Bile Salt Tolerance Research Articles

Genome assembly of a potential probiotic Bifidobacterium bifidum BB11 strain isolated from Holstein calves.

Here we report the genome sequence of Bifidobacterium bifidum BB11 strain, isolated from the Holstein bull calf's rumen samples. The strain tested positive for probiotic characteristics such as bile salt tolerance, adhesion, and acid tolerance. The genome size was 2,200,000 bp with the guanine and cytosine (G+C) content of 62.5%.

Screening for probiotic properties and potential immunogenic effects of lactobacilli strains isolated from various food products.

Deceleration of disease progression and re-establishment of microbial balance in the gut is often achieved by application of lactobacilli strains. Their beneficial effects are associated with probiotic properties, which may be accompanied by immunomodulatory action at mucosal surfaces. To single out such strains, we screened almost three hundred lactobacilli isolates from eight genera and various food sources for acid and bile salt tolerance, adhesion to mucin as well as hemolytic activity and antibiotic susceptibility. Moreover, the immunomodulatory effects of cell-free supernatant (CFS) fractions of five lactobacilli strains were assessed using an in vitro cell line model. By our rationalized selection approach, we identified thirty-five strains with probiotic potential and biosafety features. Additionally, we showed that CFS from the Lactiplantibacillus L_4 strain downregulates proinflammatory cytokines IL-8 and IL-1β. In contrast, IL-8 expression was found to increase after treatment with CFSs of Lactiplantibacillus L_2 and L_5 and IL-1β was upregulated by CFSs of Lactiplantibacillus L_1 and Lactiplantibacillus L_3. Overall, our result delineate a rational approach of selecting lactobacilli strains for probiotic development to support the gut microbiota equilibrium and reinforce the host immune system.

Antibiotic Resistance Pattern of Lactobacillus reuteri, Lactobacillus salivarius, and Enterococcus hirae Isolated from Gastrointestinal and Respiratory Tract in Commercial Broiler Chickens

Ever since antibiotics were discovered for the treatment of bacterial diseases, an increase in antibiotic-resistant bacteria has been noticed as an economic and public health concern with a high rate of morbidity and mortality. Antibiotic resistance in broiler chickens can be a great threat to public health. This research aimed to screen commercial poultry’s gastrointestinal and respiratory tract bacteria to observe the antibiotic resistance pattern. In this experiment, gastrointestinal tract (GIT) and respiratory tract (RT) bacteria were identified using 16s rRNA gene sequencing from broiler chickens in anaerobic conditions following a bile salt tolerance assay and an antibiotic susceptibility test. Lactobacillus reuteri, Lactobacillus salivarius, and Enterococcus hirae were identified as the isolates. The bacteria found in the gut had a modest degree of bile salt tolerance. The isolates were sensitive to amoxicillin, gentamicin, and streptomycin but resistant to tetracycline, levofloxacin, metronidazole, azithromycin, and erythromycin. This study ensures that the GIT and RT of broiler chicken is a hidden source of the isolated bacteria. A large part of this bacterial population is antibiotic-resistant and reported as a public health concern.

Isolation and Preliminary Characterization of Probiotic Isolates from Healthy Thai Adult Feces

Probiotics are known for health-promoting microorganisms, for examples, assisting gastrointestinal digestion and short chain fatty acid producers. Several current oral human probiotics were derived from human samples; yet, of various ethnic (genetics) and dietary patterns. As probiotics from similar ethnic (genetics) and dietary pattern might confer certain advantages (e.g., more compatibility with hosts), this study isolated and characterized probiotics from healthy Thai adults. To screen for potential probiotic isolates from healthy Thai adult feces, and preliminary characterize these probiotic isolates by biochemical tests and 16S rRNA gene sequencing, following the established Thailand’s Food and Drug Administration (FDA) and National Center for Genetic Engineering and Biotechnology (BIOTEC) guidelines. Twenty fecal samples with clinically verified healthy Thai males and females aged 22-42 years were cultivated on MRS (or M17) media along the broad probiotic screenings (pH 4.0 and 0.1% oxgall) for bacterial probiotic propagation. All derived colonies of different morphologies were continued colony PCR using universal probiotic primers (specific for genera Bifidobacterium, Enterococcus, Lactobacillus, Pediococcus and Streptococcus thermophilus), and only PCR-positive colonies from any of these probiotic primer pair were further cultivation for isolates. Then, these isolates were screened and characterized general probiotic properties that included acid tolerance (pH 2.0), bile salt tolerance (0.3% bile salt), hydrophobicity, antibiotic (ampicillin, ciprofloxacin, erythromycin and vancomycin) susceptibility, no hemolysis activity, catalase, and potassium hydroxide (KOH). The final passing isolates represented potential probiotic isolates that could perhaps maintain in gastrointestinal tract with safety, and were full-length 16S rRNA gene sequenced to identify the genus and species by BLASTN against non-redundant GenBank database. A total of 295 bacterial isolates were obtained from the cultivated on MRS (or M17) media along the broad probiotic screenings from 20 fecal samples. Then, following the general probiotic property characterizations and the full-length 16S rRNA gene sequence analysis, 7 potential isolates were finally derived: 2 Bifidobacterium animalis subsp. lactis, 1 Lactobacillus (new genus name as Limosilactobacillus) reuteri, 1 L. reuteri or L. vaginalis, and 3 Pediococcus pentosaceus. These isolates exhibited strong acid and bile salt tolerances, and relatively high cell surface hydrophobicity. These isolates were susceptible to ampicillin and erythromycin, similar to a reference probiotic control Lactobacillus casei strain Shirota; and the CU13-450 and CU13-451 were also susceptible to vancomycin, similar to a reference control Bifidobacterium breve. Catalase-negative consistent with these bacteria in intestinal tract as anaerobes or facultative anaerobes, and KOH-negative result inferred the Gram-positive bacteria consistent with the full-length 16S rRNA gene sequence identification (Bifidobacterium, Lactobacillus, and Pediococcus are Gram-positive bacteria). No hemolysis activity was observed for these isolates. These 7 probiotic isolates had general probiotic properties and with confirmed the 16S rRNA gene sequences, that follow the Thailand’s FDA and BIOTEC guidelines. However, additional beneficial function investigations, such as in vitro epithelial cell adhesion and in vivo animal model, and whole genome sequencing, should be performed to better validate their possible human health benefits and safety.

Probiotic properties, whole-genome sequence analysis, and safety assessment of BreviBacillus borstelensis S8

In previous studies, BreviBacillus borstelensis S8 exhibited favorable enzyme production characteristics. In this study, the whole genome of Br borstelenensis S8 was sequenced to analyze its genomic sequence information and functions, investigating its genotype and potential probiotic properties. The whole genome sequence of S8 was determined to be 5,559,180 bp with an average GC content of 51.5%. A total of 5406 protein-coding genes were identified and annotated using NCBI Prokaryotic Genome Annotation. By comparing it with the complete genome sequences of Bacillus cereus strains, 1911 orthologous gene families were recognized. Genome analysis revealed the absence of active antibiotic resistance genes, virulence factor genes, and toxin-encoding genes. Safety assessments included hemolytic assays, antibiotic resistance tests, and evaluations of biogenic amine production. Functional gene analysis highlighted S8's metabolic pathways based on its genomic features. The presence of adhesion-related genes, stress response genes (e.g., acid and bile salts), antimicrobial gene clusters, and genes involved in vitamin and essential amino acid synthesis in the genome showcased the probiotic potential of S8. Simultaneously, analysis of hemolytic activity, biogenic amines production ability, acid and bile salt tolerance for the strain S8 also demonstrated its probiotic traits. The genomic exploration and probiotic activity of S8 revealed its potential as a probiotic, offering valuable genomic insights and a foundation for its safe utilization in the food industry.

Probiotic Properties of Lactobacilluscasei and L. plantarum Isolated From Overripe Avocado Pear

Probiotics have been researched for their benefit to gut health. Research for sources of probiotic bacteria has moved from milk to plants, as they have been found to be an inexhaustible source of lactic acid bacteria (LAB). The probiotic properties of lactic acid bacteria isolated from Over ripe Avocado pear on selected bacterial species were assessed. Fifty Unripe Avocado pears were purchased from different markets and allowed to ferment. Enumeration of the microorganisms and pure culture isolation were done on MRS agar supplemented with1.0% CaCO3 at 37 oC for 48 hours; using the pour plate and streak plate methods respectively. The isolates were identified based on their microscopic, macroscopic, biochemical and molecular characteristics. They include Lactobacillus casei and L. plantarum. LAB isolates were tested for their phenol tolerance, bile salt tolerance, acid tolerance and antimicrobial activity. All the isolates were tolerant to simulated gastrointestinal conditions. Growth of the isolates was observed at different temperature between 15 oC to 60 oC. They exhibited tolerance to phenol ranging from 0.085±0.01% to 2.40±0.02%. Tolerance to bile salt ranged from 1.06x101± 0.96x101 to 3.04x103±1.15x102. The acid tolerance level of the LAB obtained ranged from 2.98x102±1.30x102 to 8.78x102±2.22x102. Agar-well diffusion method was used to determine the antimicrobial susceptibility of the bacterial species. The test showed that the isolates were susceptible to the LAB isolates as zones of inhibition were observed. The inhibition zones diameter obtained ranged from 8.35±1.34mm to 13.77±2.21mm. Avocado pear, asides from their high nutritional content, also have the potential to serve as source of probiotic bacteria.

Evaluation of probiotic efficacy of indigenous yeast strain, Saccharomyces cerevisiae Y-89 isolated from a traditional fermented beverage of West Bengal, India having protective effect against DSS-induced colitis in experimental mice.

Increasing awareness regarding health promotion and disease prevention has driven inclusion of fermented foods and beverages in the daily diet. These are the enormous sources of beneficial microbes, probiotics. This study aims to isolate yeast strains having probiotic potential and effectivity against colitis. Initially, ninety-two yeast strains were isolated from Haria, an ethnic fermented beverage of West Bengal, India. Primary screening was done by their acid (pH 4) and bile salt (0.3%) tolerance ability. Four potent isolates were selected and found effective against Entamoeba histolytica, as this human pathogen is responsible to cause colitis. They were identified as Saccharomyces cerevisiae. They showed luxurious growth even at 37 oC, tolerance up to 5% of NaCl, resistance to gastric juice and high bile salt (2.0%) and oro-gastrointestinal transit tolerance. They exhibited good auto-aggregation and co-aggregation ability and strong hydrophobicity. Finally, heat map and principal component analysis revealed that strain Y-89 was the best candidate. It was further characterised and found to have significant protective effects against DSS-induced colitis in experimental mice model. It includes improvement in colon length, body weight and organ indices; reduction in disease activity index; reduction in cholesterol, LDL, SGPT, SGOT, urea and creatinine levels; improvement in HDL, ALP, total protein and albumin levels; decrease in coliform count and restoration of tissue damage. This study demonstrates that the S. cerevisiae strain Y-89 possesses remarkable probiotic traits and can be used as a potential bio-therapeutic candidate for the prevention of colitis.

Application of Prebiotics and Probiotics to Improve Oral and Dental Health by Inhibiting Streptococcus mutans

Background: The most common method to combat microorganisms that cause tooth decay is the use of antibiotics. However, despite the benefits of antibiotics, their use has significant disadvantages, such as the increased resistance of microorganisms to these chemicals. Objectives: A promising and safer alternative is the use of probiotics and prebiotics, which can reduce or inhibit the growth of disease-causing oral microorganisms. Streptococcus salivarius is one such probiotic, while inulin and nisin are the prebiotics commonly recommended for this purpose. Methods: Several assays, including agar bilayer interference, agar well penetration, and microdilution, were used to assess the antibacterial activities of S. salivarius K12 and S. salivarius M18 probiotics, as well as those of the prebiotics nisin and inulin. Additionally, bile salt and low pH tolerance assessments were conducted for the probiotics, and the synthesis of bacteriocins by the probiotics was verified using proteolytic and enzymatic activity assays. Results: The minimum inhibitory concentration (MIC) assay revealed that Streptococcus mutans growth was inhibited by S. salivarius K12 and S. salivarius M18 at a concentration of 75%, and by nisin and inulin at 200 IU/mL. In the well penetration assay, S. salivarius K12 exhibited significantly better results compared to S. salivarius M18. Nisin had significantly greater inhibitory effects on S. mutans compared to inulin. A combination of all four components (S. salivarius K12, S. salivarius M18, nisin, and inulin) resulted in maximum antibacterial activity, indicating a synergistic effect. Based on the agar bilayer assay and the growth inhibition zone diameters, nisin showed the highest antibacterial activity against S. mutans, followed by S. salivarius K12, inulin, and S. salivarius M18. Enzymatic digestion and proteolytic activity assays confirmed the synthesis of bacteriocins by S. salivarius K12 and S. salivarius M18. Both probiotics demonstrated bile salt tolerance at a concentration of 0.3% and surviving rates of 36% and 33%, respectively, at pH 2. Conclusions: The most important finding of the current study is the synergistic effect between the highlighted prebiotics and probiotics, which may offer a novel and effective combination for preventing tooth decay.

Physiological and Technological Properties of Probiotic Lacticaseibacillus rhamnosus GG Encapsulated with Alginate-Chitosan Mixture and Its Incorporation in Whole Milk.

This study developed and evaluated chitosan-sodium alginate capsules containing the probiotic Lacticaseibacillus rhamnosus GG using extrusion and emulsification techniques. The encapsulated L. rhamnosus GG cells were also evaluated for technological and probiotic-related physiological functionalities, as well as when incorporated in UHT and powdered milk. Extrusion (86.01 ± 1.26%) and emulsification (74.43 ± 1.41%) encapsulation techniques showed high encapsulation efficiency and high survival rates of L. rhamnosus GG during 28days of refrigeration and room temperature storage, especially emulsification capsules (> 81%). The encapsulated L. rhamnosus GG cells showed high survival rates during exposure to simulated gastrointestinal conditions (72.65 ± 1.09-114.15 ± 0.44%). L. rhamnosus GG encapsulated by extrusion and emulsification performed satisfactorily in probiotic-related physiological (pH and bile salts tolerance) and technological properties (positive proteolytic activity, diacetyl and exopolysaccharides production, high NaCl tolerance (> 91%), besides having high heat tolerance (> 76%)). L. rhamnosus GG in extrusion and emulsification capsules had high survival rates (> 89%) and did not significantly affect physicochemical parameters in Ultra-High Temperature (UHT) and powdered milk during storage. The results demonstrate that L. rhamnosus GG can be successfully encapsulated with alginate-chitosan as a protective material through extrusion and emulsification techniques. UHT and powdered milk could serve as appropriate delivery systems to increase the intake of this encapsulated probiotic by consumers.

In vitro assessment of probiotic properties of lactic acid bacteria isolated from camel milk: enhancing sustainable foods

Probiotic properties of isolated lactic acid bacteria (LAB) from sustainable foods including camel milk are the potential research domains. For this purpose, camel milk samples (n = 20), from four different herds of Camelus dromedarius, were processed for the identification of LAB strains based on biochemical profiles followed by amplification and sequence analysis of the 16S rRNA gene. The probiotic characteristics, i.e., acids and bile salts tolerance, antimicrobial susceptibility profiles, hemolytic and antimicrobial activities, auto-aggregation assay, and adhesion to HT-29 epithelial cells were determined. Thirteen out of 20 milk samples were initially found positive for the growth of probiotics or LAB which were further confirmed as Lacticaseibacillus casei (5) and Pediococcus pentosaceus (3). The probiotics/LAB strains showed maximum survival (%) = 92.06 ± 1.82 and 81.35 ± 3.64 against acids and bile salts, respectively. The LAB strains were found sensitive to amoxicillin, ceftazidime, imipenem, linezolid, ofloxacin, tetracycline, tobramycin, and vancomycin. None of the LAB strains showed hemolytic activity. L. casei-04 strain showed a maximum zone of inhibition (15.33 ± 0.58) against multidrug-resistant E. coli AZ1 strain whereas, L. casei-05 showed a maximum zone of inhibition (16.33 ± 1.15) against methicillin-resistant S. aureus Saba-1 strain. L. casei-03 showed maximum percentage auto-aggregation (28.65 ± 1.96) at 4 h while L. casei-01 showed (41.10 ± 3.03) at 24 h of incubation. Maximum adhesion was shown by P. pentosaceus-01 (11.14%) followed by L. casei-02 (9.73%). Altogether, the current findings suggested that camel milk has significant potential of providing probiotics/LAB strains into human food chain and enabling camel milk as potential sustainable food.

Comparative genomics of Lactiplantibacillus plantarum reveals the role of the two-component system in response to bile salts stress

Lactiplantibacillus plantarum is a nomadic species that displays adaptive variations in different habitats, particularly in the intestines. Bile salts tolerance is crucial for L. plantarum to survive and exert beneficial properties in this environment. L. plantarum exhibits varying growth capacities under bile salts exposure; however, the molecular mechanisms responsible for this variation are not fully understood. In this study, comparative genomics analysis was used to elucidate the specific genes in bile salts-tolerant L. plantarum and further investigate the mechanisms that contribute to bacterial survival under bile salts. A total of eighty-seven specific genes were identified in the tolerant strains, which were primarily associated with the two-component system and carbohydrate metabolism. Notably, the mutation of a response regulator (agrA) in the two-component system decreased the growth rate of L. plantarum under bile salts exposure, whereas complementation increased tolerance. Metabolomic analysis suggested that the deletion of agrA resulted in the downregulation of pathways associated with bacterial energy metabolism. These findings indicate that L. plantarum responds to harsh environments by sensing bile salts as signaling molecules and subsequently adjusting complex metabolic pathways as a survival strategy. This provides novel insights into the molecular basis of the two-component system contributing to bile salts tolerance.

Lactic Acid Bacteria from Bombyx mori Frass: Probiotic Properties and Antagonistic Activities

Insect farming is gaining attention as a promising area for exploring probiotic bacteria, which can benefit both insect health and various industries. Silkworm farming is a key industry in Thailand; however, challenges such as disease susceptibility and optimising growth require innovative solutions for sustainable practices. Our study addresses this by assessing lactic acid bacteria (LAB) in native Thai silkworm faeces, which accumulate as natural by-products during the rearing process. We conducted biochemical tests, including those for catalase, haemolytic activity, bile salt tolerance, antimicrobial activity, antibiotic susceptibility, and cell surface hydrophobicity, along with taxonomic classification. Out of 102 isolates, eight potential probiotics were selected, with five showing strong probiotic traits like acid and bile salt tolerance and cell surface hydrophobicity, enhancing gut survivability. These isolates also displayed antagonistic activity against pathogens like Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, and Pseudomonas aeruginosa. Safety assessments confirmed their safety, with no haemolytic activity and sensitivity to antibiotics like chloramphenicol and amoxicillin. These LAB isolates (SP04, SP06, SP44, SP64, and SP67), identified as Enterococcus faecalis strain NBRC 100481, show promise as in vitro probiotics for silkworm rearing, calling for further in vivo evaluation.

Assessment of uric acid-lowering activity, safety and stress tolerance of Lactiplantibacillus plantarum MC14 based on whole gene sequencing and phenotyping experiments

With lifestyle changes, hyperuricemia has become a global epidemic. Recent studies have indicated that some probiotic strains may help control serum uric acid. This study aimed to screen probiotic strains with uric acid-lowering potential and subsequently analyze the safety and stress tolerance of the strain in food applications, utilizing phenotyping experiments and genome sequencing. Initially, we isolated a strain of Lactiplantibacillus plantarum MC14 with the best in vitro uric acid-lowering activity. The inosine and guanosine degradation rates of MC14 were 5.00 and 4.11 mg/L·min, respectively, and the XOD inhibition rate was 81.56% ± 3.87%. Gene analysis revealed that the MC14 strain encodes three nucleoside hydrolases and six nucleoside and nucleobase transporter proteins. Subsequently, safety assessments of the MC14 strain in food applications revealed no nitroreductase, amino decarboxylase, or tryptophanase activities, absence of hemolysis, and no antibiotic resistance gene transfer risk. Finally, stress tolerance tests on MC14 indicated robust tolerance to gastrointestinal fluids and bile salts, with survival rates exceeding 98% after 3 h in artificial gastric fluids and approximately 100.49% ± 0.15% after 8 h in artificial intestinal fluids. Additionally, MC14 demonstrated effective antibacterial properties and antioxidant capacity, and its genome was annotated with numerous stress tolerance-related genes, including 18 acid-base stress, 4 bile salt tolerance, 11 heat stress, 3 cold stress, 6 osmotic stress, and 28 oxidative stress. Our findings highlighted MC14 as a promising candidate for probiotic and food applications.

Probiotic Characteristics and Anti-Inflammatory Effects of Limosilactobacillus fermentum 664 Isolated from Chinese Fermented Pickles.

Limosilactobacillus fermentum (L. fermentum) is widely used in industrial food fermentations, and its probiotic and health-promoting roles attracted much attention in the past decades. In this work, the probiotic potential of L. fermentum 664 isolated from Chinese fermented pickles was assessed. In addition, the anti-inflammatory properties and mechanisms were investigated using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Results indicated that L. fermentum 664 demonstrated excellent acid and bile salt tolerance, adhesion capability, antimicrobial activity, and safety profile. L. fermentum 664 downregulated the release of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) stimulated with LPS. Moreover, L fermentum 664 inhibited the nuclear translocation of the nuclear factor κB (NF-κB) and the activation of mitogen-activated protein kinases (MAPKs) induced by LPS. This action was associated with a reduction in reactive oxygen species (ROS) levels and an enhanced expression of heme oxygenase-1 (HO-1) protein. Additionally, whole genome sequencing indicated that L. fermentum 664 contained genes that encode proteins with antioxidant and anti-inflammatory functions, including Cytochrome bd ubiquinol oxidase subunit I (CydA), Cytochrome bd ubiquinol oxidase subunit II (CydB), and NAD(P)H dehydrogenase quinone 1 (NQO1). In conclusion, our study suggested that L. fermentum 664 has the potential to become a probiotic and might be a promising strategy for the prevention of inflammation.

The Assessment of Heat-treated Turkish Type Dry Fermented Sausage (Sucuk) with Some Mixed Probiotic Lactic Acid Bacteria

T his study aims to obtain a combination of probiotic strains resistant to stress conditions, which good effect the qualitative properties of heat-treated sausage. Lactic acid bacteria (LAB) strains were identified by matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS) after phenotypic evaluation and evaluated for probiotic potential including acid and bile salt tolerance, antibiotic resistance, and antibacterial activity. All tested Lactiplantibacillus plantarum strains showed high antibacterial activity. L. plantarum (PeE2) and additionally two Enterococcus strains with the best probiotic properties were selected among the strains. Except for the control group, 6 different sucuk doughs were prepared using them as mixed cultures. The ripened sucuk doughs (18 h, 4 ºC) were subjected to fast-drying (30 min, 60 ºC), heat-treated (64 ºC of inner temperature) and rapid-drying (10 min, 75 ºC) stages. The quality characteristics of samples were determined during storage (days 7, 14, and 21). Coliform bacteria growth ≤1 log cfu/g determined on the final day of storage. The lowest TBARS value was determined in L. plantarum +E. faecium (1:1 ratio) mixed culture. Regarding L* and a* color values, lower results were observed in the control group during storage. Using probiotic culture significantly affected textural values, taste and general acceptability (p<0.05) and contributed to the quality parameters. The result of the study may support the development of a new approach for using new probiotics in meat products.

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 > 0.05). However, significant differences were noted at pH 3, 2 and 1 among the isolates (p < 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 < 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 < 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.

Probiotic functional gene explorations in the genome of Limosilactobacillus fermentum GD5MG

Limosilactobacillus fermentum is an isolate obtained from oral gingival samples of healthy human individuals. The whole genome of Lb. fermentum GD5MG is composed of a circular DNA molecule containing 1,834,134 bp and exhibits a GC content of 52.80 %. The sequencing effort produced 38.6 million reads, each 150 bp in length, resulting in a sequencing depth of 2912.48x. Our examination unveiled a total of 1961 protein-coding genes, 27 rRNA genes, 24 tRNA genes, 3 non-coding RNA genes, and 63 pseudogenes with the use of gene annotations in NCBI Prokaryotic Genome Annotation tool. RAST revealed 1863 coding genes distributed across 209 subsystems, with a predominant involvement in amino acid, carbohydrate, and protein metabolism. Phylogenetic analysis infers that the Lb. fermentum GD5MG shares 281 gene clusters. Furthermore, the genome features showed a single CRISPR locus of 45 bp in length. Three genes associated with adhesion ability (strA, dltD, and dltA) and 26 genes related to acid tolerance, digestive enzyme secretion, and bile salt resistance were identified. Numerous genes associated with oral probiotic properties, comprising adhesion, acid and bile salt tolerance, oxidative stress tolerance, and sugar metabolism, were identified in the genome. Our findings shed light on the genomic characteristics of Lb. fermentum GD5MG, which are probable probiotics with functional benefits in humans.

Encapsulation of Lactiplantibacillus plantarum CRD7 in sub-micron pullulan fibres by spray drying: Maximizing viability with prebiotic and thermal protectants

Pullulan was used as the wall material for microencapsulation of L. plantarum CRD7 by spray drying, while isomalto-oligosaccharides (IMO) was used as prebiotic. Also, the effect of different thermal protectants on survival rate during microencapsulation was evaluated. Taguchi orthogonal array design showed that pullulan at 14 % concentration, IMO at 30 % concentration and whey protein isolate at 20 % rate were the optimized wall material, prebiotic and thermal protectant, respectively for microencapsulation of L. plantarum. FESEM images revealed that the spray-dried encapsulates were fibrous similar to those produce by electrospinning, while fluorescence microscopy ascertained that most of the probiotic cells were alive and intact after microencapsulation. The adsorption-desorption isotherm was of Type II and the encapsulate had specific surface area of 1.92 m2/g and mean pore diameter of 15.12 nm. The typical amide II and III bands of the bacterial proteins were absent in the FTIR spectra, suggestive of adequate encapsulation. DSC thermogram showed shifting of melting peaks to wider temperature range due to interactions between the probiotic and wall materials. IMO at 30 % (w/w) along with WPI at 20 % concentration provided the highest storage stability and the lowest rate of cell death of L. plantarum after microencapsulation. Acid and bile salt tolerance results confirmed that microencapsulated L. plantarum could sustain the harsh GI conditions with >7.5 log CFU/g viability. After microencapsulation, L. plantarum also possessed the ability to ferment milk into curd with pH of 4.62.

Screening of a potential probiotic Lactiplantibacillus plantarum NUC08 and its synergistic effects with yogurt starter

This study aims to identify lactic acid bacteria (LAB) isolates possessing physiological characteristics suitable for use as probiotics in yogurt fermentation. Following acid and bile salt tolerance tests, Lactiplantibacillus plantarum (NUC08 and NUC101), Lacticaseibacillus rhamnosus (NUC55 and NUC201), and Lacticaseibacillus paracasei (NUC159, NUC216 and NUC351) were shortlisted based on intraspecies distribution for further evaluation. Their physiological probiotic properties, including transit tolerance, adhesion, auto-aggregation, surface hydrophobicity, biofilm formation, and antibacterial activity, were assessed. Principal Component Analysis (PCA) indicated that L. plantarum NUC08 was the preferred choice among the evaluated strains. Subsequent investigations revealed that co-culturing L. plantarum NUC08 with 2 yogurt starter strains resulted in a cooperative and synergistic effect, enhancing the growth of mixed strains and increasing their tolerance to simulated gastric and intestinal conditions. Additionally, when Vibrio harveyi bioluminescent reporter strain was used, the 3 cocultured strains cooperated to induce the activity of a quorum sensing (QS) molecule AI-2, hinting a potential connection between phenotypic traits and QS in the cocultured strains. Importantly, LAB viable counts were significantly higher in yogurt co-fermented with L. plantarum NUC08, consistently throughout the storage period. In conclusion, the study demonstrates that the probiotic strain L. plantarum NUC08 can be employed in synergy with yogurt starter strains, affirming its potential for use in the development of functional fermented dairy products.

Physiological properties evaluation and exopolysaccharide biosynthesis of Limosilactobacillus fermentum JL-2 from Chinese traditional fermented dairy product based on genomic insight

Limosilactobacillus fermentum JL-2 with high production of exopolysaccharide (EPS) was screened from the traditional fermented dairy product in Xinjiang district (China) in our previous work. In this study, the whole genome of L. fermentum JL-2 was sequenced and the genomic information and function were studied to excavate its genotype, safety and probiotic properties. The safety evaluation in vitro showed that L. fermentum JL-2 was not hemolytic, sensitive to common antibiotics, and it had a weak biogenic amine production capacity. The genes related to EPS biosynthesis and their relationship with EPS production were discussed. The EPS production of L. fermentum JL-2 was 553.6 mg/L. The genome of L. fermentum JL-2 consisted of a circular 1.98 Mbp chromosome. A variety of genes related to probiotic properties such as acid tolerance, bile salt tolerance, adhesion to gut mucosa and environmental stress resistance were contained. A complete EPS gene cluster was found in the genome of L. fermentum JL-2, and the genes related to EPS biosynthesis such as epsA and epsB were found in L. fermentum JL-2. It is worth noting that EPS biosynthesis protein (gene0099) seemed to play a key role in the EPS synthesis of L. fermentum JL-2. These results revealed the potential of L. fermentum JL-2 as a probiotic at the gene level. Therefore, this study laid a theoretical foundation for further research into its potential probiotic function and its application in practice.