The Development of Aspergillus flavus and Biosynthesis of Aflatoxin B1 are Regulated by the Golgi-Localized Mn2+ Transporter Pmr1.

Microorganisms rely on diverse ion transport and trace elements to sustain growth, development, and secondary metabolism. Manganese (Mn2+) is essential for various biological processes and plays a crucial role in the metabolism of human cells, plants, and yeast. In Aspergillus flavus, we confirmed that Pmr1 localized in cis- and medial-Golgi compartments was critical in facilitating Mn2+ transport, fungal growth, development, secondary metabolism, and glycosylation. In comparison to the wild type, the Δpmr1 mutant displayed heightened sensitivity to environmental stress, accompanied by inhibited synthesis of aflatoxin B1, kojic acid, and a substantial reduction in pathogenicity toward peanuts and maize. Interestingly, the addition of exogenous Mn2+ effectively rectified the developmental and secondary metabolic defects in the Δpmr1 mutant. However, Mn2+ supplement failed to restore the growth and development of the Δpmr1Δgdt1 double mutant, which indicated that the Gdt1 compensated for the functional deficiency of pmr1. In addition, our results showed that pmr1 knockout leads to an upregulation of O-glycosyl-N-acetylglucose (O-GlcNAc) and O-GlcNAc transferase (OGT), while Mn2+ supplementation can restore the glycosylation in A. flavus. Collectively, this study indicates that the pmr1 regulates Mn2+ via Golgi and maintains growth and metabolism functions of A. flavus through regulation of the glycosylation.

In-Vitro and In-Silico Investigation for the Spent-Coffee Bioactive Phenolics as a Promising Aflatoxins Production Inhibitor.

Aflatoxin, is a naturally occurring polyketide generated by Aspergillus flavus via biosynthetic pathways, including polyketide synthase (PKS) and non-ribosomal enzymes. The in vitro analysis supported by molecular dynamics (MD) techniques was used to examine the antifungal and anti-aflatoxigenic activity of spent coffee grounds (SCGs) methanol extract. The High-Performance Liquid Chromatography results revealed the presence of 15 phenolic acids and five flavonoids. (R)-(+)-Rosmarinic acid (176.43 ± 2.41 µg/g) was the predominant of the detected acids, followed by gallic acid (34.83 ± 1.05 µg/g). At the same time, apigenin-7-glucoside is the dominant flavonoid in the SCGs extract by 1717.05 ± 5.76 µg/g, and naringin (97.27 ± 1.97 µg/g) comes next. The antifungal and anti-aflatoxigenic activity of the SCGs extracts was 380 µL/mL and 460 µL/mL, respectively. The SGGs' effect of inhibiting five Aspergillus strains' growth on the agar media ranged between 12.81 ± 1.71 to 15.64 ± 1.08 mm by two diffusion assays. Molecular docking results confirmed the inhibitory action of different phenolics and flavonoids on the PKS and NPS key enzymes of the aflatoxin biosynthetic mechanism. The SCGs extract components with the highest free binding energy, naringin (-9.1 kcal/mL) and apigenin 7-glucoside (-9.1 kcal/mol), were subjected to an MD simulation study. The computational results infer the stabilizing effects on the enzymes upon ligand binding led to the impairment in its functionality. The current study represents a novel attempt to assess the anti aflatoxins mechanism of phenolics and flavonoids targeting PKS and NPS via computational approaches compared to in-vitro assays.

Glutamine Synthetase Contributes to the Regulation of Growth, Conidiation, Sclerotia Development, and Resistance to Oxidative Stress in the Fungus Aspergillus flavus.

The basic biological function of glutamine synthetase (Gs) is to catalyze the conversion of ammonium and glutamate to glutamine. This synthetase also performs other biological functions. However, the roles of Gs in fungi, especially in filamentous fungi, are not fully understood. Here, we found that conditional disruption of glutamine synthetase (AflGsA) gene expression in Aspergillus flavus by using a xylose promoter leads to a complete glutamine deficiency. Supplementation of glutamine could restore the nutritional deficiency caused by AflGsA expression deficiency. Additionally, by using the xylose promoter for the downregulation of AflgsA expression, we found that AflGsA regulates spore and sclerotic development by regulating the transcriptional levels of sporulation genes abaA and brlA and the sclerotic generation genes nsdC and nsdD, respectively. In addition, AflGsA was found to maintain the balance of reactive oxygen species (ROS) and to aid in resisting oxidative stress. AflGsA is also involved in the regulation of light signals through the production of glutamine. The results also showed that the recombinant AflGsA had glutamine synthetase activity in vitro and required the assistance of metal ions. The inhibitor molecule L-α-aminoadipic acid suppressed the activity of rAflGsA in vitro and disrupted the morphogenesis of spores, sclerotia, and colonies in A. flavus. These results provide a mechanistic link between nutrition metabolism and glutamine synthetase in A. flavus and suggest a strategy for the prevention of fungal infection.

Cytotoxic Potential of Bioactive Compounds from Aspergillus flavus, an Endophytic Fungus Isolated from Cynodon dactylon, against Breast Cancer: Experimental and Computational Approach.

Endophytic fungi are a diverse group of microorganisms that colonize the inter- or intracellular spaces of plants and exhibit mutual benefits. Their interactions with the host plant and other microbiomes are multidimensional and play a crucial role in the production of secondary metabolites. We screened bioactive compounds present in the extracts of Aspergillus flavus, an endophytic fungus isolated from the roots of the medicinal grass Cynodon dactylon, for its anticancer potential. An in vitro analysis of the Ethyl acetate extract from A. flavus showed significant cytostatic effects (IC50: 16.25 µg/mL) against breast cancer cells (MCF-7). A morphological analysis of the cells and a flow cytometry of the cells with annexin V/Propidium Iodide suggested that the extract induced apoptosis in the MCF-7 cells. The extract of A. flavus increased reactive oxygen species (ROS) generation and caused a loss of mitochondrial membrane potential in MCF-7 cells. To identify the metabolites that might be responsible for the anticancer effect, the extract was subjected to a gas chromatography-mass spectrometry (GC-MS) analysis. Interestingly, nine phytochemicals that induced cytotoxicity in the breast cancer cell line were found in the extract. The in silico molecular docking and molecular dynamics simulation studies revealed that two compounds, 2,4,7-trinitrofluorenone and 3α, 5 α-cyclo-ergosta-7,9(11), 22t-triene-6beta-ol exhibited significant binding affinities (-9.20, and -9.50 Kcal/mol, respectively) against Bcl-2, along with binding stability and intermolecular interactions of its ligand-Bcl-2 complexes. Overall, the study found that the endophytic A. flavus from C. dactylon contains plant-like bioactive compounds that have a promising effect in breast cancer.

A methanolic extract of Zanthoxylum bungeanum modulates secondary metabolism regulator genes in Aspergillus flavus and shuts down aflatoxin production.

Aflatoxin B1 (AFB1) is a food-borne toxin produced by Aspergillus flavus and a few similar fungi. Natural anti-aflatoxigenic compounds are used as alternatives to chemical fungicides to prevent AFB1 accumulation. We found that a methanolic extract of the food additive Zanthoxylum bungeanum shuts down AFB1 production in A. flavus. A methanol sub-fraction (M20) showed the highest total phenolic/flavonoid content and the most potent antioxidant activity. Mass spectrometry analyses identified four flavonoids in M20: quercetin, epicatechin, kaempferol-3-O-rhamnoside, and hyperoside. The anti-aflatoxigenic potency of M20 (IC50: 2-4 µg/mL) was significantly higher than its anti-proliferation potency (IC50: 1800-1900 µg/mL). RNA-seq data indicated that M20 triggers significant transcriptional changes in 18 of 56 secondary metabolite pathways in A. flavus, including repression of the AFB1 biosynthesis pathway. Expression of aflR, the specific activator of the AFB1 pathway, was not changed by M20 treatment, suggesting that repression of the pathway is mediated by global regulators. Consistent with this, the Velvet complex, a prominent regulator of secondary metabolism and fungal development, was downregulated. Decreased expression of the conidial development regulators brlA and Medusa, genes that orchestrate redox responses, and GPCR/oxylipin-based signal transduction further suggests a broad cellular response to M20. Z. bungeanum extracts may facilitate the development of safe AFB1 control strategies.

The metabolism and biotransformation of AFB1: Key enzymes and pathways.

Aflatoxins B1 (AFB1) is a hepatoxic compound produced by Aspergillus flavus and Aspergillus parasiticus, seriously threatening food safety and the health of humans and animals. Understanding the metabolism of AFB1 is important for developing detoxification and intervention strategies. In this review, we summarize the AFB1 metabolic fates in humans and animals and the key enzymes that metabolize AFB1, including cytochrome P450s (CYP450s) for AFB1 bioactivation, glutathione-S-transferases (GSTs) and aflatoxin-aldehyde reductases (AFARs) in detoxification. Furthermore, AFB1 metabolism in microbes is also summarized. Microorganisms specifically and efficiently transform AFB1 into less or non-toxic products in an environmental-friendly approach which could be the most desirable detoxification strategy in the future. This review provides a wholistic insight into the metabolism and biotransformation of AFB1 in various organisms, which also benefits the development of protective strategies in humans and animals.

Assessment of nanoencapsulated Cananga odorata essential oil in chitosan nanopolymer as a green approach to boost the antifungal, antioxidant and in situ efficacy.

In this study, a comparative efficacy of Cananga odorata EO (CoEO) and its nanoencapsulated formulation into chitosan nanoemulsion (CoEO-CsNe) against a toxigenic strain of Aspergillus flavus (AF-M-K5) were investigated for the first time in order to determine its efficacy in preservation of stored food from fungal, aflatoxin B1 (AFB1) contamination and lipid peroxidation. GC and GC-MS analysis of CoEO revealed the presence of linalool (24.56%) and benzyl acetate (22.43%) as the major components. CoEO was encapsulated into chitosan nanoemulsion (CsNe) through ionic-gelation technique and characterized by High Resolution-Scanning Electron Microscopy (HR-SEM), Fourier Transform Infrared spectroscopy (FTIR), and X-Ray Diffraction (XRD) analysis. The CoEO-CsNe during in vitro investigation against A. flavus completely inhibited the growth and AFB1 production at 1.0 µL/mL and 0.75 µL/mL, respectively. Additionally, CoEO-CsNe showed improved antioxidant activity against DPPH• and ABTS•+ with IC50 value 0.93 and 0.72 µL/mL, respectively. Further, CoEO-CsNe suppressed fungal growth, AFB1 secretion and lipid peroxidation in Arachis hypogea L. during in situ investigation without causing any adverse effect on seed germination. Overall results demonstrated that the CoEO-CsNe has potential of being utilized as a suitable plant based antifungal agent to improve the shelf-life of stored food against AFB1 and lipid peroxidation mediated biodeterioration.

High voltage atmospheric cold plasma treatment inactivates Aspergillus flavus spores and deoxynivalenol toxin.

Fungal contamination is a concern for the food industry. Fungal spores resist food sterilization treatments and produce mycotoxins that are toxic for animals and humans. Technologies that deactivate spores and toxins without impacting food quality are desirable. This study demonstrates the efficiency of a high voltage atmospheric cold plasma (HVACP) technology using air to generate reactive oxygen (ROS) and nitrogen (RNS) species for the degradation of Aspergillus flavus cultures and the deoxynivalenol (DON) mycotoxin. Optical emission and absorption spectroscopy demonstrate ionization of hydroxyl groups, atomic oxygen and nitrogen, and confirm production of ROS and RNS, e.g. O3, NO2, NO3, N2O4, and N2O5. Fungal cultures show a depletion in pigmentation and an ~50% spore inactivation after 1-min treatments. Treated spores show surface ablation and membrane degradation by scanning electron microscopy. Twenty-minute direct HVACP treatments of 100 µg of DON in one mL aqueous suspensions resulted in a greater than 99% reduction in DON structure and rescued over 80% of Caco-2 cell viability; however, the same treatment on 100 µg of powdered DON toxin only showed a 33% reduction in DON and only rescued 15% of cell viability. In summary, HVACP air treatment can inactivate both fungal spores and toxins in minutes.

Modelling the effect of temperature and water activity on the growth rate of Aspergillus flavus and aflatoxin production in peanut meal extract agar.

Aspergillus flavus is the predominant species that produce aflatoxins in stored peanuts under favourable conditions. This study aimed to describe the growth and aflatoxin production by two A. flavus strains isolated from imported raw peanuts and to model the effects of temperature and aw on their colony growth rate as a function of temperature and aw in Peanut Meal Extract Agar (PMEA). A full factorial design with seven aw levels (0.85-0.98 aw) and five temperature levels (20-40 °C) was used to investigate the growth and aflatoxin production. Colony diameter was measured daily for 28 days while AFB1 and total aflatoxin were determined on day 3, 7, 14, and 21. The maximum colony growth rate, µmax (mm/day) was estimated by using the primary model of Baranyi, and the µmax was then fitted to the secondary model; second-order polynomial and linear Arrhenius-Davey to describe the colony growth rate as a function of temperature and aw. The results indicated that both strains failed to grow at temperature of 20 °C with aw <0.94 and aw of 0.85 for all temperatures except 30 °C. The highest growth rate was observed at 30 °C, with 0.98 aw for both strains. The analysis of variance showed a significant effect of strain, temperature, and aw on the fungal growth and aflatoxin production (p < 0.05). Furthermore, both secondary models were in good agreement with the observed µmax. However, the polynomial model was found to be a better predictor of the experimental data. A similar pattern was observed in aflatoxin production but in a narrower range of temperature (25-35 °C) and aw (0.92-0.98 aw). The highest production of aflatoxins was observed on day 21 at 30 °C with the aw level of 0.98 for both strains. Overall, the current findings may help in improving the mycotoxin management and intervention strategies in peanuts, especially during storage.

Reduction of Aflatoxin B1 in Corn by Water-Assisted Microwaves Treatment and Its Effects on Corn Quality.

Aflatoxin B1 (AFB1) is one of the most commonly found mycotoxin in corn, which is highly toxic, carcinogenic, teratogenic, and mutagenic for the health of humans and animals. In order to reduce the AFB1 in corn, corn kernels were processed with Water-assisted Microwaves Treatment (WMT) and the feasibility of WMT processing on AFB1 reduction and its effects on corn quality were analyzed. Increasing the treatment time and microwave power could increase the reduction of AFB1, and the maximum reduction rate could reach 58.6% and 56.8%, respectively. There was no significant correlation between the initial concentration of AFB1 and the reduction rate of AFB1. During WMT, the main toxigenic molds were sterilized completely, and the moisture content of corn climbed up and then declined to the initial level. WMT could obviously increase the fatty acid value and pasting temperature of corn and reduce the all paste viscosity of corn. However, it had little effect on the color of corn. The results indicated that WMT could reduce AFB1 effectively and avoid the vast appearance of heat-damaged kernels simultaneously. Undoubtedly, water played an important role in WMT. This result provides a new idea for the reduction of AFB1 by microwave.

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.

Determination of Antioxidant and Antifungal Activities in Cookies Fortified with Solar Dried Prickly Pear Peels Powder.

BACKGROUND AND OBJECTIVE: The fortification of bakery products by new materials that attain various goals is considered a challenging that finally gains useful health amelioration. This study was planned to assess the effect of incorporation of solar dried prickly pear peels powder in qaraqeesh (Egyptian cookies) with respect to increase shelf life, sensory palatability and nutritional value. Prickly pear cactus (Opuntia ficus-indica) beside distributed in arid and semiarid regions proved to have phytochemical compounds with high antioxidants capacity. MATERIALS AND METHODS: Fungi colonies were isolated from prickly pear peels. Three levels (1, 3 and 5%) of dried peels powder were added to wheat flour along with other ingredients to make cookies samples. Mycological analysis was assessed in yeast with the three concentrations of peels powder as well as the fresh peels and negative control. The total phenolics, flavonoids, tannins, anthocyanins and carotenoids as well as the antioxidant activity were evaluated in fresh and dried cactus peels. RESULTS: Findings showed that the prickly pear peels powder (PPPP) antioxidant activity was not much affected by the solar drying conditions. The effect of different extracting solvents at different polarties and pH on the phenolic and flavonoids contents of PPPP was studied. Aflatoxins production by aflatoxignicity A. flavus (ATCC 28542) was inhibited by adding different concentrations of PPPP to cookies. Sensory evaluation of fortified cookies was done. All the evaluated characteristics of cookies were given nearly the same values for all levels of dried peels powder. CONCLUSION: Addition of 5% dried cactus peel had lower overall quality and color than the control. Adding 3% of PPPP to cookies (qaraqeesh) showed the highest sensory score. Dried cactus peels may improve quality, nutritional value and shelf life of cookies.

Assessment of chemically characterised Myristica fragrans essential oil against fungi contaminating stored scented rice and its mode of action as novel aflatoxin inhibitor.

The study reports chemically characterised Myristica fragrans essential oil (MFEO) as plant based food preservative against fungal and aflatoxin B1 (AFB1) contamination of scented rice varieties. The chemical profile of MFEO revealed elemicin (27.08%), myristicine (21.29%) and thujanol (18.55%) as major components. The minimum inhibitory and minimum aflatoxin inhibitory concentrations of MFEO were 2.75 and 1.5 mg/ml, respectively. The MFEO was efficacious against a broad spectrum of food deteriorating fungi. MFEO caused decrease in ergosterol content of fungal plasma membrane and enhanced leakage of cellular ions, depicting plasma membrane as the site of action. The MFEO caused reduction in cellular methylglyoxal content, the aflatoxin inducer. This is the first report on MFEO as aflatoxin suppressor. The essential oil may be recommended as plant based food preservative after large scale trials and reduction in methylglyoxal suggests its application for development of aflatoxin resistant varieties through green transgenics.

Single Production of Kojic Acid by Aspergillus flavus and the Revision of Flufuran.

Timor Island is very hot and dry due to the high intensity of sunlight experienced throughout the year. The endophytic fungi Aspergillus flavus had been isolated from medicinal plants such as Catharanthus roseus, Annona squamosa and Curcuma xanthorisa. The endophytic fungi A. flavus from each plant was cultivated on solid rice media and then analyzed for its capability for producing kojic acid. The production of kojic acid was analyzed by HPLC; the highest amount of kojic acid was observed from the endophytic fungi A. flavus, isolated from the stem of Catharanthus roseus, followed by A. flavus from Annona squamosa and Curcuma xanthorisa. Simple VLC fractionation of the extract of A. flavus from C.roseus led to the isolation of around 11.1 g of pure kojic acid. The structure of kojic acid (1) was confirmed by NMR and MS spectroscopic data. A comparison of the NMR data with the literature supported the revision of the natural product flufuran to kojic acid. To the best of our knowledge, this is the first report of a strain of endophytic fungi producing only kojic acid without any other toxic metabolites such as alfatoxins. Therefore, this Aspergillus flavus strain can be applied as a potential producer of kojic acid for industrial use.

Antimicrobial activity of green silver nanoparticles from endophytic fungi isolated from Calotropis procera (Ait) latex.

Endophytes, a potential source of bioactive secondary metabolites, were isolated from the widely used medicinal plant Calotropis procera Ait. Approximately 675 segments from 15 Calotropis procera plants and 15 latex samples were assessed for the presence of endophytic fungi. Finally, eight fungal species were isolated and identified based on their macro- and micro-morphology. The endophytic fungal filtrates were screened for their antimicrobial activity against 11 important pathogenic micro-organisms. The filtrates of nanoparticles were from three of the eight isolated endophytic fungi, namely, Penicillium chrysogenum, Aspergillus fumigatus and Aspergillus flavus, and were highly effective against the tested bacteria, while the remaining endophytic fungal filtrates displayed low activity.

Aspergillus flavus as a Model System to Test the Biological Activity of Botanicals: An Example on Citrullus colocynthis L. Schrad. Organic Extracts.

Citrullus colocynthis L. Schrader is an annual plant belonging to the Cucurbitaceae family, widely distributed in the desert areas of the Mediterranean basin. Many pharmacological properties (anti-inflammatory, anti-diabetic, analgesic, anti-epileptic) are ascribed to different organs of this plant; extracts and derivatives of C. colocynthis are used in folk Berber medicine for the treatment of numerous diseases-such as rheumatism arthritis, hypertension bronchitis, mastitis, and even cancer. Clinical studies aimed at confirming the chemical and biological bases of pharmacological activity assigned to many plant/herb extracts used in folk medicine often rely on results obtained from laboratory preliminary tests. We investigated the biological activity of some C. colocynthis stem, leaf, and root extracts on the mycotoxigenic and phytopathogenic fungus Aspergillus flavus, testing a possible correlation between the inhibitory effect on aflatoxin biosynthesis, the phytochemical composition of extracts, and their in vitro antioxidant capacities.

Preparation, characterization and anti-aflatoxigenic activity of chitosan packaging films incorporated with turmeric essential oil.

Here, we studied the preparation, characterization, anti-aflatoxigenic activity, and molecular mechanism in vitro of chitosan packaging films containing turmeric essential oil (TEO). First, we took the mechanical properties as the evaluation Index, screened for the optimum preparation conditions of packaging films with 1.5 µL/cm2 TEO using single factor and orthogonal experiments, and characterized the film properties. We found that the addition of TEO affected the microcosmic structure of films and advanced water resistance capacity. In addition, we investigated the inhibitory effects of pure chitosan films and packaging films containing 1.5 µL/cm2 or 3.0 µL/cm2 TEO on the growth and conidial formation of Aspergillus flavus (A. flavus, CGMCC 3.4410), as well as the accumulation of aflatoxin over the course of seven days. We found that the packaging films possessed a prominent antifungal activity on A. flavus. Finally, we discuss preliminary results surrounding gene expression of packaging films which inhibit aflatoxin biosynthesis. The expressions levels of 16 genes related to aflatoxin biosynthesis were found to be either completely or almost completely inhibited. Therefore, the addition of the natural antifungal agent TEO in chitosan packaging films represent a remarkable method to significantly promote the development and application of antifungal packaging materials.

Biotransformation of 20(R)-panaxatriol by the fungus Aspergillus flavus Link AS 3.3950.

Microbial transformation of 20(R)-panaxatriol by the fungus Aspergillus flavus Link AS 3.3950 was performed. Four new (1-4), along with two previously reported metabolites (5 and 6), were obtained. Their chemical structures were elucidated on the basis of extensive spectroscopic analyses. Furthermore, the inhibitory effects of those compounds on K562/ADR, Du-145, Hela, MCF-7 and HepG2 cell lines were evaluated by MTT assay. Among them, compound 15ß-hydroxy-20(R)-panaxatriol (4) exhibited selective inhibitory effects on human leukaemic progenitor cells K562/ADR through arresting cell cycle, which was associated with obvious decrease of cyclin B1, cyclin D1 and cyclin-dependent kinase (CDK) 1/2/4/6 protein expression.

Evaluation of Protective Impact of Algerian Cuminum cyminum L. and Coriandrum sativum L. Essential Oils on Aspergillus flavus Growth and Aflatoxin B1 Production.

BACKGROUND AND OBJECTIVE: Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. The aim of this investigation was to evaluate the inhibition of growth Aspergillus flavus E73 (A. flavus E73) and AFB1 production by Cuminum cyminum L. (C. cyminum L.) and Coriandrum sativum L. (C. sativum L.) essential oils (EOs) as well their antioxidant and phytotoxicity activities. METHODOLOGY: The C. cyminum L. and C. sativum L. EOs were extracted by hydrodistillation. The chemical profile of EOs was identified by GC-MS, antifungal activity was assessed by poisoned food technique and in term Minimal Inhibitory Concentration (MIC) and minimal fungicidal concentration (MFC) and antiaflatoxin effect by broth medium. The antioxidant activity of EOs was determined by DPPH free radical scavenging assay, ß-carotene bleaching test and total phenolic content by Folin-Ciocalteu. Phytotoxicity of C. cyminum L. and C. sativum L. EOs were determined for varieties of wheat. The results were analyzed by analysis of variance (one way ANOVA). RESULTS: The GS/MS analysis showed that the major components of C. cyminum L. EO were cuminaldehyde (65.98%), o-cymene (18.40%) and C. sativum L. EO was mainly consisted of linalool (78.86%). The results showed that both the EOs could inhibit the growth of A. flavus E73 in the range of 24.27-84.90% for C. cyminum and 15.09-65.00% for C. sativum. During antiaflatoxin investigation, the oils exhibited noticeable inhibition on dry mycelium weight and synthesis of AFB1 by A. flavus E73. EOs of C. cyminum L. and C. sativum L. revealed complete inhibition of AFB1 at 1.25 and 1.5 mg mL-1, respectively. EOs exhibited inhibitory influence against some fungi. The IC50 values of C. cyminum L. and C. sativum L. EOs were 494.93 and 756.43 µg mL-1, respectively, while, ß-carotene/linoleic acid bleaching was 47.68 and 29.29% , respectively. Total phenolic content of C. cyminum L. and C. sativum L. were 10.66 and 6.2 µg mg-1. Additionally, the EOs were non-phytotoxic on the two verities of wheat seeds. CONCLUSION: The C. cyminum L. and C. sativum L EOs could be good alternative to protect foods.

Toward Enhancing the Enzymatic Activity of a Novel Fungal Polygalacturonase for Food Industry: Optimization and Biochemical Analysis.

BACKGROUND: The review of literature and patents shows that enhancing the polygalacturonase (PG) production and activity are still required to fulfill the increasing demands. METHODS: A dual optimization process, which involved Plackett-Burman design (PBD), with seven factors, and response surface methodology, was applied to optimize the production of extracellular PG enzyme produced by a novel strain of Aspergillus flavus isolated from rotten orange fruit. The fungal PG was purified and biochemically characterized. RESULTS: Three variables (harvesting time, pH and orange pomace concentration), that were verified to be significant by the PBD analysis, were comprehensively optimized via Box-Behnken design. According to this optimization, the highest PG activity (4073 U/mL) was obtained under pH 7 after 48 h using 40 g/L orange pomace as a substrate, with enhancement in PG activity by 51% compared to the first PBD optimization step. The specific activity of the purified PG was 1608 U/mg with polygalacturonic acid and its molecular weight was 55 kDa. The optimum pH was 5 with relative thermal stability (80%) at 50˚C after 30 min. The PG activity improved in the presence of Cu2+ and Ca2+, while Ba2+, Fe2+ and Zn2+ greatly inhibited the enzyme activity. The obvious Km and Vmax values were 0.8 mg/mL and 2000 µmol/min, respectively. CONCLUSION: This study is a starting point for initial research in the field of optimization and characterization of A. flavus PG. The statistical optimization of A. flavus PG and its biochemical characterization clearly revealed that this fungal strain can be a potential producer of PG which has a wide range of industrial applications.