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.

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.

Bifidobacterium mongoliense genome seems particularly adapted to milk oligosaccharide digestion leading to production of antivirulent metabolites.

BACKGROUND: Human milk oligosaccharides (HMO) could promote the growth of bifidobacteria, improving young children's health. In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulent activity against intestinal pathogens. Bovine milk oligosaccharides (BMO), structurally similar to HMO, are found at high concentration in cow whey. This is particularly observed for 3'-sialyllactose (3'SL). This study focused on enzymes and transport systems involved in HMO/BMO metabolism contained in B. crudilactis and B. mongoliense genomes, two species from bovine milk origin. The ability of B. mongoliense to grow in media supplemented with whey or 3'SL was assessed. Next, the effects of cell-free spent media (CFSM) were tested against the virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. RESULTS: Due to the presence of genes encoding ß-galactosidases, ß-hexosaminidases, α-sialidases and α-fucosidases, B. mongoliense presents a genome more sophisticated and more adapted to the digestion of BMO/HMO than B. crudilactis (which contains only ß-galactosidases). In addition, HMO/BMO digestion involves genes encoding oligosaccharide transport systems found in B. mongoliense but not in B. crudilactis. B. mongoliense seemed able to grow on media supplemented with whey or 3'SL as main source of carbon (8.3 ± 1.0 and 6.7 ± 0.3 log cfu/mL, respectively). CFSM obtained from whey resulted in a significant under-expression of ler, fliC, luxS, stx1 and qseA genes (- 2.2, - 5.3, - 2.4, - 2.5 and - 4.8, respectively; P < 0.05) of E. coli O157:H7. CFSM from 3'SL resulted in a significant up-regulation of luxS (2.0; P < 0.05) gene and a down-regulation of fliC (- 5.0; P < 0.05) gene. CFSM obtained from whey resulted in significant up-regulations of sopD and hil genes (2.9 and 3.5, respectively; P < 0.05) of S. Typhimurium, while CFSM obtained from 3'SL fermentation down-regulated hil and sopD genes (- 2.7 and - 4.2, respectively; P < 0.05). CONCLUSION: From enzymes and transporters highlighted in the genome of B. mongoliense and its potential ability to metabolise 3'SL and whey, B. mongoliense seems well able to digest HMO/BMO. The exact nature of the metabolites contained in CFSM has to be identified still. These results suggest that BMO associated with B. mongoliense could be an interesting synbiotic formulation to maintain or restore intestinal health of young children.

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.

Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.

Lactic acid bacteria (LAB) found in milk can be responsible for organoleptic defects in cheese. To identify sources of LAB that could potentially develop during cheese making, we evaluated their prevalence and abundance in milk according to the type of forage used in dairy cow feeding. Forages and bulk tank milk were sampled 3 times on 24 farms using either hay alone (control), or grass or legume silage supplemented with corn silage or not. Both types of silage were either non-inoculated or inoculated with commercial preparations containing at least a Lactobacillus buchneri strain along with Lactobacillus casei, Lactobacillus plantarum, Enterococcus faecium, or Pediococcus pentosaceus. Our results indicate that LAB viable counts in milk samples (2.56 log cfu/mL) did not differ according to the type of forage used. A total of 1,239 LAB were isolated and identified by partial 16S rRNA gene sequencing. Although inoculation increased lactobacilli abundance in grass silage by 35%, we did not observe an effect on the LAB profile of milk. Indeed, we found no significant difference in milk LAB prevalence and abundance according to the type of forage (P > 0.05). Moreover, isolates belonging to the L. buchneri group were rarely found in bulk tank milk (3 out of 481 isolates). Random amplified polymorphic DNA typing of 406 LAB isolates revealed the plausible transfer of some strains from silage to milk (~6%). Thus, forage is only a minor contributor to LAB contamination of milk.

A tyrosine phosphorylation switch controls the interaction between the transmembrane modulator protein Wzd and the tyrosine kinase Wze of Lactobacillus rhamnosus.

BACKGROUND: One proposed mechanism for assembly of secreted heteropolysaccharides by many Gram positive bacteria relies on the coordinated action of a polymerization complex through reversible phosphorylation events. The role of the tyrosine protein kinase transmembrane modulator is, however, not well understood. RESULTS: The protein sequences deduced from the wzb, wzd and wze genes from Lactobacillus rhamnosus ATCC 9595 and RW-9595 M contain motifs also found in corresponding proteins CpsB, CpsC and CpsD from Streptococcus pneumoniae D39 (serotype 2). Use of an anti-phosphotyrosine antibody demonstrated that both Wzd and Wze can be found in tyrosine phosphorylated form. When tyrosine 266 was mutated to phenylalanine, WzdY266F showed slightly less phosphorylated protein than those produced by using eight other tyrosine mutated Wzd genes, when expressed along with Wze and Wzb in Lactococcus lactis subsp. cremoris MG1363. In order to demonstrate the importance of ATP for the interactions among these proteins, native and fusion Wzb, Wzd and Wze proteins were expressed and purified from Escherichia coli cultures. The modulator protein, Wzd, binds with the phosphotyrosine kinase Wze, irrespective of its phosphorylation status. However, Wze attained a higher phosphorylation level after interacting with phosphorylated Wzd in the presence of 10 mM ATP. This highly phosphorylated Wze did not remain in close association with phosphorylated Wzd. CONCLUSION: The Wze tyrosine kinase protein of Lactobacillus rhamnosus thus carries out tyrosine phosphorylation of Wzd in addition to auto- and trans- phosphorylation of the kinase itself.

Engineering of EPA/DHA omega-3 fatty acid production by Lactococcus lactis subsp. cremoris MG1363.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35 ± 0.5 mg g(-1) cell dry weight) and EPA (0.12 ± 0.04 mg g(-1) cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.

Genome sequence of Vibrio diabolicus and identification of the exopolysaccharide HE800 biosynthesis locus.

Vibrio diabolicus, a marine bacterium originating from deep-sea hydrothermal vents, produces the HE800 exopolysaccharide with high value for biotechnological purposes, especially for human health. Its genome was sequenced and analyzed; phylogenetic analysis using the core genome revealed V. diabolicus is close to another deep-sea Vibrio sp. (Ex25) within the Harveyi clade and Alginolyticus group. A genetic locus homologous to the syp cluster from Vibrio fischeri was demonstrated to be involved in the HE800 production. However, few genetic particularities suggest that the regulation of syp expression may be different in V. diabolicus. The presence of several types of glycosyltransferases within the locus indicates a capacity to generate diversity in the glycosidic structure, which may confer an adaptability to environmental conditions. These results contribute to better understanding exopolysaccharide biosynthesis and for developing new efficient processes to produce this molecule for biotechnological applications.

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.

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.

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.

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.

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.

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1.

BACKGROUND: The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances. RESULTS: Mutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested. CONCLUSION: Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans.

Correlation of maple sap composition with bacterial and fungal communities determined by multiplex automated ribosomal intergenic spacer analysis (MARISA).

During collection, maple sap is contaminated by bacteria and fungi that subsequently colonize the tubing system. The bacterial microbiota has been more characterized than the fungal microbiota, but the impact of both components on maple sap quality remains unclear. This study focused on identifying bacterial and fungal members of maple sap and correlating microbiota composition with maple sap properties. A multiplex automated ribosomal intergenic spacer analysis (MARISA) method was developed to presumptively identify bacterial and fungal members of maple sap samples collected from 19 production sites during the tapping period. Results indicate that the fungal community of maple sap is mainly composed of yeast related to Mrakia sp., Mrakiella sp., Guehomyces pullulans, Cryptococcus victoriae and Williopsis saturnus. Mrakia, Mrakiella and Guehomyces peaks were identified in samples of all production sites and can be considered dominant and stable members of the fungal microbiota of maple sap. A multivariate analysis based on MARISA profiles and maple sap chemical composition data showed correlations between Candida sake, Janthinobacterium lividum, Williopsis sp., Leuconostoc mesenteroides, Mrakia sp., Rhodococcus sp., Pseudomonas tolaasii, G. pullulans and maple sap composition at different flow periods. This study provides new insights on the relationship between microbial community and maple sap quality.

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.

Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp. longum CRC 002.

The effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp. longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080+/-120 mg EPS l(-1) (P<0.01) after 24 h of incubation. For fructose, galactose and glucose, EPS production was similar, at 512+/-63, 564+/-165 and 616+/-93 mg EPS l(-1), respectively. The proposed repeating unit composition of the EPS is 2 galactose to 3 glucose. The effect of sugar and fermentation time on expression of genes involved in sugar nucleotide production ( galK, galE1, galE2, galT1, galT2, galU, rmlA, rmlB1 and rmlCD) and the priming glycosyltransferase ( wblE) was quantified using real-time reverse transcription PCR. A significantly higher transcription level of wblE (9.29-fold) and the genes involved in the Leloir pathway (galK, 4.10-fold; galT1, 2.78-fold; and galE2, 4.95-fold) during exponential growth was associated with enhanced EPS production on lactose compared to glucose. However, galU expression, linking glucose metabolism with the Leloir pathway, was not correlated with EPS production on different sugars. Genes coding for dTDP-rhamnose biosynthesis were also differentially expressed depending on sugar source and growth phase, although rhamnose was not present in the composition of the EPS. This precursor may be used in cell wall polysaccharide biosynthesis. These results contribute to understanding the changes in gene expression when different sugar substrates are catabolized by B. longum subsp. longum CRC 002.