Studies On The Isolation,Identification And Characteristics Of Spoilage Lactic Acid Bacteria In Storage Of Chinese Rice Wine

Storage is an important procedure of Chinese rice wine production.The newly brewed Chinese rice wine needs a certain period of storage to improve its flavor quality.However,due to the rich nutrients,Chinese rice wine often appeared spoilage problems during storage process,resulting in huge economic losses.Because of the lack of effective control measures for the spoilage microbes,the spoilage problems of Chinese rice wine had become a key limiting factor in Chinese rice wine industry.In this paper,we used the culture-independent and culture-dependent technologies to analyze the main microbes in the spoilage Chinese rice wine,and analyzed the physiological and biochemical characteristics of the spoilage microbes,and tried to put forward the control strategies to prevent the spoilage problems.The main results were shown as follows:(1)In order to solve the problem that the spoilage microbes were not yet isolated and cultivated,we used the high-throughput sequencing technology to clarify the main spoilage microbes in the spoiled Chinese rice wine for the first time.The V3 ~ V4 region of microbes in different sources of spoiled Chinese rice wine was sequenced.The results showed that the main spoilage microorganisms genus were Lactobacillus.Further analysis results showed that the main spoilage microbes at the species level may be L.acetotolerans and L.fructivorans.(2)Using the specific culture mediums and culture methods,L.acetotolerans and L.fructivorans were isolated from the spoiled yellow rice wine.And the strain numbers were CGMCC No.14059 and CGMCC No.14060.Based on the specific culture mediums,36 strains were isolated,and they were identified as L.acetotolerans or L.fructivorans by 16 s r RNA sequencing.And then we classified these 36 strains of microbes into 28 strains of L.acetotolerans and 8 strains of L.fructivorans according to specific genes sequencing based on whole genome alignment analysis.Furthermore,L.acetotolerans and L.fructivorans could increase the total acid content of the yellow rice wine,resulting in the spoilage problems of yellow rice wine.(3)In order to put forward control measures of spoilage microbes,we researched the physiological and biochemical characteristics of spoilage microbes,including nutrient demand,alcohol tolerance,optimum growth temperature,and low sugar content growth.The results showed that the spoilage microbes were the most easy to use saccharide,especially glucose.And the sugar content had effect on the growth of spoilage microbes.The lower sugar content could inhibit the growth and acid production of spoilage microbes.Spoilage microbes were more resistant to alcohol and were still able to grow in 23%(v·v-1)alcohol content.The optimum growth temperature of microbes was 25 oC ~ 30 oC,and the growth of microbes were obviously inhibited when the temperature reached 40 oC.(4)And we explored the prevention and control measures of spoilage microbes in the storage procedure of Chinese rice wine.Spoilage microbes were tracked using high-throughput sequencing technology in the brewing process.And we found that spoilage microorganisms existed in the whole process of fermentation.If the sterilization was not complete,there was the potential risk of spoilage.Based on the Real-Time PCR and culture-dependent technologies,the rapid detection methods of spoilage microbes were established,which laid the foundation for the prevention and prediction of spoilage problems.We analyzed the fermentation process,fermentation raw material and fermentation environment using the RT-PCR rapid detection method and found that the wheat Qu and the brewing environment were the main sources of the spoilage microbes.