响应面法对纳豆菌冷冻保护剂的优化研究

Optimization of Cryoprotectants for Bacillus Natto by Response Surface Methodology

首先采用单因素试验,考察5种不同保护剂对冻干前后菌种存活率的影响程度,然后采用Plackett-Burman二水平因子设计试验选出3种影响显著的保护剂,再通过最陡爬坡、响应面中心组合设计,最终得出最优组合配方。试验结果表明,五种保护剂(麦芽糊精、柠檬酸钠、甘露醇、抗坏血酸和甘油)进行的单因素试验,其中麦芽糊精、抗坏血酸和甘油的作用最为显著。采用响应面优化这三个因素,以冻干后的纳豆菌存活率为响应目标,预测最优结果为:麦芽糊精5.51%、抗坏血酸0.59%、甘油4.98%,并通过此配比组合做了3次重复试验验证,纳豆菌的平均存活率达到92.56%,与理论预测值91.72%非常接近。用此配方冻干样品时,能使纳豆菌存活率比添加单一保护剂时最少提高65.29%,对于纳豆菌冻干生物制剂的研究有很大参考价值。

The objective of this research is to improve the tolerance of Bacillus natto in freeze drying in the environment and the optimization of the protective agent. Specific method is the Pb two level factorial design experiment select significant factors, the steepest ascent, response surface central composite design combined with the method of screening and optimization of the Bacillus natto freeze drying protective agent formula. The experimental results show that with the five kinds of protective agent （maltodextrin, citric acid sodium, mannitol, ascorbic acid and glycerol） by single factor experiment, maltodextrin, ascorbic acid and glycerol are the most outstanding, which can significantly improve the cell tolerance to freezing and dry environment. To freeze dry cell survival in response to the target, response surface methodology is used to optimize the prediction results： 5.51% maltodextrin, ascorbic acid 0.59% and glycerol 4.98%, and through 3 experiments of the combination of the ratio, average survival rate of Bacillus natto reached 92.56%, and theoretical prediction 91.72% values are very close. The formula of freeze-dried samples, can make the Bacillus natto survival rate than the added protection of single agent at least 65.29% for Bacillus natto freeze-dried biological agents, which have great reference value.