• Journal of Microbiology and Biotechnology
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• v.20 no.6
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• pp.978-984
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• 2010

This paper examines the probiotic bacterium Lactobacillus rhamnosus GG, and how it reacts to the presence of mucin in its extracellular milieu. Parameters studied included cell clustering, adhesion to mucin, extracellular protein production, and formation of final metabolites. L. rhamnosus GG was found to grow efficiently in the presence of glucose, N-acetylglucosamine, or mucin (partially purified or purified) as sole carbon sources. However, it was unable to grow using other mucin constituents, such as fucose or glucuronic acid. Mucin induced noticeable changes in all the parameters studied when compared with growth using glucose, including in the formation of cell clusters, which were easily disorganized with trypsin. Mucin increased adhesion of the bacterium, and modulated the production of extracellular proteins. SDS-PAGE revealed that mucin was not degraded during L. rhamnosus GG growth, suggesting that this bacterium is able to partially use the glucidic moiety of glycoprotein. This study goes some way towards developing an understanding of the metabolic and physiological changes that L. rhamnosus GG undergoes within the human gastrointestinal tract.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.5
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• pp.751-755
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• 2006

Dadih is Indonesian traditional fermented buffalo milk believed by the natives to have beneficial effects on human health. This may be due to the probiotic properties possessed by the lactic acid bacteria (LAB) involved in its fermentation process. It was discovered that ten strains of dadih lactic isolates possessed some probiotic properties in vitro. In this study, the adhesion properties of dadih LAB, in comparison with documented probiotic strains, were investigated in vitro by using mucin extracted from human faeces and Caco-2 cells as the models for human intestinal mucosal surface and intestinal cells respectively. The adhesion results showed the distinction of Lactobacillus reuteri IS-27560 in adhering to both mucus layer and Caco-2 cells. The competition assay for adhesion to the mucus layer between dadih LAB and selected pathogens indicated the competence of Lactococcus lactis IS-16183 and Lactobacillus rhamnosus IS-7257 in significantly inhibiting the adhesion of Escherichia coli O157:H7. Accordingly, these two strains may be potential candidates for use as probiotic strains. Overall, the adhesion properties of all dadih LAB strains were relatively comparable to that of Lactobacillus casei Shirota and Lactobacillus rhamnosus GG, the documented probiotic strains.

• Korean Journal of Food Science and Technology
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• v.36 no.6
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• pp.959-967
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• 2004

To screen probiotic lactic acid bacteria with potent adhesive property on human colonic mucosa, colonic mucin-binding assay was introduced. This colonic mucin-binding assay actually measures the binding activity of surface lectin-like protein (SLP) on colonic mucin, and the optimal conditions were examined. The optimal pH for colonic mucin coating on plate wells was 4.8, and ${\times}24,000$ diluted solution of commercially available horseradish peroxidase (HRP) conjugated streptoavidin yielded good results, for rapid screening, $5.0\;{\mu}g/mL$ of biotinylated SLP from lactic acid bacteria was optimal, and optimal scintillation time of 3,3',5,5'-tetramethyl benzidine (TMB) was 10 min. These conditions were useful for both rapid selection and quantitative analysis of lactic acid bacteria that have high adhesion property to human intestinal tract. Among 50 strains of lactic acid bacteria, including 32 type culture strains and 18 isolated strains from infant feces, Lactobacillus species FSB-1 isolated from kimchi showed the highest binding activity to colonic mucin. From taxonomical viewpoints based on morphological study, physico-biochemical study, partial 16S rDNA seguencing, and phylogenetic analysis, L. species FSB-1 was identified as Lactobacillus brevis.

• Nutrition Research and Practice
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• v.9 no.4
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• pp.343-349
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• 2015

BACKGROUND/OBJECTIVES: Fermentation of dietary fiber results in production of various short chain fatty acids in the colon. In particular, butyrate is reported to regulate the physical and functional integrity of the normal colonic mucosa by altering mucin gene expression or the number of goblet cells. The objective of this study was to investigate whether butyrate modulates mucin secretion in LS174T human colorectal cells, thereby influencing the adhesion of probiotics such as Lactobacillus and Bifidobacterium strains and subsequently inhibiting pathogenic bacteria such as E. coli. In addition, possible signaling pathways involved in mucin gene regulation induced by butyrate treatment were also investigated. MATERIALS/METHODS: Mucin protein content assay and periodic acid-Schiff (PAS) staining were performed in LS174T cells treated with butyrate at various concentrations. Effects of butyrate on the ability of probiotics to adhere to LS174T cells and their competition with E. coli strains were examined. Real time polymerase chain reaction for mucin gene expression and Taqman array 96-well fast plate-based pathway analysis were performed on butyrate-treated LS174T cells. RESULTS: Treatment with butyrate resulted in a dose-dependent increase in mucin protein contents in LS174T cells with peak effects at 6 or 9 mM, which was further confirmed by PAS staining. Increase in mucin protein contents resulted in elevated adherence of probiotics, which subsequently reduced the adherent ability of E. coli. Treatment with butyrate also increased transcriptional levels of MUC3, MUC4, and MUC12, which was accompanied by higher gene expressions of signaling kinases and transcription factors involved in mitogen-activated protein kinase (MAPK) signaling pathways. CONCLUSIONS: Based on our results, butyrate is an effective regulator of modulation of mucin protein production at the transcriptional and translational levels, resulting in changes in the adherence of gut microflora. Butyrate potentially stimulates the MAPK signaling pathway in intestinal cells, which is positively correlated with gut defense.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.731-737
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• 2003

Mucinase complex from V. parahaemolyticus ATCC 17802 was purified 6-fold with 0.4% yield by two sequential steps of Q-Sepharose and Superdex 200HR column chromatographies. Partially purified mucinase complex showed at least 8 times higher mucin-degrading activity than the culture filtrates. The mucinase complex also showed gelatin-and-casein-hydrolyzing activities, which demonstrates that the protein is a complex compound containing several proteases. The optimum pH and temperature of partially purified mucinase complex for mucin degradation was 8.0 and $35^{\circ}C$, respectively. The partially purified mucinase complex showed high cytotoxic activity on vero cells when examined by MTT assay and microscopic observations. Cytotoxicity was significantly increased in proportion to the concentration of the mucinase complex. Mouse experiments revealed that the jejunum, ileum. and large intestinal tissues were damaged by the injection of the mucinase complex. In particular, the reduction of the goblet cells in the large intestine was remarkable. Collectively, these data suggest that the mucinase complex partially purified from V. parahaemolyticus ATCC 17802 contributes to the adhesion and invasion of V. parahaemolyticus into the host intestinal tract.

• Journal of Microbiology and Biotechnology
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• v.28 no.10
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• pp.1604-1613
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• 2018

Lactobacillus rhamnosus GG (LGG) is a probiotic commonly used in fermented dairy products. In this study, RNA-sequencing was performed to unravel the effects of acid stress on LGG. The transcriptomic data revealed that the exposure of LGG to acid at pH 4.5 (resembling the final pH of fermented dairy products) for 1 h or 24 h provoked a stringent-type transcriptomic response wherein stress response- and glycolysis-related genes were upregulated, whereas genes involved in gluconeogenesis, amino acid metabolism, and nucleotide metabolism were suppressed. Notably, the pilus-specific adhesion genes, spaC, and spaF were significantly upregulated upon exposure to acid-stress. The transcriptomic results were further confirmed via quantitative polymerase chain reaction analysis. Moreover, acid-stressed LGG demonstrated an enhanced mucin-binding ability in vitro, with 1 log more LGG cells (p < 0.05) bound to a mucin layer in a 96-well culture plate as compared to the control. The enhanced intestinal binding ability of acid-stressed LGG was confirmed in an animal study, wherein significantly more viable LGG cells (${\geq}2log\;CFU/g$) were observed in the ileum, caecum, and colon of acid-stressed LGG-treated mice as compared with a non-acid-stressed LGG-treated control group. To our knowledge, this is the first report showing that acid stress enhanced the intestine-binding ability of LGG through the induction of pili-related genes.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1309-1316
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• 2023

To exert their beneficial effects, it is essential for the commensal bacteria of probiotic supplements to be sufficiently protected as they pass through the low pH environment of the stomach, and effectively colonize the intestinal epithelium downstream. Here, we investigated the effect of a multilayer coating containing red ginseng dietary fiber, on the acid tolerance, and the adhesion and proliferation capacities of three Lactobacillus strains (Limosilactobacillus reuteri KGC1901, Lacticaseibacillus casei KGC1201, Limosilactobacillus fermentum KGC1601) isolated from Panax ginseng, using HT-29 cells, mucin-coated plates, and human pluripotent stem cell-derived intestinal epithelial cells as in vitro models of human gut physiology. We observed that the multilayer-coated strains displayed improved survival rates after passage through gastric juice, as well as high adhesion and proliferation capacities within the various gut epithelial systems tested, compared to their uncoated counterparts. Our findings demonstrated that the multilayer coat effectively protected commensal microbiota and led to improved adhesion and colonization of intestinal epithelial cells, and consequently to higher probiotic efficacy.

• Journal of Microbiology and Biotechnology
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• v.28 no.11
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• pp.1846-1849
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• 2018

Recent human gut microbiome studies have supported that the genus Bifidobacterium is one of the most beneficial bacteria for human intestinal health. To develop a new probiotic strain for functional food applications, fourteen fecal samples were collected from healthy Koreans and the strain BCBR-583 was newly selected and isolated from a 25-year-old Korean woman's fecal sample using the selective medium for Bifidobacterium. Subsequent fructose-6-phosphate phosphoketolase (F6PPK) test and 16S rRNA gene sequencing analysis of the strain BCBR-583 confirmed that it belongs to B. longum subsp. longum. The stress resistance tests showed that it has oxygen and heat tolerance activities (5- and 3.9-fold increase for 24 h at 60 and 120 rpm, respectively; $78.61{\pm}6.67%$ survival rate at $45^{\circ}C$ for 24 h). In addition, gut environment adaptation tests revealed that this strain may be well-adapted in the gut habitat, with gastric acid/bile salt resistance ($85.79{\pm}1.53%$, survival rate under 6 h treatments of gastric acid and bile salt) and mucin adhesion ($73.72{\pm}7.36%$). Furthermore, additional tests including cholesterol lowering assay showed that it can reduce $86.31{\pm}1.85%$ of cholesterol. Based on these results, B. longum BCBR-583 has various stress resistance for survival during food processing and environmental adaptation activities for dominant survival in the gut, suggesting that it could be a good candidate for fermented food applications as a new probiotic strain.

• Journal of Oral Medicine and Pain
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• v.24 no.3
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• pp.235-244
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• 1999

The present study was performed to investigate the binding between salivary proteins(low-molecular-weight mucin;MG2, amylase, proline-rich proteins;PRPs) and oral pathogens(Streptococcus gordonii, Actinomyces viscosus, Staphylococcus aureus) by using solid-phase assay. In the case of transferring proteins to Immobilon-P, S. gordonii binds to MG2. A. viscosus binds to MG2, amylase, and PRPs, and S. aureus binds to MG2 and amylase. On nitrocellulose membrane, S, gordonii and A. viscosus bind to MG2, amylase, and PRPs. S. aureus binds to MG2 and PRPs. However, rabbit anti-A. viscosus antisera and rabbit anti-S. aureus antisera showed cross reactivity to PRPs adsorbed to only nitrocellulose membrane in negative control experiments, which were done without bacterial overlay. The results were different according to the membrane used as solid-phase, which reflected the assay-sensitive nature of binding experiment. PRPs and amylase are known to be components of tooth enamel pellicle. In addition, there was experimental evidence that PRPs and MG2 may covalently bind to oral mucosal epithelium. Considering above facts, the results of the present study can provide information on the interactions between salivary proteins and oral bacteria on tooth and oral mucosal surfaces.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1751-1760
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• 2015

Mucin2 (MUC2), an important regulatory factor in the immune system, plays an important role in the host defense system against bacterial translocation. Probiotics known to regulate MUC2 gene expression have been widely studied, but the interactions among probiotic, pathogens, and mucin gene are still not fully understood. The aim of this study was to investigate the role of MUC2 in blocking effects of probiotics on meningitic E. coli-induced pathogenicities. In this study, live combined probiotic tablets containing living Bifidobacterium, Lactobacillus bulgaricus, and Streptococcus thermophilus were used. MUC2 expression was knocked down in Caco-2 cells by RNA interference. 5-Aza-2'-deoxycytidine (5-Aza-CdR), which enhances mucin-promoted probiotic effects through inducing production of Sadenosyl-L-methionine (SAMe), was used to up-regulate MUC2 expression in Caco-2 cells. The adhesion to and invasion of meningitic E. coli were detected by competition assays. Our studies showed that probiotic agents could block E. coli-caused intestinal colonization, bacteremia, and meningitis in a neonatal sepsis and meningitis rat model. MUC2 gene expression in the neonatal rats given probiotic agents was obviously higher than that of the infected and uninfected control groups without probiotic treatment. The prohibitive effects of probiotic agents on MUC2-knockdown Caco-2 cells infected with E44 were significantly reduced compared with nontransfected Caco-2 cells. Moreover, the results also showed that 5-Aza-CdR, a drug enhancing the production of SAMe that is a protective agent of probiotics, was able to significantly suppress adhesion and invasion of E44 to Caco-2 cells by upregulation of MUC2 expression. Taken together, our data suggest that probiotic agents can efficiently block meningitic E. coli-induced pathogenicities in a manner dependent on MUC2.