乳酸菌微包囊制备技术研究及其在酸奶连续发酵中的应用
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摘要
在我国当前的乳品工业中,继代式发酵剂和冷冻干燥型发酵剂的应用比较广泛,继代式发酵剂操作复杂,成品酸奶质量控制比较难；直投式发酵剂主要依赖进口,成本较高。针对这两种发酵剂存在的不足,本研究研制开发了一种新型发酵剂制备方法。这种方法制备的液芯微包囊发酵剂,不仅克服了现在使用的两类发酵剂的不足,简化了生产工艺,而且降低了生产成本。应用于酸奶的连续接种发酵中,不仅可以保证各批次酸奶成品的质量,而且较传统的间歇式酸奶生产方法可节约生产空间,缩短整个酸奶制作加工时间。因此,乳酸菌微包囊技术研究为传统酸奶发酵技术的革新提供了理论指导和技术支持,具有实际应用价值。
主要研究结果如下：
(1)设计并制作了一套液芯微包囊化细胞无菌制备装置,应用该装置,在无菌条件下实现了液芯微包囊化细胞制备。经过测试,该液芯微包囊化细胞无菌制备装置符合设定的工艺参数,可以满足液芯微包囊化细胞无菌制备的需求。
(2)采用中心组合设计优化了用于高强度液芯微包囊制备条件,优化的微包囊制备条件为：黄原胶2.24g/L,氯化钙3.98%,海藻酸钠0.68g/L,壳聚糖3.19g/L成膜时间48min。
(3)生物相容性研究结果显示：黄原胶对唾液链球菌嗜热亚种的生长基本没有影响,高浓度的氯化钙对嗜热链球菌的生长有明显的抑制作用,并且随浓度的增高,抑制作用越明显,甚至产生毒害作用；在制微包囊过程中以及在用液芯微包囊对唾液链球菌嗜热亚种培养过程中,都显示了良好的生物相容性,并且在液芯微包囊中固定化培养的条件下更有利于菌体的生长,菌体的数量提高了两个数量级。因此作为整个体系来说,该微包囊可用于乳酸菌的固定化培养。
(4)微包囊释放性能研究结果显示：壳聚糖乙酸溶液浓度、氯化钙浓度和成膜时间对德式乳杆菌保加利亚亚种的释放性能影响都很大；当海藻酸钠浓度满足钙离子的配位需求后,其浓度对唾液链球菌嗜热亚种的释放性能影响不大。作为整个体系来说,可以通过改变液芯微包囊组分的浓度和成膜时间来控制液芯微包囊的通透性,从而改变唾液链球菌嗜热亚种的释放性能,对实现控制培养液中菌体密度具有重要的意义
(5)采用析因设计和响应曲面设计优化了用于微包囊化德式乳杆菌保加利亚亚种高密度培养的培养基,其配方为乳清粉97.15g/L,大豆蛋白粉20g/L,酵母粉7.55g/L,碳酸钙8.03g/L,硫酸镁0.3g/L,硫酸锰0.02g/L；微包囊化唾液链球菌嗜热亚种高密度培养的培养基,其配方为乳清粉103g/L,大豆蛋白粉10g/L,酵母粉6.8g/L,碳酸钙7.2g/L,硫酸镁0.3g/L,硫酸锰0.02g/L
(6)用中心组合设计(CCD)优化了微包囊化乳酸菌培养条件,最佳的培养条件：微包囊化德式乳杆菌保加利亚亚种培养温度为41.7℃,初始pH为6.9；微包囊化唾液链球菌嗜热亚种培养温度为44℃,初始pH为6.8。在优化条件下对微包囊化乳酸菌的高密度培养,唾液链球菌嗜热亚种囊内德式乳杆菌保加利亚亚种细胞密度达到3.18×1011cfu/g;囊内唾液链球菌嗜热亚种细胞密度达到2.63×1011cfu/g。
(7)通过单因素和正交实验得到液芯微包囊发酵剂的最佳保护储存液,其组成为氯化钠0.9%,蔗糖3%,海藻糖5%,甘油8%,碳酸钙0.2%。按照此配方用去离子水配制储存保护液,置于121℃灭菌20min,然后将制备好的高密度微包囊发酵剂放入储存保护液中于4℃条件下保存,30天后对微包囊中菌体密度和微包囊强度进行检测,菌体密度为1.54×1011cfu/g,微包囊强度为85.95g。
(8)用中心组合设计对应用微包囊化乳酸菌作为发酵剂的牛奶连续接种各参数进行优化,获得了牛奶连续接种的温度、pH、长径比最优参数组合分别是：41.22℃、6.86、4.99。
(9)通过对液芯微包囊发酵剂连续接种时间对接种牛奶中菌体密度、微包囊强度、无菌柱式生物反应器中牛奶的稀释率、发酵牛奶的感官评定总分等影响的结果,结合酸奶工业生产实际需求,确定液芯微包囊发酵剂连续接种使用时间为24d
Traditional subcultural and frozen-dried yoghurt starter cultures are used widely in Chinese dairy industry nowadays. However, traditional subcultural yoghurt starters require complex preparation processes and, thus resulted in difficulties for the yoghurt quality control. Meanwhile, frozen-dried starter cultures are mainly imported and with high costs. Therefore, this study is aimed to develop a novel technique for producing starters, in order to overcome the shortcomings of traditional subcultural starters and imported starters, simplify the producing techniques, and reduce the cost. Application of this novel immobilized starter to continuous yoghourt fermentation can ensure the quality of different batches of products, save more spaces than traditional techniques, and shorten processing time.
In this thesis, a set of microencapsulated cell aseptic preparation combined system is designed and manufactured. In this system, preparation conditions, intensity, biocompatibility and releasing property were studied systematically. High-density media and culture conditions of Streptococcus thermophilu and Lactobacillus bulgaricus were optimized, reaching1011cfu/ml. The microcapsules produced were used for continuously fermentation of yoghourt. The conditions of continuous inoculation were optimized, and the quality of yoghourt reached the national standards. The results will provide foundation for the innovation of traditional yoghourt fermentation technology.
The main results are given as follows:
(1) Central composite design was used to optimize the preparation conditions of high-intensity liquid-core microcapsules. The optimum conditions for the microcapsules were CaCl23.98%, xanthan gum2.24g/L, sodium alginate0.68g/L, chitosan3.19g/L and the film-forming time48min.
(2) Biocompatibility assay indicated that xanthan gum had few effects on the growth of S. thermophilus, while CaCl2with high concentration inhibited the growth of S. thermophilus significantly. The inhibition showed a concentration-dependent matter, and may even convert to poisoning effect when concentration reached some point. But overall, good biocompatibility appeared during the preparation and culturing, and the immobilized cells in liquid-core microcapsules facilitated the growth of bacteria. The number of immobilized S. thermophilus was100times higher than that of free cultivation. Thus, microcapsules can be used for immobilized culture of LAB.
(3) Study of the releasing property indicated that chitosan concentration, CaCl2concetration and film-forming time had great effects on permeability. When the concentration of sodium alginate met the need of complexation of calcium ionic, it had little effect on the release of Streptococcus thermophilus. For the entire system, the permeabiligy can be controlled by means of changing the composition of capsules and film-forming time, which is significant to achieve the control of bacteria density in media.
(4) Factorial design and response surface design were used to optimize the media for the high-density culture of microencapsuled bacteria. The obtained compositions for L. bulgaricus was:whey powder97.15g/L, soybean powder20g/L, yeast powder7.55g/L, CaCO38.03g/L, MgSO40.3g/L, MnSO40.02g/L. The composition for S. thermophilus was: whey powder103g/L, soybean powder10g/L, yeast powder6.8g/L, CaCO37.2g/L, MgSO40.3g/L, MnSO40.02g/L.
(5) Culturing conditions were optimized by central composite design. The optimal conditions were as follows:temperature41.7℃and initial pH6.9for L.bulgaricus, and temperature44℃and initial pH6.8for S. thermophilus.
(6) Cell density of S. thermophilus and L. bulgaricus in the capsule reached3.18×1011cfu/g and2.63×1011cfu/g in the optimized media and culture conditions.
(7) The best composition of storage liquid was obtained through single factor experiment and orthogonal experiment, which showed a composition of NaCl0.9%, sucrose3%, fucose5%, glycerol8%, and CaCO30.2%. After one month in the storage liquid at4℃, the cell density remained1.54×1011cfu/g, and the intensity of capsule85.95g。
(8) Optimized parameters of continuous dairy inoculation were obtained using central composite design, with the temperature41.22℃, pH6.86, and ratio of length to diameter4.99。
(9) Considering the effects of continuous inoculation time on cell density, capsule intensity, dilution rate of dairy in bioreactor, and sensory assess of yoghourt, combined with manufacturing cost and efficiency, the utilization period of liquid-core microcapsule for continuous inoculation is24days.
主要研究结果如下：
(1)设计并制作了一套液芯微包囊化细胞无菌制备装置,应用该装置,在无菌条件下实现了液芯微包囊化细胞制备。经过测试,该液芯微包囊化细胞无菌制备装置符合设定的工艺参数,可以满足液芯微包囊化细胞无菌制备的需求。
(2)采用中心组合设计优化了用于高强度液芯微包囊制备条件,优化的微包囊制备条件为：黄原胶2.24g/L,氯化钙3.98%,海藻酸钠0.68g/L,壳聚糖3.19g/L成膜时间48min。
(3)生物相容性研究结果显示：黄原胶对唾液链球菌嗜热亚种的生长基本没有影响,高浓度的氯化钙对嗜热链球菌的生长有明显的抑制作用,并且随浓度的增高,抑制作用越明显,甚至产生毒害作用；在制微包囊过程中以及在用液芯微包囊对唾液链球菌嗜热亚种培养过程中,都显示了良好的生物相容性,并且在液芯微包囊中固定化培养的条件下更有利于菌体的生长,菌体的数量提高了两个数量级。因此作为整个体系来说,该微包囊可用于乳酸菌的固定化培养。
(4)微包囊释放性能研究结果显示：壳聚糖乙酸溶液浓度、氯化钙浓度和成膜时间对德式乳杆菌保加利亚亚种的释放性能影响都很大；当海藻酸钠浓度满足钙离子的配位需求后,其浓度对唾液链球菌嗜热亚种的释放性能影响不大。作为整个体系来说,可以通过改变液芯微包囊组分的浓度和成膜时间来控制液芯微包囊的通透性,从而改变唾液链球菌嗜热亚种的释放性能,对实现控制培养液中菌体密度具有重要的意义
(5)采用析因设计和响应曲面设计优化了用于微包囊化德式乳杆菌保加利亚亚种高密度培养的培养基,其配方为乳清粉97.15g/L,大豆蛋白粉20g/L,酵母粉7.55g/L,碳酸钙8.03g/L,硫酸镁0.3g/L,硫酸锰0.02g/L；微包囊化唾液链球菌嗜热亚种高密度培养的培养基,其配方为乳清粉103g/L,大豆蛋白粉10g/L,酵母粉6.8g/L,碳酸钙7.2g/L,硫酸镁0.3g/L,硫酸锰0.02g/L
(6)用中心组合设计(CCD)优化了微包囊化乳酸菌培养条件,最佳的培养条件：微包囊化德式乳杆菌保加利亚亚种培养温度为41.7℃,初始pH为6.9；微包囊化唾液链球菌嗜热亚种培养温度为44℃,初始pH为6.8。在优化条件下对微包囊化乳酸菌的高密度培养,唾液链球菌嗜热亚种囊内德式乳杆菌保加利亚亚种细胞密度达到3.18×1011cfu/g;囊内唾液链球菌嗜热亚种细胞密度达到2.63×1011cfu/g。
(7)通过单因素和正交实验得到液芯微包囊发酵剂的最佳保护储存液,其组成为氯化钠0.9%,蔗糖3%,海藻糖5%,甘油8%,碳酸钙0.2%。按照此配方用去离子水配制储存保护液,置于121℃灭菌20min,然后将制备好的高密度微包囊发酵剂放入储存保护液中于4℃条件下保存,30天后对微包囊中菌体密度和微包囊强度进行检测,菌体密度为1.54×1011cfu/g,微包囊强度为85.95g。
(8)用中心组合设计对应用微包囊化乳酸菌作为发酵剂的牛奶连续接种各参数进行优化,获得了牛奶连续接种的温度、pH、长径比最优参数组合分别是：41.22℃、6.86、4.99。
(9)通过对液芯微包囊发酵剂连续接种时间对接种牛奶中菌体密度、微包囊强度、无菌柱式生物反应器中牛奶的稀释率、发酵牛奶的感官评定总分等影响的结果,结合酸奶工业生产实际需求,确定液芯微包囊发酵剂连续接种使用时间为24d
Traditional subcultural and frozen-dried yoghurt starter cultures are used widely in Chinese dairy industry nowadays. However, traditional subcultural yoghurt starters require complex preparation processes and, thus resulted in difficulties for the yoghurt quality control. Meanwhile, frozen-dried starter cultures are mainly imported and with high costs. Therefore, this study is aimed to develop a novel technique for producing starters, in order to overcome the shortcomings of traditional subcultural starters and imported starters, simplify the producing techniques, and reduce the cost. Application of this novel immobilized starter to continuous yoghourt fermentation can ensure the quality of different batches of products, save more spaces than traditional techniques, and shorten processing time.
In this thesis, a set of microencapsulated cell aseptic preparation combined system is designed and manufactured. In this system, preparation conditions, intensity, biocompatibility and releasing property were studied systematically. High-density media and culture conditions of Streptococcus thermophilu and Lactobacillus bulgaricus were optimized, reaching1011cfu/ml. The microcapsules produced were used for continuously fermentation of yoghourt. The conditions of continuous inoculation were optimized, and the quality of yoghourt reached the national standards. The results will provide foundation for the innovation of traditional yoghourt fermentation technology.
The main results are given as follows:
(1) Central composite design was used to optimize the preparation conditions of high-intensity liquid-core microcapsules. The optimum conditions for the microcapsules were CaCl23.98%, xanthan gum2.24g/L, sodium alginate0.68g/L, chitosan3.19g/L and the film-forming time48min.
(2) Biocompatibility assay indicated that xanthan gum had few effects on the growth of S. thermophilus, while CaCl2with high concentration inhibited the growth of S. thermophilus significantly. The inhibition showed a concentration-dependent matter, and may even convert to poisoning effect when concentration reached some point. But overall, good biocompatibility appeared during the preparation and culturing, and the immobilized cells in liquid-core microcapsules facilitated the growth of bacteria. The number of immobilized S. thermophilus was100times higher than that of free cultivation. Thus, microcapsules can be used for immobilized culture of LAB.
(3) Study of the releasing property indicated that chitosan concentration, CaCl2concetration and film-forming time had great effects on permeability. When the concentration of sodium alginate met the need of complexation of calcium ionic, it had little effect on the release of Streptococcus thermophilus. For the entire system, the permeabiligy can be controlled by means of changing the composition of capsules and film-forming time, which is significant to achieve the control of bacteria density in media.
(4) Factorial design and response surface design were used to optimize the media for the high-density culture of microencapsuled bacteria. The obtained compositions for L. bulgaricus was:whey powder97.15g/L, soybean powder20g/L, yeast powder7.55g/L, CaCO38.03g/L, MgSO40.3g/L, MnSO40.02g/L. The composition for S. thermophilus was: whey powder103g/L, soybean powder10g/L, yeast powder6.8g/L, CaCO37.2g/L, MgSO40.3g/L, MnSO40.02g/L.
(5) Culturing conditions were optimized by central composite design. The optimal conditions were as follows:temperature41.7℃and initial pH6.9for L.bulgaricus, and temperature44℃and initial pH6.8for S. thermophilus.
(6) Cell density of S. thermophilus and L. bulgaricus in the capsule reached3.18×1011cfu/g and2.63×1011cfu/g in the optimized media and culture conditions.
(7) The best composition of storage liquid was obtained through single factor experiment and orthogonal experiment, which showed a composition of NaCl0.9%, sucrose3%, fucose5%, glycerol8%, and CaCO30.2%. After one month in the storage liquid at4℃, the cell density remained1.54×1011cfu/g, and the intensity of capsule85.95g。
(8) Optimized parameters of continuous dairy inoculation were obtained using central composite design, with the temperature41.22℃, pH6.86, and ratio of length to diameter4.99。
(9) Considering the effects of continuous inoculation time on cell density, capsule intensity, dilution rate of dairy in bioreactor, and sensory assess of yoghourt, combined with manufacturing cost and efficiency, the utilization period of liquid-core microcapsule for continuous inoculation is24days.
引文
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