减毒猪霍乱沙门氏菌C500抗冻保护剂的优化研究

doi:10.16431/j.cnki.1671-7236.2020.11.033

摘要/Abstract

摘要： 本试验旨在优化减毒猪霍乱沙门氏菌C500抗冻保护剂，提高工程菌C500的存活率。通过单因素试验考察了4种类型的抗冻保护剂（渗透性、半渗透性、非渗透性保护剂及抗氧化剂）对工程菌C500冷冻保存前后存活率的影响，各保护剂设置不同的浓度并与工程菌C500等体积混合，于液氮中保存2周后进行平板计数以计算其成活率，初步筛选出最有利于菌种保存的渗透性保护剂、半渗透性保护剂、非渗透性保护剂和抗氧化剂及其最佳保护浓度，最后将筛选出的4种保护剂进行四因素三水平的正交试验以得出最佳的组合配方。单因素试验的结果表明，渗透性保护剂中乙二醇、半渗透性保护剂中蔗糖、非渗透性保护剂中聚乙烯吡络烷酮（PVP）及抗氧化剂中甘氨酸对工程菌C500的保护作用最佳，最佳保存浓度分别为15%、6%、6%和0.25%；正交试验结果表明，各因素均对工程菌C500的存活率有显著影响，筛选出的最佳组合配方为17%乙二醇、9.0%蔗糖、6.0% PVP、0.35%甘氨酸，最佳组合在-80 ℃的保存效果最佳，此温度下保存6周存活率可达88.34%，其次为液氮组、-20 ℃组及4 ℃组。这表明本试验得到的保护剂配方极大地提高了工程菌C500的抗冻能力，使其存活率大大提高。

关键词: 抗冻保护剂; 减毒猪霍乱沙门氏菌C500; 正交试验

Abstract: The purpose of this study was to optimize the antifreeze protectant of attenuated Salmonella Cholerae C500 and improve the survival rate of C500.The effect of four types of cryoprotectant (permeating,semi-permeating,non-permeating cryoprotectants and antioxidants) on the survival rate of C500 before and after cryopreservation was investigated by single factor test.Each cryoprotectant was set at different concentrations and mixed in equal volume of C500.After two weeks of cryopreservation in liquid nitrogen,becteria plate count was carried out to calculate the survival rate,and the best one of the permeating cryoprotectant,semi-permeating cryoprotectant,non-permeating cryoprotectant and antioxidants and their optimal concentrations were beneficial to the preservation of bacteria was initially selected.Finally,the four protectors selected were tested by orthogonal test of four factors and three levels to get the optimal combination formula.The results of single factor test showed that the best permeating,semi-permeating,non-permeating cryoprotectants and antioxidants were glycol,sucrose,PVP and glycine,and the corresponding preservation concentrations were 15%,6%,6% and 0.25%,respectively.The orthogonal test showed that each factor had a significant impact on the survival rate of C500.The optimal combination formula was 17% glycol,9.0% sucrose,6.0% PVP and 0.35% glycine.The optimal combination had the optimal effect at -80 ℃,the survival rate was 88.34% in 6 weeks,followed by liquid nitrogen group,-20 ℃ group and 4 ℃ group.The results showed that the formulation of the protective agent obtained in this experiment greatly improved the antifreeze ability of C500 and its survival rate.

Key words: cryoprotectant; attenuated Salmonella Cholerae C500; orthogonal test

中图分类号:

S859.79⁺7

杨帅, 周迪, 张欣欣, 胡祥维, 杨利国. 减毒猪霍乱沙门氏菌C500抗冻保护剂的优化研究[J]. 中国畜牧兽医, 2020, 47(11): 3696-3704.

YANG Shuai, ZHOU Di, ZHANG Xinxin, HU Xiangwei, YANG Liguo. Optimization of Cryoprotectant for Attenuated Salmonella Cholerae C500[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3696-3704.

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