes with a selected % ID threshold of 90 % and the selected minimum length of 60%.

For the detection and annotation of prophage sequences within bacterial genomes, the PHASTER [19] (PHAge Search Tool Enhanced Release) (http://phaster.ca) tool was used.

The CRISPRCasFinder [20] (https: //crisprcas.i2bc.parissaclay.fr/CrisprCasFinder/Index) was also used to detect the CRISPR and Cas genes by importing the contig FASTA sequences and selecting "Perform cas detection" and leaving the rest of the parameters by default.

Hemolysis and antibiotic susceptibility

In vitro tests enabling the establishment of hemolytic activity and antibiotics susceptibility of these strains were performed according to protocols reported by Colombo et al. [21]. To evaluate, the hemolytic activity, these strains were cultured on Columbia Blood Agar Settle plate (Merck KGaA, Darmstadt, Germany). After 24h at 37°C, the plates were analyzed for microbial hemolytic activity, considering a total or β-hemolysis as clear halos around the colonies, partial or α-hemolysis as greenish halos around the colonies, and γhemolysis as the absence of hemolysis [21].

Susceptibility to antibiotics was determined using the disk diffusion assay (Oxoid), and according to the protocol reported by Colombo et al. [21]. Each strain was diluted in a solution of 0.85 % NaCl (w/v) until obtained turbidity equivalent to 0.5 McFarland standard and then was swabbed onto the surface of an MRS agar plate (where the antibiotic disks were placed). These antibiotic disks contained imipenem (carbapenem), vancomycin (glycopeptide), clindamycin (lincosamide), erythromycin (macrolide), ampicillin (penicillin), ciprofloxacin (fluoroquinolone) and amoxicillin + clavulanic acid]. The plates were incubated at 37°C for 18h. After this, the diameter of the inhibition was measured and the resistance profile was determined according to CLSI breakpoints [22].

Cytotoxicity and Inflammatory activity of Weissella strains in human cell

Cytotoxicity assay

For the cytotoxicity test, we utilized a mix of human cell lines composed of 80 % of human colon adenocarcinoma Caco2 and 20 % of HT-29. The preparation of the mix of human cell culture was realized according to Pouille et al. [23]. The mix of Caco2 cell and HT-29 line were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific), supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (FBS) (Biowest, Nuaillé, France), 100 units/mL penicillin, 100 µg/mL streptomycin (Gibco, Life Technologies, Grand Island, NY, USA) and 10 mM nonessential amino acids (Gibco, Life Technologies, Paisley, UK). Cells were cultured under standard cell culture conditions (37°C and 5 % CO2). The culture medium was changed regularly and when the cells reached sub-confluence (80-90 %), they were sub-passaged. The cells were cultured at 1.5 × 10 4 cells/mL in 96-well plates for 24 h and then exposed to the 3 different Weissella strains.

The preparation of the Weissella cultures to contact the mix of human cell cultures was realized as described below. One percent of frozen Weissella strains were propagated in MRS broth (Oxoid) for 24 h, centrifugated at 10,000 rpm for 5 min and the supernatant was removed. The obtained pellet was washed with 5 mL of phosphate buffer saline (PBS, pH 7.4), resuspended in 2 mL of DMEM without antibiotics and fetal bovine serum, and diluted until a final concentration of 10 7 CFU/mL. The suspension of cells was incubated at 37°C until contact with the mix of human cells. For the contact, the mix of human cell culture fixed in the bottom of the 96 wells plate was washed with DMEM and the suspensions of Weissella strains were added in a proportion of 1:1 and 1:10 (MOI). After the plate was incubated for 24 h at 37°C.

Cell viability of Caco2 and HT-29 after 24 h of incubation with Weissella strains was measured using a cell counting kit-8 assay (CCK-8, Dojindo Molecular Technologies, Inc.

Japan). After incubation, the 96-well plate was washed with DMEM with antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). After the washing procedure and removing all Weissella strains cells, 150 µL of CCK-8 solution (5 %) was added to each well of the 96well plate, followed by incubation for 1.5 h at 37 °C. Then, 10 mL of stop solution provided in the CCK-8 kit was added to each well. The absorbance of the mixture was measured at 450 nm using a microplate reader (Synergy H1, BioTek, USA). Cell viability was calculated based on the relative absorbance compared with the control group.

Inflammatory effects of Weissella strains

To investigate the inflammatory and anti-inflammatory effects of Weissella strains, we used the promonocytic human cell line U937 according to the protocol described by Quantikine® ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA). Medium samples were diluted 100-fold according to the kit recommendations.

Survival of Weissella cibaria strains under conditions mimicking a human gastric digestion

The survival of Weissella strains in simulated human gastric and intestinal juices was assessed as described by Fonseca et al. [24] with modifications. The composition of the simulated gastric juice was pepsin (2 g/L) (Sigma Aldrich, St Louis, MO, USA) and pH 3.5 PBS, adjusted with HCl 0.5 M. Each isolate was grown at 37 °C overnight in sterile MRS broth (Oxoid) and inoculated in the solution until the final concentration of ~ 1 × 10 10 CFU/mL and followed by incubation at 37°C for 90 min with constant shaking at 160 rpm. After incubation, the samples were taken and serially diluted, then plated on MRS agar plates and incubated at 37°C for 24 h. Then, the simulated duodenal juice was prepared by adding pancreatin (Dinâmica, Brazil) and bile salts solutions to obtain final concentrations of 0.2 % and 0.5 % (w/v), respectively, and the pH was then adjusted to 7.0 by adding 1 M sodium hydroxide (NaOH). After mixing, samples were incubated at 37 °C for 180 min with agitation (160 rpm), and then viable cell counts were determined. All sample counts were determined by plating on MRS (Oxoid) agar. The experiments were repeated three times and performed in triplicate. Results were expressed as mean log colony-forming units per mL (CFU/mL).

The rate of survival was calculated as the percentage of Weissella colonies grown on MRS agar relative to the initial bacterial concentration:

𝑆𝑢𝑟𝑣𝑖𝑣𝑎𝑙 𝑟𝑎𝑡𝑒 (%) = log 𝐶𝐹𝑈𝑁1 log 𝐶𝐹𝑈𝑁0 × 100
Where N1 is the viable count of isolates after incubation and N0 is the initial viable count.

Adhesion of Weissella cibaria strains to the human Caco-2 cell line

The adhesion capacity test for the three selected strains to the human colon adenocarcinoma cell lines (Caco-2) was performed according to Fonseca et al. [24] with slight modifications. The Caco-2 cells were sub-cultured (2 × 10 5 cells/mL) in 24-well tissue culture plates (Sarstedt, Germany) and grown at 37 °C in a humidified atmosphere of 5 % CO2 for 21 days to promote differentiation in cell media. The culture medium was changed on alternate days. For the adhesion assay, bacteria were cultured in MRS broth overnight at 32°C, and after washing twice with PBS, the cultures were resuspended in the DMEM at a concentration of approximately 10 7 CFU/mL. After the DMEM of the 24-well tissue culture plates was removed, the culture cells were washed with the same media. Then 400 µL of DMEM and 100 µL of Weissella strains were added to each well. The plate was incubated at 37°C overnight in a 5 % CO2 atmosphere. Subsequently, the cells were washed three times with 1 mL of PBS to remove non-adherent bacteria cells and then lysed with 300 µL of trypsin. After 15 min of incubation at 37°C, the solution with released bacteria cells was transferred to 1.5 mL Eppendorf and the bacteria cells were centrifugated (9,000 × g/ 5 min), the supernatant was removed and the pellet was resuspended in 500 µL of PBS. After recovering the bacteria cells were serially diluted and plated on MRS agar. The plates were incubated at 32°C for 24 h. Then, enumerations of W. cibaria strains colonies were performed. Experiments were performed in duplicate and repeated three times.

In situ inhibition of pathogenic bacteria

For this analysis, Caco-2 cell line cultures were cultivated and Weissella strains were propagated as above described. Before the experiment, the DMEM of the 24-well tissue culture plates was removed and the culture cells were washed with the same media. For the pathogen inhibition test, the exclusion assay was performed according to the protocol described by Fonseca et al. [24] with modifications. The pathogens used in this experiment were Escherichia coli 184 and Staphylococcus aureus MRSA SA1, which were propagated separately in BHI medium (Oxoid) at 37°C for 24h.

Caco-2 cells were first preincubated with 100 µL of Weissella strains (10 7 CFU/mL) for 2h at 37°C in a 5 % CO2 atmosphere and then 100 µL of E. coli (10 7 CFU/mL) or S. aureus (10 7 CFU/mL) was added to each well. The plates were incubated at 37 °C overnight in a 5 % CO2 atmosphere.

After the incubation period, the cells were washed three times with 1 mL of PBS to remove non-adherent bacteria cells and then lysed with 300 µL of trypsin. After 15 min of incubation at 37°C, the solution with released bacteria cells was transferred to 1.5 mL

Eppendorf and the bacteria cells were centrifugated (9,000 × g/ 5 min), the supernatant was removed and the pellet was resuspended in 500 µL of PBS. After recovering, the bacteria cells were serially diluted and plated. E. coli was plated in Eosin Methylene Blue agar (EMB) and S. aureus in BHI supplemented with the antibiotic erythromycin (100 µL/mL) that prevented the growth of Weissella strains. The plates were incubated at 37 °C for 24 h. After this time, enumerations of pathogen colonies were performed. Experiments were performed in duplicate and repeated three times.

Results and Discussion

This study aimed at evaluating different criteria allowing W. cibaria W21, W25, and W42 strains to come as excellent probiotic candidates. First, the safety was assessed based on their phenotypic (antibiotic resistance, hemolysis) and genotypic analyses (virulence genes including antibiotic resistance genes). Then, these strains have been confronted with the harsh conditions of the gastrointestinal tract.

Weissella cibaria W21, W25 and W42 strain are safe

Strains belonging to the Weissella genus contain traits in their genome that confer versatility. In particular, Weissella cibaria encodes several beneficial genes that are useful in biotechnological applications. Based on the ResFinder data, no acquired antimicrobial resistance genes were found in the genomes of the 3 tested Weissella strains. Moreover, the research for plasmid evaluated by plasmidFinder [14] concluded the absence of MGE, delineating the stability of the genomes.

According to the findings of CRISPRCasFinder [20] (Table 1), each of the three Weissella strains' genomes harbors a single CRISPR array, with evidence level 4 indicating that these arrays may be legitimate [20]. The CRISPR-associated protein Csn2_TypeIIA is a mandatory protein of the Type IIA system and the gene is present in the three sequenced genomes. According to Roberts and Barrangou [25], the Type II systems are rare in nature compared to types I and III, which are also more well-characterized [25]. The CRISPR-Cas system is a defense mechanism used by bacteria to protect themselves against infections caused by foreign genetic elements. Thus, strains endowed with the CRISPR-Cas system could acquire fewer sequences issued from other bacteria preventing therefore horizontal gene transfer and leading to a low level of drug resistance [26]. Related to that, the CARD analysis only located genes involved in the resistance to vancomycin, (vanY gene in the vanB cluster and vanT gene in the vanG cluster) for all 3 tested strains (Table 2), and this resistance was confirmed experimentally in vitro (Table 3), demonstrating the convergence of these two methods. In a direct line, [27] reported that 60% of Weissella species are resistant to several antibiotics [27]. Recently, Tenea and Hurtado found putative resistance genes in the genome of W. cibaria UTNGt21O [28]. Overall, the CARD and the in vitro antibiotic tests data obtained here corroborate the low level of resistance, once the three tested strains presented resistance to vancomycin, which is intrinsic for this genus [10]. Indeed, Weissella is known to be intrinsically resistant to vancomycin and has a high minimum inhibitory concentration (MIC) of ≥ 256µg/ml. However, antimicrobial susceptibilities of other species like W. confusa are not fully understood. Currently, there are no standard methods and interpretation criteria for antimicrobial susceptibilities established for Weissella spp. by the Clinical and Laboratory Standards Institute (CLSI). Intrinsic resistance to antibiotics is not redhibitory or a break [12] in the procedure of the selection of these strains for probiotic applications. The transferability of antibiotic resistance (AR) genes and plasmids present in bacteria is associated with human health. Moreover, the number of spacers in the CRISPR-Cas system represents the ability to eliminate heterologous genes, such as prophage. Thus, the more spacer sequences are present, the more the strain has a limited capacity to acquire foreign sequences like prophages [26]. Here, we only could locate using PHASTER two prophages (1 entire and 1 incomplete) in the genomes of the three strains (Table 4). Dong et al. [26] demonstrated that the correlation between spacers and prophages is negative [26]. Prophages are MGE which is considered the primary factor in genetic diversity enabling niche adaptation in bacteria [29]. As above-mentioned, two prophages were located in the genomes of the three strains W. cibaria W21, W25, and W42, one of them was intact, whereas the second was incomplete in each of the tested strains. The length of the intact prophage varies from 32 to 35.8 Kb with the most common phage:

PHAGE_Entero_phiFL1A_NC_013646 (14). The presence of intact regions could be a selective advantage and help bacteria against other prophage infections [30,31].

Despite several virulence factors detected by the VFDB web server, the results indicate that no one of the known virulence factors was present among them and the analysis of PathogenFinder [15] indicates that the three input organisms were predicted as a non-human pathogen. Therefore, these three strains of W. cibaria can be considered safe for use as probiotics. Of note, to evaluate the safety of probiotics, some guidelines consider several factors in advance, like excessive immune stimulation in sensitive individuals, systemic infection, gene transfer, or deleterious metabolic effects [35]. To the best of our knowledge, W. cibaria has not current use as a probiotic ingredient, although this species has been frequently isolated from fermented foods and human feces and is well-known for its beneficial effects.

Weissella cibaria W21, W25, and W42 strains are not cytotoxic toward

Eukaryotic cells and do not induce inflammatory effects

For the cytotoxicity test, we used a mix of human cell cultures with 80 % of Caco2 and 20 % of HT-29 5 (Figure 2A) and with the human U937 cells (Figure 2B). It is noteworthy that no cytotoxicity was found against these human lines, matching with other studies that underpinned the safety of W. cibaria strains on RAW 264.7 macrophage cell line and human mouth epithelial cell line KCLB 10017 [36,[START_REF] Kang | Effect of Weissella cibaria on Fusobacterium nucleatum -induced Interleukin-6 and Interleukin-8 Production in KB Cells[END_REF].

Interestingly, we assessed the inflammatory and anti-inflammatory capacity of Weissella strains against the human U937 cells and these strains showed neither inflammatory nor anti-inflammatory responses. Taken all together, these data strengthen the safety aspects of these strains and promote their potential in the intestinal health of humans and animals.

Similarly, Kyung-Hyo Do et al. [38] showed significant therapeutic efficacy of W. cibaria CMU and W. cibaria CMS1 in improving lung inflammation exacerbated by DEPM administration in the murine model [38].

Survival of W. cibaria strains in the simulated gastro-intestinal tract

We noted during the assessment of the survivability of Weissella strains in the gastrointestinal tract, a small loss of the viability of the cells after 1.5 h in conditions mimicking the stomach compartment, and also no survivability was observed upon 3 h of incubation in conditions simulating those of the duodenal compartment (Table 5).

ND: not detected by technique limit

The ability of bacteria to survive under gastric digestion conditions is considered a key criterion for probiotic selection [39]. To overcome this drawback, some strategies of delivery are being researched such as encapsulation, which could help the passage through the gastrointestinal tract. Encapsulation acts by protecting bacteria from acidic stress and rendering their delivery process directly in the intestine where they can act as probiotics and provide health incentives to the host [START_REF] Nacef | MALDI-TOF mass spectrometry for the identi fi cation of lactic acid bacteria isolated from a French cheese : the Maroilles[END_REF]41]. A recent study showed that the HigBA TA system of W. cibaria 018 responded to the bile salt stress, but not to acid stress, which might offer novel perspectives to understand the tolerant mechanism of probiotics in the GIT environment [42]. Assessing gastrointestinal tract survival by the agar enumeration does not take into account the viable but nonculturable cells (VBNC) [43]. Previous studies used the cytometry method, which includes both viable bacteria and VBNC and as expected, the survival percentages obtained by this method were significantly higher in comparison to the data obtained after their growth and enumeration by agar plating method [39].

Weissella cibaria W21, W25 and W42 strains adhere to Eukaryotic cells and exclude pathogens

The adhesion of W. cibaria strains to the Caco-2 cell lines was almost 100 % effective for W21 and W25 strains (Table 6). The initial inoculum was 7-8 log CFU/mL and all the W. cibaria strains W21, W25, and W42 tested strains were able to adhere in the Caco-2 cell line, with lower adhesion proportion for W42. The adhesion to the intestinal cell leads to a hurdle against enteropathogenic bacteria, avoiding their adhesion to the intestinal cells and competing with them for nutrients [44,45]. Remarkably, the data from the exclusion assays revealed that only W. cibaria W21 was able to impede completely the adhesion of S. aureus MRSA S1 to the Caco-2 cells (Table 6), suggesting intraspecific differences between these strains. In the present work, only two pathogenic bacteria were tested in situ: S. aureus MRSA S1 and E. coli 184, but these strains have a broad spectrum as they are active against L. monocytogenes and Salmonella enterica [7]. Impeding pathogens to adhere to human cells may result from different mechanisms, including the production of antimicrobial substances by LAB, competition for binding sites, and further criteria [46]. Some pathogens have different levels of adhesion and invasion on the eukaryotic cells, and notably, some pathogens are more or less stable in mucus-producing intestinal cell models (HT-29-MTX) compared to non-mucusproducing cells (Caco-2) [47]. Such ability of resilience is directly related to the expression of specific proteins, pili, fimbriae, and flagella [48]. The W. cibaria strains like strain BYL4.2 are also able to inhibit fungi like Penicillium chrysogenum via the production of organic acids [49]. This antifungal activity is relevant in the food sector, as fungi can cause spoilage and deterioration of a variety of food products like dairy products, cereals, fruits and vegetables, and grains, decreasing their quality of food and shortening the shelf life, thereby causing considerable economic losses [50]. In the pharmaceutical sector, advantages are focused on food-derived chemopreventive and anti-tumor agents or formulations with fewer side effects than chemotherapy, and food-products like kimchi containing W. cibaria strains are under consideration for this purpose [51]. It should be noted that a strain of W. cibaria (CMS51) has been commercially used in Korea as an oral care probiotic for several years for its antiviral properties [52].

Conclusion

De novo sequencing in conjunction with the functional properties enabled us to provide insightful information on Weissella species. Indeed, W. cibaria W21, W25, and W42 strains are reported as safe according to the bioinformatic analyses, as these strains do not contain acquired antimicrobial resistance genes and MGE in their genomes, which could discard horizontal gene transfer. These strains harbor, nonetheless, in their genomes a CRISPR-Cas system which constitutes a defense system against infections caused by heterologous genes. W. cibaria W21, W25, and W42 do not contain any antibiotic resistance gene, except the vancomycin resistance gene; a phenotype confirmed experimentally. Such resistance is considered intrinsic for the Weissella genus and other LAB with GRAS status.

Only one intact prophage was detected in the genome of these strains, which can also help the bacteria against other prophage infections. Finally, no one of the known virulence factors were detected in these genomes and no in vitro hemolytic activity was evidenced. Further, no cytotoxicity and no inflammatory effects were detected for W. cibaria W21, W25, and W42 strains tested in this study.

Regarding the probiotic traits of the W. cibaria W21, this strain shows higher potential as it demonstrates a high exclusion ability of S. aureus MRSA S1 in the intestinal cells.

However, none of the three strains survived through the simulated gastrointestinal tract. To overcome this further studies regarding protective strategies to help the passage of these strains to the gastric system are needed and more studies with other enteropathogenic strains should be explored.

  TEIXEIRA, Camila Gonçalves, D.Sc., Thèse soutenue en cotutelle entre l'Université de Lille et Universidade Federal de Viçosa, octobre, 2022. Propriétés antimicrobiennes des souches de Weissella cibaria isolées de Campos Das Vertentes, Minas Gerais, Brésil et son utilisation potentielle en tant que producteurs de probiotiques et de bactériocines. Directeur: Antônio Fernandes de Carvalho et Djamel Drider. Co-advisors: Luis Augusto Nero et Anca Lucau-Danila. Le genre Weissella est composé de bactéries appartenant au groupe de bactéries lactiques, sont autochtones aux environnements laitiers et non laitiers et ont la capacité de produire divers composés d'intérêt pour l'industrie alimentaire. Ce travail a débuté par deux revues de littérature concernant l'utilisation des souches Weissella pour l'industrie alimentaire et pharmaceutique et le potentiel probiotique de ce genre. Un grand nombre d'études scientifiques rapportent et soutiennent l'utilisation des souches Weissella dans ce domaine en raison de leurs propriétés bénéfiques pour la santé. Du point de vue de la technologie alimentaire, certaines souches ont un potentiel de production d'oligosaccharides non digestibles, qui dépasse leur potentiel probiotique. Ce travail se poursuit par l'étude de la biodiversité des souches isolées dans les régions productrices de fromages artisanaux au Brésil. Pour cela, les analyses PFGE et Rep-PCR ont été réalisée et il a été observé que chaque région d'étude représente une niche écologique spécifique. Après avoir sélectionné les représentants de chaque groupe, le potentiel antimicrobien de ces souches a été analysé contre les agents pathogènes d'origine alimentaire. La plupart des isolats testés ont montré une bonne inhibition des pathogènes. Aussi, le potentiel technologique de treize souches de ces cultures a été évalué. Certaines souches ont montré des résultats positifs pour la plupart des tests effectués. Après cela, nous avons sélectionné trois souches avec une bonne activité inhibitrice, la première avec laquelle nous avons travaillé était Weissella cibaria W25, nous avons séquencé et assemblé le projet de séquence du génome. Ensuite, nous avons effectué une analyse génomique comparative complète avec W. cibaria 110, connu pour produire la bactériocine weissellicine 110, et quatre autres souches de W. cibaria non productrices de bactériocine. Les analyses ont montré que la souche W25 possède ses gènes de groupe de protéines uniques qui peuvent être liés aux gènes de bactériocines indiqués par AntiSmash confirmant la possibilité de produire deux bactériocines différentes. Sur la base de ces résultats, nous avons approfondi le génome des trois souches de W. cibaria (W21, W25 et W42). Ensuite, nous avons étudié la présence de gènes pour les métabolites secondaires dans le génome des souches et avons découvert qu'elles avaient une région commune dans le génome avec une bactériocine putative identifiée comme Bacteriocin_IIc. De plus, nous avons comparé les nucléotides et les acides aminés de cette bactériocine putative dans la plateforme NCBI et il n'y avait aucune similitude avec autre bactériocine décrite jusqu'à présent du genre Weissella. Enfin, nous analysons la sécurité d'utilisation de ces trois souches pour l'alimentation humaine et/ou animale et leur capacité à être utilisées comme probiotiques. Les trois souches n'ont pas de gènes acquis de résistance aux antimicrobiens ni d'éléments mobiles dans le génome. Les souches étaient aucune activité hémolytique et aucun facteur de virulence connu. Ils possèdent le système de défense CRISPR-Cas. De plus, ils n'ont présenté aucune capacité d'inflammation ou de cytotoxicité et les trois souches de Weissella n'étaient pas censées être des agents pathogènes humains. W21 démontre d'excellents résultats d'adhésion aux cellules intestinales et d'exclusion de S. aureus. Cependant, aucune des trois souches n'a survécu complètement à travers le tractus gastro-intestinal simulé, mais certaines méthodes de protection peuvent être étudiées plus avant. En conclusion, ce travail nous a permis de comprendre la diversité et la distribution des espèces de Weissella présentes dans les différentes régions productrices de fromages artisanaux au Brésil. Elle a également montré que certains d'entre eux ont un potentiel technologique et de sécurité favorable pour des utilisations futures dans l'industrie alimentaire ou comme culture probiotique. Resumo TEIXEIRA, Camila Gonçalves, D.Sc., Tese em cotutela entre l'Université de Lille e Universidade Federal de Viçosa, outubro de 2022. Propriedades Antimicrobianas de Estirpes de Weissella cibaria Isoladas dos Campos das Vertentes, Minas Gerais, Brasil e seu Potencial uso como Probióticos e Produtoras de Bacteriocinas. Orientadores: Antônio Fernandes de Carvalho e Djamel Drider. Co-orientadores: Luis Augusto Nero e Anca Lucau-Danila.

CHAPTER II:

  Biodiversity and technological features of Weissella isolates obtained from Brazilian artisanal cheese-producing regions This part aimed to identify the microbial biodiversity and evaluate the technological potential of Weissella spp. isolates obtained from different Brazilian regions that produce artisanal cheeses, such as Campo das Vertentes (Minas Gerais), Southern Pará, Guanambi (Bahia), and Marajó Island. The contents of this chapter are published as "Biodiversity and Technological Features of Weissella Isolates Obtained from Brazilian Artisanal Cheese-Producing Regions" in LWT -Food Science and Technology. CHAPTER III: Weissella cibaria W25: Comparative genomic analyses This part of the thesis aimed to announce and analyze the sequencing and annotation of the Weissella cibaria W25 genome and carry out a comprehensive comparative genomic analysis with W. cibaria 110, known to produce the weissellicin 110 bacteriocin, and four other non-bacteriocin-producing W. cibaria strains. The contents of this chapter are published as "Genomic Analyses of Weissella Cibaria W25, a Potential Bacteriocin-Producing Strain Isolated from Pasture in Campos Das Vertentes, Minas Gerais, Brazil." In Microorganisms.

  more precise phylogenetic techniques, such as the evaluation of rDNA (Fakruddin & Mannan, 2013, Gandra, Gandra, de Mello & da Godoi, 2008). In 1993, using the rDNA technique, Collins found that strains with very close phenotypic characteristics, previously classified as Leuconostoc, presented profiles of bands very different from others belonging to the same genus. This was discovered after the study of atypical Leuconostoc cultures present in fermented and dried sausages produced in Greece. These isolated microorganisms resembled the bacterium Leuconostoc sp. for producing D (-) lactic acid but differed in several biochemical tests. The name of this genus was proposed by Collins et al. (Collins, Samelis, Metaxopoulos & Wallbanks, 1993) in honor of the German microbiologist Norbert Weiss due to his many contributions in the field of research related to LAB. Collins et al. (1993) used genetic sequencing of the 16S rDNA to investigate the relationship between isolated bacteria and recognized strains of the genus Leuconostoc.

Figure 1 .

 1 Figure 1. Phylogenetic tree showing the proximity of the genus Weissella with other genera of lactic acid bacteria.

Figure 2 .

 2 Figure 2. Metabolic pathways used by bacteria of the genus Weissella.

  probiotic by[START_REF] Immerzeel | Extraction of water-soluble xylan from wheat bran and utilization of enzymatically produced xylooligosaccharides by Lactobacillus, Bifidobacterium and Weissella spp[END_REF], were studied concerning the use of xylooligosaccharides (XOS) as carbon source, since these are considered prebiotic. The study showed that strains absorbed XOS, both xylobiose, and xylotriose, and gave an increase in the production of lactic acid when xylan hydrolyzed were used. Not only but some stains can also use xylooligosaccharides (XOS) to produce short-chain fatty acids a feature only observed in Leuconostoc lactis and a few strains of the established probiotics Lactobacillus[START_REF] Månberger | Taxogenomic assessment and genomic characterisation of Weissella cibaria strain 92 able to metabolise oligosaccharides derived from dietary fibres[END_REF].Adesulu-Dahunsi et al.(2018) suggest that EPS produced by W. cibaria GA44 may be a commercial alternative for the food industry once it presents strong properties as an antioxidant when compared to commercial antioxidant ascorbic acid, especially scavenging of superoxide anions and hydroxyl radicals.

  cibaria MG1 was developed and the concentration of EPS (40 mg/L) ensured the high-water retention capacity and viscosity (0.57 mPa s) of the final product (Zaninni, Jeske, Lynch & Arendt, 2018).

Fig 1 . 2 . 2 . Biodiversity determination using molecular cluster analysis 2 . 2 . 1 .

 122221 Fig 1. Strains, biotope, and geographical origins and identification of Weissella spp. isolates obtained from Brazilian artisanal cheese-producing regions.

  et al. (2010) using a single universal primer (GTG)5 (5′-GTGGTGGTGGTGGTG-3′ ). PCR reactions contained 12.5 μL of Go Taq Green Master Mix 2x (Promega), 0.5 μL of the primer (100 mol/L), 100 ng DNA, and ultra-pure water (Promega) for a final volume of 25 μL. PCR amplification was carried out in a thermal cycler, with the following conditions: 95 °C for 5 min as an initial step, 95 °C for 30 s, annealing at 40 °C for 30 s, then 65 °C for 8 min for the next 30 cycles, and 65 °C for 16 min to conclude the amplification.

30 °C for 10

 10 min: the formation of a red ring at the top of the tubes indicated diacetyl production[START_REF] Franciosi | Biodiversity and technological potential of wild lactic acid bacteria from raw cows' milk[END_REF]. Lactococcus lactis subsp. lactis bv. diacetylactis ATCC 13675 was used as a positive control. Milk coagulation patterns were assessed by inoculating 0.1 mL aliquots of the cell suspension of the cultures into 10 mL of sterile reconstructed skim milk (10% w/v, Nestlé), followed by incubation at 30 °C for 24 h. Based on the formed clot characteristics, the coagulation patterns were described using empirical analysis and classified as: uniform, uniform with the presence of serum, uniform and fragile (appearance), broken with the presence of serum, and absence of clot (Fusieger, Martins, de Freitas, Nero, & de Carvalho, 2020). L. lactis subsp. lactis bv. diacetylactis Q1C5 was used as a positive control. A screening assay was conducted to identify the presumed extracellular proteolytic activity of the isolates as described by Franciosi et al. (2009). Two μL aliquots of bacterial cultures were spotted on the surface of a Plate Count Agar (PCA, HiMedia, Mumbai, MH, India) added to skim milk (10% w/v, Nestlé), and the cultures were incubated at 30 °C for 4 days. Presumed proteolytic activity was indicated by a clear zone around the colonies and Pseudomonas fluorescens 07A (Alves, Salgado, Eller, Vidigal, & Carvalho, 2016) was used as a positive control. EPS production from lactose present in milk was evaluated according to the protocol described by Dal Bello et al. (2012). 0.1 mL aliquots of selected cell suspension of the cultures were inoculated into 10 mL of sterile reconstituted skim milk (10% v/v, Nestlé) and incubated at 30 °C for 48 h. Then, the EPS production was determined by the quality and degree of stringiness: isolates were characterized as EPS producers when the coagulated cultures could be tied to a string with an inoculating loop. Acidifying activity was assessed by adding 100 μL of the selected cell suspension of the cultures to skim milk (10% w/v, Nestlé), followed by incubation at 30 °C for 24 h pH values were measured at the time of inoculation (T0), then after 6 (T6), 12 (T12), and 24 h (T24) using a digital pHmeter (Hanna Instruments, São Paulo, SP, Brazil). Also, titratable acidity was expressed as lactic acid[START_REF] Dal Bello | Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in Cottage cheese[END_REF]. Results have been shown as means of the triplicates and repetitions.

Fig 2 .

 2 Fig 2. Dendrograms based on UPGMA clustering of SmaI PFGE profile of Weissella spp. isolates obtained from Brazilian artisanal cheese-producing regions.

Fig 3 .

 3 Fig 3. Dendrograms generated after cluster analysis of rep-PCR fingerprints of Weissella spp. obtained from Brazilian artisanal cheese-producing regions.

  [START_REF] Freitas | Biodiversity of dairy Propionibacterium isolated from dairy farms in Minas Gerais, Brazil[END_REF] compared PFGE to random amplified polymorphic DNA (RAPD), another molecular technique, and concluded that PFGE provides better heterogeneity resolution between strains of the same species. Thus, in this study, the PFGE technique was chosen to screen select samples from each cluster for antimicrobial activity assays.The three Weissella species studied were present in almost all types of original samples including milk, cheese, pasture, and utensils. This demonstrates the high diversity of strains found in the regions studied. Fuka et al. (2013) and others have also identified Weissella strains in dairy environments and isolated W. paramesenteroides and W. hellenica in Croatian raw sheep's milk cheese. Masoud et al. (2012) have also isolated the same Weissella species in raw milk and raw milk cheeses. The regions studied represent specific ecological niches as isolated strains from different regions did not present similar PFGE restriction profiles (100% similarity). Freitas et al. (2015) in the research on Propionibacterium biodiversity obtained the same results with each of the farms they studied in Campo das Vertentes, representing a different bacterial ecological niche.

(

  [START_REF] Bounaix | Characterization of dextran-producing Weissella strains isolated from sourdoughs and evidence of constitutive dextransucrase expression[END_REF] Lynch et al., 2014). According toLynch et al. (2015), W. cibaria MG1 possesses all the necessary genes for the phosphoketolase pathway and metabolizes galactose via the Leloir pathway. Milk coagulation is important in the production of fermented products such as yogurt and cheeses. Coagulation occurs when milk pH is lowered by LAB acid production. When the caseins' isoelectric point is reached, this adds to the effects of the expelled serum to form yogurt gels and cheese curds[START_REF] Robinson | Dairy microbiology handbook[END_REF][START_REF] Tamime | Tamime and Robinson's yoghurt -science and technology[END_REF].

Figure 1 .

 1 Figure 1. Tree inferred with FastME 2.1.6.1[28] from GBDP distances calculated from 16S rDNA gene sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60 % from 100 replications, with an average branch support of 67.6 %. The tree was rooted at the midpoint [29].

Figure 2 .

 2 Figure 2. Tree inferred with FastME 2.1.6.1 [28] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers

  [START_REF] Li | Comparative genomic analysis of bacteriocin-producing Weissella cibaria 110[END_REF][25] also compared W. cibaria 110 with four other strains (W. cibaria MG1, W. cibaria AB3b, W. cibaria ff3PR, and W. cibaria KACC11862) and the comparative genomic analysis also showed the presence of unique genes that encoded the novel bacteriocin weissellicin 110 and defense system.

Figure 3 .

 3 Figure 3. Venn diagram showing the protein-coding genes or pseudogenes in each sequenced of W. cibaria W25, W. cibaria 110, and W. cibaria JMC 12495. Overlapped regions represent shared proteins and the numbers in the non-overlapped regions indicate the unique protein-coding genes or pseudogenes.

Figure 4 .

 4 Figure 4. Venn diagrams showing the protein-coding genes or pseudogenes of six W. cibaria strains. Overlapped regions represent shared proteins and the numbers in the non-overlapped regions indicate the unique protein-coding genes or pseudogenes. A: normal Venn diagram; B: Venn diagram highlighting the cluster between W. cibaria W25 and W. cibaria 110; C: Venn diagram highlighting the clusters between W. cibaria W25 and the non-bacteriocin-producing strains.

Figure 5 .

 5 Figure 5. Comparative genome map generated using CGView server, showing a full circular map obtained using BLAST (A) and an expanded view of the weissellicin 110 region (B). The rings in (A) and (B) green, purple, yellow, red, and brown indicate the W. cibaria strains W25, JCM 12495, MG1, ff3PR, and AB3b, subsequently, that were compared with the W. cibaria 110. The blue ring in (B) indicates the coding sequences (CDS) of the genome from W. cibaria 110.

  bacteriocins offer interesting prospects (Hernández-González et al., 2021). Bacteriocins are ribosomally synthesized antimicrobial peptides/proteins produced by Gram-positive, Gram-negative, and Archaea (Hammami et al., 2010; Nes, 2011) with the aim to defeat their congeners living in the ecological niches (Soltani et al., 2021).

  food and pharmaceutical sectors[START_REF] Field | Developing bacteriocins of lactic acid bacteria into next generation biopreservatives[END_REF]. In addition, bacteriocins are non-toxic to cells from the Eukarya domain and can be used against many food spoilage microbes and pathogenic bacteria(Belguesmia et al., 2011; Field et al., 2018). Additionally, bacteriocins are expected to play a role in editing-microbiomes (Heilbronner et al., 2021, Drider et al., 2021) based on their mode of action, which include different natural targets (Heilbronner et al., 2021) and even liposomes (Naghmouchi et al., 2007).

Figure 1 .

 1 Figure 1. Tree inferred with FastME 2.1.6.1 (Lefort et al., 2015) from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60 % from 100 replications,

  detected one core biosynthetic gene named biosynthetic (rule-based-clusters) hydroxymethylglutaryl-CoA synthase, and one additional biosynthetic gene identified as biosynthetic-additional (rule-based-clusters) Flavoprotein, one transport-related gene identified as transport SMCOG1184: major facilitator transporter and 29 putative genes.

Figure 2 .

 2 Figure 2. In silico prediction of promoters (-10 and -35) and terminator region for the bacteriocin coding gene. Figure adapted from AntiSMASH results.

Figure 3 .

 3 Figure 3. Antimicrobial activity of Weissella strains supernatants (Non-treated, Neutralized, and Neutralized, and treated with proteinase K) against the targets E. coli, K. rhizophila, L. innocua, and S. enterica. The analysis was in duplicate.

  Multiple cytokines in culture supernatants of the U937 cell line were detected with a customized Milliplex Map kit (Human High Sensitivity T Cell Magnetic Bead Panel, Millipore, Billerica, MA), following the manufacturer's instructions. Briefly, antibody-immobilized beads for detection of interleukin -1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, tumor necrosis factor-alpha (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-γ (IFNγ) were incubated with 5-times-diluted culture supernatants of U937. The beads were subsequently treated with detection antibodies as well as PEconjugated streptavidin and the quantification was carried out using the Luminex® 100/200 (Luminex Corporation, Austin, TX, USA) system and the Luminex xPONENT® for LX100/200 software. The cytokine IL-8 was quantified using a human IL-8/CXCL8

Figure 2 .

 2 Figure 2. Effect of W. cibaria strains on cytotoxicity of 80 % of Caco2 + 20 % of HT-29 cells (A); and U937 cells (B).

  , W. thailandensis (Tanasupawat, Shida, Okada, & Komagata, 2000), W. soli (Magnusson, Jonsson, Schnurer & Roos, 2002), W. cibaria (Björkroth, K. J. et al., 2002), W. koreensis (Lee, et al., 2002), W. ghanensis (De Bruyne, Camu, Lefebvre, De Vuyst & Vandamme, 2008), W. beninenses (Padonou, et al. 2010), W. fabaria (De Bruyne, Camu, De Vuyst, & Vandamme, 2010), W. ceti (Vela et al., 2011), W. fabalis (Snauwaert, Papalexandratou, De Vuyst, & Vandamme, 2013), W. oryzae

Table 1 .

 1 Occurrence of Weissella species in different ecosystems.

	Species	Habitat or source	Reference
		Orange, pineapple, banana	Endo, et al., 2009
		Tomato	Di Cagno, et al., 2009
	W. cibaria	Wheat flour	Alfonzo, et al., 2013
		Blackberry, papaya	Di Cagno, Minervini, Rizzello, De Angelis & Gobbetti, 2011
	W. confuse	Rhizosphere of the olive trees and surrounding soil	Fhoula et al., 2013
		Red and yellow raw pepper	Di Cagno, et al., 2009
	W. halotolerans	Rhizosphere of the olive trees and surrounding soil	Alfonzo, et al., 2013
		Fermented sausage	Tenea & Lara, 2019
	W. hellenica	Vegetable forage crops Croatian cheese fermented from raw milk	Tohno, Uegaki & Cai, 2012 Kobayashi, Fuka, et al., 2013	Nomura,
	W. kandleri	Desert plants	Holzapfel & Van Wyk, 1982
	W. uvarum	Wine grapes	Nisiotou, et al., 2014
	W. sagaensis	Chinese yogurt	Li, et al., 2020
	W.	Raw milk cheeses	Masoud, et al., 2012
	paramesenteroides	Fermented sausage	Juárez-Castelán, et al., 2019
		Source: Own authorship (2021).

Table 2 .

 2 Bacteriocins produced by Weissella strains.

	Bacteriocin	Producing species	Reference
	Weissellicin 110	W. cibaria 110	Srionnual, Yanagida, Lin, Hsiao, & Chen, 2007
	Weissellin A	W. paramesenteroides DX	Di Cagno, Minervini, Rizzello, De Angelis, M. & Gobbetti, 2011
	Weissellicin L	W. hellenica 4-7	Leong, K. H. et al., 2013
	Weissellicin D	W. hellenica D1501	Chen, et al., 2014
	Weissellicin M	W. hellenica QU 13	Masuda, et al., 2012
	Weissellicin Y	W. hellenica QU 13	Kariyawasam, et al., 2019
		Source: Own authorship (2021).
	Kariyawasam et al. (2019), used the strain W. cibaria D30 as an adjunct culture in
	cottage cheese manufacture and the strain increases the prevention of the growth of Listeria
	monocytogenes and ensures the microbial safety of ready-to-eat soft cheeses. Nam, Ha,
	Bae & Lee (2014), showed that W. confusa has antagonistic activity against the pathogen
	Helicobacter pylori a gram-negative microorganism that causes gastritis and gastric
	carcinoma, infects through the intake of food, and attaches to gastric and duodenal mucous
	membranes. W. confusa strain PL9001 inhibited the binding of H. pylori to human gastric-
	cell line MKN-45 cells by more than 90%. The results suggest that Weissella strains can be
	used as probiotics added in fermented milk, for example, to fight H. pylori. Besides that, W.
	confusa DD-A7 has antagonistic activity against the multidrug-resistant Escherichia coli
	which is resistant to almost all antibiotics used for its treatment. The strain W. confuse DD-
	A7 was capable to trigger an oxidative attack and limiting the growth of the pathogen (Dey,
	Khan & Kang, 2019).

Table 3 .

 3 Exopolysaccharides produced by Weissella strains.

	Species		Product		Reference
	Weissella cibaria RBA12	Dextran		Baruah, Maina, Katina, Juvonen & Goyal, 2017
	W. confusa		Dextran		Kajala I. et al., 2016
	Weissella KR780676	confusa	Linear exopolysaccharide galactan	Devi, Kavitake & Shetty, 2016, Kavitake, Devi & Shetty, 2016
			Dextran,	fructan	from
	W. confusa and W. cibaria	sucrose, ropy capsular polysaccharide, levan, and	Malang, Maina, Schwab, Tenkanen & Lacroix, 2015
			inulin
	W. confusa XG-3		Dextran		Zhao, et al., 2021
					Lakra,, Ramatchandirane,
	W. cibaria MD2		Fructan		Kumar, Suchiang & Arul,
					2021
		Source: Own authorship (2021).

Table 1 .

 1 Weissella species with antimicrobial effects and the respective pathogens that each one inhibits.

	Species	Pathogen	Reference
	W. viridescens	L. monocytogenes	Stratakos et al. [60]
	W. paramesenteroides	Giardia lamblia	Fonseca et al. [56]
		E. coli	Pabari et al. [62]
		S. aureus
	W. cibaria	Porphyromonas gingivalis	Do et al. [57]
		Prevotella intermedia
		Fusobacterium nucleatum
		S. enterica subsp. enterica	Tenea et al. [108]
		Escherichia coli
	W. confusa	S. enterica Typhi	Pelyuntha et al. [109]
		E. coli	Dey et al. [29]
		Candida albicans	Rosca et al. [80]
		S. enterica	Lakra et al. [61]
		S. enterica Typhi
		L. monocytogenes
		S. aureus
		L. monocytogenes	Dey et al., [27]
		S. aureus
		S. enterica Typhimurium
		B. cereus
		E. coli

Table 2 .

 2 In vitro and in vivo evaluation of Weissella species as a probiotic candidate.

  Although Brazil, the USA, and European Union countries do not have specific legislation for the production of new types of yeast, they do have recommendations. In the USA, for example, some cultures are classified as safe and suitable for human consumption while others are classified as GRAS and must apply for approval from the FDA (Food and Drug

	Administration) [104].
	In 2011, some Weissella species, including W. confusa, were added to the
	International Dairy Federation (IDF) inventory, a list of microorganisms allowed in food

Weissella strains have only been associated with "weissellosis," a disease that affects rainbow trout (Oncorhynchus mykiss). The disease leads to septicemia in the fish and can cause important economic losses on fish farms. "Weissellosis" is caused by a specific species, W. ceti

[START_REF] Figueiredo | Caracteristicas do leite cru e do queijo Minas artesanal do Serro em diferentes meses[END_REF][START_REF] Ortega | Isolation characterization, virulence potential of Weissella ceti responsible for weissellosis outbreak in rainbow trout (Oncorhynchus mykiss) cultured in Mexico[END_REF][100]

. Because certain Weissella strains are resistant to antibiotics such as vancomycin, teicoplanin, ceftazidime, and sulfamethoxazole, human infections remain a possibility. These would primarily affect immunocompromised patients [3, 101]. It should be noted, however, that even generally recognized probiotic strains may cause health complications. Lacticaseibacillus rhamnosus GG and L. helveticus are two examples of this [102, 103]. Even though there are few cases of this genus causing disease in humans or animals, recent studies have evaluated the safety of Weissella use in foods. Any strain considered for use in fermented foods must be approved by government regulatory commissions. fermentation processes. The German Committee for Biological Agents has also placed W. confusa in Risk Group 1 for microorganisms that are unlikely to cause human diseases [105]. W. confusa's important technological potential as a source of probiotics and other health benefits has led to two studies that address the microorganism's safety [105, 106].

  Weissella spp. are a LAB known for lactic acid The genus Weissella is made up of 25 validated species, most of which have already been identified in fermented foods such as vegetables, sausages, and dairy products, Weissella spp. isolates obtained from different Brazilian regions that produce artisanal cheeses. Weissella spp. isolates were obtained from the InovaLeite (Laboratory of Milk and Dairy Products, Universidade Federal de Viçosa) bacterial culture collection and included in this study. The isolates were taken from samples from four different Brazilian regions that produce artisanal cheeses (Supplementary Table1). The isolates have been previously identified by 16S rRNA sequencing(Felske, Rheims,

	2. Material and methods
	production acid through carbohydrate fermentation and for its phylogenetic position
	characterized as Gram-positive, catalase-negative, non-spore-forming, with coccoid 2.1. Microorganisms and culture conditions
	morphology or short bacilli (Bj¨orkroth, Dicks, & Holzapfel, 2009; Collins, Samelis, Fifty-seven (n = 57)
	Metaxopoulos, & Wallbanks, 1993).

particularly raw milk cheeses (Jans, Bugnard, Njage, Lacroix, & Meile, 2012; Masoud et al., 2012; Pereira, San Rom˜ao, Lolkema, & Barreto Crespo, 2009; Wouters, Ayad, Hugenholtz, & Smit, 2002). In Brazil, Weissella spp. were previously been isolated from artisanal cheeses produced in Par´a, Minas Gerais, and Paraíba states (Martins et al., 2018; Medeiros et al., 2016; Perin, Savo Sardaro, Nero, Neviani, & Gatti, 2017). Weissella species present many positive attributes for the food industry. Indeed, its wide array of characteristics has been attributed to the strains of this genus. Studies have shown that Weissella spp. Strains can be used as starter cultures and to produce antimicrobial substances and exopolysaccharides (Ayeni et al., 2011; Choi, Kim, Hwang, Kim, & Yoon, 2012; Srionnual, Yanagida, Lin, Hsiao, & Chen, 2007; Zannini et al., 2013). Specifically, W. paramesenteroides and W. hellenica, usually found in dairy products, have been widely studied for their antimicrobial potential, since certain strains have already been characterized as bacteriocinogenic (Chen et al., 2014; Leong et al., 2013; Masuda et al., 2012; Papagianni & Papamichael, 2011). W. hellenica was detected in dairy products such as raw milk Croatian cheese, while W. paramesenteroides was previously isolated from raw milk artisanal cheeses from different countries, thus demonstrating the relevance of this genus for dairy production (Coppola et al., 2006; Fuka et al., 2013; Gerasi, Litopoulou-Tzanetaki, & Tzanetakis, 2003; Masuda et al., 2012). Although the phenotypic characteristics of Weissella spp. have been studied and Weissella spp. strains have been isolated and identified in artisanal cheeses, there is little information on this genus found in Brazilian artisanal cheeses. Weissella spp. function in such products and this genus biodiversity from region to region in Brazil remain unknown. Thus, the present study aimed to identify microbial biodiversity and evaluate the technological potential of Wolterink, Stackebrandt, & Akkermans, 1997) as W. cibaria (n = 38), W. paramesenteroides

(n = 11) and W. confusa (n = 8) (Fig.

1

). The isolates obtained were stored at -60 °C in de Man Rogosa and Sharpe (MRS) broth (Oxoid Ltd., Basingstoke, England) and supplemented with 30% (v/v) glycerol (Synth, São Paulo, SP, Brazil). Prior to analysis, stock cultures (1% v/v) were transferred to MRS broth (Oxoid) and incubated at 30 °C for 14 h.

  we had observed for the PFGE results, rep-PCR allowed the isolates with identical genetic profiles from different samples to be identified. As examples, see: samples 25, 29, 35, 39, and 54, from the soil, pasture, and cow milk (W. cibaria); 20, 22, and 37, from soil and pasture (W. cibaria); 24 and 38, from pasture and soil (W. cibaria); 26, 43 and 45, from soil and pasture (W. cibaria), and 2, 5 and 7, from fermented curd, cow milk, and utensils (W.

Table 2

 2

	). Two demonstrated homogeneous

homogeneous clots, but only after 48 h of incubation. The other isolates did not coagulate milk.

Table 1 .

 1 Inhibitory potential of the cell-free supernatant of Weissella spp. isolates against target strains.

	Codes Identification	L. monocytogenes ATCC 15313	S. aureus ATCC 6538	S. Typhimurium ATCC 14028	E. coli ATCC 11229	pH
	W. confusa	89.2	53.0	80.6	67.5	4.12
	W. confusa	88.1	37.0	77.3	71.1	4.35
	W. confusa	88.7	86.4	78.1	86.1	4.03
	W. confusa	85.3	85.2	76.6	87.9	3.95
	W. paramesenteroides	62.4	25.0	50.9	29.2	4.03
	W. paramesenteroides	89.4	85.1	81.9	84.1	4.04
	W. paramesenteroides	90.3	89.2	83.0	85.6	3.94
	W. paramesenteroides	87.1	79.8	77.5	83.6	4.04
	W. paramesenteroides	88.0	86.6	79.5	87.3	4.01
	W. paramesenteroides	60.1	45.0	26.7	49.5	4.74
	W. paramesenteroides	31.8	36.8	43.4	46.3	4.36
	W. paramesenteroides	88.0	84.0	79.5	85.4	4.00
	W. paramesenteroides	88.1	68.8	78.1	65.6	4.17
	W. paramesenteroides	88.6	88.1	82.0	86.1	3.94
	W. cibaria	98.5	87.1	76.8	82.8	4.79
	W. cibaria	39.4	39.9	15.2	63.4	4.49
	W. cibaria	44.0	22.2	49.9	18.6	4.41
	W. cibaria	48.7	28.9	26.3	40.2	4.67
	W. cibaria	26.6	37.0	4.2	21.8	4.71
	W. cibaria	86.8	65.9	74.7	70.0	4.20
	W. cibaria	89.1	88.5	80.3	86.2	3.92
	W. cibaria	85.2	27.4	0.7	20.6	4.72
	W. cibaria	86.7	63.8	78.4	74.0	4.01
	W. cibaria	11.4	4.0	8.5	2.2	4.46
	W. cibaria	78.5	30.5	70.7	28.0	4.75
	W. cibaria	87.9	48.4	77.2	56.6	4.39

Table 2 .

 2 Technological features of Weissella spp. isolates obtained from Brazilian artisanal cheese-producing regions Based on the profiles obtained by PFGE and rep-PCR (Figs.2 and 3), the studied isolates presented high variability; the two techniques grouped the isolates differently, which is to be expected because the approaches used are totally different.Nisiotou, Dourou,

							Acidifying activity
	Codes Identification	Diacetyl	Coagulation		Proteolysis	EPS	pH		% lactic acid
							T0	T6	T12 T24	T0	T6	T12 T24
	W. confusa	-	absence clot	of	+	-	6.85 6.50 6.44 6.15	0.14 0.15 0.13 0.19
	W. confusa	-	absence clot	of	+	-	6.85 6.59 6.26 6.15	0.14 0.11 0.16 0.19
	W. paramesenteroides -	absence clot	of	+	-	6.85 6.62 6.40 6.33	0.14 0.13 0.11 0.20
	W. paramesenteroides +	absence clot	of	+	-	6.85 6.60 6.42 5.46	0.14 0.13 0.13 0.23
	W. paramesenteroides +	absence clot	of	+	-	6.85 6.63 6.45 6.27	0.14 0.12 0.14 0.18
	W. paramesenteroides +	absence clot	of	+	-	6.85 6.62 6.43 6.36	0.14 0.13 0.17 0.16
	W. paramesenteroides +	uniform		+	-	6.85 5.41 4.65 4.60	0.14 0.37 0.57 0.77
	W. paramesenteroides -	uniform		+	-	6.85 6.53 6.14 5.84	0.14 0.12 0.20 0.27
	W. paramesenteroides -	absence clot	of	+	-	6.85 6.69 6.53 6.37	0.14 0.12 0.13 0.17
	W. paramesenteroides -	absence clot	of	+	-	6.85 6.57 6.40 6.24	0.14 0.13 0.15 0.18
	W. cibaria	+	uniform		+	-	6.85 6.30 5.78 5.35	0.14 0.17 0.29 0.38
	W. cibaria	+	uniform		-	-	6.85 6.47 5.58 5.00	0.14 0.15 0.35 0.60
	W. cibaria	-	absence clot	of	-	-	6.85 6.77 6.73 6.66	0.14 0.12 0.13 0.12
	Results: +, positive; -, negative

Table 1 )

 1 [START_REF] Martins | Novel sequence types of Lactococcus lactis subsp. lactis obtained from Brazilian dairy production environments[END_REF] used PFGE to compare 23 isolates of L. lactis subsp. lactis isolated from the dairy environment in four different regions in Brazil and showed that isolates presented identical profiles by PFGE. Here, the isolates belonged to the same region and similar samples.The high susceptibility levels of L. monocytogenes to Weissella supernatants (were justified because antimicrobial activity among related microorganisms is common in Gram-positive bacteria. E. coli was used as a Gram-negative target microorganism and it was also shown to be highly inhibited by supernatants. Ortolani, Yamazi, Moraes, Viçosa, and Nero (2010) also reported LAB antagonist activity for foodborne pathogens L.

	However, W. paramesenteroides DX is able to produce the bacteriocin weissellin A when
	grown in M17 + 2% glucose broth at 30 °C for 60 h, according to Papagianni and
	Papamichael (2011). Srionnual et al. (2007) also incubated W. cibaria 110 in MRS broth at
	30 °C and obtained bacteriocin weissellicin 110.

monocytogenes and S. aureus when they studied isolates taken from raw milk and soft cheese. When Espeche, Otero, Sesma, and Nader-Macias (2009) isolated 6 strains of W. paramesenteroides from bovine milk samples, one sample also showed antimicrobial activity for Streptococcus dysgalactiae subsp. dysgalactiae ATCC 27957 and pathogenic E. coli. It should be noted that only cells were removed from Weissella sp. supernatants in this test, and the cells were obtained under optimum growth conditions, which does not prevent the production of more than one antimicrobial compound. LAB can produce a variety of antimicrobial compounds including hydrogen peroxide, organic acids (lactic acid, acetic acid, formic acid, for example), carbon dioxide, bacteriocins, ethanol, and diacetyl

[START_REF] Leroy | Lactic acid bacteria as functional starter cultures for the food fermentation industry[END_REF]

. The bacteriocin production test revealed that the isolates did not demonstrate any production capacity under the growth conditions (data not shown).

Table 1 .

 1 Comparison of the genomic feature of W. cibaria W25 with a bacteriocin-producing W. cibaria strains 110, and four other non-bacteriocin-producing strains.

	Genome feature	W25	110	AB3b	ff3PR	JCM 12495 T	MG1
	Accession a JAFNKE000 000000	LRRC00 000000	JWHT000 00000	JWHV0000 0000	BJEF00000 000	JWHU000 00000
	Reference This study	[25]	[26]	[26]	[27]	[26]
	Contigs	41	18	88	60	25	44
	Size (pb)	2,412,435	2,347,04 9	2,465,158 2,357,128 2,323,953 2,436,232
	GC content (%) b	45.04	44.9	44.7	44.9	45.1	44.7
	CDS	2,190	2,209	2,348	2,228	2,124	2,284
	tRNA	84	76	67	70	77	62
	rRNA	15	5	7	5	9	4
	tmRNA	1	1	1	1	1	1
	Repeat region	1	1	0	0	0	0

a GenBank accession number; b using RAST program.

  , W. paramesenteroides and W. hellenica (Chen et al., 2014; Di Cagno et al., 2011; Kariyawasam et al., 2019; Leong et al., 2013; Masuda et al., 2012; Srionnual et al., 2007).

[START_REF] Venegas-Ortega | Production of Bioactive Peptides from Lactic Acid Bacteria: A Sustainable Approach for Healthier Foods[END_REF]

.

Within the LAB group, strains of the Weissella genus are known to produce bacteriocins such as weissellicin 110, MBF, L, D, M, Y, and weissellicin A from strains of W. cibaria

Table 1 .

 1 Genome assembly and annotation statistics for W. cibaria W21, W25, and W42 strains. Results according to Teixeira et al.[19]; a The total number of uncalled bases in the assembly.

	Parameters	W25 1	Strains W21	W42
	Contigs	41	50	42
	Total length (bp)	2,412,435	2,461,013	2,455,505
	Largest contig	331,445	290,433	290,388
	N50	202,649	141,851	159,842
	N a	40	434	424
	G+C content (%)	45.04	44.96	44.95
	CDS	2,190	2,244	2,237
	rRNA	15	19	18
	tRNA	84	87	86
	tmRNA	1	1	1
	Repeat region	1	1	1
	1

  TOF analysis is utilized for the identification and confirmation of the LAB bacteria genus and species based on its recognized robustness and appropriateness for LAB (Abdelkader et al., 2021; Nacef et al., 2017) including those belonging to the Weissella genus (Abdelkader et al., 2021; Kim et al., 2021; Ogunremi et al., 2022). Besides that, it has been proven to be a fast method of identification of bacteria at the species and genus level and can be utilized for routine analysis due to its effectiveness (Nacef et al., 2017; Zidour et al., 2017). This technique has been already used to identify Weissella genus and species (Abdelkader et al., 2021; Kim et al., 2021; Ogunremi et al., 2022).

	al., 2014;
	Larsen et al., 2014) and the MALDI-TOF are in agreement with the molecular identification
	based on the 16S rRNA/DNA sequencing realized by Teixeira et al. (Teixeira et al., 2021).
	The MALDI-

  Weissella genus such as Weissellicin 110, MBF, L, D, M, Y, and Weissellin A from strains of W. cibaria, W. paramesenteroides, and W. hellenica (Chen et al., 2014; Di Cagno et al., 2011; Kariyawasam et al., 2019; Leong et al., 2013; Masuda et al., 2012; Srionnual et al., 2007).

  Cell supernatants were collected on ice and centrifuged (1,500 rpm 5 min) to eliminate cell debris. The supernatants were aliquoted and stored at -80°C until further analysis. Weissella strains samples were prepared as described above, resuspended in not supplemented RPMI, and added to each well in 1:10 MOI (multiplicity of infection). The

	Pouille et al. [23]. The cell line U937 was cultured in Roswell Park Memorial Institute 1640 Medium (RPMI, Gibco, Thermo Fisher Scientific) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM glutamine in a humidified 5 % CO2 atmosphere at 37 °C. The culture medium was replaced regularly and when the cells reached sub-confluence (80-90 %), they were sub-passaged. For the differentiation of the cell line in macrophages, U937 cells were seeded at approximately 1 × 10 inflammatory control is U937 differentiated with LPS (50 µg/mL) and the anti-inflammatory control is U937 differentiated with LPS (50 µg/mL) and dexamethasone (20 µM).

5

cells/well in 96-well plates with 60 ng/mL of phorbol-12-myristate-13-acetate (PMA, Thermo Fisher Scientific) for 48 h. The adherent cells were washed with PBS and incubated for 2 h with LPS (10 µg/mL; LPS from E. coli O26:B6, Millipore Corporation) and Weissella strains.

Table 1 .

 1 CRISPR and Cas genes founded in genomes of the 3 Weissella cibaria strains isolated from Campos das Vertentes, Minas Gerais, Brazil.

	CRISPR region

Table 2 .

 2 Antibiotic resistance genes identified in Weissella cibaria strains using CARD (Comprehensive Antibiotic Resistance Database) webserver.

	Strain	RGI Criteria	ARO Term	Detection Criteria	AMR Gene Family	Drug Class	Resistance Mechanism	% Identity of Matching Region	% Length of Reference Sequence
	W21	Strict	vanY gene in vanB cluster	Protein homolog model	vanY, gene cluster Glycopeptide resistance	Glycopeptide antibiotic	alteration Antibiotic Target	34.95	97.39
	W25 W42	Strict	vanT gene in the vanG cluster	protein homolog model	Glycopeptide resistance gene cluster, Van T	Glycopeptide antibiotic	Antibiotic alteration target	33.33	69.38

Table 3 .

 3 Results from in vitro antibiotic resistance test.

	Code			Antibiotics spectrum (mm)
			Amoxicillin
		Ampicillin	+ clavulanic	Imipenem ciprofloxacin Vancomycin Erythromycin Clindamycin
			acid
	W21	29	29	35	24	0	24	24
	W25	31	31	33	20	0	27	26
	W42	28	32	32	22	0	27	26
	Results are expressed in mm of inhibition halo from antibiotics.
	Interpretation according to CLSI breakpoints [18].

Table 4 .

 4 Prophage regions identified in Weissella cibaria strains genome using the PHASTER webserver.

	Strain Region	Length (kb)	Completeness*	Score	GC % Most Common Phage
	W21	1 2	17.9 32	Incomplete Intact	20 110	41.52 42.64	PHAGE_Lactoc_PLgT_ 1_NC_031016(3) PHAGE_Entero_phiFL 1A_NC_013646(14)
	W42	1 2	17.9 35.8	Incomplete Intact	20 110	41.51 42.30	PHAGE_Lactoc_PLgT_ 1_NC_031016(3) PHAGE_Entero_phiFL 1A_NC_013646(14)
	W25	1 2	17.9 35.8	Incomplete Intact	20 110	41.54 42.30	PHAGE_Lactoc_PLgT_ 1_NC_031016(3) PHAGE_Entero_phiFL 1A_NC_013646(14)

*Intact (score > 90); Questionable (score 70-90); Incomplete (score < 70)

Table 5 .

 5 Survival of Weissella cibaria strains under conditions simulating gastric digestion.

	Strains				Time			Survival rate (%) of time 1.5h
			0h		1.5h		4.5h
		pH	log10 (CFU/mL)	pH	log10 (CFU/mL)	pH	log10 (CFU/mL)
	W25	3.5	10.04	3.5	9.68	7	ND	96.4
	W42	3.5	9.80	3.5	9.37	7	ND	95.6
	W21	3.5	9.80	3.5	9.60	7	ND	98.0

Table 6 .

 6 Adhesion of Weissella cibaria strains to Caco-2 cells and exclusion of pathogenic bacteria by W. cibaria strains.

		Adhesion	Exclusion
		log10(CFU/mL)	log10(CFU/mL)
	Strains	S. aureus MRSA SA1	E. coli 184
	Control		6.53228176	6.46542394
	W25	7.335792102	6.6515239	6.49991994
	W42	6.33243846	6.85370046	6.42275553
	W21	7.33243846	ND	6.44388297
	ND: not detected by technique limit

Acknowledgments

We are thankful for the financial support provided by the Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnólogico (CNPq, Brasília, DF, Brazil), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Belo Horizonte, MG, Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, DF, Brazil, Financial code 001).

Acknowledgments

We are thankful for the financial support provided by the Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, DF, Brazil), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Belo Horizonte, MG, Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, DF, Brazil, Financial code 001).

Statements and Declarations

Funding: This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, DF, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, DF, Brazil), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Belo Horizonte, MG, Brazil). Research at Lille University is supported by LAI-SAMBA grant accorded by Lille university 2021-2026.

Annex 4. License to reuse the research article (ref. chapter: 3).

CHAPTER I: Literature Review

PART B: Weissella: An Emerging Bacterium with Promising Health Benefits

The content of this part has been published in: Table 3. Weissella species and its EPS products.

Species EPS produced References

W. confusa EPS Benhouna et al. [28] Dextran Heperkan et al. [41] Rosca et al. [START_REF] Rosca | Biosynthesis of dextran by Weissella confusa and its in vitro functional characteristics[END_REF] Tang et al. [START_REF] Tang | Hydration, water distribution and microstructure of gluten during freeze thaw process: Role of a high molecular weight dextran produced by Weissella confusa QS813[END_REF] Galactan Devi et al. [START_REF] Devi | Physico-chemical characterization of galactan exopolysaccharide produced by Weissella confusa KR780676[END_REF] Kavitake et al. [START_REF] Kavitake | Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food[END_REF] Mannan Lakra et al. [110] Lactose-and cellobiose-derived branched trisaccharides Isomelezitose Shi et al. [START_REF] Shi | Lactose-and cellobiose derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase[END_REF] Homopolysaccharide with glucose monomers Adesulu-Dahunsi et al. [33] Wild and mutant W. confusa EPS with eight sugar moieties Adebayo-Tayo et al. [51] W. cibaria Dextran Xu et al. [START_REF] Xu | Interactions between fava bean protein and dextrans produced by Leuconostoc pseudomesenteroides DSM 20193 and Weissella cibaria Sj 1b[END_REF] Zannini et al. [START_REF] Zannini | Development of novel quinoa-based yoghurt fermented with dextran producer Weissella cibaria MG1[END_REF] Baruah et al. [START_REF] Baruah | Purification and characterization of dextransucrase from Weissella cibaria RBA12 and its application in in vitro synthesis of prebiotic oligosaccharides in mango and pineapple juices[END_REF][START_REF] Baruah | Functional food applications of dextran from Weissella cibaria RBA12 from pummelo (Citrus maxima)[END_REF] Kanimozhi et al. [START_REF] Kanimozhi | Optimization of dextran production by Weissella cibaria NITCSK4 using response surface methodology-genetic algorithm based technology[END_REF] Yu et al. [START_REF] Yu | Production, characterization and antibacterial activity of exopolysaccharide from a newly isolated Weissella cibaria under sucrose effect[END_REF] Linear dextran Ye et al. [START_REF] Ye | Purification and characterization of exopolysaccharide produced by Weissella cibaria YB-1 from pickle Chinese cabbage[END_REF] Dextran Oligosaccharides Hu, Gänzle [START_REF] Hu | Effect of temperature on productionof oligosaccharides and dextran by Weissella cibaria 10 M[END_REF] Heteropolysaccharides Zhu et al. [START_REF] Zhu | Purification, characterization and antioxidant activity of the exopolysaccharide from Weissella cibaria SJ14 isolated from Sichuan paocai[END_REF] Isomaltooligosaccharide Baruah et al. [START_REF] Baruah | Purification and characterization of dextransucrase from Weissella cibaria RBA12 and its application in in vitro synthesis of prebiotic oligosaccharides in mango and pineapple juices[END_REF][START_REF] Baruah | Functional food applications of dextran from Weissella cibaria RBA12 from pummelo (Citrus maxima)[END_REF] Rolim et al. [START_REF] Rolim | Sensory analysis of juice blend containing isomaltooligosaccharides produced by fermentation with Weissella cibaria[END_REF] EPS mainly composed of glucose and rhamnose sugar units Adesulu-Dahunsi et al. [26] Of the LAB Weissella species, W. cibaria and W. confusa are the most important EPS producers, which is why research has focused on them and their production, purification, and characterization [41,[START_REF] Hu | Effect of temperature on productionof oligosaccharides and dextran by Weissella cibaria 10 M[END_REF][START_REF] Kanimozhi | Optimization of dextran production by Weissella cibaria NITCSK4 using response surface methodology-genetic algorithm based technology[END_REF][START_REF] Devi | Physico-chemical characterization of galactan exopolysaccharide produced by Weissella confusa KR780676[END_REF][START_REF] Ye | Purification and characterization of exopolysaccharide produced by Weissella cibaria YB-1 from pickle Chinese cabbage[END_REF][START_REF] Yu | Production, characterization and antibacterial activity of exopolysaccharide from a newly isolated Weissella cibaria under sucrose effect[END_REF][START_REF] Zhu | Purification, characterization and antioxidant activity of the exopolysaccharide from Weissella cibaria SJ14 isolated from Sichuan paocai[END_REF]. Some studies have proposed ways to optimize EPS production and test different parameters, such as the effects of different temperatures and substrates. Hu and Ganzel [START_REF] Hu | Effect of temperature on productionof oligosaccharides and dextran by Weissella cibaria 10 M[END_REF] showed that W. cibaria strains grew faster at 30 °C, but

CHAPTER II:

Biodiversity and technological features of Weissella isolates obtained from Brazilian artisanal cheese-producing regions Presumed extracellular proteolytic activity was observed in eleven Weissella isolates (Table 2); five of these isolates were obtained from artisanal cheeses (Southern Pará). EPS production was determined qualitatively and all strains proved to be negative (Table 2). Most samples showed some ability to acidify skim milk, producing a weak reduction of pH after 24 h of incubation, except for isolate 16 (W. paramesenteroides) which lowered milk pH by over 2.0 pH units after 12 h (Table 2). The titratable acidity results expressed as lactic acid content confirm the pH measurement results. (Table 2).

Genomic analyses of Weissella cibaria W25, a potential bacteriocinproducing strain isolated from pasture in Campos das Vertentes, Minas Gerais, Brazil

The content of this part has been published in:

Microorganisms

Camila Gonçalves Teixeira, Rafaela da Silva Rodriguesa, Ricardo Seiti Yamatogi, Anca Lucau-Danila, Djamel Drider, Luís Augusto Nero, Antônio Fernandes de Carvalho

The license to reuse this content in this thesis is in Annex 4

Abstract

Weissella is a genus containing Gram-positive, heterofermentative bacteria belonging to the lactic acid bacteria (LAB) group. These bacteria are endowed with promising technological and antimicrobial attributes. Weissella cibaria W25 was isolated from a dairy environment where raw milk cheeses are produced. Therefore, we sequenced and assembled the W25 draft genome sequence, which consists of 41 contigs totaling ~2.4Mbp, with a G+C content of 45.04%. Then we carried out comprehensive a comparative genomic analysis with W. cibaria 110, known to pro-duce the weissellicin 110 bacteriocin, and four other nonbacteriocin-producing W. cibaria strains.

Keywords: Weissella; genome sequencing; bacteriocins genes proteolytic. Of note, the strain presented antimicrobial activity against a panel of Grampositive and Gram-negative foodborne pathogens [7].

Genome sequencing and assembly

The whole genome of W. cibaria W25 was sequenced with Nextera technology by a whole-genome shotgun strategy using the MiSeq v3 machine and Solexa platform (Il-lumina, USA) by Neoprospecta (Florianópolis, SC, Brazil). The trimming was performed using Trimmomatic v.0.36 [8] and the Phred value > 20. The raw read files were trimmed of adapter sequences, and low-quality reads. After trimming, sequence reads were checked for quality using the fastQC v.0.11.5 [9] and then used for de novo genome assembly. Genome assembly was conducted by using MIRA Assembler v.4.9.6 [10], mode "genome, accurate".

The assembling quality was determined with QUAST v.5.0.2 [11], and ContEst16S was used to check contamination [12].

Genome annotation and analysis

Gene prediction and annotation were performed using the Rapid Prokaryotic Genome Annotation (PROKKA) v.1.14.5 [13], executed with default parameters and also performed by RAST automated web server [14]. To identify secondary metabolite biosynthetic gene clusters and bacteriocins, we used the antiSMASH v.6.0 [15] and the BAGEL4 webserver [16]. Moreover, the research for plasmid was evaluated by plasmidFinder [17] and the web tool PathogenFinder [18] was used to check the presence of potential virulence factors.

Phylogenetic analyses

The identification of the genus and species was carried out using KmerFinder [19,20] and the Type (Strains) Genome Server (TYGS) [21]. The phylogenetic trees were visualized and edited using the online tool iTol v.6 (https://doi.org/10.1093/nar/gkw290).

Comparative genomic

For establishing the relationship between W. cibaria W25 and other members of this species in the bacteriocin production context we selected four published non-bacteriocinsproducing W. cibaria strains and W. cibaria 110 known to produce the weissellicin 110 bacteriocin (Table 1). The genomes obtained from the GenBank were annotated using Prokka before subjecting to analyses in order to standardize the annotations.

All the strains were submitted to the Type (Strains) Genome Server (TYGS) to confirm genus and species. Moreover, to establish the genetic similarity between all strains, analysis was done with Digital DDH (DNA-DNA hybridization) similarities based on the GGDC (Genome-to-Genome Distance Calculator) web server, version 3.0 [22]. The core genome of each group was determined with OrthoVenn2 (e-value of 10-5) [23] and the CGView Server [24] was used to comparative genome analysis using BLAST with default parameters.

Availability of nucleotide sequence data

This Whole Genome Shotgun project was deposited at DDBJ/ENA/GenBank under the accession JAFNKE000000000. The version described in this paper is version JAFNKE010000000. The raw sequencing data were submitted to Sequence Read Archive (SRA) database under accession number SRR16076638.

Results and Discussion

Genome sequencing, annotation, and analysis

The

Bacterial Species Confirmation

The identification of the genus and species was carried out using KmerFinder Bruker. MALDI-MS profiles were acquired in positive linear mode across the m/z range of 2-20 kDa using the manufacturer's automatic method MBT_FC.par (Bruker Daltonics).

Antimicrobial Potential

To identify secondary metabolite biosynthetic gene clusters and bacteriocins, we used the antiSMASH v. strains we can see that after the treatment with proteinase K, there was no difference in the antimicrobial activity which means that the inhibitory activity from W21 is not related to the production of bacteriocins. This can be confirmed by the AntiSMASH results, once in this genome, the region that has the lasso peptide has no transport-related gene as in the genome of W25 and W42. According to AntiSMASH results, W. cibaria W25 and W42 possess one putative lasso peptide and both of them have the same genes in the region containing this bacteriocin. Also, these two strains are the ones in the antimicrobial test that maintained some antimicrobial activity after neutralizing and lost it after being treated with proteinase K.

Conclusions

The three W. cibaria strains studied in this work present high antimicrobial inhibitory activity of the non-treated supernatant against all the target strains tested. Besides that, the genome of all of them presents the putative gene for bacteriocin identified as bacteriocin_IIc by the AntiSMASH webserver. However, the strain W. cibaria W21 did not present the transport-related gene presented in the others two. This fact was confirmed by the in vitro test with the supernatant neutralized and with the supernatant neutralized treated with proteinase K. Once the supernatant was neutralized it lost antimicrobial activity, however when the supernatant of W25 and W42 was treated with proteinase K, the remaining antimicrobial activity was lost and for W21 there was no difference between the tested supernatants against all targets. This is an indication of the production of bacteriocin.

Moreover, the bacteriocin identified has no similarity with the well-known bacteriocin produced by W. cibaria nor with the other strains from this genus being a potential new bacteriocin for the food and pharmaceutical industries.

Data Availability

Draft genome sequence of the W. cibaria strains W21 and W42 was submitted to NCBI under GenBank accession numbers JALRNM000000000 and JALRNN000000000, respectively.

Conflict of Interest

The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.

Several genes were predicted as putative virulence factors by VFDB (data not shown). However, no one of the well-known virulence genes gelE (gelatinase), hyl (hyaluronidase), asa1 (aggregation substance), esp (enterococcal surface protein), cylA (cytolysin), efaA (endocarditis antigen), and ace (adhesion of collagen), were detected in the genomes of W. cibaria W21, W25, and W42 strains. Besides, most of the genes detected by the VFDB webserver are related to other cellular functions such as genes for putative enolase (eno) phosphopyruvate hydratase. Enolase is a key glycolytic enzyme in the cytoplasm of prokaryotic essential for the degradation of carbohydrates via glycolysis [32].

Some researchers reported the presence of some virulence-encoded genes such as collagen adhesin proteins and genes associated with toxin production systems (including botulinum neurotoxin homolog) in some Weissella species like W. cebi, W. confusa [33] and W. oryzae [34], respectively. None of these genes were found in our W. cibaria strains tested. Also, Tenea and Hurtado [28] report two genes (hlyD, hlyIII) in some strains of W. cibaria [28], however in our study no genes involved in hemolysis activity were observed in the genomes of the W. cibaria strains W21, W25, and W42; and this was phenotypically confirmed by assessing this activity on the blood-agar medium (Figure 1). Therefore, slight hemolysis that could be graded as an α-hemolysis was detected.

Conflict of Interest:

The authors declare that they have no competing interest.

Microorganisms of the genus Weissella have a wide range of applications in food products, since they are capable of producing a wide variety of compounds. However, few studies have been performed yet and the industrial application of Weissella is not yet a reality.

In addition, this work allowed us to understand the diversity of Weissella species found in the different regions producing artisanal cheeses in Brazil, comparing two molecular techniques used for clustering analyzes. It also allowed the understanding of how the isolated strains are distributed in the environments that produce artisanal cheese, besides helping to understand the role they play in the artisanal cheese itself. It also showed that some of them have a favorable technological potential for future uses in the food industry as starter cultures and for the study of cheese defect prevention.

Besides that our work culminated in a possible new bacteriocin from the Weissella genus leaving the perspective of further investigation on the producing of this bacteriocin.

Also with this work we have a panorama of the possibility of using different strains of Weissella for human or animal consumption and as food ingredients. We also notice the relative importance of studies technologies to help the use of this strains as probiotics.