Aspergillus versicolor and application thereof

Abstract

The present application relates to a strain of Aspergillus versicolor and its application, belonging to the technical field of microbial fermentation. The Aspergillus versicolor SC007 provided by the present application is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.41829, a preservation date of March 12, 2025, a preservation address of Beijing, China, and a classification name of Aspergillus versicolor Aspergillus versicolor The Aspergillus versicolor SC007 and Aspergillus oryzae provided by the present application are combined for koji making of soybean paste and soy sauce, which can significantly improve the protease activity of the koji, the improvement of the koji enzyme activity makes the decomposition of protein in the raw material more sufficient, thereby improving the content of amino acid nitrogen in the mash, and further greatly improving the raw material utilization rate of soy sauce and soy paste, and also playing an improving role on the quality and flavor of the product, and the Aspergillus versicolor SC007 will not inhibit the growth and enzyme production of Aspergillus oryzae.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, in particular to a strain of Aspergillus versicolor and its application. BACKGROUND

[0002] Traditional fermentation of soy sauce or sauce is generally carried out by Aspergillus oryzae for koji making, and the enzyme system is relatively simple, and the enzyme activity of important enzyme systems is also not high, such as protease activity, which results in relatively low utilization degree of soy sauce raw materials, not only affecting the flavor of the product, but also increasing the production cost, which is a common problem in the industry.

[0003] With the development of technology, the industry has begun to use Aspergillus niger and Aspergillus oryzae for compound koji making to improve enzyme activity, make up for the deficiency of Aspergillus oryzae koji making, and achieve the purpose of improving the utilization rate of raw materials, improving the flavor, and reducing the cost of soy sauce, but the enzyme system of Aspergillus niger is relatively simple, and there is a natural defect in improving the utilization rate of raw materials. SUMMARY

[0004] The purpose of the present application is to provide a strain of Aspergillus versicolor, which is used for soy sauce or sauce koji making together with Aspergillus oryzae, and can significantly improve the utilization rate of raw materials of soy sauce or sauce.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a strain of Aspergillus versicolor, which is deposited in the China General Microbiological Culture Collection Center, has a deposit number of CGMCC No. 41829, a deposit date of March 12, 2025, a deposit address of Beijing, China, and is named Aspergillus versicolor Aspergillus versicolor .

[0006] The present application also provides a compound microbial agent comprising Aspergillus oryzae and the Aspergillus versicolor.

[0007] The present application also provides the application of the Aspergillus versicolor or the compound microbial agent in the preparation of a condiment.

[0008] In a preferred embodiment, the condiment is a fermented condiment.

[0009] In a preferred embodiment, the fermented condiment comprises soybean paste and soy sauce.

[0010] The present application also provides a method for preparing a condiment, which comprises the step of using the Aspergillus versicolor or the compound microbial agent to prepare the condiment.

[0011] In a preferred embodiment, the method for preparing a condiment comprises the following steps:

[0012] 1) Koji making: inoculating the Aspergillus versicolor and Aspergillus oryzae in koji making raw materials for koji making to obtain koji;

[0013] 2) fermentation: the koji and salt water are mixed to make a mash, and the mash is fermented to obtain the condiment.

[0014] In a preferred scheme, the inoculation ratio of the Aspergillus versicolor is 0.01%-0.5% of the koji raw material weight.

[0015] In a preferred scheme, the condiment is a fermented condiment.

[0016] In a preferred scheme, the fermented condiment includes bean sauce and soy sauce.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The Aspergillus versicolor SC007 provided by the present application is used for koji making of bean sauce and soy sauce in combination with Aspergillus oryzae, which can significantly improve the protease activity of the koji, and the improved enzyme activity of the koji makes the decomposition of protein in the raw material more sufficient, thereby improving the content of amino acid nitrogen in the mash, and greatly improving the raw material utilization rate of the soy sauce / bean sauce; at the same time, the high-efficiency enzyme system of the Aspergillus versicolor SC007 can also reduce the total number of colonies of the finished product, avoid the use of higher sterilization parameters, thereby reducing the loss of flavor of the finished product, improve the quality and flavor of the product, and the Aspergillus versicolor SC007 will not inhibit the growth and enzyme production of Aspergillus oryzae itself. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A process flow chart for soy sauce preparation in the embodiments of the present application;

[0020] Figure 2 A transparent circle state of the strains SC007 and SC008 in the embodiments of the present application;

[0021] Figure 3 A transparent circle state of the spore bacteria in the embodiments of the present application;

[0022] Figure 4 A transparent circle state of the lactic acid bacteria in the embodiments of the present application;

[0023] Figure 5 A transparent circle state of the Aspergillus oryzae in the embodiments of the present application. DETAILED DESCRIPTION

[0024] The technical solutions of the present application are described below in conjunction with the drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0025] In the following examples, the raw materials used are commercially available, and the processes used are conventional processes, unless otherwise specified.

[0026] In the following examples, the koji fermentation of soybean paste and soy sauce is achieved by the following steps:

[0027] Soy sauce

[0028] 1) Raw material pretreatment and koji preparation process:

[0029] Non-genetically modified defatted soybeans are used as the main protein raw material. The defatted soybeans are steamed in a pot using hot water at a temperature of 80-100°C, and the steaming is rotated for 5-10 min. Wheat is used as the main carbon source raw material, which is roasted and crushed, and then mixed with the defatted soybeans. The roasting temperature of the wheat is controlled at above 100°C, and the time is 3-10 min.

[0030] The pretreated defatted soybeans and roasted wheat are mixed in a ratio of (1-2):1, and the moisture content of the mixture is 40%-50%. When the temperature drops below 40°C, Aspergillus oryzae and Aspergillus versicolor are inoculated. The inoculation ratio of Aspergillus oryzae and Aspergillus versicolor is 0.01%-0.5% of the dry mixture (defatted soybeans and wheat before pretreatment). The thickness of the mixture in the koji room is preferably 30-40 cm. The koji is turned over at 16 h and 30 h, respectively. The growth stage temperature of the koji should be controlled at 28-35°C, and the maximum temperature should not exceed 38°C. At 30 h, the temperature of the koji should be quickly reduced to below 24°C. The koji preparation is completed in 36-48 h, and the soy sauce koji is obtained.

[0031] Aspergillus versicolor can also be prepared into a crude enzyme solution to participate in koji preparation. The addition ratio of the crude enzyme solution is 0.1-5% of the dry mixture, and the addition period is 0-24 h during the koji preparation process. Then, the normal koji preparation process is followed.

[0032] 2) Fermentation:

[0033] The soy sauce koji is mixed with brine to make mash. The salt content of the brine used is 19-25 g / 100 mL, and the koji and brine are mixed in a weight ratio of 2:(2.5-3). During the fermentation process, the temperature needs to be controlled at 15-18°C for the first 30 days, and the tank is stirred every 3 days. The temperature is maintained at 28-30°C for the subsequent period, and the fermentation period is 6 months.

[0034] The process flow of soy sauce preparation is shown in Figure 1 .

[0035] Soybean paste

[0036] 1) Koji preparation process:

[0037] The pretreatment of soybean includes: after the soybean is washed, the temperature is controlled at 98-120°C, and the soybean is boiled for 8-10 minutes; the pretreatment of wheat flour includes: the temperature is controlled at 98-120°C, and the wheat flour is boiled for 20-30 seconds, and the moisture is controlled at 22%-24%;

[0038] The pretreated soybean and wheat flour are mixed at a ratio of 1:10-1:1, inoculated with Aspergillus oryzae and Aspergillus versicolor at the same ratio as soy sauce, and then fermented at 28-35°C for 38-45 hours to obtain brewing koji.

[0039] 2) Fermentation:

[0040] The brewing koji and brine are mixed to prepare mash, the concentration of the brine used is 20.5%-23.5%, and the amount of brine added is 0.9-1.5 times the weight of the brewing koji. The fermentation temperature is maintained at 25-35°C, and the fermentation time is 70-100 days.

[0041] Example 1: Target strain screening

[0042] (I) Preliminary screening

[0043] 1. Isolation source: soy sauce / koji, fermentation period 7d-15d mash;

[0044] 2. Preliminary screening method: 25g of koji / mash is weighed and added to 225g of sterile physiological saline, homogenized to obtain a test sample solution, 1mL of the test sample solution is inoculated into 9mL of sterile physiological saline as a 10-fold dilution, and gradient dilution is performed, 1mL of 2-3 appropriate dilutions is taken and inoculated into sterile empty petri dishes, 15% PDA medium and 15% salt concentration NA medium are poured and shaken uniformly, then allowed to solidify, and after solidification, placed in a 30°C incubator, observed after 3 days;

[0045] 3. Isolation and purification: the single colonies obtained from the preliminary screening plate are streaked onto 15% PDA and NA plates, placed in a 30°C incubator, taken out after 3 days, and repeated 3 times for standby use.

[0046] (II) Rescreening

[0047] 1. The obtained pure culture points are inoculated into casein medium plates, cultured at 30°C for 3 days, taken out, and the transparent circle situation is observed and recorded, and the K value is used as the standard to compare the abilities of the strains, the K value is the transparent circle diameter (D) / colony diameter (d), the composition of the casein medium is shown in Table 1-1, and the transparent circle comparison results are shown in Table 1-2:

[0048] Table 1-1 Composition of casein medium

[0049]

[0050] Table 1-2 Comparison of K values of each colony

[0051]

[0052] The transparent circle state of the strains SC007 and SC008 with the highest K value is shown in Figure 2 .

[0053] Meanwhile, the transparent circle size of the existing strains Bacillus sp., Lactobacillus sp. and Aspergillus oryzae under the same culture conditions as strains SC007 and SC008 was determined, and the transparent circle state of Bacillus sp., Lactobacillus sp. and Aspergillus oryzae is shown in Figures 3-5 The comparison results are shown in Table 1-3:

[0054] Table 1-3 Comparison of K values of SC007, SC008 and existing colonies

[0055]

[0056] According to the above table, the K values of strains SC007, SC008 and Aspergillus oryzae are greater than those of Bacillus sp. and Lactobacillus sp., and it is believed that they have a good effect on improving enzyme activity in koji making.

[0057] 2. Enzyme activity determination

[0058] The two strains SC007 and SC008 with the highest K values above were selected to prepare Daqu in combination with Aspergillus oryzae, and the enzyme activity of the finished koji was determined according to the method of “GB / T 23527-2009 National Standard Protease Preparations”, and the results are shown in Table 2:

[0059] Table 2 Comparison of enzyme activity determination of strains SC007 and SC008 in finished koji

[0060]

[0061] According to the determination results, the enzyme activity of the finished koji inoculated with strain SC007 is significantly superior, and therefore strain SC007 is selected for subsequent strain identification and testing.

[0062] (Three) Strain identification

[0063] The ITS sequence of strain SC007 was determined, and the sequencing results were compared and analyzed in the NCBI database, and the results showed that strain SC007 was Aspergillus versicolor Aspergillus versicolor . The strain was preserved in the China General Microbiological Culture Collection Center, Beijing, China, and the preservation number was CGMCC No. 41829.

[0064] (Four) Detection of Aspergillus versicolor toxin

[0065] The results of measuring the heterocutin produced by strain SC007 prepared into koji by high performance liquid chromatography-mass spectrometry method were ND, indicating that heterocutin SC007 did not produce heterocutin and was safe and applicable.

[0066] After evaluating the safety and application effect of heterocutin SC007, it was determined to carry out subsequent application research.

[0067] Example 2 Fermentation of soy sauce mixed koji preparation

[0068] Aspergillus oryzae and strain SC007 were inoculated into moist bran, respectively, and cultured at 30°C for 2-3d to obtain seed koji, which was taken out for standby.

[0069] Experimental group: According to the koji preparation process of soy sauce, steamed defatted soybeans and fried wheat flour were prepared, and the dry weight ratio of the two was 2:1. The inoculation amounts of Aspergillus oryzae and strain SC007 were 0.5% and 0.1%, respectively. Alternatively, the seed koji and fried wheat flour were mixed first and then mixed with the steamed defatted soybeans. After the materials were mixed and put into the koji room, the koji was turned over at 16h and 30h, respectively. The temperature control during the growth stage of the koji was 28-33°C. At 30h, the temperature of the koji material needed to be quickly reduced to below 24°C. The koji was taken out at 38h. The koji and brine were mixed uniformly according to the ratio of dry material:brine=2:2.5. The salt content of the brine was 19g / 100mL. During the fermentation process, the temperature was controlled at 18°C for the first 30d, and the tank was stirred every 3d. The subsequent temperature was maintained at 28-30°C, and the fermentation period was 6 months.

[0070] Control group 1 (mixed with Aspergillus oryzae and Aspergillus niger): The koji preparation and fermentation process of this control group was consistent with that of the experimental group described above. The difference was that strain SC007 was replaced by Aspergillus niger, and koji fermentation was carried out by mixing with Aspergillus oryzae.

[0071] Control group 2: Inoculation of Aspergillus oryzae for koji preparation as a control group.

[0072] The moisture content, enzyme activity indicators and original juice FN (amino acid nitrogen) indicators of the experimental group and the two control groups were determined, respectively. The protease activity detection referred to “GB / T 23527-2009 National Standard Protease Preparation”, and the amino acid nitrogen content detection referred to “GB 5009.235-2016 Food Safety National Standard Determination of Amino Acid Nitrogen in Food”. At the same time, the total number of colonies in the original oil of the experimental group and control group 2 was determined. The original oil colony number detection referred to “GB 4789.2-2022 Food Safety National Standard Food Microbiology Examination Determination of Total Number of Colonies”. The specific determination data are shown in Table 3:

[0073] Table 3 Related indicators of experimental group and control group

[0074]

[0075] According to the detection results:

[0076] 1) Compared with the control group 2, the koji enzyme activity of the experimental group was significantly improved, and the FN was increased by 8%, and the raw material utilization rate was greatly improved. It is proved that inoculating Aspergillus oryzae at the same time and mixed inoculating Aspergillus versicolor SC007 can decompose soy sauce raw materials more thoroughly, thereby significantly improving the utilization rate of soy sauce raw materials. After the soy sauce crude oil obtained in the experimental group was prepared into soy sauce products, 15 professional tasters evaluated it and believed that it had bright and lustrous color, sufficient salty and fresh flavor, rich soy sauce aroma and thick ester aroma. Therefore, the mixed koji making of Aspergillus oryzae and Aspergillus versicolor SC007 does not inhibit the growth and enzyme production of Aspergillus oryzae, and does not cause great changes to the existing soy sauce preparation process to increase the process cost.

[0077] 2) In the control group 1, Aspergillus niger and Aspergillus oryzae were mixed to make koji, which was significantly improved in acid protease activity compared with control group 2, and the activities of neutral protease and alkaline protease were comparable to control group 2. The final amino acid nitrogen of the fermented original juice was about 0.8% higher than that of control group 2. At the same time, the original juice was preliminarily evaluated by 15 professional tasters, and it was believed that the umami and soy sauce aroma were not significantly improved compared with control group 2.

[0078] 3) In the experimental group, the total number of colonies of the original oil obtained by mixed inoculating Aspergillus oryzae and Aspergillus versicolor SC007 was about 1 log lower than that of the original oil obtained by using Aspergillus oryzae alone in control group 2. The high-efficiency enzyme system produced by Aspergillus versicolor SC007 can seize the nutrients in the koji first, thereby inhibiting the growth of other bacteria with the same needs. The reduction of the total number of colonies in the original oil can avoid high sterilization process parameters, reduce the loss of product flavor, save costs, and prolong the shelf life of the product.

[0079] Example 3 Fermentation of soy sauce by adding crude enzyme solution for koji making

[0080] Aspergillus oryzae and strain SC007 were inoculated into moist bran, respectively, and cultured at 30°C for 2-3 days to obtain seed koji, which was taken out for standby.

[0081] Experimental group: Add an equal amount of tertiary water to the SC007 seed koji, and extract at room temperature for 1-2 hours. Filter the residue to obtain the supernatant, concentrate it by membrane to one time, and obtain the SC007 crude enzyme solution. According to the koji making process in Example 2, Aspergillus oryzae koji making and fermentation were carried out. Add the SC007 crude enzyme solution according to 0.5% of the dry weight of the material, mix the crude enzyme solution and the material evenly through the koji turning process, filter the juice after fermentation is completed.

[0082] Control group: The koji making by inoculating Aspergillus oryzae alone was used as the control group.

[0083] The moisture content, enzyme activity index and original juice FN (amino acid nitrogen) index of the experimental group and the control group were measured, wherein the protease activity detection referred to the national standard GB / T23527-2009, the amino acid nitrogen content detection referred to the national standard GB 5009.235-2016, and the specific measurement data were shown in Table 4:

[0084] Table 4 Related indexes of the experimental group and the control group

[0085]

[0086] After the soy sauce raw oil obtained in the above experimental group was prepared into soy sauce products, 15 professional taste evaluation personnel evaluated that the color was bright and lustrous, the salty and fresh flavor was sufficient, the soy sauce aroma was rich, and the ester aroma was mellow.

[0087] According to the detection results, the koji enzyme activity of the experimental group was significantly improved compared with the control group, and the FN was improved by 6.6%. The raw material utilization rate was greatly improved. In this embodiment, the crude enzyme liquid was directly used to participate in the koji preparation process, and compared with the inoculation of the strain, the mechanism was more clear, the metabolic pathway was more clear, and the relationship between the koji enzyme activity and the improvement of the amino acid nitrogen was more accurate.

[0088] Example 4 Fermentation of soybean paste mixed koji preparation

[0089] In this embodiment, the adding method of Aspergillus oryzae and strain SC007 in the experimental group was the same as that in the soy sauce koji preparation of example 3, the soybean paste koji preparation and fermentation were prepared according to the prior art, the fermentation period of the soybean paste was 90 days, and the fermentation temperature was kept at 25-28℃. The koji preparation by inoculating only Aspergillus oryzae was used as the control group.

[0090] The moisture content, enzyme activity index and original juice FN (amino acid nitrogen) index of the experimental group and the control group were measured, wherein the protease activity detection referred to the national standard GB / T23527-2009, the amino acid nitrogen content detection referred to the national standard GB 5009.235-2016, and the specific measurement data were shown in Table 4:

[0091] Table 5 Related indexes of the experimental group and the control group

[0092]

[0093] According to the detection results, the koji enzyme activity of the experimental group is significantly improved compared with the control group, and the FN is improved by 9.4%, and the raw material utilization rate is greatly improved. Similarly, the total number of bacterial colonies in the experimental group is reduced by about 1 log compared with the control group, which prolongs the shelf life of the product and improves the product quality during the shelf life. After preparing the fermented sauce obtained by the experimental group into bean sauce products and evaluating by 15 professional evaluators, it is considered that the salty and fresh flavor is sufficient, the sauce aroma is rich, and the ester aroma is mellow.

[0094] From the above embodiments, it can be seen that the combination of Aspergillus versicolor SC007 and Aspergillus oryzae for koji making of soybean paste or soy sauce can significantly improve the protease activity of the koji, and the improvement of the koji enzyme activity makes the decomposition of protein in the raw material more sufficient, thereby improving the amino acid nitrogen content in the koji, and greatly improving the raw material utilization rate of soy sauce / bean paste, while also reducing the total number of colonies in the finished product, reducing the loss of flavor of the finished product, improving the quality and flavor of the product, and the Aspergillus versicolor SC007 does not inhibit the growth and enzyme production of Aspergillus oryzae.

[0095] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above embodiments, and the above is only a preferred example of the present application and is not intended to limit the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. Aspergillus versicolor, characterized in that, The Aspergillus versicolor is preserved in the China General Microbiological Culture Collection Center, the preservation number is CGMCC No. 41829, the preservation date is March 12, 2025, the preservation address is Beijing, China, and the classification name is Aspergillus versicolor Aspergillus versicolor .

2. A complex microbial agent, characterized in that, Aspergillus oryzae and the Aspergillus versicolor of claim 1.

3. Use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce.

4. A method of preparing a condiment, characterized by, The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce.

5. The method of preparing a condiment according to claim 4, characterized in that, The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce. The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce. The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce.

6. The method of preparing a condiment according to claim 5, wherein The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce. The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce. The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce. The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce. The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment, which is a bean sauce or a soy sauce. The use of the Aspergillus versicolor of claim 1 or the complex microbial agent of claim 2 in the preparation of a condiment

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