Inhibitory activity of cheese whey fermented with kefir grains.

We investigated the chemical and microbiological compositions of three types of whey to be used for kefir fermentation as well as the inhibitory capacity of their subsequent fermentation products against 100 Salmonella sp. and 100 Escherichia coli pathogenic isolates. All the wheys after fermentation with 10% (wt/vol) kefir grains showed inhibition against all 200 isolates. The content of lactic acid bacteria in fermented whey ranged from 1.04 × 10(7) to 1.17 × 10(7) CFU/ml and the level of yeasts from 2.05 × 10(6) to 4.23 × 10(6) CFU/ml. The main changes in the chemical composition during fermentation were a decrease in lactose content by 41 to 48% along with a corresponding lactic acid production to a final level of 0.84 to 1.20% of the total reaction products. The MIC was a 30% dilution of the fermentation products for most of the isolates, while the MBC varied between 40 and 70%, depending on the isolate. The pathogenic isolates Salmonella enterica serovar Enteritidis 2713 and E. coli 2710 in the fermented whey lost their viability after 2 to 7 h of incubation. When pathogens were deliberately inoculated into whey before fermentation, the CFU were reduced by 2 log cycles for E. coli and 4 log cycles for Salmonella sp. after 24 h of incubation. The inhibition was mainly related to lactic acid production. This work demonstrated the possibility of using kefir grains to ferment an industrial by-product in order to obtain a natural acidic preparation with strong bacterial inhibitory properties that also contains potentially probiotic microorganisms.

Lactobacillus delbrueckii subsp lactis (strain CIDCA 133) resists the antimicrobial activity triggered by molecules derived from enterocyte-like Caco-2 cells.

AIMS: The aim of the present study was to assess the ability of a potentially probiotic strain to resist, in vitro, the effect of intestinal antimicrobial molecules. METHODS AND RESULTS: Strain CIDCA 133 of Lactobacillus delbrueckii subsp lactis was studied. Lactobacillus delbrueckii subsp bulgaricus as well as other gram-positive and gram-negative bacteria were used for comparison purposes. The effect of different antimicrobial extracts was determined by diffusion assays, viable counts and growth kinetics. Human-defensins (h beta D1 and h beta D2) were also included in the study. Two types of cellular fractions from Caco-2 cells were tested: (i) cytosolic fractions, obtained by sonication of cultured human enterocytes and (ii) cationic fraction, obtained by batch extraction of the cytosolic fraction with a weak cation exchange resin. In addition, the effect of Caco-2-secreted factors was studied. Strain CIDCA 133 was neither inhibited by Caco-2 secreted, cytosolic nor cationic fractions. Of note, human-defensins were inactive against strain CIDCA 133. In contrast, a related lactobacilli: Lactobacilli delbrueckii subsp bulgaricus (strain CIDCA 331) and other species of gram-positive or gram-negative bacteria were strongly inhibited. CONCLUSIONS: Strain CIDCA 133 is able to survive and grow in the presence of enterocyte-derived antimicrobial molecules. This ability is not a general property of lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Results could provide a new insight into the mechanisms of the probiotic effect and encourage further studies on this field. Resistance to antimicrobial peptides can be relevant to understand the interaction of potentially probiotic strains with the host's immune system. This ability can be also relevant as a selection criterion for new probiotic strains.

Preservation of probiotic strains isolated from kefir by spray drying.

AIMS: This work aims to investigate the survival of Lactobacillus kefir CIDCA 8348, Lactobacillus plantarum CIDCA 83114 and Saccharomyces lipolytica CIDCA 812, all isolated from kefir, during spray drying and subsequent storage. METHODS AND RESULTS: Micro-organisms were grown in De Man, Rogosa, Sharpe (MRS) or yeast medium (YM) medium and harvested in the stationary phase of growth. The thermotolerance in skim milk (D and Z values), the survival of spray drying at different outlet air temperatures and subsequent storage in different conditions during 150 days were studied. The resistance to the heat treatments was higher in Lact. plantarum compared to Lact. kefir and S. lipolytica. The three micro-organisms studied varied considerably in their ability to survive to spray drying processes. Lactobacillus plantarum showed the highest survival rate for all the tested outlet air temperatures and also to the further storage in the dried state. The survival rates of Lact. kefir and S. lipolytica through drying and subsequent storage in the dried state decreased when the drying outlet air temperatures increased. CONCLUSIONS: Spray drying is a suitable method to preserve micro-organisms isolated from kefir grains. A high proportion of cells were still viable after 80 days of storage at refrigerated temperatures. SIGNIFICANCE AND IMPACT OF STUDY: It is the first report about spray-dried probiotic strains isolated from kefir grain and contributes to the knowledge about these micro-organisms for their future application in novel dehydrated products.

Lactobacilli antagonize biological effects of enterohaemorrhagic Escherichia coli in vitro.

AIMS: To assess the effect of two lactobacilli on the biological activity of enterohaemorrhagic Escherichia coli (EHEC) in vitro. METHODS AND RESULTS: Strains CIDCA 133 (Lactobacillus delbrueckii subsp. lactis) and CIDCA 83114 (Lactobacillus plantarum) were studied. Hep-2 cells were used as an in vitro model to assess the biological effect of a clinical isolate of EHEC. Preincubation of cell monolayers with lactobacilli before EHEC prevented detachment of eukaryotic cells and minimizes both F-actin rearrangements and morphological alterations. Interestingly, the protective effect could not be ascribed to pathogen exclusion. In addition, viability of the lactobacilli was not necessary for protection and other species of the genus Lactobacillus failed to protect eukaryotic cells. CONCLUSIONS: Our results suggest that lactobacilli are antagonizing virulence mechanisms of EHEC either by modification of the microenvironment or by interfering with the signalling cascades triggered by the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings give a rationale basis for the use of specific probiotic strains for the prophylaxis and prevention of intestinal infections due to EHEC.

Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis.

Eight Lactobacillus kefir strains isolated from different kefir grains were tested for their ability to antagonize Salmonella enterica serovar Enteritidis (Salmonella enteritidis) interaction with epithelial cells. L. kefir surface properties such as autoaggregation and coaggregation with Salmonella and adhesion to Caco-2/TC-7 cells were evaluated. L. kefir strains showed significantly different adhesion capacities, six strains were able to autoaggregate and four strains coaggregated with Salmonella. Coincubation of Salmonella with coaggregating L. kefir strains significantly decreased its capacity to adhere to and to invade Caco-2/TC-7 cells. This was not observed with non coaggregating L. kefir strains. Spent culture supernatants of L. kefir contain significant amounts of S-layer proteins. Salmonella pretreated with spent culture supernatants (pH 4.5-4.7) from all tested L. kefir strains showed a significant decrease in association and invasion to Caco-2/TC-7 cells. Artificially acidified MRS containing lactic acid to a final concentration and pH equivalent to lactobacilli spent culture supernatants did not show any protective action. Pretreatment of this pathogen with spent culture supernatants reduced microvilli disorganization produced by Salmonella. In addition, Salmonella pretreated with S-layer proteins extracted from coaggregating and non coaggregating L. kefir strains were unable to invade Caco-2/TC-7 cells. After treatment, L. kefir S-layer protein was detected associated with Salmonella, suggesting a protective role of this protein on association and invasion.

Characterization of liposomes coated with S-layer proteins from lactobacilli.

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.

Lactobacillus delbrueckii subsp lactis strain CIDCA 133 inhibits nitrate reductase activity of Escherichia coli.

The aim of the present work was to investigate the effect of strain CIDCA 133 on the nitrate reductase activity of a non-pathogenic Escherichia coli strain. Suspensions containing different ratios of the strains under study were coincubated in MRS or MRS without glucose. In some experiments lactobacilli were killed by UV treatment. The nitrate reductase activity was determined by using a diazotization reaction for nitrite. Presence of live lactobacilli leads to a dose-response diminution in the specific nitrate reductase of E. coli even when no acidification occurred. Killing of lactobacilli by UV treatment completely abolished the anti-nitrate reductase effect. In addition, the effect was only partially observed with filtered spent culture supernatants of lactobacilli. Lactobacillus delbrueckii subsp lactis strain CIDCA 133 is able to antagonize the nitrate reductase activity of E. coli. This effect is neither due to a diminution of the viability of E. coli nor is depending on the acidification of the medium by the lactobacilli. Viability is needed for maximal anti-nitrate reductase activity. Modulation of undesirable enzymatic activities of intestinal microorganisms by means of selected microorganisms constitutes a further insight on the mechanisms by which probiotics lead to beneficial effects. Administration of probiotic strains able to modulate microbial intestinal activities could lead to a protection of the host against harmful effects of some members of the intestinal microflora.

Enterococcus faecium SF68 enhances the immune response to Giardia intestinalis in mice.

We studied the ability of the probiotic organism Enterococcus faecium SF68 to antagonize Giardia intestinalis infection in mice. Oral feeding of E. faecium strain SF68 starting 7 d before inoculation with Giardia trophozoites significantly increased the production of specific anti-Giardia intestinal IgA and blood IgG. This humoral response was mirrored at the cellular level by an increased percentage of CD4(+) T cells in the Peyer's patches and in the spleens of SF68-fed mice. The improvement of specific immune responses in probiotic-fed mice was associated with a diminution in the number of active trophozoites in the small intestine as well as decreased shedding of fecal Giardia antigens (GSA65 protein). The ability of SF68 to stimulate the immune system at both mucosal and systemic levels highlights mechanisms by which this probiotic might antagonize pathogens in vivo. Taken together, the data demonstrate the strong potential of strain SF68 to prevent protozoa from causing intestinal infections.

Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration.

AIM: This work determines the efficiency of trehalose on the preservation by heat or osmotic drying of a strain of Lactobacillus delbrueckii ssp. bulgaricus. Cell recovery at different trehalose concentrations during drying correlated with the surface properties and osmotic response of cells after rehydration. METHODS AND RESULTS: Bacteria were dried in the presence of glycerol, trehalose, sucrose at 70 degrees C and at 20 degrees C. Trehalose attenuates the loss of viability at 0.25 m. At this concentration, the osmotic response and zeta potential of the bacteria were comparable with the nondried ones. CONCLUSIONS: Trehalose diminishes significantly the damage produced by dehydration both when the bacteria are dried by heating or subjected to osmotic dehydration. This effect appears related to the preservation of the permeability to water and the surface potential of the bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Dehydration occurring during heating or during osmosis appears to have similar effects. As dehydration-induced damage is in correlation with osmotic response recovery and is hindered or buffered by the presence of trehalose, it may be related to water eliminated from biological structures involved in water permeation.

Effect of bile on the lipid composition and surface properties of bifidobacteria.

AIM: The changes produced on the bacterial surface of Bifidobacteria cells when they are grown in bile were compared with those provoked by bile added to bacteria grown in the absence of bile. METHODS AND RESULTS: The adhesive properties, the zeta potential and the lipid composition of Bifidobacterial strains, isolated from human faeces and grown in MRS medium, were determined. Bacteria grown in MRS with bile showed a loss of adherence and autoaggregation in correlation with a decrease in the surface hydrophobicity in comparison to those grown in MRS without bile, concomitant with the absence of two glycolipids, the increase of sugar content and minor changes in fatty acid composition. The surface changes caused by bile shock on bacteria grown in bile-free medium were much less pronounced and, in addition, no effect on the lipid composition was apparent. CONCLUSIONS: The comparison of the results indicates that bile action on surface properties is related to metabolic changes. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term exposure of bacteria to bile may cause metabolic changes affecting their adhesive properties irreversibly. This may be taken as a criterion to define the probiotic properties of different strains.

Inhibition of Giardia intestinalis by extracellular factors from Lactobacilli: an in vitro study.

The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G(1) phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.

Chemical and microbiological characterisation of kefir grains.

Chemical and microbiological composition of four Argentinean kefir grains from different sources as well as characteristics of the corresponding fermented milk were studied. Kefir grains CIDCA AGK1, AGK2 and AGK4 did not show significant differences in their chemical and microbiological composition. In contrast, protein and yeast content of AGK3 was higher than in the other grains. Although grain microflora comprised lactobacilli, lactococcus, acetic acid bacteria and yeast, we found an important difference regarding species. Lactococcus lactis subsp. lactis, Lactobacillus kefir, Lactobacillus plantarum, Acetobacter and Saccharomyces were present in all types of kefir grain. While Leuconostoc mesenteroides was only isolated from grains CIDCA AGK1 and Lactococcus lactis subsp. lactis biovar diacetylactis, Lactobacillus parakefir and Kluyveromyces marxianus were only isolated from CIDCA AGK2 grains. All grains produced acid products with pH between 3.5 and 4.0. The apparent viscosity of AGK1 fermented milk was greater than the product obtained with AGK4. All fermented milks had inhibitory power towards Escherichia coli but AGK1 and AGK2 supernatants were able to halt the bacterial growth for at least 25 h. Grain weight increment in AGK1, AGK2 and AGK3 during growth in milk did not show significant differences. Despite their fermenting activity, AGK4 grains did not increase their weight.

Enzyme-based most probable number method for the enumeration of Bifidobacterium in dairy products.

An enzyme-based assay in combination with the most probable number (MPN) technique was developed for the enumeration of bifidobacteria. The assay employs the detection of fructose-6-phosphate phosphoketolase (F6PPK) activity as an indicator of the presence of bifidobacteria. The method was validated against viable counts and optimized with respect to selective media in order to quantitatively assess bifidobacteria in dairy products and other probiotic preparations. Several commercial products and homemade fermented milks were analyzed. Counts of bifidobacteria ranged from 10(7) to 10(8) MPN/ml in commercial products and homemade fermented milks. Commercial starters provided by Argentinean industries had between 10(7) and 10(11) MPN/ml. The results obtained in this study suggest that the combination of F6PPK activity determination and the MPN methodology allows an accurate determination of Bifidobacterium in pure cultures, dairy products, and other probiotic preparations.

DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based polymerase chain reaction.

DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.

Fatty acid composition and freeze-thaw resistance in lactobacilli.

The fatty acid composition and freeze-thaw resistance of eight strains of thermophilic lactobacilli were studied. Seven of these contained the same polar and neutral lipids, the five major components making up 90% of the cellular fatty acid pool being 14:0, 16:0, 16:1, 18:1 and C19 cyclopropane (cyc19:0). Strain comparison by means of cluster analysis based on the fatty acid ratios using the overlap coefficient revealed two well defined clusters. One was formed by three strains of species Lactobacillus delbrueckii subsp. lactis and Lb. delbrueckii subsp. delbrueckii, the other included five strains of the species Lb. delbrueckii subsp. bulgaricus, Lb. acidophilus and Lb. helveticus. Resistance of strains with a high content of unsaturated fatty acids (66-70%) decreased with increasing cyc19:0 concentrations. In contrast, in strains with a low concentration of unsaturated fatty acids (42-49%), increasing cyc19:0 levels were associated with increased freeze-thaw resistance.

Technological properties of milks fermented with thermophilic lactic acid bacteria at suboptimal temperature.

In the present work the synergistic relationship between different strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus was studied at optimal (44 degrees C) and suboptimal temperatures (30 degrees C). Acidification, viscosity, whey syneresis, and bacterial concentration of the final product were evaluated on single-strain and mixed cultures after 24 h at 30 degrees C and 6 h at 44 degrees C. Three pairs of strains (LBB + CP2, LBP + CP2, and LBR + CP2) showed synergistic effect, which was reflected by the viscosity and syneresis of the coagulum. These results were more significant when cultures were incubated at 30 degrees C, reaching apparent viscosity values of 19 to 28 mPa x s. On the other hand, lactobacilli cultures enhanced the growth of two streptococci strains (CP2 and CP4). These results were confirmed by cultures of streptococci supplemented with supernatants of culture of lactobacilli. Those supernatants stimulate the viscosity produced by CP2 and CP4 strains and reduce the syneresis of all cultures of streptococci. Neither the increase of viscosity nor reduction of syneresis could be attributed to a decrease of pH.

Inhibitory power of kefir: the role of organic acids.

Milk and MRS broth fermented with kefir grains from different households were examined for inhibitory activity toward gram-negative and gram-positive strains. Fermented milk obtained with 10 g per 100 ml of inoculum (final pH 3.32 to 4.25) and MRS broth fermented with 1 and 10 g per 100 ml of inocula (final pH 4.18 to 5.25) had inhibitory power demonstrated by spot test and agar well diffusion assay. This inhibitory effect could be assigned to the undissociated form of lactic and acetic acid produced during the fermentation process. Kefir supernatants inhibited the growth of Escherichia coli 3 in nutrient broth at 37 degrees C for 24 h. However, supernatants of yogurt or milk artificially acidified with lactic and acetic acids allowed the growth of E. coli 3 in the same conditions. A bacteriostatic effect of milk fermented with kefir grains over E. coli 3 was also demonstrated.

An enzymatic-colorimetric assay for the quantification of Bifidobacterium.

An enzymatic-colorimetric assay for the quantification of Bifidobacterium was developed. The method, based upon the standard detection of fructose-6-phosphate phosphoketolase activity, was optimized with respect to bacterial cell pretreatment, time of incubation, and substrate concentration. The relationship between bacterial biomass and phosphoketolase activity was linear in a wide spectrum of bacterial densities. Higher sensitivity over the standard method was achieved by using 0.25% Triton X-100 in the reaction mixture to pretreat the bacterial cells. Because autoaggregation is a frequent feature among Bifidobacterium strains, this simple and reproducible method offers good advantage over viable plate count and turbidimetric techniques. The methodology can also be applied to the assessment of adherent Bifidobacterium strains to human epithelial cells.

Screening of bile resistance and bile precipitation in lactic acid bacteria and bifidobacteria.

A modification of the ecometric method was developed for a rapid screening of bile resistance in lactic acid bacteria and bifidobacteria. Validation of the MEM bile assay (modified ecometric method) was performed comparing the bile resistance index (RIbile) and the bile resistance ratio (R%). Most Bifidobacterium strains assayed were bile sensitive (83.3%), while only 62.1% of lactobacilli showed that behavior. Some bifidobacterial strains (55.6%) showed a crystalline precipitate when grown on solid medium supplemented with 0.5% ox bile. The crystalline structures produced by B. pseudolongum CIDCA 531 were isolated and analyzed by optical and scanning electron microscopy, thin-layer chromatography, melting point, and specific cholesterol reactions. Those studies confirmed the presence of cholesterol in these crystalline structures. On the other hand, none of the lactobacilli and streptococci studied had the ability to produce crystalline precipitates.