高产硝酸盐还原酶Staphylococcus simulans ZSJ6的复合诱变选育及其酶学性质研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-1270

摘要/Abstract

摘要：

硝酸盐还原酶可以将天然肉制品中的硝酸盐还原为亚硝酸盐，避免了直接添加亚硝酸盐导致其含量骤增，降低产生亚硝胺的概率，对天然肉制品的品质和安全非常重要。以腊肠中筛选出的模仿葡萄球菌（Staphylococcus simulans D14）为出发菌株，探究了单一诱变条件包括紫外、微波、氯化锂诱变，以及紫外-微波-氯化锂联合诱变的两种复合诱变方式对菌株的不同影响，以期获得硝酸盐还原酶高产菌株，并通过摇瓶发酵初步探究其酶学性质，以期为天然肉制品的发酵剂提供菌株选择。确定了紫外、微波和氯化锂诱变的最佳条件：紫外照射60 s，微波辐照80 s，氯化锂浓度1.5%。筛选出诱变菌株Staphylococcus simulans ZSJ6，菌株酶活力可达603.29 U/mg蛋白，是出发菌株酶活力的3.59倍，且突变菌株连续传代8次后，其酶活力并无显著变化（P>0.05），表明其遗传稳定性较好。酶学性质结果显示，其最适作用温度为30℃，最适作用pH为7.5，且稳定性良好，在pH 7.5下孵育2 h，酶活力仍保留90%以上。Mg²⁺、Ca²⁺、K⁺均能促进酶活力，其中Ca²⁺对酶活力的促进作用最大，相对于空白组酶活力提升了1.63倍。Cu²⁺、Fe²⁺、Hg²⁺、Mn²⁺均能抑制酶活，其中Cu²⁺和Hg²⁺对酶活力的抑制作用最大，酶活力均被抑制到30%以下。研究结果为天然肉制品中硝酸盐的转化，以及生产初期使用亚硝酸盐带来的亚硝酸盐浓度过高等问题提供了解决思路，具有研究价值和应用潜力。

关键词: 模仿葡萄球菌, 硝酸盐还原酶, 天然肉制品, 复合诱变, 酶学性质, 亚硝酸盐, 硝酸盐

Abstract:

Nitrate reductase can reduce the nitrate in natural meat products to nitrite, avoiding a sudden increase in its content due to direct addition of nitrite and reducing the chance of producing nitrosamines, which is very important for the quality and safety of natural meat products. Staphylococcus simulans D14 screened from sausage as the starting strain, and the different effects of single mutagenesis condition including UV, microwave, and lithium chloride mutagenesis, and two compound mutagenesis methods of UV and microwave combined with lithium chloride on the strains were investigated to obtain high-yield strains of nitrate reductase, and their enzymatic properties were initially explored by shake flask fermentation in order to provide strain selection for fermenters of natural meat products. The optimal conditions for UV, microwave and lithium chloride mutagenesis were determined: UV irradiation for 60 s, microwave irradiation for 80 s and lithium chloride concentration of 1.5%. The mutagenic strain S. simulans ZSJ6 was screened, and the enzyme activity of the strain reached 603.29 U/mg protein, which was 3.59 times higher that of the starting strain, and there was no significant change in the enzyme activity of the mutagenic strain after 8 consecutive passages（P>0.05）, indicating that its genetic stability was good. From the results of enzymatic properties, its optimal action temperature was 30℃, the optimal action pH was 7.5, and its stability was excellent, and the enzyme activity retained more than 90% after incubation at pH 7.5 for 2 h. Mg²⁺, Ca²⁺, and K⁺ all promoted enzyme activity, of which Ca²⁺ had the largest role in promoting the enzyme activity, which was 1.63 times higher than that blank group. Cu²⁺, Fe²⁺, Hg²⁺ and Mn²⁺ suppressed enzyme activity, among which Cu²⁺and Hg²⁺ had the maximum inhibitory action on the enzyme activity, and the enzyme activity was all inhibited to less than 30%. The above results provide a solution for the transformation of nitrate in natural meat products and the problem of high nitrite concentration brought by the use of nitrite at the initial stage of production, which has research value and potential for application.

Key words: Staphylococcus simulans, nitrate reductase, natural meat products, compound mutagenesis, enzymatic properties, nitrite, nitrate

赵赛赛, 张小丹, 贾晓妍, 陶大炜, 刘可玉, 宁喜斌. 高产硝酸盐还原酶Staphylococcus simulans ZSJ6的复合诱变选育及其酶学性质研究[J]. 生物技术通报, 2023, 39(4): 103-113.

ZHAO Sai-sai, ZHANG Xiao-dan, JIA Xiao-yan, TAO Da-wei, LIU Ke-yu, NING Xi-bin. Investigation on the Complex Mutagenesis Selection of High-yield Nitrate Reductase Strain Staphylococcus simulans ZSJ6 and Its Enzymatic Properties[J]. Biotechnology Bulletin, 2023, 39(4): 103-113.

图/表 14

图1 S. simulans D14的生长和产酶曲线

Fig. 1 Growth and enzyme production curve of S. simulans D14

图2 紫外照射对S. simulans D14的致死率和正突变率的影响

Fig. 2 Effects of UV irradiation on the lethality rate and positive mutation rate of S. simulans D14

表1 紫外诱变菌株的筛选结果

Table 1 Screening results of UV mutagenic strains

菌株编号Strain No.	酶活力Enzyme activity/（U·mg^-1 protein）
UV1-3	201.33
UV1-5	191.73
UV1-6	175.64
UV1-15	366.42
UV1-21	275.12
UV2-7	288.01
UV2-11	264.37
UV2-14	305.47
UV2-15	318.25
UV2-18	187.72

图3 微波诱变的致死率和正突变率

Fig. 3 Lethality rate and positive mutation rate of microwave mutagenesis

表2 微波诱变菌株的筛选结果

Table 2 Screening results of microwave mutagenic strains

菌株编号Strain No.	酶活力Enzyme activity/（U·mg^-1 protein）
MW1-1	370.39
MW1-9	394.12
MW1-13	401.93
MW1-14	383.56
MW1-17	451.06
MW2-3	377.64
MW2-7	385.17
MW2-8	396.72
MW2-11	421.38
MW2-18	435.76

图4 氯化锂诱变的致死率和正突变率

Fig. 4 Lethality rate and positive mutation rate induced by lithium chloride

表3 氯化锂诱变菌株的筛选结果

Table 3 Screening results of mutagenic strains induced by lithium chloride

菌株编号Strain No.	酶活力Enzyme activity/（U·mg^-1 protein）
LiCl2	483.52
LiCl4	505.21
LiCl5	476.32
LiCl7	490.18
LiCl11	511.97
LiCl12	489.15
LiCl16	521.75
LiCl17	518.72
LiCl20	463.74
LiCl21	470.19

表4 复合诱变菌株的筛选结果

Table 4 Screening results of compound mutagenic strains

菌株编号Strain No.	酶活力Enzyme activity/（U·mg^-1 protein）
ZSJ1	473.36
ZSJ4	485.12
ZSJ6	603.29
ZSJ17	501.12
ZSJ18	556.71
ZSJ19	578.83
ZSJ23	511.97
ZSJ26	529.31
ZSJ30	536.75
ZSJ35	562.33

表5 突变株ZSJ6各代产硝酸盐还原酶活力

Table 5 The activity of nitrate reductase produced by mutant ZSJ6 in each generation

传代数 Passage number	酶活力 Enzyme activity/（U·mg^-1protein）	显著性 Significant level
1	603.29±0.01	P>0.05
2	604.88±0.02
3	602.19±0.03
4	604.18±0.02
5	599.56±0.02
6	600.12±0.01
7	603.73±0.02
8	601.17±0.01

图5 温度对S. simulans ZSJ6所产硝酸盐还原酶活力的影响

Fig. 5 Effects of temperature on the activity of nitrate reductase produced by S. simulans ZSJ6

图6 温度对S. simulans ZSJ6所产硝酸盐还原酶稳定性的影响

Fig. 6 Effects of temperature on the stability of nitrate reductase produced by S. simulans ZSJ6

图7 pH对S. simulans ZSJ6所产硝酸盐还原酶活力的影响

Fig. 7 Effects of pH on the activity of nitrate reductase produced by S. simulans ZSJ6

图8 pH对S. simulans ZSJ6所产硝酸盐还原酶稳定性的影响

Fig. 8 Effects of pH on the stability of nitrate reductase produced by S. simulans ZSJ6

图9 金属离子对硝酸盐还原酶活力的影响 *表示与对照组有显著性差异P<0.05，未标注则无显著性差异

Fig. 9 Effects of metal ions on the nitrate reductase activity * indicates a significant difference from the control group (P<0.05), no significant difference if not marked

参考文献 35

[1]	孔令杰, 邓洁莹, 吴莹, 等. 肉制品中替代亚硝酸盐发色微生物的作用机理及其应用研究进展[J]. 食品科学, 2022. http://kns.cnki.net/kcms/detail/11.2206.TS.20220621.1749.058.html.
	Kong LJ, Deng JY, Wu Y, et al. Mechanism and application of chromogenic microorganisms replacing nitrite in meat products[J]. Food Sci, 2022. http://kns.cnki.net/kcms/detail/11.2206.TS.20220621.1749.058.html.
[2]	陈梦婷, 罗秉俊, 杨芳芳. 食品行业控制硝酸盐及亚硝酸盐含量的重要性及相关研究[J]. 广东化工, 2022, 49(8): 72-73, 105.
	Chen MT, Luo BJ, Yang FF. The importance and related research of controlling nitrate and nitrite content in food industry[J]. Guangdong Chem Ind, 2022, 49(8): 72-73, 105.
[3]	杜娟, 王青华, 刘利强. 亚硝酸盐在肉制品中应用的危害分析及其替代物的研究[J]. 食品科技, 2007, 32(8): 166-169.
	Du J, Wang QH, Liu LQ. Nitrite application harmful analysis and its substitute research in meat product[J]. Food Sci Technol, 2007, 32(8): 166-169.
[4]	汪杨峻杰. 亚硝酸盐及其对人体的危害[J]. 化工管理, 2017(5): 118, 120.
	Wang YJJ. Nitrite and its harm to human body[J]. Chem Enterp Manag, 2017(5): 118, 120.
[5]	Vasavada MN, Cornforth DP. Evaluation of milk mineral antioxidant activity in beef meatballs and nitrite-cured sausage[J]. J Food Sci, 2005, 70(4): C250-C253. doi: 10.1111/j.1365-2621.2005.tb07168.x URL
[6]	陈瑶, 刘成国, 罗扬, 等. 亚硝酸盐在腊肉加工中的作用及其替代物的研究进展[J]. 肉类研究, 2010, 24(5): 32-36.
	Chen Y, Liu CG, Luo Y, et al. The effect of nitrite in processing of cured meat and the progress of its substitute[J]. Meat Res, 2010, 24(5): 32-36.
[7]	Skibsted LH. Nitric oxide and quality and safety of muscle based foods[J]. Nitric Oxide, 2011, 24(4): 176-183. doi: 10.1016/j.niox.2011.03.307 pmid: 21605822
[8]	赵亚娟, 郇延军, 孙冬梅, 等. 木糖葡萄球菌和肉糖葡萄球菌的生理特性及其转化硝酸盐影响因素的研究[J]. 食品工业科技, 2012, 33(5): 63-66.
	Zhao YJ, Huan YJ, Sun DM, et al. Study on physiological, biochemical characteristics and their influencing factors of nitrate reduction of Staphylococcus xylosus and Staphylococcus carnosus[J]. Sci Technol Food Ind, 2012, 33(5): 63-66.
[9]	董竞, 冯美琴, 周超, 等. 侗族酸肉中硝酸盐还原菌的分离筛选及其特性研究[J]. 食品科学, 2009, 30(13): 241-244. doi: 10.7506/spkx1002-6630-200913055
	Dong J, Feng MQ, Zhou C, et al. Isolation and identification of nitrate reducing bacteria from traditionally fermented meat product “nanx wudl”[J]. Food Sci, 2009, 30(13): 241-244.
[10]	Casaburi A, Blaiotta G, Mauriello G, et al. Technological activities of Staphylococcus carnosus and Staphylococcus simulans strains isolated from fermented sausages[J]. Meat Sci, 2005, 71(4): 643-650. doi: 10.1016/j.meatsci.2005.05.008 URL
[11]	Jin SK, Choi JS, Yang HS, et al. Natural curing agents as nitrite alternatives and their effects on the physicochemical, microbiological properties and sensory evaluation of sausages during storage[J]. Meat Sci, 2018, 146: 34-40. doi: 10.1016/j.meatsci.2018.07.032 URL
[12]	Wang H, Xu JH, Liu Q, et al. Effect of the protease from Staphylococcus carnosus on the proteolysis, quality characteristics, and flavor development of Harbin dry sausage[J]. Meat Sci, 2022, 189: 108827. doi: 10.1016/j.meatsci.2022.108827 URL
[13]	Sun MJ, Ning XB. Screening and optimization of a nitrate reductase-producing Staphylococcus simulans UV-11 and its application[J]. J Food Meas Charact, 2021, 15(3): 2458-2468. doi: 10.1007/s11694-021-00829-6
[14]	Danz RBN, Gibis M, Schmidt H, et al. Nitrate reductase activity of Staphylococcus carnosus affecting the color formation in cured raw ham[J]. Food Res Int, 2016, 85: 113-120. doi: 10.1016/j.foodres.2016.04.021 URL
[15]	卢承蓉, 叶美芝, 上官文丹, 等. 高产胞外多糖乳酸菌的诱变育种及其益生特性[J]. 食品与发酵工业, 2020, 46(12): 14-20.
	Lu CR, Ye MZ, Shangguan WD, et al. Mutation breeding for high-yield exopolysaccharide lactic acid bacteria and evaluation of its probiotic properties[J]. Food Ferment Ind, 2020, 46(12): 14-20.
[16]	黄玉, 尼玛扎西, 薛正莲, 等. ARTP与紫外线复合诱变选育高性能绿僵菌菌株[J]. 食品工业科技, 2021, 42(4): 60-64, 70.
	Huang Y, Nimazhaxi, Xue ZL, et al. Breeding of high performance Metarhizium anisopliae strain by ARTP/UV mutagenesis[J]. Sci Technol Food Ind, 2021, 42(4): 60-64, 70.
[17]	梅林, 陈芳, 阚睿, 等. 枯草芽孢杆菌凝乳酶高产菌株的微波诱变[J]. 食品与发酵工业, 2012, 38(10): 120-122.
	Mei L, Chen F, Kan R, et al. The breeding of the rennet high-producing strain from Bacillus subtilis and studies on conditions for microwave irradiation[J]. Food Ferment Ind, 2012, 38(10): 120-122.
[18]	王陶, 谢平进, 董玉玮, 等. 氯化锂诱变选育3-羟基丙酸高产菌株[J]. 工业微生物, 2017, 47(2): 1-6.
	Wang T, Xie PJ, Dong YW, et al. Breeding of 3-hydroxypropnic acid high-producing strain by lithium chloride mutagenesis[J]. Ind Microbiol, 2017, 47(2): 1-6.
[19]	李文, 王陶, 李同祥. 氯化锂诱变黑曲霉原生质体选育高产植酸酶菌株[J]. 食品与发酵工业, 2012, 38(2): 69-73.
	Li W, Wang T, Li TX. Breeding of phytase high-producing Aspergillus niger using protoplasts by lithium chloride mutagenesis[J]. Food Ferment Ind, 2012, 38(2): 69-73.
[20]	王陶, 储渊明, 陈宏伟, 等. 氯化锂诱变蛹虫草菌株液体发酵富集微量元素锌[J]. 食品科学, 2017, 38(6): 74-80. doi: 10.7506/spkx1002-6630-201706012
	Wang T, Chu YM, Chen HW, et al. Zinc enrichment of Cordyceps militaris cultured in liquid medium: optimization of medium components and culture conditions and strain improvement by LiCl mutagenesis[J]. Food Sci, 2017, 38(6): 74-80.
[21]	李文, 董明盛. 发酵鹰嘴豆乳产γ-氨基丁酸乳酸菌的复合诱变选育[J]. 食品科学, 2018, 39(16): 147-153. doi: 10.7506/spkx1002-6630-201816022
	Li W, Dong MS. Improving γ-aminobutyric acid production of lactic acid bacteria in chickpea milk by compound mutagenesis[J]. Food Sci, 2018, 39(16): 147-153. doi: 10.1111/jfds.1974.39.issue-1 URL
[22]	李杨, 蔡海莺, 赵敏洁, 等. 高产耐高温脂肪酶生产菌的筛选与鉴定[J]. 生物技术通报, 2015, 31(1): 144-150. doi: 10.13560/j.cnki.biotech.bull.1985.2015.01.022
	Li Y, Cai HY, Zhao MJ, et al. Screening and identification of high-yield thermostable lipase producing microorganisms[J]. Biotechnol Bull, 2015, 31(1): 144-150. doi: 10.13560/j.cnki.biotech.bull.1985.2015.01.022
[23]	蒋雨鹤, 康大成, 周光宏, 等. 两株发酵乳杆菌体外抗氧化活性研究[J]. 南京农业大学学报, 2017, 40(5): 915-920.
	Jiang YH, Kang DC, Zhou GH, et al. Antioxidant activity in vitro of two strains of Lactobacillus fermentum[J]. J Nanjing Agric Univ, 2017, 40(5): 915-920.
[24]	郑怀忠. 产亚硝酸还原酶菌株发酵特性及酶在肉制品中的应用[D]. 厦门: 集美大学, 2009.
	Zheng HZ. The study on the strain fermentation of nitrite reductase and its application in cooking sausage[D]. Xiamen: Jimei University, 2009.
[25]	赵璐, 何婷, 丁文欢, 等. 考马斯亮兰法(Bradford法)测定驼乳中蛋白质的含量[J]. 应用化工, 2016, 45(12): 2366-2368, 2372.
	Zhao L, He T, Ding WH, et al. Determination of protein from camel milk by Bradford[J]. Appl Chem Ind, 2016, 45(12): 2366-2368, 2372.
[26]	Gøtterup J, Olsen K, Knöchel S, et al. Relationship between nitrate/nitrite reductase activities in meat associated staphylococci and nitrosylmyoglobin formation in a cured meat model system[J]. Int J Food Microbiol, 2007, 120(3): 303-310. pmid: 17920151
[27]	Tan W, Shao ZH, Zhao GP. In vitro nitrate reductase activity assay of Mycolicibacterium smegmatis crude extract[J]. Bio-protocol, 2021, 11(14): e4098.
[28]	陶大炜, 张小丹, 宁喜斌, 等. 复合诱变选育高产α-环糊精葡萄糖基转移酶的菌株及产酶条件优化[J]. 食品与发酵工业, 2021, 47(19): 63-70.
	Tao DW, Zhang XD, Ning XB, et al. Strain breeding for high-yielding α-cyclodextrin glucosyltransferase and optimization of the enzyme formation conditions[J]. Food Ferment Ind, 2021, 47(19): 63-70.
[29]	王敏. 四株葡萄球菌的产酶能力及蛋白降解机制研究[D]. 扬州: 扬州大学, 2013.
	Wang M. Study on enzyme production ability and protein degradation mechanism of four strains of staphylococci[D]. Yangzhou: Yangzhou University, 2013.
[30]	汪淼, 徐铮铮, 郭明亮, 等. 如皋火腿中肉糖葡萄球菌RG-10产硝酸盐还原酶条件的优化[J]. 食品科技, 2014, 39(11): 130-138.
	Wang M, Xu ZZ, Guo ML, et al. Optimization of nitrate reductase-producing condition for Staphylococcus carnosus RG-10 isolated from Rugao ham[J]. Food Sci Technol, 2014, 39(11): 130-138.
[31]	Filimonenkov AA, Zvyagilskaya RA, Tikhonova TV, et al. Isolation and characterization of nitrate reductase from the halophilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens[J]. Biochemistry(Mosc), 2010, 75(6): 744-751.
[32]	Antipov AN, Morozkina EV, Sorokin DY, et al. Characterization of molybdenum-free nitrate reductase from haloalkalophilic bacterium Halomonas sp. strain AGJ 1-3[J]. Biochemistry(Mosc), 2005, 70(7): 799-803.
[33]	李静, 张剑, 赵永祥. 金属离子对蛋白酶作用的研究进展[J]. 日用化学工业, 2017, 47(6): 345-351.
	Li J, Zhang J, Zhao YX. Progress in research work field with respect to effects of metal ions on protease[J]. China Surfactant Deterg ＆ Cosmet, 2017, 47(6): 345-351.
[34]	邱昌恩. 重金属对绿球藻硝酸还原酶活性的影响[J]. 微生物学杂志, 2008, 28(6): 40-43.
	Qiu CG. Effects of four heavy metal ions on nitrate reductase activity in chlorococcumsp[J]. J Microbiol, 2008, 28(6): 40-43.
[35]	赵改名, 李珊珊, 崔文明, 等. 不同来源腊肉中细菌菌群结构与风味相关性分析[J]. 食品与发酵工业, 2021, 47(13): 246-253.
	Zhao GM, Li SS, Cui WM, et al. Correlation analysis of bacterial community structure and flavor in different Chinese bacon[J]. Food Ferment Ind, 2021, 47(13): 246-253.