A strain of Aspergillus oryzae ZA304 and its application

Abstract

The present invention relates to the field of industrial microbial technology, and specifically discloses a strain of Aspergillus oryzae and its application. The present invention provides a strain of Aspergillus oryzae (Aspergillus oryzae) ZA304, which can produce high-yield protease, and its acidic protease, neutral protease, aminopeptidase, and total protease activity are significantly improved compared to Aspergillus oryzae Shanghai Brew 3.042. The strain can be used to prepare a composite protease preparation, which is used for enzymatic hydrolysis of aquatic products, and can effectively increase the amino nitrogen content in the enzymatic hydrolysis solution, improve the enzymatic hydrolysis efficiency, and improve the utilization rate of raw materials; it can also significantly reduce the content of main fishy substances such as 1-octenyl-3-ol, 1-pentenyl-3-ol, 3-octanone in aquatic products, and at the same time improve the content of aroma active compounds such as aldehydes, esters, and furans, effectively reduce the fishy smell of aquatic product enzymatic hydrolysis solution, and improve the sensory flavor.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an Aspergillus oryzae ZA304 and an application thereof. Background Art

[0002] More than 80% of the organisms on Earth live in water. These aquatic products such as fish, crustaceans, shellfish and seaweed living in the ocean and freshwater are an important source of human food. Aquatic products are rich in high-quality proteins, active lipids, active polysaccharides, vitamins and minerals, and other nutrients necessary for the human body. Aquatic product processing is a key link in achieving high-quality development of fisheries, and plays an important role in ensuring the high-quality and safe supply of aquatic products all year round. In recent years, in the development and utilization of aquatic product processing, the high-value utilization of aquatic products and their by-products through protease enzymolysis technology has become an important direction and a large number of products that can be comprehensively utilized have been developed. However, aquatic products themselves contain fishy substances such as 1-octen-3-ol, 1-penten-3-ol, and 3-octanone, which give aquatic products a unique seafood smell. However, the current development of special enzymes for aquatic products is seriously insufficient. During the hydrolysis process, many commercial enzymes cannot effectively reduce these fishy smells, and may even aggravate this unpleasant odor. In addition, the enzymatic hydrolysis rate is not ideal, which seriously restricts the application of aquatic product hydrolysates.

[0003] The prior art has adopted different technical solutions to solve the above problems. For example, patent CN117770415A proposes to use alkaline protease, papain, Bacillus subtilis neutral protease and deaminase for compound step-by-step enzymolysis, and add edible mushroom enzymolysis liquid and fructose syrup for further reaction after enzymolysis to achieve increased production and deodorization. The enzymolysis process of this method is cumbersome and the addition of edible mushroom enzymolysis liquid will increase the smell of soil. Patent CN118058445A increases production and deodorizes by the following steps: (1) subjecting the oyster meat slurry to an ultrasonic combined heat treatment to obtain oyster meat liquid; (2) adding a composite enzyme preparation, protamine and chili slurry to the oyster meat liquid for an enzymolysis, and standing and stratifying after the completion of the enzymolysis to obtain a supernatant and a precipitate; (3) subjecting the precipitate to a second ultrasonic combined heat treatment and then adding a composite enzyme preparation for a second enzymolysis to obtain a second enzymolysis liquid; (4) mixing the supernatant and the second enzymolysis liquid and heating them, and then concentrating and centrifuging them to obtain an oyster juice base. This method has a complex process, and the addition of chili slurry will affect the characteristic taste of the oyster juice. Patent CN109401983A removes the fishy smell by adding Aspergillus oryzae fermentation liquid to oyster juice. This method has limited fishy smell removal effect and is difficult to stabilize product quality. Patent CN111671072A provides a method for removing the fishy smell of oyster juice, comprising the following steps: (1) contacting oyster juice with organic acid to obtain a mixed solution; (2) heating the mixed solution for heat treatment to obtain a pretreatment solution 1; (3) contacting the pretreatment solution 1 with monosaccharides and / or disaccharides, and after heat treatment, obtaining a pretreatment solution 2; (4) contacting the pretreatment solution 2 with sodium glutamate and heat treatment. This method is complex in process, and the addition of chemical reagents such as organic acids will affect the quality of oyster juice. Patent CN108634148A proposes adding resveratrol to the clam substrate, and adding modified β-cyclodextrin for encapsulation and adsorption after enzymatic hydrolysis to remove the fishy smell. This method is complicated to operate and some nutrients will be lost after encapsulation. In summary, the above methods all use existing commercial enzymes to conduct enzymatic hydrolysis and deodorization research on aquatic products such as oysters and clams, and there is no innovation based on the source of protease. Not only is the process complicated, but it also creates great difficulties in maintaining the stability of product quality, and it is difficult to provide stronger support for the industry to improve the flavor quality of oyster sauce.

[0004] The development of special protease preparations in aquatic product processing is crucial to achieve high-value bioprocessing of aquatic products. Ideal special enzymes should not only have efficient hydrolysis capabilities, but also be able to effectively reduce the content of fishy substances in aquatic products, thereby improving their flavor characteristics. Aspergillus oryzae is an important microorganism in the brewing industry and one of the important sources of proteases. It can secrete enzymes during its growth, especially the proteases produced by metabolism, which can degrade macromolecular proteins into peptides and free amino acids, playing an important role in food industry production. 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 a strain of Aspergillus oryzae ZA304, which has the characteristics of high protease production and can be used to prepare composite proteases for enzymatic hydrolysis of aquatic products, thereby improving the utilization rate of raw materials, and can also significantly reduce the fishy and bitter taste of aquatic products and enhance the aroma, and has great application potential in high-value processing of aquatic products.

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

[0007] In a first aspect, an embodiment of the present application provides an Aspergillus oryzae ZA304, characterized in that the Aspergillus oryzae ZA304 has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with a deposit number of GDMCC No: 64926.

[0008] In a second aspect, the present invention provides a fermentation bacterial agent, characterized in that the fermentation bacterial agent comprises the above-mentioned Aspergillus oryzae ZA304.

[0009] In a third aspect, the present invention provides the use of the above-mentioned Aspergillus oryzae ZA304 or fermentation agent in aquatic product processing.

[0010] In a fourth aspect, the present invention provides the use of the above-mentioned Aspergillus oryzae ZA304 or fermentation agent in the preparation of composite protease.

[0011] In a fifth aspect, the present invention provides a method for preparing a composite protease, the method comprising the steps of extracting and filtering a fermented koji material, wherein the fermented koji material is obtained by fermenting the above-mentioned Aspergillus oryzae ZA304 or a fermentation agent.

[0012] In a sixth aspect, the present invention provides a composite protease, which is prepared by the method of the present invention.

[0013] In a seventh aspect, the present invention provides application of the above-mentioned composite protease in aquatic product processing.

[0014] In an eighth aspect, the present invention provides a method for processing aquatic products, comprising the step of treating the aquatic products with the above-mentioned composite protease.

[0015] Furthermore, the method also comprises the steps of crushing and homogenizing the aquatic product.

[0016] Furthermore, the aquatic products are oysters, mussels, and clams.

[0017] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0018] 1. The Aspergillus oryzae ZA304 provided by the present invention is obtained by artificial mutagenesis screening of Aspergillus oryzae Shanghai Niang 3.042 strain, can produce high protease, and compared with Aspergillus oryzae Shanghai Niang 3.042, its acidic protease activity is increased by 46.75%, neutral protease activity is increased by 25.18%, aminopeptidase activity is increased by 63.89%, and total protease activity is increased by 18.74%, and the improvement effect is significant.

[0019] 2. The present invention provides a method for preparing a composite protease, which has a simple preparation process and can be used for enzymatic hydrolysis of aquatic products without further compounding and combination. The degree of hydrolysis is 7.5%-28.5% higher than that of commercial proteases, which can effectively increase the content of amino nitrogen in the enzymatic hydrolysis solution, improve the enzymatic hydrolysis efficiency, and improve the utilization rate of raw materials.

[0020] 3. The composite protease preparation prepared by Aspergillus oryzae ZA304 provided by the present invention is used for enzymatic hydrolysis of aquatic products, which can significantly reduce the content of fishy substances such as 1-octen-3-ol, 1-penten-3-ol, and 3-octanone, while increasing the content of aroma-active compounds such as aldehydes, esters, and furans, effectively reducing the fishy smell of the enzymatic hydrolyzate and increasing the fragrance, thereby improving the sensory flavor of the enzymatic hydrolyzate of aquatic products.

[0021] 4. The composite protease preparation prepared by Aspergillus oryzae ZA304 provided by the present invention can directly improve the flavor of the enzymatic hydrolysate of aquatic products through an enzymatic hydrolysis process. Compared with other methods, it is simpler to operate, more environmentally friendly and safer, and also helps to reduce production costs, improve economic benefits, and meet the needs of high-value processing of aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The hydrolysis degree and amino nitrogen content of oyster juice treated with different enzymes;

[0023] Figure 2 The hydrolysis degree and amino nitrogen content of mussels treated with different enzymes;

[0024] Figure 3 Degree of hydrolysis and amino nitrogen content of clams treated with different enzymes;

[0025] The Aspergillus oryzae ZA304 provided by the present invention has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and a collection number of GDMCC No: 64926; the strain was received and registered by the collection center on July 31, 2024, and was detected as a surviving strain by the collection center on July 31, 2024. DETAILED DESCRIPTION

[0026] In order to facilitate the 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.

[0027] 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.

[0028] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional food-grade reagents, methods and equipment in the art.

[0029] The terms used in the specification 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.

[0030] Example 1 Screening of strains

[0031] 1. ARTP mutagenesis of Aspergillus oryzae 3.042:

[0032] (1) ARTP mutagenesis operation steps: Aspergillus oryzae Shanghai Niang 3.042 (purchased from Yishui Jinrun Biotechnology Co., Ltd.) was inoculated onto YPD plate medium using the spot seeding method and cultured at 30°C until the spores matured. 2 mL of physiological saline was added to the medium, and the spores were scraped off using an L-shaped spreader stick to suspend and collect in a 2 mL centrifuge tube. The spore suspension was diluted to an appropriate multiple, and the spore concentration in the stock solution was diluted to 2 × 10 after counting using a hemocytometer. 8 CFU / mL. Prepare sterile 10% (V / V) glycerol and spore suspension in a ratio of 1:1 and mix thoroughly. Pipette 10 μL of the mixed liquid and apply it on a sterile slide, then transfer the slide to a sterile carrier.

[0033] (2) 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.

[0034] 2. Preliminary screening of high-yield protease strains:

[0035] The induced spore suspension was diluted in series to make the concentrations 1×10 4 CFU / mL, 1×10 3 CFU / mL and 1×10 2CFU / mL, 100 μL of each was spread on casein medium, and cultured at 30°C for 72 h, and the growth of the strain was observed. The diameter of the mutant strain (D) and the corresponding transparent circle diameter (H) were recorded, and the mutant strain with a larger H / D ratio was screened for rescreening. The results are recorded in Table 1.

[0036] Casein culture medium: casein 10g, beef extract 3g, disodium hydrogen phosphate 2g, sodium chloride 5g, agar 18g, distilled water 1000mL, heat to dissolve, sterilize at 121℃ for 15min.

[0037] Table 1: Results of initial screening of ARTP mutagenesis of Aspergillus oryzae Shanghai Brewing 3.042

[0038]

[0039] From the results in Table 1, it can be seen that a total of 5 mutant strains have H / D ratios higher than the control group strain Shanghai Niang 3.042 that has not been treated with ARTP. Among them, the colony diameters of mutant strains 210-1 and 240-2 are smaller than Shanghai Niang 3.042, indicating that the colony growth of these two mutant strains is not as good as that of the control group; while the colony growth of the remaining three mutant strains is good. Among them, the colony diameter of strain 240-13 after ARTP mutagenesis is 0.951 cm, the transparent zone diameter reaches 2.197 cm, and the H / D ratio reaches 2.311, which is significantly higher than the control group Shanghai Niang 3.042. Therefore, the mutagenic strain 240-13 may have higher protease activity, and this strain was selected for rescreening.

[0040] 3. Rescreening of high-yield protease strains:

[0041] The mutant strain 240-13 obtained in the initial screening was inoculated into the koji-making medium and cultured at 30°C for 72 hours. 1.0 g of solid koji material was weighed and extracted with a phosphate buffer solution with a pH of 7.2 for 1 hour. The crude enzyme solution was filtered and the activities of acid protease, neutral protease, aminopeptidase and composite protease were determined. The results are recorded in Table 2.

[0042] Koji making medium: bran 16g, soybean meal 4g, distilled water 16mL, sterilize at 121℃ for 15min.

[0043] Acidic protease activity detection method: The determination method refers to GB / T 28715-2012. 1g Daqu hydrolyzes casein at 40℃ and pH 3.0 to produce 1μg tyrosine per minute, which is defined as 1 neutral protease activity unit (U / g).

[0044] Neutral protease activity detection method: The determination method refers to GB / T 28715-2012. 1g Daqu hydrolyzes casein at 40℃ and pH7.2 to produce 1μg tyrosine per minute, which is defined as 1 neutral protease activity unit (U / g).

[0045] Alkaline protease activity detection method: The determination method refers to GB / T23527-2009. 1g Daqu hydrolyzes casein at 40℃ and pH9.0 to produce 1μg tyrosine per minute, which is defined as 1 neutral protease activity unit (U / g).

[0046] Aminopeptidase activity detection method: Under the conditions of 40°C and pH 7.2, the amount of enzyme required for aminopeptidase to hydrolyze leucine p-nitroaniline to generate 1 μg p-nitroaniline (pNA) per minute is one enzyme activity unit, expressed as U / g or U / mL.

[0047] Table 2 Results of rescreening of Aspergillus oryzae 3.042 ARTP mutagenesis strains

[0048]

[0049] As shown in the results in Table 2, compared with the original strain Hu Niang 3.042, at the end of shallow plate koji making, the activity of acidic protease in the koji material of the mutant strain 240-13 increased by 46.75%, the activity of neutral protease increased by 25.18%, the activity of aminopeptidase increased by 63.89%, and the activity of total protease increased by 18.74%, with significant improvement. The strain was identified as Aspergillus oryzae by 16S rDNA sequencing, and the strain was named ZA304, which was deposited in Guangdong Microbiological Culture Collection Center on July 31, 2024, with the deposit number GDMCC No: 64926.

[0050] Example 2 Application of Aspergillus oryzae ZA304 in enzymatic hydrolysis of oysters

[0051] 1. Preparation of compound protease

[0052] Prepare fermentation medium: 9kg bran, 1kg rice husk, add 10kg water, sterilize at 121℃ for 15min and set aside.

[0053] 3 g of Aspergillus oryzae ZA304 spore powder was inoculated into the fermentation medium and cultured at 30°C for 72 h. 1.0 g of solid koji was weighed and extracted with a phosphate buffer solution with a pH of 7.2 for 1 h. The crude enzyme solution of the composite protease was obtained after filtration.

[0054] 4% diatomaceous earth was added to the crude enzyme solution of composite protease, and the extract was clarified by filtration after stirring. The enzyme solution was ultrafiltered using a 20 kDa hollow fiber membrane, and the retentate was retained to concentrate the enzyme solution by 10 times to obtain a concentrated enzyme solution of composite protease.

[0055] 2. Compound protease is used to hydrolyze oysters to prepare oyster juice

[0056] Preparation of raw oyster homogenate: Frozen oyster meat was slowly thawed at 4°C. After thawing, the oyster meat was thoroughly crushed using a grinder. Subsequently, deionized water was added to adjust the solid-liquid ratio to 1:2 (w:v), and then a homogenizer was used at 8000 rpm for 1 minute to obtain raw oyster homogenate, and the total nitrogen content of the raw oyster homogenate was determined.

[0057] Enzymatic hydrolysis of oysters: Take the above-mentioned composite protease powder, add Novozymes neutral protease, Novozymes alkaline protease, and Novozymes flavor protease to the raw oyster homogenate, and the addition amount is 2% of the substrate mass. Keep the enzymatic hydrolysis temperature at 55°C and shake the enzymatic hydrolysis at 300 rpm for 4 hours. After the enzymatic hydrolysis is completed, place the enzymatic hydrolyzate in boiling water for 10 minutes to inactivate the enzyme.

[0058] Sampling and testing: Determine the amino nitrogen content of different enzymatic hydrolysates and calculate the degree of hydrolysis of each enzymatic hydrolysate; at the same time, use the GC-MS method to measure the content of the main odorous substances and aroma substances in different enzymatic hydrolysates for comparative analysis. The results are recorded in Figure 1 And Table 3.

[0059] Calculation of hydrolysis degree: hydrolysis degree (%) = amino nitrogen content of enzymatic hydrolyzate / total nitrogen content of raw oyster homogenate × 100%.

[0060] Table 3 Comparison of key flavor substances in different oyster sauces

[0061]

[0062] like Figure 1 As shown in the results, the oyster juice prepared by the composite protease produced by the Aspergillus oryzae ZA304 strain provided in the present application has a hydrolysis degree of 34.71±0.12% and an amino nitrogen content of 0.278±0.001g / 100g, which are higher than the oyster juice prepared by the commercially available neutral protease, alkaline protease and flavor protease. This shows that in the enzymatic hydrolysis of oysters at natural pH, the composite protease prepared by Aspergillus oryzae ZA304 shows better enzymatic hydrolysis efficiency than the other three enzymes, resulting in more extensive peptide bond cleavage and amino acid release, and the hydrolysis degree is 9.67%-22.21% higher than the three commercially available enzymes.

[0063] The results of flavor substance analysis are shown in Table 3. The composite protease produced by the Aspergillus oryzae ZA304 strain provided in this application can significantly reduce the content of key fishy substances 1-octen-3-ol and 3-octanone in raw oyster homogenate, with a reduction ratio of 8.37% and 43.14%, respectively, which is better than commercially available neutral protease, alkaline protease and flavor protease. At the same time, the content of aroma active compounds such as 2-n-pentylfuran and 2-methylbutyraldehyde in oyster juice prepared by composite protease has also been improved, which is 8.55 times and 35.46 times higher than that of raw oyster homogenate, and its content is also significantly higher than that of oyster juice made by commercially available neutral protease, alkaline protease and flavor protease, enhancing the fruity flavor of oyster juice. In summary, compared with commercially available test enzymes, the composite protease prepared by Aspergillus oryzae ZA304 can reduce the content of fishy substances in the raw oyster homogenate to a greater extent while increasing the content of aroma substances in the hydrolyzate, thereby masking the fishy and bitter taste and enhancing the fruity aroma, thereby comprehensively improving the sensory flavor of oyster juice made from enzymatically hydrolyzed oysters.

[0064] Example 3 Application of Aspergillus oryzae ZA304 in enzymatic hydrolysis of mussels

[0065] Preparation of raw mussel homogenate: The frozen mussel meat was slowly thawed in an environment of 4°C. After thawing, the raw mussel meat was thoroughly crushed using a grinder. Subsequently, deionized water was added to adjust the solid-liquid ratio to 1:2 (w:v), and then a homogenizer was used at a speed of 8000 rpm for 1 minute to obtain raw mussel homogenate, and the total nitrogen content of the raw mussel homogenate was determined.

[0066] Enzymatic hydrolysis of mussels: The composite protease powder prepared in Example 2 was added to the raw mussel homogenate with Novozymes neutral protease, Novozymes alkaline protease, and Novozymes flavor protease, respectively, and the addition amount was 2% of the substrate mass, and the enzymatic hydrolysis temperature was maintained at 55°C, and the enzymatic hydrolysis was performed at a speed of 300 rpm for 4 hours. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzate was placed in boiling water to inactivate the enzyme for 10 minutes.

[0067] Sampling and testing: Determine the amino nitrogen content of different enzymatic hydrolysates and calculate the degree of hydrolysis of each enzymatic hydrolysate; at the same time, use the GC-MS method to measure the content of the main odorous substances and aroma substances in different enzymatic hydrolysates for comparative analysis. The results are recorded in Figure 2 And Table 4.

[0068] Calculation of hydrolysis degree: hydrolysis degree (%) = amino nitrogen content of enzymatic hydrolyzate / total nitrogen content of raw mussel homogenate × 100%.

[0069] Table 4 Comparison of key flavor substances in different mussel hydrolysates

[0070]

[0071]

[0072] like Figure 2 As shown in the results, the mussel hydrolysate prepared by the composite protease produced by the Aspergillus oryzae ZA304 strain provided in the present application has a hydrolysis degree of 35.73±0.23% and an amino nitrogen content of 0.293±0.0021g / 100g, which are higher than the hydrolysates prepared by the commercially available neutral protease, alkaline protease and flavor protease. This indicates that when enzymolyzing mussels at natural pH, the composite protease prepared by Aspergillus oryzae ZA304 exhibits better enzymolysis efficiency than the other three enzymes, and the hydrolysis degree is 7.51%-23.63% higher than the three commercially available enzymes.

[0073] The results of flavor substance analysis are shown in Table 4. The composite protease produced by the Aspergillus oryzae ZA304 strain provided in the present application can significantly reduce the content of the key fishy substance 1-octen-3-ol in the raw mussel homogenate, with a reduction ratio of 25.95%, which is better than the commercially available neutral protease, alkaline protease and flavor protease. At the same time, the aroma active compounds such as 2-ethylfuran and ethyl citrate in the enzymatic solution prepared by the composite protease have also been improved, which are 7.95 times and 7.25 times higher than those in the raw mussel homogenate, respectively, and their contents are also significantly higher than those prepared by the commercially available neutral protease, alkaline protease and flavor protease. In short, compared with the commercially available test enzyme, after the composite protease prepared by Aspergillus oryzae ZA304 hydrolyzes mussels, the content of fishy substances in the raw mussel homogenate can be reduced to a greater extent while increasing the content of aroma substances in the enzymatic solution, thereby comprehensively improving the sensory flavor of the mussel enzymatic solution.

[0074] Example 4 Application of Aspergillus oryzae ZA304 in enzymatic hydrolysis of clams

[0075] Preparation of raw clam homogenate: The frozen clam meat was slowly thawed at 4°C. After thawing, the clam meat was thoroughly crushed using a grinder. Subsequently, deionized water was added to adjust the solid-liquid ratio to 1:2 (w:v), and then the raw clam homogenate was obtained by homogenization at 8000 rpm for 1 minute, and the total nitrogen content of the raw clam homogenate was determined.

[0076] Enzymatic hydrolysis of clams: The composite protease powder prepared in Example 2 was added to the raw clam homogenate with Novozymes neutral protease, Novozymes alkaline protease, and Novozymes flavor protease, respectively, and the addition amount was 2% of the substrate mass, and the enzymatic hydrolysis temperature was maintained at 55°C, and the enzymatic hydrolysis was performed at a speed of 300 rpm for 4 hours. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzate was placed in boiling water for 10 minutes to inactivate the enzyme.

[0077] Sampling and testing: Determine the amino nitrogen content of different enzymatic hydrolysates and calculate the degree of hydrolysis of each enzymatic hydrolysate; at the same time, use the GC-MS method to measure the content of the main odorous substances and aroma substances in different enzymatic hydrolysates for comparative analysis. The results are recorded in Figure 3And Table 5.

[0078] Calculation of hydrolysis degree: hydrolysis degree (%) = amino nitrogen content of enzymatic hydrolyzate / total nitrogen content of raw clam homogenate × 100%.

[0079] Table 5 Comparison of key flavor substances in different clam enzymatic hydrolysates

[0080]

[0081] like Figure 3 As shown in the results, the clam hydrolysate prepared by the composite protease produced by the Aspergillus oryzae ZA304 strain provided in the present application has a hydrolysis degree of 36.76±0.13% and an amino nitrogen content of 0.297±0.0012g / 100g, which are higher than the hydrolysates prepared by the commercially available neutral protease, alkaline protease and flavor protease. This indicates that the composite protease prepared by Aspergillus oryzae ZA304 exhibits better enzymatic efficiency than the other three enzymes in the enzymatic hydrolysis of mussels at natural pH, and the hydrolysis degree is 10.21%-28.57% higher than the three commercially available enzymes.

[0082] The results of flavor substance analysis are shown in Table 5. The composite protease produced by the Aspergillus oryzae ZA304 strain provided in the present application can significantly reduce the content of the key fishy substances 1-octen-3-ol and 1-penten-3-ol in the raw clam homogenate, with a reduction ratio of 41.44% and 27.01%, respectively, which is better than the commercially available neutral protease, alkaline protease and flavor protease. At the same time, the content of aroma active compounds such as 2-n-pentylfuran and 2-ethylfuran in the enzymatic solution prepared by the composite protease has also been improved, which is 8.76 times and 5.08 times higher than that of the raw clam homogenate, respectively, and its content is also significantly higher than that of the commercially available neutral protease, alkaline protease and flavor protease. In short, compared with the commercially available test enzyme, after the composite protease prepared by Aspergillus oryzae ZA304 hydrolyzes clams, it can also reduce the content of fishy substances in the raw mussel homogenate to a greater extent while increasing the content of aroma substances in the enzymatic solution, and comprehensively improve the sensory flavor of the clam enzymatic solution.

[0083] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain of Aspergillus oryzae ( Aspergillus oryzae ) ZA304, characterized in that, The deposit number of Aspergillus oryzae ZA304 is GDMCC No:64926.

2. A fermentation agent, characterized in that: The fermentation bacteria agent comprises the Aspergillus oryzae ZA304 as claimed in claim 1.

3. Use of the Aspergillus oryzae ZA304 according to claim 1 or the fermentation agent according to claim 2 in enzymatic hydrolysis of oysters, mussels and clams.

4. Use of the Aspergillus oryzae ZA304 according to claim 1 or the fermentation agent according to claim 2 in the preparation of a composite protease.

5. A method for preparing a composite protease, characterized in that: The method comprises the steps of extracting and filtering the fermented koji material, wherein the fermented koji material is obtained by fermenting the Aspergillus oryzae ZA304 described in claim 1 or the fermentation agent described in claim 2.

6. A composite protease, characterized in that The composite protease is prepared by the method described in claim 5.

7. Use of the composite protease as claimed in claim 6 in enzymatic hydrolysis of oysters, mussels and clams.

8. A method for enzymatic processing of aquatic products, characterized in that: The method comprises the step of treating aquatic products with the composite protease according to claim 6, wherein the aquatic products are oysters, mussels, and clams.

9. The method according to claim 8, characterized in that The method also comprises the steps of crushing and homogenizing the aquatic product.

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