Draft genome sequences of 53 Lactococcus and Leuconostoc strains isolated from two undefined DL-starter cultures.

This study presents the complete genomes of 53 strains of Lactococcus and Leuconostoc isolated from two undefined DL-starter cultures originating from Denmark, Tistrup, and P. The genomes were reconstructed using long-read, nanopore-based DNA sequencing, delivering comprehensive data set for comparative genomics and taxonomic classification, with potential utility in dairy fermentation processes.

Randomised, Placebo-Controlled Investigation of the Impact of Probiotic Consumption on Gut Microbiota Diversity and the Faecal Metabolome in Seniors.

Aging has been associated with a changed composition and function of the gut microbiota (GM). Here, we investigate the effects of the multi-strain probiotic HOWARU® Restore on GM composition and function in seniors. Ninety-eight healthy adult volunteers aged ≥75 years were enrolled in a randomised, double-blinded intervention (NCT02207140), where they received HOWARU Restore (1010 CFU) or the placebo daily for 24 weeks, with 45 volunteers from each group completing the intervention. Questionnaires monitoring the effects on gastro-intestinal discomfort and bowel movements were collected. Faecal samples for GM characterisation (qPCR, 16S rRNA gene amplicon sequencing) and metabolomics (GC-FID, 1H NMR) were collected at the baseline and after 24 weeks. In the probiotic group, self-reported gastro-intestinal discomfort in the form of flatulence was significantly decreased during the intervention. At the baseline, 151 'core species' (present in ≥95% of samples) were identified. Most core species belonged to the Lachnospiraceae and Ruminococcaceae families. Neither alpha diversity nor beta diversity or faecal metabolites was affected by probiotic intake. On the contrary, we observed high intra-individual GM stability, with 'individual' accounting for 72-75% of variation. In conclusion, 24 weeks of HOWARU Restore intake reduced gastro-intestinal discomfort in the form of flatulence in healthy seniors without significantly influencing GM composition or activity.

Dietary seaweed (Saccharina latissima) supplementation in pigs induces localized immunomodulatory effects and minor gut microbiota changes during intestinal helminth infection.

Brown seaweeds have a rich bioactive content known to modulate biological processes, including the mucosal immune response and microbiota function, and may therefore have the potential to control enteric pathogens. Here, we tested if dietary seaweed (Saccharina latissima) supplementation could modulate pig gut health with a specific focus on parasitic helminth burdens, gut microbiota composition, and host immune response during a five week feeding period in pigs co-infected with the helminths Ascaris suum and Oesophagostomum dentatum. We found that inclusion of fermented S. latissima (Fer-SL) at 8% of the diet increased gut microbiota α-diversity with higher relative abundances of Firmicutes, Tenericutes, Verrucomicrobia, Spirochaetes and Elusimicrobia, and lower abundance of Prevotella copri. In the absence of helminth infection, transcription of immune-related genes in the intestine was only moderately influenced by dietary seaweed. However, Fer-SL modulated the transcriptional response to infection in a site-specific manner in the gut, with an attenuation of infection-induced gene expression in the jejunum and an amplification of gene expression in the colon. Effects on systemic immune parameters (e.g. blood lymphocyte populations) were limited, indicating the effects of Fer-SL were mainly localized to the intestinal tissues. Despite previously documented in vitro anti-parasitic activity against pig helminths, Fer-SL inclusion did not significantly affect parasite egg excretion or worm establishment. Collectively, our results show that although Fer-SL inclusion did not reduce parasite burdens, it may modify the gut environment during enteric parasite infection, which encourages continued investigations into the use of seaweeds or related products as novel tools to improve gut health.

Cesarean section induced dysbiosis promotes type 2 immunity but not oxazolone-induced dermatitis in mice.

Delivery by cesarean section (CS) is associated with an altered gut microbiota (GM) colonization and a higher risk of later chronic inflammatory diseases. Studies investigating the association between CS and atopic dermatitis (AD) are contradictive and often biased by confounding factors. The aim of this study was therefore to provide experimental evidence for the association between CS and AD in a mouse model and clarify the role of the GM changes associated with CS. It was hypothesized that CS-delivered mice, and human CS-GM transplanted mice develop severe dermatitis due to early dysbiosis. BALB/c mice delivered by CS or vaginally (VD) as well as BALB/c mice transplanted with GM from CS or VD human donors were challenged with oxazolone on the ear. The severity of dermatitis was evaluated by ear thickness and clinical and histopathological assessment which were similar between all groups. The immune response was assessed by serum IgE concentration, local cytokine response, and presence of immune cells in the draining lymph node. Both CS-delivered mice and mice inoculated with human CS-GM had a higher IgE concentration. A higher proportion of Th2 cells were also found in the CS-GM inoculated mice, but no differences were seen in the cytokine levels in the affected ears. In support of the experimental findings, a human cohort analysis from where the GM samples were obtained found that delivery mode did not affect the children's risk of developing AD. In conclusion, CS-GM enhanced a Th2 biased immune response, but had no effect on oxazolone-induced dermatitis in mice.

Dietary non-starch polysaccharides impair immunity to enteric nematode infection.

BACKGROUND: The influence of diet on immune function and resistance to enteric infection and disease is becoming ever more established. Highly processed, refined diets can lead to inflammation and gut microbiome dysbiosis, whilst health-promoting dietary components such as phytonutrients and fermentable fibres are thought to promote a healthy microbiome and balanced mucosal immunity. Chicory (Cichorium intybus) is a leafy green vegetable rich in fibres and bioactive compounds that may promote gut health. RESULTS: Unexpectedly, we here show that incorporation of chicory into semisynthetic AIN93G diets renders mice susceptible to infection with enteric helminths. Mice fed a high level of chicory leaves (10% dry matter) had a more diverse gut microbiota, but a diminished type-2 immune response to infection with the intestinal roundworm Heligmosomoides polygyrus. Furthermore, the chicory-supplemented diet significantly increased burdens of the caecum-dwelling whipworm Trichuris muris, concomitant with a highly skewed type-1 immune environment in caecal tissue. The chicory-supplemented diet was rich in non-starch polysaccharides, particularly uronic acids (the monomeric constituents of pectin). In accordance, mice fed pectin-supplemented AIN93G diets had higher T. muris burdens and reduced IgE production and expression of genes involved in type-2 immunity. Importantly, treatment of pectin-fed mice with exogenous IL-25 restored type-2 responses and was sufficient to allow T. muris expulsion. CONCLUSIONS: Collectively, our data suggest that increasing levels of fermentable, non-starch polysaccharides in refined diets compromises immunity to helminth infection in mice. This diet-infection interaction may inform new strategies for manipulating the gut environment to promote resistance to enteric parasites.

Used Nasogastric Feeding Tubes from Neonates Contain Infant-Specific Bacterial Profiles.

Nasogastric feeding tubes (NG-tubes) from neonates contain potentially pathogenic bacteria. Using culture-based techniques, we have previously determined that the usage duration of NG-tubes did not impact the colonization of the nasogastric tubes. In the present study, we performed 16S rRNA gene amplicon sequencing to evaluate the microbial profile of 94 used nasogastric tubes collected from a single neonatal intensive care unit. Using culture-based whole genome sequencing, we as-sessed whether the same strain persisted in NG-tubes collected from the same neonate across different time-points. We found that the most commonly occurring Gram-negative bacteria were Enterobacteriaceae, Klebsiella and Serratia, while the most common Gram-positive bacteria were staphylococci and streptococci. The microbiota of the NG-feeding tube was overall infant-specific, rather than dependent on the duration of use. Furthermore, we determined that reoccurring species from the individual infant represented the same strain and that several strains were common for more than one infant. Our findings indicate that bacterial profiles found in NG-tubes of neonates are host-specific, not dependent on the duration of use and strongly influenced by the environment.

Complete Genome Sequences of Three Lactiplantibacillus plantarum Strains Isolated from Traditional Iranian Raw Milk Motal Cheese.

The complete genome sequences, as determined by a combination of short- and long-read sequencing, of three Lactiplantibacillus plantarum strains (M8, M17, and M19) that were isolated from Iranian motal cheese are reported. The genome sizes were estimated to be 3.3, 3.3, and 3.5 Mbp, respectively, with GC contents of approximately 44.5%.

Bacterial community development and diversity during the first year of production in a new salmon processing plant.

The bacterial diversity and load on equipment in food processing facilities is constantly influenced by raw material, water, air, and staff. Despite regular cleaning and disinfection, some bacteria may persist and thereby potentially compromise food quality and safety. Little is known about how bacterial communities in a new food processing facility gradually establish themselves. Here, the development of bacterial communities in a newly opened salmon processing plant was studied from the first day and during the first year of operation. To focus on the persisting bacterial communities, surface sampling was done on strategical sampling points after cleaning and disinfection. To study the diversity dynamics, isolates from selected sampling and time points were classified by Oxford Nanopore Technology-based rep-PCR amplicon sequencing (ON-rep-seq) supplemented by 16S rRNA gene or rpoD gene sequencing (for Pseudomonas). An overall increase in bacterial numbers was only observed for food-contact surfaces in the slaughter department, but not in filleting department, on non-food contact surfaces or on the fish. Changes in temporal and spatial diversity and community composition were observed and our approach revealed highly point-specific bacterial communities.

Prevotella abundance and salivary amylase gene copy number predict fat loss in response to wholegrain diets.

Background: Salivary amylase (AMY1) gene copy number (CN) and Prevotella abundance in the gut are involved in carbohydrate digestion in the upper and lower gastrointestinal tract, respectively; and have been suggested as prognostic biomarkers for weight loss among overweight individuals consuming diets rich in fiber and wholegrains. Objective: We hypothesized that Prevotella abundance would be linked to greater loss of body fat after wholegrain consumption among individuals with low AMY1 CN, but not in those with high AMY1 CN. Methods: We reanalyzed data from two independent randomized ad libitum wholegrain interventions (fiber intake ∼33 g/d for 6-8 weeks), to investigate the relationship between baseline Prevotella abundance and body fat loss among healthy, overweight participants stratified into two groups by median AMY1 CN. Individuals with no detected Prevotella spp. were excluded from the main analysis. Results: In both studies, individuals with low AMY1 CN exhibited a positive correlation between baseline Prevotella abundance and fat loss after consuming the wholegrain diet (r > 0.5, P < 0.05), but no correlation among participants with high AMY1 CN (P ≥ 0.6). Following consumption of the refined wheat control diets, there were no associations between baseline Prevotella abundance and changes in body fat in any of the AMY1 groups. Conclusion: These results suggest that Prevotella abundance together with AMY1 CN can help predict fat loss in response to ad libitum wholegrain diets, highlighting the potential of these biomarkers in personalized obesity management.

Controlling the uncontrolled variation in the diet induced obese mouse by microbiomic characterization.

Group sizes in an animal study are calculated from estimates on variation, effect, power and significance level. Much of the variation in glucose related parameters of the diet-induced obese (DIO) mouse model is due to inter-individual variation in gut microbiota composition. In addition, standard tandem repeats (STRs) in the non-coding DNA shows that inbred mice are not always homogenic. C57BL/6NTac (B6NTac) mice from Taconic and C57BL/6NRj (B6NRj) mice from Janvier Labs were fed a high calorie diet and treated with liraglutide. The fecal microbiota was sequenced before high-calorie feeding (time 1) and after diet-induced obesity instantly before liraglutide treatment (time 2) and mice were divided into clusters on the basis of their microbiota. Although liraglutide in both sub-strains alleviated glucose intolerance and reduced body weight, in a one-way ANOVA a borderline reduction in glycosylated hemoglobin (HbA1c) could only be shown in B6NTac mice. However, if the microbiota clusters from time 1 or time 2 were incorporated in a two-way ANOVA, the HbA1c effect was significant in B6NTac mice in both analyses, while this did not change anything in B6NRj mice. In a one-way ANOVA the estimated group size needed for a significant HbA1c effect in B6NTac mice was 42, but in two-way ANOVAs based upon microbiota clusters of time 1 or time 2 it was reduced to 21 or 12, respectively. The lowering impact on glucose tolerance was also powered by incorporation of microbiota clusters of both times in both sub-strains. B6NRj had up to six, while B6NTac had maximum three alleles in some of their STRs. In B6NRj mice in 28.8% of the STRs the most prevalent allele had a gene frequency less than 90%, while this was only 6.6% in the B6NTac mice. However, incorporation of the STRs with the highest number of alleles or the most even distribution of frequencies in two-way ANOVAs only had little impact on the outcome of data evaluation. It is concluded that the inclusion of microbiota clusters in a two-way ANOVA in the evaluation of the glucose related effects of an intervention in the DIO mouse model might be an efficient tool for increasing power and reducing group sizes in mouse sub-strains, if these have a microbiota, which influences these parameters.

Effect of gluten-free diet and antibiotics on murine gut microbiota and immune response to tetanus vaccination.

The purpose of this study was to compare the effect of a gluten-free diet and/or antibiotics on tetanus vaccine induced immunoglobulin G titers and immune cell levels in BALB/c mice. The gluten-free diet was associated with a reduced anti-tetanus IgG response, and it increased the relative abundance of the anti-inflammatory Bifidobacterium significantly in some of the mice. Antibiotics also led to gut microbiota changes and lower initial vaccine titer. After a second vaccination, neither gluten-free diet nor antibiotics reduced the titers. In the spleen, the gluten-free diet significantly increased regulatory T cell (Treg) fractions, CD4+ T cell activation, and tolerogenic dendritic cell fractions and activation, which extend the downregulating effect of the Treg. Therefore, the systemic effect of the gluten-free diet seems mainly tolerogenic. Antibiotics reduced the fractions of CD4+ T and B cells in the mesenteric lymph nodes. These results suggest that vaccine response in mice is under influence of their diet, the gut microbiota and the interplay between them. However, a gluten-free diet seems to work through mechanisms different from those induced by antibiotics. Therefore, diet should be considered when testing vaccines in mice and developing vaccines for humans.

Dietary proanthocyanidins promote localized antioxidant responses in porcine pulmonary and gastrointestinal tissues during Ascaris suum-induced type 2 inflammation.

Proanthocyanidins (PAC) are dietary polyphenols with putative anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated if diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type-2 biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. Whilst PAC had only minor effects on pulmonary immune responses, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function and antioxidant responses in the gut of both naïve and A. suum-infected animals. A. suum infection and dietary PAC induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC consumption substantially increased the abundance of Limosilactobacillus reuteri. In vitro experiments with porcine macrophages and intestinal epithelial cells supported a role for both PAC polymers and PAC-derived microbial metabolites in regulating oxidative stress responses in host tissues. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, while having a limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract.

Gluten-free diet reduces autoimmune diabetes mellitus in mice across multiple generations in a microbiota-independent manner.

Experimental and clinical data suggest that a gluten-free diet attenuates the development of type 1 diabetes. A gluten-free diet changes the gut microbiota composition, and such microbial changes are expected to reduce the autoimmune responses. However, in experiments with laboratory mice, a gluten-free diet changes the gut microbiota differently under varying experimental settings, questioning the specific role of the gut microbes. Here we show that a maternal gluten-free diet until weaning of their pups, delayed type 1 diabetes in both dams (parent generation) and offspring (F1 generation) of untreated non-obese diabetic (NOD) mice and in mice treated with a full cocktail of antibiotics that eradicates most of the existing microbiota. Breeding a second (F2) generation of NOD mice, never exposed to the gluten-free diet or the associated microbial changes, also demonstrated a preventative effect on type 1 diabetes even though their parents (the F1 generation) had only been on a gluten-free diet very early in life. Collectively, the experimental data, thus, points towards microbiota-independent dietary protection. Furthermore, both the perinatal gluten-free diet and antibiotic treatment reduced inflammation in the salivary glands and improved glucose challenged beta cell function in the F1 offspring. However, in contrast to the autoimmune response in the pancreas, those changes appeared to be microbiota dependent, as they were missing in the antibiotic treated mice, and do, therefore, not seem to be related to the preventative effect on type 1 diabetes. Interestingly, adoptive transfer of splenocytes from gluten-free fed mice protected NOD.SCID mice from developing diabetes, demonstrating that the anti-diabetic effect of a gluten-free diet was based on early life changes in the evolving immune system. In particular, genes involved in regulation of lymphocyte activation, proliferation, and cell adhesion were highly expressed in the spleen in gluten-free fed mice at weaning compared to control fed mice of the F1 generation, which suggested that gluten promotes autoimmunity by inhibiting immune regulation, though the involvement of the specific genes needs further investigation. In conclusion, gluten-free diet reduces autoimmune inflammation in salivary glands and pancreas in NOD mice in a microbiota-dependent and -independent manner respectively, and has preventative effect on type 1 diabetes by modulating the systemic immune system.

Gut colonization in preterm infants supplemented with bovine colostrum in the first week of life: An explorative pilot study.

BACKGROUND: In the first weeks after birth, enteral feeding and bacterial colonization interact to influence gut maturation in preterm infants. Bovine colostrum (BC) has been suggested as a relevant supplementary diet when own mother's milk (MM) is insufficient or absent. This pilot trial tests whether the supplement type, BC or donor human milk (DM), affects gut colonization in preterm infants during the first week of life. METHODS: On day 7, fecal samples were collected from preterm infants (n = 24) fed BC or DM as a supplement to MM. The gut microbiome (GM) was analyzed by 16S ribosomal RNA amplicon sequencing. Correlations between the relative abundance of specific bacterial taxa and blood chemistry variables, including amino acids, were explored. RESULTS: BC-supplemented infants showed a lower relative abundance of the families Lactobacillaceae and Enterococcaceae than DM infants. Planococcaceae were more abundant in infants delivered by cesarean birth vs vaginally. The relative abundance of bacterial families, specifically Enterobacteriaceae, correlated negatively with plasma levels of multiple essential and nonessential amino acids (valine, isoleucine, lysine, histidine, and arginine). CONCLUSION: The nature of nutrition supplements (BC or DM) just after birth may affect GM development and nutrient metabolism in the neonatal period of preterm infants. The exploratory nature of our study calls for confirmation of these results and their possible long-term clinical implications for preterm infants.

The phytonutrient cinnamaldehyde limits intestinal inflammation and enteric parasite infection.

Phytonutrients such as cinnamaldehyde (CA) have been studied for their effects on metabolic diseases, but their influence on mucosal inflammation and immunity to enteric infection are not well documented. Here, we show that consumption of CA in mice significantly down-regulates transcriptional pathways connected to inflammation in the small intestine, and alters T-cell populations in mesenteric lymph nodes. During infection with the enteric helminth Heligomosomoides polygyrus, CA treatment attenuated infection-induced changes in biological pathways connected to cell cycle and mitotic activity, and tended to reduce worm burdens. Mechanistically, CA did not appear to exert activity through a prebiotic effect, as CA treatment did not significantly change the composition of the gut microbiota. Instead, in vitro experiments showed that CA directly induced xenobiotic metabolizing pathways in intestinal epithelial cells and suppressed endotoxin-induced inflammatory responses in macrophages. Collectively, our results show that CA down-regulates inflammatory pathways in the intestinal mucosa and can limit the pathological response to enteric infection. These properties appear to be largely independent of the gut microbiota, and instead connected to the ability of CA to induce antioxidant pathways in intestinal cells. Our results encourage further investigation into the use of CA and related phytonutrients as functional food components to promote intestinal health in humans and animals.

ON-rep-seq as a rapid and cost-effective alternative to whole-genome sequencing for species-level identification and strain-level discrimination of Listeria monocytogenes contamination in a salmon processing plant.

Identification, source tracking, and surveillance of food pathogens are crucial factors for the food-producing industry. Over the last decade, the techniques used for this have moved from conventional enrichment methods, through species-specific detection by PCR to sequencing-based methods, whole-genome sequencing (WGS) being the ultimate method. However, using WGS requires the right infrastructure, high computational power, and bioinformatics expertise. Therefore, there is a need for faster, more cost-effective, and more user-friendly methods. A newly developed method, ON-rep-seq, combines the classical rep-PCR method with nanopore sequencing, resulting in a highly discriminating set of sequences that can be used for species identification and also strain discrimination. This study is essentially a real industry case from a salmon processing plant. Twenty Listeria monocytogenes isolates were analyzed both by ON-rep-seq and WGS to identify and differentiate putative L. monocytogenes from a routine sampling of processing equipment and products, and finally, compare the strain-level discriminatory power of ON-rep-seq to different analyzing levels delivered from the WGS data. The analyses revealed that among the isolates tested there were three different strains. The isolates of the most frequently detected strain (n = 15) were all detected in the problematic area in the processing plant. The strain level discrimination done by ON-rep-seq was in full accordance with the interpretation of WGS data. Our findings also demonstrate that ON-rep-seq may serve as a primary screening method alternative to WGS for identification and strain-level differentiation for surveillance of potential pathogens in a food-producing environment.

The effect of early probiotic exposure on the preterm infant gut microbiome development.

Premature birth, especially if born before week 32 of gestation, is associated with increased risk of neonatal morbidity and mortality. Prophylactic use of probiotics has been suggested to protect preterm infants via supporting a healthy gut microbiota (GM) development, but the suggested strains and doses vary between studies. In this study, we profiled the GM of 5, 10 and 30-day fecal samples from two cohorts of preterm neonates (born <30 weeks of gestation) recruited in the same neonatal intensive care unit. One cohort (n = 165) was recruited from September 2006 to January 2009 before probiotics were introduced in the clinic. The second cohort (n = 87) was recruited from May 2010 to October 2011 after introducing Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis ssp. lactis BB-12 supplementation policy. Through V3-V4 region 16S rRNA gene amplicon sequencing, a distinct increase of L. rhamnosus and B. animalis was found in the fecal samples of neonates supplemented with probiotics. During the first 30 days of life, the preterm GM went through similarly patterned progression of bacterial populations. Staphylococcus and Weissella dominated in early samples, but was gradually overtaken by Veillonella, Enterococcus and Enterobacteriaceae. Probiotic supplementation was associated with pronounced reduction of Weissella, Veillonella spp. and the opportunistic pathogen Klebsiella. Potential nosocomial pathogens Citrobacter and Chryseobacterium species also gradually phased out. In conclusion, probiotic supplementation to preterm neonates affected gut colonization by certain bacteria, but did not change the overall longitudinal bacterial progression in the neonatal period.Abbreviations: GM: Gut microbiota; ASV: Amplicon sequence variant; NEC: Necrotizing enterocolitis; DOL: Days of life; NICU: Neonatal intensive care unit; ESPGHAN: European Society for Pediatric Gastroenterology, Hepatology and Nutrition; Db-RDA: Distance-based redundancy analysis; PERMANOVA: Permutational multivariate analysis of variance; ANCOM: Analysis of compositions of microbiomes; LGG: Lacticaseibacillus (former Lactobacillus) rhamnosus GG; BB-12: Bifidobacterium animalis ssp. lactis BB-12; DGGE: Denaturing Gradient Gel Electrophoresis.

Supplementation of a lacto-fermented rapeseed-seaweed blend promotes gut microbial- and gut immune-modulation in weaner piglets.

BACKGROUND: The direct use of medical zinc oxide in feed will be abandoned after 2022 in Europe, leaving an urgent need for substitutes to prevent post-weaning disorders. RESULTS: This study investigated the effect of using rapeseed-seaweed blend (rapeseed meal added two brown macroalgae species Ascophylum nodosum and Saccharina latissima) fermented by lactobacilli (FRS) as feed ingredients in piglet weaning. From d 28 of life to d 85, the piglets were fed one of three different feeding regimens (n = 230 each) with inclusion of 0%, 2.5% and 5% FRS. In this period, no significant difference of piglet performance was found among the three groups. From a subset of piglets (n = 10 from each treatment), blood samples for hematology, biochemistry and immunoglobulin analysis, colon digesta for microbiome analysis, and jejunum and colon tissues for histopathological analyses were collected. The piglets fed with 2.5% FRS manifested alleviated intraepithelial and stromal lymphocytes infiltration in the gut, enhanced colon mucosa barrier relative to the 0% FRS group. The colon microbiota composition was determined using V3 and V1-V8 region 16S rRNA gene amplicon sequencing by Illumina NextSeq and Oxford Nanopore MinION, respectively. The two amplicon sequencing strategies showed high consistency between the detected bacteria. Both sequencing strategies indicated that inclusion of FRS reshaped the colon microbiome of weaned piglets with increased Shannon diversity. Prevotella stercorea was verified by both methods to be more abundant in the piglets supplied with FRS feed, and its abundance was positively correlated with colonic mucosa thickness but negatively correlated with blood concentrations of leucocytes and IgG. CONCLUSIONS: FRS supplementation relieved the gut lymphocyte infiltration of the weaned piglets, improved the colon mucosa barrier with altered microbiota composition. Increasing the dietary inclusion of FRS from 2.5% to 5% did not lead to further improvements.

An Oligosaccharide Rich Diet Increases Akkermansia spp. Bacteria in the Equine Microbiota.

Some oligosaccharides induce growth of anti-inflammatory bacterial species and induce regulatory immunity in humans as well as animals. We have shown that the equine gut microbiota and the immune-microbial homeostasis largely stabilize within the first 50 days of life. Furthermore, we have previously established that certain bacterial species in the equine gut correlated with regulatory immunity. Accordingly, we hypothesized that an oligosaccharide rich diet fed to foals during the first 50 days would increase the abundance of bacterial species associated with regulatory immunity, and that this would influence immune responses in the foals. Eight pregnant mares and their foals were fed an oligosaccharide rich diet from 4 weeks before expected parturition until 49 days post-partum. Six mares and foals served as control. Fecal microbiota from mares and foals was characterized using 16S rRNA gene amplicon high throughput sequencing. On Day 49 the test foals had significantly higher abundances of Akkermansia spp. Blood sampled from the foals in the test group on Day 7, 28, and 49 showed non-significant increases in IgA, and decreases in IgG on Day 49. In BALB/cBomTac mice inoculated with gut microbiota from test and control foals we found increased species richness, increased relative abundance of several species identified as potentially anti-inflammatory in horses, which were unclassified Clostridiales, Ruminococcaceae, Ruminococcus, Oscilospira, and Coprococcus. We also found increased il10 expression in the ileum if inoculated with test foal microbiota. We conclude that an oligosaccharide diet fed to foals in the "window of opportunity," the first 50 days of life, increases the abundance of anti-inflammatory species in the microbiota with potentially anti-inflammatory effects on regulatory immunity.

Histamine-forming ability of Lentilactobacillus parabuchneri in reduced salt Cheddar cheese.

Lentilactobacillus parabuchneri, a member of the non-starter microbiota in cheese, was recently associated with fast and effective histamine-formation ability, a safety issue. The present study was performed to investigate Lentilactobacillus parabuchneri KUH8, a histamine-producer (HP) in reduced-salt Cheddar cheese. Four cheeses were manufactured: 1) normal-salt (NS); 2) reduced-salt (RS); 3) normal-salt with HP (NS+HP); 4) reduced-salt with HP (RS+HP). Two replicates were produced with milk from the same batch, and the cheeses ripened at 10 and 15 °C. Cheeses were sampled immediately after manufacture and after 1, 3 and 6 months of ripening. Ultra-high-performance-liquid chromatography indicated that with the HP, histamine reached higher levels in reduced-salt cheeses (3.5-3.7% S/M) at 15 °C (86, 1112, 2149 and 3149 mg kg-1), compared to normal-salt cheeses (5.4-6.3% S/M) at 10 °C (78, 584, 593 and 1389 mg kg-1), at each respective cheese-sampling point. Higher salt-content reduced the growth rate of non-starter microbiota, but after six months the levels in all cheeses were similar, according to the ripening temperature: at 10 °C (8.05-8.30 log10 cfu g-1), and at 15 °C (6.00-6.94 log10 cfu g-1). A correlation between increased histamine levels, non-starter-cell development and pH was found. This study highlights the importance of normal-salt content and low-ripening temperature as measures to control histamine-formation and to improve safety in cheese.