JP7775438B2 - Aspergillus oryzae strain ZA205 and its uses - Google Patents

Description

GDMCC GDMCC 61669

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.

Aspergillus oryzae is a core strain in the traditional brewing food industry. During fermentation, Aspergillus oryzae can secrete proteolytic and carbohydrate hydrolytic enzymes. 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 product's flavor.

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

The most widely used strain in the seasoning industry is Aspergillus oryzae Huniang 3.042 (also known as Aspergillus oryzae As3.951). The main characteristics of this strain are fast growth, good resistance to contaminating microorganisms, abundant spore formation, and high activity of neutral protease, the main enzyme secreted during koji production, but a low pH during koji production and low glutaminase activity. The lack of glutaminase is mainly reflected in insufficient secretion and poor salt tolerance.

In the related art, for example, improved strains of Aspergillus oryzae with improved glutaminase activity have been reported. However, while the glutaminase activity has been improved, other properties of the strain have not been significantly improved, and in some cases have even been significantly reduced.

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.

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

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, and is not only 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 soy sauce's pungent flavor. The optimal pH for the environment in which lactic acid bacteria convert citric acid is around 7. Therefore, the more neutral the pH of the koji, the more easily lactic acid bacteria can convert the acidic, pungent citric acid into organic acids, such as lactic acid, which has a mellow flavor, further enhancing the flavor of soy sauce.

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 pH of the koji;
(4) Higher neutral protease activity.

The Aspergillus oryzae of the present invention simultaneously possesses several of the beneficial properties described above, 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.

Aspergillus oryzae ZA205 was obtained through mutagenesis and selective breeding of 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 or raising the pH of the medium; phenol red detects changes in pH, and the more alkaline the pH, the darker the phenol red color 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 phenol red color change, salt-tolerant strains that produce high amounts of glutaminase can be selected.

In some aspects, the present disclosure provides 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.

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

In some embodiments, the koji obtained by koji production for the above-mentioned uses is used to produce soy sauce.

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

In some embodiments, the fermented food is obtained by fermenting raw materials including beans and/or grains.

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

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

In some embodiments, the koji is obtained by mixing a proteinaceous material and/or a starchy material with Aspergillus oryzae ZA205 and producing the koji.

In some embodiments, the protein material comprises one or more selected from the group consisting of soybeans, black beans, broad beans, and the like. 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, and the like. In some embodiments, the starchy material comprises one or more selected from the group consisting of wheat, wheat flour, wheat bran, and the like.

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

In some embodiments, the fermented food is obtained by fermenting raw materials including beans and/or grains.

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

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

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-mentioned Aspergillus oryzae ZA205 on raw materials including beans and/or grains.

In some embodiments, the fermentation is carried out by a method for high-salt dilute-state fermentation.

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.

In some embodiments, the nutrients necessary 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 necessary for the growth of the strain, and combinations thereof.

In some embodiments, the L-glutamine content is between 0.5% and 2%.

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

In some embodiments, the NaCl content is 2% to 5%.

In some aspects, the present disclosure provides:
- inoculating the medium according to claim 10 with a plurality of strains of Aspergillus oryzae;
- comparing the degree and/or speed of discoloration of the medium adjacent to multiple Aspergillus oryzae strains;
- A method for screening Aspergillus oryzae, which comprises selecting an Aspergillus oryzae strain based on the comparison results, is provided.

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 covered with Aspergillus oryzae, which contains various enzymes that greatly accelerate the fermentation and maturation process of the diluted mash.

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

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

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

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

Unless otherwise specified, % refers to % by weight.

In any embodiment, "comprising," "including," or "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, or such as 100%. When the content is 100%, "comprising," "including," or "containing" is equivalent to "consisting of."

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 pH of the koji;
(4) Higher neutral protease activity.

Description of the Deposit of Biological Materials 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.

FIG. 1 shows the colony characteristics of Aspergillus oryzae ZA205 after culturing it in a soy milk medium for 96 hours. 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. 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. 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.

The following describes aspects of the present invention 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 construed as limiting 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 reagents and equipment used are not listed, all are commercially available conventional products.

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

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

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

In the following examples, glutaminase activity was measured as follows: at 40°C and pH 7.2, glutaminase in 1 g of soy sauce koji catalyzed the decomposition of L-glutamine to produce 1 μmol of L-glutamic acid per minute, 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.

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 known as amino nitrogen or AAN) and total acid were measured using the potentiometric titration method in accordance with GB/T 5009.39-2003, Method for analysis of hygienic standard of soybean sauce.

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

The mutation breeding method for the Aspergillus oryzae ZA205 strain of the present invention was as follows:
1.1 Initial Screening and Rescreening Mutations were induced using Aspergillus oryzae As3.951 as the starting strain using ambient pressure room temperature plasma (ARTP). The mutagenesis conditions were as follows: mutagenesis power: 120 W, aeration: 10 L/min, and mutagenesis time: 120 s. The mutagenesis spores were uniformly spread on a high-salt phenol red color-developing medium (10 g L-glutamine, 5 g yeast extract, 1 _{g K2HPO4} , 0.1 _{g KH2PO4} , 0.5 g MgSO4, _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 minutes). After 2-3 days of cultivation, colonies that grew rapidly, discolored quickly, and were dark in color were selected as preliminary screening strains. The preliminary screening strains were inoculated onto phenol red color-developing plates for secondary screening, yielding six secondary screening strains, i.e., GM191, GM192, GM193, GM194, GM195, and ZA205. These secondary screening strains were 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.

1.2 Enzyme activity analysis of koji in Erlenmeyer flask 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 an Erlenmeyer flask. One spore ring of the Aspergillus oryzae strain described in 1.1 was collected and inoculated into the fermentation medium in an Erlenmeyer flask. The mixture was cultured at 32 °C for 96 hours to prepare koji in an Erlenmeyer flask.

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

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

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, and poured into an Erlenmeyer flask. The mixture was sterilized at 121°C for 30 minutes to prepare a 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.

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 obtained enzyme extract to achieve NaCl concentrations of 0%, 5%, 10%, and 17%, and the extract was allowed to stand at 4°C for 0, 1, 3, 5, and 8 hours, after which 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 for strains ZA205, As3.951, and GM195 in NaCl solutions of various concentrations, respectively.

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

1.4 Small-scale koji production and fermentation experiments Aspergillus oryzae ZA205, GM195, and As3.951 were successively inoculated onto slant culture media to activate them, and then expanded in Erlenmeyer flasks. Small-scale koji production and fermentation experiments were then carried out (koji production media were prepared from raw materials (e.g., soybeans, wheat, etc.), and the ratios of raw materials, water for humidification, and preparation and fermentation parameters were all based on Soy Sauce Science and Brewing Technology).

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.

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.

As these results show, the physical and chemical indicators of small-scale koji made with ZA205 were significantly better than those made with As3.951, with the pH in particular being closer to neutral. The physical and chemical indicators of crude soy sauce made with ZA205 were significantly better than those made with As3.951 and GM195.

1.5 Detection of aflatoxin The novel strain ZA205 obtained by screening was assayed for aflatoxin. No aflatoxin was detected.

1.6 Passage experiment A 10-generation passaging experiment was also conducted for ZA205, and the passed strain was verified in a small-scale soy sauce koji production experiment. The results showed that the indicators for fermentation amino nitrogen and glutamic acid were stable, indicating the stable genetic performance of ZA205.

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 koji production scale was 20 tons, and the crude soy sauce produced was 30 tons.

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

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

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.

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, it had a high lactic acid content and a low citric acid content.

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.

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] Aspergillus oryzae ZA205 strain, 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 according to [1] in koji production.
[3] The use according to [2], wherein the koji obtained by the koji production is used for producing soy sauce.
[4] Use of the Aspergillus oryzae ZA205 strain according to [1] in the production of a fermented food,
Preferably, the fermented food is a fermented food obtained by fermenting a raw material containing beans and/or grains;
Preferably, the fermented food is a paste or soy sauce.
[5] Koji produced from the Aspergillus oryzae ZA205 strain according to [1],
Preferably, the koji is obtained by mixing a raw material 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 according to [1] onto a protein material and/or a starch material and culturing the strain.
[8] A method for producing soy sauce or paste, comprising koji-making and fermentation steps, wherein the koji-making step comprises inoculating a protein material and/or a starchy material with Aspergillus oryzae ZA205, which is the strain described in [1], and culturing the resulting mixture.
[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 according to [10] with an 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 (11)

Aspergillus oryzae ZA205 strain, 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. Use of the Aspergillus oryzae ZA205 strain described in claim 1 in koji production. The use according to claim 2, wherein the koji obtained by the koji production is used to produce soy sauce. Use of the Aspergillus oryzae ZA205 strain described in claim 1 in the production of fermented foods. The use according to claim 4, wherein the fermented food is obtained by fermenting raw materials containing beans and/or grains. The use according to claim 5, wherein the fermented food is a paste or soy sauce. Koji produced from the Aspergillus oryzae ZA205 strain described in claim 1. The koji according to claim 7, which is obtained by mixing raw materials containing beans and/or grains with the Aspergillus oryzae ZA205 strain to produce koji. A method for producing koji, comprising inoculating a proteinaceous material and/or a starchy material with the Aspergillus oryzae ZA205 strain described in claim 1 and culturing the resulting mixture. A method for producing soy sauce or paste, comprising koji-making and fermentation steps, wherein the koji-making process comprises inoculating a proteinaceous material and/or a starchy material with Aspergillus oryzae ZA205, a strain of the fungus described in claim 1, and culturing the strain. 11. The method of claim 10 , wherein the fermentation is carried out by a method for high salt dilute condition fermentation.