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Natural occurrence of deoxynivalenol in soy sauces consumed in China
Food Control 29 (2013) 71e75
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Natural occurrence of deoxynivalenol in soy sauces consumed in China Haifeng Zhao a, Yaqin Wang a, Yang Zou a, Mouming Zhao a, b, * a b
College of Light Industry and Food Sciences, South China University of Technology, Wushan Road 381, Tianhe District, Guangzhou 510640, PR China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2012 Received in revised form 19 May 2012 Accepted 26 May 2012
A total of 40 samples of domestic and imported soy sauces with the greatest diversity were investigated for the presence and contents of deoxynivalenol (DON) by using GCeMS after pre-column derivatisation. Results showed that 97.5% of samples investigated were contaminated with DON, and there was a remarkable variation in the DON contents across soy sauce samples. The average incidence rate of DON in domestic soy sauces was 97.1% (range 4.5e1245.6 mg/l, average value 141.5 mg/l), while for those imported from Japan was 100% (range 30.5e238.3 mg/l, average value 113.7 mg/l). Manufacturing process and quality grade were found to have significant influences on the levels of DON in soy sauces. Generally, domestic soy sauces produced with low salt solid state fermentation and with higher quality grade exhibited higher DON contents. The present results also indicated that the exposure to DON from the consumption of soy sauces is at a very low risk level. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Deoxynivalenol Soy sauce Manufacturing process Quality grade
1. Introduction Trichothecenes are a group of agriculturally important mycotoxins produced by certain species of Fusarium, Myrothecium, Trichoderma, Cephalosporium, and Stachybotrys, causing a significant economic impact on both cereal and grain crops each year (McCormick, Stanley, Stover, & Alexander, 2011). Deoxynivalenol (DON) is one of type B trichothecenes related to human health. The presence of DON is mainly associated with Fusarium graminearum and Fusarium culmorum, both of them are considered as important plant pathogens and are the causes of wilts, blights, and ear rots in grains (JECFA, 2001). DON is the least toxic among the trichothecenes, but it is the most prevalent one throughout the world. DON appears predominantly in wheat, corn, barley, soybean and other ska-Traczyk, Kiecana, Perkowski, cereals (Barros et al., 2008; Krysin & Dutkiewicz, 2001; Ok, Choi, Chang, Chung, & Chun, 2011; Setyabudi, Nuryono, Wedhastri, Mayer, & Razzazi-Fazeli, 2012), and its occurrence is also considered to be an indicator of the possible presence of other more toxic trichothecenes (Jaji c, Juri c, & Abramovi c, 2008). DON has been suggested as a clastogenic and potential in vivo tumor-promoting toxin, and thus exposure to DON can cause feed refusal, immunological problems, vomiting, skin dermatitis, and hemorrhagic lesions (McCormick et al., 2011;
* Corresponding author. College of Light Industry and Food Sciences, South China University of Technology, Wushan Road 381, Tianhe District, Guangzhou 510640, PR China. Tel./fax: þ86 20 87113914. E-mail address: [email protected] (M. Zhao). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.066
Moazami & Jinap, 2009). Nowadays, several countries including USA, Austria, Canada, Germany, Russia and China have established tolerance values for DON in cereals (GB 2715-2005, 2005; Omurtag lu, 2007), and the Commission of the European Commu& Beyog nities has also established a tolerance values for DON in cereals and cereal-based products (European Commission, 2006). Although different countries have different regulations, the maximum permitted levels for DON are generally 1000 mg/kg (FAO, 1997). Moreover, maximum tolerated levels for DON commonly detected in food commodities are usually between 500 and 1000 mg/kg, and the Tolerable Daily Intake (TDI) for DON is estimated to be 3 mg/kg for adults and 1.5 mg/kg for infants (Moazami & Jinap, 2009). However, there is a lack of legislation on the acceptable levels of DON in fermented food. Therefore, investigating the occurrence of DON in food, particularly fermented food, would be significance to eliminate the threat to human health because of exposure to DON. Soy sauce is one of the world’s oldest condiments and has been used in China for more than 2500 years. Nowadays, it is widely used as a seasoning or condiment in eastern Asia, and its popularity in the Western part of the world is growing due to its unique taste and aroma (Gao et al., 2011). The annual production of soy sauce in China is approximately 6 million tons, accounting for over 65% of the total world production. The wide distribution of manufacturers and varied manufacturing process led to the considerable variations in safety, quality, flavor and nutritional values of soy sauce. Therefore, its safety and quality standards were under consideration from member countries of FAO/WHO Codex Commission (Li, Zhao, Zhao, & Cui, 2010). In China, soy sauce is produced by fermenting soybeans and wheat flour or wheat bran with Aspergillus
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H. Zhao et al. / Food Control 29 (2013) 71e75
Table 1 Characteristics and levels of DON contamination of soy sauces consumed in China.a Samples
Grade
Manufacturing process
Original of country
DON (mg/l)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Premium Premium Premium Premium Premium Premium Premium Premium Premium Premium Premium Premium 1 1 1 1 1 2 2 2 2 3 3 3 3 3 3 e e e e e e e e e e e e e
HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF LSF LSF LSF LSF LSF LSF Blended Blended e e e e e
China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China Japan Japan Japan Japan Japan
17.7 70.9 106.8 72.8 225.4 232.7 17.0 75.8 71.5 134.1 107.8 108.5 81.7 61.2 29.5 93.1 54.6 10.9 10.1 4.5 46.7 93.1 56.1 42.8 51.3 55.8 53.1 97.9 1230.3 1245.6 79.2 62.1 96.3 ND 14.4 30.5 98.7 238.3 152.2 48.7
a HLF, high salt liquid state fermentation; LSF, low salt solid state fermentation; ND, not detected.
oryzae or Aspergillus sojae molds (solid state fermentation or koji fermentation process), along with water and salt. After the fermentation, fermented soybean paste was pressed to yield soy sauce. Thus, the soy sauce could be contaminated with DON due to the high incidence of DON in soybeans and wheat flour (wheat bran) and the natural multi-strains fermentation of soy sauce (Pacin, Resnik, Neira, Moltó, & Martínez, 1997; Valenta, Dänicke, & Blüthgen, 2002). Moreover, although the consumption of soy sauce has been increasing in recent years, there is little information available on the general occurrence of DON in soy sauce. Furthermore, the maximum permitted level and TDI of DON in soy sauce have not yet been established in China. Therefore, the first objective of this study was to examine the occurrence and levels of DON in soy sauces consumed in China. The second objective was to elucidate the relationship between DON contents in soy sauce and manufacturing process as well as quality grade. The last objective was to estimate the exposure to DON through the consumption of soy sauce in China. 2. Materials and methods 2.1. Soy sauce samples Forty soy sauce samples including 35 domestic and 5 imported soy sauces were purchased from local markets (Guangzhou,
Guangdong province). Among 35 domestic soy sauce samples, 27 samples were manufactured by high salt liquid state fermentation (HLF), and 6 samples were brewed by low salt solid state fermentation (LSF) and the other 2 samples were blended soy sauces. In 27 HLF soy sauce samples, the samples in premium grade, grade 1, 2 and 3 were 12, 5, 4 and 6, respectively (Table 1). All samples were stored in a refrigerator at 4 C until analysis. 2.2. Chemicals DON standard (200 mg/ml in ethyl acetateemethanol, 95:5, v/v) and Mirex were supplied by Supelco (Pennsylvania, USA) and stored at 20 C. LC grade solvent (isooctane, methanol) and sodium bicarbonate were purchased from Merck (Darmstadt, Germany). 1-(Trimethylsilyl)imidazole (TMSI) and Chlorotrimethylsilane (TMCS) were obtained from Fluka (Milwaukee, USA). All other chemicals and solvents were of the highest commercial grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 2.3. Extraction and clean-up The extraction and clean-up procedure were carried out by Prom, Horsley, Steffenson, and Schwarz (1999) with slight modifications. Five milliliters of soy sauce samples were placed into tubes containing a 40 ml of acetonitrileewater (84:16, v/v) mixture. Samples were extracted by placing the tubes on a shaker 30 min at 120 r/min. Then, the extract was blown to dryness under water-free nitrogen at 55 C. The extract residue was then dissolved in 20 ml of petroleum ether and 10 ml of methanolewater (4:1, v/v), and completely dried again under nitrogen at 55 C. The dry extract was dissolved in 2 ml water, loaded on the LC18 column and forced slowly through the cartridge. DON was slowly eluted with 6 ml of 40% methanol after washing the column twice with 3 ml of distilled water. The eluate was evaporated to dryness for further derivatisation. 2.4. Derivatisation The derivatisation of DON was according the method of Mateo, Llorens, Mateo, and Jiménez (2001) with some changes. A 100 ml derivatisation mixture of TMSIeTMCS (10:1, v/v) was placed into a vial containing the dry residue. The mixture was shaken gently on Vortex for 5 min at room temperature. One milliliter of isooctane containing 0.5 mg/ml Mirex and 1 ml 3% sodium bicarbonate solution were added without removing derivatisation agents. The vial was shaken on Vortex for 2 min and layers were allowed to separate. Transfer the upper layer (isooctane) into an autosampler vial and analyzed by GCeMS. 2.5. Analysis by GCeMS Analysis of the DON in soy sauce was performed using Trace GCeMS system equipped with an Ultra GC, a TriPlus autosampler and a quadrupole DSQ II MS (Thermo Finnigan, San Jose, CA, USA) according the method described by González-Osnaya, Cortés, Soriano, Moltó, and Mañes (2011) with some modifications. The capillary column was a TR-5 MS phase (30 m 0.25 mm, film thickness 0.25 mm) (J&W Scientific, USA). The carrier gas was helium (1.0 ml/min), and the temperature of the injection port was 280 C. The injection volume was 1 ml and the injection mode was splitless. The column temperature program was: 150 C for 1 min, 10 C/min to 220 C, 3 C/min to 260 C and then 10 C/min to 280 C held for 5 min. The temperature of the transfer line was adjusted to 300 C. The mass spectrometer was operated in the
H. Zhao et al. / Food Control 29 (2013) 71e75
electron impact (EI) mode. The selected ion monitoring (SIM) mode was utilized in order to have greater sensitivity. Quantification was performed on SIM using multiple ions m/z: 422, 512 for DON and 237, 272 for internal standard Mirex. 2.6. Estimated daily intake (EDI) The exposure of DON through soy sauce consumption was calculated using the information about soy sauce intake and body weight according to the formula proposed by Moazami and Jinap (2009). EDI in mg/kg body weight ¼ mean concentration of DON (mg/l) multiplied by the amount of soy sauce consumed/day (l) and divided by the average weight of an individual (50 kg). 2.7. Statistical analysis All of experiments were carried out in triplicate. Data were reported as means standard deviation (SD) for triplicate determinations. Analysis of variance and significant difference tests were conducted to identify differences among means by one-way ANOVA using SPSS software (version 17.0 for Windows, SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Occurrence of DON in soy sauce samples consumed in China An analytical method for determination of DON in soy sauce was developed by solid phase extraction and GCeMS. A typical chromatogram of selected ion mass for DON and Mirex in soy sauce sample is given in Fig. 1. The limit of detection (LOD, S/N ¼ 3) and the limit of quantitation (LOQ, S/N ¼ 10) for developed method were 16.7 and 5.0 mg/l, respectively. At the spiked levels of 50.0e1500.0 mg/l, the average recoveries were 82.0e106.1% with the relative standard deviations of 4.3e7.8%. All these results indicated that the proposed method is suitable to detect DON in soy
73
sauce due to its high sensitivity and recovery. The levels of DON in 40 soy sauce samples are presented in Table 1. As can be seen, 97.5% of soy sauces investigated were contaminated with DON at the test concentrations, and the DON content in only one blended soy sauce sample was below the limit of detection. The present study revealed a wide variation in DON contents among the 40 samples, ranging from 4.5 to 1245.6 mg/l, with an average value of 137.9 mg/l. The results were in agreement with the report by Schollenberger et al. (2007) that two out of four soy sauce samples were contaminated with DON, with the values of 25 and 14 mg/kg, respectively. Moreover, results from Fig. 2 also showed that 80% of soy sauce samples (n ¼ 32) exhibited DON contents in the range of 0e125.0 mg/l. While 12.5% of soy sauce samples (n ¼ 5) were observed to be contaminated with DON ranging from 125.0 to 250.0 mg/l. Only two soy sauce samples produced by LSF exhibited the DON contents in the range of 1000e1250 mg/l, which higher than values considered as dangerous for health. All these results indicated that raw materials and manufacturing process might have significant impacts on DON contents of final soy sauce. Indeed, Bensassia, Zaied, Abid, Hajlaoui, and Bacha (2010) found that 83% durum wheat samples in Tunisia showed DON contamination with averages of 12.8e30.5 mg/g exceeding the maximum permitted limit of 1.75 mg/g set by the European Commission in wheat intended for human and animal consumption. Out of 40 soybean samples, two samples were contaminated with DON at levels of 1.6 mg/g and 0.9 mg/g, respectively (Barros, Oviedo, Ramirez, & Chulze, 2011). It should be noted that a guideline for the mycotoxins DON, aflatoxins B1, zearalenone and ochratoxin A appears only for the cereal and cereal products in China (GB 2715-2005, 2005), but there is a lack of legislation on the acceptable levels of DON in soy sauce. According to the ANOVO, there were no significant differences in the levels of DON for soy sauces from China and Japan. The average incidence rate of DON in domestic soy sauces was 97.1% (concentration range 4.5e1245.6 mg/l, average value 141.5 mg/l), while for those imported from Japan was 100% (concentration
Fig. 1. A typical chromatogram of selected ion mass (m/z 422/512, 237/272) for DON and Mirex in soy sauce sample.
74
H. Zhao et al. / Food Control 29 (2013) 71e75
an acid medium and results showed that selected strains were capable of removing up to 55% DON due to binding, not biodegradation effects (Niderkorn, Boudra, & Morgavi, 2006). A total decontamination of 53% of DON and T-2 toxin was observed when malt contaminated with these toxins was fermented using Saccharomyces cerevisiae (Garda et al., 2005). Moreover, A. oryzae and Rhizopus oryzae also showed significant degradation capacity to DON during submerged fermentation, and both cultures caused the largest decrease in DON at around 90% in 240 h and 74% in 96 h (Garda-Buffon & Badiale-Furlong, 2010). In addition, HLF soy sauces are made by mixing soybeans and wheat flour, whereas LSF soy sauces are manufactured by mixing soybeans and wheat bran. Previous studies also proved that most of the DON is recovered in the bran fraction and the lowest concentration is found in the flour during milling (Lancova et al., 2008). This could explain partly the high level of DON in LSF soy sauces. 3.3. Effects of quality grade on the levels of DON in HLF soy sauces Fig. 2. The distribution of the DON contents in soy sauce samples.
range 30.5e238.3 mg/l, average value 113.7 mg/l). Although domestic brands have been considered as low-priced products compared to Japanese soy sauce, the results obtained herein suggested that the quality of both groups, in terms of DON content, is appropriately the same. 3.2. Effects of manufacturing process on the levels of DON in domestic soy sauces In China, soy sauce can be classified into fermented soy sauce (HLF and LSF soy sauces) and blended soy sauce according to manufacture process (GB 18186-2000, 2000; SB 10336-2000, 2000). Different types of soy sauces might be significant differences in their DON contents due to various raw materials and manufacturing process employed. The levels of DON in different types of soy sauces are shown in Table 2. As shown in Table 2, the DON contents in fermented soy sauces were significantly higher than those in blended soy sauces, the average values of DON for HLF, LSF and blended soy sauces were 73.5, 468.6 and 14.4 mg/l, respectively. This might be that blended soy sauce was manufactured by blending fermented soy sauce, hydrolyzed vegetable protein, salt, caramel color and additives with sweet or umami tastes. Thus the low proportion of fermented soy sauces in blended soy sauces resulted in a low content of DON in blended soy sauce due to the dilution. Moreover, HLF soy sauces exhibited markedly lower DON contents than LSF soy sauces, which was probably due to the difference in manufacturing technique and raw materials. HLF process is carried out by an indigenous fermentation procedure at 28e35 C and having long aging period, while LSF process by fermentation using pure cultures at relatively higher temperature (40e50 C) and having very short aging (Li et al., 2010; Lu et al., 2009). Generally, the multiple-strains fermentation process is considered as a bio-detoxification process. Twenty-nine strains of lactic acid bacteria were tested for their ability to remove DON from
Soy sauce manufactured by HLF method is generally regarded as traditional Chinese-type soy sauce. Total nitrogen, amino nitrogen and non-salt soluble solids are the important indices for classifying the quality grade of soy sauce in China (GB 18186-2000, 2000). Twenty-seven HLF soy sauces samples were divided into four grades: premium grade, grade 1, grade 2 and grade 3 according to the quality indices described above. As shown in Fig. 3, the DON contents of different quality grades of soy sauces decreased in the following order: premium grade > grade 1 > grade 3 > grade 2. Generally, soy sauces with higher quality grade had higher DON content, while DON content was low in samples with lower grade level except for soy sauces with grade 3. This might be that soy sauce with premium grade or grade 1 made from the first pressing of the soybeans, thus the most DON contaminated in raw materials could dissolve in soy sauce by the effect of enzyme or microorganism during this stage. The higher levels of DON in soy sauces of grade 3 might be due to the differences in raw materials. 3.4. An assessment of DON exposure in humans The annual production of soy sauce in China is approximately 6 million tons, and the demand for soy sauce consumption is increasing at a rate of about 5e10% yearly. The daily intake of DON
Table 2 Natural occurrence of DON in soy sauces manufactured with different process.a Manufacturing No. of No. of positive DON content (mg/l) process samples samples (%) Average Range HLF LSF Blended a
27 6 2
27 (100) 6 (100) 1 (50)
Median
73.5 55.8 4.5e232.7 61.2 468.6 596.1 62.1e1245.6 97.1 14.4 14.4 14.4
HLF, high salt liquid state fermentation; LSF, low salt solid state fermentation.
Fig. 3. The distribution of the DON contents in HLF soy sauces according to quality grade. HLF, high salt liquid state fermentation; Vertical bars represent the standard deviation of each data point.
H. Zhao et al. / Food Control 29 (2013) 71e75 Table 3 Estimated DON exposure level from soy sauce in China.a Exposure (mg/kg BW) Soy sauce a
0.034
BW: body weight.
from soy sauce depends on not only the DON content in soy sauce, but also the amount of soy sauce consumed. Moreover, the consumption of soy sauce may vary considerably from one individual to another. Thus it is importance to estimate the exposure to DON by consumption of soy sauce. The EDI value was calculated by assuming that a 50 kg individual will consume a 12.20 ml soy sauce serving per day. The results are summarized in Table 3 and implied a low exposure to DON by consumption soy sauce in China, which is far below the Provisional Tolerable Maximum Daily Intake (PTMDI) according to the JECFA of 1 mg/kg body weight/day (Moazami & Jinap, 2009). However, it is should keep in mind exposure to all other foods that could be contaminated with DON because the soy sauce is indispensable condiments in China. 4. Conclusion The present study clearly showed that almost all samples of soy sauce consumed in China contained detectable quantities of DON. There were also considerable variations in DON contents of commercial soy sauces across different samples. Moreover, manufacturing process and quality grade had significant impacts on DON contents in soy sauces. Soy sauces brewed with LSF technology and with higher quality grade generally exhibited higher levels of DON. The results from assessment of DON exposure in humans suggested that human risk to DON exposure from the consumption of soy sauces is very low in China. This research was part of our continuous efforts to improve the safety of soy sauce production by screening raw materials and optimizing the manufacturing process. On the basis of results obtained from this study, further works on clarifying the fate of DON in soy sauce production are in progress. Acknowledgments The authors gratefully acknowledge the National ScienceTechnology Supporting Project for 12th Five-Year Plan (2012BAK17B11) and the National High Technology Research and Development Program of China (863 Program) (No. SS2012AA022502) and the Key Technology R&D Program of Guangdong Province (Nos. 2009Z52 and 2011A020102001) for their financial supports. References Barros, G., García, D., Oviedo, S., Ramirez, M. L., Torres, A., & Chulze, S. (2008). Deoxynivalenol and nivalenol analysis in soybean and soy flour. World Mycotoxin Journal, 1, 263e266. Barros, G., Oviedo, M. S., Ramirez, M. L., & Chulze, S. (2011). In T.-B. Ng (Ed.), Soybean. Biochemistry, chemistry and physiology (pp. 7e20). Rijeka, Croatia: Intech Open Access Publisher.
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Bensassia, F., Zaied, C., Abid, S., Hajlaoui, M. R., & Bacha, H. (2010). Occurrence of deoxynivalenol in durum wheat in Tunisia. Food Control, 21, 281e285. European Commission. (2006). Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuff. Official Journal of the European Union L, 364, 5e24. FAO. (1997). Worldwide regulations for mycotoxins 1995. A compendium. Rome: Food and Agriculture Organization of the United Nations. Gao, X., Cui, C., Ren, J., Zhao, H., Zhao, Q., & Zhao, M. (2011). Changes in the chemical composition of traditional Chinese-type soy sauce at different stages of manufacture and its relation to taste. International Journal of Food Science and Technology, 46, 243e249. Garda, J., Macedo, R. M., Faria, R., Bernd, L., Dors, G. C., & Badiale-Furlong, E. (2005). Alcoholic fermentation effects on malt spiked with trichothecenes. Food Control, 16, 423e428. Garda-Buffon, J., & Badiale-Furlong, E. (2010). Kinetics of deoxynivalenol degradation by Aspergillus oryzae and Rhizopus oryzae in submerged fermentation. Journal of the Brazilian Chemical Society, 21, 710e714. GB 18186-2000. (2000). China national standard: Fermented soy sauce. Beijing: Chinese Standard Publishing House. GB 2715-2005. (2005). China national standard: Hygienic standard for grain. Beijing: Chinese Standard Publishing House. González-Osnaya, L., Cortés, C., Soriano, J. M., Moltó, J. C., & Mañes, J. (2011). Occurrence of deoxynivalenol and T-2 toxin in bread and pasta commercialized in Spain. Food Chemistry, 124, 156e161. Jaji c, I., Juri c, V., & Abramovi c, B. (2008). First survey of deoxynivalenol occurrence in crops in Serbia. Food Control, 19, 545e550. JECFA e Joint Expert Committee on Food Additives. (2001). Safety evaluation of certain mycotoxins in food (pp. 419e555). FAO food and nutrition paper 74. WHO food additives, series 47. ska-Traczyk, E., Kiecana, I., Perkowski, J., & Dutkiewicz, J. (2001). Levels of Krysin fungi and mycotoxins in samples of grain and grain dust collected on farms in eastern Poland. Annals of Agricultural and Environmental Medicine, 8, 269e274. Lancova, K., Hajslova, J., Kostelanska, M., Kohoutkova, J., Nedelnik, J., Moravcova, H., et al. (2008). Fate of trichothecene mycotoxins during the processing: milling and baking. Food Additives and Contaminants: Part A, 25, 650e659. Li, Y., Zhao, H., Zhao, M., & Cui, C. (2010). Relationships between antioxidant activity and quality indices of soy sauce: an application of multivariate analysis. International Journal of Food Science and Technology, 45, 133e139. Lu, Y., Chen, X., Jiang, M., Lv, X., Nurgul, R., Dong, M., et al. (2009). Biogenic amines in Chinese soy sauce. Food Control, 20, 593e597. Mateo, J. J., Llorens, A., Mateo, R., & Jiménez, M. (2001). Critical study of and improvements in chromatographic methods for the analysis of type B trichothecenes. Journal of Chromatography A, 918, 99e112. McCormick, S. P., Stanley, A. M., Stover, N. A., & Alexander, N. J. (2011). Trichothecenes: from simple to complex mycotoxins. Toxins, 3, 802e814. Moazami, E. F., & Jinap, S. (2009). Natural occurrence of deoxynivalenol (DON) in wheat based noodles consumed in Malaysia. Microchemical Journal, 93, 25e28. Niderkorn, V., Boudra, H., & Morgavi, D. P. (2006). Binding of Fusarium mycotoxins by fermentative bacteria in vitro. Journal of Applied Microbiology, 101, 849e856. Ok, H. E., Choi, S. W., Chang, H. J., Chung, M. S., & Chun, H. S. (2011). Occurrence of five 8-ketotrichothecene mycotoxins in organically and conventionally produced cereals collected in Korea. Food Control, 22, 1647e1652. lu, D. (2007). Occurrence of deoxynivalenol (vomitoxin) in Omurtag, G. Z., & Beyog beer in Turkey detected by HPLC. Food Control, 18, 163e166. Pacin, A. M., Resnik, S. L., Neira, M. S., Moltó, G., & Martínez, E. (1997). Natural occurrence of deoxynivalenol in wheat, wheat flour and bakery products in Argentina. Food Additives and Contaminants, 14, 327e331. Prom, L. K., Horsley, R. D., Steffenson, B. J., & Schwarz, P. B. (1999). Development of Fusarium head blight and accumulation of deoxynivalenol in barley sampled at different growth stages. Journal of the American Society of Brewing Chemists, 57, 60e63. SB 10336-2000. (2000). China food industry standard: Blended soy sauce. Beijing: Chinese Standard Publishing House. Schollenberger, M., Müller, H. M., Rüfle, M., Terry-Jara, H., Suchy, S., Plank, S., et al. (2007). Natural occurrence of Fusarium toxins in soy food marketed in Germany. International Journal of Food Microbiology, 113, 142e146. Setyabudi, F. M. C. S., Nuryono, N., Wedhastri, S., Mayer, H. K., & Razzazi-Fazeli, E. (2012). Limited survey of deoxynivalenol occurrence in maize kernels and maize-products collected from Indonesian retail market. Food Control, 24, 123e127. Valenta, H., Dänicke, S., & Blüthgen, A. (2002). Mycotoxins in soybean feedstuffs used in Germany. Mycotoxin Research, 18, 208e211.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Natural occurrence of deoxynivalenol in soy sauces consumed in China Haifeng Zhao a, Yaqin Wang a, Yang Zou a, Mouming Zhao a, b, * a b
College of Light Industry and Food Sciences, South China University of Technology, Wushan Road 381, Tianhe District, Guangzhou 510640, PR China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2012 Received in revised form 19 May 2012 Accepted 26 May 2012
A total of 40 samples of domestic and imported soy sauces with the greatest diversity were investigated for the presence and contents of deoxynivalenol (DON) by using GCeMS after pre-column derivatisation. Results showed that 97.5% of samples investigated were contaminated with DON, and there was a remarkable variation in the DON contents across soy sauce samples. The average incidence rate of DON in domestic soy sauces was 97.1% (range 4.5e1245.6 mg/l, average value 141.5 mg/l), while for those imported from Japan was 100% (range 30.5e238.3 mg/l, average value 113.7 mg/l). Manufacturing process and quality grade were found to have significant influences on the levels of DON in soy sauces. Generally, domestic soy sauces produced with low salt solid state fermentation and with higher quality grade exhibited higher DON contents. The present results also indicated that the exposure to DON from the consumption of soy sauces is at a very low risk level. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Deoxynivalenol Soy sauce Manufacturing process Quality grade
1. Introduction Trichothecenes are a group of agriculturally important mycotoxins produced by certain species of Fusarium, Myrothecium, Trichoderma, Cephalosporium, and Stachybotrys, causing a significant economic impact on both cereal and grain crops each year (McCormick, Stanley, Stover, & Alexander, 2011). Deoxynivalenol (DON) is one of type B trichothecenes related to human health. The presence of DON is mainly associated with Fusarium graminearum and Fusarium culmorum, both of them are considered as important plant pathogens and are the causes of wilts, blights, and ear rots in grains (JECFA, 2001). DON is the least toxic among the trichothecenes, but it is the most prevalent one throughout the world. DON appears predominantly in wheat, corn, barley, soybean and other ska-Traczyk, Kiecana, Perkowski, cereals (Barros et al., 2008; Krysin & Dutkiewicz, 2001; Ok, Choi, Chang, Chung, & Chun, 2011; Setyabudi, Nuryono, Wedhastri, Mayer, & Razzazi-Fazeli, 2012), and its occurrence is also considered to be an indicator of the possible presence of other more toxic trichothecenes (Jaji c, Juri c, & Abramovi c, 2008). DON has been suggested as a clastogenic and potential in vivo tumor-promoting toxin, and thus exposure to DON can cause feed refusal, immunological problems, vomiting, skin dermatitis, and hemorrhagic lesions (McCormick et al., 2011;
* Corresponding author. College of Light Industry and Food Sciences, South China University of Technology, Wushan Road 381, Tianhe District, Guangzhou 510640, PR China. Tel./fax: þ86 20 87113914. E-mail address: [email protected] (M. Zhao). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.066
Moazami & Jinap, 2009). Nowadays, several countries including USA, Austria, Canada, Germany, Russia and China have established tolerance values for DON in cereals (GB 2715-2005, 2005; Omurtag lu, 2007), and the Commission of the European Commu& Beyog nities has also established a tolerance values for DON in cereals and cereal-based products (European Commission, 2006). Although different countries have different regulations, the maximum permitted levels for DON are generally 1000 mg/kg (FAO, 1997). Moreover, maximum tolerated levels for DON commonly detected in food commodities are usually between 500 and 1000 mg/kg, and the Tolerable Daily Intake (TDI) for DON is estimated to be 3 mg/kg for adults and 1.5 mg/kg for infants (Moazami & Jinap, 2009). However, there is a lack of legislation on the acceptable levels of DON in fermented food. Therefore, investigating the occurrence of DON in food, particularly fermented food, would be significance to eliminate the threat to human health because of exposure to DON. Soy sauce is one of the world’s oldest condiments and has been used in China for more than 2500 years. Nowadays, it is widely used as a seasoning or condiment in eastern Asia, and its popularity in the Western part of the world is growing due to its unique taste and aroma (Gao et al., 2011). The annual production of soy sauce in China is approximately 6 million tons, accounting for over 65% of the total world production. The wide distribution of manufacturers and varied manufacturing process led to the considerable variations in safety, quality, flavor and nutritional values of soy sauce. Therefore, its safety and quality standards were under consideration from member countries of FAO/WHO Codex Commission (Li, Zhao, Zhao, & Cui, 2010). In China, soy sauce is produced by fermenting soybeans and wheat flour or wheat bran with Aspergillus
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H. Zhao et al. / Food Control 29 (2013) 71e75
Table 1 Characteristics and levels of DON contamination of soy sauces consumed in China.a Samples
Grade
Manufacturing process
Original of country
DON (mg/l)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Premium Premium Premium Premium Premium Premium Premium Premium Premium Premium Premium Premium 1 1 1 1 1 2 2 2 2 3 3 3 3 3 3 e e e e e e e e e e e e e
HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF LSF LSF LSF LSF LSF LSF Blended Blended e e e e e
China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China China Japan Japan Japan Japan Japan
17.7 70.9 106.8 72.8 225.4 232.7 17.0 75.8 71.5 134.1 107.8 108.5 81.7 61.2 29.5 93.1 54.6 10.9 10.1 4.5 46.7 93.1 56.1 42.8 51.3 55.8 53.1 97.9 1230.3 1245.6 79.2 62.1 96.3 ND 14.4 30.5 98.7 238.3 152.2 48.7
a HLF, high salt liquid state fermentation; LSF, low salt solid state fermentation; ND, not detected.
oryzae or Aspergillus sojae molds (solid state fermentation or koji fermentation process), along with water and salt. After the fermentation, fermented soybean paste was pressed to yield soy sauce. Thus, the soy sauce could be contaminated with DON due to the high incidence of DON in soybeans and wheat flour (wheat bran) and the natural multi-strains fermentation of soy sauce (Pacin, Resnik, Neira, Moltó, & Martínez, 1997; Valenta, Dänicke, & Blüthgen, 2002). Moreover, although the consumption of soy sauce has been increasing in recent years, there is little information available on the general occurrence of DON in soy sauce. Furthermore, the maximum permitted level and TDI of DON in soy sauce have not yet been established in China. Therefore, the first objective of this study was to examine the occurrence and levels of DON in soy sauces consumed in China. The second objective was to elucidate the relationship between DON contents in soy sauce and manufacturing process as well as quality grade. The last objective was to estimate the exposure to DON through the consumption of soy sauce in China. 2. Materials and methods 2.1. Soy sauce samples Forty soy sauce samples including 35 domestic and 5 imported soy sauces were purchased from local markets (Guangzhou,
Guangdong province). Among 35 domestic soy sauce samples, 27 samples were manufactured by high salt liquid state fermentation (HLF), and 6 samples were brewed by low salt solid state fermentation (LSF) and the other 2 samples were blended soy sauces. In 27 HLF soy sauce samples, the samples in premium grade, grade 1, 2 and 3 were 12, 5, 4 and 6, respectively (Table 1). All samples were stored in a refrigerator at 4 C until analysis. 2.2. Chemicals DON standard (200 mg/ml in ethyl acetateemethanol, 95:5, v/v) and Mirex were supplied by Supelco (Pennsylvania, USA) and stored at 20 C. LC grade solvent (isooctane, methanol) and sodium bicarbonate were purchased from Merck (Darmstadt, Germany). 1-(Trimethylsilyl)imidazole (TMSI) and Chlorotrimethylsilane (TMCS) were obtained from Fluka (Milwaukee, USA). All other chemicals and solvents were of the highest commercial grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 2.3. Extraction and clean-up The extraction and clean-up procedure were carried out by Prom, Horsley, Steffenson, and Schwarz (1999) with slight modifications. Five milliliters of soy sauce samples were placed into tubes containing a 40 ml of acetonitrileewater (84:16, v/v) mixture. Samples were extracted by placing the tubes on a shaker 30 min at 120 r/min. Then, the extract was blown to dryness under water-free nitrogen at 55 C. The extract residue was then dissolved in 20 ml of petroleum ether and 10 ml of methanolewater (4:1, v/v), and completely dried again under nitrogen at 55 C. The dry extract was dissolved in 2 ml water, loaded on the LC18 column and forced slowly through the cartridge. DON was slowly eluted with 6 ml of 40% methanol after washing the column twice with 3 ml of distilled water. The eluate was evaporated to dryness for further derivatisation. 2.4. Derivatisation The derivatisation of DON was according the method of Mateo, Llorens, Mateo, and Jiménez (2001) with some changes. A 100 ml derivatisation mixture of TMSIeTMCS (10:1, v/v) was placed into a vial containing the dry residue. The mixture was shaken gently on Vortex for 5 min at room temperature. One milliliter of isooctane containing 0.5 mg/ml Mirex and 1 ml 3% sodium bicarbonate solution were added without removing derivatisation agents. The vial was shaken on Vortex for 2 min and layers were allowed to separate. Transfer the upper layer (isooctane) into an autosampler vial and analyzed by GCeMS. 2.5. Analysis by GCeMS Analysis of the DON in soy sauce was performed using Trace GCeMS system equipped with an Ultra GC, a TriPlus autosampler and a quadrupole DSQ II MS (Thermo Finnigan, San Jose, CA, USA) according the method described by González-Osnaya, Cortés, Soriano, Moltó, and Mañes (2011) with some modifications. The capillary column was a TR-5 MS phase (30 m 0.25 mm, film thickness 0.25 mm) (J&W Scientific, USA). The carrier gas was helium (1.0 ml/min), and the temperature of the injection port was 280 C. The injection volume was 1 ml and the injection mode was splitless. The column temperature program was: 150 C for 1 min, 10 C/min to 220 C, 3 C/min to 260 C and then 10 C/min to 280 C held for 5 min. The temperature of the transfer line was adjusted to 300 C. The mass spectrometer was operated in the
H. Zhao et al. / Food Control 29 (2013) 71e75
electron impact (EI) mode. The selected ion monitoring (SIM) mode was utilized in order to have greater sensitivity. Quantification was performed on SIM using multiple ions m/z: 422, 512 for DON and 237, 272 for internal standard Mirex. 2.6. Estimated daily intake (EDI) The exposure of DON through soy sauce consumption was calculated using the information about soy sauce intake and body weight according to the formula proposed by Moazami and Jinap (2009). EDI in mg/kg body weight ¼ mean concentration of DON (mg/l) multiplied by the amount of soy sauce consumed/day (l) and divided by the average weight of an individual (50 kg). 2.7. Statistical analysis All of experiments were carried out in triplicate. Data were reported as means standard deviation (SD) for triplicate determinations. Analysis of variance and significant difference tests were conducted to identify differences among means by one-way ANOVA using SPSS software (version 17.0 for Windows, SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Occurrence of DON in soy sauce samples consumed in China An analytical method for determination of DON in soy sauce was developed by solid phase extraction and GCeMS. A typical chromatogram of selected ion mass for DON and Mirex in soy sauce sample is given in Fig. 1. The limit of detection (LOD, S/N ¼ 3) and the limit of quantitation (LOQ, S/N ¼ 10) for developed method were 16.7 and 5.0 mg/l, respectively. At the spiked levels of 50.0e1500.0 mg/l, the average recoveries were 82.0e106.1% with the relative standard deviations of 4.3e7.8%. All these results indicated that the proposed method is suitable to detect DON in soy
73
sauce due to its high sensitivity and recovery. The levels of DON in 40 soy sauce samples are presented in Table 1. As can be seen, 97.5% of soy sauces investigated were contaminated with DON at the test concentrations, and the DON content in only one blended soy sauce sample was below the limit of detection. The present study revealed a wide variation in DON contents among the 40 samples, ranging from 4.5 to 1245.6 mg/l, with an average value of 137.9 mg/l. The results were in agreement with the report by Schollenberger et al. (2007) that two out of four soy sauce samples were contaminated with DON, with the values of 25 and 14 mg/kg, respectively. Moreover, results from Fig. 2 also showed that 80% of soy sauce samples (n ¼ 32) exhibited DON contents in the range of 0e125.0 mg/l. While 12.5% of soy sauce samples (n ¼ 5) were observed to be contaminated with DON ranging from 125.0 to 250.0 mg/l. Only two soy sauce samples produced by LSF exhibited the DON contents in the range of 1000e1250 mg/l, which higher than values considered as dangerous for health. All these results indicated that raw materials and manufacturing process might have significant impacts on DON contents of final soy sauce. Indeed, Bensassia, Zaied, Abid, Hajlaoui, and Bacha (2010) found that 83% durum wheat samples in Tunisia showed DON contamination with averages of 12.8e30.5 mg/g exceeding the maximum permitted limit of 1.75 mg/g set by the European Commission in wheat intended for human and animal consumption. Out of 40 soybean samples, two samples were contaminated with DON at levels of 1.6 mg/g and 0.9 mg/g, respectively (Barros, Oviedo, Ramirez, & Chulze, 2011). It should be noted that a guideline for the mycotoxins DON, aflatoxins B1, zearalenone and ochratoxin A appears only for the cereal and cereal products in China (GB 2715-2005, 2005), but there is a lack of legislation on the acceptable levels of DON in soy sauce. According to the ANOVO, there were no significant differences in the levels of DON for soy sauces from China and Japan. The average incidence rate of DON in domestic soy sauces was 97.1% (concentration range 4.5e1245.6 mg/l, average value 141.5 mg/l), while for those imported from Japan was 100% (concentration
Fig. 1. A typical chromatogram of selected ion mass (m/z 422/512, 237/272) for DON and Mirex in soy sauce sample.
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H. Zhao et al. / Food Control 29 (2013) 71e75
an acid medium and results showed that selected strains were capable of removing up to 55% DON due to binding, not biodegradation effects (Niderkorn, Boudra, & Morgavi, 2006). A total decontamination of 53% of DON and T-2 toxin was observed when malt contaminated with these toxins was fermented using Saccharomyces cerevisiae (Garda et al., 2005). Moreover, A. oryzae and Rhizopus oryzae also showed significant degradation capacity to DON during submerged fermentation, and both cultures caused the largest decrease in DON at around 90% in 240 h and 74% in 96 h (Garda-Buffon & Badiale-Furlong, 2010). In addition, HLF soy sauces are made by mixing soybeans and wheat flour, whereas LSF soy sauces are manufactured by mixing soybeans and wheat bran. Previous studies also proved that most of the DON is recovered in the bran fraction and the lowest concentration is found in the flour during milling (Lancova et al., 2008). This could explain partly the high level of DON in LSF soy sauces. 3.3. Effects of quality grade on the levels of DON in HLF soy sauces Fig. 2. The distribution of the DON contents in soy sauce samples.
range 30.5e238.3 mg/l, average value 113.7 mg/l). Although domestic brands have been considered as low-priced products compared to Japanese soy sauce, the results obtained herein suggested that the quality of both groups, in terms of DON content, is appropriately the same. 3.2. Effects of manufacturing process on the levels of DON in domestic soy sauces In China, soy sauce can be classified into fermented soy sauce (HLF and LSF soy sauces) and blended soy sauce according to manufacture process (GB 18186-2000, 2000; SB 10336-2000, 2000). Different types of soy sauces might be significant differences in their DON contents due to various raw materials and manufacturing process employed. The levels of DON in different types of soy sauces are shown in Table 2. As shown in Table 2, the DON contents in fermented soy sauces were significantly higher than those in blended soy sauces, the average values of DON for HLF, LSF and blended soy sauces were 73.5, 468.6 and 14.4 mg/l, respectively. This might be that blended soy sauce was manufactured by blending fermented soy sauce, hydrolyzed vegetable protein, salt, caramel color and additives with sweet or umami tastes. Thus the low proportion of fermented soy sauces in blended soy sauces resulted in a low content of DON in blended soy sauce due to the dilution. Moreover, HLF soy sauces exhibited markedly lower DON contents than LSF soy sauces, which was probably due to the difference in manufacturing technique and raw materials. HLF process is carried out by an indigenous fermentation procedure at 28e35 C and having long aging period, while LSF process by fermentation using pure cultures at relatively higher temperature (40e50 C) and having very short aging (Li et al., 2010; Lu et al., 2009). Generally, the multiple-strains fermentation process is considered as a bio-detoxification process. Twenty-nine strains of lactic acid bacteria were tested for their ability to remove DON from
Soy sauce manufactured by HLF method is generally regarded as traditional Chinese-type soy sauce. Total nitrogen, amino nitrogen and non-salt soluble solids are the important indices for classifying the quality grade of soy sauce in China (GB 18186-2000, 2000). Twenty-seven HLF soy sauces samples were divided into four grades: premium grade, grade 1, grade 2 and grade 3 according to the quality indices described above. As shown in Fig. 3, the DON contents of different quality grades of soy sauces decreased in the following order: premium grade > grade 1 > grade 3 > grade 2. Generally, soy sauces with higher quality grade had higher DON content, while DON content was low in samples with lower grade level except for soy sauces with grade 3. This might be that soy sauce with premium grade or grade 1 made from the first pressing of the soybeans, thus the most DON contaminated in raw materials could dissolve in soy sauce by the effect of enzyme or microorganism during this stage. The higher levels of DON in soy sauces of grade 3 might be due to the differences in raw materials. 3.4. An assessment of DON exposure in humans The annual production of soy sauce in China is approximately 6 million tons, and the demand for soy sauce consumption is increasing at a rate of about 5e10% yearly. The daily intake of DON
Table 2 Natural occurrence of DON in soy sauces manufactured with different process.a Manufacturing No. of No. of positive DON content (mg/l) process samples samples (%) Average Range HLF LSF Blended a
27 6 2
27 (100) 6 (100) 1 (50)
Median
73.5 55.8 4.5e232.7 61.2 468.6 596.1 62.1e1245.6 97.1 14.4 14.4 14.4
HLF, high salt liquid state fermentation; LSF, low salt solid state fermentation.
Fig. 3. The distribution of the DON contents in HLF soy sauces according to quality grade. HLF, high salt liquid state fermentation; Vertical bars represent the standard deviation of each data point.
H. Zhao et al. / Food Control 29 (2013) 71e75 Table 3 Estimated DON exposure level from soy sauce in China.a Exposure (mg/kg BW) Soy sauce a
0.034
BW: body weight.
from soy sauce depends on not only the DON content in soy sauce, but also the amount of soy sauce consumed. Moreover, the consumption of soy sauce may vary considerably from one individual to another. Thus it is importance to estimate the exposure to DON by consumption of soy sauce. The EDI value was calculated by assuming that a 50 kg individual will consume a 12.20 ml soy sauce serving per day. The results are summarized in Table 3 and implied a low exposure to DON by consumption soy sauce in China, which is far below the Provisional Tolerable Maximum Daily Intake (PTMDI) according to the JECFA of 1 mg/kg body weight/day (Moazami & Jinap, 2009). However, it is should keep in mind exposure to all other foods that could be contaminated with DON because the soy sauce is indispensable condiments in China. 4. Conclusion The present study clearly showed that almost all samples of soy sauce consumed in China contained detectable quantities of DON. There were also considerable variations in DON contents of commercial soy sauces across different samples. Moreover, manufacturing process and quality grade had significant impacts on DON contents in soy sauces. Soy sauces brewed with LSF technology and with higher quality grade generally exhibited higher levels of DON. The results from assessment of DON exposure in humans suggested that human risk to DON exposure from the consumption of soy sauces is very low in China. This research was part of our continuous efforts to improve the safety of soy sauce production by screening raw materials and optimizing the manufacturing process. On the basis of results obtained from this study, further works on clarifying the fate of DON in soy sauce production are in progress. Acknowledgments The authors gratefully acknowledge the National ScienceTechnology Supporting Project for 12th Five-Year Plan (2012BAK17B11) and the National High Technology Research and Development Program of China (863 Program) (No. SS2012AA022502) and the Key Technology R&D Program of Guangdong Province (Nos. 2009Z52 and 2011A020102001) for their financial supports. References Barros, G., García, D., Oviedo, S., Ramirez, M. L., Torres, A., & Chulze, S. (2008). Deoxynivalenol and nivalenol analysis in soybean and soy flour. World Mycotoxin Journal, 1, 263e266. Barros, G., Oviedo, M. S., Ramirez, M. L., & Chulze, S. (2011). In T.-B. Ng (Ed.), Soybean. Biochemistry, chemistry and physiology (pp. 7e20). Rijeka, Croatia: Intech Open Access Publisher.
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