Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Up-regulation of sortase-dependent pili in Bifidobacterium longum BBMN68 in response to bile stress enhances its adhesion to HT-29 cells.

Adhesion to gastrointestinal tract is crucial for bifidobacteria to exert their probiotic effects. Our previous work found that bile salts significantly enhance the adhesion ability of Bifidobacterium longum BBMN68 to HT-29 cells. In this study, trypsin-shaving and LC-MS/MS-based surface proteomics were employed to identify surface proteins involved in bile stress response. Among the 829 differentially expressed proteins, 56 up-regulated proteins with a fold change >1.5 were subjected to further analysis. Notably, the minor pilin subunit FimB was 4.98-fold up-regulated in response to bile stress. In silico analysis and RT-PCR confirmed that gene fimB, fimA and srtC were co-transcribed and contributed to the biosynthesis of sortase-dependent pili Pil1. Moreover, scanning electron microscopy and immunogold electron microscopy assays showed increased abundance and length of Pil1 on BBMN68 under bile stress. As the major pilin subunit FimA serves as adhesion component of Pil1, an inhibition assay using anti-FimA antibodies further confirmed the critical role of Pil1 in mediating the adhesion of BBMN68 to HT-29 cells under bile stress. Our findings suggest that the up-regulation of Pil1 in response to bile stress enhances the adhesion of BBMN68 to intestinal epithelial cells, highlighting a novel mechanism of gut persistence in B. longum strains.

A Ropy Exopolysaccharide-Producing Strain Bifidobacterium pseudocatenulatum Bi-OTA128 Alleviates Dextran Sulfate Sodium-Induced Colitis in Mice.

Inflammatory bowel disease (IBD) is a chronic disease associated with overactive inflammation and gut dysbiosis. Owing to the beneficial effects of bifidobacteria on IBD treatment, this study aimed to investigate the anti-inflammation effects of an exopolysaccharide (EPS)-producing strain Bifidobacterium pseudocatenulatum Bi-OTA128 through a dextran sulfate sodium (DSS)-induced colitis mice model. B. pseudocatenulatum treatment improved DSS-induced colitis symptoms and maintained intestinal barrier integrity by up-regulating MUC2 and tight junctions' expression. The oxidative stress was reduced after B. pseudocatenulatum treatment by increasing the antioxidant enzymes of SOD, CAT, and GSH-Px in colon tissues. Moreover, the overactive inflammatory responses were also inhibited by decreasing the pro-inflammatory cytokines of TNF-α, IL-1ß, and IL-6, but increasing the anti-inflammatory cytokine of IL-10. The EPS-producing strain Bi-OTA128 showed better effects than that of a non-EPS-producing stain BLYR01-7 in modulating DSS-induced gut dysbiosis. The Bi-OTA128 treatment increased the relative abundance of beneficial bacteria Bifidobacterium and decreased the maleficent bacteria Escherichia-Shigella, Enterorhabuds, Enterobacter, and Osillibacter associated with intestinal inflammation. Notably, the genera Clostridium sensu stricto were only enriched in Bi-OTA128-treated mice, which could degrade polysaccharides to produce acetic acid and butyrate in the gut. This finding demonstrated a cross-feeding effect induced by the EPS-producing strain in gut microbiota. Collectively, these results highlighted the anti-inflammatory effects of the EPS-producing strain B. pseudocatenulatum Bi-OTA128 on DSS-induced colitis, which could be used as a candidate probiotic supporting recovery from ongoing colitis.

bglG Regulates the Heterogeneity Driven by the Acid Tolerance Response in Lacticaseibacillus paracasei L9.

The acid tolerance of lactic acid bacteria is crucial for their fermentation and probiotic functions. Acid adaption significantly enhances the acid tolerance of strains, and the phenotypic heterogeneity driven by the acid tolerance response (ATR) contributes to this process by providing a selective advantage in harsh environments. The mechanism of heterogeneity under the ATR is not yet clear, but individual gene expression differences are recognized as the cause. In this study, we observed four heterogeneous subpopulations (viable, injured, dead, and unstained) of Lacticaseibacillus paracasei L9 (L9) induced by acid adaption (pH 5.0, 40 min) using flow cytometry. The viable subpopulation represented a significantly superior acid tolerance to the injured subpopulation or total population. Different subpopulations were sorted and transcriptomic analysis was performed. Five genes were found to be upregulated in the viable subpopulation and downregulated in the injured subpopulation, and bglG (LPL9_RS14735) was identified as having a key role in this process. Using salicin (glucoside)-inducing gene expression and gene insertion mutagenesis, we verified that bglG regulated the heterogeneity of the acid stress response and that the relevant mechanisms might be related to activating hsp20. This study provides new evidence for the mechanism of the ATR and may contribute to the theoretical basis of improving the acid tolerance of Lacticaseibacillus paracasei L9.

An underlying mechanism for MleR activating the malolactic enzyme pathway to enhance acid tolerance in Lacticaseibacillus paracasei L9.

Tolerance to acid stress is a crucial property of probiotics against gastric acids. The malolactic enzyme pathway is one of the most important acid resistance systems in lactic acid bacteria. It has been reported that the malolactic enzyme pathway was regulated by the transcriptional regulator, MleR. However, regulatory mechanisms underlying malolactic enzyme pathway to cope with acid stress remain unknown. In this study, the acid tolerance ability of the ΔmleR deletion strain was significantly lower than that of the wild-type strain, and the complementation of the mleR gene into the ΔmleR strain restored the acid tolerance of the ΔmleR strain, indicating that MleR was involved in acid tolerance response of Lacticaseibacillus paracasei L9. Real-time quantitative PCR and transcriptional fusion experiments confirmed MleR-activated transcription of the mleST gene cluster. Furthermore, MleR was confirmed to directly bind to the promoter region of the mleST operon using ChIP assays and EMSAs. The transcription start site G of the mleST operon was located at position -198 relative to the start codon of the mleS gene. The region from -80 to -61 upstream of the transcription start site was determined to be essential for MleR binding. Moreover, L-malic acid acted as an effector for MleR to activate the transcription of the mleST operon in a dose-dependent manner. These results revealed the regulatory mechanism behind MleR-mediated activation of the malolactic enzyme pathway to enhance acid tolerance in Lc. paracasei L9. IMPORTANCE Lacticaseibacillus paracasei is extensively used as probiotics in human health and fermented dairy production. Following consumption, Lc. paracasei is exposed to a variety of physico-chemical stresses, such as low pH in the stomach and bile salts in the intestines. The high acidity of the stomach severely inhibits bacterial metabolism and growth. Therefore, the acid tolerance response is critical for Lc. paracasei to survive. It has been reported that the malolactic enzyme (MLE) pathway plays an important role for LAB to resist acid stress. However, the regulatory mechanism has not yet been investigated. In this study, we determined that the LysR-type regulator MleR positively regulated the MLE pathway to enhance acid tolerance by binding -80 to -61 upstream of the transcription start site of the mleST operon. Further, L-malic acid acts as a co-inducer for MleR transcriptional regulation. Our study provides novel insights into acid tolerance mechanisms in LAB.

Biofilm-Forming Capacity and Drug Resistance of Different Gardnerella Subgroups Associated with Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common infection of the lower reproductive tract among women of reproductive age. Recurrent infections and antibiotic resistance associated with biofilms remain significant challenges for BV treatment. Gardnerella species are commonly found in women with and without BV, indicating that genetic differences among Gardnerella isolates may distinguish pathogenic from commensal subgroups. This study isolated 11 Gardnerella strains from vaginal samples obtained from women with BV before or after treatment. The biofilm formation ability of each strain was examined by crystal violet staining. Eight strains were selected using phylogenetic analysis of the cpn60 sequences and classified as subgroups A (6/8), B (1/8), and D (1/8). The biofilm formation ability and antibiotic resistance profile of these strains was compared among the subgroups. Subgroup D had the strongest biofilm formation ability. Six of the planktonic strains exhibited resistance to the first-line BV drug, metronidazole, and one to clindamycin. Moreover, biofilm formation in vitro increased strain resistance to clindamycin. Two strains with strong biofilm ability, S20 and S23, and two with weak biofilm ability, S24 and S25, were selected for comparative genomic analysis. S20 and S23 were found to contain four key genes associated with biofilm formation and more genes involved in carbohydrate synthesis and metabolism than S24 and S25. Identifying differences in the expression of virulence factors between Gardnerella subgroups could inform the development of novel treatments for BV.

Molecular Organization and Functional Analysis of a Novel Plasmid-Borne cps Gene Cluster from Lactiplantibacillus plantarum YC41.

Capsular polysaccharide (CPS) can tightly attach to bacterial surfaces and plays a critical role in protecting microorganisms from environmental stresses. However, the molecular and functional properties of some plasmid-borne cps gene clusters are poorly understood. In this study, comparative genomics of the draft genomes of 21 Lactiplantibacillus plantarum strains revealed that the specific gene cluster for CPS biosynthesis was observed only in the 8 strains with a ropy phenotype. Furthermore, the complete genomes showed that the specific gene cluster cpsYC41 was located on the novel plasmid pYC41 in L. plantarum YC41. In silico analysis confirmed that the cpsYC41 gene cluster contained the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the wzx gene. The insertional inactivation of the rmlA and cpsC genes abolished the ropy phenotype and reduced the CPS yields by 93.79% and 96.62%, respectively, in L. plantarum YC41 mutants. These results revealed that the cpsYC41 gene cluster was responsible for CPS biosynthesis. Moreover, the survival rates of the YC41-rmlA- and YC41-cpsC- mutants under acid, NaCl, and H2O2 stresses were decreased by 56.47 to 93.67% compared to that of the control strain. Furthermore, the specific cps gene cluster was also confirmed to play a vital role in CPS biosynthesis in L. plantarum MC2, PG1, and YD2. These findings enhance our understanding of the genetic organization and gene functions of plasmid-borne cps gene clusters in L. plantarum. IMPORTANCE Capsular polysaccharide is well known to protect bacteria against various environmental stresses. The gene cluster for CPS biosynthesis is typically organized in the chromosome in bacteria. It is worth noting that complete genome sequencing showed that a novel plasmid pYC41-borne cpsYC41 gene cluster was identified in L. plantarum YC41. The cpsYC41 gene cluster included the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the wzx gene, which was verified by the significantly decreased CPS yield and the absent ropy phenotype in the corresponding mutants. The cpsYC41 gene cluster plays an important role in bacterial survival under environmental stress, and the mutants had decreased fitness under stress conditions. The vital role of this specific cps gene cluster in CPS biosynthesis was also confirmed in other CPS-producing L. plantarum strains. These results advanced a better understanding of the molecular mechanisms of plasmid-borne cps gene clusters and the protective functionality of CPS.

Prevalence, antimicrobial susceptibility, and antibiotic resistance gene transfer of Bacillus strains isolated from pasteurized milk.

Pasteurization is carried out in dairy industries to kill harmful bacteria present in raw milk. However, endospore-forming bacteria, such as Bacillus, cannot be completely eliminated by pasteurization. In this study, a total of 114 Bacillus strains were isolated from 133 pasteurized milk samples. Antibiotic susceptibility tests showed that the percentage of Bacillus with intrinsic resistance to ampicillin and penicillin were 80 and 86%, respectively. Meanwhile, some Bacillus isolates had acquired resistance, including trimethoprim-sulfamethoxazole resistance (10 isolates), clindamycin resistance (8 isolates), erythromycin resistance (2 isolates), and tetracycline resistance (1 isolate). To further locate these acquired resistance genes, the plasmids were investigated in these 16 Bacillus strains. The plasmid profile indicated that Bacillus cereus BA008, BA117, and BA119 harbored plasmids, respectively. Subsequently, the Illumina Novaseq PE150 was applied for the genomic and plasmid DNA sequencing. Notably, the gene tetL encoding tetracycline efflux protein was found to be located on plasmid pBC46-TL of B. cereus BA117. In vitro conjugative transfer indicated that pBC46-TL can be transferred into Bacillus invictae BA142, Bacillus safensis BA143, and Bacillus licheniformis BA130. The frequencies were of 1.5 × 10-7 to 1.7 × 10-5 transconjugants per donor cells. Therefore, Bacillus strains with acquired antibiotic resistance may represent a potential risk for the spread of antibiotic resistance between Bacillus and other clinical pathogens via horizontal gene transfer.

The plasmid-encoded lactose operon plays a vital role in the acid production rate of Lacticaseibacillus casei during milk beverage fermentation.

Lacticaseibacillus casei is used extensively in the fermented milk-beverage industry as a starter culture. Acid production capacity during fermentation is the main criterion for evaluating starters although it is strain-dependent. In this study, the acid production rates of 114 L. casei strains were determined and then classified into high acid (HC), medium acid (MC), and low acid (LC) groups. Comparative genomics analysis found that the lac operon genes encoding the phosphoenolpyruvate-lactose phosphotransferase system (PTSLac) were located on plasmids in the HC strains; however, it is notable that the corresponding operons were located on the chromosome in LC strains. Real-time PCR analysis showed that the copy numbers of lac operon genes in HC strains were between 3.1 and 9.3. To investigate the relationship between copy number and acid production rate, the lac operon cluster of the HC group was constitutively expressed in LC strains. The resulting copy numbers of lac operon genes were between 15.8 and 18.1; phospho-ß-galactosidase activity increased by 1.68-1.99-fold; and the acid production rates increased by 1.24-1.40-fold, which enhanced the utilization rate of lactose from 17.5 to 42.6% in the recombinant strains. The markedly increased expression of lac operon genes increased lactose catabolism and thereby increased the acid production rate of L. casei.

Unraveling the mechanism of raffinose utilization in Ligilactobacillus salivarius Ren by transcriptomic analysis.

In the gastrointestinal tract, some dietary carbohydrates, such as xylose, raffinose and arabinose, are able to stimulate the growth of Lactobacillus and Bifidobacterium. In this study, the growth rate of Ligilactobacillus salivarius Ren in raffinose was 0.91 ± 0.03 h-1, which was higher than that in glucose (0.83 ± 0.01 h-1). However, limited information is available on specific transporters and glycoside hydrolases responsible for raffinose uptake and catabolism in L. salivarius. Transcriptomic analysis revealed the differential expression of 236 genes (∣log2FoldChange∣ > 0.8) in response to raffinose, which were mainly associated with raffinose transport, raffinose hydrolysis, galactose metabolism and pyruvate metabolism. Notably, gene rafP encoding lactose/raffinose permease was 101.86-fold up-regulated. Two α-galactosidase gene galA1 and galA2 were 117.82-fold and 2.66-fold up-regulated, respectively. To further investigate the role of these genes in raffinose utilization, insertional inactivation was performed using the pORI28-pTRK669 system. The growth assay of these mutants in modified MRS containing 2% (w/v) raffinose indicated that RafP played an important role in raffinose transport and GalA1 was the primary enzyme involved in raffinose hydrolysis. To our knowledge, this is the first report on the molecular mechanism of raffinose utilization in L. salivarius. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03280-6.

L-Malic Acid Protects Lacticaseibacillus paracasei L9 from Glycodeoxycholic Acid Stress via the Malolactic Enzyme Pathway.

Bile stress tolerance is a crucial characteristic of probiotics for surviving in the human gastrointestinal tract. The mechanism underlying the effect of l-malic acid on enhancing the glycodeoxycholic acid (GDCA) tolerance of Lacticaseibacillus paracasei L9 was investigated herein. Bile tolerance specificity assays revealed that Lc. paracasei L9 was more sensitive to GDCA than to taurocholic acid, glycocholic acid, and taurodeoxycholic acid. Notably, l-malic acid significantly enhanced the GDCA tolerance of Lc. paracasei L9 by increasing the pH of the medium. The role of the malolactic enzyme pathway in enhancing GDCA resistance was investigated using molecular techniques. Confocal laser scanning and scanning electron microscopy revealed that l-malic acid preserved membrane permeability and cellular morphology, thereby protecting bacterial cells from GDCA stress-induced damage. The study also demonstrated that l-malic acid enhanced bile tolerance in different species of lactobacilli. These findings provide a novel protective mechanism for coping with bile stress in lactobacilli.

Anti-inflammatory effect of Bifidobacterium animalis subsp. lactis A6 on DSS-induced colitis in mice.

AIMS: This study aimed to investigate the protective effect of Bifidobacterium animalis subsp. lactis A6 on dextran sodium sulphate (DSS)-induced colitis in C57BL/6J mice. METHODS AND RESULTS: Mice were randomly divided into three groups (n = 8 per group). Each group was administered with PBS (Control and DSS group) or B. lactis A6 with a dosage of ~4.0 × 109  CFU day-1 (DSS + A6 group) for 21 consecutive days. The DSS and DSS + A6 group mice were ad libitum drinking 2.5% DSS water during day 15-21, while the Control group mice were given normal water. The administration of B. lactis A6 significantly inhibited DSS-induced bodyweight loss and colon shortening (p < 0.001), but showed no significant influence on the spleen enlargement (p > 0.05). The intestinal barrier integrity was improved by reducing colonic damage, recovering mucus layer loss and enhancing tight junction expression including ZO-1, occludin and claudin-1. In addition, B. lactis A6 attenuated the oxidative stress by decreasing MDA and increasing SOD and GSH levels in colon tissues. Moreover, B. lactis A6 suppressed DSS-induced inflammatory responses via downregulating TNF-α, IL-1ß and IL-6 levels and upregulating IL-10 level in colon tissues. CONCLUSION: B. lactis A6 effectively alleviated DSS-induced colitis by maintaining intestinal barrier integrity, reducing oxidative stress and inhibiting inflammatory responses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. lactis A6 could act as a candidate probiotic for UC treatment.

Transcriptome analysis revealed growth phase-associated changes of a centenarian-originated probiotic Bifidobacterium animalis subsp. lactis A6.

BACKGROUND: The physiology and application characteristics of probiotics are closely associated with the growth phase. Bifidobacterium animalis subsp. lactis A6 is a promising probiotic strain isolated from the feces of a healthy centenarian in China. In this study, RNA-seq was carried out to investigate the metabolic mechanism between the exponential and the stationary phase in B. lactis A6. RESULTS: Differential expression analysis showed that a total of 815 genes were significantly changed in the stationary phase compared to the exponential phase, which consisted of 399 up-regulated and 416 down-regulated genes. The results showed that the transport and metabolism of cellobiose, xylooligosaccharides and raffinose were enhanced at the stationary phase, which expanded carbon source utilizing profile to confront with glucose consumption. Meanwhile, genes involved in cysteine-cystathionine-cycle (CCC) pathway, glutamate dehydrogenase, branched-chain amino acids (BCAAs) biosynthesis, and Clp protease were all up-regulated in the stationary phase, which may enhance the acid tolerance of B. lactis A6 during stationary phase. Acid tolerance assay indicated that the survival rate of stationary phase cells was 51.07% after treatment by pH 3.0 for 2h, which was 730-fold higher than that of 0.07% with log phase cells. In addition, peptidoglycan biosynthesis was significantly repressed, which is comparable with the decreased growth rate during the stationary phase. Remarkably, a putative gene cluster encoding Tad pili was up-regulated by 6.5 to 12.1-fold, which is consistent with the significantly increased adhesion rate to mucin from 2.38% to 4.90% during the transition from the exponential phase to the stationary phase. CONCLUSIONS: This study reported growth phase-associated changes of B. lactis A6 during fermentation, including expanded carbon source utilizing profile, enhanced acid tolerance, and up-regulated Tad pili gene cluster responsible for bacterial adhesion in the stationary phase. These findings provide a novel insight into the growth phase associated characteristics in B. lactis A6 and provide valuable information for further application in the food industry.

Lactobacillus rhamnosus GG Derived Extracellular Vesicles Modulate Gut Microbiota and Attenuate Inflammatory in DSS-Induced Colitis Mice.

Ulcerative colitis (UC) is a relapsing and remitting inflammatory disease. Probiotics have a potential beneficial effect on the prevention of UC onset and relapse in clinical trials. Lactobacillus rhamnosus GG (L. rhamnosus GG) have shown clinical benefits on UC patients, however, the precise mechanisms are unknown. The aim of this study is to explore the effect of extracellular vesicles released from L. rhamnosus GG (LGG-EVs) on dextran sulfate sodium (DSS)-induced colitis and propose the underlying mechanism of LGG-EVs for protecting against colitis. The results showed that LGG-EVs could prevent colonic tissue damage and shortening of the colon (p < 0.01), and ameliorate intestinal inflammation by inhibiting TLR4-NF-κB-NLRP3 axis activation. Consistently, the pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-2) were suppressed effectively upon LGG-EVs treatment (p < 0.05). The 16S rRNA sequencing showed that LGG-EVs administration could reshape the gut microbiota in DSS-induced colitis mice, which further alters the metabolism pathways of gut microbiota. These findings propose a novel perspective of L. rhamnosus GG in attenuating inflammation mediated by extracellular vesicles and offer consideration for developing oral gavage of LGG-EVs for colitis therapies.

Homologous Over-Expression of Chain Length Determination Protein EpsC Increases the Molecular Weight of Exopolysaccharide in Streptococcus thermophilus 05-34.

In Streptococcus thermophilus, EpsC is a polysaccharide co-polymerase which is involved in determining the chain length of EPS synthesized by the Wzx/Wzy-dependent pathway. Our previous study found that there was a positive correlation between transcription level of epsC and molecular weight of EPS in S. thermophilus 05-34. To further investigate the effects of EpsC on EPS biosynthesis, this gene was over-expressed in S. thermophilus 05-34 in this study. Reverse transcription qPCR and Western blotting confirmed the successful transcription and translation of epsC in 05-34, respectively. The yield of EPS was not affected by the over-expression of EpsC. Gas chromatography-mass spectrometry (GC-MS) showed that the monosaccharide composition was still composed of galactose and glucose in a molar ratio of 1.0:0.8, whereas high performance gel permeation chromatography (HPGPC) indicated that the molecular weight of EPS was increased from 4.62 × 105 Da to 9.17 × 105 Da by the over-expression of EpsC. In addition, S. thermophilus 05epsC which could produce higher molecular weight EPS improved the viscoelasticity and water-holding capacity of yogurt, but significantly reduced the level of syneresis in yogurt. In summary, these results indicated that homologous over-expression of EpsC in S. thermophilus could increase the molecular weight of EPS and improve the microrheological or physical properties of yogurt.

Correlation in Causality: A Progressive Study of Hierarchical Relations within Human and Organizational Factors in Coal Mine Accidents.

It has been revealed in numerous investigation reports that human and organizational factors (HOFs) are the fundamental causes of coal mine accidents. However, with various kinds of accident-causing factors in coal mines, the lack of systematic analysis of causality within specific HOFs could lead to defective accident precautions. Therefore, this study centered on the data-driven concept and selected 883 coal mine accident reports from 2011 to 2020 as the original data to discover the influencing paths of specific HOFs. First, 55 manifestations with the characteristics of the coal mine accidents were extracted by text segmentation. Second, according to their own attributes, all manifestations were mapped into the Human Factors Analysis and Classification System (HFACS), forming a modified HFACS-CM framework in China's coal-mining industry with 5 categories, 19 subcategories and 42 unsafe factors. Finally, the Apriori association algorithm was applied to discover the causal association rules among external influences, organizational influences, unsafe supervision, preconditions for unsafe acts and direct unsafe acts layer by layer, exposing four clear accident-causing "trajectories" in HAFCS-CM. This study contributes to the establishment of a systematic causation model for analyzing the causes of coal mine accidents and helps form corresponding risk prevention measures directly and objectively.

Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pseudomonas fluorescens in Raw Milk.

Raw milk is susceptible to microbial contamination during transportation and storage. Pseudomonas fluorescens producing heat-resistant enzymes have become the most common and harmful psychrophilic microorganisms in the cold chain logistics of raw milk. To rapidly detect P. fluorescens in raw milk, the protease gene aprX was selected as a detection target to construct a set of primers with strong specificity, and a loop-mediated isothermal amplification (LAMP) assay was established. The detection thresholds of the LAMP assay for pure cultured P. fluorescens and pasteurized milk were 2.57 × 102 and 3 × 102 CFU/mL, respectively. It had the advantages over conventional method of low detection threshold, strong specificity, rapid detection, and simple operation. This LAMP assay can be used for online monitoring and on-site detection of P. fluorescens in raw milk to guarantee the quality and safety of dairy products.

A Novel Major Pilin Subunit Protein FimM Is Involved in Adhesion of Bifidobacterium longum BBMN68 to Intestinal Epithelial Cells.

Adhesion to the gastrointestinal tract is considered to be important for bifidobacteria to colonize the human gut and exert their probiotic effects. Some cell surface proteins of bifidobacteria, known as adhesins, play critical roles in the binding to host cells or the extracellular matrix (ECM). To elucidate the mechanisms associated with the adhesion of Bifidobacterium longum BBMN68, a centenarian originated potential probiotic, PSORTdb was employed to identify putative extracellular localized proteins in the B. longum BBMN68. Of the 560 predicted extracellular proteins, 21 were further identified as putative adhesion proteins using the conserved domain database of NCBI, and four were successfully overexpressed in the heterologous host, Lactococcus lactis NZ9000. Notably, a recombinant strain expressing FimM showed a significantly increased adhesive affinity for both HT-29 and mucus-secreting LS174T goblet cells (2.2- and 5.4-fold higher than that of the control strain, respectively). Amino acid sequence alignment showed that FimM is a major pilin subunit protein containing a Cna-B type domain and a C-terminal LPKTG sequence. However, in silico analysis of the fimM-coding cluster revealed that BBMN68_RS10200, encoding a pilus-specific class C sortase, was a pseudogene, indicating that FimM may function as a surface adhesin that cannot polymerize into a pili-like structure. Immunogold electron microscopy results further confirmed that FimM localized to the surface of L. lactis NZfimM and B. longum BBMN68 but did not assemble into pilus filaments. Moreover, the adhesive affinity of L. lactis NZfimM to fibronectin, fibrinogen, and mucin were 3.8-, 2.1-, and 3.1-fold higher than that of the control. The affinity of FimM for its attachment receptors was further verified through an inhibition assay using anti-FimM antibodies. In addition, homologs of FimM were found in Bifidobacterium bifidum 85B, Bifidobacterium gallinarum CACC 514, and 23 other B. longum strains by sequence similarity analysis using BLASTP. Our results suggested that FimM is a novel surface adhesin that is mainly present in B. longum strains.

Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli.

Candida albicans is an important fungal pathogen that causes a wide variety of human infections, ranging from mucocutaneous infections to life-threatening systemic infections. Phospholipase B1 (PLB1) has been reported to be directly responsible for C. albicans pathogenicity and is likely to be involved in the early steps of host invasion. Therefore, PLB1 could be a potential marker for diagnosis of C. albicans infection. In this study, PLB1 was expressed using an Escherichia coli expression system. Recombinant PLB1 is found in inclusion bodies and constitutes up to 38.4% of total insoluble protein. After refolding in a GSH/GSSG redox system, GST-tagged PLB1 was purified by GST-sepharose 4B affinity chromatography and then cleaved with thrombin to remove the GST-tag. The recombinant PLB1 was further purified by anion-exchange chromatography and reverse phase HPLC. The final yield of purified PLB1 was approximately 15.6 mg from 100 mL of bacterial cell culture, and its concentration was 784 µg/µL. The recombinant PLB1 could form a white precipitation zone on egg yolk agar plate, suggesting its phospholipase activity. Moreover, the maximum activity of PLB1 was 68 IU/mg at pH 6.0, 37 °C. Therefore, recombinant PLB1 has potential application in structural analytical studies, or diagnosis of C. albicans infection.

Combined transcriptomic and proteomic analysis of the response to bile stress in a centenarian-originated probiotic Lactobacillus salivarius Ren.

Tolerance to bile stress is a crucial property for probiotics to survive in the gastrointestinal tract and exert their beneficial effects. In this work, transcriptomic analysis combined with two-dimensional electrophoresis revealed that the transcript levels of 129 genes and the abundance of 34 proteins were significantly changed in Lactobacillus salivarius Ren when exposed to 0.75 g/L ox-bile. Notably, carbohydrate metabolism shifted to the utilization of maltose and glycerol for energy production, suggesting that L. salivarius Ren expanded carbon sources profile for gut adaptation in response to bile. Moreover, the enzymes involved in cell surface charge modification and the cell envelope-located hemolysin-like protein were overproduced, which was supposed to hinder the penetration of bile. Then, the up-regulated ABC transporters could contribute to the extrusion of bile accumulated in the cytoplasm. Additionally, proteolytic system was activated to provide more amino acids for the synthesis and repair of proteins damaged by bile. Finally, γ-glutamylcysteine with antioxidant activity and oxidoreductases for redox homeostasis were increased to cope with the bile-induced oxidative stress. These findings provide new insights into the molecular mechanisms involved in bile stress response and adaptation in L. salivarius.