Biogenic amines in Chinese soy sauce

Food Control 20 (2009) 593–597

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Biogenic amines in Chinese soy sauce Lu Yongmei a, Chen Xiaohong a, Jiang Mei a, Lv Xin a, Nurgul Rahman b, Dong Mingsheng a,*, Gujun Yan c a b c

College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, PR China College of Life Science and Chemistry, Xinjiang Normal University, Wulumuqi 830053, Xinjiang, PR China School of Plant Biology (MO84), Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, Perth, WA 6009, Australia

a r t i c l e

i n f o

Article history: Received 5 May 2008 Received in revised form 20 August 2008 Accepted 26 August 2008

Keywords: Biogenic amine Chinese soy sauce Amino nitrogen

a b s t r a c t The biogenic amine contents in 40 samples of Chinese soy sauce from different manufactures were determined using HPLC after pre-column derivatization with dansyl chloride. Also, the amino nitrogen levels and pH values of these samples were analyzed. Of the five biogenic amines under study, tyramine was the most prevailing amine and was detected in 97.5% of the samples, followed by spermidine (95%), histamine (92.5%), cadaverine (82.5%) and spermine (80%). The total content for the five biogenic amines in these samples was 497 mg/l with a range from 41.7 to 1357 mg/l. The pH and amino nitrogen levels in soy sauce samples were between 3.86 and 4.98 and between 0.25 and 1.26 mg/100 g, respectively. In general, total content for the five biogenic amines was high in samples that had high amino nitrogen levels. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Biogenic amines are low molecular weight organic bases that possess biological activity. High concentrations have been found in fermented foods as a result of a contaminating microflora exhibiting amino acid decarboxylase activity (Silla-Santos, 1996). The presence of biogenic amines in food constitutes a potential public health concern due to their physiological and toxicological effects. Furthermore, some biogenic amines can react with nitrite to form carcinogenic nitrosamines (Warthesen, Scanlan, Bills, & Libbey, 1975). Biogenic amines do not usually represent any health hazard to individuals unless large amounts are ingested or the natural mechanism for the catabolism of the amines is inhibited or genetically deficient. High amounts of certain amines may be found in food as a consequence of the use of poor quality raw materials, contamination and inappropriate conditions during food processing and storage (Brink, Damink, Joosten, & Huis in´t Veld, 1990; Halász, Baráth, Simon-Sarkadi, et al., 1994). As the microbial spoilage of food may be accompanied by the increased production of decarboxylases, the presence of biogenic amines might serve as a useful indicator of food spoilage. For these reasons, it is important to monitor biogenic amines levels in food. Soy sauce, a Chinese traditional fermented condiment, is made from soy bean and wheat flour. During the manufacturing process of soy sauce, soy sauce koji is traditionally prepared by growing the koji mold such as Aspergillus oryzae or Aspergillus sojae on the raw material containing a mixture of steam-cooked defatted soybean

* Corresponding author. Fax: +86 25 84399090. E-mail address: [email protected] (D. Mingsheng). 0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2008.08.020

and roasted wheat flour. Soy sauce mash obtained by mixing the finished koji with brine solution is then subjected to various periods of aging (Whitaker, 1978). During the fermentation of soy sauce, proteins in the raw materials are hydrolyzed into small molecular weight peptides, amino acids and ammonia by the proteases produced by A. oryzae or A. sojae (Whitaker, 1978). During the fermentation and ageing, the flavor may develop gradually. Meanwhile, soy sauce contains relatively high amounts of free amino acid, which could be potentially the sources of biogenic amines. The objective of this paper is searching for the existence of a relationship between biogenic amine concentrations in Chinese soy sauces and the kinds of fermentation and also the levels of amino nitrogen contents.

2. Materials and methods 2.1. Samples and reagents Forty soy sauce samples covered 15 different brands from three basic soy sauce making regions of China (Shanghai, Jiangsu and Guangdong province) were purchased from local supermarkets. Twenty-four samples were brewed by high salt liquid state fermentation (HLF) and 16 samples were brewed by low salt solid state fermentation (LSF). The samples were carried to the laboratory and stored in a refrigerator at 20 °C until analysis. Biogenic amines standards (cadaverine, histamine, tyramine, spermidine and spermine) and dansyl chloride (Dns-Cl) were from Sigma Chemical Co.; acetonitrile and acetone for HPLC from Merck; the other reagents used in this study were HPLC-grade.

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2.2. Biogenic amines determination Biogenic amine contents of soy sauce samples were determined according to the methods previously described by us (Lu et al., 2007). Briefly, Biogenic amines were extracted from 5 ml samples with 0.4 M perchloric acid and were pipetted into a screw-capped vial. To the vial, 0.2 ml of 2 M NaOH solution, 0.3 ml of saturated NaHCO3 solution and 1.0 ml of dansyl chloride solution (10 mg/ ml) were added and the mixture was stirred in dark at 40 °C for 0.5 h. Dansyl chloride was then precipitated with ammonia. The supernatant was placed in a volumetric flask and the volume was made up to 50 ml with acetonitrile. The complete extract was then filtered through a Millipore filter (pore size 0.45 lm) before HPLC analysis. The gradient elution system was a mixture of acetonitrile (A) and water was used (B). The gradient elution procedure was 35% A + 65% B at 1 min, 20% A + 80% B at 5 min, 10% A + 90% B at 6 min and 8% A + 92% B at 16 min. The biogenic amine contents in soy sauces were carried out by Agilent 1100 HPLC system, using C18 column, 7 lm, 4.6 cm  150 mm (E. Merck, Damstadt, Germany), with diode-array detector (set at 254 nm). A series

of diluted standard solutions containing cadaverine, histamine, tyramine, spermidine and spermine (in 0.4 M perchloric acid) were prepared from the standard stock solutions and used to obtain the standard curve for each biogenic amine. 2.3. Determination of pH and amino nitrogen The pH of the samples was determined by directly using a digital pH meter (HM-5 S; TOA Electric Industrial Co. Ltd., Tokyo, Japan). Amino acid nitrogen was determined by titration method (Cai & Yuan, 1982). Briefly, diluted samples (20 ml) were mixed with 60 ml distilled water and titrated to pH 9.6 with 0.1 N NaOH, before 20 ml formalin solution (40%) was added. The volume of consumed NaOH was recorded in order to determine amino nitrogen content. 2.4. Statistical analysis Statistical Package for Social Sciences (SPSS) version 11.5 for Microsoft Windows was used to perform all statistical analyses.

Table 1 Biogenic amine contents (mg/l) and amino nitrogen levels (g/100 ml) and pH values in tested soy sauces Samplesa

Cadaverineb

Histamine

Tyramine

Spermidine

Spermine

Total contentc

Amino nitrogen contentsd

pH

Brewing processd

Shanghai 01LC 02LC 03LC 04LC 05LC 01TTL 02TTL 01TD 02TD 01QWL

nde nd 138 ± 0.26 nd 14.1 ± 0.23 23.1 ± 0.19 5.66 ± 0.43 38.1 ± 0.33 30.0 ± 0.32 46.1 ± 0.53

nd nd 13.1 ± 0.45 32.6 ± 0.35 65.5 ± 0.21 36.7 ± 0.13 33.2 ± 0.23 136 ± 0.54 371 ± 0.04 19.9 ± 0.42

591 ± 0.13 262 ± 0.19 10.9 ± 0.25 386 ± 0.33 8.4 ± 0.11 16.2 ± 0.17 20.3 ± 0.15 329 ± 0.09 673 ± 028 61.4 ± 0.22

18.8 ± 0.33 10.1 ± 0.23 nd 11.4 ± 0.43 152 ± 0.25 25.5 ± 0.06 22.9 ± 0.27 19.5 ± 0.45 46.5 ± 0.24 22.4 ± 0.33

39.7 ± 0.17 57.8 ± 0.18 8.87 ± 0.51 127 ± 0.31 7.5 ± 0.45 54.2 ± 0.53 47.0 ± 0.45 25.2 ± 0.32 16.3 ± 0.14 145 ± 0.05

649 330 171 557 248 156 129 548 1136 295

0.64 0.38 0.40 0.43 0.43 0.39 0.38 0.62 0.76 0.40

4.33 4.28 4.01 4.24 4.00 4.63 4.69 4.31 4.32 4.47

LSF LSF LSF LSF LSF LSF LSF HLF HLF LSF

Jiangsu 01JL 02JL 03JL 04JL 05JL 06JL 07JL 08JL 01TY 02TY 03TY 01SH 01YH 01THY 01HS

181 ± 0.42 70.2 ± 0.12 42.8 ± 0.39 35.6 ± 0.38 7.3 ± 0.42 55.3 ± 0.17 85.3 ± 0.35 20 ± 0.11 11.4 ± 0.28 5.7 ± 0.12 550 ± 0.14 50.2 ± 0.24 59.2 ± 0.50 11.4 ± 0.23 nd

278 ± 0.43 279 ± 0.27 85.9 ± 0.29 6.7 ± 0.32 20.9 ± 0.05 13 ± 0.35 51.6 ± 0.29 340 ± 0.13 29.8 ± 0.13 14.3 ± 0.30 143 ± 0.14 86.4 ± 0.35 42.1 ± 0.56 77.4 ± 0.40 24.4 ± 0.26

20.8 ± 0.12 274 ± 0.36 161 ± 0.17 61.1 ± 0.27 55.2 ± 0.38 90.4 ± 0.14 48.2 ± 0.32 21.9 ± 0.03 18.9 ± 0.171 14.9 ± 0.08 20.3 ± 0.23 521 ± 0.46 469 ± 0.25 324 ± 0.36 80.8 ± 0.03

197 ± 0.32 24.2 ± 0.32 14.2 ± 0.43 20.7 ± 0.28 7.1 ± 0.28 14.5 ± 0.24 11.8 ± 0.36 15.3 ± 0.25 73.8 ± 0.35 33.7 ± 0.43 235 ± 0.50 24.7 ± 0.34 32.4 ± 0.55 30.1 ± 0.53 8.5 ± 0.47

20.4 ± 0.12 21.5 ± 0.45 22.2 ± 0.35 61.6 ± 0.39 21.2 ± 0.29 5.8 ± 0.27 7.0 ± 0.35 19.9 ± 0.25 9.3 ± 0.14 6.2 ± 0.24 43.1 ± 0.25 25.0 ± 0.14 19.8 ± 0.14 20.7 ± 0.13 6.8 ± 0.45

697 669 326 186 112 179 204 417 143 74.8 993 708 623 464 121

0.65 0.71 0.60 0.37 0.36 0.44 0.25 0.65 0.73 0.61 0.61 0.58 0.45 0.4 0.38

4.40 4.43 4.50 4.49 4.49 4.34 4.35 4.28 4.51 4.47 4.62 4.58 4.43 4.54 4.39

HLF HLF LSF LSF LSF LSF HLF HLF HLF HLF HLF LSF LSF LSF LSF

Guangdong 01LJJ 02LJJ 03LJJ 01HT 02HT 03HT 04HT 05HT 06HT 07HT 08HT 09HT 01GZH 01MJ 01XH

287 ± 0.14 nd nd 53.5 ± 0.34 118 ± 0.14 87.1 ± 0.42 18.4 ± 0.24 73.8±0.24 197 ± 0.43 136 ± 0.49 13.9 ± 0.53 35.9 ± 0.36 nd 252 ± 0.53 332 ± 0.45

259 ± 0.35 nd 295 ± 0.24 546 ± 0.22 592 ± 0.43 230 ± 0.15 181 ± 0.53 129 ± 0.32 88.7 ± 0.14 64.4 ± 0.28 4.5 ± 0.24 155 ± 0.28 81.9 ± 0.07 432 ± 0.30 418 ± 0.13

47.6 ± 0.04 35.6 ± 0.32 35.9 ± 0.24 201 ± 0.20 522 ± 0.10 245 ± 0.16 19.7 ± 0.42 197 ± 0.35 nd 143 ± 0.25 26.6 ± 0.07 20.1 ± 0.50 478 ± 0.06 365 ± 0.24 256 ± 0.43

nd 6.1 ± 0.22 7.8 ± 0.11 21.8 ± 0.24 21.6 ± 0.34 33.9 ± 0.49 407 ± 0.37 10 ± 0.46 486 ± 0.07 98.2 ± 0.25 5.1 ± 0.25 180 ± 0.38 11.1 ± 0.04 212 ± 0.34 242 ± 0.24

nd nd nd nd nd nd nd nd 48.2 ± 0.05 31.1 ± 0.42 49 ± 0.25 38.7 ± 0.26 25.3 ± 0.02 85.7 ± 0.40 109 ± 0.43

595 41.7 339 822 1253 596 626 409 820 473 99 431 597 1346 1358

0.61 0.46 0.43 1.26 1.21 0.97 0.85 1.00 1.18 0.4 0.48 0.39 1.03 0.72 1.07

4.79 4.81 4.80 4.82 4.84

HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF HLF

a b c d e

Letters in the latter signify brands of soy sauce of each region. Numbers in the former signify different samples. Values represent the average of duplicates (n = 3) ± standard deviation. Total contents of the five biogenic amines including histamine, tyramine, cadaverine, spermidine and spermine. HLF, high salt liquid state fermentation and LSF, low salt solid state fermentation. nd, not detected or below detection limits; mean values were calculated by using zero for nd.

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All data were expressed as means ± SD (mean of at least three determinations for each sample, n = number of samples). 3. Results and discussion Biogenic amine contents in 40 soy sauce samples are shown in Table 1. The result showed that each kind of soy sauce contained at least two biogenic amines and the content of biogenic amines was quite different in the tested samples. Tyramine was detected in 39 of 40 samples (97.5% of the soy sauces). Spermidine was detected in 95% of the soy sauces, while histamine, cadaverine and spermine occurred in 92.5%, 82.5% and 80% of the soy sauces examined, respectively. The total content for the five biogenic amines in these samples was 497 mg/l with a range from 41.7 to 1357 mg/l. The levels for each of the five amines were 0–673 mg/l for tyramine, 0–592 mg/l for histamine, 0–550 mg/l for cadaverine, 0–486 mg/l for spermidine and 0–145 mg/l for spermine, respectively. In agreement with the publications of Mower and Bhagavan (1989) and Stute, Petridis, Steinhart, and Biernoth (2002), tyramine was the dominating biogenic amine in soy sauces, which is formed at the beginning of the fermentation mainly by Enterococcus faecum (Stute et al., 2002). Amino nitrogen content is an important index for classifying the quality grades of soy sauce in China. Amino nitrogen content of Chinese soy sauces was determined to be between 0.25 and 1.26 (Table 1). Based on our data, the 40 soy sauce samples under study were divided into three groups (Fig. 1): low contents (<0.50 g/ 100 ml, n = 19), moderate contents (0.50–0.9 g/100 ml, n = 9) and high contents (>0.70 g/100 ml, n = 12), which intervals of amino nitrogen content are correlated with quality grades (good, very good and excellent) of Chinese soy sauces. As shown in Fig. 1, total five biogenic amine content was generally high in samples that had high amino nitrogen level, while soy sauce samples containing low amino nitrogen level had low total biogenic amine contents. Availability of free amino acids is one of prerequisites for biogenic amine formation by microorganisms, although not always leading

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to amine production (Marklinder & Lönner, 1992). In view of this fact, we suggest that there is a possible relationship between biogenic amine content and amino nitrogen level. Fig. 2 shows that the average amounts of cadaverine, histamine, spermidine and total content of the five biogenic amines in 24 samples by HLF process was much higher than that of 16 samples by LSF process, which was probably due to the difference in manufacturing technique. HLF process is carried out by an indigenous fermentation procedure and having long aging period, while LSF process by fermentation using pure cultures and having very short aging. Similar result had reported by Nout, Ruikes, Bouwmeester, and Beljaars (1993), who showed that tyramine, putrescine and cadaverine concentrations in tempeh were low or high depending on the applied manufacturing process, including soaked soybeans, type of fermentative microorganisms, boiling, home cooking by stewing or frying in oil and storage temperature. Biogenic amines in foods were extensively studied; however, the determination of the exact toxicity threshold of biogenic amines in individuals is extremely difficult, since the toxic dose is strongly dependent on the efficiency of the detoxification mechanisms of each individual and the presence of other amines (Halász et al., 1994). Nout (1994) suggested that an intake of over 40 mg biogenic amines per meal has been considered potentially toxic. Askar and Treptow (1993) indicated that histamine at a concentration of 500 mg/kg in food to be hazardous for human health. On the other hand, Brink et al. (1990) reported that 100–800 mg/kg of tyramine in foods are toxic; while Silla-Santos (1996) suggested that more than 1000 mg/kg (total amines in food) was dangerous for health. As shown in Table 2, the concentrations of histamine in two of Chinese soy sauces tested were higher than values considered as dangerous for health. The histamine, tyramine and total amine contents in sample of Guangdong 02HT were simultaneously high (Table 1). Some biogenic amines, e.g. histamine and tyramine, are considered as anti-nutritional compounds. For sensitive individuals they represent a health risk. Poisoning by histamine with its allergy-like

Fig. 1. Biogenic amine contents (mg/l) versus amino acid nitrogen levels (g/100 ml) in soy sauces. Amino nitrogen levels (g/100 ml): low (<0.5, n = 19), moderate (0.5–0.7, n = 9) and high (>0.7, n = 12).

L. Yongmei et al. / Food Control 20 (2009) 593–597

Biogenic amine contents (mg/L)

596

LSF

HL F Brewing Process

Fig. 2. Biogenic amine contents (mg/l) in soy sauces produced by different brewing process. HLF, high salt liquid state fermentation (n = 24) and LSF, low salt solid state fermentation (n = 16).

Table 2 Distribution of histamine, tyramine and total biogenic amine contents (mg/l) in the 40 tested soy sauces Biogenic amine contents (mg/l)

<49 50–249 250–499 500–999 >1000

Number of soy sauce samples Histamine

Tyramine

Total

16 13 9 2 0

17 10 9 4 0

1 12 9 14 4

symptoms (expressed as headaches, respiratory distress, heart palpitation, hypertension or hypotension and several allergy-related disorders) is usually related to the consumption of scombroid fish such as tuna or mackerel (Nogue Veciana, Marine Font, & Vidal Carou, 1997; Wu, Yang, Yang, Ger, & Deng, 1997). Chinese restaurant syndrome is a combination of symptoms that include feelings of burning, flushing, tingling and tightness – symptoms that some people experience after eating a meal from a Chinese restaurant. Our results suggest that the high level of biogenic amines in soy sauce may hasten the Chinese restaurant syndrome at a certain extent. People suffer the symptoms may be resulted by intake of toxic level of biogenic amines per meal, especially when people eating a meal with Kraut, Chinese rice wines, sausages or other fermented foods. More carefully designed studies are needed in order to identify all the causes of this syndrome. 4. Conclusion As a result, tyramine, histamine and spermidine are the most important biogenic amines for Chinese soy sauces. However, there

were significant variations in biogenic amine contents of soy sauce. Variation in biogenic amine levels of could be due to the manufacturing techniques and the type of fermentation. Generally, total five amine content was high in samples that had high amino nitrogen levels. Acknowledgement This work was supported by Hi-Tech R&D program of China, Project No. 2006AA10Z343.

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