应用响应面法优化副猪嗜血杆菌冻干保护剂

Optimization of lyoprotectant for Haemophilus parasuis based on Box-Behnken response surface methodology

旨在对副猪嗜血杆菌HS1712冻干保护剂进行优化,提高菌株在冷冻干燥后的存活率。以冻干存活率为评价指标,对配方中甘油、海藻糖、甘露醇、聚乙烯吡咯烷酮(PVP)、牛血清白蛋白(BSA)等5种成分缺失,考察其对副猪嗜血杆菌冻干存活率的影响。在此基础之上,结合单因素变量试验,应用Box-Behnken响应面法对脱脂奶粉、甘油、海藻糖的添加比例进一步优化。结果表明,配方中甘油、海藻糖缺失后,副猪嗜血杆菌冻干存活率显著下降(P<0.05)。通过对Box-Behnken试验结果进行回归模型拟合,得到最佳添加比例为脱脂奶粉6.8%、甘油2.2%、海藻糖2.7%,具体配方成分及添加比例为脱脂奶粉6.8%、甘油2.2%、海藻糖2.7%、PVP 0.5%。使用优化后配方进行冻干验证,副猪嗜血杆菌HS1712平均冻干存活率为79.20%,与预测值(80.87%)无显著性差异(P>0.05),表示模型可靠。上述结果表明,优化后的冻干保护剂可显著提高副猪嗜血杆菌HS1712冷冻干燥后的存活率。

This study aimed to optimize the lyoprotectant to improve the viability of Haemophilus parasuis(H.parasuis)HS1712under lyophilized conditions.Taking the freeze-drying survival rate of H.parasuis HS1712as the experiment indexes,the effects of ingredients in the formulation were investigated by lacking glycerol,trehalose,mannitol,L-ascorbate sodium,bovine albumin(BSA)and polyvinylpyrrolidone(PVP),respectively.On this basis,combined with single-factor experiment,Box-Behnken response surface method was applied to further optimize the coefficient proportion of skim milk powder,glycerin and trehalose.The results showed that the viability of H.parasuis HS1712under lyophilized conditions was significantly decreased when glycerin and trehalose were missing in the formulation(P<0.05).Through the regression model fitting of the Box-Behnken experimental results,the coefficient optimum proportion was obtained as skim milk powder 6.8%,glycerin 2.2%,trehalose 2.7%,the whole ingredients of formulation were skim milk powder 6.8%,glycerin 2.2%,trehalose 2.7%,PVP 0.5%.Using the optimized formulation for freeze-drying,the average freeze-drying survival rate of H.parasuis HS1712was 79.20%,which was not significantly different from the predicted value(80.87%)(P>0.05),indicating that the model was reliable.These results indicated that the optimized lyoprotectant could significantly improve the viability of H.parasuis HS1712under lyophilized conditions.