Aspergillus oryzae AT21 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses an Aspergillus oryzae AT21 strain and application thereof, and the Aspergillus oryzae AT21 provided by the invention realizes comprehensive improvement of indexes such as glutamic acid, amino nitrogen and total nitrogen of crude oil on the premise of not influencing the spore production performance of the strain. Experiments show that compared with the Huniang 3.042 strain, the aspergillus oryzae AT21 strain has no obvious change in spore number in the propagation stage, and the germination rate is increased by 5%; compared with a Huniang 3.042 strain, the Aspergillus oryzae AT21 strain has the advantages that the amino acid nitrogen content of fermented crude oil is increased by 13.5%, the total nitrogen content is increased by 21.5%, the glutamic acid content is increased by 28.6%, the glutaminase activity is increased by 74.9%, and the neutral protease activity is increased by 28.0%; the sense of crude oil, the salty taste and the strong sense are obviously improved, and the comprehensive taste is improved.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular discloses a strain of Aspergillus oryzae AT21 and its applications. Background Technology

[0002] Soy sauce is a condiment with a unique color, aroma, and flavor, made primarily from soybeans (or their products) and wheat (or their products) through microbial fermentation. A crucial step in soy sauce production is the solid-state fermentation process using Aspergillus oryzae to produce the koji (fermentation starter). Aspergillus oryzae plays a key role in the physicochemical properties, flavor, and aroma of soy sauce during production.

[0003] In recent years, soy sauce production has faced the problem of rising raw material prices. Under the same raw material input conditions, how to improve the formation of key flavor substances such as amino acid nitrogen, glutamic acid, and total nitrogen in soy sauce is also the key to improving the cost-effectiveness of soy sauce production. Factors affecting the formation of key flavor substances such as amino acid nitrogen, glutamic acid, and total nitrogen in soy sauce include raw material ratios and production processes. However, the limitations of microbial strains determine the upper limit of process adjustments. Modifying the microbial strains can raise the fermentation level of soy sauce from the source. Superimposing processes on strains with high fermentation levels will better improve the yield of soy sauce flavor substances per unit of raw material.

[0004] Currently, strains that simultaneously improve the total nitrogen, amino nitrogen, and glutamic acid levels in crude oil are relatively few. Among strains that can improve glutamic acid, amino nitrogen, and total nitrogen, publication number CN113308383A discloses a strain of Aspergillus oryzae ZA223 and its application. Compared with Hu Niang 3.042, the highest improvement of Aspergillus oryzae is an 8.6% increase in amino acid nitrogen, a 19.6% increase in total nitrogen, and a 15.9% increase in glutamic acid. However, the strain has problems such as delayed sporulation time, a 25% decrease in sporulation yield compared to Hu Niang 3.042, prolonged pre-cultivation time, and low spore output per unit of inoculum culture machine. Publication number CN113293105A discloses a strain of Aspergillus oryzae ZA173 and its application. Compared with Hu Niang 3.042, the highest improvement of Aspergillus oryzae is a 7.5% increase in amino acid nitrogen, a 12.0% increase in total nitrogen, and an 11.0% increase in glutamic acid, which is insufficient. Summary of the Invention

[0005] Current strains used to improve crude oil quality suffer from limitations in simultaneously increasing glutamic acid, amino nitrogen, and total nitrogen, or only to a limited extent. Furthermore, high-yielding strains often exhibit prolonged sporulation time and decreased sporulation yield. This application aims to overcome these shortcomings by providing an Aspergillus oryzae AT21 strain and its applications. This application provides a leading Aspergillus oryzae strain (Aspergillus oryzae AT21) that achieves a comprehensive improvement in crude oil's glutamic acid, amino nitrogen, and total nitrogen levels without affecting the strain's sporulation performance.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a strain of Aspergillus oryzae AT21, which was deposited on April 30, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66245, classification number Aspergillus oryzae, and address: Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0007] The Aspergillus oryzae AT21 strain provided in this application showed a 13.5% increase in amino acid nitrogen, a 21.5% increase in total nitrogen, a 28.6% increase in glutamic acid, a 74.9% increase in glutaminase activity, and a 28.0% increase in neutral protease activity in the fermented crude oil compared to the Hu Niang 3.042 strain. The crude oil also showed a significant improvement in saltiness and richness, resulting in an overall enhanced taste.

[0008] This application obtained a strain of Aspergillus oryzae AT21 by combining ARTP mutagenesis technology, high-throughput screening technology and traditional plate clear zone screening method.

[0009] In some specific embodiments, the mutagenesis screening of Aspergillus oryzae AT21 includes the following steps: 1. Using strain Hu Niang 3.042 as the starting strain, the known ARTP technology was used to mutagenesis of the starting strain to obtain Aspergillus oryzae AT21. The specific steps included: (1) Take mature Hu Niang 3.042 strain spores to prepare 1×10 8 cells / mL; (2) The mutagenesis conditions for ARTP are 120W power, 10SLM gas volume, and 300s mutagenesis time.

[0010] 2. Strains were screened using a combination of glutamic acid, total nitrogen, and amino nitrogen: (1) High-throughput screening: After the strain is mutated, droplets are taken and fluorescent conversion is used to achieve high-throughput rapid screening of glutamic acid, total nitrogen and amino nitrogen (100 droplets / second rapid screening can be achieved).

[0011] (2) Casein plate re-selection: The strains screened by high throughput were diluted and spread on a known casein detection medium (Coollab MM6051), and incubated at 30°C for 3 days. The diameter of the clear zone and the diameter of the colony were measured, and the K value (diameter of clear zone / colony diameter) was calculated. Strains with large K values ​​were screened.

[0012] The strain exhibited a hydrolysis zone diameter of 7.5 mm, a colony diameter of 3.0 mm, and a K value of 2.5. After preservation, the strain was cultured on CYA medium at 25°C for 7 days, resulting in colonies with a diameter of 65 mm. The sporulation structure was yellowish-green, with white hyphae and a velvety texture. The reverse side of the colonies was yellowish-brown, and exudate was produced; no soluble pigments were generated. When cultured on AFPA medium at 30°C for 48 hours, the reverse side of the colonies turned orange.

[0013] This application also provides the application of the aforementioned Aspergillus oryzae AT21 in food fermentation.

[0014] The Aspergillus oryzae AT21 strain provided in this application can simultaneously and significantly improve indicators such as glutamic acid, amino nitrogen, and total nitrogen in crude oil, solving the problem that current strains used to improve crude oil quality cannot simultaneously improve glutamic acid, amino nitrogen, and total nitrogen, or the improvement is only minimal. Furthermore, the Aspergillus oryzae AT21 strain of this application does not exhibit prolonged sporulation time or decreased sporulation yield.

[0015] Sporulation tests conducted in Erlenmeyer flasks showed that the spore production of Aspergillus oryzae AT21 was close to that of Hu Niang 3.042, with a certain improvement in germination rate.

[0016] Therefore, the Aspergillus oryzae AT21 provided in this application can ferment better to obtain seasonings.

[0017] In a preferred embodiment of the application described in this application, the food is a condiment.

[0018] In a preferred embodiment of the application described in this application, the condiment is soy sauce or sauce.

[0019] This application also provides the application of Aspergillus oryzae AT21 in koji making.

[0020] The Aspergillus oryzae AT21 provided in this application can be used in the koji-making process, which helps to significantly improve the glutamic acid, amino nitrogen, and total nitrogen content of crude oil.

[0021] As a preferred embodiment of the application described in this application, the koji obtained from the koji-making process is used to prepare soy sauce or broth.

[0022] This application also provides the use of the above-mentioned Aspergillus oryzae AT21 in improving glutaminase activity and / or neutral protease activity.

[0023] This application also provides the application of the above-mentioned Aspergillus oryzae AT21 in simultaneously increasing total nitrogen content, amino acid nitrogen content, or glutamic acid content.

[0024] Fermentation experiments showed that, compared with strain 3.042 from Huniang, strain AT21 of Aspergillus oryzae did not show significant changes in spore number during the expansion stage, but the germination rate increased by 5%; glutaminase activity increased by 75% and neutral protease activity increased by 28% during the koji-making stage; and total nitrogen of crude oil increased by 22%, amino acid nitrogen increased by 14%, and glutamic acid content increased by 29% during the fermentation stage, while the oil yield remained unchanged.

[0025] Therefore, compared with strain 3.042 of Huniang, strain AT21 of Aspergillus oryzae has a high input-output ratio. With the same input of materials, it has significantly improved the total nitrogen content, amino acid nitrogen content and glutamic acid content of crude oil per unit, and has a high economic conversion rate.

[0026] This application also provides a method for producing a condiment, using the aforementioned Aspergillus oryzae AT21 as the fermentation strain.

[0027] In a preferred embodiment of the production method of the condiment described in this application, the condiment includes soy sauce or sauce.

[0028] When the above-mentioned Aspergillus oryzae AT21 was applied in production, the number of spores in the expansion stage of Aspergillus oryzae AT21 strain was no different from that of Hu Niang 3.042 strain, but the spore germination rate was increased by 5%; the amino acid nitrogen of fermented crude oil increased by 13.5%, total nitrogen by 21.5%, and glutamic acid by 28.6%.

[0029] Furthermore, the Aspergillus oryzae AT21 strain showed a significant improvement in the saltiness and richness of the crude oil compared to the Hu Niang 3.042 strain, resulting in an overall improved taste.

[0030] Compared with the prior art, this application has the following beneficial effects: This application provides a leading Aspergillus oryzae strain (Aspergillus oryzae AT21) that comprehensively improves the glutamic acid, amino nitrogen, and total nitrogen content of crude oil without affecting the sporulation performance of the strain. Experiments show that compared to the Hu Niang 3.042 strain, the Aspergillus oryzae AT21 strain showed no significant change in spore number during the expansion stage, but a 5% increase in germination rate. The Aspergillus oryzae AT21 strain, compared to the Hu Niang 3.042 strain, increased amino acid nitrogen by 13.5%, total nitrogen by 21.5%, glutamic acid by 28.6%, glutaminase activity by 74.9%, and neutral protease activity by 28.0% in the fermented crude oil. Sensory improvements in the crude oil include a significant enhancement in saltiness and richness, resulting in an overall improved taste. Attached Figure Description

[0031] Figure 1 This image shows the morphological results of Aspergillus oryzae AT21 on CYA medium. Detailed Implementation

[0032] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0033] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified, and the raw materials used in each parallel experiment are the same.

[0034] Example 1: Screening of bacterial strains through mutagenesis 1. Using Hu Niang 3.042 strain as the starting strain, the known ARTP technology was used to induce mutagenesis in the starting strain.

[0035] (1) Take mature Hu Niang 3.042 strain spores to prepare 1×10 8 cells / mL; (2) The mutagenesis conditions for ARTP are 120W power, 10SLM gas volume, and 300s mutagenesis time.

[0036] 2. The strain screening method uses a combination of glutamic acid, total nitrogen, and amino nitrogen.

[0037] (1) High-throughput screening: After the strain is mutated, droplets are taken and fluorescent conversion is used to achieve high-throughput rapid screening of glutamic acid, total nitrogen and amino nitrogen (100 droplets / second rapid screening can be achieved).

[0038] (2) Casein plate re-selection: The strains screened by high throughput were diluted and spread on a known casein detection medium (Coollab MM6051), and incubated at 30°C for 3 days. The diameter of the clear zone and the diameter of the colony were measured, and the K value (diameter of clear zone / colony diameter) was calculated. Strains with large K values ​​were screened.

[0039] (3) The casein plate selection data are shown in Table 1.

[0040] Table 1 The above-mentioned strain was deposited and named Aspergillus oryzae AT21. This strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 30, 2025, with accession number GDMCC No: 66245, classification number Aspergillus oryzae, and deposit address: Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0041] This strain, cultured on CYA medium at 25°C for 7 days, produced colonies with a diameter of 65 mm; sporulation structures were yellowish-green with white hyphae; the texture was velvety; the reverse side of the colonies was yellowish-brown; exudate was produced; no soluble pigments were produced. The results are as follows: Figure 1 As shown.

[0042] When this strain was cultured on AFPA medium at 30°C for 48 hours, the reverse side of the colonies turned orange.

[0043] Example 2: Sporulation and Fermentation Level Test 1. Sporulation test in Erlenmeyer flasks: Aspergillus oryzae AT21 and Hu Niang 3.042 were inoculated into Erlenmeyer flask culture medium and propagated using the conventional Erlenmeyer flask method. The culture medium was prepared using a conventional formula of wheat bran, soybean flour, and water. The medium was incubated at 34℃ for 3 days, with loosening of the mold as per standard Erlenmeyer flask cultivation methods. After maturation, spore count and spore germination rate were measured. The measured indicators are as follows (in Table 2, the level of each indicator for Hu Niang 3.042 strain is 1.00, and the mutagenized strains are converted to the corresponding ratios): Table 2 Based on the data from the front-end expansion culture, the spore production of Aspergillus oryzae AT21 is close to that of Hu Niang 3.042, and the germination rate has been improved to a certain extent.

[0044] 2. Test of koji-making and fermentation levels: Aspergillus oryzae AT21 and Hu Niang 3.042 were inoculated onto malt extract agar slants for activation and cultured at 34℃ for 3 days. After maturation, they were inoculated onto a soy sauce koji culture medium prepared by a wheat-to-soybean ratio of 3:7. A small-scale koji-making process was conducted using the following method: culturing at 40℃ for 13 hours, then releasing the starter; culturing at 32℃ for 20 hours, then releasing the starter; and continuing culturing at 32℃ for 40 hours. The fermentation process used 18°Bé brine, which was sterilized and added to the fermentation flasks. The inner walls were thoroughly rinsed, the flasks were tightly capped, and fermentation was carried out at 30℃ and 70% RH. The koji-making and fermentation conditions were kept completely consistent throughout the small-scale test to obtain crude oil. The indicators of the mature crude oil were determined as follows (Table 3 uses Hu Niang 3.042 strain as the level of each indicator as 1.00, and the mutagenized strains are converted to the corresponding ratios): Table 3 Moisture determination method: Take 20g and dry at 37℃ for 2 days. Measure the weight before and after drying, and calculate according to (formula: moisture = 1 - (weight after drying / weight before drying)).

[0045] The method for determining neutral protease activity is described in SB / T 10317-1999, "Protein Activity Assay".

[0046] The method for determining glutaminase activity is based on GB 5009.295-2023, the national standard for food safety, which specifies the general rules for the validation of chemical analysis methods.

[0047] The total nitrogen, amino acid nitrogen, glutamic acid, and oil yield of crude oil were analyzed, as shown in Table 4.

[0048] Table 4 In summary, compared with strain 3.042 from Hu Niang, strain AT21 of Aspergillus oryzae showed the following: no significant change in spore number during the propagation stage, but a 5% increase in germination rate; a 75% increase in glutaminase activity and a 28% increase in neutral protease activity during the koji-making stage; and a 22% increase in total nitrogen, a 14% increase in amino acid nitrogen, and a 29% increase in glutamic acid content in the crude oil during the fermentation stage, while maintaining the same oil yield.

[0049] Therefore, compared with strain 3.042 of Huniang, strain AT21 of Aspergillus oryzae has a high input-output ratio. With the same input of materials, it has significantly improved the total nitrogen content, amino acid nitrogen content and glutamic acid content of crude oil per unit, and has a high economic conversion rate.

[0050] Example 3: Production Application 1. Aspergillus oryzae AT21 strain and Hu Niang 3.042 strain were cultured under the conditions described in Example 2 to obtain starter culture. The spore count and spore germination rate of the starter culture were measured to determine the optimal level of strain expansion for actual production applications (Table 5 uses Hu Niang 3.042 strain as the base value of each indicator, and the mutagenic strains are converted to corresponding ratios): Table 5 2. Aspergillus oryzae strain AT21 was inoculated with Hu Niang strain 3.042 koji, and the koji-making conditions of Example 2 were used to obtain mature koji. The mature koji from the discs containing Aspergillus oryzae strain AT21 and Hu Niang strain 3.042 was then subjected to high-salt, low-temperature fermentation with 18°Bé brine. The fermentation period was 90 days, taking into account actual weather conditions. After fermentation, the fermented crude oil was obtained by pressing, and the amino acid nitrogen, glutamic acid, and total nitrogen content of the crude oil were analyzed (as shown in Table 6). Table 6 The number of spores in the propagation stage of Aspergillus oryzae strain AT21 was not different from that of strain Hu Niang 3.042, but the spore germination rate was increased by 5%; the amino acid nitrogen of the fermented crude oil increased by 13.5%, total nitrogen by 21.5%, and glutamic acid by 28.6%.

[0051] The obtained crude oil underwent blind sensory evaluation. Twenty professionally trained sensory evaluators scored the samples on a scale of 1-5. The average score of these 20 evaluators was taken; a higher score indicated more outstanding sensory performance. The sensory evaluation scores are shown in Table 7. Table 7 Sensory evaluation results showed that the Aspergillus oryzae AT21 strain had a significantly improved saltiness and richness compared to the Hu Niang 3.042 strain, resulting in an overall improved taste.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A strain of Aspergillus oryzae AT21, characterized in that, The Aspergillus oryzae AT21 was deposited on April 30, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 66245, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

2. The application of Aspergillus oryzae AT21 as described in claim 1 in food fermentation.

3. The application as described in claim 2, characterized in that, The food in question is a condiment.

4. The application as described in claim 3, characterized in that, The condiment is soy sauce or sauce.

5. The application of Aspergillus oryzae AT21 as described in claim 1 in koji making.

6. The application as described in claim 5, characterized in that, The koji obtained from the koji-making process is used to prepare soy sauce or broth.

7. The use of Aspergillus oryzae AT21 as described in claim 1 in enhancing glutaminase activity and / or neutral protease activity.

8. The application of Aspergillus oryzae AT21 as described in claim 1 in simultaneously increasing total nitrogen content, amino acid nitrogen content, or glutamic acid content.

9. A method for producing a condiment, characterized in that, Aspergillus oryzae AT21 as described in claim 1 was used as the fermentation strain.

10. The production method as described in claim 9, characterized in that, The condiments include soy sauce or sauce.

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