WO2023283937A1 - 一株米曲霉za205及其应用 - Google Patents

一株米曲霉za205及其应用 Download PDF

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WO2023283937A1
WO2023283937A1 PCT/CN2021/106797 CN2021106797W WO2023283937A1 WO 2023283937 A1 WO2023283937 A1 WO 2023283937A1 CN 2021106797 W CN2021106797 W CN 2021106797W WO 2023283937 A1 WO2023283937 A1 WO 2023283937A1
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aspergillus oryzae
koji
fermented food
soy sauce
raw materials
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PCT/CN2021/106797
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English (en)
French (fr)
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周斌
周其洋
童星
王静
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佛山市海天(高明)调味食品有限公司
佛山市海天调味食品股份有限公司
广东海天创新技术有限公司
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Application filed by 佛山市海天(高明)调味食品有限公司, 佛山市海天调味食品股份有限公司, 广东海天创新技术有限公司 filed Critical 佛山市海天(高明)调味食品有限公司
Priority to CN202180096180.6A priority Critical patent/CN117157385A/zh
Priority to PCT/CN2021/106797 priority patent/WO2023283937A1/zh
Publication of WO2023283937A1 publication Critical patent/WO2023283937A1/zh

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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
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/50—Soya sauce
    • 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
    • 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
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae

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  • the invention relates to the technical fields of food processing and microbial fermentation. Specifically, the present invention relates to a strain of Aspergillus oryzae and its application in soy sauce and miso brewing.
  • Aspergillus oryzae is the core strain of traditional brewed food industry. Aspergillus oryzae can secrete proteolytic enzymes and sugar hydrolytic enzymes during the fermentation process. These enzymes degrade raw materials such as soybeans and wheat into small molecules such as short peptides, amino acids, oligosaccharides, and monosaccharides. These small molecules are what constitute product flavor. source of.
  • umami is the key taste component.
  • the umami taste mainly comes from the amino acids and polypeptides formed by enzymatic hydrolysis of soybean and wheat protein.
  • Glutamate is the main amino acid responsible for umami.
  • Soy protein contains about 16% glutamic acid, and about 46% of glutamic acid exists in the form of glutamine.
  • Glutamine itself has no umami taste, and only when it is enzymatically converted into glutamic acid by glutaminase can it fully exert its umami taste function.
  • Aspergillus oryzae Shanghai Niang 3.042 (also known as Aspergillus oryzae As3.951) is the most widely used in the condiment industry.
  • the main characteristics of this strain are fast growth, good resistance to impurities, abundant spore production, and the main enzyme system secreted by koji making.
  • the activity of neutral protease is high, but the pH of koji making is low, and the activity of glutaminase is not high at the same time.
  • the lack of glutaminase is mainly reflected in two points: one is insufficient secretion, and the other is not strong salt tolerance.
  • the inventors realized that salt tolerance is a key factor affecting whether glutaminase can fully function in high-salt fermented foods. Therefore, improving the salt tolerance of Aspergillus oryzae glutaminase is of great help to increasing the glutamic acid content in soy sauce and fermented sauce.
  • the inventor also realized that glutaminase can decompose glutamine in the raw material into glutamic acid and ammonia; the more ammonia, the higher the koji-making pH. At the same time, the higher the pH of koji making, the secretion of glutaminase can be further promoted, so there is a strong correlation between glutaminase and the pH of koji making.
  • soy sauce lactic acid bacteria convert sugars into organic acids after the soy sauce proliferates, and simultaneously convert the organic acids in the raw materials into other organic acids, wherein lactic acid is
  • the representative of organic acids with mild flavor is not only the taste component in soy sauce, which makes the strong salty taste of soy sauce soft, but also related to the aroma of soy sauce.
  • excessive citric acid content is an important source of soy sauce that stimulates the taste.
  • the optimum pH environment for lactic acid bacteria to convert citric acid is around 7. Therefore, the more neutral the pH of the fermented koji, the more it helps lactic acid bacteria to convert sourness.
  • the stimulating citric acid is converted into organic acids such as lactic acid with a mild flavor, further enhancing the flavor of soy sauce.
  • the present invention provides Aspergillus oryzae ZA205, which has the following multiple beneficial properties:
  • the synergistic effect of these beneficial properties is based on the fact that the fermented food obtained by the Aspergillus oryzae has significantly improved physical and chemical indicators and significantly improved sensory evaluation.
  • Aspergillus oryzae ZA205 is obtained from Aspergillus oryzae As3.951 (Shanghai Niang 3.042) by mutagenesis.
  • L-glutamine was added to the screening medium as a decomposition substrate of glutaminase, phenol red was used as an acid-base indicator, and 4 % NaCl as a screening sieve for salt tolerance.
  • Glutaminase decomposes the substrate L-glutamine to produce alkaline ammonia, which can increase the pH of the medium, or the characteristics of Aspergillus oryzae itself change, alkaline substances increase, and the pH of the medium is improved; phenol red senses pH changes , the more alkaline the pH, the darker the color of phenol red. The better the salt tolerance of synchronously secreted glutaminase, the faster the color change of phenol red. According to the speed and depth of the color change of phenol red, strains with high yield and salt-tolerant glutaminase can be screened out.
  • the present disclosure provides Aspergillus oryzae ZA205 (Aspergillus oryzae ZA205), which was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 18, 2021, with a preservation number of GDMCC NO: 61669 and a preservation address of Guangzhou, China. Building 59, Compound, No. 100 Middle Road.
  • the present disclosure provides the use of Aspergillus oryzae ZA205 in koji making.
  • the koji material obtained from koji making is used to prepare soy sauce.
  • the disclosure provides uses of Aspergillus oryzae ZA205 in the preparation of fermented food products
  • the fermented food is a fermented food obtained by fermenting a raw material containing beans and/or grains.
  • the fermented food is miso or soy sauce.
  • the present disclosure provides a koji material prepared by the above-mentioned Aspergillus oryzae ZA205;
  • the koji material is obtained by mixing protein material and/or starchy material with Aspergillus oryzae ZA205.
  • the protein raw material includes one or more of soybeans, black beans, broad beans, and the like.
  • soybean is soybean, scientific name: Glycine max (Linn.) Merr.).
  • soybeans are one or more of defatted soybeans, soybean meal, and the like.
  • the starchy raw material includes one or more of wheat, wheat flour, wheat bran, and the like.
  • the present disclosure provides a fermented food prepared from any one of the above koji ingredients.
  • the fermented food is a fermented food obtained by fermenting a raw material containing beans and/or grains.
  • the fermented food is miso or soy sauce.
  • the present disclosure provides a method for preparing koji material, which comprises inoculating and culturing the above-mentioned Aspergillus oryzae ZA205 into a raw material containing beans and/or grains.
  • the present disclosure provides a method for preparing soy sauce or miso, which includes the steps of koji making and fermentation, wherein the koji making includes inoculating the above-mentioned Aspergillus oryzae ZA205 into raw materials containing beans and/or grains and To cultivate.
  • the fermentation is performed using a high-salt dilute fermentation method.
  • the present disclosure provides a culture medium for screening bacterial strains, comprising:
  • acid-base indicators such as phenol red
  • the medium also contains nutrients required for the growth of the strain.
  • the nutrient components required for the growth of the bacterial strain are, for example, selected from: nitrogen source, carbon source, phosphorus source, trace elements required for the growth of the bacterial strain, or a combination thereof.
  • the percentage of L-glutamine is 0.5%-2%.
  • the percentage content of the acid-base indicator is 0.01 ⁇ -0.5 ⁇ .
  • the percentage of NaCl is 2%-5%.
  • the present disclosure provides a method of screening Aspergillus oryzae, comprising
  • Koji refers to the product of bean or grain-containing raw materials mixed with Aspergillus oryzae after culturing for several days.
  • the surface of the bean-containing raw material is covered with Aspergillus oryzae, which contains various enzymes, which greatly promotes the fermentation and maturation process of dilute fermented glutinous rice.
  • High-Salt and dilute state fermentaion (high-Salt and dilute state fermentaion), for example, can refer to "High-salt dilute state fermented soy sauce brewing process regulations SB/T 10312-1999"
  • “soy sauce” can refer to "GB 2717-2018 National Food Safety Standard Soy Sauce” or "GB/T 18186-2000 Brewed Soy Sauce”.
  • the "paste” can be soybean paste or wheat paste, for example, refer to "GB/T 24399-2009 soybean paste” or SB/T 10296-2009 sweet noodle paste.
  • % means % by weight.
  • “comprising”, “including” and “containing” may refer to a content greater than zero, such as more than 1%, such as more than 10%, such as more than 20%, such as more than 30%, such as more than 40%, such as 50% Above, such as above 60%, such as above 70%, such as above 80%, such as above 90%, such as 100%. When the content is 100%, the meaning of “comprising", “including” and “containing” is equivalent to “consisting of”.
  • Figure 1 shows the colony characteristics of Aspergillus oryzae ZA205 after 96 hours of culture in soybean juice medium.
  • Fig. 2 shows aspergillus oryzae ZA205, As3.951 and GM195 in 5% NaCl solution glutaminase activity relative retention rate curve with time;
  • Fig. 3 shows aspergillus oryzae ZA205, As3.951 and GM195 in 10% NaCl solution glutaminase activity relative retention rate curve with time;
  • Fig. 4 shows the relative retention rate of glutaminase activity of Aspergillus oryzae ZA205, As3.951 and GM195 in 17% NaCl solution as a function of time.
  • the present invention relates to the following biological materials that have been preserved in the Guangdong Microbial Culture Collection Center:
  • Aspergillus oryzae ZA205 (Aspergillus oryzae ZA205), the strain was preserved in the Guangdong Microbial Culture Collection Center on May 18, 2021, the preservation number is GDMCC NO: 61669, and the preservation address is No. 59, Compound No. 100, Xianlie Middle Road, Guangzhou, China building.
  • Huniang 3.042 also known as Aspergillus oryzae As3.951
  • Huniang 3.042 also known as Aspergillus oryzae As3.951
  • L-glutamine used in the following examples purity>99%, produced by Sigma-Aldrich (Sigma-Aldrich); glutamic acid detection kit: produced by R-Biopharm, Germany; other reagents: All are analytically pure and prepared with deionized water.
  • the assay of protease activity is assayed according to the method in SB/T 10317-1999.
  • the determination method of glutaminase activity is as follows: under the conditions of 40°C and a pH value of 7.2, the glutaminase in every 1g of soy sauce koji catalyzes the decomposition of L-glutamine to generate 1 ⁇ mol L- Glutamic acid is one unit of enzyme activity, expressed in U/g. L-glutamic acid was detected using a glutamic acid detection kit from R-Biopharm, Germany.
  • the determination of total nitrogen is with reference to the determination of protein in GB/T 5009.5-2003 food; determined by the titrator method.
  • Fig. 1 shows the colony photograph of Aspergillus oryzae ZA205 of the present invention after being cultivated in soybean milk medium for 96 hours.
  • the characteristics of the colony are as follows: the diameter of the colony reaches 58 mm, the hyphae are abundant, the spores are abundant, and the color is yellow-green.
  • the mutagenesis selection method of Aspergillus oryzae ZA205 bacterial strain of the present invention is as follows:
  • mutagenesis power 120W was used for mutagenesis, and the mutagenesis conditions were as follows: mutagenesis power 120W, gas flow rate 10L/min, and mutagenesis time 120s.
  • phenol red chromogenic medium L-glutamine 10g, yeast extract 5g, K 2 HPO 4 1g, KH 2 PO 4 0.1g, MgSO 4 0.5g, phenol Red 0.015g, NaCl 40g, agar 20g, add deionized water to dissolve, dilute to 1000mL, sterilize at 115°C for 20min), after culturing for 2-3 days, select colonies with fast growth, fast color change and deep color as the initial Screen strains.
  • high-salt phenol red chromogenic medium L-glutamine 10g, yeast extract 5g, K 2 HPO 4 1g, KH 2 PO 4 0.1g, MgSO 4 0.5g, phenol Red 0.015g, NaCl 40g, agar 20g, add deionized water to dissolve, dilute to 1000mL, sterilize at 115°C for 20min), after culturing for 2-3 days, select colonies with fast growth, fast color change and deep color as the initial Screen strains.
  • the primary screened strains were inoculated on the phenol red chromogenic plate medium for rescreening, and six rescreened strains were obtained, GM191, GM192, GM193, GM194, GM195, and ZA205. Transfer the obtained rescreened strains to soybean juice slant medium (KH 2 PO 4 1g, MgSO 4 0.5g, (NH 4 ) 2 SO 4 0.5g, soluble starch 20g, agar 20g, soybean juice 1000mL, sterilized at 115°C for 20min ), and routinely preserved after cultured and matured.
  • soybean juice slant medium KH 2 PO 4 1g, MgSO 4 0.5g, (NH 4 ) 2 SO 4 0.5g, soluble starch 20g, agar 20g, soybean juice 1000mL, sterilized at 115°C for 20min
  • the koji materials of GM195 and ZA205 have obvious advantages in pH value and glutaminase activity.
  • the pH value is closer to 7, and the glutaminase activity is higher.
  • the Erlenmeyer koji material of ZA205 also had higher activity of neutral protease.
  • the salt tolerance stability test of glutaminase was carried out on the koji material, and the specific test method was as follows: take an appropriate amount of koji material and add twice the pH7.2 phosphate buffer solution, and soak it in a refrigerator at 4°C overnight to obtain an extraction enzyme solution. Use NaCl to adjust the extracting enzyme solution to 0%, 5%, 10%, 17% concentration, and place it in an environment of 4°C for 0, 1, 3, 5, 8h, and detect the relative residual enzyme activity of glutaminase , and the results are shown in Table 2.
  • Figure 2, Figure 3 and Figure 4 respectively show the salt tolerance stability of the glutaminase activities of ZA205, As3.951 and GM195 strains in different concentrations of NaCl solutions.
  • the expansion culture was carried out in the triangular flask, and then the koji making and fermentation experiments were continued (the koji making medium was prepared from soybeans, wheat and other raw materials, the ratio of raw materials, moistening water, etc. , cooking, and fermentation parameters are all carried out with reference to "Soy Sauce Science and Brewing Technology").
  • ZA205 has better physical and chemical indicators than As3.951 in small trial production, especially the pH value is closer to neutral.
  • the physical and chemical indexes of fermented crude oil of ZA205 are significantly better than those of As3.951 and GM195.
  • the new strain ZA205 obtained through screening was tested for aflatoxin, and the results showed that aflatoxin was not detected.
  • the new strain ZA205 and the original strain As3.951 were simultaneously launched for large-scale production and use of soy sauce.
  • the scale of koji making is 20 tons
  • the scale of crude oil preparation is 30 tons.
  • the koji material prepared by Aspergillus oryzae ZA205 has a significantly increased glutaminase activity, which is closer to the pH of 7, and the neutral protease activity is also higher.
  • the soy sauce prepared by Aspergillus oryzae ZA205 has significantly increased amino acid nitrogen content, glutamic acid content, and glucose content; in addition, the content of lactic acid is higher and the content of citric acid is lower.
  • the soy sauce prepared by Aspergillus oryzae ZA205 had improved color, aroma and umami taste, showing a significantly improved overall taste.

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Abstract

本发明涉及一株米曲霉ZA205及其应用。该米曲霉ZA205同时具有：(1)显著提高的谷氨酰胺酶活力；(2)显著提高的谷氨酰胺酶耐盐性；(3)曲料pH更接近中性；(4)较高的中性蛋白酶活力。

Description

一株米曲霉ZA205及其应用 技术领域
本发明涉及食品加工及微生物发酵技术领域。具体而言，本发明涉及一株米曲霉，以及其在酱油及酱酿造中的应用。
背景技术
米曲霉是传统酿造食品行业核心菌种。米曲霉在发酵过程中能够分泌蛋白水解酶和糖类水解酶，这些酶将大豆和小麦等原料降解成短肽、氨基酸、寡糖、单糖等小分子物质，这些小分子物质是构成产品风味的源泉。
对于酱油及酱类产品来说，鲜味是其关键的味道组成。鲜味主要来源于大豆、小麦蛋白经酶解之后形成的氨基酸、多肽类物质。谷氨酸是呈现鲜味的主要氨基酸。大豆蛋白中含有约16％谷氨酸，约46％的谷氨酸是以谷氨酰胺的形式存在。谷氨酰胺本身不具有鲜味，只有当其被谷氨酰胺酶酶解转化为谷氨酸后，才能充分发挥鲜味功能。
在调味品行业使用最为广泛的是米曲霉沪酿3.042(也称米曲霉As3.951)，该菌株特性主要是生长速度快、抗杂性好、产孢丰富、且制曲分泌的主要酶系-中性蛋白酶活力高，但制曲pH较低，同时谷氨酰胺酶活力不高。对于谷氨酰胺酶的不足，主要体现在两点：一是分泌量不够，二是耐盐性不强。
相关技术记载了一些改进的米曲霉，比如提高了米曲霉的谷氨酰胺酶酶活力，但是在谷氨酰胺酶酶活获得提升的同时，菌株的其他性能没有获得显著提升，甚至明显下降。
发明内容
发明人认识到，耐盐性却是谷氨酰胺酶能否在高盐发酵食品中充分发挥作用的关键影响因素。因此提高米曲霉谷氨酰胺酶的耐盐性，对提升酱油及发酵酱中谷氨酸含量有巨大帮助。
发明人还认识到，谷氨酰胺酶能将原料中的谷氨酰胺分解为谷氨酸和氨；氨越多，制曲pH就越高。同时制曲pH越高，也能进一步促进谷氨酰胺酶分泌，因此谷氨酰胺酶与制曲pH有极强关联性。
发明人还认识到酱油的酸味是以风味柔和的有机酸为主，酱油乳酸菌在酱醪增殖后，将糖类转换成有机酸，同时将原料中的有机酸转化成其他有机酸，其中乳酸是风味柔和有机酸的代表，其不仅是酱油中的呈味成分，使得酱油强烈盐味变得柔和，也关乎到酱油的香气。然而过量的柠檬酸含量是导致酱油刺激口感的重要来源，乳酸菌将柠檬酸转化的最适pH值环境为7左右，因此发酵曲料pH越是偏向于中性，越有助于乳酸菌将酸爽刺激的柠檬酸转化成风味柔和的乳酸等有机酸，进一步提高酱油风味。
为了实现上述目的，本发明提供米曲霉ZA205，其同时具有如下多项有益性能：
(1)显著提高的谷氨酰胺酶耐盐性；
(2)显著提高的谷氨酰胺酶活力；
(3)曲料pH更接近中性；
(4)较高的中性蛋白酶活力。
由于本发明米曲霉同时具有上述多项有益性能，这些有益性能协同作用，是基于该米曲霉获得的发酵食品具有显著改善的理化指标和显著提高的感官评价。
米曲霉ZA205是以米曲霉As3.951(沪酿3.042)为出发菌株，经诱变选育获得。为了筛选获得高制曲pH、高产、耐盐谷氨酰胺酶的菌株，在筛选培养基中加入L-谷氨酰胺作为谷氨酰胺酶的分解底物、酚红作为酸碱指示剂，加入4％的NaCl作为耐盐特性筛选筛子。谷氨酰胺酶分解底物L-谷氨酰胺产生碱性氨，可提高培养基的pH，或米曲霉自身的特性发生改变，碱性物质增多，培养基的pH得到提升；酚红感受pH变化，pH越碱，酚红颜色越深。同步分泌的谷氨酰胺酶耐盐性越好，酚红颜色变化越快。通过酚红的颜色变化快慢及深浅，就能从中筛选出高产及耐盐谷氨酰胺酶的菌株。
在一些方面，本公开提供米曲霉ZA205(Aspergillus oryzae ZA205)，该菌株于2021年05月18日保藏于广东省微生物菌种保藏中心，保藏号为GDMCC NO：61669，保藏地址为中国广州市先烈中路100号大院59号楼。
在一些方面，本公开提供米曲霉ZA205在制曲中的用途。
在一些实施方案中，对于上述用途，其中制曲获得的曲料用于制备酱油。
在一些方面，本公开提供米曲霉ZA205在制备发酵食品中的用途；
在一些实施方案中，所述发酵食品是对含豆和/或谷的原料发酵获得的发酵食品。
在一些实施方案中，所述发酵食品是酱或酱油。
在一些方面，本公开提供一种曲料，其由上述的米曲霉ZA205制备获得；
在一些实施方案中，所述曲料是将蛋白质原料和/或淀粉质原料与米曲霉ZA205混合制曲获得。
在一些实施方案中，蛋白质原料是包括：黄豆、黑豆、蚕豆等一种或多种。在一个实施方案中，黄豆是大豆，学名：Glycine max(Linn.)Merr.)。在一个实施方案中，大豆是脱脂大豆、豆粕等一种或多种。在一些实施方案中，淀粉质原料包括小麦、小麦粉、麦麸等一种或多种。
在一些方面，本公开提供一种发酵食品，其由上述任一项的曲料制备获得。
在一些实施方案中，所述发酵食品是对含豆和/或谷的原料发酵获得的发酵食品。
在一些实施方案中，所述发酵食品是酱或酱油。
在一些方面，本公开提供一种制备曲料的方法，其包括将上述的米曲霉ZA205接种到含豆和/或谷的原料中并进行培养。
在一些方面，本公开提供一种制备酱油或酱的方法，其包括制曲和发酵的步骤，其中，所述制曲包括将上述的米曲霉ZA205接种到含豆和/或谷的原料中并进行培养。
在一些实施方案中，使用高盐稀态发酵法进行所述发酵。
在一些方面，本公开提供一种用于筛选菌株的培养基，含有：
L-谷氨酰胺；
酸碱指示剂(例如酚红)；和
NaCl；
可选地，所述培养基还含有菌株生长所需营养成分。
在一些实施方案中，菌株生长所需营养成分例如选自：氮源、碳源、磷源、菌株生长所需微量元素、或其组合。
在一些实施方案中，L-谷氨酰胺的百分含量为0.5％-2％。
在一些实施方案中，酸碱指示剂的百分含量为0.01‰-0.5‰。
在一些实施方案中，NaCl的百分含量为2％-5％。
在一些方面，本公开提供一种筛选米曲霉的方法，包括
-将多个米曲菌接种于权利要求10所述的培养基；
-比较所述多个米曲菌附近培养基的颜色变化程度和/或速度；
-根据比较结果筛选米曲霉。
术语说明：
曲料(koji)是指混有米曲霉的含豆或谷类原料培养若干天后的产物。含豆原料的表面覆盖着米曲霉，它含有各种酶，大大促进了稀态醪糟发酵和成熟过程。
“高盐稀态法”(high-Salt and diluted state fermentaion)例如可以参照“高盐稀态发酵酱油酿造工艺规程SB/T 10312-1999”
“酱油”(soy sauce)例如可以参照“GB 2717-2018食品安全国家标准酱油”或“GB/T 18186-2000酿造酱油”。
“酱”可以是豆酱(soybean paste)或面酱(wheat paste)，例如可以参照“GB/T 24399-2009黄豆酱”或SB/T 10296-2009甜面酱。
“干基”:曲料在105℃烘箱条件下，进行水分烘干，直至水分保持恒定不变的物料。
除非特别说明，％是指重量％。
在任一实施方案中，“包含”“包括”“含有”可以是指含量大于零，例如1％以上，例如10％以上，例如20％以上，例如30％以上，例如40％以上，例如50％以上，例如60％以上，例如70％以上，例如80％以上，例如90％以上，例如100％。当含量为100％时，“包含”“包括”“含有”的含义相当于“由…构成”。
有益效果
本公开一项或多项技术方案具有以下一项或多项有益效果：
(1)显著提高的谷氨酰胺酶耐盐性；
(2)显著提高的谷氨酰胺酶活力；
(3)曲料pH更接近中性；
(4)较高的中性蛋白酶活力。
附图说明
图1示出米曲霉ZA205在豆汁培养基培养96h后的菌落特征。
图2示出米曲霉ZA205、As3.951和GM195在5％NaCl溶液中谷氨酰胺酶活力相对保持率随时间变化曲线；
图3示出米曲霉ZA205、As3.951和GM195在10％NaCl溶液中谷氨酰胺酶活力相对保持率随时间变化曲线；
图4示出米曲霉ZA205、As3.951和GM195在17％NaCl溶液中谷氨酰胺酶活力相对保持率随时间变化曲线。
关于生物材料保藏的说明
本发明涉及下列已在广东省微生物菌种保藏中心进行保藏的生物材料：
米曲霉ZA205(Aspergillus oryzae ZA205)，该菌株于2021年05月18日保藏于广东省微生物菌种保藏中心，保藏号为GDMCC NO：61669，保藏地址为中国广州市先烈中路100号大院59号楼。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用药品或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
以下实施例使用的沪酿3.042(也称米曲霉As3.951)海天公司保藏。
以下实施例使用的L-谷氨酰胺:纯度>99％，美国西格玛奥德里奇公司(Sigma-Aldrich)生产；谷氨酸检测试剂盒:德国拜发公司(R-Biopharm)生产；其他试剂:均为分析纯，采用去离子水配制。
以下实施例中，蛋白酶活力的测定根据SB/T 10317-1999中的方法进行测定。
以下实施例中，谷氨酰胺酶活测定方法如下：采用在40℃，pH值为7.2的条件下，每1g酱油曲中的谷氨酰胺酶每1min催化L-谷氨酰胺分解生成1μmol L-谷氨酸，即为1个酶活力单位，以U/g表示。L-谷氨酸采用德国拜发公司(R-Biopharm)的谷氨酸检测试剂盒进行检测。
以下实施例中，全氮测定参照GB/T 5009.5-2003食品中蛋白质的测定；氨基酸态氮(简称氨基氮或AAN)和总酸采用GB/T 5009.39-2003酱油卫生标准的分析方法中的电位滴定仪方法测定。
图1示出本发明米曲霉ZA205在豆汁培养基培养96h后的菌落照片。其菌落特征如下：菌落直径达58mm，菌丝丰富，孢子丰富、色泽黄绿色。
本发明的米曲霉ZA205菌株的诱变选育方法如下：
1.1初筛和复筛
以米曲霉As3.951为出发菌株，采用常压室温等离子体(ARTP)进行诱变，诱变条件如下：诱变功率120W，气流量为10L/min，诱变时间为120s。将诱变后的孢子均匀涂布到高盐酚红显色培养基(L-谷氨酰胺10g，酵母浸膏5g，K 2HPO 4 1g，KH 2PO 4 0.1g，MgSO 4 0.5g，酚红0.015g，NaCl 40g，琼脂20g，加入去离子水溶解后，定容到1000mL，于115℃灭菌20min)，培养2-3天后，挑选生长快、颜色变化快、色泽深的菌落作为初筛菌株。将初筛菌株接种到酚红显色平板培养基进行复筛，获得6株复筛菌株，GM191，GM192，GM193，GM194，GM195，ZA205。将获得复筛菌株转接到豆汁斜面培养基(KH 2PO 4 1g，MgSO 4 0.5g，(NH 4) 2SO 4 0.5g，可溶性淀粉20g，琼脂20g，豆汁1000mL，于115℃灭菌20min)，培养成熟后进行常规保藏。
1.2三角瓶曲料-酶活力分析
提供非转基因大豆和小麦的混合物(质量比，大豆:小麦＝1:1)，润水1.2倍，混合拌匀，分装至三角瓶，121℃灭菌30min，制成三角瓶发酵培养基。从1.1中的米曲霉菌种挑取1环孢子，接种于三角瓶发酵培养基中，32℃培养96h，制得三角瓶曲料。
对三角瓶曲料进行pH、谷氨酰胺酶、中性蛋白酶进行检测，结果如下表所示。
表1、三角瓶曲料的理化指标
Figure PCTCN2021106797-appb-000001
如上所示，GM195和ZA205的三角瓶曲料在pH值、谷氨酰胺酶活具有明显优势，其pH值更接近7，谷氨酰胺酶活更高。ZA205的三角瓶曲料还具有较高的中性蛋白酶活。
1.3三角瓶曲料-耐盐能力分析
提供非转基因大豆和小麦的混合物(质量比，大豆:小麦＝1:1)，润水1.2倍，混合 拌匀，分装至三角瓶，121℃灭菌30min，制成三角瓶发酵培养基。从GM195、ZA205、As3.951米曲霉斜面菌种挑取1环孢子，接种于三角瓶发酵培养基中，32℃培养96h，制得三角瓶曲料。
对曲料进行谷氨酰胺酶耐盐稳定性测试，具体测试方法如下：取适量曲料加入两倍的pH7.2的磷酸盐缓冲液，在4℃冰箱中浸泡过夜，获得浸提酶液。使用NaCl将浸提酶液调节到0％、5％、10％、17％浓度后，并在4℃的环境中放置0、1、3、5、8h，检测谷氨酰胺酶相对残留酶活，结果如表2所示。图2、图3和图4分别示出了，ZA205、As3.951和GM195菌株的谷氨酰胺酶活在不同浓度NaCl溶液中的耐盐稳定性。
表2、不同菌株的谷氨酰胺酶在NaCl溶液中的相对残留酶活
Figure PCTCN2021106797-appb-000002
结果显示，在不同的NaCl浓度下，ZA205的谷氨酰胺酶相对残留酶活均显著高于As3.951和GM195，同时菌株的保留时间也显著长于As3.951和GM195。ZA205的谷氨酰胺酶耐盐稳定性改善到达了预料不到的程度。
1.4制曲小试和发酵小试
将米曲霉ZA205、GM195与As3.951依次转接斜面活化后，进行三角瓶扩大培养，然后继续进行制曲和发酵小试(制曲培养基采用大豆、小麦等原料制备，原料比例、润水、 蒸煮、发酵参数均参考《酱油科学与酿造技术》进行)。
发酵小试制备了曲料0.4吨，检测制曲曲料水分、pH值、中性蛋白酶、谷氨酰胺酶，检测结果见表3。
发酵小试制备了发酵原油(酱油)0.6吨，检测发酵原油总酸、氨基氮、谷氨酸、葡萄糖等指标，检测结果见表4。
表3、小试制曲的理化指标
Figure PCTCN2021106797-appb-000003
*标准化是指以As3.951指标为1.00
表4、发酵原油的理化指标
Figure PCTCN2021106797-appb-000004
如上所示，如上所示，ZA205的小试制曲理化指标显著优于As3.951，特别是pH值更接近中性。ZA205的发酵原油理化指标显著优于As3.951和GM195。
1.5黄曲霉素检测
将筛选获得新菌株ZA205进行黄曲霉毒素检测，结果显示黄曲霉毒素未检出。
1.6传代实验
同步对ZA205进行10代传代试验，并将传代菌株进行酱油制曲小试验证，结果显示发酵氨基氮及谷氨酸指标稳定，表明ZA205遗传性能稳定。
表5、ZA205传代稳定性验证
代数 氨基氮(g/100mL) 谷氨酸(g/L)
原代 1.12 14.0
2代 1.11 13.9
6代 1.13 14.6
10代 1.12 14.2
1.7规模化生产
将新菌株ZA205与出发菌株As3.951同步展开规模化酱油、生产使用。规模化生产中，制曲规模为20吨，制备原油规模为30吨。
制曲结束后抽样检测规模化生产制曲理化指标，结果如表6。发酵结束后检测规模化生产原油的理化指标，结果如表7。对规模化生产的酱油原油进行加热灭菌等后处理后，进行感官评测，结果如表8。
表6、规模化生产的曲料的理化指标
Figure PCTCN2021106797-appb-000005
表7、规模化生产的酱油原油的理化指标
Figure PCTCN2021106797-appb-000006
表8、规模化生产的酱油原油的感官分析
菌株 色泽 香气 体态 鲜味 咸味 酸味 综合口感
ZA205 4.5 4.5 3.5 4.5 3.5 2.5 4.5
As3.951 3.5 3.5 3.5 3 4.5 3 3
由表6～表8所示，米曲霉ZA205用于规模化生产，曲料的理化指标，酱油的理化指标，酱油的感官分析，均优于米曲霉As3.951。
如表6所示，米曲霉ZA205制备的曲料具有显著提高的谷氨酰胺酶活，更接近于7的pH，中性蛋白酶活也较高。
如表7所示，米曲霉ZA205制备的酱油具有显著提高的氨基酸态氮含量、谷氨酸含量、葡萄糖含量；另外其乳酸的含量更高，柠檬酸的含量更低。
如表8所示，米曲霉ZA205制备的酱油具有提高的色泽、香气和鲜味，表现出显著改善的综合口感。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 米曲霉ZA205(Aspergillus oryzae ZA205)，该菌株于2021年05月18日保藏于广东省微生物菌种保藏中心，保藏号为GDMCC NO：61669，保藏地址为中国广州市先烈中路100号大院59号楼。
  2. 权利要求1所述的米曲霉ZA205在制曲中的用途。
  3. 权利要求2所述的用途，其中制曲获得的曲料用于制备酱油。
  4. 权利要求1所述的米曲霉ZA205在制备发酵食品中的用途；
    优选地，所述发酵食品是对含豆和/或谷的原料发酵获得的发酵食品；
    优选地，所述发酵食品是酱或酱油。
  5. 一种曲料，其由权利要求1所述的米曲霉ZA205制备获得；
    优选地，所述曲料是将含豆和/或谷的原料与米曲霉ZA205混合制曲获得。
  6. 一种发酵食品，其由权利要求5所述的曲料制备获得；
    优选地，所述发酵食品是对蛋白质原料和/或淀粉质原料发酵获得的发酵食品；
    优选地，所述发酵食品是酱或酱油。
  7. 一种制备曲料的方法，其包括将权利要求1所述的米曲霉ZA205接种到蛋白质原料和/或淀粉质原料中并进行培养。
  8. 一种制备酱油或酱的方法，其包括制曲和发酵的步骤，其中，所述制曲包括将权利要求1所述的米曲霉ZA205接种到蛋白质原料和/或淀粉质原料中并进行培养。
  9. 权利要求8所述的方法，其中，使用高盐稀态发酵法进行所述发酵。
  10. 一种用于筛选菌株的培养基，含有：
    L-谷氨酰胺；
    酸碱指示剂(例如酚红)；和
    NaCl；
    可选地，所述培养基还含有菌株生长所需营养成分。
  11. 根据权利要求10所述的培养基，其特征在于以下一项或多项
    L-谷氨酰胺的百分含量为0.5％-2％
    酸碱指示剂的百分含量为0.01‰-0.5‰；
    NaCl的百分含量为2％-5％。
  12. 一种筛选米曲霉的方法，包括
    -将多个米曲菌接种于权利要求10所述的培养基；
    -比较所述多个米曲菌附近培养基的颜色变化程度和/或速度；
    -根据比较结果筛选米曲霉。
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