The winey yeast JM037 produces a rich and GABA-rich mulberry wine.

Explore the changes in flavor substances during the fermentation preparation of mulberry wine by producing gamma-aminobutyric acid, GABA winey yeast JM037Using high-efficiency liquid chromatography and top-air solid phase micro-extraction-gas chromatography-mass spectrometry usage, the changes of organic acids and volatile flavor substances in the fermentation process were analyzed, and the GABA content in mulberry wine was evaluatedThe results showed that there were 7 kinds of organic acids detected during fermentation, and the content of them showed a trend of rising and then decreasing, and 54 kinds of volatile flavor substances were detected, of which 19 were esters, 16 were alcohols, 8 were aldehydes, ketones 4, phenols were 3 and 4 other categories, and the relative content of high to low alcohols was class, ester, ketone, aldehyde and phenol, and the types of volatile flavor substances were also changed with the relative change of the fermentation processThe analysis of the main ingredients showed that the aldehydes, ketones and phenols in mulberry provide precursors for other flavor ingredients in fermented mulberry wineAn evaluation of GABA content found that the GABA quality concentration in mulberry wine reached 1,180 mg/Lmulberry (MB), also known as the mulberry plant, its fruit is deep fuchsia, containing a large number of natural red pigment, its main pigment is anthocyanins, with the inhibition of small platelet coagulation, prevention of heart disease, blood clots, delay aging and other physiological effectsChina's Ministry of Health has listed mulberry as one of the agricultural products "both food and medicine"Therefore, mulberry is a kind of medicine-eating health fruit with great potential and economic benefitsAt present, mulberry mainly fresh food, less used for deep processing, because of its seasonal strength, short preservation time, not easy to store, high transport costs, so that the sale of fresh fruit is greatly restricted, seriously restricting the development of mulberry industryIn recent years, under the guidance of advocating the diversified development of fruit industry, the development of mulberry wine can not only reflect the flavor characteristics of mulberry fruit, but also maintain some of the nutritional and health benefits of its fruitTherefore, the study of mulberry wine has a positive significance for the sustainable development of mulberry industrygamma-aminobutyric acid, or GABA, is a non-protein amino acid with a variety of physiological functions, such as lowering blood pressure, diureticing, and enhancing memoryWith the improvement of people's living standard and the enhancement of health care consciousness, functional fermented drinks rich in GABA have become a hot topic in research and developmentIn the development of fermented drinks, flavor is one of the important evaluation indicators, the quality of flavor directly affects the quality of drinkHowever, the unique flavor and quality of fermented drinks are affected by many factors, which are particularly closely related to fermented microorganismsLi Yan et alstudied the influence and correlation of 3 kinds of winey yeast on aroma composition in the process of dry red wine, and the results showed that there were significant differences in the relative content of the main aroma composition in the three yeast fermented wines; Kang Mingli et al analyzed the changes of active dry yeast in the volatile flavor substances in the citrus wine brewing process At present, the study of the change and correlation of flavor substances in the process of producing GABA's brewing yeast fermentation and preparation of functional mulberry wine has not been reported this study to mulberry as raw material, using the laboratory pre-screening obtained by the production of GABA brewing yeast JM037 fermentation preparation functional mulberry wine, through the fermentation process of flavor material changes, correlation and GABA content analysis, for the brewing yeast and its fermentation preparation functional mulberry wine industry development to provide a certain theoretical reference 1 Materials and Methods 1.1 Materials and Reagents produced GABA Brewing Yeast JM037, the laboratory separated from Sichuan kimchi obtained strains, NCBI login number: KU761835, commercial Angel ican high temperature wine Angel active dry yeast purchased in Yeast Co., Ltd The strain is preserved with a malt agar medium slope with 5 g/L-L-glutamate (L-monosodium glutamate, L-MSG) mulberry (big ten), purchased from Sichuan Otsuka mulberry base (sugar content 7 g/100mL); GABA chromatography pure (content of 99%), Shanghai Golden Spike Biotech Co., Ltd.; phthalates (ortho-Phthalaldehyde, OPA), Chengdu Huaxia Chemical Reagent Co., Ltd.; 1.2 Instruments and Equipment Waters2695 High-Efficiency Liquid ChromaTography (with Waters2998 UV Detector), Waters Corporation, USA; GC-MS QP2010 Plus Gas Chromatography-Mass Spectrometry, Japan' Mizuno Corporation; Extraction needle 75 sm CAR/PDMS, SPME manual sample handle, Supelco, United States; 7820A gas chromatography (with hydrogen flame ionization detector), Agilent Technology Co., Ltd.; pHS-3C acidity meter, Chengdu Century Ark Technology Co., Ltd 1.3 method 1.3.1 brewing process reference pan-tinsean wine brewing process, select and completely crush mulberry wine brewing process, after selection and complete lysave, sucrose sugar regulation to 19%, add a heavy potassium sulphate, so that the effective SO2 content of 60 mg/L, and add L-MSG to 5 g/L In contrast to Angel's high-temperature brewing high-activity dry yeast (AQ) for comparison, according to 6 lg CFU/mL inoculation of the amount of wine yeast JM037, the temperature is controlled at (22 x 2) to 4 g/L, after the residual sugar quality concentration is reduced to 4 g/L, to 30 mg/L sulphate seal tank, to stop fermentation, and reduce the temperature to 0 -5 degrees C, to clarify 1.3.2 Analysis of Yeast Count: Using diluted coating YPD plate to determine the change in the number of brewery yeast JM037 and commercially active dry yeast during fermentation; pH determination: PH change during saphration fermentation using pHS-3C acidity meter; The determination of total acid titration, the determination of sugar content: by reference to GB 5009.7-2016 "Measured sugar reduction in food" method, ethanol content determination: gas chromatography method, reference to Xiewen and so on, using methanol dilution method for ethanol yield detection, while not inoculated AQ and JM037 yeast of mulberry juice detection as an experimental blank group 1.3.3 Analysis of the organic acid content of the fermentation process of mulberry wine using HPLC method analysis, by reference to the organic acid detection method of Shang Jingmin, etc , to determine the organic acid content during the fermentation process of mulberry wine, chromatography conditions: chromatography for resin column HPX-87H (hpX-87H) 300 mm x 7.8 mm, Bio-Rad), flow phase 0.057 mol/L H2SO4, flow rate of 0.6 mL/min, detection wavelength of 210 nm, column temperature of 60 degrees C, sample volume 20 sL 1.3.4 GC-MS analysis reference to PAULO and so on, based on HS-SPME/GC-MS analysis of volatile substances of different mulberry varieties, using top-air solid phase micro-extraction techniques and gas-phase chromatography-mass spectrometry methods to analyze the process of flavor ingenuity and differences in different fermentation stages 1.3.5 The GABA content determination in mulberry wine using the method of phthalate diformph derivation, etc to determine the yield of GABA after fermentation of mulberry wine, HPLC analysis conditions: the column is Isauin-GL IN ERTSIL ODS-3 (4.6 mm x 150 mm, 5 sm); UV detection wavelength of 254 nm; column temperature of 30 ?C; sample intake 20 ?L; flow phase A is methanol, flow Phase B is sodium acetate (pH 6.2): methanol: tetrahydrofuran (84:25:1, V/V); min, 80% - 50%; 6 -9 min, 50% - 20%; 9 - 10 min, 20% - 0%; 10 - 11 min, maintain edt; 11 - 15 min, return 80% 1.3.6 Experimental data analysis experimental data using IBM SPSS Statistics 20 statistical software for single-factor aNOVA, multiple comparison (LSD), and principal component analysis (principal analysis, PCA) The line and hot-spot charts are drawn using Origin 8.0 and Heml 1.0.3.3-Heatmapat Illustror, respectively Each set of experiments has 5 levels, and all results are represented by mean averages and standard deviations 2 Results and Analysis 2.1 Analysis of the physiological and chemical index of the fermentation process of mulberry wine the results of the chemical and chemical index in the process of brewing yeast JM037 and commercially active dry yeast fermentation preparation of mulberry wine are shown in Table 1 note Note: "MB" is a sample treated with the adjustment of acid and sugar in mulberry juice and the use of ultra-high temperature instantaneous sterilization technology; showed from Table 1 that the pH decreased at the beginning of fermentation and then increased slightly, while the change in the titration acid content was not significant (p.gt; 0.05) This may be associated with the initial fermentation of yeast formation of acids such as acetic acid, oxalic acid (see Figure 1 (A)), resulting in a decrease in pH, but then the yeast into the alcohol fermentation stage, its secretion of protease hydrolysis protein spawning amino acids, amino acid desanosisto act to produce NH3, resulting in a rise in pH In addition, the changeof the residual sugar and ethanol content in the fermentation process of mulberry wine can be seen, with the fermentation of the brewing yeast JM037 fermentation, the residual sugar is significantly reduced (p-lt;0.05), and the ethanol content is significantly higher, indicating that the yeast strain has good ethanol conversion ability 2.2 changes in organic acids in the fermentation process of mulberry wine using HPLC to determine the organic acid content of mulberry wine in different fermentation periods, the results are shown in Figure 1 (A) Apple acid (malic acid, MLA), citric acid (citric acid, CTA), talic acid (tartaric acid, TTA), succinic acid, SCA, lactic acid (lactic acid, LT) were detected in mulberry juice A) and oxalic acid (OXalic acid, OLA) 6 organic acids with a total organic acid mass concentration of 7.00 g/L, of which the content of malic acid and citric acid is higher, at 2.73 mg/L and 2.24 mg/L, respectively Slightly different from THE results of the determination of the organic acid content in the mulberry juice by KOYUNCU, it may be due to the difference in mulberry varieties can be seen from Figure 1 (A), inoculated wine yeast JM037 fermentation 1 day, 6 kinds of organic acid content are increased, and a small amount of acetic acid production, in the initial fermentation, yeast fermentation using substrate fermentation to produce a variety of organic acids As fermentation progresses, malic acid-lactic acid fermentation is inhibited by alcohol fermentation, resulting in a rapid reduction in lactic acid, malic acid and citric acid The trend and amplitude of organic acid changes in the fermentation liquid of commercially active dry yeast are approximately the same, except for lactic acid content remains stable, and the remaining organic acids basically show a trend of rising and then decreasing, and produce a large number of propionic acid (propionic acid, PPA) and acetic acid (acetic acid, ATA) Main Component Analysis (PCA) is a commonly used mathematical statistical analysis method, which can be de-dimensionalizing in multiple indicators of multiple indicators, and filter out key features and useful information Figure 1 (B) can be seen, the first 2 main components of the winey yeast JM037 and commercialactive dry yeast, respectively, accounted for 73.82 percent and 24.62 percent of the total variance, the two main ingredients have basically represented the main information characteristics of the sample, can be used to analyze the wine fermentation process of wine yeast JM037 and commercial active dry yeast and fermented sanwine organic acid correlation At the forward end of PC1, malic acid and citric acid are associated with the fermentation process of mulberry juice and mulberry wine At the forward end of PC2, malic acid, citric acid, rosacea and oxalic acid are associated with mulberry juice, commercially active dry yeast fermentation 1 d and wine yeast JM037 fermentation 1 d; 2.3 HS-SPME/GC-MS detection of volatile substances in the fermentation process of mulberry wine using HS-SPME/GC-MS method sorcine fermentation process of volatile substances extraction and detection, the results are shown in Table 2 27 types of volatile substances were measured in mulberry juice, 1 d, 5 d, 9 d fermentation of the brewery yeast was 23, 27, 34, respectively, while commercially active dry yeast fermentation 1 d, 5 d, 9 d had 20, 24 and 26 volatile ingredients, respectively As can be seen from Table 3, the relative content of alcohols, aldehydes and phenols in mulberry juice is higher, divided.