Study On The Preparation Of Lactic Acid Bacteria Microcapsule And Its Application To The Continuous Milk Fermentation

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.