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Sequencing-based analysis of the bacterial and fungal composition of kefir grains and milks from multiple sources.

ABSTRACT: Kefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists.

SUBMITTER: Marsh AJ

PROVIDER: S-EPMC3716650 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Sequencing-based analysis of the bacterial and fungal composition of kefir grains and milks from multiple sources.

Marsh Alan J AJ O'Sullivan Orla O Hill Colin C Ross R Paul RP Cotter Paul D PD

PloS one 20130719 7

Kefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicat ...[more]

PMID: 23894461

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Project description:Kefir grains consist of complex symbiotic mixtures of bacteria and yeasts, and are reported to impart numerous health-boosting properties to milk and water kefir beverages. The objective of this work was to investigate the microbial communities in kefir grains, and explore the possibility of deriving useful probiotic strains from them. A total of 158 microbial strains, representing six fungal and 17 bacterial species, were isolated from milk and water kefir grains collected from a Singapore-based homebrewer. Based on 16S rRNA sequencing, isolated genera included Lactobacillus, Liquorilactobacillus, Lacticaseibacillus, Lentilactobacillus, Leuconostoc, Lactococcus, Acetobacter, Gluconobacter, Oenococcus, Clostridium, Zymomonas, Saccharomyces, Kluyveromyces, Pichia, Lachancea, Candida, and Brettanomyces. To characterize these isolates, a funnel approach, involving numerous phenotypic and genomic screening assays, was applied to identify kefir-derived microbial strains with the highest probiotic potential. Particular focus was placed on examining the pathogen inhibitory properties of kefir isolates toward enteric pathogens which pose a considerable global health burden. Enteric pathogens tested include species of Bacillus, Salmonella, Vibrio, Clostridium, Klebsiella, Escherichia, and Staphylococcus. Well diffusion assays were conducted to determine the propensity of kefir isolates to inhibit growth of enteric pathogens, and a competitive adhesion/exclusion assay was used to determine the ability of kefir isolates to out-compete or exclude attachment of enteric pathogens to Caco-2 cells. Seven bacterial strains of Lentilactobacillus hilgardii, Lacticaseibacillus paracasei, Liquorilactobacillus satsumensis, Lactobacillus helveticus, and Lentilactobacillus kefiri, were ultimately identified as potential probiotics, and combined to form a "kefir probiotics blend." Desirable probiotic characteristics, including good survival in acid and bile environments, bile salt hydrolase activity, antioxidant activity, non-cytotoxicity and high adhesion to Caco-2 cells, and a lack of virulence or antimicrobial resistance genes. In addition, vitamin and γ-aminobutyric acid (GABA) synthesis genes, were identified in these kefir isolates. Overall, probiotic candidates derived in this study are well-characterized strains with a good safety profile which can serve as novel agents to combat enteric diseases. These kefir-derived probiotics also add diversity to the existing repertoire of probiotic strains, and may provide consumers with alternative product formats to attain the health benefits of kefir.

Project description:The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) isolated from Tibetan kefir grains. Three Lactobacillus isolates identified as Lactobacillus acidophilus LA15, Lactobacillus plantarum B23 and Lactobacillus kefiri D17 that showed resistance to acid and bile salts were selected for further evaluation of their probiotic properties. The 3 selected strains expressed high in vitro adherence to Caco-2 cells. They were sensitive to gentamicin, erythromycin and chloramphenicol and resistant to vancomycin with MIC values of 26 µg/ml. All 3 strains showed potential bile salt hydrolase (BSH) activity, cholesterol assimilation and cholesterol co-precipitation ability. Additionally, the potential effect of these strains on plasma cholesterol levels was evaluated in Sprague-Dawley (SD) rats. Rats in 4 treatment groups were fed the following experimental diets for 4 weeks: a high-cholesterol diet, a high-cholesterol diet plus LA15, a high-cholesterol diet plus B23 or a high-cholesterol diet plus D17. The total cholesterol, triglyceride and low-density lipoprotein cholesterol levels in the serum were significantly (P<0.05) decreased in the LAB-treated rats compared with rats fed a high-cholesterol diet without LAB supplementation. The high-density lipoprotein cholesterol levels in groups B23 and D17 were significantly (P<0.05) higher than those in the control and LA15 groups. Additionally, both fecal cholesterol and bile acid levels were significantly (P<0.05) increased after LAB administration. Fecal lactobacilli counts were significantly (P<0.05) higher in the LAB treatment groups than in the control groups. Furthermore, the 3 strains were detected in the rat small intestine, colon and feces during the feeding trial. The bacteria levels remained high even after the LAB administration had been stopped for 2 weeks. These results suggest that these strains may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.

Project description:Research backgroundKefir is a natural probiotic drink traditionally produced by milk fermentation using kefir grains. Kefir grains are composed of a complex population of bacteria and yeasts embedded in a polysaccharide-protein matrix. The geographic origin of kefir grains may largely influence their microbial composition and the associated kefir drink properties. Although the detailed bacterial composition of kefir grains from several geographic regions has been reported, to date, analogous data about the microbiome of Greek kefir are lacking. Hence, the aim of this study is to investigate the structure and the diversity of the bacterial community of Greek kefir grains.Experimental approachThe bacterial community structure and diversity of two different kefir grains from distant geographic regions in Greece were examined via high-throughput sequencing analysis, a culture-independent metagenomic approach, targeting the 16S rRNA V4 variable region, in order to gain a deeper understanding of their bacterial population diversities.Results and conclusionsFirmicutes (a phylum that includes lactic acid bacteria) was strikingly dominant amongst the identified bacterial phyla, with over 99% of the sequences from both kefir grains classified to this phylum. At the family level, Lactobacillaceae sequences accounted for more than 98% of the operational taxonomic units (OTUs), followed by Ruminococcaceae, Lahnospiraceae, Bacteroidaceae and other bacterial families of lesser abundance. Α relatively small number of bacterial genera dominated, with Lactobacillus kefiranofaciens being the most abundant in both kefir grains (95.0% of OTUs in kefir A and 96.3% of OTUs in kefir B). However, a quite variable subdominant population was also present in both grains, including bacterial genera that have been previously associated with the gastrointestinal tract of humans and animals, some of which are believed to possess probiotic properties (Faecalibacterium spp., Bacteroides spp., Blautia spp.). Differences among the bacterial profiles of the two grains were very small indicating a high homogeneity despite the distant geographic origin.Novelty and scientific contributionThis is the first study to deeply explore and report on the bacterial diversity and species richness of Greek kefir.

Dataset Information

Sequencing-based analysis of the bacterial and fungal composition of kefir grains and milks from multiple sources.

Publications

Sequencing-based analysis of the bacterial and fungal composition of kefir grains and milks from multiple sources.

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