Bacteroidota polysaccharide utilization system for branched dextran exopolysaccharides from lactic acid bacteria.

Dextran is an α-(1→6)-glucan that is synthesized by some lactic acid bacteria, and branched dextran with α-(1→2)-, α-(1→3)-, and α-(1→4)-linkages are often produced. Although many dextranases are known to act on the α-(1→6)-linkage of dextran, few studies have functionally analyzed the proteins involved in degrading branched dextran. The mechanism by which bacteria utilize branched dextran is unknown. Earlier, we identified dextranase (FjDex31A) and kojibiose hydrolase (FjGH65A) in the dextran utilization locus (FjDexUL) of a soil Bacteroidota Flavobacterium johnsoniae and hypothesized that FjDexUL is involved in the degradation of α-(1→2)-branched dextran. In this study, we demonstrate that FjDexUL proteins recognize and degrade α-(1→2)- and α-(1→3)-branched dextrans produced by Leuconostoc citreum S-32 (S-32 α-glucan). The FjDexUL genes were significantly upregulated when S-32 α-glucan was the carbon source compared with α-glucooligosaccharides and α-glucans, such as linear dextran and branched α-glucan from L. citreum S-64. FjDexUL glycoside hydrolases synergistically degraded S-32 α-glucan. The crystal structure of FjGH66 shows that some sugar-binding subsites can accommodate α-(1→2)- and α-(1→3)-branches. The structure of FjGH65A in complex with isomaltose supports that FjGH65A acts on α-(1→2)-glucosyl isomaltooligosaccharides. Furthermore, two cell surface sugar-binding proteins (FjDusD and FjDusE) were characterized, and FjDusD showed an affinity for isomaltooligosaccharides and FjDusE for dextran, including linear and branched dextrans. Collectively, FjDexUL proteins are suggested to be involved in the degradation of α-(1→2)- and α-(1→3)-branched dextrans. Our results will be helpful in understanding the bacterial nutrient requirements and symbiotic relationships between bacteria at the molecular level.

Comparative genomics of four lactic acid bacteria identified with Vitek MS (MALDI-TOF) and whole-genome sequencing.

Lactic acid bacteria (LAB) can be used as a probiotic or starter culture in dairy, meat, and vegetable fermentation. Therefore, their isolation and identification are essential. Recent advances in omics technologies and high-throughput sequencing have made the identification and characterization of bacteria. This study firstly aimed to demonstrate the sensitivity of the Vitek MS (MALDI-TOF) system in the identification of lactic acid bacteria and, secondly, to characterize bacteria using various bioinformatics approaches. Probiotic potency-related genes and secondary metabolite biosynthesis gene clusters were examined. The Vitek MS (MALDI-TOF) system was able to identify all of the bacteria at the genus level. According to whole genome sequencing, the bacteria were confirmed to be Lentilactobacillus buchneri, Levilactobacillus brevis, Lactiplantibacillus plantarum, Levilactobacillus namurensis. Bacteria had most of the probiotic potency-related genes, and different toxin-antitoxin systems such as PemIK/MazEF, Hig A/B, YdcE/YdcD, YefM/YoeB. Also, some of the secondary metabolite biosynthesis gene clusters, some toxic metabolite-related genes, and antibiotic resistance-related genes were detected. In addition, Lentilactobacillus buchneri Egmn17 had a type II-A CRISPR/Cas system. Lactiplantibacillus plantarum Gmze16 had a bacteriocin, plantaricin E/F.

Genomics-based analysis of four porcine-derived lactic acid bacteria strains and their evaluation as potential probiotics.

The search for probiotics and exploration of their functions are crucial for livestock farming. Recently, porcine-derived lactic acid bacteria (LAB) have shown great potential as probiotics. However, research on the evaluation of porcine-derived LAB as potential probiotics through genomics-based analysis is relatively limited. The present study analyzed four porcine-derived LAB strains (Lactobacillus johnsonii L16, Latilactobacillus curvatus ZHA1, Ligilactobacillus salivarius ZSA5 and Ligilactobacillus animalis ZSB1) using genomic techniques and combined with in vitro tests to evaluate their potential as probiotics. The genome sizes of the four strains ranged from 1,897,301 bp to 2,318,470 bp with the GC contents from 33.03 to 41.97%. Pan-genomic analysis and collinearity analysis indicated differences among the genomes of four strains. Carbohydrate active enzymes analysis revealed that L. johnsonii L16 encoded more carbohydrate active enzymes than other strains. KEGG pathway analysis and in vitro tests confirmed that L. johnsonii L16 could utilize a wide range of carbohydrates and had good utilization capacity for each carbohydrate. The four strains had genes related to acid tolerance and were tolerant to low pH, with L. johnsonii L16 showing the greatest tolerance. The four strains contained genes related to bile salt tolerance and were able to tolerate 0.1% bile salt. Four strains had antioxidant related genes and exhibited antioxidant activity in in vitro tests. They contained the genes linked with organic acid biosynthesis and exhibited antibacterial activity against enterotoxigenic Escherichia coli K88 (ETEC K88) and Salmonella 6,7:c:1,5, wherein, L. johnsonii L16 and L. salivarius ZSA5 had gene clusters encoding bacteriocin. Results suggest that genome analysis combined with in vitro tests is an effective approach for evaluating different strains as probiotics. The findings of this study indicate that L. johnsonii L16 has the potential as a probiotic strain among the four strains and provide theoretical basis for the development of probiotics in swine production.

Prebiotic effect of galacto-N-biose on the intestinal lactic acid bacteria as enhancer of acetate production and hypothetical colonization.

Galacto-N-biose (GNB) is an important core structure of glycan of mucin glycoproteins in the gastrointestinal (GI) mucosa. Because certain beneficial bacteria inhabiting the GI tract, such as bifidobacteria and lactic acid bacteria, harbor highly specialized GNB metabolic capabilities, GNB is considered a promising prebiotic for nourishing and manipulating beneficial bacteria in the GI tract. However, the precise interactions between GNB and beneficial bacteria and their accompanying health-promoting effects remain elusive. First, we evaluated the proliferative tendency of beneficial bacteria and their production of beneficial metabolites using gut bacterial strains. By comparing the use of GNB, glucose, and inulin as carbon sources, we found that GNB enhanced acetate production in Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Lactobacillus gasseri, and Lactobacillus johnsonii. The ability of GNB to promote acetate production was also confirmed by RNA-seq analysis, which indicated the upregulation of gene clusters that catalyze the deacetylation of N-acetylgalactosamine-6P and biosynthesize acetyl-CoA from pyruvate, both of which result in acetate production. To explore the in vivo effect of GNB in promoting acetate production, antibiotic-treated BALB/cA mice were administered with GNB with L. rhamnosus, resulting in a fecal acetate content that was 2.7-fold higher than that in mice administered with only L. rhamnosus. Moreover, 2 days after the last administration, a 3.7-fold higher amount of L. rhamnosus was detected in feces administered with GNB with L. rhamnosus than in feces administered with only L. rhamnosus. These findings strongly suggest the prebiotic potential of GNB in enhancing L. rhamnosus colonization and converting L. rhamnosus into higher acetate producers in the GI tract. IMPORTANCE: Specific members of lactic acid bacteria, which are commonly used as probiotics, possess therapeutic properties that are vital for human health enhancement by producing immunomodulatory metabolites such as exopolysaccharides, short-chain fatty acids, and bacteriocins. The long residence time of probiotic lactic acid bacteria in the GI tract prolongs their beneficial health effects. Moreover, the colonization property is also desirable for the application of probiotics in mucosal vaccination to provoke a local immune response. In this study, we found that GNB could enhance the beneficial properties of intestinal lactic acid bacteria that inhabit the human GI tract, stimulating acetate production and promoting intestinal colonization. Our findings provide a rationale for the addition of GNB to lactic acid bacteria-based functional foods. This has also led to the development of therapeutics supported by more rational prebiotic and probiotic selection, leading to an improved healthy lifestyle for humans.

Decarboxylase activity of the non-starter lactic acid bacterium Loigolactobacillus rennini gives crack defects in Gouda cheese through the production of γ-aminobutyric acid.

Ten Gouda cheese wheels with an age of 31 weeks from six different batch productions were affected by a crack defect and displayed an unpleasant off-flavor. To unravel the causes of these defects, the concentrations of free amino acids, other organic acids, volatile organic compounds, and biogenic amines were quantified in zones around the cracks and in zones without cracks, and compared with those of similar Gouda cheeses without crack defect. The Gouda cheeses with cracks had a significantly different metabolome. The production of the non-proteinogenic amino acid γ-aminobutyric acid (GABA) could be unraveled as the key mechanism leading to crack formation, although the production of the biogenic amines cadaverine and putrescine contributed as well. High-throughput amplicon sequencing of the full-length 16S rRNA gene based on whole-community DNA revealed the presence of Loigolactobacillus rennini and Tetragenococcus halophilus as most abundant non-starter lactic acid bacteria in the zones with cracks. Shotgun metagenomic sequencing allowed to obtain a metagenome-assembled genome of both Loil. rennini and T. halophilus. However, only Loil. rennini contained genes necessary for the production of GABA, cadaverine, and putrescine. Metagenetics further revealed the brine and the rennet used during cheese manufacturing as the most plausible inoculation sources of both Loil. rennini and T. halophilus.IMPORTANCECrack defects in Gouda cheeses are still poorly understood, although they can lead to major economic losses in cheese companies. In this study, the bacterial cause of a crack defect in Gouda cheeses was identified, and the pathways involved in the crack formation were unraveled. Moreover, possible contamination sources were identified. The brine bath might be a major source of bacteria with the potential to deteriorate cheese quality, which suggests that cheese producers should regularly investigate the quality and microbial composition of their brines. This study illustrated how a multiphasic approach can understand and mitigate problems in a cheese company.

A multifaceted investigation of lactococcal strain diversity in undefined mesophilic starter cultures.

The dairy fermentation industry relies on the activity of lactic acid bacteria in robust starter cultures to accomplish milk acidification. Maintenance of the composition of these starter cultures, whether defined or undefined, is essential to ensure consistent and high-quality fermentation end products. To date, limited information exists regarding the microbial composition of undefined starter culture systems. Here, we describe a culture-based analysis combined with a metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, we describe a qPCR-based genotype detection assay, which is capable of discerning nine distinct lactococcal genotypes to characterize these undefined starter cultures, and which can be applied to monitor compositional changes in an undefined starter culture during a fermentation. IMPORTANCE: This study reports on the development of a combined culture-based analysis and metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, a novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes (based on lactococcal cell wall polysaccharide biosynthesis gene clusters), was used to monitor compositional changes in an undefined starter culture following phage attack. These analytical approaches facilitate a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important QC aspect due to the increasing demand for consistent and high-quality dairy products.

Deciphering the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice fermentation using phenotypic and transcriptional analysis.

In the context of sustainable diet, the development of soy-based yogurt fermented with lactic acid bacteria is an attractive alternative to dairy yogurts. To decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice (SJ) fermentation, the whole genome of the strain CIRM-BIA865 (Ld865) was sequenced and annotated. Then Ld865 was used to ferment SJ. Samples were analyzed throughout fermentation for their cell number, carbohydrate, organic acid, free amino acid, and volatile compound contents. Despite acidification, the number of Ld865 cells did not rise, and microscopic observations revealed the elongation of cells from 3.6 µm (inoculation) to 36.9 µm (end of fermentation). This elongation was observed in SJ but not in laboratory-rich medium MRS. Using transcriptomic analysis, we showed that the biosynthesis genes of peptidoglycan and membrane lipids were stably expressed, in line with the cell elongation observed, whereas no genes implicated in cell division were upregulated. Among the main sugars available in SJ (sucrose, raffinose, and stachyose), Ld865 only used sucrose. The transcriptomic analysis showed that Ld865 implemented the two transport systems that it contains to import sucrose: a PTS system and an ABC transporter. To fulfill its nitrogen needs, Ld865 probably first consumed the free amino acids of the SJ and then implemented different oligopeptide transporters and proteolytic/peptidase enzymes. In conclusion, this study showed that Ld865 enables fast acidification of SJ, despite the absence of cell division, leads to a product rich in free amino acids, and also leads to the production of aromatic compounds of interest. IMPORTANCE: To reduce the environmental and health concerns related to food, an alternative diet is recommended, containing 50% of plant-based proteins. Soy juice, which is protein rich, is a relevant alternative to animal milk, for the production of yogurt-like products. However, soy "beany" and "green" off-flavors limit the consumption of such products. The lactic acid bacteria (LAB) used for fermentation can help to improve the organoleptic properties of soy products. But metabolic data concerning LAB adapted to soy juice are lacking. The aim of this study was, thus, to decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during fermentation of a soy juice, based on a multidisciplinary approach. This result will contribute to give tracks for a relevant selection of starter. Indeed, the improvement of the organoleptic properties of these types of products could help to promote plant-based proteins in our diet.

The effect of seed germination and Bacillus spp. on the ripening of plant cheese analogs.

Consumer demand for plant cheeses is increasing, but challenges of improving both flavor and quality remain. This study investigated the microbiological and physicochemical impact of seed germination and fermentation with Bacillus velezensis and Bacillus amyloliquefaciens on the ripening of plant cheese analogs. Chlorine treatment or addition of Lactiplantibacillus plantarum and Lactococcus lactis controlled microbial growth during seed germination. Lp. plantarum and Lc. lactis also served as starter cultures for the acidification of soy and lupine milk and were subsequently present in the unripened plant cheese as dominant microbes. Acidification also inhibited the growth and metabolic activity of bacilli but Bacillus spores remained viable throughout ripening. During plant cheese ripening, Lc. lactis was inactivated before Lp. plantarum and the presence of bacilli during seed germination delayed Lc. lactis inactivation. Metagenomic sequencing of full-length 16S rRNA gene amplicons confirmed that the relative abundance of the inoculated strains in each ripened cheese sample exceeded 99%. Oligosaccharides including raffinose, stachyose, and verbascose were rapidly depleted in the initial stage of ripening. Both germination and the presence of bacilli during seed germination had impact on polysaccharide hydrolysis during ripening. Bacilli but not seed germination enhanced proteolysis of plant cheese during ripening. In conclusion, the use of germination with lactic acid bacteria in combination with Bacillus spp. exhibited the potential to improve the quality of ripened plant cheeses with a positive effect on the reduction of hygienic risks. IMPORTANCE: The development of novel plant-based fermented food products for which no traditional templates exist requires the development of starter cultures. Although the principles of microbial flavor formation in plant-based analogs partially overlap with dairy fermentations, the composition of the raw materials and thus likely the selective pressure on the activity of starter cultures differs. Experiments that are described in this study explored the use of seed germination, the use of lactic acid bacteria, and the use of bacilli to reduce hygienic risks, to acidify plant milk, and to generate taste-active compounds through proteolysis and fermentative conversion of carbohydrates. The characterization of fermentation microbiota by culture-dependent and culture-independent methods also confirmed that the starter cultures used were able to control microbial communities throughout 90 d of ripening. Taken together, the results provide novel tools for the development of plant-based analogs of fermented dairy products.

Effects of Neolamarckia cadamba leaves extract on microbial community and antibiotic resistance genes in cecal contents and feces of broilers challenged with lipopolysaccharides.

The effects of Neolamarckia cadamba leaves extract (NCLE), with effective ingredients of flavonoids, on antibiotic resistance genes (ARGs) and relevant microorganisms in cecal contents and feces of broilers treated with or without lipopolysaccharide stimulation (LPS) were investigated. LPS stimulation increased (P < 0.05) the relative abundance of ARGs and mobile genetic elements (MGEs), such as tet(W/N/W), APH(3')-IIIa, ErmB, tet (44), ANT (6)-Ia, tet(O), tet (32), Vang_ACT_CHL, myrA, ANT (6)-Ib, IncQ1, tniB, and rep2 in cecal contents. However, the difference disappeared (P > 0.05) when NCLE was added at the same time. These differential ARGs and MGEs were mainly correlated (P < 0.01) with Clostridiales bacterium, Lachnospiraceae bacterium, and Candidatus Woodwardibium gallinarum. These species increased in LPS-stimulated broilers and decreased when NCLE was applied at the same time. In feces, LPS stimulation decreased (P < 0.05) the relative abundance of tet(Q), adeF, ErmF, Mef(En2), OXA-347, tet (40), npmA, tmrB, CfxA3, and ISCrsp1, while the LPS + NCLE treated group showed no significant effect (P > 0.05) on these ARGs. These differential ARGs and MGEs in feces were mainly correlated (P < 0.01) with Clostridiales bacterium, Pseudoflavonifractor sp. An184, Flavonifractor sp. An10, Ruminococcaceae bacterium, etc. These species increased in LPS-stimulated broilers and increased when NCLE was applied at the same time. In conclusion, LPS stimulation and NCLE influenced microbial communities and associated ARGs in both cecal contents and feces of broilers. NCLE alleviated the change of ARGs and MGEs in LPS-induced broilers by maintaining the microbial balance.IMPORTANCEAntibiotics showed a positive effect on gut health regulation and growth performance improvement in livestock breeding, but the antimicrobial resistance threat and environment pollution problem are increasingly severe with antibiotics abuse. As alternatives, plant extract containing bioactive substances are increasingly used to improve immunity and promote productivity. However, little is known about their effects on diversity and abundance of ARGs. Here, we investigated the effects of NCLE, with effective ingredients of flavonoids, on ARGs and relevant microorganisms in cecal contents and feces of broilers treated with or without lipopolysaccharide stimulation. We found that NCLE reduced the abundance of ARGs in cecal contents of lipopolysaccharide-induced broilers by maintaining the microbial balance. This study provides a comprehensive view of cecal and fecal microbial community, ARGs, and MGEs of broiler following LPS stimulation and NCLE treatment. It might be used to understand and control ARGs dissemination in livestock production.

Potential role of salivary lactic acid bacteria in pathogenesis of oral lichen planus.

BACKGROUND: Emerging evidence emphasized the role of oral microbiome in oral lichen planus (OLP). To date, no dominant pathogenic bacteria have been identified consistently. It is noteworthy that a decreased abundance of Streptococcus, a member of lactic acid bacteria (LAB) in OLP patients has been commonly reported, indicating its possible effect on OLP. This study aims to investigate the composition of LAB genera in OLP patients by high-throughput sequencing, and to explore the possible relationship between them. METHODS: We collected saliva samples from patients with OLP (n = 21) and healthy controls (n = 22) and performed 16 S rRNA gene high-throughput sequencing. In addition, the abundance of LAB genera was comprehensively analyzed and compared between OLP and HC group. To verify the expression of Lactococcus lactis, real time PCR was conducted in buccal mucosa swab from another 14 patients with OLP and 10 HC. Furthermore, the correlation was conducted between clinical severity of OLP and LAB. RESULTS: OLP and HC groups showed similar community richness and diversity. The members of LAB, Lactococcus and Lactococcus lactis significantly decreased in saliva of OLP cases and negatively associated with OLP severity. In addition, Lactococcus and Lactococcus lactis showed negative relationship with Fusobacterium and Aggregatibacter, which were considered as potential pathogens of OLP. Similarly, compared with healthy controls, the amount of Lactococcus lactis in mucosa lesion of OLP patients was significantly decreased. CONCLUSIONS: A lower amount of Lactococcus at genus level, Lactococcus lactis at species level was observed in OLP cases and associated with disease severity. Further studies to verify the relationship between LAB and OLP, as well as to explore the precise mechanism is needed.

The predominant lactic acid bacteria and yeasts involved in the spontaneous fermentation of millet during the production of the traditional porridge Hausa koko in Ghana.

Spontaneous fermentation of cereals like millet involves a diverse population of microbes from various sources, including raw materials, processing equipment, fermenting receptacles, and the environment. Here, we present data on the predominant microbial species and their succession at each stage of the Hausa koko production process from five regions of Ghana. The isolates were enumerated using selective media, purified, and phenotypically characterised. The LAB isolates were further characterised by 16S rRNA Sanger sequencing, typed using (GTG)5 repetitive-PCR, and whole genome sequencing, while 28S rRNA Sanger sequencing was performed for yeast identification. The pH of the millet grains ranged from mean values of 6.02-6.53 to 3.51-3.99 in the final product, depending on the processors. The mean LAB and yeast counts increased during fermentation then fell to final counts of log 2.77-3.95 CFU/g for LAB and log 2.10-2.98 CFU/g for yeast in Hausa koko samples. At the various processing stages, the counts of LAB and yeast revealed significant variations (p < 0.0001). The species of LAB identified in this study were Limosilactobacillus pontis, Pediococcus acidilactici, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Pediococcus pentosaceus, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Schleiferilactobacillus harbinensis, and Weissella confusa. The yeasts were Saccharomyces cf. cerevisiae/paradoxus, Saccharomyces cerevisiae, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis. The identification and sequencing of these novel isolates and how they change during the fermentation process will pave the way for future controlled fermentation, safer starter cultures, and identifying optimal stages for starter culture addition or nutritional interventions. These LAB and yeast species are linked to many indigenous African fermented foods, potentially acting as probiotics in some cases. This result serves as the basis for further studies into the technological and probiotic potential of these Hausa koko microorganisms.

Characterization of the broad-spectrum antibacterial activity of bacteriocin-like inhibitory substance-producing probiotics isolated from fermented foods.

Antimicrobial peptides, such as bacteriocin, produced by probiotics have become a promising novel class of therapeutic agents for treating infectious diseases. Selected lactic acid bacteria (LAB) isolated from fermented foods with probiotic potential were evaluated for various tests, including exopolysaccharide production, antibiotic susceptibility, acid and bile tolerance, antibacterial activity, and cell adhesion and cytotoxicity to gastric cell lines. Six selected LAB strains maintained their high viability under gastrointestinal conditions, produced high exopolysaccharides, showed no or less cytotoxicity, and adhered successfully to gastric cells. Furthermore, three strains, Weissella confusa CYLB30, Lactiplantibacillus plantarum CYLB47, and Limosilactobacillus fermentum CYLB55, demonstrated a strong antibacterial effect against drug-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica serovar Choleraesuis, Enterococcus faecium, and Staphylococcus aureus. Whole genome sequencing was performed on these three strains using the Nanopore platform; then, the results showed that all three strains did not harbor genes related to toxins, superantigens, and acquired antimicrobial resistance, in their genome. The bacteriocin gene cluster was found in CYLB47 genome, but not in CYLB30 and CYLB55 genomes. In SDS-PAGE, the extract of CYLB30 and CYLB47 bacteriocin-like inhibitory substance (BLIS) yielded a single band with a size of less than 10 kDa. These BLIS inhibited the growth and biofilm formation of drug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA), causing membrane disruption and inhibiting adhesion ability to human skin HaCaT cells. Moreover, CYLB30 and CYLB47 BLIS rescued the larvae after being infected with P. aeruginosa and MRSA infections. In conclusion, CYLB30 and CYLB47 BLIS may be potential alternative treatment for multidrug-resistant bacteria infections.

Lactococcus lactis CNCM I-5388 versus NCDO2118 by its GABA hyperproduction ability, counteracts faster stress-induced intestinal hypersensitivity in rats.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by its main symptom, visceral hypersensitivity (VH), which is aggravated by stress. Gut-brain interactions and gut bacteria may alleviate IBS symptoms, including VH. γ-amino butyric acid (GABA), produced notably by lactic acid bacteria (LAB), shows promising result in IBS symptoms treatment. In bacteria, GABA is generated through glutamate decarboxylase (GAD) metabolism of L-glutamic acid, maintaining intracellular pH. In mammals, GABA acts as an inhibitory neurotransmitter, modulating pain, stress, and anxiety. Therefore, utilizing GABA-producing LAB as a therapeutic approach might be beneficial. Our previous work showed that a GABA-producing Lactococcus lactis strain, NCDO2118, reduced VH induced by acute stress in rats after a 10-day oral treatment. Here, we identified the strain CNCM I-5388, with a four-fold higher GABA production rate under the same conditions as NCDO2118. Both strains shared 99.1% identical GAD amino acid sequences and in vitro analyses revealed the same optimal pH for GAD activity; however, CNCM I-5388 exhibited 17 times higher intracellular GAD activity and increased resistance to acidic pH. Additionally, in vivo experiments have demonstrated that CNCM I-5388 has faster anti-VH properties in rats compared with NCDO2118, starting from the fifth day of treatment. Finally, CNCM I-5388 anti-VH effects partially persisted after 5-day treatment interruption and after a single oral treatment. These findings highlight CNCM I-5388 as a potential therapeutic agent for managing VH in IBS patients.

Detection and elimination of trace d-lactic acid in lignocellulose biorefining chain: Generation, flow, and impact on chiral lactide synthesis.

High chiral purity of lactic acid is a crucial indicator for the synthesis of chiral lactide as the primary intermediate chemical for ring-open polymerization of high molecular weight polylactic acid (PLA). Lignocellulose biomass is the most promising carbohydrate feedstock for commercial production of PLA, but the presence of trace d-lactic acid in the biorefinery chain adversely affects the synthesis and quality of chiral lactide. This study analyzed the fingerprint of trace  d-lactic acid in the biorefinery chain and found that the major source of  d-lactic acid comes from lignocellulose feedstock. The naturally occurring lactic acid bacteria and water-soluble carbohydrates in lignocellulose feedstock provide the necessary conditions for  d-lactic acid generation. Three strategies were proposed to eliminate the generation pathway of  d-lactic acid, including reduction of moisture content, conversion of water-soluble carbohydrates to furan aldehydes in pretreatment, and conversion to  l-lactic acid by inoculating engineered  l-lactic acid bacteria. The natural reduction of lactic acid content in lignocellulose feedstock during storage was observed due to the lactate oxidase-catalyzed oxidation of  l- and  d-lactic acids. This study provided an important support for the production of cellulosic  l-lactic acid with high chiral purity.

Isolation and characterization of lactic acid bacteria producing a potent anti-listerial bacteriocin-like inhibitory substance (BLIS) from chhurpi, a fermented milk product.

Nowadays, the bacteriocin industries have seen significant growth, supplanting chemical preservatives in its ability to improve the shelf-life and safety of food. The increasing customer desire to use natural preservatives has fueled advancing bacteriocin research. The objective of this study was to identify lactic acid bacteria (LAB) that produce bacteriocin-like inhibitory substance (BLIS) and have strong anti-listerial activity. We have identified and analyzed a LAB obtained from chhurpi samples, a popular milk-derived product in the Himalayan regions of India and Nepal. The strain was studied and identified based on its morphological, biochemical, and physiological characteristics. Furthermore, the molecular 16s-rDNA analysis suggests that the strain was Lactococcus sp. RGUAM1 (98.2% similar to Lactococcus lactis subsp. hordniae NBRC 100931T). The isolated strain can produce a potent BLIS, which has shown efficacy against three gram-positive bacteria responsible for food spoilage, such as Listeria monocytogenes (MTCC 657), Staphylococcus aureus subsp. aureus (MTCC 87), Lactobacillus plantarum (MTCC 1407), Lactobacillus paraplantarum (MTCC 12904). The scanning electron microscope (SEM) image illustrates that the crude cell-free supernatant (CFS) disrupts the cell envelope, leading to the release of cellular contents and the clustering of cells. In addition, this BLIS can easily withstand a wide range of pH (2-12), temperature (up to 100 °C for 15 min), bile salt (0.3% W/V), salinity (4% W/V), and enzyme activity of 1600 AU/ml against Listeria monocytogenes. Our research offers a robust framework and valuable insights into bio-preservation and its potential applications in diverse food products.

Microbial characterization of Sichuan Baoning vinegar: lactic acid bacteria, acetic acid bacteria and yeasts.

Sichuan Baoning vinegar, a typical representative of Sichuan bran vinegar, is a famous traditional fermented food made from cereals in China. At present, there are few studies on microbial characterization of culturable microorganisms in solid-state fermentation of Sichuan bran vinegar. To comprehensively understand the diversity of lactic acid bacteria, acetic acid bacteria and yeasts, which play an important role in the fermentation of Sichuan bran vinegar, traditional culture-dependent methods combined with morphological, biochemical, and molecular identification techniques were employed to screen and identify these isolates. A total of 34 lactic acid bacteria isolates, 39 acetic acid bacteria isolates, and 48 yeast isolates were obtained. Lactic acid bacteria were dominated by Enterococcus durans, Leuconostoc citreum, Lactococcus lactis, and Lactiplantibacillus plantarum, respectively. Latilactobacillus sakei was the first discovery in cereal vinegar. Acetic acid bacteria were mainly Acetobacter pomorum and A. pasteurianus. The dominant yeast isolates were Saccharomyces cerevisiae, in addition to four non-Saccharomyces yeasts. DNA fingerprinting revealed that isolates belonging to the same species exhibited intraspecific diversity, and there were differences between phenotypic and genotypic classification results. This study further enriches studies on cereal vinegar and lays a foundation for the development of vinegar starters.

Resuscitation of baijiu pit mud bacteria based on Rpf protein of Umezawaea beigongshangensis.

The pit mud in the Baijiu fermentation cellar is an abundant microbial resource that is closely related to the quality of baijiu. However, many naturally existing species might be in a viable but nonculturable (VBNC) state, posing challenges to the isolation and application of functional species. Herein, a previously isolated strain from baijiu mash, Umezawaea beigongshangensis, was found to contain the rpf gene that encodes resuscitation promotion factor (Rpf). Therefore, the gene was cloned and heterologously expressed, and the recombinant protein (Ub-Rpf 2) was purified. Ub-Rpf 2 was found to significantly promote the growth of resuscitated VBNC state Corynebacterium beijingensis and Sphingomonas beigongshangensis. To determine the resuscitation effect of Ub-Rpf 2 on real ecological samples, the protein was supplemented in pit mud for enrichment culture. Results revealed that specific families and genera were enriched in abundance upon Ub-Rpf 2 incubation, including a new family of Symbiobacteraceae and culturable Symbiobacterium genus. Furthermore, 14 species belonging to 12 genera were obtained in Ub-Rpf 2 treated samples, including a suspected novel species. This study lays a foundation for applying Rpf from U. beigongshangensis to exploit microbial resources in baijiu pit mud.

Impact of lactic acid bacteria strains against Listeria monocytogenes biofilms on various food-contact surfaces.

Listeria monocytogenes is one of the most important foodborne pathogens, causing listeriosis, a disease characterized by high mortality rates. This microorganism, commonly found in food production environments and transmitted to humans by consuming contaminated food, has the ability to form biofilms by attaching to a wide variety of surfaces. Traditional hygiene and sanitation procedures are not effective enough to completely remove L. monocytogenes biofilms from food-contact surfaces, which makes them a persistent threat to food safety. Alternative approaches to combating Listeria biofilms are needed, and the use of lactic acid bacteria (LAB) and their antimicrobial compounds shows promise. The present study investigated the effect of Lactobacillus strains, previously isolated from various foods and known to possess antimicrobial properties, on the biofilm formation of L. monocytogenes on three different food-contact surfaces. To study L. monocytogenes IVb ATCC 19115 type, culture was preferred to represent serotype IVb, which is responsible for the vast majority of listeriosis cases. The results demonstrated that cell-free supernatants (CFSs) of LAB strains inhibited biofilm formation by up to 51.57% on polystyrene, 60.96% on stainless steel, and 30.99% on glass surfaces. Moreover, these CFSs were effective in eradicating mature biofilms, with reductions of up to 78.86% on polystyrene, 73.12% on stainless steel, and 72.63% on glass surfaces. The strong inhibition rates of one strain of L. curvatus (P3X) and two strains of L. sakei (8.P1, 28.P2) used in the present study imply that they may provide an alternate technique for managing Listeria biofilms in food production environments.

Caproicibacterium argilliputei sp. nov., a novel caproic acid producing anaerobic bacterium isolated from pit clay.

An anaerobic, Gram-positive, rod-shaped, motile and spore-forming bacterium, designated strain ZCY20-5T, was isolated from pit clay of Chinese strong-aroma type Baijiu (Chinese liquor). Phylogenetic analyses based on 16S rRNA gene and genome sequences showed that strain ZCY20-5T belonged to the genus Caproicibacterium, family Oscillospiracheae, but it showed low similarity to the type species Caproicibacterium amylolyticum LBM18003T (98.00 %) and Caproicibacterium lactatifermentans LBM19010T (95.67 %). In anaerobic yeast extract medium, growth was observed at 20-45 °C (optimum, 35-40 °C), at pH 4.0-9.0 (optimum, pH 6.5-7.0) and with 0.0-2.0 % NaCl (w/v). The predominant fatty acids were C16 : 0, C14 : 0, C13 3-OH and C16 3-OH, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, three phospholipids of unknown structure containing glucosamine and two unidentified phospholipids. Strain ZCY20-5T exhibited an 81.32 % pairwise average nucleotide identity value, a 78.98 % average amino acid identity value and a 22.30 % digital DNA-DNA hybridization value compared to its closest relative C. amylolyticum LBM18003T. Based on morphological, physiological, biochemical, chemotaxonomic, genotypic and phylogenetic results, strain ZCY20-5T represents a novel species of Caproicibacterium, and the type strain is ZCY20-5T (=MCCC 1A19399T=KCTC 25590T).

Metagenomic analysis revealed the potential of lactic acid bacteria in improving natural saline-alkali land.

Management and improving saline-alkali land is necessary for sustainable agricultural development. We conducted a field experiment to investigate the effects of spraying lactic acid bacteria (LAB) on the cucumber and tomato plant soils. Three treatments were designed, including spraying of water, viable or sterilized LAB preparations to the soils of cucumber and tomato plants every 20 days. Spraying sterilized or viable LAB could reduce the soil pH, with a more obvious effect by using viable LAB, particularly after multiple applications. Metagenomic sequencing revealed that the soil microbiota in LAB-treated groups had higher alpha-diversity and more nitrogen-fixing bacteria compared with the water-treated groups. Both viable and sterilized LAB, but not water application, increased the complexity of the soil microbiota interactive network. The LAB-treated subgroups were enriched in some KEGG pathways compared with water or sterilized LAB subgroups, such as environmental information processing-related pathways in cucumber plant; and metabolism-related pathways in tomato plant, respectively. Redundancy analysis revealed association between some soil physico-chemical parameters (namely soil pH and total nitrogen) and bacterial biomarkers (namely Rhodocyclaceae, Pseudomonadaceae, Gemmatimonadaceae, and Nitrosomonadales). Our study demonstrated that LAB is a suitable strategy for decreasing soil pH and improving the microbial communities in saline-alkali land.