Characterization of the Bacterial Composition of 47 Fermented Foods in Sweden

Fermentation has long been utilized to preserve and enhance the flavor and nutritional value of foods. Recently, fermented foods have gained popularity, reaching new consumer groups due to perceived health benefits. However, the microbial composition of many fermented foods re-mains unknown. Here, we characterized the bacterial composition, diversity, and richness of 47 fermented foods available in Sweden, including kombucha, water kefir, milk kefir, yogurt, plant-based yogurt alternatives, kimchi, sauerkraut, and fermented vegetables. Via 16S rRNA gene sequencing, we identified 2497 bacteria (amplicon sequence variants). The bacterial composition was strongly associated with the type of fermented food, and lactic acid bacteria and/or acetic acid bacteria dominated most samples. However, each fermented food had a unique composition, with kombucha and water kefir having the highest diversity across and within samples. Few bacteria were abundant in multiple foods and food groups. These were Streptococcus thermophilus in yogurts and plant-based yoghurts; Lactococcus lactis in milk kefirs and one water kefir; and Lactiplantibacillus plantarum in kimchi, sauerkraut, and fermented cucumber. The broad range of fermented foods included in this study and their diverse bacterial communities warrant further investigation into the implications of microbial compositions for product traits and potential impact on human health

Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells

Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus-specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell-cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens

Differential Responders to a Mixed Meal Tolerance Test Associated with Type 2 Diabetes Risk Factors and Gut Microbiota-Data from the MEDGI-Carb Randomized Controlled Trial

The global prevalence of type 2 diabetes mellitus (T2DM) has surged in recent decades, and the identification of differential glycemic responders can aid tailored treatment for the prevention of prediabetes and T2DM. A mixed meal tolerance test (MMTT) based on regular foods offers the potential to uncover differential responders in dynamical postprandial events. We aimed to fit a simple mathematical model on dynamic postprandial glucose data from repeated MMTTs among participants with elevated T2DM risk to identify response clusters and investigate their association with T2DM risk factors and gut microbiota. Data were used from a 12-week multi-center dietary intervention trial involving high-risk T2DM adults, comparing high- versus low-glycemic index foods within a Mediterranean diet context (MEDGICarb). Model-based analysis of MMTTs from 155 participants (81 females and 74 males) revealed two distinct plasma glucose response clusters that were associated with baseline gut microbiota. Cluster A, inversely associated with HbA1c and waist circumference and directly with insulin sensitivity, exhibited a contrasting profile to cluster B. Findings imply that a standardized breakfast MMTT using regular foods could effectively distinguish non-diabetic individuals at varying risk levels for T2DM using a simple mechanistic model

Similarity in milk microbiota in replicates

Receiving the same results from repeated analysis of the same sample is a basic principle in science. The inability to reproduce previously published results has led to discussions of a reproducibility crisis within science. For studies of microbial communities, the problem of reproducibility is more pronounced and has, in some fields, led to a discussion on the very existence of a constantly present microbiota. In this study, DNA from 44 bovine milk samples were extracted twice and the V3-V4 region of the 16S rRNA gene was sequenced in two separate runs. The FASTQ files from the two data sets were run through the same bioinformatics pipeline using the same settings and results from the two data sets were compared. Milk samples collected maximally 2 h apart were used as replicates and permitted comparisons to be made within the same run. Results show a significant difference in species richness between the two sequencing runs although Shannon and Simpson's diversity was the same. Multivariate analyses of all samples demonstrate that the sequencing run was a driver for variation. Direct comparison of similarity between samples and sequencing run showed an average similarity of 42%-45% depending on whether binary or abundance-based similarity indices were used. Within-run comparisons of milk samples collected maximally 2 h apart showed an average similarity of 39%-47% depending on the similarity index used and that similarity differed significantly between runs. We conclude that repeated DNA extraction and sequencing significantly can affect the results of a low microbial biomass microbiota study

Supplementation of Lactobacillus early in life alters attention bias to threat in piglets

Gut microbes play an important role in regulating brain processes and influence behaviour, cognition and emotional states in humans and rodents. Nevertheless, it is not known how ingestion of beneficial microbes modulates emotional states in piglets and whether it can improve welfare. Here we use an attention bias task to assess the effects of Lactobacillus reuteri ATCC-PTA-6475 and Lactobacillus plantarum L1-6 supplementation early in life on emotional states in 33 piglets compared to 31 placebo supplemented piglets. We hypothesized that Lactobacillus supplementation would reduce vigilance behaviour (head at shoulder height or higher) and attention (head oriented towards the threat) in response to an auditory threat. The results showed that the control group increased vigilance behaviour in response to the threat, but there was no increase in the probiotics group. Despite the increased vigilance, the control group paid less attention to the threat. One explanation may be that control piglets avoided looking in the direction of the threat just because they perceived it as more threatening, but further research is necessary to confirm this. In conclusion, Lactobacillus supplementation may be a suitable tool to reduce anxiety, promote a more appropriate response to a challenge and so improve welfare

Chemical composition and physical characteristics of faeces in horses with and without free faecal liquid - two case-control studies

Background Free faecal liquid (FFL) is a condition in horses characterised by two-phase (one solid and one liquid) separation of faeces. Causes of the condition are unknown, but disturbed hindgut fermentation has been suggested as it may alter biochemical composition and appearance of faeces in equines. However, information on faecal composition in horses with FFL is scarce. Faecal chemical composition (dry matter, osmolality, ash, macro minerals, short-chain fatty acids (SCFA) and pH) and physical characteristics (free liquid, sand, water holding capacity and particle size distribution) were compared in horses with (case) and without (control) FFL in two sub-studies. In sub-study I, faeces from 50 case-control horse pairs in Sweden and Norway were sampled in three sampling periods (SP1-SP3). In sub-study II, faeces from 32 case-control horse pairs in Germany were sampled on one occasion. Results In sub-study I, faecal concentration and proportion of lactic acid (of total short-chain fatty acids, SCFA) and water holding capacity was lower in case compared to control horses. Other variables (content of dry matter, ash, sodium, calcium, phosphorous, magnesium, sulphur, and concentrations of i-butyric, n-valeric and total SCFA, ammonia-N as proportion of total N, and pH) were similar in faeces from case and control horses. In sub-study II, all analysed variables were similar in faecal samples from case and control horses. Faecal particle size distribution was similar in case and control horses, but the proportion of larger particles (2 and 1 mm) were lower and proportion of smaller particles (< 1 mm) was higher in sub-study I compared to in sub-study II. Conclusions To the authors' knowledge, this is the first study to investigate faecal chemical composition and physical characteristics in horses with FFL. Case and control horses had similar total SCFA, pH and osmolality, indicating that hindgut fermentation was similar. However, small differences in concentration and proportion (of total SCFA) of lactic acid and water holding capacity of faeces were shown and are of interest for further studies of horses with FFL

Untangling the role of environmental and host-related determinants for on-farm transmission of verotoxin-producing Escherichia coli O157

Background: Cattle colonised by the zoonotic pathogen verotoxin-producing Escherichia coli of serotype O157 (VTEC O157) can shed high levels of the pathogen in their faeces. A suggested key for controlling VTEC O157 is preventing colonisation of individuals. Aim: In this study the role of individual super-shedders and factors related to susceptibility and environmental exposure in the transmission of VTEC O157 among dairy calves are explored. Methods: The association between sex, age, pen hygiene, pen type and stocking density and colonisation of individual calves, established by recto-anal mucosal swabs, on farms where pathogenic VTEC O157 had been confirmed was investigated. In a follow-up sampling, the consistency of previously identified risk factors and the role of shedding pen mates was assessed by studying the risk of new/re-colonisation. Results: The results suggest an important role of stocking density that decreases with age, possibly due to increased resistance to colonisation following exposure. However, previous colonisation did not influence the risk of being colonised in the second sampling. Super-shedders (shedding >103 colony forming units/g faeces) significantly increased the risk of colonisation in peers (OR = 10, CI 4.2–52). In addition, environmental factors associated with survival of the bacteria, affected risk. Conclusion: The results confirm the suggested importance of super-shedders but also emphasises the importance of considering the combined exposure from peers and the environment

The microbiota of ensiled forages and of bulk tank milk on dairy cattle farms in northern Sweden

Factors contributing to variations in the quality and microbiota of ensiled forages and in bulk tank microbiota milk from cows fed different forages were investigated. Nutritional quality, fermentation parameters and hygiene quality of forage samples and corresponding bulk tank milk samples collected in 3 periods from 18 commercial farms located in northern Sweden were compared. Principal coordinates analysis revealed that the microbiota forage and bulk milk, analyzed using 16S rRNA gene based amplicon sequencing, were significantly different. The genera Lactobacillus, Weissella, and Leuconostoc dominated in forage samples, whereas Pseudomonas, Staphylococcus, and Streptococcus dominated in bulk milk samples. Forage quality and forage-associated microbiota were affected by ensiling method and by use silage additive. Forages stored in bunker and tower silos (confounded with use of additive) were associated with higher levels of acetic and lactic acid and Lactobacillus. Forage ensiled as bales (confounded with no use of additive) was associated with higher DM content, watersoluble carbohydrate content, pH, yeast count, and the genera Weissella, Leuconostoc, and Enterococcus. For bulk tank milk samples, milking system was identified as the major factor affecting the microbiota and type forage preservation had little effect. Analysis of common amplicon sequence variants (ASV) suggested that forage was not the major source of Lactobacillus found in bulk tank milk

A meta-analysis revealing the technical, environmental, and host-associated factors that shape the gut microbiota of Atlantic salmon and rainbow trout

Salmonids, specifically Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), are commonly farmed and their gut microbiota plays important roles for optimal growth, health, and physiology. However, differences in experimental design, technical factors and bioinformatics make it challenging to compare the results from different studies and draw general conclusions about their influence on the fish gut microbiota. For a more comprehensive understanding of the gut microbiota, we collected all the publicly accessible 16S rRNA gene sequencing data with clearly stated sample metadata from freshwater Atlantic salmon and rainbow trout intestinal contents and mucosa sequenced on the Illumina MiSeq platform. A total of 783 samples from 19 published studies were included in this meta-analysis to test the impact of the technical, environmental, and host-accociated factors. This meta-analysis revealed that all the tested factors significantly influenced the alpha and beta diversities of the gut microbiota of salmon and trout. Technical factors, especially target region and DNA extraction kit, affected the beta diversity to a larger extent, while host-associated and environmental factors, especially diet and initial fish weight, had a higher impact on the alpha diversity. Salmon had a higher alpha diversity and higher abundance of Enterococcus and Staphylococcus than trout, which had higher abundance of Weissella and Mycoplasma. The results of this meta-analysis fill in a critical knowledge gap that demonstrate technical methodologies must be standardized and factors associated with host and environment need to be accounted for in the future design of salmonid gut microbiota experiments.A systematic meta-analysis of 19 studies found that technical factors (target region and DNA extraction kit) had the most significant effects on the fish gut microbiota followed by environmental factors (diet and rearing system) and host factors (initial weight, intestinal region and fish species). Images from Biorender.com. imag

In vitro fermentation of substrates from Saccharina latissima by broiler chicken's caecal microbiota

This study investigated seasonal variations in the composition of the kelp species Saccharina latissima cultivated on the Swedish west coast and, using an in vitro fermentation system, evaluated the effect of fermented whole S. latissima biomass or biomass components on the gut microbiota of broiler chickens. Caecal contents of six Ross 308 broilers fed a standard wheat and soy -based diet were retrieved on two different occasions, with three birds sampled per occasion. Two in vitro fermentation batches (A, B) were established using caecal contents as inoculum, and whole S. latissima, ethanol -washed S. latissima, laminarin extract or inulin (control) as substrate. Total short -chain fatty acid (SCFA) content, gas production, pH and changes in microbiota composition were studied after 6, 12 and 24 h of fermentation. Analysis of seasonal variations revealed that S. latissima harvested in June had the highest laminarin content and lowest ash and crude protein content. Broiler microbiota, SCFA profile, gas production and pH mainly varied depending on fermentation inoculum, but there were also some variations depending on substrate. For instance, uncultured bacterium from Clostridiales_vadinBB60_group showed higher relative abundance (RA) in batch A, while Faecalibacterium showed higher RA in batch B. Whole and ethanol -washed S. latissima resulted in highest RA of unclassified Ruminococcaceae and Tyzzerella for both batches, while ethanol -washed S. latissima and laminarin gave highest RA of Erysipelatoclostridium. Inulin resulted in highest RA for the genus Subdoligranulum. Acetic, nbutyric and propionic acid were the main fermentation products and total SCFA level was higher in batch B. Within batch B, inulin and laminarin generated higher levels of acetic and butyric acid. When using inulin and laminarin, gas production was lower in batch B compared with batch A. In summary, this study showed that S. latissima is a good source of laminarin, especially when harvested in summer. Within the in vitro system, the microbiota was affected by different substrates, but inoculum source was identified as an important contributor to microbial community development during fermentation