Residual nitrite and biogenic amines of traditional northeast sauerkraut in China

ABSTRACT

The safety of northeast sauerkraut (NS) has always been a concern in China. In the present study, the nitrite and biogenic amine (BA) contents in 378 NS, collected or purchased from Chinese families and markets (including morning markets and local supermarkets), were measured. The results showed that the nitrite and BA contents of samples from Chinese families and some morning markets were higher than those from local supermarkets. The highest level of nitrites was 38.5 ± 5.2 mg/kg in unpackaged NS from Chinese families, and the lowest was 17.2 ± 2.1 mg/kg in packaged NS from local supermarkets. The highest levels of tyramine and tryptamine were 203 ± 3 and 28.1 ± 4 mg/kg in unpackaged NS from Chinese families, respectively. The highest levels of histamine, putrescine, and cadaverine were mainly found in Chinese families and morning markets, and the lowest level of BAs was from local supermarkets. The results suggest that improving the safety of NS by controlling environmental sanitation and strengthening standardised production is a top priority at present in China.

KEYWORDS:

• Biogenic amines
• China
• Nitrite
• Northeast sauerkraut
• Safety assessment

Introduction

Northeast sauerkraut (NS) is a traditional pickled vegetable in northeast China that uses Chinese cabbage as a raw material. The method of fermenting NS in China has not changed for more than 3,000 years.^[1] The production process of traditional NS is as follows: first, fresh Chinese cabbage is washed with water, drained, and immersed in 3%–6% NaCl solution (w/v) in earthenware jars before being airproofed with a waterproof membrane. In the end, the jars with Chinese cabbage are stored at the fermentation temperature (20°C–25°C) for 30–40 days for maturation.^[2] The fermentation and processing of NS differ from those of another Chinese sauerkraut, Sichuang paocai.^[1]

Traditional sauerkraut is produced by spontaneous fermentation. Therefore, the quality of products is highly dependent on the lactic acid bacteria on the surface of the raw materials.^[3,4] These bacteria include Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus brevis, Lactobacillus sakei, and Lactobacillus reuteri. The product quality is also influenced by environmental factors, such as the concentration of salty water, fermentation time, and fermentation temperature. The fermentation of NS can be hampered due to bacterial contamination. It is difficult to ensure the safety of the final fermented products in a large-scale processing environment. High amounts of residues of harmful substances, such as nitrite^[5,6] and Bas,^[7,8] are common problems that arise during spontaneous fermentation.

Nitrite may react with amines and amides to produce N-nitroso compounds, which are related to increased risks of gastric, esophageal, nasopharyngeal, and bladder cancer.^[9–11] Nitrite is formed in NS mainly due to the transformation of nitrate. The concentration of nitrate is dependent on many factors, including the biological properties of the plant culture, culture conditions (light intensity, type of soil, temperature, and humidity), picking conditions (plant maturity, vegetation period, harvesting time, and vegetable size), and utilisation of nitrogen fertiliser during planting.^[12]

BAs are organic bases with aliphatic, aromatic, or heterocyclic structures that primarily emerge via the microbial decarboxylation of amino acids and can be found in a number of foodstuffs,^[13,14] especially during spontaneous fermentation.^[15] Many factors may alter the BA contents in fermented food, including temperature, pH, salt content, the microbial load, and the storage conditions.^[16,17] BAs may lead to some symptoms, such as nausea, discomfort, hot flashes, cold sweat, palpitations, headaches, red rash, and abnormally high or low blood pressure.^[9,10]

The World Health Organization (WHO) has listed pickled vegetables, such as cultured vegetables, sauerkraut, and kimchi, as possible carcinogens.^[18] For these reasons, the contents of nitrite and BAs in NS should be evaluated. In the present study, 278 samples from Chinese families, local supermarkets, and morning markets were collected or purchased, and the nitrite and BA contents in the samples were analysed.

Materials and methods

Samples

Sauerkraut samples were collected or purchased from Chinese families and markets in Harbin, China: 130 unpackaged samples were collected from 130 Chinese families; 118 packaged samples were purchased from 21 local supermarkets; and 130 samples were purchased from 10 morning markets. Among the last set of samples, 67 were unpackaged, and 63 were packaged. Plastic film was employed to seal the sauerkrauts using a vacuum, and packaged sauerkrauts were stored in refrigerated conditions. The unpackaged products remained unsealed and were exposed to air at room temperature. All collected or purchased samples prior to analysis were stored at a refrigerated temperature.

Nitrite content

The content of nitrite in samples was determined using the Griess reaction method.^[19] A Shimadzu UV2401 spectrophotometer (Shimadzu Inc., Tokyo, Japan) was used for analysis. First, 5 g of samples was crushed, deproteinated, and defatted by precipitation with 10 mL of 0.42 mol/L ZnSO₄ followed by filtration. Then, 1 mL of each of the three color development reagents, including 0.2% sulfanilamide, 0.1% N-1-naphtyethylene diamine dihydrochloride, and 44.5% HCl, was added sequentially to the filtrates. The mixtures were kept at room temperature for 5 min under dark conditions. The optical density (OD) value of the coloured mixtures was measured at 538 nm against the reagent blank. A standard curve in the range 0–2000 mg/L of NaNO₂ solutions was constructed and subjected to similar colour development and OD measurements. The above test was repeated three times. All reagents were analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

BA contents

The BA contents in the samples were determined according to the method reported by Frias et al.^[20] with some modifications. Briefly, 10 g of each sample was crushed, extracted with 15 mL of 0.1 mol/L HCl, and vortexed for 1 min. The resulting sample was centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected, and the remaining residue was re-extracted under the same conditions. Both extracts were combined and filtered through a qualitative filter paper (Whatman No. 1), and the volume was increased to 100 mL with 0.1 mol/L HCl. Subsequently, 1 mol/L NaOH was added to 1 mL of the diluted extract to adjust the pH to 6.7–7.0 for a better derivatisation condition, and 0.5 mL of o-Phthalaldehyde (OPA) (Sinopharm Chemical Reagent Co., Ltd, China) solution was added after 2 min. The mixture was kept in darkness for 2 min. The sample was then extracted twice with 1 mL of ethyl acetate. Both extracts were combined and filtered by 0.45 μm Millipore filter before HPLC analysis.

BA standards (histamine [him], tyramine [tym], tryptamine [trpm], putrescine [put], and cadaverine [cad]) were purchased from Sigma Chemical Co. (Shenyang, China). A stock standard aqueous solution of BAs was prepared by placing an accurately weighed amount of each standard (ca. 50 mg) in a 25 mL volumetric flask. Standards were processed and derivatised for samples, as described previously.

The quantification of BAs was performed by HPLC method, using an Alliance Separation Module 2695 (Waters, Milford, MA, USA), a Photodiode Array detector 2996 at 254 nm (Waters, Milford, USA) and a computer running the Empower 2 for Microsoft Windows chromatographic software (Waters). Then, 10 μL of each sample were injected into a Hypersil ODS2 column (4.6 mm × 250 mm, 5 μm). The column oven was set at 35°C. A mixture of 0.02 mol/L KH₂PO₄ buffer (pH 3.5) and methyl alcohol (30:70, v/v) was used as the mobile phase with a flow rate of 0.8 ml/min.

Statistical analysis

The SPSS software 19.0 was used to perform all statistical analyses. All data were expressed as the means ± SD (mean of at least three determinations for each sample, n = number of samples). A value of P < 0.05 was used to indicate significant differences.

Results and discussion

Nitrite content in sauerkrauts

In China, the maximum nitrite content in pickled vegetables is 20 mg/kg. ^[21] In this study, the nitrite content of 313 samples (83%) was detected to be less than 20 mg/kg among 378 samples from three different sources (Table 1). The number of samples with a nitrite content below 20 mg/kg was 113 (96%), 95 (73%), and 106 (812%) for local supermarkets, Chinese families, and morning markets, respectively. The number of samples with a nitrite content above 20 mg/kg was 5 (4%), 35 (27%), and 24 (18%) for local supermarkets, Chinese families, and morning markets, respectively. The average nitrite contents found in samples from local supermarkets, Chinese families, and morning markets were 17.2 ± 2.1, 38.5 ± 5.2, and 25.6 ± 3.4 mg/kg and ranged from 5.8 to 32.4, 10.8 to 80.1, and 5.6 to 88.1 mg/kg, respectively.

Table 1. The content of nitrite in northeast sauerkrauts from three different sources.

Different source of samples		Number (n)	Nitrite content (mg/kg)
			≤20	>20	Mean ± SD	Min	Max
Local supermarkets	packaged	118	113	5	17.2 ± 2.1^a	5.8	32.4
Chinese families	unpackdged	130	95	35	38.5 ± 5.2^b	10.8	80.1
Morning markets	packaged	63	55	8	18.2 ± 2.2	6.2	78.4
	unpackaged	67	51	16	33.2 ± 5.6	5.6	88.1
	Total*	130	106	24	25.6 ± 3.4^c	5.6	88.1

*, the total content of nitrite of samples from morning markets, including packaged and unpackaged sauerkrauts.

a–c, lowercase letters indicate a significant difference among samples in the three sources (P < 0.05).

The accumulation of nitrite is a common problem for vegetable fermentation.^[22] There are remarkable differences in the nitrite contents of NS from different sources. The nitrite content of NS from Chinese families was higher than those from local supermarkets and morning markets. The possible reason for this is that the fermentation temperature and NaCl concentration of NS from each Chinese family are different and that the uniformity of NS processing is difficult to control. NS processing involves several steps, such as washing, peeling, and low-temperature blanching, which may affect the final content of nitrite. Leszczynska et al.^[23] also found higher nitrate contents of NS from Chinese families. During the spontaneous fermentation of NS, some gram-negative bacteria (such as Enterobacteria) are nitrate-reducing bacteria that exist predominantly at the initial stage of fermentation, and these bacteria may be overproduced^[5] due to poor processing conditions, such as low salt concentration. Poor processing conditions may lead the nitrate to change into nitrite. In general, the nitrite content in fresh leafy vegetables is less than 2 mg/kg under proper storage conditions.^[24,25] The nitrate content in plant tissue was excessive due to the abuse of nitrogen fertiliser when cultivation was carried out in China. During fermentation, a massive amount of nitrate was converted to nitrite, especially when the fermentation conditions were poor. Therefore, the nitrite content of samples from Chinese families and morning markets may have been higher due to the poor processing conditions. The fermentation time of traditional NS was approximately 30–35 d at 20°C–25°C. The peak value of nitrite content in NS appears between 5 and 7 d and then decreases. During fermentation, lactic acid bacteria from NS may degrade the nitrite concentration.^[26] However, adverse post-harvest storage conditions could result in the growth of harmful bacteria, which could contribute to the increasing accumulation of high nitrite content as a result of bacterial contamination and endogenous nitrate reductase action.^[25,27] This result will also cause NS quality to be unstable, even in the presence of lactic acid bacteria.

Biogenic amine contents in NS

In general, BAs can be found in various foods and beverages, such as seafood, meat products, dairy products, vegetables, fruits, nuts, chocolate, wine, and beer.^[28] The production of BAs in food depends on the presence of precursors (i.e., amino acids) and microorganisms that possess decarboxylation activity. Gerbaux et al.^[29] reported that pH is one of the most important enological factors influencing the production of BAs, particularly him, tym, and put. Nevertheless, in the present study, no significant correlation was found between pH and the amount of any of the amines detected. It is possible that the pH values of NS, which ranged from 3.45 to 3.56, changed in a similar manner. In China, the BA contents in pickled vegetables have no official limitations, whereas him is the only biogenic amine that is officially limited in fish products; it is regulated to be below 50 mg/kg by the US Food and Drug Administration^[30] and below 100 mg/kg by the European Community.^[31] BAs are ubiquitous constituents of foods.^[32] The determination of BAs has received extensive attention due to their disadvantageous effects on human beings.^[9] In the present study, the range of BA contents in samples from different sources was 22.9–31.6 mg/kg for him, 88.9–206.3 mg/kg for tym, 10.4–31.6 mg/kg for trpm, 95.4–162.4 mg/kg for put, and 37.6–79.5 mg/kg for cad. There is a remarkable difference in BA content among the three different sources (Table 2). For him, put, and cad, the BA contents of samples were not different between Chinese families and morning markets but were higher than those from local supermarkets. For tym and trpm, the BA contents of samples from Chinese families were higher than those from the other two sources. Among 278 samples, the BAs contents from highest to lowest were tym, put, cad, him, and trpm. Špička et al.^[33] also indicated that there were higher levels of put, cad, and tym in sauerkraut compared to the remaining biogenic amines. However, there are no legal upper limits for BAs in sauerkraut. Kunsch et al.^[34] recommended maximum values of 10, 20, 50, 25, and 5 mg/kg for him, tym, put, cad, and 2-phenylethylamine, respectively, for good-quality sauerkraut, whereas Nout^[35] proposed 50–100, 100–800, <30, and 100–200 mg/kg as acceptable ranges for him, tym, 2-phenylethylamine, and total biogenic amines (TBA), respectively, in fermented vegetables.

Table 2. The content of biogenic amines in northeast sauerkrauts from three different sources.

			Biogenic amine content (mg/kg)
Different source of samples		Number (n)	his	tym	trpm	put	cad
Local supermarkets	packaged	118	25.0 ± 2.1^a	91.6 ± 2.7^a	12.5 ± 2.1^a	98.3 ± 2.9^a	41.1 ± 3.5^a
Chinese families	unpackdged	130	29.1 ± 2.5^b	203.7 ± 2.6^b	28.1 ± 3.5^b	128.3 ± 2.3^b	67.6 ± 2.3^b
Morning markets	packaged	63	26.1 ± 2.7	99.9 ± 2.0	16.3 ± 2.1	100.6 ± 3.5	45.9 ± 3.9
	unpackaged	67	32.6 ± 2.9	107.9 ± 2.3	27.3 ± 3.8	159.6 ± 2.8	76.9 ± 2.6
	Total	130	29.4 ± 2.8^b	103.9 ± 2.2^c	21.8 ± 2.9^c	130.1 ± 3.2^b	61.4 ± 3.3^b

a–c, lowercase letters indicate a significant difference among samples in the three sources (P < 0.05).

NS is traditionally made at home, and the sanitary conditions, fermented way, salt concentration, and fermentation temperature are different for each Chinese family. Therefore, it is difficult to control the process conditions of NS. The results of Mehta et al.^[36] showed that the growth and metabolic activities of lactic acid bacteria under controlled processing conditions during fermentation can improve the quality and safety of traditionally fermented vegetables. Vegetable-fermenting lactic acid bacteria are generally non-toxic and nonpathogenic. However, some LAB strains, such as Lactobacillus buchneri, Lactobacillus plantarum, Lactobacillus curvatus, and Lactobacillus bavaricus, exhibit BA production.^[4,37] Unfavourable conditions may result in spoilage bacteria and change the microflora in traditional fermented vegetable products. The difference in microflora appearing in fermented vegetable products from different regions was probably related to chemical and physical factors, including the substrates, NaCl concentration, and fermentation temperature.^[38] Therefore, the viewpoint that microbiological spoilage cannot occur in salted products is incorrect because BA accumulation can occur before salting.^[39] Therefore, BAs have not been reported in kimchi, and kimchi is considered to have low levels of BAs because its major ingredients (i.e., cabbage, radish, garlic, ginger, and red pepper powder) do not contain high levels of biogenic amine precursors (i.e., amino acids and microorganisms that possess decarboxylation activity).^[40]

The contents of nitrite and BAs in packaged and unpackaged NS were determined in this study (Figs. 1 and 2). The contents of nitrite and total BAs of the unpackaged samples from Chinese families and some morning markets were significantly higher than those of the packaged products (P < 0.05). This may be attributed to differences in the storage conditions. Chung et al.^[25] also indicated that poor storage could result in bacterial growth, which can, in turn, contribute to the increasing nitrite levels. The nitrite and total BA contents differed between the packaged and unpackaged products, which may also be attributed to the fact that the ‘storage’ conditions were not the same. Because the packaged NSs were mainly kept under refrigerated storage, the nitrite and BA accumulation tended to be inhibited.^[25] The unpackaged NSs were mainly family-produced and were exposed to air in the production process; therefore, the processing conditions cannot be strictly controlled, and the pickling time remains insufficient.^[41] Exposure to aerobic bacteria might allow the continuous conversion of nitrate to nitrite.^[5] NS from morning markets may be processed in two different ways: one way involves industrial production, and the other way resembles the same process employed by Chinese families. Therefore, the nitrite and BA contents of samples were higher than those from Chinese families. The vast majority of unpackaged vegetables did not have a clear pickling time or experience a complete pickling rhythm.^[2]

Figure 1. The average nitrite contents of packaged and unpackaged sauerkraut from the northeast region of China.

Figure 2. The total BA contents of packaged and unpackaged sauerkraut from the northeast region of China.

Conclusions

The nitrite and BA contents of 278 NS samples from the northeast region of China were studied. The results of the safety assessment of NS were very poor, especially those made in household and small-scale processes. At the same time, unsuitable packaging affected the nitrite and BA contents in NS. The industrial production of NS is increasing along with the increasing demand for commercialisation due to public health requirements, especially in regions where sauerkraut is eaten frequently as part of a daily diet. Therefore, it is reasonable to recommend that manufacturers establish an effective quality control system to improve the safety of NS by decreasing the nitrite and BA contents of sauerkraut when planting the raw materials and processing the final product.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31301519, 31671874) and Heilongjiang Postdoctoral Grant (LBH-Q13029).