Bile Salt Hydrolase Activity Research Articles

Effect of different drying methods on the functional properties of probiotics encapsulated using prebiotic substances.

Probiotics and prebiotics together work synergistically as synbiotics and confer various health benefits. Many studies on synbiotic foods only focus on the survival of probiotics but fail to evaluate their functional properties. The impact on functional properties should be explored to better understand its therapeutic efficacy. In this work, probiotics (Lactiplantibacillus plantarum NCIM 2083) were encapsulated with prebiotics (fructooligosaccharide + whey protein + maltodextrin) using spray-drying (SD), freeze-drying (FD), spray-freeze-drying (SFD), and refractance window-drying (RWD) techniques. Aggregation, intestinal adhesion, antagonistic activity, and bile salt hydrolase (BSH) activity of probiotics were studied before and after the encapsulation process. The SFD probiotics showed better aggregation ability (79% at 24-h incubation), on par with free cells (FC) (81% at 24-h incubation). The co-aggregation ability of encapsulated probiotics has drastic variations with each pathogenic strain. The adhesion ability of probiotics in chicken intestinal mucus was assessed by the crystal violet method, indicating no significant variations between FC and SFD probiotics. Also, encapsulated probiotics exhibit antagonistic activity (zone of inhibition in mm) against gut pathogens E. coli (11.33 to 17.34), S. faecalis (8.83 to 15.32), L. monocytogenes (13.67 to 18), S. boydii (12.17 to 15.5), and S. typhi (2.17 to 6.86). Overall, these studies confirm the significance and impact of various drying techniques on the functionality of encapsulated probiotics in synbiotic powders. KEY POINTS: • Understanding the relevance of processing effects on the functionality of probiotics. • Spray-freeze-dried probiotics showed superior functional properties. • The encapsulation process had no significant impact on bile salt hydrolase activity.

Profiling rhythmicity of bile salt hydrolase activity in the gut lumen with a rapid fluorescence assay.

Diurnal rhythmicity of cellular function is key to survival for most organisms on Earth. Many circadian functions are driven by the brain, but regulation of a separate set of peripheral rhythms remains poorly understood. The gut microbiome is a potential candidate for regulation of host peripheral rhythms, and this study sought to specifically examine the process of microbial bile salt biotransformation. To enable this work, an assay for bile salt hydrolase (BSH) that could work with small quantities of stool samples was necessary. Using a turn-on fluorescence probe, we developed a rapid and inexpensive assay to detect BSH enzyme activity with concentrations as low as 6-25 μM, which is considerably more robust than prior approaches. We successfully applied this rhodamine-based assay to detect BSH activity in a wide range of biological samples such as recombinant protein, whole cells, fecal samples, and gut lumen content from mice. We were able to detect significant BSH activity in small amounts of mouse fecal/gut content (20-50 mg) within 2 h, which illustrates its potential for use in various biological/clinical applications. Using this assay, we investigated the diurnal fluctuations of BSH activity in the large intestine of mice. By using time restricted feeding conditions, we provided direct evidence of 24 h rhythmicity in microbiome BSH activity levels and showed that this rhythmicity is influenced by feeding patterns. Our novel function-centric approach has potential to aid in the discovery of therapeutic, diet, or lifestyle interventions for correction of circadian perturbations linked to bile metabolism.

Comparative evaluation of the probiotic and antioxidant potential of indigenous Lactic Acid Bacteria

This study was undertaken to assess the probiotic and antioxidant potential of five indigenous cultures of lactic acid bacteria viz, Lactobacillus acidophilus, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, Lactobacillus bulgaricus and Streptococcus thermophilus. The probiotic properties in terms of acid and bile tolerance, cell surface hydrophobicity, autoaggregation, coaggregation and bile salt hydrolase activity were assessed in vitro. The DPPH assay indicated highest antioxidant activity of 45.33 per cent for Streptococcus thermophilus. All five cultures exhibited significant antioxidant potential. Lacticaseibacillus rhamnosus, Lactobacillus acidophilus and Lactiplantibacillus plantarum showed higher probiotic potential in terms of acid and bile tolerance, cell surface hydrophobicity, autoaggregation and coaggregation. None of the cultures exhibited bile salt hydrolase activity.

Characterization of Lactic Acid Bacteria from Fermented Fish (pla-paeng-daeng) and Their Cholesterol-lowering and Immunomodulatory Effects.

The cholesterol-lowering and immunomodulatory effects and probiotic properties of 25 lactic acid bacteria (LAB) isolated from fermented fish (pla-paeng-daeng) in Thailand were examined in the present study. Based on their phenotypic and genetic characteristics, LAB were identified as Lactiplantibacillus pentosus (Group I, 6 isolates), Lactiplantibacillus argentoratensis (Group II, 1 isolate), Limosilactobacillus fermentum (Group III, 2 isolates), Companilactobacillus pabuli (Group IV, 4 isolates), Companilactobacillus farciminis (Group V, 5 isolates), Companilactobacillus futsaii (Group VI, 6 isolates), and Enterococcus lactis (Group VII, 1 isolate). Lactiplantibacillus pentosus PD3-1 and PD9-2 and Enterococcus lactis PD3-2 exhibited bile salt hydrolase (BSH) activities. The percentage of cholesterol assimilated by all isolates ranged between 21.40 and 54.07%. Bile salt hydrolase-producing isolates tolerated acidic and bile conditions and possessed adhesion properties. They also exerted immunomodulatory effects that affected the production of interleukin-12 (IL-12), interferon-γ (IFN-γ), human β-defensin-2 (hBD-2), and nitric oxide (NO). These isolates meet standard probiotic requirements and exert beneficial effects.

Caracterização probiótica de bactérias láticas de queijo de leite de cabra artesanal para desenvolvimento de produtos lácteos funcionais

Abstract The increasing interest in functional foods has encouraged the search for new lactic acid bacteria (LAB) with singular characteristics, including technological and probiotic properties, present in natural sources. The present research aimed to isolate and in vitro characterize the probiotic potential of LAB isolated from artisanal goat cheese. In addition, the acidifying and proteolytic capacity of the strains were evaluated in order to develop a functional dairy product. Fifty strains, presumptively identified as LAB, were isolated from artisanal goat cheese. From them, seventeen strains were selected for identification through Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry (MS) and 16S gene sequence. None of the tested strains showed hemolytic ability, while all strains showed at least 76% of survival to low pH and conjugated bile salts. Hydrophobicity and auto-aggregation values varied among LAB with ranges between 15.5 ± 1.8 to 84.7 ± 3.2, and 4.72% to 59.2%, respectively. Selected LAB were capable of adhering to intestinal mucus in a range of 5.08 to 6.90 Log CFU/mL. Remarkably, eight strains showed high bile salt hydrolase activity. Autochthons strains were able to grow and acidify milk after 24 h. Besides, proteolytic activity showed a range between 10 and 151 mmol/L amino acids. PCA analysis and technological properties allow us to propose Lactiplantibacillus plantarum CB5, CB8 and Lentilactobacillus parabuchneri CB12 strains as potential probiotic starter cultures. The isolation of new LAB strains with demonstrated functional properties is of interest to academic institutions and the food industry as it allows the design of indigenous dairy cultures with added probiotic and technological properties.

Distribution, cholesterol-lowering and immunomodulation effects of lactic acid bacteria from fermented mussel (Hoi-dong)

Forty-eight lactic acid bacteria (LAB) isolated from fermented mussels in Thailand were evaluated for their probiotic properties, bile salt hydrolase (BSH), cholesterol assimilation and immunomodulatory effects. They were identified as Companilactobacillus formosensis (Group I, 10 isolates), Lentilactobacillus buchneri (Group II, 8 isolates), Lactiplantibacillus plantarum subsp. plantarum (Group III, 16 isolates), Lacticaseibacillus rhamnosus (Group IV, 1 isolate), Pediococcus pentosaceus (Group V, 5 isolates) and P. acidilactici (Group V, 1 isolate), Enterococcus thailandicus (Group VI, 2 isolates), En. hirae (Group VII, 1 isolate), En. durans (Group VI, 1 isolate), Lactococcus lactis subsp. lactis (Group VII, 1 isolate), Lc. lactis subsp. hordinae (Group VII, 1 isolate), and Leuconostoc lactis (Group VIII, 1 isolate), based on their phenotypic and genetic characteristics. Seven isolates, L. plantarum subsp. plantarum LM6-1, LM6-2, LM7-2-2B, LM12-1, LM14-1, LM15-1P and LM15-2 expressed bile salt hydrolase activity. All isolates assimilated cholesterol ranging from 20.73 to 79.40%. BSH-producing isolates were tolerant to acidic and bile conditions and showed the adhesion ability to Caco-2 cells. The BSH-producing and selected isolates showed the immunomodulatory effects to stimulate interleukin-12 (IL-12), interferon-gamma (IFN-γ), human beta defensin-2 (hBD-2) and nitric oxide (NO) production at various levels. Therefore, these results indicated that the isolates meet the standard probiotic criteria and beneficial effects.

Newly characterized Lactiplantibacillus plantarum strains isolated from raw goat milk as probiotic cultures with potent cholesterol-lowering activity

One of the most promising aspects of lactic acid bacteria (LAB) in foodstuffs with probiotic characteristics is cholesterol-lowering action. This study aimed to examine the bile salt hydrolase (BSH) positive strains; out of 50 LAB isolates, Lactiplantibacillus plantarum Lpb01 and Lpb02 were selected. Both Lpb01 and Lpb02 were more bile-tolerant strains, surviving in 1% bile for 3 h at up to 7.27 and 7.14 log CFU/mL, respectively. Lactiplantibacillus plantarum Lpb01 and Lpb02 deconjugated considerably more sodium glycocholate, releasing 5.50 ± 0.12 mM and 5.23 ± 0.41 mM cholic acid, respectively, compared with 4.32 ± 0.17 mM cholic acid released by ATCC 8014. In addition, Lpb. plantarum Lpb01 and Lpb02 deconjugated a considerably higher quantity of sodium glycocholate than ATCC 8014. Compared to ATCC 8014, Lactiplantibacillus plantarum Lpb01 and Lpb02 demonstrated a higher level of BSH activity. The tested strains Lactiplantibacillus plantarum Lpb01 and Lpb02 resulted in a 63–68% reduction in the cholesterol level in broth media, whereas the commercial strain Lpb. plantarum ATCC 8014 showed a 51% reduction. In the case of survivability during in vitro gastrointestinal digestion, they did not lyse in the presence of lysozyme, and they survived more than 96% of the time when exposed to pepsin and low acidity (pH 3) for 1.5 h. Finally, the LAB strains were resistant to bile salts and pancreatin, with an overall viability rate of 94.0–99.4% after exposure to small intestinal conditions. This suggests that the isolates can live in the GI tract's severe environment. As a result, these strains with demonstrated in vitro capabilities are promising candidates for novel probiotic-containing formulations and could be incorporated into functional dairy foods. • The Lactiplantibacillus plantarum Lpb01and Lpb02 strains have shown high bile salt hydrolase activity. • The strains Lpb01 and Lpb02 resulted in remarkably higher cholesterol reductions of 63 and 68%, respectively. • The evaluated strains have a cholesterol removal ability of 12–17% higher than commercial strain ATCC 8014. • Biomimetic digestion model assessed that both strains had more than 96% survivability.

Clostridioides difficile bile salt hydrolase activity has substrate specificity and affects biofilm formation

The Clostridioides difficile pathogen is responsible for nosocomial infections. Germination is an essential step for the establishment of C. difficile infection (CDI) because toxins that are secreted by vegetative cells are responsible for the symptoms of CDI. Germination can be stimulated by the combinatorial actions of certain amino acids and either conjugated or deconjugated cholic acid-derived bile salts. During synthesis in the liver, cholic acid- and chenodeoxycholic acid-class bile salts are conjugated with either taurine or glycine at the C24 carboxyl. During GI transit, these conjugated bile salts are deconjugated by microbes that express bile salt hydrolases (BSHs). Here, we surprisingly find that several C. difficile strains have BSH activity. We observed this activity in both C. difficile vegetative cells and in spores and that the observed BSH activity was specific to taurine-derived bile salts. Additionally, we find that this BSH activity can produce cholate for metabolic conversion to deoxycholate by C. scindens. The C. scindens-produced deoxycholate signals to C. difficile to initiate biofilm formation. Our results show that C. difficile BSH activity has the potential to influence the interactions between microbes, and this could extend to the GI setting.

Lactobacillus gasseri RW2014 Ameliorates Hyperlipidemia by Modulating Bile Acid Metabolism and Gut Microbiota Composition in Rats

Hyperlipidemia is a leading risk of cardiovascular and cerebrovascular disease. Dietary supplementation with probiotics has been suggested as an alternative intervention to lower cholesterol. In the current study, we isolated a strain of Lactobacillus gasseri RW2014 (LGA) from the feces of a healthy infant fed with breast milk, and it displayed bile salt hydrolase (BSH) activity. Using this strain we determined its cholesterol-lowering and fatty liver-improving functions. SD rats were randomly divided into four groups. The control rats were fed a commercial chow diet and the other three groups were fed a high-fat diet (HFD) for a 7-week experiment period. After two weeks of feeding, the rats in PBS, simvastin, and LGA group were daily administered through oral gavage with 2 mL PBS, simvastin (1 mg/mL), and 2 × 109 CFU/mouse live LGA in PBS, respectively. After five weeks of such treatment, the rats were euthanized and tissue samples were collected. Blood lipid and inflammatory factors were measured by ELISA, gut microbiota was determined by 16S rRNA sequencing, and bile acids profiles were detected by metabolomics. We found that LGA group had lower levels of blood cholesterol and liver steatosis compared to the simvastin group. LGA also significantly reducedthe levels of inflammatory factors in the serum, including TNFα, IL-1β, MCP-1, IL-6, and exotoxin (ET), and increased the levels of short-chain fatty acids in feces, including isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid. In addition, LGA altered the compositions of gut microbiota as manifested by the increased ratio of Firmicutes/Bacteroides and the relative abundance of Blautia genus. Targeted metabolomics results showed that bile acids, especially free bile acids and secondary bile acids in feces, were increased in LGA rats compared with the control rats. Accordingly, the rats administrated with LGA also had a higher abundance of serum bile acids, including 23-norcholic acid, 7-ketolithocholic acid, β-muricholic acid, cholic acid, and deoxycholic acid. Together, this study suggests that LGA may exert a cholesterol-lowering effect by modulating the metabolism of bile acids and the composition of gut microbiota.

The Morphological and Functional Properties of Lactiplantibacillus plantarum B411 Subjected to Acid, Bile and Heat Multi-Stress Adaptation Process and Subsequent Long-Term Freezing

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.

Assessing the clinical value of faecal bile acid profiling to predict recurrence in primary Clostridioides difficile infection.

Factors influencing recurrence risk in primary Clostridioides difficile infection (CDI) are poorly understood, and tools predicting recurrence are lacking. Perturbations in bile acids (BAs) contribute to CDI pathogenesis and may be relevant to primary disease prognosis. To define stool BA dynamics in patients with primary CDI and to explore signatures predicting recurrence METHODS: Weekly stool samples were collected from patients with primary CDI from the last day of anti-CDI therapy until recurrence or, otherwise, through 8 weeks post-completion. Ultra-high performance liquid chromatography-mass spectrometry was used to profile BAs. Stool bile salt hydrolase (BSH) activity was measured to determine primary BA bacterial deconjugation capacity. Multivariate and univariate models were used to define differential BA trajectories in patients with recurrence versus those without, and to assess faecal BAs as predictive markers for recurrence. Twenty (36%) of 56 patients (median age: 57, 64% male) had recurrence; 80% of recurrences occurred within the first 9 days post-antibiotic treatment. Principal component analysis of stool BA profiles demonstrated clustering by recurrence status and post-treatment timepoint. Longitudinal faecal BA trajectories showed recovery of secondary BAs and their derivatives only in patients without recurrence. BSH activity increased over time only among non-relapsing patients (β= 0.056; likelihood ratio test p= 0.018). A joint longitudinal-survival model identified five stool BAs with area under the receiver operating characteristic curve >0.73 for predicting recurrence within 9 days post-CDI treatment. Gut BA metabolism dynamics differ in primary CDI patients between those developing recurrence and those who do not. Individual BAs show promise as potential novel biomarkers to predict CDI recurrence.

In Vitro Assessment of Probiotic and Technological Properties of Lactic Acid Bacteria Isolated from Indigenously Fermented Cereal-Based Food Products

The present study concerns the isolation and characterization of potential probiotic bacteria isolated from indigenously fermented cereal-based products commonly produced by tribal people of the Aravali hills region of India and the documentation of their unexplored probiotic attributes. The isolated strains were evaluated for probiotic attributes, such as bile salt and acid tolerance, lysozyme and phenol tolerance, antagonistic and antifungal activity, cell autoaggregation, cell-surface hydrophobicity, simulated gastric and pancreatic digestion, antioxidative potential, bile salt hydrolase activity, and H2O2 production. The safety of isolates was assessed by antibiotic sensitivity, hemolytic activity, DNase activity, and biogenic amine production assays, while technological properties, such as fermenting ability, amylolytic activity, and EPS production, were also evaluated. A total of 70 LAB isolates were screened initially, and 6 strains showed good potential as probiotic candidates in in vitro assessments. The efficient strains were identified using phenotyping and biochemical characterization, which results were further confirmed and recognized at the strain level using phylogenetic analysis and 16S rDNA sequencing. The current study has shown that Lactiplantibacillus plantarum KMUDR7 isolated from “Makka ki Raab” has excellent probiotic attributes and could be a potential probiotic for product preparation. However, other strains, Lactobacillus delbrueckii subsp. bulgaricus KMUDR1 and Lacticaseibacillus rhamnosus KMUDR9, showed good properties, while KMUDR14, -17, and -20 also have comparable probiotic attributes.

Technological characterisation and probiotic traits of yeasts isolated from Sha'a, a Cameroonian maize-based traditional fermented beverage

The current trend in starter selection is to combine both technological and probiotic properties to standardise and make functional artisanal fermented beverages such as Sha'a whose properties are very variable due to the lack of a known starter. The objective of this work was to study technological and probiotic properties of yeasts isolated from Sha'a sold in Bamenda, Bafoussam, Bonabérie, Dschang, Foumbot, Mbouda and Njombé (Cameroon). The isolated yeasts were studied for their ability to produce CO2 from glucose, to grow in the presence of 8% ethanol, 20% glucose and pH 3, to assimilate maltose and to produce ethanol. Then, the survival of the pre-selected isolates was assessed in simulated gastric (pH 2 and 3) and intestinal juices, followed by self-aggregation, co-aggregation, hydrophobicity, haemolysin, gelatinase, biogenic amine production, antibiotic and antifungal susceptibility, bile salt hydrolase and antiradical activity. The selected isolates were identified by sequencing the 5.8S/28S rRNA gene. From the 98 isolates obtained, 66 produced CO2 from glucose and 16 were then selected for their ability to grow in the presence of 8% ethanol, 20% glucose, pH 3 and maltose. The overall survival of isolates ranged from 4.12 ± 1.63 to 104.25 ± 0.19% (LT16) and from 0.56 ± 0.20 to 96.74 ± 1.60% (LT66) at pH 3 and pH 2 respectively. All of them have remarkable surface hydrophobicity properties. Based on principal component analysis, 5 isolates were selected as the best. However, only 3 of them, LT16 (the most promising), LT25 identified as Saccharomyces cerevisiae and LT80 as Nakaseomyces delphensis, do not produce a virulence factor. The latter can deconjugate bile salts with a maximum percentage of 60.54 ± 0.12% (LT16) and the highest inhibition of DPPH° radicals was 55.94 ± 1.14% (LT25). In summary, the yeast flora of Sha'a contains yeasts capable of fermenting and producing ethanol while producing bioactive compounds that would benefit the consumer.

Screening bile salt hydrolase activity of Lactobacillus isolated from Vietnamese human origins

Hypercholesterolemia is a major cause of cholesterol build-up in the coronary arteries, which can subsequently lead to heart disease or atherosclerosis. Cholesterol levels can be lowered by cholesterol-lowering drugs but some of these drugs may have harmful side effects, while supplementation of Lactobacillus has shown the potential to reduce serum cholesterol levels by virtue of bile salt hydrolase (bsh) activity. In this study, Lactobacillus plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14, had been isolated from Vietnamese healthy adults, were able to deconjugate glycodeoxycholate (GDC) on MRS plates and MRS broth supplemented with GDC. In addition, deconjugating activity of L. plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14 were also found in cell-free extract as expressed by amount of glycine that released in the supernatant. Four bsh genes including bsh1, bsh2, bsh3, and bsh4 have been identified by PCR in these strains. In addition, L. plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14 also showed high ability to resist bile salts and low pH. The results of 16S rRNA gene analyses showed that L. plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14 and had high similarity scores with L. plantarum ZZU 23 (100%), L. rhamnosus JCM 1136 (99%) and L. plantarum S7 (98.65%), respectively. This study suggests that L. plantarum VFE-04, L. rhamnosus VFE-08, and L. plantarum VFE-14 have the potential to be explored as probiotics in the management of hypercholesterolaemia in near future.

Deciphering Beneficial Health Promoting Properties of Lactic Acid Bacteria Isolated From Infant Gut Microbiome

Aim and Objective: To evaluate the probiotic potenties along with safety of probiotics within infant faecal origin. Methods and Results: The present study isolates lactic acid bacteria from infant feces were characterized and identified by 16S rRNA sequencing. On evaluation of in vitro probiotic capability, the test isolates exhibited in vitro probiotic potency, antagonistic property and better cell surface properties. The Bile salt hydrolase activity, cholesterol assimilation, antioxidant activity and angiotensin converting enzyme inhibitory action of the test isolates proves their health conferring potency. Standard protocols were used for the estimation of lymphocyte proliferation rate, phagocytosis rate, cytokine response and faecal enzyme levels. Animal experiment using different doses of L. plantarum (MBTU-S1B) substantiated its immunotolerance against inhibitory effects of supraoptimal concentration of both T and B cell mitogens with significant reduction on β-Glucosidase and Nitroreductase activity with p< 0.05, while contrasting effect was noticed for β-Glucuronidase. Findings of the in vivo study proved higher dose of probiotic would not provide any additional benefit on host body. Conclusions: The test isolates possesed in vitro probiotic potency and the in vivo study with MBTU- S1B proved their applicability in nutraceutical and pharmaceutical industries. Significance and Impact of Study: To conflict disease outbreaks by improving general immunity via probiotics.

In vitro assessment of probiotic properties and gamma-aminobutyric acid production of selected lactic acid bacteria strains

This study aimed to perform an in vitro assessment of probiotic properties and to select lactic acid bacteria (LAB) strains that can produce the bioactive compound, gammaaminobutyric acid (GABA). A total of seven LAB strains of various species were examined for probiotic properties. These strains produced antimicrobial substances to inhibit the growth of target pathogenic bacteria. All selected LAB strains could withstand GI tract conditions. The strains showed bile salt hydrolase (BSH) activities. An antibiotic susceptibility test revealed that all examined strains were susceptible to ampicillin, chloramphenicol, erythromycin, gentamycin, penicillin and tetracycline, whereas they were resistant to kanamycin, norfloxacin, streptomycin and vancomycin. All strains of LAB in the current study gave high adhesion to Caco-2 cells ranging from 23.32-75.48%. Moreover, all tested LAB cells could inhibit Salmonella enterica serovar Enteritidis invasion of Caco-2 cells. Additionally, the tested LAB cells and cell-free supernatants (CFS) showed low cytotoxicity to inhibit the proliferation of Caco-2 cells. Moreover, three LAB strains, L. curvatus FJNP12, L. plantarum SKKL1 and W. cibaria SFM5, clearly showed GABA production based on the TLC results. Additionally, these strains also yielded high amounts of GABA according to an HPLC method, 15.66, 11.76 and 5.62 g/L, respectively. The open reading frame (ORF) of the gadB gene of L. plantarum SKKL1 revealed that it was 1,407 bp in length, encoding a polypeptide of 469 amino acids, with a molecular weight of 53.56 kDa and an isoelectric point (pI) of 5.58 predicted using the ExPASy program. These strains could be used in starter cultures for value-added agricultural products.

L-Theanine regulates lipid metabolism by modulating gut microbiota and bile acid metabolism.

l-Theanine (LTA) is a biologically active ingredient in tea that shows great potential for regulating lipid metabolism. Bile acids (BA), an important end-product of cholesterol catabolism, participate in the regulation of lipid metabolism and gut microbiota. Here, we investigated the effect of LTA on lipid metabolism and the mechanism by which it regulates BA metabolism and gut microbiota. Male BALB/c mice were treated with LTA for 28 days. Daily LTA doses of 100 and 300 mg kg-1 d-1 altered the gut microbiota in mice, predominantly by decreasing Lactobacillus, Streptococcus, Bacteroides, Clostridium and Enterorhabdus microbes associated with bile-salt hydrolase (BSH) activity, thereby decreasing the activity of BSH and increasing the levels of ileum conjugated BA (such as glycocholic acid (GCA) and lithocholic acid), thereby inhibiting the intestinal farnesoid X receptor (FXR)-fibroblast growth factor 15 (FGF15) signaling pathway. Inhibition of FXR-FGF15 signaling was accompanied by upregulation of cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein expression and increased hepatic production of cholic acid, deoxycholic acid, GCA, glycine cholic acid and glycine ursodeoxycholic acid. Meanwhile, increasing hepatic unconjugated BA upregulated the mRNA and protein expression of liver 3-hydroxy-3-methylglutaryl-CoA reductase and downregulated the mRNA and protein expression of stearoyl-CoA desaturase-1, liver low-density lipoprotein receptor and type B scavenger receptor. Therefore, the serum levels of cholesterol and triglycerides decreased. Our findings indicate that LTA regulates lipid metabolism by modulating the gut microbiota and BA metabolism via the FXR-FGF15-CYP7A1 pathway. © 2022 Society of Chemical Industry.

Characterization of potential probiotic bacteria Enterococcus faecium MC-5 isolated from the gut content of Cyprinus carpio specularis

The goal of this study was to determine the unique characteristics of Enterococcus faecium MC-5, a probiotic bacteria isolated from the intestine of a fish, Cyprinus carpio specularis, collected from Dal Lake in Srinagar, Kashmir, India. For this, the important valuable probiotic attributes, some functional properties, and safety assessments were analyzed in-vitro for the strain MC-5. The strain E. faecium MC-5 exhibited high resistance to low pH, high bile salt, lysozyme, and phenol. The strain MC-5 showed excellent auto- and co-aggregation properties and displayed remarkable hydrophobicity towards various tested hydrocarbons which suggested that the strain possesses venerable adhesion properties. Apart from these, the cell-free supernatant (CFS) of strain MC-5 exhibited phenomenal antimicrobial activity against the tested pathogens. A scanning electron microscope (SEM) image revealed strain MC-5 finely adhered to human colon adenocarcinoma cells (HCT-15 cells). The strain MC-5 showed high bile salt hydrolase activity and excellent cholesterol removal ability of 70.27%. The intact cells of strain MC-5 also showed strong DPPH scavenging activity. The EPS produced by E. faecium MC-5 inhibited the adhesion of Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica on HCT-15 cells with maximum inhibition rates of 41.82, 40.34, and 55.51%, respectively for displacement assay, which was higher as compared to exclusion (26.06, 26.11, and 39.23%) and competition assays (30.06, 26.7, and 41.20%). Strain MC-5 did not exhibit hemolysis and was also found susceptible to vancomycin and other clinically important antibiotics. When evaluating all the results from the present study, it is propounded that strain MC-5 has enviable probiotic characteristics and thus can be used as bio-protective cultures and/or bio-shield in food and pharmaceutical industries.

Bile Salt Hydrolases with Extended Substrate Specificity Confer a High Level of Resistance to Bile Toxicity on Atopobiaceae Bacteria.

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.

Astragaloside IV ameliorates diet-induced hepatic steatosis in obese mice by inhibiting intestinal FXR via intestinal flora remodeling

BackgroundNonalcoholic fatty liver disease (NAFLD) is a major clinical and public health burden worldwide with no established pharmacological therapy. Changes in the intestinal flora and associated metabolite bile acids (BAs) have been described in NAFLD. Astragaloside IV (AS-IV) is a low drug permeability saponin with protective effects against multiple diseases. However, the specific mechanism underlying the involvement of AS-IV in the regulation of NAFLD is yet to be clarified. PurposeThis study aimed to investigate the effect of AS-IV on NAFLD and explore whether intestinal flora was involved. MethodsThe effect of AS-IV was evaluated on high-fat diet-fed mice. Real-time PCR, immunohistochemistry, immunofluorescence, and biochemical analyses were performed. 16S rRNA gene sequencing and UPLC-TQMS were used to determine the alterations in the intestinal flora and concentration of BAs. Fecal microbiota transplantation (FMT) and intestine-specific farnesoid X receptor (FXR) knockout were also performed. ResultsAS-IV treatment alleviated diet-induced metabolic impairments, particularly hepatic steatosis. These changes occurred in the setting of decreased intestinal bile salt hydrolase (BSH)-expressing flora. Further analysis showed that the reduced BSH activity increased intestinal tauro-β-muricholic acid levels, an inhibitor of intestinal FXR. Inhibition of intestinal FXR signaling by AS-IV was accompanied by decreased expression of intestinal fibroblast growth factor 15 and subsequent hepatic FXR activation as well as increased glucagon-like peptide-1 and decreased ceramide production, all of which contribute to the inhibition of sterol regulatory element-binding protein-1c-mediated hepatic steatosis. Furthermore, intestine-specific Fxr knockout and FMT further demonstrated an FXR- and intestinal flora-dependent preventive effect of AS-IV on hepatic steatosis. ConclusionThese results show that the changes in intestinal flora and BAs serve an essential role in the remission of hepatic steatosis by AS-IV, thereby suggesting that AS-IV may be used as a prebiotic agent to provide viable treatment for NAFLD.