Preservation of Mimosa tenuiflora Antiaflatoxigenic Activity Using Microencapsulation by Spray-Drying.

Mimosa tenuiflora aqueous extract (MAE) is rich in phenolic compounds. Among them, condensed tannins have been demonstrated to exhibit a strong antioxidant and antiaflatoxin B1 activities in Aspergillus flavus. Since antioxidant capacity can change with time due to environmental interactions, this study aimed to evaluate the ability of encapsulation by spray-drying of Mimosa tenuiflora aqueous extract to preserve their biological activities through storage. A dry formulation may also facilitate transportation and uses. For that, three different wall materials were used and compared for their efficiency. Total phenolic content, antioxidant activity, antifungal and antiaflatoxin activities were measured after the production of the microparticles and after one year of storage at room temperature. These results confirmed that encapsulation by spray-drying using polysaccharide wall materials is able to preserve antiaflatoxin activity of Mimosa tenuiflora extract better than freezing.

Protective Effect of Nigella sativa and Nigella damascena Fixed Oils Against Aflatoxin Induced Mutagenicity in the Classical and Modified Ames Test.

The antioxidant and mutagenic/antimutagenic activities of the fixed oils from Nigella sativa (NSO) and Nigella damascena (NDO) seeds, obtained by cold press-extraction from the cultivar samples, were comparatively investigated for the first time. The antimutagenicity test was carried out using classical and modified Ames tests. The fatty acid composition of the fixed oils was characterized by gas chromatography-mass spectrometry (GC-MS) while the quantification of thymoquinone in the fixed oils was determined by UPC2 . The main components of the NSO and NDO were found to be linoleic acid, oleic acid, and palmitic acid. The results of the Ames test confirmed the safety of NSO and NDO from the viewpoint of mutagenicity. The results of the three antioxidant test methods were correlated with each other, indicating NDO as having a superior antioxidant activity, when compared to the NSO. Both NSO and NDO exhibited a significant protective effect against the mutagenicity induced by aflatoxin B1 in Salmonella typhimurium TA98 and TA100 strains. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, a significant increase in antimutagenic activity was observed, suggesting that the degradation of aflatoxin B1 epoxides by these oils may be a possible antimutagenic mechanism. It is worthy to note that this is the first study to assess the mutagenicity of NSO and NDO according to the OECD 471 guideline and to investigate antimutagenicity of NDO in comparison to NSO against aflatoxin.

Bacillus amyloliquefaciens B10 inhibits aflatoxin B1-induced cecal inflammation in mice by regulating their intestinal flora.

Aflatoxin B1 is a mycotoxin that widely exists in feed and has a great impact on human and animal health. This study aimed to examine whether Bacillus amyloliquefaciens B10 protected against aflatoxin B1-induced cecal inflammation in mice. It was found that Bacillus amyloliquefaciens B10 could significantly improve the effects of AFB1 on body weight and intestinal inflammation of mice and enhance the expression of tight-junction protein. Compared with the CON group, the combination of AFB1 and B10 significantly increased the abundance of Actinobacteria and Bacilli in a collaborative manner, and significantly reduced the abundance of Ruminococcae, Lactobacillaceae and Clostridia. Meanwhile, the results showed that the abundance of Bacterides and Bacterdia in AFB1 + B10 group was significantly lower than that of AFB1 group, and the Firmicutes increased significantly. Bacillus amyloliquefaciens B10 can be used as a feed additive and alleviate cecal inflammation induced by AFB1 in mice by regulating intestinal flora.

Mimosa tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that contaminates numerous crops pre- and post-harvest. To protect foods and feeds from such toxins without resorting to pesticides, the use of plant extracts has been increasingly studied. The most interesting candidate plants are those with strong antioxidative activity because oxidation reactions may interfere with AFB1 production. The present study investigates how an aqueous extract of Mimosa tenuiflora bark affects both the growth of Aspergillus flavus and AFB1 production. The results reveal a dose-dependent inhibition of toxin synthesis with no impact on fungal growth. AFB1 inhibition is related to a down-modulation of the cluster genes of the biosynthetic pathway and especially to the two internal regulators aflR and aflS. Its strong anti-oxidative activity also allows the aqueous extract to modulate the expression of genes involved in fungal oxidative-stress response, such as msnA, mtfA, atfA, or sod1. Finally, a bio-guided fractionation of the aqueous extract demonstrates that condensed tannins play a major role in the anti-aflatoxin activity of Mimosa tenuiflora bark.

Untangling the multi-regime molecular mechanism of verbenol-chemotype Zingiber officinale essential oil against Aspergillus flavus and aflatoxin B1.

Aflatoxin B1 (AFB1), the natural polyketide produced by Aspergillus flavus, has a potent carcinogenic effect on humans as well as animals. In the present study, the antifungal and anti-aflatoxigenic B1 activity of chemically characterized Zingiber officinale essential oil (ZOEO) was investigated via in vitro analysis aided with molecular dynamics (MD) approaches. The GC-MS results revealed verbenol (52.41%) as the major component of oil. The antifungal and anti-aflatoxigenic activity of ZOEO was found to be 0.6 µl/ml and 0.5 µl/ml respectively. In-vitro analysis targeting the cell membrane, mitochondria and carbohydrate catabolism elucidated the probable antifungal mode of action. Further, docking and MD simulation results confirmed the inhibitory action of verbenol on the structural gene products (Nor-1, Omt-1, and Vbs) of aflatoxin biosynthetic machinery. Biochemical assays revealed the fungitoxic potential of the ZOEO while, computational results infers the stabilizing effects on the gene products upon verbenol binding leads to the impairment in its functionality. This is the first attempt to assess the multi-regime anti-AFB1 mechanism of verbenol chemotype-ZOEO targeting the Nor-1, Omt-1, and Vbs via computational approaches.

Evaluation of berberine efficacy in reducing the effects of aflatoxin B1 and ochratoxin A added to male broiler rations.

Many types of mycotoxins are found in food sources contaminated with fungi, and if these are ingested in large quantities or over a long period, they can affect the health of humans and domestic animals. Berberine (BBR) is a plant alkaloid with multiple pharmacological functions. This study aimed to investigate the effect of different levels of the plant alkaloid BBR on reducing toxic effects of aflatoxin B1 (AFB) and ochratoxin A (OTA) in broilers by examining performance characteristics, blood biochemistry, antioxidant systems, ileum morphology, and histopathology of the liver. The experiment was performed with 288 Ross 308 broilers reared in floor pens for 42 d in a randomized design with 9 treatments. Each treatment was replicated 4 times, and each replicate contained 8 chicks. Experimental treatments included (1) negative control diet with no additives (NC); (2) NC + 2 ppm AFB (positive control AFB; PCAFB); (3) NC + 2 ppm OTA (positive control OTA; PCOTA); (4) PCAFB + 200 mg/kg BBR; (5) PCAFB + 400 mg/kg BBR; (6) PCAFB + 600 mg/kg BBR; (7) PCOTA + 200 mg/kg BBR; (8) PCOTA + 400 mg/kg BBR; and (9) PCOTA + 600 mg/kg BBR. Compared with NC, feeding PCAFB and PCOTA diets reduced average daily feed intake, weight gain, serum concentrations of superoxide dismutase, glutathione peroxidase, and the length and width of ileum villi (P < 0.05). At the same time, these parameters increased in birds fed PCAFB or PCOTA diets supplemented with 600 mg/kg of BBR (P < 0.05). Feeding PCAFB and PCOTA diets increased feed conversion ratio (FCR), serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) activities, serum urea, and liver lesions compared with NC. By contrast, compared with PCAFB and PCOTA, adding 600 mg/kg BBR decreased FCR, AST, LDH, ALT, and GGT activities, urea, and liver lesions (P < 0.05). Overall, supplementation with 600 mg/kg BBR may improve growth performance, liver function, and antioxidant status of broilers fed diets contaminated with AFB and OTA.

Eugenol loaded chitosan nanoemulsion for food protection and inhibition of Aflatoxin B1 synthesizing genes based on molecular docking.

The present investigation entails the fabrication and characterization of nanometric emulsion of eugenol (Nm-eugenol) encompassed into chitosan for assessing bio-efficacy in terms of in vitro antifungal actions, antiaflatoxigenic potential, and in situ preservative efficacy against Aspergillus flavus infestation and aflatoxin B1 (AFB1) mediated loss of dietary minerals, lipid triglycerides and alterations in composition of important macronutrients in stored rice. Nm-eugenol characterized by SEM, XRD, and FTIR exhibited biphasic burst release of eugenol. Reduction in ergosterol and methylglyoxal (AFB1-inducer) content after Nm-eugenol fumigation depicted biochemical mechanism of antifungal and antiaflatoxigenic activities. In silico 3D homology docking of eugenol with Ver-1 gene validated molecular mechanism of AFB1 inhibition. Further, significant protection of rice seeds from fungi, AFB1 contamination and preservation against loss of rice minerals, macronutrients and lipids during storage suggested deployment of chitosan as a biocompatible wall material for eugenol encapsulation and application as novel green preservative for food protection.

Nanostructured Pimpinella anisum essential oil as novel green food preservative against fungal infestation, aflatoxin B1 contamination and deterioration of nutritional qualities.

Application of synthetic preservatives to control the contamination of stored food commodities with aflatoxin B1 causing considerable loss in nutritional value is a major challenge. However, employment of essential oils for protecting food commodities is much limited due to high volatility, and increased susceptibility to oxidation. Therefore, objective of the present investigation was encapsulation of Pimpinella anisum essential oil in chitosan nanobiopolymer (CS-PAEO-Nm) to improve its bioefficacy, and sensorial suitability for application in food system. The synthesized CS-PAEO-Nm was characterized through SEM, FTIR, and XRD and evaluated for improved biological activity. The CS-PAEO-Nm exhibited improved antifungal (minimum inhibitory concentration = 0.08 µL/mL) and antiaflatoxigenic (minimum aflatoxin inhibitory concentration = 0.07 µL/mL) activities. CS-PAEO-Nm treatment significantly inhibited ergosterol, enhanced leakage of ions and induced impairment in defense enzymes (p < 0.05). In situ minerals and macronutrient preservation, and acceptable sensorial characteristics suggested possible recommendation of nanoencapsulated PAEO as potential safe green food preservative.

Fabrication of volatile compounds loaded-chitosan biopolymer nanoparticles: Optimization, characterization and assessment against Aspergillus flavus and aflatoxin B1 contamination.

The study demonstrates the use of chitosan as a carrier agent of designed antifungal formulation (CME 4:1:1) based on a combination of plant compounds such as trans- cinnamaldehyde (C), methyl eugenol (M), and estragole (E). The formulation was encapsulated inside the chitosan biopolymer nanomatrix (Ne-CME) and characterized by SEM, FTIR, and XRD. The Ne-CME exhibited enhanced antifungal and aflatoxin B1 inhibitory effect compared to the individual compounds and unencapsulated form. Ne-CME (0.04 µl/ml) caused significant protection of Piper longum fruit from fungal (90.05%) and aflatoxin B1 (100%) contamination and had no significant negative effects on its nutritional properties. In addition, the probable antifungal mechanism of Ne-CME was investigated using in-silico (effect on Omt-1 and Vbs structural genes of AFB1 biosynthesis) and biochemical (perturbances in the cell membrane, carbohydrate catabolism, methyl-glyoxal, mitochondrial membrane potential, and antioxidant defense system) assay.

Assessing the antifungal and aflatoxin B1 inhibitory efficacy of nanoencapsulated antifungal formulation based on combination of Ocimum spp. essential oils.

The aim of the study was to explore the antifungal and aflatoxin B1 inhibitory efficacy of nanoencapsulated antifungal formulation. Mixture design response surface methodology (RSM) was utilized to design the antifungal formulation (SBC 4:1:1) based on the combination of chemically characterized Ocimum sanctum (S), O. basilicum (B), and O. canum (C) against Aspergillus flavus. The SBC was incorporated inside the chitosan nanomatrix (Ne-SBC) using an ultrasonic probe (40 kHz) and interactions were confirmed by SEM, FTIR and XRD analysis. The results showed that the Ne-SBC possessed enhanced antifungal and aflatoxin B1 inhibitory effect over the free form of SBC. The biochemical and in silico results indicate that the antifungal and aflatoxin B1 inhibitory effect was related to perturbance in the plasma membrane function (ergosterol biosynthesis and membrane cation) mitochondrial membrane potential, C-sources utilization, antioxidant defense system, and the targeted gene products Erg 28, cytochrome c oxidase subunit Va, and Nor-1. In-situ observation revealed that Ne-SBC effectively protects the Avena sativa seeds from A. flavus and AFB1 contamination and preserves its sensory profile. The findings suggest that the fabrication of SBC inside the chitosan nano-matrix has promising use in the food industries as an antifungal agent.

Improvement of in vitro and in situ antifungal, AFB1 inhibitory and antioxidant activity of Origanum majorana L. essential oil through nanoemulsion and recommending as novel food preservative.

Origanum majorana essential oil (OmEO) encapsulated into chitosan nanoemulsion is being reported as a novel preservative of stored food items against fungi, aflatoxin B1 (AFB1) contamination and lipid peroxidation. The major component of OmEO identified through GC-MS was terpinen-4-ol (28.92%). HR-SEM, FTIR and XRD analyses confirmed successful encapsulation of OmEO into chitosan nanoemulsion (OmEO-CsNe). The results showed remarkable improvement in efficacy after nanoencapsulation, since OmEO-CsNe completely inhibited the growth and AFB1 production by Aspergillus flavus at 1.0 µL/mL, which was 2.5 and 1.5 µL/mL, respectively for OmEO. The inhibition of ergosterol followed by release of cellular ions and 260 and 280 nm absorbing materials demonstrated plasma membrane as possible antifungal target. Inhibition of methylglyoxal confirmed antiaflatoxigenic mode of action. OmEO-CsNe showed enhanced antioxidant activity (IC50 = 14.94 and 5.53 µL/mL for DPPH and ABTS, respectively) and caused in situ inhibition of lipid peroxidation and AFB1 production in maize (third most important staple crop after wheat and rice) without altering their sensory attributes and presented safety profile (LD50 = 11,889 µL/kg) when tested on mice. The findings indicate that the encapsulation considerably enhances the performance of OmEO, therefore can be recommended as a promising antifungal agent to extend the shelf-life of food items.

Saccharomyces cerevisiae as a probiotic agent and a possible aflatoxin B1 adsorbent in simulated fish intestinal tract conditions / Saccharomyces cerevisiae como agente probiótico e possível adsorvente de aflatoxina B1 em condições simuladas do trato intestinal de peixes

The aim of this study was to evaluate in vitro the probiotic potential and absorption of Saccharomyces cerevisiae for the aflatoxin B1 in simulated fish intestinal tract conditions. Three yeast strains were used, two from brewery: S. cerevisiae RC1 and S. cerevisiae RC3 and one from a fish farming environment: S. cerevisiae A8L2. The selected yeasts were subjected to the following in vitro tests: homologous inhibition, self-aggregation, co-aggregation, antibacterial activity, gastrointestinal conditions tolerance and adsorption of AFB1. All S. cerevisiae strains showed good capability of self-aggregation and co-aggregation with pathogenic bacteria. All yeast strains were able to survive the gastrointestinal conditions. In acidic conditions, the factors (strain vs. time) had interaction (P=0.0317), resulting in significant variation among the strains tested in the time periods analyzed. It was observed that there was also interaction (P=0.0062) in intestinal conditions, with an increased number of cells in the 12-hour period for all strains tested. In the adsorption test, the A8L2 strain was statistically more effective (P<0.005) for both AFB1 concentrations evaluated in this study (10 and 25ng/mL). Thus, it was observed that the strains of S. cerevisiae have potential probiotic and adsorbent of AFB1.(AU)

Objetivou-se, com esta pesquisa, avaliar in vitro o potencial probiótico e adsorvente de Saccharomyces cerevisiae para aflatoxina B1 em condições simuladas do trato intestinal de peixes. Foram utilizadas três cepas de leveduras, sendo duas provenientes de cervejaria: S. cerevisiae RC1 e S. cerevisiae RC3, e uma de ambiente de piscicultura: S. cerevisiae A8L2. As leveduras selecionadas foram submetidas aos seguintes testes in vitro: inibição homóloga, autoagregação, coagregação, atividade antibacteriana, viabilidade às condições gastrointestinais e adsorção de AFB1. Todas as estirpes de S. cerevisiae mostraram boa capacidade de autoagregação e coagregação com bactérias patogênicas. Todas as estirpes de levedura foram capazes de sobreviver às condições gastrointestinais. Em condições ácidas, os fatores (cepa x tempo) tiveram interação (P=0,0317), resultando em variações significativas entre as cepas testadas nos períodos de tempo analisados. Observou-se que também houve interação (P=0,0062) em condições intestinais, havendo um aumento do número de células no período de 12h para todas as cepas avaliadas. No ensaio de adsorção, a estirpe A8L2 foi a mais eficaz estatisticamente (P<0,005), para as duas concentrações de AFB1 avaliadas neste estudo (10 e 25ng. mL-1). Dessa forma, conclui-se que as cepas de Saccharomyces cerevisiae possuem potencial probiótico e adsorvente de AFB1.(AU)

Unravelling the antifungal and anti-aflatoxin B1 mechanism of chitosan nanocomposite incorporated with Foeniculum vulgare essential oil.

The study reports the antifungal and aflatoxin B1 inhibitory efficacy of chemically characterized chitosan-based nanoencapsulated Foeniculum vulgare Mill. essential oil (Ne-FvEO), and its effect on sensory and nutritional properties of Sorghum bicolor. The Ne-FvEO was characterized through SEM, FTIR and XRD. The Ne-FvEO demonstrated superior antifungal (0.4 µl/ml) and aflatoxin B1 inhibitory (0.3 µl/ml) performance than the free FvEO. The biochemical studies reveal the significant alteration in ergosterol content, ions leakage, mitochondrial membrane potential, C-sources utilization and antioxidant defense system in A. flavus exposed to Ne-FvEO. The 3D modeling of the Nor-1 gene product was done using the I-TASSER server and validated by the Ramachandran plot. The in-situ result reveals that the Ne-FvEO significantly preserved the nutritional and sensory characteristics of S. bicolor seeds. Therefore, Ne-FvEO could be used as a green preservative agent to enhance the shelf-life of the food commodities.

Effects of zinc chelating nutrients on Aflatoxin production in Aspergillus flavus.

Aflatoxins are carcinogenic metabolites produced by Aspergillus and Penicillium spp. Aflatoxin contamination of food is a serious health hazard. Some metal ions (such as Zn2+) affect Aspergillus growth and aflatoxin biosynthesis. Presence of zinc in the growth medium incites aflatoxin production. This study investigates the effect of zinc binding amino acids and peptides on aflatoxin synthesis in indigenous toxigenic Aspergillus species isolated from agro-ecological zones in Northern Iran. Zinc (II) chelating nutrients (such as Histidine (His), Cysteine (Cys), Histidine-Cysteine (His-Cys), and triple peptide (Asn-Cys-Ser) were added to the growth medium of toxigenic Aspergillus isolates and incubated at temperature range of 25-40 °C. Aflatoxin production on different culture media was tested using ELISA. Addition of cysteine to Sabouraud dextrose broth (SDB) medium significantly reduced aflatoxin production, which could be related to its zinc chelating property. Aflatoxin production was drastically restrained at high concentration of His, especially in combination with Cys, at high pH values and incubation temperature (pH = 7.5, temperature = 40 °C). Aflatoxin production was low in presence of triple peptide (Asn-Cys-Ser) at concentration of 500 mg/L. From the application perspective, natural zinc chelators can be used as harmless aflatoxin-production inhibitors.

Effects of Melatonin and Flavonoid-Rich Fractions of Chromolaena odorata on the Alteration of Proinflammatory Cytokines and Nitric Oxide Induced by Aflatoxin B1 in the Gastric Mucosa of Wistar Rats.

In this study, we investigated serum interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) after ingestion of aflatoxin B1 (AFB1) in rats. We also studied the effects of nitric oxide (NO) on the stomach after consumption of AFB1. Therefore, we hypothesized that a standard anti-inflammatory agent-melatonin (MEL), and the flavonoid-rich fractions from Chromolaena odorata (FRFC) could counteract the deleterious effects of IL-1ß, TNF-α, and NO after consumption of AFB1. Thirty-five Wistar rats (211.86 ± 27.23 g) were randomly selected into 5 groups, with 7 rats in each group. Group A (control); all rats in groups B, C, D, and E received 2.5 mg/kg AFB1 each orally on day 5, whereas those of groups C, D, and E received oral administration of 10 mg/kg MEL, 50 mg/kg FRFC1, and 100 mg/kg FRFC2, respectively, for 7 days. All of them were killed on the 8th day, 24 h after last treatment. Serum samples were analyzed for IL-1ß and TNF-α, whereas stomach tissue was evaluated for NO level. Significant (P < 0.5) increase in serum IL-1ß and TNF-α in rats given AFB1 only was recorded when compared with those in the control group. Conversely, we observed significant reduction in serum IL-1ß and TNF-α in all the groups that received MEL, FRFC1, and FRFC2 after pretreatment with AFB1 when compared with those that were given AFB1 only. In addition, there was a significant increase in NO in rats given AFB1 only when compared with control, whereas reduction in NO was significant in the groups C, D, and E that were given MEL, FRFC1, and FRFC2, respectively, when compared with AFB1 group. MEL and FRFC may be responsible for the prevention of increased gastric mucosal NO and inflammatory effects of proinflammatory cytokines induced by AFB1.

Chemically characterised Pimenta dioica (L.) Merr. essential oil as a novel plant based antimicrobial against fungal and aflatoxin B1 contamination of stored maize and its possible mode of action.

The chemical characterisation of Pimenta dioica essential oil (PDEO) revealed the presence of 50 components, amongst which α-Terpineol (30.31%) was the major component followed by ß-Linalool (6.75%) and γ-Terpinene (4.64%). The oil completely inhibited the growth of aflatoxin B1 secreting strain Aspergillus flavus LHP-VS-8 and aflatoxin B1 production at 2.5 µL/mL and 1.5 µL/mL, respectively. The oil caused dose dependent reduction of methylglyoxal (an AFB1 inducer), enhanced leakage of Ca2+, Mg2+ and K+ ions and significantly reduced ergosterol content of fungal plasma membrane. During in situ experiments, PDEO exhibited complete protection of fumigated maize cob slices from fungal infestation without affecting seed germination. The chemically characterised PDEO is recommended as a plant based preservative and shelf life enhancer of food commodities by preventing fungal growth, AFB1 production and lipid peroxidation. This is the first report on PDEO as inhibitor of AFB1 secretion and methylglyoxal biosynthesis.

Dietary Supplementation with sodium bentonite and coumarin alleviates the toxicity of aflatoxin B1 in rabbits.

Eighty-four male New Zealand White rabbits with average body weight 778 ±â€¯65 g were blocked into four groups to evaluate the ability of sodium bentonite and coumarin in alleviating the toxicity of aflatoxin B1. The first group was fed on a diet without any treatment (CON), while the remaining three diets were added with aflatoxin B1 at 0.25 ppm diet. Diet fed to the third and fourth group of rabbits were further supplemented with sodium bentonite at 5 g/kg (SOB) and coumarin at 5 g/kg (COU) of the diet, respectively. Feeding aflatoxin-contaminated diet (AFL) caused necrosis of liver tissue and reduced the weight gain, average daily gain, feed conversion ratio, nutrient digestibility coefficients, and nitrogen balance of rabbits. This, in turn, was reflected as a reduction in carcass characteristics. The serum collected from rabbits fed aflatoxin-contaminated diet showed decreased levels of total protein, albumin, globulin, glucose, total cholesterol, and triglycerides, and increased concentrations of urea, creatinine, and liver enzymes. Further, aflatoxin diet increased the cecal pH, and decreased the ammonia nitrogen, total volatile fatty acids, and individual fatty acids proportion of cecal fluid. Supplementing sodium bentonite and coumarin at 5 g/kg diet reduced the negative effects of aflatoxin B1 on growth performance, digestibility of nutrients, biochemical parameters, carcass characteristics, and cecal fermentation profile. Furthermore, the coumarin-supplemented group showed better body weight gains and carcass weights compared to the rabbits fed with diets containing sodium bentonite. In conclusion, both sodium bentonite and coumarin supplementation was beneficial in ameliorating the toxicity of aflatoxin B1. Further, the increased body weight gains and better-feed conversion in coumarin-supplemented rabbits project the coumarin as a better anti-aflatoxigenic supplement.

Nanoencapsulated plant-based bioactive formulation against food-borne molds and aflatoxin B1 contamination: Preparation, characterization and stability evaluation in the food system.

A novel synergistic formulation (TML) based on the combination of thymol (T), methyl cinnamate (M), and linalool (L) has been prepared using the mixture design assay. Nanoencapsulation of developed formulation TML (Ne-TML) was prepared and characterised by SEM, XRD and FTIR. The Ne-TML was assessed for its antifungal and anti-aflatoxin B1 potential in vitro and in the food systems (Pennisetum glaucum L.), and also examined its effects on organoleptic properties. The Ne-TML cause complete inhibition of growth and AFB1 production at 0.3 µl/ml and 0.2 µl/ml. In-situ results revealed that Ne-TML exhibited maximum protection from fungal (75.40%) and aflatoxin B1 contamination (100%) at 0.3 µl/ml during six months of storage. The speculated antifungal mode of action of Ne-TML was related to the decrease in ergosterol content, membrane ions leakage, impairment in carbon-source utilization, mitochondrial functioning, anti-oxidative defence system (SOD, CAT, and GR) and Ver-1 gene of aflatoxin B1 biosynthesis.

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.

Fusion expression and anti-Aspergillus flavus activity of a novel inhibitory protein DN-AflR.

The regulatory gene (aflR) encodes AflR, a positive regulator of transcriptional pathway that activates aflatoxin biosynthesis. It has been demonstrated in our laboratory that L-Asp-L-Asn (DN) extracted from Bacillus megaterium inhibited the growth of Aspergillus flavus. We fused gene encoding DN with the gene encoding specific dinuclear zinc finger cluster protein of AflR, then fusion protein competed with the AflS-AflR complex for the AflR binding site and significantly improved anti-A. flavus activity (growth of A. flavus and biosynthesis of aflatoxin B1) of DN. The fusion gene dn-aflR was cloned into pET32a and recombinant plasmid was introduced into Escherichia coli BL21. The highest expression was observed after 10 h induction and fusion protein was purified by affinity chromatography column. Compared with DN, the novel fusion protein DN-AflR significantly inhibited the growth of A. flavus and biosynthesis of aflatoxin B1 (P < 0.05). This study promoted the use of competitive inhibition of fusion proteins to reduce the expression of regulatory genes in the biosynthetic pathway of aflatoxin. Moreover, it provided more supports for deep research and industrialization of such novel anti-A. flavus bio-inhibitors and biological control of microbial contamination.