Probiotic Properties of Lactic Acid Bacteria Isolated From Neera: A Naturally Fermenting Coconut Palm Nectar.

Probiotic bacteria were isolated from different traditional fermented foods as there are several such foods that are not well explored for their probiotic activities. Hence, the present study was conducted to find the potential of lactic acid bacteria (LAB) as probiotics that were isolated from the sap extract of the coconut palm inflorescence - Neera, which is a naturally fermented drink consumed in various regions of India. A total of 75 isolates were selected from the Neera samples collected aseptically in the early morning (before sunrise). These isolates were initially screened for cultural, microscopic, and biochemical characteristics. The initial screening yielded 40 Gram-positive, catalase-negative isolates that were further subjected to acid - bile tolerance with resistance to phenol. Among 40 isolates, 16 survived screening using analysis of cell surface hydrophobicity, auto aggregation with adhesion to epithelial cells, and gastric-pancreatic digestion for gastrointestinal colonization. The isolates were also assessed for antimicrobial, antibiotic sensitivity, and anti-oxidative potential. The safety of these isolates was evaluated by their hemolytic and deoxyribonuclease (DNase) activities. Based on these results, seven isolates with the best probiotic attributes were selected and presented in this study. These LAB isolates, with 51.91-70.34% survival at low pH, proved their resistance to gastric conditions. The cell surface hydrophobicity of 50.32-77.8% and auto aggregation of 51.02-78.95% represented the adhesion properties of these isolates. All the seven isolates exhibited good antibacterial and antifungal activity, showing hydroxyl-scavenging activity of 32.86-77.87%. The results proved that LAB isolated from Neera exhibited promising probiotic properties and seem favorable for use in functional fermented foods as preservatives.

Antiaflatoxigenic Potential of Cell-Free Supernatant from Lactobacillus plantarum MYS44 Against Aspergillus parasiticus.

The study aims to evaluate the cell-free supernatant (CFS) from Lactobacillus plantarum strain MYS44 against the growth and aflatoxin production by Aspergillus parasiticus MTCC 411. Standard in vitro techniques revealed the potential antifungal activity of CFS of LpMYS44. In poison food technique, it was observed that 6% CFS of LpMYS44 retarded maximum growth. The inhibition of A. parasiticus on peanuts confirmed the ability of CFS of LpMYS44 for biopreservation. Further, CFS of LpMYS44 was purified by chromatography and analyzed by GC-MS. The major antifungal compounds were oleic acid, octanoic acid, butanamide, and decanoic acid derivatives. Twofold concentrated 80 µL of CFS was found to be minimum inhibitory concentration (MIC) of CFS of LpMYS44. CFS of LpMYS44 suppressed the germination and growth of the spores of A. parasiticus. Microscopic observation showed that CFS of LpMYS44 severely affected the hyphal wall of A. parasiticus by the leakage of cytoplasmic content leading to complete destruction. Acidic condition is favorable for CFS of LpMYS44 activity. In poultry feed sample, CFS of LpMYS44 reduced the aflatoxin B1 content by 34.2%, reflecting its potentiality to use as detoxification agent. The multiple antifungal components in CFS of LpMYS44 exhibited antifungal properties against aflatoxigenic A. parasiticus resulted in causing overall morphological changes. Furthermore, we also observed the biopreservative ability of CFS of LpMYS44 against A. parasiticus and AFB1 reduction in for poultry feed. This study makes a contribution to using CFS of LpMYS44 and their applications in food and feed as pretreatment against aflatoxigenic A. parasiticus to reduce or eliminate AFB1 and maybe other aflatoxins, produced by other Aspergillus spp.

Lactobacillus plantarum MYS6 Ameliorates Fumonisin B1-Induced Hepatorenal Damage in Broilers.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium species is a predominant Group 2B carcinogen occurring in maize and maize-based poultry feeds. It is shown to be nephrotoxic, hepatotoxic, neurotoxic, and immunosuppressing in animals. In this study, we report the ameliorating effects of a probiotic strain, Lactobacillus plantarum MYS6 on FB1-induced toxicity and oxidative damage in broilers. A 6-week dietary experiment consisting of 48 broilers was performed in six treatment groups. Probiotic treatment (109 cells/mL) involved pre-colonization of broilers with L. plantarum MYS6 while co-administration treatment involved supplementation of probiotic and FB1-contaminated diet (200 mg/Kg feed) simultaneously. At the end of the treatment period, growth performance, hematology, serum biochemistry, and markers of oxidative stress in serum and tissue homogenates were evaluated in all the broilers. The histopathological changes in hepatic and renal tissues were further studied. The results demonstrated that administration of L. plantarum MYS6 efficiently improved the feed intake, body weight and feed conversion ratio in broilers. It mitigated the altered levels of hematological indices such as complete blood count, hemoglobin, and hematocrit. Serum parameters such as serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, creatinine, cholesterol, triglycerides, and albumin were significantly restored after administering the probiotic in FB1-intoxicated broilers. Additionally, L. plantarum MYS6 alleviated the levels of oxidative stress markers in serum and tissue homogenate of liver. The histopathological data of liver and kidney further substantiated the overall protection offered by L. plantarum MYS6 against FB1-induced cellular toxicity and organ damage in broilers. Our results indicated that co-administration of probiotic along with the toxin had better effect in detoxification compared to its pre-colonization in broilers. Collectively, our study signifies the protective role of L. plantarum MYS6 in ameliorating the FB1-induced toxicity in the vital organs and subsequent oxidative stress in broilers. The probiotic L. plantarum MYS6 can further be formulated into a functional feed owing to its anti-fumonisin attributes and role in mitigating FB1-induced hepatorenal damage.

Effect of γ-irradiation on fumonisin producing Fusarium associated with animal and poultry feed mixtures.

Contamination of animal and poultry feeds by Fusarium and the mycotoxin Fumonisin B1 is frequent in the feed supply chain. The present study evaluated the prevalence of fumonisin B1 producing Fusarium among irradiated and non-irradiated animal and poultry feed mixtures. Further, the efficiency of γ-rays (2.5, 5.0, 7.5, and 10.0 kGy) to minimize Fusarium growth and biosynthesis of fumonisin B1 in artificially inoculated feed was evaluated. A total of 108 feed samples were collected in which 45.37% of feed mixtures were contaminated with Fusarium species. Among the contaminated samples, the frequency levels of F. verticillioides and F. proliferatum were 42.59 and 24.07%, respectively. Out of the 98 Fusarium isolates from feed samples, 84.7% of F. verticillioides and 64.28% of F. proliferatum were positive for FUM1 set of primers. Fumonisin B1 biosynthesis by the FUM1 positive isolates in feed was confirmed by LC/MS which recorded 0.1-45 µg/g of feed. Fungal growth and viable count of Fusarium in PDA medium and feed decreased with increasing irradiation dosage. Interestingly, fumonisin content was 11 µg/g of feed in 2.5 kGy irradiated sample as compared to 5 µg/g of feed in non-irradiated control. Ionizing radiation at 7.5 kGy was found lethal for fungal growth and fumonisin production. Our findings suggest that γ-radiation above 7.5 kGy effectively prevented fungal growth in feed mixtures and minimized the exposure of animal and human life to the potential risk of mycotoxin. Also it is necessary to maintain proper storage system for feeds until consumption.

Antifungal Attributes of Lactobacillus plantarum MYS6 against Fumonisin Producing Fusarium proliferatum Associated with Poultry Feeds.

Fumonisins, being common in occurrence in maize-based feeds, pose a great threat to animal and human health. The present study is aimed at determining the antifungal activity of Lactobacillus plantarum MYS6 against a fumonisin producing fungus, Fusarium proliferatum MYS9. The isolate was subjected to standard tests for determining its probiotic attributes and antifungal properties. L. plantarum MYS6 thrived well at pH 3.0 and 6.0, and exhibited strong resistance up to 3% bile. The isolate showed a high degree of cell surface hydrophobicity corresponding to its strong adhesion to chicken crop epithelial cells. Co-inoculation with the fungus on modified de Man Rogosa Sharpe medium revealed the inhibitory effect of L. plantarum MYS6 on fungal growth and biomass. Observation using scanning electron microscopy showed distortion of hyphal structures, swollen tips and disrupted conidia. Conidia germination inhibition assay restrained germination and showed deformed hyphae. The bioprotective feature of the isolate was evident by the inhibition of fungal development in maize-kernel treated with the cell free supernatant of L. plantarum MYS6. Both the isolate and its extracellular metabolites lowered fumonisin content in feed model up to 0.505 mg/Kg of feed and 0.3125 mg/Kg of feed respectively when compared to the level of 0.870 mg/Kg of feed in control. The major antifungal compounds produced by the isolate were 10-Octadecenoic acid, methyl ester; palmitic acid, methyl ester; heptadecanoic acid, 16-methyl ester; stearic acid and lauric acid. L. plantarum MYS6 reduced 61.7% of fumonisin possibly by a binding mechanism. These findings suggest the application of L. plantarum MYS6 as an efficient probiotic additive and biocontrol agent in feed used in poultry industry. Additionally, the antifungal metabolites pose a conspicuous inhibition of Fusarium growth and fumonisin production.