黄酒贮存酸败关键微生物的分离鉴定

Identification and characterization of spoilage microorganisms in aging Chinese rice wine by culture-independent and culture-dependent technologies

摘要:

【背景】贮存陈酿是黄酒生产的重要工艺环节，但由于黄酒中营养物质含量丰富，贮存陈酿过程中时常出现酸败变质的现象，尤其是在大罐的陈酿过程中酸败的发生对黄酒行业造成较大的经济损失。【目的】解析黄酒贮存陈酿过程中造成黄酒酸败的关键微生物，为黄酒贮存酸败微生物的控制提供依据。【方法】采用高通量测序技术分析不同来源酸败黄酒中的主要微生物种类；设计针对性培养基分离培养酸败黄酒中的难培养微生物；设计微生物特异性引物，对16S rRNA基因高度相似的酸败微生物进行区分鉴定；将分离的黄酒酸败微生物接入到未发生酸败的黄酒中验证其生酸能力。【结果】高通量测序技术分析结果显示，酸败黄酒中污染微生物主要为乳酸杆菌属(丰度>95%)微生物，在种水平上分析比对结果显示两种难培养微生物[耐酸乳杆菌(Lactobacillus acetotolerans)和食果糖乳杆菌(Lactobacillus fructivorans)]的相对丰度达到了82%以上；采用改进的分离培养基分离出酸败黄酒中的难培养污染微生物36株；利用耐酸乳杆菌recA基因和食果糖乳杆菌tuf基因的特异性引物准确鉴定出这些微生物菌株为 28株耐酸乳杆菌和8株食果糖乳杆菌；将两种主要酸败微生物接入到未发生酸败的黄酒中，培养2周后能够显著提高黄酒酸度。【结论】基于高通量测序的未培养技术可作为食品中难培养污染微生物快速分析的有效方法。基于未培养技术和可培养技术相结合，首次解析并验证黄酒贮存过程中造成黄酒酸败的主要微生物为耐酸乳杆菌和食果糖乳杆菌。

关键词:陈酿黄酒，酸败微生物，高通量测序，难培养微生物，耐酸乳杆菌，食果糖乳杆菌

Abstract:

[Background] Storage is an important procedure of Chinese rice wine production. However, due to the rich nutrients, Chinese rice wine often appeared spoilage problems during storage process, resulting in huge economic losses. [Objective] The purpose of this study was to analyze the key microorganisms in spoiled Chinese rice wine by culture-independent and culture-dependent methods. [Methods] High-throughput sequencing technique was used to analyze the key microorganisms in the aging yellow rice wine of different origins. Specific culture medium was used to isolate and cultivate spoilage microorganisms in rice wine. Specific primers (Lactobacillus acetotolerans and Lactobacillus fructivorans according to the recA and tuf, respectively) were designed to identify the spoilage microorganisms. The isolated species were inoculated into non-spoiled rice wine to verify the spoilage ability. [Results] High-throughput sequencing results showed that Lactobacillus was the dominant genus (abundance>95%). And the abundance of two species (L. acetotolerans and L. fructivorans) was more than 82% at the species level. Thirty-six strains were successfully isolated on the designed culture media from spoiled rice wine. Twenty-eight strains of L. acetotolerans and eight strains of L. fructivorans were specifically identified by the specific primers of recA and tuf genes. The two main spoilage microbes were then inoculated into non-spoiled rice wine, and the acidity of the wine was improved significantly after two weeks. [Conclusion] High-throughput sequencing technology is a quick method for analyzing hard-to-culture microorganisms in food. Based on the combination of culture-independent and culture-dependent methods, we found that the key microorganisms that caused yellow rice wine spoiled were L. acetotolerans and L. fructivorans.

Key words:Aging Chinese rice wine, Spoilage microorganisms, High-throughput sequencing, Hard-to-culture microbes, Lactobacillus acetotolerans, Lactobacillus fructivorans