Isolation, Identification and Enzyme Activity Analysis of Dominant Fermentation Strains from Traditional Pickled and Dried Mustard in Ningbo
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摘要: 以宁波地区梅干菜半成品和成品为试验材料，采用十倍稀释法分离其中优势发酵菌株，从半成品中得到7株细菌，分别命名为MGCB1~MGCB7，从成品中得到3株细菌，分别命名为MGC1~MGC3。通过形态学观察、生理生化试验及16S rRNA序列分析对菌种进行鉴定，结果表明，MGCB1~MGCB7分别为枯草芽孢杆菌属（Bacillus subtilis）、空气芽孢杆菌属（Bacillus aerius）、高山芽孢杆菌属（Bacillus altitudinis）、短小芽孢杆菌属（Bacillus pumilus）、琥珀葡萄球菌属（Staphylococcus succinus）、琥珀葡萄球菌属 （Staphylococcus succinus）和解淀粉芽孢杆菌属（Bacillus amyloliquefaciens），MGC1~MGC3分别为芽孢杆菌属（Bacillus sp.）、枝芽孢杆菌属（Virgibacillus halodenitrificans）、哥特氏芽孢杆菌属（Cytobacillus gottheilii）。MGCB1和MGCB7产酶能力相对较高，在37 ℃培养96 h后，蛋白酶活分别为16.39±0.79和13.45±0.46 U/mL，纤维素酶活分别为3.27±0.13和1.60±0.02 U/mL。研究结果可为梅干菜纯种发酵剂的开发及工业化生产提供有益参考。Abstract: With the semi-finished and finished products of pickled and dried mustard in Ningbo as experimental materials, the dominant bacteria strains were isolated by ten-fold dilution method. The results showed that seven and three bacteria were obtained from the semi-finished and finished products, respectively, which were named as strain MGCB1~MGCB7 and strain MGC1~MGC3. The ten strains were identified by morphological observation, physiological and biochemical tests, and 16S rRNA sequence analysis. They were likely belong to Bacillus subtilis (MGCB1), Bacillus aerius (MGCB2), Bacillus altitudinis (MGCB3), Bacillus pumilus (MGCB4), Staphylococcus succinus (MGCB5), Staphylococcus succinus (MGCB6) and Bacillus amyloliquefaciens (MGCB7), Bacillus sp.
(MGC1), Virgibacillus halodenitrificans (MGC2), and Cytobacillus gottheilii (MGC3), respectively. The protease activities of MGCB1 and MGCB7 were 16.39±0.79 and 13.45±0.46 U/mL, and the cellulase activities of MGCB1 and MGCB7 were 3.27±0.13 and 1.60±0.02 U/mL, respectively, after cultured at 37 ℃ for 96 h. These findings provide a useful reference for developing appropriate starter cultures to improve the quality of traditional pickled and dried mustard. -
图 3 待测菌株基于16S rRNA的系统发育树
Figure 3. The phylogenetic tress of the tested strains based on 16S rRNA
表 1 菌落形态特征
Table 1. Characteristics of colony morphological
菌株 直径（mm） 外观 颜色 边缘 黏着性 MGCB1 20.0±1.6 圆形，表面有褶皱 灰白色 凹凸不平 很强 MGCB2 3.0±0.1 圆形，表面呈丝状凸起 乳白色 整齐 弱 MGCB3 3.1±0.1 圆形，表面呈丝状 乳白色 较整齐 弱 MGCB4 9.4±3.7 圆形，中心呈丝状 淡黄色 呈花边状 弱 MGCB5 17.6±0.5 不规则圆形，表面光滑湿润 黄色 不规则凸起 弱 MGCB6 3.4±0.1 圆形，表面光滑湿润 奶油色 呈锯齿状 弱 MGCB7 25.3±4.1 圆形，表面颗粒状 白色 整齐 较强 MGC1 10.0±0.8 圆形，表面呈细丝状 乳白色 较整齐 弱 MGC2 4.0±0.2 圆形，表面光滑 淡粉色 规则有晕边 弱 MGC3 7.6±1.2 圆形，表面光滑 淡黄色 规则有晕边 弱 
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表 2 生理生化试验结果
Table 2. Results of physiological and biochemical tests
特征 菌株 MGCB1 MGCB2 MGCB3 MGCB4 MGCB5 MGCB6 MGCB7 MGC1 MGC2 MGC3 氧化酶 + + + + − − + + − − 接触酶 − + + + + + − + + + 糖发酵 + + + + + + + + + + 甲基红试验 − − + + − − + + − − V-P试验 + + + + − − + + − − 苯丙氨基酸脱氨酶 − − − − − − − − − − 吲哚试验 − − − − − − − − − − 硝酸盐还原 + + − − − − + − + + 纤维素分解 − − − − − − − − − − 3-酮基乳糖 − − − − − − − − − − 脲酶 + − − − + + + − − − 色氨酸脱氨酶 − − − − − − − − − − 明胶液化 + + − − + + + + − − 精氨酸双水解酶 + + + + + + + + + − 乙酸氧化 − − − − + + − − − − 七叶灵水解 + + + + + + + + + + 耐
渗
透
压氯化钠 4% + + + + + + + + + + 8% + + + + + + + + + + 12% − + − + + + + − − − 16% − − − − − − + − − − 蔗糖 2% + + + + + + + + + − 10% + + + + + + + + − − 20% + + + + + + + + − − 40% + − − − − − − − − − 耐
酸
耐
碱pH 3.5 − − − − − − − − − − 5.5 + + + + + + + + − − 7.5 + + + + + + + + − − 9.5 + + + + + + + + + + 11.5 + + + + + + + + − − 微生物分类 芽孢杆菌属 芽孢杆菌属 芽孢杆菌属 芽孢杆菌属 葡萄球菌属 葡萄球菌属 芽孢杆菌属 芽孢杆菌属 芽孢杆菌属 芽孢杆菌属 注：“+”表示反应呈阳性或生长；“−”表示反应呈阴性或不生长。
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表 3 菌株的16S rRNA序列同源性比对结果
Table 3. Homologous alignment analysis of 16S rRNA sequences of strains
菌株
编号BLAST结果 序列大小（bp） 覆盖百分率（%） 同源性（%） 菌株登录号 MGCB1 枯草芽孢杆菌属（Bacillus subtilis） 1449 99 100.00 MK521066.1 MGCB2 空气芽孢杆菌属（Bacillus aerius） 797 86 98.70 MT225712.1 MGCB3 高山芽孢杆菌属（Bacillus altitudinis） 1450 99 99.93 KU877629.1 MGCB4 短小芽孢杆菌属（Bacillus pumilus） 1449 99 99.86 JQ308586.1 MGCB5 琥珀葡萄球菌属（Staphylococcus succinus） 1450 98 100.00 MZ674190.1 MGCB6 琥珀葡萄球菌属（Staphylococcus succinus） 1452 99 99.59 MN461567.1 MGCB7 解淀粉芽孢杆菌属（Bacillus amyloliquefaciens） 1451 99 99.86 MH373537.1 MGC1 芽孢杆菌属（Bacillus sp. ） 1433 100 99.93 MH475934.1 MGC2 枝芽孢杆菌属（Virgibacillus halodenitrificans） 1468 99 99.45 MN756670.1 MGC3 哥特氏芽孢杆菌属（Cytobacillus gottheilii） 1390 99 99.86 CP071709.1 注：以上菌株E值均为0。
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表 4 不同菌株的产酶能力
Table 4. Enzyme production capacity of different bacterial strains
菌株 蛋白酶活（U/mL） 纤维素酶活（U/mL） MGCB1枯草芽孢杆菌属 16.39±0.79a 3.27±0.13a MGCB2空气芽孢杆菌属 8.40±0.13cd 2.00±0.08b MGCB3高山芽孢杆菌属 9.20±0.71c 1.32±0.01de MGCB4短小芽孢杆菌属 8.76±0.42c 1.31±0.02e MGCB5琥珀葡萄球菌属 0.87±0.12e ND MGCB6琥珀葡萄球菌属 0.75±0.09e ND MGCB7解淀粉芽孢杆菌属 13.45±0.46b 1.60±0.02c MGC1芽孢杆菌属 7.82±0.36d 1.42±0.01 de MGC2枝芽孢杆菌属 ND 1.19±0.06f MGC3哥特氏芽孢杆菌属 0.29±0.05e 1.44±0.06d 注：ND表示未检测到酶活；同列不同小写字母表示差异显著（P<0.05）。
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