CN114181843B - Fermented pichia pastoris Z9Y-3 and fruit wine aroma enhancement brewing process thereof - Google Patents
Fermented pichia pastoris Z9Y-3 and fruit wine aroma enhancement brewing process thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
- C12G3/024—Preparation of other alcoholic beverages by fermentation of fruits other than botanical genus Vitis
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- C12N1/16—Yeasts; Culture media therefor
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention relates to the technical field of wine and fruit wine brewing, in particular to a fermented pichia pastoris Z9Y-3 and a fruit wine aroma-enhancing brewing process thereof. The fermented pichia pastoris Z9Y-3 is preserved in the China center for type culture collection in 2019, 10 months and 28 days, and the preservation number is CCTCC NO: m2019857 and the fermented pichia pastoris Z9Y-3 flavor enzyme extracting solution are respectively applied to the fruit wine fermentation process, so that the release of variety aroma substances is promoted, the generation of fruit aroma substances is improved, and the fruit aroma and flower aroma characteristics of the fruit wine are enhanced.
Description
Technical Field
The invention relates to the technical field of wine and fruit wine brewing, in particular to a fermented pichia pastoris Z9Y-3 and a fruit wine aroma enhancement brewing process thereof.
Background
For a long time, the wine brewing technology of China is in accordance with the European and American brewing technology standard, and wine grapes are almost introduced from European and American countries, so that most of wine products in China are seriously homogenized, and the typical fruit aroma and flavor are not prominent. In addition, other fruit brewing technologies in China are single, the wine fermentation technology is carried out, the commercial saccharomyces cerevisiae is used for single alcohol fermentation, the product quality is generally not high, particularly the typical aroma of the fruit is weak, and the market competitiveness is lacking.
The aroma is an important evaluation aspect of the quality of fruit wine products and is also a main embodiment aspect of the flavor typicality of the fruit wine products. The aroma substances in the fermented fruit wine are various, and about 1000 kinds of volatile substances in the wine and the fruit wine are detected by the existing instruments. However, not all volatile components can present fragrance, only those substances whose content exceeds their olfactory threshold can be sensed by the human olfactory organ, and the odor activity value is used scientifically and domestically to evaluate the fragrance potential of volatile substances. Odour Activity Value (OAV), i.e. the ratio of the concentration of a substance to the olfactory threshold. The olfactory threshold is the minimum concentration of a substance in solution that is sensed by the panelist's olfactory senses. According to the OAV value and the aroma characteristics, terpenes and C in the aroma substances of the variety 13 The norisoprenoid is an important component of typical aroma characteristics of wine, and the two aroma components generally have lower olfactory threshold, so that the aroma characteristics of the wine can be obviously influenced. The variety fragrance substance mainly comes from fruit wine and fruit without volatile componentsHair glycoside fragrance materials which release active fragrance components under the action of various glycoside hydrolases. Glycosidases in wine are generally derived from various saccharomyces cerevisiae microorganisms in the fermentation process, such as saccharomyces cerevisiae, which produce glycosidases with low activity and low tolerance to the wine brewing environment, and the consistency of the fermentation performance of the yeasts reduces the variety characteristics and regional typicality of wine. Esters in the fermented aroma substances are important contributors to fruity aroma in the fruit wine, and main fruity aroma esters comprise ethyl acetate, isobutyl acetate, isoamyl acetate, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl phenylacetate and phenethyl acetate. The final concentration of the fruity ester substances in the alcoholic fermentation process of the fruit wine depends on the balance of enzymatic synthesis and esterase hydrolysis reaction, so that the content of the fruity ester substances in the wine can be influenced by the enzyme activity of esterase and ester synthetase released by brewing microorganisms in the alcoholic fermentation process.
At present, researches find that non-saccharomyces cerevisiae can secrete glucoside hydrolase with higher activity and stronger tolerance, and mixed fermentation with the saccharomyces cerevisiae greatly contributes to increasing the aroma complexity of wine. In addition, researchers propose that the enzyme activity of esterase and ester synthetase in the alcohol fermentation process can be remarkably promoted by using the preferred non-saccharomyces cerevisiae and saccharomyces cerevisiae mixed fermentation, and the content of fruity aroma substances in the wine is improved. And the fruit wine fragrance and fruit fragrance characteristics can be enhanced by adding the preferred yeast flavor enzyme extracting solution at different stages of single fermentation of the saccharomyces cerevisiae.
Disclosure of Invention
The invention provides a strain of fermented Pichia pastoris Z9Y-3 and a fruit wine aroma enhancement brewing process thereof, and through respectively applying a strain of preferably fermented Pichia pastoris Z9Y-3 and a flavor enzyme extracting solution thereof to a fruit wine fermentation process, the release of variety aroma substances is promoted, the generation of fruit aroma substances is improved, and the fruit aroma and flower aroma characteristics in fruit wine are enhanced.
In order to achieve the purpose, the invention adopts the technical scheme that: a strain of fermented pichia pastoris Z9Y-3 is characterized in that: the fermented Pichia pastoris (Pichia pastoris) Z9Y-3 is preserved in the China center for type culture Collection in 2019, 10 months and 28 days, and the preservation number is CCTCC NO: m2019857.
The aroma-enhancing brewing process of the fermented pichia pastoris Z9Y-3 fruit wine comprises the following steps:
1) Rejuvenating, expanding culture and fermentation culture of a fermentation pichia pastoris Z9Y-3 strain;
inoculating dry powder/glycerol preservation solution of the preferred fermentation pichia pastoris Z9Y-3 into 5 mL-10 mL YEPD culture medium, wherein the inoculation amount is 10 6 cfu/mL, activating for 24-48 hours in a constant temperature incubator at 28 ℃, then inoculating the activated culture solution into 50-100 mL YEPD culture medium with the inoculum size of 10% in volume ratio, and carrying out amplification culture for 24-48 hours at 28 ℃ and 180rpm/min in a shaking table; then inoculating the fermentation pichia pastoris Z9Y-3 after the amplification culture into a fermentation culture medium in an inoculation amount of 10 percent in volume ratio, and performing fermentation culture for 72 to 96 hours at the temperature of 28 ℃ and at the speed of 180 r/min;
2) Extraction of fermented pichia pastoris Z9Y-3 flavor enzyme
Placing the fermented and cultured pichia pastoris Z9Y-3 bacterial liquid into a cell disruption instrument, utilizing glass beads to disrupt yeast cells for 30min under the condition of 65HZ, centrifuging the yeast cells in a centrifuge at the rotating speed of 8000rpm/min for 15min, taking supernatant, and concentrating the supernatant through a PEG 2000 dialysis bag to obtain a fermented pichia pastoris flavor enzyme extracting solution;
3) The flavor enzyme of the fermented pichia pastoris Z9Y-3 is applied to flavor enhancement brewing of fruit wine;
taking ripe fruit, quickly removing stems and crushing at low temperature, and adding 50mg/L SO while crushing 2 Adding 20mg/L pectase, cold soaking the fermented mash at 4 deg.C for 24 hr, and inoculating 10 6 cfu/mL commercial saccharomyces cerevisiae, in the vigorous alcoholic fermentation period, starting fermentation for 0-48, and then adding the yeast flavor enzyme extracting solution in the step 2), wherein the fermentation temperature is controlled to be 20-25 ℃, and during the alcoholic fermentation period, if the sugar content of fruit juice is insufficient, adding cane sugar in the vigorous fermentation period until the target alcohol content reaches 11 +/-1% vol; adjusting the acid content of the fermented juice to 5-8g/L by adding tartaric acid or calcium carbonate to reduce acid, monitoring temperature and specific gravity every day, pressing peel and residue cap, soaking in the juice to promote soaking, separating peel and residue when specific gravity is reduced to about 1.015,continuing to monitor the fermentation temperature and specific gravity of the fermented mash, detecting reducing sugar when the specific gravity is reduced to 0.992-0.996, when the content is lower than 2g/L, indicating that the fermented mash is fermented to be dry, separating upper clear wine, putting the upper clear wine into a clean tank, and simultaneously adding about 60mg/L SO 2 Stopping fermentation, filling the tank, sealing and storing, keeping the temperature at about 15 ℃, and then carrying out conventional clarification and stabilization processes on the fruit wine.
The aroma-enhancing brewing process of the fermented pichia pastoris Z9Y-3 fruit wine comprises the following steps:
1) Rejuvenating and expanding culture of the fermentation pichia pastoris Z9Y-3 strain;
inoculating dry powder/glycerol preservation solution of preferably fermented Pichia pastoris Z9Y-3 into YEPD culture medium, wherein the inoculation amount is 10 6 cfu/mL, activating for 24-48 hours in a constant temperature incubator at 28 ℃, then inoculating the activated culture solution into 50-100 mL YEPD culture medium with the inoculum size of 10% in volume ratio, and carrying out amplification culture for 24-48 hours in a shaking table at 28 ℃ and 180 rpm/min;
2) Flavoring of fermented pichia pastoris Z9Y-3 in alcohol fermentation process
Taking ripe fruit, quickly removing stems and crushing at low temperature, and adding 50mg/L SO while crushing 2 Adding 20mg/L pectase, cold soaking the fermented mash at 4 deg.C for 24 hr, and inoculating the enlarged culture solution (10) of fermented Pichia pastoris Z9Y-3 after the fermented mash is returned to temperature 6 cfu/mL) and Saccharomyces cerevisiae (10) 6 cfu/mL), the fermentation temperature is controlled at 20-25 ℃, during the alcoholic fermentation period, if the sugar content of the juice is insufficient, white granulated sugar is added during the vigorous fermentation period until the target alcohol content reaches 11 +/-1 percent by volume; adjusting the acid content of the fermented juice to 5-8g/L by adding tartaric acid or calcium carbonate for reducing acid, monitoring the temperature and specific gravity every day, pressing a peel residue cap to immerse the fruit juice to promote the immersion of fruit peel, separating peel residue when the specific gravity is reduced to about 1.015, continuously monitoring the fermentation temperature and specific gravity of the fermented mash until the specific gravity is reduced to 0.992-0.996, detecting reducing sugar, separating the upper clear wine into a clean tank when the content is less than 2g/L, and simultaneously adding about 60mg/L of SO 2 Stopping fermentation, sealing and storing the fruit wine in a full tank, keeping the temperature at about 15 ℃, and then carrying out conventional clarification and stabilization processes of the fruit wine.
Furthermore, the sugar content of the fruits is more than or equal to 150g/L, and is calculated by glucose; the acid content is more than or equal to 4g/L in terms of tartaric acid.
Compared with the prior art, the invention has the following advantages and effects:
1) The fermented pichia pastoris Z9Y-3 can produce flavor enzymes such as glycosidase, esterase, ester synthetase and the like at high yield, promote the generation of terpene substances and fruity ester substances in fruit wine, enhance the fruity and floral aroma characteristics of the wine and improve the quality of the wine;
2) Aiming at the substrate specificity of the flavor enzyme, the enzyme activity is detected by using different substrates, the operation method is simple, and the detection result is accurate;
3) According to the invention, the fermented pichia pastoris Z9Y-3 and the flavor enzyme extracting solution thereof are respectively applied to the alcoholic fermentation process of the fruit wine, so that not only are the release of various aroma substances promoted, but also the generation of fruit aroma ester substances is improved, the shaping of the aroma profile of the fruit wine is facilitated, and the quality of the fruit wine is further improved.
Drawings
FIG. 1 is a technical flow diagram of the present invention;
FIG. 2 shows colony morphology (28 ℃,72 h) and cell microscopic morphology (10X 100 fold) of a WL medium of a strain of fermented Pichia pastoris;
FIG. 3 shows the variation of the cumulative amount of C2-C10 esterase enzyme activity during the alcoholic fermentation process; in the figure, deltaSC is single fermentation of saccharomyces cerevisiae; e1, fermenting for 0 hour, and adding yeast flavor enzyme extract; ■ ES2, fermenting for 48 hours, and adding yeast flavor enzyme extract; ● ES3, fermenting 120 alcohol with yeast flavor enzyme extract; o SP, fermenting the saccharomyces cerevisiae and pichia pastoris in a mixed manner;
FIG. 4 shows the cumulative amount of C2-C10 ester-synthesizing enzyme during alcoholic fermentation, wherein the cumulative amount of C2-C10 ester-synthesizing enzyme during alcoholic fermentation is shown in the figure (DeltaSC: single fermentation of Saccharomyces cerevisiae; ES1: addition of yeast flavor enzyme extract after 0 hour of fermentation; ■ ES2: addition of yeast flavor enzyme extract after 48 hours of fermentation; ● ES3:
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The fermented Pichia pastoris (Pichia pastoris) Z9Y-3 is preserved in the China center for type culture collection in 2019, 10 months and 28 days, and the preservation number is CCTCC NO: m2019857 is obtained by screening from Sichuan liquor cellar, and compared with other saccharomyces cerevisiae, the fermentation pichia pastoris Z9Y-3 can secrete higher glycosidase enzyme activity, especially beta-D-glucosidase enzyme activity, has stronger tolerance to brewing environment, and can promote the release of various aroma substances. Meanwhile, the fermented pichia pastoris Z9Y-3 and the saccharomyces cerevisiae are subjected to mixed fermentation in different inoculation ratios, or Z9Y-3 flavor enzyme extracting solutions are added at different stages of single fermentation of the saccharomyces cerevisiae, so that the enzyme activity accumulation of esterase and ester synthase is improved, the generation of fruit-flavor esters is promoted, and the fruit-flavor aroma characteristic of the fruit wine is enhanced.
As shown in figure 2 (a), the artificially screened excellent fermentation pichia pastoris Z9Y-3 strain is cultured in a YEPD culture medium at 28 ℃ for 48h, a bacterial liquid is diluted to prepare a temporary tablet for microscopic observation, as shown in the figure, the fermentation pichia pastoris cell is lemon-shaped or rod-shaped, the size of the fermentation pichia pastoris cell is (2.7-5.3) × (2.8-6.4) mu m, and the fermentation pichia pastoris cell appears singly or in pairs under the microscopic observation, most of the fermentation pichia pastoris cell is budding and breeding, and no ascospore is observed. As shown in FIG. 2 (b), the bacterial colony of the strain on the WLAN culture medium is circular, the edge is irregular, the surface is not smooth and has bulges, the section is raised, the phenomenon of film generation is obvious, and the strain is easy to pick up. Meanwhile, the strain has higher enzyme activities of glycosidase, esterase and ester synthetase, and is a potential strain for producing fruity ester substances in quantity.
The first embodiment is as follows:
the aroma-enhancing brewing process for fermenting the pichia pastoris Z9Y-3 fruit wine comprises the following steps:
1) Rejuvenation, enlarged culture and fermentation culture of a fermentation pichia pastoris Z9Y-3 strain;
inoculating dry powder/glycerol preservation solution of preferably fermented Pichia pastoris Z9Y-3 into 5mL of YEPD medium, wherein the inoculation amount is 10 6 cfu/mL, activated in a constant temperature incubator at 28 ℃ for 48 hours, followed by inoculating the activated culture broth at an inoculum size of 10% (v/v) in 100mL YEPD mediumCarrying out amplification culture in a shaking table at 28 ℃ and 180rpm/min for 24 hours; then inoculating the fermentation pichia pastoris Z9Y-3 after the expanded culture into a fermentation culture medium in an inoculation amount of 10% (v/v), and performing fermentation culture for 72 hours at the temperature of 28 ℃ and at the speed of 180 r/min;
2) Extraction of fermented pichia pastoris Z9Y-3 flavor enzyme
Adding 2mL of fermentation pichia pastoris Z9Y-3 fermentation culture solution and 2mL of 0.45mm glass beads into a 5mL centrifuge tube, placing the centrifuge tube into a cell crusher, crushing yeast cells for 30min under the condition of 65HZ, carrying out ice-bath on ice for 30s every 3min when the cells are crushed, repeating the whole operation for 10 times until the cell crushing rate reaches more than 95%, then centrifuging the cells in a centrifuge at the rotation speed of 8000rpm/min at 4 ℃ for 15min, taking supernatant, concentrating the supernatant through a PEG 2000 dialysis bag, and concentrating the volume by 20 times to obtain the fermentation pichia pastoris flavor enzyme extract. Measuring glycosidase, esterase and ester synthetase enzyme activities by using an ultraviolet spectrophotometer and a gas chromatography-mass spectrometer (GC-MS) respectively;
3) The flavor enzyme of the fermented pichia pastoris Z9Y-3 is applied to flavor enhancement brewing of fruit wine;
taking ripe grapes, quickly removing stems and crushing at low temperature, and adding 50mg/L SO while crushing 2 Adding 20mg/L pectase, cold soaking the fermented mash at 4 deg.C for 24 hr, and inoculating 10 6 cfu/mL commercial saccharomyces cerevisiae, in the vigorous fermentation period of alcohol, after starting fermentation for 0-48, adding the yeast flavor enzyme extracting solution (with the enzyme activity of glycosidase or esterase) in the step 2), controlling the fermentation temperature at 20-25 ℃, and during the alcohol fermentation, if the sugar content of fruit juice is insufficient, adding cane sugar in the vigorous fermentation period until the target alcohol content reaches 11 +/-1 vol; adjusting the acid content of the fermented juice to 5-8g/L by adding tartaric acid or calcium carbonate for reducing acid, monitoring the temperature and specific gravity every day, pressing a peel residue cap to immerse the peel residue cap in the juice to promote immersion, separating peel residue when the specific gravity is reduced to about 1.015, continuously monitoring the fermentation temperature and specific gravity of the fermented mash until the specific gravity is reduced to 0.992-0.996, detecting reducing sugar, when the content is less than 2g/L, indicating that the fermented mash is dry, separating the upper clear wine into a clean tank, and simultaneously adding about 60mg/L of SO 2 Terminating fermentation, filling the tank, sealing, storing at 15 deg.C, and keepingCarrying out conventional clarification and stabilization process of the fruit wine.
Example two:
the non-saccharomyces cerevisiae Z9Y-3 fruit wine aroma enhancement brewing process comprises the following steps:
1) Rejuvenating and expanding culture of the fermentation pichia pastoris Z9Y-3 strain;
rejuvenation, enlarged culture and fermentation culture of the fermentation pichia pastoris Z9Y-3 strain: inoculating dry powder/glycerol preservation solution of preferably fermented Pichia pastoris Z9Y-3 into 5mL of YEPD medium, wherein the inoculation amount is 10 6 cfu/mL, activated in a constant temperature incubator at 28 ℃ for 48 hours, then inoculated with 10% inoculum size (v/v) in 100mL YEPD medium, expanded and cultured in a shaker at 28 ℃ for 24 hours at 180 rpm/min;
2) Aroma enhancement of fermented pichia pastoris Z9Y-3 in alcohol fermentation process
Taking ripe grapes, quickly removing stems and crushing at low temperature, and adding 50mg/L SO while crushing 2 Adding 20mg/L pectase, cold soaking at 4 deg.C for 24 hr, and inoculating 10 mg/L pectase 6 Amplification culture solution and 10 for cfu/mL fermentation of pichia pastoris Z9Y-3 6 cfu/mL commercial saccharomyces cerevisiae, the fermentation temperature is controlled at 20-25 ℃, during the alcoholic fermentation, if the sugar content of the juice is insufficient, white granulated sugar is added during the vigorous fermentation period until the target alcohol content reaches 11 +/-1 vol percent; adjusting the acid content of the fermented juice to 5-8g/L by adding tartaric acid or calcium carbonate for reducing acid, monitoring the temperature and specific gravity every day, pressing a peel residue cap to immerse the fruit juice to promote the immersion of fruit peel, separating peel residue when the specific gravity is reduced to about 1.015, continuously monitoring the fermentation temperature and specific gravity of the fermented mash until the specific gravity is reduced to 0.992-0.996, detecting reducing sugar, separating the upper clear wine into a clean tank when the content is less than 2g/L, and simultaneously adding about 60mg/L of SO 2 Stopping fermentation, sealing and storing the fruit wine in a full tank, keeping the temperature at about 15 ℃, and then carrying out conventional clarification and stabilization processes of the fruit wine.
The sugar content of the fruits in the two embodiments is more than or equal to 150g/L, and is calculated by glucose; the acid content is more than or equal to 4g/L in terms of tartaric acid.
In the early stage of the experiment, the ultraviolet spectrophotometer and GC-MS are utilized to measure the enzyme activities of different flavor enzymes in the fermentation pichia pastoris and the saccharomyces cerevisiae, and the enzyme activities are shown in the table 1:
TABLE 1 enzymatic Activity of different flavors in fermented Pichia and Saccharomyces cerevisiae (mU/mL)
The data results show that Pichia pastoris ferments with β -D-glucosidase (+ 68%) and α -L-arabinosidase (+ 128%) significantly higher than Saccharomyces cerevisiae, especially β -D-glucosidase, which is considered to be the key enzymes for the hydrolysis of non-volatile aroma substances in wine. Therefore, the yeast pichia pastoris is preferably used as the yeast for producing glycosidase with high yield, and can obviously promote the release of aroma substances of varieties when applied to fruit wine fermentation and improve the fragrance of wine aroma. The esterase activity in yeast is obviously higher than that of ester synthetase, and the enzyme activity of C2 esterase (+ 32%), C4 esterase (+ 5%) and C6 esterase (+ 4%) in fermented Pichia pastoris Z9Y-3 is higher than that of saccharomyces cerevisiae, especially the enzyme activity of C2 esterase; the enzyme activity of the ester synthetase in Z9Y-3 is higher than that of saccharomyces cerevisiae, wherein the enzyme activities of C2 ester synthetase (+ 118%), C8 ester synthetase (+ 92%) and C10 ester synthetase (+ 389%) are remarkably different.
TABLE 2 aroma content (mg/L) of fermented Pichia pastoris and its flavor enzyme extract, respectively, and Saccharomyces cerevisiae mixed fermented variety
In the early stage of the experiment, the Elegar grape juice is used as a raw material, and fermentation pichia pastoris Z9Y-3 and Z9Y-3 flavor enzyme extracts are fermented with saccharomyces cerevisiae respectively. The SPME-GC-MS was used to detect the aroma content of the wine (Table 2). As can be seen from Table 2, the variety aroma substances mainly comprise C6 compounds, terpenes and C13-norisoprenoid substances, and compared with single fermentation of the saccharomyces cerevisiae, the release of the terpenes and the C6 compounds in the variety aroma compounds can be remarkably promoted by respectively mixing and fermenting the flavor enzyme extracts of the pichia pastoris Z9Y-3 and Z9Y-3 with the saccharomyces cerevisiae. Wherein, the terpene content is respectively improved by 14 percent and 37 percent, the C6 compound content is respectively improved by 2 percent and 19 percent, and the promotion effect of the Z9Y-3 flavor enzyme extract on the two compounds is larger than the mixed fermentation effect of the Z9Y-3 and the saccharomyces cerevisiae in the single fermentation process of the saccharomyces cerevisiae. However, the hydrolysis effect of Z9Y-3 and the flavor enzyme extract thereof on the C13-nor-isoprenoid precursor is not obvious, and the content of free aroma substances is lower than that of single fermentation of saccharomyces cerevisiae. In general, the fermented pichia pastoris Z9Y-3 and Z9Y-3 flavor enzyme extracts are applied to fruit wine fermentation, so that the release of fruit wine variety fragrance can be promoted, and the fragrance and fruit fragrance characteristics of the fruit wine can be enhanced.
TABLE 3 fermentation of Pichia pastoris and its flavor enzyme extract with Saccharomyces cerevisiae mixed fermentation volatile fermentation aroma content (mg/L)
And then taking simulated grape juice as a raw material, fermenting the pichia pastoris Z9Y-3 and the flavor enzyme extract thereof with saccharomyces cerevisiae respectively, monitoring the enzyme activity changes of esterase and ester synthase by using an ultraviolet spectrophotometer and a gas chromatography-mass spectrometer (GC-MS) respectively in the fermentation process, and drawing a change curve according to the enzyme activity cumulant (as shown in figures 3 and 4). As can be seen from FIG. 3, the cumulative amount of C2-C10 esterase activity is in the range of 90-160U, which is significantly higher than the cumulative amount of ester synthase activity (6-70U) in FIG. 4. Compared with single fermentation of saccharomyces cerevisiae, the mixed fermentation of the fermented pichia pastoris and the saccharomyces cerevisiae can obviously promote the enzyme activities of C2, C4, C6 esterase and C2 ester synthetase, and the improvement ratios are 28.18%,7.67%,116.77% and 59.94% respectively. And after the pichia pastoris flavor enzyme extract is added, the cumulative activity of C2, C4, C6, C8 esterase and C2 ester synthetase is respectively improved by 5.74%, 71.29%, 91.19%, 35.83% and 48.93%. SPME-GC-MS is used for detecting the content of volatile fermentation aroma substances in the simulated grape juice (Table 3), and data results show that the generation amounts of ethyl acetate and higher alcohol acetate obtained by single fermentation of saccharomyces cerevisiae are respectively 24.3mg/L and 1.7mg/L, the contents of the ethyl acetate and the higher alcohol acetate obtained by mixed fermentation of pichia pastoris and saccharomyces cerevisiae are respectively 28.9mg/L and 3.4mg/L, the mixed fermentation obviously promotes the generation of acetate, and the yield of the higher alcohol acetate is doubled. And the content of acetate is improved by adding the yeast flavor enzyme extract at different time, and the content of acetic acid higher alcohol in the yeast flavor enzyme extract added in 0 hour is the highest (3.5 mg/L), which indicates that the effect of adding the fermented pichia pastoris flavor enzyme extract in the alcohol fermentation process on improving the content of acetate can be the same as that of mixed fermentation.
As for fatty acid ethyl ester, short-chain fatty acid ethyl ester has no obvious difference between different treatments, mixed fermentation has no obvious promotion effect on the generation of the short-chain fatty acid ethyl ester, the content of medium-chain fatty acid ethyl ester in mixed fermentation is 7.3mg/L, the content of single fermentation of saccharomyces cerevisiae is 6.8mg/L, and the mixed fermentation also promotes the generation of the medium-chain fatty acid ethyl ester. Meanwhile, fermentation pichia pastoris flavor enzyme extracts are added in different stages of fermentation, which is also beneficial to the generation of ethyl acetate, higher alcohol acetate and medium-chain fatty acid ethyl ester. The data result shows that the optimized fermentation pichia pastoris and the flavor enzyme thereof are applied to the alcoholic fermentation process, so that the release of various aroma compounds can be remarkably promoted, the generation of fruit-flavor esters is facilitated, and the fruit-flavor aroma characteristics and aroma quality of the wine are improved.
Further, pearson correlation analysis revealed that the relationship between the enzyme activities of different esterases and ester synthases and the fruity esters was different (Table 4). The C2, C4 and C6 esterases and the fruity ester substances present obvious positive correlation, the C8 and C10 esterases are negatively correlated with acetate and short-chain fatty acid ethyl ester, and the total esterase enzyme activity and the medium-chain fatty acid ethyl ester present obvious positive correlation, so that the improvement of the esterase enzyme activity is beneficial to the generation of the fruity ester substances, particularly the medium-chain fatty acid ethyl ester (0.473). The correlation between the fruity ester substances and the enzyme activities of the ester synthetases with different carbon chain lengths is not obvious, the correlation between the fruity ester substances and the esterase is positive, the correlation coefficients with the short-chain fatty acid ethyl ester and the medium-chain fatty acid ethyl ester are respectively 0.429 and 0.375, and the correlation is obvious positive. Therefore, the enzyme activity of esterase and ester synthetase can be promoted by adding the yeast flavor enzyme extract in the process of mixed fermentation or fermentation, so that the generation of fruity ester substances such as ethyl acetate, higher alcohol acetate, fatty acid ethyl ester and the like is promoted, and the fruity aroma characteristic of the fruit wine is enhanced.
TABLE 4 Pearson correlation coefficients between enzyme activities of different esterases, ester synthases and fruity esters
| Influencing factor | Ethyl acetate | Higher alcohol acetate | Short-chain fatty acid ethyl ester | Medium chain fatty acid ethyl ester |
| C 2 Esterase | 0.419 * | 0.392 * | 0.355 | 0.334 |
| C 4 -esterase | 0.421 * | 0.454 * | 0.522 ** | 0.567 ** |
| C 6- Esterase | 0.348 | 0.422 * | 0.196 | 0.449 * |
| C 8 Esterase | -0.007 | -0.144 | -0.016 | 0.259 |
| C 10 Esterase | 0.081 | -0.131 | -0.008 | 0.317 |
| Total esterase activity | 0.340 | 0.304 | 0.354 | 0.473 ** |
| C 2 -ester synthases | 0.365 * | 0.227 | 0.083 | 0.209 |
| C 4 -ester synthases | 0.191 | 0.188 | 0.207 | 0.267 |
| C 6 -ester synthases | 0.292 | 0.330 | 0.300 | 0.258 |
| C 8 -ester synthases | 0.345 | 0.354 | 0.455 * | 0.403 * |
| C 10 -ester synthases | 0.330 | 0.351 | 0.487 ** | 0.386 * |
| Total ester synthase enzyme Activity | 0.323 | 0.345 | 0.429 * | 0.375 * |
The detection method of the fruit wine aroma contribution substances refers to Kong, li, jin, zhu, and Tao (2019), and the contents of variety aroma substances and fermentation volatile substances are detected by SPME-GC-MS. The fermentation pichia pastoris Z9Y-3 is used as a non-saccharomyces cerevisiae for high yield of glycosidase enzyme activity, can promote the release of variety aroma substances by mixed fermentation with saccharomyces cerevisiae, can improve the enzyme activity of the flavor enzymes such as esterase, ester synthetase and the like in the alcohol fermentation process, and is further favorable for the generation of fruity ester substances. The flavor enzyme extract of the saccharomyces cerevisiae is added in a single fermentation process of the saccharomyces cerevisiae, so that the generation of ethyl acetate, higher alcohol acetate, short-chain fatty acid ethyl ester and medium-chain fatty acid ethyl ester can be obviously improved, and the aroma characteristic of the fruit wine is enhanced. Therefore, the brewing process of applying the fermented pichia pastoris Z9Y-3 or Z9Y-3 flavor enzyme extracting solution to fruit wine fermentation is effective in enhancing fruit aroma and flower aroma characteristics of the fruit wine.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention.
Claims (4)
1. A strain of fermented pichia pastoris Z9Y-3 is characterized in that: the fermented pichia pastoris (A)Pichia fermentans) Z9Y-3 is preserved in the China center for type culture Collection in 2019, 10 and 28 months, and the preservation number is CCTCC NO: m2019857.
2. The method for flavoring and brewing pichia pastoris Z9Y-3 fruit wine according to claim 1, which is characterized in that: the method comprises the following steps:
1) Rejuvenation, enlarged culture and fermentation culture of a fermentation pichia pastoris Z9Y-3 strain;
inoculating dry powder/glycerol preservation solution of the preferred fermentation pichia pastoris Z9Y-3 into 5 mL-10 mL YEPD culture medium, wherein the inoculation amount is 10 6 cfu/mL, activating for 24 to 48 hours in a constant temperature incubator at 28 ℃, then inoculating the activated culture solution into 50 to 100mL YEPD culture medium with the inoculation amount of 10 percent in volume ratio, and carrying out amplification culture for 24 to 48 hours in a shaking table at 28 ℃ and 180 rpm/min; then inoculating the fermentation pichia pastoris Z9Y-3 after the expanded culture into the strain with the volume ratio of 10 percentFermenting and culturing for 72-96 hours in a fermentation culture medium under the conditions of 28 ℃ and 180 r/min;
2) Extraction of fermented pichia pastoris Z9Y-3 flavor enzyme
Placing the fermented and cultured pichia pastoris Z9Y-3 bacterial liquid into a cell disruption instrument, utilizing glass beads to disrupt yeast cells for 30min under the condition of 65HZ, centrifuging the yeast cells in a centrifuge at the rotating speed of 8000rpm/min for 15min, taking supernatant, and concentrating the supernatant through a PEG 2000 dialysis bag to obtain a fermented pichia pastoris flavor enzyme extracting solution;
3) The flavor enzyme of the fermented pichia pastoris Z9Y-3 is applied to flavor enhancement brewing of fruit wine;
taking ripe grapes, quickly removing stalks and crushing at low temperature, and adding 50mg/L SO while crushing 2 Then 20mg/L pectinase is added, 24h is cold-dipped under the condition of 4 ℃ of the fermented fruit, and then 10 is inoculated 6 cfu/mL commercial saccharomyces cerevisiae, in the vigorous alcoholic fermentation period, starting fermentation for 0-48, and then adding the yeast flavor enzyme extracting solution in the step 2), wherein the fermentation temperature is controlled to be 20-25 ℃, and during the alcoholic fermentation period, if the sugar content of fruit juice is insufficient, adding cane sugar in the vigorous fermentation period until the target alcohol content reaches 11 +/-1% vol; adjusting the acid content of the fermented juice to 5-8g/L by adding tartaric acid or calcium carbonate for reducing acid, monitoring the temperature and specific gravity every day, pressing a peel residue cap to immerse the peel residue cap in the fruit juice to promote immersion, separating peel residue when the specific gravity is reduced to about 1.015, continuously monitoring the fermentation temperature and specific gravity of the fermented mash until the specific gravity is reduced to 0.992 to 0.996, detecting reducing sugar, when the content is less than 2g/L, indicating that the fermentation is dry, separating the upper clear wine into a clean tank, and simultaneously adding about 60mg/L of SO 2 Stopping fermentation, sealing and storing the fruit wine in a full tank, keeping the temperature at about 15 ℃, and then carrying out conventional clarification and stabilization processes of the fruit wine.
3. The pichia pastoris Z9Y-3 fruit wine aroma enhancement brewing method according to claim 1, which is characterized by comprising the following steps: the method comprises the following steps:
1) Rejuvenating and expanding culture of the fermentation pichia pastoris Z9Y-3 strain;
inoculating dry powder/glycerol stock of preferably fermented Pichia pastoris Z9Y-3 into YEPDThe inoculation amount in the culture medium is 10 6 Activating cfu/mL in a constant-temperature incubator at 28 ℃ for 24-48 hours, then inoculating the activated culture solution into 50-100 mL YEPD culture medium with the inoculation amount of 10% by volume ratio, and carrying out amplification culture in a shaking table at 28 ℃ for 24-48 hours at 180 rpm/min;
2) Flavoring of fermented pichia pastoris Z9Y-3 in alcohol fermentation process
Taking ripe grapes, quickly removing stalks and crushing at low temperature, and adding 50mg/L SO while crushing 2 Then 20mg/L pectinase is added, 24h is cold-dipped under the condition that the mash is at 4 ℃, and 10 inoculation is carried out simultaneously after the mash is cooled back to the temperature 6 Amplification culture solution and 10 for cfu/mL fermentation of pichia pastoris Z9Y-3 6 controlling the fermentation temperature of the cfu/mL saccharomyces cerevisiae at 20-25 ℃, and adding white granulated sugar to the target alcohol content to 11 +/-1 vol% in the vigorous fermentation period if the sugar content of the juice is insufficient during the alcoholic fermentation period; adjusting the acid content of the fermented juice to 5-8g/L by adding tartaric acid for increasing acid or calcium carbonate for reducing acid, monitoring the temperature and specific gravity every day, pressing a peel slag cap to immerse into the fruit juice to promote the impregnation of the peel, separating peel and slag when the specific gravity is reduced to about 1.015, continuously monitoring the fermentation temperature and specific gravity of the fermented mash until the specific gravity is reduced to 0.992 to 0.996, detecting reducing sugar, separating the upper clear wine into a clean tank when the content is lower than 2g/L, and simultaneously adding about 60mg/L of SO 2 Stopping fermentation, sealing and storing the fruit wine in a full tank, keeping the temperature at about 15 ℃, and then carrying out conventional clarification and stabilization processes of the fruit wine.
4. The pichia pastoris Z9Y-3 fruit wine aroma enhancement brewing method according to claim 2 or 3, which is characterized in that: the glucose content of the grapes is more than or equal to 150g/L, and is measured by glucose; the acid content is more than or equal to 4g/L in terms of tartaric acid.
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