Phylogenetic variation in raw cow milk microbiota and the impact of forage combinations and use of silage inoculants.

Introduction: The microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk. Methods: We identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells. Results and discussion: Three community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

Taxonomy, Sequence Variance and Functional Profiling of the Microbial Community of Long-Ripened Cheddar Cheese Using Shotgun Metagenomics.

Shotgun metagenomic sequencing was used to investigate the diversity of the microbial community of Cheddar cheese ripened over 32 months. The changes in taxa abundance were compared from assembly-based, non-assembly-based, and mOTUs2 sequencing pipelines to delineate the community profile for each age group. Metagenomic assembled genomes (MAGs) passing the quality threshold were obtained for 11 species from 58 samples. Although Lactococcus cremoris and Lacticaseibacillus paracasei were dominant across the shotgun samples, other species were identified using MG-RAST. NMDS analysis of the beta diversity of the microbial community revealed the similarity of the cheeses in older age groups (7 months to 32 months). As expected, the abundance of Lactococcus cremoris consistently decreased over ripening, while the proportion of permeable cells increased. Over the ripening period, the relative abundance of viable Lacticaseibacillus paracasei progressively increased, but at a variable rate among trials. Reads attributed to Siphoviridae and Ascomycota remained below 1% relative abundance. The functional profiles of PMA-treated cheeses differed from those of non-PMA-treated cheeses. Starter rotation was reflected in the single nucleotide variant profiles of Lactococcus cremoris (SNVs of this species using mOTUs2), while the incoming milk was the leading factor in discriminating Lacticaseibacillus paracasei/casei SNV profiles. The relative abundance estimates from Kraken2, non-assembly-based (MG-RAST) and marker gene clusters (mOTUs2) were consistent across age groups for the two dominant taxa. Metagenomics enabled sequence variant analysis below the bacterial species level and functional profiling that may affect the metabolic interactions between subpopulations in cheese during ripening, which could help explain the overall flavour development of cheese. Future work will integrate microbial variants with volatile profiles to associate the development of compounds related to cheese flavour at each ripening stage.

Farm management practices and season dependent factors affect the microbial community and chemical profile of corn and grass-legume silages of farms in Ontario, Québec, and Northern New York.

The effects of farm management practices and seasonal variation on the microbial community and chemical composition of corn and grass-legume silage are largely understudied due to the advantages of controlled mini-silo experiments. This study aims to investigate the effects that some key farm factors (use of an inoculant, farm region, and bunker or tower silo) and seasonal variations have on corn and grass-legume silage from farms across Ontario, Quebec, and New York. The silage was either treated with a commercial inoculant (Lallemand Biotal Buchneri 500® or Chr Hansen SiloSolve FC®) or left untreated. The bacterial communities of silage were compared to those of raw bulk tank milk from the same farm to determine if they were similarly affected by management practices or seasonal variations. Family level analysis of the 16S rRNA V3-V4 gene amplicon bacterial community, the ITS1 amplicon fungal community, NMR water soluble metabolome, and mycotoxin LC-MS were performed on silage over a two-year period. Chemical compounds associated with the use of inoculants in corn and grass-legume silage were higher in inoculated corn (acetate, propane-1,2-diol, γ-aminobutyrate; p < 0.001) and grass-legume (propionate; p = 0.011). However, there was no significant difference in the relative abundance (RA) of Lactobacillaceae in either silage type. Leuconostocaceae was higher in non-inoculated corn (p < 0.001) and grass-legume (p < 0.001) silage than in inoculated silage. Tower silos had higher RA of Leuconostocaceae (p < 0.001) and higher pH (p < 0.001) in corn and grass-legume silage. The one farm that used liquid manure with no other fertilizer type had higher RA of Clostridiaceae (p = 0.045) and other rumen/fecal (p < 0.006) bacteria in grass-legume silage than all other farms. Seasonal variation affected most of the key silage microbial families, however the trends were rarely visible across both years. Few trends in microbial variation could be observed in both silage and bulk tank milk: two farms had higher Moraxellaceae (p < 0.001) in milk and either corn or grass-legume silage. In farms using an inoculant, lower Staphylococcaceae was observed in the raw bulk tank milk.

Comparative analysis of exopolysaccharide-producing Lactiplantibacillus plantarum with ropy and non-ropy phenotypes on the gel properties and protein conformation of fermented milk.

In this study, we evaluated the impact of Lactiplantibacillus plantarum (L. plantarum) with ropy and non-ropy phenotypes on gel structure and protein conformation of fermented milk. Ropy L. plantarum (T1 & CL80) secreted EPS with high molecular weight (1.41 × 106, 1.19 × 106 Da) and intrinsic viscosity (486.46, 316.32 mL/g), effectively enhances fermented milk viscosity and water holding capacity (WHC) (65.4%, 84.6%) by forming a dense gel structure. Non-ropy L. plantarum (CSK & S-1A) fermented milk gel's high surface hydrophobicity and free sulfhydryl content caused high hardness and low WHC. Raman spectroscopy combined with circular dichroism analysis showed that high levels of α-helix (29.32-30.31%) and random roil (23.06-25.36%) protein structures are the intrinsic factors that contribute to the difference among fermented milk gels of ropy and non-ropy strains. This study provides a basis for understanding the structural variability of fermented milk gels using ropy or non-ropy lactic acid bacteria.

The digestive fate of beef versus plant-based burgers from bolus to stool.

Ultra-processed, plant-based burgers (PB) and traditional comminuted-beef burgers (BB) share similar organoleptic characteristics, yet a knowledge gap exists in understanding how consumption of these divergent physical structures alters the lipemic response and gut microbiota. PB, comprised of highly refined ingredients, is formulated with no intact whole food structure, while BB entraps lipids throughout the myofibrillar protein network. PB presented significantly higher free fatty acid (FFA) bioaccessibility (28.2 ± 4.80 %) compared to BB (8.73 ± 0.52 %), as obtained from their FFA release profiles over digestion time after characterizing them with a modified logistic model (SLM), using the simulated TIM Gastro-Intestinal Model (TIM-1). Additionally, the rate of lipolysis, k, obtained from the SLM for PB (90% CI [0.0175, 0.0277] min-1) was higher than for BB (90% CI [0.0113, 0.0171] min-1). Using the Simulated Human Intestinal Microbial Ecosystem (SHIME®), the Firmicutes to Bacteroidetes ratio (F/B ratio) was significantly higher for PB than BB; and linear discriminant analysis effect size (LEfSe) showed Clostridium and Citrobacter were more highly represented in the microbial community for the PB feed, whereas BB feed differentially enriched Megasphaera, Bacteroides, Alistipes, and Blautia at the genus level. Additionally, short-chain fatty acid (SCFA) production was altered (p < 0.05) site-specifically in each colon vessel, which could be attributed to the available substrates and changes in microbial composition. Total SCFAs were significantly higher for PB in the ascending colon (AC) and descending colon (DC) but higher for BB only in the transverse colon (TC). This research illustrates the crucial role of meat analog physical structure in modulating nutritional aspects beyond food composition alone.

Microorganisms Special Issue "How Do Food and Probiotics Influence the Composition and Activity of the Gut Microbiota?"

We are a product of the foods we chronically consume, and life expectancy correlates with the quality of our diet [...].

Dynamics of Starter and Non-Starter Lactic Acid Bacteria Populations in Long-Ripened Cheddar Cheese Using Propidium Monoazide (PMA) Treatment.

The microbial community of industrially produced Canadian Cheddar cheese was examined from curd to ripened cheese at 30-32 months using a combination of viable plate counts of SLAB (GM17) and NSLAB (MRSv), qPCR and 16S rRNA gene amplicon sequencing. Cell treatment with propidium monoazide excluded DNA of permeable cells from amplification. The proportion of permeable cells of both Lactococcus spp. and Lacticaseibacillus spp. was highest at 3-6 months. While most remaining Lacticaseibacillus spp. cells were intact during later ripening stages, a consistent population of permeable Lactococcus spp. cells was maintained over the 32-month period. While Lactococcus sequence variants were significant biomarkers for viable cheese curd communities at 0-1 m, Lacticaseibacillus was identified as a distinctive biomarker for cheeses from 7 to 20 months. From 24 to 32 months, Lacticaseibacillus was replaced in significance by four genera (Pediococcus and Latilactobacillus at 24 m and at 30-32 m, Secundilactobacillus and Paucilactobacillus). These results underscore the importance of monitoring potential defects in cheeses aged over 24 months, which could be diagnosed early through microbial DNA profiling to minimize potential waste of product. Future perspectives include correlating volatile flavor compounds with microbial community composition as well as the investigation of intra-species diversity.

Graduate Student Literature Review: Farm management practices: Potential microbial sources that determine the microbiota of raw bovine milk.

Environmental and herd-associated factors such as geographical location, climatic conditions, forage types, bedding, soil, animal genetics, herd size, housing, lactation stage, and udder health are exploited by farmers to dictate specific management strategies that ensure dairy operation profitability and enhance the sustainability of milk production. Along with milking routines, milking systems, and storage conditions, these farming practices greatly influence the microbiota of raw milk, as evidenced by several recent studies. During the past few years, the increased interest in high-throughput sequencing technologies combined with culture-dependent methods to investigate dairy microbial ecology has improved our understanding of raw milk community dynamics throughout storage and processing. However, knowledge is still lacking on the niche-specific communities in the farm environment, and on the factors that determine bacteria transfer to the raw milk. This review summarizes findings from the past 2 decades regarding the effects of farm management practices on the diversity of bacterial species that determine the microbiological quality of raw cow milk.

Modulation of Virulence Gene Expression in Salmonella enterica subsp. enterica typhimurium by Synthetic Milk-Derived Peptides.

The hydrolysis of milk proteins produces valuable bioactive peptides, some of which show antivirulence activity. In this study, five synthetic milk-derived peptides (ß-LG f(9-18), ß-CN f(5-15), ß-CN f(17-27), ß-CN f(94-106), and ß-CN f(129-137)) were shown to decrease the expression of virulence genes in Salmonella enterica subsp. enterica typhimurium when tested at four concentrations (0.02, 0.05, 0.1, and 0.2 mg/ml). A mixture of these synthetic peptides at concentrations of 0.02 and 0.2 mg/ml each significantly downregulated the expression of both hilA and ssrB virulence genes in Salmonella typhimurium after a 3-h incubation. Individually, ß-CN f(17-27) at 0.02 mg/ml caused a significant decrease in both hilA and ssrB gene expressions. These results suggest a synergistic interaction between bioactive peptides. Depending on dose and amino acid sequence, these five peptides were able to affect the expression of some virulence genes in Salmonella typhimurium.

Salmonella enterica subsp. enterica virulence potential can be linked to higher survival within a dynamic in vitro human gastrointestinal model.

Salmonella enterica subsp. enterica is one of the leading causes of human foodborne infections and several outbreaks are now associated with the consumption of fresh fruit and vegetables. This study aims at evaluating whether Salmonella virulence can be linked to an enhanced ability to survive successive digestive environments. Thirteen S. enterica strains were selected according to high and low virulence phenotypes. Lettuce inoculated separately with each S. enterica strain was used as food matrix in the TNO gastrointestinal model (TIM-1) of the human upper gastrointestinal tract. During the passage in the stomach, counts determined using PMA-qPCR were 2-5 logs higher than the cultivable counts for all strains indicating the presence of viable but non-cultivable cells. Bacterial growth was observed in the duodenum compartment after 180 min for all but one strain and growth continued into the ileal compartment. After passage through the simulated gastrointestinal tract, both virulent and avirulent S. enterica strains survived but high virulence strains had a significantly (p = 0.004) better average survival rate (1003 %-3753 %) than low virulence strains (from 25 % to 3730%). The survival rates of S. enterica strains could be linked to the presence of genes associated with acid and bile resistance and their predicted products. The presence of single nucleotide polymorphisms may also impact the function of virulence associated genes and play a role in the resulting phenotype. These data provide an understanding of the relationship between measured virulence potential and survival of S. enterica during dynamic simulated gastrointestinal transit.

Effect of two thermoresistant non-starter lactic acid bacteria strains on volatilome profile during Cheddar ripening simulation.

Dairy farm management practices can modify milk microbiota and therefore modulate non-starter lactic acid bacteria (NSLAB) found in cheese. These NSLAB can cause organoleptic defects. This study aimed to investigate the impact of two potential NSLAB in Cheddar cheesemaking: Lactiplantibacillus plantarum RKG 2-212 a strain isolated both in corn silage and raw milk, and Lactobacillus delbrueckii RKG R10, a strain isolated after pasteurisation of milk from a farm using grass and legume silage, and corn silage. The whole genome of these two lactobacilli was first sequenced. Then, the thermoresistance was evaluated after treatment at 60 °C for 5 min and compared to reference strains. Both lactobacilli were highly thermoresistant compared to other three lactic acid bacteria which are Lactococcus lactis subsp. cremoris ATCC 19257 and SK11, and L. plantarum ATCC 14917 (P < 0.0001). They lost less than 1 log cfu/mL (Δlog) and their genome contained a great number of copy number of genes coding for heat shock protein. During a Pearce test activity simulating Cheddar cheesemaking, the two lactobacilli did not show interaction with the starter Lcc. lactis subsp. cremoris SK11, and their population remained stable. During a ripening simulation, L. delbrueckii RKG R10 had a slight loss in viability in cheese slurry samples incubated at 30 °C for 12 d. However, L. plantarum RKG 2-212 had considerable growth, from 6.51 to 8.3 log cfu/g. This growth was associated with the acidification of the slurries (P < 0.0001). The presence of the lactobacilli modified the profile of volatile compounds evaluated by gas chromatography-mass spectrometry, accounting for 10.7% of the variation. The strain L. plantarum RKG 2-212 produced volatile compounds in greater quantity that could be associated with organoleptic defects such as acetic acid and 2-methylbutyraldehyde. Therefore, silage can be a vector of thermoresistant lactic acid bacteria for milk which can lead to flavor defects in cheese.

Effect of Bifidobacterium crudilactis and 3'-sialyllactose on the toddler microbiota using the SHIME® model.

The important changes in diet during the first years of life strongly modulate the intestinal microbiota of young children. Among in vitro digestive models, the simulator of human intestinal microbial ecosystem (SHIME®) model, seems particularly adapted to study the effects of prebiotics and/or probiotics on the dynamic microbiota of toddlers. The main purpose of this study was to investigate different formulations with prebiotic (3'-sialyllactose: 3'SL) and probiotic (Bifidobacterium crudilactis FR/62/b/3) effects on young child microbiota using the SHIME® model. The ascending (AC), transverse (TC) and descending (DC) colons of the SHIME® model were inoculated with feces from 3 donors aged between 1 and 2 years, in three separate vessels. After two weeks of microbiota stabilization, four treatments of one week (prebiotic, probiotic, synbiotic and cell-free spent media from the synbiotic) were administered. In all the colon vessels, the short chain fatty acid analyses, determined using high-performance liquid chromatography highlighted a ratio acetate/propionate/butyrate proportion of 5:19:6, situated between infant and adult normal values. As already observed in other validated studies focusing on the SHIME® model, the 16S rDNA sequencing highlighted a low richness and diversity in the AC, while the microbial communities in the TC and the DC remained similar to each other. Although some bacteria involved in biofilm development have been identified (Stenotrophomonas, Megasphera and Enterobacter), specific bacterial populations, proper to each colon were developed. Some bacteria associated to the upper intestinal tract, such as Lactobacillus and Veillonella genera, seemed to grow easily in the AC. The quantitative polymerase chain reaction (qPCR) targeting the hsp60 gene confirmed the ability of bifidobacteria to survive in this toddler model. In addition, the synbiotic treatment tended to a bifidogenic effect (P < 0.1). On the other hand, the feces of the donors and the content of the three colon vessels were filtered and placed in contact with Escherichia coli O157:H7 ATCC 43890 to evaluate the modulation of virulence gene expression using reverse transcription PCR. Finally, filtered supernatants from donor feces significantly up-regulated the expression of the luxS gene of E. coli O157:H7 (P = 0.013). In conclusion, despite the presence of biofilms, the toddler SHIME® model used in his study shared characteristics found both in adults and infants. Although additional investigations should be performed, combining 3'SL and B. crudilactis FR/62/b/3 could lead to a beneficial effect on infant microbiota by favoring bifidobacterial presence. Finally, the filtrated supernatant from young child feces could be able to modulate the quorum sensing mechanism for E. coli O157:H7.

Arabinogalactan Utilization by Bifidobacterium longum subsp. longum NCC 2705 and Bacteroides caccae ATCC 43185 in Monoculture and Coculture.

Arabinogalactan (AG) has been studied as a potential prebiotic in view of stimulating bifidobacteria presence in the gut microbiota. However, bifidobacteria prefer fermentation of oligosaccharides to that of polysaccharides. The contribution of other gut bacteria may allow better growth of bifidobacteria on AG. ß-galactanases and ß-galactosidases are the main enzymes for the degradation of AG. Additional enzymes such as α-L-arabinofuranosidase and ß-L-arabinopyranosidase are required to remove the arabinose side chains. All of these predicted functions are encoded by the genomes of both Bifidobacterium longum subsp. longum NCC 2705 and Bacteroides caccae ATCC 43185. However, neither strain was able to grow significantly on AG, with 25% (B. longum subsp. longum NCC 2705) and 39% (Bac. caccae ATCC 43185) of AG degraded after 48-h fermentation, respectively. In this study, the ß-galactanase, ß-galactosidase, α-L-arabinofuranosidase, and ß-L-arabinopyranosidase from both strains were investigated. The extracellular ß-galactosidases of both B. longum subsp. longum NCC 2705 and Bac. caccae ATCC 43185 were able to cleave the ß-1,3; 1,4 and 1,6 linkages. However, the ß-galactosidase activity of B. longum subsp. longum NCC 2705 was weaker for the ß-1,4 linkage, compared with the ß-1,3 and 1,6 linkages. The arabinose side chains of AG inhibited the cleavage of ß-1,3 and 1,6 linkages by the endo-ß-galactanase from both strains, and partially inhibited the cleavage of ß-1,4 linkages by the endo-ß-1,4 galactanase from Bac. caccae ATCC 43185. The α-L-arabinofuranosidase and ß-L-arabinopyranosidase from both strains were unable to cleave arabinose from AG under the conditions used. These results show limited breakdown of AG by these two strains in monoculture. When cocultured with Bac. caccae ATCC 43185, B. longum subsp. longum NCC 2705 grew significantly better than in monoculture on AG after 6 h of fermentation (p < 0.05). The coculture showed 48% AG degradation after 48 h of fermentation, along with reduced pH. Furthermore, compared to monoculture of Bac. caccae ATCC 43185, the concentration of succinate significantly increased from 0.01 ± 0.01 to 4.41 ± 0.61 mM, whereas propionate significantly decreased from 13.07 ± 0.37 to 9.75 ± 2.01 mM in the coculture (p < 0.05). These results suggest that the growth and metabolic activities of Bac. caccae ATCC 43185 were restrained in the coculture, as the pH decreased due to the metabolism of B. longum subsp. longum NCC 2705.

Effect of enzymatic treatment of citrus by-products on bacterial growth, adhesion and cytokine production by Caco-2 cells.

Citrus by-products are inexpensive sources of polyphenols, important bioactive compounds with wide pharmaceutical and food applications. This study aimed to investigate the effect of enzymatic treatment of citrus by-products on the polyphenolic profile of extracts and assess the influence of extracts on the growth and adhesion of probiotics and foodborne pathogenic bacteria and on the inflammatory response of epithelial cells. Enzyme-assisted extraction altered the polyphenolic profile (as assessed by HPLC-DAD), increasing the content of aglycone flavanones (naringenin and hesperetin). Enzymatic extracts and aglycone flavanones exhibited higher antibacterial and prebiotic activities than non-enzymatic extracts and glycoside flavanones. However, a higher content of aglycones was not associated with higher anti-adhesion activity. Citrus extracts significantly (P ≤ 0.05) decreased the inflammatory response of Caco-2 cells to Salmonella Typhimurium adhesion. These results support the sustainable reuse of citrus agroindustrial wastes and indicate the potential of citrus extracts in preventing infection by foodborne pathogenic bacteria and inducing proliferation of probiotics in foods and the gut environment.

Invited review: Starter lactic acid bacteria survival in cheese: New perspectives on cheese microbiology.

The importance of starter cultures to cheese manufacture and ripening is well known. Starters are inoculated into cheese milk at a level of ∼106 cfu/mL either from a bulk culture or using commercial direct-to-vat cultures. Before ripening, starters grow in the milk to reach populations of 107 to 109 cfu/g of curd depending on processing variables such as cook temperature, inclusion of washing steps, degree of partitioning with curds and whey, and importantly salt addition rate. Inherent strain-related properties also determine final populations in the curd following manufacture and include temperature sensitivity, salt sensitivity, presence of prophage, autolytic and permeabilization properties (which are influenced by processing steps), presence and type of cell envelope proteinase, and metabolic activity. Ripening of important industrial cheese varieties such as Cheddar, Dutch, Swiss, and Italian-type cheese varieties is characterized by extended storage under temperature-controlled conditions enabling characteristic flavor and texture development to occur. Over ripening, microbiological, biochemical and enzymatic changes occur with a decline in starter viability, release of intracellular enzymes, hydrolysis of proteins, carbohydrates and lipids, and formation of a range of volatile and nonvolatile flavor components. Recent reports suggest that starter strains may be present during the later stages of ripening and therefore their potential role needs to be reconsidered. This review will focus on our current understanding of starter viability and vitality during cheese ripening and will also review the area of starter permeabilization, autolysis, and enzyme release.

Detection of spore forming Paenibacillus macerans in raw milk.

Paenibacillus macerans can cause spoilage of milk during extended storage. However, the natural milk microbiota interferes with the enumeration of Paenibacillus species in raw milk. In this study, a qualitative SYBR Green real-time PCR assay based on the groEL gene was developed for detecting P. macerans (PMassay) in raw milk and compared with one designed for total Paenibacillus detection (TPassay). The specificity of the PMassay was confirmed against a panel of dairy-related spore forming isolates. In the presence of background DNA substituted up to 95%, P. macerans DNA could still be detected by the PMassay although interference occurred as non-target DNA substitution increased. The PMassay was sensitive (detection limit of 2 log CFU/ml in milk) and specific as non-P. macerans isolates gave a Ct > 30. After enrichment of raw milk for 7 days at 37 °C in Reinforced Clostridial Medium with D-cycloserine (RCM-D) under anaerobiosis, Paenibacillus was detected in 10 of the 16 raw milk samples tested. Enrichment in RCM-D yielded about 0.5 to 5.8 log CFU/ml total Paenibacillus and 0.3 to 4.6 log CFU/ml P. macerans in the samples. The assay could be useful in commercial settings, allowing a sensitive detection of P. macerans.

Draft Genome Sequences of Five Paenibacillus Species of Dairy Origin.

Paenibacillus species are important spoilage organisms in the dairy industry. The genomes of five Paenibacillus species which were isolated from dairy products in Canada were sequenced using the Illumina MiSeq platform. Draft genomes ranging from 5.1 to 7.1 Mb with GC contents of 49 to 53% were generated.

A toddler SHIME® model to study microbiota of young children.

The 'first 1000 days of life' determine the gut microbiota composition and can have long-term health consequences. In this study, the simulator of the human intestinal microbial ecosystem (SHIME®) model, which represents the main functional sections of the digestive tract, was chosen to study the microbiota of young children. The aim of this study was to reproduce the digestive process of toddlers and their specific colonic environment. The ascending, transverse and descending colons of SHIME® model were inoculated with feces from three donors aged between 1 and 2 years-old, in three separate runs. For each run, samples from colon vessels were collected at days 14, 21 and 28 after microbiota stabilization period. Short chain fatty acid concentrations determined by HPLC showed that microbiota obtained in SHIME® model shared characteristics between adults and infants. In addition, microbial diversity and bacterial populations determined by 16S rRNA amplicon sequencing were specific to each colon vessel. In conclusion, the SHIME® model developed in this study seemed well adapted to evaluate prebiotic and probiotic impact on the specific microbiota of toddlers, or medicine and endocrine disruptor metabolism. Moreover, this study is the first to highlight some biofilm development in in vitro gastrointestinal modelling systems.

Fermentation of Wheat Bran and Whey Permeate by Mono-Cultures of Lacticaseibacillus rhamnosus Strains and Co-culture With Yeast Enhances Bioactive Properties.

The aim of this work was to obtain a bioingredient (BI) with bioactive properties through the solid fermentation of a wheat bran-whey permeate (WB/WP) mixture with three strains of Lacticaseibacillus rhamnosus (R0011, ATCC 9595, and RW-9595M) in mono or co-culture with Saccharomyces cerevisiae. The choice of these strains was based on their capacity to produce the same exopolysaccharide (EPS), but at different yields. The solid fermentation of WB/WP revealed a similar growth pattern, sugar utilization and metabolite production between strains and types of culture. Lactic acid, soluble protein, free amino acid and phenolic compound content in BI were compared to NFWB. Water soluble polysaccharides (including EPS) were significantly increased in co-culture for (44%) ATCC 9595, (40%) R0011 and (27%) RW-9595M. The amount of bound Total Phenolic Content (TPC) as well as the antioxidant activity in BI were higher after fermentation. The free phenolic acid content was higher after fermentation with ATCC 9595 (53-59%), RW-9595M (45-46%), and R0011 (29-39%) compared to non-fermented NFWB. Fermentation by these strains increased the amounts of free caffeic acid and 4-hydroxybenzoic acid in both types of culture. The bound phenolic acid content was enhanced in co-culture for the BI obtained from the highest EPS producer strain RW-9595M which was 30% higher than NFWB. After in vitro digestion, bioaccessibility of free total phenolic acids was improved by more than 40% in BI compared to NFWB. The co-culture increased recovery of TPC (%) and antioxidant activity compared to monoculture for the strains in digested product. In contrast, the recovery of bound total phenolic acids in co-culture was 33 and 38% lower when compared to monoculture for R0011 and RW-9595M. Our findings provide new insights into the impact of LAB/yeast co-culture on the bioactive properties of fermented wheat bran.