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![Bile salt hydrolase activity in Lactobacillus gasseri JCM1131 T . Full-grown culture of L. gasseri JCM1131 T was streaked on an MRS agar plate (A) or an MRS agar plate supplemented with 0.25% taurodeoxycholic acid (B). The plates were anaerobically incubated at 37 °C for 5 days. The visible Bile salt hydrolase activity in Lactobacillus gasseri JCM1131 T . Full-grown culture of L. gasseri JCM1131 T was streaked on an MRS agar plate (A) or an MRS agar plate supplemented with 0.25% taurodeoxycholic acid (B). The plates were anaerobically incubated at 37 • C for 5 days. The visible halo surrounding colonies and the white precipitates with colonies are the well-known indicator of bacterial BSH activity [19,30].](publication/351455621/figure/fig1/AS:1021903955320835@1620652297825/Bile-salt-hydrolase-activity-in-Lactobacillus-gasseri-JCM1131-T-Full-grown-culture-of.png)
Bile salt hydrolase activity in Lactobacillus gasseri JCM1131 T . Full-grown culture of L. gasseri JCM1131 T was streaked on an MRS agar plate (A) or an MRS agar plate supplemented with 0.25% taurodeoxycholic acid (B). The plates were anaerobically incubated at 37 °C for 5 days. The visible Bile salt hydrolase activity in Lactobacillus gasseri JCM1131 T . Full-grown culture of L. gasseri JCM1131 T was streaked on an MRS agar plate (A) or an MRS agar plate supplemented with 0.25% taurodeoxycholic acid (B). The plates were anaerobically incubated at 37 • C for 5 days. The visible halo surrounding colonies and the white precipitates with colonies are the well-known indicator of bacterial BSH activity [19,30].
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Lactobacillus gasseri is one of the most likely probiotic candidates among many Lactobacillus species. Although bile salt resistance has been defined as an important criterion for selection of probiotic candidates since it allows probiotic bacteria to survive in the gut, both its capability and its related enzyme, bile salt hydrolase (BSH), in L. g...
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The harshest conditions of the human gastrointestinal tract were simulated in order to study probiotic bacteria in their intended environment. Eight Lactobacillus strains were cultivated in MRS broth with added bile in different concentrations and their growth was monitored as optical density. The gathered data was used to determine the MIC of bile...
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... 21252) and Salmonella enterica serovar Typhimurium 1344 (CGMCC no. 1.1174) served as positive-and negative-control strains (26), respectively. Animals and experimental regime. ...
As a core microbe of the human gut ecosystem, Bacteroides vulgatus has been linked to multiple aspects of metabolic disorders in a collection of associative studies, which, while indicative, warrants more direct experimental evidence to verify. In this study, we experimentally demonstrated that oral administration of B. vulgatus Bv46 ameliorated the serum lipid profile and systemic inflammation of high-fat diet-induced hyperlipidemic rats in a microbiome-regulated manner, which appears to be associated with changes of bile acid metabolism, short-chain fatty acid biosynthesis, and serum metabolomic profile.
... The ability of cells to prevent damage on their cell membrane and DNA during exposure to high level of NaDC also plays important roles to survive in such condition (Merritt & Donaldson 2009). According to Kusada et al. (2021) enzyme activity (bile salt hydrolase activity) that converts the physicochemical properties of the bile salt reduces toxicity of this compound to the cells. ...
In the last two decades the use of yeasts as new probiotics has increased significantly. Therefore, our current research was focused on the investigation of yeasts for novel probiotic development in Bali. The main objectives of this research were to isolate and characterize yeasts isolated from ragi tape (dried mix cultures of microorganisms normally used in the fermentation of rice or cassava in Indonesia) and tape ketan (fermented sticky rice) for possible use as yeast-based novel probiotics, with capability to assimilate cholesterol in vitro. In this study, the potential yeast isolates were evaluated for survival at low pH conditions (pH 2, 3, or 4) and in high levels of sodium deoxicholic (NaDC), at concentrations of 0.2, 0.4, or 0.6 mM. In addition, the yeast isolates were also evaluated for their ability to assimilate cholesterol in vitro and to elucidate biotransformation of cholic acid into deoxycholic acid. This study led to 10 isolates that were resistant to pH levels of 2, 3, or 4 and to NaDC at concentration of higher than 0.4 mM. Most of those isolates were also found to assimilate cholesterol in vitro at the rate of between 18% and 76% in 24 hours incubation. In the biotransformation test, none of those isolates
transformed cholic acid into deoxycholic acid, indicating that they are safe and have potential to be developed into novel probiotics, either for human or cattle.
... The de-conjugation of bile salts through the activity of the inducible bsh gene is regarded as one of the ways by which bacteria acquire bile stress tolerance [18,39]. The presence of a putative BSH activity was linked to bile salt resistance and human intestinal survival of Lactobacillus gasseri JCM1131 T as its purified protein showed enhanced deconjugation activity for taurocholic and taurochenodeoxycholic acids [40]. The expression of BSH activity improved bile tolerance of Lactococcus lactis [18]. ...
... Possibly, the superior bile tolerance of non-adapted cells without BSH activity could be attributed to these systems. Although BSH activity did not result in increased bile salt resistance, this trait is still desirable because bile salt hydrolysis can reduce serum cholesterol levels [40,43] and is involved in regulation of lipid absorption, glucose metabolism and energy homeostasis [40]. Unfortunately, the stress adaptation process induced transient BSH expression by L. plantarum B411 cells as long-term freezing of the stress adapted cells allowed them to revert to their original phenotype of negative BSH activity. ...
... Possibly, the superior bile tolerance of non-adapted cells without BSH activity could be attributed to these systems. Although BSH activity did not result in increased bile salt resistance, this trait is still desirable because bile salt hydrolysis can reduce serum cholesterol levels [40,43] and is involved in regulation of lipid absorption, glucose metabolism and energy homeostasis [40]. Unfortunately, the stress adaptation process induced transient BSH expression by L. plantarum B411 cells as long-term freezing of the stress adapted cells allowed them to revert to their original phenotype of negative BSH activity. ...
The preadaptation of probiotics to sub-lethal levels of multiple stress factors boosts their survival and stability. However, little is known about how long-term cold storage affects the properties of such preadapted probiotics. This study examined the impact of long-term freezing on structural and functional properties of multi-stress (acid, bile and heat) adapted Lactiplantibacillus plantarum B411. Cell morphology was investigated using scanning electron microscopy, and then their selected functional (bile salt hydrolase (BSH) activity, surface hydrophobicity, auto-aggregative and antimicrobial) properties were evaluated. Furthermore, the survival of L. plantarum B411 cells in yoghurt and juices during storage and under simulated gastrointestinal (GIT) conditions was evaluated. Long-term freezing negatively affected the morphology, auto-aggregation ability, BSH and antimicrobial activities of L. plantarum B411. The viability of freshly adapted and old adapted L. plantarum B411 cells in foods was similar. Under simulated GIT conditions, the viability of the stress adapted cells from the freezer diminished more than that of freshly adapted cells. Prolonged freezing compromised some functional properties of stress adapted cells and their stability under simulated GIT conditions. Care should thus be taken to ensure that a method used to preserve stress adapted cells does not cause them to lose beneficial properties, nor revert to their pre-adaptation status.
... BSH enzymes have been known to confer further beneficial effects on human health, including antimicrobial [15][16][17] and cholesterol-lowering activities [12]. In general, BSH enzymes have been identified in probiotic candidates within the phylum Firmicutes (i.e., genera Clostridium [18], Christensenella [19], Enterococcus [20], Lacticaseibacillus [21], Lactiplantibacillus [22], Lactobacillus [23][24][25], Ligilactobacillus [26], Listeria [27], and Lysinibacillus [28]). Although gut bacteria belonging to the phylum Actinobacteria have also been widely recognized as beneficial probiotic candidates [29], previous research related to BSH has been limited to members of the genus Bifidobacterium within the family Bifidobacteriaceae [30][31][32][33][34]. ...
... We performed heterologous gene expression and protein purification experiments, as described in our previous studies [23][24][25]54]. In brief, the constructed plasmids (pET28b-lcBSH and pColdII-gfBSH) were transformed into competent E. coli BL21 (DE3) Champion TM 21 cells. ...
... The purified recombinant proteins (LcBSH and GfBSH) were treated with 4× premixed sample buffer solution (Bio-Rad, Hercules, CA, USA) and heat-denatured at 95 • C for 5 min using a thermal cycler (TaKaRa). The resulting protein solutions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad), according to the methods described in previous studies [23][24][25]54]. The gels were stained overnight with QC Colloidal Coomassie Stain (Bio-Rad), with gentle agitation, then decolorized with deionized water. ...
The bile resistance of intestinal bacteria is among the key factors responsible for their successful colonization of and survival in the mammalian gastrointestinal tract. In this study, we demonstrated that lactate-producing Atopobiaceae bacteria (Leptogranulimonas caecicola TOC12T and Granulimonas faecalis OPF53T) isolated from mouse intestine showed high resistance to mammalian bile extracts, due to significant bile salt hydrolase (BSH) activity. We further succeeded in isolating BSH proteins (designated LcBSH and GfBSH) from L. caecicola TOC12T and G. faecalis OPF53T, respectively, and characterized their enzymatic features. Interestingly, recombinant LcBSH and GfBSH proteins exhibited BSH activity against 12 conjugated bile salts, indicating that LcBSH and GfBSH have much broader substrate specificity than the previously identified BSHs from lactic acid bacteria, which are generally known to hydrolyze six bile salt isomers. Phylogenetic analysis showed that LcBSH and GfBSH had no affinities with any known BSH subgroup and constituted a new BSH subgroup in the phylogeny. In summary, we discovered functional BSHs with broad substrate specificity from Atopobiaceae bacteria and demonstrated that these BSH enzymes confer bile resistance to L. caecicola TOC12T and G. faecalis OPF53T.
... While in the intestinal tract of birds little is known about this parameter, the intestinal concentration of bile acids in the human tract is approximately 0.3% [26]. While in the present study, we did not evaluate the BSH activity, in a recent study, it was demonstrated that BSH is responsible for the bile-salt resistance in Lactobacillus gasseri JCM1131T strain, supporting the importance of the typical lactic acid bacterium as probiotics [27]. The growth of LP-40 at high concentrations of bile salts could be associated with the ability of Lactobacillus salivarius to produce the BSH enzyme intracellularly [28]. ...
The objective of the present study was to characterize the probiotic potential of nine strains of Lactobacillus spp. isolated from the gastrointestinal tract of Creole hens through in vitro and in vivo tests. The following parameters were evaluated in vitro: (1) growth at four pH levels, (2) resistance to four bile salt concentrations, (3) tolerance to four NaCl concentrations, (4) growth capacity in the culture medium, and (5) the antimicrobial activity against Escherichia coli, Salmonella, Klebsiella, and Staphylococcus aureus. The candidate LP-40 had significantly (p < 0.05) increased resistance to pH 3 and 0.3% bile salts and elevated antimicrobial activity in vitro compared to the other strains evaluated. This strain was identified as Lactobacillus salivarius by 16S rRNA sequencing. An in vivo experiment was conducted to assess the effect of LP-40 supplementation in the drinking water on 42-day performance parameters in broiler chickens as compared to a non-treated control or dietary treatment with enrofloxacin. The administration of LP-40 in the drinking water significantly (p < 0.05) improved body weight, feed conversion compared to the antibiotic-treated control. Thus, the in vitro and in vivo results suggest that Lactobacillus salivarius LP-40 demonstrates probiotic potential and, perhaps, could be utilized as an alternative to antibiotic treatment.
... The bile salt hydrolyzing activity of the purified protein was evaluated as described previously (Allain et al., 2018b;Kusada et al., 2021). The purified LpBSH (100 µg/100 µl) was mixed with eight different conjugated bile salts (each 0.24 mg/100 µl) and incubated at 37 • C. Each bile salt solution treated with buffer (instead of LpBSH) was used as a negative control. ...
... The optimum enzymatic conditions of LpBSH were determined according to previous studies (Rani et al., 2017;Kusada et al., 2021) as follows. Taurodeoxycholic acid (TDCA) was selected as a representative substrate and mixed with 100 µg of purified LpBSH at various temperatures (10-90 • C) and pH (pH 3.0-10.0) ...
Bile salt hydrolase (BSH) is a well-characterized probiotic enzyme associated with bile detoxification and colonization of lactic acid bacteria in the human gastrointestinal tract. Here, we isolated a putative BSH (LpBSH) from the probiotic bacterium Lactobacillus paragasseri JCM 5343T and demonstrated its bifunctional activity that allows it to degrade not only bile salts but also the antibiotic (penicillin). Although antibiotic resistance and bile detoxification have been separately recognized as different microbial functions, our findings suggest that bifunctional BSHs simultaneously confer ecological advantages to host gut bacteria to improve their survival in the mammalian intestine by attaining a high resistance to bile salts and β-lactams. Strain JCM 5343T showed resistance to both bile salts and β-lactam antibiotics, suggesting that LpBSH may be involved in this multi-resistance of the strain. We further verified that such bifunctional enzymes were broadly distributed among the phylogeny, suggesting that the bifunctionality may be conserved in other BSHs of gut bacteria. This study revealed the physiological role and phylogenetic diversity of bifunctional enzymes degrading bile salts and β-lactams in gut bacteria. Furthermore, our findings suggest that the hitherto-overlooked penicillin-degrading activity of penicillin acylase could be a potential new target for the probiotic function of gut bacteria.
... To date, several genes encoding BSH have been isolated from Lacticaseibacillus casei (Zhang et al., 2009), Limosilactobacillus fermentum (Kumar et al., 2013), Lactiplantibacillus plantarum (Christiaens et al., 1992;Lambert et al., 2008;Ren et al., 2011;Dong et al., 2012Dong et al., , 2013Gu et al., 2014), Limosilactobacillus reuteri (Taranto et al., 1999), Lacticaseibacillus rhamnosus (Kaya et al., 2017), Ligilactobacillus salivarius (Wang et al., 2012;Bi et al., 2013), and some Lactobacillus spp. including Lactobacillus acidophilus (McAuliffe et al., 2005), Lactobacillus gasseri (Rani et al., 2017;Kusada et al., 2021), Lactobacillus johnsonii Savage, 1990, 1992;Elkins et al., 2001;Oh et al., 2008;Chae et al., 2013), and Lactobacillus paragasseri (Kusada et al., submitted). The substrate specificity of BSH enzymes may be strain specific; however, most of the characterized BSH enzymes prefer to hydrolyze glycine-conjugated bile salts rather than taurine-conjugated ones (Dong and Lee, 2018). ...
... The BSH assay was performed using the purified LapBSH protein as described previously (Allain et al., 2018;Kusada et al., 2021). In brief, the purified LapBSH protein (100 µg/100 µl) was mixed with 0.24 mg/100 µl of the selected conjugated bile salts and incubated at 37 • C. The bile salt hydrolysis reaction was terminated by adding 15% trichloroacetic acid solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and the proteins were removed by centrifugation at 10,000 × g for 15 min at 20 • C. The supernatant was then mixed with 0.3 M borate buffer with 1% SDS (pH 9.5) and 0.3% 2,4,6trinitrobenzenesulfonic acid solution (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan). ...
... Studies on the optimum enzymatic condition of LapBSH were performed as described previously (Rani et al., 2017;Kusada et al., 2021). We selected TDCA as a representative substrate and mixed it with purified LapBSH (100 µg/100 µl) at various temperatures (10-90 • C, in intervals of 10 • C) and pH values (pH 3.0-10.0, in intervals of pH 1.0). ...
Bile salt hydrolase (BSH) enzymes produced by intestinal Lactobacillus species have been recognized as major targets for probiotic studies owing to their weight-loss and cholesterol-lowering effects. In this study, we isolated a highly thermostable BSH with broad substrate specificity, designed as LapBSH (BSH from a probiotic bacterium, Lactobacillus paragasseri JCM 5343 T ). The recombinant LapBSH protein clearly hydrolyzed 12 different substrates, including primary/secondary, major/minor, and taurine/glycine-conjugated bile salts in mammalian digestive tracts. Intriguingly, LapBSH further displayed a highly thermostable ability among all characterized BSH enzymes. Indeed, this enzyme retained above 80% of its optimum BSH activity even after 6 h of incubation at 50–90°C. LapBSH also exerted a functionally stable activity and maintained above 85% of its original activity after pre-heating at 85°C for 2 h. Therefore, LapBSH is a very unique probiotic enzyme with broad substrate specificity and high thermostability. The strain itself, JCM 5343 T , was also found to exhibit high heat-resistance ability and could form colonies even after exposure to 85°C for 2 h. As thermostable enzyme/bacterium offers industrial and biotechnological advantages in terms of its productivity and stability improvements, both thermostable LapBSH and thermotolerant L. paragasseri JCM 5343 T could be promising candidates for future probiotic research.
The accumulation of organic selenium (Se) by lactic acid bacteria (LAB) showed excellent biosafety and absorption efficiency. Identification of Se-enriched strains and the production of Se-enriched products by fermentation are popular research areas. However, there are few studies that focus on the distribution characteristics of Se in cells. In this study, a Se-enriched dominant Lactobacillus Paracasei 20241 was selected to characterize the distribution of Se on the cell surface and inside the cell. The results showed that L. paracei can convert sodium selenite into zero-valent Se and organic Se. The former was then released outside the cell and the latter was combined with cell components. The content of organic Se in the cells was 2144.99 ± 123.16 μg/g, accounting for 52.73% of the total Se. Compared with cellular polysaccharide and nucleic acid, protein exhibited the highest Se binding ability of 864.03 ± 26.04 μg/g, indicating that the main form of organic Se was selenoprotein. Thus, Lactobacillus Paracasei 20241 is expected to be used as a dietary supplement for Se-enrich biomass probiotics.
The gut microbiome is increasingly recognized as a vital organ that participates in nutrient acquisition, energy regulation and maintenance of human health. Targeted manipulation of the gut microbiota by dietary supplements has been validated as an effective approach to improve human health. Among various gut microbiome-host interactions, bile acids (BAs) are intensively metabolized by the gut microbes and control a variety of metabolic processes such as energy homeostasis, glucose and lipid metabolism by activation of farnesoid X receptor (FXR). How dietary supplements regulate FXR signaling and promote human health is thus attracting broad interest and well-reviewed. However, the exact effects of BAs on FXR activity and host metabolism are compound-specific. Different BAs are metabolized by the gut microbes into varied BA derivatives and then impose distinct modulation of FXR activity and host health. As the gateway enzymes, bile salt hydrolases (BSHs), produced by the gut microbiota, control the first step of BA transformation in the gut. Therefore, BSH is a key mediator linking the food supplements' modulation to the gut microbiota and BAs-FXR signaling. In this review, we generalized the relationship between BAs, gut microbial BSHs and FXR, and summarized the dietary regulators of BSH and FXR activities, aiming to rationalize the dietary management of human health via dietary supplement-gut microbiota-FXR signaling axis.
