WO2017215355A1 - 一株低产尿素、产风味的异常威克汉姆酵母菌株及其在食品生产中的应用 - Google Patents

一株低产尿素、产风味的异常威克汉姆酵母菌株及其在食品生产中的应用 Download PDF

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WO2017215355A1
WO2017215355A1 PCT/CN2017/082103 CN2017082103W WO2017215355A1 WO 2017215355 A1 WO2017215355 A1 WO 2017215355A1 CN 2017082103 W CN2017082103 W CN 2017082103W WO 2017215355 A1 WO2017215355 A1 WO 2017215355A1
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wickerhamomyces anomalus
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吴群
徐岩
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • C12G3/022—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn of botanical genus Oryza, e.g. rice
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
    • C12H6/00—Methods for increasing the alcohol content of fermented solutions or alcoholic beverages
    • C12H6/02—Methods for increasing the alcohol content of fermented solutions or alcoholic beverages by distillation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungi isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention relates to a strain of low-yield urea and flavor-producing Wickham's yeast strain and its application in food production, and belongs to the field of wine brewing and food safety.
  • ethyl carbamate Traditional foods inevitably produce a metabolic by-product during the fermentation process, namely ethyl carbamate (EC).
  • urethane is formed by the spontaneous reaction of urea, citrulline, etc., which are produced by the metabolism of microorganisms during fermentation or from the raw materials, with ethanol.
  • the precursors that form EC are mainly urea, cyanide and citrulline, and urea is currently recognized as the main precursor of EC formation in alcoholic beverages, such as rice wine and wine.
  • the main precursor substance formed by EC was also confirmed to be urea, and its content in the wine cellar was 26-80 mg/kg.
  • Urea is mainly produced by yeast metabolism of arginine. Because most of the foreign alcoholic beverages are single-fermentation fermentation or a small number of strains participate in the fermentation, Saccharomyces cerevisiae plays a major role in the fermentation process. Therefore, most of the research focused on Saccharomyces cerevisiae and concluded that Saccharomyces cerevisiae is the main strain of urea produced in beverage wines. However, many fermented foods are multi-strain fermentations, in addition to Saccharomyces cerevisiae, and non-Saccharomyces cerevisiae participates and plays an important role in the fermentation process.
  • Wickerhamomyces anomalus which is characterized by the production of various esters during fermentation, is an important precursor for the formation of various wines, and Wickerhamomyces anomalus is an important strain in food production. During the fermentation process, the flavor of the food can be improved by adding the Wickerhamomyces anomalus strain. For example, in the production of sesame-flavored bran liquor, yeast bran will improve flavor by adding abnormal Wickham's yeast.
  • Wickerhamomyces anomalus is an important urea-producing strain, and the large amount of urea produced during the fermentation process leads to the formation of EC during fermentation, which creates safety problems. Therefore, Wickerhamomyces anomalus, which has low-yield urea and has a flavor-producing function and can adapt to various food production environments, is particularly important for the production of fermented foods using yeast as a main functional microorganism, and is an important contribution to food safety.
  • the present invention screened a low-yield urea-like Wickerhamomyces anomalus CGMCC NO. 12416, which was screened from a variety of flavored liquor fermentation environments (Daqu).
  • the strain is obtained from the Chinese liquor brewing process and has the characteristics of flavor, alcohol production and acid resistance. It is an excellent brewing functional strain and can be effectively applied in the fields of brewing wine, distilled liquor, mixed liquor and other foods to reduce the fermentation process of food.
  • the formation of urea and EC to solve food safety problems.
  • a first object of the present invention is to provide a strain of Wickerhamomyces anomalus CGMCC No. 12416 which was deposited on May 6, 2016 at the General Microbiology Center of the China Collection of Microorganisms Collections, under the accession number CGMCC No. 12416.
  • the Wickerhamomyces anomalus CGMCC NO. 12416 strain was isolated and screened from the natural fermentation process of liquor (Daqu).
  • the Wickerhamomyces anomalus CGMCC No. 12416 strain has low yield of urea and has a flavor-producing function and can adapt to a food fermentation environment.
  • the Wickerhamomyces anomalus CGMCC No. 12416 strain can be cultured by all medium suitable for yeast growth (in which colonies are grown on WL solid medium as shown in Figure 1).
  • the culture method is selected from static culture or shaking culture at a temperature of 4-46 ° C, and the culture time is sufficient for the growth of microorganisms.
  • the Wickerhamomyces anomalus CGMCC NO.12416 strain was fermented in sorghum medium for 48 hours, and the alcohol in the flavor substance reached 11365.08 ⁇ g/L, the ester reached 23905.69 ⁇ g/L, the ketone reached 259.32 ⁇ g/L, and the aldehyde reached 176.95 ⁇ g. /L, benzene ring up to 4623.65 ⁇ g / L, acid up to 165.23 ⁇ g / L, terpene up to 150.23 ⁇ g / L.
  • the content of alcohols and esters is the most.
  • alcohols such as: 3-methyl-1-butanol, phenylethyl alcohol, 2-methyl-1-propanol; esters: the highest ethyl acetate content, followed by 2-phenethyl acetate , ethyl propionate, ethyl hexanoate and the like.
  • the Wickerhamomyces anomalus CGMCC NO.12416 strain has a growth temperature range of 4-46 ° C and a suitable temperature of 25-42 ° C; a growth pH range of 2.3-12.0, preferably 4.0-8.0, which may contain 60% glucose, 2.0, respectively. Environment growth of %KCl and 12% ethanol.
  • a second object of the present invention is to provide a microbial agent comprising the Wickerhamomyces anomalus CGMCC No. 12416 strain.
  • the microbial inoculant comprises living cells of Wickerhamomyces anomalus CGMCC NO. 12416 cells, freeze-dried Wickerhamomyces anomalus CGMCC NO. 12416 dried cells, immobilized Wickerhamomyces anomalus CGMCC NO. 12416 cells, a liquid fungus of Wickerhamomyces anomalus CGMCC No. 12416, a solid fungus of Wickerhamomyces anomalus CGMCC No. 12416, or a Wickerhamomyces anomalus CGMCC NO. 12416 strain present in any other form.
  • Wickerhamomyces anomalus CGMCC NO.12416 strain can be cultured at an ethanol concentration of 0-14%, a temperature of 10-42 ° C, a sugar concentration of 0-60% and a pH of 2.3-11, and can produce flavor.
  • the microbial inoculant further contains any strain that can be applied to any species of food or food preparation, such as Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, and the like.
  • the microbial agent further comprises any carrier that can be used in foods.
  • the liquid microbial agent of the Wickerhamomyces anomalus CGMCC No. 12416 is prepared by inoculating the Wickerhamomyces anomalus CGMCC No. 12416 strain in a liquid yeast culture medium and incubating at 30 ° C for 24 hours.
  • the liquid yeast agent medium formula is A or B; wherein
  • A in g/L, containing yeast extract 10, peptone 20, glucose 20, and the rest being water;
  • the raw material used for brewing liquor is the medium component. After the raw materials are pulverized, the ratio of raw materials to water is mixed at a ratio of 1:1 to 1:4 w/v, and the mixture is cooked for 1-5 hours. After cooling, saccharification enzyme is added. 50 units / g of raw materials, kept at 40-80 ° C for 2-10h, filtered, centrifuged to obtain a filtrate to adjust the sugar content of 10-150Bx, pH of 4-6; wherein the raw materials are sorghum, barley, wheat, peas, bran Any one or more of a mixture in any ratio.
  • the solid bacterial agent of the Wickerhamomyces anomalus CGMCC No. 12416 is prepared by inoculating the activated strain in a solid yeast culture medium at a 10% inoculation rate, and incubating at 30 ° C. 24-36h; the liquid yeast agent medium formula is C or D; wherein
  • the raw material is any one or more of sorghum, barley, wheat, pea, and bran mixed in any ratio; after the raw material is pulverized, the ratio of the raw material to water is 1:0.5-1:2w/v. After mixing in proportion, cooking at 80-100 ° C for 30 min;
  • a third object of the present invention is to provide the use of the Wickerhamomyces anomalus CGMCC No. 12416 strain, which is applied in the field of food technology, especially in the field of fermented foods.
  • the application is applied to brewing wine, distilled wine, etc., such as white wine, wine, rice wine, fruit wine, especially sesame-flavored bran liquor.
  • the application is to add Wickerhamomyces anomalus CGMCC NO. 12416 strain to the brewing process of liquor, wine, rice wine or fruit wine.
  • the application is to add Wickerhamomyces anomalus CGMCC NO. 12416 strain to seeds (curved or seed culture), such as to yeast bran.
  • a fourth object of the present invention is to provide a method of reducing EC by adding Wickerhamomyces anomalus CGMCC No. 12416 strain to a food preparation process.
  • the invention obtains a strain of Wickerhamomyces anomalus with low yield of urea, flavor, alcohol and acid resistance, and the use of the yeast in the fields of brewing wine, distilled spirits and other foods can reduce the formation of urea and EC to some extent.
  • the strain of the present invention can be grown in an environment containing 60% glucose, 2.0% KCl and 12% ethanol, respectively, and the urea yield is far lower than other existing Wickerhamomyces anomalus strains, and the flavoring ability is strong.
  • the strain of the invention is fermented
  • the maximum value of urea production during the process is 351.23 ⁇ g / L, which is much lower than other strains of the same species.
  • the maximum value of the model strain is 3335.15 ⁇ g / L.
  • Figure 1 Colony morphology of Wickerhamomyces anomalus JZ420 yeast on WL medium
  • FIG. 1 Wickerhamomyces anomalus JZ420 yeast 26S rDNA amplification gel electrophoresis; A is the model strain Wickerhamomyces anomalus 21886 (China General Microorganisms Collection) amplification results, B is Wickerhamomyces anomalus JZ420 amplification results, M is 2000DLmaker.
  • Example 1 Screening and Identification of Yeast Wickerhamomyces anomalus with Low Yield of Urea
  • WL medium yeast extract 4.0g/L, trypsin 5.0g/L, glucose 50.0g/L, potassium dihydrogen phosphate 0.55g/L, potassium chloride 0.425g/L, calcium chloride 0.125g/L , magnesium sulfate 0.125g / L, ferric chloride 0.0025g / L, manganese sulfate 0.0025g / L, agar 20g / L, olfactory phenol green 22mg / L.
  • Example 2 Function of strain metabolism to produce flavor
  • Seed medium A 25 ml test tube was taken, and 5 ml of sorghum medium was placed, and the strain Wickerhamomyces anomalus JZ420 obtained in the above Example 1 was inoculated, and the natural pH was 30 ° C, 200 rpm, and aerobic culture was carried out for 48 hours.
  • Fermentation medium The cultured seed culture was inoculated into a 250 ml flask containing 50 ml of sorghum medium at a natural pH of 5%, 30 ° C, 200 rpm, and fermented for 48 hours.
  • Volatile products were analyzed by headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS). 8 mL samples were taken and placed in a headspace vial containing 3 g of NaCl. 10 ⁇ L of 4-methyl-2-pentanol having a concentration of 42.60 mg/L was used as an internal standard. The headspace flask was extracted at 50 ° C for 45 min at a constant temperature, and after extraction, GC-MS analysis was performed.
  • HS-SPME headspace solid phase microextraction
  • GC-MS gas chromatography-mass spectrometry
  • the content of alcohols and esters is the most.
  • the alcohols 3-methyl-1-butanol 4432.18 ⁇ g/L, phenylethyl alcohol 2653.32 ⁇ g/L, 2-methyl-1-propanol 1232.68 ⁇ g/L, n-propanol 1564.12 ⁇ g/L, different Pentanol 3641.32 ⁇ g / L, ethanol 2976.32 ⁇ g / L; ester: ethyl acetate content is the highest, is 147117.51 ⁇ g / L, 2-phenylethyl acetate 31234.96 ⁇ g / L, ethyl propionate 2269.86 ⁇ g / L Ethyl hexanoate 4569.32 ⁇ g / L.
  • Seed medium A 25 ml test tube was taken, and 5 ml of sorghum medium was placed, and the strain obtained in the above Example 1 was inoculated, and the aerobic culture was carried out for 48 hours at a natural pH of 30 ° C, 200 rpm.
  • Fermentation medium The cultured seed culture was inoculated into a 250 ml flask containing 50 ml of sorghum medium (500 mg/L of arginine precursor and 2% ethanol) in an inoculum of 5%, 30 ° C. At 200 rpm and fermentation for 96 h, the yeast growth (OD 600 ) and urea production during the fermentation were determined. The strain was found to enter a stable phase (OD 600 of 1.8) during the fermentation process, and the maximum urea yield reached 227.42 ⁇ g / L (48 h) during the fermentation process.
  • Urea detection The content of urea in the fermentation broth was determined by pre-column derivatization high performance liquid chromatography with fluorescence detector (HPLC-FLD). The specific operation is as follows: 500 ⁇ l of the fermentation broth is added with 500 ml of absolute ethanol, 400 ⁇ l of 9-hydroxy ton solution and 100 ⁇ l of 0.1 M hydrochloric acid solution, shaken, and derivatized at room temperature for 30 min in the dark.
  • Seed medium 25 ml tube was taken, 5 ml sorghum medium was added, and the strain obtained in the above Example 1 and the model strain Wickerhamomyces anomalus 21886 (China General Microorganisms Collection, (Strain No. 2.1886)), natural pH, were inoculated. Aerobic incubation was carried out for 48 h at 30 ° C, 200 rpm.
  • Fermentation medium The cultured seed culture solution was inoculated into a 250 ml flask containing 50 ml of sorghum medium (500 mg/L arginine, 2% ethanol) in an inoculum amount of 5%, 30 ° C, 200 rpm. After fermentation for 96 h, the yeast growth (OD 600 ) and urea production during fermentation were determined. It was found that the ability of the Wickerhamomyces anomalus JZ420 strain of the present invention to produce urea during fermentation was significantly less than that of the model strain Wickerhamomyces anomalus 21886. The maximum value of urea produced by the strain of the present invention during fermentation was 227.42 ⁇ g/L, while the urea of the model strain during fermentation was urea. The maximum value produced was 3541.34 ⁇ g/L.
  • Urea detection The content of urea in the fermentation broth was determined by pre-column derivatization high performance liquid chromatography with fluorescence detector (HPLC-FLD). The specific operation is as follows: 500 ⁇ l of the fermentation broth is added with 500 ml of absolute ethanol, 400 ⁇ l of 9-hydroxy ton solution and 100 ⁇ l of 0.1 M hydrochloric acid solution, shaken, and derivatized at room temperature for 30 min in the dark.
  • strains included in this example are Saccharomycopsis fibuligera, Millerozyma farinosa, Trichosporon asahii, Hanseniaspora osmophila, Pichiafermentans, Pichia membranifaciens and Clavispora lusitaniae, and the strain Wickerhamomyces anomalus JZ420 of the present invention. These seven strains are all derived from the liquor brewing environment.
  • Seed medium 25 ml tube, 5 ml sorghum medium, and inoculated with Wickerhamomyces anomalus JZ420 strain and Wickerhamomyces anomalus, Saccharomycopsis fibuligera, Millerozymafarinosa, Saccharomyces cerevisiae, Trichosporon asahii, Hanseniaspora osmophila, Pichiafermentans, Pichia membranifaciens and Clavispora lusitaniae, natural pH, 30 Aerobic culture for 48 h at °C, 200 rpm.
  • Fermentation medium The cultured seed culture solution was inoculated into a 250 ml flask containing 50 ml of sorghum medium (500 mg/L arginine, 2% ethanol) in an inoculum amount of 5%, 30 ° C, 200 rpm. After fermentation for 96 h, the yeast growth (OD 600 ) and urea production during fermentation were determined.
  • the growth temperature ranges from 4 to 48 ° C, the suitable temperature is 25-33 ° C; the growth pH ranges from 2.3-12.0, preferably 4.0-8.0, and can be grown in an environment containing 60% glucose, 2.0% KCl and 12% ethanol, respectively. .
  • the Wickerhamomyces anomalus JZ420 strain obtained in the above Example 1 was inoculated into YPD liquid medium, cultured at 30 ° C for 24 h, then diluted with sterile physiological saline to an OD 600 of 1, and 0.5 ml of the bacterial solution was taken up to 50 ml of the tolerant medium. in.
  • Temperature-tolerant medium YPD medium, static culture for 48 h at 30 ° C, 37 ° C, 40 ° C, 42 ° C and 46 ° C, respectively.
  • the results show that the Wickerhamomyces anomalus JZ420 strain of the present invention can be grown at a temperature ranging from 28 to 46 °C.
  • Acidity Tolerance Medium YPD medium, pH of the medium was adjusted with 0.1 M lactic acid to a pH of 2.9, 2.7, 2.5, 2.4, 2.3, respectively, and static culture was carried out at 30 ° C for 48 h.
  • the results show that the Wickerhamomyces anomalus JZ420 strain of the present invention can be grown at a pH ranging from 2.9 to 2.3.
  • Alcohol tolerance culture YPD medium, add alcohol, make the alcohol content of 0,4%, 6%, 8%, 10%, 12%, 14%, static culture at 30 °C for 48h .
  • the results show that the Wickerhamomyces anomalus JZ420 strain of the present invention can grow in an alcohol range of 0-12%.
  • Chroma tolerance medium YPD medium, adding different masses of glucose to a glucose concentration of 1%, 40%, 50%, 60%, 65%, 70%, and standing culture at 30 ° C for 48 h .
  • the results show that the Wickerhamomyces anomalus JZ420 strain of the present invention is capable of growing at a glucose concentration ranging from 0 to 60%.
  • Osmotic pressure tolerance medium YPD medium, adding different masses of KCl, the KCl concentration is 0, 0.7mol / L, 1mol / L, 1.4mol / L, 1.7mol / L, 2mol / L, The cells were cultured at 30 ° C for 48 h. The results show that the Wickerhamomyces anomalus JZ420 strain of the present invention can be grown in the KCl range of 0-2 mol/L.
  • the strain Wickerhamomyces anomalus JZ420 of the present invention is prepared into a liquid microbial preparation by inoculating the Wickerhamomyces anomalus JZ420 strain in a liquid yeast culture medium and culturing at 30 ° C for 24 hours; the liquid yeast culture medium formula is A Or B; where
  • A in g/L, containing yeast extract 10, peptone 20, glucose 20, and the rest being water;
  • the raw material used for brewing liquor is the medium component. After the raw materials are pulverized, the ratio of raw materials to water is mixed at a ratio of 1:1 to 1:4 w/v, and the mixture is cooked for 1-5 hours. After cooling, saccharification enzyme is added. 50 units / g of raw materials, kept at 40-80 ° C for 2-10h, filtered, centrifuged to obtain a filtrate to adjust the sugar content of 10-150Bx, pH of 4-6; wherein the raw materials are sorghum, barley, wheat, peas, bran Any one or more of a mixture in any ratio.
  • the liquid microbial agent is added into the raw material prepared from the sesame fragrant white wine bran, and after the bran is finished, the wine is added to the wine cellar for fermentation, and after the fermentation is completed, the content of urea and EC in the wine cellar and the original wine in the late fermentation stage is detected. It was found that the addition of Wickerhamomyces anomalus JZ420 bran was significantly reduced in wine cellars and raw wines during production. The urea is 20-50%, and the EC reduction is 15 ⁇ 45%. The EC reduction in the original wine is 15%-35%, indicating that the strain added by the right one can actually reduce the urea and EC content in the food.

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Abstract

本发明公开了一株低产尿素、产风味的异常威克汉姆酵母菌株及其在食品生产中的应用，属于酒类酿造以及食品安全领域。本发明的异常威克汉姆酵母是从白酒发酵环境（大曲）中分离得到，命名为Wickerhamomyces anomalus JZ420，于2016年5月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.12416。本发明的菌株具有低产尿素、产风味，耐酒精、耐酸的特征，是优良的酿酒功能菌株，可应用于酿造酒、蒸馏酒及其它食品领域，保证食品安全性。

Description

一株低产尿素、产风味的异常威克汉姆酵母菌株及其在食品生产中的应用 技术领域
本发明涉及一株低产尿素、产风味的异常威克汉姆酵母菌株及其在食品生产中的应用，属于酒类酿造以及食品安全领域。
背景技术
传统食品在发酵过程中会不可避免地产生一种代谢副产物，即氨基甲酸乙酯(ethyl carbamate，EC)。氨基甲酸乙酯的产生是由于发酵过程中微生物代谢所产生的或来自原料当中的尿素、瓜氨酸等与乙醇自发发生化学反应所形成的。形成EC的前体物质主要有尿素，氰化物以及瓜氨酸，而尿素是目前公认的酒精饮料当中EC形成的主要前体物质，如黄酒，葡萄酒等。在中国白酒发酵过程中，EC形成的主要前体物质也被证实是尿素，其在酒醅中的含量为26～80mg/kg。
尿素主要是由酵母代谢精氨酸所产生的，由于国外的酒精饮料大多是单菌发酵或者少数菌株共同参与的发酵，而酿酒酵母在发酵过程中发挥主体作用。因此，大多数的研究集中在酿酒酵母，并得出酿酒酵母是饮料酒中尿素产生的主要菌株。然而，许多发酵食品属于多菌种发酵，除酿酒酵母外，还有非酿酒酵母的参与，并在发酵过程中发挥重要作用。异常威克汉姆酵母(Wickerhamomyces anomalus，)在发酵过程具有产多种酯类物质的特点，是各种酒形成的重要前体，并且在食品生产过程中，Wickerhamomyces anomalus是重要的菌株，因此，在发酵过程中，可通过添加Wickerhamomyces anomalus菌株来改善食品的风味。如在芝麻香型麸曲白酒生产中，酵母麸曲会通过添加异常威克汉姆酵母，改善风味。
最近研究发现，Wickerhamomyces anomalus为重要的尿素产生菌株，其在发酵过程当中所产生的大量尿素会导致发酵过程中EC的形成，从而产生安全问题。因此，获取低产尿素且具有产风味功能并能适应多种食品生产环境的Wickerhamomyces anomalus，对于以酵母作为主要功能微生物的发酵食品的生产就显得尤为重要，对于食品安全具有重要贡献。
发明内容
为了解决上述问题，本发明筛选得到了一株低产尿素的异常威克汉姆酵母Wickerhamomyces anomalus CGMCC NO.12416，是从多种香型白酒发酵环境(大曲)当中筛选的。该菌株是从中国白酒酿造过程中获得，具有产风味、产酒精、耐酸等特性，是优良的酿酒功能菌株，能够有效应用于酿造酒、蒸馏酒、配制酒及其它食品领域，降低食品发酵过程尿素和EC的形成，从而解决食品安全问题。
本发明的第一个目的是提供一株Wickerhamomyces anomalus CGMCC NO.12416菌株，于2016年5月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.12416。
所述Wickerhamomyces anomalus CGMCC NO.12416菌株是从白酒自然发酵过程中(大曲)分离筛选得到的。
所述Wickerhamomyces anomalus CGMCC NO.12416菌株，低产尿素且具有产风味功能并能适应食品发酵环境。
所述Wickerhamomyces anomalus CGMCC NO.12416菌株，可通过所有适合酵母生长的的培养基进行培养(其中在WL固体培养基上生长菌落如图1)。培养方式选用静置培养或振荡培养，温度4-46℃，培养时间足够微生物的生长即可。
所述Wickerhamomyces anomalus CGMCC NO.12416菌株，经高粱培养基发酵48h后代谢风味物质中醇类达11365.08μg/L、酯类达23905.69μg/L、酮类达259.32μg/L、醛类达176.95μg/L、苯环类达4623.65μg/L、酸类达165.23μg/L、萜烯类达150.23μg/L。其中，各主要风味物质中，由以醇类和酯类含量为最多。其中，醇类中如：3-甲基-1-丁醇、苯乙醇、2-甲基-1-丙醇；酯类中：乙酸乙酯含量最高，其次为2-苯乙基乙酸酯、丙酸乙酯、己酸乙酯等。
所述Wickerhamomyces anomalus CGMCC NO.12416菌株，生长温度范围为4-46℃，适温为25-42℃；生长pH范围为2.3-12.0，优选4.0-8.0，可在分别含60％的葡萄糖、2.0％KCl以及12％乙醇的环境生长。
本发明的第二个目的是提供含有所述Wickerhamomyces anomalus CGMCC NO.12416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Wickerhamomyces anomalus CGMCC NO.12416菌体的活细胞、冷冻干燥得到的Wickerhamomyces anomalus CGMCC NO.12416干菌体、固定化的Wickerhamomyces anomalus CGMCC NO.12416细胞、Wickerhamomyces anomalus CGMCC NO.12416的液体菌剂、Wickerhamomyces anomalus CGMCC NO.12416的固体菌剂，或者以其他任何形式存在的Wickerhamomyces anomalus CGMCC NO.12416菌株。这些形式的Wickerhamomyces anomalus CGMCC NO.12416菌株，均能在乙醇浓度0-14％，温度10-42℃，糖浓度0-60％及pH 2.3-11进行培养，并能产风味。
在本发明的一种实施方式中，所述微生物菌剂中还含有任何可以应用于食品或者食品制备的任意种属的菌株，比如地衣芽孢杆菌、酿酒酵母、枯草芽孢杆菌等等。
在本发明的一种实施方式中，所述微生物菌剂中还含有任意能用于食品的载体。
在本发明的一种实施方式中，所述Wickerhamomyces anomalus CGMCC NO.12416的液体菌剂的制作方法，是将Wickerhamomyces anomalus CGMCC NO.12416菌株接种于液体酵母菌剂培养基中，于30℃培养24h；所述液体酵母菌剂培养基配方为A或者B；其中，
A：以g/L计，含有酵母膏10、蛋白胨20、葡萄糖20、其余为水；
B：以白酒酿造所用的原料为培养基成分，原料经粉碎后，按原料与水的比例为1:1-1:4w/v的比例混合，蒸煮1-5h，冷却后加入糖化酶10-50单位/g原料，于40-80℃保持2-10h，过滤，离心所得滤液调节糖度为10-150Bx、pH为4-6；其中，原料为高粱、大麦、小麦、豌豆、麸皮中的任意一种或者多种以任意比例的混合。
在本发明的一种实施方式中，所述Wickerhamomyces anomalus CGMCC NO.12416的固体菌剂的制作方法，是将活化后的菌株以10％接种比例接种于固体酵母菌剂培养基中，30℃培养24-36h；所述液体酵母菌剂培养基配方为C或D；其中，
C：原料为高粱、大麦、小麦、豌豆、麸皮中的任意一种或者多种以任意比例的混合；原料经粉碎后，按原料与水的比例为1:0.5-1：2w/v的比例混合后，80-100℃蒸煮30min；
D：在高粱、大麦、小麦、豌豆、麸皮中的任意一种或者多种以任意比例的混合得到的发酵原料，于40-80℃保持2-10h，并加入0.1-1mol/LNaOH溶液调节酸度为0-5。
本发明的第三个目的是提供所述Wickerhamomyces anomalus CGMCC NO.12416菌株的应用，是应用于食品技术领域，尤其是发酵食品技术领域。
所述应用，是应用于酿造酒、蒸馏酒等方面，比如白酒、葡萄酒、黄酒、果酒方面，尤其是芝麻香型麸曲白酒。
所述应用，是将Wickerhamomyces anomalus CGMCC NO.12416菌株添加到白酒、葡萄酒、黄酒或者果酒酿造过程中。
所述应用，是将Wickerhamomyces anomalus CGMCC NO.12416菌株添加到种子(曲或者种子培养液)中，比如添加到酵母麸曲中。
本发明的第四个目的是提供一种降低EC的方法，所述方法是将Wickerhamomyces anomalus CGMCC NO.12416菌株添加到食品制备过程中。
本发明的有益效果：
本发明得到了一株低产尿素、产风味、耐酒精、耐酸的Wickerhamomyces anomalus菌株，利用该酵母在酿造酒、蒸馏酒及其它食品领域的应用可在一定程度上减少尿素和EC的形成。本发明的菌株，可在分别含60％的葡萄糖、2.0％KCl以及12％乙醇的环境生长，尿素产量远远低于现有的其他Wickerhamomyces anomalus菌株，而且产风味能力强。本发明菌株在发酵 过程中尿素产生的最大值为351.23μg/L，远低于其他同种的菌株，比如模式菌株的最大值为3339.15μg/L。
生物材料保藏
异常威克汉姆酵母，分类学命名为异常威克汉姆酵母Wickerhamomyces anomalus，于2016年5月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.12416。
附图说明
图1：Wickerhamomyces anomalus JZ420酵母在WL培养基上的菌落形态；
图2：Wickerhamomyces anomalus JZ420酵母26S rDNA扩增凝胶电泳图；其中A为模式菌株Wickerhamomyces anomalus 21886(中国普通微生物菌种保藏中心)扩增结果，B为Wickerhamomyces anomalus JZ420扩增结果，M为2000DLmaker。
具体实施方式
实施例1：低产尿素的酵母Wickerhamomyces anomalus的筛选及鉴定
取10g大曲溶于100ml无菌生理盐水中，摇床振荡30分钟后进行梯度稀释，涂布WL固体平板，根据平板上菌落形态特征(图1)，挑取Wickerhamomyces anomalus酵母单菌落进行高通量液体发酵，对获得的潜在的低产尿素Wickerhamomyces anomalus酵母进行分子生物学鉴定，利用酵母特异分类鉴定引物NL1和NL4分别扩增酵母的26S rDNA的片段，凝胶电泳检测(图2)。随后进行测序比对，确定所筛选得低产尿素的酵母在分类学上都属于Wickerhamomyces anomalus酵母。最终所得到一株低产尿素的异常威克汉姆酵母，命名为Wickerhamomyces anomalus JZ420。将Wickerhamomyces anomalus JZ420保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.12416。
WL培养基：酵母浸粉4.0g/L，胰蛋白陈5.0g/L，葡萄糖50.0g/L，磷酸二氢钾0.55g/L，氯化钾0.425g/L，氯化钙0.125g/L，硫酸镁0.125g/L，氯化铁0.0025g/L，硫酸锰0.0025g/L，琼脂20g/L，嗅甲酚绿22mg/L。
实施例2：菌株代谢产风味的功能
种子培养基：取25ml试管，装入5ml高粱培养基，并接种上述实施例1获得的菌株Wickerhamomyces anomalus JZ420，自然pH，30℃，200rpm，进行好氧培养48h。
发酵培养基：将培养好的种子培养液接种在装有50ml高粱培养基的250ml三角瓶中，自然pH，接种量为5％，30℃，200rpm，发酵48h。
运用顶空固相微萃取技术(HS-SPME)和气相色谱-质谱(GC-MS)方法对挥发性产物进行分析，取8mL样品，放入装有3g NaCl的顶空进样瓶中，加入10μL浓度为42.60mg/L的4-甲基-2-戊醇为内标。将顶空瓶于50℃恒温萃取45min，萃取完后进行GC-MS分析。
上述实施例1获得的菌株，其发酵48h后代谢产风味的能力如表1所示。
表1 风味物质含量(μg/L)
Figure PCTCN2017082103-appb-000001
各主要风味物质中，以醇类和酯类含量为最。其中，醇类中：3-甲基-1-丁醇4324.18μg/L、苯乙醇2653.32μg/L、2-甲基-1-丙醇1232.68μg/L，正丙醇1564.12μg/L、异戊醇3641.32μg/L、乙醇2976.32μg/L；酯类中：乙酸乙酯含量最高，为147117.51μg/L、2-苯乙基乙酸酯31234.96μg/L、丙酸乙酯2693.86μg/L、己酸乙酯4569.32μg/L。
实施例3：Wickerhamomyces anomalus JZ420酵母低产尿素
种子培养基：取25ml试管，装入5ml高粱培养基，并接种上述实施例1获得的菌株，自然pH，30℃，200rpm，进行好氧培养48h。
发酵培养基：将培养好的种子培养液接种在装有50ml高粱培养基(添加500mg/L的精氨酸前体物质以及2％乙醇)的250ml三角瓶中，接种量为5％，30℃，200rpm，发酵96h，测定发酵过程中酵母生长情况(OD600)和尿素生成情况。发现该菌株在发酵过程24h进入稳定期(OD600为1.8)，发酵过程中尿素产量达到的最大值为227.42μg/L(48h)。
尿素检测：采用柱前衍生高效液相色谱荧光检测器(HPLC-FLD)进行测定发酵液尿素的含量。具体操作为：500μl发酵液加入500ml的无水乙醇，400μl 9-羟基吨溶液以及100μl的0.1M盐酸溶液，摇匀，室温避光衍生30min。
实施例4：Wickerhamomyces anomalus JZ420与Wickerhamomyces anomalus 21886产尿素情况对比
种子培养基：取25ml试管，装入5ml高粱培养基，并接种上述实施例1获得的菌株以及模式菌株Wickerhamomyces anomalus 21886(中国普通微生物菌种保藏中心，(菌种编号2.1886))，自然pH，30℃，200rpm，进行好氧培养48h。
发酵培养基：将培养好的种子培养液接种在装有50ml高粱培养基(添加500mg/L的精氨酸，2％乙醇)的250ml三角瓶中，接种量为5％，30℃，200rpm，发酵96h，测定发酵过 程中酵母生长情况(OD600)以及尿素生成情况。发现本发明的Wickerhamomyces anomalus JZ420菌株在发酵过程中尿素产生的能力明显小于模式菌株Wickerhamomyces anomalus21886，本发明的菌株在发酵过程中尿素产生的最大值为227.42μg/L，而模式菌株在发酵过程中尿素产生的最大值为3451.34μg/L。
尿素检测：采用柱前衍生高效液相色谱荧光检测器(HPLC-FLD)进行测定发酵液尿素的含量。具体操作为：500μl发酵液加入500ml的无水乙醇，400μl 9-羟基吨溶液以及100μl的0.1M盐酸溶液，摇匀，室温避光衍生30min。
实施例5：Wickerhamomyces anomalus JZ420与其他酵母产尿素的情况
本实施例所包括的菌株有Saccharomycopsis fibuligera，Millerozyma farinosa，Trichosporon asahii，Hanseniaspora osmophila，Pichiafermentans，Pichia membranifaciens和Clavispora lusitaniae，以及本发明的菌株Wickerhamomyces anomalus JZ420。这7种菌株都来自白酒酿造环境。
种子培养基：取25ml试管，装入5ml高粱培养基，并接种Wickerhamomyces anomalus JZ420菌株以及Wickerhamomyces anomalus，Saccharomycopsisfibuligera，Millerozymafarinosa，Saccharomyces cerevisiae，Trichosporon asahii，Hanseniaspora osmophila，Pichiafermentans，Pichia membranifaciens和Clavispora lusitaniae，自然pH，30℃，200rpm，进行好氧培养48h。
发酵培养基：将培养好的种子培养液接种在装有50ml高粱培养基(添加500mg/L的精氨酸，2％乙醇)的250ml三角瓶中，接种量为5％，30℃，200rpm，发酵96h，测定发酵过程中酵母生长情况(OD600)以及尿素生成情况。通过比较发酵6h尿素的产量与OD600，发现这8株酵母的在发酵6h时单位时间内尿素的产量分别为：Wickerhamomyces anomalus JZ420100-500μg/OD，而Millerozymafarinose和Trichosporon asahii 1300-2500μg/OD；其余的菌株其产量均大于3000μg/OD。
这说明本发明的菌株Wickerhamomyces anomalus JZ420的尿素产生的能力明显小于其他从白酒发酵环境当中分离得到的酵母。
实施例6：菌株生理生化性质
生长温度范围为4-48℃，适温为25-33℃；生长pH范围为2.3-12.0，优选4.0-8.0，可在分别含60％的葡萄糖、2.0％KCl以及12％乙醇的环境生长中。
将上述实施例1获得的Wickerhamomyces anomalus JZ420菌株，接种于YPD液体培养基 中，30℃培养24h，然后用无菌生理盐水稀释至OD600为1，吸取0.5ml菌液至50ml耐受性培养基中。
温度耐受性培养基：YPD培养基，分别于30℃，37℃，40℃，42℃以及46℃进行静止培养48h。结果显示本发明的Wickerhamomyces anomalus JZ420菌株能够在温度范围为28-46℃进行生长。
酸度耐受性培养基：YPD培养基，用0.1M乳酸调节培养基pH，使其成pH分别为2.9，2.7，2.5，2.4，2.3，于30℃进行静置培养48h。结果显示本发明的Wickerhamomyces anomalus JZ420菌株能够在pH范围为2.9-2.3进行生长。
酒精度耐受性培养：YPD培养基，加入酒精，使其成酒精度含量分别为0，4％，6％，8％，10％，12％，14％，于30℃进行静置培养48h。结果显示本发明的Wickerhamomyces anomalus JZ420菌株能够在酒精度范围为0-12％进行生长。
糖度耐受性培养基：YPD培养基，加入不同质量的葡萄糖，使其成葡萄糖浓度分别为1％,40％，50％，60％，65％，70％，于30℃进行静置培养48h。结果显示本发明的Wickerhamomyces anomalus JZ420菌株能够在葡萄糖浓度范围为0-60％进行生长。
渗透压耐受性培养基：YPD培养基，加入不同质量的KCl，使其KCl浓度分别为0，0.7mol/L，1mol/L，1.4mol/L，1.7mol/L，2mol/L，于30℃进行静置培养48h。结果显示本发明的Wickerhamomyces anomalus JZ420菌株能够在KCl范围为0-2mol/L进行生长。
实施例7 Wickerhamomyces anomalus JZ420在芝麻香白酒中的应用
将本发明的菌株Wickerhamomyces anomalus JZ420制作成液体菌剂，其制作方法是将Wickerhamomyces anomalus JZ420菌株接种于液体酵母菌剂培养基中，于30℃培养24h；所述液体酵母菌剂培养基配方为A或者B；其中，
A：以g/L计，含有酵母膏10、蛋白胨20、葡萄糖20、其余为水；
B：以白酒酿造所用的原料为培养基成分，原料经粉碎后，按原料与水的比例为1:1-1:4w/v的比例混合，蒸煮1-5h，冷却后加入糖化酶10-50单位/g原料，于40-80℃保持2-10h，过滤，离心所得滤液调节糖度为10-150Bx、pH为4-6；其中，原料为高粱、大麦、小麦、豌豆、麸皮中的任意一种或者多种以任意比例的混合。
将液体菌剂添加进入芝麻香白酒麸曲制作的原料当中，等麸曲制作完成之后，加入酒醅当中进行发酵，发酵完成后，检测白酒生产发酵后期酒醅及原酒里面尿素与EC的含量，发现添加Wickerhamomyces anomalus JZ420的麸曲，在进行生产时，能显著减少酒醅和原酒中 尿素20～50％，同时，EC减少量为15～45％，原酒里面EC的减少量为15％～35％，说明添加权利一所述的菌株确实能达到减少食品当中尿素及EC的含量。
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 (13)

  1. 一株异常威克汉姆酵母Wickerhamomyces anomalus CGMCC NO.12416菌株，于2016年5月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.12416。
  2. 一种微生物菌剂，其特征在于，所述微生物菌剂含有Wickerhamomyces anomalus CGMCC NO.12416菌体的活细胞、冷冻干燥得到的Wickerhamomyces anomalus CGMCC NO.12416干菌体、固定化的Wickerhamomyces anomalus CGMCC NO.12416细胞、Wickerhamomyces anomalus CGMCC NO.12416的液体菌剂、Wickerhamomyces anomalus CGMCC NO.12416的固体菌剂，或者以其他任何形式存在的Wickerhamomyces anomalus CGMCC NO.12416菌株。
  3. 根据权利要求2所述的微生物菌剂，其特征在于，所述微生物菌剂中还含有任何能够应用于食品或者食品制备的任意种属的菌。
  4. 根据权利要求2所述的微生物菌剂，其特征在于，所述微生物菌剂中还含有任意能用于食品的载体。
  5. 根据权利要求2所述的微生物菌剂，其特征在于，所述Wickerhamomyces anomalus CGMCC NO.12416的液体菌剂的制作方法，是将Wickerhamomyces anomalus CGMCC NO.12416菌株接种于液体酵母菌剂培养基中，于30℃培养24h；所述液体酵母菌剂培养基配方为A或者B；其中，
    A：以g/L计，含有酵母膏10、蛋白胨20、葡萄糖20、其余为水；
    B：以白酒酿造所用的原料为培养基成分，原料经粉碎后，按原料与水的比例为1:1-1:4w/v的比例混合，蒸煮1-5h，冷却后加入糖化酶10-50单位/g原料，于40-80℃保持2-10h，过滤，离心所得滤液调节糖度为10-150Bx、pH为4-6；其中，原料为高粱、大麦、小麦、豌豆、麸皮中的任意一种或者多种以任意比例的混合。
  6. 根据权利要求2所述的微生物菌剂，其特征在于，所述Wickerhamomyces anomalus CGMCC NO.12416的固体菌剂的制作方法，是将活化后的菌株以10％接种比例接种于固体酵母菌剂培养基中，30℃培养24-36h；所述液体酵母菌剂培养基配方为C或D；其中，
    C：原料为高粱、大麦、小麦、豌豆、麸皮中的任意一种或者多种以任意比例的混合；原料经粉碎后，按原料与水的比例为1:0.5-1：2w/v的比例混合后，80-100℃蒸煮30min；
    D：在高粱、大麦、小麦、豌豆、麸皮中的任意一种或者多种以任意比例的混合得到的发酵原料，于40-80℃保持2-10h，并加入0.1-1mol/LNaOH溶液调节酸度为0-5。
  7. 权利要求1所述Wickerhamomyces anomalus CGMCC NO.12416菌株或者权利要求2所述的微生物菌剂在食品技术领域的应用，尤其是发酵食品方面的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述应用是应用于酿造酒、蒸馏酒。
  9. 根据权利要求8所述的应用，其特征在于，是应用于白酒、葡萄酒、黄酒、果酒方面。
  10. 根据权利要求8所述的应用，其特征在于，所述应用是将Wickerhamomyces anomalus CGMCC NO.12416菌株添加到白酒、葡萄酒、黄酒或者果酒酿造过程中。
  11. 根据权利要求8所述的应用，其特征在于，所述应用是将Wickerhamomyces anomalus CGMCC NO.12416菌株添加到种子中。
  12. 根据权利要求7所述的应用，其特征在于，所述应用是将Wickerhamomyces anomalus CGMCC NO.12416菌株的液体菌剂添加进入芝麻香白酒麸曲制作的原料当中，等麸曲制作完成之后，加入酒醅当中进行发酵。
  13. 一种降低EC的方法，其特征在于，所述方法是将权利要求1的Wickerhamomyces anomalus CGMCC NO.12416菌株添加到食品制备过程中。
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