Breeding Of Acid Tolerant Acetic Bacteria And High-acid Fruit Vinegar Fermentation

The high acidity of vinegar has so many advantages, such as strongbactericidal power, low cost of storage and transportation. The vinegar not onlywas used as condiment, but also played an important role in food processing,medicine, household and cosmetic fields. The fruit vinegar was fermented withfruit or the by-product of fruit processing as main materials. The fruit vinegarincluded the nutrition of vinegar and fruit, so is a new and health beverage.Compared with conventional vinegar, high acidity fruit vinegar was made of fruitinstead of grain, which could not only make full use the abundant fruit resourcesto solve the difficult of fruit transportation and storage, but also save a largeamount of grain. In this paper, we mainly separated and improved the acidtolerant acetic acid bacteria, optimized the conditions of fruit vinegarfermentation and high-acid fruit vinegar flask-shaking fed batch fermentation.A bacterial strain was isolated from the natural fermented vinegar in theliquid of apricot, namely Acetic bacteria AFA-01. The characters wereinvestigated include ethanol resistance, acid resistance, thermo-resistant,easter-producing, genetic stability. The acid producing ability of AFA-01wasabout5.00g/100mL. According to the morphology, physiological andbiochemical characteristics, AFA-01was preliminarily identified as AcetobacterBeijerinck.Plackett-Burman design and response surface methodology were employedto optimize the acetic fermentation conditions for production of acetic acid byAFA-01using grape pomace as material. A Plackett-Burman design of fivefactors was applied to evaluate the effects of different fermentation conditions.Initial alcohol concentrarion, shaker rotate speed and fermentation time werefound to be significant components influencing the acetic acid production. The optimal levels of three factors were investigated by response surfacemethodology using Box-Benhnken design. The final fermentation conditionsoptimized was as follows: initial alcohol concentrarion7%, shaker rotate speed147r/min and fermentation time132h. Under these conditions.The maximumtotal acidity (measured by the amount of acetic acid) obtained was5.11g/100mL.The compound mutation of hydroxylamine hydrochloride and microwaveirradiation was performed to improve the acetic acid-producing ability ofAFA-01. The mutant strain AFA-WH3was obtained with higher yeild of aceticacid and alcohol-dehydrogenase(ADH) activity. Using the strain, total acidity ofproduced vinegar was increased by43.55%, up to a concentration of7.35g/100mL. Moreover ADH activity reached2786u/mL, increased by94.83%.Biochemical tests followed by16S rDNA were employed for identification ofAFA-WH3and phylogenic tree was constructed. In phylogenetic trees based on16S rDNA gene sequences, WFA-WH3was located in the lineage of Acetobacterpasteurianus and had100%sequence similarity to the type strain of Acetobacterpasteurianus.The technology for making apricot vinegar was researched via alcoholicfermentation and then acetic fermentation. The best processing condition wasthat apricot with the material and water ratio of1:1.5was enzyme-treated by0.5g/L pectase concentration at44℃, pH4.4for four hours. The alcoholicfermentation was optimized by means of L49(3) orthogonal test based onmono-factor experiments. The optimal conditions were set at the Brix16°Bx,inoculation volume of yeast3%and fermentation temperature30℃for threedays. These factors that influenced liquid fermentation of AFA-WH3aceticfermentation were analyzed, and technological parameters were optimized byusing the four factors quadratic currency rotational composite experiment. Theoptimal fermentation parameters were obtained as follows: initial alcoholconcentrarion6.7%,13%inoculation volume of acetic bacteria, fermentationtemperature34℃and shaker rotate speed153r/min for132hours. Under theseconditions, total acidity of apricot vinegar was up to7.11g/100mL, more than13.8%compared to the unoptimization, and volatile acid was up to6.23g/100mL. Reversed-phase high performance liquid chromatography was used to takequalitative and quantitative analysis of oxalic acid, tartaric acid, pyruvic acid,malic acid, lactic acid, acetic acid, citric acid, fumaric acid and succinic acid infruit wine and fruit vinegar. The experiment results demonstrated that the abovenine kind of organic acids and unknown organic acids were contained in apricotwine, among which the content of citric acid was highest. Acetic acid was themajor acid in fermented apricot vinegar and grape pomace vinegar. There was nopyruvic acid in the above fruit vinegars. The difference between apricot vinegarand grape pomace vinegar lied in the content of tartaric acid, lactic acid, citricacid. So kinds and contents of organic acids were different owing to differentfermented materials and different metabolism.Based on batch fermentation of apricot vinegar, flask-shaking fed batchfermentation adjusting initial alcohol concentration to4%was carried out byrespectively supplementing apricot wine with1%alcohol concentration at48h,60h,72h. It was indicated that final acidity reached9.02g/100mL, increased by8.4%contrasted to that in batch fermentation.