Aspergillus oryzae strain ZA205 and its applications

Abstract

The present invention relates to a strain of Aspergillus oryzae (Koji mold) ZA205 and its use. Aspergillus oryzae ZA205 simultaneously exhibits: (1) significantly increased glutaminase activity; (2) significantly improved glutaminase salt tolerance; (3) koji material with a more neutral pH; and (4) higher neutral protease activity.

Description

[Technical Field]

[0001] The present 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 use in brewing soy sauce and paste. [Background technology]

[0002] Aspergillus oryzae is a core strain in the traditional brewing food industry. Aspergillus oryzae can secrete proteolytic enzymes and carbohydrate hydrolytic enzymes during fermentation. These enzymes break down raw materials (e.g., soybeans and wheat) into small molecules (e.g., short peptides, amino acids, oligosaccharides, and monosaccharides). These small molecules are the source of the flavor of the product.

[0003] Umami is an important flavor component for soy sauce and paste products. Umami is derived from amino acids and peptides formed mainly after enzymatic hydrolysis of soybean and wheat proteins. Glutamic acid is the main amino acid responsible for umami. Soybean protein contains about 16% glutamic acid, and about 46% of glutamic acid exists in the form of glutamine. Glutamine itself does not exhibit umami flavor. It can only exert its full umami function when it is enzymatically converted into glutamic acid by glutaminase.

[0004] The most widely used strain in the seasoning industry is Aspergillus oryzae Huniang 3.042 (also known as Aspergillus oryzae As3.951). This strain's key characteristics include fast growth, good resistance to contaminating microorganisms, abundant spore formation, and high activity of neutral protease, the main enzyme secreted during koji production. However, it also exhibits low pH during koji production and low glutaminase activity. The lack of glutaminase is primarily reflected in its insufficient secretion and poor salt tolerance.

[0005] In the related art, for example, improved Aspergillus oryzae strains with improved glutaminase activity have been reported. However, while the glutaminase activity is improved, other properties of the strain are not significantly improved, and in some cases are significantly reduced. Summary of the Invention

[0006] The inventors have recognized that salt tolerance is an important factor that affects whether glutaminase can fully exert its role in high-salt fermented foods. Therefore, improving the salt tolerance of glutaminase derived from Aspergillus oryzae will be of great help in increasing the glutamic acid content of soy sauce and fermented sauces.

[0007] The inventors also recognized that glutaminase can decompose glutamine in raw materials into glutamic acid and ammonia; the more ammonia, the higher the pH during koji-making. At the same time, a higher pH during koji-making may promote the secretion of more glutaminase. Therefore, there is a strong correlation between glutaminase and pH during koji-making.

[0008] The inventors also recognized that the sourness of soy sauce is primarily due to organic acids with a mild flavor. When lactic acid bacteria grow in soy sauce mash, sugars are converted into organic acids, and at the same time, organic acids in the raw materials are converted into other organic acids. Among these, lactic acid is a representative organic acid with a mild flavor. It not only serves as a flavor component that softens the strong saltiness of soy sauce, but also contributes to its aroma. However, excessive citric acid content is an important cause of the pungent flavor of soy sauce. The optimal pH for lactic acid bacteria to convert citric acid is approximately 7. Therefore, the more neutral the koji pH, the more easily lactic acid bacteria can convert the acidic, pungent citric acid into organic acids, such as lactic acid, which has a mild flavor, further enhancing the flavor of soy sauce.

[0009] To achieve the above object, the present invention provides Aspergillus oryzae ZA205, which simultaneously possesses the following beneficial properties: (1) Significantly improved glutaminase salt tolerance; (2) significantly improved glutaminase activity; (3) a more neutral koji pH; (4) Higher neutral protease activity.

[0010] The Aspergillus oryzae of the present invention simultaneously possesses several of the above-mentioned beneficial properties, and these beneficial properties act synergistically, so that fermented foods obtained based on this Aspergillus oryzae have significantly improved physical and chemical indicators and significantly improved sensory evaluation.

[0011] Aspergillus oryzae ZA205 was obtained by mutagenesis and selective breeding from the starting strain Aspergillus oryzae As3.951 (Huniang 3.042). To screen for strains with high pH, ​​high yield, and salt-tolerant glutaminase during koji production, L-glutamine was added to the screening medium as a substrate for glutaminase degradation, phenol red was used as an acid-base indicator, and 4% NaCl was used as a sieve to screen for salt tolerance. Glutaminase decomposes the substrate L-glutamine to produce alkaline ammonia, which can increase the pH of the medium, or the properties of Aspergillus oryzae itself change, increasing alkaline substances and raising the pH of the medium; phenol red detects changes in pH, and the more alkaline the pH, the darker the phenol red becomes. At the same time, the higher the salt tolerance of the secreted glutaminase, the faster the color change of phenol red. Based on the speed and intensity of the color change of phenol red, salt-tolerant strains that produce high amounts of glutaminase can be selected.

[0012] In some aspects, the present disclosure provides Aspergillus oryzae ZA205, which was deposited at the Guangdong Provincial Microbial Culture Collection Center, Building 59, Dayuan, No. 100, Xianlie Middle Road, Guangzhou, China, on May 18, 2021, under accession number GDMCC NO: 61669.

[0013] In some aspects, the present disclosure provides the use of Aspergillus oryzae ZA205 in koji production.

[0014] In some embodiments, the koji obtained by koji production for the above uses is used in the production of soy sauce.

[0015] In some aspects, the present disclosure provides the use of Aspergillus oryzae ZA205 in the production of fermented foods.

[0016] In some embodiments, the fermented food is a fermented food obtained by fermenting ingredients including beans and / or grains.

[0017] In some embodiments, the fermented food is a paste or soy sauce.

[0018] In some aspects, the present disclosure provides koji prepared from the above-mentioned Aspergillus oryzae ZA205.

[0019] In some embodiments, the koji is obtained by mixing a proteinaceous material and / or a starchy material with Aspergillus oryzae ZA205 to produce koji.

[0020] In some embodiments, the protein material comprises one or more selected from the group consisting of soybeans, black beans, fava beans, etc. In some embodiments, the soybeans are soybeans of the scientific name Glycine max (Linn.) Merr. In some embodiments, the soybeans are one or more selected from the group consisting of defatted soybeans, soybean meal, etc. In some embodiments, the starchy material comprises one or more selected from the group consisting of wheat, wheat flour, wheat bran, etc.

[0021] In some aspects, the present disclosure provides a fermented food product prepared from any one of the koji described above.

[0022] In some embodiments, the fermented food is a fermented food obtained by fermenting ingredients including beans and / or grains.

[0023] In some embodiments, the fermented food is a paste or soy sauce.

[0024] In some aspects, the present disclosure provides a method for producing koji, comprising inoculating and culturing the above-described Aspergillus oryzae ZA205 on a raw material including beans and / or grains.

[0025] In some aspects, the present disclosure provides a method for producing soy sauce or paste, comprising the steps of koji production and fermentation, wherein the koji production comprises inoculating and culturing the above-described Aspergillus oryzae ZA205 on raw materials including beans and / or grains.

[0026] In some embodiments, the fermentation is carried out by a method for high salt dilute condition fermentation.

[0027] In some aspects, the present disclosure provides: L-glutamine; Acid-base indicators (e.g., phenol red); and NaCl A medium for screening a bacterial strain, comprising: Optionally, a culture medium is provided which further contains nutrients necessary for the growth of the strain.

[0028] In some embodiments, the nutrients required for the growth of the strain are selected from the group consisting of, for example, a nitrogen source, a carbon source, a phosphorus source, trace elements required for the growth of the strain, and combinations thereof.

[0029] In some embodiments, the L-glutamine content is 0.5% to 2%.

[0030] In some embodiments, the content of the acid-base indicator is 0.01‰ to 0.5‰.

[0031] In some embodiments, the NaCl content is between 2% and 5%.

[0032] In some aspects, the present disclosure provides: - inoculating the medium according to claim 10 with a plurality of Aspergillus oryzae strains; - Comparing the degree and / or speed of discoloration of the medium adjacent to multiple strains of Aspergillus oryzae; - Selecting Aspergillus oryzae strains based on the comparison results The present invention provides a method for screening Aspergillus oryzae, comprising:

[0033] Glossary Koji is a product of soybean or grain ingredients mixed with Aspergillus oryzae, which has been cultivated for several days. The surface of the soybean ingredients is coated with Aspergillus oryzae, which contains various enzymes that greatly accelerate the fermentation and maturation process of the diluted mash.

[0034] For the "high-salt dilution state fermentation", see, for example, Technological regulations for soy sauce with process high-salt-diluted state fermentation, SB / T 10312-1999.

[0035] "Soy sauce" can refer to, for example, GB 2717-2018, National Food Safety Standard, Soy Sauce or GB / T 18186-2000, Fermented soy sauce.

[0036] The "paste" may be soybean paste or wheat paste, which may refer to, for example, GB / T 24399-2009, Soybean paste or SB / T 10296-2009, Wheat paste.

[0037] "Waterless base": The koji is dried in an oven at 105°C until the moisture content is constant.

[0038] Unless otherwise specified, % refers to % by weight.

[0039] In any embodiment, "comprising", "including", "containing" may mean an amount greater than zero, such as 1% or more, such as 10% or more, such as 20% or more, such as 30% or more, such as 40% or more, such as 50% or more, such as 60% or more, such as 70% or more, such as 80% or more, such as 90% or more, such as 100%. When the content is 100%, "comprising", "including", "containing" is equivalent to "consisting of".

[0040] Beneficial effects One or more technical solutions of the present disclosure have one or more of the following beneficial effects: (1) Significantly improved glutaminase salt tolerance; (2) significantly improved glutaminase activity; (3) a more neutral koji pH; (4) Higher neutral protease activity.

[0041] Instructions for depositing biological material The present invention relates to the following biological materials deposited at the Guangdong Provincial Microbial Culture Collection Center: Aspergillus oryzae ZA205. This strain was deposited on May 18, 2021, at the Guangdong Provincial Microbial Culture Collection Center, Building 59, Dayuan, No. 100, Xianlie Middle Road, Guangzhou, China, under accession number GDMCC NO:61669. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 shows the colony characteristics of Aspergillus oryzae ZA205 after culturing it in soy milk medium for 96 hours. [Figure 2] FIG. 2 shows the relative residual rate curves of glutaminase activity over time for Aspergillus oryzae ZA205, As3.951, and GM195 in a 5% NaCl solution. [Figure 3] FIG. 3 shows the relative residual rate curves of glutaminase activity over time for Aspergillus oryzae ZA205, As3.951, and GM195 in a 10% NaCl solution. [Figure 4] FIG. 4 shows the relative residual rate curves of glutaminase activity over time for Aspergillus oryzae ZA205, As3.951, and GM195 in a 17% NaCl solution. DETAILED DESCRIPTION OF THE INVENTION

[0043] Hereinafter, the embodiments of the present invention will be described in detail with reference to examples. However, those skilled in the art will understand that the following examples are used only to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific conditions are not specified in the examples, the conditions are assumed to be conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents and instruments used are not listed, they are all commercially available conventional products.

[0044] Huniang 3.042 (also known as Aspergillus oryzae As3.951) used in the following examples was deposited by the Haitian Company.

[0045] The L-glutamine used in the following examples was >99% pure, manufactured by Sigma-Aldrich, USA; the glutamic acid detection kit used was manufactured by R-Biopharm, Germany; all other reagents used were analytical grade and prepared with deionized water.

[0046] In the following examples, protease activity was measured according to the method of SB / T 10317-1999.

[0047] In the following examples, glutaminase activity was measured as follows: glutaminase in 1 g of soy sauce koji catalyzed the degradation of L-glutamine to produce 1 μmol of L-glutamic acid per minute at 40°C and pH 7.2, which was defined as 1 unit of enzyme activity expressed in U / g. L-glutamic acid was detected using a glutamic acid detection kit obtained from R-Biopharm, Germany.

[0048] In the following examples, total nitrogen was measured with reference to GB / T 5009.5-2003, Determination of protein in foods; amino acid nitrogen (also called amino nitrogen or AAN) and total acid were measured using the potentiometric titration method of GB / T 5009.39-2003, Method for analysis of hygienic standard of soybean sauce.

[0049] 1 shows a photograph of a colony of Aspergillus oryzae ZA205 of the present invention after 96 hours of culture in a soy milk medium. The colony characteristics were as follows: a maximum colony diameter of 58 mm, abundant hyphae, abundant spores, and yellow-green color.

[0050] The mutation breeding method for the Aspergillus oryzae ZA205 strain of the present invention was as follows: 1.1 Initial Screening and Rescreening Aspergillus oryzae As3.951 was used as the starting strain and mutations were induced using ambient pressure room temperature plasma (ARTP). The mutagenesis conditions were as follows: mutagenesis power: 120 W, aeration rate: 10 L / min, and mutagenesis time: 120 s. The mutated spores were uniformly spread on a high-salt phenol red medium (10 g L-glutamine, 5 g yeast extract, 1 g KHPO, 0.1 g KHPO, 0.5 g MgSO, 0.015 g phenol red, 40 g NaCl, and 20 g agar, dissolved in deionized water, adjusted to a volume of 1000 mL, and sterilized at 115°C for 20 min). After 2–3 days of incubation, colonies with rapid growth, rapid discoloration, and deep color were selected as preliminary screening strains. For secondary screening, the pre-screened strains were inoculated onto phenol red-colored plates to obtain six secondary screened strains, i.e., GM191, GM192, GM193, GM194, GM195, and ZA205. These secondary screened strains were then inoculated onto soy milk slants (1 g KH2PO4, 0.5 g MgSO4, 0.5 g (NH4)2SO4, 20 g soluble starch, 20 g agar, and 1000 mL soybean juice, sterilized at 115°C for 20 minutes), matured, and then stored as usual.

[0051] 1.2 Enzyme activity analysis of koji in Erlenmeyer flasks A mixture of non-GMO soybeans and wheat (soybeans: wheat = 1:1, mass ratio) was prepared, moistened with 1.2 times the amount of water, mixed well, filled into an Erlenmeyer flask, and sterilized at 121°C for 30 minutes to prepare a fermentation medium in the Erlenmeyer flask. One spore ring of the Aspergillus oryzae strain (1.1) was collected and inoculated into the fermentation medium in the Erlenmeyer flask. The mixture was then cultured at 32°C for 96 hours to prepare koji in the Erlenmeyer flask.

[0052] The pH, glutaminase and neutral protease of the koji in the Erlenmeyer flask were measured, and the results are shown in the table below.

[0053] [Table 1]

[0054] As shown in Table 1, koji GM195 and ZA205 in Erlenmeyer flasks showed clear advantages in pH and glutaminase activity, with a pH closer to 7 and higher glutaminase activity. Koji ZA205 in Erlenmeyer flasks also showed higher neutral protease activity.

[0055] 1.3 Salt tolerance analysis of koji in Erlenmeyer flasks A mixture of non-GMO soybeans and wheat (mass ratio, soybeans:wheat = 1:1) was prepared, moistened with 1.2 times the amount of water, mixed well, filled into an Erlenmeyer flask, and sterilized at 121°C for 30 minutes to prepare the fermentation medium. One spore ring of Aspergillus oryzae strains GM195, ZA205, and As3.951 was collected and inoculated into the fermentation medium in an Erlenmeyer flask. The mixture was then cultured at 32°C for 96 hours to prepare koji.

[0056] The salt-tolerance stability of glutaminase in koji was measured. The specific measurement method was as follows: an appropriate amount of koji was collected, and a double volume of pH 7.2 phosphate buffer was added to soak the koji. The koji was then stored overnight in a refrigerator at 4°C to obtain an enzyme extract. NaCl was added to the resulting enzyme extract to achieve NaCl concentrations of 0%, 5%, 10%, and 17%. The extract was then allowed to stand at 4°C for 0, 1, 3, 5, and 8 hours, and the relative residual enzyme activity of glutaminase was measured. The results are shown in Table 2. Figures 2, 3, and 4 show the salt-tolerance stability of glutaminase activity in various concentrations of NaCl solutions for strains ZA205, As3.951, and GM195, respectively.

[0057] [Table 2]

[0058] The results showed that the relative residual enzyme activity of glutaminase from ZA205 was significantly higher than that of As3.951 and GM195 at various NaCl concentrations, and the retention time of the strain was also significantly longer than that of As3.951 and GM195. The salt tolerance of glutaminase from ZA205 was improved beyond expectations.

[0059] 1.4 Small-scale koji making and fermentation experiments Aspergillus oryzae ZA205, GM195, and As3.951 were successively inoculated onto slant medium to activate them, and then expanded in Erlenmeyer flasks. Small-scale koji-making and fermentation experiments were then conducted (the koji-making medium was prepared from raw materials (e.g., soybeans, wheat, etc.), and the ratio of raw materials, water for humidification, and preparation and fermentation parameters were all based on Soy Sauce Science and Brewing Technology).

[0060] In the small-scale koji production experiment, 0.4 tons of koji was prepared, and the moisture content, pH, neutral protease activity, and glutaminase activity of the koji were measured. The results are shown in Table 3.

[0061] In the small-scale fermentation experiment, 0.6 tons of fermented crude soy sauce was prepared, and the total acid, amino nitrogen, glutamic acid, glucose and other indicators of the fermented crude soy sauce were measured. The results are shown in Table 4.

[0062] [Table 3]

[0063] [Table 4]

[0064] These results show that the physical and chemical indices of small-scale koji made with ZA205 were significantly better than those of As3.951, especially the pH, which was closer to neutral. The physical and chemical indices of fermented crude soy sauce made with ZA205 were significantly better than those of As3.951 and GM195.

[0065] 1.5 Aflatoxin detection The novel strain ZA205 obtained by screening was tested for aflatoxin, and no aflatoxin was detected.

[0066] 1.6 Passage experiment ZA205 was also subcultured for 10 generations, and the subcultured strain was verified in a small-scale soy sauce koji production experiment. The results showed that the fermentation amino nitrogen and glutamic acid indicators were stable, indicating the genetic stability of ZA205.

[0067] [Table 5]

[0068] 1.7 Large-scale production Using the new strain ZA205 and the starting strain As3.951, large-scale soy sauce production and use was initiated. The large-scale production involved 20 tons of koji and 30 tons of crude soy sauce.

[0069] After the koji production was completed, the physical and chemical indices of the large-scale koji were measured by sampling, and the results are shown in Table 6. After fermentation, the physical and chemical indices of the large-scale crude soy sauce were measured, and the results are shown in Table 7. After post-treatment (e.g., heating and sterilization), the large-scale crude soy sauce was subjected to sensory evaluation, and the results are shown in Table 8.

[0070] [Table 6]

[0071] [Table 7]

[0072] [Table 8]

[0073] As shown in Tables 6 to 8, when Aspergillus oryzae ZA205 was used for large-scale production, the physical and chemical indicators of koji, the physical and chemical indicators of soy sauce, and the sensory analysis of soy sauce were all superior to those of Aspergillus oryzae As3.951.

[0074] As shown in Table 6, koji prepared with Aspergillus oryzae ZA205 had significantly improved glutaminase activity, a pH close to 7, and higher neutral protease activity.

[0075] As shown in Table 7, the soy sauce prepared with Aspergillus oryzae ZA205 had significantly increased amino acid nitrogen content, glutamic acid content, and glucose content; in addition, the lactic acid content was high and the citric acid content was low.

[0076] As shown in Table 8, the soy sauce prepared with Aspergillus oryzae ZA205 had significantly improved color, aroma, and umami, and the overall taste was significantly improved.

[0077] Although specific embodiments of the present invention have been described in detail, it is understood that those skilled in the art can make various modifications and changes to the details based on all the teachings disclosed, and these modifications fall within the scope of protection of the present invention. The full scope of the present invention is also given by the claims and their equivalents. The following claims as originally filed in this application are appended as embodiments. [1] Strain of Aspergillus oryzae ZA205, deposited at the Guangdong Provincial Microbial Culture Collection Center, Building 59, Dayuan, No. 100, Xianlie Middle Road, Guangzhou, China, on May 18, 2021, under accession number GDMCC NO:61669. [2] Use of the Aspergillus oryzae ZA205 strain described in [1] in koji production. [3] The use according to [2], wherein the koji obtained by the koji production is used for the production of soy sauce. [4] Use of the Aspergillus oryzae ZA205 strain according to [1] in the production of fermented foods, Preferably, the fermented food is a fermented food obtained by fermenting raw materials comprising beans and / or cereals; Preferably, the fermented food is a paste or soy sauce. [5] Koji produced from the Aspergillus oryzae ZA205 strain described in [1], Preferably, the koji is obtained by mixing raw materials containing beans and / or grains with the strain of Aspergillus oryzae ZA205 and producing the koji. [6] A fermented food prepared from the koji according to [5], Preferably, the fermented food is a fermented food obtained by fermenting a proteinaceous material and / or a starchy material; Preferably, the fermented food is a paste or soy sauce. [7] A method for producing koji, comprising inoculating the Aspergillus oryzae ZA205 strain described in [1] onto a protein material and / or a starch material and culturing it. [8] A method for producing soy sauce or paste, comprising koji-making and fermentation steps, wherein the koji-making step comprises inoculating a proteinaceous material and / or a starchy material with Aspergillus oryzae ZA205, the strain described in [1], and culturing the same. [9] The method according to [8], wherein the fermentation is carried out by a method for high-salt dilution fermentation.

[10] L-glutamine; Acid-base indicators (e.g., phenol red); and NaCl A medium for screening a bacterial strain, comprising: Optionally, the medium further comprises nutrients necessary for the growth of the strain.

[11] The L-glutamine content is 0.5% to 2%; The content of the acid-base indicator is 0.01‰ to 0.5‰; The NaCl content is 2% to 5% The medium according to

[10] , characterized by one or more of the following:

[12] - Inoculating the medium described in

[10] with Aspergillus oryzae strain; - comparing the degree and / or speed of discoloration of the medium adjacent to the Aspergillus oryzae strain; - Selecting strains of Aspergillus oryzae based on the comparison results A method for screening strains of Aspergillus oryzae, comprising:

Claims

1. Strain of Aspergillus oryzae ZA205, deposited at Guangdong Provincial Microbial Culture Collection Center, Building 59, Dayuan, No. 100, Xianlie Middle Road, Guangzhou, China, on May 18, 2021, under accession number GDMCC NO:61669.

2. Use of the strain of Aspergillus oryzae ZA205 according to claim 1 in koji production.

3. The use according to claim 2, wherein the koji obtained by the koji production is used for the production of soy sauce.

4. Use of the strain of Aspergillus oryzae ZA205 according to claim 1 in the production of fermented foods.

5. The use according to claim 4, wherein the fermented food is a fermented food obtained by fermenting raw materials containing beans and / or grains.

6. The use according to claim 5, wherein the fermented food is a paste or soy sauce.

7. 2. Koji produced from the strain of Aspergillus oryzae ZA205 according to claim 1.

8. The koji according to claim 7, which is obtained by mixing raw materials containing beans and / or grains with the strain of Aspergillus oryzae ZA205 to produce koji.

9. A method for producing koji, comprising inoculating the strain of Aspergillus oryzae ZA205 according to claim 1 onto a protein material and / or a starch material, and culturing the resulting mixture.

10. 1. A method for producing soy sauce or paste, comprising koji-making and fermentation steps, wherein the koji-making step comprises inoculating a proteinaceous material and / or a starchy material with Aspergillus oryzae ZA205, the strain of claim 1, and culturing the resulting material.

11. 11. The method of claim 10, wherein the fermentation is carried out by a method for high salt dilute condition fermentation.

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