| Sichuan bran vinegar is one of the most famous vinegar in China with deep color and fruity vinegar and mellow acidity and lingering aftertaste. Also, it has some pharmacological effects which other kinds of vinegar don’t have. However, in the traditional solid-state fermentation process the starch utilization and the acetic acid content was low, the production cycle was long, and it was difficult to achieve the highly staged and mechanise production. In this article, acetic acid bacteria with high acid producing capacity were screened from the normal grains of Sichuan bran vinegar; The screened acetic acid bacteria and bran leachate were added in the process of liquid fermentation of vinegar; The fermentation conditions were optimized by response surface methodology; The influence of new fermentation process affected on the flavor of vinegar were investigated by measuring total acid and flavor substances in liquid bran vinegar. The main results are as follows:1.104 strains were isolated and purified from the normal fermented grains, among them 78 strains were Gram-negative; 69 strains were proved to be able to produce acetic acid by qualitative test of producing acetic acid; Acid quantitative test results showed that a total of 15 strains was higher than the strain 20064 (32.40g/L) on acid production, and 2 strains were higher than the strain 7015 (39.00g/L); Five of the high-yielding strains (C9-4, C12-4, A12-7, A30-16, A30-14-2) were identified by morphological, physiological and biochemical characteristics,16S rDNA genetic identification and they were all identified as Acetobacter pasteurianus.2. The growth characteristics of the five high-yielding acetic acid bacteria were studied. The results of alcohol concentration adaptive tests showed that the highest acid production of strain A30-16 (55.51g/L) were the ethanol content was 7%; the highest acid production of strain C9-4 and C12-4 and A12-7 were achieved when the ethanol content was 6%, and the acid production were respectively 45.51g/L, 42.98g/L,39.46g/L; the highest acid production of strain A30-14-2(41.54g/L) were achieved when the ethanol content was 5%. The results of ethanol addition tests showed the highest acid production was produced by strain C9-4 for 43.60g/L, which means having strong ability to transform ethanol to acid continuously. The results of bottom acid concentration adaptive tests showed when bottom acid concentration was lower than 4%, the acid production of strain A30-16 were increased with bottom acid concentration increases while the acid production of other strains increased at the first but reduced in the end, and the highest value were reached when bottom acid concentration was 3.5%. The results of temperature adaptive tests showed that in the range of 28-40 ℃, the acid production of the strain C12-4 and strain A30-14-2 continued decreasing with temperature increased; the acid production of the strain C9-4, A12-7 and A30-16 increased at the first but reduced in the end and reached the highest value at 34℃; addition, in the range of 28-40℃, the fluctuation of acid production of strain C9-4, A30-14-2 and A30-16 was small; in particularly, strain C9-4 and A30-16 can withstand the high temperatures of 40℃ and produce acid. The measurement of growth curve showed that the logarithmic growth phase of strain A30-16 was 6h-36h, and strain C9-4 was 6h-44h.3. The acid producing characteristics of the five high-yielding acetic acid bacteria were studied. The acid production curve showed the environmental adaptability of strain A30-14-2, C9-4 and A30-16 to broth with 7% alcohol concentration were very well, a rapid increase in acid production were found in 0-5d, and then it was slowly in 5-10d; however, the environmental adaptability of strain C12-4 and A12-7 was poor, they had a trend of "S", there was a delay stage for 3 days, the acid production didn’t increase at the first 3 days, a rapid increase in acid production were found in 3-6d, and then slowed down in 6-10d. The results of organic acids teste showed that more than five kinds of organic acids were detected in fermentation broth, but lactic acid was not detected; strain C12-4 produced more tartaric acid and strain A30-16 produced more acetic acid than other strains; the order of the total amount of organic acids were:A30-16> C12-4> C9-4> A30-14-2> A12-7.4. Strains A30-16 and C9-4 were selected to ferment liquid bran vinegar for their high alcohol concentration adaptability, strong resistance to bottom acid concentration, wide temperature adaptive range, fast and continuous fermentation ability and high organic acid production. The results of single factor test for proportion of strains showed that the difference of acid producton in fermentation broth with different ratio was significant, when the inoculum was 10%, the highest amount of acid production was up to 65.08g/L with the ratio C9-4:A30-16= 6:4. The analysis results of variance for single factor tests showed the addition of bran koji, the inoculum of acetic acid bacteria and the temperature of acetic acid fermentation exerted significant influence on acid production. The regression equation: Y=66.99-1.07X1+3.81X2+3.71X3-4.3X1X2+1.33XiX3-1.15X2X3-5.42X12-5.21X22-4.8 5X32 (where, X1, X2 and X3 respectively represent the coded level of the addition of bran koji, the inoculum of acetic acid bacteria and the temperature of acetic acid fermentation) was acquired by Response Surface Box-Behnken experimental design. The optimal conditions were predicted combined with regression model:the addition of bran koji was 1.11%, the inoculum of acetic acid bacteria was 13.71% and the temperature of acetic fermentation was 31.50 ℃. Under this conditions, the result of verification test was 67.97g/L, fitting the predicted values (68.4817g/L) well.5. In liquid fermentation bran vinegar,20 kinds of Volatile flavor substances and 17 kinds of amino acids were detected, and the total amino acids was 805.29mg/100mL; the quality of liquid fermentation bran vinegar was superior to the liquid vinegar produced in factory (11 kinds of Volatile flavor substances and 12 kinds of free amino acid were detected, and the total amino acid was 3.38mg/100 ml), but still had certain gap when compared to solid-state fermentation bran vinegar (33 kinds of Volatile flavor substances and 17 kinds of free amino acid were detected, and the total amino acid 1283.55mg/100 ml).The results of this article can not only provide excellent acetic acid bacteria to vinegar brewing, but also provide technical guidance for the fermentation of liquid bran vinega, thus posessing theoretical significance and practical value. |
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