Raw milk kefir: microbiota, bioactive peptides, and immune modulation.

This study aims to characterize the microbiota and peptidomic composition of raw milk kefir, and to address the potential anti-allergic effects of raw milk kefir using validated research models for food allergy. Raw milk kefir was produced after incubation with a defined freeze-dried starter culture. Kefir was sampled during fermentation at seven time intervals. For comparison, kefir was also prepared from heat-treated milk. Peptide compositions were determined for the raw and heated milk, and kefir end products made from these milks. In a murine food allergy model, the two kefir end products were investigated for their allergy modulating effects. In both kefirs, we identified amplicon sequence variants identical to those in the starter culture, matching the bacteria Lactococcus lactis, Streptococcus thermophilus, Leuconostoc and the yeast Debaryomyces. In raw milk kefir, additional sequence variants of Lactococcus lactis and the yeasts Pichia and Galactomyces could be identified, which were absent in heated milk kefir. Analysis of peptide compositions in both kefirs indicated that the number and intensity of peptides drastically increased after fermentation. Heating of the milk negatively affected the diversity of the peptide composition in kefir. Only raw milk kefir suppressed the acute allergic skin response to the food allergen ovalbumin in sensitised mice. These effects coincided with differences in the T-cell compartment, with lower percentages of activated Th1 cells and IFNg production after treatment with kefir made from heated milk. The results of this study indicate specific properties of raw milk kefir that may contribute to its additional health benefits.

Normative cognition and the effects of a probiotic food intervention in first grade children in Côte d'Ivoire.

The cognitive skills critical for success have largely been studied in Western populations, despite the fact that children in low- and middle-income countries are at risk to not reach their full developmental potential. Moreover, scientists should leverage recent discovery to explore means of boosting cognition in at-risk populations. This semi-randomized controlled trial examined normative cognitive development and whether it could be enhanced by consumption of a probiotic food in a sample of 251 4- to 7-year-old children in urban schools in Côte d'Ivoire. Participants completed executive functioning measures at baseline (T1) and 5 months later (T2). After T1, children in one school received a probiotic (N = 74) or placebo (N = 79) fermented dairy food every day they were in school for one semester; children in the other school (N = 98) continued their diet as usual. Children improved on all tests across time (Cohen's d = 0.08-0.30). The effects of probiotic ingestion were inconclusive and are interpreted with caution due to socio-political factors affecting daily administration. Given the general feasibility of the study, we hope that it will serve as an inspiration for future research into child development and sustainable (health-promoting) interventions for school children in developing nations.

Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina.

Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatuspulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

The Two-Way Interaction between the Molecules That Cause Vaginal Malodour and Lactobacilli: An Opportunity for Probiotics.

Vaginal malodour is a sign of dysbiosis. The biogenic amines (BAs) cadaverine, putrescine and tyramine are known to be causative compounds. Recent reports suggest these compounds produced by pathogens might have a role beyond causing malodour; namely inhibiting the growth of lactobacilli bacteria that are crucial in the maintenance of vaginal homeostasis. The aim of this study was to identify whether certain lactobacilli strains could reduce BAs and to evaluate how Lactobacillus species were affected by these compounds. Using LC-MS and HPLC-UV, five Lactobacillus crispatus strains were identified as being capable of significantly reducing BAs from the media under in vitro conditions. Through 16S rRNA gene sequencing of vaginal swabs exposed to Bas, cadaverine was found to reduce the relative abundance of lactobacilli. When L. crispatus was exposed to media supplemented with BAs with an HCl adjusted lower pH, its growth was enhanced, demonstrating the relevance of the maintenance of an acidic vaginal environment. If strains are to be developed for probiotic application to alleviate bacterial vaginosis and other conditions affecting large numbers of women worldwide, their ability to adapt to Bas and regulate pH should be part of the experimentation.

Project Earthrise: Proceedings of the Ninth Annual Conference of inVIVO Planetary Health.

The "Earthrise" photograph, taken on the 1968 Apollo 8 mission, became one of the most significant images of the 20th Century. It triggered a profound shift in environmental awareness and the potential for human unity-inspiring the first Earth Day in 1970. Taking inspiration from these events 50 years later, we initiated Project Earthrise at our 2020 annual conference of inVIVO Planetary Health. This builds on the emergent concept of planetary health, which provides a shared narrative to integrate rich and diverse approaches from all aspects of society towards shared solutions to global challenges. The acute catastrophe of the COVID-19 pandemic has drawn greater attention to many other interconnected global health, environmental, social, spiritual, and economic problems that have been underappreciated or neglected for decades. This is accelerating opportunities for greater collaborative action, as many groups now focus on the necessity of a "Great Transition". While ambitious integrative efforts have never been more important, it is imperative to apply these with mutualistic value systems as a compass, as we seek to make wiser choices. Project Earthrise is our contribution to this important process. This underscores the imperative for creative ecological solutions to challenges in all systems, on all scales with advancing global urbanization in the digital age-for personal, environmental, economic and societal health alike. At the same time, our agenda seeks to equally consider our social and spiritual ecology as it does natural ecology. Revisiting the inspiration of "Earthrise", we welcome diverse perspectives from across all dimensions of the arts and the sciences, to explore novel solutions and new normative values. Building on academic rigor, we seek to place greater value on imagination, kindness and mutualism as we address our greatest challenges, for the health of people, places and planet.

Interstrain Variability of Human Vaginal Lactobacillus crispatus for Metabolism of Biogenic Amines and Antimicrobial Activity against Urogenital Pathogens.

Lactobacillus crispatus is the dominant species in the vagina of many women. With the potential for strains of this species to be used as a probiotic to help prevent and treat dysbiosis, we investigated isolates from vaginal swabs with Lactobacillus-dominated and a dysbiotic microbiota. A comparative genome analysis led to the identification of metabolic pathways for synthesis and degradation of three major biogenic amines in most strains. However, targeted metabolomic analysis of the production and degradation of biogenic amines showed that certain strains have either the ability to produce or to degrade these compounds. Notably, six strains produced cadaverine, one produced putrescine, and two produced tyramine. These biogenic amines are known to raise vaginal pH, cause malodour, and make the environment more favourable to vaginal pathogens. In vitro experiments confirmed that strains isolated from women with a dysbiotic vaginal microbiota have higher antimicrobial effects against the common urogenital pathogens Escherichia coli and Enterococcus faecium. The results indicate that not all L. crispatus vaginal strains appear suitable for probiotic application and the basis for selection should not be only the overall composition of the vaginal microbiota of the host from which they came, but specific biochemical and genetic traits.

Model Selection Reveals the Butyrate-Producing Gut Bacterium Coprococcus eutactus as Predictor for Language Development in 3-Year-Old Rural Ugandan Children.

INTRODUCTION: The metabolic activity of the gut microbiota plays a pivotal role in the gut-brain axis through the effects of bacterial metabolites on brain function and development. In this study we investigated the association of gut microbiota composition with language development of 3-year-old rural Ugandan children. METHODS: We studied the language ability in 139 children of 36 months in our controlled maternal education intervention trial to stimulate children's growth and development. The dataset includes 1170 potential predictors, including anthropometric and cognitive parameters at 24 months, 542 composition parameters of the children's gut microbiota at 24 months and 621 of these parameters at 36 months. We applied a novel computationally efficient version of the all-subsets regression methodology and identified predictors of language ability of 36-months-old children scored according to the Bayley Scales of Infant and Toddler Development (BSID-III). RESULTS: The best three-term model, selected from more than 266 million models, includes the predictors Coprococcus eutactus at 24 months of age, Bifidobacterium at 36 months of age, and language development at 24 months. The top 20 four-term models, selected from more than 77 billion models, consistently include C. eutactus abundance at 24 months, while 14 of these models include the other two predictors as well. Mann-Whitney U tests suggest that the abundance of gut bacteria in language non-impaired children (n = 78) differs from that in language impaired children (n = 61). While anaerobic butyrate-producers, including C. eutactus, Faecalibacterium prausnitzii, Holdemanella biformis, Roseburia hominis are less abundant, facultative anaerobic bacteria, including Granulicatella elegans, Escherichia/Shigella and Campylobacter coli, are more abundant in language impaired children. The overall predominance of oxygen tolerant species in the gut microbiota was slightly higher in the language impaired group than in the non-impaired group (P = 0.09). CONCLUSION: Application of the all-subsets regression methodology to microbiota data established a correlation between the relative abundance of the anaerobic butyrate-producing gut bacterium C. eutactus and language development in Ugandan children. We propose that the gut redox potential and the overall bacterial butyrate-producing capacity in the gut are important factors for language development.

Salivary biomarkers of stress and inflammation in first graders in Côte d'Ivoire: Effects of a probiotic food intervention.

This semi-randomized controlled trial examined the effects of a probiotic food supplement on cortisol and C-reactive protein (CRP) in a sample of 262 four-to seven-year-old children (56% girls) in two economically-disadvantaged schools in an urban setting in Côte d'Ivoire. For one semester, children in one school were randomized to receive a probiotic (N = 79) or placebo (N = 85) fermented dairy food each day they attended school; one child (due to medical reasons) and all children in the other school (N = 98) continued their diets as usual. Children provided two saliva samples at 11:30 on consecutive days at the end of the study. Analyses revealed that the probiotic group had lower cortisol than the placebo or diet-as-usual groups (p = .015); CRP levels were comparable across groups (p = .549). Exploratory analyses suggested that dose and regularity of consumption may impact the biomarkers as well. This study provides the first evidence that a probiotic milk product may lower cortisol in a sample of young, economically-disadvantaged children.

Different Resource Allocation in a Bacillus subtilis Population Displaying Bimodal Motility.

To cope with sudden changes in their environment, bacteria can use a bet-hedging strategy by dividing the population into cells with different properties. This so-called bimodal or bistable cellular differentiation is generally controlled by positive feedback regulation of transcriptional activators. Due to the continuous increase in cell volume, it is difficult for these activators to reach an activation threshold concentration when cells are growing exponentially. This is one reason why bimodal differentiation is primarily observed from the onset of the stationary phase, when exponential growth ceases. An exception is the bimodal induction of motility in Bacillus subtilis, which occurs early during exponential growth. Several mechanisms have been put forward to explain this, including double-negative feedback regulation and the stability of the mRNA molecules involved. In this study, we used fluorescence-assisted cell sorting (FACS) to compare the transcriptomes of motile and nonmotile cells and noted that expression of ribosomal genes is lower in motile cells. This was confirmed using an unstable green fluorescent protein (GFP) reporter fused to the strong ribosomal rpsD promoter. We propose that the reduction in ribosomal gene expression in motile cells is the result of a diversion of cellular resources to the synthesis of the chemotaxis and motility systems. In agreement with this, single-cell microscopic analysis showed that motile cells are slightly shorter than nonmotile cells, an indication of slower growth. We speculate that this growth rate reduction can contribute to the bimodal induction of motility during exponential growth. IMPORTANCE To cope with sudden environmental changes, bacteria can use a bet-hedging strategy and generate different types of cells within a population-so-called bimodal differentiation. For example, a Bacillus subtilis culture can contain both motile and nonmotile cells. In this study, we compared the gene expression between motile and nonmotile cells. It appeared that motile cells express fewer ribosomes. To confirm this, we constructed a ribosomal promoter fusion that enabled us to measure expression of this promoter in individual cells. This reporter fusion confirmed our initial finding. The reallocation of cellular resources from ribosome synthesis toward synthesis of the motility apparatus results in a reduction in growth. Interestingly, this growth reduction has been shown to stimulate bimodal differentiation.

Fermented foods in a global age: East meets West.

Fermented foods and alcoholic beverages have long been an important part of the human diet in nearly every culture on every continent. These foods are often well-preserved and serve as stable and significant sources of proteins, vitamins, minerals, and other nutrients. Despite these common features, however, many differences exist with respect to substrates and products and the types of microbes involved in the manufacture of fermented foods and beverages produced globally. In this review, we describe these differences and consider the influence of geography and industrialization on fermented foods manufacture. Whereas fermented foods produced in Europe, North America, Australia, and New Zealand usually depend on defined starter cultures, those made in Asia and Africa often rely on spontaneous fermentation. Likewise, in developing countries, fermented foods are not often commercially produced on an industrial scale. Although many fermented products rely on autochthonous microbes present in the raw material, for other products, the introduction of starter culture technology has led to greater consistency, safety, and quality. The diversity and function of microbes present in a wide range of fermented foods can now be examined in detail using molecular and other omic approaches. The nutritional value of fermented foods is now well-appreciated, especially in resource-poor regions where yoghurt and other fermented foods can improve public health and provide opportunities for economic development. Manufacturers of fermented foods, whether small or large, should follow Good Manufacturing Practices and have sustainable development goals. Ultimately, preferences for fermented foods and beverages depend on dietary habits of consumers, as well as regional agricultural conditions and availability of resources.

A Comparative Interrupted Times Series on the Health Impact of Probiotic Yogurt Consumption Among School Children From Three to Six Years Old in Southwest Uganda.

Introduction: Following a school milk feeding program in Southwest Uganda, we initiated a probiotic yogurt school feeding program in the same region in 2018. In order to investigate the potential health benefits from probiotic yogurt we conducted an observational study, where we compared the effect of the consumption of locally produced probiotic yogurt containing Lactobacillus rhamnosus yoba 2012 to milk in pre-primary schoolchildren from different schools on the occurrence of respiratory tract infections (common cold) and skin infections (e.g., tinea capitis). Method: A comparative interrupted time series over a period of 3 weeks of baseline followed by 9 weeks of 100 ml of probiotic yogurt or milk consumption for 5 days per week. In total 584 children attending five different schools were followed during consumption of probiotic yogurt and 532 children attending five other schools during consumption of milk. Incidences of respiratory tract infection symptoms and skin infection symptoms, changes in anthropometric indicators and absenteeism were recorded. Results: Over the course of the study period the incidence rate for common cold symptoms decreased faster in the yogurt group than in the milk group (p = 0.09) resulting in a final RR of 0.85 (95% CI: 0.5-1.4) at the end of the observational period. The incidence rate of skin infection related symptoms also reduced faster in the yogurt group compared to the milk group (p < 0.0001) resulting in a relative risk factor (RR) of 0.6 (CI: 0.4-0.9) at the end of the observational period. Anthropometric indicators and level of absenteeism did not show significant differences between yogurt and milk. Conclusion: Notwithstanding the observed positive trend and effect of probiotic yogurt on the incidences of common cold and skin infections, respectively, we consider the results of this comparative interrupted time series inconclusive due to differences in the recorded health parameters between the probiotic yogurt and milk control groups at base line, and fluctuations over the course of the intervention period. An improved study design, with more uniform study groups, a longer intervention period and a third control group without yogurt or milk is required to draw definitive conclusions.

Aflatoxins: Occurrence, Exposure, and Binding to Lactobacillus Species from the Gut Microbiota of Rural Ugandan Children.

Chronic exposure of children in sub-Saharan Africa to aflatoxins has been associated with low birth weight, stunted growth, immune suppression, and liver function damage. Lactobacillus species have been shown to reduce aflatoxin contamination during the process of food fermentation. Twenty-three Lactobacillus strains were isolated from fecal samples obtained from a cohort of rural Ugandan children at the age of 54 to 60 months, typed by 16S rRNA gene sequencing, and characterized in terms of their ability to bind aflatoxin B1 in vitro. Evidence for chronic exposure of these children to aflatoxin B1 in the study area was obtained by analysis of local foods (maize flour and peanuts), followed by the identification of the breakdown product aflatoxin M1 in their urine samples. Surprisingly, Lactobacillus in the gut microbiota of 140 children from the same cohort at 24 and 36 months showed the highest positive correlation coefficient with stunting among all bacterial genera identified in the stool samples. This correlation was interpreted to be associated with dietary changes from breastfeeding to plant-based solid foods that pose an additional risk for aflatoxin contamination, on one hand, and lead to increased intake of Lactobacillus species on the other.

Eighth Annual Conference of inVIVO Planetary Health: From Challenges to Opportunities.

inVIVO Planetary Health (inVIVO) is a progressive scientific movement providing evidence, advocacy, and inspiration to align the interests and vitality of people, place, and planet. Our goal is to transform personal and planetary health through awareness, attitudes, and actions, and a deeper understanding of how all systems are interconnected and interdependent. Here, we present the abstracts and proceedings of our 8th annual conference, held in Detroit, Michigan in May 2019, themed "From Challenges, to Opportunities". Our far-ranging discussions addressed the complex interdependent ecological challenges of advancing global urbanization, including the biopsychosocial interactions in our living environment on physical, mental, and spiritual wellbeing, together with the wider community and societal factors that govern these. We had a strong solutions focus, with diverse strategies spanning from urban-greening and renewal, nature-relatedness, nutritional ecology, planetary diets, and microbiome rewilding, through to initiatives for promoting resilience, positive emotional assets, traditional cultural narratives, creativity, art projects for personal and community health, and exploring ways of positively shifting mindsets and value systems. Our cross-sectoral agenda underscored the importance and global impact of local initiatives everywhere by contributing to new normative values as part of a global interconnected grass-roots movement for planetary health.

Bacterial community dynamics in lait caillé, a traditional product of spontaneous fermentation from Senegal.

Spontaneously fermented food products contain a complex, natural microbial community with potential probiotic activity. The addition of a health-promoting, probiotic bacterium to these products ensures the delivery of that probiotic activity to consumers. Here, we assess the microbial community of a traditional Senegalese milk product produced by spontaneous fermentation, called lait caillé. We produced the lait caillé in a traditional way and added a probiotic starter containing Lactobacillus rhamnosus yoba 2012 to the traditional process. We found various species that are known for their ability to ferment milk, including species from the genera Lactobacillus, Acetobacter, Lactococcus, and Streptococcus. Our results show that the addition of L. rhamnosus to the inoculum, can result in detectable levels of this strain in the final product, ranging between 0.2 and 1 percent of the total bacterial population. Subsequent rounds of fermentation using passive back-slopping without the addition of new L. rhamnosus led to a loss of this strain from the community of fermenting bacteria. Our results suggest that the addition of probiotic strains at every fermentation cycle can enrich the existing complex communities of traditionally fermented lait caillé while traditional bacterial strains remain dominant in the bacterial communities.

Child development, growth and microbiota: follow-up of a randomized education trial in Uganda.

BACKGROUND: Undernutrition impairs child development outcomes and growth. In this follow-up study of an open cluster-randomized intervention trial we examined the effects of an education package delivered to mothers in rural Uganda on their children's development, growth and gut microbiota at 36 months of age. METHODS: The parental trial included 511 mother-child pairs recruited when the children were 6-8 months. In that trial, a nutrition, stimulation and hygiene education was delivered to mothers in the intervention group while the control group received routine health care. A follow-up sample of 155 pairs (intervention n = 77, control n = 78) were re-enrolled when the children were 24 months. Developmental outcomes were assessed with the Bayley Scales of Infant and Toddler Development (BSID-III) composite scores for cognitive (primary endpoint), language and motor development. Development outcomes were also evaluated using the Ages and Stages Questionnaire (ASQ) and the Mullen Scales of Early Learning (MSEL). Other outcomes included growth and gut microbiota composition. RESULTS: The demographic characteristics were not different (P > 0.05) between the intervention and control groups and similar to those of the parental study. The intervention group had higher BSID-III scores than controls, with mean difference 10.13 (95% confidence interval (CI): 3.31-17.05, P = 0.002); 7.59 (1.62-13.66, P = 0.01); 9.00 (2.92-15.40, P = 0.005), for cognitive, language and motor composite scores, respectively. An improvement in the intervention compared to the control group was obtained for both the ASQ and the MSEL scores. The mean difference in height-for-age z-score was higher in the intervention compared to the control group: 0.50 (0.25-0.75, P = 0.0001). Gut microbiota composition did not differ significantly between the two study groups. CONCLUSIONS: The maternal education intervention had positive effects on child development and growth at three years, but did not alter gut microbiota composition. This intervention may be applicable in other low-resource settings. TRIAL REGISTRATION: ClinicalTrials.gov registration number NCT02098031.

Comparative genomics of human Lactobacillus crispatus isolates reveals genes for glycosylation and glycogen degradation: implications for in vivo dominance of the vaginal microbiota.

BACKGROUND: A vaginal microbiota dominated by lactobacilli (particularly Lactobacillus crispatus) is associated with vaginal health, whereas a vaginal microbiota not dominated by lactobacilli is considered dysbiotic. Here we investigated whether L. crispatus strains isolated from the vaginal tract of women with Lactobacillus-dominated vaginal microbiota (LVM) are pheno- or genotypically distinct from L. crispatus strains isolated from vaginal samples with dysbiotic vaginal microbiota (DVM). RESULTS: We studied 33 L. crispatus strains (n = 16 from LVM; n = 17 from DVM). Comparison of these two groups of strains showed that, although strain differences existed, both groups degraded various carbohydrates, produced similar amounts of organic acids, inhibited Neisseria gonorrhoeae growth, and did not produce biofilms. Comparative genomics analyses of 28 strains (n = 12 LVM; n = 16 DVM) revealed a novel, 3-fragmented glycosyltransferase gene that was more prevalent among strains isolated from DVM. Most L. crispatus strains showed growth on glycogen-supplemented growth media. Strains that showed less-efficient (n = 6) or no (n = 1) growth on glycogen all carried N-terminal deletions (respectively, 29 and 37 amino acid deletions) in a putative pullulanase type I protein. DISCUSSION: L. crispatus strains isolated from LVM were not phenotypically distinct from L. crispatus strains isolated from DVM; however, the finding that the latter were more likely to carry a 3-fragmented glycosyltransferase gene may indicate a role for cell surface glycoconjugates, which may shape vaginal microbiota-host interactions. Furthermore, the observation that variation in the pullulanase type I gene is associated with growth on glycogen discourages previous claims that L. crispatus cannot directly utilize glycogen.

Probiotic Enrichment and Reduction of Aflatoxins in a Traditional African Maize-Based Fermented Food.

Fermentation of food products can be used for the delivery of probiotic bacteria and means of food detoxification, provided that probiotics are able to grow, and toxins are reduced in raw materials with minimal effects on consumer acceptability. This study evaluated probiotic enrichment and detoxification of kwete, a commonly consumed traditional fermented cereal beverage in Uganda, by the use of starter culture with the probiotic Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106. Probiotic kwete was produced by fermenting a suspension of ground maize grain at 30 °C for a period of 24 h, leading to a decrease of the pH value to ≤ 4.0 and increase in titratable acidity of at least 0.2% (w/v). Probiotic kwete was acceptable to the consumers with a score of ≥6 on a 9-point hedonic scale. The products were stable over a month's study period with a mean pH of 3.9, titratable acidity of 0.6% (w/v), and Lactobacillus rhamnosus counts >108 cfu g-1. HPLC analysis of aflatoxins of the water-soluble fraction of kwete indicated that fermentation led to an over 1000-fold reduction of aflatoxins B1, B2, G1, and G2 spiked in the raw ingredients. In vitro fluorescence spectroscopy confirmed binding of aflatoxin B1 to Lactobacillus rhamnosus with an efficiency of 83.5%. This study shows that fermentation is a means to enrich with probiotics and reduce widely occurring aflatoxin contamination of maize products that are consumed as staple foods in sub-Saharan Africa.