WO2018214330A1 - 一株高产β-苯乙醇的酿酒酵母菌株及其应用 - Google Patents

一株高产β-苯乙醇的酿酒酵母菌株及其应用 Download PDF

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WO2018214330A1
WO2018214330A1 PCT/CN2017/100446 CN2017100446W WO2018214330A1 WO 2018214330 A1 WO2018214330 A1 WO 2018214330A1 CN 2017100446 W CN2017100446 W CN 2017100446W WO 2018214330 A1 WO2018214330 A1 WO 2018214330A1
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wine
fermentation
phenylethanol
fermented food
strain
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PCT/CN2017/100446
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French (fr)
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毛健
刘双平
姬中伟
韩笑
徐新彪
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江南大学
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Priority claimed from CN201710378178.3A external-priority patent/CN107177520B/zh
Priority claimed from CN201710379232.6A external-priority patent/CN107164250B/zh
Application filed by 江南大学 filed Critical 江南大学
Priority to KR1020197001330A priority Critical patent/KR102139018B1/ko
Priority to JP2018566974A priority patent/JP6946358B2/ja
Publication of WO2018214330A1 publication Critical patent/WO2018214330A1/zh
Priority to US16/226,622 priority patent/US10982295B2/en

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Definitions

  • the invention relates to a strain of Saccharomyces cerevisiae with high yield of ⁇ -phenylethanol and application thereof, and belongs to the technical field of industrial microorganisms.
  • Phenylethanol is an aromatic alcohol with a rose flavor. It is naturally found in essential oils such as jasmine and rose. It is also an important flavor and fragrance ingredient, and is widely used in cosmetics, tobacco and daily chemical products. As a flavor substance in the fermented food, ⁇ -phenylethyl alcohol can improve the flavor and overall quality of the fermented product. However, at present, the chemical synthesis of ⁇ -phenylethanol will have difficult to remove by-products, which may cause cancer risk and seriously affect product quality. Although physical extraction from natural plants can obtain non-toxic and harmless quality ⁇ -phenylethanol For the production of food or other products, but the production cycle is long, the output is low, the price is high, and it is difficult to meet the market demand.
  • the ⁇ -phenylethyl alcohol in the fermented food can be produced by microbial metabolism, and the ⁇ -phenylethyl alcohol content in the fermented food can be improved by microbial fermentation, and the obtained product belongs to natural food. Saccharomyces cerevisiae produces ⁇ -phenylethanol through the Ehrlich pathway and other metabolic pathways during fermentation.
  • the ⁇ -phenylethanol content in fermented foods such as rice wine can reach about 100 mg/L, although the concentration is already high, Further improvement of ⁇ -phenylethanol is significant for enhancing the flavor of yellow wine.
  • the content of ⁇ -phenylethyl alcohol in wine can reach about 60 mg/L, it is necessary to further improve the ⁇ -phenylethanol content for the development of the characteristics of rose aromatic wine, so it is necessary to screen yeast with excellent performance.
  • non-Saccharomyces cerevisiae since non-Saccharomyces cerevisiae has low ethanol production capacity, it cannot be used as a main strain for the fermentation of fermented foods such as alcohol and vinegar.
  • Rafael et al. (Overproduction of 2-phenylethanol by industrial yeasts to improve organoleptic properties of bakers' products, International Journal of Food Microbiology, 2014, 180(1): 7-12.) reported a high-yield ⁇ -phenylethanol baker's yeast in Application in baked goods, but did not report on the alcohol-producing properties of the reported yeast, which is applied to baked goods that do not require high concentrations of alcohol.
  • Saccharomyces cerevisiae Although there have been many reports on the production of certain concentrations of Saccharomyces cerevisiae for brewing foods, it is unclear whether the strain has a high alcohol production capacity. Since the stress ability of ⁇ -phenylethanol to yeast is significantly higher than that of ethanol, the ability of ethanol producing high-yield ⁇ -phenylethanol to produce ethanol is usually reduced, so the alcoholic production of Saccharomyces cerevisiae with high yield of ⁇ -phenylethanol is low. Therefore, in the case of not adding exogenous precursor compounds, Saccharomyces cerevisiae capable of producing ⁇ -phenylethyl alcohol and ethanol can be brewed. It has high application value in industry.
  • the present invention provides a strain of Saccharomyces cerevisiae which has high yield of ⁇ -phenylethanol and has excellent alcohol-producing properties without adding exogenous amino acids, and is used in rice wine, cooking wine, brewing vinegar, soy sauce, white wine, and fruit wine.
  • the strain has the ability to produce ⁇ -phenylethyl alcohol and the high-yield flavor substance acetate-2-phenylethyl ester, and has good fermentation performance, can significantly improve the ⁇ -phenylethanol content in the fermented product, improve the quality of the fermented product, and has wide application prospects. .
  • a first object of the present invention is to provide a strain of Saccharomyces cerevisiae having high yield of ⁇ -phenylethanol, which was deposited with the China Center for Type Culture Collection on December 26, 2016, and deposited at Wuhan University, Wuhan, China. The number is CCTCC NO: M 2016785.
  • the Saccharomyces cerevisiae strain of the present invention is a starting strain of Saccharomyces cerevisiae selected from the yellow wine sputum liquid, and is subjected to ultraviolet mutagenesis to screen for fluorophenylalanine resistance, and then screening a strain with good growth to be inoculated with 10% ethanol.
  • the alcohol tolerance screening and the yellow wine simulated liquid fermentation screening were carried out, and the strain with relatively high ⁇ -phenylethanol production was used as the starting strain for normal temperature isobaric plasma mutagenesis, and the strain after the second mutagenesis was carried out. Fluorophenylalanine resistance screening, and screening of fermentation characteristics, yielded high-yield ⁇ -phenylethanol Saccharomyces cerevisiae.
  • the ⁇ -phenylethyl alcohol content of the rice wine obtained by fermentation can reach 410mg/L, the content of 2-phenylethyl acetate is 56 ⁇ g/L, and the alcohol content is 17% (v/v);
  • the ⁇ -phenylethyl alcohol content in the wine obtained by fermentation can reach 450mg/L, the content of 2-phenylethyl acetate is 50 ⁇ g/L, and the alcohol content is 15% (v/v);
  • the obtained wine cellar is fermented by acetic acid, and the content of ⁇ -phenylethyl alcohol in the vinegar is 300 mg/L, and the ethyl acetate is 2-phenylethyl acetate.
  • the content is 45 ⁇ g / L;
  • the Saccharomyces cerevisiae is inoculated into a soy sauce fermentation system, the ⁇ -phenylethanol content in the soy sauce obtained by fermentation is 200 mg / L;
  • the brewing yeast is inoculated into the liquor fermentation system, the content of ⁇ -phenylethyl alcohol in the distilled liquor is 110 mg/L, the content of 2-phenylethyl acetate is 64 ⁇ g/L, and the alcohol content is up to 65% (v/v);
  • the colonies are white, round or elliptical with neat edges.
  • a second object of the present invention is to provide the microbial agent containing the Saccharomyces cerevisiae CCTCC NO: M 2016785 strain.
  • the microbial inoculum contains live cells of Saccharomyces cerevisiae CCTCC NO: M 2016785 cells, freeze-dried Saccharomyces cerevisiae CCCCC NO: M 2016785 dry cells, immobilized Saccharomyces cerevisiae CCCCC NO: M 2016785 cells, liquid yeast of Saccharomyces cerevisiae CCTCC NO: M 2016785, solid fungus of Saccharomyces cerevisiae CCTCC NO: M 2016785, or Saccharomyces cerevisiae CCTCC NO: M 2016785 strain present in any other form.
  • a third object of the present invention is to provide the use of the Saccharomyces cerevisiae strain or the microbial agent.
  • the application refers to the manufacture of a fermented food.
  • the application refers to the field of brewing technology.
  • a fourth object of the present invention is to provide a fermented food obtained by fermenting Saccharomyces cerevisiae CCTCC NO: M2016785 as a starter or a main starter.
  • the fermented food is a brewed food.
  • the brewed food is an alcohol, vinegar or soy sauce.
  • the wine includes, but is not limited to, rice wine, cooking wine, white wine, and the like.
  • the brewed food is yellow wine, and the brewing yeast is used as a wine.
  • the rice wine is brewed by adding the brewing yeast as a wine master to the cooked or gelatinized raw material in an amount of 5% to 10%, and fermenting, pressing, frying, and aging. , filtering, sterilization and filling to get yellow wine.
  • the brewing of the rice wine is specifically: preparing the wine master by cultivating the brewing yeast, and then adding the koji according to a total volume of 4%, adding the wine to the glutinous rice which is high temperature gelatinized according to the total volume of 10%. Stir well, then fermented, pressed, decocted, aged, filtered, sterilized and filled to obtain yellow wine.
  • the brewed food is cooking wine.
  • the cooking wine is brewed by first using the brewing yeast as a wine to obtain a yellow wine, and then using the obtained yellow wine to prepare a raw wine.
  • the brewed food is vinegar.
  • the vinegar is brewed by first using the Saccharomyces cerevisiae as a wine to produce a yellow wine, and then using the obtained yellow wine as a raw material for acetic acid fermentation to brew vinegar.
  • the vinegar is brewed by solid state fermentation or liquid fermentation.
  • the brewed food is soy sauce.
  • the soy sauce is brewed using a high salt lean fermentation or a low salt solid state fermentation to prepare the soy sauce.
  • the high-salt dilute fermentation preparation of the soy sauce is: mixing soybean meal and wheat with steam, inoculating Aspergillus oryzae, adding salt water to make the salt content of the sauce is 18%, and the water content is 65%. Stir and mix; then culture the CCTCC NO:M 2016785 Yeast access part of steamed and cooled soybean meal and wheat, add water, culture to make CCTCC NO: M 2016785 wine master, waiting to be added to the sauce; when the temperature of the sauce is raised to 20 °C during the fermentation process When accessing CCTCC NO: M 2016785 wine master; fermentation time is 5 months; after the fermentation, the sauce is pressed, filtered, and clarified to obtain soy sauce.
  • the low-salt solid-state fermentation preparation of the soy sauce is specifically: the soybean meal and the wheat are mixed and steamed, and the amount of Aspergillus oryzae is inoculated, and the salt content of the sauce is 7%, the water content is 40%, and the mixture is stirred and mixed. Then, the cultured CCTCC NO:M 2016785 yeast is partially added to the steamed and cooled soybean meal and wheat, and the water is added to form CCTCC NO:M2016785 wine master, which is connected to the sauce fermentation system, and the temperature is controlled at 40. °C; fermentation time is 15d; the sauce after the end of the fermentation removes impurities and precipitates, and clarifies by filtration to obtain soy sauce.
  • the brewed food is white wine.
  • the liquor is brewed by additionally adding the brewing yeast when the liquor is fermented into the pond for fermentation.
  • the additional amount of Saccharomyces cerevisiae in the brewing of the liquor is 1%.
  • a fifth object of the present invention is to provide a method for enhancing the aroma of fruit wine, particularly a method for enhancing the aroma of rose wine, which utilizes the Saccharomyces cerevisiae CCTCC NO: M 2016785 of the present invention as a fermentation strain.
  • Saccharomyces cerevisiae is a lyophilized powder of CCTCC NO: M 2016785 strain.
  • the method comprises adding the freeze-dried Saccharomyces cerevisiae CCTCC NO: M 2016785 dry cells to the juice in an amount of 2 ⁇ .
  • the fruit wine is obtained by mixing and fermenting one or more of mulberry juice, hawthorn wine, bayberry juice, and cherry juice.
  • the fruit wine is mulberry wine, hawthorn wine, bayberry wine, cherry berry wine, and the like.
  • the method includes the following steps:
  • the present invention obtains a strain of Saccharomyces cerevisiae which has high yield of ⁇ -phenylethanol and has excellent alcohol-producing properties without adding exogenous amino acids.
  • the Saccharomyces cerevisiae strain of the present invention can be used for the brewing of rice wine, cooking wine, vinegar, soy sauce, and white wine; when used in the brewing of these products, not only can produce high concentration of ⁇ -phenylethyl alcohol, but also have high alcohol production. Ability, and can effectively increase the content of other flavor ingredients or beneficial ingredients, such as the content of 2-phenylethyl acetate.
  • Figure 1 is a growth curve of the starting yeast strain in Example 1;
  • Example 2 is a graph showing the ultraviolet irradiation lethality curve of the starting yeast strain in Example 1;
  • Figure 3 is a graph showing the lethality ratio of the starting strain to fluorophenylalanine in Example 1;
  • Figure 4 is a graph showing the change in alcoholic degree of rice wine fermentation in Example 2.
  • Figure 5 is a graph showing the acidity change of rice wine fermentation in Example 2.
  • Figure 6 is a graph showing the pH change of rice wine fermentation in Example 2.
  • Figure 7 is a diagram showing the colony morphology of BYC3 Saccharomyces cerevisiae in Example 2;
  • Figure 8 is a graph showing the ⁇ -phenylethanol production of the Saccharomyces cerevisiae strain in Example 2 in YPD.
  • YPD liquid medium yeast extract 10 g / L, fish meal peptone 20 g / L, glucose 20 g / L.
  • YPD solid medium yeast extract 10g / L, fish meal peptone 20g / L, glucose 20g / L, nutrient agar 20 g/L.
  • Fig. 1 The experimental results are shown in Fig. 1 : the yeast OD600 value increased significantly when cultured for 3-5 h, at which time the yeast growth was in the exponential growth phase, and the wild strain was in the mid-exponential growth phase when the shaker was cultured for 4 h. Therefore, a yeast cultured for 4 hours on a shaker was selected as the mutagenic starting strain.
  • the yeast strain bacterial solution is obtained by mutagenesis.
  • UV radiation first open the UV lamp for 20 minutes to stabilize the light wave. Pipette 4.5 mL of the above bacterial suspension into a sterile Petri dish with a diameter of 9 cm using a 5 mL sterile pipette, and add a sterile pin to the Petri dish. Place the culture dish containing the bacterial suspension on a magnetic stirrer, place it vertically under the UV lamp, illuminate for 20 s, open the lid under dark conditions (ensure that the UV lamp is evenly illuminated), and expose to ultraviolet light (15W UV lamp, Distance 30cm), time is 40s, 60s, 80s, 100s, 120s.
  • the mutagenized yeast suspension is diluted by a 10-fold dilution method under a red light or in a dark condition by 4 gradients 10 -1 , 10 -2 , 10 -3 , 10 -4 200 ⁇ L of coated YPD plates were taken for each gradient and wrapped in tin foil to protect from light.
  • Unmutated yeast suspension Dilute 5 gradients 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 by 10 - fold dilution , and take 200 ⁇ L of coated YPD for each gradient Plate, as a control.
  • the control group was made in three parallels and cultured at 30 ° C for 48 h.
  • YNBP solid medium 6.7% YNB, 20g/L glucose, 10g/L proline, and additional p-fluorophenylalanine, the concentration is 0 (control group), 0.04g / L, 0.05g / L, 0.06 g/L, 0.07 g/L, 0.08 g/L, 0.09 g/L, and 0.1 g/L.
  • the exponential growth medium-stage bacterial suspension was diluted with 10 gradients of 10 -1 , 10 -2 , 10 -3 , and 10 -4 by 10 -fold dilution method, and 200 ⁇ L of 10 -4 gradient bacterial suspension was applied to coat the YNBP plate.
  • Mortality rate (number of colonies in the control group - number of colonies in the mutagenized group) / number of colonies in the control group
  • the lethality curve of fluorophenylalanine is shown in Figure 3. As the concentration of fluorophenylalanine on the YNBP plate increases, the yeast lethality increases and the concentration of p-fluorophenylalanine increases to 0.09 g/ The yeast was all lethal at L, so the minimum lethal concentration of p-fluorophenylalanine was determined to be 0.09 g/L.
  • the UV-induced mutagenized strain was obtained by ultraviolet mutagenesis in step 2, and the total ultraviolet mutagenesis time was 110s, 130s, 150s, respectively.
  • Formulation medium for fluorophenylalanine resistance screening 6.7% YNB, 20 g/L glucose, 10 g/L valine, p-fluorophenylalanine 0.09 g/L, nutrient agar 20 g/L.
  • UV mutagen suspension 200 ⁇ L was applied to a p-fluorophenylalanine resistance screening plate, and wrapped in tin foil to protect from light. 3 plates were prepared for each lethality and cultured at 30 ° C for 72 h.
  • Alcohol screening medium formula: yeast extract 10g / L, fish meal peptone 20g / L, glucose 20g / L, sterile ethanol 10%.
  • a 96-well plate was added with 20 ⁇ L of LYPD liquid medium per well, and the mutant strains selected for the resistance to fluorophenylalanine resistance were inoculated into the wells, and cultured at 30 ° C for 24 hours.
  • a 96-well plate was added with 200 ⁇ L of alcohol screening medium per well.
  • the seed solution of each well in the previous step was inoculated to the well plate at 5%, and cultured at 30 ° C. OD600 was measured with a microplate reader at 12 h and 24 h, respectively.
  • Preparation of rice wine simulating liquid 1kg steamed rice (water content: 70%), adding 1L of water, 0.05kg of koji, stirring evenly, incubating at 60°C for 8h, centrifuging at 4500r/min for 5min, taking supernatant at 115°C for 15min. .
  • the content of ⁇ -phenylethanol in the simulated fermentation broth of rice wine was determined by high performance liquid chromatography.
  • the average content of ⁇ -phenylethyl alcohol in the 1-e4 mutant strain was relatively high at 185.032 mg/L (as shown in Table 1). This strain was used as the starting strain for the next round of isothermal isostatic plasma mutagenesis.
  • Example 2 Normal temperature isostatic plasma mutagenesis and screening
  • the selected strain 1-e4 was used as a starting strain for normal temperature isobaric plasma mutagenesis.
  • 1-e4 strain YPD shake flask was cultured at 30 ° C for 24 h, and the bacterial suspension with OD600 of 0.6-0.8 was made with physiological saline, and subjected to isothermal isostatic plasma mutagenesis.
  • the mutagenesis time was 60 s, the power was 100 w, and the strain was mutagenized.
  • YNBP plate grows good strain for yellow wine simulation solution Fermentation screening.
  • the content of ⁇ -phenylethanol in rice wine simulating liquid was determined by high performance liquid chromatography, and the strain with relatively high ⁇ -phenylethanol content was screened.
  • the content of ⁇ -phenylethyl alcohol in the simulated fermentation broth of the rice wine after 8 days of fermentation was determined by high performance liquid chromatography. 3-c10, 4-c7, 5-f5
  • the average content of ⁇ -phenylethyl alcohol was relatively high, which was 217.192 mg/L, 257.388 mg/L and 337.168 mg/L, respectively.
  • the three strains were named BYC1, BYC2 and BYC3.
  • the production of ⁇ -phenylethanol in Saccharomyces cerevisiae increased. Although the ethanol production of Saccharomyces cerevisiae decreased slightly after mutagenesis, the obtained Saccharomyces cerevisiae could still be fermented well.
  • Preparation of wine master 50mLYPD shake flask was inoculated into yeast strain, cultured at 30 ° C, 200r / min for 24h. 1 kg of steamed rice (water content: 70%) was added with 1 L of water and 0.05 kg of koji, stirred uniformly, and kept at 60 ° C for 4 h. After cooling, the yeast solution was inoculated at 5%, and cultured at 30 ° C, 200 r / min for 16 h.
  • Steamed rice (water content 70%), add equal weight of water, 2% koji, 5% wine, stir well.
  • the fermentation temperature was 28 ° C, and the mixture was stirred and sampled at 18 h, 24 h, 30 h, 42 h, 54 h, 78 h, 126 h after the completion of the ingredients.
  • the supernatant sample was obtained by centrifugation at 5000 r/min for 10 min for index detection.
  • the samples were taken at 18h, 24h, 30h, 42h, 54h, 78h and 126h to determine the total acid (as lactic acid), alcohol content and pH.
  • the changes of the three indexes were observed and determined by high performance liquid chromatography in 126h samples. --Phenylethanol content.
  • the content of ⁇ -phenylethanol in the fermentation broth of rice wine is as shown in Table 3 below.
  • the rice wine was fermented with BYC1, BYC2 and BYC3 strains.
  • the ⁇ -phenylethanol content was 219.08, 254.91, 365.70 mg/L.
  • the fermentation of the starting strain was only ⁇ -phenylethyl alcohol production.
  • the yield of ⁇ -phenylethanol of the three strains was 188.07 mg/L, which was 1.16 times, 1.35 times and 1.94 times of that of the original strain, respectively, and had good ability to produce ⁇ -phenylethanol.
  • Isobutanol, isoamyl alcohol, 2-phenylethyl acetate and glycerol in the fermentation broth of rice wine can be used as flavor substances to improve the overall quality of yellow wine.
  • the content of the wine is shown in Table 4.
  • the acetic acid-2-phenethyl acetate of BYC3 strain And the glycerol content is significantly higher than the starting bacteria Strain and BYC1 and BYC2.
  • BYC3 has a high yield of ⁇ -phenylethanol, and isobutanol, isoamyl alcohol, and 2-phenylethyl acetate are also high.
  • the BYC3 strain was streaked onto a YPD solid medium plate and cultured at 30 ° C for 24 h. The colony morphology was as shown in Fig. 7.
  • the BYC3 strain was deposited with the China Center for Type Culture Collection under the accession number CCTCC NO: M 2016785.
  • the BYC3 strain was inoculated into the rice wine fermentation system, and the alcohol fermentation performance was good.
  • the ⁇ -phenylethyl alcohol content in the obtained rice wine was 365.70 mg/L.
  • BYC3 strain (ie CCTCC NO: M 2016785), wild strain, commercial active dry yeast, Saccharomyces cerevisiae model strain W303 (genotype Mat a/a, ura3-1, leu2-3, 112, trp1-1, his3-11, 15 , ade2-1, can1-100), Saccharomyces cerevisiae model strain S288c (genotype MAT ⁇ SUC2gal2mal2melflo1flo8-1hap1ho bio1bio6) was subjected to comparative fermentation experiments. The five strains were activated by YPD plate and then connected to YPD shake flask, and then sequentially connected to YPD shake flask and YPD shake flask (add 1g/L phenylalanine) according to 5%.
  • Example 3 Application of high-yield ⁇ -phenylethanol yeast in rice wine
  • Fermentation and agitation The fermentation temperature was 28 ° C, the timing was started after the blanking was completed, and the stirring was performed every 8 hours, and the mixture was stirred 6 times at 48 hours. Fermentation for 5-7 days, the fermentation is terminated when the alcohol index is no longer elevated.
  • Sterilization and filling over-sterilization machine, sterilized at 85 ° C for 30 min, hot filling.
  • the obtained product was detected by high performance liquid phase method with ⁇ -phenylethanol content up to 410 mg/L, alcohol content of 17% (v/v), ethyl acetate content of 24 mg/L, and acetic acid 2-phenylethyl ester content of 56 ⁇ g/L. .
  • the obtained product was detected by high performance liquid phase method with ⁇ -phenylethanol content up to 185 mg/L, alcohol content of 14% (v/v), ethyl acetate content of 14 mg/L, and acetic acid 2-phenylethyl ester content of 24 ⁇ g/L. .
  • BYC3 strain wild strain, commercial active dry yeast, Saccharomyces cerevisiae model strain W303 (genotype Mat a/a, ura3-1, leu2-3, 112, trp1-1, his3-11, 15, ade2-1, can1-100)
  • Saccharomyces cerevisiae model strain S288c Genotype MAT ⁇ SUC2gal2mal2melflo1flo8-1hap1ho bio1bio6 was subjected to comparative fermentation experiments.
  • the rice wine brewing method of the five strains is as in the above-mentioned yellow wine brewing process of the present embodiment.
  • ⁇ -phenylethanol production (410 mg/L) of Saccharomyces cerevisiae strain BYC3 was significantly higher than that of wild strain (82 mg/L), commercial active dry yeast (87 mg/L), W303 (81 mg/L) and S288c (73 mg/). L).
  • the alcoholic degree of Saccharomyces cerevisiae strain BYC3 reached 17% (v/v), and the commercial alcohol dry yeast yeasts BYC3, W303, and S288c were 16.5% (v/v) 12.4% (v/v) and 13.1%, respectively. /v).
  • Example 3 According to the yellow wine brewing process in Example 3, the rice wine was obtained, 10% of the salt was added, and the decocting machine was sterilized at 85 ° C for 30 minutes. Hot filling.
  • the obtained product was detected by high performance liquid phase method with ⁇ -phenylethanol content up to 450 mg/L, alcohol content of 15% (v/v), ethyl acetate content of 20 mg/L, and acetic acid 2-phenylethyl ester content of 50 ⁇ g/L. .
  • the yellow wine was obtained, and 10% of the salt was added, and the sterilization was performed by sterilizing at 85 ° C for 30 minutes.
  • the content of ⁇ -phenylethanol is as high as 140 mg/L, the alcohol content is 12% (v/v), the ethyl acetate content is 10 mg/L, and the ethyl acetate content is 20 ⁇ g/L.
  • Example 5 Application of high-yield ⁇ -phenylethanol yeast in solid fermentation of brewing vinegar
  • the rice wine was fermented by the process of Example 3 as a raw material for acetic acid fermentation.
  • Acetic acid fermentation adopts solid-state fermentation process: mix the big glutinous rice, bran and yellow wine in a ratio of 1:4:10, and connect to 5% vinegar vinegar, and tweeted from the surface of the material every day for 1-2 days after inoculation, the temperature is 35- 40 ° C. Turn to the bottom of the material by 6-8 days. On days 8-12, the temperature is naturally lowered from the bottom every day. The vinegar was separated from the vinegar and aged for 12 months after sterilization at 85 ° C for 30 min. Hot-filled after high temperature sterilization before filling.
  • the obtained solid-state fermentation brewing vinegar has an acetic acid content of 60 g/L, a ⁇ -phenylethanol content of up to 300 mg/L, and a 2-phenylethyl acetate content of 45 ⁇ g/L.
  • Example 6 Application of high-yield ⁇ -phenylethanol yeast in liquid fermentation of brewing vinegar
  • the rice wine was fermented by the process of Example 3 as a raw material for acetic acid fermentation.
  • the acetic acid fermentation adopts the liquid fermentation process: after the rice wine is diluted 4 times with the water, it is connected to the 5% cultured acetic acid bacteria liquid, and the oxygen is 1 L/min and stirred.
  • the alcohol content in the fermentation system is less than 1%
  • the rice wine is added in batches, the alcohol content in the acetic acid fermentation system is controlled at 1%-4%, and the acetic acid content in the fermentation system is about 80g/L, and the liquid vinegar is obtained by centrifugation. Hot-filled after high temperature sterilization.
  • the content of ⁇ -phenylethyl alcohol in the obtained liquid fermented vinegar was 100 mg/L, and the content of 2-phenylethyl acetate was 36 ⁇ g/L.
  • Example 7 Application of High Yield ⁇ -Phenylethanol Yeast in High Salt Dilute Fermentation of Brewed Soy Sauce
  • the brewed soy sauce is fermented by high-salt method, and the soybean meal and wheat are mixed and steamed in a ratio of 1:1.
  • the inoculum of Aspergillus oryzae was 10%
  • the control temperature was 30 °C
  • the salt water of 2 times the mass of the material was added.
  • the salt content of the sauce was 18%
  • the water content was 65%
  • the mixture was stirred and mixed.
  • BYC3 yeast was cultured in YPD shake flask, and was added to partially steamed and cooled soybean meal and wheat by 5% inoculation. Add 2 times volume of water, and culture at 30 °C, 200r/min for 24h to make BYC3 wine, wait for adding sauce. .
  • the initial fermentation temperature of the sauce was 15 ° C, and the temperature increased to 15-35 ° C with the fermentation, and the BYC 3 was introduced when the temperature was raised to 20 ° C.
  • the fermentation time is 5 months.
  • the sauce is pressed through the plate frame to remove the sauce. After the end of the pressing, diatomaceous earth filtration and membrane filtration are carried out. In addition to precipitation.
  • the clarified soy sauce was filtered and sterilized by heat at 85 ° C for 30 min.
  • the high-yield ⁇ -phenylethanol yeast was used for high-salt dilute fermentation, and the obtained soy sauce product contained ⁇ -phenylethyl alcohol 200 mg/L.
  • Example 8 Application of high-yield ⁇ -phenylethanol yeast in low-salt solid fermentation of brewing soy sauce
  • the brewed soy sauce is fermented with low-salt solid state, and the soybean meal and wheat are mixed and steamed in a ratio of 1:1.
  • the inoculum amount of Aspergillus oryzae is 10%
  • the control temperature is 30 ° C
  • 2 times the mass of the salt water is added
  • the salt content of the sauce is 7%
  • the water content is 40%
  • the mixture is stirred and mixed.
  • BYC3 yeast was cultured in YPD shake flask, and it was connected to partially steamed and cooled soybean meal and wheat according to 5% inoculation amount. Add 1 volume of water and incubate at 30 °C, 200r/min for 24 hours to make BYC3 wine wine into sauce. Fermentation system, the temperature of the product is controlled at 40 °C. The fermentation time was 15d.
  • the sauce after the fermentation is finished to remove impurities and precipitates.
  • the clarified soy sauce was filtered and sterilized by heat at 85 ° C for 30 min.
  • the high-yield ⁇ -phenylethanol yeast is used for high-salt dilute fermentation, and the obtained soy sauce product contains ⁇ -phenylethyl alcohol 50 mg/L.
  • Two-round fermentation method was adopted. After the first round of fermentation, the sorghum was steamed, and the air was cooled to 28 ° C, 4% Aspergillus oryzae was added, and cultured at 28 ° C for 24 hours. Adding rice husk 10%, koji 15%, bran 8%, 1% to BYD3 yeast cultured in YPD shake flask, steamed wine after 30 days of closed fermentation. At the time of secondary fermentation, 10% of medium-temperature Daqu was added, and 1% of BYC3 yeast cultured in YPD shake flask was added, and the wine was steamed after 15 days of fermentation. The two wines were blended into an alcohol content of 65% (v/v), a ⁇ -phenylethanol content of 110 mg/L, and a 2-phenylethyl acetate content of 64 ⁇ g/L.
  • the content of ⁇ -phenylethyl alcohol was 50 mg/L, and the content of 2-phenylethyl acetate was 26 ⁇ g/L.
  • Example 10 A brewing method for enhancing the aroma of raspberry wine
  • the amount of white sugar is determined according to the fermentation condition, and the mixture is evenly stirred.
  • CCTCC NO: M 2016785 was statically cultured in YPD medium at 28 ° C for 12 to 24 hours, and the juice containing 50 g / L of sugar was inoculated in a 5% inoculation amount at 28 ° C for more than 16 h.
  • the fermentation liquid diatomaceous earth was filtered, and 0.04 g/L of sulfur dioxide was added to the fermentation liquid to be transferred and fermented.
  • the post-fermentation temperature is controlled below 20 °C, and the 2 barium bentonite clarifying agent is added and thoroughly mixed for about 20 days.
  • the content of ⁇ -phenylethanol was determined by liquid chromatography.
  • the ⁇ -phenylethyl alcohol content of Anqi commercial Saccharomyces cerevisiae was 61 mg/L, while the content of ⁇ -phenylethyl alcohol in the fermentation of Saccharomyces cerevisiae CTCCC M 2016785 was as high as 350mg/L.
  • Example 11 A brewing method for enhancing the aroma of fruit wine using Saccharomyces cerevisiae CCTCC NO: M 2016785 freeze-dried powder
  • the activated CCTCC NO: M 2016785 seed liquid is used; in this embodiment, the 2 ⁇ CCTCC NO:M 2016785 lyophilized powder is added, and the dry cells are fully hydrated at 38 ° C for 30 min-60 min.
  • the defoaming purpose is achieved, and the other steps are the same as in the embodiment 10.
  • the obtained fruit wine had a ⁇ -phenylethanol content of 325 mg/L.
  • Example 10 The results of Example 10 and Sakura Berry wine fermented with different strains are shown in Table 5.
  • Example 12 A brewing method for enhancing the aroma of hawthorn wine
  • the pectin content of hawthorn fruit is as high as 3% to 7%, and the fruit is in a gel state after being broken. Therefore, in step 10 of step 10, 40 to 50 °C warm water can be added, and the water quantity is controlled within 1 time of the amount of hawthorn; heating After boiling for 5 minutes, dip the extract juice, and adjust the sugar content and acidity of the pulp according to the alcohol content of the finished wine during the boiling process. After the end of the dip-extracted juice, the temperature of the pulp was lowered to about 40 ° C, pectinase and SO 2 were added, and the device was stirred to be used as a juice for fermentation. The other steps were the same as in Example 10.
  • the obtained hawthorn wine has a ⁇ -phenylethanol content of 243 mg/L and a wine content of up to 13% (v/v).
  • Example 13 A brewing method for enhancing the aroma of mulberry wine
  • the raw material was changed to mulberry in Example 10, and the other steps were the same as in Example 10.
  • the obtained mulberry wine has a ⁇ -phenylethanol content of 305 mg/L and an alcohol content of 13.5% (v/v).
  • Example 14 A brewing method for enhancing the aroma of bayberry wine
  • Example 10 the raw material was changed to bayberry, and the other steps were the same as in Example 10.
  • the obtained arbutin has a ⁇ -phenylethanol content of 212 mg/L and an alcohol content of 14.5% (v/v).

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Abstract

提供了一株高产β-苯乙醇的酿酒酵母菌株及其应用。该高产β-苯乙醇酿酒酵母菌株，于2016年12月26日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2016785。该菌株具有高产β-苯乙醇能力，在黄酒发酵、料酒发酵、酿造食醋、酱油发酵、白酒发酵中应用，β-苯乙醇含量分别可达410mg/L、450mg/L、300mg/L、200mg/L、110mg/L、370mg/L。

Description

一株高产β-苯乙醇的酿酒酵母菌株及其应用 技术领域
本发明涉及一株高产β-苯乙醇的酿酒酵母菌株及其应用，属于工业微生物技术领域。
背景技术
β-苯乙醇是一种具有玫瑰风味的芳香醇，天然存在于茉莉、玫瑰等植物精油中，同时作为一种重要的香精香料成分，在化妆品、烟草及日化用品中也广泛应用。β-苯乙醇作为发酵食品中特征的风味物质，可以提高发酵产品风味和整体品质。但目前，化学法合成β-苯乙醇过程中会存在难以去除的副产物，存在致癌风险，严重影响产品质量；虽然从天然植物中物理提取可以获得无毒无害、质量上乘的β-苯乙醇，用于食品或其他产品生产，但是生产周期长、产量低、价格高，难以满足市场需求。
发酵食品中β-苯乙醇可由微生物代谢产生，通过微生物发酵能够提高发酵食品中β-苯乙醇含量，所获产品属于天然食品。酿酒酵母(Saccharomyces cerevisiae)在发酵过程中通过艾利希途径和其他代谢途径产生β-苯乙醇，在黄酒等发酵食品中β-苯乙醇含量可以达到100mg/L左右，虽然浓度已经较高，但是进一步提高β-苯乙醇对于提升黄酒风味显著。虽然在葡萄酒中β-苯乙醇含量可以达到60mg/L左右，但是对于玫瑰芳香性果酒的特征开发，有待于进一步提高β-苯乙醇含量，所以有必要筛选具有优良性能的酵母。
虽然外源添加L-苯丙氨酸等前体化合物可以促进酿酒酵母产生β-苯乙醇提高发酵食品中β-苯乙醇，但是成本相对较高，并且改变了食品原有的生产配方。另外，少数非酿酒酵母类的酵母如库德里阿紫威毕赤酵母(Pichia kudriavzevii)、马克斯克鲁维酵母(Marx Kluyveromyces)可以在不外源添加L-苯丙氨酸的情况下，产生一定浓度的β-苯乙醇，用于提高发酵食品中β-苯乙醇浓度，但是由于非酿酒酵母产乙醇能力较低，不能够作为主要菌株用于酒类及食醋醋等发酵食品的酿造。Rafael等(Overproduction of2-phenylethanol by industrial yeasts to improve organoleptic properties of bakers'products,International Journal of Food Microbiology,2014,180(1):7-12.)中报道了一株高产β-苯乙醇面包酵母在烘焙食品中的应用，但并未对所报酵母的产酒精特性做出报道，其所应用范围为不需要产生高浓度酒精的烘焙食品。虽然已有多个具有产生一定浓度的酿酒酵母应用于酿造食品的报道，但是菌株是否具有较高的酒精生产能力尚不清楚。由于β-苯乙醇对酵母的胁迫能力显著高于乙醇，高产β-苯乙醇的酿酒酵母产乙醇的能力通常会有降低，所以已有高产β-苯乙醇的酿酒酵母的酒精生产能力较低，因此在不外源添加前体化合物情况下，能够高产β-苯乙醇和乙醇的酿酒酵母在酿造 工业中具有较高应用价值。
发明内容
为了解决上述问题，本发明提供了一株不添加外源氨基酸而具有高产β-苯乙醇特性且产酒精特性良好的酿酒酵母菌株，及其在黄酒、料酒、酿造食醋、酱油、白酒、果酒中的应用。该菌株具有高产β-苯乙醇能力和高产风味物质乙酸-2-苯乙酯的能力，且发酵性能良好，能够明显提高发酵产品中β-苯乙醇含量，改善发酵产品品质，具有广泛的应用前景。
本发明的第一个目的是提供一株高产β-苯乙醇的酿酒酵母菌株(Saccharomyces cerevisiae)，于2016年12月26日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC NO:M 2016785。
本发明所述酿酒酵母菌株是从黄酒醪液筛选出的酿酒酵母作为出发菌株，经紫外诱变后进行对氟苯丙氨酸抗性筛选，然后筛选长势良好的菌株接种在含有10％乙醇的YPD液体培养基中，进行酒精耐受性筛选以及黄酒模拟液发酵筛选，得到的β-苯乙醇产量相对较高的菌株作为常温等压等离子诱变出发菌株，对二次诱变后菌株进行对氟苯丙氨酸抗性筛选，以及发酵特性筛选，得到高产β-苯乙醇酿酒酵母。
本发明的酿酒酵母，具有如下特性：
(1)应用于黄酒发酵体系，发酵所得黄酒中β-苯乙醇含量可达410mg/L，乙酸-2-苯乙酯含量为56μg/L，酒精度17％(v/v)；
(2)应用于料酒发酵体系，发酵所得料酒中β-苯乙醇含量可达450mg/L，乙酸-2-苯乙酯含量为50μg/L，酒精度15％(v/v)；
(3)在酿造食醋发酵中的应用，使用本发明的酿酒酵母代替酒母，所得酒醪再经醋酸发酵，酿造食醋中β-苯乙醇含量为300mg/L，乙酸-2-苯乙酯含量为45μg/L；
(4)在酱油中的应用，将所述酿酒酵母接种于酱油发酵体系，发酵所得酱油中β-苯乙醇含量为200mg/L；
(5)在白酒中的应用，将所述酿酒酵母接种于白酒发酵体系，蒸馏白酒中β-苯乙醇含量为110mg/L，乙酸-2-苯乙酯含量为64μg/L，酒精度可达65％(v/v)；
(6)应用于果酒(桑葚酒、山楂酒、杨梅酒、樱莓酒)发酵体系，纯种发酵所得果酒中β-苯乙醇含量可达350mg/L，乙酸-2-苯乙酯含量为50μg/L，酒精度14.5％(v/v)；
(7)菌落为白色，圆形或者椭圆形，边缘整齐。
本发明的第二个目的是提供所述含有所述酿酒酵母CCTCC NO:M 2016785菌株的微生物菌剂。
在本发明的一种实施方式中，所述微生物菌剂含有酿酒酵母CCTCC NO:M 2016785菌体的活细胞、冷冻干燥得到的酿酒酵母CCTCC NO:M 2016785干菌体、固定化的酿酒酵母CCTCC NO:M 2016785细胞、酿酒酵母CCTCC NO:M 2016785的液体菌剂、酿酒酵母CCTCC NO:M 2016785的固体菌剂，或者以其他任何形式存在的酿酒酵母CCTCC NO:M 2016785菌株。
本发明的第三个目的是提供所述酿酒酵母菌株或者微生物菌剂的应用。
在一种实施方式中，所述应用是指用于发酵食品的制作。
在一种实施方式中，所述应用是指用于酿造技术领域。
本发明的第四个目的是提供一种发酵食品，所述发酵食品是酿酒酵母CCTCC NO:M2016785为发酵剂或者主要发酵剂发酵得到的。
在一种实施方式中，所述发酵食品为酿造食品。
在一种实施方式中，所述酿造食品为酒类、食醋或酱油。所述酒类包括但不限于黄酒、料酒、白酒等。
在一种实施方式中，所述酿造食品为黄酒，使用所述的酿酒酵母作为酒母。
在一种实施方式中，所述黄酒的酿造是将所述酿酒酵母作为酒母，按照5％-10％的添加量添加到蒸煮或糊化好的原料中，经发酵、压榨、煎酒、陈酿、过滤、灭菌灌装得到黄酒。
在一种实施方式中，所述黄酒的酿造具体是：先将所述酿酒酵母培养制备酒母，然后按照总体积4％加入麦曲、按总体积10％加入酒母到高温糊化好的糯米中，搅拌均匀，然后经发酵、压榨、煎酒、陈酿、过滤、灭菌灌装得到黄酒。
在一种实施方式中，所述酿造食品为料酒。
在一种实施方式中，所述料酒的酿造是先利用所述酿酒酵母作为酒母酿造得到黄酒，再利用得到的黄酒制备成料酒。
在一种实施方式中，所述酿造食品为食醋。
在一种实施方式中，所述食醋的酿造是先利用所述酿酒酵母作为酒母酿造得到黄酒，再利用得到的黄酒作为醋酸发酵原料来酿造食醋。
在一种实施方式中，所述食醋是采用固态发酵或者液态发酵的方法酿造。
在一种实施方式中，所述酿造食品为酱油。
在一种实施方式中，所述酱油的酿造是采用高盐稀态发酵或者低盐固态发酵制备酱油。
在一种实施方式中，所述高盐稀态发酵制备酱油具体是：将豆粕和小麦混匀蒸熟，接种米曲霉，加入盐水使酱醪含盐量为18％、含水量为65％，搅拌混匀；然后将培养好的CCTCC  NO:M 2016785酵母接入部分蒸熟冷却后的豆粕和小麦中，加入清水，培养制成CCTCC NO:M 2016785酒母，等待加入酱醪中；当酱醅温度在发酵过程中升高到20℃时接入CCTCC NO:M 2016785酒母；发酵时间为5个月；发酵结束后的酱醪经过压榨、过滤、澄清，得到酱油。
一种实施方式中，所述低盐固态发酵制备酱油具体是：豆粕和小麦混匀蒸熟，接种米曲霉量，加入盐水使酱醪含盐量为7％、含水量为40％，搅拌混匀；然后将培养好的CCTCC NO:M 2016785酵母接入部分蒸熟冷却后的豆粕和小麦中，加入清水，培养制成CCTCC NO:M2016785酒母，接入酱醪发酵体系，品温控制在40℃；发酵时间为15d；发酵结束后的酱醪去除杂质和沉淀、过滤澄清得到酱油。
一种实施方式中，所述酿造食品为白酒。
一种实施方式中，所述白酒的酿造，是在白酒发酵入池发酵时额外添加所述酿酒酵母。
一种实施方式中，所述白酒酿造时酿酒酵母额外添加量为1％。
本发明的第五个目的是提供一种增强果酒芳香的方法，尤其是增强果酒玫瑰芳香的方法，所述方法是利用本发明的酿酒酵母CCTCC NO:M 2016785作为发酵菌株。
在一种实施方式中，所述酿酒酵母为CCTCC NO:M 2016785菌株冻干粉。
在一种实施方式中，所述方法，是将冷冻干燥得到的酿酒酵母CCTCC NO:M 2016785干菌体按2‰添加量添加到果汁中。
在一种实施方式中，所述果酒是以桑葚汁、山楂酒、杨梅汁、樱莓汁中的一种或者两种以上按一定比例混合发酵得到的。
在一种实施方式中，所述果酒为桑葚酒、山楂酒、杨梅酒、樱莓酒等。
在一种实施方式中，所述方法包括如下步骤：
(1)清洗果实原料，进行破碎与压榨，分离得到果汁；
(2)补加白砂糖，搅拌均匀；
(3)流加焦亚硫酸钾160mg/L，果胶酶100mg/L，入罐搅拌均匀；
(4)将酿酒酵母CCTCC M 2016785进行种子液活化，采用YPD培养基(1％酵母膏，2％蛋白胨，2％葡萄糖)培养12h以上；或者取2‰CCTCC NO:M 2016785冻干粉，在38℃下搅拌30min-60min让干菌体充分吸水，得到菌液；
(5)将上一步活化好的种子液或者冻干粉的菌液接种入发酵罐中，进行前发酵；接种后，控温23-25℃，12-24h后启酵，启酵后控温20℃-23℃；
(6)在发酵过程中，根据发酵情况确定是否需要补加白砂糖及补加量；前发酵时间8-12d；
(7)前发酵结束后，将发酵液中的酵母分离，在发酵液中添加一定浓度二氧化硫转入后 发酵；
(8)后发酵温度控制20℃以下，添加澄清剂，充分混合，发酵时间为16～24d；
(9)过滤：将发酵罐底部酒脚放掉，上部液体进行过滤。
本发明的优点和效果：
(1)本发明获得了一株不添加外源氨基酸而具有高产β-苯乙醇特性且产酒精特性良好的酿酒酵母菌株。
(2)本发明的酿酒酵母菌株可以用于黄酒、料酒、食醋、酱油、白酒的酿造；应用于这些产品的酿造时，不仅可以产生高浓度的β-苯乙醇、具有较高的酒精生产能力，而且可有效提高其他风味成分或者有益成分的含量，比如乙酸-2-苯乙酯的含量。
(3)利用CCTCC NO:M 2016785制备果酒，成品果酒中β-苯乙醇的含量达350mg/L，比普通酵母菌种酿造的果酒提高571％左右，显著增强果酒芳香，并且产酒精特性良好，酒精度可达14.5％(v/v)。
生物材料保藏
一株酿酒酵母菌株，分类学命名为酿酒酵母BYC3Saccharomyces cerevisiae BYC3，于2016年12月26日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC NO:M 2016785。
附图说明
图1是实施例1中出发酵母菌株生长曲线；
图2是实施例1中出发酵母菌株紫外照射致死率曲线；
图3是实施例1中出发菌株对氟苯丙氨酸致死率曲线；
图4是实施例2中黄酒发酵酒精度变化曲线；
图5是实施例2中黄酒发酵酸度变化曲线；
图6是实施例2中黄酒发酵pH变化曲线；
图7是实施例2中BYC3酿酒酵母菌落形态；
图8是实施例2中酿酒酵母菌株在YPD中β-苯乙醇产量。
具体实施方案
下面是对本发明进行具体描述。
实施例1：紫外诱变与筛选
YPD液体培养基：酵母提取物10g/L、鱼粉蛋白胨20g/L、葡萄糖20g/L。
YPD固体培养基：酵母提取物10g/L、鱼粉蛋白胨20g/L、葡萄糖20g/L，营养琼脂20 g/L。
1、诱变出发菌株的获得
(1)从甘油保藏管中取黄酒生产用酿酒酵母(以下称为野生菌株)的菌液200ul，涂布YPD平板，30℃培养24h。
(2)用接种环挑取单菌落到含100ml YPD液体培养基的摇瓶，30℃、200r/min培养24h。
(3)取5ml所得菌液，接种到含100ml YPD液体培养基的摇瓶，30℃、200r/min培养，每隔1h测定菌液的OD600，酵母指数增长期结束后每隔3h测定OD600，每次取三个样品。绘制生长曲线，确定出发菌株指数增长中期时间即是紫外诱变开始时间，此时的菌株即为诱变出发菌株。
实验结果如附图1中所示：培养到3-5h时酵母OD600值增长显著，此时酵母生长处于指数生长期，而摇床培养4h时野生菌株处于指数生长中期。因此选择摇床培养4h的酵母作为诱变出发菌株。
2、紫外诱变时间的确定
(1)如步骤1，获得诱变出发酵母菌株菌液。
(2)取10ml诱变出发菌株菌悬液，6000r/min离心5min后去上清，加入50ml生理盐水震荡混匀，6000r/min离心5min后去上清，加入50ml生理盐水震荡混匀得到菌悬液。
(3)紫外线照射：先将紫外灯打开预热20min，以稳定光波。用5mL无菌移液管移取上述菌悬液4.5mL于无菌的直径为9cm的培养皿中，培养皿中加入无菌大头针。将装有菌悬液的培养皿放置于磁力搅拌器上，垂直放置于紫外灯下，照射20s，黑暗条件下打开皿盖(确保紫外灯照射均匀)，暴露紫外光下照射(15W紫外灯，距离30cm)，时间为40s、60s、80s、100s、120s。
(4)照射完毕后，在红光灯下或者黑暗条件下，将诱变后的酵母菌悬液以10倍稀释法稀释4个梯度10-1、10-2、10-3、10-4，每个梯度各取200μL涂布YPD平板，用锡纸包好以避光。每个照射时间下三个平行，30℃培养48h。
(5)未经诱变的酵母菌悬液以10倍稀释法稀释5个梯度10-1、10-2、10-3、10-4、10-5，每个梯度各取200μL涂布YPD平板，作为对照。对照组做三个平行，30℃培养48h。
(6)平板计数，记录表格，计算致死率，绘制致死率曲线。分别确定紫外致死率为70％-80％、80％-90％、90％-100％时紫外照射时间。致死率＝(对照组菌落数一诱变组菌落数)/对照组菌落数。
实验结果：诱变出发菌株进行紫外诱变，涂布不同浓度梯度菌悬液到YPD平板，根据生长菌落数，绘制致死率曲线，结果如附图2。根据致死率曲线图，致死率70-80％、80-90％、90-100％时照射时间分别为110s、130s、150s。
3、对氟苯丙氨酸最低全部致死浓度的确定
YNBP固体培养基：6.7％YNB、20g/L葡萄糖、10g/L脯氨酸，额外的加入对氟苯丙氨酸，浓度分别为0(对照组)、0.04g/L、0.05g/L、0.06g/L、0.07g/L、0.08g/L、0.09g/L、0.1g/L。
(1)指数增长中期菌悬液，10倍稀释法稀释4个梯度10-1、10-2、10-3、10-4，取10-4梯度菌悬液200μL涂布YNBP平板。
(2)30℃培养48-72h。记录菌落数，绘制对氟苯丙氨酸致死率曲线。
致死率＝(对照组菌落数一诱变组菌落数)/对照组菌落数
实验结果：对氟苯丙氨酸致死率曲线如附图3，随着YNBP平板上对氟苯丙氨酸浓度的增加，酵母致死率不断增加，对氟苯丙氨酸浓度增加到0.09g/L时酵母全部致死，因此对氟苯丙氨酸最低全部致死浓度确定为0.09g/L。
4、紫外诱变
如步骤2获得紫外诱变出发菌株并对其进行紫外照射，紫外诱变总时间分别为110s、130s、150s。
5、对氟苯丙氨酸抗性筛选和酒精耐受性筛选
(1)对氟苯丙氨酸抗性筛选
对氟苯丙氨酸抗性筛选培养基配方：6.7％YNB、20g/L葡萄糖、10g/L脯氨酸、对氟苯丙氨酸0.09g/L，营养琼脂20g/L。
分别取紫外诱变菌悬液200μL涂布到对氟苯丙氨酸抗性筛选平板，用锡纸包好以避光。每个致死率下做3个平板，30℃培养72h。
(2)酒精耐受性筛选
酒精筛选培养基配方：酵母提取物10g/L、鱼粉蛋白胨20g/L、葡萄糖20g/L，无菌乙醇10％。
1)96孔板每孔加入20μLYPD液体培养基，将对氟苯丙氨酸抗性筛选后的突变菌株分别接种到孔中，30℃培养24h。
2)96孔板每孔加入200μL酒精筛选培养基，将上一步每孔的种子液按5％接种到此孔板，30℃静止培养。分别在12h和24h用酶标仪测定OD600。
3)计算OD60012h、OD60024h以及OD60024h-OD60012h值，挑选数值相对较高的诱变酵母菌共计22株。
6、黄酒模拟液发酵筛选
黄酒模拟液的制备：1kg蒸熟米饭(含水率为70％)中加入水1L、麦曲0.05kg，搅拌均匀，60℃保温8h，4500r/min离心5min，取上清液115℃灭菌15min。
(1)将22株突变菌株，分别划线到YPD平板上，30℃培养24h。
(2)分别挑取单菌落，接种到含有10ml YPD的50ml离心管中，30℃、200r/min培养12h。
(3)移取5％菌液，接种到含有20ml黄酒模拟液的50离心管中，30℃，静止发酵7d，每组三个平行。
(4)高效液相色谱法测定黄酒模拟液中β-苯乙醇含量，筛选β-苯乙醇含量相对较高的菌株。
高效液相色谱分析：
1)将2mL样品放入2mL离心管中离心去除菌体，离心条件为12000rpm、1min。
2)取上清液1mL过0.22μm水系膜，移入液相进样瓶中备用。
3)选用X-bridge C18柱，流动相为甲醇:纯水＝1:1，在30℃条件下以1mL/min的流速进样，进样量为10ul。
高效液相色谱测定黄酒模拟发酵液中β-苯乙醇含量，1-e4突变菌株的β-苯乙醇平均含量相对较高为185.032mg/L(如表1所示)，产酒精能力优良，因此将此菌株作为下一轮常温等压等离子诱变的出发菌株。
表1紫外诱变后黄酒模拟发酵液中β-苯乙醇含量
Figure PCTCN2017100446-appb-000001
实施例2：常温等压等离子诱变与筛选
1、常温等压等离子诱变与黄酒模拟液发酵筛选
第一轮紫外诱变后所选菌株1-e4作为常温等压等离子诱变出发菌株。1-e4菌株YPD摇瓶30℃培养24h，用生理盐水制成OD600为0.6-0.8的菌悬液，进行常温等压等离子诱变，诱变时间为60s，功率为100w，将诱变后菌株重悬成菌液，稀释涂布于YPD平板，30℃培养48h，将单菌落划线到YNBP平板(对氟苯丙氨酸浓度为0.5g/L)，YNBP平板长势良好菌株进行黄酒模拟液发酵筛选。高效液相色谱法测定黄酒模拟液中β-苯乙醇含量，筛选β-苯乙醇含量相对较高的菌株。
高效液相色谱法测定发酵8d后的黄酒模拟发酵液中β-苯乙醇含量。3-c10、4-c7、5-f5突 变菌株β-苯乙醇平均含量相对较高，分别为217.192mg/L、257.388mg/L、337.168mg/L，将该三株菌株分别命名为BYC1、BYC2、BYC3。酿酒酵母β-苯乙醇产量增加，虽然诱变后酿酒酵母乙醇产量略有下降，但是所得酿酒酵母仍可良好进行酒精发酵。
表2常温等压等离子诱变黄酒模拟发酵液中β-苯乙醇含量
Figure PCTCN2017100446-appb-000002
2、黄酒发酵筛选
酒母制备：50mLYPD摇瓶接种酵母菌株，30℃、200r/min培养24h。1kg蒸熟米饭(含水率为70％)中加入水1L、麦曲0.05kg，搅拌均匀，60℃保温4h，冷却后按5％接种酵母菌液，30℃、200r/min培养16h。
(1)配料
蒸熟米饭(含水率为70％)加入等重量清水，2％麦曲、5％酒母，搅拌均匀。
(2)发酵与搅拌
发酵温度为28℃，在配料完成后18h、24h、30h、42h、54h、78h，126h搅拌并取样。5000r/min离心10min得到上清液样品，用于指标检测。
(3)指标检测
18h、24h、30h、42h、54h、78h、126h时所取样品测定其总酸(以乳酸计)、酒精度、pH，观察三项指标变化情况，并用高效液相色谱法测定126h时样品中β-苯乙醇含量。
用本轮诱变出发菌株和三株筛选后突变酵母菌株发酵黄酒，产酒精能力结果如图4：三株正突变菌株和出发菌株相比，产酒精性能差别较小，酒精度都达13％(v/v)以上，酒精发酵良好。三株酵母在前60h时酒精发酵迅速，在60h后酒精含量基本稳定。酸度变化结果如图5：三株突变酵母发酵黄酒酸度在3-4g/L之间，随着发酵进行酸度略显上升且趋于平缓。pH变化如图6：pH在3-4.5之间，酿酒酵母可以良好的进行酒精发酵。
黄酒发酵液中β-苯乙醇含量如下表3所示，用BYC1、BYC2、BYC3菌株发酵黄酒，β-苯乙醇含量为219.08、254.91、365.70mg/L，出发菌株发酵黄酒β-苯乙醇产量仅为188.07mg/L，三株菌株β-苯乙醇产量分别为出发菌株的1.16倍、1.35倍、1.94倍，有良好的高产β-苯乙醇能力。
表3常温等压等离子诱变黄酒发酵液中β-苯乙醇含量
Figure PCTCN2017100446-appb-000003
黄酒发酵液中异丁醇、异戊醇、乙酸-2-苯乙酯、甘油作为风味物质，可以提高黄酒整体质量，其含量见表4，通过比较，BYC3菌株的乙酸-2-苯乙酯以及甘油含量明显高于出发菌 株以及BYC1及BYC2。
表4黄酒发酵液中异丁醇、异戊醇、乙酸-2-苯乙酯、甘油含量表
Figure PCTCN2017100446-appb-000004
综上比较结果，BYC3的β-苯乙醇产量高，且异丁醇、异戊醇、乙酸-2-苯乙酯含量也较高。BYC3菌株划线到YPD固体培养基平板，30℃培养24h，菌落形态如图7。将BYC3菌株于保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2016785。将BYC3菌株接种于黄酒发酵体系，酒精发酵性能良好，所得黄酒中β-苯乙醇含量为365.70mg/L。
3、YPD培养基中高产β-苯乙醇酵母菌株对照实验
用BYC3菌株(即CCTCC NO:M 2016785)、野生菌株、商业活性干酵母、酿酒酵母模式菌株W303(基因型Mat a/a,ura3-1,leu2-3,112,trp1-1,his3-11,15,ade2-1,can1-100)、酿酒酵母模式菌株S288c(基因型MATαSUC2gal2mal2melflo1flo8-1hap1ho bio1bio6)进行对比发酵实验。五株菌株用YPD平板活化后接入YPD摇瓶，再分别按5％依次接入YPD摇瓶和YPD摇瓶(加入1g/L苯丙氨酸)。
实验结果如图8，酿酒酵母菌株BYC3的β-苯乙醇产量明显高于野生菌株、商业活性干酵母、W303和S288c，BYC3酵母在YPD培养基中β-苯乙醇产量达31.6mg/L，是添加1g/L苯丙氨酸时的50.9％(而野生酵母为21％，商业酵母为27％、W303为22％、S288c为20％)，酵母BYC3高产β-苯乙醇性能显著，明显高于现有酿酒酵母菌株。
实施例3：高产β-苯乙醇酵母在黄酒中的应用
1、黄酒酿造工艺一
(1)酒母制作：50mLYPD摇瓶接种酵母菌株，30℃、200r/min培养24h。1kg蒸熟米饭(含水率为70％)中加入水1L、麦曲0.05kg，搅拌均匀，60℃保温4h，冷却后按5％接种酵母菌液，30℃、200r/min培养16h；酵母选用高产β-苯乙醇酿酒酵母BYC3(即CCTCC NO:M 2016785)。
(2)蒸熟米饭(含水率为40％)，加入等重量清水，15％麦曲、5％酒母，搅拌均匀。
(3)发酵与搅拌：发酵温度为28℃，落料完成后开始计时，每隔8h进行搅拌，至48h时共搅拌6次。发酵5-7天，检测酒精度指标不再升高时结束发酵。
(4)压榨：发酵结束后，发酵醪液过板框过滤机进行压榨得到清酒。
(5)煎酒：清酒过煎酒灭菌机85℃灭菌30min。
(6)陈酿：煎酒后清酒入陈酿罐陈酿6个月。
(7)过滤：陈酿后清酒用硅藻土过滤机和膜过滤机过滤除去杂菌及杂质。
(8)灭菌灌装：过灭菌机，85℃灭菌30min，热灌装。
所得产品用高效液相法检测β-苯乙醇含量高达410mg/L，酒精度为17％(v/v)，乙酸乙酯含量为24mg/L，乙酸-2-苯乙酯含量为56μg/L。
2、黄酒酿造工艺二
(1)酒母制作：如本实施例上述工艺一。
(2)糯米粉碎，加入糯米质量2.5倍的清水，高温淀粉酶110℃高温糊化40min，降温到35℃加入糖化酶糖化40min，降温到28℃，按总体积4％加入麦曲、按总体积10％加入酒母，搅拌均匀。
(3)其余步骤如本实施例的工艺一所述。
所得产品用高效液相法检测β-苯乙醇含量高达185mg/L，酒精度为14％(v/v)，乙酸乙酯含量为14mg/L，乙酸-2-苯乙酯含量为24μg/L。
3、黄酒发酵醪中高产β-苯乙醇酵母菌株对照实验
用BYC3菌株、野生菌株、商业活性干酵母、酿酒酵母模式菌株W303(基因型Mat a/a,ura3-1,leu2-3,112,trp1-1,his3-11,15,ade2-1,can1-100)、酿酒酵母模式菌株S288c(基因型MATαSUC2gal2mal2melflo1flo8-1hap1ho bio1bio6)进行对比发酵实验。五株菌株的黄酒酿造方法如本实施例上述黄酒酿造工艺一。
结果如下：酿酒酵母菌株BYC3的β-苯乙醇产量(410mg/L)明显高于野生菌株(82mg/L)、商业活性干酵母(87mg/L)、W303(81mg/L)和S288c(73mg/L)。酿酒酵母菌株BYC3的酒度达到17％(v/v)，商业活性干酵母酵母BYC3、W303、S288c的酒度分别为16.5％(v/v)12.4％(v/v)、13.1％(v/v)。说明菌株菌株BYC3高产β-苯乙醇性能显著，明显高于现有酿酒酵母菌株，同时也具有较强的酒精生产能力。酵母菌株BYC3产生的乙酸-2-苯乙酯浓度达到56μg/L，是野生菌株的4.5倍，商业活性干酵母的4.3倍，W303的5倍，S288c的5.7倍。酵母菌株BYC3产生的异戊醇浓度是是野生菌株的1.7倍，商业活性干酵母的1.8倍，W303的2.1倍，S288c的2.4倍。
实施例4：高产β-苯乙醇酵母在料酒中的应用
1、料酒酿造工艺一
按实施例3中黄酒酿造工艺一获得黄酒，加入食盐10％，过煎酒灭菌机85℃灭菌30min 热灌装。
所得产品用高效液相法检测β-苯乙醇含量高达450mg/L，酒精度为15％(v/v)，乙酸乙酯含量为20mg/L，乙酸-2-苯乙酯含量为50μg/L。
2、黄酒酿造工艺二
按实施例3中黄酒酿造工艺二获得黄酒，加入食盐10％，过灭菌机85℃灭菌30min热灌装。β-苯乙醇含量高达140mg/L，酒精度为12％(v/v)，乙酸乙酯含量为10mg/L，乙酸-2-苯乙酯含量为20μg/L。
实施例5：高产β-苯乙醇酵母在酿造食醋固态发酵中的应用
以实施例3中工艺一发酵黄酒，作为醋酸发酵原料。
醋酸发酵采用固态发酵工艺：将大糠、麸皮、黄酒按照1:4:10的比例拌匀，接入5％醋醅，接种后1-2天内每天从物料表面翻醅，温度为35-40℃。到6-8天时翻到物料底部。第8-12天，每天从底部翻醅，温度自然下降。从醋醅中分离后得生醋，在经过85℃灭菌30min后陈酿12个月。在灌装前经过高温灭菌后热灌装。
所得固态发酵酿造食醋中醋酸含量为60g/L，β-苯乙醇含量高达300mg/L，乙酸-2-苯乙酯含量为45μg/L。
实施例6：高产β-苯乙醇酵母在酿造食醋液态发酵中的应用
以实施例3中工艺一发酵黄酒，作为醋酸发酵原料。
醋酸发酵采用液态发酵工艺：将黄酒用清水稀释4倍后，接入5％培养好的醋酸菌菌液，通氧1L/min并进行搅拌，待发酵体系中酒精含量少于1％时，分批次加入黄酒，醋酸发酵体系中酒精含量控制在为1％-4％，发酵体系中醋酸含量约80g/L时离心分离，得到液态食醋。经过高温灭菌后热灌装。所得液态发酵酿造食醋中β-苯乙醇含量达100mg/L，乙酸-2-苯乙酯含量为36μg/L。
实施例7：高产β-苯乙醇酵母在酿造酱油高盐稀态发酵中的应用
酿造酱油选用高盐稀态法发酵，豆粕和小麦按1:1比例混匀蒸熟。米曲霉接种量为10％，控制温度为30℃，加入2倍物料质量的盐水，酱醪含盐量为18％、含水量为65％，搅拌混匀。BYC3酵母用YPD摇瓶培养，按5％接种量接入部分蒸熟冷却后的豆粕和小麦中，加入2倍体积清水，30℃、200r/min培养24h制成BYC3酒母，等待加入酱醪中。酱醅起始发酵温度为15℃，随着发酵进行温度升高为15-35℃，在温度升高到20℃时接入BYC3酒母。发酵时间为5个月。
发酵结束后的酱醪经过板框压榨，去除酱醅。压榨结束后进行硅藻土过滤和膜过滤，去 除沉淀。过滤澄清的酱油经过85℃灭菌30min热灌装。高产β-苯乙醇酵母用于高盐稀态法发酵，所得酱油产品中含有β-苯乙醇200mg/L。
实施例8：高产β-苯乙醇酵母在酿造酱油低盐固态发酵中的应用
酿造酱油选用低盐固态发酵，豆粕和小麦按1:1比例混匀蒸熟。米曲霉接种量为10％，控制温度为30℃，加入2倍物料质量的盐水，酱醪含盐量为7％、含水量为40％，搅拌混匀。BYC3酵母用YPD摇瓶培养，按5％接种量接入部分蒸熟冷却后的豆粕和小麦中，加入1倍体积的清水，30℃、200r/min培养24h，制成BYC3酒母接入酱醪发酵体系，品温控制在40℃。发酵时间为15d。
发酵结束后的酱醪去除杂质和沉淀。过滤澄清的酱油经过85℃灭菌30min热灌装。高产β-苯乙醇酵母用于高盐稀态法发酵，所得酱油产品中含有β-苯乙醇50mg/L。
实施例9：高产β-苯乙醇酵母在白酒中的应用
1、白酒酿造工艺一
采用两轮发酵法，第一轮发酵时高粱蒸熟后，风冷降温至28℃，添加4％米曲霉，28℃培养24h。添加稻壳10％、曲15％、麸皮8％、按1％接入YPD摇瓶培养的BYC3酵母，密闭发酵30天后蒸酒。二次发酵时添加中温大曲10％、按1％接入YPD摇瓶培养的BYC3酵母，发酵15天后蒸酒。两种酒勾兑成酒精度65％(v/v)，β-苯乙醇含量为110mg/L，乙酸-2-苯乙酯含量为64μg/L。
2、白酒酿造工艺二
高粱40％、小麦10％、玉米5％、大米25％、糯米20％蒸熟后，风冷后温度25℃，大糠使用量20％、曲20％、水分30％，按1％接入YPD摇瓶培养的BYC3酵母。温度在20℃，湿度在70％，发酵60天，蒸馏获得38％白酒。β-苯乙醇含量为50mg/L，乙酸-2-苯乙酯含量为26μg/L。
实施例10：一种增强樱莓酒芳香的酿造方法
(1)清洗野樱莓果实，进行破碎与压榨，碟片分离得到樱莓果汁。
(2)根据樱莓果汁含糖量补糖，按按17g/L糖转化为1度酒精的换算关系，根据发酵情况确定补加白砂糖量，搅拌均匀。
(3)流加焦亚硫酸钾160mg/L，果胶酶100mg/L，入罐搅拌均匀。
(4)种子液活化：在YPD培养基中于28℃静止培养CCTCC NO:M 2016785达到12～24h，以5％接种量接种含有50g/L糖的果汁中28℃静止培养16h以上。
(5)将活化好的种子液接种入发酵罐中，进行前发酵。接种后，控温23-25℃，12-24h 后启酵，启酵后控温20℃-23℃。每隔24h测定酒精度、残糖、酸度、pH。
(6)待残糖小于60g/L时，为使果汁发酵酒精度达到要求，按17g/L糖转化为1度酒精的换算关系，根据发酵情况确定是否需要补加白砂糖及补加量；待残糖量为4g/L左右，酒体表面平静，气泡较少，上层酒液清澈时，前酵结束，前发酵时间10d左右。
(7)前发酵结束后，将发酵液硅藻土过滤，在发酵液中添加0.04g/L的二氧化硫转入后发酵。
(8)后发酵温度控制20℃以下，添加2‰皂土澄清剂，充分混合，20d左右。
(9)过滤：静置澄清结束后，将发酵罐底部酒脚放掉，上部液体进行硅藻土过滤，在硅藻土过滤后用膜过滤器进行微滤。
发酵完毕后采用液相色谱法测定β-苯乙醇含量，其中安琪商业酿酒酵母发酵β-苯乙醇含量为61mg/L，而利用酿酒酵母CCTCC M 2016785发酵的樱莓酒β-苯乙醇含量高达350mg/L。
实施例11：一种使用酿酒酵母CCTCC NO:M 2016785冻干粉增强果酒芳香的酿造方法
在实施例10中是采用活化的CCTCC NO:M 2016785种子液；本实施例中为添加2‰CCTCC NO:M 2016785冻干粉，在38℃下搅拌30min-60min让干菌体充分吸水，同时达到消泡目的，其他步骤同实施例10。所得果酒β-苯乙醇含量为325mg/L。
其中，实施例10和采用不同菌株发酵的樱莓果酒的结果，如表5所示。
表5不同菌株发酵的樱莓果酒的结果对比
Figure PCTCN2017100446-appb-000005
实施例12：一种增强山楂酒芳香的酿造方法
山楂果实果胶含量高达3％～7％，果实破碎后呈胶着状态，因此在实施例10中步骤(1)破碎时可加入40～50℃温水，水量控制在山楂量的1倍以内；加热沸腾5min后，浸提取汁，在煮沸过程中可按照成品酒的酒精度要求调整果浆糖度、酸度。浸提取汁结束后使果浆温度降到40℃左右，加入果胶酶和SO2，搅拌均匀装置，作为发酵用果汁，其他步骤同实施例10。所得山楂酒β-苯乙醇含量为243mg/L，酒度高达13％(v/v)。
实施例13：一种增强桑葚酒芳香的酿造方法
在实施例10中将原料换为桑葚，其他步骤同实施例10。所得桑葚酒β-苯乙醇含量为305mg/L，酒精度可达13.5％(v/v)。
实施例14：一种增强杨梅酒芳香的酿造方法
在实施例10中将原料换为杨梅，其他步骤同实施例10。所得杨梅酒β-苯乙醇含量为212mg/L，酒精度可达14.5％(v/v)。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (12)

  1. 一株高产β-苯乙醇的酿酒酵母菌株(Saccharomyces cerevisiae)，于2016年12月26日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC NO:M 2016785。
  2. 含有权利要求1所述酿酒酵母菌株的微生物菌剂。
  3. 权利要求1所述酿酒酵母菌株或者权利要求2所述微生物菌剂的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述应用是用于发酵食品的制作。
  5. 一种发酵食品，其特征在于，所述发酵食品是以权利要求1所述的酿酒酵母菌株或者权利要求2所述的微生物菌剂为发酵剂或者主要发酵剂发酵得到的。
  6. 根据权利要求5所述的发酵食品，其特征在于，所述发酵食品为酒类、食醋或酱油。
  7. 根据权利要求6所述的发酵食品，其特征在于，所述酒类为黄酒、料酒、白酒或者果酒。
  8. 根据权利要求5或6任一所述的发酵食品，其特征在于，所述发酵食品为黄酒，使用权利要求1所述的酿酒酵母作为酒母。
  9. 根据权利要求5或6任一所述的发酵食品，其特征在于，所述发酵食品为料酒，是先利用权利要求1的所述酿酒酵母作为酒母酿造得到黄酒，再利用得到的黄酒制备成料酒。
  10. 根据权利要求5或6任一所述的发酵食品，其特征在于，所述发酵食品为食醋，先利用权利要求1所述的酿酒酵母作为酒母酿造得到黄酒，再利用得到的黄酒作为醋酸发酵原料来酿造食醋。
  11. 根据权利要求5或6任一所述的发酵食品，其特征在于，所述发酵食品为白酒，在白酒发酵入池发酵时额外添加权利要求1所述的酿酒酵母。
  12. 根据权利要求5或6任一所述的发酵食品，其特征在于，所述发酵食品为果酒，所述果酒是以桑葚汁、山楂酒、杨梅汁、樱莓汁中的一种或者两种以上按一定比例混合发酵得到的。
PCT/CN2017/100446 2017-05-25 2017-09-05 一株高产β-苯乙醇的酿酒酵母菌株及其应用 WO2018214330A1 (zh)

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CN111733041A (zh) * 2020-05-11 2020-10-02 陈君伟 一种用于酿造黄酒的白曲及其生产方法
CN111909862A (zh) * 2020-09-08 2020-11-10 天津科技大学 一种高产2-苯乙醇的基因工程菌及其应用
CN113462585A (zh) * 2021-05-28 2021-10-01 华南理工大学 一种提升酱油中乙酯类化合物含量的耐盐酵母菌及其应用
CN113462585B (zh) * 2021-05-28 2023-02-21 华南理工大学 一种提升酱油中乙酯类化合物含量的耐盐酵母菌及其应用
CN113702535A (zh) * 2021-08-31 2021-11-26 四川省绵阳市丰谷酒业有限责任公司 一种大曲质量评价方法及酿酒工艺
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CN114410546B (zh) * 2022-02-25 2022-10-11 中国水产科学研究院南海水产研究所 一株产酯增香的威尼斯不动杆菌及其在海水鱼发酵制品中的应用
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