| Zhenjiang aromatic vinegar (ZAV) is a representive of traditional Chinese fermentedvinegar (TCVs), which was produced by “multiple-layer culture”solid-state fermentation andexhibited a good taste and uniquesmell. ZAV contained the major productacetic acid and someother organic acids, which made it display a soft taste of “acidic but not astringent”. However,the flavors of ZAV including organic acid components, their ratio and the contribution havenot been studied intensively. The previous ZAV National Standard “geography signs products”characterized the quality only using total organic acids as the indicator, which led to seriousfraud on the market and limited the healthy development of ZAV. In this study, analysismethod for nine organic acids of TCVs was optimized, and the optimal method was used toanalyze organic acids of TCVs from seven different regions. The organic acid compositionand its effect on the sense of ZAV were studied. Secondly, lactate-producing functionalmicrobes were isolated from vinegar Pei by culture-dependent method. Furthermore, theacetic acid fermentationprocess of ZAV were bioaugmented by lactate-producing functionalmicrobe, which resulted in the increase of lactic acid content in ZAV. The main conclusionsare as follows:Effect of sample pretreatment on HPLC analysis of organic acids in traditional Chinesevinegars was studied. The results showed that macromolecules such as proteins andpolysaccharides in vinegars were precipitated by potassiumferrocyanide and zincsulfate, andmicromoleculessuch as pigment and tannin were absorbed by C18solid phase-extractioncartridges.The method of quantitative determination of9organic acids in traditional Chinesevinegars by HPLC was optimized. The procedure was carried out on a reversed-phase C18column, using Monopotassium phosphate as mobile phase. The flow rate was0.9mL min-1and the detection wavelength was210nm. The analysis was completed within14min.According to the practical applications, this method is proven simple, accurate and suitablefor the determination of organic acids in traditional Chinese vinegars. This method wasadopted by the newly revised National Standard-Zhenjiang aromatic vinegar(GB/T18623-2011), and it became a standard determination approach.With the optimal method, organic acid composition of50vinegar samples from7differentregions (Zhenjiang, Beijing, Fujian, Shanxi, Shanghai, GuangdongandZhejiang) was analyzed.The results showed that the percentages of acetic acid in the total organic acids of solid-statefermented vinegars were58.1%,79.2%and97.4%, while the percentages of lactic acid were28.8%,16.6%and0.1%, respectively. Lactic acid content in solid-state fermented vinegarswas significantly higher than that of liquid fermented vinegars and prepared vinegars. Lacticacid and acetic acid are the main organic acids of fermented vinegar. Citric acid andpyroglutamic acid were characteristic organic acids to discriminate ZAV from viengars of6other regions.Results of HPLC analysis showed that organic acid content of22ZAV samples from13different vinegar factories are different, which was related with the seasons and fermentationtechniques.Senses of22samples of ZAV were evaluated, and the results showed that sensory scores were not related with the contents of organic acids. Organic acid content can not beused to evaluate the sense of ZAV. Flavor intensities of actic acid, lactic acid, citric acid,succinic acid and pyroglutamic acid were the highest in those of9organic acids, which wereimportant organic acids, influenced the sense of ZAV. However, citric acid was not listed aslandmark organic acid due to its low cost and easiness to add. Thus, the newly revisedNational Standard selected “actic acid, lactic acid,succinic acid and pyroglutamic acid” asthelandmark organic acids and clearly stated that the actic acid content should be lower than65%of the total organic acids, while the lactic acid should be higherthan10%of the total organicacids. Further sensory study showed a positive relation between total acidity andflavor ofZhenjiang aromatic vinegar when the totoal vinegar acidity was4.5-5.5g100mL-1. Thesensory score of vinegar decreased while relative content of acetic acid increased. Theincrease of relative content of lactic acid will raise the sensory score of Zhenjiang aromaticvinegar. Otherwise, nonvolatile acid content of ZAV produced in winter was significantlylower than that produced in summer.By culture-dependent method, acid-producing microbes were isolated from vinegar Pei inthe acetic acid fermentation of ZAV. Acetic acid bacteria (AAB), lactic acid bacteria (LAB)and bacillus were the main categoaries of isolated microbes, and LAB produced much morelactic acid than AAB and bacillus. Thus, the diversity of LAB in vinegar Pei were studied,and30strains of LAB with diffenent morphological, physiological and biochemical featureswere isolated, which belonged to7species of Lactobacillus were isolated. Among them, thestrain of L. helveticus M3-1which possessed highest lactate-producing ability (701.2mg100mL-1in flask) was screened. Meanwhile, L. helveticus M3-1could synthezed glycine,glutamine, promine,3-hydroxy-butanoic acid, ethyl ester (6.6?g100mL-1), and2-methyl-butanoic acid (11.3?g100mL-1).L. helveticus M3-1, a lactic acid high-producing strain, was cultured in a large scale and tobioaugment the acetic acid fermentation of Zhenjiang aromatic vinegar. The results showedthat relative content of lactic acid bacteria and content of lactic acid in the vinegar culturewere increased by the bioagumentation of L. helveticus M3-1. Relative content of lactic acidwas increased by3.4%in bioaugmented group while the content of acetic acid showed noobvious changes. Sensory evaluation results demonstrated that the bioaugmentation did notinfluence the odor scores, but increased the flavour scores. Therefore, L. helveticus M3-1bioaugmentation could maintain sensory scores and further it also accelerated fermentationrate, increased relative content of lactic acidand improved the flavor of ZAV. |
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