The synthesis of soluble and volatile bioactive compounds by selected brewer's yeasts: Antagonistic effect against enteropathogenic bacteria and food spoiler - toxigenic Aspergillus sp.

Contamination by Aspergillus sp. and the accumulation of its mycotoxins in food and beverages have a high impact on human health and food safety. This investigation inquires the ability of brewer's yeasts discarded after fermentation (brewing fermentation residue, BFR) to synthesize bioactive compounds and to biocontrol Aspergillus sp. BFRs of Saccharomyces cerevisiae MBELGA62 and Pichia kudriavzevii MBELGA61 proved to have bacteriostatic properties and to be efficient in fungal growth reduction, decreasing the growth rate of Aspergillus flavus and Aspergillus parasiticus up to 37.8% and 42.5%, respectively. Fungal mycelium degradation along with absentia of conidia was detected near the yeast inoculum. Moreover, the yeasts synthesize volatile bioactive compounds that extend Aspergillus sp. lag phase above 100% and decrease fungal growth rates from 20% towards 44%, along with the complete inhibition of conidia synthesis. These results indicate the potential of this residue to be used in biocontrol applications in the food industry.

Changes in Microbiota During Multiple Fermentation of Kefir in Different Sugar Solutions Revealed by High-Throughput Sequencing.

Kefir is a fermented beverage produced through the activity of its grains, which is constituted by lactic acid and acetic acid bacteria and yeasts. We studied the bacterial succession during multiple fermentation of Argentinian kefir in brown sugar, purified molasses or high-test molasses, using 16S high-throughput sequencing. Firmicutes was dominant (up to 98% of total population) in grains and beverages made from various sugar substrates, except in high-test molasses beverage, which was dominated by Proteobacteria (up to 78% of total population). Major bacterial species in Firmicutes were Liquorilactobacillus nagelii, Lentilactobacillus hilgardii/diolivorans and Lacticaseibacillus casei/paracasei, which are active in lactic acid fermentation. Proteobacteria comprised Acetobacter lovaniensis and Gluconobacter oxydans/roseus as major species, which are presumably responsible for the acetic acid formation in sugary kefir beverages. Bacteria differ in abundance depending on the sugar type, as revealed by the competitive dominances between L. nagelii and A. loveniensis. Purified molasses led to scarce acetic acid bacteria during fermentation, indicating that it is not a suitable substrate for their growth. Our results suggest that acetic acid (and/or ethanol) in sugary kefir modulates the succession and dominance of specific lactic acid bacteria. This study will provide valuable information for designing more sophisticated non-dairy fermented beverages with stable microbial properties.

Lactobacillus strains inhibit biofilm formation of Salmonella sp. isolates from poultry.

Lactic acid bacteria (LAB) exert a strong antagonistic activity against many microorganisms including food spoilage organisms and may be used as an alternative to control biofilm formation of pathogens in food industries. The objective of this work was to investigate the ability of fifteen Salmonella strains isolated from poultry environment to form biofilms on different surfaces. In addition, the effect of Lactobacillus kefiri strains 8321 and 83113 and Lactobacillus plantarum 83114 and their surface proteins on biofilm development of Salmonella Enteritidis 115 was studied. The relationship between surface properties of bacteria (hydrophobicity, autoaggregation and coaggregation with lactobacilli) and biofilm formation was also investigated. Most of Salmonella strains were hydrophilic and five strains were moderately hydrophobic. In general, Salmonella strains showed high aggregation abilities (27-54%). S. Enteritidis 106 and S. Typhimurium 102 and 108 showed the highest percentages of autoaggregation. All Salmonella strains tested showed aggregation abilities with the three lactobacilli studied, but the percentage of coaggregation proved to be strain-specific. When comparing stainless steel, glass and polystyrene surfaces, higher levels of biofilm formation occurred on polystyrene plate than on glass surfaces or stainless steel. S. Enteritidis 115 exhibited the greatest attachment to polyestyrene surface. The preincubation or coincubation with the three lactobacilli strains significantly reduced (about 1 log CFU/ml of reduction) the ability of S. Enteritidis 115 to form biofilm compared to the control without lactobacilli. These results were confirmed by confocal microscopy. In the same way, when surface proteins extracted from lactobacilli strains were preincubated or coincubated with S. Enteritidis 115, biofilm formation of this strain was significantly decreased compared to the control. The results obtained showed that these Lactobacillus strains and their surface proteins can be used as alternatives for control of biofilm formation by Salmonella in the poultry industry.

Suitability of kefir powder production using spray drying.

Spray drying was applied for the production of kefir powder. The survival of microorganisms after drying, storage and simulated gastrointestinal (GI) conditions was investigated. Kefir was obtained by fermentation of milk and whey permeate, and was dehydrated directly (traditional kefir) or using different carriers (skim milk, whey permeate and maltodextrin). Low survival (5.5 log and <2 log CFU/g for lactic acid bacteria and yeast respectively) of microorganisms was achieved when kefir was dehydrated without thermoprotectants (carriers). In contrast, survival of the microorganisms was significantly improved in the presence of different carriers. When skim milk (SM) was used as the carrier medium, lactic acid bacteria (LAB) survival was above 9 log CFU/g. In contrast, viability of yeast was dramatically reduced after spray drying in these conditions. When whey permeate was used as the carrier medium, LAB survival was 8 log CFU/g and yeast survival was above 4 log CFU/g. LAB in the kefir powder survived better the simulated GI conditions when spray drying was conducted in SM. LAB in kefir powder sample dehydrated in SM and SM plus maltodextrin remained stable for at least 60 days at 4 °C. Our results demonstrated that spray drying of kefir is a suitable approach to obtain a concentrated kefir-derived product.

Antifungal effect of kefir fermented milk and shelf life improvement of corn arepas.

Fungal contamination negatively affects the production of cereal foods such as arepa loaf, an ancient corn bread consumed daily in several countries of Latin-America. Chemical preservatives such as potassium sorbate are applied in order to improve the arepa's shelf life and to reduce the health risks. The use of natural preservatives such as natural fermented products in food commodities is a common demand among the consumers. Kefir is a milk fermented beverage obtained by fermentation of kefir grains. Its antibacterial and probiotic activity has been exhaustively demonstrated. Our objectives were to determine the antifungal effect of kefir fermented milk on Aspergillus flavus AFUNL5 in vitro and to study if the addition of kefir fermented milk to arepas could produce shelf life improvement. We determined the antifungal effect on solid medium of kefir cell-free supernatants (CFS) obtained under different fermentation conditions. Additionally, we compared the antifungal effect of kefir CFS with that obtained with unfermented milk artificially acidified with lactic plus acetic acids (lactic and acetic acids at the same concentration determined in kefir CFS) or with hydrochloric acid. Finally, kefir was added to the corn products either in the loaf recipe (kefir-baked arepas) or sprayed onto the baked-loaf surface (kefir-sprayed arepas). The loaves' resistance to natural and artificial fungal contamination and their organoleptic profiles were studied. The highest fungal inhibition on solid medium was achieved with kefir CFS produced by kefir grains CIDCA AGK1 at 100 g/L, incubated at 30 °C and fermented until pH 3.3. Other CFS obtained from different fermentation conditions achieved less antifungal activity than that mentioned above. However, CFS of milk fermented with kefir grains, until pH 4.5 caused an increase of growth rates. Additionally, CFS produced by kefir grains CIDCA AGK1 at 100 g/L, incubated at 30 °C and fermented until pH 3.3 achieved higher antifungal activity than CFS from artificially acidified milk with organic acids (CFS L + A) at the same concentration of kefir CFS. Besides, CFS from milk acidified with hydrochloric acid (CFS HCl) showed no fungal inhibition. On the other hand, kefir-baked arepas exhibited significant resistance to natural and artificial fungal contamination. Finally, both kefir-baked and kefir-sprayed arepas retained the organoleptic characteristics of the traditional corn product, but with certain tastes imparted by the kefir fermentation. This work constitutes the first study on fungal inhibition by kefir-fermented milk extending to the protection of corn products of mass-consumption and the possible application as a food preservative.

Whey permeate fermented with kefir grains shows antifungal effect against Fusarium graminearum.

The objective of the work reported here was to study the antifungal capability of cell-free supernatants obtained from whey permeates after fermentation by the kefir grains CIDCA AGK1 against Fusarium graminearum growth and zearalenone (ZEA) production. The assays were performed in order to study the conidial germination inhibition -in liquid media- and the effect on fungal growth rate and the Latency phase -in solid media. We observed that fermented supernatants of pH 3·5 produced the highest percentages of inhibition of conidial germination. The dilution and, particularly, alkalinisation of them led to the gradual loss of antifungal activity. In the fungal inhibition assays on plates we found that only the highest proportion of supernatant within solid medium had significant antifungal activity, which was determined as fungicidal. There was no ZEA biosynthesis in the medium with the highest proportion of supernatant, whereas at lower concentrations, the mycotoxin production was strain-dependent. From the results obtained we concluded that kefir supernatants had antifungal activity on the F. graminearum strains investigated and inhibited mycotoxin production as well, but in a strain-dependent fashion. The present work constitutes the first report of the effect of the products obtained from the kefir-grain fermentation of whey permeates - a readily available by-product of the dairy industry - on F. graminearum germination, growth, and toxin production.

Novel Functional Whey-Based Drinks with Great Potential in the Dairy Industry.

This work focuses on the production of liquid whey protein concentrates by ultrafiltration followed by thermal denaturation and homogenization of the ultrafiltrated concentrate, as well as on the production of ultrafiltrated permeates concentrated by reverse osmosis. Kefir grains (fresh and thawed) and/or commercial probiotic bacteria were inoculated in both liquid whey protein concentrates and concentrated ultrafiltrated permeates and grown at 25 °C for 24 h for the manufacture of fermented drinks. The physicochemical characterization (pH, titratable acidity, viscosity, and content of total solids, ash, fat and proteins) of the obtained drinks was then assessed and compared. Enumeration of viable microorganisms was carried out immediately after inoculation (at 0 h), during the fermentation period (at 12 and 24 h) and during refrigerated storage (at 48, 168 and 336 h). The fermented drinks showed acceptable physicochemical and sensorial properties, and contained above 7 log CFU/mL of lactococci and lactobacilli and 6 log CFU/mL of yeasts after 14 days of refrigerated storage, which is in agreement with the standards required by international organizations like European Food Safety Authority (EFSA) and Food and Drug Administration (FDA) for products containing probiotics. In summary, the strategy developed in this work contributes to the expansion of the applications of products derived from whey fractionation for the design of novel functional foods.

Safety characterization and antimicrobial properties of kefir-isolated Lactobacillus kefiri.

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused α- or ß-hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(-) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 10(8) CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.

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.

Adhesion properties of potentially probiotic Lactobacillus kefiri to gastrointestinal mucus.

We investigated the mucus-binding properties of aggregating and non-aggregating potentially probiotic strains of kefir-isolated Lactobacillus kefiri, using different substrates. All the strains were able to adhere to commercial gastric mucin (MUCIN) and extracted mucus from small intestine (SIM) and colon (CM). The extraction of surface proteins from bacteria using LiCl or NaOH significantly reduced the adhesion of three selected strains (CIDCA 8348, CIDCA 83115 and JCM 5818); although a significant proportion (up to 50%) of S-layer proteins were not completely eliminated after treatments. The surface (S-layer) protein extracts from all the strains of Lb. kefiri were capable of binding to MUCIN, SIM or CM, and no differences were observed among them. The addition of their own surface protein extract increased adhesion of CIDCA 8348 and 83115 to MUCIN and SIM, meanwhile no changes in adhesion were observed for JCM 5818. None of the seven sugars tested had the ability to inhibit the adhesion of whole bacteria to the three mucus extracts. Noteworthy, the degree of bacterial adhesion reached in the presence of their own surface protein (S-layer) extract decreased to basal levels in the presence of some sugars, suggesting an interaction between the added sugar and the surface proteins. In conclusion, the ability of these food-isolated bacteria to adhere to gastrointestinal mucus becomes an essential issue regarding the biotechnological potentiality of Lb. kefiri for the food industry.

Administration of kefir-fermented milk protects mice against Giardia intestinalis infection.

Giardiasis, caused by the protozoan Giardia intestinalis, is one of the most common intestinal diseases worldwide and constitutes an important problem for the public health systems of various countries. Kefir is a probiotic drink obtained by fermenting milk with 'kefir grains', which consist mainly of bacteria and yeasts that coexist in a complex symbiotic association. In this work, we studied the ability of kefir to protect mice from G. intestinalis infection, and characterized the host immune response to this probiotic in the context of the intestinal infection. Six- to 8-week-old C75BL/6 mice were separated into four groups: controls, kefir mice (receiving 1 : 100 dilution of kefir in drinking water for 14 days), Giardia mice (infected orally with 4×10(7) trophozoites of G. intestinalis at day 7) and Giardia-kefir mice (kefir-treated G. intestinalis-infected mice), and killed at 2 or 7 days post-infection. Kefir administration was able to significantly reduce the intensity of Giardia infection at 7 days post-infection. An increase in the percentage of CD4(+) T cells at 2 days post-infection was observed in the Peyer's patches (PP) of mice belonging to the Giardia group compared with the control and kefir groups, while the percentage of CD4(+) T cells in PP in the Giardia-kefir group was similar to that of controls. At 2 days post-infection, a reduction in the percentage of B220-positive major histocompatibility complex class II medium cells in PP was observed in infected mice compared with the other groups. At 7 days post-infection, Giardia-infected mice showed a reduction in RcFcε-positive cells compared with the control group, suggesting a downregulation of the inflammatory response. However, the percentages of RcFcε-positive cells did not differ from controls in the kefir and Giardia-kefir groups. An increase in IgA-positive cells was observed in the lamina propria of the kefir group compared with controls at 2 days post-infection. Interestingly, the diminished number of IgA-positive cells registered in the Giardia group at 7 days post-infection was restored by kefir feeding, although the increase in IgA-positive cells was no longer observed in the kefir group at that time. No significant differences in CXCL10 expression were registered between groups, in concordance with the absence of inflammation in small-intestinal tissue. Interestingly, a slight reduction in CCL20 expression was observed in the Giardia group, suggesting that G. intestinalis might downregulate its expression as a way of evading the inflammatory immune response. On the other hand, a trend towards an increase in TNF-α expression was observed in the kefir group, while the Giardia-kefir group showed a significant increase in TNF-α expression. Moreover, kefir-receiving mice (kefir and Giardia-kefir groups) showed an increase in the expression of IFN-γ, the most relevant Th1 cytokine, at 2 days post-infection. Our results demonstrate that feeding mice with kefir reduces G. intestinalis infection and promotes the activation of different mechanisms of humoral and cellular immunity that are downregulated by parasitic infection, thus contributing to protection.

Protective effect of a mixture of kefir-isolated lactic acid bacteria and yeasts in a hamster model of Clostridium difficile infection.

The objective of this work was to test the protective effect of a mixture (MM) constituted by kefir-isolated microorganisms (Lactobacillus plantarum, Lactobacillus kefir, Lc. lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae) in a hamster model of infection with Clostridium difficile, an anaerobic Gram-positive bacterium that causes diarrhoea. Placebo or MM was administered ad libitum in drinking water from day 0 to the end of treatment. Hamsters received orally 200 µg of clyndamicin at day 7 and then were infected with 1 × 10(8) CFU of C. difficile by gavage. Development of diarrhoea and death was registered until the end of the protocol. Surviving animals were sacrificed at day 16, and a test for biological activity of clostridial toxins and histological stainings were performed in caecum samples. Six of seven infected animals developed diarrhoea and 5/7 died at the end of the experimental protocol. The histological sections showed oedema and inflammatory infiltrates with neutrophils and crypt abscesses. In the group of animals infected and treated with MM1/1000, only 1 of 7 hamsters showed diarrhoea and none of them died. The histological sections showed only a slight thickening of the mucosa with presence of lymphocytic infiltrate. These results demonstrate that an oral treatment with a mixture of kefir-isolated bacteria and yeasts was able to prevent diarrhoea and enterocolitis triggered by C. difficile.

Protective effect of bifidobacteria in an experimental model of Clostridium difficile associated colitis.

The aim of this study was to evaluate the ability of Bifidobacterium strains to prevent the effects associated with Clostridium difficile infection in a hamster model of enterocolitis. After clindamycin treatment (30 mg/kg), animals were infected intragastrically with C. difficile (5×108 CFU per animal). Seven days prior to antibiotic administration, probiotic treatment was started by administering bacterial suspensions of bifidobacteria in drinking water. Strains CIDCA 531, CIDCA 5310, CIDCA 5316, CIDCA 5320, CIDCA 5323 and CIDCA 5325 were used. Treatment was continued during all the experimental period. Development of diarrhoea, enterocolitis and mortality were evaluated. All the infected animals belonging to the placebo group developed enterocolitis (5/5) and only two dead (2/5) whereas in the group administered with Bifidobacterium bifidum strain CIDCA 5310 the ratio of animals with enterocolitis or dead decreased significantly (1/5 and 0/5 respectively). Biological activity of caecum contents was evaluated in vitro on Vero cells. Animals treated with strain CIDCA 5310 presented lower biological activity than those belonging to the placebo group. The present study shows the potential of selected strains of bifidobacteria to antagonise, in vivo, the virulence of C. difficile.

Kefir-isolated Lactococcus lactis subsp. lactis inhibits the cytotoxic effect of Clostridium difficile in vitro.

Kefir is a dairy product obtained by fermentation of milk with a complex microbial population and several health-promoting properties have been attributed to its consumption. In this work, we tested the ability of different kefir-isolated bacterial and yeast strains (Lactobacillus kefir, Lb. plantarum, Lactococcus lactis subps. lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) or a mixture of them (MM) to antagonise the cytopathic effect of toxins from Clostridium difficile (TcdA and TcdB). Cell detachment assays and F-actin network staining using Vero cell line were performed. Although incubation with microbial cells did not reduce the damage induced by C. difficile spent culture supernatant (SCS), Lc. lactis CIDCA 8221 and MM supernatants were able to inhibit the cytotoxicity of SCS to Vero cells. Fraction of Lc. lactis CIDCA 8221 supernatant containing components higher than 10 kDa were responsible for the inhibitory activity and heating of this fraction for 15 min at 100 °C completely abrogated this ability. By dot-blot assay with anti-TcdA or anti-TcdB antibodies, concentration of both toxins seems to be reduced in SCS treated with Lc. lactis CIDCA 8221 supernatant. However, protective effect was not affected by treatment with proteases or proteases-inhibitors tested. In conclusion, we demonstrated that kefir-isolated Lc. lactis CIDCA 8221 secreted heat-sensitive products able to protect eukaryotic cells from cytopathic effect of C. difficile toxins in vitro. Our findings provide new insights into the probiotic action of microorganisms isolated from kefir against virulence factors from intestinal pathogens.

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.

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.

Surface proteins from Lactobacillus kefir antagonize in vitro cytotoxic effect of Clostridium difficile toxins.

In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer antibodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD.

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.

Development of a method based on chemometric analysis of Raman spectra for the discrimination of heterofermentative lactobacilli.

In this work, a method based on Raman spectroscopy in combination with Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) has been developed for the rapid differentiation of heterofermentative related lactobacilli. In a first approach, Lactobacillus kefir strains were discriminated from other species of heterofermentative lactobacilli: Lb. parakefir and Lb. brevis. After this first approach, PCA allowed for a clear differentiation between Lb. parakefir and Lb.brevis. For the first level of discrimination, PCA was performed on the whole spectra and also on delimited regions, defined taking into consideration the loading values. The best regions allowing a clear differentiation between Lb. kefir and non-Lb. kefir strains were found to be: the 1700-1500 cm(-1), 1500-1185 cm(-1) and 1800-400 (whole spectrum) cm(-1) Raman ranges. In order to develop a classification rule, PLS-DA was carried out on the mentioned regions. This method permitted the discrimination and classification of the strains under study in two groups: Lb. kefir and non-Lb. kefir. The model was further validated using lactobacilli strains from different culture collections or strains isolated from kefir grains previously identified using molecular methods. The second approach based on PCA was also performed on the whole spectra and on delimited regions, being the regions 1700-1500 cm(-1), 1500-1185 cm(-1) and 1185-1020 cm(-1), i.e., those allowing the clearest discrimination between Lb. parakefir and Lb. brevis. The results obtained in this work, allowed a clear discrimination within heterofermentative lactobacilli strains, proteins being the biological structures most determinant for this discrimination.

Effect of freeze-drying on viability and in vitro probiotic properties of a mixture of lactic acid bacteria and yeasts isolated from kefir.

The effect of freeze-drying on viability and probiotic properties of a microbial mixture containing selected bacterial and yeast strains isolated from kefir grains (Lactobacillus kefir, Lactobacillus plantarum, Lactococcus lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) was studied. The microorganisms were selected according to their potentially probiotic properties in vitro already reported. Two types of formulations were performed, a microbial mixture (MM) suspended in milk and a milk product fermented with MM (FMM). To test the effect of storage on viability of microorganisms, MM and FMM were freeze-dried and maintained at 4°C for six months. After 180 days of storage at 4°C, freeze-dried MM showed better survival rates for each strain than freeze-dried FMM. The addition of sugars (trehalose or sucrose) did not improve the survival rates of any of the microorganisms after freeze-drying. Freeze-drying did not affect the capacity of MM to inhibit growth of Shigella sonnei in vitro, since the co-incubation of this pathogen with freeze-dried MM produced a decrease of 2 log in Shigella viability. The safety of freeze-dried MM was tested in mice and non-translocation of microorganisms to liver or spleen was observed in BALB/c mice feed ad libitum during 7 or 20 days. To our knowledge, this is the first report about the effect of freeze-drying on viability, in vitro probiotic properties and microbial translocation of a mixture containing different strains of both bacteria and yeasts isolated from kefir.