三华李果坯发酵液中腐败真菌分离鉴定、相关抑菌剂效价评定及发酵工艺优化

Isolation and Identification of Spoilage Fungi in the Fermentation Broth of Sanhua Plum, Evaluation of the Potency of Related Antibacterial Agents and Optimization of Fermentation Technology

为了探究三华李果坯接种乳酸菌发酵异常的原因,改善发酵工艺,以表面长有白色菌膜的异常发酵液为分离源,对其中的腐败真菌进行分离纯化,通过形态学特征观察和ITS序列鉴定,得到3株优势腐败真菌,分别为Candida stellimalicola、Pichia kudriavzevii和Pichia occidentalis;用牛津杯法评定了山梨酸、纳他霉素及二甲基二碳酸盐的抑菌效价,发现3种抑菌剂作用于上述3株腐败真菌,均出现了明显抑菌圈,二甲基二碳酸盐对发酵所用的干酪乳杆菌略有抑制作用,抑菌圈直径为(10.45±0.11)mm,说明3种抑菌剂可以用于抑制腐败真菌的生长繁殖但不会影响发酵的正常进行;用棋盘稀释法测定了抑菌剂的联合抑菌效力,山梨酸与纳他霉素联合作用于Pichia kudriavzevii的部分抑菌浓度指数为0.50;山梨酸与二甲基二碳酸盐联合作用于Candida stellimalicola和Pichia occidentalis的部分抑菌浓度指数分别为0.50和0.38,均表现出协同抑菌作用;采用响应面法优化发酵防腐工艺,显示在发酵液中按果重添加质量浓度0.040%山梨酸、0.010%纳他霉素和0.015%二甲基二碳酸盐,室温密封发酵30 d后,测得发酵液中霉菌酵母活菌数为3.212 lg CFU/mL,远低于旧工艺发酵液中霉菌酵母活菌数,发酵液表面不再出现白膜。新工艺简单有效、效果稳定,解决了发酵异常导致的果坯软化、产生异味等问题。

In order to explore the causes of abnormal fermentation of Sanhua plum fruit by inoculating lactic acid bacteria and improve the fermentation process,the spoilage fungi were isolated and purified from the abnormal fermentation broth with white bacterial membrane on the surface,through morphological observation and ITS sequence identification,three dominant spoilage fungi were obtained,namely Candida stellimalicola,Pichia kudriavzevii and Pichia occidentalis;the Oxford cup method was used to evaluate antibacterial activity of sorbic acid,natamycin and dimethyldicarbonate,it was found that the three kinds of antibacterial agents had obvious inhibition zones on three strains of spoilage fungi,dimethyldicarbonate had slight inhibition on Lactobacillus casei used in fermentation,the diameter of inhibition zone was 10.45±0.11 mm,which indicated that the three antimicrobial agents could be used to inhibit the growth and reproduction of spoilage fungi without affecting the normal fermentation;the combined bacteriostatic effect of antibacterial agents was determined by chessboard dilution method,natamycin on Pichia kudriavzevii was 0.50;the combined inhibitory effect of sorbic acid and dimethyldicarbonate on Candida stellimalicola and Pichia occidentalis was 0.50 and 0.38,respectively,all of them showed synergistic antibacterial effect;the fermentation process was optimized by response surface methodology;the results showed that 0.040%sorbic acid,0.010%natamycin and 0.015%dimethyldicarbonate were added to the fermentation broth according to the fruit weight,fermentation time was 30 days at room temperature,the number of viable yeast in the fermentation broth was 3.212 lg CFU/mL,which was far lower than the number of viable yeast and mold in the fermentation broth of the old process,there was no white film on the surface of the fermentation broth.The new process was simple,effective and stable,the problems of softening and peculiar smell caused by abnormal fermentation were solved.