冻干乳酸菌菌种的研究
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摘要
发酵剂是影响发酵乳制品生产的关键，也是其质量优劣的决定因素。嗜热链球菌和保加利亚乳杆菌是发酵乳制品生产中最常用的菌种。近年来，直接使用的超浓缩冷冻干燥乳酸菌发酵剂因其活力强、用量少、污染低、品种多、便于运输保藏等诸多优点而倍受人们青睐，但国内尚无此类成品，均为国外进口，此方面科学研究处于零散的实验阶段，这严重制约了我国乳品企业的发展。
本课题通过6个试验，系统研究了真空冷冻干燥乳酸菌发酵剂生产的最佳工艺条件，优选最佳工艺参数，为大规模工业化生产奠定基础。
试验Ⅰ采用传统的平板划线、分离分纯方法从实验室保藏菌种中选择性状优良的菌株，用于实验。
试验Ⅱ采用正交实验，在常规培养基的基础上优化碳源、氮源和酵母粉的添加量，Streptococuus thermophilus和Lactobacillus bulgaricus的最佳基础培养基配方分别为胰蛋白胨(0.5％)、葡萄糖(1％)、酵母粉(0.7％)和鱼肉蛋白胨(0.5％)、乳糖(1％)、酵母粉(0.3％)，培养12h和15h后OD值分别为0.533和0.473，菌数达2.2×10~7个／mL和2.5×10~6个／mL。
试验Ⅲ用无菌的乳酸溶液调节培养基的初始pH值分别为5.6、5.8和6.0，不同温度、不同氧气条件下培养，结论是球菌的最佳培养条件为初始pH值5.6，于普通生化培养箱中41℃培养，而杆菌应在pH值5.8，CO_2培养箱中37℃培养。
试验Ⅳ确定两株菌各四种效果较好的增殖因子，在此基础上应用正交试验，优选出的最佳增菌培养基配方分别为平菇浸汁14％、胡萝卜汁10％、玉米浆0.6％、Vc0.2％和啤酒10％、番茄汁10％、胡萝卜汁15％、平菇浸汁16％，培养9h和15h后菌数分别达2.9×10~9个／mL和3.5×10~8个／mL。
试验Ⅴ综合采用四种菌体浓缩方法，超滤后，球菌和杆菌的OD值和pH值分别为0.858、1.825和4.78、4.07；添加磷酸盐缓冲溶液后，OD值分别比空白组增加50％和20％多；生长过程中流加20％的Na_2CO_3溶液以中和产生的酸，至生长曲线的最高点菌数分别为10~(10)和10~9个／mL；优选球菌和杆菌最佳离心条件均为6000rpm。
试验Ⅵ为成品发酵试验，冻干菌粉制作酸奶的总体品质与液体发酵剂无太大差别，凝乳时间较长，但基本无乳清析出，组织状态良好，均匀细腻，粘度较小，适当调整菌粉比例，风味应该能够有所改善。
试验用菌种经多次活化使用，性状稳定，可用于冷冻干燥乳酸菌发酵剂的连续生产。基础培养基及增殖因子价格便宜、来源广泛，制备工艺简单可行，增菌效果较好。培养条件要求不高，菌体浓缩冻干后制作的酸奶组织状态良好。
The starter is one of the key factors influenced the fermented dairy products and quality. Streptococcus thermophilus (S.t) and Lactobacillus bulgaricus (L.b) were the two important bacteria, ultra-concentration and freeze-drying LAB starter has such advantages as high activity, less dosage and polluted, plenty kinds, easy transportition and preservation. However, they are all commercially imported, study on this is not very fast and there is not a systematic research report yet, this certainly will bring heavier financial burden to dairy enterprise of our country.
Six trials were conducted in the experiment, the objective is to exam the best LAB and ascertain the optimum industrilization conditions and this will have great ecnomical and far-reaching social benefits.
The first experiment select the fine bacterial strains from own lab preserved and get the single colony after a lot of activations and purifications, then incubate repeatedly.
In the second one, three sources of carbon, nitrogen and yeast extract powder were optimized with others same, it showed that the medium components of S.t and L.b were pancreas peptone ( 0.5% ) , glucose( 1 %) , yeast extract powder ( 0.7% ) and polypeptdn ( 0.5% ), lactose(l %), yeast extract powder ( 0.3%) , absorbance at 600nm of S.t and L.b were 0.5325 and 0.4732, colony were 2.2 +1 07mL-1 and 2.5 +106mL-1 respectively after incubation.
The third one showed that the optimal condition of S.t is in common incubation, 41 C, pH5.6 and the L.b is in CO2 incubation, 37 C, pH5.8.
In the fourth one, single factor experiment confirmed four better growing factors of either, on this basis, orthogonal design was used to study the interact influnce, S.t grows well in mushroom juice (14%), carrot juice (10%), maize fluid (0.6%), Vc (0.2%) and L.b in haerbin beer (10%), tomato juice (10%), carrot juice (15%), mushroom juice (16%) ,the colony of S.t and L.b were 2.9 + 109mL-1 and 3.5 + 108mL-1' in this medium.
The four methods were used at the same time in the fifth experiment Absorbance at 600nm and pH of S.t and L.b were 0.858, 1.825 and 4.78, 4.07 after ultrafiltration; OD600 was increased 50% and 20% respectively after adding phosphate buffer, 20% Na2CO3 was added in
the incubation, the colony were 1010 and 109mL-1, the best centrifugation condition was 6000rpm.
Cryoprotective agents (1000ml) consist of skimmed milk powder100g, monosodium glutamate10g, glycerol 10g, oligosaccharide 25g, lactose 12.5g, sucrose 12.5g, cystine 1g, Vc3g, after freeze drying, the products were used to ferment milk in the sixth trial, the quality of youghurt was nearlly the same as the liquid stater one, but the concreting time was longer,
the state was good and viscosity was low. The flavor should be improved after regulating ratio of the freeze-drying LAB powder.
Masterate student: Zhang Jianyou Major: Food Science Supervisor: Prof. Huo Guicheng
本课题通过6个试验，系统研究了真空冷冻干燥乳酸菌发酵剂生产的最佳工艺条件，优选最佳工艺参数，为大规模工业化生产奠定基础。
试验Ⅰ采用传统的平板划线、分离分纯方法从实验室保藏菌种中选择性状优良的菌株，用于实验。
试验Ⅱ采用正交实验，在常规培养基的基础上优化碳源、氮源和酵母粉的添加量，Streptococuus thermophilus和Lactobacillus bulgaricus的最佳基础培养基配方分别为胰蛋白胨(0.5％)、葡萄糖(1％)、酵母粉(0.7％)和鱼肉蛋白胨(0.5％)、乳糖(1％)、酵母粉(0.3％)，培养12h和15h后OD值分别为0.533和0.473，菌数达2.2×10~7个／mL和2.5×10~6个／mL。
试验Ⅲ用无菌的乳酸溶液调节培养基的初始pH值分别为5.6、5.8和6.0，不同温度、不同氧气条件下培养，结论是球菌的最佳培养条件为初始pH值5.6，于普通生化培养箱中41℃培养，而杆菌应在pH值5.8，CO_2培养箱中37℃培养。
试验Ⅳ确定两株菌各四种效果较好的增殖因子，在此基础上应用正交试验，优选出的最佳增菌培养基配方分别为平菇浸汁14％、胡萝卜汁10％、玉米浆0.6％、Vc0.2％和啤酒10％、番茄汁10％、胡萝卜汁15％、平菇浸汁16％，培养9h和15h后菌数分别达2.9×10~9个／mL和3.5×10~8个／mL。
试验Ⅴ综合采用四种菌体浓缩方法，超滤后，球菌和杆菌的OD值和pH值分别为0.858、1.825和4.78、4.07；添加磷酸盐缓冲溶液后，OD值分别比空白组增加50％和20％多；生长过程中流加20％的Na_2CO_3溶液以中和产生的酸，至生长曲线的最高点菌数分别为10~(10)和10~9个／mL；优选球菌和杆菌最佳离心条件均为6000rpm。
试验Ⅵ为成品发酵试验，冻干菌粉制作酸奶的总体品质与液体发酵剂无太大差别，凝乳时间较长，但基本无乳清析出，组织状态良好，均匀细腻，粘度较小，适当调整菌粉比例，风味应该能够有所改善。
试验用菌种经多次活化使用，性状稳定，可用于冷冻干燥乳酸菌发酵剂的连续生产。基础培养基及增殖因子价格便宜、来源广泛，制备工艺简单可行，增菌效果较好。培养条件要求不高，菌体浓缩冻干后制作的酸奶组织状态良好。
The starter is one of the key factors influenced the fermented dairy products and quality. Streptococcus thermophilus (S.t) and Lactobacillus bulgaricus (L.b) were the two important bacteria, ultra-concentration and freeze-drying LAB starter has such advantages as high activity, less dosage and polluted, plenty kinds, easy transportition and preservation. However, they are all commercially imported, study on this is not very fast and there is not a systematic research report yet, this certainly will bring heavier financial burden to dairy enterprise of our country.
Six trials were conducted in the experiment, the objective is to exam the best LAB and ascertain the optimum industrilization conditions and this will have great ecnomical and far-reaching social benefits.
The first experiment select the fine bacterial strains from own lab preserved and get the single colony after a lot of activations and purifications, then incubate repeatedly.
In the second one, three sources of carbon, nitrogen and yeast extract powder were optimized with others same, it showed that the medium components of S.t and L.b were pancreas peptone ( 0.5% ) , glucose( 1 %) , yeast extract powder ( 0.7% ) and polypeptdn ( 0.5% ), lactose(l %), yeast extract powder ( 0.3%) , absorbance at 600nm of S.t and L.b were 0.5325 and 0.4732, colony were 2.2 +1 07mL-1 and 2.5 +106mL-1 respectively after incubation.
The third one showed that the optimal condition of S.t is in common incubation, 41 C, pH5.6 and the L.b is in CO2 incubation, 37 C, pH5.8.
In the fourth one, single factor experiment confirmed four better growing factors of either, on this basis, orthogonal design was used to study the interact influnce, S.t grows well in mushroom juice (14%), carrot juice (10%), maize fluid (0.6%), Vc (0.2%) and L.b in haerbin beer (10%), tomato juice (10%), carrot juice (15%), mushroom juice (16%) ,the colony of S.t and L.b were 2.9 + 109mL-1 and 3.5 + 108mL-1' in this medium.
The four methods were used at the same time in the fifth experiment Absorbance at 600nm and pH of S.t and L.b were 0.858, 1.825 and 4.78, 4.07 after ultrafiltration; OD600 was increased 50% and 20% respectively after adding phosphate buffer, 20% Na2CO3 was added in
the incubation, the colony were 1010 and 109mL-1, the best centrifugation condition was 6000rpm.
Cryoprotective agents (1000ml) consist of skimmed milk powder100g, monosodium glutamate10g, glycerol 10g, oligosaccharide 25g, lactose 12.5g, sucrose 12.5g, cystine 1g, Vc3g, after freeze drying, the products were used to ferment milk in the sixth trial, the quality of youghurt was nearlly the same as the liquid stater one, but the concreting time was longer,
the state was good and viscosity was low. The flavor should be improved after regulating ratio of the freeze-drying LAB powder.
Masterate student: Zhang Jianyou Major: Food Science Supervisor: Prof. Huo Guicheng
引文
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72 Robinson R.K. Dairy Microbiology. Microbiology of starter culture 1985.113-156
73 Rumian, N. etal Comparative investigation on activity of lactobacillus bulgaricus during lyophilization. Cryobiology. 1993 (30) :438-442
74 Sheu. T. Y. et al. Improving survival of culture bacteria in frozen desserts by microentrapment.. J. Dairy Sci.. 1993 (76) :1902-1907
75 Speckman C.A.Sandine. W.E.and Elliker. P.R.. Lyophilized lactic acid starter culture:Preparation and use inoculation of vat milk for cheddar and cottage cheese. J.Dairy Sci.,1973.57 (2) :165-172
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77 Vinderola,-C.G,Costa,-G.A. Influence of compounds associated with fermented dairy products on the growth of lactic acid starter and probiotic bacteria. Int-dairy-j. 2002. v. 12 (7) p. 579-589..
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73 Rumian, N. etal Comparative investigation on activity of lactobacillus bulgaricus during lyophilization. Cryobiology. 1993 (30) :438-442
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75 Speckman C.A.Sandine. W.E.and Elliker. P.R.. Lyophilized lactic acid starter culture:Preparation and use inoculation of vat milk for cheddar and cottage cheese. J.Dairy Sci.,1973.57 (2) :165-172
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77 Vinderola,-C.G,Costa,-G.A. Influence of compounds associated with fermented dairy products on the growth of lactic acid starter and probiotic bacteria. Int-dairy-j. 2002. v. 12 (7) p. 579-589..
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