生物保鲜乳酸菌的筛选鉴定及培养条件的优化
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摘要
乳酸菌及其代谢物质，以其抗菌性强、无毒性等优势，成为开发研究生物保鲜剂的热点；冷却肉以其外观新鲜、口感好等优势成为消费趋势，而其保质期成为束缚其市场发展的瓶颈，因此筛选具生物保鲜价值的乳酸菌，并将其应用于冷却肉保鲜成为本试验的内容和目的。
本试验从多年收集保藏的菌种中，筛选得到5株对大肠杆菌、荧光假单胞菌、蜡状芽孢杆菌、单核细胞增生李氏杆菌和金黄色葡萄球菌均有良好抑菌效果的乳酸菌，分别是11118、11050、11052、10171和11095。经过生理生化试验和16SrDNA同源性比对，鉴定11118、10171和11095为植物乳杆菌(Lactobacillus planatarum)，11050和11052为干酪乳杆菌(Lactobacillus casei)。
筛出5株菌株的代谢产物经121℃高温处理20min，仍保持80％左右的抑菌活性，但只在酸性条件下(pH4左右)才表现良好的抑菌效果。在排除酸性末端产物和过氧化氢干扰后，用蛋白酶k等多种蛋白酶处理其代谢产物粗品，发现它们的抑菌活性都会有所降低，甚至是丧失。因此，其代谢产物中均含有一定的蛋白质类物质。
通过单因素试验和正交设计，对这5株菌产抑菌物质的发酵条件进行了优化。初始pH6，37℃培养16h，筛出各乳酸菌的最佳接种量和培养基成分因菌株不同而表现出一定的差异性：
(1) 11118最佳接种量为2％，最佳培养基成分为蔗糖2.5％、大豆蛋白胨2.5％、乙酸钠0.7％、K_2HPO_4 0.2％、吐温-80 1 mL、蒸馏水1000mL；
(2) 11050最佳接种量为3％，最佳培养基成分为乳糖2％、大豆蛋白胨3％、乙酸钠0.5％、K_2HPO_4 0.1％、吐温-80 1 mL、蒸馏水1000mL；
(3) 11052最佳接种量为4％，最佳培养基成分为葡萄糖2.5％、蛋白胨1.6％、酵母粉为0.8％、乙酸钠0.5％、K_2HPO_4 0.3％、吐温-80 1 mL、蒸馏水1000mL；
(4) 10171最佳接种量为4％，最佳培养基成分为蔗糖2％、大豆蛋白胨3％、乙酸钠0.5％、K_2HPO_4 0.1％、吐温-80 1 mL、蒸馏水1000mL；
(5) 11095最佳接种量是4％，最佳培养基成分为麦芽糖2％、大豆蛋白胨2％、乙酸钠0.5％、K_2HPO_4 0.3％、吐温-80 1 mL、蒸馏水1000mL。
将这5株菌的代谢产物应用于冷却肉保鲜，以细菌总数作为评价保鲜效果的指标。4℃时，经筛出乳酸菌代谢产物处理的冷却肉较参照组的菌落总数低一个数量级左右，可延长保质期1d以上；处理组在28℃保持8h后，菌落总数仍低于10~5CFU／g，而参照组高于10~6CFU／g。10171较其它菌株对冷却肉表现出更好的保鲜效果。
Lactic acid bacteria and its fermentation production for fresh-keeping usage attract attentions of many researchers because of its nontoxicity and stronge inhibitory activity against many bacerias associate with food spoilage; while chilled meat is becoming a consume trend because of its fresh appearance and good taste.However, the preservation of chilled meat has been the chock point of its marketing development. So the object of this study was to screen lactic acid bacterias as natural food preservtives and apply them to chilled meat.
5 stains of lactic acid bacteria was screened from the collection of ACCC, the supernate of which exhibited strong inhibitory acticity against Escherichia coli、Staphylococcus aureus、Bacillus cereus、Listeria monocytogenes and Pseudomomas fluorescens. According to the morphological features, physiological and biochemical features with 16S rDNA sequence analyse of the strains, 11118, 10171 and 11095 were identified as LactobacilIus plantarum, while 11050 and 11052 were identified as Lactobucillus casei.
The supernate of the 5 strains screened was steady in acid conditions (about pH4) and remained about 80% baceriostasis activity after boiled at 121℃for 20 minutes. Excluded hydrogen peroxide and lactic acid, the inhibitive activity of them was decreased and even totally lost after treatment with papain, which showed that inhibitory materials included the contents with the the features of protein, could be classed as bactericin.
Different factors were investigated such as the ingredients of medium, temperature, pH and inoculating amount on inhibitory activity of the supernate in order to optimize the fermentation condition. The optimum incubation time was 16h, the optimum temperature was 37℃, the optimum broth initial pH was about 6, while the optimum inoculating amount and medium component varied from strains to strains:
1. The optimum inoculating amount of 11118 was 2% and its optimum medium component was: sucrose 2.5%, soybean peptone 2.5%, NaAc 0.7%, K_2HPO_4 0.2%, Tween-80 1mL, H_2O 1000 mL;
2. The optimum inoculating amount of 11050 was 3% and its optimum medium component was: lactose 2%, soybean peptone 3%, NaAc 0.5%, K_2HPO_4 0.1%, Tween-80 1mL, H_2O 1000 mL;
3. The optimum inoculating amount of 11052 was 4% and its optimum medium component was: glucose 2.5%, peptone 1.6%, yeast extract 0.8%, NaAc 0.5%, K_2HPO_4 0.3%, Tween-80 1mL, H_2O 1000 mL;
4. The optimum inoculating amount of 10171 was 4% and its optimum medium component was: sucrose 2%, soybean peptone 3%, NaAc 0.5%, K_2HPO_4 0.1%, Tween-80 1mL, H_2O 1000 mL;
5. The optimum inoculating amount of 11095 was 4% and its optimum medium component was: maltose 2%, soybean peptone 2%, NaAc 0.5%, K_2HPO_4 0.3%, Tween-80 1mL, H_2O 1000 mL.
The fermented production of the 5 strains was applied as fresh-keeping agent on chilled pork, with the total bacteria as the parameter to value the effects of storing. When stored at 4℃, the total bacteria of the comparision group was about 10 times more than that of the groups treated with the supernate of the 5 strains, in which situation the shelf life of chilled pork could be extended about 1d; while after stored at 28℃for 8h, the total bacteria of the comparision group was more than 10~6CFU/g and the other groups were less than 10~5CFU/g. The fermented production of 10171 exhibited the best effect on the preservation of chilled pork.
本试验从多年收集保藏的菌种中，筛选得到5株对大肠杆菌、荧光假单胞菌、蜡状芽孢杆菌、单核细胞增生李氏杆菌和金黄色葡萄球菌均有良好抑菌效果的乳酸菌，分别是11118、11050、11052、10171和11095。经过生理生化试验和16SrDNA同源性比对，鉴定11118、10171和11095为植物乳杆菌(Lactobacillus planatarum)，11050和11052为干酪乳杆菌(Lactobacillus casei)。
筛出5株菌株的代谢产物经121℃高温处理20min，仍保持80％左右的抑菌活性，但只在酸性条件下(pH4左右)才表现良好的抑菌效果。在排除酸性末端产物和过氧化氢干扰后，用蛋白酶k等多种蛋白酶处理其代谢产物粗品，发现它们的抑菌活性都会有所降低，甚至是丧失。因此，其代谢产物中均含有一定的蛋白质类物质。
通过单因素试验和正交设计，对这5株菌产抑菌物质的发酵条件进行了优化。初始pH6，37℃培养16h，筛出各乳酸菌的最佳接种量和培养基成分因菌株不同而表现出一定的差异性：
(1) 11118最佳接种量为2％，最佳培养基成分为蔗糖2.5％、大豆蛋白胨2.5％、乙酸钠0.7％、K_2HPO_4 0.2％、吐温-80 1 mL、蒸馏水1000mL；
(2) 11050最佳接种量为3％，最佳培养基成分为乳糖2％、大豆蛋白胨3％、乙酸钠0.5％、K_2HPO_4 0.1％、吐温-80 1 mL、蒸馏水1000mL；
(3) 11052最佳接种量为4％，最佳培养基成分为葡萄糖2.5％、蛋白胨1.6％、酵母粉为0.8％、乙酸钠0.5％、K_2HPO_4 0.3％、吐温-80 1 mL、蒸馏水1000mL；
(4) 10171最佳接种量为4％，最佳培养基成分为蔗糖2％、大豆蛋白胨3％、乙酸钠0.5％、K_2HPO_4 0.1％、吐温-80 1 mL、蒸馏水1000mL；
(5) 11095最佳接种量是4％，最佳培养基成分为麦芽糖2％、大豆蛋白胨2％、乙酸钠0.5％、K_2HPO_4 0.3％、吐温-80 1 mL、蒸馏水1000mL。
将这5株菌的代谢产物应用于冷却肉保鲜，以细菌总数作为评价保鲜效果的指标。4℃时，经筛出乳酸菌代谢产物处理的冷却肉较参照组的菌落总数低一个数量级左右，可延长保质期1d以上；处理组在28℃保持8h后，菌落总数仍低于10~5CFU／g，而参照组高于10~6CFU／g。10171较其它菌株对冷却肉表现出更好的保鲜效果。
Lactic acid bacteria and its fermentation production for fresh-keeping usage attract attentions of many researchers because of its nontoxicity and stronge inhibitory activity against many bacerias associate with food spoilage; while chilled meat is becoming a consume trend because of its fresh appearance and good taste.However, the preservation of chilled meat has been the chock point of its marketing development. So the object of this study was to screen lactic acid bacterias as natural food preservtives and apply them to chilled meat.
5 stains of lactic acid bacteria was screened from the collection of ACCC, the supernate of which exhibited strong inhibitory acticity against Escherichia coli、Staphylococcus aureus、Bacillus cereus、Listeria monocytogenes and Pseudomomas fluorescens. According to the morphological features, physiological and biochemical features with 16S rDNA sequence analyse of the strains, 11118, 10171 and 11095 were identified as LactobacilIus plantarum, while 11050 and 11052 were identified as Lactobucillus casei.
The supernate of the 5 strains screened was steady in acid conditions (about pH4) and remained about 80% baceriostasis activity after boiled at 121℃for 20 minutes. Excluded hydrogen peroxide and lactic acid, the inhibitive activity of them was decreased and even totally lost after treatment with papain, which showed that inhibitory materials included the contents with the the features of protein, could be classed as bactericin.
Different factors were investigated such as the ingredients of medium, temperature, pH and inoculating amount on inhibitory activity of the supernate in order to optimize the fermentation condition. The optimum incubation time was 16h, the optimum temperature was 37℃, the optimum broth initial pH was about 6, while the optimum inoculating amount and medium component varied from strains to strains:
1. The optimum inoculating amount of 11118 was 2% and its optimum medium component was: sucrose 2.5%, soybean peptone 2.5%, NaAc 0.7%, K_2HPO_4 0.2%, Tween-80 1mL, H_2O 1000 mL;
2. The optimum inoculating amount of 11050 was 3% and its optimum medium component was: lactose 2%, soybean peptone 3%, NaAc 0.5%, K_2HPO_4 0.1%, Tween-80 1mL, H_2O 1000 mL;
3. The optimum inoculating amount of 11052 was 4% and its optimum medium component was: glucose 2.5%, peptone 1.6%, yeast extract 0.8%, NaAc 0.5%, K_2HPO_4 0.3%, Tween-80 1mL, H_2O 1000 mL;
4. The optimum inoculating amount of 10171 was 4% and its optimum medium component was: sucrose 2%, soybean peptone 3%, NaAc 0.5%, K_2HPO_4 0.1%, Tween-80 1mL, H_2O 1000 mL;
5. The optimum inoculating amount of 11095 was 4% and its optimum medium component was: maltose 2%, soybean peptone 2%, NaAc 0.5%, K_2HPO_4 0.3%, Tween-80 1mL, H_2O 1000 mL.
The fermented production of the 5 strains was applied as fresh-keeping agent on chilled pork, with the total bacteria as the parameter to value the effects of storing. When stored at 4℃, the total bacteria of the comparision group was about 10 times more than that of the groups treated with the supernate of the 5 strains, in which situation the shelf life of chilled pork could be extended about 1d; while after stored at 28℃for 8h, the total bacteria of the comparision group was more than 10~6CFU/g and the other groups were less than 10~5CFU/g. The fermented production of 10171 exhibited the best effect on the preservation of chilled pork.
引文
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2．陈秀珠，何松，龙力红．乳链菌肽高产菌株AL2的发酵条件研究．微生物学通报，1995，27(4)：215-218
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