High Bile Salt Concentrations Research Articles

Characterisation of the lipolytic enzymatic activities of fungal rizoenzymes from Rhizopus oryzae in comparison to pancreatin from pigs

Abstract Aim In case of exocrine pancreatic insufficiency (EPI), the replacement of digestive enzymes with, for example porcine pancreatin or fungal rizoenzymes, is unavoidable under certain conditions. Current guidelines indicate that preparations from porcine pancreas have more advantageous physicochemical properties compared to those from fungi, especially at high bile salt concentrations, and that the latter can, therefore, only be used clinically to a limited extent. Since rizoenzymes are increasingly used in clinical practice, the present in vitro study investigated efficiency of enzymatic activity of rizoenzymes in comparison to pancreatin under various physiological and partly extreme environmental conditions. Materials and Methods The lipolytic properties of two typical preparations containing digestive enzymes from porcine pancreatin and rizoenzymes were compared (same dosage of activity units) at different pH values (pH 3–9), with the addition of different bile salt concentrations (0–15 mmol/L sodium taurocholate). Lipolytic activity was measured by quantifying the fatty acids released from olive oil substrate by liquid chromatography–tandem mass spectrometry after enzymatic digestion. Results For both enzyme preparations, the lipolytic activity maximum was reached at pH 7 with comparable fatty acid conversion rates (57% pancreatin, 58% rizoenzymes). However, in contrast to pancreatin, rizoenzymes were already active for certain fatty acids from pH 3 to 4. At a bile salt concentration up to 10 mmol/L taurocholate, there was an increase in activity of both enzyme preparations (rizoenzymes 69% vs. pancreatin 58% enzymatic conversion). Only at rather unphysiological concentration of 15 mmol/L during EPI, there was a slight decrease in activity (to 56%) for the rizoenzymes. Conclusions Rizoenzymes are an alternative therapeutic option for EPI. Relevant differences between rizoenzymes and pancreatin with regard to lipolytic activity under different physicochemical conditions could not be demonstrated in this study, whereas a potential advantage in favor of rizoenzymes activity at already low pH values was observed.

PROBIOTIC LACTIC ACID BACTERIA FROM ADUWA KO JAANR, A LESSER-KNOWN FERMENTED GINGER BEVERAGE FROM EASTERN HIMALAYAN REGION

The Eastern Himalayan natives enjoy drinking Aduwa ko jaanr, a traditionally fermented mild-alcoholic ginger brew. Studies on manufacturing process and microbiological dynamics are infrequent for Aduwa ko jaanr. Current study analyzed the probiotic properties of Lactic Acid Bacteria (LAB) associated with Aduwa ko jaanr, using in-vitro method, simulating the conditions of intestinal tract. Three LAB isolates were screened and selected for preliminary characterization (morphological and biochemical). Isolates were identified as Lactiplantibacillus argentoratensis AKJ(W), Lactiplantibacillus argentoratensis AKJ(Y), and Levilactobacillus brevis AKJ(P) by 16S rRNA sequencing. Studies on growth kinetics of stationary phase and formation times of secondary metabolites with probiotic effects were conducted. Probiotic tests, namely acid-bile tolerance, phenol tolerance, cell surface hydrophobicity, auto-aggregation property, adherence to mammalian cell line, antibiotic susceptibility, antimicrobial activity and radical scavenging activity were performed. Results indicated the isolates to have health-benefiting properties with antibiotic resistance and inhibiting human pathogens. Isolates are predicted to survive in gut environment as they tolerated high bile salt concentration (0.5%), low pH (pH2) and high phenol concentration (0.6%). Results of cell surface hydrophobicity, auto-aggregation, and mammalian cell line (HEK 293) adherence tests suggested that isolates could adhere and possibly colonize intestine's inner wall. High radical scavenging activity of isolates suggested overall antioxidant property of the fermented beverage. MAR index showed isolates’ resistance to different antibiotics, indicating that their action is unaffected even when administered during illness. The present study suggests that LAB in Aduwa ko jaanr has potential probiotic properties which makes it an appropriate traditional beverage conferring health benefits.

Effect of bile salts on intestinal epithelial function in gilthead seabream (Sparus aurata)

In the context of modern aquaculture, the effort to reduce the reliance on fishmeal/marine ingredients in fish diets has led to the exploration of plant-based protein sources as potential substitutes, a dietary shift that disrupts the bile acid profile in fish. Therefore, bile salts are being sought as additives. However, artificially increased intestinal levels of bile acids may significantly impact mucosal function. Therefore, here, we explored the regulatory role in the intestine of gilthead sea bream (Sparus aurata) of (i) chenodeoxycholic acid (CDC), (ii) a mixture formed by two bile acids, 3% cholic acid and 97% deoxycholic acid (MIX), and (iii) a conjugated bile salt sodium taurocholate (TC) in Ussing chambers with the epithelial voltage clamp technique. We tested the bile salts in a 50–500 μg/ml concentration range, and all of them promoted ion absorption. Yet, clear concentration-dependent and more pronounced effects on the ion transport were observed in the posterior intestine. On the other hand, bile salts had no or minor effects on tissue resistance. However, there are indications that the MIX could have adverse effects at high concentrations (500 μg/ml), promoting a threefold increase in tissue permeability measured using FITC-dextran (4 kD) regardless of the intestinal region, thus suggesting an alteration in intestinal permeability at high bile salt concentrations. The findings from our study emphasize the importance of considering intestinal function when contemplating the possible use of a particular bile salt as a dietary supplement. It appears that bile salts, whether acting individually or in combination, play a pivotal role in orchestrating nutrient absorption by influencing the function of epithelial ion transport. However further research is needed to fully grasp the region-dependent nuances of bile salt effects on ion transport and the ultimate consequences for nutrient absorption in the context of fish aquaculture.

Isolation and Identification of Chicken-Derived Lactic Acid Bacteria: In Vitro Probiotic Properties and Antagonistic Effects against Salmonella pullorum, Staphylococcus aureus, and Escherichia coli.

The development of probiotics capable of quickly colonizing the intestines of animals is important in promoting the healthy growth of livestock. The aim of this study was to screen lactic acid bacteria (LAB) from the intestinal microbiota of chickens with potential applications, and to evaluate their probiotic properties and antagonistic abilities against Salmonella pullorum, Staphylococcus aureus, and Escherichia coli. The results showed that a total of 79 strains with the characteristics of LAB were isolated from the chicken cecum microbiota, of which 7 strains exhibited strong inhibitory activity against S. pullorum, S. aureus, and E. coli. Performing 16s rDNA sequencing revealed that these seven strains were Lactiplantibacillus pentosus (n = 1), Lactiplantibacillus plantarum (n = 3), Lactiplantibacillus paraplantarum (n = 1), Lactiplantibacillus argentoratensis (n = 1), and Lactiplantibacillus fabifermentans (n = 1). Among them, L. pentosus R26 and L. plantarum R32 exhibited superior antibacterial activity. These two strains demonstrated high lactic acid production ability, with survival rates of 86.29% and 87.99% after 3 h of treatment at pH 1.5, 86.66% and 85.52% after 3 h of treatment with 0.5% bile salts, 90.03% and 88.16% after 2 h of treatment with simulated gastric fluid, and 98.92% and 98.22% after 2 h of treatment with simulated intestinal fluid, respectively. Co-cultivation with L. pentosus R26 for 24 h resulted in 50% of the pathogens being antagonized, while almost complete inhibition was observed following 72 h of co-cultivation. In conclusion, L. pentosus R26 and L. plantarum R32 exhibited high antibacterial activity and acid production capability, while also demonstrating satisfactory tolerance to low pH values and high concentrations of bile salts and digestive fluid. The probiotic characteristics and stress resistance of L. pentosus R26 were slightly superior to those of L. plantarum R32, indicating its potential for development as a probiotic.

Bacillus halotolerans SW207 alleviates enterotoxigenic Escherichia coli-induced inflammatory responses in weaned piglets by modulating the intestinal epithelial barrier, the TLR4/MyD88/NF-κB pathway, and intestinal microbiota.

Enterotoxigenic Escherichia coli (ETEC) is one of the major pathogens contributing to piglet diarrhea, with significant implications for both piglet health and the economic aspects of the livestock industry. SW207 is an isolate of Bacillus halotolerans isolated from the cold- and disease-resistant Leixiang pigs in Northeastern China. We have discovered that SW207 can survive in the pig's gastrointestinal fluid and under conditions of high bile salt concentration, displaying potent antagonistic activity against ETEC. In this study, we established a weaned piglet diarrhea model infected with ETEC to investigate the role of SW207 in preventing diarrhea and improving intestinal health. Results indicate that SW207 upregulates the expression of tight junction proteins, including claudin-1, occludin, and zonula occludens-1, at both the transcriptional and translational levels. Furthermore, SW207 reduces serum endotoxin, D-lactic acid, and various oxidative stress markers while enhancing piglet mechanical barrier function. In terms of immune barrier, SW207 suppressed the activation of the TLR4/MyD88/NF-κB pathway, reducing the expression of various inflammatory factors and upregulating the expression of small intestine mucosal sIgA. Concerning the biological barrier, SW207 significantly reduces the content of E. coli in the intestines and promotes the abundance of beneficial bacteria, thereby mitigating the microbiota imbalance caused by ETEC. In summary, SW207 has the potential to prevent weaned piglet diarrhea caused by ETEC, alleviate intestinal inflammation and epithelial damage, and facilitate potential beneficial changes in the intestinal microbiota. This contributes to elucidating the potential mechanisms of host-microbe interactions in preventing pathogen infections.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC) has consistently been one of the significant pathogens causing mortality in weaned piglets in pig farming. The industry has traditionally relied on antibiotic administration to control ETEC-induced diarrhea. However, the overuse of antibiotics has led to the emergence of drug-resistant zoonotic bacterial pathogens, posing a threat to public health. Therefore, there is an urgent need to identify alternatives to control pathogens and reduce antibiotic usage. In this study, we assessed the protective effect of a novel probiotic in a weaned piglet model infected with ETEC and analyzed its mechanisms both in vivo and in vitro. The study results provide theoretical support and reference for implementing interventions in the gut microbiota to alleviate early weaned piglet diarrhea and improve intestinal health.

Tofu Whey-Based Media for Probiotic Lactiplantibacillus plantarum D4 as a Halal Starter Culture

In recent years, the development of probiotic-based fermented products with halal status has been a concern. The use of growth medium has relied on de Man Rogosa and Sharpe (MRS) as a relatively expensive standard medium, and its halal status is still uncertain. Extensive research has been carried out to investigate the development of low-cost halal alternative media for the cultivation of probiotic lactic acid bacteria (LAB). This study aimed to develop a probiotic halal and low-cost culture medium using a tofu whey-based medium. This study used three tofu whey-based media – A (tofu whey 100%), B (tofu whey 94.5%, molasses 3%, skim milk 2.5%), C (tofu whey 92.5%, molasses 3%, cheese whey 2.5%, tomato extract 2%), and MRS broth as a standard medium. Bacterial populations, total sugars utilized, total lactic acids produced, low pH (2.0) tolerance, and high bile salt concentration (oxgall 1.5%) were assayed. The highest bacterial population after 48 h of incubation was shown by medium B compared to medium MRS (12.34 ± 0.87 and 11.48 ± 0.3 log CFU/mL). Total sugars utilized by 48.28 ± 2.89, 38.89 ± 6.94, 39.14 ± 4.24, and 76.00 ± 1.41 %; on the other hand, total lactic acids produced by 0.16 ± 0.12, 0.03 ± 0.04, 0.31 ± 0.03, and 2.25 ± 1.48 % in A, B, C, and MRS, respectively. Probiotic tolerance at low pH and the presence of bile salts of Lactiplantibacillus plantarum D4 consistently showed a high survival rate in medium B compared to MRS. Based on these results, the components and proportions used in medium B were suitable for the growth of L. plantarum D4 as a halal probiotic starter candidate.

Selection and evaluation of lactic acid bacteria from chicken feces in Thailand as potential probiotics.

Lactic acid bacteria (LAB) are widely used as probiotics in poultry production due to their resilience to low pH and high bile salt concentrations, as well as their beneficial effects on growth performance and antagonistic activity against enteric pathogens. However, the efficacy of probiotics depends on strain selection and their ability to colonize the host's intestine. This study aimed to select, identify, and evaluate LAB strains isolated from chicken feces in Thailand for potential use as probiotics in the chicken industry. LAB strains were isolated from 58 pooled fresh fecal samples collected from chicken farms in various regions of Thailand, including commercial and backyard farms. Gram-positive rods or cocci with catalase-negative characteristics from colonies showing a clear zone on MRS agar supplemented with 0.5% CaCO3 were identified using MALDI-TOF mass spectrometry. The LAB isolates were evaluated for acid (pH 2.5 and pH 4.5) and bile salt (0.3% and 0.7%) tolerance. Additionally, their cell surface properties, resistance to phenol, antimicrobial activity, hemolytic activity, and presence of antimicrobial resistance genes were determined. A total of 91 LAB isolates belonging to the Pediococcus, Ligilactobacillus, Limosilactobacillus, and Lactobacillus genera were obtained from chicken feces samples. Backyard farm feces exhibited a greater LAB diversity compared to commercial chickens. Five strains, including Ligilactobacillus salivarius BF12 and Pediococcus acidilactici BF9, BF14, BYF20, and BYF26, were selected based on their high tolerance to acid, bile salts, and phenol. L. salivarius BF12 and P. acidilactici BF14 demonstrated strong adhesion ability. The five LAB isolates exhibited significant cell-cell interactions (auto-aggregation) and co-aggregation with Salmonella. All five LAB isolates showed varying degrees of antimicrobial activity against Salmonella strains, with P. acidilactici BYF20 displaying the highest activity. None of the LAB isolates exhibited beta-hemolytic activity. Whole genome analysis showed that L. salivarius BF12 contained ermC, tetL, and tetM, whereas P. acidilactici strains BF9 and BF14 carried ermB, lnuA, and tetM. The selected LAB isolates exhibited basic probiotic characteristics, although some limitations were observed in terms of adhesion ability and the presence of antibiotic resistance genes, requiring further investigation into their genetic location. Future studies will focus on developing a probiotic prototype encapsulation for application in the chicken industry, followed by in vivo evaluations of probiotic efficacy.

Evaluating Enterococcus faecium9 N-2 as a probiotic candidate from traditional village white cheese.

In this study, various functional and probiotic attributes of the Enterococcus faecium 9 N-2 strain isolated from village-style white cheese were characterized, while also assessing its safety. To achieve this, we conducted an invitro analysis of several key probiotic properties exhibited by the 9 N-2 strain. Notably, this strain demonstrated robust resilience to low pH, high bile salt concentrations, lysozyme, pepsin, pancreatin, and phenol. Furthermore, this strain displayed exceptional auto-aggregation capabilities and moderate co-aggregation tendencies when interacting with Escherichia coli. The cell-free supernatant derived from strain 9 N-2 exhibited significant antimicrobial activity against the tested pathogens. The strain exhibited resistance to gentamicin, meropenem, and bacitracin, while remaining susceptible to vancomycin and various other antibiotics. Furthermore, it was found that E. faecium 9 N-2 possessed the capacity to produce the phytase enzyme. When all the results of this study are evaluated, it is thought that 9 N-2 strain has superior probiotic properties, and therefore it can be used as probiotic in food, medicine, and animal feed in the future. In addition, further invivo tests should be performed to fully understand its effects and mechanisms of action and to confirm its safety and probiotic effects. Further research and clinical trials are also needed to identify new strains with potential probiotic properties.

Survivability of probiotics in Pickering emulsion gels stabilized by salmon by-product protein / sodium alginate soluble complexes at neutral pH

Adequate amounts of live probiotics reaching the gut are necessary to maintain host health. However, the harsh environment during processing, the low pH of human gastric acid, and the high concentration of bile salts in the gut can significantly reduce survivability of probiotics. In this work, we propose a simple Pickering emulsion gels strategy to encapsulate Lactobacillus plantarum Lp90 into oil droplets filled in calcium alginate gels to improve its viability under pasteurization and gastrointestinal conditions. The emulsion gels were stabilized by the soluble complexes of salmon by-product protein (SP) and sodium alginate (ALG), and the aqueous phase was solidified by the addition of calcium. The interaction between SP and ALG and the effect of ALG concentration on emulsifying ability and emulsion stability were studied. The results from optical imaging, nuclear magnetic resonance, and rheological properties showed that the stability and viscosity of the emulsions gradually increased with the increased ALG concentration, while the droplet size of the emulsions and the content of free water in the system decreased significantly. Especially when the concentration of ALG was 1 %, the emulsion system was stable under the environment of high temperature and high ionic strength, and the water holding capacity was the highest. Through pasteurization and gastrointestinal digestion experiments, it was found that the survival rate of probiotics encapsulated in emulsion gels was significantly higher than that encapsulated in emulsions or hydrogels, which benefited from the dual action of oil droplets and calcium alginate gels network. These results provide a new strategy for the processing of probiotics and the high-value utilization of marine fish by-products.

Effect of Digestion on Ursolic Acid Self-Stabilized Water-in-Oil Emulsion: Role of Bile Salts.

Exploring the effect of bile salts on the properties of emulsion carriers containing hydrophobic bioactive compounds is particularly critical to understanding the stability and bioavailability of these hydrophobic bioactive compounds in the digestive process. In this study, the effects of bile salts on the stability and digestive characteristics of the ursolic acid (UA) self-stabilized water-in-oil (W/O) emulsion were investigated via static and dynamic (with or without enzyme) in vitro simulated digestive systems. The results showed that under the static system, the basic conditions had less interference, while the bile salts had a significant effect on the appearance and microstructure of the emulsion. The primary mechanism of emulsion instability is hydrophobic binding and depletion flocculation. Under the dynamic condition, it was found that the low concentrations of bile salts can promote the release amount and the rate of free fatty acids via displacement, while high concentrations of bile salts inhibit the decomposition of lipid, which may be related to the secondary coverage formed at the interface by the bile salts. These findings provide a theoretical basis for understanding the digestive behavior of the UA emulsion and its interaction with bile salts, which are conducive to developing and designing new emulsions to improve the bioaccessibility of UA.

Isolation and characterization of probiotic bacteria from fruit pulp, screening of probiotic properties and production of bacteriocin and probiotic curd

The present study was investigated to isolate probiotic lactic acid bacteria from Actinidia deliciosa (kiwi fruit) pulp. A total of eight isolates were found and two of them were probiotic LAB strains. The both A2 and A5 isolates strains were Gram-positive, catalase and oxidase negative, non-spore forming, non-motile, rod or short cocci shaped bacteria. The A2 and A5 strains both survived well in conditions of low pH, 0.4% phenol and high bile salt concentrations. They could grow at different temperatures and were resistant to different NaCl concentrations. A study was also conducted on the various probiotic and safety attributes of isolates. Bacteriocins, a kind of ribosomal protein produced by LAB, were estimated using the Folin-Lowery method. The formation of bacteriocins was influenced by various physical and chemical factors. The result demonstrates that the optimum conditions for the production of bacteriocins by the A2 strain were glucose as carbon source, pH 6.5, 2% NaCl and 1% bile salts. For the A5 strain, sucrose as a carbon source, pH 6.5, 1% NaCl and 1% bile salt were optimum conditions for bacteriocin production. Study was also conducted on exopolysaccharide (EPS) production, adhesion characteristics, antagonistic activity and antibiotic sensitivity. Probiotic curd (Dahi) formation was done and several parameters of curd like pH, total lactic acid concentration and water-holding capacity were all investigated.

Microencapsulation of riboflavin-producing Lactiplantibacillus Plantarum MTCC 25,432 and Evaluation of its Survival in Simulated Gastric and Intestinal Fluid.

Microencapsulation is an optimistic method for the delivery of live microbial cells through different food products. In this study, riboflavin-producing probiotic strain Lactiplantibacillus plantarum MTCC 25,432 was encapsulated using a spray drying technique with different wall materials including Inulin, maltodextrin (MD), and MD + Inulin (1:1). The obtained spray dried powder was investigated for probiotic viability, encapsulation efficiency, particle size, water activity, moisture content, hygroscopicity, bulk and tapped densities, storage stabilities, Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). Besides this, the viability of the free and encapsulated probiotic cells was tested under simulated gastric and intestinal fluid conditions. In the results, microcapsules produced with the combination of MD + Inulin showed higher dry powder yield (36.5%) and viability of L. plantarum MTCC 25,432 (7.4 log CFU / g) as compared with individual coating materials. Further characterization revealed that MD + Inulin microcapsules are spherical (3.50 ± 1.61μm in diameter) in shape with concavities, showed the highest encapsulation efficiency (82%), low water activity (0.307), moisture content (3.67%) and good survival ability at low pH (pH 2.0 and 3.0), high bile salt concentrations (1.0% and 2.0%), and long storage conditions. No differences in FTIR spectra were observed among the tested samples. However, TGA showed enhanced thermal stability of probiotic-loaded microcapsules when MD + Inulin was used together. In conclusion, MD + Inulin could be a potential encapsulation material for riboflavin-producing probiotic bacteria L. plantarum MTCC 25,432.

Contribution of non-ionic interactions on bile salt sequestration by chitooligosaccharides: Potential hypocholesterolemic activity

Chitooligosaccharides have been suggested as cholesterol reducing ingredients mostly due to their ability to sequestrate bile salts. The nature of the chitooligosaccharides-bile salts binding is usually linked with the ionic interaction. However, at physiological intestinal pH range (6.4 to 7.4) and considering chitooligosaccharides pKa, they should be mostly uncharged. This highlights that other type of interaction might be of relevance. In this work, aqueous solutions of chitooligosaccharides with an average degree of polymerization of 10 and 90 % deacetylated, were characterized regarding their effect on bile salt sequestration and cholesterol accessibility. Chitooligosaccharides were shown to bind bile salts to a similar extent as the cationic resin colestipol, both decreasing cholesterol accessibility as measured by NMR at pH 7.4. A decrease in the ionic strength leads to an increase in the binding capacity of chitooligosaccharides, in agreement with the involvement of ionic interactions. However, when the pH is decreased to 6.4, the increase in charge of chitooligosaccharides is not followed by a significant increase in bile salt sequestration. This corroborates the involvement of non-ionic interactions, which was further supported by NMR chemical shift analysis and by the negative electrophoretic mobility attained for the bile salt-chitooligosaccharide aggregates at high bile salt concentrations. These results highlight that chitooligosaccharides non-ionic character is a relevant structural feature to aid in the development of hypocholesterolemic ingredients.

Spray-dried Probiotic adjunct with in vitro acid and bile salt tolerance

Probiotic Lactobacillus acidophilus LA1 was encapsulated using spray drying with concentrated skim milk with total solids (17%), and maltodextrin (25%), and gum acacia(15%) as carriers. The encapsulated probiotics were assessed for viability following drying at an inlet temperature of 170°C ±3°C and an outlet temperature of 80°C ± 3°C. The viable countenumerated after drying was 108 log cfu/g. The prepared probiotic adjunct was further assessed for viability through in vitro acidand bile salt exposure assay. The results indicated that the cellcount decreased at lower pH (1, 2) and high incubation periods(1 hr and 2 hr). A similar trend was observed with bile. However, they showed better survivability for different incubation periods at low pH and in high bile salt concentrations. The spray-dried probiotic adjunct was kept for storage at three different temperatures i.e., 8 °C, 25 °C, and 37 °C for eight weeks, and assessed weekly for cell viability. An inverse relationship between cell survival and storage temperature was observed during storage.

Bile components affect the functions and transcriptome of the rainbow trout intestinal epithelial cell line RTgutGC

The concomitant increase in cultivation of fish and decrease in supply of marine ingredients, have greatly increased the demand for new nutrient sources. This also regards so-called functional ingredients which may benefit health and welfare of the fish. In vitro cell line-based intestinal epithelial barrier models may serve as tools for narrowing down the broad range of ingredient options, to identify the most promising candidates before in vivo feeding trials are run. In vivo, differentiation of the various epithelial cells in the fish intestine, from the multipotent stem cells, takes place in the presence of a variety of substances from dietary and endogenous origin. Among these, bile salts have recently received attention as regulators of epithelial function in health and disease but have not, until now, been included in the medium when culturing fish gut epithelial cells in vitro. As bile salts are present at high levels in the chyme of the fish intestine, in particular in salmon and rainbow trout, mostly as taurocholate (>90%), their role for effects of diet ingredients on the in vitro gut cell model should be understood.With this study, we wanted to investigate whether inclusion of bile from rainbow trout or pure taurocholate in the culture media would modulate functions of the RTgutGC epithelial cells. Here, we demonstrated that the rainbow trout intestinal epithelial cell line RTgutGC responded significantly to the presence of bile components. Treatment with rainbow trout bile taken from the gall bladder (RTbile) or pure taurocholate (TC) at taurocholate concentrations of ≤0.5 mg/mL retained normal cell morphology, cell viability as in cell oxidation-reduction metabolic activity and membrane integrity, and barrier features, while high concentrations of bile salts (≥1 mg/mL) were cytotoxic to the cells. After long-term (4 days) bile treatment, transcriptome responses showed how bile salts play important roles in intestinal epithelial cell metabolism. qPCR data demonstrated that barrier function genes, brush border enzyme genes and immune genes were significantly affected. Although similar trends were seen, treatment with bile salt as a component of rainbow trout bile or pure taurocholate, induced somewhat different effects. In conclusion, this study clearly indicates that bile salts should be included in the cell medium when running in vitro studies of gut cell functions, not at least immune functions, preferably at the level of ∼0.5 mg/mL supplemented as pure taurocholate to ensure reproducibility.

Psychobiotic Potential of Gamma-Aminobutyric Acid–Producing Marine Enterococcus faecium SH9 from Marine Shrimp

Psychobiotics are a novel class of probiotics with potential to confer mental wellness via production of neuroactive compounds such as gamma-aminobutyric acid (GABA). The demand for new biological sources of GABA has increased steadily. Therefore, the current study reports the isolation of 17 presumptive lactic acid bacteria (LAB) from marine samples and their screening for GABA synthesis from monosodium glutamate (MSG) using thin-layer chromatography (TLC). The isolate SH9 was selected as a high GABA producing strain. The GABA content of SH9 cell free supernatant (CFS) was quantitatively determined by high performance liquid chromatography (HPLC) to be 0.97 g/L. SH9 was identified biochemically and molecularly as Enterococcus faecium (identity 99%). Moreover, SH9 demonstrated promising probiotic potentials; it gave no signs of hemolysis and could survive at low pH values and high bile salt concentrations. It also exhibited antimicrobial activity against highly pathogenic strains and the ability to grow at 6.5% NaCl. In addition, SH9 CFS showed anti-inflammatory and antioxidant properties. The glutamate decarboxylase (GAD) gene was detected in SH9 by using specific primers. Product of 540 bp was obtained, sequenced, and analyzed (accession number: MW713382). The inferred amino acid sequence was 99.3% identical to Lactobacillus plantarum M-6 gadB gene. The findings of this study suggest that the marine isolate E. faecium SH9 could be used as a novel psychobiotics in the development of GABA rich healthy products.

Physiological properties, survivability and genomic characteristics of Pediococcus pentosaceus for application as a starter culture

There is an increasing demand for the use of Pediococcus pentosaceus in the dairy industry due to its remarkable abilities as a probiotic or starter culture bacterium. This study determines the physiological, survivability and genomic properties of P. pentosaceus to evaluate its potential as a starter culture. The strain showed great tolerance in low pH and high concentrations of bile salts. A good coaggregation ability against pathogens ranging between 71% and 84%, as well as strong biofilm formation ability and remarkable resistance to antibiotics. Whole‐genome sequence results reveal the presence of various genes responsible for stress tolerance, adhesion, biofilm formation, acids, and bile salts tolerance. The findings of this study can be used as essential criteria for selecting P. pentosaceus as a starter culture.

The probiotic potential of Lactobacillus plantarum strain RW1 isolated from canine faeces.

To evaluation the probiotic potential of Lactobacillus plantarum strain RW1 isolated from healthy dogs for its further utilization as a dietary supplement for dogs. This study aimed to evaluate the probiotic potential of L. plantarum strain RW1 isolated from canine faeces. After confirming by conventional and then by 16S rRNA sequencing, the identified strain RW1 was in vitro screened for its survivability in simulated gastrointestinal conditions, low pH, bile salts and adhesion to gut epithelial tissues, growth inhibitory effects on common pathogens and anti-inflammatory potential by measuring the mRNA expression level of IL-6, IL-8, IL-1β in Salmonella-infected MODE-K cells. Furthermore, the effects on epithelial barrier function and host defensin peptide (beta-defensin 3) was studied by measuring the mRNA expression level of tight junction protein (occludin) and beta-defensin 3 in MODE-K cells. The strain RW1 showed a considerable potential to survive in simulated gastrointestinal environmental conditions, low pH and high bile salt concentrations along with good adhesion to MODE-K cell line. Pathogenic bacterial growth and their adhesion to MODE-K cell line were significantly inhibited by the strain RW1. Real-time PCR analyses demonstrated that the strain RW1 inhibited Salmonella-induced pro-inflammatory cytokines (IL-6, IL-8 and IL-1β) production and reinforced the expression of tight junction protein (occludin). The strain RW1 did not induce mRNA expression of beta-defensin 3. Based on in vitro results, the strain RW1 has the potential to be used as a probiotic supplement in dogs. However, further study involving in vivo health effects is needed. Antibiotics have many side effects and nowadays the probiotics are considered as a potential alternative to antibiotics. This study evaluates the probiotic potential of dog isolated L. plantarum strain RW1 to use it as a dietary supplement in dogs feeding to control infectious diseases.

Cytoprotective Effects of Lactobacilli on Mouse Epithelial Cells during Salmonella Infection

Treatment of common pathogens, such as Salmonella species, Escherichia coli, Staphylococcus aureus, etc., is a big challenge for a practitioner. Antibiotics’ side effects during their application for the treatment of infectious diseases should not be underestimated as they have many issues, such as the transfer of antibiotics-resistant genes, dysbiosis, and antibiotic-resistant strains, which is the main hurdle in the eradication of diseases. To avoid these antibiotics complications, in modern countries, the interest of using probiotics in feed supplementation to promote health and prevent or treat intestinal infectious diseases has been increasing. The purpose of the present study was to evaluate the probiotic potential of three Lactobacilli strains isolated from clinically healthy dogs for their further utilization as a dietary supplement for dogs to avoid pathogenic and antibiotic complication. After 16SrRNA sequencing, in vitro tests were conducted to assess the survival potential of Lactobacilli under simulated gastrointestinal conditions and adhesion ability to the MODE-K cell line, effects on epithelial barrier function, anti-inflammatory activities, effects on host defensin peptides (beta-defensin 3), and inhibitory effects on common pathogens. Lactobacilli showed considerable potential to survive in simulated gastrointestinal environmental conditions, low pH, and high bile salt concentrations along with good adhesion properties with MODE-K cells. Pathogenic bacterial growth and their adhesion to MODE-K cells were significantly inhibited by Lactobacilli. Real-time PCR analyses further demonstrated that the L. acidophilus strain AR1 and AR3 inhibit Salmonella-induced proinflammatory cytokine (IL-6, IL-8, IL-1β) production and reinforce the expression of tight junction protein (occludin). None of the strains induce mRNA expression of beta-defensin 3 in MODE-K cells. Based on the in vitro results, the L. acidophilus strain AR1 has the potential to be supplemented in canine feed. However, further in vivo studies investigating health-promoting effects are awaited.

Effects of Bacillus amyloliquefaciens TL106 Isolated from Tibetan Pigs on Probiotic Potential and Intestinal Microbes in Weaned Piglets.

ABSTRACTBacillus amyloliquefaciens is a nonpathogenic microorganism whose highly active amylase is widely isolated from soil and plants. TL106 is an isolate of Bacillus amyloliquefaciens isolated from cold- and disease-resistant Tibetan pigs in Linzhi, Tibet. Here, we report that TL106 not only could survive in acidic environments, high bile salt concentrations, and high-temperature conditions but also was resistant to antibiotics. It significantly improved the growth performance of weaned piglets, especially in the prevention of diarrhea. The crude fiber and crude ash digestibility in weaned piglets after TL106 administration was considerably higher than that in other groups. The results of 16S rRNA sequencing conveyed that TL106 stabilized gut microbiota that was disturbed by the weaning process with an increased level of Lachnospiraceae, Peptococcaceae.rc4_4, Erysipelotrichaceae.L7A_E11, and Mollicutes.RF39. Hence, this study proved that Bacillus amyloliquefaciens TL106 might be a candidate for antibiotics in Duroc×Landrace×Yorkshire weaned piglets.IMPORTANCE Antibiotics are often used to promote animal growth and prevent diarrhea in weanling piglets. Nevertheless, intestinal pathogenic bacterial resistance and drug residues caused by antibiotic overuse are worthy of concern and demand an urgent solution. Bacillus amyloliquefaciens TL106 has been isolated from cold- and disease-resistant Tibetan pigs in Linzhi, Tibet. It significantly improved the growth performance, decreased diarrhea, increased the absorption of crude substances, and regulated the gut flora homeostasis in Duroc×Landrace×Yorkshire weaned piglets. As an antibiotic candidate, TL106 perfectly displayed its probiotic potential and pollution-free properties.