产乳酸菌素乳酸菌的筛选及其在肉制品和碎鲜辣椒发酵中的应用研究
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摘要
乳酸菌(LAB，Lactic acid bacteria)是一类能使可发酵性碳水化合物转化成乳酸的细菌的通称。乳酸菌在其生长代谢过程中，能产生有机酸(organic acid)、细菌素(bacteriocin)、过氧化氢(hydrogen peroxide)和丁二酮(diacetyl)等多种拮抗物质。研究表明：乳酸菌除了对人体具有许多特殊生理功能以外，乳酸菌及其代谢产物对某些腐败菌如单核细胞增生的李斯特氏菌、金黄色葡萄球菌、蜡样芽孢杆菌、肉毒梭状芽孢杆菌、产气夹膜梭菌等还具有抑制作用，同时，乳酸菌产生的乳酸菌素作为一种纯天然的无毒防腐剂可以部分取代化学防腐剂，因而，利用乳酸菌对果蔬进行纯种发酵，既可以提高产品的营养和风味，又可以防腐和降低产品亚硝酸盐的含量。随着科技的发展，乳酸菌及其代谢产物乳酸菌素因具有广泛的用途和潜在的应用领域，越来越受到重视，筛选高性能的乳酸菌素产生菌具有非常重要的意义。
本试验以自然发酵辣椒和泡菜为分离源，选育出了3株具有较强抑菌作用的产乳酸菌素菌株，对菌株进行了鉴定，并对其发酵性能、代谢产物的理化性质、防腐保鲜作用及其在碎鲜辣椒发酵中的应用等方面进行了研究。本试验的主要研究结果如下：
(1)被选菌株的鉴定和抑菌作用研究：3株菌株经菌种鉴定为乳酸菌，分别标为Lact.1(乳球菌)、Lact.2(短乳杆菌)和Lact.3(植物乳杆菌)。3株乳酸菌对金黄色葡萄球菌、李斯特氏菌有明显的抑制作用，每1ml Lact.1、Lact.2和Lact.3的10倍发酵浓缩液对利斯特氏菌的抑菌直径分别为12.8mm、10.1 mm和10.8 mm，对金黄色葡萄球菌的抑菌直径分别为15.6mm、12.2 mm和13.6mm，对大肠杆菌、产气杆菌、蜡样芽孢杆菌也有一定程度的抑制作用。
(2)被选菌株的性能研究：Lact.1、Lact.2和Lact.3的最佳生长温度范围是30℃-35℃；Lact.1、Lact.2和Lact.3均具有较快的产酸速度，发酵前12h，Lact.1发酵液的pH值由6.5迅速降至3.9，Lact.2和Lact.3的发酵液pH值从6.5降到4.2左右，发酵液的pH值最终均稳定在3.5左右；食盐对Lact.1、Lact.2和Lact.3的生长有明显的抑制作用，但Lact.1、Lact.2和Lact.3在食盐的浓度为8％左右的MRS培养基中均尚能生长，若食盐的浓度大于10％，则3株乳酸菌的生长都受到一定程度的抑制。
(3) Lact.1、Lact.2和Lact.3的抑菌成分研究：对Lact.1、Lact.2和Lact.3的抑菌成分进行分离、电泳和理化性质研究，结果表明起抑菌作用的物质均为细菌素，分别命名为Bact.1、Bact.2和Bact.3，大致分子量分别为5.7kDa、6.0kDa和3.4kDa。
(4) Bact.1、Bact.2和Bact.3的细菌素的生物学特性研究：Bact.1、Bact.2和Bact.3在80℃左右进行热处理，其抑菌活性基本不变；经100℃、15min热处理后，Bact.1、Bact.2和Bact.3对金黄色葡萄球菌的抑菌直径分别下降19％、9％、20％左右；经121℃、15min热处理后，Bact.1、Bact.2和Bact.3对金黄色葡萄球菌的抑菌直径分别下降45％、10％h和36％左右，这说明Bact.2的热稳定性比Bact.1、和Bact.3好，属于耐热性质蛋白；Bact.1、Bact.2和Bact.3具有较好热稳定性能，作为食品生物防腐剂具有良好的应用潜力；Bact.1、Bact.3在pH4-7的范围内均表现出较高的抑菌活性，在pH6.0左右时抑菌活性最高；Bact.2在pH2-7范围内均表现出较高的抑菌活性，在pH5.0左右时抑菌活性最高
(5)用Bact.1、Bact.2和Bact.3对肉制品防腐保鲜作用研究：通过对Bact.1、Bact.2和Bact.3在肉制品中的防腐保鲜作用的研究，结果表明处理组比对照组的微生物总数的数量级要低2-3个；Bact.1、Bact.2和Bact.3作为防腐剂在肉制品中使用时，均能抑制金黄色葡萄球菌的生长，对金黄色葡萄球菌的抑制效果与金黄色葡萄球菌的浓度有关，浓度越大，抑菌效果越差。
(6) Lact.1、Lact.2和Lact.3在碎鲜辣椒接种发酵中的应用研究：通过正交试验，得出了碎鲜辣椒接种发酵的最佳乳酸菌接种量和食盐的添加量，Lact.1、Lact.2和Lact.3的接种量分别是1％、1％、0.5％(细胞浓度均为10~8 cfu·ml~(-1))，食盐的添加量为10％。通过比较测定，自然发酵碎鲜辣椒与接种发酵碎鲜辣椒的风味物质组成与含量无明显差异，但接种发酵碎鲜辣椒的发酵周期比自然发酵碎鲜辣椒的发酵周期短5d左右。
(7) Lact.1、Lact.2和Lact.3对亚硝酸盐降解作用研究：Lact.1、Lact.2和Lact.3具有较强的降解亚硝酸盐能力，在72h之内将检验降解亚硝酸盐能力培养基中的亚硝酸盐含量从125.00mg／l分别降为46.20mg／l、38.32mg／l和35.26mg／l，其降解能力是植物乳杆菌1.555和干酪乳杆菌1.29的3-4倍。自然发酵碎鲜辣椒发酵过程中会出现亚硝酸盐含量明显增加的现象，其峰值为25.06μg／g；用Lact.1、Lact.2和Lact.3对碎鲜辣椒进行纯种发酵，避免了碎鲜辣椒发酵过程中亚硝酸盐含量明显增加的现象的出现，且降低了成品中亚硝酸盐的含量，自然发酵碎鲜辣椒与接种发酵碎鲜辣椒产品中亚硝酸盐的含量分别为3.1μg／g和0.1μg／g。
Lactic acid bacteria (LAB) are a group of bacteria that can fermentcarbohydrate and produce a great number of lactic acid. They can produce organic acid,bacteriocin, hydrogen peroxide and diacetyl in fermentation. Studies have shown lacticacid bacteria not only have a lot of special physiological functions to human body, theyand their metabolites can inhibit the growth of Bacillus cereus, clostridium botulinum,clostridium perfringens, Listeria monocytogenes and Staphylococcus aureus. At thesame time, bacteriocin as one pure natural nonpoisonous preservative can replacechemical preservative partly. So the method that ferments fruits and vegetables withlactic acid bacteria not only can improve the nutrition and flavor of the products butalso can preserve and reduce the content of nitrate. With the development of technology,lactic acid bacteria and their metabolites have received the extensive attention ofvarious countries because of their uses and potential applications in many fields. Soselecting antibacterial LAB has important significance.
Three strains with stronger antibacterial function were isolated from fermentedpepper and pickles. Their fermented performance, physical and chemical characters ofmetabolites, preservative function and the application in fermenting pepper werestudied in this study. The following was the main results:
(1) The identification and antibacterial function of the three strains were studied.Three strains were identified to be LAB and called Lact.1(L.Lactis), Lact.2(L.brevis)and Lact.3(L.plantarum). Their metabolites had obvious inhibition to Staphylococcusaureus and Listeria monocytogenes. The antibacterial diameter of the three strains toListeria monocytogenes was 12.8mm, 10.1 mm and 10.8 mm, and to Staphylococcusaureus was 15.6mm, 12.2mm and 13.6 mm respectively. Their metabolites also hadsome inhibition to Escherichia coli, Bacillus cereus and Clostridium perfringens.
(2) The fermented performance of the three strains was studied in this paper. Thebest growth temperature of the three strains was between 30℃-35℃. The speed of acidproducing was very fast. The pH value of fermented liquid of Lact.1 was to be 3.9 from 6.5 in 12h, and the pH value of fermented liquid of Lact.2 and Lact.3 was to be 4.2from 6.5 in 12h.The last pH value of fermented liquid of three strains was to be 3.5 orso; The salt content had obvious inhibition on the growth of the three strains, but thethree strains can grow in MRS with 8% salt or so, and inhibited when salt concentrationwas more then 10%.
(3) The three kinds of metabolites were proved to be bacteriocin afterelectrophoresis and study of physical and chemical characters. The three kinds ofmetabolites were called Bact.1, Bact.2 and Bact.3 respectively. The molecular weightwas 5.7kda, 6.0kda and 3.4kda or so respectively.
(4) The three kinds of metabolites had good heat stability when heated at 80℃Heated at 121℃for 15min, the antibacterial diameter of the three metabolitesreduced by 19% and 9% and 20% respectively. But when heated at 121℃for 15min,the antibacterial diameter of the three metabolites reduced by 45% and 10% and 36%respectively which could prove that Bact.2 was a kind of heat-stable protain. Becauseof their heat-stable characters, they had good potential applications. Bact. 1 and Bact.3had the highest antibacterial activity at pH6 or so, and showed high activity at pH4-7;Bact.2 had the highest antibacterial activity at pH5 or so, and showed high activity atpH2-7.
(5) Through studying preservative function of three kinds of bacteriocin in meat,the result was that the order of magnitude of total microorganism in the group with oneof the three kinds of bacteriocin was 2-3 orders of magnitude lower than those of thegroup without bacteriocin. Bact.1 and Bact.2 and Bact.3 can inhibit the growth ofStaphylococcus aureus. The inhibition effect had relation with the concentration ofStaphylococcus aureus.
(6) The best parameter of fermented pepper was that the inoculating amount ofLact.1, Lact.2 and Lact.3 was 1%, 1% and 0.5% respectively and adding amount of saltwas 10%. Through comparing the composition and content of the flavor materials,there was no obvious difference between naturally and inoculated fermented pepper.But the production cycle of inoculated fermented pepper was 5d shorter then that ofnaturally.
(7) Lact.1, Lact.2 and Lact.3 can make the nitrate concentration of MRS from125.00mg/l to 20mg/l, 38.32mg/l and 35.26mg/l respectively in 72h. The ability was3-4 times of other two strains preserved in lab. The maximum nitrate content innaturally and inoculated fermented pepper was 25.06μg/g and 1.56μg/g respectivelyduring the fermentation. The nitrate content of naturally fermented product was 3.1μg/g;but the nitrate content of inoculated fermented product was only 0.1μg/g.
本试验以自然发酵辣椒和泡菜为分离源，选育出了3株具有较强抑菌作用的产乳酸菌素菌株，对菌株进行了鉴定，并对其发酵性能、代谢产物的理化性质、防腐保鲜作用及其在碎鲜辣椒发酵中的应用等方面进行了研究。本试验的主要研究结果如下：
(1)被选菌株的鉴定和抑菌作用研究：3株菌株经菌种鉴定为乳酸菌，分别标为Lact.1(乳球菌)、Lact.2(短乳杆菌)和Lact.3(植物乳杆菌)。3株乳酸菌对金黄色葡萄球菌、李斯特氏菌有明显的抑制作用，每1ml Lact.1、Lact.2和Lact.3的10倍发酵浓缩液对利斯特氏菌的抑菌直径分别为12.8mm、10.1 mm和10.8 mm，对金黄色葡萄球菌的抑菌直径分别为15.6mm、12.2 mm和13.6mm，对大肠杆菌、产气杆菌、蜡样芽孢杆菌也有一定程度的抑制作用。
(2)被选菌株的性能研究：Lact.1、Lact.2和Lact.3的最佳生长温度范围是30℃-35℃；Lact.1、Lact.2和Lact.3均具有较快的产酸速度，发酵前12h，Lact.1发酵液的pH值由6.5迅速降至3.9，Lact.2和Lact.3的发酵液pH值从6.5降到4.2左右，发酵液的pH值最终均稳定在3.5左右；食盐对Lact.1、Lact.2和Lact.3的生长有明显的抑制作用，但Lact.1、Lact.2和Lact.3在食盐的浓度为8％左右的MRS培养基中均尚能生长，若食盐的浓度大于10％，则3株乳酸菌的生长都受到一定程度的抑制。
(3) Lact.1、Lact.2和Lact.3的抑菌成分研究：对Lact.1、Lact.2和Lact.3的抑菌成分进行分离、电泳和理化性质研究，结果表明起抑菌作用的物质均为细菌素，分别命名为Bact.1、Bact.2和Bact.3，大致分子量分别为5.7kDa、6.0kDa和3.4kDa。
(4) Bact.1、Bact.2和Bact.3的细菌素的生物学特性研究：Bact.1、Bact.2和Bact.3在80℃左右进行热处理，其抑菌活性基本不变；经100℃、15min热处理后，Bact.1、Bact.2和Bact.3对金黄色葡萄球菌的抑菌直径分别下降19％、9％、20％左右；经121℃、15min热处理后，Bact.1、Bact.2和Bact.3对金黄色葡萄球菌的抑菌直径分别下降45％、10％h和36％左右，这说明Bact.2的热稳定性比Bact.1、和Bact.3好，属于耐热性质蛋白；Bact.1、Bact.2和Bact.3具有较好热稳定性能，作为食品生物防腐剂具有良好的应用潜力；Bact.1、Bact.3在pH4-7的范围内均表现出较高的抑菌活性，在pH6.0左右时抑菌活性最高；Bact.2在pH2-7范围内均表现出较高的抑菌活性，在pH5.0左右时抑菌活性最高
(5)用Bact.1、Bact.2和Bact.3对肉制品防腐保鲜作用研究：通过对Bact.1、Bact.2和Bact.3在肉制品中的防腐保鲜作用的研究，结果表明处理组比对照组的微生物总数的数量级要低2-3个；Bact.1、Bact.2和Bact.3作为防腐剂在肉制品中使用时，均能抑制金黄色葡萄球菌的生长，对金黄色葡萄球菌的抑制效果与金黄色葡萄球菌的浓度有关，浓度越大，抑菌效果越差。
(6) Lact.1、Lact.2和Lact.3在碎鲜辣椒接种发酵中的应用研究：通过正交试验，得出了碎鲜辣椒接种发酵的最佳乳酸菌接种量和食盐的添加量，Lact.1、Lact.2和Lact.3的接种量分别是1％、1％、0.5％(细胞浓度均为10~8 cfu·ml~(-1))，食盐的添加量为10％。通过比较测定，自然发酵碎鲜辣椒与接种发酵碎鲜辣椒的风味物质组成与含量无明显差异，但接种发酵碎鲜辣椒的发酵周期比自然发酵碎鲜辣椒的发酵周期短5d左右。
(7) Lact.1、Lact.2和Lact.3对亚硝酸盐降解作用研究：Lact.1、Lact.2和Lact.3具有较强的降解亚硝酸盐能力，在72h之内将检验降解亚硝酸盐能力培养基中的亚硝酸盐含量从125.00mg／l分别降为46.20mg／l、38.32mg／l和35.26mg／l，其降解能力是植物乳杆菌1.555和干酪乳杆菌1.29的3-4倍。自然发酵碎鲜辣椒发酵过程中会出现亚硝酸盐含量明显增加的现象，其峰值为25.06μg／g；用Lact.1、Lact.2和Lact.3对碎鲜辣椒进行纯种发酵，避免了碎鲜辣椒发酵过程中亚硝酸盐含量明显增加的现象的出现，且降低了成品中亚硝酸盐的含量，自然发酵碎鲜辣椒与接种发酵碎鲜辣椒产品中亚硝酸盐的含量分别为3.1μg／g和0.1μg／g。
Lactic acid bacteria (LAB) are a group of bacteria that can fermentcarbohydrate and produce a great number of lactic acid. They can produce organic acid,bacteriocin, hydrogen peroxide and diacetyl in fermentation. Studies have shown lacticacid bacteria not only have a lot of special physiological functions to human body, theyand their metabolites can inhibit the growth of Bacillus cereus, clostridium botulinum,clostridium perfringens, Listeria monocytogenes and Staphylococcus aureus. At thesame time, bacteriocin as one pure natural nonpoisonous preservative can replacechemical preservative partly. So the method that ferments fruits and vegetables withlactic acid bacteria not only can improve the nutrition and flavor of the products butalso can preserve and reduce the content of nitrate. With the development of technology,lactic acid bacteria and their metabolites have received the extensive attention ofvarious countries because of their uses and potential applications in many fields. Soselecting antibacterial LAB has important significance.
Three strains with stronger antibacterial function were isolated from fermentedpepper and pickles. Their fermented performance, physical and chemical characters ofmetabolites, preservative function and the application in fermenting pepper werestudied in this study. The following was the main results:
(1) The identification and antibacterial function of the three strains were studied.Three strains were identified to be LAB and called Lact.1(L.Lactis), Lact.2(L.brevis)and Lact.3(L.plantarum). Their metabolites had obvious inhibition to Staphylococcusaureus and Listeria monocytogenes. The antibacterial diameter of the three strains toListeria monocytogenes was 12.8mm, 10.1 mm and 10.8 mm, and to Staphylococcusaureus was 15.6mm, 12.2mm and 13.6 mm respectively. Their metabolites also hadsome inhibition to Escherichia coli, Bacillus cereus and Clostridium perfringens.
(2) The fermented performance of the three strains was studied in this paper. Thebest growth temperature of the three strains was between 30℃-35℃. The speed of acidproducing was very fast. The pH value of fermented liquid of Lact.1 was to be 3.9 from 6.5 in 12h, and the pH value of fermented liquid of Lact.2 and Lact.3 was to be 4.2from 6.5 in 12h.The last pH value of fermented liquid of three strains was to be 3.5 orso; The salt content had obvious inhibition on the growth of the three strains, but thethree strains can grow in MRS with 8% salt or so, and inhibited when salt concentrationwas more then 10%.
(3) The three kinds of metabolites were proved to be bacteriocin afterelectrophoresis and study of physical and chemical characters. The three kinds ofmetabolites were called Bact.1, Bact.2 and Bact.3 respectively. The molecular weightwas 5.7kda, 6.0kda and 3.4kda or so respectively.
(4) The three kinds of metabolites had good heat stability when heated at 80℃Heated at 121℃for 15min, the antibacterial diameter of the three metabolitesreduced by 19% and 9% and 20% respectively. But when heated at 121℃for 15min,the antibacterial diameter of the three metabolites reduced by 45% and 10% and 36%respectively which could prove that Bact.2 was a kind of heat-stable protain. Becauseof their heat-stable characters, they had good potential applications. Bact. 1 and Bact.3had the highest antibacterial activity at pH6 or so, and showed high activity at pH4-7;Bact.2 had the highest antibacterial activity at pH5 or so, and showed high activity atpH2-7.
(5) Through studying preservative function of three kinds of bacteriocin in meat,the result was that the order of magnitude of total microorganism in the group with oneof the three kinds of bacteriocin was 2-3 orders of magnitude lower than those of thegroup without bacteriocin. Bact.1 and Bact.2 and Bact.3 can inhibit the growth ofStaphylococcus aureus. The inhibition effect had relation with the concentration ofStaphylococcus aureus.
(6) The best parameter of fermented pepper was that the inoculating amount ofLact.1, Lact.2 and Lact.3 was 1%, 1% and 0.5% respectively and adding amount of saltwas 10%. Through comparing the composition and content of the flavor materials,there was no obvious difference between naturally and inoculated fermented pepper.But the production cycle of inoculated fermented pepper was 5d shorter then that ofnaturally.
(7) Lact.1, Lact.2 and Lact.3 can make the nitrate concentration of MRS from125.00mg/l to 20mg/l, 38.32mg/l and 35.26mg/l respectively in 72h. The ability was3-4 times of other two strains preserved in lab. The maximum nitrate content innaturally and inoculated fermented pepper was 25.06μg/g and 1.56μg/g respectivelyduring the fermentation. The nitrate content of naturally fermented product was 3.1μg/g;but the nitrate content of inoculated fermented product was only 0.1μg/g.
引文
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