ارزیابی برخی ازآفلاتوکسین ها در ترکیبات خوراک دام و طیور با روش HPLC : یک مطالعه محلی در استان کرمانشاه

Introduction

Mycotoxins and aflatoxins contaminate more than 25% of the agricultural products and as a result a lot of economic losses occur. These toxins cause damage to agricultural products worldwide each year (de Oliveira et al., 2014; Gummadidala et al., 2019). Mycotoxins and aflatoxins are responsible for many different toxic effects on both animal and human species and are considered the most dangerous problem for feed and food (Faghihi et al., 2019; Serdar et al., 2020). Aflatoxins are toxic secondary metabolites produced by two species of the Aspergillus genus, including Aspergillus flavus, and Aspergillus parasiticus (Escrivá et al., 2015). There is a growing concern regarding the recurrent observation of aflatoxins in the milk and animal products (Khadivi et al., 2019). Aflatoxins are metabolites produced by the growth of fungi on foodstuffs, which may occur during the food storage and food processing (Chiewchan et al., 2015; Karlovsky et al., 2016). They contaminate a wide variety of foods and mixed foods. Studies show that mycotoxins and aflatoxins are mostly found in cereals (Hernandez-Martinez et al., 2010). Aflatoxins have adverse effects on growth performance, blood profiles, hepatic histopathology, intestinal morphology, relative weight of digestive organs, activity of digestive enzymes, and biochemical index of intestinal development in animals (He et al., 2013; Feng et al., 2017). Chronic aflatoxin poisoning in farm animals seems to weaken the immune system, impairing the metabolism of proteins and vital substances in the body. These compounds have toxic, carcinogenic, and mutagenic properties (Sirajudeen et al., 2011; Kumar et al., 2017; Theumer et al., 2018). Intake of feeds contaminated with aflatoxins in the long-term exerts adverse effects on the organs, including liver tissue injury and immune disorders (Fan et al., 2015). The consumption of aflatoxin-contaminated foodstuffs by humans and animals in unauthorized or continuous amounts can cause acute or chronic poisoning and cancer (Authority 2013; Ostry et al., 2017). One of the most important and predisposing factors for the contamination of feedstuffs of farm animals and poultry is their moisture content (Kana et al., 2013; Abdallah et al., 2015). Although the presence of molds in foods does not always imply the harmful levels of aflatoxin, it may account for a significant risk. Aspergillus can colonize and contaminate food during the storage, especially following prolonged exposure to high humidity environment conditions (Mannaa et al., 2017; Reed et al., 2018). The risk of aflatoxin production is also higher in severe droughts, where soil moisture is below normal and temperature is high (Ostry et al., 2017). Codex Alimentarius Commission (CAC), the central part of the Joint FAO/WHO Food Standards Programme has determined the maximum levels of aflatoxins (B₁, B₂, G₁, G₂, and M1) in foodstuffs (Kotinagu et al., 2015). Recently, this commission has announced the maximum accepted/residue levels of aflatoxins in animal feeds as 0.02 mg/kg, that is 20 ppb in all feed materials and complementary feedstuffs for cattle, sheep, goats, pigs, and poultry; while it is 0.005 mg/kg in feeding stuffs for dairy animals and 0.01 mg/kg in complete feeding stuffs for calves and lambs (Bakırdere et al., 2012; EFSA Panel on Food Additives and Nutrient Sources added to Food, 2014; Hamed et al., 2017). High-performance liquid chromatography (HPLC) is a technique in analytical chemistry, which is used to separate, identify, and quantify aflatoxin in a mixture (Wacoo et al., 2014). Considering the dangerous effects of aflatoxins on the health of livestock, poultry, and subsequently, humans, it is important to measure the levels of aflatoxins B₁, B₂, G₁, and G₂, as well as total aflatoxin as critical health indicators in feedstuffs (Wu et al., 2008; Wu 2014). Accordingly, the main purposes of this study were to measure the aflatoxin levels using the HPLC in feedstuffs of three factories of Kermanshah province and to evaluate the effect of season on feed ingredients of livestock and poultry.

Materials and Methods

A: Sampling and Analytical Instrument

Sampling was done according to the method of Iranian national standards for the accurate determination of mycotoxin level in foods and agricultural products [INSO, No 12004] (Mazaheri et al., 2018). Ninety-three samples of barley, wheat, soybean, rice, and maize were taken from the factories of Kermanshah province (three major factories in four seasons) and analyzed using the method of high-performance liquid chromatography (HPLC) in the central laboratory of Provincial Veterinary Office (Campos et al., 2017). The HPLC system (Agilent Technologies -1260 Infinity, USA) was equipped with a photochemical reaction device (PHRED, Aura industries, NY, USA). Based on the Food Analysis Performance Assessment Scheme (FAPAS), in the cereal-based animal feed sample using aflatoxin standard, all testing methods include three steps: 1: extraction, 2: purification, and 3: determination of toxin content (Sykes et al., 2017).

B: Chemicals and Method Validation

All the reagents were of HPLC standard grade. Standard aflatoxins were purchased from Sigma Chemical Company (Germany). They were diluted with methanol to different concentrations and used as a calibration standard. The stock standard solution of aflatoxins (a concentration of 10 mg/mL in methanol of each aflatoxin species) was prepared and wrapped in aluminum foil at -20°C. Then the aflatoxin standard mixture was prepared in methanol at 0.4 ng/mL for aflatoxins G1 and B1 and 0.08 ng/mL for aflatoxins G2 and B2. The analytical grade solvents were obtained from Merck (Sigma-Aldrich, Germany). The pure water used in the analysis was prepared by Micro Siemens water purification system using millipore filters (0.45 µ) (TDS, Chemistry Danshvar Co; Iran). PBS: Phosphate Buffer Saline (0.2 g potassium chloride, 0.2 g potassium dihydrogen phosphate, 1.16 g hydrogen; 8 g sodium chloride phosphate, and 900 mL deionized water at pH 7.4) was also used. Fifty grams of samples were weighted. Acetonitrile and methanol were at HPLC grade (Sigma-Aldrich,) and other solvents were up to analytical purity grade. The working solutions of aflatoxins were diluted in the same solvent and stored in glass-stoppered tubes at 0°C. The CH0-3817 column (5 µm C18 UG 120 A, LC column 250 x 4.6 mm) was used (Figure 1). Immunoaffinity columns (IAC) were also used for the purification of samples (China: Huaan Magnech Bio-Tech Co., Ltd.).

Figure 1. HPLC chromatogram of Aflatoxin standard mixture: AFG₂ and AFB₂ at 0.08 ng/mL; AFB₂ AFG₁ and AFB₁ 0.4 ng/mL; Chromatographic conditions: CH0-3817; C18 UG 120 A column (5 µM; 250 × 4.6 mm); mobile phase water methanol and acetonitrile solution (60:30:20, v/v/v); flow rate; mL/min; fluorescence detector (λ ex = 365 nm and λ em = 435nm). Note: Retention time (min) for G₂, G₁, B₂ & B₁ peaks are seen left to right, respectively.

The method validation in terms of sensitivity and precision was performed according to the guidelines set by the International Union of Pure and Applied Chemistry (Yakubu et al., 2020).

C: Samples Preparation and Analysis

The samples were ground evenly and kept in the refrigerator. The samples were kept away from other materials to avoid secondary contamination. The extraction of the samples was done as per the standard methods of analysis that ensure the safety and integrity of foods and other products impacting public health (AOAC international method) and was quantified with the reference standard of High-performance thin-layer chromatography (HPTLC). Aflatoxin analyses were performed using 50 g of each sample. For extractions, 200 mL of methanol 80% was used and the mixture was stirred for 3 min at high speed. Then each aliquot was passed through a filter paper, diluted in water, and filtered again using a glass microfiber filter. IACs were used to clean up the aflatoxin sample (AflaOchra). First, 10 mL phosphate buffer saline (PBS) was passed through each IAC. Then 70 mL of the filtrate was passed through the IAC at a flow rate of 1 drop/sec. The IAC was washed with 10 mL of water and dried by applying mild vacuum. Finally, the aflatoxins were eluted with 1.5 mL methanol and then with 1.5 mL pure water and were analyzed by HPLC. To do it, the samples (100 μL) were injected into the HPLC column and heated to 40ºC. The mobile phase was a mixture of water, methanol, and acetonitrile (60:30:20, v/v/v). For the mobile phase preparation, 120 mg of potassium bromide and 350 μL of 4 M nitric acid were added to one liter of the mixture. The aflatoxins were detected with an excitation wavelength of 365 nm and emission wavelength of 435 nm fluorescence intensity. In addition, analysis at the maximum permitted limits in feeds was done based on the guidelines of Iranian National Standard (maximum tolerated levels of mycotoxins in food and feed, ICS: 67.020. ISIRI. 5925, Amendment No. 1). The limit of detection (LOD) of aflatoxin mass fraction was 0.4 for B₁ and G₁, and 0.08 for B₂ and G₂. The quantification of separated aflatoxins was performed by comparing it with standard aflatoxins using the formula:

The concentration of aflatoxins in ppm: Standard peak height× Sample peak height / Standard peak height ×Final volume of sample

D: Statistical Analysis

Data were analyzed according to the statistical method of a complete randomized block design (samples and seasons as blocks). Data were analyzed using the SPSS software version 23 (SPSS Inc., Chicago, IL., USA) with the general linear model (GLM) procedure. The statistical model of data analysis is as follows: Yijk = µ + Ti + Sj + eijk. Yijk: the values of each observation; µ: total average; Ti: the effect of treating (barley, corn, etc.); Sj: the season of the year; and eijk: residual effects. Comparisons between mean values were performed using Duncan's multiple range test (at 5% and 1% levels).

Results

The aflatoxin levels for 93 samples and the significance level of interactions between the positive samples and the season are shown in Table 1. As seen, there was no significant relationship between the total aflatoxin values of the samples (0.862) and the total of the seasons (0.919). Measurement and evaluation of B1, B2, G1, G2, and total aflatoxin values in different samples did not show significant differences in impermissible levels (Figure 2, Table 2). The positive samples were related to aflatoxin B1 and were seen mainly in winter. In total, out of 93 samples, six samples (6.45%) were positive for B1 aflatoxin. Out of a total of 27 wheat samples, one sample was positive and related to the winter sampling. Moreover, out of the total 23 soybean samples, one positive sample was related to winter and the other one sample was related to autumn. Based on the evaluation of the total 26 samples of corn, two samples were positive, one sample related to spring, another to winter. In addition, out of the total 10 barley samples, one sample was positive and related to summer. In rice samples evaluated, no positive cases were observed in terms of impermissible levels of aflatoxin (Table 2).

Table 1. Amount of aflatoxin B₁, B₂, G₁& G₂ by HPLC in raw material of livestock and poultry factories based on seasons and type of samples

*Comparisons between mean treatments were performed using Duncan's comparison (at 5% and 1% levels). There was no significant difference between positive and negative samples in terms of aflatoxins (B₁, B₂, G₁, G₂ and Total)

** 6 of 93 samples were positive for aflatoxin B₁.

*** Based on ppb, the positive samples were mainly in cold seasons

Table 2. Number of tested samples and positive samples of raw materials of livestock and poultry factories of Kermanshah province based on seasons and type of samples

*Comparisons between mean treatments were performed using Duncan's comparison (at 5% and 1% levels). There was no significant difference between positive and negative samples in terms of aflatoxins (B₁, B₂, G₁, G₂ and Total)

** 6 of 93 samples were positive for aflatoxin B₁.

*** Based on ppb, the positive samples were mainly in cold seasons.

Figures 2. Measurement and evaluation Aflatoxin B₁, B₂, G₁& G₂ in different samples by HPLC

*Comparisons between mean treatments were performed using Duncan's comparison (at 5% and 1% levels). No was significant difference between positive and negative samples in terms of aflatoxins (B₁, B₂, G₁, G₂ and Total)

** Y axis based on ppb

Discussion

The presence of mycotoxins in feeds pose serious health problems (He et al., 2013; Feng et al., 2017). Among mycotoxins, aflatoxins have immense toxicity and have been associated with various health and disease risks in livestock (Joint et al., 2017; Serdar et al., 2020). Aflatoxins are the secondary metabolites produced by certain species of fungi on grains and animal feeds. The mycotoxin poisoning increases losses of animal productivity, immunosuppression, damage to vital organs, and animal death (Kumar et al., 2017). The infestation of toxigenic fungi can occur before or after harvesting the food crops, grains, and seeds, resulting in serious human and animal health consequences (Al-Faragi et al., 2014; Escrivá et al., 2015). Aflatoxins have been widely studied compared to other mycotoxins because of their acute toxicity (Chiewchan et al., 2015; Karlovsky et al., 2016). Despite controlling production methods, aflatoxins are considered unavoidable contaminants in feeds. Therefore, regulatory agencies have developed specific guidelines on acceptable levels of aflatoxins in human and animal feeds. Studies have shown contamination with different types of aflatoxin in field conditions. The US Food and Drug Administration (FDA) has set an aflatoxin exposure limit of 20 ppb at the lowest possible and acceptable level for animal feed. In European Union (EU), aflatoxin B1 is the only toxin with a legal limit for presence in animal feeds (Sirajudeen et al., 2011; Theumer et al., 2018). One of the most important and predisposing factors for the contamination of feedstuffs of farm animals and poultry is their moisture content (Kana et al., 2013; Abdallah et al., 2015). The results of various studies indicate that Aspergillus and aflatoxin contamination are directly related to the moisture content in stored feedstuffs (Qazi et al., 2006; Torres et al., 2014; Hussain et al., 2015). Products with high humidity are not suitable for storage, as they lead to contamination during harvesting or storing (Waliyar et al., 2015). Therefore, climatic conditions and storage quality are two important determinants of mycotoxin contamination (Mannaa et al., 2017). According to the WHO estimates, a quarter of the products are affected by fungal toxins annually (Amanloo et al., 2014; Adeyeye et al., 2016). Aflatoxins and mycotoxins have adverse effects on livestock products and increase food risk factors (Wu et al., 2008; Adeyeye et al., 2016). Furthermore, the milk, eggs, and meat of these animals can contain residues of fungal toxins (Ren et al., 2007; Markov et al., 2013). On the other hand, aflatoxin contamination is identified as a pressing issue in food hygiene and public health (Richard et al., 2007; Frazzoli et al., 2017). The amount of aflatoxin and its duration of use determine the clinical consequences of poisoning (Niu et al., 2021). According to the Iran National Standards Organization, the maximum tolerance of aflatoxin B1 is 5 ng/g, and the total aflatoxin is 20 ng/g or ppb (Hedayati et al., 2016). The results of the present study showed that aflatoxin contamination was not significantly different in all five feedstuffs and the aflatoxin ranges were 0.452 to 0.648, 0.500 to 0.697, 0.579 to 1.688, 0.859 to 0.797, and 2.071 to 3.699 ppb for G2, G1, B1, B2, and total, respectively. Moreover, the effect of different seasons on these types of aflatoxins was investigated, which showed that although the positive cases were related to cold seasons, statistically no significant difference was observed in various seasons. Accordingly, changes in moisture levels and the ambient temperature had no effect on the contamination rate with this type of toxin in the study area. These results may be related to the lack of long-term storage of materials in factory warehouses and the timely consumption of feed. Our findings are in agreement with the previous studies where aflatoxins (especially B1) were found in most analyzed samples. This variation of percentage of the contamination may be due to differences in the types of substrates and handling processes from the time of harvesting to the time of consumption. In a study by Pourlemi et al (2013) on determining the rate of aflatoxin contamination in feed, industrial and local eggs in the western regions of Mazandaran in Iran, 40 industrial eggs from seven different poultry feed areas with 5 repetitions (corn, barley, chopped diet, and soybean meal) were contaminated with aflatoxin B1 in ELISA sampling; their results showed that the rate of aflatoxin contamination in local eggs was higher than that in industrial samples (Pourelmi et al., 2013). In the study by Mayahi (2001) on thin-layer chromatography (TLC) on silica gel coated plates of 100 poultry feed samples (25 samples of each item of corn, wheat, soybean, and fish meal), 86% of the samples were contaminated with aflatoxin (Mayahi et al., 2001). Mayahi (2007) also showed Aspergillus and aflatoxin contamination in major constituents of 75 samples in bird feed (soybean, corn, and fish powder); and that 25 samples with the highest rate of contamination were from fish powder (aflatoxin B1 was 15 μg/kg). These researchers also found that soybeans, corn, and fish powder were contaminated during the storage. Therefore, controlling and training proper food storage can reduce or eliminate aflatoxin contamination in these materials (Mayahi et al., 2007). In a similar study, the levels of aflatoxins B1, B2, G1, and G2 were measured in farms, in domestic animal feed. The results showed that two of the 19 tested samples had aflatoxin G2 higher than the standard level; however, other aflatoxins were negligible (López Grío et al., 2010). Hashemi et al (2016) also studied feed of 144 dairy cattle in Fars province. They found that in 36 cases, the level of aflatoxin B1 was higher than its standard level (Hashemi et al., 2016). The aflatoxin-contaminated food consumed by livestock is absorbed into the muscles and other organs and subsequently into their products (Dashti et al., 2009; Magnussen et al., 2013). Various studies have shown that consumption of aflatoxin-contaminated food by poultry has caused aflatoxin toxication with disorders of weight gain and food intake, reduced egg production, reduced food efficiency, damage to the digestive tract, blood disorders, liver lesions, and weakened immune system (He et al., 2013; da Rocha et al., 2014). Aflatoxin-contaminated foods also increase susceptibility to environmental stresses, microbes, neurological abnormalities, and mortality (Ogodo et al., 2016). The permitted limit for aflatoxin B1 by FAO is 1-20 ppb. This value is 20 ppb in Iran (Sani et al., 2014; Mahfouz et al., 2015). Most of the food samples (87 samples) measured in this study had not aflatoxin higher than these values. In our study, the aflatoxin B1 levels were lower than the limit in most samples. In a similar study conducted in Turkey to measure the aflatoxin levels in 41 wheat samples, 59% of the samples contained aflatoxin. In positive samples, the share of each of the aflatoxins B2, B1, G1, and G2 was 42, 12, 37, and 12%, respectively. In this study, the total aflatoxin in the tested samples was between 10.4 and 643.5 ng/g. The results of this study differ from our results. As abovementioned, there is a significant difference between the amounts of aflatoxins. Because aflatoxin-producing fungi respond differently to several conditions, therefore, these differences may be due to the storage conditions, sampling area, geographical area, ambient temperature, and weather conditions. Different concentrations of fungal toxins have been reported in different geographical regions following seasonal climate change. As a result, the differences observed in the values of the present study can be ascribed to sampling in several seasons and specific climatic conditions of the study area.

Conclusion

Aflatoxins are dangerous toxins in livestock and poultry feed and can occur in different climatic conditions. HPLC assures good recovery and precision in the quantitative determination of aflatoxin extracted from livestock compound feed and feed ingredients. Our study showed that aflatoxin B₁ contamination was present in the feed ingredients. No significant difference was observed for aflatoxins in the samples of animal feed in the factories of Kermanshah province, Iran. However, the aflatoxin B₁ levels in animal concentrate feed were found higher during rainy seasons compared to the summer season. Therefore, continuous surveillance of aflatoxins is required in animal feeds to reduce animal and consequently human exposure. On the other hand, due to the various factors that affect the amount of aflatoxins in food, further studies are needed for identifying the source of contamination and executing control measures.

Acknowledgments

This research was related to the implementation of a student graduate project of Veterinary medicine with the No: 2547723, Date: 12.10. 2019, Faculty of Veterinary Medicine, Razi University, Iran. We also thank the experts from both the Veterinary and Medical Laboratory for helping us conduct the tests for this study.

Ethical Approval

The ethical code approved with NO: 2547723, 12.10. 2019 by the research council of Razi University.

Informed Consent

Only the authors are responsible for the content of the paper.

Funding

This study was funded by the faculty of veterinary medicine, Razi University.

Conflict of Interest

The authors declare that they have no conflict of interest.