Bile Salt Concentration Research Articles

Evaluation of Probiotic Potential and Functional Properties of Lactobacillus Strains Isolated from Dhan, Traditional Algerian Goat Milk Butter

Goat milk butter, locally known as “Dhan,” from the Sfisfa region of Algeria, holds significant cultural and economic value. This study investigates the probiotic properties of lactic acid bacteria (LAB) present in Dhan, focusing particularly on Lactobacillus strains. Molecular identification using 16S rRNA revealed a dominance of Levilactobacillus brevis and Lactiplantibacillus plantarum, forming a substantial part of the bacterial profile. Three LAB isolates (DC01-A, DC04, and DC06) were selected from fresh samples, and rigorous analyses were performed to evaluate their probiotic properties. Safety assessments confirmed the absence of gelatinase, DNase, and haemolytic activities in all isolates. The isolates demonstrated high tolerance to bile salts and acidic conditions, along with the ability to survive simulated gastrointestinal digestion. Notably, strain DC06 exhibited exceptional survival at low pH (1.5) and high bile salt concentrations (0.15–0.3%). All isolates showed substantial growth in MRS medium with 2% phenol, although growth was significantly decreased at 5% phenol. Furthermore, our strains exhibited high adhesion rates to various solvents, demonstrating their potential for strong interaction with cell membranes. Specifically, adhesion to chloroform was observed at 98.26% for DC01-A, 99.30% for DC04, and 99.20% for DC06. With xylene, the adhesion rates were 75.94% for DC01-A, 61.13% for DC04, and 76.52% for DC06. The LAB strains demonstrated impressive growth in ethanol concentrations up to 12%, but their tolerance did not exceed this concentration. They also exhibited robust growth across temperatures from 10 °C to 37 °C, with strains DC04 and DC06 able to proliferate at 45 °C, though none survived at 50 °C. Additionally, the isolates showed significant resistance to oxidative stress induced by hydrogen peroxide (H2O2) and displayed medium to high autolytic activity, with rates of 50.86%, 37.53%, and 33.42% for DC01-A, DC04, and DC06, respectively. The cell-free supernatant derived from strain DC04 exhibited significant antimicrobial activity against the tested pathogens, while strain DC06 demonstrated moderate antioxidant activity with the highest DPPH scavenging rate at 68.56%, compared to the probiotic reference strain LGG at 61.28%. These collective findings not only suggest the probiotic viability of LAB strains found in Dhan but also highlight the importance of traditional food practises in contributing to health and nutrition. Consequently, this study supports the potential of traditional Dhan butter as a functional food and encourages further exploration of its health benefits.

Isolation of yeast from some Ethiopian traditional fermented beverages and in vitro evaluation for probiotic traits

Traditional fermented foods and beverages are important sources of probiotic microbes. The purpose of this study was to isolate yeast from Ethiopian fermented beverages and assess their probiotic activity in an in vitro setting. Yeast isolation, identification, and in vitro probiotic trait screening were conducted in accordance with established protocols. Eleven isolates were obtained. Of them, GB1D5, RTj3D3 and DMTD2 were low hydrogen sulfide producers and were selected. The D1/2 genotyping of selected isolates revealed that they were strains of Saccharomyces cerevisiae. All strains grew well at low pH, body temperature, bile salt concentrations (0.3–0.6 (w/v)) and survived at simulated gastrointestinal conditions with survival percentages of 12.8 ± 4.9 to 14.4 ± 5.0 % and 5.3 ± 1.7–5.9 ± 1.8 %, respectively. They demonstrated surface hydrophobicity ranging from 61.3 to 68.7 %; and 80.7–86 % auto-aggregation percentages after 24 h of incubation. They also showed hydroxyl radical scavenging activity ranging between 91.6 and 92.3 % and mild inhibitory activity against Escherichia coli (ATCC 893614) and Staphylococcus aureus (ATCC 892760). The PCA revealed that two strains (DMTD2 and RTj3D3) have a strong association with most probiotic properties, which affirms their promising candidacy. Safety assessments indicated that they were resistant to antibacterial antibiotics, susceptible to antifungals, and negative for protease, gelatinase, biogenic amine production, and hemolytic activity. All these suggest that they are promising candidates for the production of food containing probiotics. Examining their performance in vivo circumstances is recommended.

Functional and molecular characterization of millet associated probiotic bacteria

The lactic acid bacteria are one of the sustainable ways of food production. As the native lactic acid bacteria (LAB) easily manipulate the substrate, helps in production of health essential probiotics with enhancing the bioavailability of the substrate. Here also, in present study, the native LAB isolates isolated from the millets and characterize them for the functional analysis for the human health association. In the present study, fermented millet-associated lactic acid bacteria were screened and characterized for their probiotic potential, safety evaluation and antimicrobial activity. A total of 33 isolates were purified as lactic acid bacteria based on colony shape and biochemical assays. However, only 13 isolates were found to be catalase-negative. Among the 13 isolates, 5 isolates exhibited optimum growth at 6.5% and 9.5% of salt concentrations, pH of 4.5 to 8.5 and 17 °C to 40 °C of the temperature. The probiotic properties of the five isolates exhibited that the survival rates in acid and bile salt concentration ranged from 56.2 to 73.7% and 55.3 to 70.3%, respectively. Similarly, the surface hydrophobicity of the isolates was 41–75%. Antibiotic assay revealed that all five isolates were resistant to Amoxicillin, Cloxacillin, and Penicillin-V. Interestingly, all the isolates except ME26 displayed susceptibility towards Penicillin (2 units) and Tetracycline (10 µg). Further, the four isolates (ME25, ME26, ME9, and ME2) had more antifungal activity against Aspergillus flavus. However, only three, except ME1 and ME2, showed maximum antibacterial activity and produced more antimicrobial compounds compared to reference strain L. plantarum Pb3. The potential probiotic isolates were identified as Weisella cibaria ME9, Weisella cibaria ME26, and Weisella confusa ME25.

Understanding the Impact of Lipids on the Solubilizing Capacity of Human Intestinal Fluids.

Lipids in human intestinal fluids (HIF) form various structures, resulting in phase separation in the form of a lipid fraction and a micellar aqueous fraction. Currently used fed state simulated intestinal fluids (SIF) lack phase separation, highlighting the need for a deeper understanding of the effect of these fractions on intestinal drug solubilization in HIF to improve simulation accuracy. In this study, duodenal fluids aspirated from 21 healthy volunteers in fasted, early fed, and late fed states were used to generate 7 HIF pools for each prandial state. The apparent solubility of seven lipophilic model drugs was measured across these HIF pools, differentiating between the micellar fraction and the total sample (including both micellar and lipid fractions). The solubilizing capacities of these fluids were analyzed in relation to their composition, including total lipids, bile salts, phospholipids, total cholesterol, pH, and total protein. The solubility data generated in this work demonstrated that current fed state SIF effectively predicted the average solubility in the micellar fraction of HIF but failed to discern the considerable variability between HIF pools. Furthermore, the inclusion of a lipid fraction significantly enhanced the solubility of fed state HIF pools, resulting on average in a 13.9-fold increase in solubilizing capacity across the seven model compounds. Although the average composition of the fluids was consistent with previous studies, substantial variability was observed in micellar lipid concentrations, despite relatively stable total lipid concentrations. This variability is critical, as evidenced by the strong correlations between the solubilizing capacity of the micellar fraction and its micellar lipid concentrations. Additionally, this study identified that fluctuations in bile salt concentrations and pH contributed to the observed variability in micellar lipid concentration. In summary, the influence of the lipid fraction on solubility was 2-fold: it enhanced the solubility of lipophilic drugs in the total fluid, and contributed to the variability in the solubilizing capacity of the micellar fraction.

Exploring Fresh Lettuce (Lactuca sativa) as a Dairy-Free Probiotic Source

Probiotics are living microorganisms that, when given in appropriate amounts, have optimistic effects on body. This study explores the potential of fresh lettuce (Lactuca sativa) leaves as a probiotic source for individuals with dairy allergies. Gram staining and the catalase test were used to identify the bacteria that were recovered from lettuce leaves as gram-positive, catalase-negative strains. The resistance to antibiotics and their capacity to tolerate low pH and bile salt concentrations both essential for surviving the digestive tract were evaluated. The bacteria showed a high tolerance to bile salts and low pH, but they were susceptible to ampicillin, streptomycin, gentamycin, chloramphenicol, and neomycin. Tests for gas production in the presence of glucose revealed no gas production. Molecular techniques such as PCR and 16S rRNA gene sequencing revealed the presence of Enterococcus lactis, Enterococcus durans, Lactobacillus paracasei, and Lactobacillus casei

Interfacial behaviour of human bile and its substitution for in vitro lipolysis studies

This study examined the interfacial evolution of individual bile salts (BSs) and their blends with phosphatidylcholine (BS/PC) to simulate the complex behaviour of human bile (HB) during lipolysis at the triglyceride/water interface. Using adsorption and desorption cycles, mimicking exposure to small intestinal fluids, we demonstrate that the interfacial behaviour of real HB can be replicated using simple mixtures of BSs and PC. Interfacial tension (IFT) measurements after lipolysis and desorption showed no significant differences (P > 0.05) between HB samples and BS/PC mixtures across the total BS concentrations analysed (2.23–7.81 mM). However, individual BSs without PC yielded significantly different IFT results (P < 0.01) compared to HB, highlighting the importance of phospholipids. Dilatation rheology further emphasised the need for accurate phospholipid representation in bile models. Our results suggest that phospholipids in HB and in BS/PC systems enhance resistance to desorption, potentially affecting lipolysis. This is important, as current in vitro digestion models often replicate only intestinal BS concentrations to mimic the behaviour of HB in the intestinal lumen. Furthermore, the specific composition of BSs in HB appears less critical than the overall BS and phospholipid contents, suggesting that the kinetics of triglyceride digestion is influenced by the combined luminal concentrations of these components. These findings have significant implications for understanding the role of bile in digestion and offer insights for designing more accurate in vitro models to study the gastrointestinal behaviour of food emulsions and lipid-based delivery systems.

Evaluation of the probiotic ability of Cellulolytic Bacillus subtilis SBMP4 (FSP20) isolated from waste-dump site in FIIRO, Lagos Nigeria

BACKGROUND: Current invention in the field of biotechnology includes research into the probiotic potentials of enzyme-producing bacteria species. OBJECTIVE: This study evaluated the probiotic ability of cellulolytic Bacillus subtilis SBMP4 isolated from waste dump site. METHODS: Isolated Bacillus species was screened for cellulolytic ability on carboxyl methylcellulose agar (CMCA), and hydrolysis efficiency was calculated. Probiotic ability of cellulolytic Bacillus species was carried out as follows: pathogenic potentials, bacteriocin producing ability, antibiotic susceptibility testing, gastric acid, and bile tolerance. Molecular identification was done by means of 16S rRNA gene sequencing, blast search analysis and phylogenetic analysis. RESULTS: Zone of clearance of 30 ± 2.08 mm on CMCA with hydrolysis efficiency of 114 % depicted the isolate’s cellulolytic potential. Bacillus subtilis SBMP4 showed γ-hemolytic phenotype with negative lecithinase activity. Antibacterial activity of bacteriocin observed against Staphylococcus aureus ATCC 25923 reference culture, had maximum inhibition zone of 14±1.0 mm at 72 hour of incubation. Substantial susceptibility towards 9 out of 10 commonly used antibiotics further depicted the isolate’s probiotic ability. Bacillus subtilis SBMP4 exhibited good gastric acid tolerance (pH 2.5) of 5.857 Log cycle/ml and good tolerance to 5 % and 10 % bile salt concentrations with 6.134 and 6.301 Log cycle/ml of viable bacteria cells. Molecular identification of the selected bacteria species revealed it to be Bacillus subtilis SBMP4 with RefSeq accession number NR_118383, showing maximum sequence similarity of 98%. CONCLUSION: Cellulolytic Bacillus subtilis SBMP4 possesses good probiotic ability and has the potentials of being applied as additive in livestock feeding to enhance overall productivity. KEYWORDS: Cellulolytic, Probiotic, CelA gene, Bacteriocin, Bacillus subtilis

Influence of bile salts on the gastrointestinal digestion of agar-casein hybrid systems and the nanoassembly of their digestion products

This manuscript reports on the effect of different bile salts concentration on the gastrointestinal digestion of casein and casein-agar hybrid systems and evaluates the effect on the nanostructural assembly of the digestion products through the application of advanced small angle X-ray scattering analysis (SAXS).The results showed that bile salts promote the hydrolysis of micellar casein upon in vitro gastrointestinal digestions. It is noteworthy that in the presence of agar, bile salts interact with the polysaccharide, leading to a reduction in their proteolytic activity. While structured agar-casein hydrogels were shown to be able to limit the proteolysis during the gastric phase, increasing concentrations of bile salts promoted the diffusion of casein from the gel network during the intestinal phase, hence leading to higher degree of hydrolysis. The released digestion products were seen to interact with bile salts, forming ordered lamellar/micellar nanostructures. In particular, the presence of solubilized agar in the digesta was seen to promote the formation of these nanostructures. These nanostructures were formed at 10 mM bile salts for the hydrogels, and across all concentrations for agar-casein blends. Thus, this work demonstrates the great relevance of bile salts in the nanostructural assembly of protein digestion products, which is expected to have a great relevance in bioavailability and metabolic responses induced by protein-rich foods.

Probiotic Potential of Bacillus Strains Isolated from Traditional Cassava Ferments (Manihot esculenta Crantz)

ABSTRACT: Probiotic potentials of Bacillus strains isolated from traditional cassava ferments, notably their growth at different pH, bile salts, temperatures, NaCl and antibiotics, their inhibition of pathogenic bacteria, non-production of hemolytic enzymes as well as the formation of biofilm were studied. These different probiotic parameters were determined according to the referenced methods. The results obtained showed that all Bacillus strains resisted acidic pH with the highest growths (7.29± 1.40) × 108 CFU/mL and (5.14±0.15) × 108 CFU /mL obtained with Bacillus toyonensis respectively at pH 2 for 24 hours and at pH 3 for 4 hours. All Bacillus strains grow well at 37°C and 44°C, despite their optimum growth temperature of 30°C. They also showed good growth at different bile salt concentrations and were multi-resistance to antibiotics Ciprofloxacin (100%), as was B. subtilis to Rifampicin (100%) but they were multi-sensitive (100%) to Amoxicillin, Imipenem, Gentamycin, Penicillin, Vancomycin, Chloramphenicol and Rifampicin except B. pumilus and B. methylotrophicus which were sensitive to Rifampicin (77%). Bacillus strains inhibited more than half of the pathogens (80.27% with S. aureus and 65.09% with E. coli). No strain showed hemolytic activity but rather a good capacity to form a biofilm (optical density ranging from 0.663 ± 00 to 3.15 ± 02 nm).

The Particle Drifting Effect: A Combined Function of Colloidal and Drug Properties.

The particle drifting effect, where nanosized colloidal drug particles overcome the diffusional resistance of the aqueous boundary layer adjacent to the intestinal wall and increase drug absorption rates, is drawing increasing attention in pharmaceutical research. However, mechanistic understanding and accurate prediction of the particle drifting effect remain lacking. In this study, we systematically evaluated the extent of the particle drifting effect affected by drug and colloidal properties, including the size, number, and type of the moving species using biphasic diffusion experiments combined with computational fluid dynamics simulations and mass transport analyses. The results showed that the particle drifting effect is a sequential reaction of particle dissolution/dissociation in the diffusional boundary layer, followed by absorption of the free drug. Therefore, factors affecting the rate-limiting step, which can be either process or both under different circumstances, alter the particle drifting effect. Experimental results also agree with the theory that the particle dissolution rate is dependent on particle size, concentration, and drug solubility. In addition, rapid bile micelle dissociation and bile salt absorption facilitated drug absorption by the particle drifting effect. Our findings explain the highly dynamic nature of the particle drifting effect and will contribute to rational formulation development and better bioavailability prediction for formulations containing colloidal particles.

Impact of Physiological Characteristics on Chylomicron Pathway-Mediated Absorption of Nanocrystals in the Pediatric Population.

Nanocrystals exhibit significant advantages in improving the oral bioavailability of poorly soluble drugs. However, the complicated absorption properties of nanocrystals and the differences in physiological characteristics between children and adults limit pediatric applications of nanocrystals. To elucidate the absorption differences and the underlying mechanisms between children and adults, the pharmacokinetics and tissue distribution of aprepitant crystals with different particle sizes (NC200, NC500, and MC2.5) in rats and mice at different ages were studied, and their absorption mechanisms were investigated in Caco-2 cells, mice, and rats. It was found that childhood animals demonstrated higher bioavailability compared with adolescent and adult animals, which was related to higher bile salt concentration and accelerated drug dissolution in the intestine of childhood animals. The majority of nanocrystals were dissolved and formed micelles under the influence of bile salts. Compared with intact nanocrystals, the bile salt micelle-associated aprepitant was absorbed through the chylomicron pathway, wherein Apo B assisted in the reassembling of the aprepitant micelles after endocytosis. Higher bile salt concentration and Apo B expression in the intestines of childhood animals are both responsible for the higher chylomicron transport pathways. Elucidation of the chylomicron pathway in the varied absorption of nanocrystals among children, adolescents, and adults provides strong theoretical guidance for promoting the rational and safe use of nanocrystals in pediatric populations.

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 potential of lactic acid bacteria isolated from Ethiopian traditional fermented Cheka beverage

Introduction: Lactic acid bacteria (LAB) are a cluster of microbes distributed in a variety of environments and have potential probiotic activity to improve human well-being. This study was aimed at assessing the probiotic potential of LAB isolated from Cheka, an Ethiopian traditionally fermented beverage.MethodPure isolates obtained from 16 Cheka samples from Konso (n = 8) and Derashe (n = 8) were characterized morphologically, biochemically, and physiologically by considering basic criteria to identify the LAB. The probiotics properties of the LAB were evaluated in vitro at low pH values (2.0 and 3.0), and two bile salt concentrations (0.3 and 0.5%) for 3 and 6 h. The 16 S rRNA gene sequencing was done using an ABI 3730xl sequencer, and the gene sequences were aligned.ResultsOf the 27 pure isolates, 11 isolates were proven to be LAB with non-motile, negative for catalase, and non-spore former characteristics. Based on cultural characteristics and sugar fermentation ability, the 11 isolates were assembled into the genera Lactobacillus (55%), Lactococcus (18%), Pediococcus (18%), and Leuconostoc (9%). At pH 3.0 and a bile salt concentration of 0.3%, isolate ChK-11 showed a better survival rate (97 and 94%) than other isolates [ChK-7 (93 and 80%) > ChD-5 (84 and 76%) > ChD-8 (46 and 36%) > ChK-4 (41 and 34%)] for 6 and 3 h, respectively. According to 16 S rRNA sequencing results, isolates ChK-11 and ChK-7 were found to be Weissella paramesenteroides and Leuconostoc pseudomesenteroides with sequence similarity of 99 and 91%, respectively.ConclusionsIn the present study, probiotic LAB (Weissella paramesenteroides and Leuconostoc pseudomesenteroides) was successfully isolated and sequenced from Cheka samples. The findings of this in vitro study indicated that fermented beverages like Cheka are a source of the LAB with probiotic functional properties. Overall, Weissella paramesenteroides and Leuconostoc pseudomesenteroides isolates, which showed promising probiotic properties under in vitro conditions, can be used for starter culture development for the Cheka fermentation process.

Efficiency of Encapsulation of Thioflavin T (ThT) into Polar and Nonpolar Environments of Different Bile Salt Aggregates: A Femtosecond Fluorescence Study.

The binding of Thioflavin T (ThT) with various bile salts, a potential host molecule, has been analyzed by steady-state and time-resolved fluorescence spectroscopy. A comparative study has been executed to investigate the influence of confinement of different bile salts, namely, sodium cholate (NaCh), sodium taurocholate (NaTC), and sodium deoxycholate (NaDC) on binding and excited state torsional motion of ThT molecules. The changes in absorption and emission properties of probe molecules were found to be sensitive to increasing bile salt concentration in aqueous 0.2 (M) NaCl solutions. The photophysics of ThT mainly depends on hydrophobicity, morphology, and size of bile salt aggregates in solution. In the presence of bile salts, the emission intensity and emission lifetime of ThT increase significantly, indicating encapsulation of dye. Moreover, we have also investigated the effect of the ionic strength of the medium by sodium chloride (NaCl) on the spectroscopic properties of ThT in the restricted surroundings of aqueous bile salts. It is observed that the fluorescence lifetime of ThT in bile salts increases significantly in the presence of NaCl. The encapsulation efficiency of ThT in bile salt aggregates has been assessed by iodide (I-) as an external ionic quencher. We found that NaDC aggregates are more efficient in the modulation of photophysical properties of ThT and also provide better protection efficiency to decrease the nonradiative deactivation processes.

Cholesterol-lowering effect of Pediococcus strains isolated from mother’s milk in mice

PurposeThe Pediococcus strains belong to the group of lactic acid bacteria (LAB). This study aims to isolate and identify the Pediococcus spp. from mother’s milk, and investigate their tolerance to low pH and bile salts, antibacterial activity, attachment to HT-29 cells and effect on cholesterol-lowering and digestive enzymes in mice.Design/methodology/approachPediococcus species were isolated from mother’s milk, then tested for tolerance to acid and bile salts, their antibacterial effect and attachment to HT-29 cell line. In mice experiment, the levels of triglyceride (TG), total cholesterol (TC), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C) and digestive enzymes such as amylase and lipase in serum of mice were measured after administration of 108 CFU/mL bacterial suspension.FindingsA total of 24 Pediococcus spp. including 16 isolates of Pediococcus pentosaceus and 8 isolates of Pediococcus acidilactici were isolated. Fifteen isolates (62.5%) were able to survive in low pH and bile salt concentration. Ten isolates (41.6%) exhibited the most powerful inhibitory effects against both Gram-positive and Gram-negative bacterial pathogens. A total of four Pediococcus isolates (16.67%) showed strong attachment to HT-29 cells. In contrast to HDL-C and amylase, the serum levels of TC, TG and LDL-C were reduced and lipase was elevated significantly following administration of mother milk-derived Pediococcus isolates.Originality/valueThe Pediococcus isolates demonstrated good probiotic properties in particular cholesterol-lowering ability in mice. Thus, after more studies they can be considered as probiotic strain(s).

Enhanced resistance of Lactiplantibacillus plantarum by expression of albumin.

Human serum albumin (HSA) is the most abundant protein in plasma, playing crucial roles in regulating osmotic pressure and maintaining protein homeostasis. It is widely applied in the clinical treatment of various diseases. HSA can be purified from plasma or produced using recombinant DNA technology. Due to the improved efficiency and reduced costs, a growing body of research has focused on enhancing albumin production through bacterial strain overexpression. However, there have been few studies on the effect of albumin on the characteristics of the overexpressing-strain itself, particularly stress resistance. In this study, we utilized Lactiplantibacillus plantarum (L. plantarum) AR113 as the expression host and successfully constructed the albumin overexpression strain AR113-pLLY01 through gene editing technology. The successful expression of albumin was achieved and subsequently compared with the wild-type strain AR113-pIB184. The results demonstrated that the survival rate of AR113-pLLY01 was also significantly better than that of AR113-pIB184 after lyophilization. In addition, AR113-pLLY01 exhibited a significantly better protective effect than AR113-pIB184 at pH 3, indicating that albumin possesses a certain tolerance to acidic stress. At bile salt concentrations higher than 0.03%, both strains showed limited growth, but at a concentration of 0.02%, AR113-pLLY01 had a significant protective effect. This study suggest that albumin can improve strain tolerance, which has significant implications for future applications. © 2024 Society of Chemical Industry.

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.

In Vitro Evaluation of Gastrointestinal Stability of Pediococcus pentosaceus Isolated from Fermented Maize and Pearl Millet for Possible Novel Chicken Probiotic Development

Research has identified certain bio-based products, such as probiotics, as alternatives to antibiotics for use in animal feed. They are capable of controlling, preventing or minimizing the colonization of the gastrointestinal tract by pathogenic bacteria. To isolate Pediococcus spp. and assess its technological properties for possible probiotic development, maize and pearl millet were used. The cereals were steeped and wet milled after 48 h of fermentation. The milled cereals were kneaded into dough for 24 h, after which a 10% slurry was prepared for tenfold serial dilution to enumerate the LAB by employing pour plate techniques using MRS Agar. Based on the cell morphology of the isolated bacteria, eight isolates (four from maize and four from millet) that were selected for identification using MALDI-TOF MS showed that five were Pediococcus pentosaceus (P. pentosaceus), one was Pediococcus acidilactici, and two did not match any organism. Subsequently, the six isolates were labeled as MZ1, MZ2, MZ3, MZ4 for the maize isolate and MLT5 and MLT7 for the millet isolate. The six Pediococcus spp. were assessed in vitro for acid and bile salt tolerance, gastric juice and intestinal fluid tolerance and antibiotic resistance and antimicrobial susceptibility tests were performed, and the survivability rate of the strains was calculated. With regard to the mean count, there was a reduction in log10 CFU/mL under the lower pH conditions and their duration of exposure with regard to time. Among the isolates, no differences were noted at the various periods of exposure (0 h, 1 h, 2 h and 3 h) at pH 4 (p &gt; 0.05). However, significant differences were noted at pH 3, 2 and 1 among the isolates (p &lt; 0.05). The percentage survival of MZ4 and MLT7 at pH 1 was higher compared to the other isolates at 0 h. Significant differences were observed among the isolated at pH 2, 3 and 4 across the various periods. The mean count of the isolates in gastric juice was similar at 0 and 1 h, but significant differences were noted at 2 and 3 h, where MLT7 was highest (p &lt; 0.05). A similar trend was observed for percentage survival. The mean count and the percentage survival of isolates under different concentrations of bile salt were similar. Significant differences were noted among isolates in both mean count and percentage survival when exposed to intestinal fluid (p &lt; 0.05). All of the isolates were highly tolerant to the antibiotics tested and possessed antibacterial properties against the selected pathogens. The LABs proved to be good probiotic materials, according to the results obtained. However, the Pediococcus strain MLT7 proved to be the LAB of choice; therefore, its molecular identity was verified using the 16S rRNA sequence and was labeled as Pediococcus pentosaceus GT001 after it was discovered to have 100% similarity with some strains of Pediococcus pentosaceus.

Identification of novel probiotic lactic acid bacteria from soymilk waste using the 16s rRNA gene for potential use in poultry.

In-feed antibiotics have been used as antibiotic growth promoters (AGPs) to enhance the genetic potential of poultry. However, the long-term use of AGPs is known to lead to bacterial resistance and antibiotic residues in poultry meat and eggs. To address these concerns, alternatives to AGPs are needed, one of which is probiotics, which can promote the health of livestock without having any negative effects. In vitro probiotic screening was performed to determine the ability of lactic acid bacteria (LAB) isolated from soymilk waste to be used as a probiotic for livestock. Four LAB isolates (designated F4, F6, F9, and F11) isolated from soymilk waste were used in this study. In vitro testing was performed on LAB isolates to determine their resistance to temperatures of 42°C, acidic pH, bile salts, hydrophobicity to the intestine, and ability to inhibit pathogenic bacteria. A promising isolate was identified using the 16S rRNA gene. All LAB isolates used in this study have the potential to be used as probiotics. On the basis of the results of in vitro testing, all isolates showed resistance to temperatures of 42°C and low pH (2.5) for 3 h (79.87%-94.44%) and 6 h (76.29%-83.39%), respectively. The survival rate at a bile salt concentration of 0.3% ranged from 73.24% to 90.39%, whereas the survival rate at a bile salt concentration of 0.5% ranged from 56.28% to 81.96%. All isolates showed the ability to attach and colonize the digestive tract with a hydrophobicity of 87.58%-91.88%. Inhibitory zones of LAB against pathogens ranged from 4.80-15.15 mm against Staphylococcus aureus, 8.85-14.50 mm against Salmonella enteritidis, and 6.75-22.25 mm against Escherichia coli. Although all isolates showed good ability as probiotics, isolate F4 showed the best probiotic ability. This isolate was identified as Lactobacillus casei strain T22 (JQ412731.1) using the 16S rRNA gene. All isolates in this study have the potential to be used as probiotics. However, isolate F4 has the best probiotic properties and is considered to be the most promising novel probiotic for poultry.