Article pubs.acs.org/JAFC

Safety Assessment of the Biogenic Amines in Fermented Soya Beans and Fermented Bean Curd Juan Yang, Xiaowen Ding,* Yingrui Qin, and Yitao Zeng College of Food Science, Chongqing Key Lab of Agricultural Product Processing, Southwest University, Chongqing 400716, People’s Republic of China S Supporting Information *

ABSTRACT: To evaluate the safety of biogenic amines, high performance liquid chromatography (HPLC) was used to evaluate the levels of biogenic amines in fermented soya beans and fermented bean curd. In fermented soya beans, the total biogenic amines content was in a relatively safe range in many samples, although the concentration of histamine, tyramine, and βphenethylamine was high enough in some samples to cause a possible safety threat, and 8 of the 30 samples were deemed unsafe. In fermented bean curd, the total biogenic amines content was more than 900 mg/kg in 19 white sufu amples, a level that has been determined to pose a safety hazard; putrescine was the only one detected in all samples and also had the highest concentration, which made samples a safety hazard; the content of tryptamine, β-phenethylamine, tyramine, and histamine had reached the level of threat to human health in some white and green sufu samples, and that may imply another potential safety risk; and 25 of the 33 samples were unsafe. In conclusion, the content of biogenic amines in all fermented soya bean products should be studied and appropriate limits determined to ensure the safety of eating these foods. KEYWORDS: fermented soya beans,fermented bean curd, biogenic amines, food safety

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smoked fish products; the U.S. Food and Drug Administration set the stricter acceptable histamine value of 50 mg/kg for scombroid-like fishes.16,17 Tryptamine, β-phenethylamine, and tyramine can induce hypertension and migraines.18−20 Among these, tryptamine can cause hypertension, through making blood pressure increase, and hepatotoxicity;21 β-phenethylamine can eliminate norepinephrine in the nervous system, and also cause migraines by making blood pressure increase;19,20 tyramine can cause migraines when more than 100 mg is ingested orally and can induce severe poisoning symptoms when more than 1080 mg/kg is ingested. The maximum allowable concentration range for tyramine is 100−800 mg/kg in food.18 Finally, cadaverine, putrescine, spermidine, and spermine are aliphatic compounds, and have no obvious toxicity, relatively. However, they can seriously influence food flavor and quality, may interact with nitrite to form carcinogenic nitrosamine, and can inhibit other toxic BAs’ metabolism to make their toxicity increase.22 Among these, putrescine can promote tumor growth;22 putrescine and cadaverine can react with nitrite to generate nitrosamines;23 cadaverine and tyramine can inhibit intestinal oxidase and the activity of histamine-N-methyl transferase, which could boost the toxicity of histamine.24 Moreover, putrescine and spermidine have an acute oral toxicity of 2000 mg/kg body weight and 600 mg/kg body weight, respectively; the no-observed-adverse-effect level is 180 mg/kg body weight/day for tyramine, cadaverine, and putrescine, 83 mg/kg body weight/day for spermidine and 19 mg/kg body weight/day for spermine.25

INTRODUCTION Biogenic amines (BAs) are organic nitrogenous compounds produced in food via microbial decarboxylation of amino acids.1−3 The amount and types of biogenic amines are influenced by microbial flora, food composition, and fermentation conditions.3 Common BAs in food include aliphatic (e.g., putrescine, cadaverine, spermidine, and spermine), aromatic (e.g., tyramine and β-phenethylamine) and heterocyclic (e.g., histamine and tryptamine).4 The existence of biogenic amines not only indicates the extent of microbial contamination, but also makes food a potential toxic hazard for humans.5,6 BAs are toxic substances that can cause disease in humans, although the toxic effects of BAs differ. As a whole, scholars believe that total BA content in food should not be more than 900 mg/kg,7,8 Santos9 also asserts that they cause great harm to human health when the total BA content reaches 1000 mg/kg in food. However, it is very difficult to set a content limit of total BAs in food, for the toxicity of total BAs is affected by many factors, including the type of BAs, the synergy between them, the activity of ammonia oxidase in human body, and individual intestinal function.10 Histamine, for example, is a highly toxic heterocyclic compound, which might cause slight, intermediate, or intensive symptoms of poisoning when ingested at varying levels (8−40 mg, 40−100 mg, and higher than 100 mg, respectively), so the maximum allowable concentration range is 50−100 mg/kg in food.11 In addition, histamine could also cause damage to the liver, kidneys, and nervous system, acting through four different G-proteincoupled receptors: H1R, H2R, H3R, and H4R.12−15 For these reasons, the Council Directive of the European Union regulated that the concentration of histamine should not exceed 200 mg/kg in fresh fish, and should not exceed 400 mg/kg in © 2014 American Chemical Society

Received: Revised: Accepted: Published: 7947

April 18, 2014 July 3, 2014 July 16, 2014 July 16, 2014 dx.doi.org/10.1021/jf501772s | J. Agric. Food Chem. 2014, 62, 7947−7954

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Table 1. Type, Origin and Trade Name of the Samplesa no. 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

a

type bacteria-type douchi

aspergillus-type douchi

mucor-type douchi

origin

trade name

no.

Hunan Yunnan Guizhou Guizhou Chongqing Chongqing Yunnan Sichuan Yunnan Guangzhou Guangzhou Guangzhou Guangzhou Guangzhou Guangzhou Guangzhou Guangzhou Guangzhou Hunan Guangxi Guangxi Guangxi Shandong Yongchuan,Chongqing Yongchuan,Chongqing Yongchuan,Chongqing Yongchuan,Chongqing Yongchuan,Chongqing Yongchuan,Chongqing Yongchuan,Chongqing

LinSanLin YunZhiNan ShuiXiangZi LaoGanMa # # SanChuanBan XinFan ShanLiXiang YangFan YangFan YangFan YangFan YangFan YangJiang YangJiang YangJiang YangJiang LiuYang HuangYaoGuZhen HuangYaoGuZhen HuangYaoGuZhen WeiYiZhai WaiZuMu QinMei AnJun WuJianFang JunYi JiaTai YongLai

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

type gray sufu

red sufu

White sufu

origin

trade name

Beijing Sichuan Hunan Hunan Chongqing Guizhou Beijing Beijing Beijing Beijing Chongqing Chongqing Sichuan Guangxi Chongqing Chongqing Chongqing Chongqing Chongqing Sichuan Sichuan Sichuan Beijing Chongqing Sichuan Beijing Chongqing Beijing Yunnan Chongqing Chongqing Beijing Chongqing

WangZhiHe LinJiangSi # # # LaoGanMa WangZhiHe WangZhiHe WangZhiHe LaoCaiChen ZhongZhou DouXiaoDe LinJiangSi GuRong ZhongZhou XianJia ZhongZhou GanShui DouXiaoDe WeiDuiWei WeiDuiWei QiaoPai LaoCaiChen GanShui QiaoPai WangZhiHe GanShui WangZhiHe ShiFangZhi ZhuJiangQiao DouXiaoDe LaoCaiChen XianJia

Among them, the same brands are composed of different flavor; “#” expressing home-fermented.

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Fermented soya beans (or douchi) and fermented bean curd (or sufu) are the most typical traditional fermented soy products. Both are made by microbial fermentation beginning with soybean as raw material. On the basis of the microorganisms used, fermented soya beans can be classified into aspergillus-type douchi, mucor-type douchi, and bacterial-type douchi. Similarly, fermented bean curd can be divided into three main types according to the differences of the dressing mixture, which are white sufu, gray sufu, and red sufu, respectively. During the fermentation process both of fermented soya beans and fermented bean curd, microorganisms are involved, such as some type of yeast, lactic acid bacteria (bending Lactobacillus casei and lactobacillus), bacillus, streptococcus, Staphylococcus aureus, etc., some of which could produce decarboxylase.26,27 In short, because of the existence of biogenic amine precursors, the process is bound to generate biogenic amines in an appropriate environment. Therefore, this study seeks to determine what biogenic amines occur in commercially available fermented soy products, and to evaluate the safety of biogenic amines. We hope to also provide advice so people can safely eat fermented soy products.

MATERIALS AND METHODS

Samples and Standards of BAs. Thirty fermented soya bean samples (9 bacteria-type douchi, 10 aspergillus-type douchi, 11 mucortype douchi.) and 33 fermented bean curd samples (5 gray sufu, 5 red sufu, and 23 white sufu) were purchased from retail markets and supermarkets in China. Specific descriptions of the samples (region of origin, types, and trade names) are listed in Table 1. Standard amines, including putrescine (Put, ≥98%), cadaverine (Cad, 98%), spermidine (Spd, ≥98%), tryptamine (Try, ≥98%), β-phenethylamine (β-Phe, ≥98%), histamine (His, ≥99%), tyramine (Tyr, ≥98%) and spermine (Spm, ≥98%) were purchased from the Sigma-Aldrich Corporation in the United States. Dansyl chloride (≥98%) was bought from Tedia Company in the United States. Apparatus. High performance liquid chromatograph (Agilent1260), with UV-detector and Agilent XDB-C18 (250 × 4.6 mm2, 5 μm) column; an ultrasonic bath KQ5200DB; an Eppendorf centrifuge table high speed; an ultrapure water Milli-Q; water bath temperature oscillator, and a syringe filter 0.22 μm. Sample and Standard Preparation. Five grams of each sample was extracted with approximately 20 mL of 0.4 M perchloric acid (Sigma), placed in the ultrasonic bath for 30 min, and then centrifuged for 5 min at 5000 rpm, taking supernatants on standby. Standard stock solutions of the eight biogenic amines were prepared separately at 1 mg/mL concentration in 0.4 M HClO4. Working solutions, containing all the amines at 3.0−100 mu g/mL concentrations, were prepared by diluting the standard stock solution.28 7948

dx.doi.org/10.1021/jf501772s | J. Agric. Food Chem. 2014, 62, 7947−7954

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Figure 1. Chromatogram of derivatives of biogenic amines standards (70 μg/mL). (1) Tryptamine, (2) β-phenylethylamine, (3) putrescine, (4) cadaverine, (5) histamine, (6) tyramine, (7) spermidine, and (8) spermine. The peaks that are omitted have no meaning. As shown, the peaks of the 8 standard derivatives are shown later and flagged as 1, 2, 3, 4, 5, 6, 7, and 8.

Figure 2. Chromatogram of derivatives of biogenic amines in one of the fermented soya beans. (1) β-Phenylethylamine, (2) putrescine, (3) cadaverine, (4) histamine, and (5) tyramine.

Figure 3. Chromatogram of derivatives of biogenic amines in one of the fermented bean curd samples. (1) tryptamine, (2) β-phenylethylamine, (3) putrescine, (4) cadaverine, (5) histamine, and (6) tyramine. Derivatization. One milliliter of a sample solution was mixed with 200 μL of 2 M sodium hydroxide and 300 μL of saturated sodium carbonate. Under the alkaline conditions, the mixture was derivatized by the addition of 2 mL dansyl chloride solution (10 mg/mL in acetone). After incubation for 30 min at 40 °C in darkness, 100 mL of aqueous ammonia was added to remove any residual Dansyl chloride. The volume was adjusted to 5 mL with acetonitrile. Finally, the supernatant was filtered through 0.22 μm pore-size filters prior to HPLC analysis. Chromatographic Conditions. Column temperature = 30 °C, flow rate = 1.0 mL/min, injected volume = 5 μL, detection wavelength (λ) = 254 nm were used. The mobile phase was a gradient elution program with a binary mixture of acetonitrile and H2O. Each HPLC

run took about 30 min, and the column was conditioned again with a mixture of 65% solvent A and 35% solvent B. Statistical Analysis. All the tests were repeated three times, using Excel 2007 version for analysis and data processing.

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RESULTS

Chromatograms of Standard BAs and Sample. Through the elution procedure, each BAs was detected in turn. The representative HPLC chromatogram and peak identification recorded for mixer of standard BAs, a fermented soya bean sample, and a fermented bean curd are shown in Figures 1, 2, and 3, respectively. 7949

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Table 2. Contents of Biogenic Amines in Samplesa biogenic amines (mg·kg−1) Trp 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 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 69.56 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 463.45 147.20 124.82 163.57 394.61 70.41 118.71 118.82 126.11 237.63 256.08 185.28 240.81 91.51 246.74 228.33 54.47 121.80 51.95

Phe

± 1.87

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.60 4.86 1.58 0.60 0.77 0.89 4.10 6.20 3.59 3.77 4.58 2.47 8.99 5.49 5.01 10.64 2.71 0.68 0.66

ND ND ND ND ND ND ND ND ND 16.77 ND 28.37 ND 79.00 ND ND ND ND ND 736.64 25.14 135.66 ND 17.56 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 238.88 125.34 136.98 8.73 245.84 13.89 341.03 100.77 52.38 66.69 27.83 9.52 36.14 25.27 52.62 39.24 53.35 51.90 38.66

Put

± 0.88 ± 0.63 ± 4.36

± 26.32 ± 2.25 ± 8.37 ± 0.40

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.62 2.97 4.53 0.17 2.88 0.39 11.26 3.05 0.48 1.13 0.12 0.80 1.45 1.29 4.22 1.63 2.81 3.68 0.39

32.12 26.92 ND 27.56 15.67 34.94 25.27 14.08 21.11 51.58 34.82 139.85 44.36 120.10 28.27 26.49 29.84 36.00 38.03 276.03 249.01 267.59 36.71 83.14 48.93 54.75 48.77 48.97 54.30 39.42 124.32 25.16 234.12 123.79 205.57 11.38 19.93 13.16 15.95 13.57 907.79 922.98 542.16 911.26 1024.10 361.81 580.05 475.68 919.57 1147.32 920.93 585.06 764.70 411.40 1075.92 172.90 316.86 471.18 224.34

Cad

± 0.30 ± 0.31 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.03 0.74 1.07 2.13 1.03 0.55 4.39 9.11 3.98 1.77 1.54 0.62 1.53 2.68 1.64 1.58 8.41 8.10 9.32 2.42 10.20 4.50 1.79 1.10 0.79 3.77 1.10 6.49 1.11 2.37 1.26 1.11 0.18 0.55 0.23 2.37 0.84 9.35 24.19 8.63 24.12 5.33 11.37 11.12 37.69 23.54 17.45 25.61 8.50 26.95 21.53 9.74 6.21 2.44 9.34 2.60

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 672.33 222.94 243.08 18.30 ND ND 10.28 ND ND ND 5.14 19.33 10.01 23.11 34.33 32.12 ND 5.47 11.00 ND 14.36 235.12 308.44 47.33 102.14 242.31 12.92 72.56 137.17 ND 88.72 32.28 76.67 26.46 36.64 82.61 3.37 260.86 51.40 152.61

± ± ± ±

His

51.46 2.81 13.07 1.53

± 0.99

± ± ± ± ± ±

0.49 0.36 0.75 1.11 2.67 1.26

± 0.07 ± 0.15 ± ± ± ± ± ± ± ± ±

0.31 3.39 3.03 1.06 5.09 1.06 0.83 0.84 10.35

± ± ± ± ± ± ± ± ± ±

1.40 1.76 5.07 1.49 2.58 3.26 0.29 21.87 2.33 0.20 7950

ND ND ND ND ND ND ND ND ND 152.42 100.17 255.36 122.63 478.77 14.76 18.94 24.47 18.45 19.03 52.80 83.87 102.09 20.62 ND ND ND ND ND ND ND 16.85 174.36 120.78 12.11 17.43 ND ND ND 7.82 ND 41.39 45.95 14.28 145.70 68.82 6.07 23.18 34.28 304.16 161.68 312.24 164.59 160.45 9.42 312.11 20.30 164.30 415.38 6.08

± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ±

Tyr

4.60 1.86 12.91 4.51 8.79 1.01 0.57 1.48 1.51 1.10 4.03 1.64 2.27 1.22

0.67 3.43 1.06 1.41 2.77

± 0.30 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.90 4.03 0.75 0.94 1.26 0.20 0.15 3.27 13.39 4.51 6.93 4.03 6.68 0.78 16.51 1.38 16.03 6.15 0.46

ND ND ND ND ND ND ND ND ND 180.14 159.38 183.06 139.93 252.08 ND ND 57.85 ND ND 250.21 86.77 170.12 ND ND ND 20.04 ND 35.91 ND 17.90 15.58 34.44 26.00 12.79 14.55 ND ND ND 39.07 ND 330.12 370.38 357.50 167.91 431.27 265.37 213.82 336.07 388.15 270.32 410.72 549.34 269.23 239.75 388.41 222.01 223.56 380.62 152.27

± ± ± ± ±

Spd

5.97 6.64 3.44 2.30 1.27

± 1.63

± 3.24 ± 7.65 ± 5.16

± 0.83 ± 2.76 ± ± ± ± ± ±

1.33 0.84 0.92 2.11 1.66 1.26

± 2.63 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7.05 13.70 29.90 13.59 33.30 22.99 6.55 14.17 25.29 10.10 8.24 19.63 20.11 13.43 22.39 12.50 11.59 15.20 6.79

ND ND 19.51 ND ND ND ND 74.92 ND ND ND ND ND ND ND ND ND ND ND ND ND ND 23.47 ND ND ND ND ND ND 16.15 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

± 1.43

± 1.66

± 1.34

± 0.27

Spm

total content

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

32.12 26.92 19.51 27.56 15.67 34.94 25.27 89.00 42.22 400.91 294.37 606.64 306.92 929.97 43.03 45.43 112.17 54.45 57.07 2057.56 667.74 918.53 99.10 100.70 48.93 80.82 48.77 84.87 54.30 83.74 176.10 243.96 304.01 183.02 269.67 11.38 25.41 24.16 62.83 27.93 2216.76 1920.29 1223.08 1499.29 2406.95 730.48 1349.36 1202.80 1790.37 1972.36 1960.07 1570.46 1497.79 813.99 2158.41 686.14 1073.39 1492.27 625.92

dx.doi.org/10.1021/jf501772s | J. Agric. Food Chem. 2014, 62, 7947−7954

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Table 2. continued biogenic amines (mg·kg−1) Trp 60 61 62 63

322.56 70.13 208.83 188.23

± ± ± ±

Phe 24.98 3.53 16.18 7.59

73.28 73.00 6.84 47.62

± ± ± ±

Put 5.30 1.31 0.41 2.12

907.02 409.78 578.23 671.36

± ± ± ±

Cad 61.64 2.81 42.55 13.99

35.23 210.94 88.08 71.38

± ± ± ±

His 2.97 3.38 7.72 3.37

265.48 58.85 54.39 32.58

± ± ± ±

Tyr 19.98 3.77 4.08 2.15

257.59 361.47 33.11 330.57

± ± ± ±

Spd 14.96 20.83 2.24 6.60

ND ND ND ND

Spm

total content

ND ND ND ND

1861.16 1184.17 969.48 1341.74

Trp: tryptamine, Phe: β-phenethylamine, Put: Putrescine, Cad: cadaverine, His: histamine, Tyr: tyramine, Spd: spermidine, and Spm: spermine. 1− 30: fermented soya bean, 31−63: fermented bean curd, 1−9: Bacterial-type douchi, 10−19: Aspergillus-type douchi, 20−30: Mucor-type douchi, 31−35: Gray sufu, 36−40: Red sufu, 41−63: White sufu, and ND expressed not detected. a

in gray sufu (the 31st∼35th samples). Second, there were a little difference in red sufu (the 36th∼40th samples), the 36th sample only detected putrescine; the 37th, 38th, and 40th samples detected putrescine and cadaverine; the 39th sample detected putrescine, histamine, and tyramine. At last, there were tryptamine, β-phenethylamine, putrescine, cadaverine, histamine, and tyramine detected in all white sufu samples except the 49th sample, and the 49th sample detected tryptamine, β-phenethylamine, putrescine, histamine, and tyramine. As above, there were a few differences of BAs’ types among gray, red, and white sufu samples. It could be speculated that white sufu was most dangerous among the sufu samples. Safety of the BAs in the Samples. Safety of the Total BAs in Samples. As shown in Table 2, the total BAs content was between 15.67−89.00, 43.03−929.97, and 48.77−2057.56 mg/kg, the median was 27.56, 203.27, and 84.87 mg/kg in bacterial-type, aspergillus-type, and mucor-type douchi samples, respectively. The total BAs content was more than 900 mg/kg only in the 14th, 20th, and 22nd samples. As mentioned before, the total BAs content in food should not be more than 900 mg/ kg,7,8 and when the total BAs content reached 1000 mg/kg in food, they would cause great harm to human health.9 So the sample would be considered unsafe when the total BAs content were more than 900 mg/kg. In conclusion, it was observed that the total BAs content was within the relative safety range in most fermented soya bean samples except the 14th, 20th, and 22nd sample. However, the total BAs content range was 625.92−2406.95, 11.38−62.83, and 176.10−404.01 mg/kg, the median was 1492.27, 25.41, and 243.96 mg/kg in white, red, and gray sufu samples, respectively. The total BAs content was less than 900 mg/kg in gray and red sufu, while which was more than 900 mg/kg in 19 white sufu samples (all samples except the 46th, 54th, 56th, and 59th sample), accounting for 82.61% of the total number of white sufu samples. According to the total BAs content in samples and the related knowledge of toxicology. It is worth noting that the total BAs content was in a relatively safe range in gray and red sufu samples, but was in a dangerous concentration range in part white sufu samples, which could cause a safety risk.8−10 Safety of Histamine in the Samples. Histamine was detected in 10 aspergillus-type and 4 mucor-type douchi samples, respectively. The content was 14.76−478.77 and 20.62−102.09 mg/kg, the median was 62.32 and 68.33 mg/kg, the detection rate was 100% and 36.36%, respectively. Histamine’s content was more than 50 mg/kg in 5 aspergillus-type (the 10th−14th sample) and 1 mucor-type (the 22th sample) douchi sample, accounting for 50% and 25%, respectively. As mentioned before, the maximum allowable

As shown in Figure 1, all BAs were detected in 30 min. The effect of the useful peak’s separation was good. The retention time of tryptamine, β-phenethylamine, putrescine, cadaverine, histamine, tyramine, spermine, and spermine were 8.374, 9.994, 10.606, 11.536, 12.257, 20.595, 12.257, and 20.595 min, respectively. In addition, at the beginning of the chromatogram, a few peaks were omitted, whose value were so high that the useful ones were not present. Type of BA in the Samples. Through the HPLC method, all samples were tested, the results showed in Table 2. The types of BAs can decide whether the food is safety, so this part had introduced the types of BAs, to evaluate the safety of the samples in brief. Tryptamine, β-phenethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine were detected in fermented soya bean samples, the content range was 69.56, 16.77−736.64, 14.08−276.03, 5.14−672.33, 14.76−478.77, 17.90−252.08, and 16.15−74.92 mg/kg, respectively. The types of BA was no obvious regularity, and had nothing to do with the fermentation type. The 1st, 2nd, 4th∼7th, 9th, 25th, 27th, and 29th samples only detected putrescine; the 3rd sample only detected spermidine; the 8th sample detected putrescine and spermidine; the 10th, 12th, and 14th samples detected β-phenethylamine, putrescine, histamine, and tyramine; the 11th, 13th, and 17th samples detected putrescine, histamine, and tyramine; the 15th, 16th, 18th, and 19th samples detected putrescine and histamine; the 20th sample detected six kinds of BAs except spermidine and spermine; the 21st and 22nd samples detected five kinds of BAs excepte tryptamine, spermidine, and spermine; the 23th sample detected putrescine, cadaverine, histamine, and spermidine; the 24th sample detected βphenethylamine and putrescine; the 26th sample detected putrescine, cadaverine and tyramine; the 28th sample detected putrescine and tyramine; the 30th sample detected putrescine, cadaverine, tyramine, and spermidine. In conclusion, the BAs’ types were different in fermented soya beans, and the BAs’ types were less than three there were in 22 samples. Among these, only putrescine existed in all samples, histamine, and tyramine were detected in 14 and 12 samples, respectively, closely following putrescine. Therefore, putrescine, histamine, and tyramine might be regard as safety indexes of fermented soya bean samples contaminated by BAs. Alternatively, tryptamine, β-phenethylamine, putrescine, cadaverine, histamine, and tyramine were detected in fermented bean curd, the content range was 51.95−463.45, 6.84−341.03, 11.38−1147.32, 3.37−308.44, 6.07−415.38, and 12.76−549.34 mg/kg, respectively. Compared with fermented soya beans, the type of BA was more regular in fermented bean curd, and had something with the differences of the dressing mixture. First, putrescine, cadaverine, histamine, and tyramine were detected 7951

dx.doi.org/10.1021/jf501772s | J. Agric. Food Chem. 2014, 62, 7947−7954

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concentration of histamine were 50−100 mg/kg in food.11 So the sample would be considered unsafe when the total content of BAs were more than 50 mg/kg. Therefore, the high histamine’s concentration in part aspergillus-type and mucortype douchi samples could cause a potential safety hazard.15−17 Similarly, histamine was detected in 23 white, 5 gray, and 1 red sufu samples, the content range was 6.07−415.38, 12.11− 174.36, and 12.11 mg/kg, the median was 58.85, 17.43, and 7.82 mg/kg, respectively. Among the samples, histamine’s content was more than 50 mg/kg in two gray (the 32nd and 33rd samples) and 13 white (the 44th, 45th, 49th∼53rd, 55th, 57th, 58th, and 60th∼62nd sample) sufu samples, accounting for 40% and 56.52%, respectively. According to the related knowledge of toxicology, the content of histamine was high in some white and gray sufu samples, resulting in health problems.15−17 Safety of Tryptamine, β-Phenethylamine, and Tyramine in Samples. Tyramine was detected in 6 aspergillus-type and 6 mucor-type douchi samples, the content range was 57.85− 252.08 and 17.90−250.21 mg/kg, the median was 169.76 and 61.34 mg/kg, the detection rate was 60% and 54.54%, respectively. Tyramine’s content was more than 100 mg/kg in 5 aspergillus-type (the 10th∼14th samples) and 2 mucortype (the 20th and 22nd samples) douchi samples, accounting for 83.33% and 33.33%, respectively. β-Phenethylamine was detected in 3 aspergillus-type and 4 mucor-type douchi samples, the content was 16.77−79.00 and 17.56−736.64 mg/kg, the median was 28.37 and 80.40 mg/kg, the detection rate was 30% and 36.36%, respectively. As mentioned before, Tyramine could cause migraines or induce severe poisoning symptoms when ingested more than 100 mg orally or 1080 mg/kg respectively, the maximum allowable range was 100− 800 mg/kg in food.18 So the sample would be considered unsafe when the content of tyramine was more than 100 mg/ kg. Obviously, the content of tyramine and β-phenethylamine were a little high in part aspergillus-type and mucor-type douchi samples, which would pose a safety risk. Besides, tryptamine and β-phenethylamine were detected in 23 white sufu samples, the content range were 51.95−463.45 and 6.84−341.03 mg/kg, the median were 163.57 and 52.38 mg/kg, and the detection rate were both 76.67%, respectively. Tyramine was detected in one red, all white, and all gray sufu samples, the contents were ispermidinen the ranges 33.11− 549.34, 39.07, and 12.79−34.44 mg/kg, the median was 330.12, 39.07, and 15.58 mg/kg, respectively. Tyramine’s content was more than 100 mg/kg in 22 white sufu samples (except the 62nd sample). As a result, the detection of tyramine, βphenethylamine, and tyramine made them some certain safety hidden danger in white sufu samples.18−20 Safety of Putrescine, Cadabverine, and Spermidine in Samples. Putrescine was detected in all samples except the third sample, the content range was 26.49−139.85, 39.42− 276.03, and 39.42−42.22 mg/kg, the median was 37.02, 54.30, and 27.24 mg/kg in aspergillus-type, mucor-type, and bacterialtype douchi samples, respectively; cadaverine was detected in 6 mucor-type douchi samples, the content range was 6.03− 672.33 mg/kg, the median was 120.62 mg/kg; spermine was detected in 2 mucor-type and 2 bacterial-type douchi samples, the content was 16.15−23.47 and 19.51−74.92 mg/kg, the median was 19.81 and 47.21 mg/kg, respectively. As mentioned above, the toxicity of cadaverine, putrescine, spermidine, and spermine is not obvious relatively, but they can interact with nitrite to form carcinogenic nitrosamine, and can inhibit other

toxic BAs’ metabolism to make their toxicity higher.22 Furthermore, putrescine’s detection rate was the highest among all the BAs, and the concentration was also highest in most of the samples, in consideration of the addition of the synergy between BAs, it was still considered that the existence of putrescine was a potential safety hazard in fermented soya beans. Similarly, the cadaverine and spermidine were considered in a safe range concentration in fermented soya bean samples. However, putrescine was detected in all fermented bean curd samples, the content range was 172.90−1147.32, 11.38−19.93 and 25.16−234.12 mg/kg, the median was 585.06, 13.57, and 124.32 mg/kg in white, red, and gray sufu samples, respectively. The detection rate was the highest among all the BAs, and the concentration was also the highest in most samples. Meanwhile, putrescine’s concentration was more than 900 mg/kg in 9 white sufu samples (the 41st, 42nd, 44th, 45th, 49th∼51st, 55th, and 60th sample), which made the total BAs content out of the safe limit. Similarly, cadaverine was detected in 22 white, 3 red, and 5 gray sufu samples, the content was 3.37−308.44, 5.47−14.36, and 10.01−34.33 mg/kg, the median was 79.64, 11.00, and 23.11 mg/kg, respectively, the detection rate was just lower than putrescine. As a result, the existence of putrescine and cadaverine was a certain safety hidden danger in some white and gray sufu samples. Safety of the Samples. In summary, because of BAs, several fermented soya bean and fermented bean curd samples had some potential safety risk. On the one hand, the content of histamine and tyramine had exceeded their limit level for the 10th∼14th aspergillus-type douchi samples, and the total BAs content of the 14th sample was more than 900 mg/kg, therefore it could be considered that the 10th∼14th samples had some obvious safety risk; the 20th and 22nd samples were unsafe because the content of total BAs, histamine, and tyramine were more than their limit level; histamine’s content was 83.87 mg/kg in the 21th sample, which could pose safety risk. In conclusion, there were 8 samples (the 10th−14th, and 20th−22nd samples) unsafe for fermented soya beans. On the other hand, first, the red sufu samples (the 36th∼40th samples) were relatively safe. Second, among the gray sufu samples (the 31st∼35th samples), the 32nd and 33rd samples were unsafe for their high histamine concentration. Finally, as mentioned above, all the white sufu samples (the 41st∼63rd samples) had some unsafe factors. Namely, the total BAs content had exceeded 900 mg/kg in 19 samples except the 46th, 54th, 56th, and 59th samples; the tyramine’s content was more than 100 mg/kg in all except the 62th samples; the histamine’s content was more than 50 mg/kg in 13 samples (the 44th, 45th, 49th∼53rd, 55th, 57th, 58th, and 60th∼62nd samples). So all of the white sufu samples were unsafe. As a whole, there were 21 samples (the 32nd, 33rd, and 41st∼63rd samples) unsafe for fermented bean curds.

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DISCUSSION Safety of Fermented Soya Products. Looking from the results, there were 8 samples unsafe for fermented soya beans. The 32nd and 33rd gray sufu sample were unsafe for their high histamine’s concentration, and all the white sufu samples were unsafe. Therefore, the content of BAs should be limited in all the fermented soy products. However, the experimental results were just a preliminary evaluation; there still needs to be a more in-depth study to 7952

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(6) Onal, A. A review: Current analytical methods for the determination of biogenic amines in foods. Food Chem. 2007, 103 (4), 1475−1486. (7) ten Brink, B.; Damink, C.; Joosten, H. M. L. J. Occurrence and formation of biologically active amines in foods. Int. J. Food Microbiol. 1990, 11, 73−84. (8) Spanjer, M. C.; van Roode, B. A. S. W. Towards a regulatory limit for biogenic amines in fish, cheese and sauerkraut. DeWare(n)Chemicus 1991, 21, 139−167. (9) Santos, M. H. S. Biogenic amines in foods. Int. J. Food Microbiol. 1996, 29, 213−231. (10) Shukla, S.; Park, H. K.; Kim, J. K.; Kim, M. Determination of biogenic amines in Korean traditional fermented soybean paste. Food Chem. Toxicol. 2010, 48, 1191−1199. (11) Maijala, R.; Eerola, S. Contaminant lactic acid bacteria of dry sausages produce histamine and tyramine. Meat Sci. 1993, 35, 387− 395. (12) Tripathi, T.; Khan, A. A.; Shahid, M.; Khanc, H. M.; Siddiquia, M.; Khand, R. A.; Mahdie, A. A.; Malikc, A. Biochemical and histopathological evaluation of histamine receptors (H1R, H2R, H3R, and H4R)-agonist in rabbits. Exp. Toxicol. Pathol. 2013, 65, 271−275. (13) Wu, T. S.; Gan, X. L.; Zhou, S. L.; Ge, M.; Zhang, Z.; Hei, Z. Q. Histamine at low concentrations aggravates rat liver BRL-3A cell injury induced by hypoxia/reoxygenation through histamine H2 receptor in vitro. Toxicol. In Vitro 2013, 27, 3778−3786. (14) Tripathi, T.; Shahid, M.; Khan, H. M.; Khan, R. A.; Siddiqui, M.; Mahdi, A. A. The Influence of histamine H1-receptor on liver functions in immunized rabbits. Saudi J. Biol. Sci. 2011, 18, 411−418. (15) Keles, N.; Arican, R. Y.; Coskun, M.; Elpek, G. O. Histamine induces the neuronal hypertrophy and increases the mast cell density in gastrointestinal tract. Exp. Toxicol. Pathol. 2012, 64, 713−716. (16) Tang, T.; Qian, K.; Shi, T. Y.; Wang, F.; Li, J. Q.; Cao, Y. S.; Hu, Q. B. Monitoring the contents of biogenic amines in sufu by HPLC with SPE and pre-column derivatization. Food Control 2011, 22, 1203−1208. (17) Shalaby, A. R. Significance of biogenic amines to food safety and human health. Food Research International. 1996, 29, 675−690. (18) Nout, M. J. R. Fermented foods and food safety. Food Res. Int. 1994, 27, 291−298. (19) Mazumder, M. K.; Rajib, P.; Anupom, B. β-Phenethylamine-A Phenylalanine Derivative in Brain-Contributes to Oxidative Stress by Inhibiting Mitochondrial Complexes and DT-Diaphorase: An In Silico Study. CNS Neurosci. Ther. 2013, 19, 596−602. (20) Ma, G. Y.; Bavadekar, S. A.; Schaneberg, B. T.; Khan, I. A.; Feller, D. R. Effects of Synephrine and β-Phenethylamine on Human α-Adrenoceptor Subtypes. Planta Medica 2010, 76, 981−986. (21) Shalaby, A. R. Significance of biogenic amines to food safety and human health. Food Res. Int. 1996, 29, 675−690. (22) Rauscher-Gabernig, E.; Gabernig, R.; Brueller, W.; Grossgut, R.; Bauer, F.; Paulsen, P. Dietary exposure assessment of putrescine and cadaverine and derivation of tolerable levels in selected foods consumed in Austria. Eur. Food Res. Technol. 2012, 235, 209−220. (23) Tasic, T.; Ikonic, P.; Mandic, A.; Jokanovic, M.; Tomovic, V.; Savatic, S.; Petrovic, L. Biogenic amines contents in traditional dry fermented sausage petrovska klobasa as possible indicator of good manufacturing practice. Food Control 2012, 23, 107−112. (24) Zhai, H. L.; Yang, X. Q.; Lai, H.; Li, L. H.; Xia, G. B.; Cen, J. W.; Hui, H. H.; Hao, S. X. Biogenic amines in commercial fish and fish products in southern China. Food Control 2012, 25, 303−308. (25) Til, H. P.; Falke, H. E.; Prinsen, M. K.; Willems, M. I. Acute and subacute toxicity of tyramine, spermidine, spermine, putrescine and cadaverine in rats. Food Chem. Toxicol. 1997, 35, 337−348. (26) Halásza, A.; Barátha, Á .; Simon-Sarkadib, L.; Holzapfel, W. Biogenic amines and their production by microorganisms in food. Trends Food Sci. Technol. 1994, 5, 42−49. (27) Wang, R. Z. Fremented Bean Curd in China (In Chinese); Chinese Light Industry Press: Beijing, 2009, pp 1−10.

realize the safety limit of each BA in the fermented soya products, like the histamines in fish products. Safety of Other Foods. The BAs have been detected in many foods, such as cheese, fish, fish products, dairy products, pork, and sausages, and so forth. From the related research, most of the BAs content were in a relatively safe range in pork and sausages;29−36 the histamines content was more than 100 mg/kg in some sausages,29,31 which could cause a safety hazard; putrescine and cadaverine could be not only used as the spoilage indicators of chilled pork, but also might reflect the spoilage degree in pork;34 some BAs content had reached the level of threat to human health in some fish products,37−41 and the highest content of tryptamine, β-phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine was 517.2,40 219.0,40 420,37 969.4,40 2500,37 456.2,40 51.98,41 and 53.3041 mg/kg respectively, which could pose a safety risk; almost all the BAs content had reached the level of threat to human health in cheese,42−46 the highest contents of tryptamine, β-phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine were 312.2,45 312.2,46 986.0,43 2127.6,43 1157.9,45 1771.3,43 340.0,46 and 449.646 mg/kg respectively, which were obviously dangerous factors. As above, the content of BAs has exceeded the safety limit in these foods, and there were very few scholars having evaluated the safety of them. So it must be emphasized that the foods which contain a large number of BAs should be detected to evaluate whether they are safe, and if not, the content of BAs should be limited. Toxicity of BAs. The toxicity of BAs was not discussed in the current study, but there still needs to be a more in-depth study to realize the toxicology of BAs deeply and comprehensively, such as the toxic effect of each BA, the safety range of each BA, and the synergy among them, and so forth.

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ASSOCIATED CONTENT

S Supporting Information *

Elution gradient of mobile phase for separation of derivatives of biogenic amines (Table S1); regression equations, correlation coefficients, linearity range, detection limits, and recovery of biogenic amines derivatives (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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