Polyphasic characterization of the lactic acid bacteria in kefir

The lactic acid bacteria of kefir were isolated and characterized using phenotypical, biochemical, and genotypical methods. Polyphasic analyses of results permitted the identification of the microflora to the strain level. The genus Lactobacillus was represented by the species Lb. kefir and Lb. kefiranofaciens. Both subspecies of Lactococcus lactis (lactis and cremoris) were isolated. Leuconostoc mesenteroides subsp. cremoris was also found. The kefir studied contained few species of lactic acid bacteria but showed a high number of different strains. We found that the polyphasic analysis approach increases the confidence in strain determination. It helped confirm strain groupings and it showed that it could have an impact on the phylogeny of the strains.     

Kefir alleviates obesity and hepatic steatosis in high-fat diet-fed mice by modulation of gut microbiota and mycobiota: targeted and untargeted community analysis with correlation of biomarkers

Kefir is a probiotic beverage containing over 50 species of lactic acid bacteria and yeast. In this study, the anti-obesity and anti-non-alcoholic fatty liver disease (NAFLD) effects of kefir were comprehensively addressed along with targeted and untargeted community analysis of the fecal microbiota in a high-fat diet (HFD)-induced obese mouse model. HFD-fed C57BL/6 mice were orally administrated either kefir or milk (control) once a day for 12 weeks, and body and organ weight, fecal microbiota and mycobiota, histopathology, blood cholesterol and cytokines and gene expressions were analyzed. Compared to the control, mice in the kefir group exhibited a significantly lower body weight (34.18 g vs. 40.24 g; p=0.00004) and histopathological liver lesion score (1.13 vs. 3.25; p=0.002). Remarkably, the kefir-fed mice also harbored more Lactobacillus/Lactococcus (7.01 vs. 6.32 log CFU/g), total yeast (6.07 vs. 5.01 log CFU/g) and Candida (5.56 vs. 3.88 log CFU/g). Kefir administration also up-regulated genes related to fatty acid oxidation, PPARα and AOX, in both the liver and adipose tissue (PPARα, 2.95- and 2.15-fold; AOX, 1.89- and 1.9-fold, respectively). The plasma concentration of IL-6, a proinflammatory marker, was significantly reduced following kefir consumption (50.39 pg/ml vs. 111.78 pg/ml; p=0.03). Strikingly, the populations of Lactobacillus/Lactococcus, total yeast and Candida were strongly correlated with PPARα gene expression in adipose and hepatic tissue (r=0.599, 0.580 and 0.562, respectively). These data suggest that kefir consumption modulates gut microbiota and mycobiota in HFD-fed mice, which prevents obesity and NAFLD via promoting fatty acid oxidation.     

Lactic acid bacteria and yeasts in kefir grains and kefir made from them

In an investigation of the changes in the microflora along the pathway: kefir grains (A)→kefir made from kefir grains (B)→kefir made from kefir as inoculum (C), the following species of lactic acid bacteria (83–90%) of the microbial count in the grains) were identified: Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp. pseudoplantarum and Lactobacillus brevis. Yeasts (10–17%) identified were Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, Candida inconspicua and Candida maris. In the microbial population of kefir grains and kefir made from them the homofermentative lactic streptococci (52–65% and 79–86%, respectively) predominated. Within the group of lactobacilli, the homofermentative thermophilic species L. delbrueckii subsp. bulgaricus and L. helveticus (70–87% of the isolated bacilli) predominated. Along the pathway A→B→C, the streptococcal proportion in the total kefir microflora increased by 26–30% whereas the lactobacilli decreased by 13–23%. K. marxianus var. lactis was permanently present in kefir grains and kefirs, whereas the dominant lactose-negative yeast in the total yeast flora of the kefir grains dramatically decreased in kefir C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 1–6 DOI: 10.1038/sj/jim/7000186 Received 02 August 2000/ Accepted in revised form 15 July 2001     

Whey-cheese production using freeze-dried kefir culture as a starter

AIMS: The aim of the present study was to evaluate the use of a freeze-dried kefir culture in the production of a novel type of whey-cheese similar to traditional Greek Myzithra-cheese, to achieve improvement of the quality characteristics of the final product and the extension of shelf-life. METHODS AND RESULTS: The use of kefir culture as a starter led to increased lactic acid concentrations and decreased pH values in the final product compared with whey-cheese without starter culture. The effect of the starter culture on production of aroma-related compounds responsible for cheese flavour was also studied using the solid phase microextraction gas chromatography/mass spectrometry technique. Spoilage in unsalted kefir-whey-cheese was observed on the thirteenth and the twentieth day of preservation at 10 and 5 degrees C, respectively, while the corresponding times for unsalted whey-cheese preservation were 11 and 14 days. CONCLUSIONS: The cheeses produced were characterized as high-quality products during the preliminary sensory evaluation. An indication of increased preservation time was attributed to the freeze-dried kefir culture, which also seemed to suppress growth of pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggested the use of kefir culture as a means to extend the shelf-life of dairy products with reduced or no salt content.     

Effects of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> MA2 isolated from Tibet kefir on lipid metabolism and intestinal microflora of rats fed on high-cholesterol diet

The objective of this study was to evaluate the effects of Lactobacillus plantarum MA2, an isolate from Chinese traditional Tibet kefir, on cholesterol-lowering and microflora of rat in vivo. Rats were fed on cholesterol-enriched experimental diet, supplemented with lyophilized L. plantarum MA2 powder, with a dose of 10¹¹ cells/day per mice. The results showed that L. plantarum MA2 feeding significantly lowered serum total cholesterol, low-density lipoprotein cholesterol, and triglycerides level, while there was no change in high-density lipoprotein cholesterol. In addition, liver total cholesterol and triglycerides was also decreased. However, fecal cholesterol and triglycerides was increased significantly (P < 0.05) in comparison with the control. Also, L. plantarum MA2 increased the population of lactic acid bacteria and bifidobacteria in the fecal, but it did not change the number of Escherichia coli as compared to control. Moreover, pH, moisture, and organic acids in the fecal were also measured. The present results indicate the probiotic potential of the L. plantarum MA2 strain in hypocholesterolemic effect and also increasing the probiotic count in the intestine.     

Kefir micro-organisms: their role in grain assembly and health properties of fermented milk

Kefir is a homemade viscous and slightly effervescent beverage obtained by milk fermentation with kefir grains, which are built up by a complex community of lactic acid and acetic acid bacteria and yeasts confined in a matrix of proteins and polysaccharides. The present review summarizes the role of kefir micro-organisms in grain assembly and in the beneficial properties attributed to kefir. The use of both culture-dependent and independent methods has made possible to determine the micro-organisms that constitute this ecosystem. Kefir consumption has been associated with a wide range of functional and probiotic properties that could be attributed to the micro-organisms present in kefir and/or to the metabolites synthetized by them during milk fermentation. In this context, the role of micro-organisms in kefir health promoting properties is discussed with particular attention to the contribution of yeast as well as bioactive metabolites such as lactic and acetic acid, exopolysaccharides and bioactive peptides. Even though many advances on the knowledge of this ancient fermented milk have been made, further studies are necessary to elucidate the complex nature of the kefir ecosystem.     

High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain

Lacticin 3147 is a two-peptide broad spectrum lantibiotic produced by Lactococcus lactis DPC3147 shown to inhibit a number of clinically relevant Gram-positive pathogens. Initially isolated from an Irish kefir grain, lacticin 3147 is one of the most extensively studied lantibiotics to date. In this study, the bacterial diversity of the Irish kefir grain from which L. lactis DPC3147 was originally isolated was for the first time investigated using a high-throughput parallel sequencing strategy. A total of 17 416 unique V4 variable regions of the 16S rRNA gene were analysed from both the kefir starter grain and its derivative kefir-fermented milk. Firmicutes (which includes the lactic acid bacteria) was the dominant phylum accounting for > 92% of sequences. Within the Firmicutes, dramatic differences in abundance were observed when the starter grain and kefir milk fermentate were compared. The kefir grain-associated bacterial community was largely composed of the Lactobacillaceae family while Streptococcaceae (primarily Lactococcus spp.) was the dominant family within the kefir milk fermentate. Sequencing data confirmed previous findings that the microbiota of kefir milk and the starter grain are quite different while at the same time, establishing that the microbial diversity of the starter grain is not uniform with a greater level of diversity associated with the interior kefir starter grain compared with the exterior.     

Intestinal transit of an orally administered streptomycin-rifampicin-resistant variant of Bifidobacterium longum SBT2928: its long-term survival and effect on the intestinal microflora and metabolism

AIMS: The objectives of this study are to investigate the fate of a streptomycin-rifampicin-resistant variant of Bifidobacterium longum SBT2928 (BL2928SR) and the influence of its oral administration on the composition and metabolism of the intestinal microflora. METHODS AND RESULTS: Intestinal passage of BL2928SR was monitored by a combination of selection with antibiotics and identification by a randomly amplified polymorphic DNA (RAPD)-PCR method. Intestinal microflora was analysed by the method developed by Mitsuoka et al. (1965, 1974). Long-term survival of orally administered BL2928SR in the human intestine was confirmed. BL2928SR ingestion specifically lowered faecal populations of Enterobacteriaceae and clostridia, including lecithinase-positive Clostridium spp. CONCLUSION: BL2928SR and its parent strain, BL2928, are considered to be appropriate candidates for probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: It is clarified that BL2928SR has the ability for long-term survival in the human gastrointestinal tract, and alters the composition and metabolism of the intestinal microflora.     

Microbiological and Biochemical Study of Coffee Fermentation

The coffee fermentation microflora were rich and mainly constituted of aerobic Gram-negative bacilli, with Erwinia and Klebsiella genuses at the highest frequencies. The best population increase was observed with lactic acid bacteria and yeasts, whereas those microorganisms that counted on a pectin medium remained constant during the fermentation step. Qualitatively, lactic acid bacteria belonged mainly to Leuconostoc mesenteroides species but the others microflora were relatively heterogeneous. The microorganisms isolated on pectin medium were Enterobacteriaceae, identified as Erwinia herbicola and Klebsiella pneumoniae, not reported as strong pectolytic strains. Throughout coffee fermentation, 60% of the simple sugars were degraded by the total microflora and not specifically by pectolytic microorganisms. Received: 21 August 2000 / Accepted: 25 September 2000     

Survival of Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3 in different yogurt and kefir combinations as prefermentation contaminant

AIMS: To compare microbiological safety of yogurt, kefir and different combinations of yogurt and kefir samples by using three foodborne pathogenic strains (Escherichia coli O157:H7, Listeria monocytogenes 4b and Yersinia enterocolitica O3) as indicators. METHODS AND RESULTS: Fresh yogurt and kefir drinks were added to pasteurized milk at a 5% rate either separately or together, and then incubated at different temperatures (43 degrees C for yogurt and 30 degrees C for kefir), depending on appropriate growth temperature of their starter microflora. While traditional yogurt was found to be the least suppressive on the three pathogenic micro-organisms, samples obtained from two subsequent fermentation process (samples fermented at 43 degrees C for 3 h and at 30 degrees C for 21 h) were more suppressive than that of traditional kefir. There was no significant survival difference between E. coli O157:H7 and L. monocytogenes 4b in samples tested (P > 0.05), but Y. enterocolitica O3 was more susceptible than other two test strains (P < 0.05). CONCLUSIONS: The microbiological safety of the dairy product fermented at two consecutive periods was superior than that of traditional yogurt or kefir alone. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when yogurt and kefir starter micro-organisms are combined in a milk fermentation process with different time and temperature periods.     

Characterization of lactobacilli towards their use as probiotic adjuncts in poultry

AIMS: A total of 112 strains of lactic acid bacteria of duck origin were studied for their use as a probiotic feed supplement. METHODS AND RESULTS: In vitro studies included aggregation, co-aggregation, cell surface hydrophobicity and adhesion activities on poultry crop cells and human Hep2-cells. Additionally, growth with bile acids (chicken bile, ox gall and taurocholic acid) and tolerance to acidic pH were tested. Among all the isolates, two strains (Lactobacillus animalis TMW 1.972 and Lactobacillus salivarius TMW 1.992) were selected for a survival test in poultry. Monitoring and differentiation of these strains was achieved by selective detection as rifampicin and erythromycin double-resistant mutants. After a single feed administration, both micro-organisms were shown to persist in the crop and caecum of ducks for a period of 18 and 22 days, respectively. For identification of Lact. animalis and Lact. salivarius, two specific PCRs targeted against 16S rDNA were developed. CONCLUSIONS: Within the autochtoneous microflora of ducks, two strains of lactobacilli exhibited strong potential as probiotic adjuncts. The results indicate that the natural gut microflora of poultry serves as an excellent source for optimal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: A general strategy for the selection of probiotic strains is presented. The suggested sequence of tests allows identification of the most promising candidates within complex ecosystems or large strain collections with minimal expenditure.     

A lactic acid bacterium isolated from kimchi ameliorates intestinal inflammation in DSS-induced colitis

Some species of lactic acid bacteria have been shown to be beneficial in inflammatory bowel disease (IBD). In the present study, a strain of lactic acid bacterium (Lactobacillus paracasei LS2) was isolated from the Korean food, kimchi, and was shown to inhibit the development of experimental colitis induced by dextran sulfate sodium (DSS). To investigate the role of LS2 in IBD, mice were fed DSS in drinking water for seven days along with LS2 bacteria which were administered intragastrically to some of the mice, while phosphate-buffered saline (PBS) was administered to others (the controls). The administration of LS2 reduced body weight loss and increased survival, and disease activity indexes (DAI) and histological scores indicated that the severity of colitis was significantly reduced. The production of inflammatory cytokines and myeloperoxidase (MPO) activity also decreased. Flow cytometry analysis showed that the number of Th1 (IFN-γ) population cells was significantly reduced in the LS2-administered mice compared with the controls. The administration of LS2 induced the increase of CD4⁺FOXP3⁺ Treg cells, which are responsible for IL-10. Numbers of macrophages (CD11b⁺ F4/80⁺), and neutrophils (CD11b⁺ Gr-1⁺) among lamina propria lymphocytes (LPL) were also reduced. These results indicate that LS2 has an anti-inflammatory effect and ameliorates DSS-induced colitis.     

Administration of different Lactobacillus strains in fermented oatmeal soup: in vivo colonization of human intestinal mucosa and effect on the indigenous flora.

In vivo colonization by different Lactobacillus strains on human intestinal mucosa of healthy volunteers was studied together with the effect of Lactobacillus administration on different groups of indigenous bacteria. A total of 19 test strains were administered in fermented oatmeal soup containing 5 x 10(6) CFU of each strain per ml by using a dose of 100 ml of soup per day for 10 days. Biopsies were taken from both the upper jejunum and the rectum 1 day before administration was started and 1 and 11 days after administration was terminated. The administration significantly increased the Lactobacillus counts on the jejunum mucosa, and high levels remained 11 days after administration was terminated. The levels of streptococci increased by 10- to 100-fold in two persons, and the levels of sulfite-reducing clostridia in the jejunum decreased by 10- to 100-fold in three of the volunteers 1 day after administration was terminated. In recta, the anaerobic bacterium counts and the gram-negative anaerobic bacterium counts decreased significantly by the end of administration. Furthermore, a decrease in the number of members of the Enterobacteriaceae by 1,000-fold was observed on the rectal mucosa of two persons. Randomly picked Lactobacillus isolates were identified phenotypically by API 50CH tests and genotypically by the plasmid profiles of strains and by restriction endonuclease analysis of chromosomal DNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and identification of lactic acid bacteria from soil using an enrichment procedure

AIMS: To survey, and identify and classify the ecological distribution of lactic acid bacteria from soil in Japan and Taiwan. METHODS AND RESULTS: Acid-producing bacteria were isolated from 68 soil samples, collected from Japan and Taiwan, in the rhizospheres of fruit trees, from the floor of a henhouse and around a horse farm. All isolates were identified by physiological and genetic tests. Thirty-two of the 54 isolates were identified as lactic acid bacteria (LAB), 16 as spore-forming lactic acid bacteria, five as Clostridium and one as Bacillus. These lactic acid bacteria represent five genera: Lactobacillus, Lactococcus, Enterococcus, Leuconostoc and Weissella. CONCLUSIONS: A high rate of isolating lactic acid bacteria was obtained from soil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that soil may be a common source for the isolation of lactic acid bacteria.     

Fermentation properties of gentio-oligosaccharides

AIMS: To investigate the fermentation properties of gentio-oligosaccharides (GOS), as compared to fructo-oligosaccharides (FOS) and maltodextrin in mixed faecal culture. METHODS AND RESULTS: The substrates were incubated in 24 h batch culture fermentations of human faecal bacteria. Fluorescent in situ hybridization was used to determine changes in populations of bifidobacteria, lactobacilli, clostridia, bacteroides, streptococci and Escherichia coli. Gas and short-chain fatty acid (SCFA) production was also measured. GOS gave the largest significant increases in bifidobacteria, lactobacilli and total bacterial numbers during the incubations. However, FOS appeared to be a more selective prebiotic as it did not significantly stimulate growth of bacterial groups which were not probiotic in nature. GOS and maltodextrin produced the highest levels of SCFA. Lowest gas production was seen with GOS and highest with FOS. CONCLUSIONS: GOS possessed bifidogenic activity in vitro. Although fermentation of GOS was not as selective as FOS, gas production was lower. Gas production is often seen as an undesirable side effect of prebiotic consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided the first data on fermentation of GOS in mixed faecal culture. The study has also used molecular microbiology methods (FISH) to quantify bacterial groups. The data extend our knowledge of the selectivity of fermentation of oligosaccharides by the gut microflora.     

Population dynamics and antagonistic potential of enterococci colonizing the phyllosphere of grasses

AIMS: To investigate the spatial and temporal dynamics of enterococci colonizing forage grass and their ability to produce bacteriocins. Methods and RESULTS: Enterococci could be detected on above-ground plant parts throughout the growing season, with high continuity but low cell numbers (2.60 x 101-6.16 x 104 cfu g-1 fresh matter). A total of 750 strains were isolated and identified by their whole-cell protein patterns as Enterococcus faecalis (7.9%), Ent. mundtii (7.9%), Ent. casseliflavus (5.5%), Ent. faecium (5.2%) and Ent. sulfureus (0.1%). The vast majority of the strains (69.7%) formed a homogeneous 16S rDNA genotype that differed from those of known enterococci. A screening for antagonistic activity using an agar spot test revealed that 18.4% of all isolates were potential antagonists. Partially-purified proteins extracted from cell-free culture supernatant fluids of various species were characterized as pH- and heat-stable bacteriocins active against a wide range of lactic acid bacteria, clostridia and Listeria. The producing strains were antagonistically active even on 'phylloplane agar' at temperatures between 4 and 37 degrees C. CONCLUSION: Enterococci are a common part of the epiphytic microflora of grasses, displaying probably some antagonistic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide new information on the distribution, species diversity and antagonistic potential of enterococci in the phyllosphere.     

Antagonistic activity of lactic acid bacteria as probiotics against selected bacteria of the Enterobaceriacae family in the presence of polyols and their galactosyl derivatives

Probiotic lactic acid bacteria were grown on erythritol, xylitol, sorbitol or lactitol and produced various derivatives: gal-erythritol, gal-xylitol, and gal-sorbitol as prebiotics. Galactosyl derivatives of erythritol, xylitol and sorbitol were metabolised by Lactobacillus spp. This resulted in their antagonistic activity against the test microflora. No activity was observed in the presence of xylitol and erythritol. Gal-sorbitol obtained by enzymatic transglycosylation from lactose had the same abilities of inducing the antagonistic activity of lactic acid bacteria that lactitol had.     

Isolation of a novel strain of Butyrivibrio fibrisolvens that isomerizes linoleic acid to conjugated linoleic acid without hydrogenation, and its utilization as a probiotic for animals

AIM: Isolation of a new strain of Butyrivibrio fibrisolvens possessing great capacity to produce conjugated linoleic acid (CLA) in order to utilize as a probiotic for animals. METHODS AND RESULTS: A novel strain (MDT-5) was isolated from the goat rumen, which exclusively converted linoleic acid (LA) to CLA, because of its high LA isomerase activity with virtually no CLA reductase activity. MDT-5 also converted linolenic acid to conjugated linolenic acid that may be more bioactive than CLA. The oral administration of MDT-5 every other day to mice for 2 weeks resulted in increased amounts of CLA in the contents of the large intestine (2.5-fold), as well as in adipose tissue (threefold). Feeding a high-LA diet, as well as prolonging the period of MDT-5 administration, further increased the CLA content in body fat. CONCLUSIONS: MDT-5 has by far greater ability to produce CLA than any other known bacteria. Administration of MDT-5 to mice increases CLA production in the large intestine, which results in increased CLA absorption. SIGNIFICANCE AND IMPACT OF THE STUDY: MDT-5 may be useful in pet animals as a probiotic to provide CLA continuously.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.     

The Effects of Dietary Ferric Iron and Iron Deprivation on the Bacterial Composition of the Mouse Intestine

The influence of dietary ferric iron on the intestinal microbiota of mice was investigated with a view to promoting benign lactic acid bacteria (which have minimal iron requirements) in order to enhance colonization-resistance potential. Three groups of eight mice received a diet differing only in iron content, for a period of 12 weeks. Dietary iron deprivation resulted in overall increased small intestinal bacterial populations, including lactic acid bacteria, but these differences were generally not significant (p > 0.05). With the exception of coliforms, all examined bacterial groups (anaerobes, micro-aerophiles, lactobacilli, and enterococci) were significantly (p < 0.05) elevated in the colons of iron-deprived mice. The relatively low numbers of total anaerobes in the colons of iron-replete and iron-overloaded mice suggested that, as well as promotion of bacteria under iron-deprived condition, provision of ferric iron suppressed bacteria, probably by oxidation of normally reduced environments. Received: 13 October 2000 / Accepted: 20 December 2000