Investigation of the Biogenic Amine Content in Jiangxi-made Sufu and Isolation and Identification of Amine-producing Bacteria
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摘要: 采用高效液相色谱法对江西产腐乳生物胺含量水平进行调查分析，通过微生物学分离结合高效液相色谱确认，筛选出生物胺产生菌，并对其进行鉴定。结果表明，12种腐乳样品中总生物胺含量范围为68.5～1084.0 mg/kg，色胺、腐胺、组胺和酪胺是主要生物胺，其中腐胺和酪胺的含量较高。筛选出的9株产胺菌产生物胺的含量范围为93.8～369.2 mg/L。对产胺量较高的6株菌进行表型鉴定和基因型16S rDNA序列测定，得到耐热芽孢杆菌（Bacillus sporothermodurans）1株，鲁梅利杆菌（Rummeliibacillus stabekisii）1株，耐硼赖氨酸芽孢杆菌（Lysinacillus boronitolerans）3株和皮脂葡萄球菌（Staphylococcus piscifermentans）1株。研究结果对腐乳食用安全性评价提供了理论依据，为进一步探清如何抑制腐乳中产胺菌的活性提供了理论研究基础。Abstract: The biogenic amine Level in Jiangxi-made sufu was analyzed by high performance liquid chromatography (HPLC) and amine-producing bacteria were isolated and identified by microbiological methods combined with HPLC confirmation. The results showed that total biogenic amine level ranged from 68.5 to 1084.0 mg/kg in sufu samples. Major biogenic amines were tryptamine, putrescine, histamine and tyramine, among which putrescine and tyramine were of the highest level. Biogenic amine level produced by 9 amine-producing bacteria ranged from 93.8 to 369.2 mg/L. For six bacteria with relatively high amine productivity, phenotype and genotype 16S rDNA sequence determination had found one Bacillus sporothermodurans strain, one Rummeliibacillus stabekisii strain, three Lysinacillus boronitolerans strains and one Staphylococcus piscifermentans strain. The current research offered theoretical evidence for edible safety evaluation of sufu and served as research basis to investigate how to inhibit activity of amine-producing bacteria in sufu.
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Key words:
- sufu /
- biogenic amine /
- amine-producing bacteria /
- isolation /
- identification
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表 1 样品信息
Table 1. Sample information
编号 名称 产地 配料 1 豆腐乳 江西南昌 黄豆、辣椒、植物油、味精、食用盐、白酒、白砂糖、香辛料、食品添加剂：硫酸钠、脱氢乙酸钠 2A 茶油腐乳香辣味 江西吉安 水、黄豆、茶油、食用植物油、食用盐、辣椒粉、香辛料 3A 农家腐乳猛辣型 江西吉安 水、黄豆、植物油、食用盐、辣椒粉、香辛料 4B 红油腐乳香辣味 江西吉安 水、大豆、菜籽油、食用盐、白酒、辣椒、八角、食品添加剂：谷氨酸钠 5B 白腐乳原味 江西吉安 水、大豆、食用盐、白酒、食品添加剂：脱氢乙酸钠 6C 香辣腐乳 江西南昌 黄豆、植物油、辣椒、食盐、酒、香辛料 7C 茶油腐乳 江西南昌 黄豆、菜籽精炼油、山茶油、辣椒、食盐、酒、香辛料 8D 家乡豆腐乳 江西南昌 黄豆、水、食用大豆油、麻油、食用碘盐、辣椒、白酒、生姜、味精、香辛料 9D 豆腐乳家乡味 江西南昌 黄豆、水、食用大豆油、食用碘盐、辣椒、白酒、生姜、味精、香辛料 10 山茶油腐乳 江西吉安 黄豆、饮用水、山茶油、辣椒、食用盐、白酒、香辛料 11E 红油腐乳王 江西吉安 水、大豆、菜籽油、食用盐、辣椒、白酒、香辛料、味精 12E 茶油腐乳香辣味 江西吉安 水、大豆、菜籽油、山茶油、食用盐、辣椒、白酒、香辛料、味精 注：编号中的相同大写字母表示同一品牌不同系列样品；表4~表7同。 
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表 2 梯度洗脱程序
Table 2. Gradient elution procedures
时间（min） 0 22 25 32 32.01 34 流动相A（%） 58 78 90 90 58 58 流动相B（%） 42 22 10 10 42 42
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表 3 生物胺的保留时间、回归方程和相关系数
Table 3. Retention time, the regression equations and correlation coefficients of biogenic amines
生物胺 保留时间（min） 回归方程 相关系数 色胺 10.722 Y=0.5257x+0.0016 0.9999 苯乙胺 13.057 Y=0.6156x+0.0063 0.9999 腐胺 14.176 Y=1.7395x+0.0107 0.9999 尸胺 15.569 Y=1.3790x+0.0003 0.9999 组胺 16.369 Y=0.7710x+0.0186 0.9990 章鱼胺 17.840 Y=0.7449x+0.0158 0.9998 酪胺 23.545 Y=1.0711x−0.0127 0.9998 亚精胺 25.448 Y=1.4346x+0.0008 0.9999 精胺 30.937 Y=1.3006x−0.0058 0.9999
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表 4 腐乳样品中生物胺含量 （mg/kg）
Table 4. Contents of biogenic amines in sufu samples (mg/kg)
编号 色胺 苯乙胺 腐胺 尸胺 组胺 章鱼胺 酪胺 亚精胺 精胺 总生物胺 1 66.7±1.7g 15.1±1.1ef 181.8±4.7i 14.6±0.8d 99.8±3.7a ND 208.4±3.7f ND ND 586.8±10.0f 2A 160.2±1.3a 42.3±1.7b 370.2±10.4d 5.7±0.7e 78.0±1.2d ND 431.7±11.7a ND ND 1084.0±22.8a 3A 89.3±2.9f 23.5±1.4d 411.5±8.4b 7.6±1.3e 71.1±1.6e ND 414.4±4.2b ND ND 1013.6±8.4b 4B 152.3±2.1b 35.8±3.4c 326.7±9.8f 5.1±0.6e 82.9±2.9c ND 192.5±3.2g ND ND 797.1±20.9d 5B 110.6±4.6d 13.7±1.9f 353.8±9.3e 18.5±1.7c 85.4±2.5c ND 385.7±6.9c ND ND 967.9±11.3c 6C ND ND 33.4±2.5j 24.8±2.2b ND ND 10.3±0.9k ND ND 68.5±5.6i 7C ND 6.4±0.9g 446.4±12.1a 41.4±2.5a 37.5±1.7g ND 50.2±2.0i ND 12.9±1.5 595.9±13.0f 8D 105.1±3.5e 25.3±2.2d 316.9±6.8f 6.0±0.6e 37.6±1.5g ND 343.4±9.7d ND ND 824.5±17.2d 9D 59.1±4.3h 18.0±2.3e 279.0±13.5g 7.2±1.4e 43.7±1.7f ND 328.3±8.4e ND ND 735.6±14.8e 10 ND ND 12.5±1.4k ND ND ND 119.4±2.3h ND ND 131.7±4.4h 11E 118.2±4.8c 45.8±2.7a 220.1±9.8h 40.2±2.1a 90.5±2.8b ND 26.7±1.9j ND ND 534.4±20.3g 12E 84.8±4.4f 23.2±1.1d 387.1±8.0c 14.8±1.7d 94.5±4.5b ND 421.7±21.8ab ND ND 1022.5±35.7b 注：ND表示未检出；同列的不同小写字母表示有显著性差异，P<0.05；表5同。
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表 5 菌株的生物胺生成情况 （mg/L）
Table 5. Biogenic amine contents produced by strains (mg/L)
编号 色胺 苯乙胺 腐胺 尸胺 组胺 章鱼胺 酪胺 亚精胺 精胺 总生物胺 F1-3A 3.8±0.6c ND 17.8±0.8d ND ND ND ND ND ND 21.5±0.6f F2-2A ND 80.1±2.1a 66.4±2.2a ND ND ND 222.7±5.1a ND ND 369.2±8.8a F3-2A ND 28.5±0.9c ND ND 37.9±1.7c ND 177.8±2.9b ND ND 244.1±2.9b F4-5B 3.3±0.6c ND 23.0±1.5bc ND ND ND ND ND ND 26.3±2.0ef F5-12E 25.8±1.7b ND 20.6±1.3d ND 47.3±1.1b ND ND ND ND 93.8±1.5d F6-12E 85.3±3.9a 56.8±2.9b ND ND ND ND 14.6±0.7c ND ND 156.7±7.5c F7-3A 6.1±0.6c ND 10.6±1.3e ND ND ND 14.1±0.9c ND ND 30.8±1.8e F8-3A 23.8±1.0b ND 24.2±0.4b ND 48.0±2.0b ND ND ND ND 96.0±1.0d F9-5B 23.1±2.0b ND 21.1±1.4cd ND 54.3±1.8a ND ND ND ND 98.5±3.4d 注：编号中“−”后面的序号表示来源于哪种腐乳；表6~表7同。
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表 6 菌株的形态学鉴定
Table 6. Morphological identification of strains
编号 TSA平板菌落形态 革兰氏染色结果 F2-2A 乳白色，近圆形，表面平坦，有光泽 革兰氏阳性芽孢杆菌 F3-2A 乳白色，近圆形，略隆起，边缘整齐 革兰氏阳性杆菌 F5-12E 微黄色，不规则，表面干燥有褶皱，边缘略不整齐 革兰氏阳性芽孢杆菌 F6-12E 乳白色，圆形，表面光泽湿润，边缘整齐 革兰氏阳性葡萄球菌 F8-3A 微黄色，不规则，表面干燥有褶皱，边缘略不整齐 革兰氏阳性芽孢杆菌 F9-5B 微黄色，不规则，表面干燥有褶皱，边缘略不整齐 革兰氏阳性芽孢杆菌
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表 7 16S rDNA 序列 Blast 比对
Table 7. Blast analysis of 16S rDNA sequences
菌株编号 鉴定结果 匹配度（%） F2-2A 耐热芽孢杆菌（Bacillus sporothermodurans） 97.57 F3-2A 鲁梅利杆菌（Rummeliibacillus stabekisii） 99.65 F5-12E 耐硼赖氨酸芽孢杆菌（Lysinacillus boronitolerans） 99.24 F6-12E 皮脂葡萄球菌（Staphylococcus piscifermentans） 99.72 F8-3A 耐硼赖氨酸芽孢杆菌（Lysinibacillus boronitolerans） 97.07 F9-5B 耐硼赖氨酸芽孢杆菌（Lysinibacillus boronitolerans） 99.24
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[1] 凃婧, 李笑梅. 腐乳相关研究及开发进展[J]. 大豆科技,2016(4):39−44. [TU J, LI X M. Related research and development progress in fermented bean curd[J]. Soybean Science & Technoloyg,2016(4):39−44. doi: 10.3969/j.issn.1674-3547.2016.04.013TU J, LI X M. Related research and development progress in fermented bean curd[J]. Soybean Science & Technoloyg, 2016(4): 39-44. doi: 10.3969/j.issn.1674-3547.2016.04.013 [2] SHAO Y, WANG L, CHEN C S, et al. Antioxidant capacity of fermented soybeans and their protective effect on protein oxidation in largemouth bass (Micropterus salmoides) during repeated freezing-thawing (FT) treatments[J]. Lwt-Food Science and Technology,2018,91(5):213−221. [3] SANJUKTA S, RAI A K. Production of bioactive peptides during soybean fermentation and their potential health benefits[J]. Trends in Food Science & Technology,2016,50:1−10. [4] KUNG H F, LEE Y H, CHANG S C, et al. Histamine contents and histamine-forming bacteria in sufu products in Taiwan[J]. Food Control,2007,18(5):381−386. doi: 10.1016/j.foodcont.2006.02.012 [5] PARK Y K, LEE J H, MAH J H. Occurrence and reduction of biogenic amines in traditional Asian fermented soybean foods: A review[J]. Food Chemistry,2019,278:1−9. doi: 10.1016/j.foodchem.2018.11.045 [6] LIU K F, KUO H W, CHANG C C, et al. The intracellular signaling pathway of octopamine upregulating immune resistance functions in Penaeus monodon[J]. Fish & Shellfish Immunology,2019,92(67):188−195. [7] FATIH Ö, HAMED I. The importance of lactic acid bacteria for the prevention of bacterial growth and their biogenic amines formation: A review[J]. Critical Reviews in Food Science and Nutrition,2018,58(10):1660−1670. doi: 10.1080/10408398.2016.1277972 [8] BODMER S, IMARK C, KNEUBUHL M. Biogenic amines in foods: Histamine and food processing[J]. Inflammation Research,1999,48(6):296−300. doi: 10.1007/s000110050463 [9] YANG B, TAN Y, KAN J Q. Regulation of quality and biogenic amine production during sufu fermentation by pure Mucor strains[J]. LWT-Food Science Technology,2020,117:108637. doi: 10.1016/j.lwt.2019.108637 [10] SANG X, MA X X, HAO H S, et al. Evaluation of biogenic amines and microbial composition in the Chinese traditional fermented food grasshopper sub shrimp paste[J]. LWT,2020,134(12):134−141. [11] TEN B B, DAMINK C, JOOSTEN H M, et al. Occurrence and formation of biologically active amines in foods[J]. International Journal of Food Microbiology,1990,11(1):73−84. doi: 10.1016/0168-1605(90)90040-C [12] 李大伟, 李丹丹, 梁静静, 等. 高效液相色谱法测定市售腐乳中生物胺的含量[J]. 食品研究与开发,2018(16):120−124,129. [LI D W, LI D D, LIANG J J, et al. Determination of biogenic amines in commercially sufu by high performance liquid chromatography[J]. Food Research and Development,2018(16):120−124,129. doi: 10.3969/j.issn.1005-6521.2018.16.022LI D W, LI D D, LIANG J J, et al. Determination of biogenic amines in commercially sufu by high performance liquid chromatography[J]. Food Research and Development, 2018(16): 120-124, 129. doi: 10.3969/j.issn.1005-6521.2018.16.022 [13] QIU S, WANG Y, CHENG Y Q, et al. Reduction of biogenic amines in sufu by ethanol addition during ripening stage[J]. Food Chem,2018,239:1244−1252. doi: 10.1016/j.foodchem.2017.07.056 [14] 朱晓娟. 黄酒酒曲中生物胺产生菌的分离鉴定和分子检测[D]. 天津: 天津科技大学, 2018.ZHU X J. Isolation, indentification and molecule detection biogenic amines producing bacteria of rice wine QU [D]. Tianjin: Tianjin University of Science & Technology, 2018. [15] 郝淑贤, 魏涯, 周婉君, 等. 鲭鱼生物胺生成菌的分离与鉴定[J]. 食品科学,2015,36(7):97−100. [HAO S X, WEI Y, ZHOU W J, et al. Isolation and identification of biogenic amine-producing bacteria in mackerel[J]. Food Science,2015,36(7):97−100. doi: 10.7506/spkx1002-6630-201507018HAO S X, WEI Y, ZHOU W J, et al. Isolation and identification of biogenic amine-producing bacteria in mackerel [J]. Food Science, 2015, 36(7): 97-100. doi: 10.7506/spkx1002-6630-201507018 [16] 张凤杰, 王德良, 薛洁, 等. 黄酒酿造过程中优势细菌产生物胺的检测与评价[J]. 酿酒科技,2016(4):17−20,24. [ZHANG F J, WANG D L, XUE J, et al. Detection of biogenic amines produced by predominant bacteria in yellow rice wine brewing process[J]. Liquor-Making Science & Technology,2016(4):17−20,24.ZHANG F J, WANG D L, XUE J, et al. Detection of biogenic amines produced by predominant bacteria in yellow rice wine brewing process[J]. Liquor-Making Science & Technology, 2016 (4): 17-20, 24. [17] RAFFAELLA P, SIMONE V, GIULIANA V. Biogenic amine pro-files and antioxidant properties of Italian red wines from different price categories[J]. Journal of Food Composition and Analysis,2016,46:7−14. doi: 10.1016/j.jfca.2015.09.014 [18] JING S G, TONG J L, FAI Z A, et al. Biogenic amines content and assessment of bacterial and fungal diversity in stinky tofu –A traditional fermented soy curd[J]. LWT-Food Science and Technology,2018,88:26−34. doi: 10.1016/j.lwt.2017.08.085 [19] 刘振锋. 腐乳和臭干中生物胺的研究[D]. 杭州: 浙江大学, 2011.LIU Z F. Studies on biogenic amines in fermented soybean products: Sufu (Furu) and stinky tofu (Chougan)[D]. Zhejiang: Zhejiang University, 2011. [20] SANTOS M H S. Biogenic amines: Their importance in foods[J]. International Journal of Food Microbiology,1996,29(2-3):213−231. doi: 10.1016/0168-1605(95)00032-1 [21] ORDÓEZ J L, TRONCOSO A M, GARCÍA-PARRILLA M D C, et al. Recent trends in the determination of biogenic amines in fermented beverages-a review[J]. Analytica Chimica Acta,2016,939(10):10−25. [22] 涂婷. 腐乳中生物胺调查及生产过程中变化研究[D]. 长沙: 湖南农业大学, 2013.TU T. Research on biogenic amines in fermented tofu and their variation during production procedure[D]. Changsha: Hunan Agricultural University, 2013. [23] MAIJALA R, EEROLA S. Contaminant lactic acid bacteria of drysausages produce histamine and tyramine[J]. Meat Science,1993,35(3):387−395. doi: 10.1016/0309-1740(93)90043-H [24] 宁伟杰, 曹传波. 酸碱指示剂在氨氮在线测量中的应用研究[J]. 分析仪器,2017(6):145−148. [NING W J, CAO C B. Research and application of acid-base indicator in online measurement of ammonia nitrogen[J]. Analytical Instrumentation,2017(6):145−148. doi: 10.3969/j.issn.1001-232x.2017.06.025NING W J, CAO C B. Research and application of acid-base indicator in online measurement of ammonia nitrogen [J]. Analytical Instrumentation, 2017(6): 145-148. doi: 10.3969/j.issn.1001-232x.2017.06.025 [25] 赵中辉. 水产品贮藏中生物胺的变化及组胺形成机制的研究[D]. 青岛: 中国海洋大学, 2011.ZHAO Z H. Studies on changes of biogenic amines during the storage and mechanism of histamine formation in seafood[D]. Qingdao: Ocean University of China, 2011. [26] 胡鹏. 传统毛霉型豆豉发酵过程中生物胺的变化及组胺生成机理的初步研究[D]. 重庆: 西南大学, 2014.HU P. Study on the biogenic amine content of China traditional fermented Douchi and formation mechanism research of histamine[D]. Chongqing: Southwest University, 2014. [27] FADHLAOUI- ZIDJOSÉ K, CURIEL J A, LANDETA G, et al. Biogenic amine production by bacteria isolated from ice-preserved sardine and mackerel[J]. Food Control,2012,25(1):89−95. doi: 10.1016/j.foodcont.2011.10.032 [28] 李颖畅, 曹娜娜, 韩笑, 等. 贮藏过程中阿根廷鱿鱼生物胺的变化及产胺菌的分离鉴定[J]. 中国食品学报,2021,21(4):236−245. [LI Y C, CAO N N, HAN X, et al. Changes of biogenic amines in Illexargentinus during storage and isolation and identification of amine bacteria[J]. Journal of Chinese Institute of Food Science and Technology,2021,21(4):236−245.LI Y C, CAO N N, HAN X, et al. Changes of biogenic amines in Illexargentinus during storage and isolation and identification of amine bacteria[J]. Journal of Chinese Institute of Food Science and Technology, 2021, 21 (4): 236-245. [29] 张黎明, 章祎, 赵云松, 等. 腌鱼中产生物胺菌株的筛选、鉴定及其特性研究[J]. 中国食品学报,2021,21(7):291−299. [ZHANG L M, ZHANG Y, ZHAO Y S, et al. Screening, identification and characteristics of biogenic amines producing strains in salted fish[J]. Journal of Chinese Institute of Food Science and Technology,2021,21(7):291−299.ZHANG L M, ZHANG Y, ZHAO Y S, et al. Screening, identification and characteristics of biogenic amines producing strains in salted fish[J]. Journal of Chinese Institute of Food Science and Technology, 2021, 21 (7): 291-299. [30] 樊明涛. 发酵食品工艺学[M]. 北京: 科学出版社, 2017.FAN M T. Fermented food technology[M]. Beijing: Science Press, 2017. -
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