Dietary Exposure and Risk Assessment of Multi-Mycotoxins (AFB1, AFM1, OTA, OTB, DON, T-2 and HT-2) in the Lebanese Food Basket Consumed by Adults: Findings from the Updated Lebanese National Consumption Survey through a Total Diet Study Approach.

Mycotoxins have been linked to adverse health impacts, including liver cancer and kidney diseases. The objectives of the current study were to evaluate the dietary exposure of Lebanese adults to multi-mycotoxins (aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), ochratoxin A (OTA), ochratoxin B (OTB), deoxynivalenol (DON), T-2 and HT-2) and to assess their associated health risks. Hence, a nationally representative sample of 449 participants aged 18-64 years old were interviewed to obtain their socio-demographic characteristics, food consumption data and exposure estimates. A food frequency questionnaire and 24 h-recall were used to collect data. The concentration of mycotoxins in all foods consumed by the participants was collected from previous national published studies. The estimated daily intake (EDI), the hazard quotient (HQ) and the margin of exposure (MOE) were calculated. The total exposure to AFB1, AFM1, OTA and DON was 1.26, 0.39, 4.10 and 411.18 ng/kg bw/day, respectively. The MOE to AFB1, AFM1, OTA and DON in the Lebanese food basket was 316, 1454, 3539 and 510, respectively, indicating high health-related risks. Per food items, the MOE to AFB1 was below 10,000 in cereals (466.5), mainly in rice (827.9) and Burgul (4868.5). Similarly, the MOE to OTA in cereals was 1439, in which bread (4022), rice (7589) and bulgur (7628) were considered unsafe. Moreover, the MOE to DON in cereals (605) is alarming, especially in bread (632) and manakesh (6879). The MOE to AFM1 in dairy products was 1454, indicating health-related risks with a focus on yogurt (9788) and labneh (8153). As for the herbs/spices group and traditional dishes, the MOE to AFB1 was relatively lower than 10,000 (3690 and 1625, respectively), with a focus on thyme (2624) and kishik (3297), respectively. It is noteworthy that the MOE to DON and the MOE to OTA in traditional foods and coffee were lower than 10,000 (8047 and 8867, respectively). All hazard quotient (HQ) values were below 1, except the HQ value of milk and dairy products (1.96). The intake of some food groups varied between age categories, corresponding to differences in EDI between them. Thus, it is essential to put control measures in place to decrease the contamination and exposure to mycotoxins by Lebanese consumers.

Aflatoxins and Ochratoxin A in Tea Sold in Lebanon: Effects of Type, Packaging, and Origin.

Tea is among the oldest and most-known beverages around the world, and it has many flavors and types. Tea can be easily contaminated in any of its production steps, especially with mycotoxins that are produced particularly in humid and warm environments. This study aims to examine the level of ochratoxin A (OTA) and total aflatoxin (AF) contamination in black and green tea sold in Lebanon, evaluate its safety compared to international standards, and assess the effect of different variables on the levels of OTA and AFs. For this, the Lebanese market was screened and all tea brands (n = 37; 24 black and 13 green) were collected twice. The Enzyme-Linked Immunoassay (ELISA) method was used to determine OTA and AFs in the samples. AFs and OTA were detected in 28 (75.7%) and 31 (88.6%) samples, respectively. The average of AFs in the positive (above detection limit: 1.75 µg/kg) samples was 2.66 ± 0.15 µg/kg, while the average of OTA in the positive (above detection limit: 1.6 µg/kg) samples was 3.74 ± 0.72 µg/kg. The mean AFs in black and green tea were 2.65 ± 0.55 and 2.54 ± 0.40 µg/kg, respectively, while for OTA, the mean levels were 3.67 ± 0.96 and 3.46 ± 1.09 µg/kg in black and green tea samples, respectively. Four brands (10.8%) contained total aflatoxin levels above the EU limit (4 µg/kg). As for OTA, all samples had OTA levels below the Chinese limit (5 µg/kg). No significant association (p > 0.05) was found between OTA and tea type, level of packaging, country of origin, country of packing, and country of distribution. However, AF contamination was significantly (p < 0.05) higher in unpacked tea, and in brands where the country of origin, packing, and distributor was in Asia. The results showed that the tea brands in Lebanon are relatively safe in terms of AFs and OTA.

Prevalence and Antibiotic Resistance of Staphylococcus aureus and Escherichia coli Isolated from Bovine Raw Milk in Lebanon: A study on Antibiotic Usage, Antibiotic Residues, and Assessment of Human Health Risk Using the One Health Approach.

The emergence, persistence, and spread of antibiotic-resistant microbes is a tremendous public health threat that is considered nowadays a critical One Health issue. In Lebanon, the consumption of raw bovine milk has been recently reported as a result of the financial crisis. The objectives of the current study were (1) to evaluate raw bovine milk samples in a comprehensive manner for the types of antibiotics used and their residues, (2) to determine the presence of mesophilic bacteria, extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA), and (3) to determine the associated human health risk caused by drinking raw milk with antibiotic residues among all age categories. LC-MS-MS was used to carry out the analysis. From 200 milk samples, 30 (15%) were found contaminated with four major antibiotics. The highest average concentration detected was for oxytetracyline 31.51 ± 13.23 µg/kg, followed by 5.5 ± 0.55 µg/kg for gentamicin, 4.56 ± 0.73 µg/kg for colistin, and 4.44 ± 0.89 µg/kg for tylosin. The mean contamination among most samples was below the maximum residue limits (MRLs). Upon comparison with the acceptable daily intake (ADI), the estimated daily intake (EDI) across all age groups was acceptable. The hazard quotient (HQ) was also below 1 across all age groups, signifying the absence of associated health risks for the Lebanese consumers. On the other hand, all milk samples were found exceeding the maximum tolerable value of mesophilic flora. Antibiotic-resistant bacteria (ARB) were detected and represented by ESBL-producing E. coli and MRSA isolates. Thus, the greatest threat of antibiotic use in Lebanon does not fall under antibiotic residues but rather the proliferation of antibiotic resistance in potentially pathogenic bacteria. In this study, the virulence profile of detected bacteria was not investigated; thus their pathogenicity remains unknown. Therefore, to mitigate this health threat in Lebanon, a "One Health" action plan against ABR is required. It will provide a framework for continued, more extensive action to reduce the emergence and spread of ABR in Lebanon.

Inhibition of Aflatoxin B1 Synthesis in Aspergillus flavus by Mate (Ilex paraguariensis), Rosemary (Rosmarinus officinalis) and Green Tea (Camellia sinensis) Extracts: Relation with Extract Antioxidant Capacity and Fungal Oxidative Stress Response Modulation.

Plant extracts may represent an ecofriendly alternative to chemical fungicides to limit aflatoxin B1 (AFB1) contamination of foods and feeds. Mate (Ilex paraguariensis), rosemary (Romarinus officinalis) and green tea (Camellia sinensis) are well known for their beneficial properties, which are mainly related to their richness in bioactive phenolic compounds. AFB1 production is inhibited, with varying efficiency, by acetone/water extracts from these three plants. At 0.45 µg dry matter (DM)/mL of culture medium, mate and green tea extracts were able to completely inhibit AFB1 production in Aspergillus flavus, and rosemary extract completely blocked AFB1 biosynthesis at 3.6 µg DM/mL of culture medium. The anti-AFB1 capacity of the extracts correlated strongly with their phenolic content, but, surprisingly, no such correlation was evident with their antioxidative ability, which is consistent with the ineffectiveness of these extracts against fungal catalase activity. Anti-AFB1 activity correlated more strongly with the radical scavenging capacity of the extracts. This is consistent with the modulation of SOD induced by mate and green tea in Aspergillus flavus. Finally, rutin, a phenolic compound present in the three plants tested in this work, was shown to inhibit AFB1 synthesis and may be responsible for the anti-mycotoxin effect reported herein.

Phenolic Compounds Recovery from Blood Orange Peels Using a Novel Green Infrared Technology Ired-Irrad®, and Their Effect on the Inhibition of Aspergillus flavus Proliferation and Aflatoxin B1 Production.

The intensification of total phenolic compound (TPC) extraction from blood orange peels was optimized using a novel green infrared-assisted extraction technique (IRAE, Ired-Irrad®) and compared to the conventional extraction using a water bath (WB). Response surface methodology (RSM) allowed for the optimization of ethanol concentration (E), time (t), and temperature (T) in terms of extracted TPC and their antiradical activity, for both WB extraction and IRAE. Using WB extraction, the multiple response optimums as obtained after 4 h at 73 °C and using 79% ethanol/water were 1.67 g GAE/100 g for TPC and 59% as DPPH inhibition percentage. IRAE increased the extraction of TPC by 18% using 52% ethanol/water after less than 1 h at 79 °C. This novel technology has the advantage of being easily scalable for industrial usage. HPLC analysis showed that IRAE enhanced the recovery of gallic acid, resveratrol, quercetin, caffeic acid, and hesperidin. IR extracts exhibited high bioactivity by inhibiting the production of Aflatoxin B1 by 98.9%.

Aflatoxin B1 Occurrence in Children under the Age of Five's Food Products and Aflatoxin M1 Exposure Assessment and Risk Characterization of Arab Infants through Consumption of Infant Powdered Formula: A Lebanese Experience.

Aflatoxin M1 (AFM1) is a salient metabolite that can be used to assess Aflatoxin B1 (AFB1) exposure in humans and animals. The carcinogenic potency of AFB1 and AFM1 was severely reported. The aims of this study were (1) to survey the contamination level of AFM1 in the most traded infant powdered formula brands (IPF) (n = 42) along with the AFB1 level in under 5's children food brands (biscuits, cornflakes, and cereals) (n = 42) and (2) to assess the estimated daily intake (EDI), the hazard quotient (HQ) and the margin of exposure (MOE) of AFM1 among infants (0-12 months) in Lebanon. All of the samples were analyzed using ELISA technique. AFB1 was below detection limit in all of the children's food brands samples. Out of 42 IPF samples 9.5% were AFM1-positive in the range of 29.54-140.16 ng/L and exceeded the maximum tolerable limit (MTL) set by the European commission (25 ng/kg). The overall average contamination level was 5.72 ± 0.014 ng/L. The EDI of AMF1 for male was in the range of 0.37-0.78 ng/kg/b.w./day and 0.40-0.87 ng/kg/b.w./day for females. Similarly, the HQ calculation resulted in an average of 3.05 for males and 3.28 for females. MOE calculations were far lower from 10,000 in both genders which indicates a high risk of genotoxicity and carcinogenicity. Our findings show that AFM1's EDI, HQ and MOE scored high among Lebanese infants. As infants consume more IPF relative to their body weight, the persistence of IPF with high AFM1 levels threatens their health. Thus, infant's exposure risk to AFM1 in IPF should be a continuous focus of attention.

Aflatoxin B1 in Rice: Effects of Storage Duration, Grain Type and Size, Production Site, and Season.

ABSTRACT: Our study evaluated aflatoxin B1 (AFB1) levels in packed rice marketed in Lebanon and determined the exposure to this toxin from rice consumption. A total of 105 packed white, parboiled, and brown rice bags were collected. Enzyme-linked immunosorbent assay was used to measure AFB1. A comprehensive food frequency questionnaire was completed by 500 participants to determine patterns of rice consumption and, subsequently, the exposure levels to AFB1 from rice consumption in Lebanon. AFB1 was detected in all rice samples (100%). The average concentration ± standard deviation of AFB1 was 0.5 ± 0.3 µg/kg. Contamination ranged between 0.06 and 2.08 µg/kg. Moisture content in all rice samples was below the recommended percentage (14%). Only 1% of the samples had an AFB1 level above the European Union limit (2 µg/kg). Brown rice had a significantly higher AFB1 level than white and parboiled rice (P = 0.02), while a significant difference was found between both collections for the same brands (P = 0.016). Packing season, packing country, country of origin, presence of a food safety management certification, grain size, and time between packing and purchasing had no significant effect. Exposure to AFB1 from rice consumption in Lebanon was calculated as 0.1 to 2 ng/kg of body weight per day.

Single, Subsequent, or Simultaneous Treatments to Mitigate Mycotoxins in Solid Foods and Feeds: A Critical Review.

Mycotoxins in solid foods and feeds jeopardize the public health of humans and animals and cause food security issues. The inefficacy of most preventive measures to control the production of fungi in foods and feeds during the pre-harvest and post-harvest stages incited interest in the mitigation of these mycotoxins that can be conducted by the application of various chemical, physical, and/or biological treatments. These treatments are implemented separately or through a combination of two or more treatments simultaneously or subsequently. The reduction rates of the methods differ greatly, as do their effect on the organoleptic attributes, nutritional quality, and the environment. This critical review aims at summarizing the latest studies related to the mitigation of mycotoxins in solid foods and feeds. It discusses and evaluates the single and combined mycotoxin reduction treatments, compares their efficiency, elaborates on their advantages and disadvantages, and sheds light on the treated foods or feeds, as well as on their environmental impact.

Public health risk associated with the co-occurrence of aflatoxin B1 and ochratoxin A in spices, herbs, and nuts in Lebanon.

Background: Aflatoxin B1 and ochratoxin A are mycotoxins produced by filamentous fungi that attack crops on field and storage. Both mycotoxins present a risk on public health since aflatoxin B1 is a hepatotoxic and hepatocarcinogenic agent while ochratoxin A can be nephrotoxic. Those mycotoxins can be found in several food items including spices, herbs, and nuts. Objectives: In Lebanon, few studies address aflatoxin B1 and ochratoxin A contamination in spices, herbs, and nuts. So, the aim of this study is to investigate the concentrations of those two mycotoxins particularly in spices and herbs and the concentration of aflatoxin B1 in nuts, and to determine the dietary exposure of the Lebanese population and their possible attribution to liver cancer and renal damage. Methods: In this work, a total of 198 samples of spices, herbs, and nuts were collected from different sites. Aflatoxin B1 and ochratoxin A were quantified using immune-affinity columns. A food frequency questionnaire was used to quantify the consumption of spices, herbs, and nuts in Lebanon. Exposure to aflatoxin B1 and ochratoxin A was calculated accordingly and liver and kidney cancer risks were evaluated. Results: Aflatoxin B1 was respectively found in 100, 20.4, and 98.6% of the spices, herbs, and nuts samples, while ochratoxin A was found in 100 and 44.4% of spices and herbs, respectively. Aflatoxin B1 was found at mean concentration of 0.97, 0.27, and 0.40 µg/kg in spices, herbs, and nuts, respectively while ochratoxin A was found at mean concentrations of 38.8 and 1.81 µg/kg in spices and herbs, respectively. Aflatoxin B1 occurrence was shown to be associated in this study with 0.017 additional cancer cases per 100,000 persons per year, and ochratoxin A weekly exposure was shown to be 5.04 ng/kg bw less than the Provisional Tolerable Weekly Intake of 100 ng/kg bw which indicates low risk of renal damage from spices and herbs consumption. Conclusion: The consumption of spices, herbs, and nuts in Lebanon could lead to an increase in health risks associated with aflatoxin B1 and ochratoxin A, specifically spices. The reported occurrence may be directly related to poor storage conditions.

Aflatoxin B1 and ochratoxin A in imported and Lebanese wheat and -products.

AFB1 and OTA are produced in food products due to climatic conditions like temperature and humidity. In Lebanon, few studies address AFB1 and OTA contamination in wheat and wheat products. In this study, a total of 312 samples of wheat and wheat products were collected from different sites, including port, silos, mills, and supermarkets were analysed. Wheat and wheat products consumption in Lebanon was quantified using a food frequency questionnaire. Exposure to AFB1 and OTA was calculated and liver and kidney cancer risk were evaluated. Results showed that AFB1 and OTA were, respectively, found in 65.7% and 100% of the samples, with 0.6% and 17.6% exceeding the maximum limit set by the European Commission. AFB1 was shown to be associated with 0.076 additional cancer cases per 100,000 persons per year and OTA weekly exposure was shown to be 53.2 ng/kg bw, which is less than the Provisional Tolerable Weekly Intake of 100 ng/kg bw.

Antifungal and anti-aflatoxigenic properties of organs of Cannabis sativa L.: relation to phenolic content and antioxidant capacities.

Aflatoxin B1 is a carcinogenic mycotoxin that frequently contaminates crops worldwide. Current research indicates that the use of natural extracts to combat mycotoxin contamination may represent an eco-friendly, sustainable strategy to ensure food safety. Although Cannabis sativa L. has long been known for its psychoactive cannabinoids, it is also rich in many other bioactive molecules. This study examines extracts from various organs of Cannabis sativa L. to determine their ability to limit aflatoxin production and growth of Aspergillus flavus. The results indicate that flower extract is most effective for limiting the synthesis of aflatoxin B1, leading to an almost-complete inhibition of toxin production at a concentration of 0.225 mg dry matter per gram of culture medium. Since flower extract is rich in phenolic compounds, its total antioxidant ability and radical-scavenging capacity are determined. Compared with other anti-aflatoxigenic extracts, the anti-oxidative potential of Cannabis sativa L. flower extract appears moderate, suggesting that its anti-mycotoxin effect may be related to other bioactive compounds.

Co-Occurrence of Aflatoxin B1 and Ochratoxin A in Lebanese Stored Wheat.

ABSTRACT: The aim of the study was to determine the levels of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in durum wheat samples collected over six periods of time from two official warehouses (A and B) in Lebanon. The concentrations of AFB1 in wheat samples taken from both warehouses ranged from 1.05 to 7.36 µg/kg. Results showed that 23.3 and 25.3% of samples taken from warehouse A and warehouse B, respectively, had AFB1 levels >2 µg/kg. For OTA, the contamination level of wheat samples taken from warehouse A and warehouse B was 52.0 and 44.6%, respectively, with concentrations ranging from 0.51 to 9.71 µg/kg. The percentage of samples with high detectable OTA levels (>3 µg/kg) was 28.6 and 25.3% for wheat samples taken from warehouse A and warehouse B, respectively. Thus, the consumption of wheat and derived products in Lebanon can be considered hazardous and may present a significant risk to the health of the Lebanese population regarding the overall daily exposure to these mycotoxins.

Prediction of Salmonella serovars isolated from clinical and food matrices in Lebanon and genomic-based investigation focusing on Enteritidis serovar.

Salmonella enterica subsp. enterica serovars are considered major causes of food poisoning and we performed this study because Salmonella is a burden in Lebanon. The present study investigated the ability of genomic information to predict serovar using a collection of Salmonella isolates from infected humans (n = 24) and contaminated food (n = 63) in Lebanon. Further, the phylogenomic relationships of the serovar the predominated in Lebanon (i.e., S. Enteritidis; n = 25) were investigated in comparison with isolates from other countries (n = 130) based on coregenome single nucleotide polymorphisms (SNPs). Genetic elements, specifically Salmonella pathogenicity islands (SPIs), plasmid replicons, and antibiotic-resistance genes were screened in S. Enteritidis genomes (n = 155). Our results revealed that the Salmonella serovars identification by seroagglutination from the samples isolated in Lebanon (n = 87) was highly correlated with the genomic-based prediction of serovars (80.4-85.0% with SeqSero1 and 93.1-94.2% with SeqSero2). The Salmonella serovars isolated from human and food samples in Lebanon were mainly Enteritidis (28.7%) and Infantis (26%). To a rare extent, other serovars included Amager, Anatum, Bredeney, Chincol, Heidelberg, Hofit, Kentucky, Montevideo, Muenster, Newport, Schwarzengrund, Senftenberg and Typhimurium. In comparison with other countries, S. Enteritidis samples isolated in Lebanon (56 ± 27 intra-group pairwise SNP differences) presented a strong phylogenomic relativeness at the coregenome level with samples, as for example with samples isolated from Syria (65 ± 31 inter-group pairwise SNP differences). Most of the studied S. Enteritidis genomes encoded 10 SPIs involved in survival in immune cells (i.e. SPIs 1, 2, 3, 4, 5, 12, 13, 14, 16 and 17). The plasmid replicons IncFIB (S)_1 and IncFII (S)_1 encoding elements involved in virulence were identified in the majority of the S. Enteritidis genomes (94% and 96%, respectively), the majority exhibiting aminoglycosides (gene aac(6')-Iaa_1). The IncI_1_Alpha replicon responsible for ampicillin-resistance was only detected in 2 of 25 S. Enteritidis Lebanese strains. Genomic-based risk assessment of Salmonella serovars in Lebanon showed that food imported from Syria might be an origin of the S. Enteritidis human cases in Lebanon. The detection of several SPIs involved in the survival, plasmid replicons involved in virulence, and aminoglycoside-resistance genes, emphasizes that S. Enteritidis is of paramount importance for public health in Lebanon and other countries.

Assorted Methods for Decontamination of Aflatoxin M1 in Milk Using Microbial Adsorbents.

Aflatoxins (AF) are carcinogenic metabolites produced by different species of Aspergillus which readily colonize crops. AFM1 is secreted in the milk of lactating mammals through the ingestion of feedstuffs contaminated by aflatoxin B1 (AFB1). Therefore, its presence in milk, even in small amounts, presents a real concern for dairy industries and consumers of dairy products. Different strategies can lead to the reduction of AFM1 contamination levels in milk. They include adopting good agricultural practices, decreasing the AFB1 contamination of animal feeds, or using diverse types of adsorbent materials. One of the most effective types of adsorbents used for AFM1 decontamination are those of microbial origin. This review discusses current issues about AFM1 decontamination methods. These methods are based on the use of different bio-adsorbent agents such as bacteria and yeasts to complex AFM1 in milk. Moreover, this review answers some of the raised concerns about the binding stability of the formed AFM1-microbial complex. Thus, the efficiency of the decontamination methods was addressed, and plausible experimental variants were discussed.

A novel technique for aflatoxin M1 detoxification using chitin or treated shrimp shells: in vitro effect of physical and kinetic parameters on the binding stability.

This study aimed to investigate the ability of chitin and heat-treated shrimp shells to bind aflatoxin M1 (AFM1) in liquid matrix. Several concentrations of chitin or shrimp shells (grinded and ungrinded) were incubated in AFM1-contaminated phosphate-buffered saline (PBS) at different incubation times. The stability of the formed adsorbent-AFM1 complex was also tested in milk at different incubation times and temperatures. The unbound AFM1 was quantified by HPLC. Thereby, the percentages of the initial bounded AFM1 varied between 14.29 and 94.74%. Interestingly, in milk, an increase in incubation time coupled with a decrease in temperature affected positively the amount of bounded AFM1 to chitin and negatively those bounded to ungrinded shells. Results also revealed a partial reversibility in the binding of AFM1 to these adsorbents. These findings provided strong evidence on ability of chitin or shrimp shells by-product to bind AFM1 in milk and in PBS.

Secondary metabolism in Penicillium expansum: Emphasis on recent advances in patulin research.

The plant pathogenic fungus Penicillium expansum is a major concern of the global food industry due to its wide occurrence and ability to produce various mycotoxins, of which the most significant is patulin. Relatively less highlighted in the literature, in comparison with the other food-borne mycotoxins, patulin is one of the main factors in economic losses of vegetables and fruits. Otherwise, patulin is a health hazard which results in both short-term and long-term risks. This review includes knowledge on the biosynthetic mechanisms used for secondary metabolite production in P. expansum, with special emphasis on patulin biosynthesis. The abiotic factors triggering the production of patulin and the strategies developed to reduce or prevent the contamination by this mycotoxin are comprehensively discussed. The database presented in this review would be useful for the prioritization and development of future research.

Antifungal and Antiochratoxigenic Activities of Essential Oils and Total Phenolic Extracts: A Comparative Study.

This study is intended to prevent ochratoxin A (OTA) production by Aspergillus carbonarius S402 using essential oils (EOs) and total phenolic compounds extracted from plants and herbs. The EOs used in this study are the following: bay leaves, cumin, fenugreek, melissa, mint, and sage. As for the phenolic compounds, they were extracted from bay leaves, cumin, fenugreek, melissa, mint, sage, anise, chamomile, fennel, rosemary, and thyme. The experiments were conducted on Synthetic Grape Medium (SGM) medium at 28 °C for 4 days. OTA was extracted from the medium with methanol and quantified using HPLC (High Performance Liquid Chromatography). Results showed that EOs had a greater impact than the total phenolic extracts on the OTA production. Reduction levels ranged between 25% (sage) and 80% (melissa) for the EOs at 5 µL mL-1, and 13% (thyme) and 69% (mint) for the phenolic extracts. Although they did not affect the growth of A. carbonarius, total phenolic extracts and EOs were capable of partially reducing OTA production. Reduction levels depended on the nature of the plants and the concentration of the EOs. Reducing OTA with natural extracts could be a solution to prevent OTA production without altering the fungal growth, thus preserving the natural microbial balance.

Identification of the Anti-Aflatoxinogenic Activity of Micromeria graeca and Elucidation of Its Molecular Mechanism in Aspergillus flavus.

Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B1 (AFB1), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these toxic fungal metabolites. In this study, we demonstrated that an aqueous extract of the medicinal plant Micromeria graeca-known as hyssop-completely inhibits aflatoxin production by Aspergillus flavus without reducing fungal growth. The molecular inhibitory mechanism was explored by analyzing the expression of 61 genes, including 27 aflatoxin biosynthesis cluster genes and 34 secondary metabolism regulatory genes. This analysis revealed a three-fold down-regulation of aflR and aflS encoding the two internal cluster co-activators, resulting in a drastic repression of all aflatoxin biosynthesis genes. Hyssop also targeted fifteen regulatory genes, including veA and mtfA, two major global-regulating transcription factors. The effect of this extract is also linked to a transcriptomic variation of several genes required for the response to oxidative stress such as msnA, srrA, catA, cat2, sod1, mnsod, and stuA. In conclusion, hyssop inhibits AFB1 synthesis at the transcriptomic level. This aqueous extract is a promising natural-based solution to control AFB1 contamination.

PCR-RFLP for Aspergillus Species.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is the most simple method for single-nucleotide change detection. It is widely used in the detection and differentiation between mycotoxigenic species. It is based on PCR amplification of a target region containing the variant site of the studied species followed by restriction endonuclease digestion and gel electrophoresis to visualize the RFLP patterns. In this method primers are designed to flank the polymorphic site and positioned in such a way as to create unequally sized fragments upon restriction endonuclease cleavage of the PCR products. Here, we describe the protocol of PCR-RFLP developed for the detection and differentiation between Aspergillus flavus and A. parasiticus by amplifying a 674 bp fragment of the aflR-aflJ intergenic region followed by restriction endonuclease analysis using BglII to obtain RFLP patterns.