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

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