Aspergillus oryzae ZA307 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to an Aspergillus oryzae ZA307 strain and application of the Aspergillus oryzae ZA307 strain. The aspergillus oryzae ZA307 high-yield protease and aminopeptidase provided by the invention cooperate with aspergillus oryzae Huniang 3.042 for starter propagation, so that the activity of the protease, aminopeptidase, saccharifying enzyme and amylase of a starter material can be remarkably improved, and furthermore, the content of ammonia nitrogen and total nitrogen and the variety and content of volatile flavor substances in a product are remarkably improved. The soy sauce or soybean paste prepared and fermented by double-strain starter propagation is obviously improved in the aspects of sauce fragrance, ester fragrance, delicate flavor and harmonious taste, and the quality of the product is comprehensively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an Aspergillus oryzae ZA307 and applications thereof. Background Art

[0002] Soy sauce is a traditional fermented condiment made from soybeans, flour, or wheat. Made through koji production with Aspergillus oryzae and fermented in a brine system, it boasts a rich, sweet, and intensely flavorful sauce aroma, making it a popular choice among consumers. Traditional soy sauce production often utilizes a single strain of Aspergillus oryzae for koji production, resulting in a single enzyme source, insufficient enzyme diversity, and limited raw material conversion efficiency. Different strains of Aspergillus oryzae typically have varying enzyme profiles. Scientifically combining these components can enhance the enzyme richness of the koji, resulting in a more flavorful soy sauce.

[0003] In recent years, there have also been studies on improving the quality of brewed soy sauce by making koji with two or more strains. For example, patent CN113801795A discloses a process for making koji with multiple strains for soy sauce brewing. Aspergillus oryzae, Aspergillus niger and Aspergillus 7801 are made into koji separately and then mixed and fermented. The amino acid nitrogen is greatly improved. However, although the mode of making koji independently and then mixed fermenting avoids the antagonism of strains, it fails to achieve effective synergy of the enzyme system. Patent CN106616856A proposes a phased inoculation strategy, introducing Aspergillus oryzae and Monascus purpureus at the early stage of koji making, and supplementing Aspergillus niger liquid in the loosening stage to form a dynamic enzyme system synergy. However, in actual production, there are problems such as uneven strain distribution and complex equipment operation.

[0004] While composite microbial agents can theoretically improve the quality of soy sauce koji, practical research and application face challenges such as biological antagonism, weak enzyme complementarity, cumbersome processes, and increased manufacturing costs. Therefore, breeding high-quality Aspergillus oryzae strains that can overcome competitive inhibition and compensate for the limited raw material conversion efficiency of existing Aspergillus oryzae strains, and establishing a composite microbial agent koji production method that achieves efficient and synergistic enzyme enhancement, are crucial for improving the flavor quality of soy sauce. Summary of the Invention

[0005] Based on the above technical problems, the main purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide an Aspergillus oryzae strain ZA 307, which has a high yield of protease and aminopeptidase. When used in synergistic koji making with Aspergillus oryzae Shanghai Niang 3.042, it can significantly improve the enzyme activity of the prepared koji material, thereby significantly increasing the ammonia nitrogen and total nitrogen content and the types and contents of volatile flavor substances in the product, thereby achieving an overall improvement in product quality.

[0006] To achieve the above objectives, the inventors conducted in-depth research and, after repeated research and demonstration, completed the present invention, which is as follows:

[0007] In a first aspect, the present invention provides an Aspergillus oryzae ZA307, the deposit number of which is GDMCC No: 65504.

[0008] The strain was deposited in the Guangdong Microbial Culture Collection Center on November 19, 2024, at the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0009] In a second aspect, the present invention provides a fermentation agent, which comprises the above-mentioned Aspergillus oryzae ZA 307, and preferably further comprises Aspergillus oryzae Shanghai Niang 3.042.

[0010] In a third aspect, the present invention provides an application of the above-mentioned fermentation agent in fermented food processing, preferably a fermented condiment. The condiment mainly refers to a fermented condiment that produces flavor by fermentation using microorganisms and their enzymes, such as soy sauce, sauce, vinegar, fermented black beans or fermented bean curd, preferably soy sauce or sauce.

[0011] In a fourth aspect, the present invention provides a method for preparing soy sauce or paste, comprising the step of fermenting using the above-mentioned fermentation agent, preferably the fermentation agent is used in the koji-making step.

[0012] The preparation method can be carried out using a fermentation method commonly used in the art, such as high-salt dilute fermentation, high-salt dilute natural sun-dried fermentation, low-salt solid-state fermentation, and low-salt heat preservation fermentation. The process parameters of the fermentation method can be adjusted according to actual production conditions.

[0013] Furthermore, the added amount of the fermentation agent is 0.1% spore powder of the koji weight.

[0014] Furthermore, the ratio of Aspergillus oryzae ZA 307 and Aspergillus oryzae Shanghai Niang 3.042 in the fermentation bacteria agent is 3:1-1:1, preferably 3:1.

[0015] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0016] 1. The Aspergillus oryzae ZA 307 strain provided in the present application was obtained by artificially inducing mutagenesis of the Shanghai Niang 3.042 strain using the ARPT mutagenesis technique and then screening; compared with Shanghai Niang 3.042, the aminopeptidase activity in the fermentation koji of Aspergillus oryzae ZA 307 reached a maximum of 1563.92±31.02 U / g, an increase of 16.03% over Shanghai Niang 3.042, and the total protease activity reached 3720.18 U / g, an increase of 17.82% over Shanghai Niang 3.042, with a significant improvement effect.

[0017] 2. The Aspergillus oryzae ZA 307 strain provided by the present invention, when mixed with the Shanghai Niang 3.042 strain to make koji, can significantly improve the enzyme activity of the prepared koji. Compared with Shanghai Niang 3.042, the protease activity of the prepared koji is increased by 28%, the aminopeptidase activity is increased by 71%, the saccharification enzyme activity is increased by 23%, and the amylase activity is increased by 13%.

[0018] 3. The composite fermentation agent provided by the present invention is used for soy sauce fermentation. Compared with Shanghai Brewing 3.042, the total acid content of the fermented crude oil is increased by 6%, the amino acid nitrogen content is increased by 16%, and the umami amino acid and sweet amino acid contents are increased by 14% and 17%, respectively. The content of volatile flavor compounds in the fermented crude oil is significantly increased. Among the volatile compounds related to the aroma of soy sauce, the content of alcohols is increased by 233%, aldehydes by 23%, esters by 109%, phenols by 380%, and aromatic heterocyclic compounds by 57%. The total content of volatile flavor compounds is increased by 96%. Among them, pleasant higher alcohols such as 1-octene-3- The content of 2-methyl-1-propanol, 3-octanol increased by 48%-305% compared with Shanghai Brewing 3.042, the content of 2-methyl-butyraldehyde with malt aroma increased by 63%, the content of ethyl acetate and ethyl lactate that can give crude oil a sweet floral and fruity aroma increased by 81%-445%, the content of maltol and guaiacol with smoky aroma increased by 42%-1678%, the content of aromatic heterocyclic compounds such as furans with caramel aroma and pyrazines with nutty and baked aroma increased by 6%-127%; the fermented soy sauce has been significantly improved in the aroma of soy sauce, ester aroma, umami taste and harmonious taste, which comprehensively improves the quality of soy sauce.

[0019] 5. The composite fermentation agent provided by the present invention is used for soybean paste fermentation. Compared with Shanghai Brewing 3.042, the amino acid nitrogen content is increased by 15%, the umami amino acid content and the sweet amino acid content are increased by 15% and 14% respectively; the content of volatile flavor substances is significantly increased. Among the volatile compounds related to the aroma of soybean paste, the alcohol content is increased by 142%, the aldehyde content is increased by 7%, the ester content is increased by 189%, the phenol content is increased by 271%, and the aromatic heterocyclic compound content is increased by 23%. In addition, the overall volatile flavor substance content is increased by 86%. %; Among them, the content of higher alcohols that can bring people pleasure, such as 1-octen-3-ol, 2-methyl-1-butanol, and 3-methyl-1-butanol, increased by 23%-204% compared with Shanghai Brewing 3.042; ethyl acetate and ethyl propionate that can impart sweet floral and fruity aroma increased by 90%-481%; maltol and guaiacol with smoky aroma increased by 25%-374%; fermented soybean paste is superior to Shanghai Brewing 3.042 in aroma and taste, significantly improving the flavor and overall quality of soybean paste, and showing a more balanced performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The Aspergillus oryzae ZA 307 provided by the present invention was deposited in the Guangdong Microbial Culture Collection Center on November 19, 2024, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the deposit number GDMCC NO: 65504; the strain was received and registered by the collection center on November 19, 2024, and was detected as a viable strain by the collection center on November 19, 2024. DETAILED DESCRIPTION

[0021] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional food-grade reagents, methods and equipment in the art.

[0024] The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0025] The enzyme activity determination method used in the present invention is:

[0026] 1) Acidic protease activity is determined in accordance with the "Determination of the activity of acidic and neutral proteases in feed additives - Spectrophotometric method GB / T 28715-2012". One unit of acidic protease activity (U / g) is defined as the amount by which 1 g of Daqu hydrolyzes casein at 40°C and pH 3.0 to produce 1 μg of tyrosine per minute.

[0027] 2) Neutral protease activity is also tested in accordance with the National Standard of the People's Republic of China: Determination of the Activity of Acidic and Neutral Proteases in Feed Additives - Spectrophotometric Method (GB / T 28715-2012). This means that 1 g of Daqu hydrolyzes casein at 40°C and pH 7.2 to produce 1 μg of tyrosine per minute, which is 1 unit of neutral protease activity (U / g).

[0028] 3) Alkaline protease activity is tested in accordance with the National Standard of the People's Republic of China: Protease Preparations (GB / T23527-2022). This specifies that 1 g of Daqu hydrolyzes casein at 40°C and pH 9.0 to produce 1 μg of tyrosine per minute, corresponding to 1 unit of alkaline protease activity (U / g).

[0029] 4) In the aminopeptidase activity assay, the amount of enzyme required for the composite protease to hydrolyze leucine p-nitroaniline to produce 1 μg of p-nitroaniline (pNA) per minute at 40°C and pH 7.2 is one unit of enzyme activity, expressed in U / g or U / mL.

[0030] 5) Amylase activity assay: The assay method was based on the National Standard of the People's Republic of China: α-amylase preparations (GB / T 24401-2009). One amylase activity unit was defined as the liquefaction of 1 mg of soluble starch per minute at 60°C and pH 6.0.

[0031] 6) Detection method of saccharifying enzyme activity: The determination method refers to the "General Method for Industrial Enzyme Preparations (QB / T 1803-2022)". At 60°C and pH 4.6, 1 mg of glucose is produced per minute by hydrolyzing soluble starch, which is defined as 1 unit of saccharifying enzyme activity.

[0032] 7) Cellulase activity assay: The assay method refers to the National Standard of the People's Republic of China: Determination of Feed Cellulase Activity - Filter Paper Method (GB / T 23881-2009). One unit of enzyme activity is defined as the amount of enzyme required to hydrolyze carboxymethyl cellulose (CMC) to produce 1 mg of glucose per minute at 50°C and pH 4.8.

[0033] Example 1 Screening of target strains

[0034] 1. ARTP mutagenesis of Aspergillus oryzae Shanghai Brewing 3.042

[0035] Aspergillus oryzae Shanghai Niang 3.042 was inoculated onto YPD plate medium using the spot seeding method and cultured at 30°C for 48 h until spores matured. 2 mL of physiological saline was added to the medium, and spores were scraped off using an L-shaped spreader stick to suspend the spores and collected in a 2 mL centrifuge tube. The spore suspension was diluted to an appropriate multiple, counted using a hemocytometer, and the spore concentration in the stock solution was diluted to 2 × 10 8 CFU / mL. Prepare sterile 10% (v / v) glycerol and spore suspension at a ratio of 1:1 and mix thoroughly. Pipette 10 μL of the mixture onto a sterile slide and transfer the slide to a sterile platform.

[0036] ARPT mutagenesis conditions: helium was used as the working gas, the distance between the slide and the plasma emitter was 2 mm, the power was 120 W, the gas flow rate was 10 SLM, and the mutagenesis time was 150 s.

[0037] 2. Preliminary screening of bacterial strains

[0038] The induced spore suspension was diluted in series to make the concentrations of 1×10 4 CFU / mL, 1×10 3 CFU / mL and 1×10 2 CFU / mL, 100 μL of each was spread on casein medium and incubated at 30°C for 72 h. The growth of the strains was observed, and the diameter (D) of the mutant strains and the diameter (H) of the corresponding clearing zone of the mutant strains were recorded. Mutant strains with a large H / D ratio were selected for rescreening. The results are recorded in Table 1.

[0039] Casein culture medium: 10 g casein, 3 g beef extract, 2 g disodium hydrogen phosphate, 5 g sodium chloride, 18 g agar, 1000 mL distilled water, heat to dissolve, and sterilize at 121°C for 15 min.

[0040] Table 1 Results of the initial screening of ARTP mutagenesis of Aspergillus oryzae Shanghai Brewing 3.042

[0041]

[0042] As shown in Table 1, a total of 4 mutant strains had H / D ratios higher than that of the control strain Shanghai Niang 3.042 that had not been treated with ARTP. Among them, the colony diameter of the mutant strain 243-6 was smaller than that of Shanghai Niang 3.042, indicating that the colony growth of this mutant strain was not as good as that of the control group; while the colony growth of the other three mutant strains was good. Among them, the colony diameter of strain 243-9 after ARTP mutagenesis was 0.948 cm, the transparent zone diameter reached 2.099 cm, and the H / D ratio reached 2.215, which was significantly higher than that of the control group Shanghai Niang 3.042. Therefore, the mutant strain 243-9 may have higher protease activity, and this strain was selected for rescreening.

[0043] 3. Strain Rescreening

[0044] A secondary screening was conducted using shallow plate koji making to measure the protease and aminopeptidase activities in samples after koji making. Spore powder of the mutant strain identified in the initial screening was inoculated into koji making medium (0.1%) and incubated at 30°C for 72 hours. 1.0 g of solid koji material was then extracted with phosphate buffer (pH 7.2) for 1 hour. The crude enzyme solution was filtered and assayed for enzyme activity. The results are recorded in Table 2.

[0045] Koji making medium: 16 g bran, 4 g soybean meal, 16 mL distilled water, sterilized at 121°C for 15 min.

[0046] Table 2 Results of rescreening of Aspergillus oryzae Shanghai Brewing 3.042 ARTP mutagenesis strain

[0047]

[0048] At the end of shallow-plate koji making of mutant strain 243-9, the aminopeptidase activity in the koji reached a maximum of 1563.92±31.02U / g, an increase of 16.03% compared with Shanghai Brewing 3.042; the total protease activity reached 3720.18 U / g, an increase of 17.82% compared with Shanghai Brewing 3.042, with a significant improvement effect.

[0049] The strain 243-9 obtained from the above screening was named Aspergillus oryzae ZA 307 and was deposited in Guangdong Provincial Microbiological Culture Collection on November 19, 2024, with the deposit number GDMCC NO: 65504.

[0050] Example 2 Dual strain combined koji making effect test

[0051] Soybeans and wheat were used as raw materials. Wheat was roasted and soybeans were steamed and then cooled to obtain the koji-making raw materials. Then, spore powder of Aspergillus oryzae Shanghai Niang 3.042, Aspergillus oryzae ZA 307 and three different mixing ratios (Aspergillus oryzae ZA 307: Shanghai Niang 3.042 were 1:3, 1:1 and 3:1 respectively) were added to the koji-making raw materials and mixed evenly. The spore powder addition amount was 1%.

[0052] The initial culture temperature for making koji is 32℃-33℃. After 11-12 hours of culture, the first loosening of the koji is carried out after a large amount of koji materials used for making koji are agglomerated. After the first loosening of the koji, the culture temperature is kept at 30℃, and water is sprayed every 2 hours. The second loosening of the koji is carried out at 17-18 hours according to the agglomeration of the koji materials. After loosening of the koji, the culture temperature is kept at 26-28℃, and the third loosening of the koji is carried out at 24-26 hours. After that, the culture temperature is kept unchanged. The koji is taken out after 48 hours of making koji to obtain the finished koji.

[0053] Five types of koji prepared with Aspergillus oryzae Shanghai Niang 3.042, Aspergillus oryzae ZA 307, and a dual-strain composite inoculum (Aspergillus oryzae ZA 307: Shanghai Niang 3.042 ratios of 1:3, 1:1, and 3:1, respectively) were measured for enzyme activities and spore counts. The results are recorded in Table 3.

[0054] Table 3 Comparison of enzyme activity and spore count of mature koji

[0055]

[0056] The results are shown in Table 3. Although the protease and aminopeptidase activities of the koji produced by Aspergillus oryzae ZA 307 were significantly improved compared to those of Shanghai Niang 3.042, the amylase activity and spore count were far lower than those of Shanghai Niang 3.042. The koji produced by the dual-strain mixture with a 3:1 ratio showed a 28% increase in protease activity, a 71% increase in aminopeptidase activity, a 23% increase in saccharification activity, and a 13% increase in amylase activity compared to Shanghai Niang 3.042. The differences in cellulase activity and spore count were not significant compared to Shanghai Niang 3.042. The koji performance was outstanding across all koji parameters and was superior to koji produced by the other two strain combinations. Therefore, the dual-strain mixture with a 3:1 ratio of Aspergillus oryzae ZA 307:Shanghai Niang 3.042 performed best and had the best enzyme synergy.

[0057] Example 3 Application of dual-strain combined koji production in soy sauce fermentation

[0058] The three kojis prepared from Aspergillus oryzae Shanghai Niang 3.042, Aspergillus oryzae ZA 307, and a dual-species composite inoculant (Aspergillus oryzae ZA307: Shanghai Niang 3.042 in a ratio of 3:1) in Example 2 were mixed in a mass ratio of 1:2 between the koji and the brine, with a brine concentration of 18%. The mixtures were fermented in an incubator at 32°C for 60 days. After the fermentation was completed, the crude oil was filtered through a filter cloth to obtain the crude oil.

[0059] The amino acid nitrogen, total acid, total nitrogen, and free amino acid contents of the three crude oils were measured, and the volatile flavor compound contents in the crude oils were measured using the GC-MS method. The results are recorded in Tables 4-6.

[0060] Table 4 Comparison of physical and chemical indicators of fermented crude oil

[0061]

[0062] As shown in Table 4, the crude oil fermented with Aspergillus oryzae ZA 307 had a 3% increase in amino acid nitrogen content compared to Shanghai Niang 3.042, while the total acid and total nitrogen contents were slightly insufficient, showing no advantage. The crude oil fermented with a dual-strain mixed koji had a 6% increase in total acid, a 16% increase in amino acid nitrogen, a 10% increase in total nitrogen content, and a 14% and 17% increase in umami amino acids and sweet amino acids, respectively, compared to Shanghai Niang 3.042. This indicates that the crude oil prepared by this method has significant advantages in quality, especially the significant increase in ammonia nitrogen content and umami and sweet amino acid content, which can bring a rich and sweet taste.

[0063] Table 5 Comparison of types and contents of volatile flavor compounds

[0064]

[0065] As shown in Table 5, the content of volatile flavor compounds in the soy sauce crude oil produced by the dual-strain mixture was significantly improved compared with that of Shanghai Brewing 3.042. Among the volatile compound types related to the aroma of soy sauce, the content of alcohols increased by 233%, aldehydes increased by 23%, esters increased by 109%, phenols increased by 380%, and aromatic heterocyclic compounds increased by 57%. In addition, the overall content of volatile flavor compounds increased by 96%, indicating that dual-strain mixed fermentation can significantly increase the content of volatile flavor compounds in soy sauce.

[0066] Table 6 Comparison of the contents of some volatile flavor compounds

[0067]

[0068] As shown in Table 6, the dual-strain mixed fermentation of soy sauce can enhance the richness and diversity of soy sauce aroma compared with Shanghai Brewing 3.042. Among them, the content of higher alcohols that can bring pleasure, such as 1-octen-3-ol, 2-methyl-1-propanol, and 3-octanol, increased by 48%-305% compared with Shanghai Brewing 3.042; 2-methyl-butyraldehyde with malt aroma increased by 63%; ethyl acetate and ethyl lactate, which can give crude oil a sweet floral and fruity aroma, increased by 81%-445%; maltol and guaiacol with smoky aroma increased by 42%-1678%, and 4-ethylguaiacol increased by about 16 times; at the same time, aromatic heterocyclic compounds such as furans with caramel aroma and pyrazines with nutty and baked aroma increased by 6%-127%.

[0069] Test Example 1: Soy Sauce Sensory Evaluation

[0070] 15 professional R&D personnel who had undergone sensory training were selected to conduct a sensory evaluation of the soy sauce prepared in Example 3, and the intensity of the soy sauce aroma, ester aroma, umami flavor, and rich taste was scored on a scale of 1-5, where the scores from 1 to 5 represented weak (1 point), relatively weak (2 points), medium (3 points), relatively strong (4 points), and strong (5 points) taste sensory levels, respectively. The arithmetic mean of the scores of the 15 professional R&D personnel was taken for comparison, and the results are recorded in Table 7.

[0071] Table 7 Sensory evaluation of fermented crude oil

[0072]

[0073] The results of the sensory evaluation experiment show that the soy sauce fermented with dual strains is superior to Shanghai Brewing 3.042 in terms of sauce aroma, ester aroma, umami taste and richness. It can significantly improve the flavor and overall quality of soy sauce and has the potential for application in soy sauce production.

[0074] In summary, the combined koji production with Aspergillus oryzae ZA 307 and Shanghai Niang 3.042 can significantly enhance the enzyme activity of the koji, thereby significantly increasing the contents of ammonia nitrogen, total nitrogen, umami-sweet amino acids, and the types and contents of volatile flavor substances in soy sauce. In particular, the contents of 2-methyl-1-propanol, ethyl lactate, ethyl phenylacetate, 4-ethyl-guaiacol, thymol, 2,5-dimethylfuran, and 2-pentylfuran are significantly increased. The increase of these compounds can significantly enhance the flavor, taste, and aroma coordination of soy sauce, and significantly improve its sauce aroma, ester aroma, umami flavor, and richness, thereby achieving an overall improvement in the quality of soy sauce.

[0075] Example 4 Application of dual-strain combined koji production in fermented soybean paste

[0076] The three kinds of koji prepared by Aspergillus oryzae Shanghai Niang 3.042, Aspergillus oryzae ZA 307 and a dual-species composite inoculant (Aspergillus oryzae ZA307: Shanghai Niang 3.042 is 3:1) in Example 2 were mixed according to a mass ratio of 1:1.5 between the koji and the brine. The concentration of the brine was 18%, and the mixture was fermented in an incubator at 30°C for 60 days. After the fermentation, the amino acid nitrogen, total acid, total nitrogen and free amino acids of the three kinds of mash were measured, and the content of volatile flavor substances in the soybean paste was measured by GC-MS. The results are recorded in Tables 8-10.

[0077] Table 8 Comparison of physical and chemical indicators of fermented soybean paste

[0078]

[0079] As shown in Table 8, the soybean paste fermented with a double-strain mixed koji had a 3% increase in total acid, a 15% increase in amino acid nitrogen content, a 2% increase in total nitrogen content, and a 15% and 14% increase in umami amino acid content and sweet amino acid content, respectively, compared with Shanghai Brewing 3.042, indicating that the soybean paste prepared by it had better quality.

[0080] Table 9 Comparison of types and contents of volatile flavor compounds

[0081]

[0082] As shown in Table 9, the soybean paste made by the dual-strain mixture has a significantly higher content of volatile flavor substances than Shanghai Brewing 3.042. Among the volatile compound types related to the aroma of soybean paste, the content of alcohols increased by 142%, aldehydes increased by 7%, esters increased by 189%, phenols increased by 271%, and aromatic heterocyclic compounds increased by 23%. In addition, the overall content of volatile flavor substances increased by 86%, indicating that dual-strain mixed fermentation can significantly increase the content of volatile flavor substances in soybean paste.

[0083] Table 10 Comparison of the contents of some volatile flavor compounds

[0084]

[0085] As shown in Table 10, the dual-strain mixed fermentation of soybean paste can enhance the richness and diversity of soybean paste aroma compared with Shanghai Brewing 3.042. Among them, the content of higher alcohols that can bring pleasure, such as 1-octen-3-ol, 2-methyl-1-butanol, and 3-methyl-1-butanol, increased by 23%-204% compared with Shanghai Brewing 3.042; ethyl acetate and ethyl propionate that can impart sweet floral and fruity aroma increased by 90%-481%; maltol and guaiacol with smoky aroma increased by 25%-374%.

[0086] Test Example 2: Sensory Evaluation of Soybean Paste

[0087] 15 professional R&D personnel who have undergone sensory training were selected to conduct a sensory evaluation of the soybean paste prepared in Example 4, and the intensity of the soybean paste aroma, color and taste was scored on a scale of 1-5. The scoring criteria are as follows:

[0088] Aroma identification standards: significant aroma defects (1 point); obvious koji flavor, no sauce flavor (2 points); slightly weak aroma / slightly koji flavor / slightly other flavors (3 points); strong sauce flavor, no foreign smell (4 points); strong sauce flavor, rich aroma (5 points).

[0089] Color identification standards: beans are obviously dark and dull (1 point); beans are slightly dark and dull (2 points); beans are slightly reddish brown, slightly shiny, and partially dark (3 points); beans are relatively brownish red, shiny, and not dark (4 points); beans are brownish red as a whole, with obvious luster and no dark (5 points);

[0090] Taste identification standards: taste too salty / has an unusual smell / obviously inharmonious (1 point); taste slightly sour or salty / inharmonious (2 points); taste slightly sour or salty, slightly harmonious (3 points); taste with a sauce flavor, sweet and fresh taste, and a strong feeling (4 points); taste with a sauce flavor, prominent sweet and fresh taste, and a strong feeling (5 points).

[0091] The arithmetic mean of the scores of 15 professional R&D personnel was taken for comparison, and the results are recorded in Table 11.

[0092] Table 11 Sensory evaluation of fermented soybean paste

[0093]

[0094] The results of the sensory evaluation experiment show that the soybean paste fermented with double strains is superior to Shanghai Brewing 3.042 in aroma and taste, and is very close to Shanghai Brewing 3.042 in color. It can significantly improve the flavor and overall quality of soybean paste, and perform more balanced.

[0095] In summary, the combined koji production of Aspergillus oryzae ZA 307 and Shanghai Brewing 3.042 can significantly enhance the enzyme activity of the koji, thereby significantly increasing the ammonia nitrogen content, umami-sweet amino acid content, and the types and contents of volatile flavor compounds in soybean paste. In particular, the contents of 2,3-butanediol, ethyl propionate, ethyl phenylacetate, 4-vinyl-guaiacol, and thymol are significantly increased. The increase of these compounds can significantly enhance the flavor, mouthfeel, and aroma coordination of soy sauce, giving it a prominent aroma layering and rich ester aroma, while also containing a distinct and rich spicy and smoky flavor, and significantly enhancing the umami and body, thereby comprehensively improving the quality of soybean paste.

[0096] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A strain of Aspergillus oryzae ( Aspergillus oryzae ) ZA 307, characterized in that, The deposit number of the Aspergillus oryzae ZA 307 is GDMCC No: 65504.

2. A fermentation agent, characterized in that: The fermentation bacteria agent comprises the Aspergillus oryzae ZA 307 according to claim 1.

3. The fermentation agent according to claim 2, wherein The fermentation agent also includes Aspergillus oryzae Shanghai Niang 3.

042.

4. Use of the fermentation bacteria agent as claimed in claim 3 in fermented seasonings.

5. The use according to claim 4, characterized in that The fermented seasoning is soy sauce or paste.

6. A method for preparing soy sauce or paste, characterized in that: The method comprises the step of fermenting using the fermentation bacteria agent according to claim 3.

7. The preparation method according to claim 6, wherein The fermentation bacteria agent is used in the koji making step.

8. The preparation method according to claim 6, wherein The added amount of the fermentation agent is 0.1% of the spore powder by weight of the koji material.

9. The preparation method according to claim 6, wherein The ratio of Aspergillus oryzae ZA 307 to Aspergillus oryzae Shanghai Niang 3.042 in the fermentation bacteria agent is 3:1-1:

1.

10. The preparation method according to claim 9, characterized in that The ratio of Aspergillus oryzae ZA 307 and Aspergillus oryzae Shanghai Niang 3.042 in the fermentation bacteria agent is 3:1.

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