Growth Of Aspergillus Parasiticus Research Articles

Inhibitory Effects of Bacillus subtilis Isolated from Meju (Fermented Soybean Brick) on the Growth of Aspergillus parasiticus

Background: Meju is a base material for making soy sauce, soybean paste, and red chili pepper paste, which are representative ingredients of Korean cuisine. Objectives: This study aimed to isolate a predominant bacterial strain of B. subtilis from meju and to observe its inhibitory effects on an aflatoxigenic mold. Methods: We used yellow soybeans (Glycine max (L.) Merr.) grown in South Korea, and meju was produced according to the recommended methods of the Korea Food Research Institute. The identification of the strain was conducted based on its morphological and biochemical characteristics and 16S rDNA sequence. Evaluation of the bacterial effect against A. parasiticus ATCC 15517 was done in yeast extract–sucrose broth at 28 °C. Its inhibitory effect was evaluated using two approaches: mycelial weight and aflatoxin production. Aflatoxins were determined using high-performance liquid chromatography. Results: In the meju samples fermented for three months, a B. subtilis strain, K-0924 was identified. At the end of the incubation period of A. parasiticus, dry mycelial weight was significantly reduced by more than 80% (p < 0.01) and total aflatoxin production was inhibited by more than 63% (p < 0.05) in the presence of B. subtilis. Conclusions: These results indicate that B. subtilis K-0924 inhibits the growth and aflatoxin production of toxigenic Aspergillus, which can be contaminated with meju. We could expect more inhibition by other bacteria related to fermentation of meju, and further examination is necessary on species other than B. subtilis.

Evaluation of antifungal activity of natural compounds on growth and aflatoxin B1 production of Aspergillus parasiticus and Aspergillus flavus.

Aspergillus species cause broad spectrum infections especially invasive lethal infections in immunocompromised patients. This study aimed to assess the antifungal activity of plants and compounds including Aloe vera, Thyme, carvacrol, and nano-encapsulation of carvacrol on the growth and production of aflatoxin B1 production by Aspergillus parasiticus and Aspergillus flavus. Minimum inhibitory concentrations of extracts Aloe vera, Thyme, carvacrol, and nanocarvacrol, and fluconazole as a control were determined according to Clinical and Laboratory Standards Institute by serial microdilution protocol. Then, the effect of inhibitory concentrations of these compounds on the aflatoxin B1 production level was evaluated by real-time PCR and high-performance liquid chromatography. Our results indicate that the Aspergillus parasiticus and Aspergillus flavusare sensitive to selected plants and compounds. Our findings showed that the compounds are appropriate alternative candidates against growth and production of aflatoxin of Aspergillus spp.

Yeast suppresses Aspergillus parasiticus growth and aflatoxins synthesis: Finding a new fungal antagonistic strain based on stored marinade

Aspergillus parasiticus is prone to producing toxic and carcinogenic aflatoxins (AFs) and posing risks to food safety. This study aims to explore the effect of yeasts isolated from marinade on the growth of A. parasiticus and to evaluate the inhibitory effects of these yeasts on the biosynthesis of AFs and their ability to remove AFs. Six yeast strains were screened through 18S rDNA and ITS rDNA gene sequence analysis, and among them, Candida tropicalis, Pichia kudriavzevii, Trichosporon asahii, Lodderomyces elongisporus and Pichia fermentans showed vigorous growth activity in the stored marinade. P. fermentans and L. elongisporus culture solutions significantly inhibited the growth and mycelial differentiation of A. parasiticus and effectively removed AFB1. The volatile compounds of P. fermentans and L. elongisporus significantly inhibited the growth of A. parasiticus, with inhibition rates of 72.10% and 67.03%, respectively. In addition, P. fermentans and L. elongisporus were co-cultured with A. parasiticus in marinade for seven days. The inhibition rates of AFB1 synthesis were 69.06% and 61.78%, while the inhibition rates of AFG1 were 65.89% and 60.02%, respectively. Hence, P. fermentans and L. elongisporus can be used as fungal antagonistic strains to help reduce AFs contamination in the marinade and its products.

Antifungal effects of selected menthol and eugenol in vapors on green coffee beans during long-term storage

Nowadays, coffee (Coffea Arabica L.) is among the most significant agricultural products of the world and drinking coffee has become one of the most popular habits in the world. The main contamination of stored coffee beans is related with the mycotoxin produced by the toxigenic fungi belonging the genus Aspergillus. Fungal infection followed by mycotoxin biosynthesis in coffee results in notable financial losses. subsequent mycotoxin biosynthesis in coffee leads to major economic losses. Complications ranging from mild to severe can be caused by the mycotoxins produced by this genus. The aim of this investigation was to determine the effect of menthol and eugenol on Aspergillus parasiticus (CBS 100926T) growth, spore germination, and their potential use as green coffee beans preservative during long-term storage (12 months). The minimum inhibitory concentrations (MICs) values of the menthol and eugenol were recorded to completely inhibit the growth of A. parasiticus in 400 μg/ml and 300 μg/ml, respectively. Both reduced spore germination by 9.33% and 5.66% at 300 μg/ml and 200 μg/ml, respectively. They showed efficacy in fumigated green coffee beans sample during the storage for up to 12 months providing an increase in the protection level of 62.5% for menthol and 73.21% for eugenol against the A. parasiticus contamination. This suggests that menthol and eugenol could be used as good alternatives for decreasing the deteriorations due to the fungal infections in green coffee beans during long-term storage.

Comparing the effects of essential oils and methanolic extracts on the inhibition of Aspergillus flavus and Aspergillus parasiticus growth and production of aflatoxins.

The antifungal and antiaflatoxigenic effects of four distinct plant species against Aspergillus flavus and Aspergillus parasiticus were investigated. Essential oils and methanolic extracts were prepared from aerial parts of Lippia javanica, Ocimum gratissimum, Satureja punctata, and stem barks of Toddalia asiatica by hydro-distillation and maceration, respectively. The poisoned food method was used to confirm the antifungal activity of essential oils and methanolic extracts from four different plant species against Aspergillus flavus and Aspergillus parasiticus, and high-performance liquid chromatography was used to quantify the antiaflatoxigenic activity. The essential oils of Satureja punctata and Lippia javanica showed the highest antiaflatoxigenic activity against the fungi strains tested at concentrations of 1.25, 2.5, and 5 µL/mL, followed by Ocimum gratissimum essential oil while Toddalia asiatica essential oil exerted moderate antiaflatoxigenic activity. Meanwhile, the methanolic extracts showed a wide spectrum of low to high antifungal and antiaflatoxigenic activities at concentrations of 125, 250, and 500µg/mL against A. flavus and A. parasiticus. This study has indicated that the essential oils of Satureja punctate, Lippia javanica, and Ocimum gratissimum had substantial antifungal and antiaflatoxigenic activities compared to their methanolic extracts, while Toddalia asiatica methanolic extract had a moderate antifungal activity compared to its essential oil.

Production of Aflatoxin B1 by Aspergillus parasiticus Grown on a Novel Meat-Based Media.

The aim of the present study was to develop meat-based media with compositions similar to those of dry-fermented meat products and to evaluate their use in studying the growth of Aspergillus parasiticus and the kinetics of aflatoxin B1 (AFB1) production. In our previous experiments, we found that the strain A. parasiticus ŽMJ7 produced a high amount of AFB1. Cooked meat agar (CMA2) was used as a novel complex meat-based medium with four variations: CMA2G (CMA2 supplemented with 1% glucose), CMA2YE (CMA2 supplemented with 0.2% yeast extract), and CMA2GYE (CMA2 supplemented with 1% glucose and 0.2% yeast extract). Media were inoculated with an A. parasiticus spore suspension (105 spores/mL) and incubated at 25 °C for up to 15 days. The A. parasiticus lag phase lasted less than 1 day, irrespective of the growth medium, with the exception of control medium CMA1 (cooked meat agar) as an already known meat-based medium. The highest mean colony growth rate was observed on CMA2 and CMA2G. Reversed-phase UPLC-MS/MS analysis was performed to determine the AFB1 concentration in combination with solid phase extraction (SPE). The highest AFB1 concentration in meat-based media was detected in CMA2GYE after 15 days of incubation (13,502 ± 2367 ng/mL media). The results showed that for studying AFB1 production in dry-fermented meat products, novel suitable media such as CMA2-based media are required. This finding could represent a potential concern with regard to the production of dry-fermented meat products.

Lactic Acid Bacteria as Potential Agents for Biocontrol of Aflatoxigenic and Ochratoxigenic Fungi.

Aflatoxins (AF) and ochratoxin A (OTA) are fungal metabolites that have carcinogenic, teratogenic, embryotoxic, genotoxic, neurotoxic, and immunosuppressive effects in humans and animals. The increased consumption of plant-based foods and environmental conditions associated with climate change have intensified the risk of mycotoxin intoxication. This study aimed to investigate the abilities of eleven selected LAB strains to reduce/inhibit the growth of Aspergillus flavus, Aspergillus parasiticus, Aspergillus carbonarius, Aspergillus niger, Aspergillus welwitschiae, Aspergillus steynii, Aspergillus westerdijkiae, and Penicillium verrucosum and AF and OTA production under different temperature regiments. Data were treated by ANOVA, and machine learning (ML) models able to predict the growth inhibition percentage were built, and their performance was compared. All factors LAB strain, fungal species, and temperature significantly affected fungal growth and mycotoxin production. The fungal growth inhibition range was 0-100%. Overall, the most sensitive fungi to LAB treatments were P. verrucosum and A. steynii, while the least sensitive were A. niger and A. welwitschiae. The LAB strains with the highest antifungal activity were Pediococcus pentosaceus (strains S11sMM and M9MM5b). The reduction range for AF was 19.0% (aflatoxin B1)-60.8% (aflatoxin B2) and for OTA, 7.3-100%, depending on the bacterial and fungal strains and temperatures. The LAB strains with the highest anti-AF activity were the three strains of P. pentosaceus and Leuconostoc mesenteroides ssp. dextranicum (T2MM3), and those with the highest anti-OTA activity were Leuconostoc paracasei ssp. paracasei (3T3R1) and L. mesenteroides ssp. dextranicum (T2MM3). The best ML methods in predicting fungal growth inhibition were multilayer perceptron neural networks, followed by random forest. Due to anti-fungal and anti-mycotoxin capacity, the LABs strains used in this study could be good candidates as biocontrol agents against aflatoxigenic and ochratoxigenic fungi and AFL and OTA accumulation.

Review of lignolytic fungi invading wood, and leather and control of selected fungi by using volatile essential oil of plant origin

Several cultural and historical objects are made up of wood and leather that are prone to the attack of wood- and leather-degrading microorganisms. Lignolytic fungi have a huge potential to degrade such historical objects that are made up of wood and leather. To protect these types of objects from lignolytic fungi, several chemical agents are being used which have a significant impact on the environment that necessitates sustainable approaches to control these microorganisms. In the present study, seven lignolytic fungi (four from wood and three from leather) were isolated and were identified as Colletotrichum capsici, Phoma spp., Aspergillus niger, Fusarium oxysporum, Aspergillus flavus, Aspergillus paraciticus and Colletotrichum capsici-1. All these fungi were able to produce a significant quantity of lignin peroxidase enzyme which confirms that these do have the potential to degrade wood and leather objects. 1200 mg/L was found to be the minimum inhibitory concentration for Aspergillus flavus, Colletotrichum capsici, Fusarium oxysporum, Colletotrichum capsici-1 and Aspergillus parasiticus. However, growth of Aspergillus niger and Phoma spp. was invisible at a concentration of 1400 mg/L, which is the minimum inhibitory concentration of Eucalyptus oil for Aspergillus niger and Phoma spp. At a concentration of 1200 mg/L of Eucalyptus oil, growth of Aspergillus flavus, Colletotrichum capsici-1, Fusarium oxysporum, Colletotrichum capsici and Aspergillus parasiticus was completely inhibited, while 100% growth inhibition was found for Aspergillus niger and Phoma spp. at 1400 mg/L of Eucalyptus oil. This study revealed that these lignolytic fungi may causes degradation of wood and leather made historical objects due to the presence of several organic compounds in Eucalyptus oil that have high anti-fungal activity. It was found that Eucalyptus oil has huge potential to control growth of lignolytic fungi including C. capsici, Phoma spp., A. niger, F. oxysporum, A. flavus, A. paraciticus and C. capsici-1 to protect wood and leather made historical objects in a sustainable and environmentally friendly way.

Inhibition of Aspergillus Parasiticus Growth and Aflatoxins Production by Natural Essential Oils and Phenolic Acids.

Aflatoxins represent a significant risk to food safety, and strategies are being implemented to reduce their entry into the food chain. The aim of this study was to evaluate the in vitro effect of four essential oils (EOs) (lavandins Grosso and Abrial, Origanum virens, and Rosmarinus officinalis) and four natural phenolic acids (PAs) (caffeic, chlorogenic, ferulic, and p-coumaric) on the growth and aflatoxins (B1, B2, G1, and G2) production by Aspergillus parasiticus. Minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC) were determined by the broth macrodilution method. Additionally, the mycelia weight was determined at concentration levels lower than MIC. The antiaflatoxigenic activity was evaluated in the two concentrations of the EOs right before MIC and at concentrations below the MIC value for the PAs. To this end, in-house validated methodology based on high-performance liquid chromatography with post-column photochemical derivatization and fluorescence detection (HPLC-PHRED-FLD) was used. EOs of O. virens and lavandins (Grosso and Abrial) completely inhibited mold growth. In addition, a significant reduction in mycelial mass (p < 0.05) was observed for all EOs and PAs at different concentrations. In all cases except for lavandin Abrial, EO concentrations just before the MIC value strongly reduced (p < 0.05) aflatoxins synthesis. Aflatoxins production was completely inhibited by all PAs at a concentration of 20 mM; although at low concentrations, mycotoxin production was stimulated in some cases. The present study provides a scientific basis for further study of the inhibiting mechanisms.

Effects of climatic conditions and hermetic storage on the growth of Aspergillus parasiticus and aflatoxin B1 contamination in basmati rice

Climate change has influenced the growth of Aspergillus species and subsequent aflatoxins (AFs) production. In this study, the combined effect of temperature (20–40 °C) with respect to moisture content (MC = 7–25%) and water activity (aw = 0.86–0.98) on the growth of A. parasiticus and aflatoxin B1 (AFB1) occurrence was studied in basmati rice. The effect of hermetic storage on AFB1 occurrence was also investigated. The growth of A. parasiticus and the AFB1 production were found between 15 and 25% MC and 0.92–0.98 aw at 25–35 °C. The optimum production of these entities with 25% MC or 0.95 aw at 30 °C was noticed. However, the lower temperature (<20 °C), MC (<10%) and aw (<0.86) doesn't support the growth of A. parasiticus and AFB1 production. The production rate of the fungi and their metabolite were found in the following increasing order 30° > 35° > 25° > 20° > 40 °C at all tested MC and aw levels. The growth of A. parasiticus and AFB1 production were found as increasing trend 25% > 20% > 15% > 10% at all tested temperature. Whereas, the fungal growth and AFB1 production with respect to aw were seen in the increasing order 0.95 ˃ 0.98 ˃ 0.92 ˃ 0.89 ˃ 0.86 aw at all tested temperatures. The storage of rice samples in a hermetic bag, the significant control (less than 90% AFB1 production compared with normal conditions) the AFB1 production in rice. The results also indicated that the growth of A. parasiticus and AFB1 production were significantly (p <0.05) affected by the temperature, MC and aw, as well as the storage conditions in normal and hermetic bags as analyzed using ANOVA. In conclusion, hermetic storage is experimentally proved as the best way of storing cereals and grains for a long time.

Combinational inhibitory action of essential oils and gamma irradiation for controlling Aspergillus flavus and Aspergillus parasiticus growth and their aflatoxins biosynthesis in vitro and in situ conditions.

The purpose of this study was to investigate the effects of certain essential oils (star anise, lemon leaves, marjoram, fennel, and lavender) on the fungal growth of Aspergillus flavus and Aspergillus parasiticus and their production of aflatoxin B1 (AFB1). The degree of suppression of the aflatoxigenic strains' growth and their production of AFB1 is mainly affected by the kind and the concentration of the tested essential oils (EOs). Star anise essential oil had the lowest minimum inhibitory concentration (0.5 and 1.0 μL/mL) against A. flavus and A. parasiticus, respectively, so it was the best among the five different oils. The study of liquid chromatography with tandem mass spectrometry revealed that star anise EO resulted in a 98% reduction in AFB1 without a breakdown of AFB1 products after treatment thus the complete removal of AFB1 was done without any toxic residues. The combination showed a synergistic effect, the combinational treatment between γ-irradiation at a low dose (2 kGy) and star anise EO at concentrate 0.5 μL/g destroyed A. flavus and A. parasiticus inoculated (individually) in sorghum and peanut, respectively throughout the storage period (8 weeks).

Modelling the effect of environmental factors on the growth of Aspergillus parasiticus and mycotoxin production in paddy during storage

The simulated experiment of A. parasiticus isolated from the paddy was carried out during the paddy storage for 20 days. The growth and mycotoxin data were collected for constructing kinetic and probability models of moulds. The Baranyi and Gompertz model was employed as the primary model and estimated the lag phase and maximum specific growth rate. Secondary models, such as polynomial, Davey and Gibson model were used and completely evaluated under different conditions. The polynomial equation was highly rated compared with Gibson and Davey model and gave realistic temperatures and aw for mould growth. Logistic model showed promising results on the prediction of growth boundary and AFB1 production. Employed models showed promising predicted results, indicating that it is an effective tool for describing and predicting the growth of moulds under different temperatures and aw. The results can be applied to develop the optimal strategy to prevent fungal spoilage and aflatoxin production during paddy storage.

Detoxification of Aflatoxin B1 and Ochratoxin A Using Salvia farinacea and Azadirachta indica Water Extract and Application in Meat Products.

Seventy-five samples of selected meat products, including luncheon, beef burger, sausage, basterma, and kofta, were collected from Alexandria and New Borg El-Arab cities (Egypt). The samples were subjected to mycological examination as well as for detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) residues. Besides, the study evaluated the effect of aqueous leaf extracts from mealycup sage (Salvia farinacea) and neem (Azadirachta indica), individually and in combination, on the growth of human pathogens Aspergillus parasiticus and Aspergillus flavus producing AFB1, as well as Aspergillus ochraceus and Aspergillus niger which produce OTA. The obtained results revealed that sausage samples had the highest mould count with a mean value of 13.20×102/g, followed by basterma samples 12.05×102/g, then beef burger 7.39×102/g. In contrast, luncheon and kofta samples had the lowest count with a mean value of 5.51×102/g and 2.82×102/g. The findings revealed the antifungal potential of tested extracts. The total inhibition of A. parasitcus and A. niger growth was observed at 2 mg/mL of the combined extract. Salvia farinacea extract had the highest total phenolic content and total flavonoid content with a value of 174.1 and 52.6 mg g-1, respectively. Rutin was the major phenolic component in neem and combined extracts, accounting for 19123 and 8882 μg/g, respectively. Besides, the study investigated detoxification of AFB1 and OTA using combined extract in albino rats. The results confirmed the convenient and safe use of Salvia farinacea and Azadirachta indica extract and their combination as natural antifungal and antioxidant agents. The combined extract could be used as a natural preservative in food processing to control or prevent contamination.

Effect of Lactic Acid Bacteria Strains on the Growth and Aflatoxin Production Potential of Aspergillus parasiticus, and Their Ability to Bind Aflatoxin B1, Ochratoxin A, and Zearalenone in vitro.

The increased consumption of plant-based foods has intensified the concern related to mycotoxin intoxication. This study aimed to investigate the effect of selected lactic acid bacteria (LAB) strains on the growth of Aspergillus parasiticus NRRL 2999 and its production of aflatoxin (AF). The ability of the heat-killed (100°C for 1 h) LAB strains to bind aflatoxin M1 (AFM1) in milk and aflatoxin B1 (AFB1), ochratoxin A (OTA), and zearalenone (ZEN) in potassium phosphate buffer (PPB) was also evaluated in vitro. Ten LAB strains were tested individually, by inoculating them simultaneously with the fungus or after incubation of the fungus for 24 or 48 h at 25°C. Double layer yeast extract sucrose (YES) agar, de Man Rogosa and Sharpe (MRS) agar, and YES broth were incubated for 7 days at 25°C to follow the development of the fungus. Levilactobacillus spp. 3QB398 and Levilactobacillus brevis 2QB422 strains were able to delay the growth of A. parasiticus in YES broth, even when these strains were inoculated 24 h after the fungus. The inhibitory effect of these LAB strains was confirmed by the reduction of fungus colony size, suggesting dominance of LAB by competition (a Lotka-Voltera effect). The production of AFB1 by A. parasiticus was inhibited when the fungus was inoculated simultaneously with Lactiplantibacillus plantarum 3QB361 or L. plantarum 3QB350. No AFB1 was found when Levilactobacillus spp. 2QB383 was present, even when the LAB was inoculated 48 h after the fungus. In binding studies, seven inactivated LAB strains were able to promote a reduction of at least 50% the level of AFB1, OTA, and ZEN. This reduction varied depending on the pH of the PPB. In milk, however, only two inactivated LAB strains were able to reduce AFM1, with a reduction of 33 and 45% for Levilactobacillus spp. 3QB398 (Levilactobacillus spp.) and L. brevis 2QB422, respectively. Nevertheless, these results clearly indicate the potential of using LAB for mycotoxin reduction.

Growth Inhibition of Aflatoxigenic Molds and Biodegradation of Aflatoxin B1 by Certain Bacterial Isolates

AFLATOXIN B1 (AFB1) is mutagenic, carcinogenic, and harmful to humans and animals. Different physical and chemical methods have been proposed to reduce mycotoxins but few have been approved for practical application. This study investigated the possible use of certain bacterial species were isolated from different agricultural soils as biological control agents against the growth of toxigenic Aspergillus flavus and Aspergillus parasiticus isolated from sorghum and peanuts as well as biodegradation of aflatoxin B1 (AFB1). Out of these 30 bacterial isolates, 6 isolates showed that have antagonistic effects against the aflatoxigenic fungi but with varying efficiencies. The six different isolates were screened for their ability to degrade AFB1. Out of 6 isolates, one isolate showed high reduction activity of AFB1. Maximum antifungal activities were observed in one isolate was identified as Staphylococcus lentus. S. lentus completely prevented the A. flavus and A. parasiticus growth and completely degraded AFB1 in Liquid Culture. LC-MS/MS analysis revealed that S. lentus resulted in a 96.54% degradation of AFB1, but no products has been detected. In practical application, it was found that S. lentus at concentration of 10 and 25mL kg-1 were completely prevented the growth of A. flavus and A. parasiticus in sorghum and peanuts, respectively, consequently no aflatoxin were produced. According to our information, this is the first study to prove that Staphylococcus lentus has the capability to inhibit the growth of Aspergillus flavus and Aspergillus parasiticus by 100% and degrading AFB1 by 96%.

Synthesis and Evaluation of Lipid-based Nanoparticle Containing Ginger Extract against Aspergillus Species

Introduction: Loading the active ingredients of medicinal plants in lipid nanoparticles reduces the reaction of the active substance with the surrounding environment, such as water and oxygen, and reduces the intensity of transmission or evaporation to the external environment. In this study, intended to enhance efficacy of ginger extract, encapsulation in nanoliponiosome synthesized by thin-film hydration method were done and their antifungal effect on the growth of Aspergillus flavus and Aspergillus parasiticus were studied.&#x0D; Methods: In this experimental laboratory study, derivation was done using Soxhlet extractor method. Antifungal activity of ginger extract was specific by disc diffusion and microplate dilution methods. The inhibitory effect of extract was investigated. Physiochemical characteristics and structural characterization of nanoparticle were evaluated from the perspective of in vitro efficiency, drug release, nanoparticle size, zeta potential, surface morphology and FTIR (Fourier-transform infrared spectroscopy), DLS (Dynamic light scattering) and finally SEM (Scanning electron microscope) spectra.&#x0D; Results: FTIR investigations showed ginger extract and nanoliponiosome had no chemical interaction leading to change the functional groups. SEM microscope showed the spherical mprphology of particles and average particles size of 73nm. Ginger extract was loaded into the nanoliponiosome with a yield of 71%. It was also found out that ginger extract had a stronger antifungal effect against Aspergillus flavus fungus compared to the Aspergillus parasiticus fungus. At both 37°C and 42°C, the release of ginger extract was higher at pH of 4.5 compared to neutral pH (7.4).&#x0D; Conclusion: Nanoliponiosomes containing ginger extract with good physicochemical properties, increased drug stability and good release control can be promising antifungal agents with high antifungal effects and low side effects.

Inhibitory effect of Capsicum chinense and Piper nigrum fruits, capsaicin and piperine on aflatoxins production in Aspergillus parasiticus by downregulating the expression of aflD, aflM, aflR, and aflS genes of aflatoxins biosynthetic pathway

Aflatoxins produced by Aspergillus parasiticus are toxic and carcinogenic metabolites. The biosynthesis of this mycotoxins is a complex process and involves at least 30 genes clustered within an approximately 82 kB gene cluster. In the present study, the effect of Capsicum chinense and Piper nigrum fruits on Aspergillus parasiticus growth and aflatoxin production were studied in relation to the expression of aflD, aflM, aflR, and aflS four; key genes of aflatoxins biosynthesis pathway. GC-EIMS analysis identified capsaicin (66,107 µg g−1) and piperine (1,138 µg g−1) as the most abundant compounds in C. chinense and P. nigrum fruits, respectively. The antifungal and anti-aflatoxigenic assays showed that C. chinense, P. nigrum, capsaicin, and piperine inhibited A. parasiticus growth and aflatoxins production in a dose-dependent manner. The piperine at 300 µg mL−1 produced higher radial growth inhibition (89%) and aflatoxin production inhibition (69%). The expression of aflatoxin biosynthetic genes was evaluated by quantitative real-time PCR (qRT-PCR) and revealed that aflatoxin inhibition occurring via downregulating the aflS and aflR, and subsequently aflD and aflM genes. These results will improve our understanding of the mechanism of aflatoxin regulation by C. chinense, P. nigrum, capsaicin, and piperine, and provides a reference for further study.

The effect of Propolis on inhibition of Aspergillus parasiticus growth, aflatoxin production and expression of aflatoxin biosynthesis pathway genes.

Aflatoxins are one of the most important mycotoxins, which have been classified as Group I carcinogenic compounds by the International Agency for Research on Cancer. This investigation aimed to examine the effect of Propolis on inhibition of the Aspergillus parasiticus growth, aflatoxin production and expression of aflatoxin biosynthesis pathway genes. A standard strain of Aspergillus parasiticus (ATCC 15517) was used to perform antifungal susceptibility test, using a microdilution method in accordance with the CLSI M38-A2 guidelines. The aflatoxin concentrations in the control and treated media were determined by HPLC. Also, the quantitative changes in the level of nor-1, ver-1 and omtA genes expression in aflatoxin biosynthetic pathway were analyzed using Real-Time PCR method. The results showed that the minimum inhibitory concentrations (MIC) of propolis was 100 µg/ml. The results showed that total levels of aflatoxin decreased from 386.1 ppm to 3.01 ppm at 50 µg/ml of propolis. In addition, quantitative real-time PCR analysis showed that the level of nor-1, ver-1 and omtA genes expression was significantly decreased after treatment with propolis extract. The findings reveal that propolis extract, have a significant inhibitory effect on important genes for aflatoxin biosynthesis pathway in aflatoxin production.

Antifungal Effect of Triglycerol Monolaurate Synthesized by Lipozyme 435-Mediated Esterification.

This study was designed to synthesize triglycerol monolaurate (TGML) with Lipozyme 435 as the catalyst, and explore its effects on the growth of Aspergillus parasiticus (A. parasiticus) and Aspergillus flavus (A. flavus) and the secretion of aflatoxin b1. The highest content of TGML (49.76%) was obtained at a molar ratio of triglycerol to lauric acid of 1.08, a reaction temperature of 84.93°C, a reaction time of 6 h and an enzyme dosage of 1.32%. After purification by molecular distillation combined with the washes with ethyl acetate and water, the purity of TGML reached 98.3%. Through characterization by electrospray-ionization mass spectrometry, infrared spectrum and nuclear magnetic resonance, the structure of TGML was identified as a linear triglycerol combined with lauroyl at the end. Finally, the inhibitory effects of TGML on the growths of A. parasiticus and A. flavus and the secretion of aflatoxin b1 were evaluated by measuring the colony diameter, the inhibition rate of mycelial growth and the content of mycotoxin in the media. The results indicated that TGML had a stronger inhibitory effects on colony growth and mycelial development of both toxic molds compared to sodium benzoate and potassium sorbate, and the secretions of toxins from A. parasiticus and A. flavus were completely suppressed when adding TGML at 10 and 5 mM, respectively. Based on the above results, TGML may be used as a substitute for traditional antifungal agents in the food industry.

In Vitro Anti-Mycotoxigenic and Anti-Aflatoxigenic Properties of Probiotic Bacteria; Lactobacillus plantarum and L. paracasei

The present study was designed to examine the effects of Lactobacillus plantaraum and L. paracasei on the growth and aflatoxin-producing ability of Aspergillus parasiticus. In these experiments, direct and indirect interactions of two different probiotic lactobacilli with A. parasiticus was examined.A co-culture system which supported both fungal growth and probiotic bacteria showed the inhibitory actions of the lactobacilli on A. parasiticus growth. Disk Diffusion Assay was developed to examine the anti-fungal action of the probiotic cell free extract. The probiotic extracts were also applied in a micro-dilution assay to evaluate the anti-fungal and anti-aflatoxin properties of the probiotic extracts. The results showed that direct interaction of the bacteria with A. parasiticus in a spot-culture method (two-layer semi-solid/solid culture) caused a clear zone of inhibition of A. parasiticus growth. Fungal growth inhibition was found to be dependent on the concentration of the probiotic extracts added to A. parasiticus culture (micro dilution assay). The inhibitory effects of the probiotic extracts was further confirmed using Disc Diffusion Assay by showing that discs loaded with 50 or 100 µl of extracts resulted in a significant (4-5 folds) inhibition in A. parasiticus growth. The extract prepared from the probiotics inhibited the fungal growth and at higher levels. Accordingly, the probiotic extract (50% v/v) could significantly suppress aflatoxin levels in mycelia and fungal culture media. In conclusion, probiotic bacteria and their cell-free extracts produced under optimized condition exhibit anti-fungal and anti-mycotoxin effects provided that these bacteria undergo screening and selection prior totheir application.