| Vacuum freeze drying is one of the key drying technologies for the preparation of lactic acid bacteria preparations at home and abroad,which can retain the maximum biological activity of the bacteria while removing more water.Pre-freezing is a key step in freeze-drying,and proper pre-freezing can effectively reduce lyophilization damage of the bacterium.In this thesis,Lactobacillus helveticus H11 was used as the object of study to prepare lactic acid bacteria preparations by vacuum freeze-drying technology,and single-factor and response surface analysis were conducted to determine the optimal pre-freezing process parameters for the survival rate of the bacteria under different pre-freezing process parameters.The morphological characteristics,membrane integrity changes,survival rate changes during storage and key enzyme activity differences of the freeze-dried bacteria under the parameters were observed to evaluate the stability of the bacteria and storage after process optimization,and the results of the study were as follows:(1)The results of single-factor test for the preparation of Lactobacillus helveticus H11 by vacuum freeze-drying method showed that the effects on the lyophilization survival rate of the bacteria were material thickness,p H of bacterial suspension,pre-freezing temperature and pre-freezing time in descending order.After optimization by response surface method,the best vacuum freezing process conditions were determined as-35℃,3h pre-freezing time,6.5 p H of bacterial suspension,and 0.37 cm thickness of material,and the freeze-drying survival rate was 81.57%.(2)The morphology of the vacuum freeze-dried powder was observed by scanning electron microscopy,and the morphology of the bacteriophage after the optimization of the vacuum freezing process was more complete than that before the optimization,without obvious damage and wrinkling,which effectively reduced the leakage of intracellular material and significantly improved the viability of the bacteriophage(from 62.84% to81.57%,P<0.05).The changes in membrane integrity of the bacterium before and after optimization were compared by flow cytometry,and it was found that the percentage of damage to the membrane of the bacterium decreased significantly(P < 0.05)and the percentage of intact cell membrane increased significantly(P < 0.05)after optimization of the vacuum freezing process,indicating that the optimized process significantly reduced the damage to the cell membrane of the bacterium during the freeze-drying process.(3)The survival rate and key enzyme activity of Lactobacillus helveticus H11 were analysed before and after the optimisation of the vacuum freeze-drying process at 4℃ and25℃ for 14 d.The survival rates of the optimised powder at 4℃ and 25℃ were 94.23%and 90.26%,which were significantly higher than those of the control group at 92.15% and86.42%(P<0.05),indicating that the optimised freeze-drying process better The optimized lyophilization process was found to be better in maintaining the activity of the bacterial powder.The overall trend of lactate dehydrogenase activity decreased,but the inactivation rate of enzyme activity in the control group was significantly higher than that in the experimental group(P<0.05).β-galactosidase leakage in the experimental group was significantly lower than that in the control group(P<0.05).The results show that the optimised powder has a higher survival rate,which is conducive to stable storage and better maintenance of activity.The aim of this study is to optimize the pre-freezing process,determine the optimal process parameters and analyse the cell membrane damage of Lactobacillus helveticus H11 powder and the trend of freeze-dried survival and activity during storage,in order to provide a theoretical basis and technical support for the industrial production of Lactobacillus helveticus H11 related lactic acid bacteria preparations. |
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