直投式酸奶发酵剂的产业化研究
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摘要
为提高我国酸奶的品质和生产水平，缩小与发达国家在直投式酸奶发酵剂的差距，本实验对酸奶菌种的大量菌株进行选育，从工业化生产的角度研究了菌株的生理特性、增殖培养基、保护剂、冷冻干燥和最佳工艺条件。
采用4株嗜热链球菌、8株保加利亚乳杆菌作交叉拮抗试验，筛选出8对具有共生作用，适合于酸乳加工的保加利亚乳杆菌(Lactobacillus delbrueckii subsp．bulgaricus)与嗜热链球菌(Streptococcus thermophilus)菌株组合，即ST1+LB1、ST1+LB2、ST1+LB3、ST1+LB4、ST1+LB5、ST1+LB6、ST1+Y-5、ST1+Y-6；进一步对这些组合的酸度、粘度、乙醛、氨基酸、活菌数等技术指标进行了检测，筛选出3对适于发酵生产用的菌株组合，即ST1+LB2、ST1+LB1以及ST1+Y-6。
通过四因素三水平正交试验，获得保加利亚乳杆菌LB2、LB1和Y6的最佳培养基组成分别为番茄汁6.0％、酵母膏0.6％、乳清粉2％和11％麦芽汁；番茄汁6.0％、酵母膏0.4％、乳清粉1％和10％麦芽汁；番茄汁7.5％、酵母膏0.6％、乳清粉3％和10％麦芽汁，在37℃，培养16h，其活菌数分别为1.76×10~9cfu／mL、0.97×10~9cfu／mL、1.88×10~9cfu／mL。嗜热链球菌ST1的最佳培养基组成为番茄汁7.0％、酵母膏0.3％、碳酸钙0.9％和2.0％蛋白胨，在37℃，培养16h，其活菌数达到1.05×10~9cfu／mL。
通过四因素三水平正交试验，获得保加利亚乳杆菌和嗜热链球菌LB2+ST1、Y6+ST1、LB1+ST1最佳培养基组成分别为番茄汁4.0％、乳糖0.0％、酵母膏0.8％和1.0％蛋白胨、番茄汁2.5％、乳糖为0.0％、酵母膏为0.5％和1.5％蛋白胨、番茄汁1.0％、乳糖0.0％、酵母膏0.3％和0.5％蛋白胨。在37℃，培养16h，其活菌数分别为1.59×10~9cfu／mL、1.22×10~9cfu／mL、1.64×10~9cfu／mL。在42℃培养，LB2+ST1、Y6+ST1、LB1+ST1最佳培养基组成分别为番茄汁1.0％、乳糖1.0％、酵母膏0.5％和1.0％蛋白胨、番茄汁2.5％、乳糖为0.0％、酵母膏为0.5％和1.5％蛋白胨、番茄汁1.0％、乳糖0.0％、酵母膏0.3％和0.5％蛋白胨。6h时其活菌数分别为1.64×10~9cfu／mL、1.34×10~9cfu／mL、2.04×10~9cfu／mL。
通过四因素三水平正交试验，获得了嗜热链球菌ST1、保加利亚乳杆菌LB2、嗜热链球菌和保加利亚乳杆菌LB2+ST1的最佳保护剂，其组成分别为海藻糖1.0％、甘油0.5％、谷氨酸钠为15％和1.0％吐温80、海藻糖2.0％、甘油0.3％、谷氨酸钠5％和0.5％吐温80、海藻糖0.5％、甘油0.3％、谷氨酸钠5％和0.3％吐温80。经冷冻干燥后，产品活菌数分别为4.72×10~(11)cfu／g、1.34×10~(11)cfu／g、3.57×10~(11)cfu／g。
在摇瓶实验中保加利亚乳杆菌LB2在转速为40rpm／min时活菌数最高，为1.08×10~9cfu／mL，LB1在转速为60rpm／min时活菌数最高，为6.5×10~8cfu／mL，Y6在转速为80rpm／min时活菌数最高，为2.8×10~8cfu／mL；嗜热链球菌ST1在转速为40rpm／min时活菌数最高；嗜热链球菌和保加利亚乳杆菌LB2+ST1、LB1+ST1和Y6+ST1在转速为80rpm／min时的活菌数最高，分别为1.27×10~9cfu／mL、1.22×10~9cfu／mL和1.21×10~9cfu／mL。
在上述实验基础上，确定了10L发酵实验的最佳工艺参数组合，采用此组合所获得产品
的活菌数分别为:保加利亚乳杆菌1 .34xl0‘’c细启、嗜热链球菌4.72X10”。细g、嗜热链球菌
和保加利亚乳杆菌3.57xlo”e彻g。
在10L发酵实验的基础上，确定了300L发酵实验的最佳工艺参数组合，采用此组合所
得产品的活菌数分别为:保加利亚乳杆菌0.85 X10’‘c创g、嗜热链球菌1 .50 xlo‘’c创g、嗜热
链球菌和保加利亚乳杆菌1 .30 xlo”。创g。达到了国外同类产品的水准。
在42OC恒温培养6h，在不同时间测定酸度，表明在4h罗地亚公司产品、丹麦汉森产品、
本实验产品混合菌株、单株复配的酸度分别为650T、750T、700T、800T，6h酸度分别为900T、
100OT、93.“T、950T，适宜作为直投式发酵剂用于发酵乳的生产。
Strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus isolated from yogurt products were screened for commercial direct-vat-starters (DVS). The physiology of yogurt strains, media for their proliferation, protectants for the viability of cells, and the optimal technique for producing lyophilized cells were investigated to improve the quality of commercial yogurt starters. One of the purposes of the present study was to stop a gap between China and developed countries in the production of direct-inoculated-starter cultures used for yogurt products.
The interactions, including the symbiosis and antagonism between 4 Streptococcus thermophilus strains and 8 Lactobacillus delbrueckii subsp. bulgaricus strains were studied. The results proved that among the tested strains, ST1 and LB1, STland LB2, ST1 and LB3, ST1and LB4, ST1 and LB5, ST1 and LB6, ST1 and Y-5, and ST1 and Y-6 were symbiotic in media. The strains of streptococci and lactobacilli were paired for further screening based on their abilities to produce lactic acid, viscosity, viable counts, and the levels of amino acid and acetaldehyde. It was seen that the paired strains ST1 and LB1, STland LB2, ST1 and Y-6 were more suitable for the production of yogurt.
The optimal media for growing the above-mentioned yogurt strains were investigated. It was found that the strain LB2 required the following ingredients for the optimal growth: that is 6.0% of tomato extract, 0.6% of yeast extract, 2% of whey powder, and 11% of malt extract. The optimal medium for the growth of strain LB1 was tomato extract (6.0%), yeast extract (0.4%), whey power (1%) and malt extract (10%), respectively. The optimal medium supporting strain Y-6 was 7.5% of tomato extract, 0.6% of yeast extract, 3% of whey power, and 10% of malt extract. When incubated at 37℃ for 16 h, the populations of the 3 lactobacilli strains reached 1.76 x 109cfu/mL, 0.97 x 109cfu/mL, and 1.88 x 109cfu/mL, respectively. Meanwhile, medium supporting the optimal growth of streptococci strain ST1 was tomato extract (7.0%), yeast extract (0.3%), CaCO3 (0.9%) and peptone (2.0%). When incubated at 37℃ for 16 h, the live cells of the strain ST1 were 1.05 x 109cfu/mL.
The optimal medium for the paired yogurt strains LB2-ST1 was tomato extract (4.0%), yeast extract (0.8%) and peptone (1.0%). That for the paired yogurt strains LB1-ST1 was tomato extract (2.5%), yeast extract (0.5%) and peptone (1.5%). That for the paired yogurt strains Y-6-ST1 was tomato extract (1.0%), yeast extract (0.3%) and peptone (0.5%). The live cells of these yogurt strains, when grown in their respective medium for incubation at 37℃ for 16 h, were 1.59 x 109cfu/mL, 1.22 x 109cfu/mL, and 1.64 x 109cfu/mL, respectively.
If they were cultivated at 42℃ for 6 h, the 3 yogurt starters required different components in media to support their maximum growth. For strains LB2-ST1, the best medium was 1.0% of tomato extract, 1.0% of lactose, 0.5% of yeast extract and 1.0% of
peptone. For strains LB1-ST1, that was 2.5% of tomato extract, 0.5% of yeast extract and 1.5% of peptone. For strains Y-6-ST1 that was 1.0% of tomato extract, 0.3% of yeast extract and 0.5% of peptone. The cell population was up to 1.64×109cfu/mL, 1.34×109cfu/mL, and 2.04×109cfu/mL, respectively.
The optimal cryoprotectants to improve the viability of strains ST1, LB2 and LB2-ST1 were investigated. It was seen that the combination of trehalose (1.0%), glycerin (0.5%), glutamate-Na (15%) and 1.0% of sorbitan mono-oleate offered the optimal protection to the viability of the strain STL The mixture of trehalose (2.0%), glycerin (0.3%), glutamate-Na (5%) and Sorbitan mono-oieate (0.5%) favored the cells of strain LB2 to survive best in stress conditions. A combination consisting of trehalose (0.5%), glycerin (0.3%), glutamate-Na (5%) and Sorbitan mono-oleate (0.3%) produced a very positive effect on promoting the viability of the paired strains LB2-STI. Their viable cells after freeze-drying were 4.72 x10 11cfu/g, 1.34×10 11cfu/g and 3.57×10 11cfu/g, re
采用4株嗜热链球菌、8株保加利亚乳杆菌作交叉拮抗试验，筛选出8对具有共生作用，适合于酸乳加工的保加利亚乳杆菌(Lactobacillus delbrueckii subsp．bulgaricus)与嗜热链球菌(Streptococcus thermophilus)菌株组合，即ST1+LB1、ST1+LB2、ST1+LB3、ST1+LB4、ST1+LB5、ST1+LB6、ST1+Y-5、ST1+Y-6；进一步对这些组合的酸度、粘度、乙醛、氨基酸、活菌数等技术指标进行了检测，筛选出3对适于发酵生产用的菌株组合，即ST1+LB2、ST1+LB1以及ST1+Y-6。
通过四因素三水平正交试验，获得保加利亚乳杆菌LB2、LB1和Y6的最佳培养基组成分别为番茄汁6.0％、酵母膏0.6％、乳清粉2％和11％麦芽汁；番茄汁6.0％、酵母膏0.4％、乳清粉1％和10％麦芽汁；番茄汁7.5％、酵母膏0.6％、乳清粉3％和10％麦芽汁，在37℃，培养16h，其活菌数分别为1.76×10~9cfu／mL、0.97×10~9cfu／mL、1.88×10~9cfu／mL。嗜热链球菌ST1的最佳培养基组成为番茄汁7.0％、酵母膏0.3％、碳酸钙0.9％和2.0％蛋白胨，在37℃，培养16h，其活菌数达到1.05×10~9cfu／mL。
通过四因素三水平正交试验，获得保加利亚乳杆菌和嗜热链球菌LB2+ST1、Y6+ST1、LB1+ST1最佳培养基组成分别为番茄汁4.0％、乳糖0.0％、酵母膏0.8％和1.0％蛋白胨、番茄汁2.5％、乳糖为0.0％、酵母膏为0.5％和1.5％蛋白胨、番茄汁1.0％、乳糖0.0％、酵母膏0.3％和0.5％蛋白胨。在37℃，培养16h，其活菌数分别为1.59×10~9cfu／mL、1.22×10~9cfu／mL、1.64×10~9cfu／mL。在42℃培养，LB2+ST1、Y6+ST1、LB1+ST1最佳培养基组成分别为番茄汁1.0％、乳糖1.0％、酵母膏0.5％和1.0％蛋白胨、番茄汁2.5％、乳糖为0.0％、酵母膏为0.5％和1.5％蛋白胨、番茄汁1.0％、乳糖0.0％、酵母膏0.3％和0.5％蛋白胨。6h时其活菌数分别为1.64×10~9cfu／mL、1.34×10~9cfu／mL、2.04×10~9cfu／mL。
通过四因素三水平正交试验，获得了嗜热链球菌ST1、保加利亚乳杆菌LB2、嗜热链球菌和保加利亚乳杆菌LB2+ST1的最佳保护剂，其组成分别为海藻糖1.0％、甘油0.5％、谷氨酸钠为15％和1.0％吐温80、海藻糖2.0％、甘油0.3％、谷氨酸钠5％和0.5％吐温80、海藻糖0.5％、甘油0.3％、谷氨酸钠5％和0.3％吐温80。经冷冻干燥后，产品活菌数分别为4.72×10~(11)cfu／g、1.34×10~(11)cfu／g、3.57×10~(11)cfu／g。
在摇瓶实验中保加利亚乳杆菌LB2在转速为40rpm／min时活菌数最高，为1.08×10~9cfu／mL，LB1在转速为60rpm／min时活菌数最高，为6.5×10~8cfu／mL，Y6在转速为80rpm／min时活菌数最高，为2.8×10~8cfu／mL；嗜热链球菌ST1在转速为40rpm／min时活菌数最高；嗜热链球菌和保加利亚乳杆菌LB2+ST1、LB1+ST1和Y6+ST1在转速为80rpm／min时的活菌数最高，分别为1.27×10~9cfu／mL、1.22×10~9cfu／mL和1.21×10~9cfu／mL。
在上述实验基础上，确定了10L发酵实验的最佳工艺参数组合，采用此组合所获得产品
的活菌数分别为:保加利亚乳杆菌1 .34xl0‘’c细启、嗜热链球菌4.72X10”。细g、嗜热链球菌
和保加利亚乳杆菌3.57xlo”e彻g。
在10L发酵实验的基础上，确定了300L发酵实验的最佳工艺参数组合，采用此组合所
得产品的活菌数分别为:保加利亚乳杆菌0.85 X10’‘c创g、嗜热链球菌1 .50 xlo‘’c创g、嗜热
链球菌和保加利亚乳杆菌1 .30 xlo”。创g。达到了国外同类产品的水准。
在42OC恒温培养6h，在不同时间测定酸度，表明在4h罗地亚公司产品、丹麦汉森产品、
本实验产品混合菌株、单株复配的酸度分别为650T、750T、700T、800T，6h酸度分别为900T、
100OT、93.“T、950T，适宜作为直投式发酵剂用于发酵乳的生产。
Strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus isolated from yogurt products were screened for commercial direct-vat-starters (DVS). The physiology of yogurt strains, media for their proliferation, protectants for the viability of cells, and the optimal technique for producing lyophilized cells were investigated to improve the quality of commercial yogurt starters. One of the purposes of the present study was to stop a gap between China and developed countries in the production of direct-inoculated-starter cultures used for yogurt products.
The interactions, including the symbiosis and antagonism between 4 Streptococcus thermophilus strains and 8 Lactobacillus delbrueckii subsp. bulgaricus strains were studied. The results proved that among the tested strains, ST1 and LB1, STland LB2, ST1 and LB3, ST1and LB4, ST1 and LB5, ST1 and LB6, ST1 and Y-5, and ST1 and Y-6 were symbiotic in media. The strains of streptococci and lactobacilli were paired for further screening based on their abilities to produce lactic acid, viscosity, viable counts, and the levels of amino acid and acetaldehyde. It was seen that the paired strains ST1 and LB1, STland LB2, ST1 and Y-6 were more suitable for the production of yogurt.
The optimal media for growing the above-mentioned yogurt strains were investigated. It was found that the strain LB2 required the following ingredients for the optimal growth: that is 6.0% of tomato extract, 0.6% of yeast extract, 2% of whey powder, and 11% of malt extract. The optimal medium for the growth of strain LB1 was tomato extract (6.0%), yeast extract (0.4%), whey power (1%) and malt extract (10%), respectively. The optimal medium supporting strain Y-6 was 7.5% of tomato extract, 0.6% of yeast extract, 3% of whey power, and 10% of malt extract. When incubated at 37℃ for 16 h, the populations of the 3 lactobacilli strains reached 1.76 x 109cfu/mL, 0.97 x 109cfu/mL, and 1.88 x 109cfu/mL, respectively. Meanwhile, medium supporting the optimal growth of streptococci strain ST1 was tomato extract (7.0%), yeast extract (0.3%), CaCO3 (0.9%) and peptone (2.0%). When incubated at 37℃ for 16 h, the live cells of the strain ST1 were 1.05 x 109cfu/mL.
The optimal medium for the paired yogurt strains LB2-ST1 was tomato extract (4.0%), yeast extract (0.8%) and peptone (1.0%). That for the paired yogurt strains LB1-ST1 was tomato extract (2.5%), yeast extract (0.5%) and peptone (1.5%). That for the paired yogurt strains Y-6-ST1 was tomato extract (1.0%), yeast extract (0.3%) and peptone (0.5%). The live cells of these yogurt strains, when grown in their respective medium for incubation at 37℃ for 16 h, were 1.59 x 109cfu/mL, 1.22 x 109cfu/mL, and 1.64 x 109cfu/mL, respectively.
If they were cultivated at 42℃ for 6 h, the 3 yogurt starters required different components in media to support their maximum growth. For strains LB2-ST1, the best medium was 1.0% of tomato extract, 1.0% of lactose, 0.5% of yeast extract and 1.0% of
peptone. For strains LB1-ST1, that was 2.5% of tomato extract, 0.5% of yeast extract and 1.5% of peptone. For strains Y-6-ST1 that was 1.0% of tomato extract, 0.3% of yeast extract and 0.5% of peptone. The cell population was up to 1.64×109cfu/mL, 1.34×109cfu/mL, and 2.04×109cfu/mL, respectively.
The optimal cryoprotectants to improve the viability of strains ST1, LB2 and LB2-ST1 were investigated. It was seen that the combination of trehalose (1.0%), glycerin (0.5%), glutamate-Na (15%) and 1.0% of sorbitan mono-oleate offered the optimal protection to the viability of the strain STL The mixture of trehalose (2.0%), glycerin (0.3%), glutamate-Na (5%) and Sorbitan mono-oieate (0.5%) favored the cells of strain LB2 to survive best in stress conditions. A combination consisting of trehalose (0.5%), glycerin (0.3%), glutamate-Na (5%) and Sorbitan mono-oleate (0.3%) produced a very positive effect on promoting the viability of the paired strains LB2-STI. Their viable cells after freeze-drying were 4.72 x10 11cfu/g, 1.34×10 11cfu/g and 3.57×10 11cfu/g, re
引文
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[23] Brian J B Wood，徐岩译．发酵食品微生物学(第二版)[M]．北京：中国轻工业出版社，2001，250～257．
[24] Boyaval N Y P. Concentrated starter productions in a two stage recycled bioreactor[J]. Plant Biotecnologyletters, 1992, 14: 589～592.
[25] Rurta S. Freezing of islated thylakiod membranes, in complex media[J]. Cryobiology, 1996, 33: 118～126.
[26] Wolff E. Free-drying of streptococcus thermophilus: a comparison between the vacuum and the atmospheric method [J]. Cryobiology, 1990, 27: 569～575.
[27] 张伦照．生物制品生产的基本技术(兽医生物制品)[M]．北京：农业出版社，1996，257～372．
[28] Kim W S, Khunajakr N, Dunn N W. Effect of cold shock on protein synthesis and on cryotolerance of cells frozen for long periods in Lactococcus lactis[J]. Cryobilogy, 1998, 37: 86～91.
[29] 谢继志，顾瑞霞．酸奶混合菌种连续继代培养对发酵剂稳定性的影响[J]．中国乳品工业，1995，23(1)：10～13．
[30] 于景华．喷涂法生产酸奶粉基料的研究[D]．东北农业大学，1992．
[31] 徐心军．高效嗜酸菌发酵剂的研制[J]．中国乳品工业，1990，18：237～238．
[32] 乔发东，南庆贤，兰风英．浓缩乳酸菌发酵剂冷冻干燥介质的选择[J]．中国乳品工业，
1998，26(4)：13～15．
[33] 刘宇峰，王金英．直接使用型酸奶发酵剂的研制[J]．中国乳品工业，1995，23(6)：274～279．
[34] 吕兵，张国农，肖光辉．新型酸奶发酵剂的研究[J]．中国乳品工业，1999，27(5)：28～32．
[35] 刘会平．高校浓缩乳酸菌发酵剂[J]．中国畜产与食品，1997，134～135．
[36] 张以芳．乳酸菌发酵的意义及应用现状[J]．微生物学研究与应用，1993，3：15～17．
[37] Sutherland I W. Bacterial exopolysaccharides[J]. Adv. Microbiol. Physiol., 1972, 8: 143～212.
[38] 谢继志．液态乳制品科学与技术[M]．北京：轻工业出版社，2000．
[39] 张建友，李艳武，赵群波，等．冻干乳酸菌菌种增菌培养基的优化[J]．中国乳品工业，2002，30(5)：14～17．
[40] 李志成，徐怀德，王敏，等．嗜热链球菌和保加利亚乳杆菌增菌培养研究[J]．西北农林科技大学学报，2002，30：50～52．
[41] 史媛英，肖冬光．酸奶发酵剂高浓度培养的研究[J]．天津轻工业学院学报，1999，1：20～25．
[42] 房兴利，晋津．酸奶生产液体发酵剂的研究[J]．中国乳品工业，1994，22(5)：207～208．
[43] 黄良昌，吕晓玲，姚秀玲，等．保加利亚乳杆菌浓缩培养的研究[J]．中国乳品工业，2002，30(1)：12～15．
[44] 吕加平，骆承庠．乳酸菌发酵剂冻干保护剂筛选及应用的研究[J]．东北农业大学学报，1998，29(4)：371～379．