Mycotoxins in Chinese Traditional Cured Meats: A Review

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  Abstract Chinese cured meats are popular in the world, which are not only meat products but also cultures in China. Fungi can grow on the surface of the traditional cured meats easily during the processing and storage. Some fungi contribute to the specific flavors, color and sensory characteristics of cured meats, while others produce mycotoxins like aflatoxins, ochratoxins, and deoxynivalenol, which will lead to cancer. Although the mycotoxins limits in food have been published by national standard in China, the limits in cured meats are still in blank. Therefore, it is reasonable to identify and reduce the mycotoxins in Chinese cured meats and meat products. This review analyzed the causes of mycotoxins, identification and control methods for Chinese cured meats.
  Key words Fungus; Mycotoxin; Causes; Identification; Reduction
  Chinese traditional meats like cured meats, sausages and hams are famous in the world. Most of these products are normally processed in the twelfth month of the lunar year (la month), thats why we called them "La rou" in China[1]. It is the best way to store and enhance the flavor of the meat in the old time[2]. With the spices and local microorganisms in the proper temperature, the meat proteins produce a lot of flavor and the color becomes dark red. The microorganisms play an important role in this process. The reducing of pH, water activity and other change of physical and chemical index are directly related to them[3-4]. However, due to the particularity of the manufacturing process, like salt-curing and air-drying directly outside, cured meats are contaminated by fungal toxins easily[5]. Chinese people usually clean the surface and then boil the meats in the water to remove fungi. However, mycotoxins in meats cannot be removed and thus hazard of food safety still exists.
  Mycotoxins are secondary metabolites of filamentous fungi, which are toxic to vertebrates[6-7]. Fusarium spp., Penicillium spp. and Aspergillus spp are found in food and feed[8-9] and they produce mycotoxins like ochratoxin A (OTA), aflatoxin (AFT) and tentoxin (T-2) toxin and so on[5]. Studies have shown that these molecules could be nephrotoxic, hepatotoxic, neurotoxic, teratogenic and immunotoxic[10-12]. OTA was often found in various foodstuffs, and it had been classified into the 2B Group of possible human carcinogens by the International Agency for Research on Cancer (IARC)[13]. Pigs are particularly sensitive to OTA through their diet among all the farm animals[13], and the OTA could remain in the pork, the most commonly consumed  meat in China[14], which leads to the growth of fungi in the cured meat during ripening[15-16]. AFT is another dangerous mycotoxin in food, which has immunosuppressive and hepatotoxic properties and has been classified as carcinogenic to humans (group 1 A) by IARC[17-18]. AFT is usually found in corns, peanuts, grains and legumes. Recent studies have highlighted the mycotoxins in meat products[17,19].   Thus, the aim of the present review was to analyze the mycotoxin problems in Chinese traditional cured meats and the way to eliminate these food risks.
  Mycotoxin Problem in Chinese Cured Meats
  Manufacturing process factors
  Chinese traditional cured meats are usually made with the temperature ranging from 0-10 ℃, humidity below 50% in winter. However, the processing is different in different areas (Table 1)[1]. The characteristics of each category are associated with the variety of pig reared in particular areas, the distinctive processing of products, climate, and dietary habit of most local residents[20], but pickling and hanging to decrease the activity water are the same processes. All the manufacturing processes are performed in open space. When the meats are ripened, they will be hanged on beams, cool and ventilated place without package until eaten. The optimum temperature and aw for growth of most molds was 16-31 ℃ and 0.82-0.99, respectively[21]. aw of ripened Chinese cured meats is lower than 0.85 and it will increase when the air humidity is high. Molds will grow on cured meats especially from May to July, which are the wettest months called plum rain season. The molds in the air are stochastic grown on the surface[22]. They formed a protective film on the cured meat, which will slow down the diffusion of aroma components. Meanwhile, the carbon-dioxide produced by molds can effectively inhibit the growth of other microorganisms. However, some studies have shown that Penicillium, Aspergillus, Blastocladiella, Rhizopus, and Lternri are mainly detected in the cured meats[17,23], which are prone to producing mycotoxins. Traditional manufacturing process and the storage have more risk of mycotoxins.
  Traditional food storage factors
  In Chinese countryside, the feeding of farm animals is natural. The feed materials are foodstuffs, like corn, bran and vegetables. The outdoor dry feed materials are gathered in the storage house from the harvest season to the next one for a year. They will mildew when the temperature and the humidity were high in the raining season. The most important mycotoxins in corn worldwide are aflatoxins, fumonisins, trichothecenes (especially deoxynivalenol) and zearalenone[24-26]. The mycotoxins pollute the feed material and are carried by the animal meats, and then become a potential safety hazard in the production of cured meats. Meanwhile, cured meats might be polluted by the spores in the environment when molds grow in the foodstuffs. Allyl isothiocyanate[25], antifungal peptides derived from pepper seeds, and organic acids[27] have used to prevent mildewing. Current scientific knowledge and improvements in production and storage techniques have not led to any major developments in the prevention of these molds, the presence of these MT cannot be entirely eliminated from the food chain[28].   Mycotoxin Detection in Chinese Cured Meats
  Aflatoxin (AFT)
  Aflatoxin is mainly found in nuts, Chinese herbs, edible oils and other foods. Aflatoxin including aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and M1 (AFM1) frequently contaminating food[29-32]. The Chinese national standard GB/T 5009.23-2003 for detection of aflatoxins B1, B2, G1, G2 in foods included two methods: thin-layer chromatography (TLC) and microcolumn screening. They could also be detected by high performance liquid chromatography (HPLC) and ELISA[33]. TLC can be completed in ordinary laboratory with low cost, but it is low in sensitivity and harmful to the operators. Test strip for fluorescence quantitative detection and rapid detection kit of aflatoxin B1 have already produced by companies[34]. Chinese researchers have reported that the content of AFB1 in Suzhou traditional cured meats was 0.031 μg/kg on 90th d using the rapid detection kit[35]. The terahertz spectroscopy with chemometric methods[36], high-performance liquid chromatography with fluorescence detection[37], and near infrared spectroscopy (NIRS), mid-infrared spectroscopy (MIRS), conventional imaging techniques (color imaging (CI) and hyperspectral imaging (HSI)), and fluorescence spectroscopy and imaging (FS/FI)[38] have already been used to detect the aflatoxins in food products such as maize, wheat, rice and soybean oil[36]. Nondestructive testing techniques are the direction of future development.
  Ochratoxins
  Ochratoxins are a group of toxic metabolites from several species of Aspergillus and Penicillium, classified into A, B, C, D, etc. Ochratoxin A (OTA) is the most virulent of the ochratoxins. The methods of OTA detection in food of animal origin include TLC[39], HPLC[33], liquid chromatography-tandem mass spectrometry (LC-MS/MS)[40], enzyme linked immunosorbent assay (ELISA)[41] and gold immune chromatography assay (GICA)[11,42]. These methods are included in Chinese national standards, GB 5009.96-2016 National food safety standards: Detection of OTA in foods. TLC method is still in use because of its simple requirements for equipment and easy popularization, but the sample pretreatment is complicate, and the extraction and purification effect are not ideal. ELISA is a simple and fast method, but it is prone to false positive because of impurity interference in matrix due to unclear extraction and treatment. There are many impurities in the extract, which affect the fluorescence intensity of the spots. In recent years, immunoaffinity column has been widely used in the purification of mycotoxins, which improves the detection efficiency[43-45]. Researchers have used ELISA to investigate the content and risk assessment of OTA in Shenzhen area. The results showed that OTA was detected in 9 samples of cured meats in the total of 40, and the average pollution level was 0.27 μg/kg. Eight samples of cured pork were detected to contain OTA in the total of 25. The pollution level was 0-1.07 μg/kg[46]. This showed a low risk of OTA in Chinese cured meats.   Other mycotoxins
  Deoxynivalenol (DON) has been found in wheat, barley, oats and corn[47]. Patulin (PAT) is widely found in various mouldy fruits and silage[48]. Zearalenone (F-2) is the main pollution of grain[49]. Chinese researchers determined the DON and T-2 toxin in fresh meat in Chongqing by HPLC-MS/MS. 66 meat samples were analyzed, in which DON was lower than 0.5 μg/kg, and T-2 toxin was lower than 0.5 μg/kg[50]. 30 meat product samples in supermarket were analyzed by HPLC and GC, in which the contents of DON were lower than detection line[51]. 8.2 and 13.6 μg/kg citrinin (CIT) were found in 2 sausage samples by HPLC in Zhejiang[52]. The CIT content in dry meat products is 0.68 mg/kg by HPLC-FLC. The mycotoxin effects are more and more known by the customers, but the references of mycotoxins in Chinese cured meats are still scarce, suggesting that researchers in China have to pay much more attention on this.
  Mycotoxin Elimination in Chinese Cured Meats
  The national limit standard
  The mycotoxins in food have been brought into focus by the government with the development of China. The limit of AFB1 was first set in 1977, and replaced in 1981. Then appeared limit standard of DON in 1996, those of AFM1 and PAT in 2003, respectively. The mycotoxin limit standards in food were published in 2005 and replaced in 2011 and 2017, respectively. National Health Commission and Food and Drug Administration of China published the limit of mycotoxins in food in 2017. The maximal residue limits of AFB1, AFM1, DON, PAT and F-2 were set in this standard (Table 2). These standards were used to control mycotoxins contained in food to protect the consumers health. But the mycotoxins in cured meats were not mentioned for now. We can only judge the degree of damage and risk of mycotoxins in meat and meat products by referring to the standards of developed countries. The European Union (EU) stipulated that the residue amount of AFB1 in food should be<2 μg/kg, and the total amount of aflatoxin should be<4 μg/kg[53]. Aflatoxin cannot be detected in all foods in Japan. Most countries require OTA residues in foods<5 μg/kg[44]. The national limit standard can guide enterprises and government to control the mycotoxin content in cured meats. Due to the current research on mycotoxin of cured meats is not in-depth. Therefore, the government has not issued corresponding limit standard. This needs to be taken seriously by relevant departments in China.   Modernization of traditional technology
  With the fast developing of meat processing industry, the two major constraints of traditional meat product quality and safety control were appeared increasingly[55]. To protect traditional cured meat and its culture, many companies use modern equipments to produce cured meats. The vacuum rolling and kneading equipment has been used to replace the curing process, which increases the color of lightness by 2.1% and decreases redness 12.2%. And the color of cured meats is much better, and the content of cocci and molds that enhance flavor increase[56]. With the automatic air drying equipment replacing the natural air drying environment, the number of molds can be controlled in the safe level[57-58]. Vacuum and low temperature storage reduces the chance of contact with mold (Fig. 1)[59]. These improvements not only reduce mycotoxins in the cured meats, but also extend shelf life and improved product quality.
  Biological control
  The mechanism of biological control includes three aspects. The first is microbial adsorption, because many microorganisms can be adsorbed through the cell wall. The second aspect lies in microbial antagonism depending on the antagonism of microorganisms. The last is micro-biological degradation based on the biotransformation of microorganisms, the metabolism of which can destroy the structure of toxins, so that the fungal toxins can be degraded or transformed into low-toxic or non-toxic substances[44,60-61].
  The selected microorganisms as starter culture are added to cured meats to enrich the initial amount of bacteria in meats. The activity water would be consumed in the ripening procedure, which is the important condition for microbial growth. Dominant species are artificially controlled by reducing water activity, pH, etc., to avoid the possibility of contaminated bacteria and mold growth. The fermentation agents (Pediococcus pentosaceus, Staphylococcus xylosus, S. carnosus, Lactobacillus sakei and Debaromyces hansenii) added at 0.25 g/kg in cured meats could increase the cocci, lactobacillus and reduce the mold content compared to the control one[62-63]. This was a popular research direction in China about cured meats.
  Biocontrol by antagonistic microorganisms is another way to use microorganisms and has been proposed for controlling toxigenic moulds in foodstuffs, including fruits, wheat, dairy and meat products[64]. Ochratoxin A content in meat portions is significantly reduced when yeasts are co-inoculated with P. nordicum. D. hansenii 147 shows the greatest antagonistic activity and is proven to be effective in the investigated conditions[65]. Using yeasts D. hansenii could significantly reduce the growth rates of A. parasiticus when grown in a meat-model system[17]. This kind of research has not been reported in Chinese cured meats. Microbiological control method cannot meet the requirements of industrialization at the high cost level, only limited to the laboratory trial scale research. But the easy handle, no pollution advantage will have a wide application prospect.   Application of natural mildew inhibitor
  The natural mildew inhibitors are extracted metabolite from plant, animal and microorganism. They are natural and safe, and used in cured meats efficiently to prevent from mold pollution. Some condiment extracts like clove[40], cinnamon[66], garlic[61] are found to be efficient in inhibiting the growth of mold. The Chinese herb extracts have also been investigated. The inhibition rate of xu changqing on penicillium spore was close to 100%, flos lonicerae, bupleurum, thrips and cortex phellodendri were 56.7%-81.8%[61]. However, the condiment extracts and herbal medicine will affect the flavor and color of the meat products, and the use of them is limited. The natural mildew inhibitors with small amount have significant effects in inhibiting mold and have been more concerned by researchers. 0.1% natamycin wine sodium could keep the Hunan cured meats in normal temperature for 180 d in vacuum package, and 30 d without package[67]. After soaking cured meats with 0.06% curcumine water for 1 mim, the number of mold spores in the washing solution under the microscope was 0 on 72th h[68]. However, the cost of natural mildew inhibitors is usually higher than the chemical ones, and the combination of several natural mildew inhibitors can achieve satisfactory results. Therefore, the use of natural mildew inhibitors is not extensive.
  Conclusions
  Chinese traditional cured meats are not only a product but a culture in China. It is very important to prevent and control the contamination of cured meats from mycotoxins. From the information presented in this review, it can be inferred that, mycotoxins have been found in cured meats. However, there is no limit standard for them in cured meats, and rare research about this. Consumers believe that it is normal for cured meats to grow mold, which can be guaranteed by washing and cooking. The effect of nitrite in preserved meats on food safety is emphasized, but not enough attention is paid to mycotoxin in cured meats. In future, modern processing of traditional cured meats, nondestructive and rapid detection and natural control technology would be the important subjects.
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