Toxigenic Aspergillus Flavus Research Articles

Sodium alumino silicate (SAS) neem nanocomposite – A novel seed storage tool against toxigenic Aspergillus flavus causing deterimental effects on seeds health and quality of rice

Infection of rice grains in the field during harvest and storage by Aspergillus flavus, a toxigenic mold causes significant losses to market acceptability of seeds due to its detrimental effects on seed health and quality parameters. Production of aflatoxin by A. flavus interferes with physiological factors affecting the quality of seeds during storage in addition to nutritional losses to the grains. Chemical seed treatments are generally not recommended during storage due to their adverse effects on seed germination and vigor. Leaves of the neem tree (Azadirachta indica) are well known for antifungal properties against phytopathogenic fungi such as A. flavus. The paper presents the effect of neem leaf extract adsorbed onto sodium alumino-silicate (SAS) nanoparticles against A. flavus in rice seeds during storage. SAS-Neem nanocomposite, a novel unexplored seed storage material against toxigenic A. flavus, was synthesized for augmented antitoxin and seed invigorating potential. The nanocomposite was characterized using an array of microscopic and spectroscopic methods to evaluate the adsorption of neem leaf extract components on SAS nanoparticles. The antifungal mechanism of the nanocomposite was evaluated via scanning electron microscope, phase contrast, and fluorescence microscopy. The toxin analysis of pathogen-inoculated seeds was carried out at monthly intervals for 6 months during storage of two rice varieties, i.e. PR114 and Pusa-Basmati 1121. The results indicated that the nSAS-Neem composite used as seed coating material maintained significantly higher seed germination (86.23 and 81.49%) and minimized seed rot (6.70 and 5.30%, respectively) in the two varieties under study than the control after 6 months of storage. There was a notable decrease in toxin concentration from 20.58 to 11.19 μg/kg and 19.16 to 12.58 μg/kg in seeds of the two varieties i.e. PR114 and Pusa Basmati 1121, respectively. The nano-composite proved effective against toxigenic A. flavus during seed storage of rice without compromising the seed quality parameters.

An atoxigenic Aspergillus flavus PA67 from Shandong province exhibits potential in biocontrol against toxigenic Aspergillus flavus, Sclerotium rolfsii, and Fusarium proliferatum

Peanuts and corn are susceptible to various soil-borne fungi, leading to significant economic losses. Atoxigenic Aspergillus flavus have been widely used as biocontrol agents for managing aflatoxin contamination because of their minimal environmental impact, strong competitive ability, and sustained inhibition effect. After multiple identifications and cluster amplification pattern (CAP) analysis, three atoxigenic A. flavus PA04, PA10 and PA67 were isolated from peanut samples in Shandong Province, which can reduce aflatoxin levels by up to 90 %. Our study revealed that atoxigenic A. flavus also competed vigorously with Sclerotium rolfsii and Fusarium proliferatum for nutrition and space, achieving notable inhibition rates of up to 90.4 % and 90.6 %, respectively. The supernatants of atoxigenic A. flavus also inhibited the growth of S. rolfsii and F. proliferatum, with PA67 demonstrating the most significant effect. Whole genome sequencing revealed that PA67 contains multiple glycoside hydrolases and metabolites with antifungal activity. The kojic acid production of PA67 was higher than that of PA04 and PA10, reaching 17.48 g/L, which has a significant inhibition on sclerotia germination. PA67 supernatant significantly inhibited the hyphae growth of S. rolfsii and F. proliferatum, and down-regulated genes related to sclerotia and fumonisin formation. This study demonstrates the biocontrol potential of PA67 against three soil-borne fungi and is the first investigation of atoxigenic A. flavus to inhibit S. rolfsii and F. proliferatum.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

The impact of fullerenol nanoparticles on the growth of toxigenic Aspergillus flavus and aflatoxins production in vitro and in corn flour.

Antifungal and antimycotoxigenic activity of fullerenol nanoparticles (FNPs) were investigated on Aspergillus flavus growth isolated from a real food sample and aflatoxins (AFs) (AFB1 and AFB2 ) production. The final FNPs concentrations in in vitro and in commercial corn flour after the stationary incubation period of 7 and 14 days were in the range 0.16-80µg/mL and 0.16-80µg/g, respectively. Nanocharacterization of FNPs revealed an average size of 5-20nm and a zeta potential of -35mV. The highest degree of A. flavus mycelium growth inhibition (28%) after 7 days was observed for applied FNP concentration of 8.0µg/mL, while after 14 days FNP concentration of 0.32µg/mL led to the maximal inhibition of A. flavus mycelium growth (36%). Spearman's correlations analysis revealed a strong positive correlation between AFB1 and AFB2 concentrations in YES broth after 7 (R=0.994, p<0.05) and 14 days (R=0.976), as well as between AFs concentrations and A. flavus mycelium mass after 7 (R=0.786 for AFB1 and R=0.766 for AFB2 ) and 14 days (R=0.810 for AFB1 and R=0.833 for AFB2 ). Paired samples t-test showed the existence of a statistically significant difference (p<0.05) between the produced AFs concentrations after the incubation of 7 and 14 days. Regarding the artificially inoculated corn flour the lower applied FNP concentrations (0.16-0.8µg/g) achieved a reduction of AFB1 up to 42% and 60% after 7 and 14 days, respectively.

INHIBITION OF AFLATOXIGENIC FUNGI BY LACTIC ACID BACTERIA ISOLATED FROM FERMENTED MAIZE SLURRY

Toxigenic fungi are persistent contaminants of food and food products. This study aims at the biocontrol of toxigenic fungi. The inhibitory activity of Lactic acid bacteria (LAB) isolated from fermented cereal slurry against toxigenic Aspergillus isolated from maize was investigated. The antagonistic compounds produced by LAB were identified using gas chromatography-mass spectrometry (GCMS). Toxigenic Aspergillus flavus and A. parasiticus were isolated from maize after screening with an ammonium hydroxide test, and their aflatoxin levels were quantified to be 877 and 797 ng g-1 respectively. Lactobacillus fermentum and Lactobacillus paraplantarum were isolated and their culture-free supernatants (CFS) had a mean inhibitory zone of 15 ± 0.4 to 18 ± 0.6 mm. They were sensitive to pH but slight loss of inhibitory activity when treated with proteolytic enzymes. The inhibitory CFS was subjected to GC-MS. Among the compounds detected, lactic acid, benzenepropanoic acid, 4-hydroxy, pyrrolo [1, 2a] pyrazine 1,4 dione and fatty acids especially hexadecanoic acid, were the main compounds with potential antifungal activity identified. Lactic acid bacteria inhibited the growth of aflatoxigenic Aspergilli, hereby reducing food intoxication.

Antifungal activity of Lysinibacillus macroides against toxigenic Aspergillus flavus and Fusarium proliferatum and analysis of its mycotoxin minimization potential

BackgroundToxigenic fungi (Aspergillus and Fusarium) and their metabolites represent the major cause of corn and corn-based products contamination and consequently lead to severe economic and health issues.AimOur current study aimed to investigate the efficacy of using L. macroides Bac6 as a biological control agent against the toxigenic fungi; A. flavus f10 and F. proliferatum f30 and their mycotoxins.ResultsThe results illustrated that A. flavus f10 produced the aflatoxins AFB1 and AFG2 with concentrations of 21.239 and 13.593 ppb, respectively. While F. proliferatum f30 produced fumonisin B1 (9600 ppb). Furthermore, L. macroides showed a high potential for inhibition of toxigenic fungal growth using a dual culture method. F. proliferatum f30 and A. flavus f10 were found to be inhibited by a percentage of 80 and 62.5%, respectively. The results were confirmed using the scanning electron microscope. The antagonistic bacteria, L. macroides, showed chitinase productivity and activity of 26.45 U/L and 0.12 U/mL/min, respectively, which illustrates its potential application as a biocontrol agent. The GC-MS analysis revealed an abundance of Pyrrolo[1,2-a] pyrazine-1,4-dione, Hexahydro in the bacterial supernatant that exhibited antifungal characteristics. L. macroides had a significant reduction of AFB1 and AFG2 produced by A. flavus f10, recording 99.25% and 99% inhibition, respectively. It also showed strong inhibition of fumonisin B1 (90% inhibition) produced by F. proliferatum f30. Conclusion: Thus, the current study is a prospective study evaluating for the first time the potential impact of L. macroides Bac6 against the toxigenic fungi and their toxins.

Control of Toxigenic strain, Aspergillus flavus and Mycotoxin by the extracts of Andrographis paniculata in Maize seeds (Zea mays L.)

Control of Toxigenic strain, Aspergillus flavus and Mycotoxin by the extracts of Andrographis paniculata in Maize seeds (Zea mays L.)

MOLECULAR CHARACTERIZATION OF Aspergillus flavus TOXIGENICITY IN AGRICULTURAL COMMODITIES IN INDONESIA

Toxigenic Aspergillus flavus is a primary producer of aflatoxin in Indonesia, and its presence can lead to the contamination of agricultural commodities. This contamination poses a risk to export-targeted commodities, potentially resulting in their rejection. Therefore, this study aims to characterize the molecular profile of nativeA. flavus isolated from several Indonesian agricultural products, with a major focus on its toxigenicity and toxin production. A total of 18 A. flavus collections were isolated from nutmeg, ground peanut, cacao, coffee bean, corn, white pepper, and soil peanut plantation. Species identification was carried out using molecular and morphological approaches. The toxigenicity of isolates was characterized based on the amplification of aflatoxin gene clusters, while toxin production was assessed through growth simulation on a 10% coconut broth media followed by HPLC quantification. The result showed that all isolates were confirmed as A. flavus based on the morphological and sequence analysis of the ITS region. A total of 11 isolates (61%) were confirmed as toxigenic and produced 1-2 types of aflatoxin, in varying concentrations of high, moderate, or low levels of AFB1. High levels of AFB1 produced by seven isolates namely BIO3313, BIO33212, BIO3361, BIO33404, BIO3338, BIO3352, and BIO3344, had concentration levels ranging from 76.78 to 2241.06 µg/kg, while three isolates (BIO3314, BIO3312, and BIO3381) produced AFB1 below 1 µg/kg. Twenty-nine pairs of aflatoxin gene-specific sequences were successfully amplified as a single band, while some produced non-specific patterns in several low toxigenic and non-toxigenic isolates. Based on the results, it was concluded that completed gene clusters and variations of gene deletion were observed in both toxigenic and non-toxigenic isolates. However, no specific target gene could effectively distinguish the two groups. Two non-toxigenic isolates namely BIO3393 and BIO33403 exhibited a large deletion and could be potential candidates for biocontrol agents.

Discrimination of toxigenic and non-toxigenic Aspergillus flavus in wheat based on nanocomposite colorimetric sensor array

A novel method was developed for the early detection of wheat infected with Aspergillus flavus (A. flavus) using a nanocomposite colorimetric sensors array (CSA). LC-MS analysis revealed the presence of Aflatoxin B1 (AFB1) and Aflatoxin B2 (AFB2) on day seven, demonstrating mycotoxin variabilities in infected wheat. HS-SPME-GC–MS analysis detected 2-methylbutyral, a gas exclusively associated with toxigenic A. flavus. The CSA was modified using three nanoparticles of MOF and successfully used to detect the wheat infected with A. flavus. Discrimination of different types of infected wheat samples was achieved using the RGB difference map and Principal Component Analysis (PCA) model. Additionally, the Linear Discriminant Analysis (LDA) model accurately predicted the presence of toxigenic A. flavus at various stages of infection. These findings highlight the promising capabilities of nanocomposite CSA for early-stage detection of A. flavus infection in wheat.

Laurus nobilis essential oil nanoemulsion-infused chitosan: A safe and effective antifungal agent for masticatory preservation

The present study reports the first time investigation on encapsulation of Laurus nobilis essential oil into chitosan nanoemulsion (CS-Ne-LNEO) and assessment of its efficacy to inhibit fungal infestation and aflatoxin B1 (AFB1) contamination in stored masticatories food system. Gas chromatography mass spectrometry (GC-MS) analysis revealed m-Eugenol (46.23%) and D-Limonene (8.89%) as the most abundant components of LNEO. The CS-Ne-LNEO was physico-chemically characterized through scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), and x-ray diffractometry (XRD) analyses. The CS-Ne-LNEO exhibited broad range of antifungal activity against food contaminating fungi including inhibition of toxigenic Aspergillus flavus (AF-LHP-PE-4) and AFB1 production at lower concentrations as compared to unencapsulated LNEO. The CS-Ne-LNEO caused impairment in ergosterol biosynthesis and enhancement in leakage of Ca2+, Mg2+, K+ ions and 260, 280 nm absorbing materials along with inhibition of methylglyoxal production suggesting the antifungal and antiaflatoxigenic mechanism of action. The DPPH antioxidant activity of CS-Ne-LNEO was noted with IC50 value of 0.004 µL/mL. In addition, the CS-Ne-LNEO caused complete protection of stored Phyllanthus emblica (model masticatories) fruit samples against fungal and AFB1 contamination without altering their sensory characteristics and exhibited high LD50 value (13,504 µL/Kg body weight) mammalian system. Overall, these results indicated that LNEO loaded chitosan nanoemulsion could be promoted as an eco-friendly preservative for complete protection of stored plant masticatories against fungal and AFB1 contamination.

A Low-Cost, Portable Device for Detecting and Sorting Aflatoxin-Contaminated Maize Kernels.

Aflatoxin contamination of maize is a major food safety issue worldwide. The problem is of special significance in African countries because maize is a staple food. This manuscript describes a low-cost, portable, non-invasive device for detecting and sorting aflatoxin-contaminated maize kernels. We developed a prototype employing a modified, normalized difference fluorescence index (NDFI) detection method to identify potentially aflatoxin-contaminated maize kernels. Once identified, these contaminated kernels can be manually removed by the user. The device consists of a fluorescence excitation light source, a tablet for image acquisition, and detection/visualization software. Two experiments using maize kernels artificially infected with toxigenic Aspergillus flavus were implemented to evaluate the performance and efficiency of the device. The first experiment utilized highly contaminated kernels (71.18 ppb), while mildly contaminated kernels (1.22 ppb) were used for the second experiment. Evidently, the combined approach of detection and sorting was effective in reducing aflatoxin levels in maize kernels. With a maize rejection rate of 1.02% and 1.34% in the two experiments, aflatoxin reduction was achieved at 99.3% and 40.7%, respectively. This study demonstrated the potential of using this low-cost and non-invasive fluorescence detection technology, followed by manual sorting, to significantly reduce aflatoxin levels in maize samples. This technology would be beneficial to village farmers and consumers in developing countries by enabling safer foods that are free of potentially lethal levels of aflatoxins.

Detection of wheat toxigenic Aspergillus flavus based on nano-composite colorimetric sensing technology

Volatile organic compounds (VOCs) are an important indicator for fungal-infected wheat identification. This work proposes a novel approach for toxigenic Aspergillus flavus infected wheat identification through characteristic VOCs analyzed by nano-composite colorimetric sensors. Nanoparticles of poly styrene-co-acrylic acid (PSA), porous silica nanoparticles (PSN), and metal–organic framework (MOF) were combined with boron dipyrromethene (BODIPY) to fabricate nano-composite colorimetric sensors. The combination mechanisms for nanoparticles and the information extracted from nano-colorimetric sensors by digital images were analyzed in the current work. Furthermore, linear discriminant analysis (LDA) and k-nearest neighbor (KNN) were used comparatively to analyze the data from images, and toxigenic Aspergillus flavus infected wheat samples could be 100.00% correctly identified when using the optimal KNN model. This research contributes to the practical analysis of VOCs and the detection of toxigenic Aspergillus flavus infected wheat.

Assessment of toxin-producing genes in Aspergillus species from traditional herbal products in Khon Kaen province, Thailand using molecular method

A large number of traditional herbal products from medicinal plants are available in local markets. However, the risk of fungal contamination especially toxigenic Aspergillus species, Aspergillus niger and Aspergillus flavus was potentially possible. Thus, this study targeted to investigate the prevalence of A. niger and A. flavus and the occurrence of genes encoding toxins, aflatoxins and ochratoxin A among the fungi using the molecular method in 33 herbal products randomly collected from local markets in Khon Kaen province, Thailand. Based on the identification using DNA sequences of the internal transcribed spacer region (ITS), there were two Aspergillus species, A. niger and A. flavus, with prevalence rates of 73.6% and 0.1% respectively. The genes related to aflatoxins and ochratoxin A were undetectable in all isolates. This study suggested the products were naturally contaminated with the fungi, but they were safe from the toxigenic A. niger and A. flavus.

Fungal Contamination and Characterization of Aspergillus flavus on Poultry Feeds and their Ingredients in North Sumatera

Poultry feeds mainly consist of cereal grains and crops that are vulnerable to fungal infection. Improper handling of the feeds increases spoilage and contamination by aflatoxigenic fungi. The aim of this study was to determine fungal contamination including characterization of toxigenic and non-toxigenic Aspergillus flavus strains in poultry feed and its ingredients. We collected 200 composite samples of compound poultry feeds and their ingredients sold by retailers at traditional markets. The fungal population was enumerated using a dilution followed by the pour plate in dichloran 18% glycerol agar medium. All isolated A. flavus were identified by culture technique and molecular approach. Aflatoxin production was determined in 10% coconut agar medium with thin layer chromatography. Regulatory (aflR) and structural (nor-1, ver-1, omt-1) genes of A. flavus strains were isolated using four sets of primers (aflR, nor-1, ver-1, omt-1). We found that all feeds were infected by fungi. Aspergillus chevalieri was the most found in all feeds, particularl on chick starter. Of the 200 samples of compound and poultry feed ingredients, 57 isolates of A. flavus were examined and 16 of them (28%) were toxigenic, each strain producing aflatoxin B1 from &lt;3.01 to 35.50 ppb. Among feeds, layer finisher, pellet starter and grain corn were the most infected by toxigenic A. flavus, respectively. The contamination occurred on feeds that contained corn as the main compound. Routine analysis of compound poultry feeds and their ingredients for aflatoxigenic fungi particularly A. flavus is compulsory

Evaluation of the antagonistic effect of some species of lactic acid bacteria isolated from fermented food against toxigenic Aspergillus flavus

The presence of mycotoxigenic molds in food potentially poses hazards to healthy living and needs to be controlled. There is a greater risk that the resistance phenomenon will increase in the future due to the frequent use of antibiotics and preservatives, unlike biopreservation. Lactic acid bacteria are capable of producing secondary metabolites with antimicrobial properties. This study was conducted to evaluate the antagonistic effect of lactic acid bacteria isolates against toxigenic mold, Aspergillus flavus. Lactic acid bacteria were isolated from palm wine, ogi, and yogurt samples, and their antagonistic activity against toxigenic mold was investigated using the agar overlay method. Streptomyces spp. had the highest counts of 15.1×104 cfu/ml, while the least count of 2.0×104 cfu/ml from Lactobacillus plantarum was obtained. Pediococcus damnosus and Lactobacillus plantarum were present in both yogurt and palm wine samples. Similarly, Streptococcus spp. was identified in both yogurt and pap (ogi) samples. Aspergillus flavus was susceptible to half of the lactic acid bacterial isolates with varying degrees of inhibition. Total inhibition of toxigenic mold was observed on the control plate and plates inoculated with L. lactis, S. thermophilus, Leuconostoc mesenteriodes, P. damnosus, and L. bulgaricus. The antifungal activity of lactic acid bacteria suggests their biocontrol efficacy against A. flavus. Also, the antimicrobial properties of lactic acid bacteria can make them a suitable candidate in food preservation. Furthermore, this study can stand as a preliminary step in multistep research to investigate the anti-fungal and anti-aflatoxigenic potential of lactic acid bacteria from fermented foods against toxigenic A. flavus.

Co-encapsulation of Pimpinella anisum and Coriandrum sativum essential oils based synergistic formulation through binary mixture: Physico-chemical characterization, appraisal of antifungal mechanism of action, and application as natural food preservative

The present study aimed to co-encapsulate binary synergistic formulation of Pimpinella anisum and Coriandrum sativum (PC) essential oils (0.75:0.25) into chitosan nanoemulsion (Nm-PC) with effective inhibition against fungal proliferation, aflatoxin B1 (AFB1) secretion, and lipid peroxidation in stored rice. Physico-chemical characterization of Nm-PC by SEM, FTIR, and XRD confirmed successful encompassment of PC inside the chitosan nanomatrix with efficient interaction by functional groups and reduction in crystallinity. Nm-PC showed superior antifungal, antiaflatoxigenic, and antioxidant activities over unencapsulated PC. Reduction in ergosterol biosynthesis and enhanced leakage of Ca2+, K+, Mg2+ ions and 260, 280 nm absorbing materials by Nm-PC fumigation confirmed irreversible damage of plasma membrane in toxigenic Aspergillus flavus cells. Significant diminution of methylglyoxal in A. flavus cells by Nm-PC fumigation illustrated biochemical mechanism for antiaflatoxigenic activity, suggesting future exploitation for development of aflatoxin resistant rice varieties through green transgenic technology. In silico findings indicated specific stereo-spatial interaction of anethole and linalool with Nor-1 protein, validating molecular mechanism for AFB1 inhibition. In addition, in situ investigation revealed effective protection of stored rice against fungal occurrence, AFB1 biosynthesis, and lipid peroxidation without affecting organoleptic attributes. Moreover, mammalian non-toxicity of chitosan entrapped PC synergistic nanoformulation could provide exciting potential for application as eco-smart safe nano-green food preservative.

Aflatoxin Reduction and Retardation of Aflatoxin Production by Microorganisms in Doenjang during a One-Year Fermentation.

Meju, a raw material for doenjang preparation, is highly vulnerable to aflatoxin-producing fungi. The aim of this study was to evaluate the effect of a one-year fermentation on aflatoxins and aflatoxin-producing fungi in doenjang spiked with aflatoxins B1, G1, B2, and G2 and inoculated with toxigenic Aspergillus flavus. A significant reduction in aflatoxins was observed after a year of fermentation, measuring 92.58%, 100%, 98.69%, and 100% of B1, G1, B2, and G2, respectively. After a year of fermentation, 6.95 ± 3.64 µg/kg of total aflatoxin was detected, which represents a 97.88% reduction in the total aflatoxin compared with the initial value (328.83 ± 36.60 µg/kg). Several aflatoxin-degrading fungi (Aspergillus versicolor, Cladosporium subcinereum, Aspergillus ochraceus) and bacteria (Bacillus albus, Bacillus velezensis) isolated from doenjang were identified as the major contributors to the reduction of aflatoxin. Furthermore, it was observed that most of the aflatoxin contamination in doenjang occurred during the meju stage, and this stage was found to be most susceptible to A. flavus contamination and growth. These findings reveal that native microorganisms mediate aflatoxin clean-up in doenjang during fermentation and support the use of such microorganisms as a starter culture for the preparation of aflatoxin-free doenjang.

Antifungal Activity and Major Bioactive Compounds of Water Extract of Pangium edule Seed against Aspergillus flavus.

Pangium edule seeds are widely used as spices in Southeast Asia in a fresh and fermented form and are reported to have active compounds for food preservation. However, scientific data on the active compounds of P. edule seed that can prevent the growth of toxigenic Aspergillus flavus have not been widely reported. This research subjected to determine the antifungal activity and identify the active compounds of water extract of old and fermented seed of P. edule against A. flavus. The water extract was compared to the extracts obtained by multilevel maceration using 50% ethanol, ethyl acetate, and n-hexane as solvents. Alkaloid, saponin, phenolic compound, flavonoid, triterpenoid, and glycoside were detected qualitatively in the crude extracts. The water extract showed the best activity to suppress the growth of A. flavus, determined by the agar dilution method, with the minimum inhibitory concentration (MIC) of 12.5 and 25 mg/mL for old and fermented seed, respectively. The water extracts showed a moderate toxicity with LC50 of 100-500 μg/mL, determined by the brine-shrimp toxicity test. After fractionation using 3 kDa molecular-weight (MW) cut-off ultrafiltration membrane, two fractions, i.e., fraction with MW < 3 kDa and >3 kDa, were obtained. The fraction with MW < 3 kDa showed the best antifungal activity with the MIC of 6.25 and 12.5 mg/mL for old and fermented seed, respectively. LC-MS/MS profile showed that different compounds belong to fatty acid, amino acid, glycoside, and peptide were found as major active compounds in the fractionated water extract. The principal compounds and partial least-square analysis, however, suggested that fatty acid and glycoside are responsible for the antifungal activity. Hence, this study concluded that the water extract of P. edule seed had promising antifungal activity against A. flavus which was due to presence of particular compounds belong to fatty acid and glycoside.

Effect of in-package gas composition on growth and aflatoxin production of Aspergillus flavus in culture medium and red pepper

In this study, the effect of in-package gas composition on growth and aflatoxin production of toxigenic Aspergillus flavus was investigated. For this purpose, semi-dried red pepper samples and potato dextrose agar media with different pH values (3.5, 5.6, 7.0 and 9.0) were inoculated with A. flavus and packaged under different gas atmospheres (air, 100% N2, 70% N2 + 30% CO2 and 100% CO2). The growth of the fungus was monitored during storage at 25 °C for 8 days and the aflatoxin levels produced were determined at the end of storage. The highest growth rate was observed in the medium at pH 9.0 and the highest toxin production was in the medium at pH 5.6. Slowdowns up to 55.6% and 28.0% in the growth of A. flavus in the culture medium and red pepper were observed when packaging under 100% N2 was employed. It was found that 70% N2 + 30% CO2 atmosphere was more effective than 100% N2 in inhibiting the growth of A. flavus in red peppers; however, the difference between the aflatoxin levels produced in the samples packaged under these gas compositions was not significant (p > 0.05). CO2 concentrations higher than 90% in the package were quite effective against A. flavus resulting in 60% and 99% reductions in the growth and aflatoxin production of the fungus in red pepper.

Chemically characterized nanoencapsulated Homalomena aromatica Schott. essential oil as green preservative against fungal and aflatoxin B1 contamination of stored spices based on in vitro and in situ efficacy and favorable safety profile on mice.

Present study deals with the efficacy of nanoencapsulated Homalomena aromatica essential oil (HAEO) as a potent green preservative against toxigenic Aspergillus flavus strain (AF-LHP-NS 7), storage fungi, AFB1, and free radical-mediated deterioration of stored spices. GC-MS analysis revealed linalool (68.51%) as the major component of HAEO. HAEO was encapsulated into chitosan nanomatrix (CS-HAEO-Ne) and characterized through SEM, FTIR, and XRD. CS-HAEO-Ne completely inhibited A. flavus growth and AFB1 biosynthesis at 1.25 μL/mL and 1.0 μL/mL, respectively in comparison to unencapsulated HAEO (1.75 μL/mL and 1.25 μL/mL, respectively). CS-HAEO-Ne caused significant reduction in ergosterol content in treated A. flavus and provoked leakage of cellular ions (Ca+2, Mg+2, and K+) as well as 260 nm and 280 nm absorbing materials. Depletion of methylglyoxal level in treated A. flavus cells illustrated the novel antiaflatoxigenic efficacy of CS-HAEO-Ne. CS-HAEO-Ne exhibited superior antioxidant efficacy (IC50 (DPPH) = 4.5 μL/mL) over unencapsulated HAEO (IC50 (DPPH) = 15.9 μL/mL) and phenolic content. CS-HAEO-Ne depicted excellent in situ efficacy by inhibiting fungal infestation, AFB1 contamination, lipid peroxidation, and mineral loss with acceptable sensorial profile. Moreover, broad safety paradigm (LD50 value = 7150.11 mg/kg) of CS-HAEO-Ne also suggests its application as novel green preservative to enhance shelf life of stored spices.