In vitro safety assessment of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 for probiotics use.

The current study aimed to validate the safety of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 in accordance with guidelines of FAO/WHO and ICMR/DBT. In vitro assays such as mucin degradation, hemolysis of blood cells, antimicrobial susceptibility pattern, possession of virulence factors, biogenic amine, and ammonia production were assessed. In results, the cross-streak and co-culture techniques revealed that CRD7 and CRD11 were compatible in vitro. Upon visual inspection through scanning electron and fluorescence microscopy, the integrity of bacterial cell membrane was confirmed even after the encapsulation process. CRD7 and CRD11 were non-hemolytic and showed negative responses to gelatinase, urease, and DNase activities. Non-mucinolytic activity of CRD7 and CRD11 was verified by measuring cell growth rate (p < 0.05) in different modified media followed by SDS-PAGE. High-performance liquid chromatography analysis revealed that both the strains did not produce biogenic amines (putrescine, cadaverine, histamine, and tyramine). Neither of the Lactobacillus strains produced ammonia after growing in BHI broth for 5 days at 37 °C. L-lactate production was highest (p < 0.05) in CRD11 (8.83 g/L), followed by CRD7 (8.16 g/L), whereas the lowest (p < 0.05) D-lactate was registered for encapsulated CRD11 (0.33 g/L) and CRD7 (0.49 g/L). The antibiogram profile determined through minimum inhibitory concentration showed that CRD7 and CRD11 were sensitive to key antibiotics suggested by EFSA except for gentamycin and kanamycin. PCR data on virulence genes demonstrated that both strains were safe for probiotic use. Moreover, CRD7 and CRD11 strains caused insignificant (p > 0.05) changes in the cell viability of Caco-2 cells as estimated by MTT (98.94-99.50%) and NR uptake (95.42-97.03%) assays and showed sensitivity to human serum. According to the results of these evaluated attributes, it is concluded that L. plantarum CRD7 and L. rhamnosus CRD11 are safe, non-toxic to human epithelial cells, and thus may be potentially suitable for various food/feed applications.

Techno-functional characterization of fecal lactobacilli isolates of Bos indicus calves for probiotic properties.

In this study, 105 bacterial colonies were isolated from the feces of newborn healthy Bos indicus calves and 37 isolates were confirmed using morphological, biochemical tests, and genus-specific PCR as lactobacilli. 11 isolates were then short-listed for in vitro probiotic testing based on their ability to dwell under acid and bile stress. Species-level identification using 16S rRNA gene sequencing revealed that they were Ligilactobacillus salivarius. These isolates flourished in 0.4% phenol, depicting resistance in adverse conditions encountered in the gastrointestinal tract. The results of cell surface hydrophobicity were found to be 74.50% for RBL12 and 62.62% for RBL09 in hexadecane and xylene, respectively, and that of auto-aggregation was highest in RBL26 (58.92%). These isolates also produced digestive enzymes like amylase, protease, and ß-galactosidase. Further assays reiterated their antimicrobial and coaggregation potential against diarrhea-causing pathogens like Escherichia coli ATCC-25922 and Salmonella arizonae ATCC-13314. Biosafety assessment revealed that none of the tested isolates were hemolytic and mucinolytic in nature. Furthermore, the antioxidant potential of the isolates was also confirmed using 1,1­diphenyl­2­picrylhydrazyl (DPPH) and ferric ion-reducing antioxidant power (FRAP) assay. Along with efficient utilization of inulin, isolates showed promising adhesion ability to the HT-29 cell line. The current findings hence conclude that these Lactobacillus isolates can be exploited as animal probiotics for potential application in young calves to foster gut health and immunity.

Screening and characterization of Sahiwal cattle calves-origin lactic acid bacteria based on desired probiotic attributes for potential application.

Probiotics are living microorganisms that confer health benefits to host when administered in adequate amounts. To develop novel host-specific probiotic for their application as feed additive, the present study was undertaken to isolate and characterize probiotic strains of indigenous cattle-calves origin. A total of 55 colonies were isolated from 12 healthy calves, with 34 of the isolates being Gram-positive, catalase-negative and vancomycin-resistant. Furthermore, eleven isolates showed tolerance to acid (pH 2.0) and thirteen isolates tolerated bile salts (0.3%). Seven common acid and bile tolerance strains were further investigated for other probiotic attributes and displayed higher (p< 0.05) auto-aggregation and cell surface hydrophobicity values. Moreover, all seven isolates had potent antibacterial activity against pathobiont E. coli as well as significant co-aggregation capacity and enzyme activity. In vitro biosafety assessment revealed that all seven isolates were non-hemolytic, negative for mucin degradation and susceptible to most of the antibiotics. Based on the obtained findings, heatmap and principal component analysis identified four highly effective probiotic candidates confirmed by 16S rDNA sequencing as Limosilactobacillus reuteri SW23, Limosilactobacillus reuteri SW26, Limosilactobacillus reuteri SW27 and Enterococcus faecium SW28, respectively. Further studies on biosafety aspect are warranted for the application of these strains in animal as potential probiotics.HIGHLIGHTSL. reuteri SW23, L. reuteri SW26, L. reuteri SW28 and Enterococcus faecium SW28 were successfully isolated and identified from indigenous calves' feces.These microbes were characterized for potential probiotics attributes.Heatmap analysis and principal component analysis (PCA) was used along with probiotic attributes to select highly effective probiotic candidates.

Dietary supplementation of Aloe vera extract modulates rumen microbes and improves the functional food value of milk by altering phenolic content, antioxidant capacity, and fatty acid profile in lactating goats.

Twenty-four cross-breed (Alpine × Beetal) lactating goats (average body weight: 37.5 ± 2.7 kg and milk yield: 1.78 ± 0.31 kg/day) were chosen for the ninety days duration of the experiment and divided into three groups following a completely randomized design. Group I acted as control (T0) and received only a basal diet as per requirement, whereas group II (T1) and group III (T2) received basal diet added with Aloe vera extract at 2% and 4% of dry matter intake (DMI), respectively. Total phenolic compounds (TPC) and milk antioxidant status were higher (p < 0.05) in T1 and T2 than T0. Relative abundance of methanogen, protozoa, Butyrivibrio proteoclasticus, and Ruminococcus flavefaciens in the rumen were lower, while Butyrivibrio fibrisolvens population was higher (p < 0.05) in T2 and T1 compared to T0. Saturated fatty acids levels in milk were lower, whereas different polyunsaturated fatty acids levels were higher (p < 0.05) in T1 and T2 than T0. Nutritional indices of milk increased in both supplemented groups. Overall, it may be concluded that dietary supplementation of Aloe vera extract at 2% and 4% of DMI may enhance the functional characteristics of milk by boosting TPC and antioxidant status, as well as the FA profile and nutritional quality indices of milk fat.

Host-specific probiotics feeding influence growth, gut microbiota, and fecal biomarkers in buffalo calves.

The current study is aimed to evaluate the effect of host-specific probiotics on the gut microbiome, performance, and select fecal biomarkers of gut health in preruminant buffalo calves. Eight Murrah buffalo calves (3-5 days old; 32.52 ± 0.43 kg average body weight (BW)) were randomly allocated into two groups as follows; 1) Group I (n = 4) fed basal diet alone (CON); 2) Group II (n = 4) supplemented with a lyophilized probiotic formulation at a dose rate of 1 g/day/head (1 × 109 CFU/g) having Limosilactobacillus reuteri BF-E7 and Ligilactobacillus salivarius BF-17 along with basal diet (PF) for 30 days. Results revealed that final BW (kg), average daily gain (g/day), average dry matter intake (g/day), and structural growth measurements were significantly (P < 0.05) increased in the probiotics supplemented group (PF) compared to the control (CON). Fecal pH, fecal moisture, and fecal score were reduced (P < 0.05) in PF than in CON. Moreover, levels of fecal propionate, lactate, and ammonia altered positively in PF compared with CON. The relative abundance of Firmicutes tended to be higher (P = 0.10) in the probiotics fed group than CON. However, the relative abundance of Proteobacteria was significantly lower (P = 0.03) for calves fed probiotics on day 15. A trend was observed in Bacteroides (P = 0.07) and Lactobacillus (P = 0.08) abundances in the feces of the PF than in CON. Overall, it can be concluded that the administration of probiotic formulations significantly improved the performance and gut health of buffalo calves via modulating the gut microbiota composition.

Autochthonous Limosilactobacillus reuteri BFE7 and Ligilactobacillus salivarius BF17 probiotics consortium supplementation improves performance, immunity, and selected gut health indices in Murrah buffalo calves.

Probiotics have emerged as biotherapeutic adjuncts to combat neonatal calf gastrointestinal disorders. Therefore, they are considered a suitable alternative to antibiotics for maintaining a healthy and balanced gut microbiota. Hence, the current investigation was carried out to evaluate the effect of autochthonous probiotics on Murrah buffalo calves. Sixteen calves (5-7 days of age) were randomly divided into four groups. Group I served as control (CT), fed a basal diet with no supplementation. Groups II (LR), III (LS), and IV (CS) were supplemented with Limosilactobacillus reuteri BF-E7, Ligilactobacillus salivarius BF-17, and a consortium of both probiotic strains at a rate of 1x108 CFU/g/calf per day along with the basal diet, respectively. Two previously isolated potential probiotic strains, Limosilactobacillus reuteri BF-E7 and Ligilactobacillus salivarius BF-17, were found to be compatible in vitro. Dietary supplementation of probiotics for sixty days significantly increased (P<0.05) dry matter intake (DMI, g/d), average daily gain (ADG, g/d), net body weight gain (kg), feed conversion efficiency (FCE), and structural growth measurements as compared to control. Furthermore, a considerable (P<0.05) increase in the abundance of beneficial intestinal microbiota (lactobacilli and bifidobacteria) was observed along with improvement in fecal biomarkers like lactate and ammonia, immune status, and reduced fecal score. Upon comparative analysis among treatment groups, the results were found to be better in the probiotic consortium fed group compared to the LR and LS treated groups. The present findings conclusively deduced that autochthonous probiotic consortium might serve as potential candidate for fostering performance, immunity, and gut health biomarkers in Murrah buffalo calves.