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
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摘要: 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。Abstract: 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±0.11mm, 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.
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图 1 棋盘稀释法加抑菌剂示意图
Figure 1. Schematic diagram of bacteriostatic agent added by checkerboard dilution method
图 2 发酵液中优势腐败真菌的菌落形态
Figure 2. Colony morphology of dominant spoilage fungi in fermentation broth
注：（a）C2菌株；（b）6H7菌株；（c）6H8菌株。
图 3 优势酵母菌镜检下的细胞形态（×800）
Figure 3. Cell morphology of dominant yeasts under microscope（×800）
注:（a）C2菌株；（b）6H7菌株；（c）6H8菌株。
图 4 优势腐败真菌PCR扩增产物琼脂糖凝胶电泳结果
Figure 4. Agarose gel electrophoresis results of PCR amplification products of dominant spoilage fungi
图 5 基于C2菌株ITS序列及其相似菌株的系统发育树
Figure 5. Phylogenetic tree based on the ITS sequence of C2 strain and its similar strains
图 6 基于6H7菌株ITS序列及相似菌株的系统发育树
Figure 6. Phylogenetic tree based on the ITS sequence of 6H7 strain and similar strain
图 7 基于6H8菌株ITS序列及相似菌株的系统发育树
Figure 7. Phylogenetic tree based on ITS sequence of 6H8 strain and similar strains
表 1 Box-Behnken试验因素水平设计表
Table 1. Box-Behnken test factors level design table
水平 因素 A 山梨酸（%） B 纳他霉素（%） C 二甲基二碳酸盐（%） −1 0.020 0.010 0.015 0 0.030 0.020 0.020 1 0.040 0.030 0.025 注：抑菌剂添加量按果重百分比计。 
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表 2 不同抑菌剂对供试菌的抑菌效力
Table 2. Antibacterial efficacy of different antibacterial agents on the tested bacteria
菌株 抑菌圈直径（mm） 二甲基二碳酸盐 纳他霉素 山梨酸 C2 25.35±0.36a 14.35±0.54c 20.80±0.71b 6H7 24.21±0.74a 19.39±0.71b 13.75±0.92c 6H8 24.16±0.31b 26.71±1.00a 13.22±0.24c 植物乳杆菌 × × × 干酪乳杆菌 10.45±0.11 × × 肠膜明串珠菌 × × × 注：“×”表示无抑菌圈；字母表示不同抑菌剂处理同一菌株的显著性差异，P<0.05。
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表 3 山梨酸对腐败酵母菌的MIC和MBC值
Table 3. MIC and MBC values of sorbic acid on spoilage yeast
菌株 MIC（μg/mL） MBC（μg/mL） C2 62.50 125.00 6H7 125.00 250.00 6H8 125.00 250.00
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表 4 纳他霉素对腐败酵母菌的MIC和MBC值
Table 4. MIC and MBC values of natamycin on spoilage yeast
菌株 MIC（μg/mL） MBC（μg/mL） C2 7.81 31.25 6H7 3.91 31.25 6H8 7.81 31.25
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表 5 二甲基二碳酸盐对腐败酵母菌的MIC和MBC值
Table 5. MIC and MBC values of dimethyl dicarbonate on spoilage yeast
菌株 MIC（μg/mL） MBC（mg/mL） C2 125.00 40.00 6H7 125.00 20.00 6H8 250.00 40.00
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表 6 山梨酸与纳他霉素对三株供试酵母菌联合抑菌的结果
Table 6. The combined antibacterial results of sorbic acid and natamycin on the three tested yeasts
菌株 MIC单独（μg/mL） MIC联合（μg/mL） FICI 作用类型 山梨酸 纳他霉素 山梨酸 纳他霉素 C2 62.50 7.81 1.95 3.91 0.53 部分协同或相加 6H7 125.00 3.91 31.25 0.98 0.50 协同 6H8 125.00 7.81 3.91 3.91 0.53 部分协同或相加
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表 7 山梨酸与二甲基二碳酸盐对三株供试酵母菌联合抑菌的结果
Table 7. The combined antibacterial results of sorbic acid and dimethyl dicarbonate on the three tested yeasts
菌株 MIC单独（μg/mL） MIC联合（μg/mL） FICI 作用类型 山梨酸 二甲基二碳酸盐 山梨酸 二甲基二碳酸盐 C2 62.50 125.00 15.63 31.25 0.50 协同 6H7 125.00 125.00 3.91 62.50 0.53 部分协同或相加 6H8 125.00 250.00 15.63 62.50 0.38 协同
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表 8 纳他霉素与二甲基二碳酸盐对三株供试酵母菌联合抑菌的结果
Table 8. The combined antibacterial results of natamycin and dimethyl dicarbonate on the three tested yeasts
菌株 MIC单独（μg/mL） MIC联合（μg/mL） FICI 作用类型 纳他霉素 二甲基二碳酸盐 纳他霉素 二甲基二碳酸盐 C2 7.81 125.00 3.91 62.50 1.00 部分协同或相加 6H7 3.91 125.00 1.95 62.50 1.00 部分协同或相加 6H8 7.81 250.00 0.98 125.00 0.63 部分协同或相加
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表 9 响应面方案设计及试验结果
Table 9. Response surface scheme design and test results
试验序号 A B C 霉菌酵母
活菌数（lg CFU/mL）1 −1 −1 0 4.021 2 1 −1 0 4.040 3 −1 1 0 5.151 4 1 1 0 4.796 5 −1 0 −1 5.841 6 1 0 −1 4.040 7 −1 0 1 5.349 8 1 0 1 6.060 9 0 −1 −1 4.526 10 0 1 −1 5.088 11 0 −1 1 4.128 12 0 1 1 4.123 13 0 0 0 4.841 14 0 0 0 4.510 15 0 0 0 4.458
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表 10 回归模型方差分析
Table 10. Analysis of variance of regression model
方差来源 平方和 自由度 均方 F值 P值 显著性 模型 5.90 11 0.54 10.23 0.0404 * A 0.25 1 0.25 4.85 0.1150 B 0.08 1 0.08 1.48 0.3106 C 0.46 1 0.46 8.85 0.0588 AB 0.03 1 0.03 0.67 0.4742 AC 1.58 1 1.58 30.06 0.0119 * BC 0.08 1 0.08 1.53 0.3036 A2 0.53 1 0.53 10.03 0.0506 B2 0.85 1 0.85 16.14 0.0277 * C2 0.43 1 0.43 8.23 0.0641 A2B 0.22 1 0.22 4.20 0.1328 A2C 1.05 1 1.05 19.94 0.0209 * 残差 0.16 3 0.05 失拟项 0.07 1 0.07 1.65 0.3280 不显著 纯误差 0.09 2 0.04 矫正总和 6.06 14 注：*表示P<0.05，影响显著。
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