| Vinegar is one of the most common condiments in people’s daily lives,and brewing vinegar is produced with a microbial fermentation process of grain raw materials that consists primarily of solid-state fermentation and liquid-state fermentation.Compared to solid-state fermentation,liquid-state fermentation has numerous advantages,including a shorter fermentation cycle,greater productivity,mechanization,and automation.However,because the current liquid fermentation process is essentially pure fermentation of Saccharomyces cerevisiae and Acetobacter,the fermentation process is not similar to the solid fermentation process in which rich microorganisms are involved;these microorganisms are the primary "contributors" to the flavor substances of vinegar;therefore,liquid vinegar contains fewer amino acids,organic acids,and flavor substances than solid vinegar,and its flavor and aroma are inferior.This study begins with the isolation and screening of key functional microorganisms in different fermentation stages and then systematically investigates the method of strengthening functional microorganisms to develop multi-strain compound pure-strain fermentation technology to improve the flavor quality of liquid vinegar.The main research results are as follows:1)Isolation and identification of functional microbial strains.3 strains of mold,12 strains of yeast,and 6 strains of bacteria were isolated from the traditional Jiuqu by the microbial isolation method,combined with the strains’ cellular morphology and colony morphology.The enzyme-producing ability of mold strains,the acid-producing ability of bacterial strains,and the aroma-producing ability of yeast strains were examined,and 2strains of yeast,2 strains of mould,and 1 strain of bacteria were obtained by screening.ITS and 16 S r DNA sequence analysis indicated that one yeast strain,HQ3,was identified as Clavispora lusitaniae and one yeast strain,HQ9,as Candida glabrata;the bacterial strain HB8 showed 99% similarity with Lactobacillus plantarum;the mold strains ZSM1 and ZSM2 showed similarity with Monascus and Aspergillus Niger.2)The fermentation performance in terms of alcohol and ester production and flavor substance metabolism of the two yeast strains,as well as the tolerance of the strains under different environmental stresses,were systematically investigatced.The results demonstrated that both yeast HQ3 and HQ9 have the capacity to produce aroma(ester)and alcoholic fermentation.In medium with 15% glucose and shaker fermentation at30°C for 72 h,the total ester and alcohol production of strain HQ3 were 6.95±0.5 g/L and61.08±0.69 g/L,and those of strain HQ9 were 7.46±0.87 g/L and 65.02±0.81 g/L,respectively.In the metabolites of strain HQ3,alcohols predominated,while esters predominated in the metabolites of strain HQ9.Both strains are able to grow and metabolise normally in an environment containing 30% glucose and 20% ethanol,indicating that they can adapt well to the environmental conditions of the liquid vinegar production.The feasibility of using pure cultures of Monascus ZSM1 and Aspergillus Niger ZSM2 as saccharifying agents instead of liquefaction and saccharification of starch for sugar production in current liquid-state fermentation was investigated.Using rice as the main raw material,the fermentation was carried out by shaking at 30°C and 180 r/min for48 h.The strain ZSM1 produced higher amylase activity of 124 U/m L and protease activity of 10.05 U/m L,while the strain ZSM2 reached higher enzyme activity with amylase activity of 281 U/m L and protease activity of 14.84 U/m L at 24 h of fermentation.The growth and metabolic characteristics of Lactobacillus plantarum HB8 were studied,and the results showed that strain HB8 has a strong organic acid production capacity,and the lactic acid production reached 39.2 g/L at 24 h of static fermentation in MRS medium at 30°C and has the characteristics of resistance to 20% glucose hypertonicity and 14%(V/V)high alcoholic strength.3)The research established the process of functional microorganisms and pure strain fortified fermentation of liquid vinegar.The effect of fortifying fermented rice vinegar with yeast HQ9 to enhance the flavor quality of the product was investigated.Using HQ9 instead of traditional alcoholic yeast for liquid vinegar fermentation,the total acid production reached 42.6±0.3 g/L,and the content and types of organic acids increased significantly,including oxalic acid 1.24±0.17 g/L,citric acid 1.65±0.05 g/L,and malic acid 0.43±0.08 g/L.The fingerprinting of volatile flavor compounds of rice vinegar showed that the esters,acids,and ketones in rice vinegar were more abundant,among which the abundant isoamyl acetate,butyl acetate,and ethyl acetate constituted the unique aroma of rice vinegar.The acetic acid was not astringent,and the taste was moderately sweet and sour.In the saccharification stage,the ethanol production rate was not significantly different from that of saccharification with double enzymes using the liquid cultures of ZSM1 and ZSM2 mixed in the ratio of 4:1 as saccharifying agents and saccharification production substrate,and the ethanol yield reached 11.42 g/100 m L,but the vinegar was richer in aromatic compounds such as propyl acetate,2-octanone,and2,3-pentanedione.The content of organic acids in rice vinegar was significantly increased by adding Lactobacillus plantarum HB8 in the alcoholic fermentation stage,and the content of lactic acid reached 1.43 g/L and the content of other non-volatile organic acids increased.4)Effect of liquid fermentation of functional microbial combinations of pure strains on the flavor quality of vinegar products.The functional microbial strains obtained by screening were used to intensify the fermentation of liquid rice vinegar and to study their effects on the formation of physicochemical indicators and flavor substances during the fermentation process.The results showed that the use of ZSM1 and ZSM2 liquid cultures metabolising a dual enzyme saccharifying agent in the saccharification stage and access to strains HQ9 and HB8 in the alcohol fermentation stage resulted in a fermentation alcohol yield of 11.13 g/100 m L and a total acid yield of 4.32 g/100 m L.Compared with the traditional liquid vinegar process,the amino acid content in the alcohol mash is richer and the composition is more ideal,among which the sweet and umami amino acids are significantly improved,the glutamic acid content is up to 175.94 mg/g,and the alanine content is 71.10 mg/g.Compared with the control group using Wuyi Qu as a saccharifier,the content of the bitter amino acids arginine,leucine,and valine was significantly reduced.In vinegar samples,compared with the control group of red yeast rice,the contents of citric acid and malic acid in the total acid were significantly increased,while the difference between lactic acid and succinic acid was not significant.However,compared with the existing liquid fermented vinegar,the organic acid content is significantly improved,the composition is more reasonable,the content of flavor substances such as 2,3-butanedione,propyl butyrate,γ-butyrolactone in the volatile aroma components is significantly increased,the acetic acid is sweet and mellow,with a strong vinegar aroma and a light fragrance,the acetic acid is weak in irritation,the taste is soft,and the flavor quality of the product is improved.The functional microorganisms obtained from the screening and the liquid fermentation process of pure strains of functional microorganism combinations established by the research lay the foundation for improving the quality of liquid vinegar and promoting the technological progress of liquid vinegar production,which has good prospects for promotion and application. |
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