| Ethyl hexanoate is one of the main characteristic flavor substances of strong-flavor Baijiu,and the content of ethyl hexanoate directly affects the quality of strong-flavor Baijiu.Caproic acid is the precursor of the synthesis of ethyl hexanoate in Baijiu.The study of caproic acid producing strains in pit mud is crucial to the improvement of the quality of strong-flavor Baijiu.This study mainly studied the microbial structure and quantity in the pit mud through dilution and marking method.Based on this,the caproic acid producing strain btq-7 was isolated and optimized for the response surface of the culture medium during the card jar expansion stage.The most practical application of the caproic acid producing strain in artificial organic pit mud was achieved.The specific research content includes:(1)Taking the pit mud of the new and old plant areas of the strong-Fragrance Workshop of Jinan Baotu Spring Brewing Co.,Ltd as the research object,the structure and abundance of clostridium microbial community in different types of pit mud were explored through high-throughput sequencing combined with DGGE(denaturing gradient gel electrophoresis).Clostridium bacteria detected in the pit mud at the genus level were: Alkaliphilus,Vibrio,clostridium,thermophilus,clostridium ruminalis,fecococcus,sedimentibacter Caldicoprobacter,Tepidimicrobium,Tisserella,Sporanaerobacter,Sulfate-reducing bacteria,Lutisea and Clostridiisalibacter.(2)Through high-throughput sequencing analysis,689 bacterial genera were detected in the pit mud of strong-flavor Baijiu at the bacterial genus level.The new and old factory pit mud has complex fungi and strong differences in genus classification level.The cellar mud in both the new and old factory areas contains caproic acid producing bacteria Capriciproducen,Clostridium,and Bacillus.The caproic acid producing bacteria in the cellar mud in the old factory area are mainly Capriciproducens and Clostridium.The caproic acid producing bacteria in the cellar mud in the new factory area are mainly Bacillus.(3)The microorganism in the pit mud of strong-flavor Baijiu was separated and purified by the dilution and lineation method,and a total of 8 strains of bacteria were screened.Through the preliminary screening of copper sulfate color reaction and the full analysis and re screening of gas chromatography,a target strain btq-7 with the largest acid production was obtained,and the content of caproic acid of the target strain was determined to be 5.2 g/L.DNA extraction,PCR amplification,agarose gel electrophoresis and 16 S r DNA sequencing analysis of the target strain showed that the 16 S r DNA of the strain was 99.56 % similar to that of Clostridium kluyveri,indicating that the target strain btq-7 was Clostridium kruyveri.For C The tolerance performance test of Kluyveri btq-7 showed that the target strain grew and propagated well within the p H range of 5.5-6.5 and temperature range of 25-35 ℃,while also exhibiting good ethanol and glucose tolerance.The higher the liquid content,the better the growth and reproduction of the target strain and gradually approaching stability.This indicates that the target strain has good adaptability and can be used for subsequent practical applications.(4)Based on the raw materials and by-products in the production process of strong-flavor Baijiu,the culture medium for caproic acid bacteria in the expansion tank stage was prepared.Through single factor experiments and response interviews,the process optimization of hexanoic acid expansion solution was carried out,and the experimental data was summarized and analyzed.The order of influence on the content of hexanoic acid in the pit mud culture medium was as follows: tail addition amount(A)>cultivation time(D)>cultivation temperature(C)>ratio of wine culture waste to water(B).The optimal process for producing hexanoic acid in hexanoic acid expansion solution was predicted through regression models,The optimal conditions after improvement are as follows: the addition amount of wine tail is 11.80 %,the ratio of wine culture waste to water is 5.20,the cultivation temperature is 37 ℃,the cultivation time is 37 days,and the output value of hexanoic acid in the hexanoic acid expansion solution is 6.88 g/L.(5)To be screened from the pit mud After expanding the cultivation of C.Kluyveri btq-7,different inoculation ratios of caproic acid bacteria suspension and yellow water esterification solution were mixed and inoculated into the preparation process of artificial organic pit mud.The changes in physical and chemical properties of the pit mud during the cultivation process were tracked and monitored.Combined with sensory evaluation,a comprehensive comparison was made between each group of pit mud,and the optimal inoculation ratio was determined to be a 1:1 ratio of caproic acid producing bacteria suspension and yellow water esterification solution.After that,by rejuvenating and strengthening the degenerated pits of strong-flavor Baijiu,the physical and chemical indexes of pit mud before and after the rejuvenation of the pits,the aroma components of the original liquor,and the sensory evaluation of the original liquor were analyzed.The results showed that,on average,compared with the degenerated pits 5-12 maintained by CK in the control group,the contents of pit mud water,effective potassium,effective phosphorus,humus,ammonia nitrogen,and ethyl hexanoate in the original liquor in pits 5-5 were significantly increased,The water content increased from34.54 % to 40.94 %;Effective potassium increased from 25.08 mg/100 g dry soil to 42.85mg/100 g dry soil;The effective phosphorus increased from 216.20 mg/100 g to 670.23mg/100g;The ammonia nitrogen content increased from 111.77 mg/100 g dry soil to 337.48mg/100g;The average humus increased from 7.98 % to 13.11 %;The content of ethyl caproate in the original wine increased from 1.73 g/L to 3.47 g/L;The sensory aspect of pit mud also changes from grayish brown to dark gray;The p H is closer to 7.This indicates that group A artificial pit mud has a significant promoting effect on the improvement of pit quality.Sensory analysis of the original wine before and after maintenance in the degraded cellar revealed that the enhanced organic cellar mud effectively improved the quality of the original wine and significantly improved its aroma characteristics.The original wine produced from the organic pit mud 5-5 pit in Group A had significantly higher sensory circumference(fineness,pit aroma,coordination,and fullness)than the control group CK 5-12 pit mud original wine.This experiment shows that after the rejuvenation of group A organic pit mud,the quality of the pit mud has been improved,and the overall style of the original wine is more rich,with a more typical strong aroma style. |
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