| Vinegar is one of the traditional fermentation foods in China. Zhenjiang aromatic vinegar, which is produced from sticky rice through traditional solid-state fermentation, is highly prized as one of the four famous China-style vinegars due to its unique cycle-inoculation style, which the fermentation culture of the 7th day is used as seed (starter) for the next fermentation round inoculation. Since no additional microbe is supplied during the fermentation process, the microbial community and quality of each fermentation circle is supposed to be comparatively stable. Because of the limitation of research method (culture-dependent approaches and analytical methods), the knowledge of microbial community and chemical substance change during the vinegar fermentation process are uncertain. Process control of Chinese traditional vinegar production is mainly based on experience, so it seriously hinders the development of Chinese traditional vinegar industry. In the present study, PCR-DGGE and modern analysis apparatuses were applied to investigate the microbial community and chemical substance change during the acetic acid fermentation process of Zhenjiang aromatic vinegar.Firstly, basic physicochemical indexes (temperature, moisture content, total acid and pH) and flavor substance (organic acid, amino acid, volatile substance and ligustrazine) were analyzed. A total of 7 organic acids (acetic acid, lactic acid, succinic acid, tartaric acid, oxalic acid,α-ketoglutaric acid and citric acid) were determined by HPLC. Thereinto, acetic acid, lactic acid and succinic acid were the most abundant three kinds of characteristic organic acid which the relative content maintained above 90%, the total content of these 3 organic acids increased rapidly during the fermentation process, from 1.86 g/100 g dry culture in the fresh culture without inoculation to 21.68 g/100 g dry culture when the fermentation ended, which increased about 11 times. A total of 17 amino acids (AAs) were analyzed by amino acid analyzer, the results showed that the total content of 17 AAs increased from 1834.71 mg/100 g dry culture to 13058.35 mg/100 g dry culture during the fermentation process, which increased about 7 times. The relative content of the most abundant 5 AAs (glutamic acid, arginine, alanine, valine and leucine) maintained above 60%. Volatile substances were analyzed by GC-MS, and 30 esters were attained, which the total content increased from 229.18μg/g dry culture to 796.79μg/ g dry culture. Total 10 kinds of carboxylic acid were attained, which the total content increased from 14.75μg/g dry culture to 39.94μg/g dry culture. A total of 11 alcohols were attained, which the total content decreased from 844.51μg/g dry culture to 92.02μg/g dry culture when the fermentation ended. A total of 7 carbonyl compounds were attained, which the total content increased from 5.22μg/g dry culture to 57.68μg/g dry culture. Total 2 kinds of pyrazine compound were attained, which the total content increased from 0μg/g dry culture to 64.43μg/g dry culture during the fermentation process.Microbial community and transition throughout the acetic acid fermentation process were studied by DGGE and Real-time PCR analysis. Meanwhile, a culture-dependent approach was applied to screen the strains which had good ability for organic acid producing characteristics, and 10 strains were attained. Thereinto, 1 strain belonged to genus Acetobacter, 2 strains belonged to genus Lactobacillus, 4 strains belonged to genus Bacillus, 2 strains belonged to genus Paenibacillus, and 1 strain belonged to genus Staphylococcus. On this basis, the fermentation characteristics of 10 isolated strains mentioned above were studied. In the result of organic acid analysis, a total of 8 organic acids (acetic acid, lactic acid, malic acid, succinic acid, pyroglutamic acid,α-ketoglutaric acid, tartaric acid and pyruvate) with the contents between 0 and 1755.89 mg/100 mL were determined, and at least 2 organic acids were produced by each strain. In the result of volatile substances analysis, at least 5 volatile substances (esters, carboxylic acids, alcohols, carbonyl, and aromatic compounds) were attained. At the same time, we found that these 10 strains also had good ability for pyrazine compounds production. A total of 10 pyrazine compounds were determined, and at least 1 pyrazine compound which the content was between 0.12 and 86.40μg/100 mL could be produced by 9 strains except for Lactobacillus helveticu.The data of organic acid, amino acid and biomass were analyzed by principal component analysis (PCA). The result showed that the acetic acid fermentation process of Zhenjiang aromatic vinegar might be divided into 5 steps, namely the under layer culture for the first 5 days of acetic acid fermentation, the upper layer culture for the first 7 days, the culture from the 7th day to the 12th day, the culture from the 12th day to fermentation ended, and the culture from fermentation ended to culture sealing ended. On the basis, the mechanisms of organic acid and amino acid formation in Zhenjiang aromatic vinegar were also preliminary discussed.Based on the previous studies, 3 functional strains which had better organic acid and ligustrazine producing abilities were picked out for further bioaugmentation experiment. The result showed that bioaugmentation of the 3 functional strains could accelerate the pre-heat time, improve the conversion efficiency of raw materials, and then shorten the acetic acid fermentation period. The relative content of three primary organic acids (acetic acid, lactic acid and succinic acid) could be raised at the range of 10% and 15% through bioaugmentation. The relative content of three primary amino acids (alanine, valine and leucine) could be also raised at the range of 10% and 35% through bioaugmentation. By analysis of the raw vinegar, we found that the vinegar rate might be raised about 7% to 25% through bioaugmentation. |
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