Probiotic cell-free supernatant as effective antimicrobials against Klebsiella pneumoniae and reduce antibiotic resistance development.

This study evaluated the antimicrobial activity, resistance development, and synergistic potential of cell-free supernatant (CFSs) derived from Levilactobacillus brevis (Lb-CFS) and Lactiplantibacillus plantarum (Lp-CFS) against Klebsiella pneumoniae. Both CFSs exhibited potent growth inhibition, with minimum inhibitory concentrations (MICs) of 128 µg/mL and 64 µg/mL for Lb-CFS and Lp-CFS, respectively, and demonstrated dose-dependent bactericidal activity, achieving complete bacterial eradication at minimum bactericidal concentrations (MBC) within 6 h. The CFSs suppressed the expression of virulence genes (galF, wzi, and manC) and biofilm formation in a dose-dependent manner. Synergistic interactions were observed when combining CFSs with antibiotics, resulting in 2- to fourfold reductions in antibiotic MICs and MBCs. Notably, adaptive evolution experiments revealed significantly slower resistance development in K. pneumoniae against CFSs (twofold MIC/MBC increase) compared to antibiotics (16- to 128-fold increase) after 21 days. Furthermore, CFS-adapted strains exhibited increased antibiotic susceptibility, while antibiotic-adapted strains displayed cross-resistance to multiple antibiotics. No cross-resistance occurred between Lb-CFS and Lp-CFS, suggesting distinct adaptive mechanisms. These findings highlight the potential of probiotic-derived CFSs as effective antimicrobials with a lower propensity for inducing rapid resistance compared to conventional antibiotics, suggesting their promise in combating multidrug-resistant infections.

The effect of heat-killed Lactobacillus brevis SBL88 on improving selective hepatic insulin resistance in non-alcoholic fatty liver disease mice without altering the gut microbiota.

BACKGROUND AND AIM: There have been several reports that some probiotics improve non-alcoholic fatty liver disease (NAFLD); however, many studies have involved cocktail therapies. We evaluated whether heat-killed Lactobacillus brevis SBL88 (L. brevis SBL88) monotherapy improves the clinical features of NAFLD. METHODS: The NAFLD model was induced in mice fed a high-fat diet (HFD) (HFD mice) or HFD + 1% heat-killed L. brevis SBL88 (SBL mice) for 16 weeks. Histopathological liver findings were analyzed. To evaluate the gut microbiota, a modified terminal restriction fragment length polymorphism analysis of the feces was performed. RNA sequencing in the liver was performed with Ion Proton™. To investigate the direct effects of heat-killed L. brevis SBL88, an in vitro study was performed. RESULTS: Histopathological findings revealed that fat droplets in the liver were significantly reduced in SBL mice; however, terminal restriction fragment length polymorphism did not show alterations in the gut microbiota between HFD mice and SBL mice. RNA sequencing and pathway analysis revealed that the regulation of lipid and insulin metabolism was affected. The mRNA expression of insulin receptor substrate 2 (IRS-2) was significantly higher in SBL mice, whereas the expression of IRS-1 was not significantly different. Phospho-IRS-2 expression was also significantly increased in SBL mice. In addition, an in vitro study revealed significant alterations in IRS-2 and forkhead box protein O1 expression levels. CONCLUSION: SBL mice exhibited partially improved selective hepatic insulin resistance. Our data suggest that heat-killed L. brevis SBL88 could attenuate the clinical features of NAFLD that are not mediated by alterations in the gut microbiota.

Ability of Lactobacillus brevis 47f to Alleviate the Toxic Effects of Imidacloprid Low Concentration on the Histological Parameters and Cytokine Profile of Zebrafish (Danio rerio).

In the present article, the possible mitigation of the toxic effect of imidacloprid low-concentration chronic exposure on Danio rerio by the probiotic strain Lactobacillus brevis 47f (1 × 108 CFU/g) was examined. It was found that even sublethal concentration (2500 µg/L) could lead to the death of some fish during the 60-day chronic experiment. However, the use of Lactobacillus brevis 47f partially reduced the toxic effects, resulting in an increased survival rate and a significant reduction of morphohistological lesions in the intestines and kidneys of Danio rerio. The kidneys were found to be the most susceptible organ to toxic exposure, showing significant disturbances. Calculation of the histopathological index, measurement of morphometric parameters, and analysis of principal components revealed the most significant parameters affected by the combined action of imidacloprid and Lactobacillus brevis 47f. This effect of imidacloprid and the probiotic strain had a multidirectional influence on various pro/anti-inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8). Therefore, the results suggest the possibility of further studying the probiotic strain Lactobacillus brevis 47f as a strain that reduces the toxic effects of xenobiotics. Additionally, the study established the possibility of using imidacloprid as a model toxicant to assess the detoxification ability of probiotics on the kidney and gastrointestinal tract of fish.

Ketonization of Ginsenoside C-K by Novel Recombinant 3-ß-Hydroxysteroid Dehydrogenases and Effect on Human Fibroblast Cells.

BACKGROUND AND OBJECTIVE: The ginsenoside compound K (C-K) (which is a de-glycosylated derivative of major ginsenosides) is effective in the treatment of cancer, diabetes, inflammation, allergy, angiogenesis, aging, and has neuroprotective, and hepatoprotective than other minor ginsenosides. Thus, a lot of studies have been focused on the conversion of major ginsenosides to minor ginsenosides using glycoside hydrolases but there is no study yet published for the bioconversion of minor ginsenosides into another high pharmacological active compound. Therefore, the objective of this study to identify a new gene (besides the glycoside hydrolases) for the conversion of minor ginsenosides C-K into another highly pharmacological active compound. METHODS AND RESULTS: Lactobacillus brevis which was isolated from Kimchi has showed the ginsenoside C-K altering capabilities. From this strain, a novel potent decarboxylation gene, named HSDLb1, was isolated and expressed in Escherichia coli BL21 (DE3) using the pMAL-c5X vector system. Recombinant HSDLb1 was also characterized. The HSDLb1 consists of 774 bp (258 amino acids residues) with a predicted molecular mass of 28.64 kDa. The optimum enzyme activity was recorded at pH 6.0-8.0 and temperature 30 °C. Recombinant HSDLb1 effectively transformed the ginsenoside C-K to 12-ß-hydroxydammar-3-one-20(S)-O-ß-D-glucopyranoside (3-oxo-C-K). The experimental data proved that recombinant HSDLb1 strongly ketonized the hydroxyl (-O-H) group at C-3 of C-K via the following pathway: C-K → 3-oxo-C-K. In vitro study, 3-oxo-C-K showed higher solubility than C-K, and no cytotoxicity to fibroblast cells. In addition, 3-oxo-C-K induced the inhibitory activity of ultraviolet A (UVA) against matrix metalloproteinase-1 (MMP-1) and promoted procollagen type I synthesis. Based on these expectations, we hypothesized that 3-oxo-C-K can be used in cosmetic products to block UV radiations and anti-ageing agent. Furthermore, we expect that 3-oxo-C-K will show higher efficacy than C-K for the treatment of cancer, ageing and other related diseases, for which more studies are needed.

RNA-seq-based transcriptomic analysis of AHL-induced biofilm and pyocyanin inhibition in Pseudomonas aeruginosa by Lactobacillus brevis.

Quorum sensing (QS) regulates hundreds of genes in Pseudomonas aeruginosa, and many of which encode extracellular virulence factors. Lactobacillus as a probiotic has been verified to inhibit pathogenesis in P. aeruginosa via quenching QS. The objective of this study was to investigate mechanism of the QS quenching function of Lactobacillus via analyzing the gene expression by transcriptome. We previously isolated a Lactobacillus brevis strain 3M004 from an aquafeed and identified the strain has the function of degrading QS molecular AHL (OC12-HSL). The result showed that 3M004 cells/lysate inhibited biofilm and pyocyanin production of P. aeruginosa PA002. The biofilm inhibition rates were 16.92% and 33.0% after treatment by 1 and 2 mg/mL 3M004 lysate, respectively, and the rates for pyocyanin inhibition were 25.16% and 30.75%, respectively. Transcriptomic analysis showed that down-regulation of genes of LasA and LasB in PA002 was essential in regulating the QS system. The biofilm decrease of PA002 seems not only resulted from gene biosynthesizing of polysaccharides but also from other genes regulating components biosynthesis. Pyocyanin biosynthesis appears to be inhibited by down-regulating the key gene of PhzAB on the nonreversing action from chorismite to pyocyanin.

Oral administration of Lactobacillus brevis 23017 combined with ellagic acid attenuates intestinal inflammatory injury caused by Eimeria infection by activating the Nrf2/HO-1 antioxidant pathway.

The aim of this study was to investigate whether oral administration of Lactobacillus brevis 23017 (LB) alone and in combination with ellagic acid inhibits ChTLR15/ChNLRP3/ChIL-1ß by activating the Nrf2/HO-1 pathway to attenuate intestinal inflammatory injury. Two animal experiments were performed. In Experiment 1, chickens were allocated into 7 groups: PBS, and low, medium and high dosages of live and heat-killed LB, named L/LB(+), M/LB(+) and H/LB(+), and L/LB(-), M/LB(-) and H/LB(-), respectively. In Experiment 2, chickens were divided into 5 groups: PBS, challenge control, and low, medium and high dosages of ellagic acid combined with LB(+), named L/EA + L/LB(+), M/EA + M/LB(+) and H/EA + H/LB(+), respectively. Chickens were gavaged with LB with or without ellagic acid once a day. Then, the mRNA and protein levels of the components of the Nrf2/HO-1 pathway found in the caecal tissues were quantified. On Day 7 post-infection with E. tenella, the levels of the components of the ChTLR15/NLRP3/IL-1ß pathway in the caeca were again quantified, and the anticoccidial effects were assessed. The results showed that the levels of the genes in the Nrf2/HO-1 pathway in the chickens in the LB(+) groups were higher than those in the LB(-) groups (p < 0.001); those in the H/LB(+) group were higher than those in the M/LB(+) and L/LB(+) groups (p < 0.001); and those in the H/EA + H/LB(+) group showed the highest expression levels compared with the other groups (p < 0.001). After challenge, the chickens in the H/LB(+) group displayed less inflammatory injury than those in the M/LB(+) and L/LB(+) groups (p < 0.05), and the chickens in the H/EA + H/LB(+) group showed stronger anti-inflammatory effects than the other groups (p < 0.05). Thus, these protective effects against infection were consistent with the above results. Overall, significant anti-inflammatory effects were observed in chickens orally gavaged with high dosages of live L. brevis 23017 and ellagic acid, which occurred by regulation of the ChTLR15/NLRP3/IL-1ß pathway.

Activation of galactose utilization by the addition of glucose for the fermentation of agar hydrolysate using Lactobacillus brevis ATCC 14869.

OBJECTIVE: To investigate the application of carbon catabolite repression (CCR) relaxed Lactobacillus brevis ATCC 14869 in the utilization of agar hydrolysate to produce bioethanol and lactic acid through fermentation. RESULTS: As a single carbon source, galactose was not metabolized by L. brevis. However, L. brevis consumed galactose simultaneous to glucose and ceased cell growth after depletion of glucose. For complete use of galactose from agar hydrolysis, glucose need to be periodically replenished into the growth medium. Overall, L. brevis successfully used agar hydrolysate and produced 17.2 g/L of ethanol and 31.9 g/L of lactic acid. The maximum specific cell growth rate on galactose and glucose mixture was the same with the glucose-only medium at 0.12 h-1. The molar product yields from glucose for lactic acid and ethanol were 1.02 and 0.95 respectively, equal to values obtained from the simultaneous utilization of glucose and galactose. CONCLUSION: In contribution to the ongoing efforts to utilize marine biomass, the relaxed CCR in Lactobacillus brevis ATCC 14869 was herein exploited to produce bioethanol and lactic acid from red seaweed hydrolysates.

The Anti-Cancer Potential of Heat-Killed Lactobacillus brevis KU15176 upon AGS Cell Lines through Intrinsic Apoptosis Pathway.

Recent research has focused on the anti-cancer properties of Lactobacillus strains isolated from fermented foods. Their anti-cancer effects are caused by the apoptosis induction in cancer cells. However, sepsis, which can occur when cancer patients consume living organisms, can cause serious conditions in patients with reduced immunity because of cancer. Therefore, this study was conducted using heat-killed Lactobacillus brevis KU15176 (KU15176). To determine the relationship between inflammation and cancer, the anti-inflammatory effect of KU15176 was evaluated using a nitric oxide (NO) assay. Then, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was conducted to select cancer cells that showed the anti-proliferative effect of KU15176. Next, reverse transcription-polymerase chain reaction (RT-PCR), 4',6-diamidino-2-phenylindole (DAPI) staining, flow cytometry, and caspase colorimetric assay were performed. As a result, it was confirmed that KU15176 could cause the increasing expression of apoptosis-related genes (Bax, caspase-3, and caspase-9), DNA breakage, effective apoptosis rate, and increased caspase activity in the human stomach adenocarcinoma (AGS) gastric cancer cell line. In conclusion, these results suggest a potential prophylactic effect of KU15176 against cancer.

Identification of WxL and S-Layer Proteins from Lactobacillus brevis with the Ability to Bind Cellulose and Xylan.

Xylanase releases xylo-oligosaccharides from dietary xylan, which stimulate the growth of the gut bacteria lactobacilli. Many lactobacilli adhere to dietary fibers, which may facilitate the assimilation of xylo-oligosaccharides and help them gain competence in the gut, but the underlying mechanisms remain elusive. Herein we report, from the highly abundant transcripts of Lactobacillus brevis cultured in wheat arabinoxylan supplemented with a xylanase, the identification of genes encoding four putative cell-surface WxL proteins (Lb630, Lb631, Lb632, and Lb635) and one S-layer protein (Lb1325) with either cellulose- or xylan-binding ability. The repetitively occurring WxL proteins were encoded by a gene cluster, among which Lb630 was chosen for further mutational studies. The analysis revealed three aromatic residues (F30, W61, and W156) that might be involved in the interaction of the protein with cellulose. A homology search in the genome of Enterococcus faecium identified three WxL proteins with conserved counterparts of these three aromatic residues, and they were also found to be able to bind cellulose and xylan. The findings suggested a role of the cell-surface WxL and S-layer proteins in assisting the cellular adhesion of L. brevis to plant cell wall polysaccharides.

Heat-killed Lactobacillus brevis KB290 attenuates visceral fat accumulation induced by high-fat diet in mice.

AIMS: This study aimed to evaluate the anti-adiposity effect of heat-killed Lactobacillus brevis KB290 originating from traditional Japanese fermented pickles in mice fed a high-fat diet (HFD). METHODS AND RESULTS: C57BL/6J mice were fed a normal-fat diet, HFD or HFD supplemented with heat-killed KB290 for 8 weeks. Epididymal and renal adipose tissue weights, as well as areas of epididymal adipocytes, were significantly lower in the mice fed a HFD supplemented with KB290 than in those fed an unsupplemented HFD. Mice whose diets were supplemented with KB290 had elevated adiponectin and ß3-adrenergic receptor expression in epididymal adipose tissue and an accompanying higher serum free fatty acid level. Furthermore, the HFD-induced elevations in serum glucose, insulin and HOMA-IR were significantly suppressed by dietary supplementation with KB290. Amplicon sequencing of 16S rRNA genes revealed that KB290 ingestion altered the composition of the intestinal microbiota. CONCLUSIONS: Heat-killed L. brevis KB290 suppressed diet-induced visceral fat accumulation and ameliorated diet-induced metabolic symptoms and intestinal gut microbiota modifications, suggesting possibility of novel paraprobiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: Heat-killed L. brevis KB290 is useable as a material to develop functional foods that attenuate visceral fat accumulation.

Fermented date residue extract mix containing gamma-aminobutyric acid augments the immune function of mouse splenocytes.

An extract of date (fruit of a palm tree) residue plus food-grade glutamate, acetic acid, and yeast extract (date residue extract mix, DREM) has been successfully fermented with using Lactobacillus brevis JCM 1059T to produce gamma-aminobutyric acid (GABA). Here, mouse splenocytes were found to be viable when supplemented with DREM and fermented DREM containing GABA (fDREM). The addition of DREM and fDREM resulted in the secretion of tumor necrosis factor (TNF)-α from the splenocytes, fDREM being more effective than DREM. The TNF-α secretion with DREM was elevated by exogenous addition of GABA and that with fDREM was in part mediated via A-type GABA receptors. Contrary to general understanding of the suppressive effects of GABA on various biological functions, our findings suggest that GABA-containing fDREM arguments the immune function as a food and pharmaceutical material.

Lactobacillus brevis CD2 attenuates traumatic oral lesions induced by fixed orthodontic appliance: A randomized phase 2 trial.

OBJECTIVE: To evaluate the effect of the probiotic Lactobacillus brevis CD2 on the prevention of early traumatic oral lesions induced by a fixed orthodontic appliance. SETTINGS AND SAMPLE POPULATION: Twenty orthodontic patients (14-57 yo) were recruited from a private clinic. SUBJECTS AND METHODS: In a phase 2, double-blind clinical trial, all patients were randomly allocated (1:1 ratio) to a 21-day course of soluble tablets containing L brevis CD2 (4 billion colony-forming units after breakfast, lunch and dinner) or placebo, starting at the day of orthodontic appliance placement. The primary outcomes were days with oral lesions and lesion-related pain [ranging between 0 (no pain) and 10 (maximum pain)]. Oral health-related quality of life was measured using OHIP-14 before and after treatments. RESULTS: All patients completed the study. Ten were treated with L brevis (28.1 ± 13.3 yo, 70% women), and 10 received placebo (27.5 ± 9.1 yo, 60% women). The oral lesions lasted significantly less time (P = .018) in patients treated with L brevis (2.5 ± 1.0 days) than with placebo (4.9 ± 3.0 days). Pain score was significantly lower (P = .039) when L brevis was used [median (min-max): 0 (0-4) vs. 3 (0-5)]. OHIP-14 scores were not significantly different between treatments. CONCLUSIONS: Lactobacillus brevis CD2 reduced almost 50% the persistence of traumatic oral lesions in patients with fixed orthodontics. Yet, there was no improvement in quality of life compared to placebo, suggesting that such differences in persistency and pain related to oral lesions may be considered clinically irrelevant.

Effect of adzuki bean sprout fermented milk enriched in γ-aminobutyric acid on mild depression in a mouse model.

This study focused on the ability of adzuki bean (Vigna angularis) sprout fermented milk, which is rich in γ-aminobutyric acid (GABA), to relieve anxiety and mild depression. A high-yield GABA-producing strain, Lactobacillus brevis J1, from a healthy cow was screened, and its physiological and probiotic properties were evaluated. The effect of adzuki bean sprout fermented milk was investigated in vivo in a chronic depression mouse model. The results showed that Lb. brevis J1 had excellent probiotic properties, grew well at low pH and 3% NaCl, and adhered to the surface of HT-29 cells. The GABA-enriched (241.30 ± 1.62 µg/mL) adzuki bean sprout fermented milk prepared with Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus plantarum, and Lb. brevis J1 can reduce and possibly prevent mild depression-like symptoms in mice (C57/B6) by increasing social interaction and enhancing the pleasure derived from movement. The research revealed that the GABAB-cyclic AMP-protein kinase A-cAMP-response element binding protein (GABAB-cAMP-PKA-CREB) signaling pathway was related to the depression-like symptoms and that levels of 5-hydroxytryptamine, norepinephrine, and dopamine in the hippocampus of mice increased after treatment with the adzuki bean sprout fermented milk. Our results suggest that GABA-enriched dairy products have the potential to prevent or treat mild depression-like symptoms in mice, which suggests a new approach for a dietary therapy to treat chronic social stress.

Probiotic strains of Lactobacillus brevis and Lactobacillus plantarum as adjunct to non-surgical periodontal therapy: 3-month results of a randomized controlled clinical trial.

OBJECTIVES: To determine if periodontitis patients benefit from treatment with Lactobacillus brevis and Lactobacillus plantarum strains, applied into periodontal pockets as gel and thereafter taken as lozenges, as an adjunct to scaling and root planing (SRP). MATERIALS AND METHODS: In a double-blind, randomized, placebo-controlled trial, 40 patients received scaling and root planing (SRP) in two sessions within 7 days. Patients then received either probiotic gel and lozenges (n = 20) or placebo (n = 20). The primary outcome variable was the number of diseased sites (DS: PD > 4 mm + BOP) at the 3-month re-evaluation. The effects of gender, age, probiotic therapy, presence of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans, smoking, tooth being a molar and interdental location were evaluated using a multivariate multilevel logistic regression model. RESULTS: The number of DS after 3 months was similar in the test (Me = 8, IQR = 5-11) and control (Me = 5, IQR = 1-10) groups. Both groups showed substantial but equivalent improvements in periodontal parameters. The logistic regression showed higher odds for the healing of gingival bleeding (OR = 2.12, p = 0.048) and lower odds for the healing of DS (OR = 0.51; p < 0.001) in the probiotic group. CONCLUSIONS: Patients with periodontitis benefit from adjunctive use of probiotics containing L. brevis and L. plantarum in terms of reduction of gingival bleeding. However, adjunctive probiotics increase the number of persisting diseased sites with PD > 4 mm and BOP. CLINICAL RELEVANCE: The adjunctive use of probiotics containing L. brevis and L. plantarum strains in treating chronic periodontitis results in a higher number of residual diseased sites when compared with SRP + placebo; its use is therefore unfounded.

Effect of Lactobacillus brevis CD2 containing lozenges and plaque pH and cariogenic bacteria in diabetic children: a randomised clinical trial.

OBJECTIVE: The short-term effect (60 days) of Lactobacillus brevis CD2 lozenges vs placebo on variables related to caries and gingivitis in type 1 diabetic children was evaluated. MATERIAL AND METHODS: Eight diabetics (4-14 years old) were assigned to two groups (n = 34 subjects each), probiotic lozenges and placebo. Stimulated saliva for microbiological analysis and plaque pH were assessed at baseline (t0), 30 days (t1), 60 days (t2) and in the follow-up period (90 days from baseline, t3). Gingival status was assessed at t0, t2 and t3. Two-way ANOVA assessed differences between groups. RESULTS: In the probiotic group, Streptococcus mutans bacterial density mean scores dropped from 3.11 ± 1.13 at baseline to 1.82 ± 0.72 (t2) and to 2.06 ± 0.56 (t3), while in the placebo group, the scores were 3.09 ± 0.8 (t0), 2.82 ± 0.47 (t2) and 3.11 ± 0.43 (t3) (p < 0.01). Lowest and maximum pH fall increased in the probiotic group, from 5.37 ± 0.41 at baseline to 5.49 ± 0.24 at t3 (p < 0.01) and from 1.20 ± 0.46 to 0.98 ± 0.29 (p = 0.02). Bleeding score decreased significantly in both groups, showing a statistically significant lower bleeding score at t2 in the probiotic group (25.6%, 95% CI 21.5-32.7 vs 29.5%, 95% CI 25.2-34.9, p = 0.02). CONCLUSIONS: Lactobacillus brevis CD2 has shown to improve caries-related risk factors and gingival health in diabetic children. CLINICAL RELEVANCE: Lactobacillus brevis CD2 might contribute to improved oral health in type 1 diabetic children.

Physicochemical and transcriptomic responses of Lactobacillus brevis JLD715 to sodium selenite.

BACKGROUND: Elemental selenium, as a new type of selenium supplement, can be prepared by microorganisms reducing inorganic selenium. In this study, Lactobacillus brevis JLD715 was incubated in broth containing different concentrations of sodium selenite (Na2 SeO3 ). RESULTS: The results showed that the bacterial biomass of L. brevis JLD715 decreased due to the inhibition of Na2 SeO3 . The cell membrane of L. brevis JLD715 treated with Na2 SeO3 was damaged, as evidenced by the reduction of intracellular ATP concentration, depolarization of cell membrane, reduction of intracellular pH and impairment of membrane integrity. In addition, we investigated the metabolism mechanism of Na2 SeO3 by L. brevis JLD715 based on transcriptome sequencing. A total of 461 genes were significantly differentially expressed under Na2 SeO3 treatment, of which 231 genes were up-regulated and 230 genes were down-regulated. These genes were involved in pathways such as pyruvate metabolism, fatty acid biosynthesis, selenocompound metabolism and nucleotide-binding oligomerization domain-like (NOD-like) receptor signaling. Meanwhile, the genes related to sulfhydryl oxidoreductase, electron carrier proteins and transmembrane transport proteins synthesis were significantly up-regulated. CONCLUSION: To sum up, the findings of this research will contribute to providing support for the application of L. brevis JLD715 in selenium-enriched functional foods. © 2021 Society of Chemical Industry.

A Plasmid-Encoded Putative Glycosyltransferase Is Involved in Hop Tolerance and Beer Spoilage in Lactobacillus brevis.

Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species.IMPORTANCELactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.

GlnR Negatively Regulates Glutamate-Dependent Acid Resistance in Lactobacillus brevis.

Lactic acid bacteria often encounter a variety of multiple stresses in their natural and industrial fermentation environments. The glutamate decarboxylase (GAD) system is one of the most important acid resistance systems in lactic acid bacteria. In this study, we demonstrated that GlnR, a nitrogen regulator in Gram-positive bacteria, directly modulated γ-aminobutyric acid (GABA) conversion from glutamate and was involved in glutamate-dependent acid resistance in Lactobacillus brevis The glnR deletion strain (ΔglnR mutant) achieved a titer of 284.7 g/liter GABA, which is 9.8-fold higher than that of the wild-type strain. The cell survival of the glnR deletion strain was significantly higher than that of the wild-type strain under the condition of acid challenge and was positively correlated with initial glutamate concentration and GABA production. Quantitative reverse transcription-PCR assays demonstrated that GlnR inhibited the transcription of the glutamate decarboxylase-encoding gene (gadB), glutamate/GABA antiporter-encoding gene (gadC), glutamine synthetase-encoding gene (glnA), and specific transcriptional regulator-encoding gene (gadR) involved in gadCB operon regulation. Moreover, GABA production and glutamate-dependent acid resistance were absolutely abolished in the gadR glnR deletion strain. Electrophoretic mobility shift and DNase I footprinting assays revealed that GlnR directly bound to the 5'-untranslated regions of the gadR gene and gadCB operon, thus inhibiting their transcription. These results revealed a novel regulatory mechanism of GlnR on glutamate-dependent acid resistance in LactobacillusIMPORTANCE Free-living lactic acid bacteria often encounter acid stresses because of their organic acid-producing features. Several acid resistance mechanisms, such as the glutamate decarboxylase system, F1Fo-ATPase proton pump, and alkali production, are usually employed to relieve growth inhibition caused by acids. The glutamate decarboxylase system is vital for GAD-containing lactic acid bacteria to protect cells from DNA damage, enzyme inactivation, and product yield loss in acidic habitats. In this study, we found that a MerR-type regulator, GlnR, was involved in glutamate-dependent acid resistance by directly regulating the transcription of the gadR gene and gadCB operon, resulting in an inhibition of GABA conversion from glutamate in L. brevis This study represents a novel mechanism for GlnR's regulation of glutamate-dependent acid resistance and also provides a simple and novel strategy to engineer Lactobacillus strains to elevate their acid resistance as well as GABA conversion from glutamate.

Expression of two glutamate decarboxylase genes in Lactobacillus brevis during gamma-aminobutyric acid production with date residue extract.

Gamma-aminobutyric acid (GABA) is produced by Lactobacillus brevis using date residue fermentation. In this study, the GABA production method was improved, for which L. brevis strain JCM 1059T was the most efficient among the four L. brevis strains examined. This was presumably due to a difference in the expression level of the gene encoding glutamate decarboxylase that catalyzes GABA synthesis.Abbreviation: GABA: gamma-aminobutyric acid.

Optimization of n-butanol synthesis in Lactobacillus brevis via the functional expression of thl, hbd, crt and ter.

N-butanol is an important chemical and can be naturally synthesized by Clostridium species; however, the poor n-butanol tolerance of Clostridium impedes the further improvement in titer. In this study, Lactobacillus brevis, which possesses a higher butanol tolerance, was selected as host for heterologous butanol production. The Clostridium acetobutylicum genes thl, hbd, and crt which encode thiolase, ß-hydroxybutyryl-CoA dehydrogenase, and crotonase, and the Treponema denticola gene ter, which encodes trans-enoyl-CoA reductase were cloned into a single plasmid to express the butanol synthesis pathway in L. brevis. A titer of 40 mg/L n-butanol was initially achieved with plasmid pLY15-opt, in which all pathway genes are codon-optimized. A titer of 450 mg/L of n-butanol was then synthesized when ter was further overexpressed in this pathway. The role of metabolic flux was reinforced with pLY15, in which only the ter gene was codon-optimized, which greatly increased the n-butanol titer to 817 mg/L. Our strategy significantly improved n-butanol synthesis in L. brevis and the final titer is the highest achieved amongst butanol-tolerant lactic acid bacteria.