抗Nisin乳酸菌筛选及其生物学特性研究
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摘要
应用纯接种乳酸发酵技术生产发酵蔬菜是对传统自然发酵模式的一种变革，也是发酵技术现代化的一种标志。
本论文旨在筛选可用于蔬菜发酵的优良菌株，为低盐化发酵蔬菜的开发做一些基础性的研究工作。考虑到食盐在蔬菜发酵中不仅起到增进制品风味的作用，更主要的是在发酵中能够抑制杂菌的生长，所以在降低用盐量的同时需要解决杂菌抑制的问题。本研究意欲利用天然的食品防腐剂Nisin来协同食盐达到抑菌的目的。Nisin是由各种乳酸乳球菌(Lactoccus lactis subsp. lactis)产生的具有抑菌(杀菌)活性的多肽类化合物，早已进行商业化生产，因而较易获得，现已被许多国家和地区批准作为食品防腐剂用于食品生产中。但是Nisin不仅能够抑制许多革兰氏阳性食品腐败菌的生长，而且能够抑制包括明串珠菌、乳杆菌在内的多数乳酸菌的生长繁殖，因此要求所选育的优良发酵剂能够耐受一定浓度的Nisin，以便加入的Nisin既能抑制杂菌的生长，又不影响发酵的顺利完成。主要的研究结果如下：
1．经过分离筛选，从362株菌中得到两株能够耐受2000IU／ml乳链菌肽的高抗菌株，利用现代分析技术与经典分类法对其中一株菌Ls-5105进行系统细菌学鉴定，鉴定该菌株为明串珠菌属肠膜明串珠菌肠膜亚种(Leuconostoc mesenteroides subsp. mesenteroides)，另一株菌Lb-31010来源于实验室保藏的植物乳杆菌。
2．对Ls-5105、Lb-31010两菌株在含有2000IU／ml Nisin MRS培养液及不合Nisin MRS培养液中的生长、菌体繁殖(OD值)、产酸能力及发酵液pH值的变化情况进行了测定、比较，发现Nisin对菌株的生长有一定的延滞作用，但最终的变化趋势一致。另外，测定了Ls-5105及Lb-31010在不同pH值、盐浓度、温度下的生长特性，结果表明，Ls-5105生长的最适温度为25-30℃，最适pH值为6.0-7.0，能够耐受5％以下的盐浓度，3％以下的盐浓度下生长较好；Lb-31010生长的最适温度为35-37℃，最适初始pH值为5.0-7.0，能够耐受8％以下的盐浓度，3％以下的盐浓度对其生长影响不明显。
3．发酵后的残余Nisin效价试验，菌株抑菌活性代谢产物的检测，及质粒消除试验结果表明，抗Nisin菌株不分解Nisin，不产生抑菌物质，吖啶橙处理后菌株Nisin抗性无显著变化。
4．利用正交试验法确定了添加Nisin并接种耐Nisin菌株Ls-5105及Lb-31010进行蔬菜发酵的最优工艺参数：接种量5％，接种比例1：1，Nisin加入量150mg／kg，
抗Nisin乳酸菌筛选及其生物学特性研究
食盐 2见 白砂糖 3地 培养温度 3 0 C，发酵时间 5 k。另外还研究了最优发酵树下
发酵赔中细菌总数、乳酸菌、亚硝酸盐、PH值、滴定鹏等的变化情况以及发酵前
后部分风味成分的变化．
It is a revolution to traditional natural fermentation that uses pure strain inoculation in lactic acid fermentation to make fermented vegetable, and it is a symbol of fermentation technology's modernization.
In this study some strains of lactic acid bacteria would be screened for the development of fermentative vegetables with a low NaCl concentration?%). In the vegetable fermentation, NaCl not only gives products flavor, but also inhibits the growth of spoilage organism. So if NaCl concentration is lowed, some other methods must be used in order to inhibit the growth of spoilage organism. In this case , our research efforts have focused on the use of a bacteriocin nisin to inhibit them. Nisin is a bacteriocidal polypetide produced by lactococcus lactis subsp. lactis and is a readily available bacteriocin that has been approved for use in some foods in many countries /regions. But nisin is active against gram-positive bacteria including not only many spoilage organism but also lactic acid bacteria such as leuconostoc and lactobacillus species. Some lactic acid bacteria that are resistant to nisin must be isolated firstly so that nisin added not only can inhibits the growth of spoilage organism but also does not
affect the fermentative progress.
In this paper two high nisin-resistant strains were screened, and the optimum process parameters for fermentation pickle were studied. The main results were given as follows:
1. Two strains that are resistant to nisin with high levels (up to 2000IU/ml in MRS broth ) were screened from 362 strains. One strain Ls-5105 isolated from pickles was systematically identified as leuconostoc mesenteroides subsp. mesenteroides on the basis of morphology, physiological and biochemical features with modern analytical methods and classical methods. Another strain Lb-31010 was lactobacillus plantarum obtained from our laboratory.
2. The dynamics of strain Ls-5105 and Lb-31010 in MRS broth with or without 2000 IU/ml nisin including the growth, biomass, acid-producing ability, and pH value was studied. The growth of two strains were slightly inhibited at the initial stage in the presence of nisin. After 32h, the inhibition was little observed. And the effects of environment factors such as pH value,
NaCl concentration, temperature on the growth of two strains were studied. The results showed that for the strain Ls-5105 , the optinum temperature and pH were 25-30 and 6.0-7.0; no growth in 5% NaCl respectively. For the strain Lb-31010, the optinum temperature and pH was 35-37 and 5. 0-7. 0, no growth in 8% NaCl respectively.
3. The mechanism of nisin-resistance of two strains was studied with the measurment of residual nisin levels, production of the antimicrobial activities, and plasmid curing experiment. The results were given as follows: nisin degradation wasnot observed, no antimicrobial substance was produced, and the nisin-resistance of two strains after acridine organge treatment did not change clearly.
4. The effect of various factors on vegetable fermentation with nisin and nisin-resistant strains Ls-5105&Lb~31010 was studied in the experiment with orthogonal design. The optimum fermentative conditions were given as follows: inoculation load 5%, Ls-5105/Lb-31010=1:1, Nisin 150mg/kg, salt 2%, sugar 3%, kept at 30 for 5 days. Total bacterial counts , lactic acid bacteria, pH value, titrated acid coupled with the flavor after fermentation were also studied during pickle fermentation at the best fermentative condition.
本论文旨在筛选可用于蔬菜发酵的优良菌株，为低盐化发酵蔬菜的开发做一些基础性的研究工作。考虑到食盐在蔬菜发酵中不仅起到增进制品风味的作用，更主要的是在发酵中能够抑制杂菌的生长，所以在降低用盐量的同时需要解决杂菌抑制的问题。本研究意欲利用天然的食品防腐剂Nisin来协同食盐达到抑菌的目的。Nisin是由各种乳酸乳球菌(Lactoccus lactis subsp. lactis)产生的具有抑菌(杀菌)活性的多肽类化合物，早已进行商业化生产，因而较易获得，现已被许多国家和地区批准作为食品防腐剂用于食品生产中。但是Nisin不仅能够抑制许多革兰氏阳性食品腐败菌的生长，而且能够抑制包括明串珠菌、乳杆菌在内的多数乳酸菌的生长繁殖，因此要求所选育的优良发酵剂能够耐受一定浓度的Nisin，以便加入的Nisin既能抑制杂菌的生长，又不影响发酵的顺利完成。主要的研究结果如下：
1．经过分离筛选，从362株菌中得到两株能够耐受2000IU／ml乳链菌肽的高抗菌株，利用现代分析技术与经典分类法对其中一株菌Ls-5105进行系统细菌学鉴定，鉴定该菌株为明串珠菌属肠膜明串珠菌肠膜亚种(Leuconostoc mesenteroides subsp. mesenteroides)，另一株菌Lb-31010来源于实验室保藏的植物乳杆菌。
2．对Ls-5105、Lb-31010两菌株在含有2000IU／ml Nisin MRS培养液及不合Nisin MRS培养液中的生长、菌体繁殖(OD值)、产酸能力及发酵液pH值的变化情况进行了测定、比较，发现Nisin对菌株的生长有一定的延滞作用，但最终的变化趋势一致。另外，测定了Ls-5105及Lb-31010在不同pH值、盐浓度、温度下的生长特性，结果表明，Ls-5105生长的最适温度为25-30℃，最适pH值为6.0-7.0，能够耐受5％以下的盐浓度，3％以下的盐浓度下生长较好；Lb-31010生长的最适温度为35-37℃，最适初始pH值为5.0-7.0，能够耐受8％以下的盐浓度，3％以下的盐浓度对其生长影响不明显。
3．发酵后的残余Nisin效价试验，菌株抑菌活性代谢产物的检测，及质粒消除试验结果表明，抗Nisin菌株不分解Nisin，不产生抑菌物质，吖啶橙处理后菌株Nisin抗性无显著变化。
4．利用正交试验法确定了添加Nisin并接种耐Nisin菌株Ls-5105及Lb-31010进行蔬菜发酵的最优工艺参数：接种量5％，接种比例1：1，Nisin加入量150mg／kg，
抗Nisin乳酸菌筛选及其生物学特性研究
食盐 2见 白砂糖 3地 培养温度 3 0 C，发酵时间 5 k。另外还研究了最优发酵树下
发酵赔中细菌总数、乳酸菌、亚硝酸盐、PH值、滴定鹏等的变化情况以及发酵前
后部分风味成分的变化．
It is a revolution to traditional natural fermentation that uses pure strain inoculation in lactic acid fermentation to make fermented vegetable, and it is a symbol of fermentation technology's modernization.
In this study some strains of lactic acid bacteria would be screened for the development of fermentative vegetables with a low NaCl concentration?%). In the vegetable fermentation, NaCl not only gives products flavor, but also inhibits the growth of spoilage organism. So if NaCl concentration is lowed, some other methods must be used in order to inhibit the growth of spoilage organism. In this case , our research efforts have focused on the use of a bacteriocin nisin to inhibit them. Nisin is a bacteriocidal polypetide produced by lactococcus lactis subsp. lactis and is a readily available bacteriocin that has been approved for use in some foods in many countries /regions. But nisin is active against gram-positive bacteria including not only many spoilage organism but also lactic acid bacteria such as leuconostoc and lactobacillus species. Some lactic acid bacteria that are resistant to nisin must be isolated firstly so that nisin added not only can inhibits the growth of spoilage organism but also does not
affect the fermentative progress.
In this paper two high nisin-resistant strains were screened, and the optimum process parameters for fermentation pickle were studied. The main results were given as follows:
1. Two strains that are resistant to nisin with high levels (up to 2000IU/ml in MRS broth ) were screened from 362 strains. One strain Ls-5105 isolated from pickles was systematically identified as leuconostoc mesenteroides subsp. mesenteroides on the basis of morphology, physiological and biochemical features with modern analytical methods and classical methods. Another strain Lb-31010 was lactobacillus plantarum obtained from our laboratory.
2. The dynamics of strain Ls-5105 and Lb-31010 in MRS broth with or without 2000 IU/ml nisin including the growth, biomass, acid-producing ability, and pH value was studied. The growth of two strains were slightly inhibited at the initial stage in the presence of nisin. After 32h, the inhibition was little observed. And the effects of environment factors such as pH value,
NaCl concentration, temperature on the growth of two strains were studied. The results showed that for the strain Ls-5105 , the optinum temperature and pH were 25-30 and 6.0-7.0; no growth in 5% NaCl respectively. For the strain Lb-31010, the optinum temperature and pH was 35-37 and 5. 0-7. 0, no growth in 8% NaCl respectively.
3. The mechanism of nisin-resistance of two strains was studied with the measurment of residual nisin levels, production of the antimicrobial activities, and plasmid curing experiment. The results were given as follows: nisin degradation wasnot observed, no antimicrobial substance was produced, and the nisin-resistance of two strains after acridine organge treatment did not change clearly.
4. The effect of various factors on vegetable fermentation with nisin and nisin-resistant strains Ls-5105&Lb~31010 was studied in the experiment with orthogonal design. The optimum fermentative conditions were given as follows: inoculation load 5%, Ls-5105/Lb-31010=1:1, Nisin 150mg/kg, salt 2%, sugar 3%, kept at 30 for 5 days. Total bacterial counts , lactic acid bacteria, pH value, titrated acid coupled with the flavor after fermentation were also studied during pickle fermentation at the best fermentative condition.
引文
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