WO2020248962A1 - 一株高温敏感型啤酒酵母及其应用 - Google Patents

一株高温敏感型啤酒酵母及其应用 Download PDF

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WO2020248962A1
WO2020248962A1 PCT/CN2020/095044 CN2020095044W WO2020248962A1 WO 2020248962 A1 WO2020248962 A1 WO 2020248962A1 CN 2020095044 W CN2020095044 W CN 2020095044W WO 2020248962 A1 WO2020248962 A1 WO 2020248962A1
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yeast
brewer
cultured
autolysis
microbial preparation
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French (fr)
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李崎
张明芳
李昕玥
王金晶
刘春凤
钮成拓
郑飞云
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江南大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/145—Extracts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • 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, e.g. protozoa; 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • C12N1/18—Baker's yeast; Brewer's yeast
    • 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, e.g. protozoa; 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
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
    • C12R2001/85—Saccharomyces
    • 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
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention relates to a high-temperature sensitive beer yeast and its application, and belongs to the technical field of microorganisms.
  • Brewer's yeast is rich in protein (45%-60%), amino acids, B vitamins, polysaccharides, minerals, etc. It is a natural source of nutritional biologically active substances.
  • Yeast extract is a product that uses yeast autolysis to degrade the protein and nucleic acid substances in the yeast, and is concentrated together with other active ingredients into soluble nutrients. It can be used in condiments, cosmetics, health products, and medicine. And other fields. Its main components are nucleotides, amino acids, peptides, B vitamins and trace elements. Because amino acids, guanylic acid (5'-GMP) and inosinic acid (5'-IMP) have unique umami flavors, yeast extract has become an important natural flavor enhancer to replace glutamine in processed foods Sodium and nucleotides.
  • yeast autolysates The preparation of yeast autolysates is the same as that of yeast extracts, but the subsequent high temperature concentration is not carried out, which is beneficial to maintain the natural structure of peptides, amino acids and other substances, and to obtain yeast autolysates with biological activity.
  • the biologically active substances contained in yeast autolysates are ⁇ -glucan, mannan, oligosaccharide, mineral ions and polypeptides, etc., which can be used in functional foods and dietary additives. This is one of the new directions in the application of beer yeast. Therefore, providing a method for improving the dissolution and nutritional value of yeast autolysates has important application value.
  • the first purpose of the present invention is to provide a strain of Saccharomyces pastorianus, classified as Saccharomyces pastorianus, which has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on April 3, 2019, and the deposit number is CGMCC NO .17520, the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
  • the second object of the present invention is to provide a microbial preparation containing the above-mentioned beer yeast.
  • the microbial preparation contains S. cerevisiae cells with a viable cell count ⁇ 10 7 CFU/g.
  • the brewer's yeast cells comprise dry cells or wet cells of brewer's yeast.
  • the third object of the present invention is to provide a method for increasing the content of flavored amino acids in yeast extract, so that the above-mentioned brewer's yeast can autolyze at 37°C.
  • the brewer's yeast is cultured at 25-30°C for 8-16 hours, and then the culture is continued to enter the autolysis state.
  • the brewer’s yeast is cultured at 25-30°C for 8-16 hours, the cultured bacterial cells are collected, and the bacterial cells are transferred to another culture system at 150-200 rpm. min -1 , autolysis 20-50h.
  • the fourth object of the present invention is to provide a method for increasing the content of glucan in yeast extract, so that the above-mentioned brewer's yeast can autolyze at 37-50°C.
  • the brewer's yeast is cultured at 25-30°C for 8-16 hours, and then the culture is continued to enter the autolysis state.
  • the brewer’s yeast is cultured at 25-30°C for 8-16 hours, the cultured bacterial cells are collected, and the bacterial cells are transferred to another culture system at 150-200 rpm. min -1 , autolysis 20-50h.
  • the fifth object of the present invention is to provide the application of the above-mentioned beer yeast in the production of functional foods or dietary additives.
  • the dietary additives include nutritional fortifiers, flavor enhancers, and food processing aids.
  • the sixth object of the present invention is to provide the application of the above-mentioned brewer's yeast in the field of condiments, cosmetics, health products or pharmaceuticals.
  • the condiments include soy sauce, vinegar, bean paste, sweet noodle sauce, and oyster sauce.
  • the seventh object of the present invention is to provide the application of the above-mentioned microbial preparation in the production of functional foods or dietary additives.
  • the dietary additives include nutritional fortifiers, flavor enhancers, and food processing aids.
  • the eighth object of the present invention is to provide the application of the above-mentioned brewer's yeast in the field of condiments, cosmetics, health products or pharmaceuticals.
  • the condiments include soy sauce, vinegar, bean paste, sweet noodle sauce, and oyster sauce.
  • the beer yeast Pilsner is mutagenized by ARTP, and the principle that the yeast intracellular alkaline phosphatase reacts with BCIP to generate blue precipitation can be used to screen the strains according to the appearance time and depth of blue.
  • the RNA elution content of the mutagenized strains during autolysis at 37°C was compared, and a high-temperature sensitive S. cerevisiae P-510 was screened and autolyzed quickly at 37°C. In the final screening, a high-temperature sensitive brewer's yeast P-510 was obtained, which autolyzed rapidly at 37°C.
  • the autolysate obtained by autolysing the nucleotide content was 3 times that of the original bacteria, after P-510 autolyzed for 120h, the glucan content of P-510 Pilsner has a high glucan content of 40.3% and can be used for the production of brewer's yeast autolysates with high nutritional value.
  • the deposit number is CGMCC NO.17520, and the deposit address is Chaoyang, Beijing No. 3, No. 1, No. 1, Beichen West Road, District, Institute of Microbiology, Chinese Academy of Sciences.
  • Figure 1 shows the lethality curve of ARTP mutagenesis.
  • Figure 2 shows the screening of mutant bacteria.
  • Figure 3 shows the growth of P-510 at 28°C and 37°C.
  • Figure 4 shows the mortality curve of P-510 at 37°C.
  • Figure 5 shows the nucleic acid permeability (a) and protein permeability (b) curves of P-510 autolysis at 37°C.
  • Figure 6 shows the growth curves of Pilsner and P-510.
  • Figure 7 shows the content of glucan in the 37°C self-solution.
  • Figure 8 shows the DPPH clearance rate of the same bacterial concentration from the solution at 37°C.
  • the content of guanylic acid and inosinic acid in the self-solution was determined by high performance liquid chromatography. Chromatographic conditions: as shown in Table 1, flow rate 0.5mL ⁇ min -1 , wavelength 254nm, chromatographic column Waters XSELECT TM HSS T3 5 ⁇ m 4.6 ⁇ 250mm Column.
  • the dextran content in the self-solution was determined by the aniline blue method. Take 1 mL of the self-solution and centrifuge at 12000 rpm for 5 min, draw 100 ⁇ L of supernatant and add 1 mL of aniline blue working solution. Incubate the dark reaction at 50°C for 30 minutes to form fluorescent substances. Place it at room temperature for 30 minutes, mix thoroughly, and pipette 180 ⁇ L to a black 96-well plate for detection. The excitation wavelength is 405nm, the emission wavelength is 495nm, and the fluorescence value of the reaction solution is measured.
  • Aniline blue working solution 12.7mL ultrapure water, 3.70mL glycine/sodium hydroxide buffer (1.0M glycine, 1.25M sodium hydroxide) and 2.50mL dye solution (5.0g/L aniline blue) mixed and left overnight in the dark store.
  • the DPPH clearance rate is used to characterize the antioxidant capacity of the solution. Using the spectrophotometer method, 0.5072mM DPPH free radical alcohol solution was prepared with absolute ethanol, and 1 mL of DPPH free radical alcohol solution was mixed with 1 mL of the self-solution, and the OD 517 value was measured for 30 minutes in the dark.
  • the calculation formula of DPPH clearance rate is as follows:
  • OD 517 measured values Aj, ethanol samples were mixed with reaction; A0, DPPH solution and OD 517 value measured after mixing of anhydrous ethanol; Ai, DPPH solution react with the sample measured OD 517 values: formula.
  • the starting strain Pilsner was cultured to the logarithmic phase and diluted with saline until the number of cells in the bacterial solution reached about 10 7 ⁇ mL -1 .
  • the treatment time is 15s, 30s, 45s, 60s, 75s.
  • the treated slides were placed in 1 mL of sterile physiological saline. Dilute the bacteria solution reasonably and spread it on a YPD plate and place it at 28°C for culture. Plot the lethality curve. see picture 1. Since the selection of brewer's yeast needs to maintain the main flavor of the finished beer of the strain, a lethality rate of 75%-85% is generally selected, so 46s is selected as the mutagenesis time.
  • temperature-sensitive mutants can be screened according to the time and intensity of blue appearance.
  • the bacterial solution obtained by 46s mutagenesis was diluted 10-fold, spread on a YPD plate containing 40mg ⁇ L -1 BCIP, and cultured at 28°C for 36h.
  • the colony was copied to the YPD plate by plate photocopying method and placed at 28°C for culture.
  • the YPD plate containing 40mg ⁇ L -1 BCIP was placed at 37°C for 24h.
  • Autolysis was performed on a shaker at 180r ⁇ min -1 at 37°C for 30h, and the nucleic acid concentration in the supernatant was determined by NanoDrop. See Table 2.
  • P-510 is a mutant with better autolysis ability.
  • the nucleic acid and protein content of the self-solution increased, and the increase in nucleic acid content was greater than the increase in protein content.
  • the nucleic acid content of P-510 was 232.3 mg ⁇ L -1
  • the nucleic acid content of Pilsner was 83.43 mg ⁇ L -1
  • the increase rate of nucleic acid content of P-510 was greater than that of Pilsner and 3-3.
  • the increase in protein content is small, and the change is the largest in 0-24h.
  • the protein content of P-510 was 7.57 mg ⁇ L -1
  • the protein content of Pilsner was 6.02 mg ⁇ L -1 .
  • P-510 is more self-dissolving at 37°C than Pilsner and more sensitive to temperature.
  • Mutagenesis may affect the growth ability and fermentation performance of the strain, so the growth curve and fermentation ability of the mutant strain were determined.
  • P-510 and Pilsner entered the logarithmic phase at the same time. After entering the plateau phase, the bacterial concentration was slightly lower than the original bacteria, but the overall growth of the strains was consistent, indicating that the growth ability of P-510 was not affected after mutagenesis. .
  • Table 3 the alcohol content of the fermentation broth and the actual fermentation degree of Pilsner and P-510 after being fermented at 11°C for 7 days are not much different, indicating that P-510 can undergo normal fermentation.
  • the high temperature sensitive S. cerevisiae P-510 selected in Example 1 was autolyzed at 37° C., and the content of guanylic acid and inosinic acid in the self-solution was determined by high performance liquid chromatography.
  • the tasting nucleotide content (GMP+IMP) of the autolysate obtained by autolysing P-510 at 37°C for 144h is 3 times that of the original bacteria, indicating that the high temperature sensitive P-510 is at 37°C It is easier to autolyse, which is conducive to obtaining yeast extracts and autolysates with high taste nucleotide content.
  • the cell wall is under pressure, a variety of hydrolytic enzymes act on the cell wall, and then the cell wall components are dissolved, including the biologically active substance ⁇ -glucan.
  • the content of ⁇ -glucan in self-solution of Pilsner and P-510 at 37°C was determined respectively, as shown in Figure 3.
  • the glucan content of P-510 was 40.3% higher than that of Pilsner, indicating that the high temperature sensitive mutation of P-510 caused it to dissolve glucan during autolysis at 37°C.
  • the glycan content increases.
  • Yeast extracts and autolysates have certain antioxidant properties and can be used as functional food additives. It is mainly due to the active peptides that are degraded into proteins during the autolysis of yeast, and substances such as glucan and mannan dissolved out of cell wall components have antioxidant properties.
  • the DPPH clearance rates of Pilsner and P-510 from the solution at 37°C were measured respectively, as shown in Figure 8. The highest DPPH clearance rate was 79.80% when Pilsner autolyzed at 37°C for 120h, and the highest DPPH clearance rate was 86.09% at 80h for P-510, which was 6.29% higher than Pilsner.
  • Example 5 Application of high temperature sensitive brewer's yeast in preparing vinegar
  • the acetic acid bacteria are cultured in the mixed material; when the acetic acid content in the fermentation system does not increase, the vinegar mash is sealed to isolate the air, and the fermentation is continued to obtain a mature vinegar mash; the fermented mature vinegar mash is sealed and isolated from the air, and fermented at room temperature for 40 ⁇ After 45 days, add water and filter to get vinegar liquid.
  • Example 6 Application of high temperature sensitive brewer's yeast in preparing bean paste
  • soybeans, brine, and flour as raw materials, 100 kg of soybeans, 4.3 kg of flour, 24.3 to 25.7 kg of sea salt, and 0.29 to 0.43 kg of seed koji.
  • the soybeans are steamed and mixed with flour, sea salt and seed koji, and koji is made at a constant temperature of 30°C for 24 hours.
  • the high-temperature-sensitive brewer's yeast of the present invention is inoculated on the 0th day after entering the vat, and fermented at a constant temperature of 37°C until the sauce is mature; after the sauce is mature, it is heated and sterilized to obtain the finished bean paste.

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Abstract

提供一株高温敏感型酵母及其应用。通过ARTP诱变Saccharomyces pastorianus Pilsner，再利用酵母胞内碱性磷酸酶与BCIP反应生成蓝色沉淀这一原理，可根据蓝色出现时间与深浅进行高温敏感型突变菌的筛选，最终筛选得到一株高温敏感型酵母P-510，其在37℃下快速自溶。酵母P-510在37℃自溶能够促进自溶物的溶出，得到高呈味核苷酸、葡聚糖和抗氧化活性物质，可用于具有较高营养价值的酵母自溶物的生产。

Description

一株高温敏感型啤酒酵母及其应用 技术领域
本发明涉及一株高温敏感型啤酒酵母及其应用，属于微生物技术领域。
背景技术
啤酒酵母含有丰富的蛋白质(45％-60％)、氨基酸、B族维生素、多糖、矿物质等，是营养生物活性物质的天然来源。酵母抽提物是利用酵母自溶的特性将酵母菌体内的蛋白质、核酸类物质进行降解，并与其他有效成分一起被浓缩成可溶性营养物质的产品，可用于调味品、化妆品、保健品、医药等领域。其主要成分为核苷酸、氨基酸、多肽、B族维生素和微量元素。由于氨基酸、鸟苷酸(5’-GMP)和肌苷酸(5’-IMP)等成分具有独特的鲜味，酵母抽提物成为重要的天然风味增强剂，以取代加工食品中的谷氨酸钠和核苷酸。酵母自溶物的制备与酵母抽提物相同，但后续不进行高温浓缩，有利于维持多肽、氨基酸等物质的天然结构，获得具有生物活性的酵母自溶物。酵母自溶物中含有的生物活性物质有β-葡聚糖、甘露聚糖、寡糖、矿物离子和多肽等，可用于功能性食品和膳食添加剂。这是目前啤酒酵母应用的新方向之一。因此，提供一种提高酵母自溶物的溶出和营养价值的方法，具有重要的应用价值。
发明内容
本发明的第一个目的是提供一株啤酒酵母(Saccharomyces pastorianus)，分类命名为Saccharomyces pastorianus，已于2019年4月3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.17520，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
本发明的第二个目的是提供含有上述啤酒酵母的微生物制剂。
在本发明的一种实施方式中，所述微生物制剂中含有活菌数≥10 7CFU/g的啤酒酵母细胞。
在本发明的一种实施方式中，所述啤酒酵母细胞包括啤酒酵母干菌体或湿菌体。
本发明的第三个目的是提供一种增加酵母抽提物中呈味氨基酸含量的方法，使上述的啤酒酵母在37℃下进行自溶。
在本发明的一种实施方式中，将所述啤酒酵母在25～30℃培养8～16h后，继续培养，进入自溶状态。
在本发明的一种实施方式中，将所述啤酒酵母在25～30℃培养8～16h，收集培养后的菌体细胞，将菌体细胞转移到另一培养体系中，于150～200r·min -1、自溶20～50h。
本发明的第四个目的是提供一种增加酵母抽提物中葡聚糖含量的方法，使上述的啤酒酵母在37-50℃下进行自溶。
在本发明的一种实施方式中，将所述啤酒酵母在25～30℃培养8～16h后，继续培养，进入自溶状态。
在本发明的一种实施方式中，将所述啤酒酵母在25～30℃培养8～16h，收集培养后的菌体细胞，将菌体细胞转移到另一培养体系中，于150～200r·min -1、自溶20～50h。
本发明的第五个目的是提供上述的啤酒酵母在生产功能性食品或膳食添加剂中的应用。
在本发明的一种实施方式中，所述膳食添加剂包括营养强化剂、增味剂、食品加工助剂。
本发明的第六个目的是提供上述的啤酒酵母在调味品、化妆品、保健品或制药领域中的应用。
在本发明的一种实施方式中，所述调味品包括酱油、醋、豆瓣酱、甜面酱、蚝油。
本发明的第七个目的是提供上述的微生物制剂在生产功能性食品或膳食添加剂中的应用。
在本发明的一种实施方式中，所述膳食添加剂包括营养强化剂、增味剂、食品加工助剂。
本发明的第八个目的是提供上述的啤酒酵母在调味品、化妆品、保健品或制药领域中的应用。
在本发明的一种实施方式中，所述调味品包括酱油、醋、豆瓣酱、甜面酱、蚝油。
本发明通过ARTP诱变啤酒酵母Pilsner，再利用酵母胞内碱性磷酸酶与BCIP反应生成蓝色沉淀这一原理，可根据蓝色出现时间与深浅进行菌株的初筛。最后比较诱变菌株在37℃自溶时RNA溶出含量，筛选得到一株高温敏感型啤酒酵母P-510，在37℃下快速自溶。最终筛选得到一株高温敏感型啤酒酵母P-510，在37℃下快速自溶。P-510在37℃下自溶144h得到的自溶物呈味核苷酸含量(GMP+IMP)是原始菌的3倍，P-510自溶120h后，P-510的葡聚糖含量比Pilsner的葡聚糖含量高40.3％，可用于具有较高营养价值的啤酒酵母自溶物的生产。
生物材料保藏
一株啤酒酵母(Saccharomyces pastorianus)，分类命名为Saccharomyces pastorianus，已于2019年4月3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.17520，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
附图说明
图1为ARTP诱变致死率曲线。
图2为突变菌的筛选。
图3为P-510在28℃和37℃的生长情况。
图4为P-510在37℃的死亡率曲线。
图5为P-510在37℃自溶的核酸渗透率(a)和蛋白质渗透率(b)曲线。
图6为Pilsner和P-510的生长曲线。
图7为37℃自溶液中葡聚糖含量。
图8为37℃下相同菌浓自溶液DPPH清除率。
具体实施方式
(一)高效液相色谱法测定自溶液中鸟苷酸、肌苷酸含量
利用高效液相色谱法测定自溶液中鸟苷酸、肌苷酸含量，色谱条件：如表1，流速0.5mL·min -1，波长254nm，色谱柱Waters XSELECT TM HSS T3 5μm 4.6×250mm Column。
表1色谱条件
Figure PCTCN2020095044-appb-000001
(二)测定葡聚糖含量的方法
通过苯胺蓝法测定自溶液中葡聚糖含量。取1mL自溶液12000rpm离心5min，吸取上清液100μL加入1mL苯胺蓝工作液。50℃暗反应保温30min使荧光物质形成。置于室温30min,彻底混匀，吸取180μL至黑色96孔板进行检测。激发波长为405nm，发射波长为495nm，测定反应液荧光值。苯胺蓝工作液：12.7mL超纯水，3.70mL甘氨酸/氢氧化钠缓冲液(1.0M甘氨酸，1.25M氢氧化钠)和2.50mL染液(5.0g/L苯胺蓝)混合后在黑暗中过夜储存。
(三)DPPH清除率测定
以DPPH清除率为指标表征自溶液的抗氧化能力。采用分光光度计法，无水乙醇配制0.5072mM DPPH自由基醇溶液后，取1mL DPPH自由基醇溶液与1mL自溶液混合，避光反应30min测定其OD 517值。DPPH清除率计算公式如下：
Figure PCTCN2020095044-appb-000002
式中：A0，DPPH溶液与无水乙醇混合后测定的OD 517值；Ai，DPPH溶液与样品反应测得 OD 517值；Aj，无水乙醇与样品混合反应后测得OD 517值。
实施例1：高温敏感型啤酒酵母的筛选
(1)ARTP诱变时间的选择
出发菌株Pilsner培养至对数期，用生理盐水稀释至菌液细胞数达10 7个·mL -1左右。取上述菌悬液进行ARTP诱变处理，处理时间分别为15s、30s、45s、60s、75s。诱变结束后，将处理过的载片置于1mL无菌生理盐水中。取菌液合理稀释后涂布于YPD平板，置于28℃培养。绘制致死率曲线。见图1。由于啤酒酵母的选育需要保持菌株的成品啤酒主体风味，一般选择75％-85％的致死率，因此选择46s为诱变时间。
(2)突变菌的筛选
利用酵母胞内碱性磷酸酶与BCIP反应生成蓝色沉淀这一原理，可根据蓝色出现时间与深浅进行温度敏感型突变菌的筛选。将经过46s诱变所得到的菌液稀释10倍，涂布于含有40mg·L -1BCIP的YPD平板，28℃培养36h。通过平板影印法将菌落复制于YPD平板，并将其放置于28℃培养，同时将含有40mg·L -1BCIP的YPD平板放置于37℃培养24h。挑选菌落颜色变蓝的单菌落过夜培养于20mL YPD，将菌液OD 600调至0.6-0.8，同时划线于两块YPD平板，分别放置于28℃和37℃培养24h。获得16株温度敏感型突变菌，见图2。
(3)突变菌37℃自溶能力测定
菌株过夜活化，离心收集菌体，1.0g酵母泥加入10mL柠檬酸缓冲液(pH＝4.0)中同时记录细胞个数。180r·min -1、37℃摇床自溶30h，通过NanoDrop测定上清液中核酸浓度。见表2，得到P-510为自溶能力较好的突变菌。
表2突变菌株37℃自溶物RNA相对含量
Figure PCTCN2020095044-appb-000003
实施例2：高温敏感型啤酒酵母的生理性能
(1)突变菌37℃的生长状况和自溶能力
为了进一步分析P-510在37℃的生理性能，测定了其在37℃的生长状况(图3)和死亡率(图4)。P-510在37℃表现出明显的生长抑制。在37℃下培养12h后，Pilsner的死亡率变化不明显，84h后死亡率为6.5％，P-510的死亡率迅速增加，84h后至95.5％，说明P-510对高温敏感。同时测定P-510在37℃自溶过程中核酸(a)和蛋白质(b)的渗透率，如图5所示。在自溶过程中，自溶液核酸、蛋白质含量均增加，核酸含量增加量大于蛋白质含量增加量。120h后P-510核酸含量为232.3mg·L -1，而Pilsner的核酸含量为83.43mg·L -1，且P-510核酸含量增加率大于Pilsner和3-3。蛋白质含量增加幅度较小，在0-24h变化最大。120h后P-510蛋白质含量为7.57mg·L -1，而Pilsner的蛋白质含量为6.02mg·L -1。综上说明P-510在37℃较Pilsner更易自溶，对温度更敏感。
(2)生长曲线和发酵能力
诱变可能会对菌株的生长能力和发酵性能造成影响，因此测定了突变菌的生长曲线和发酵能力。如图6所示，P-510和Pilsner同时进入对数期，进入平台期后菌浓比原始菌略低，但菌株总体生长一致，说明经过诱变处理后P-510的生长能力没有受到影响。同时如表3所示，Pilsner和P-510在11℃发酵7天后发酵液的酒精度和实际发酵度差异不大，说明P-510能够进行正常发酵。
表3 Pilsner、P-510发酵液的酒精度和实际发酵度
指标 Pilsner P-510
酒精度/％v·v -1 3.7±0.1 3.62±0.1
实际发酵度/％ 55.3±1.0 54.6±1.1
实施例3：高温敏感型突变菌37℃自溶物分析
(1)呈味核苷酸含量测定
将实施例1中筛选出的高温敏感型啤酒酵母P-510在37℃自溶，利用高效液相色谱法测定自溶液中鸟苷酸、肌苷酸含量。如表4所示，P-510在37℃下自溶144h得到的自溶物呈味核苷酸含量(GMP+IMP)是原始菌的3倍，说明高温敏感型的P-510在37℃更易自溶，有利于获得高呈味核苷酸含量的酵母抽提物及自溶物。
表4 37℃自溶时相同菌浓的Pilsner、P-510自溶液中GMP、IMP含量
Figure PCTCN2020095044-appb-000004
(2)葡聚糖含量测定
自溶过程中细胞壁受到压力，多种水解酶作用于细胞壁，随后细胞壁组分溶出，其中包括生物活性物质β-葡聚糖。分别测定Pilsner和P-510在37℃自溶液中β-葡聚糖的含量，如图3所示。自溶时间越长，溶出葡聚糖含量越高。如图7所示，自溶120h后，P-510的葡聚糖含量比Pilsner的葡聚糖含量高40.3％，说明P-510的高温敏感突变性状使其在37℃下自溶时溶出葡聚糖含量增加。
(3)DPPH清除率测定
酵母抽提物及自溶物具有一定抗氧化性，可作为功能性食品添加剂。主要是由于酵母自溶过程中蛋白质降解成的活性肽，细胞壁组分溶出的葡聚糖和甘露聚糖等物质具有抗氧化性。分别测定Pilsner和P-510在37℃自溶液中DPPH清除率，如图8所示。37℃下Pilsner自溶120h时DPPH清除率最高为79.80％，P-510在80h时DPPH清除率最高为86.09％，比Pilsner高6.29％。可能是由于蛋白酶催化蛋白质中肽键水解时，温度会影响多肽链的长度、游离氨基酸的数量和氨基酸顺序，从而影响自溶液的抗氧化性。因此P-510在37℃自溶有利于获得抗氧化性较强的酵母抽提物及自溶物。
实施例4：高温敏感型啤酒酵母在制备酱油中的应用
将饼粕加水并蒸熟；在蒸熟的原料中，接入扩大培养米曲霉菌种，再接入本发明的高温敏感型啤酒酵母，充分拌匀；接种后的曲料通风培养，控制制曲温度，制得成曲；在成曲中加入盐水拌匀入发酵池，品温42～45℃维持20天左右，酱醅基本成熟；将成熟的酱醅进行后加工得到后处理酱油，后处理酱油加热灭菌，再配制(勾兑)、澄清及质量检验，得到符合质量标准的成品。
实施例5：高温敏感型啤酒酵母在制备食醋中的应用
在米中加水，进行浸渍；将浸渍后的米进行蒸煮；将蒸煮后的米饭再加热，加热后，迅速用凉水冲淋；在米饭中加入根霉菌和本发明的高温敏感型啤酒酵母，进行发酵。
将醋酸菌接入混合料中培养；当发酵体系中醋酸含量不上升时，将醋醅密封隔绝空气，继续发酵得到成熟的醋醅；将发酵成熟的醋醅进行密封隔绝空气，常温发酵40～45天，加水过滤后即得到醋液。
实施例6：高温敏感型啤酒酵母在制备豆瓣酱中的应用
以黄豆、盐水、面粉为原料，黄豆100千克、面粉4.3千克、海盐24.3～25.7千克、种曲0.29～0.43千克。将大豆进行蒸煮并与面粉、海盐、种曲混匀，在30℃恒温24h制曲。
制曲完成后，入缸第0天接种本发明的高温敏感型啤酒酵母，于37℃恒温发酵至酱醅成熟；酱醅成熟之后进行加热灭菌，即得到豆瓣酱成品。
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 (17)

  1. 一株啤酒酵母(Saccharomyces pastorianus)，已于2019年4月3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.17520，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
  2. 含有权利要求1所述啤酒酵母的微生物制剂。
  3. 如权利要求2所述的微生物制剂，其特征在于，所述微生物制剂中含有活菌数≥10 7CFU/g的啤酒酵母细胞。
  4. 如权利要求3所述的微生物制剂，其特征在于，所述啤酒酵母细胞包括啤酒酵母干菌体或湿菌体。
  5. 一种增加酵母抽提物中呈味氨基酸含量的方法，其特征在于，使权利要求1所述的啤酒酵母在37-50℃下进行自溶。
  6. 如权利要求5所述的方法，其特征在于，将权利要求1所述啤酒酵母在25～30℃培养8～16h后，继续培养，进入自溶状态。
  7. 如权利要求5所述的方法，其特征在于，将权利要求1所述啤酒酵母在25～30℃培养8～16h，收集培养后的菌体细胞，将菌体细胞转移到另一培养体系中，于150～200r·min -1、自溶20～50h。
  8. 如权利要求5～7任一所述的方法，其特征在于，所述啤酒酵母与培养体系的质量体积比为50～200:1。
  9. 一种增加酵母抽提物中葡聚糖含量的方法，其特征在于，使权利要求1所述的啤酒酵母在37-50℃下进行自溶。
  10. 如权利要求9所述的方法，其特征在于，将权利要求1所述啤酒酵母在25～30℃培养8～16h后，继续培养，进入自溶状态。
  11. 如权利要求9所述的方法，其特征在于，将权利要求1所述啤酒酵母在25～30℃培养8～16h，收集培养后的菌体细胞，将菌体细胞转移到另一培养体系中，于150～200r·min -1、自溶20～50h。
  12. 如权利要求9～11任一所述的方法，其特征在于，所述啤酒酵母与培养体系的质量体积比为50～200:1。
  13. 权利要求1所述的啤酒酵母，或权利要求2所述的微生物制剂在生产功能性食品中的应用。
  14. 权利要求1所述的啤酒酵母，或权利要求2或3所述的微生物制剂在膳食添加剂中的应用。
  15. 如权利要求14所述的应用，其特征在于，所述膳食添加剂包括营养强化剂、增味剂、 食品加工助剂。
  16. 权利要求1所述的啤酒酵母，或权利要求2所述的微生物制剂在调味品、化妆品、保健品或制药领域中的应用。
  17. 如权利要求16所述的应用，其特征在于，所述调味品包括酱油、醋、豆瓣酱、甜面酱、蚝油。
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