中草药饲料添加剂发酵工艺优化及安全性试验

Fermentation process optimization and safety test of Chinese herbal medicine feed additives

为了解复合益生菌对复方中草药饲料添加剂的最佳发酵条件,并考察发酵复方中草药饲料添加剂对小鼠的急性毒性和亚慢性毒性,试验以发酵中草药中总黄酮得率(亚硝酸钠-硝酸铝-氢氧化钠比色法测定)为指标,采用单因素试验和正交试验对混合菌种比例、菌液接种量、发酵温度、发酵时间进行了优化,并在最佳发酵工艺条件下测量发酵中草药中总黄酮质量浓度。在急性毒性试验中,将40只小鼠随机均分为高(按体重20 mg/kg灌胃)、中(按体重15 mg/kg灌胃)、低(按体重10 mg/kg灌胃)剂量组和对照组(按体重10 mL/kg灌胃无菌生理盐水),灌胃后连续观察7 d,每天记录小鼠的精神状态、饮食、行为及死亡情况;在亚慢性毒性试验中,将40只小鼠随机均分为高(按体重15 mg/kg灌胃)、中(按体重10 mg/kg灌胃)、低(按体重5 mg/kg灌胃)剂量组和对照组(按体重10 mL/kg灌胃无菌生理盐水),每天灌胃1次,连续90 d,给药结束后继续观察28 d,最后1天采集小鼠血液及器官,计算脏器系数,检测血清生化指标及观察组织病理学变化。结果表明:最佳发酵工艺条件为解淀粉芽孢杆菌与干酪乳杆菌接种比例(V/V)1∶4、接种量1.0%、发酵温度37℃、发酵时间72 h。在此工艺条件下发酵后中草药总黄酮质量浓度平均值为3.032 g/L,符合预期。在小鼠急性毒性试验中未能检测出半数致死剂量(LD50)。在亚慢性毒性试验中,在停药后第28天各组小鼠脏器系数均差异不显著(P>0.05),血清生化指标(除天冬氨酸氨基转移酶、碱性磷酸酶外)差异不显著(P>0.05)。说明发酵中草药饲料添加剂安全性好,无毒副作用。

In order to understand the optimal fermentation technology of complex probiotics on compound Chinese herbal medicine feed additives,and investigate the acute and subchronic toxicity of fermented compound Chinese herbal feed additives on mice,the yield of total flavonoids in fermented Chinese herbal medicine(determined by sodium nitrite-aluminum nitrite-sodium hydroxide method)was used as an indicator,and the proportion of mixed strains,inoculation amount of bacterial solution,fermentation temperature and fermentation time of mixed bacteria were optimized by single factor experiment and orthogonal test,and the mass concentration of total flavonoids in fermented Chinese herbal medicines was measured under the optimal process parameters.In the acute toxicity test,40 mice were randomly divided into high dose(gavage 20 mg/kg according to body weight),medium dose(gavage 15 mg/kg according to body weight),low dose(gavage 10 mg/kg according to body weight)and control group(gavage 10 mL/kg sterile saline according to body weight),which were continuously observed for 7 days after gavage.The mental state,diet,behavior and death of the mice were recorded every day.In the subchronic toxicity test,40 mice were randomly divided into high dose(gavage 15 mg/kg according to body weight),medium dose(gavage 10 mg/kg according to body weight),low dose(gavage 5 mg/kg according to body weight)and control group(gavage 10 mL/kg sterile saline according to body weight).Intragastric administration was performed once a day for 90 days.After the end of administration,the observation continued for 28 days.On the last day,blood and organs of mice were collected;organ coefficients were calculated;serum biochemical indexes were detected and histopathological changes were observed.The results showed that the optimum fermentation conditions were as follows:the ratio of Bacillus amyloliquefaciens to Lactobacillus casei was 1∶4(V/V);the inoculum amount was 1.0%;the fermentation temperature was 37℃,and the fermentation time was 72 h.The average mass contentration of total flavonoids in Chinese herbal medicine after fermentation was 3.032 g/L,which was in line with expectation.In the acute toxicity test,no median lethal dose(LD_(50))was detected.In the subchronic toxicity test,there was no significant difference in the organ coefficient and serum biochemical indexes(except for aspartate aminotransferase and alkaline phosphatase)of mice in each group at 28th day after drug withdrawal(P>0.05).The results indicated that fermented Chinese herbal feed additives was safe and harmless.