Optimization Of Technological Conditions And Metabolomics Research In The Fermentation Procecc Of Black Glutinous Rice Wine

Most of the traditional koji was used the grain as the main raw material,which is inoculated and fermented by microorganisms naturally.So there was a wide variety of microorganisms in the koji,resulted the quality instability and a serious contamination by miscellaneous bacteria.Modern wine koji is pure strain mostly,but the wine with a simple flavor,which can not meet consumers' demand.In order to solve the problem,and gives a better quality of black glutinous rice wine.In this study,using the 12 kinds of homologous substances of medicine and food as a new substrate for making koji.Inoculation amount of Rhizopus,matrix moisture content,cultivation time and culturing temperature were studied on glucoamylase and liquefaction enzyme activity about koji.On the basis of single factor experiments,the Box-Behnken Center combinatorial test design principle was adopted,and glucoamylase activity was used as the response value to optimize the optimum process conditions for koji making.The black glutinous rice wine was fermented by new matrix koji under the optimum conditions.The orthogonal experiment was designed on the basis of single factor experiment.Metabonomics method was used to track metabolites within 72 h of black glutinous rice wine.The results of the study are as follows:(1)The optimum conditions for new substrate were as follows: 0.3% Rhizopus inoculum,culturing time 40 h,matrix water content of 65 % and 28 ? of culturing temperature.Under this condition,the predicted value of glucoamylase activity was 778.65 mg/g.h.Three confirmatory tests were carried out under the optimal culture conditions.The average value of the measured values was 779.233±0.577 mg/g.h.The actual and predicted values were close to each other,indicating that this scheme was feasible.The analysis of the two koji enzyme systems showed that the experiment koji had higher enzyme activity than the traditional koji,and the glucoamylase and liquefactase activity were about 1.3 and 2.8 times than the traditional koji.Using the experimental and traditional koji brewed rice wine and a total of 33 substances were detected.The isoamyl alcohol(10.065?g/100mL/109.394?g/100 mL)and ethyl butyrate(0.867?g/100mL/187.552?g/100mL),acetic acid(135.240 ?g/100mL/560.939?g/100mL)and phenol(0.156?g/100mL/0.858 ?g/100mL)were substances with the greatest differences in the two rice wine.Based on the above results,the experimental koji's fermentation performance is fine and the brewed rice wine has richer taste which has better development prospects.(2)The results showed that the fermentation time 6 d,fermentation temperature 34 °C,koji addition amount 15%,and soaking time 7 h.Under these conditions,the sensory score of black glutinous rice wine was 86 and a total phenol content was 0.85 mg/m L.(3)Aimed to track metabolites in the pre-fermentation of black waxy rice wine for 72 h using metabolomics.The results showed that 344 metabolites were identified during the fermentation of black glutinous rice wine,including sugar,sugar alcohol,organic acid,amino acid,fatty acid,phenolic acid and unknown metabolites,and 33 were considered significantly different metabolites(SDMs)(VIP > 1 and P< 0.05).The heat map shows that the SDMs can be divided into 4 categories according to fermentation time,and the SDMs enriched at the 60 h stage of fermentation were the most.Pathway analysis indicated that the SDMs were involved in 18 pathways,including 3 significantly relevant pathways(pathway impact > 0.1and p < 0.05),including starch sucrose metabolism,alanine,aspartate and glutamate metabolism and pentose phosphate pathways.Further integrated key metabolic pathway analysis showed that starch sucrose metabolism and alanine,aspartate and glutamate metabolism mainly occurred 24 hours before the fermentation of black glutinous rice wine,and pentose phosphate pathway occurred primarily between 36-60 h of fermentation.In the 6different fermentation stages,different metabolites were obtained,but the number and species of different metabolites changed with the fermentation.Correlating the process-specific metabolites with the corresponding physicochemical factors and enzyme phenotypes,it is found that with the extension of fermentation time,the increase of acid substances,the pH decreased which was consistent with the changes of metabolites.With the increase of cellulase,pectinase and saccharase activity,the content of polysaccharide decreased and the content of monosaccharide and oligosaccharide increased.Protease activity increased,amino acid content increased.In the 6 different fermentation stages,different metabolites were obtained,but the number and species of different metabolites changed with the fermentation.Fructose was differences metabolites in the three stages and the maltose metabolites in all fermentation stages except the third stage.These two kinds of sugar were likely to impact on rice wine flavor taste greatly.