Bile Salt Hydrolase Activity Research Articles

Fermentation dynamics of bile salt hydrolase production in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012: Addressing ninhydrin assay limitations with a novel HPTLC–MS method

Bile salt hydrolase (BSH), a pivotal enzyme in cholesterol management, holds significant promise in both human and animal subjects. This study investigated the effect of fermentation dynamics in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012 to enhance BSH production. Cultivation of cultures in MRS and M17 media revealed that MRS medium enhanced BSH production by 235.98 % in H. coagulans ATCC 7050 and 147.37 % in L. plantarum ATCC 10012, compared to M 17 medium. Additionally, varying oxygen concentration levels indicated that H. coagulans ATCC 7050 exhibited its minimum doubling time of 79.8 ± 0.64 min in anaerobic conditions, whereas L.plantarum ATCC 10012 demonstrated its minimum doubling time of 85.5 ± 1.2 min under microaerophilic conditions. However, their highest BSH activity was observed during the stationary phase under anaerobic conditions, yielding 17.14 ± 0.78 U/mL by H. coagulans ATCC 7050 and 19.04 ± 0.81 U/mL by L.plantarum ATCC 10012. Furthermore, it was observed that both organisms did not retain BSH within their cells. BSH activity was assessed using ninhydrin assay that detected free taurine liberated from sodium taurocholate. However, ninhydrin can yield false–positive results owing to its interaction with other free amino acids. To subjugate this limitation, the study introduced a novel and sensitive HPTLC–MS method capable of accurately detecting taurine. By comprehending fermentation dynamics and selecting appropriate conditions, BSH production increased 2.1–fold in both organisms. These findings illuminate critical insights, offering a pathway for novel strategies to enhance the BSH–producing capabilities of these LAB strains.

Characterization of indigenous lactobacilli from dairy fermented foods of Haryana as potential probiotics utilizing multiple attribute decision-making approach

The interest in region-specific ethnic fermented foods and their functional microbiota is rising. The demands for functional foods are continuously rising, so research is going on to develop nutritious food with many beneficial attributes and low safety concerns. The present study was designed to isolate and characterize lactobacilli probiotic candidates from locally resourced fermented foods (dahi, lassi, and raabadi) to make ready-to-eat fermented functional products later. Cultures were isolated from 82 fermented food samples collected from different villages. The initial experiments of gram staining, catalase test, and carbohydrate fermentation were assessed for the morphology, purity, and primary characterization on the genus level, which was verified through molecular characterization using PCR. Seven lactobacilli strains (no. MS001-MS007) were then assessed for safety, probiotic candidacy, phytase degradation, and biofilm forming abilities. All seven bacterial cultures showed no hemolytic activity and antibiotic sensitivity against more than 14 antibiotics out of 20. All seven lactobacilli isolates were able to tolerate pH 3.0, 0.3% bile 0.5% pancreatin, lysozyme (100 mg/L to 300 mg/L) and also shown possessed phytase degradation ability. All the cultures showed antioxidative potential and biofilm formation ability. Culture MS007 showed considerably higher bile salt hydrolase activity among all the isolates, whereas MS005 possessed excellent phytate degradation ability among others. Bacterial strains were identified using 16S rRNA gene sequencing. Moreover, the order of preference of isolates was calculated using the multidimensional Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS) based on probiotic and other functional properties. The most promising attributes showing cultures were recognised as Limosilactobacillus fermentum MS005 and Lactiplantibacillus plantarum MS007, which could be further used for functional food product development.Graphical

Influence of gut microbiome composition on effect variations of anti-cholesterol treatment among individual mice

This study investigated if the variation in the effect of anti-cholesterol (AC) treatment on individual mice are related to gut microbiome composition. The bile salt hydrolase (BSH) activity of 23 commercial fermented milk products was examined to select a fermented milk product for AC treatment. Mice were fed to different diets for 6 weeks: high-fat (660 % of total calories from fat; D1), high-dietary fibre (20 % cellulose; D2), and low-fat (17.2 % of total calories from fat; D3) diets to change their gut microbiomes. Subsequently, faecal microbiome was transplanted (FMT) into mice treated with high cholesterol diet contained 2 % cholesterol, followed by AC or non-AC (sterile tap water, STW) treatments. Control groups with normal (NC) and high-cholesterol diets (PC) were prepared for both AC and STW treatment. All experimental groups were subjected to serum and liver cholesterol, cholesterol metabolism-related (CMR) gene expression, and intestinal microbiome analyses. D3-FMT mice showed the most significant enhancements in cholesterol ratio and decreased hepatic cholesterol levels with AC treatment. Moreover, upregulation of the Cyp7a1 gene expression was observed in this group. Furthermore, the intestinal microbiome analysis indicated higher abundances of BSH-producing Eubacterium, Bifidobacterium, and Parabacteroides in the D3-FMT + AC group compare to others, potentially contributing to increased bile acid synthesis.

Bile Salt Hydrolase Activity of Cholesterol-Lowering Probiotic Lactic Acid Bacteria Isolated from Indigenous Fermented Foods

Bile Salt Hydrolase Activity of Cholesterol-Lowering Probiotic Lactic Acid Bacteria Isolated from Indigenous Fermented Foods

Interplay between Bile Acids and Intestinal Microbiota: Regulatory Mechanisms and Therapeutic Potential for Infections.

Bile acids (BAs) play a crucial role in the human body's defense against infections caused by bacteria, fungi, and viruses. BAs counteract infections not only through interactions with intestinal bacteria exhibiting bile salt hydrolase (BSH) activity but they also directly combat infections. Building upon our research group's previous discoveries highlighting the role of BAs in combating infections, we have initiated an in-depth investigation into the interactions between BAs and intestinal microbiota. Leveraging the existing literature, we offer a comprehensive analysis of the relationships between BAs and 16 key microbiota. This investigation encompasses bacteria (e.g., Clostridioides difficile (C. difficile), Staphylococcus aureus (S. aureus), Escherichia coli, Enterococcus, Pseudomonas aeruginosa, Mycobacterium tuberculosis (M. tuberculosis), Bacteroides, Clostridium scindens (C. scindens), Streptococcus thermophilus, Clostridium butyricum (C. butyricum), and lactic acid bacteria), fungi (e.g., Candida albicans (C. albicans) and Saccharomyces boulardii), and viruses (e.g., coronavirus SARS-CoV-2, influenza virus, and norovirus). Our research found that Bacteroides, C. scindens, Streptococcus thermophilus, Saccharomyces boulardii, C. butyricum, and lactic acid bacteria can regulate the metabolism and function of BSHs and 7α-dehydroxylase. BSHs and 7α-dehydroxylase play crucial roles in the conversion of primary bile acid (PBA) to secondary bile acid (SBA). It is important to note that PBAs generally promote infections, while SBAs often exhibit distinct anti-infection roles. In the antimicrobial action of BAs, SBAs demonstrate antagonistic properties against a wide range of microbiota, with the exception of norovirus. Given the intricate interplay between BAs and intestinal microbiota, and their regulatory effects on infections, we assert that BAs hold significant potential as a novel approach for preventing and treating microbial infections.

Gut microbiota, dietary taurine, and fiber shift taurine homeostasis in adipose tissue of calorie-restricted mice to impact fat loss

Previously, we demonstrated that caloric restriction (CR) stimulates the synthesis, conjugation, secretion, and deconjugation of taurine and bile acids in the intestine, as well as their reuptake. Given taurine's potent antiobesogenic properties, this study aimed to assess whether the CR-induced shift in taurine homeostasis contributes to adipose tissue loss. To verify that, male C57Bl/6 mice were subjected to 20% CR or ad libitum feeding, with variations in cage bedding and gut microbiota conditions. Additional groups received taurine supplementation or were fed a low-taurine diet (LTD). The results showed that in CR animals, taurine derived from the intestine was preferentially trafficked to epididymal white adipose tissue (eWAT) over other tested organs. Besides increased levels of taurine transporter TauT, gene expression of Cysteine dioxygenase (Cdo) involved in taurine synthesis was upregulated in CR eWAT. Taurine concentration in adipocytes was inversely correlated with fat pad weight of CR mice. Different types of cage bedding did not impact eWAT taurine levels; however, the lack of bedding and consumption of a diet high in soluble fiber did. Depleting gut microbiota with antibiotics or inhibiting bile salt hydrolase (BSH) activity reduced WAT taurine concentration in CR mice. Taurine supplementation increased taurine levels in WAT and brown adipose tissue (BAT), promoting fat loss in CR animals. LTD consumption blunted WAT loss in CR animals, with negligible impact on BAT. This study provides multiple insights into taurine's role in CR-triggered fat loss and describes a novel communication path between the liver, gut, microbiota, and WAT, with taurine acting as a messenger.

Changes in the Bile Acid Pool and Timing of Female Puberty: Potential Novel Role of Hypothalamic TGR5.

The regulation of pubertal timing and reproductive axis maturation is influenced by a myriad of physiologic and environmental inputs yet remains incompletely understood. To contrast differences in bile acid isoform profiles across defined stages of reproductive maturity in humans and a rat model of puberty and to characterize the role of bile acid signaling via hypothalamic expression of bile acid receptor populations in the rodent model. Secondary analysis and pilot studies of clinical cohorts, rodent models, ex vivo analyses of rodent hypothalamic tissues. Bile acid concentrations is the main outcome measure. Lower circulatory conjugated:deconjugated bile acid concentrations and higher total secondary bile acids were observed in postmenarcheal vs pre-/early pubertal adolescents, with similar shifts observed in infantile (postnatal day [PN]14) vs early juvenile (PN21) rats alongside increased tgr5 receptor mRNA expression within the mediobasal hypothalamus of female rats. 16S rRNA gene sequencing of the rodent gut microbiome across postnatal life revealed changes in the gut microbial composition predicted to have bile salt hydrolase activity, which was observed in parallel with the increased deconjugated and increased concentrations of secondary bile acids. We show that TGR5-stimulated GnRH release from hypothalamic explants is mediated through kisspeptin receptors and that early overexpression of human-TGR5 within the arcuate nucleus accelerates pubertal onset in female rats. Bile acid isoform shifts along stages of reproductive maturation are conserved across rodents and humans, with preclinical models providing mechanistic insight for the neuroendocrine-hepatic-gut microbiome axis as a potential moderator of pubertal timing in females.

Safety evaluation of Bifidobacterium animalis subsp. lactis BLa80 under in vitro, and in vivo conditions

Bifidobacterium animalis subsp. lactis BLa80 is a new probiotic strain with extensive applications in food products both domestically and internationally. Given the rising consumption of this probiotic, its safety assessment is increasingly crucial in the food industry. This study evaluates the safety of strain BLa80 using a combination of in vitro and in vivo assays along with genomic analysis. Methods included exposing the strain to artificial gastric and intestinal fluids, as well as a medium containing bile salts, to stimulate human digestive conditions. The strain showed high tolerance to gastric fluid at pH of 2.5 and to 0.3 % bile salts. It maintained a 99.92 % survival rate in intestinal fluid. Additional tests assessed hemolytic activity, antibiotic susceptibility (revealing sensitivity to 7 antibiotics), and biogenic amine production using HPLC-ELSD, confirming the absence of histamine, and other harmful amines. Bile salt hydrolase activity was demonstrated qualitatively, and metabolic byproducts were quantitatively analyzed using a D-/l-lactic acid assay kit, showing that BLa80 produces 1.48 mg/mL of l-lactic acid and no harmful d-lactic acid. Genomic analysis confirmed the absence of virulence or pathogenicity genes, and a 90-day oral toxicity study in rats confirmed no toxic effects at various doses. Overall, these findings support the safety classification of the strain BLa80.

Unveiling the Impact of Lactic Acid Bacteria on Blood Lipid Regulation for Cardiovascular Health

Lactic acid bacteria (LAB) are a group of microorganisms which are beneficial and well-characterized with respect to the flavor and texture of food products via fermentation. The accumulated literature has suggested that dietary intake of fermented foods rich in LAB is related to different health-promoting benefits; however, in recent years, emerging evidence suggests a contribution of LAB to blood lipid regulation and cardiovascular health via certain mechanisms. Different potential mechanisms for the lipid regulatory effects of LAB may include the interaction of hydroxymethylglutaryl-CoA (HMG-CoA) reductase and bile salt hydrolase activity and bile salt metabolism; gut microbiome modulation; and regulation of mRNA expression of genes related to fat metabolism in animal models and human studies. This review comprehensively aims to answer whether/how LAB influence blood lipids in both animal models and human studies while also uncovering the underlying mechanisms linking LAB to lipid metabolism.

Ketogenic diet-induced bile acids protect against obesity through reduced calorie absorption.

The low-carbohydrate ketogenic diet (KD) has long been practiced for weight loss, but the underlying mechanisms remain elusive. Gut microbiota and metabolites have been suggested to mediate the metabolic changes caused by KD consumption, although the particular gut microbes or metabolites involved are unclear. Here, we show that KD consumption enhances serum levels of taurodeoxycholic acid (TDCA) and tauroursodeoxycholic acid (TUDCA) in mice to decrease body weight and fasting glucose levels. Mechanistically, KD feeding decreases the abundance of a bile salt hydrolase (BSH)-coding gut bacterium, Lactobacillus murinus ASF361. The reduction of L. murinus ASF361 or inhibition of BSH activity increases the circulating levels of TDCA and TUDCA, thereby reducing energy absorption by inhibiting intestinal carbonic anhydrase 1 expression, which leads to weight loss. TDCA and TUDCA treatments have been found to protect against obesity and its complications in multiple mouse models. Additionally, the associations among the abovementioned bile acids, microbial BSH and metabolic traits were consistently observed both in an observational study of healthy human participants (n = 416) and in a low-carbohydrate KD interventional study of participants who were either overweight or with obesity (n = 25). In summary, we uncover a unique host-gut microbiota metabolic interaction mechanism for KD consumption to decrease body weight and fasting glucose levels. Our findings support TDCA and TUDCA as two promising drug candidates for obesity and its complications in addition to a KD.

A gut microbiota-independent mechanism shapes the bile acid pool in mice with MASH

Background & AimsAn imbalance between primary and secondary bile acids contributes to the development of metabolic dysfunction-associated steatohepatitis (MASH). The precise mechanisms underlying changes in the bile acid pool in MASH remain to be identified. As gut bacteria convert primary bile acids to secondary bile acids, we investigated the contribution of the gut microbiota and its metabolizing activities to bile acid alterations in MASH. MethodsTo disentangle the influence of MASH from environmental and dietary factors, high fat diet fed foz/foz mice were compared to their high fat diet fed wildtype littermates. We developed functional assays (stable isotope labeling and in vitro experiments) to extend the analyses beyond a mere study of gut microbiota composition (16S rRNA gene sequencing). Key findings were confirmed in C57BL/6J mice were fed a western and high fructose diet, as an independent mouse model of MASH. ResultsWhile mice with MASH exhibited lower levels of secondary 7α-dehydroxylated bile acids (3.5-fold lower, p=0.0008), the gut microbial composition was similar in mice with and without MASH. Similar gut microbial bile salt hydrolase and 7α-dehydroxylating activities could not explain the low levels of secondary 7α-dehydroxylated bile acids. Furthermore, the 7α-dehydroxylating activity was unaffected by Clostridium scindens administration in mice with a non-standardized gut microbiota.By exploring alternative mechanisms, we identified an increased bile acid 7α-rehydroxylation mediated by liver CYP2A12 and CYP2A22 enzymes (4.0-fold higher, p<0.0001), that reduces secondary 7α-dehydroxylated bile acid levels in MASH. ConclusionsThis study reveals a gut microbiota-independent mechanism that alters the level of secondary bile acids and contributes to the development of MASH in mice. Impact and implicationsWhile changes in bile acid levels are implicated in the development of metabolic dysfunction-associated steatohepatitis (MASH), the precise mechanisms underpinning these alterations remain elusive. In this study, we investigated the mechanisms responsible for the changes in bile acid levels in mouse models of MASH. Our results support that neither the composition nor the metabolic activity of the gut microbiota can account for the alterations in the bile acid pool. Instead, we identified hepatic 7α-rehydroxylation of secondary bile acids as a gut microbiota-independent factor contributing to the reduced levels of secondary bile acids in mice with MASH. Further investigation is warranted to understand bile acid metabolism and its physiological implications in clinical MASH. Nonetheless, our findings hold promise for exploring novel therapeutic interventions for MASH.

Lactobacillus rhamnosus GG ameliorates triptolide-induced liver injury through modulation of the bile acid-FXR axis

Triptolide (TP) is the principal bioactive compound of Tripterygium wilfordii with significant anti-tumor, anti-inflammatory and immunosuppressive activities. However, its severe hepatotoxicity greatly limits its clinical use. The underlying mechanism of TP-induced liver damage is still poorly understood. Here, we estimate the role of the gut microbiota in TP hepatotoxicity and investigate the bile acid metabolism mechanisms involved. The results of the antibiotic cocktail (ABX) and fecal microbiota transplantation (FMT) experiment demonstrate the involvement of intestinal flora in TP hepatotoxicity. Moreover, TP treatment significantly perturbed gut microbial composition and reduced the relative abundances of Lactobacillus rhamnosus GG (LGG). Supplementation with LGG reversed TP-induced hepatotoxicity by increasing bile salt hydrolase (BSH) activity and reducing the increased conjugated bile acids (BA). LGG supplementation upregulates hepatic FXR expression and inhibits NLRP3 inflammasome activation in TP-treated mice. In summary, this study found that gut microbiota is involved in TP hepatotoxicity. LGG supplementation protects mice against TP-induced liver damage. The underlying mechanism was associated with the gut microbiota-BA-FXR axis. Therefore, LGG holds the potential to prevent and treat TP hepatotoxicity in the clinic.

Monitoring probiotic properties and safety evaluation of antilisterial Enterococcus faecium strains with cholesterol-lowering potential from raw Cow's milk

Enterococci are an important member of lactic acid bacteria due to their presence in fermented foods, tolerance to low pH and bile salts, bile salt hydrolase (BSH) activity, reducing cholesterol, and producing bacteriocin. However, probiotic and safety issues of enterococci are still debated. It is important to investigate probiotic properties and safety evaluation of these genus because they are intentionally or unintentionally present in foods. In this study, probiotic properties (tolerance to bile salts and simulated gastric juice, antimicrobial and BSH activity, cholesterol lowering potential, auto/co-aggregation, and cell surface hydrophobicity) and safety evaluation (antibiotic resistance, virulence factor, haemolysis and DNase activity, Galleria mellonella infection model, gelatinase activity, and biofilm formation) of Enterococcus faecium (E. faecium) BH04, BH12, BH84, and BH99 isolated raw cow's milk were determined. All strains had high tolerance to bile salts (47.70%–103.84%) and simulated gastric juice (76.80%–82.05%), and antimicrobial activity on L. monocytogenes ATCC 7644 by protein-like substances such as bacteriocin. The strains had strong BSH property and reduced cholesterol by 69.47%–95.00%. The strains were resistant only to gentamicin, kanamycin, and streptomycin and did not have haemolysis and DNase activity. Only gelE and esp genes were detected in some strains. According to the results, it is thought that the E. faecium strains could be possible probiotic candidates. Moreover, this study is the first report on Enterococcus faecium strains isolated from raw cow's milk, which tolerate to bile salt and simulated gastric juice, have BSH potential and antimicrobial activity by protein-like substances, and reduce cholesterol level.

Recombinant Bile Salt Hydrolase Enhances the Inhibition Efficiency of Taurodeoxycholic Acid against Clostridium perfringens Virulence.

Clostridium perfringens is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen C. perfringens. Assays were conducted to evaluate the inhibition of C. perfringens growth, hydrogen sulfide (H2S) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the bsh gene for cloning. The bsh gene from Bifidobacterium longum was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein in E. coli BL21 and Bacillus subtilis 168 (B-sub-BSH), respectively. His-tag-purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and a Western blot (WB) assays. Secretory BSH from B. subtilis was analyzed by a Dot-Blot. B-sub-BSH was evaluated for the inhibition of C. perfringens growth. C. perfringens growth reached 7.8 log10 CFU/mL after 24 h culture. C. perfringens growth was at 8 vs. 7.4, 7.8 vs. 2.6 and 6 vs. 0 log10 CFU/mL in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced C. perfringens H2S production and the virulence gene expression of asrA1, netB, colA, and virT. BSH activity was observed in Lactobacillus johnsonii and B. longum under anaerobe but not L. johnsonii under 10% CO2 air. After the sequence alignment of bsh from ten bacteria, bsh from B. longum was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After the subcloned bsh and amylase signal peptide sequence was inserted into pDR-BSH, B. subtilis was transformed and named B-sub-BSH. The transformation was evaluated using PCR with B. subtilis around 3 kb and B-sub-BSH around 5 kb. Secretory BSH expressed from B-sub-BSH was determined for His-tag using Dot-Blot. Importantly, C. perfringens growth was reduced greater than 59% log10 CFU/mL in the B-sub-BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, TDCA was less potent than DCA against C. perfringens virulence, and recombinant secretory BSH from B-sub-BSH reduced C. perfringens growth, suggesting a new potential intervention against the pathogen-induced chicken NE.

Antibiotic-Induced Gut Microbial Dysbiosis Reduces the Growth of Weaning Rats via FXR-Mediated Hepatic IGF-2 Inhibition.

The gut microbiota plays a crucial role in postnatal growth, particularly in modulating the development of animals during their growth phase. In this study, we investigated the effects of antibiotic-induced dysbiosis of the gut microbiota on the growth of weaning rats by administering a non-absorbable antibiotic cocktail (ABX) in water for 4 weeks. ABX treatment significantly reduced body weight and feed intake in rats. Concurrently, ABX treatment decreased microbial abundance and diversity in rat ceca, predominantly suppressing microbes associated with bile salt hydrolase (BSH) activity. Furthermore, decreased appetite may be attributed to elevated levels of glucagon-like peptide-1 (GLP-1) in the serum, along with reduced neuropeptide Y (NPY) and increased cocaine and amphetamine-regulated transcript (CART) in the hypothalamus at the mRNA level. Importantly, concentrations of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2) were decreased in the serum and liver of antibiotic-treated rats. These alterations were associated with significant down-regulation of IGF-2 mRNA in the liver and significantly decreased farnesoid X receptor (FXR) protein expression and binding to the IGF-2 promoter. These results indicate that antibiotic-induced gut microbial dysbiosis not only impacts bile acid metabolism but also diminishes rat growth through the FXR-mediated IGF-2 pathway.

Molecular Insights into Bile Salt Hydrolase-Positive Lactobacilli: A PCR-Based Identification Approach

Bile salt hydrolase (BSH)-positive Lactobacilli play a crucial role in gut health and have been associated with various physiological benefits. This study presents a comprehensive molecular investigation into the identification of BSH-positive Lactobacilli using a polymerase chain reaction (PCR)-based approach. The methodology involves the targeted amplification of specific genetic markers associated with BSH activity, enabling the precise discrimination of these beneficial bacteria within complex microbial communities. The study encompasses the design and validation of PCR primers specific to BSH-encoding genes, ensuring the accurate detection of BSH-positive Lactobacilli strains. Utilizing this molecular tool, we conducted extensive screenings across diverse environmental samples, revealing the prevalence and diversity of BSH positive strains in various ecosystems.

188 A combination of green tea and butyric acid as a potential alternative for zinc oxide improves gut microbiota composition and growth performance in weaning pigs

Abstract Antibiotic growth promotor (AGP) and zinc oxide (ZnO) has been shown to inhibit bile salt hydrolase (BSH) activity, resulting in improved animal growth performance and reduced post-weaning diarrhea but, their usage should be minimized at pig farms. The objective of this study was to evaluate the effects of green tea and butyric acid (hereafter called Gutluk) as alternative BSH inhibitors on the growth performance, nutrient digestibility, fecal score, blood profile, and gut microbiota of weaning pigs. A total of 192 weaned pigs [(Landrace × Yorkshire) × Duroc)] were divided into six groups. Dietary treatments were NC, Basal diet; PC1, Basal diet + 0.2% antibiotics (colistin); PC2, Basal diet + 2,000ppm ZnO; TRT1, Basal diet + 0.05% Gutluk; TRT2, Basal diet + 0.10% Gutluk; TRT3, Basal diet + 0.20% Gutluk. After 4 wk of the trial, fresh fecal samples were collected from the rectum for microbiome analysis. TRT3 improved average daily gain (ADG) and gain to feed ratio (G:F) at wk 3 and 4. The digestibility, white blood cell, red blood cell, lymphocyte percentage, blood urea nitrogen, fecal score and diarrhea score of weaned pigs were not affected by the addition of Gutluk. The treatment groups showed decreased TNF-α and IL-6 (P &amp;lt; 0.05) while immunoglobulin (IgA, IgG) increased (P &amp;lt; 0.05). GutLuk significantly enriched Bifidobacterium (P &amp;lt; 0.01). Conversely, GutLuk reduced the abundance of Clostridium sensu stricto 6, Marvinbryantia, and Clostridia vadinBB60 group, and marginally reducing Muribaculaceae, Enterococcus, and Staphylococcus. The correlation analysis also revealed that the abundance of secondary bile acid biosynthesis had a strong positive correlation with Marvinbryantia, Enterococcus, Clostridium sensu stricto 6, Lactobacillus, and Clostridia vadinBB60 (P &amp;lt; 0.001). The ADG of the pigs also exhibited a slight negative correlation with secondary bile acid biosynthesis. These results indicated that the supplementation of GutLuk had an inhibitory effect on the BSH activity of certain gut microbiota. Consequently, A combination of green tea and butyric acid can be effective alternatives of AGP and ZnO in early weaning pig diet to improve growth performance, immune response, oxidative stress, diarrhea score and gut microbiota.

Tong-Xie-Yao-Fang strengthens intestinal feedback control of bile acid synthesis to ameliorate irritable bowel syndrome by enhancing bile salt hydrolase-expressing microbiota

Ethnopharmacological relevanceA herbal formula Tong-Xie-Yao-Fang (TXYF) is traditionally used to treat irritable bowel syndrome (IBS), modern pharmacological evidence supports that the formula efficacy is associated with altered gut microbiota. Yet, the mechanistic role of gut microbiota in the therapy of TXYF remains unclear. We previously clarified that gut microbiota-dysregulated bile acid (BA) metabolism contribute to the pathogenesis of IBS, deriving a hypothesis that microbiota-BA metabolic axis might be a potential target of TXYF. Aim of the studyWe aim to investigate a new gut microbiota-mediated mechanism underlying anti-IBS efficacy of TXYF. Materials and methodsWe established an IBS rat model with a combination of stressors, compared the herbal efficacy in models undergone gut bacterial manipulations, also examined BA metabolism-related microbiota, metabolites, genes and proteins by 16S rRNA gene sequencing, targeted metabolomics, qPCR and multiplex immunofluorescence staining. ResultsWe observed that TXYF attenuated visceral hyperalgesia and diarrhea in IBS rats but not in those underwent gut bacteria depletion. Transferring gut microbiota from TXYF-treated donors also decreased visceral sensitivity and slightly relief diarrhea-like behaviors in IBS recipient rats. Fecal 16S rRNA gene sequencing revealed that TXYF modulated microbial β-diversity and taxonomic structure of IBS rats, with a significant increase in relative abundance of bile salt hydrolase (BSH)-expressing Bacteroidaceae. qPCR and culturing data validated that TXYF had a promotive effect on the growth and BSH activity of Bacteroides species. TXYF-reshaped microbiota upregulated the expression of intestinal Fgf15, a feedback signal to control BA synthesis in the liver. As a result, the BA synthetic and excretory levels in IBS rats were decreased by TXYF, so as that colonic BA membrane receptor Tgr5 sensing and its mediated Calcitonin gene-related peptide (Cgrp)-positive neuronal response were attenuated. ConclusionThis study poses a new microbiota-driven therapeutic action for TXYF, highlighting the potential of developing new anti-IBS strategies from the herbal formula targeting BSH-expressing gut bacteria.

Bile Salt Hydrolase Activity-Based Probes for Monitoring Gut Microbial Bile Acid Metabolism.

Bile acids are bioactive metabolites that are biotransformed into secondary bile acids by the gut microbiota, a vast consortium of microbes that inhabit the intestines. The first step in intestinal secondary bile acid metabolism is carried out by a critical enzyme, bile salt hydrolase (BSH), that catalyzes the gateway reaction that precedes all subsequent microbial metabolism of these important metabolites. As gut microbial metabolic activity is difficult to probe due to the complex nature of the gut microbiome, approaches are needed to profile gut microbiota-associated enzymes such as BSH. Here, we develop a panel of BSH activity-based probes (ABPs) to determine how changes in diurnal rhythmicity of gut microbiota-associated metabolism affects BSH activity and substrate preference. This panel of covalent probes enables determination of BSH activity and substrate specificity from multiple gut anerobic bacteria derived from the human and mouse gut microbiome. We found that both gut microbiota-associated BSH activity and substrate preference is rhythmic, likely due to feeding patterns of the mice. These results indicate that this ABP-based approach can be used to profile changes in BSH activity in physiological and disease states that are regulated by circadian rhythms.

Investigation of the antidiabetic and probiotic properties of lactic acid bacteria isolated from some ethnic fermented foods of Darjeeling District

BackgroundIndigenous communities residing in the Darjeeling Himalayan region and its adjacent hilly areas have a deeply rooted cultural tradition of consuming a diverse range of vegetable and milk-based fermented products, believed to confer various health advantages. With this traditional knowledge, lactic acid bacteria (LAB) were isolated from popular fermented foods such as Chhurpi (derived from Bos grunniens milk), Gundruk (made from Brassica juncea leaves), Sinki (derived from Raphanus sativus taproots), and Kinema (produced from Glycine max beans). This study aimed to investigate the probiotic properties of the prevalent LABs, including aggregation properties, bile salt hydrolase activities, survival under gastro-inhibitory conditions, safety evaluations, and their potential health-promoting attributes, with a specific focus on inhibiting α-amylase and α-glucosidase enzymes.ResultsFive of the LAB isolates demonstrated notable viability rates exceeding 85% when exposed to gastro-inhibitory challenges. Based on 16S rRNA gene sequencing, these isolates were identified as Pediococcus pentosaceus (isolate GAD), Lactobacillus plantarum (isolates KAD and CAD), Lactobacillus brevis (isolate SAD), and Lactiplantibacillus plantarum (isolate CMD). These LAB isolates exhibited versatile carbon source utilization, significant auto- and co-aggregation, and bile salt hydrolase (BSH) properties. Auto-aggregation capacity notably increased over time, ranging from 30 to 150 min, with percentage increments from 4.83 ± 1.92% to 67.60 ± 5.93%. L. brevis SAD displayed the highest co-aggregation increment (%) against Staphylococcus aureus, while L. plantarum KAD demonstrated potent antimicrobial activity. In vitro analyses postulated potential health benefits related to antidiabetic properties, particularly inhibiting α-amylase and α-glucosidase enzymes. L. brevis SAD exhibited the highest α-glucosidase inhibitory activity, while L. plantarum KAD displayed the most potent α-amylase inhibitory activity. Comprehensive safety assessments, including antibiotic susceptibility profiling, hemolytic activity evaluation, and in vivo acute toxicity studies, confirmed the suitability of these LAB isolates for human consumption.ConclusionsThe isolates show promising probiotic characteristics and significant potential in addressing metabolic health. These results carry substantial scientific implications, suggesting the pharmaceutical-based applications of these traditional fermented foods. Further in vivo investigation is recommended to fully elucidate and exploit the health benefits of these LAB isolates, opening avenues for potential therapeutic interventions and the development of functional foods.