Transcriptome Analysis Reveals the Role of Sucrose in the Production of Latilactobacillus sakei L3 Exopolysaccharide.

Latilactobacillus (L.) sakei is a species of lactic acid bacteria (LAB) mostly studied according to its application in food fermentation. Previously, L. sakei L3 was isolated by our laboratory and possessed the capability of high exopolysaccharide (EPS) yield during sucrose-added fermentation. However, the understanding of sucrose promoting EPS production is still limited. Here, we analyzed the growth characteristics of L. sakei L3 and alterations of its transcriptional profiles during sucrose-added fermentation. The results showed that L. sakei L3 could survive between pH 4.0 and pH 9.0, tolerant to NaCl (<10%, w/v) and urea (<6%, w/v). Meanwhile, transcriptomic analysis showed that a total of 426 differentially expressed genes and eight non-coding RNAs were identified. Genes associated with sucrose metabolism were significantly induced, so L. sakei L3 increased the utilization of sucrose to produce EPS, while genes related to uridine monophosphate (UMP), fatty acids and folate synthetic pathways were significantly inhibited, indicating that L. sakei L3 decreased self-growth, substance and energy metabolism to satisfy EPS production. Overall, transcriptome analysis provided valuable insights into the mechanisms by which L. sakei L3 utilizes sucrose for EPS biosynthesis. The study provided a theoretical foundation for the further application of functional EPS in the food industry.

Novel insight into the formation and improvement mechanism of physical property in fermented tilapia sausage by cooperative fermentation of newly isolated lactic acid bacteria based on microbial contribution.

Single starter can hardly elevate the gel property of fermented freshwater fish sausage. In this work, in order to improve the physical properties of tilapia sausage, two newly isolated strains of lactic acid bacteria (LAB), Latilactobacillus sakei and Pediococcus acidilactici were used for cooperative fermentation of tilapia sausage, followed by the revelation of their formation mechanisms during cooperative fermentation and their improvement mechanisms after comparison with natural fermentation. Both strains, especially L. sakei possessed good growth, acidification ability, and salt tolerance. The gel strength, hardness, springiness, chewiness, whiteness, acidification, and total plate count significantly elevated during cooperative fermentation with starters. Pediococcus, Acinetobacter, and Macrococcus were abundant before fermentation, while Latilactobacillus quickly occupied the dominant position after fermentation for 18-45 h with the relative abundance over 51.5 %. The influence of each genus on the physical properties was calculated through the time-dimension and group-dimension correlation networks. The results suggested that the increase of Latilactobacillus due to the good growth and metabolism of L. sakei contributed the most to the formation and improvement of gel strength, texture properties, color, acidification, and food safety of tilapia sausage after cooperative fermentation. This study provides a novel analysis method to quantitatively evaluate the microbial contribution on the changes of various properties. The cooperative fermentation of LAB can be used for tilapia sausage fermentation to improve its physical properties.

Improvement mechanism of volatile flavor in fermented tilapia surimi by cooperative fermentation of Pediococcus acidilactici and Latilactobacillus sakei: Quantization of microbial contribution through influence of genus.

Single starter can hardly improve the volatile flavor of fermented fish surimi. In this study, the changes of volatile compounds (VCs) and microbial composition during cooperative fermentation of Latilactobacillus sakei and Pediococcus acidilactici were studied by headspace solid-phase microextraction gas chromatography-mass spectrometry and 16S rRNA gene high-throughput sequencing. During cooperative fermentation, most VCs and the abundance of Latilactobacillus and Lactococcus significantly increased, while Pediococcus, Acinetobacter, and Macrococcus obviously decreased. After evaluation of correlation and abundance of each genus, Latilactobacillus and Lactococcus possessed the highest influence on the formation of volatile flavor during cooperative fermentation. Compared with the natural fermentation, cooperative fermentation with starters significantly enhanced most of pleasant core VCs (odor activity value≥1), but inhibited the production of trimethylamine and methanethiol, mainly resulting from the absolutely highest influence of Latilactobacillus. Cooperative fermentation of starters is an effective method to improve the volatile flavor in the fermented tilapia surimi.

Impact of different sugars and glycosyltransferases on the assertiveness of Latilactobacillus sakei in raw sausage fermentations.

Latilactobacillus sakei comprises a biodiversity of strains, which display different assertiveness upon their application as starter cultures in raw sausage fermentation. While the assertiveness of winning partner strains has been referred to competitive exclusion based on genomic settings enabling occupation of multiple niches of the sausage habitat, single strain assertiveness of L. sakei remained unexplained. In this study we assessed the impact of the expression of a glycosyltransferase enabling the production of a glucan from sucrose to the assertiveness of L. sakei TMW 1.411, which expresses a plasmid-encoded glycosyltransferase (gtf). In a sausage fermentation model wild type L. sakei TMW 1.411 and its plasmid-cured mutant were employed in competition with each other and with other Latilactobacillus sakei strains. To differentiate any effects resulting from general sugar utilization from those of glucan formation, the experiments were carried out with glucose, fructose, and sucrose, respectively. It was shown that the type of sugar affects the individual strains behaviour, and that the wild type was more competitive than the mutant in the presence of any of these sugars. In direct competition between wild type and mutant, a clear competitive advantage could also be demonstrated for the strain possessing the plasmid with the glycosyltransferase. Since this competitive advantage was observed with all sugars, not just sucrose, and Gtf expression has been shown as independent of the employed sugar, it is suggested that possession of the gtf gene-carrying plasmid confers a competitive advantage. It appears that the Gtf contributes to competitive exclusion and the establishment of colonization resistance, to a larger extent by an adhesive functionality of the Gtf on the cellular surface than by the production of glucan. Hence, gtf genes can be used as a possible additional marker for the selection of assertive L. sakei starter strains in sausage fermentation.

Genomic and metabolic features of the Lactobacillus sakei JD10 revealed potential probiotic traits.

Lactic acid bacteria that inhabit in the lung play important roles in maintaining the microbiome balance by interacting with the host immune system. Numerous metabolites (e.g., short chain fatty acids, bacteriocins, and hydrogen peroxide) produced by Lactobacillus sakei possess a special inhibitory spectrum against invading pathogens. In this research, the whole genome of L. sakei JD10 strain isolated from the porcine lung was sequenced and investigated. The whole size of the L. sakei JD10 chromosome was 1,989,921 bp, which encoded a total of 1951 predicted genes. Genome analyses revealed that many genes encoded carbohydrate-active enzymes (CAZymes) were predicted, which were responsible for the carbohydrate degradation and short chain fatty acids production. The metabolic profiles of short chain fatty acids in the L. sakei JD10 culture medium were measured by GC/TOFMS, and their regulatory effects on bacterial phagocytosis of RAW264.7 cells were also determined. The bacteriocin-producing genes of the L. sakei JD10 genome were also predicted, and a bacteriocin gene encoding carnocin was characterized and its molecular structure was analyzed. Two CRISPR-Cas system related genes were identified from the L. sakei JD10 genome, revealed that precise and efficient genome editing technologies could be applied for genetic engineering-manipulation. In all, investigation on the genomic features and metabolic features of L. sakei JD10 showed the potential probiotic traits to fight against pathogenic infection and regulate the host immune function.

Lactic fermentation of cooked, comminuted mussel, Perna canaliculus.

The endogenous microflora of mussels, filter feeders, can include pathogens with resulting food safety concerns. The aim was to develop a cook-then-ferment technology to extend shelf life and safety of a ready-to-eat mussels. Only after cooking to destroy the mussel's endogenous microflora could an edible product be made as determined by pH decline after fermentation and the fate of common pathogens. Perna canaliculus was bought live at retail on many dates. Fermentation was with commercial lactic cultures incubated under vacuum at 30 °C for four days. Using one culture containing Pediococcus pentosaceus and Staphylococcus carnosus as a model, pH typically declined to 4.5 to 4.7, and common pathogens, Staphylococcus aureus, Salmonella and Vibrio parahaemolyticus were absent or reduced to acceptable levels. The fate of Listeria monocytogenes was studied with five cultures. These were variably effective at inhibition with one clear success, Chr Hansen's T-SC-150 containing a specific strain of Lactobacillus sakei, and flavour-generating Staphylococcus carnosus. This culture's efficacy was confirmed with sterile extracts of LAB challenging L. monocytogenes in vitro. This culture was also the most rapid fermenter by pH fall. Cook-then-ferment technology may be applied to other novel foods to minimise a disruptive endogenous microflora.

Lactobacillus sakei Pro-Bio65 Reduces TNF-α Expression and Upregulates GSH Content and Antioxidant Enzymatic Activities in Human Conjunctival Cells.

Purpose: The study investigates the regulatory effects exhibited by lysate of Lactobacillus sakei pro-Bio65 (4%; L.SK) on the human conjunctival epithelial (HCE) cell line. Methods: Trypan blue and methylthiazol tetrazolium (MTT) methods were used to assess cell growth and viability. Mitochondrial membrane potential was assessed by JC-1 staining and cytofluorimetric detection methods. The antioxidant pattern and the intracellular reactive oxygen species (ROS) levels were analyzed by spectrophotometric and spectrofluorimetric methods. NF-κB luciferase activity was quantified by luminometric detection. NF-κB nuclear translocation, as well as mitochondrial morphology, were investigated by immunofluorescence using confocal microscopy. Cytokines and COX2 expression levels were determined by Western blot analyses. Results: This study demonstrates that L.SK exposure does not influence HCE cell proliferation and viability in vitro. L.SK paraprobiotic induces mild-low levels of intracellular ROS. It is coupled to changes in the mitochondrial membrane potential (ΔΨm), in a context of a regular mitochondrial-network organization. The negative modulation of tumor necrosis factor alpha (TNF-α) expression levels and rising antioxidant defense efficiency, mediated by the upregulation of glutathione (GSH) and increased antioxidant enzymatic activities, were observed. Conclusions: This study demonstrates that L.SK empowers the antioxidant endogenous efficiency of HCE cells, by the upregulation of the GSH content and the enzymatic antioxidant pattern, and concurrently reduces TNF-α protein expression. Translational Relevance: Although the obtained in vitro results should be confirmed by in vivo investigations, our data suggest the possibility of L.SK paraprobiotic application for promoting eye health, exploring its use as an endogen antioxidant system inducer in preventing and treating different oxidative stress-based, inflammatory, and age-related conditions.

Changes in Bacterial and Chemical Components and Growth Prediction for Lactobacillus sakei during Kimoto-Style Fermentation Starter Preparation in Sake Brewing: a Comprehensive Analysis.

Kimoto-style seed mash is a traditional preparation method for sake that takes advantage of spontaneous lactic acid fermentation before the growth of yeast. Lactic acid helps decrease the pH in seed mash and control the growth of unfavorable microorganisms. In this study, we carried out a comprehensive analysis of the change in the bacterial community and chemical composition during the lactic acid fermentation stage in kimoto-style seed mash preparation. The bacterial transitions were diverse at five sake breweries, but they exhibited three patterns. Lactobacillus sakei was the dominant species in the later stage of lactic acid fermentation in all sake breweries. This species was found to be the most important bacterium for the accumulation of lactic acid, because its average production rate of lactic acid in seed mash reached 4.44 × 10-11 mg cell-1 h-1, which is 10 times higher than those of other species. As a result of specific growth rate analysis, it was revealed that the growth rate of L. sakei was influenced by the strain, pH, and temperature. The effects of pH and temperature were explained by the square root model, and the result indicates that the strains isolated in this study were incapable of growth below pH 3.9. The growth curve predicted using the growth model fit the actual cell density in two out of five sake breweries; however, our model did not work well for the remaining three sake breweries, and we presume that the error was caused by the strain or an unknown factor.IMPORTANCE It is important to produce lactic acid in kimoto-style seed mash; however, the bacterial transition is different depending on the sake brewery. The reason why there are diverse bacterial transitions during kimoto-style seed mash preparation for each sake brewery is unclear so far, and it causes difficulty in starting kimoto-style seed mash. Our findings indicate that the changes in pH caused by lactic acid bacteria grown prior to L. sakei in seed mash influence the growth of L. sakei and are related to the diversity of the bacterial transition. This study uses comprehensive analytical methods to reveal that there is a diversity of bacterial transition and chemical compositions in kimoto-style seed mash depending on the sake brewery and to explain the differences in bacterial transition depending on the characteristics of L. sakei.

Interspecies assertiveness of Lactobacillus curvatus and Lactobacillus sakei in sausage fermentations.

Lactobacillus (L.) curvatus and L. sakei contain strains, which are assertive in sausage fermentation. Previous work has demonstrated differences in assertiveness at strain level within one species, and revealed either exclusion of competitors by complementary partner strains or their inhibition by single strains. This work addresses interspecies differences in the assertiveness of L. curvatus and L. sakei. Strain sets of L. curvatus and L. sakei were employed as starters in a fermented sausage model and their abundancy upon fermentation was determined by strain-specific MALDI-TOF MS identification. Generally, single or groups of L. sakei strains outcompeted L. curvatus strains. In multiple growth tests employing mMRS and mMSM it could be shown that assertive L. sakei strains can be predicted along their µ max in mMSM. Still, L. curvatus TMW 1.624 could suppress all L. curvatus and most L. sakei strains in competitive settings. This could be referred to its expression of several bacteriocins, which are active against all of the L. curvatus strains. Strain specific differences could be demonstrated in the susceptibility of L. sakei to bacteriocins, and in oxidative stress tolerance, which is higher in co-existing L. sakei strains than in the bacteriocin producer. This suggests that tolerance to bacteriocins and oxidative stress represent additional determinants for assertiveness, above previously reported bacteriocin production versus metabolic complementarism of partner strains.

Heme Uptake in Lactobacillus sakei Evidenced by a New Energy Coupling Factor (ECF)-Like Transport System.

Lactobacillus sakei is a nonpathogenic lactic acid bacterium and a natural inhabitant of meat ecosystems. Although red meat is a heme-rich environment, L. sakei does not need iron or heme for growth, although it possesses a heme-dependent catalase. Iron incorporation into L. sakei from myoglobin and hemoglobin was previously shown by microscopy and the L. sakei genome reveals the complete equipment for iron and heme transport. Here, we report the characterization of a five-gene cluster (from lsa1836 to lsa1840 [lsa1836-1840]) encoding a putative metal iron ABC transporter. Interestingly, this cluster, together with a heme-dependent catalase gene, is also conserved in other species from the meat ecosystem. Our bioinformatic analyses revealed that the locus might correspond to a complete machinery of an energy coupling factor (ECF) transport system. We quantified in vitro the intracellular heme in the wild type (WT) and in our Δlsa1836-1840 deletion mutant using an intracellular heme sensor and inductively coupled plasma mass spectrometry for quantifying incorporated 57Fe heme. We showed that in the WT L. sakei, heme accumulation occurs rapidly and massively in the presence of hemin, while the deletion mutant was impaired in heme uptake; this ability was restored by in trans complementation. Our results establish the main role of the L. sakei Lsa1836-1840 ECF-like system in heme uptake. Therefore, this research outcome sheds new light on other possible functions of ECF-like systems.IMPORTANCELactobacillus sakei is a nonpathogenic bacterial species exhibiting high fitness in heme-rich environments such as meat products, although it does not need iron or heme for growth. Heme capture and utilization capacities are often associated with pathogenic species and are considered virulence-associated factors in the infected hosts. For these reasons, iron acquisition systems have been deeply studied in such species, while for nonpathogenic bacteria the information is scarce. Genomic data revealed that several putative iron transporters are present in the genome of the lactic acid bacterium L. sakei In this study, we demonstrate that one of them is an ECF-like ABC transporter with a functional role in heme transport. Such evidence has not yet been brought for an ECF; therefore, our study reveals a new class of heme transport system.

Genomic and metabolic features of Lactobacillus sakei as revealed by its pan-genome and the metatranscriptome of kimchi fermentation.

The genomic and metabolic features of Lactobacillus sakei were investigated using its pan-genome and by analyzing the metatranscriptome of kimchi fermentation. In the genome-based relatedness analysis, the strains were divided into the Lb. sakei ssp. sakei and Lb. sakei ssp. carnosus lineage groups. Genomic and metabolic pathway analysis revealed that all Lb. sakei strains have the capability of producing d/l-lactate, ethanol, acetate, CO2, formate, l-malate, diacetyl, acetoin, and 2,3-butanediol from d-glucose, d-fructose, d-galactose, sucrose, d-lactose, l-arabinose, cellobiose, d-mannose, d-gluconate, and d-ribose through homolactic and heterolactic fermentation, whereas their capability of d-maltose, d-xylose, l-xylulose, d-galacturonate, and d-glucuronate metabolism is strain-specific. All strains carry genes for the biosynthesis of folate and thiamine, whereas genes for biogenic amine and toxin production, hemolysis, and antibiotic resistance were not identified. The metatranscriptomic analysis showed that the expression of Lb. sakei transcripts involved in carbohydrate metabolism increased as kimchi fermentation progressed, suggesting that Lb. sakei is more competitive during late fermentation stage. Homolactic fermentation pathway was highly expressed and generally constant during kimchi fermentation, whereas expression of heterolactic fermentation pathway increased gradually as fermentation progressed. l-Lactate dehydrogenase was more highly expressed than d-lactate dehydrogenase, suggesting that l-lactate is the major lactate metabolized by Lb. sakei.

Isolation and characterization of dextran produced by Lactobacillus sakei L3 from Hubei sausage.

An exopolysaccharide (EPS)-producing bacterial strain L3 was isolated from Hubei sausage and identified as Lactobacillus sakei via morphological, physiological, biochemical and 16S rDNA analysis. FT-IR spectroscopy and NMR revealed that L3 EPS was a dextran containing d-glucose residues with α-1,6 glycosidic linkage. Rheological studies showed that it had high viscosity at high concentration, low temperature, and acidic pH (pH 3.0). Scanning electron microscopy of the L3 dextran demonstrated a porous and branched morphology, and atomic force microscopy showed lumps of varying height on the rough surface of the L3 EPS polymer. The EPS was thermally stable up to 272°C and could coagulate sucrose-supplemented milk. Together, these results suggested that L3 EPS might have potential applications in food processing and other areas.

The application of selected ion flow tube-mass spectrometry to follow volatile formation in modified-atmosphere-packaged cooked ham.

Cooked pork products, i.e., sliced cooked hams maintained under modified-atmosphere-packaging (MAP), were analysed both microbiologically and with respect to volatile levels during storage. Three storage temperature ranges were compared (4-6 °C, 7-9 °C, and 11-13 °C), representing different refrigeration conditions at household level. The microbial loads were determined by plating samples on six different agar media, followed by (GTG)5-PCR fingerprinting of genomic DNA of selected isolates, and identification of representative isolates by 16S rRNA, pheS, and rpoA gene sequencing. Carnobacterium maltaromaticum, Lactobacillus sakei, and Serratia proteamaculans were the major bacterial species found among the 619 isolates identified. The volatiles produced during storage were followed by selected ion flow tube-mass spectrometry (SIFT-MS) and the identity of the volatiles was confirmed by headspace solid-phase microextraction combined with gas chromatography and time-of-flight mass spectrometry (HS-SPME-GC-TOF-MS). SIFT-MS analysis showed that volatiles, such as 2,3-butanediol, acetoin, and ethanol, may serve as potential markers for spoilage development. Differences in volatile production between samples were likely due to discrepancies in the initial microbial load and the effect of storage conditions. In conclusion, this study combines the use of new mass spectrometric techniques to examine volatile production during spoilage as an additional source of information during microbiological community analysis.

Comparative genomics of Lactobacillus sakei supports the development of starter strain combinations.

Strains of Lactobacillus sakei can be isolated from a variety of sources including meat, fermented sausages, sake, sourdough, sauerkraut or kimchi. Selected strains are widely used as starter cultures for sausage fermentation. Recently we have demonstrated that control about the lactic microbiota in fermenting sausages is achieved rather by pairs or strain sets than by single strains. In this work we characterized the pan genome of L. sakei to enable exploitation of the genomic diversity of L. sakei for the establishment of assertive starter strain sets. We have established the full genome sequences of nine L. sakei strains from different sources of isolation and included in the analysis the genome of L. sakei 23K. Comparative genomics revealed an accessory genome comprising about 50% of the pan genome and different lineages of strains with no relation to their source of isolation. Group and strain specific differences could be found, which namely referred to agmatine and citrate metabolism. The presence of genes encoding metabolic pathways for fructose, sucrose and trehalose as well as gluconate in all strains suggests a general adaptation to plant/sugary environments and a life in communities with other genera. Analysis of the plasmidome did not reveal any specific mechanisms of adaptation to a habitat. The predicted differences of metabolic settings enable prediction of partner strains, which can occupy the meat environment to a large extent and establish competitive exclusion of autochthonous microbiota. This may assist the development of a new generation of meat starter cultures containing L. sakei strains.

Modelling the interaction of the sakacin-producing Lactobacillus sakei CTC494 and Listeria monocytogenes in filleted gilthead sea bream (Sparus aurata) under modified atmosphere packaging at isothermal and non-isothermal conditions.

The objective of this work was to quantitatively evaluate the effect of Lactobacillus sakei CTC494 (sakacin-producing bioprotective strain) against Listeria monocytogenes in fish juice and to apply and validate three microbial interaction models (Jameson, modified Jameson and Lotka Volterra models) through challenge tests with gilthead sea bream (Sparus aurata) fillets under modified atmosphere packaging stored at isothermal and non-isothermal conditions. L. sakei CTC494 inhibited L. monocytogenes growth when simultaneously present in the matrix (fish juice and fish fillets) at different inoculation ratios pathogen:bioprotector (i.e. 1:1, 1:2 and 1:3). The higher the inoculation ratio, the stronger the inhibition of L. monocytogenes growth, with the ratio 1:3 yielding no growth of the pathogen. The maximum population density (Nmax) was the most affected parameter for L. monocytogenes at all inoculation ratios. According to the microbiological and sensory analysis outcomes, an initial inoculation level of 4 log cfu/g for L. sakei CTC494 would be a suitable bioprotective strategy without compromising the sensory quality of the fish product. The performance of the tested interaction models was evaluated using the Acceptable Simulation Zone approach. The Lotka Volterra model showed slightly better fit than the Jameson-based models with 75-92% out of the observed counts falling into the Acceptable Simulation Zone, indicating a satisfactory model performance. The evaluated interaction models could be used as predictive modelling tool to simulate the simultaneous behaviour of bacteriocin-producing Lactobacillus strains and L. monocytogenes; thus, supporting the design and optimization of bioprotective culture-based strategies against L. monocytogenes in minimally processed fish products.

Monitoring of assertive Lactobacillus sakei and Lactobacillus curvatus strains using an industrial ring trial experiment.

AIMS: In a previous study, we used a 5-day fermenting sausage model to characterize assertiveness of Lactobacillus curvatus and Lactobacillus sakei starter strains towards employ autochthonous contaminants. In this work, we probed those findings and their transferability to real sausage fermentation including the drying process in an industrial ring trial experiment. METHODS AND RESULTS: Raw fermented sausages ('salami') were produced with three L. curvatus and four L. sakei strains as starter cultures in cooperation with three manufacturers from Germany. We monitored pH, water activity and microbiota dynamics at strain level over a total fermentation and ripening time of 21 days by MALDI-TOF-MS identification of isolates. The principal behaviour of the strains in real sausage fermentations was the same as that one observed in the 5-day model system delineating single strain assertiveness of a bacteriocin producer from co-dominance of strains. CONCLUSIONS: The water activity decrease, which is concomitant with the sausage ripening process has only limited impact on the assertiveness and survival of the starter strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of a 5-day model can provide insight in the assertiveness of a specific starter strain in sausage fermentation.

Semi-Rational Screening of Probiotics from the Fecal Flora of Healthy Adults against DSS-Induced Colitis Mice by Enhancing Anti-Inflammatory Activity and Modulating the Gut Microbiota.

Ulcerative colitis (UC), a chronic inflammatory bowel disease, substantially impacts patients' health-related quality of life. In this study, an effective strategy for discovering high-efficiency probiotics has been developed. First, in order to survive in the conditions of the stomach and intestine, high bile salt-resistant and strong acid-resistant strains were screened out from the fecal flora of healthy adults. Next, the probiotic candidates were rescreened by examining the induction ability of IL-10 (anti-inflammatory factor) production in dextran sodium sulfate (DSS)-induced colitis mice, and Lactobacillus sakei 07 (L07) was identified and selected as probiotic P. In the end, fourteen bifidobacterium strains isolated from stools of healthy males were examined for their antimicrobial activity. Bifidobacterium bifidum B10 (73.75% inhibition rate) was selected as probiotic B. Moreover, the colonic IL-6 and TNF-α expression of the DSSinduced colitis mice treated with L. sakei 07 (L07) - B. bifidum B10 combination (PB) significantly decreased and the IL-10 expression was up-regulated by PB compared to the DSS group. Furthermore, Bacteroidetes and Actinobacteria decreased and Firmicutes increased in the DSS group mice, significantly. More interestingly, the intestinal flora biodiversity of DSS colitis mice was increased by PB. Of those, the level of B. bifidum increased significantly. The Bacteriodetes/Firmicutes (B/F) ratio increased, and the concentration of homocysteine and LPS in plasma was down-regulated by PB in the DSS-induced colitis mice. Upon administration of PB, the intestinal permeability of the the DSS-induced colitis mice was decreased by approximately 2.01-fold. This method is expected to be used in high-throughput screening of the probiotics against colitis. In addition, the L. sakei 07 - B. bifidum B10 combination holds potential in UC remission by immunomodulatory and gut microbiota modulation.

Robust Domination of Lactobacillus sakei in Microbiota During Traditional Japanese Sake Starter Yamahai-Moto Fermentation and the Accompanying Changes in Metabolites.

The successful production of sake (Japanese rice wine) is brought about by drastic changes in microbial flora and chemical components during fermentation. In the traditional manufacturing process of sake starter (yamahai-moto), spontaneous growth of lactic acid bacteria suppresses inappropriate microorganisms and prepares the optimum environment for the alcohol fermentative yeast. In this study, we analyzed the changes in bacterial flora and chemical components of yamahai-moto. High-throughput next-generation sequencing (NGS) of the 16S ribosomal RNA gene V4 region revealed that various kinds of bacteria, including nitrate-reducing bacteria, existed in the early fermentation stage; however, Lactobacillus sakei then increased drastically to become dominant in the middle stage. Interestingly, this result was different from that obtained in the previous year at the same manufacturer; the early-stage major bacterium was Lactobacillus acidipiscis. Lactic acid, glucose, isomaltose, and total free amino acids increased throughout the fermentation process, which was attributable to the metabolism of L. sakei and the koji mold. It is noteworthy that significant ornithine accumulation and arginine consumption were observed from the middle to late stages. Thirty-eight percent of the L. sakei isolates from yamahai-moto exhibited significant ornithine production, indicating that the arginine deiminase pathway of L. sakei was working to survive the extremely low pH environment of the moto after the middle stage. This is the first report that includes concurrent analyses of the NGS-based bacterial flora and chemical components of yamahai-moto, providing further knowledge to help understand and improve the process of sake brewing.

A peptidomic approach of meat protein degradation in a low-sodium fermented sausage model using autochthonous starter cultures.

Fermented sausage technology is currently compromised in decreasing the addition of NaCl. Use of starter cultures with peptidogenic potential could be a valuable strategy that can mask or hide off flavors produced by the use of NaCl substituents. In the present work, the peptidogenic potential of four lactic acid bacteria species was evaluated in a low-sodium beaker sausage (BS) model. Using a peptidomic approach, a total of 86 low molecular weight (LMW) peptides were accurately identified, mostly derived from myofibrillar proteins, especially actin, which generated 53 peptides. The BS inoculated with L. curvatus CRL705 generated 56 LMW peptides, followed by Enterococcus (E.) mundtii CRL35 with 43 peptides. In addition, BS inoculated with Lactobacillus (L.) plantarum and with L. sakei produced higher amino acid amounts over time as compared to the rest of BS models, highlighting the importance of both, time and sample effect on the overall free amino acid generation. The presence of each LAB strain in BS models generated a unique profile of small peptides and amino acids that could serve as a distinctive biochemical trait to differentiate specific fermented products. According to these results, E. mundtii and L. sakei, which are compatible between them, are proposed as the most efficiently adapted to low-sodium conditions. The use of selected strains during the processing of low-sodium fermented sausages could have a positive effect on the production of small peptides and free amino acids.

Characterization of multiple antilisterial peptides produced by sakacin P-producing Lactobacillus sakei subsp. sakei 2a.

Antimicrobial compounds produced by lactic acid bacteria can be explored as natural food biopreservatives. In a previous report, the main antimicrobial compounds produced by the Brazilian meat isolate Lactobacillus sakei subsp. sakei 2a, i.e., bacteriocin sakacin P and two ribosomal peptides (P2 and P3) active against Listeria monocytogenes, were described. In this study, we report the spectrum of activity, molecular mass, structural identity and mechanism of action of additional six antilisterial peptides produced by Lb. sakei 2a, detected in a 24 h-culture in MRS broth submitted to acid treatment (pH 1.5) and proper fractionation and purification steps for obtention of free and cell-bound proteins. The six peptides presented similarity to different ribosomal proteins of Lb. sakei subsp sakei 23K and the molecular masses varied from 4.6 to 11.0 kDa. All peptides were capable to increase the efflux of ATP and decrease the membrane potential in Listeria monocytogenes. The activity of a pool of the obtained antilisterial compounds [enriched active fraction (EAF)] against Listeria monocytogenes in a food model (meat gravy) during refrigerated storage (4 °C) for 10 days was also tested and results indicated that the populations of L. monocytogenes in the food model containing the acid extract remained lower than those at time 0-day, evidencing that the acid extract of a culture of Lb. sakei 2a is a good technological alternative for the control of growth of L. monocytogenes in foods.