Water kefir: Factors affecting grain growth and health-promoting properties of the fermented beverage.

Nowadays, the interest in the consumption of healthy foods has increased as well as the homemade preparation of artisanal fermented product. Water kefir is an ancient drink of uncertain origin, which has been passed down from generation to generation and is currently consumed practically all over the world. Considering the recent and extensive updates published on sugary kefir, this work aims to shed light on the scientific works that have been published so far in relation to this complex ecosystem. We focused our review evaluating the factors that affect the beverage microbial and chemical composition that are responsible for the health attribute of water kefir as well as the grain growth. The microbial ecosystem that constitutes the grains and the fermented consumed beverage can vary according to the fermentation conditions (time and temperature) and especially with the use of different substrates (source of sugars, additives as fruits and molasses). In this sense, the populations of microorganisms in the beverage as well as the metabolites that they produce varies and in consequence their health properties. Otherwise, the knowledge of the variables affecting grain growth are also discussed for its relevance in maintenance of the starter biomass as well as the use of dextran for technological application.

Changes in lifestyle behaviors during COVID-19 isolation in Argentina: A cross-sectional study.

OBJECTIVE: Our aim was to identify changes in population habits induced by COVID-19 confinement in Argentina. METHODS: An internet-based cross-sectional survey was conducted among adults in Argentina on December 2020, requesting possible changes occurring during the COVID-19 outbreak. It included 26 questions regarding general information (age, gender, location), eating habits, desire/anxiety for food or to eat between meals, weight gain, physical activity, and hours of sleep. We ran a descriptive statistical analysis of changes in habits and lifestyle during the confinement, followed by a logistic regression analysis to explore the relation between these changes and weight gain. Results: Out of 1536 survey participants, 57.1% were female, aged 38.8 ± 13.1 years. Data showed that during the outbreak, people experienced significant changes in food intake, physical activity, nutritional supplement consumption, anxiety, and sleeping disorders. These changes in behavior resulted in an elevated percentage of people (39.7%) that gained weight (average 4.8 ± 2.8 kg). Weight gain was associated with more food consumption (OR: 9.398), increased snacking between meals (OR: 1.536), anxiety about food (OR: 3.180), less practice of physical activity (OR: 0.586) and less consumption of nutritional supplements (OR: 0.762). Conclusions: COVID-19 outbreak was associated with unhealthy lifestyle changes and body weight increase. These adverse side effects could be prevented by active promotion of nutritional advice and physical activity, implementing virtual activities associated with regular mass promotion campaigns.

Inulin addition improved probiotic survival in soy-based fermented beverage.

Currently, the growing demand for non-dairy functional foods leads to the constant development of new products. The objective of the present work was to obtain a soy-based fermented beverage employing the strains Lactiplantibacillus plantarum CIDCA 8327 or Lacticaseibacillus paracasei BGP1 and to analyze the effect of post-fermentation addition of inulin of low or high average polymerization degree on the bacterial resistance. Also, the antimicrobial and antioxidant activity of the fermented soy-based beverages were analyzed. The soy-based matrix was shown to be a suitable substrate for the growth of both lactic acid bacteria, and the fermented beverages obtained presented bioactive properties such us antioxidant activity and bactericidal effect against pathogen microorganisms. The addition of inulin after the fermentation process avoid the hydrolysis and so, preserve its polymerization degree and thus the potential prebiotic effect. The incorporation of inulin to the soy-based fermented beverages increased the bacterial count after 30 days of refrigerated storage up to 8.71 ± 0.15 and 8.41 ± 0.10 log CFU/mL for L. paracasei and L. planatrum respectively. The resistance to the gastrointestinal conditions of the strain L. paracasei BGP1 in the fermented beverage was improved up to 70% when inulin of high polymerization degree was added. Meanwhile the strain L. plantarum CIDCA 8327 showed a survival of 97 and 94% in the fermented beverage added with inulin of low or high polymerization degree, respectively. These results contribute to the development of non-dairy products containing inulin and probiotics and the diversification agri-based functional foods.

Impact of growth temperature on exopolysaccharide production and probiotic properties of Lactobacillus paracasei strains isolated from kefir grains.

EPS-producing LAB are widely used in the dairy industry since these polymers improve the viscosity and texture of the products. Besides, EPS might be responsible for several health benefits attributed to probiotic strains. However, growth conditions (culture media, temperature, pH) could modify EPS production affecting both technological and probiotic properties. In this work, the influence of growth temperature on EPS production was evaluated, as well as the consequences of these changes in the probiotic properties of the strains. All Lactobacillus paracasei strains used in the study showed changes in EPS production caused by growth temperature, evidenced by the appearance of a high molecular weight fraction and an increment in the total amount of produced EPS at lower temperature. Nevertheless, these changes do not affect the probiotic properties of the strains; L. paracasei strains grown at 20 °C, 30 °C and 37 °C were able to survive in simulated gastrointestinal conditions, to adhere to Caco-2 cells after that treatment and to modulate the epithelial innate immune response. The results suggest that selected L. paracasei strains are new probiotic candidates that can be used in a wide range of functional foods in which temperature could be used as a tool to improve the technological properties of the product.

Biological activity of the non-microbial fraction of kefir: antagonism against intestinal pathogens.

Kefir is a fermented milk obtained by the activity of kefir grains which are composed of lactic and acetic acid bacteria, and yeasts. Many beneficial health effects have been associated with kefir consumption such as stimulation of the immune system and inhibition of pathogenic microorganisms. The biological activity of kefir may be attributed to the presence of a complex microbiota as well as the microbial metabolites that are released during fermentation. The aim of this work was to characterise the non-microbial fraction of kefir and to study its antagonism against Escherichia coli, Salmonella spp. and Bacillus cereus. During milk fermentation there was a production of organic acids, mainly lactic and acetic acid, with a consequent decrease in pH and lactose content. The non-microbial fraction of kefir added to nutrient broth at concentrations above 75% v/v induced a complete inhibition of pathogenic growth that could be ascribed to the presence of un-dissociated lactic acid. In vitro assays using an intestinal epithelial cell model indicated that pre-incubation of cells with the non-microbial fraction of kefir did not modify the association/invasion of Salmonella whereas pre-incubation of Salmonella with this fraction under conditions that did not affect their viability significantly decreased the pathogen's ability to invade epithelial cells. Lactate exerted a protective effect against Salmonella in a mouse model, demonstrating the relevance of metabolites present in the non-microbial fraction of kefir produced during milk fermentation.

Gelling ability of kefiran in the presence of sucrose and fructose and physicochemical characterization of the resulting cryogels.

In this work, the influence of sucrose and fructose on the gel-forming capacity of kefiran was investigated as well as the physicochemical characteristics of the resulting gels. The addition of sugar to gel-forming solutions did not alter the pseudoplastic flow properties of kefiran solutions and after one freeze-thaw cycle translucent gels with high water-holding capability were obtained. A highly porous matrix was revealed by microscopy whose pore size varied with sugar concentration. Sucrose and fructose had different effects on the rheological characteristics of sugar-kefiran gels. An increment in the strength of the gels with progressive concentrations of sucrose was evidenced by an increase in the elastic modulus (G'), indicating that sucrose reinforces the binding interactions between the polymer molecules (p ≤ 0.05). A drastic reduction in elastic modulus occurred, however, when 50.0 % w/w sucrose was added to kefiran gels, resulting in less elasticity. In contrast, when fructose was added to kefiran gels, elastic modulus decreased slightly with progressive sugar concentrations up to 10 %, thereafter increasing up to 50 % (p ≤ 0.05). Supplementation with up to 30 % sugar contributed to water retention and increased the viscous modulus. The relative increment in the elastic and viscous moduli elevated the loss tangent (tanδ) depending on the type and concentration of sugar. Sugars (sucrose, fructose) present in the matrix of the polysaccharide networks modified water-polymer and polymer-polymer interactions and consequently changed the gels' physicochemical characteristics, thus allowing the possibility of selecting the appropriate formulation through tailor-made kefiran cryogels.

Fermented whey as poultry feed additive to prevent fungal contamination.

BACKGROUND: Fungal contamination of poultry feed causes economic losses to industry and represents a potential risk to animal health. The aim of the present study was to analyze the effectiveness of whey fermented with kefir grains as additive to reduce fungal incidence, thus improving feed safety. RESULTS: Whey fermented for 24 h at 20 °C with kefir grains (100 g L(-1) ) reduced conidial germination of Aspergillus flavus, Aspergillus parasiticus, Aspergillus terreus, Aspergillus fumigatus, Penicillium crustosum, Trichoderma longibrachiatum and Rhizopus sp. Poultry feed supplemented with fermented whey (1 L kg(-1) ) was two to four times more resistant to fungal contamination than control feed depending on the fungal species. Additionally, it contained kefir microorganisms at levels of 1 × 10(8) colony-forming units (CFU) kg(-1) of lactic acid bacteria and 6 × 10(7) CFU kg(-1) of yeasts even after 30 days of storage. CONCLUSION: Fermented whey added to poultry feed acted as a biopreservative, improving its resistance to fungal contamination and increasing its shelf life.

Autochthonous Fermentation as a Means to Improve the Bioaccessibility and Antioxidant Activity of Proteins and Phenolic Compounds of Yellow Pea Flour.

This study focused on evaluating the potential of the natural fermentation of pea flour to improve the release of antioxidant compounds. Preliminary fermentations of 36.4% w/w flour dispersions were performed in tubes under different conditions (24 and 48 h, 30 and 37 °C). Finally, fermented flours (FFs) were obtained in a bioreactor under two conditions: 1: 36.4% w/w, 24 h, 30 °C (FF1); 2: 14.3% w/w, 24 h, 37 °C (FF2). The pH values decreased to 4.4-4.7, with a predominance of lactic acid bacteria. As in the fermentations in tubes, an increment in the proteolysis degree (TNBS method) (greater for FF2), polypeptide aggregation and a decrease in their solubility, an increase in <2 kDa peptides, and an increase in the Oxygen Radical Absorption Capacity (ORAC) potency of PBS-soluble fractions after fermentation were demonstrated. Also, fermentation increased the proteolysis degree after simulated gastrointestinal digestion (SGID, COST-INFOGEST) with respect to the non-fermented flour digests, with some differences in the molecular composition of the different digests. ORAC and Hydroxyl Radical Averting Capacity (HORAC) potencies increased in all cases. The digest of FF2 (FF2D) presented the greater ORAC value, with higher activities for >4 kDa, as well as for some fractions in the ranges 2-0.3 kDa and <0.10 kDa. Fermentation also increased the 60%-ethanol-extracted phenolic compounds, mainly flavonoids, and the ORAC activity. After SGID, the flavan-3-ols disappeared, but some phenolic acids increased with respect to the flour. Fermentation in condition 2 was considered the most appropriate to obtain a functional antioxidant ingredient.

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains.

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)5-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations.

Lactobacillus plantarum isolated from kefir protects vero cells from cytotoxicity by type-II shiga toxin from Escherichia coli O157:H7.

Kefir is a fermented-milk beverage originating and widely consumed in the Caucasus as well as in Eastern Europe and is a source of bacteria with potential probiotic properties. Enterohaemorrhagic Escherichia coli producing Shiga toxin is commonly associated with food-transmitted diseases; the most prevalent serotype causing epidemics is Esch. coli O157:H7. The aim of this study was to evaluate the antagonism of Lactobacillus plantarum isolated from kefir against the action on Vero cells of supernatants of the Esch. coli O157:H7 strain 69160 expressing the type-II Shiga toxin (Stx2) and to study the role of the Lactobacillus cell wall in that inhibition. Spent culture supernatants of Esch. coli O157:H7 strain 69160 led to cytotoxic effects on cultured eukaryotic cells as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide-cleavage assay or by lactate-dehyrogenase release. Lb. plantarum CIDCA 83114 reduced the cytotoxic activity of Stx present in strain-69160 supernatants, and this protection was markedly higher than those of Lactobacillus kefir CIDCA 83113 and 8348 and Lb. delbrueckii subsp. bulgaricus CIDCA 333. This antagonism of cytotoxicity was mimicked by Lb. plantarum cell walls but was reduced after heating or protease treatments, thus indicating a protein or peptide as being involved in the protection mechanism. The cell surface of the lactobacilli bound the subunit B of Stx thereby decreasing the cytotoxicity. These interactions could constitute the first step in preventing the damage induced by Esch. coli O157:H7 supernatants, thus representing a valuable means of potentially mitigating the noxious effects of this food pathogen.

Exopolysaccharide-producing Lacticaseibacillus paracasei strains isolated from kefir as starter for functional dairy products.

Exopolysaccharides (EPS) produced by lactic acid bacteria are molecules of great interest for the dairy food industry. Lacticaseibacillus paracasei CIDCA 8339, CIDCA 83123, and CIDCA 83124 are potentially probiotic strains isolated from kefir grains whose EPS-production on MRS broth is dependent on incubation temperature. The aim of the present work is to evaluate the effect of fermentation temperature on the characteristics of EPS produced in milk by L. paracasei strains and the consequent impact on the rheological properties of the fermented products. Additionally, the protective effect of these EPS against Salmonella infection was evaluated in vitro. Acid gels with each strain were obtained by milk fermentation at 20°C, 30°C, and 37°C evidencing for all the strains a reduction in growth and acidification rate at lower temperature. Lacticaseibacillus paracasei CIDCA 83123 showed low fermentation rate at all temperatures requiring between 3 and 8 days to obtain acids gels, whereas CIDCA 8339 and 83124 needed between 24 and 48 h even when the temperature was 20°C. Fermentation temperature led to changes in crude EPS characteristics of the three strains, observing an increase in the relative amount of the high molecular weight fraction when the fermentation temperature diminished. Additionally, EPS83124 and EPS83123 presented modifications in monosaccharide composition, with a reduction of rhamnose and an increase of amino-sugars as temperature rise. These changes in the structure of EPS83124 resulted in an increase of the apparent viscosity of milks fermented at 20°C (223 mPa.s) and 30°C (217 mPa.s) with respect to acid gels obtained at 37°C (167 mPa.s). In order to deepen the knowledge on EPS characteristics, monosaccharide composition of low and high molecular weight EPS fractions were evaluated. Finally, it was evidenced that the preincubation of intestinal epithelial cells Caco-2/TC-7 with EPS8339 and EPS83124 partially inhibit the association and invasion of Salmonella. In light of these results, it can be concluded that the selection of the EPS-producing strain along with the appropriate fermentation conditions could be an interesting strategy to improve the technological properties of these L. paracasei fermented milks with potential protective effects against intestinal pathogens.

Kefir grains as a starter for whey fermentation at different temperatures: chemical and microbiological characterisation.

We report here a comparative analysis of the growth, acidification capacity, and chemical and microbiologic composition between kefir grains after 20 subcultures in whey at 20, 30, and 37°C and the original kefir grains coming from milk along with a determination of the microbiological composition of the fermented whey as compared with that of traditional fermented milk. When fermentation was carried out repeatedly at 30 or 37°C, kefir grains changed their kefir-like appearance, exhibited reduced growth rates, had a lower diversity of yeasts and water content, and a higher protein-to-polysaccharide ratio compared with the original kefir grains. In contrast, at 20°C kefir grains could remain in whey for prolonged periods without altering their acidification capacity, growth rate, macroscopic appearance or chemical and microbiologic composition-with the only difference being a reduction in certain yeast populations after 20 subcultures in whey. At this incubation temperature, the presence of Lactobacillus kefiranofaciens, Lb. kefir, Lb. parakefir, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces unisporus, and Sac. cerevisiae was detected in kefir grains and in fermented whey by denaturing-gradient-gel electrophoresis (DGGE). In whey fermented at 20°C the number of lactic-acid bacteria (LAB) was significantly lower (P<0·05) and the number of yeast significantly higher (P<0·05) than in fermented milk. Since the DGGE profiles were similar for both products, at this temperature the microbiologic composition of fermented whey is similar to that of fermented milk. We therefore suggest a temperature of 20°C to preserve kefir grains as whey-fermentation starters.

Spray-Dried Infant Formula Emulsion Stability as Affected by Pre-Heat Treatment and Total Solids Content of the Feed.

Pre-spray-drying processing may affect stability after reconstitution of emulsion-based powders, such as infant formulas. This study aimed to evaluate the effects of pasteurization temperature and total solids (TS) of the feed on the stability of the emulsions obtained from the reconstituted powders. Four infant formula powders (50%-75 °C, 50%-100 °C, 60%-75 °C, and 60%-100 °C) were produced at pilot scale, from emulsions with 50 or 60% TS pasteurized at 75 or 100 °C for 18 s. Both the emulsion feeds and the emulsions from the reconstituted powders (12.5% TS) were analyzed. The results showed that feeds with 60% TS were flocculated, as indicated by the large particle size and viscosity and the pseudoplastic behavior. Light microscopy revealed that, during spray drying, the flocs were disrupted in 60%-100 °C, while the 60%-75 °C emulsion remained flocculated, reducing its stability post-reconstitution. Although all four emulsions were mainly stabilized by caseins, the presence of ß-lactoglobulin was also detected at the oil-water interface, in native state in the formulas preheated at 75 °C and aggregated in the formulas preheated at 100 °C. In conclusion, both the degree of whey protein denaturation (resulting from pasteurization) and the TS of the concentrates during infant formula production affected the emulsion stability of the reconstituted powders.

Survival of spray-dried Lactobacillus kefir is affected by different protectants and storage conditions.

Survival of two Lactobacillus kefir strains after spray drying in reconstituted skim milk with or without the addition of 12.5 g monosodium glutamate/l, 20 g sucrose/l, or 20 g fructo-oligosaccharides (FOS)/l and during subsequent storage under different conditions of temperature (20 and 30°C) and relative humidity (RH) (0, 11 and 23%) was evaluated. After being dried, L. kefir 8321 and L. kefir 8348 had a decrease in viability of 0.29 and 0.70 log cfu/ml respectively, while the addition of different protectants improved the survival of both strains significantly. During storage, bacterial survival was significantly higher under lower conditions of RH (0-11%), and monosodium glutamate and FOS proved to be the best protectants.

Physicochemical, microbiological and sensory profiles of fermented milk containing probiotic strains isolated from kefir.

A two-strain starter culture containing Lactobacillus plantarum CIDCA 83114, a potential probiotic strain isolated from kefir grains, and Streptococcus thermophilus CIDCA 321 was tested for the preparation of a fermented milk product. Kluyveromyces marxianus CIDCA 8154, a yeast with immunomodulatory properties was included to formulate a three-strain starter culture. Supernatants of enterohaemorragic Escherichia coli, shiga-toxin-producing strain, along with a two-strain or a three-strain starter culture were included in the medium of Vero-cell surface cultures. The results demonstrated that these combinations of microorganisms antagonize the cytopathic action of shiga toxins. The cell concentration of Lb. plantarum did not decrease during fermentation, indicating that the viability of this strain was not affected by low pH, nor did the number of viable bacteria change during 21 days of storage in either fermented products. The number of viable yeasts increases during fermentation and storage. Trained assessors analyzed the general acceptability of fresh fermented milks and considered both acceptable. The milk fermented with the two-strain starter culture was considered acceptable after two week of storage, while the product fermented with the three-strain starter culture remained acceptable for less than one week. The main changes in sensory attributes detected by the trained panel were in sour taste, milky taste and also in fermented attributes. The correlation between different sensory attributes and acceptability indicated that the panel was positively influenced by milky attributes (taste, odour, and flavour) as well as the intensity of flavour. In conclusion, the two-strain starter culture would be the more promising alternative for inclusion of that potential probiotic lactobacillus in a fermented milk product.

Health-Promoting Properties of Lacticaseibacillus paracasei: A Focus on Kefir Isolates and Exopolysaccharide-Producing Strains.

Among artisanal fermented beverages, kefir (fermented milk drink) and water kefir (fermented nondairy beverage) are of special interest because their grains can be considered natural reservoirs of safe and potentially probiotic strains. In the last years, several reports on Lacticaseibacillus paracasei (formerly Lactobacillus paracasei) isolated from both artisanal fermented beverages were published focusing on their health-promoting properties. Although this is not the predominant species in kefir or water kefir, it may contribute to the health benefits associated to the consumption of the fermented beverage. Since the classification of L. paracasei has been a difficult task, the selection of an adequate method for identification, which is essential to avoid mislabeling in products, publications, and some publicly available DNA sequences, is discussed in the present work. The last findings in health promoting properties of L. paracasei and the bioactive compounds are described and compared to strains isolated from kefir, providing a special focus on exopolysaccharides as effector molecules. The knowledge of the state of the art of Lacticaseibacillus paracasei from kefir and water kefir can help to understand the contribution of these microorganisms to the health benefits of artisanal beverages as well as to discover new probiotic strains for applications in food industry.

Exopolysaccharides From Lactobacillus paracasei Isolated From Kefir as Potential Bioactive Compounds for Microbiota Modulation.

Microbiota coexists in true symbiosis with the host playing pivotal roles as a key element for well-being and health. Exopolysaccharides from lactic acid bacteria are an alternative as novel potential prebiotics that increase microbiota diversity. Considering this, the aim of the present work was to evaluate the capacity of the EPS produced by two L. paracasei strains isolated from kefir grains, to be metabolized in vitro by fecal microbiota producing short chain fatty acids. For this purpose, fecal samples from healthy children were inoculated in a basal medium with EPS and incubated in anaerobiosis at 37°C for 24, 48, and 72 h. DGGE profiles and the production of SCFA after fermentation were analyzed. Additionally, three selected samples were sequenced by mass sequencing analysis using Ion Torrent PGM. EPS produced by L. paracasei CIDCA 8339 (EPS8339) and CIDCA 83124 (EPS83124) are metabolized by fecal microbiota producing a significant increase in SCFA. EPS8339 fermentation led to an increment of propionate and butyrate, while fermentation of EPS83124 increased butyrate levels. Both EPS led to a profile of SCFA different from the ones obtained with inulin or glucose fermentation. DGGE profiles of 72 h fermentation demonstrated that both EPS showed a different band profile when compared to the controls; EPS profiles grouped in a cluster that have only 65% similarity with glucose or inulin profiles. Mass sequencing analysis demonstrated that the fermentation of EPS8339 leads to an increase in the proportion of the genera Victivallis, Acidaminococcus and Comamonas and a significant drop in the proportion of enterobacteria. In the same direction, the fermentation of EPS83124 also resulted in a marked reduction of Enterobacteriaceae with a significant increase in the genus Comamonas. It was observed that the changes in fecal microbiota and SCFA profile exerted by both polymers are different probably due to differences in their structural characteristics. It can be concluded that EPS synthesized by both L. paracasei strains, could be potentially used as bioactive compound that modify the microbiota increasing the production of propionic and butyric acid, two metabolites highly associated with beneficial effects both at the gastrointestinal and extra-intestinal level.

Kefiran protects Caco-2 cells from cytopathic effects induced by Bacillus cereus infection.

The aim of this work was to evaluate the ability of kefiran to antagonize cytopathic effects triggered by Bacillus cereus strain B10502 on cultured human enterocytes (Caco-2 cells). Cell damage was evaluated by F-actin labelling, scanning electron microscopy and determination of ratios of necrotic and detached cells. To assess the interaction between kefiran and bacteria or eukaryotic cells, flow cytometric analysis was conducted with FITC-labelled kefiran. Kefiran significantly protected infected cells from cytopathic effects induced by B. cereus such as cell necrosis, F-actin disorganisation and microvilli effacement, although presence of kefiran did not modify the adhesion of microorganisms to cultured human enterocytes. Results could be ascribed to the ability of kefiran to interact with both bacteria and eukaryotic cells thus antagonizing interactions necessary for maximal biological effects. Our findings encourage further research on the use of bacterial exopolysaccharides to antagonize virulence factors associated to direct bacteria-cell interactions.

Heterogeneity of S-layer proteins from aggregating and non-aggregating Lactobacillus kefir strains.

Since the presence of S-layer protein conditioned the autoaggregation capacity of some strains of Lactobacillus kefir, S-layer proteins from aggregating and non-aggregating L. kefir strains were characterized by immunochemical reactivity, MALDI-TOF spectrometry and glycosylation analysis. Two anti-S-layer monoclonal antibodies (Mab5F8 and Mab1F8) were produced; in an indirect enzyme-linked immunosorbent assay Mab1F8 recognized S-layer proteins from all L. kefir tested while Mab5F8 recognized only S-layer proteins from aggregating strains. Periodic Acid-Schiff staining of proteins after polyacrylamide gel electrophoresis under denaturing conditions revealed that all L. kefir S-layer proteins tested were glycosylated. Growth of bacteria in the presence of the N-glycosylation inhibitor tunicamycin suggested the presence of glycosydic chains O-linked to the protein backbone. MALDI-TOF peptide map fingerprint for S-layer proteins from 12 L. kefir strains showed very similar patterns for the aggregating strains, different from those for the non-aggregating ones. No positive match with other protein spectra in MSDB Database was found. Our results revealed a high heterogeneity among S-layer proteins from different L. kefir strains but also suggested a correlation between the structure of these S-layer glycoproteins and the aggregation properties of whole bacterial cells.

"Physicochemical, immunomodulatory and safety aspects of milks fermented with Lactobacillus paracasei isolated from kefir".

The use of Lactobacillus paracasei strains isolated from kefir grains as starters for the development of functional dairy products was evaluated. The physicochemical and immunomodulatory properties of milks fermented with L. paracasei CIDCA8339, CIDCA83123 and CIDCA83124 were analyzed. The three strains produced bioactive metabolites during fermentation, since the fermented milk supernatants were able to downregulate >75% of the induced innate immune response in vitro. Although all strains presented absence of hemolytic activity and susceptibility to antibiotics, L. paracasei CIDCA8339 presented more attractive probiotic and technological properties. Mice consuming the fermented milk with L. paracasei CIDCA 8339 did not present significant modifications in sIgA levels or TNF-α, TGF-ß and IL-10 mRNA expression in ileum. Additionally, a decrease of INF-γ level in ileum and no microbiological translocation to liver and spleen was observed. These results demonstrate that L. paracasei CIDCA8339 represents a safe promising potential probiotic strain for the development of functional foods.