| Luzhou-flavor liquor is one of the most popular distilled spirits in China.Ethyl caproate is the main flavor substance,and its ester content ratio determines the quality of Luzhou-flavor liquor.The precursor of ethyl caproate is caproic acid,which plays an important role in the flavor of Luzhou-flavor liquor.The flavor characteristics of Luzhou-flavor liquor are determined by the microbial diversity.In the traditional fermentation process,the ester flavor is mainly esterified by fragrant-producing yeast,bacteria and molds with strong fermentation capacity.At present,bacteria of Clostridium,Ruminaceae,Macrospherium and Bacillus have been found to have the ability to produce caproic acid.Nowadays,these microorganisms mainly focus on the study of classification,isolation,screening and application,and are rarely reported in the study of microbial genetic modification.Tartary buckwheat is rich in many functional substances,such as flavonoids,polyphenol active substances.Tartary buckwheat can be decomposed into small molecular substances in brewing,which is one of the high quality raw materials for liquor fermentation.Its application in liquor brewing can improve the nutritional value of liquor,and its market potential is huge.In this paper,the strain isolated from Luzhouflavor Daqu was used as the starting strain,and the strain with high acid and ester production potential was selected by atmospheric pressure and room temperature plasma mutagenesis.By studying the physiological and biochemical characteristics,genetic stability and ability evaluation of the strains in artificial esterification system,the application of mutagenic bacteria in Tartary buckwheat liquor brewing was further discussed,and the optimization of fermentation process of Tartary buckwheat liquor was emphatically studied,and the quality and flavor changes of Tartary buckwheat liquor brewed by the optimized group were preliminarily analyzed and evaluated.The main research results were as follows:1.Through microbial morphological observations,physiological and biochemical reactions,and molecular biology,the starting cultures of this paper were identified as Saccharomycopsis fibuligera and Bacillus velezensis.The colony of Saccharomycopsis fibuligera is yellowish,moist,convex in the middle,smooth and opaque edges,easy to provoke,and has a candidate branching morphology after Gram color.The logarithmic growth period of Saccharomycopsis fibuligera is about 20-40 h,and neither color development with copper sulfate solution(not obvious color development)nor gas production.Bacillus velezensis colonies are yellowish,moist,smooth and opaque,not easy to provoke,and Gram color after rod-shaped and spores Bacillus Velezensis showed rod-shaped and spore after Gram staining.The logarithmic growth stage is 2-6 d,and light green precipitation is produced with copper sulfate solution,and gas is produced by Duchenne tube test.The results showed that Saccharomycopsis fibuligera did not produce caproic acid,while Bacillus velezensis produced caproic acid significantly.2.Two mutagenesis strains were performed by ARTP technology,and two mutagenic strains with high acid and ester production were screened separately.The optimum mutagenesis time of Saccharomycopsis fibuligera was 120 s,the logarithmic growth period was 25 h-50 h,the total acid and total ester were 5.60 g/L and 5.08 g/L for 5 d fermentation.The fermentation performance improved with the increase of fermentation time.After mutagenesis,pH and alcohol tolerance became stronger,and it could grow at low pH 3.2 and high alcohol concentration 5%.Secondly,the optimum culture temperature of Saccharomycopsis fibuligera was 28℃,and the addition range of glucose and NaCl was about 10% and 5%,respectively.The esterification ability increased by 6 times of that before mutagenic and was 887.05 mg/5g,and the esterification power was stable at 903.34 mg/5g after 6 generations of propagation.Finally,in the evaluation of the ability of artificial esterification system,it is found that lactic acid has great influence on the pH,alcohol,total acid and total ester of esterification system.The addition of ethanol also affects pH,total acid and total ester.The results indicated that lactic acid in the system could promote the synthesis of ester,while excessive ethanol might inhibit the propagation of strains and thus reduce the synthesis ability of Saccharomycopsis fibuligera.The optimum mutagenesis time of Bacillus velezensis was 90 s,the logarithmic growth period was 4 d,the total acid and total ester of Bacillus Velezensis were 0.36 g/L and 0.528 g/L respectively at 5 d.The fermentation performance of Bacillus Velezensis increased with the increase of fermentation time.Secondly,Bacillus velezensis showed no significant change in pH and alcohol tolerance before and after mutagenesis.The optimal culture temperature was32℃,and the addition ranges of glucose and NaCl were 25% and 10%,respectively.The esterification ability increased after mutagen,which was 7 times higher than before mutagen,and was 1028.35 mg/5g.The esterification power was stable after 6 generations of propagation,which was about 1110.2 mg/5g.Finally,the addition of four acids in the artificial esterification system can promote the consumption of ethanol to a certain extent,thus promoting the formation of esters,and the system is accompanied by the consumption and formation of acids.3.According to the preliminary study on fermentation process of Tartary buckwheat liquor and dross,the optimal fermentation process of Tartary buckwheat liquor with Saccharomycopsis fibuligera seed solution was as follows: fermentation time 25 d,bacteria solution added 4%,fermentation temperature 32℃;However,the orthogonal fermentation process of dross was not significant in terms of total flavonoids and total polyphenols,which may be due to the fact that the orthogonal optimization experiment was not designed with the single factor optimal physicochemical index of dross.When Bacillus velezensis seed solution was added,the optimal fermentation process of Tartary buckwheat white wine was as follows:fermentation time 30 days,bacterial solution content 5%,fermentation temperature 32℃.When total flavonoids and total polyphenols were used as evaluation indexes,there were two optimal fermentation processes for dross: total flavonoids(fermentation time 20 d,bacterial solution addition 3%,fermentation temperature 36℃)and total polyphenols(fermentation time 25 d,bacterial solution addition 3%,fermentation temperature 32℃).Finally,the quality and flavor substances of Tartary buckwheat liquor brewed by the optimum fermentation process were analyzed.It was found that AHS(sour taste)threshold of Tartary buckwheat liquor was significantly differentiated,and principal component analysis(PCA)showed obvious differentiation.Caproic acid and ethyl caproate could be detected in both groups.In general,the identification and flavor characterization of Tartary buckwheat liquor can be combined with sensory analysis for objective evaluation in the future,so as to make the results more accurate and objective. |
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