Aflatoxins in some food products imported from china to Mongolia: a recent overview

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National University of Mongolia

Department of Chemistry, School of Arts and Sciences

Laboratory of Professional Inspection Zamiin-Uud

Department of Chemical and Biological Engineering, School of Engineering and Applied Sciences

Aflatoxins in some food products imported from china to Mongolia: a recent overview

Saruul I., Ass. Professor

Ariuntsetseg D., Master of Chemistry

Tuyagerel B., Ass. Professor

Munkhjargal B., Ass. Professor

Ulaanbaatar, Mongolia

Abstract

Aflatoxins (AFs) are toxic fungal metabolites produced by fungi Aspergillus species, that are widely distributed in nature, and have severely contaminated humans and animal food supplies, posing in health hazards. We evaluated the aflatoxin surveillance from 2021 to 2022 in this paper. Throughout this period, a total of 1328 food samples, under the several food groups were collected for aflatoxin analysis and the results were compared against the statutory limits stipulated in the Mongolian National Standards (MNS). Depending on the type of food sample, AF contamination ranged from 3.2 to 83.3 percent, with a total AF level ranging from 0.20 to 4.1 pg-kg~¹. Despite the fact that the values did not exceed the upper limits set by MNS for food products, AFs are still frequently found in food products at levels that necessitate a greater emphasis on public health protection. AFs are "natural" contaminants of foods; their emergence is unavoidable, and it is critical to detoxify contaminated food products chemically, physically, or biologically in ways that preserve their edibility.

Keywords: Aflatoxin, food contamination, ELISA, food safety, detoxification methods, public health.

Аннотация

Саруул И., Ариунцэцэг Д., Туягэрэл Б., Мунхджаргал Б. Афлатоксины в некоторых продуктах питания, импортируемых из Китая в Монголию: недавний обзор

Афлатоксины (АФ) -- это токсичные грибковые метаболиты, вырабатываемые грибами рода Aspergillus, которые широко распространены в природе и серьезно загрязняют продукты питания людей и животных, представляя опасность для здоровья. В этой статье мы оценили надзор за афлатоксинами с 2021 по 2022 год. За этот период было собрано в общей сложности 1328 образцов продуктов питания из нескольких групп продуктов питания для анализа на афлатоксины, и результаты были сравнены с установленными нормами, предусмотренными в Монгольских национальных стандартах (МНС). В зависимости от типа образца пищи загрязнение АФ варьировалось от 3,2 до 83,3 процентов, а общий уровень АФ варьировался от 0,20 до 4,1 пг-кг~ 1. Несмотря на то, что значения не превышали верхние пределы, установленные МНС для пищевых продуктов, АФ по-прежнему часто встречаются в пищевых продуктах в количествах, которые требуют большего внимания к охране здоровья населения. АФ являются «естественными» загрязнителями пищевых продуктов; их появление неизбежно, и крайне важно детоксифицировать загрязненные продукты питания химическим, физическим или биологическим способом, чтобы сохранить их съедобность.

Ключевые слова: афлатоксин, загрязнение пищевых продуктов, ИФА, безопасность пищевых продуктов, методы детоксикации, общественное здравоохранение.

Introduction

Aflatoxin contamination in crops is a worldwide concern that impedes food safety and public health. Fungi can contaminate crops during the harvesting, storage, and transportation processes, resulting in the production of a variety of mycotoxins (1). Moreover, it has an impact on the agricultural economy and crop-dependent industries. Aflatoxins (AFs) are one of the five most important mycotoxins in agriculture. AFs are produced by 22 species of Aspergillus section Flavi, 4 species of A. section Nidulantes, and 2 species of A. section Ochraceorosei (2). Chemically, the AFs are difuran coumarin derivatives with a bifuran group attached to the coumarin nucleus and a pentanone ring (in the case of aflatoxin AFBs) or a lactone ring (in case of aflatoxin AFGs) and are one of the highly toxic secondary metabolites derived from polyketides produced by fungal species (3). Aflatoxin-contaminated foods and feeds pose a health risk to both humans and animals. Aflatoxin (B1, B2, G1 and G2) is the most toxic mycotoxin among the five major mycotoxins that are most toxic to mammals. Aflatoxin induces cancerous cell formation by forming deoxyribonucleic acid (DNA) adducts with guanine (4). In 1987, the International Agency for Research on Cancer (IARC) classified naturally occurring AFs (AFB1, AFB2, AFG1, and AFG2) as Group 1 "carcinogenic to humans" and reevaluated them in 2012. AFM1 was classified as a Group 2B substance that was "possibly carcinogenic to humans" in 1993. Additional health impacts of aflatoxins include acutely toxicity, hepatotoxicity, immunosuppression, mutagenesis, teratogenic, cytotoxicity, neurotoxicity, reproductive dysfunctions, stunted growth, and epigenetic effects (5). According to the Global Cancer Observatory's most recent data, liver disease is the sixth most prevalent cancer in men and women of all ages, with a projected total of 905,677 new cases in 2020 (6). The World Health Organization considers a global average of 8.4 cases or more of liver cancer per 100,000 populations to be very high. In Mongolia, however, this figure reached 68.4 in 2019, which is 8.1 times higher (7). AFs are estimated to account for 4.6 percent to 28.2 percent of all hepatocellular carcinomas globally (8).

Therefore, AFs must be closely monitored, and their concentrations in food must be kept as low as possible. As a result, almost every country is developing and implementing regulations aimed at reducing mycotoxins in domestic food supplies, including the issue of reliable sampling and analysis methods. The maximum permissible levels of AF for human consumption range from 4 to 30 pg-kg^-1 depending on the food type (9). The maximum allowed levels of total AFs by the EU are 2 pg-kg^-1 for AFB1 and 4 pg-kg^-1 for total AFs, but 20 pg-kg^-1 of AFs in the United States (10)

There has been reported high AF detoxification resistance to common treatment approaches such as pasteurization and sterilization, requiring the development of effective methodologies to control AF A variety of methods for decontamination/detoxification of aflatoxins are used, including physical, chemical, and biological methods (11). These methods must ensure that the degradation process preserves nutritional value, does not introduce new toxic or carcinogenic-mutagenic substances, and destroys Aspergillus spores and mycelia, preventing the proliferation and production of new toxins under favorable conditions. (12).

Thin Layer Chromatography (TLC) has been the most widely used method for quantitative estimations of aflatoxins. Developed TLC plates are examined under UV light, and aflatoxin concentrations are estimated by visual comparison of the fluorescent intensity of the spots in the sample extracts with those of the appropriate aflatoxin standards chromatographed on the same plate (13).

Coomes et al described this visual method of estimation as sensitive, and concentrations of aflatoxin as low as 3 to 4 pg per kilogram can be detected (14). However, visual estimations present problems of accuracy and precision. The coefficient of variation with this method may range from 20% to 30%. Aflatoxin can be detected and quantified using a number of reliable methods, however, because of the high cost and time-consuming sample preparation procedure, those analytical methods have been replaced by the competitive Enzyme Linked Immunosorbent Assay (ELISA) assay technique, which is a fast and easily detectable assay technique for routine analysis. The ELISA provides a specific and quick response with large-scale replication capabilities, and it is also the first choice due to its low cost, quick action, and requirement for a small sample volume (15).

Materials and Methods. Samples. Aflatoxins were determined in various food products imported from China to Mongolia. We collected 1328 different samples from food products imported through the Zamiin-Uud port, during 2021-2022 according to AOAC 1995 method in terms of size reduction and mixing. Samples were immediately transferred to the laboratory and prepared for subsequent TLC and ELISA analyses in accordance with the manufacturer's instructions in terms of sample preparation and extraction.

toxic fungal aflatoxin metabolite contamination product standard mongolia

In this paper, we evaluated the aflatoxin surveillance conducted in the Food Toxicological Laboratory of the Zamiin-Uud port of Mongolia from 2021 to 2022.

During this period, a total of 1328 imported food samples, under the several food groups namely peanut and nut products, tea, herbs and spices, fermented and pickled products, cereal and cereal products, dried fruits, were taken for aflatoxin analysis and the findings were compared against the statutory limits stipulated in the Mongolian National Standards (MNS 6361:2012) and in Food Regulations. Thus, we analyzed 431 food samples for aflatoxin type analysis using TLC and 897 food samples for total aflatoxin content by competitive ELISA methods.

The type of aflatoxin detected in food samples was determined using a TLC technique. Generally, four aflatoxin classes (AFB1, AFB2, AFG1 and AFG2) are defined based on temperature, humidity, extended drought, storage time, and other important factors that influence AF synthesis (16). According to the TLC analysis, aflatoxin was found in 27 of the 404 samples tested, accounting for 6.7 percent of the total; however, only a detectable amount of the aflatoxin B1, B2 types were found in the food samples tested for molds. Other aflatoxin types (G1 and G2) were not detected in the food samples by the TLC analysis. Aflatoxin is produced by 28 species of Aspergillus mushrooms, the most important of which being Aflatoxin B1 and B2 produced by the fungus Flavi. Furthermore, A. flavus, A. parasiticus, and A. nomius are the most prevalent species in food (2). A. flavus produces AFB1, AFB2, A. parasiticus and A. nomius produce AFB1, AFB2, AFG1 and AFG2(17). Only the B type toxins were detected, implying that imported aflatoxin-detected foods may have been infected with fungi of species that generate type B toxins, such as A. Flavus, A. Pseudotamarii, and A. Togoensis, which do not produce G type toxins(18). It could also be infected with Aspergillus-type fungi, which produce the B and G type aflatoxins, though the B type toxins probably to be more prevalent due to more resistant and favorable conditions for the growth of type B toxins. In addition, the food source, enzymes, and various environmental factors all play a role in the production of mycotoxins (9). The fungus A. flavus, which produces aflatoxin B type, has the ability to multiply under a variety of stressful conditions. Because it is the most abundant mold found in soil and has the saprobe character that allows it to grow on many organic nutrient substrates, A.flavus is the main species responsible for aflatoxin production and crop contamination(19).

Food samples from Asia (40%) and Europe (38%), according to studies, contain at least one detectable mycotoxin. It was also discovered that 38% of all mycotoxins identified included two or more mycotoxins synergistically (20). Moreover, A. flavus can grow in a wide range of temperatures ranging from 12°C to 48°C, but the optimum temperature range for its growth is 28°C to 37°C (21). The AFB production is usually higher than AFG production at high temperatures, but at low temperatures, both AFB and AFG production are equal (22). Fungi that produce aflatoxin can grow in a wide pH range (1.7-9.3), but the optimum pH range is (3-7). Lower pH (3-6) promotes the production of both fungal and aflatoxins, and a slightly lower pH (pH = 5) promotes the production of AFB, while higher pH (pH = 7) promotes the production of AFG (23). Aflatoxin production is also aided by a combination of vitamins, amino acids, and metal ions. AFB1 production was aided by arginine, glycine, glutamic acid, and aspartic acid (24). All of these physical, chemical, and biological factors indicate that aflatoxin type predominating in food products. However, because of the matrix effect of samples, TLC method is only suitable for qualitative analysis. To assess the AF levels in imported foods, an effective monitoring scheme for food safety system, screening for possible contaminated samples by TLC, and quantification of contaminated levels by ELISA, has been implemented. The ELISA method used in this study was appropriate for detecting mycotoxins at very low concentrations and Tables 1 shows total aflatoxin values by ELISA methods. The amount of AF contamination detected in dry food commodities samples ranged from 0.20 to 4.1 pg-kg^-1.

Table 1

Results on the occurrence of aflatoxin (AF) distribution (pg-kg^-1) in imported food samples during 2021-2022

< LOD limits of detection;

The obtained results of Table 1 indicated that the incidence of AFs in samples collected from imported food commodities was 55% in green tea, 50% in spicy bean sauce, 38.7% in kimchi and 30% in compote, but in biscuits 26.9%, pickled cucumber and sauce 20%, glass noodles 9.2%, yeast 6.6% and dried fruits 3.2%, respectively. The highly percentage of AFs was found in nuts (83.3%) and instant noodles (77.3%). Although nuts and instant noodles account for the highest incidence of aflatoxin" containing foods, the highest levels of aflatoxin are found in spicy bean sauce (0.32322 4.13 (ag-kg^-1), green tea (0.57-3.28 (ag-kg^-1), instant noodles (0.39-1.64 (ag-kg^-1), and glass noodles (0.36-1.42 (ag-kg^-1). Aflatoxin contamination is most common in corn, rice, spices, dried fruits, nuts, and figs and aflatoxin levels have been found to be higher in peanuts, corn, dry spices, tea, and fermentation products in other studies (2,18). The values were lower than the upper limits set by Mongolian National Standards of Maximum residue limits mycotoxins in the food and feed stuffs (MNS 6361:2012). AFs are still found frequently in food products at certain levels that are of significant concern for consumer protection, according to a review of monitoring studies on the occurrence of AF in food products (2). The highest levels of AF contamination were found in one peanut sample (156.68 (ag-kg^-1) and one pistachio sample (245.6 (ag-kg^-1) in previous studies (11).

Other than physical factors, the substrate and various nutritional factors have a large impact on aflatoxin production. In comparison to oil, a carbohydrate-rich substrate supports more production because carbohydrate readily provides carbon, which is required for good fungal growth. A total of 67.9% of maize samples, 92.9% of millet samples, and 50% of sorghum samples obtained from a storage room were found to be contaminated by aflatoxins. Because A. flavus is naturally present in soil, pre-harvest contamination of field crops is common, and post-harvest contamination by A. flavus occurs during storage because it spoils the food grains (25).

Despite the fact the values were lower than the upper limits, chronic aflatoxicosis causes cancer, immune suppression, and other pathological conditions (4); thus, the amount of aflatoxin in food should be kept to a lowest possible. Conventional techniques for mycotoxin poisoning prevention frequently require both pre- and post-harvest tactics; however, these methods are insufficient, necessitating additional processing for decontamination and detoxification of food and feed products (11). The detoxification methods must ensure that the degradation process preserves the nutritive value of food and destroys or modifies AFs. As a result, we tested a few basic methods for reducing and detoxifying the amount of aflatoxin in imported food.

Results of a study on the chemical reduction of aflatoxin content Table 2 shows the results of the analysis after 24 hours of dried plum and peanut sampling in various solvents. However, due to the negative effects on food quality and health concerns associated with chemical residues, an attempt has been made to find non-toxic alternatives to control aflatoxin contamination.

Table 2

Results of analysis after 24 hours of soaking of dried plums and peanuts in different solvents

As shown in Table 2, aflatoxin levels in food samples were significantly reduced (19.0-24.6%) in acetic acid, which was more effective than in distilled water (3.1-14.6%) and bicarbonate of soda solution (0.3-2.1%). Although there are many physical, chemical, and biological methods for detoxifying and reducing aflatoxins in food raw materials and products, many of them are expensive and can only be used in industrial settings such as ozonation and ammonization (12). To reduce the aflatoxin content, chemical methods such as sodium bisulfite, calcium hydroxide, formaldehyde, sodium hypochlorite, sodium borate, and sorbents (18) can be used, but we have conducted mitigation experiments using low-cost, low-hazard chemicals that can be used at household, such as food organic acids, water, and soda solutions, in accordance with the ALARA-safety principle (as low as reasonably achievable). The experiments were plain, accessible, and suitable for domestic use, as were the chemicals used, which would not affect food raw materials or products, and the few factors mentioned in terms of temperature and duration of the test. Some food additives, when combined with physical factors such as temperature and humidity, inhibit fungi growth and toxin production. Citric acid has been shown in studies to inhibit the growth of aflatoxin in rice plants under high temperatures and pressures. Researchers also discovered that some food preservatives, such as propionic acid, crystalline violet, p-amino benzoic acid, benzoic acid, boric acid, and sodium acetate, inhibited the growth of the fungus A. aflatoxin and the production of aflatoxin. The fact that weak citric acid, propionic acid, acetic acid, and sorbic acid reduce the aflatoxin produced by A. Flavus in peanuts is consistent with our findings (26).

Aflatoxin levels in acidic solutions decreased because aflatoxin is slightly soluble in water (10-20 g-ml^-1), well soluble in polar solvents, unstable in alkaline and acidic pH (<3;> 10), lactone rings are broken in acidic environments, so aflatoxin is detoxified, and hydrolyzed aflatoxin can be easily washed away. Washing of wheat seeds in water removed 40% of AFB1. Acids, as hydrolytic agents, oxidize the double bond of the terminal furan ring or hydrolyze and oxidize the lactone ring of AFB1(27).

Results of a study on the reduction of aflatoxin levels by physical methods

Table 3 reveals the results of tests on dried plum and peanut samples after 12 hours of exposure to UV light and sunlight. The table displays the results of experiments that examined physical methods of exposure to ultraviolet light and sunlight as one of the next possible methods of aflatoxin detoxification and removal.

Table 3

Test results of dried plum and peanut samples after exposure to UV radiation and sunlight for 12 hours

Aflatoxin levels in dried plum and peanut samples were reduced by 16.9-28.4% and 3.1-7.2%, respectively, after 12 hours of exposure to UV radiation and sunlight samples containing aflatoxin. This is consistent with the findings of previous studies that found that aflatoxins were reduced when seeds were exposed to different types of radiation, such as ultraviolet and violet radiation (28).

In addition, sunlight plays an important role in the detoxification of AFV1 in a variety of crops. Other studies, such as those on aflatoxin-infected corn and peanuts, found that exposing seeds to sunlight for 10 to 12 hours reduced mycotoxin levels by 80% and 17%, respectively (18). AFB1 absorbs ultraviolet light at three wavelengths: 222, 265, and 362 nm, with the highest absorption at 362 nm. Ultraviolet light with a wavelength of 362 nm activates AFB1 and facilitates its degradation (29).

The structural components that degrade by photolysis under the influence of ultraviolet light as well as ozone, resulting in the formation of OH radicals in the liquid media and the breaking of the double bond in the peripheral furan ring. The hydroxyl group then replaces the double bond in the pentanone (lactone) or the methyl group in the ether bond to form aflatoxin derivatives, which are less toxic compounds (11).

Conclusion

Among of the 1,328 samples surveyed, certain samples contained 0.2-4.1 pg-kg^-1 of total aflatoxin, which was less than the maximum allowable aflatoxin level. When compared to the labeled regulatory limit of allowed aflatoxins in Mongolia, the obtained results allow all tested food samples to be considered safe for human consumption. However, food samples primarily contain aflatoxin B1 type the most toxic, resistant, and widespread form of aflatoxin. Because of aflatoxin toxicity and carcinogenicity, as well as the risk of other chronic diseases, certain basic methods were tested to keep the amount of aflatoxin in food to a minimum. Consequently, it has been determined that sunlight and UV irradiation and hydrolysis by polar solvents such as acetic acid and water can reduce the amount of aflatoxin by 3.1-28.4% and 3.13-24.59%, respectively, as a result aflatoxin intake from food products was found can be lowered.

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