Bioinformatic approaches for studying the microbiome of fermented food.

High-throughput DNA sequencing-based approaches continue to revolutionise our understanding of microbial ecosystems, including those associated with fermented foods. Metagenomic and metatranscriptomic approaches are state-of-the-art biological profiling methods and are employed to investigate a wide variety of characteristics of microbial communities, such as taxonomic membership, gene content and the range and level at which these genes are expressed. Individual groups and consortia of researchers are utilising these approaches to produce increasingly large and complex datasets, representing vast populations of microorganisms. There is a corresponding requirement for the development and application of appropriate bioinformatic tools and pipelines to interpret this data. This review critically analyses the tools and pipelines that have been used or that could be applied to the analysis of metagenomic and metatranscriptomic data from fermented foods. In addition, we critically analyse a number of studies of fermented foods in which these tools have previously been applied, to highlight the insights that these approaches can provide.

Gut microbiota composition correlates with diet and health in the elderly.

Alterations in intestinal microbiota composition are associated with several chronic conditions, including obesity and inflammatory diseases. The microbiota of older people displays greater inter-individual variation than that of younger adults. Here we show that the faecal microbiota composition from 178 elderly subjects formed groups, correlating with residence location in the community, day-hospital, rehabilitation or in long-term residential care. However, clustering of subjects by diet separated them by the same residence location and microbiota groupings. The separation of microbiota composition significantly correlated with measures of frailty, co-morbidity, nutritional status, markers of inflammation and with metabolites in faecal water. The individual microbiota of people in long-stay care was significantly less diverse than that of community dwellers. Loss of community-associated microbiota correlated with increased frailty. Collectively, the data support a relationship between diet, microbiota and health status, and indicate a role for diet-driven microbiota alterations in varying rates of health decline upon ageing.

Dietary Conjugated Linoleic Acid-Enriched Cheeses Influence the Levels of Circulating n-3 Highly Unsaturated Fatty Acids in Humans.

n-3 highly unsaturated fatty acids (n-3 HUFA) directly and indirectly regulate lipid metabolism, energy balance and the inflammatory response. We investigated changes to the n-3 HUFA score of healthy adults, induced by different types and amounts of conjugated linoleic acid (CLA)-enriched (ENCH) cheeses consumed for different periods of time, compared to dietary fish oil (FO) pills (500 mg, each containing 100 mg of eicosapentaenoic and docosahexaenoic acids­EPA+DHA) or α-linolenic acid (ALA)-rich linseed oil (4 g, containing 2 g of ALA). A significant increase in the n-3 HUFA score was observed, in a dose-dependent manner, after administration of the FO supplement. In terms of the impact on the n-3 HUFA score, the intake of ENCH cheese (90 g/day) for two or four weeks was equivalent to the administration of one or two FO pills, respectively. Conversely, the linseed oil intake did not significantly impact the n-3 HUFA score. Feeding ENCH cheeses from different sources (bovine, ovine and caprine) for two months improved the n-3 HUFA score by increasing plasma DHA, and the effect was proportional to the CLA content in the cheese. We suggest that the improved n-3 HUFA score resulting from ENCH cheese intake may be attributed to increased peroxisome proliferator-activated receptor alpha (PPAR-α) activity. This study demonstrates that natural ENCH cheese is an alternative nutritional source of n-3 HUFA in humans.

Kefir4All, a citizen science initiative to raise awareness of the roles that microbes play in food fermentation.

Microorganisms are ubiquitous in nature and are central to human, animal, environmental, and planetary health. They play a particularly important role in the food chain and the production of high-quality, safe, and health-promoting foods, especially fermented foods. This important role is not always apparent to members of the public. Here, we describe Kefir4All, a citizen science project designed to provide the general public with an opportunity to expand their awareness, knowledge, and practical skills relating to microbiology, introduced through the medium of producing fermented food, i.e., milk kefir or water kefir. During the course of Kefir4All, 123 citizen scientists, from second-level school and non-school settings, participated in a study to track changes in the microbial composition of kefirs, by performing and recording details of milk kefir or water kefir fermentations they performed in their homes or schools over the 21-week project. At the start of the study, the citizen scientists were provided with milk or water kefir grains to initiate the fermentations. Both types of kefir grain are semi-solid, gelatinous-like substances, composed of exopolysaccharides and proteins, containing a symbiotic community of bacteria and yeast. The experimental component of the project was complemented by a number of education and outreach events, including career talks and a site visit to our research center (Kefir Day). At the end of the study, a report was provided to each citizen scientist, in which individualized results of their fermenting activities were detailed. A number of approaches were taken to obtain feedback and other insights from the citizen scientists. Evaluations took place before and after the Kefir4All project to gauge the citizen scientist's self-reported awareness, knowledge, and interest in microbiology and fermented foods. Further insights into the level of citizen science participation were gained through assessing the number of samples returned for analysis and the level of participation of the citizen scientists throughout the project. Notably, the survey results revealed a self-reported, increased interest in, and general knowledge of, science among the Kefir4All citizen scientists after undertaking the project and a willingness to take part in further citizen science projects. Ultimately, Kefir4All represents an example of the successful integration of citizen science into existing education and research systems.

Alterations in intestinal microbiota of elderly Irish subjects post-antibiotic therapy.

OBJECTIVES: The human intestinal microbiota composition alters naturally with age, but is unusually perturbed by antibiotic therapy. The impact of antibiotic therapy on the composition of the intestinal microbiota of a cross-section of elderly Irish subjects (n = 185, ≥ 65 years) was investigated, taking into consideration their residence location. METHODS: Forty-two of the 185 elderly subjects were treated with at least one antibiotic within 1 month prior to faecal microbiota profiling. The residence locations of the subjects varied from long-term nursing care and rehabilitation wards to day hospitals and the community. RESULTS: Culture-dependent methods indicated that faecal Bifidobacterium spp. numbers were significantly reduced following antibiotic treatment (P = 0.004, 7-fold reduction), while levels of Lactobacillus spp. and Enterobacteriaceae were unaffected. The largest decrease in Bifidobacterium spp. numbers was linked to the administration of nucleic acid synthesis inhibitors (P = 0.004, 23-fold reduction). Microbiota profiling revealed a significant compositional change across nine genera following antibiotic therapy, including a relative increase in Lactobacillus spp. (P = 0.031), as well as a decrease in the number of genera identified in the antibiotic-treated subjects (n = 58), when compared with untreated subjects (n = 79). More alterations in the intestinal microbiota were observed post-nucleic acid synthesis inhibitor therapy, most notably a decrease in relative Faecalibacterium spp. numbers (P < 0.001). CONCLUSIONS: The impact of antibiotic therapy on the intestinal microbiota in the elderly should be considered for long-term health effects, and differential susceptibility may require the development of products (e.g. prebiotics and probiotics) for at-risk subjects.

Analysis of the milk kefir pan-metagenome reveals four community types, core species, and associated metabolic pathways.

A comprehensive metagenomics-based investigation of the microorganisms present within milk kefir communities from across the globe was carried out with a view to defining the milk kefir pan-metagenome, including details relating to core and non-core components. Milk kefir samples, generated by inoculating full fat, pasteurized cow's milk with 64 kefir grains sourced from 25 different countries, were analyzed. We identified core features, including a consistent pattern of domination by representatives from the species Lactobacillus helveticus or the sub-species Lactobacillus kefiranofaciens subsp. kefiranofaciens, Lactococcus lactis subsp. lactis or Lla. cremoris subsp. cremoris in each kefir. Notably, even in kefirs where the lactococci did not dominate, they and 51 associated metabolic pathways were identified across all metagenomes. These insights can contribute to future efforts to create tailored kefir-based microbial communities for different applications and assist regulators and producers to ensure that kefir products have a microbial composition that reflects the artisanal beverage.

Modulation of the gut microbiome with nisin.

Nisin is a broad spectrum bacteriocin used extensively as a food preservative that was identified in Lactococcus lactis nearly a century ago. We show that orally-ingested nisin survives transit through the porcine gastrointestinal tract intact (as evidenced by activity and molecular weight determination) where it impacts both the composition and functioning of the microbiota. Specifically, nisin treatment caused a reversible decrease in Gram positive bacteria, resulting in a reshaping of the Firmicutes and a corresponding relative increase in Gram negative Proteobacteria. These changes were mirrored by the modification in relative abundance of pathways involved in acetate, butyrate (decreased) and propionate (increased) synthesis which correlated with overall reductions in short chain fatty acid levels in stool. These reversible changes that occur as a result of nisin ingestion demonstrate the potential of bacteriocins like nisin to shape mammalian microbiomes and impact on the functionality of the community.

Lactobacillus gasseri APC 678 Reduces Shedding of the Pathogen Clostridium difficile in a Murine Model.

Clostridium difficile is a common cause of health-care acquired diarrhea, resulting in a spectrum of disease from mild diarrhea to life-threatening illness. Sixty Lactobacillus strains were screened for anti-C. difficile activity using a co-culture method. Based on their ability to inhibit C. difficile, L. gasseri APC 678 and L. rhamnosus DPC 6111 were selected for study in a murine model of C. difficile infection. L. gasseri ATCC 33323, was included as a control. It was established that, relative to control mice not fed Lactobacillus, feeding with L. gasseri APC 678 resulted in a significant reduction by day 7 (8-fold, p = 0.017) of viable C. difficile VPI 10463 in the feces of mice. In contrast, neither L. rhamnosus DPC 6111 nor L. gasseri ATCC 33323 significantly reduced fecal C. difficile shedding. Sequencing of the cecal microbiota showed that in mice fed L. gasseri APC 678 there was a significant increase in bacterial diversity across a number of indices when compared to the control or other Lactobacillus-fed groups. There was no significant change in the relative abundance of Firmicutes or Bacteroidetes in the group fed L. gasseri APC 678 relative to the control, while the groups fed L. rhamnosus DPC 6111 or L. gasseri ATCC 33323 showed a significant decrease in the relative abundance of Firmicutes (p = 0.002 and p = 0.019, respectively) and a significant increase in Bacteroidetes (p = 0.002 and p = 0.023, respectively). These results highlight the potential of L. gasseri APC 678 as a live therapeutic agent to target C. difficile infection.

Prevalence and characterization of Clostridium perfringens from the faecal microbiota of elderly Irish subjects.

The aim of this study was to investigate the diversity and composition of the intestinal microbiota of elderly subjects using a combination of culture-dependent techniques and 16S rRNA gene amplicon sequencing. The study was performed as part of the ELDERMET project, in which 368 faecal samples were assessed for viable numbers of Bifidobacterium spp., Lactobacillus spp. and Enterobacteriaceae on selective agar. However, the Bifidobacterium selective medium used also supported the growth of Clostridium perfringens, which appeared as distinct colonies and were subsequently characterized phenotypically and genotypically. All the isolates were confirmed as toxin biotype A producers. In addition, three isolates tested also had the genetic determinants for the ß2 toxin. Of the 368 faecal samples assessed, C. perfringens was detected in 28 samples (7.6%). Moreover, C. perfringens was observed in samples from subjects in all the residence locations assessed but was most prevalent in subjects from long-stay residential care, with 71.4% of the samples (63.2% of the subjects) being from this residence location, and with a shedding level in excess of 10(6) c.f.u. (g faeces)(-1). Microbiota profiling revealed some significant compositional changes across both the family and genus taxonomic levels between the C. perfringens-positive and -negative datasets. Levels of culturable Bifidobacterium spp. and Lactobacillus spp. were significantly (P<0.05) lower in the C. perfringens-positive samples. Sequence-based methods also confirmed a significant difference in the Bifidobacterium spp. level (P<0.05) between both datasets. Taken together, these data suggest that a high viable count [>10(6) c.f.u. (g faeces)(-1)] of C. perfringens in stool samples may be indicative of a less healthy microbiota in the intestine of elderly people in long-stay residential care.

Inhibitory effect of conjugated alpha-linolenic acid from bifidobacteria of intestinal origin on SW480 cancer cells.

In this study, we assessed the ability of six strains of bifidobacteria (previously shown by us to possess the ability to convert linoleic acid to c9, t11-conjugated linoleic acid (CLA) to grow in the presence of alpha-linolenic acid and to generate conjugated isomers of the fatty acid substrate during fermentation for 42 h. The six strains of bifidobacteria were grown in modified MRS (mMRS) containing alpha-linolenic acid for 42 h at 37 degrees C, after which the fatty acid composition of the growth medium was assessed by gas liquid chromatography (GLC). Indeed, following fermentation of one of the strains, namely Bifidobacterium breve NCIMB 702258, in the presence of 0.41 mg/ml alpha-linolenic acid, 79.1% was converted to the conjugated isomer, C18:3 c9, t11, c15 conjugated alpha-linolenic acid (CALA). To examine the inhibitory effect of the fermented oils produced, SW480 colon cancer cells were cultured in the presence of the extracted fermented oil (10-50 microg/ml) for 5 days. The data indicate an inhibitory effect on cell growth (p

Metabolic activity of the enteric microbiota influences the fatty acid composition of murine and porcine liver and adipose tissues.

BACKGROUND: Recent reports suggest that the metabolic activity of the gut microbiota may contribute to the pathogenesis of obesity and hepatic steatosis. OBJECTIVE: The objective was to determine whether the fat composition of host tissues might be influenced by oral administration of commensal bifidobacteria previously shown by us to produce bioactive isomers of conjugated linoleic acid (CLA). DESIGN: Murine trials were conducted in which linoleic acid-supplemented diets were fed with or without Bifidobacterium breve NCIMB 702258 (daily dose of 10(9) microorganisms) to healthy BALB/c mice and to severe combined immunodeficient mice for 8-10 wk. To ensure that the observations were not peculiar to mice, a similar trial was conducted in weanling pigs over 21 d. Tissue fatty acid composition was assessed by gas-liquid chromatography. RESULTS: In comparison with controls, there was an increase in cis-9, trans-11 CLA in the livers of the mice and pigs after feeding with linoleic acid in combination with B. breve NCIMB 702258 (P < 0.05). In addition, an altered profile of polyunsaturated fatty acid composition was observed, including higher concentrations of the omega-3 (n-3) fatty acids eicosapentaenoic acid and docosahexaenoic acid in adipose tissue (P < 0.05). These changes were associated with reductions in the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma (P < 0.05). CONCLUSIONS: These results are consistent with the concept that the metabolome is a composite of host and microbe metabolic activity and that the influence of the microbiota on host fatty acid composition can be manipulated by oral administration of CLA-producing microorganisms.

Intestinal bifidobacteria that produce trans-9, trans-11 conjugated linoleic acid: a fatty acid with antiproliferative activity against human colon SW480 and HT-29 cancer cells.

Bifidobacterium breve species of human intestinal origin have the ability to synthesize cis-9, trans-11 (c9, t11) conjugated linoleic acid (CLA) from free linoleic acid. In this study, the ability of Bifidobacterium species to isomerize C(18) polyunsaturated fatty acids was investigated, and the antiproliferative activities of the two main microbially produced CLA isomers were assessed. Linoleic acid was converted principally to c9, t11 CLA and lesser amounts of t9, t11 CLA, whereas c9, t11 CLA was converted mainly to t9, t11 CLA. Likewise, t10, c12 CLA was converted principally to t9, t11 CLA, which was incorporated into the bacterial cell membranes. To examine the antiproliferative effect of the two main CLA isomers formed, SW480 and HT-29 human colon cancer cells were cultured in the presence of c9, t11 CLA and t9, t11 CLA. The t9, t11 CLA had a more potent antiproliferative effect than c9, t11 CLA. It is tempting to suggest that the ability of Bifidobacterium to produce such bioactive metabolites may be associated with the beneficial effects of bifidobacteria present in the human gastrointestinal tract.