Aspergillus oryzae strain ZA205 and application thereof

Abstract

The present application relates to a strain of aspergillus oryzae ZA205 and its application. The aspergillus oryzae ZA205 has the following characteristics: (1) significantly improved glutamine enzyme activity; (2) significantly improved glutamine enzyme salt tolerance; (3) koji pH closer to neutral; (4) higher neutral protease activity.

Description

Technical Field

[0001] This invention relates to the fields of food processing and microbial fermentation technology. Specifically, this invention relates to a strain of Aspergillus oryzae and its application in soy sauce and sauce brewing. Background Technology

[0002] Aspergillus oryzae is a core microbial strain in the traditional fermented food industry. During fermentation, Aspergillus oryzae can secrete proteolytic enzymes and saccharide hydrolases. 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 the source of the product's flavor.

[0003] For soy sauce and other sauce products, umami is a key flavor component. Umami primarily originates from amino acids and polypeptides formed after the enzymatic hydrolysis of soybean and wheat proteins. Glutamic acid is the main amino acid responsible for umami. Soy protein contains approximately 16% glutamic acid, of which about 46% exists in the form of glutamine. Glutamine itself does not possess umami flavor; it only fully exerts its umami function after being converted into glutamic acid by glutaminase.

[0004] The most widely used strain in the condiment industry is *Aspergillus oryzae* Hu Niang 3.042 (also known as *Aspergillus oryzae* As3.951). This strain is characterized by rapid growth, good resistance to contamination, abundant sporulation, and high activity of its main enzyme system secreted during koji production—neutral protease. However, it requires a low koji-making pH and has low glutaminase activity. The deficiencies in glutaminase are mainly reflected in two aspects: insufficient secretion and poor salt tolerance.

[0005] Related technologies document some improvements to Aspergillus oryzae, such as increasing the glutaminase activity of Aspergillus oryzae. However, while the glutaminase activity was increased, other properties of the strain were not significantly improved, or even significantly decreased. Summary of the Invention

[0006] The inventors recognized that salt tolerance is a key factor influencing whether glutaminase can function effectively in high-salt fermented foods. Therefore, improving the salt tolerance of Aspergillus oryzae glutaminase greatly helps to increase the glutamate content in soy sauce and fermented sauces.

[0007] The inventors also recognized that glutaminase can break down glutamine in raw materials into glutamate and ammonia; the more ammonia, the higher the pH of the koji-making process. At the same time, a higher pH of the koji-making process can further promote the secretion of glutaminase, thus there is a strong correlation between glutaminase and the pH of the koji-making process.

[0008] The inventors also recognized that the sourness of soy sauce is primarily due to mild-tasting organic acids. After the lactic acid bacteria proliferate in the fermentation mash, they convert sugars into organic acids, and simultaneously transform the organic acids in the raw materials into other organic acids. Lactic acid is a representative of mild-tasting organic acids; it not only contributes to the flavor of soy sauce, softening its strong saltiness, but also affects its aroma. However, excessive citric acid content is a significant source of the pungent taste in soy sauce. The optimal pH for lactic acid bacteria to convert citric acid is around 7. Therefore, the closer the pH of the fermentation starter is to neutral, the more conducive it is to the conversion of the pungent citric acid into milder lactic acid and other organic acids, further enhancing the flavor of the soy sauce.

[0009] To achieve the above objectives, the present invention provides Aspergillus oryzae ZA205, which simultaneously possesses the following beneficial properties:

[0010] (1) Significantly improved salt tolerance of glutaminase;

[0011] (2) Significantly increased glutaminase activity;

[0012] (3) The pH of the koji material is closer to neutral;

[0013] (4) High neutral protease activity.

[0014] Since the Aspergillus oryzae of the present invention possesses the above-mentioned multiple beneficial properties, these beneficial properties work synergistically, resulting in fermented foods obtained from Aspergillus oryzae exhibiting significantly improved physicochemical indicators and significantly enhanced sensory evaluation.

[0015] Aspergillus oryzae ZA205 was obtained through mutagenesis and selection using Aspergillus oryzae As3.951 (Hu Niang 3.042) as the starting strain. To screen for strains with high pH levels during koji production, high yield, and salt tolerance to glutaminase, L-glutamine was added to the screening medium as a substrate for glutaminase decomposition, phenol red was added as an acid-base indicator, and 4% NaCl was added as a screening sieve for salt tolerance. The decomposition of L-glutamine by glutaminase produces alkaline ammonia, which can increase the pH of the medium. Alternatively, changes in the properties of Aspergillus oryzae itself, leading to an increase in alkaline substances, can also raise the pH of the medium. Phenol red senses pH changes; the more alkaline the pH, the deeper the phenol red color. Synchronously secreted glutaminase exhibits better salt tolerance, and the faster the phenol red color changes. By observing the speed and intensity of the phenol red color change, strains with high yield and salt tolerance to glutaminase can be screened.

[0016] In some respects, this disclosure provides Aspergillus oryzae ZA205, which was deposited on May 18, 2021, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 61669), located at Building 59, No. 100, Xianlie Middle Road, Guangzhou, China.

[0017] In some respects, this disclosure provides the use of Aspergillus oryzae ZA205 in koji making.

[0018] In some implementations, for the above-mentioned uses, the koji obtained from koji making is used to prepare soy sauce.

[0019] In some respects, this disclosure provides the use of Aspergillus oryzae ZA205 in the preparation of fermented foods;

[0020] In some implementations, the fermented food is a fermented food obtained by fermenting raw materials containing beans and / or grains.

[0021] In some implementations, the fermented food is a sauce or soy sauce.

[0022] In some aspects, this disclosure provides a koji material prepared from the aforementioned Aspergillus oryzae ZA205;

[0023] In some embodiments, the koji material is obtained by mixing protein raw materials and / or starch raw materials with Aspergillus oryzae ZA205.

[0024] In some embodiments, the protein raw material includes one or more of the following: soybeans, black beans, broad beans, etc. In one embodiment, the soybean is soybean (scientific name: *Glycine max* (Linn.) Merr.). In another embodiment, the soybean is defatted soybean, soybean meal, etc., or one or more. In some embodiments, the starch raw material includes one or more of the following: wheat, wheat flour, wheat bran, etc.

[0025] In some respects, this disclosure provides a fermented food product prepared from the koji material of any of the above.

[0026] In some implementations, the fermented food is a fermented food obtained by fermenting raw materials containing beans and / or grains.

[0027] In some implementations, the fermented food is a sauce or soy sauce.

[0028] In some aspects, this disclosure provides a method for preparing koji, which includes inoculating the above-mentioned Aspergillus oryzae ZA205 into raw materials containing beans and / or grains and culturing them.

[0029] In some aspects, this disclosure provides a method for preparing soy sauce or sauce, 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 culturing them.

[0030] In some implementations, the fermentation is carried out using a high-salt dilute-state fermentation method.

[0031] In some aspects, this disclosure provides a culture medium for screening strains, comprising:

[0032] L-glutamine;

[0033] Acid-base indicators (e.g., phenol red); and

[0034] NaCl;

[0035] Optionally, the culture medium also contains nutrients required for the growth of the strain.

[0036] In some implementations, the nutrients required for the growth of the strain are selected from, for example, nitrogen sources, carbon sources, phosphorus sources, trace elements required for the growth of the strain, or combinations thereof.

[0037] In some implementations, the percentage of L-glutamine is 0.5%-2%.

[0038] In some implementation schemes, the percentage content of the acid-base indicator is 0.01‰-0.5‰.

[0039] In some implementations, the NaCl content is 2%-5%.

[0040] In some aspects, this disclosure provides a method for screening Aspergillus oryzae, including

[0041] - Inoculate multiple Aspergillus oryzae into the culture medium of claim 10;

[0042] - Compare the degree and / or rate of color change in the culture medium near the multiple Aspergillus oryzae;

[0043] - Screen for Aspergillus oryzae based on comparison results.

[0044] Terminology Explanation:

[0045] Koji refers to the product of culturing bean or cereal raw materials mixed with Aspergillus oryzae for several days. The surface of the bean raw materials is covered with Aspergillus oryzae, which contains various enzymes that greatly promote the fermentation and maturation process of the diluted mash.

[0046] For example, the "high-salt and diluted state fermentation" process can be referenced in the "High-salt and diluted state fermentation process for soy sauce brewing" (SB / T 10312-1999).

[0047] For example, "soy sauce" can be found in "GB 2717-2018 National Food Safety Standard for Soy Sauce" or "GB / T 18186-2000 Brewed Soy Sauce".

[0048] "Sauce" can be either soybean paste or wheat paste, for example, refer to "GB / T24399-2009 Soybean Paste" or SB / T 10296-2009 Sweet Bean Paste.

[0049] "Dry basis": The material is dried in an oven at 105℃ until the moisture content remains constant.

[0050] Unless otherwise specified, % refers to weight%.

[0051] In any implementation, "comprising," "including," or "containing" can mean a content greater than zero, such as 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%. When the content is 100%, "comprising," "including," or "containing" is equivalent to "consisting of."

[0052] Beneficial effects

[0053] One or more of the technical solutions disclosed herein have one or more of the following beneficial effects:

[0054] (1) Significantly improved salt tolerance of glutaminase;

[0055] (2) Significantly increased glutaminase activity;

[0056] (3) The pH of the koji material is closer to neutral;

[0057] (4) High neutral protease activity. Attached Figure Description

[0058] Figure 1 The colony characteristics of Aspergillus oryzae ZA205 after culturing in soybean juice medium for 96 h are shown.

[0059] Figure 2 The curves showing the relative retention rate of glutaminase activity of Aspergillus oryzae ZA205, As3.951 and GM195 in 5% NaCl solution as a function of time are shown.

[0060] Figure 3 The curves showing the relative retention rate of glutaminase activity of Aspergillus oryzae ZA205, As3.951 and GM195 in 10% NaCl solution as a function of time are shown.

[0061] Figure 4 The curves showing the relative retention rate of glutaminase activity of Aspergillus oryzae ZA205, As3.951 and GM195 in 17% NaCl solution over time are shown.

[0062] Instructions on the Preservation of Biological Materials

[0063] This invention relates to the following biological materials that have been deposited at the Guangdong Provincial Microbial Culture Collection Center:

[0064] Aspergillus oryzae ZA205 was deposited on May 18, 2021, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 61669), located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, China. Detailed Implementation

[0065] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Unless otherwise specified, the manufacturers of the pharmaceuticals or instruments used are all commercially available conventional products.

[0066] The following examples use Shanghai Brewing 3.042 (also known as Aspergillus oryzae As3.951) preserved by Haitian Company.

[0067] The L-glutamine used in the following examples was of >99% purity and manufactured by Sigma-Aldrich, USA; the glutamate detection kit was manufactured by R-Biopharm, Germany; and other reagents were all of analytical grade and prepared with deionized water.

[0068] In the following examples, the protease activity was determined according to the method in SB / T 10317-1999.

[0069] In the following examples, the method for determining glutaminase activity is as follows: Under conditions of 40°C and pH 7.2, each unit of enzyme activity is defined as the amount of glutaminase in 1g of soy sauce koji that catalyzes the decomposition of L-glutamine into 1μmol of L-glutamic acid per minute, expressed as U / g. L-glutamic acid was detected using a glutamic acid assay kit from R-Biopharm (Germany).

[0070] In the following examples, total nitrogen was determined in accordance with GB / T 5009.5-2003 Determination of Protein in Food; amino acid nitrogen (abbreviated as amino nitrogen or AAN) and total acid were determined using the potentiometric titration method in the analytical methods of GB / T 5009.39-2003 Hygienic Standard for Soy Sauce.

[0071] Figure 1The image shows a colony photograph of Aspergillus oryzae ZA205 after culturing in soybean juice medium for 96 hours. Its colony characteristics are as follows: colony diameter reaches 58 mm, with abundant hyphae, abundant spores, and a yellow-green color.

[0072] The mutagenesis and selection method of Aspergillus oryzae strain ZA205 of the present invention is as follows:

[0073] 1.1 Primary screening and secondary screening

[0074] Using Aspergillus oryzae As3.951 as the starting strain, mutagenesis was induced by ambient pressure room temperature plasma (ARTP) under the following conditions: mutagenesis power 120W, gas flow rate 10L / min, and mutagenesis time 120s. The mutagenized spores were evenly spread onto a high-salt phenol red coloring medium (10g L-glutamine, 5g yeast extract, 1g K₂HPO₄, 0.1g KH₂PO₄, 0.5g MgSO₄, 0.015g phenol red, 40g NaCl, 20g agar, dissolved in deionized water, and brought to a final volume of 1000mL, then sterilized at 115℃ for 20min). After culturing for 2-3 days, colonies with rapid growth, rapid color change, and deep color were selected as the initial screening strains. The initially screened strains were inoculated onto phenol red agar plates for secondary screening, yielding six secondary-screened strains: GM191, GM192, GM193, GM194, GM195, and ZA205. These secondary-screened strains were then transferred to soybean juice slant agar (1 g KH2PO4, 0.5 g MgSO4, 0.5 g (NH4)2SO4, 20 g soluble starch, 20 g agar, 1000 mL soybean juice, sterilized at 115℃ for 20 min), and cultured until mature before routine preservation.

[0075] 1.2 Enzyme Activity Analysis of Erlenmeyer Flask Koji Material

[0076] A mixture of non-GMO soybeans and wheat (mass ratio: soybean:wheat = 1:1) was prepared by adding 1.2 times the amount of water, mixing thoroughly, dispensing into Erlenmeyer flasks, and sterilizing at 121℃ for 30 min to obtain Erlenmeyer flask fermentation medium. One ring of spores was picked from the Aspergillus oryzae strain in section 1.1 and inoculated into the Erlenmeyer flask fermentation medium. The mixture was then incubated at 32℃ for 96 h to obtain Erlenmeyer flask koji.

[0077] The pH, glutaminase, and neutral protease levels of the Erlenmeyer flask koji were measured, and the results are shown in the table below.

[0078] Table 1. Physicochemical properties of Erlenmeyer flask koji.

[0079]

[0080] As shown above, GM195 and ZA205 Erlenmeyer flask koji have significant advantages in pH value and glutaminase activity, with their pH value being closer to 7 and their glutaminase activity being higher. ZA205 Erlenmeyer flask koji also has higher neutral protease activity.

[0081] 1.3 Analysis of Salt Tolerance of Erlenmeyer Flask Curing Material

[0082] A mixture of non-GMO soybeans and wheat (mass ratio: soybean:wheat = 1:1) was prepared by adding 1.2 times the amount of water, mixing thoroughly, and dispensing into Erlenmeyer flasks. The mixture was then sterilized at 121°C for 30 minutes to obtain the Erlenmeyer flask fermentation medium. One loop of spores was picked from the Aspergillus oryzae slant culture of GM195, ZA205, and As3.951 and inoculated into the Erlenmeyer flask fermentation medium. The mixture was incubated at 32°C for 96 hours to obtain the Erlenmeyer flask starter culture.

[0083] The salt tolerance stability of glutaminase in the koji material was tested. The specific test method is as follows: Take an appropriate amount of koji material and add twice the amount of pH 7.2 phosphate buffer. Soak overnight in a refrigerator at 4℃ to obtain the enzyme extract. Adjust the concentration of the enzyme extract to 0%, 5%, 10%, and 17% using NaCl, and place it in an environment at 4℃ for 0, 1, 3, 5, and 8 hours. The relative residual enzyme activity of glutaminase was measured, and the results are shown in Table 2. Figure 2 , Figure 3 and Figure 4 The salt tolerance of glutaminase activity in different concentrations of NaCl solution for strains ZA205, As3.951 and GM195 is shown.

[0084] Table 2. Relative residual enzyme activities of glutaminase from different strains in NaCl solution

[0085]

[0086] The results showed that, under different NaCl concentrations, the relative residual activity of glutaminase in ZA205 was significantly higher than that in As3.951 and GM195, and the retention time of the strain was also significantly longer than that of As3.951 and GM195. The improvement in the salt tolerance stability of glutaminase in ZA205 reached an unexpected level.

[0087] 1.4 Small-scale koji-making and fermentation trials

[0088] After activating Aspergillus oryzae ZA205, GM195 and As3.951 by sequentially transferring them to slant culture, they were cultured in Erlenmeyer flasks for scale-up. Then, small-scale koji making and fermentation were carried out. (The koji making culture medium was prepared from soybeans, wheat and other raw materials. The proportion of raw materials, watering, cooking and fermentation parameters were all based on "Soy Sauce Science and Brewing Technology").

[0089] A small-scale fermentation test was conducted to prepare 0.4 tons of koji material. The moisture content, pH value, neutral protease, and glutaminase of the koji material were tested. The test results are shown in Table 3.

[0090] A small-scale fermentation experiment was conducted to prepare 0.6 tons of fermented crude oil (soy sauce). The total acid, amino nitrogen, glutamic acid, glucose and other indicators of the fermented crude oil were tested. The test results are shown in Table 4.

[0091] Table 3. Physicochemical indicators of small-scale koji production

[0092]

[0093] *Standardization refers to setting the As3.951 index to 1.00.

[0094] Table 4. Physicochemical properties of fermented crude oil

[0095]

[0096] As shown above, the physicochemical properties of ZA205 in the small-scale koji production were significantly better than those of As3.951, especially the pH value, which was closer to neutral. The physicochemical properties of the fermented crude oil from ZA205 were also significantly better than those of As3.951 and GM195.

[0097] 1.5 Aflatoxin Detection

[0098] The newly obtained strain ZA205 was tested for aflatoxin, and the results showed that aflatoxin was not detected.

[0099] 1.6 Subculture Experiment

[0100] Simultaneously, ZA205 was subjected to 10 generations of subculture experiments, and the subcultured strain was used in a small-scale experiment for soy sauce koji making. The results showed that the fermentation amino nitrogen and glutamic acid levels were stable, indicating that the genetic performance of ZA205 was stable.

[0101] Table 5. Stability verification of ZA205 through passaging

[0102] Algebra Amino nitrogen (g / 100mL) Glutamic acid (g / L) original 1.12 14.0 2nd generation 1.11 13.9 6th generation 1.13 14.6 10th generation 1.12 14.2

[0103] 1.7 Large-scale production

[0104] The new strain ZA205 was simultaneously used in large-scale soy sauce production along with the original strain As3.951. In large-scale production, the koji-making scale was 20 tons, and the crude oil production scale was 30 tons.

[0105] After the koji-making process was completed, samples were taken and tested for the physicochemical indicators of the koji produced on a large scale. The results are shown in Table 6. After fermentation, the physicochemical indicators of the crude oil produced on a large scale were tested. The results are shown in Table 7. After post-treatment such as heat sterilization, the crude oil for soy sauce produced on a large scale was subjected to sensory evaluation. The results are shown in Table 8.

[0106] Table 6. Physicochemical properties of koji produced on a large scale

[0107]

[0108] Table 7. Physicochemical properties of crude oil used for large-scale production of soy sauce

[0109]

[0110] Table 8. Sensory analysis of crude soy sauce produced on a large scale

[0111] strain Color aroma posture Umami Salty sour Overall taste 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

[0112] As shown in Tables 6 to 8, when Aspergillus oryzae ZA205 is used for large-scale production, the physicochemical properties of the starter culture, the physicochemical properties of the soy sauce, and the sensory analysis of the soy sauce are all superior to those of Aspergillus oryzae As3.951.

[0113] As shown in Table 6, the koji prepared by Aspergillus oryzae ZA205 has significantly improved glutaminase activity, a pH closer to 7, and also higher neutral protease activity.

[0114] As shown in Table 7, the soy sauce prepared by Aspergillus oryzae ZA205 has significantly increased amino acid nitrogen content, glutamic acid content, and glucose content; in addition, it has higher lactic acid content and lower citric acid content.

[0115] As shown in Table 8, the soy sauce prepared by Aspergillus oryzae ZA205 has improved color, aroma and umami, and exhibits a significantly improved overall taste.

[0116] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings disclosed, and all such modifications and variations are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Aspergillus oryzae ZA205, this strain was deposited on May 18, 2021 at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC NO: 61669, deposit address: Building 59, No. 100, Xianlie Middle Road, Guangzhou, China.

2. The use of Aspergillus oryzae ZA205 as described in claim 1 in koji making.

3. The use according to claim 2, wherein the koji material obtained from koji making is used to prepare soy sauce.

4. The use of Aspergillus oryzae ZA205 as described in claim 1 in the preparation 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 sauce or soy sauce.

7. A koji material prepared from Aspergillus oryzae ZA205 as described in claim 1.

8. The koji material of claim 7, wherein the koji material is obtained by mixing raw materials containing beans and / or grains with Aspergillus oryzae ZA205 to make koji.

9. A fermented food product prepared from the koji material as described in claim 7 or 8.

10. The fermented food of claim 9, wherein the fermented food is a fermented food obtained by fermenting protein raw materials and / or starch raw materials.

11. The fermented food product of claim 10, wherein the fermented food product is a sauce or soy sauce.

12. A method for preparing koji material, comprising inoculating Aspergillus oryzae ZA205 as described in claim 1 into protein raw materials and / or starch raw materials and culturing them.

13. A method for preparing soy sauce or sauce, comprising the steps of koji making and fermentation, wherein, The koji-making process includes inoculating the Aspergillus oryzae ZA205 of claim 1 into protein raw materials and / or starch raw materials and culturing them.

14. The method of claim 13, wherein, The fermentation was carried out using a high-salt, dilute-state fermentation method.

15. A culture medium for screening bacterial strains, comprising: L-glutamine; Acid-base indicators; and NaCl.

16. The culture medium of claim 15, wherein the culture medium further contains nutrients required for the growth of the strain.

17. The culture medium of claim 15, wherein the acid-base indicator is phenol red.

18. The culture medium according to any one of claims 15-17, characterized in that... One or more of the following: The percentage content of L-glutamine is 0.5%-2%. The percentage content of the acid-base indicator is 0.01‰-0.5‰; The percentage content of NaCl is 2%-5%.

19. A method for screening Aspergillus oryzae, comprising: - Inoculate multiple Aspergillus oryzae into the culture medium according to any one of claims 15-18; - Compare the degree and / or speed of color change in the multiple Aspergillus oryzae culture media; - Screen for Aspergillus oryzae based on comparison results.

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