Aspergillus versicolor and application thereof

Abstract

The invention relates to aspergillus versicolor and application thereof, and belongs to the technical field of microbial fermentation. The aspergillus versicolor SC007 provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.41829, the preservation date is March 12, 2025, the preservation address is Beijing, China, and the aspergillus versicolor SC007 is classified and named as aspergillus versicolor. The Aspergillus versicolor SC007 and Aspergillus oryzae provided by the invention are jointly used for making koji of soybean sauce and soy sauce, the protease activity of finished koji can be remarkably improved, and the improvement of the enzyme activity of the finished koji enables protein in raw materials to be decomposed more sufficiently, so that the content of amino acid nitrogen in soy sauce mash is improved, and the utilization rate of the raw materials of the soy sauce and the soy sauce is further greatly improved; the aspergillus versicolor SC007 can improve the quality and flavor of the product, and the aspergillus versicolor SC007 cannot inhibit the growth and enzyme production of aspergillus oryzae.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, in particular to an Aspergillus versicolor strain and application thereof. Background Art

[0002] The fermentation of traditional soy sauce or paste is generally carried out by making koji using Aspergillus oryzae. The types of enzymes are relatively single, and the enzymatic activity of important enzymes, such as protease activity, is not high enough. As a result, the utilization rate of soy sauce raw materials is relatively low, which not only affects the product flavor but also increases production costs. This is a common problem in the industry.

[0003] With the development of technology, the industry has begun to use Aspergillus niger and Aspergillus oryzae to make a combined koji to enhance enzyme activity, make up for the shortcomings of Aspergillus oryzae koji production, and achieve the purpose of improving the raw material utilization rate of soy sauce, enhancing the flavor and reducing costs. However, the Aspergillus niger enzyme system is relatively single, and there are natural defects in improving the raw material utilization rate. Summary of the Invention

[0004] The invention aims to provide an Aspergillus versicolor strain, which is used together with Aspergillus oryzae to make koji for soy sauce or paste, thereby significantly improving the utilization rate of the raw materials of the soy sauce or paste.

[0005] To achieve the above object, the present invention provides the following technical solution: a strain of Aspergillus versicolor, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 41829, a deposit date of March 12, 2025, and a deposit address in Beijing, China, and is classified as Aspergillus versicolor. Aspergillus versicolor .

[0006] The present invention also provides a composite bacterial agent, comprising Aspergillus oryzae and the Aspergillus versicolor.

[0007] The present invention also provides use of the Aspergillus versicolor or the composite bacterial agent in preparing condiments.

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

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

[0010] The present invention also provides a method for preparing a seasoning, comprising the step of using the Aspergillus versicolor or the composite bacterial agent to prepare the seasoning.

[0011] In a preferred embodiment, the method for preparing the condiment comprises the following steps: 1) koji making: inoculating the Aspergillus versicolor and Aspergillus oryzae into the koji making raw materials to make koji to obtain finished koji; 2) Fermentation: mixing the koji and salt water to prepare a mash, and fermenting the mash to obtain the seasoning.

[0012] In a preferred embodiment, the inoculation ratio of Aspergillus versicolor is 0.01%-0.5% of the weight of the koji-making raw materials.

[0013] In a preferred embodiment, the condiment is a fermented condiment.

[0014] In a preferred embodiment, the fermented condiment includes soybean paste and soy sauce.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The Aspergillus versicolor SC007 provided by the present invention is used in combination with Aspergillus oryzae for making koji for soybean paste and soy sauce, and can significantly improve the protease activity of the finished koji. The improvement of the enzyme activity of the finished koji allows for more complete decomposition of proteins in the raw materials, thereby increasing the amino acid nitrogen content in the mash, and further significantly improving the raw material utilization rate of the soy sauce / paste. At the same time, the high-efficiency enzyme system of Aspergillus versicolor SC007 can also reduce the total colony count of the finished product, avoid the use of high sterilization parameters, thereby reducing the flavor loss of the finished product, and improving the quality and flavor of the product. In addition, Aspergillus versicolor SC007 does not inhibit the growth and enzyme production of Aspergillus oryzae itself. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 1 is a process flow chart for preparing soy sauce in an embodiment of the present invention; Figure 2 The transparent zone state of strains SC007 and SC008 in the examples of the present invention; Figure 3 This is the transparent circle state of the spore-forming bacteria in the embodiment of the present invention; Figure 4 This is the transparent circle state of lactic acid bacteria in the embodiment of the present invention; Figure 5 This is the transparent circle state of Aspergillus oryzae in the embodiment of the present invention. DETAILED DESCRIPTION

[0017] The following is a clear and complete description of the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] In the following examples, unless otherwise specified, the raw materials used are commercially available and the processes used are conventional processes.

[0019] In the following examples, the koji making and fermentation of soybean paste and soy sauce are achieved by the following steps: soy sauce 1) Raw material pretreatment and koji making process: Non-GMO defatted soybeans are used as the main protein source. The defatted soybeans are steamed in hot water at 80-100°C for 5-10 minutes. Wheat, the main carbon source, is roasted and crushed before being mixed with the defatted soybeans. The wheat is roasted at a temperature above 100°C for 3-10 minutes.

[0020] Mix pretreated defatted soybeans and roasted wheat in a ratio of (1-2) to 1, with a moisture content of 40% to 50%. When the temperature drops below 40°C, inoculate Aspergillus oryzae and Aspergillus versicolor at a ratio of 0.01% to 0.5% of the weight of the dry mix (pretreated defatted soybeans and wheat), respectively. After the koji enters the koji room, the mixture should be 30-40 cm thick. Turn the koji after 16 and 30 hours, respectively. The temperature during the koji growth phase should be controlled between 28 and 35°C, with a maximum temperature of no more than 38°C. By the 30th hour, the koji temperature should be rapidly lowered to below 24°C. Koji making is completed within 36-48 hours to obtain the soy sauce koji.

[0021] Aspergillus versicolor can also participate in koji making by preparing crude enzyme solution: the addition ratio of crude enzyme solution is 0.1-5% of the weight of dry mixture, and the addition period is 0-24h in the koji making process, and then the normal koji making process can be followed.

[0022] 2) Fermentation: The mash is made by mixing soy sauce koji and brine. The salt content of the brine is 19-25g / 100mL, and the koji and brine are mixed in a weight ratio of 2:(2.5-3). During the first 30 days of fermentation, the temperature should be maintained at 15-18°C, and the tank should be stirred every three days. The temperature is then maintained at 28-30°C, and the fermentation cycle is 6 months.

[0023] The process flow of soy sauce preparation is detailed in Figure 1 .

[0024] soybean paste 1) Koji making process: Soybean pretreatment includes: after washing, the soybeans are steamed at a temperature of 98-120°C for 8-10 minutes; wheat flour pretreatment includes: the temperature is controlled at 98-120°C, steamed for 20-30 seconds, and the moisture content is controlled at 22%-24%. The pretreated soybeans and wheat flour are mixed in a ratio of 1:10 to 1:1, and Aspergillus oryzae and Aspergillus versicolor are inoculated in the same ratio as soy sauce, and then koji is made at 28 to 35° C. for 38 to 45 hours to obtain brewing sauce koji.

[0025] 2) Fermentation: Mix the fermented koji and brine to make a mash. The brine concentration should be 20.5% to 23.5% and the amount of brine added should be 0.9 to 1.5 times the weight of the fermented koji. Maintain the fermentation temperature at 25 to 35°C for 70 to 100 days.

[0026] Example 1 Target strain screening (1) Initial screening 1. Separation source: soy sauce / soy sauce koji, fermentation period 7d-15d soy sauce mash; 2. Primary screening method: Weigh 25 g of koji / mash, add it to 225 g of sterile saline, homogenize to obtain the sample solution to be tested, take 1 mL of the sample solution to be tested and inoculate it into 9 mL of sterile saline as a 10-fold dilution solution, and so on, for gradient dilution, take 1 mL of 2-3 appropriate dilution solutions, each of which is inoculated into a sterile empty plate, pour in PDA medium with a salt concentration of 15% and NA medium with a salt concentration of 15%, shake evenly and wait for solidification, then put it into a 30°C incubator after solidification, take it out after 3 days and observe the phenomenon; 3. Isolation and purification: Streak the single colonies obtained from the primary screening plate onto PDA and NA plates with a salt concentration of 15%, place them in a 30°C incubator, take them out after 3 days, repeat this three times and set aside.

[0027] (2) Rescreening 1. Inoculate the obtained pure culture spots onto casein medium plates and culture at 30°C for 3 days. Observe and record the clearing zones. Use the K value as the criterion for comparing the strains' abilities. The K value is the clearing zone diameter (D) divided by the colony diameter (d). The components of casein medium are shown in Table 1-1, and the clearing zone comparison results are shown in Table 1-2: Table 1-1 Components of casein culture medium

[0028] Table 1-2 Comparison of K values ​​of various colonies

[0029] Among them, the transparent zone states of strains SC007 and SC008 with the highest K values ​​are as follows Figure 2 shown.

[0030] At the same time, the existing strains of Bacillus, Lactobacillus and Aspergillus oryzae were selected and the clear zone sizes were measured under the same culture conditions as those of strains SC007 and SC008. The clear zone states of Bacillus, Lactobacillus and Aspergillus oryzae can be found in Figure 3-Figure 5 The comparison results are shown in Table 1-3: Table 1-3 Comparison of K values ​​of SC007, SC008 and existing colonies

[0031] According to the above table, the K values ​​of strains SC007, SC008 and Aspergillus oryzae are greater than those of Bacillus and lactic acid bacteria, and it is believed that they have a better effect on improving enzyme activity in koji making.

[0032] 2. Enzyme activity determination The two strains with the highest K values, SC007 and SC008, were selected and combined with Aspergillus oryzae to prepare Daqu. The enzyme activity of the finished Daqu was determined according to the method of "GB / T23527-2009 National Standard for Protease Preparations". The results are shown in Table 2: Table 2 Comparison of kojirase activity assay between strains SC007 and SC008

[0033] According to the test results, the inoculated strain SC007 had a significant advantage in kojic acid enzyme activity, so the strain SC007 was selected for subsequent strain identification and testing.

[0034] (3) Identification of bacterial species ITS sequencing was performed on strain SC007, and the sequencing results were compared with the NCBI database. The results showed that strain SC007 was Aspergillus versicolor. Aspergillus versicolor The strain was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, Beijing, China, with the deposit number CGMCC No.41829.

[0035] (IV) Detection of Sterigmatocystin The Aspergillus sterigmatotoxin in the koji prepared by strain SC007 was determined by high performance liquid chromatography-mass spectrometry, and the result was ND, indicating that Aspergillus sterigmatotoxin was not produced by Aspergillus SC007 and it was safe to use.

[0036] After evaluating the safety and application effect of Aspergillus versicolor SC007, it was determined that it would undergo subsequent application research.

[0037] Example 2 Soy sauce mixed koji fermentation Aspergillus oryzae and strain SC007 were inoculated into moist bran respectively, cultured at 30°C for 2-3 days, and seed koji was obtained, which was taken out for later use.

[0038] Experimental Group: Following the soy sauce koji production process, steamed defatted soybeans and roasted wheat flour were prepared in a dry weight ratio of 2:1. The inoculation rates of Aspergillus oryzae and strain SC007 were 0.5% and 0.1%, respectively. Alternatively, the seed koji and roasted wheat flour were first mixed before being mixed with the steamed defatted soybeans. After the mixture entered the koji room, it was turned over at 16 and 30 hours. The temperature during the koji growth phase was controlled at 28-33°C. By the 30th hour, the koji material temperature needed to be rapidly lowered to below 24°C, and the koji was removed after 38 hours. The koji was mixed evenly with brine in a dry material: brine ratio of 2:2.5, with a salt content of 19g / 100mL. The temperature was maintained at 18°C ​​for the first 30 days of fermentation, with the tank stirred every 3 days. The temperature was then maintained at 28-30°C, and the fermentation cycle lasted for 6 months.

[0039] Control group 1 (mixture of Aspergillus oryzae and Aspergillus niger): The koji making and fermentation process of this control group was the same as that of the above experimental group, except that the strain SC007 was replaced by Aspergillus niger, and the koji was mixed with Aspergillus oryzae for fermentation.

[0040] Control group 2: The control group was only inoculated with Aspergillus oryzae to make koji.

[0041] The moisture content, enzyme activity index and FN (amino acid nitrogen) index of the finished koji of the experimental group and the two control groups were measured respectively. Among them, the protease activity test referred to the "GB / T23527-2009 National Standard Protease Preparation", and the amino acid nitrogen content test referred to the "GB 5009.235-2016 National Food Safety Standard Determination of Amino Acid Nitrogen in Food". At the same time, the total number of crude oil colonies in the experimental group and the control group 2 was measured. The crude oil colony count test referred to the "GB 4789.2-2022 National Food Safety Standard Food Microbiological Examination Determination of Total Colony Number". The specific measurement data are shown in Table 3: Table 3 Related indicators of the experimental group and the control group

[0042] According to the test results: 1) Compared to control group 2, the enzyme activity of the finished soy sauce in the experimental group was significantly increased, and the FN increased by 8%, significantly improving raw material utilization. This indicates that inoculating Aspergillus oryzae with Aspergillus versicolor SC007 simultaneously can more thoroughly decompose the soy sauce raw materials, thereby significantly improving the utilization rate of soy sauce raw materials. After the soy sauce crude oil obtained from the experimental group was prepared into finished soy sauce, 15 professional tasters evaluated it and found it to have a bright red and moist color, a rich salty and savory flavor, a strong sauce aroma, and a mellow ester aroma. Therefore, the co-production of Aspergillus oryzae and Aspergillus versicolor SC007 does not inhibit the growth and enzyme production of Aspergillus oryzae itself, nor does it require major changes to the existing soy sauce preparation process and increase process costs.

[0043] 2) In the control group 1, a mixture of Aspergillus niger and Aspergillus oryzae was used to make the koji. Compared with the control group 2, only the activity of the acidic protease was significantly improved. The activities of the neutral protease and the alkaline protease were comparable to those of the control group 2. The final amino acid nitrogen content of the fermented juice was increased by approximately 0.8% compared with that of the control group 2. At the same time, a preliminary evaluation of the fermented juice was conducted. After evaluation by 15 professional tasters, it was found that the umami and sauce flavor of the juice were not significantly improved compared with those of the control group 2.

[0044] 3) In the experimental group, the crude oil obtained by inoculating Aspergillus oryzae with Aspergillus versicolor SC007 exhibited a bacterial count reduction of approximately 1 log compared to the crude oil in the control group (control group 2) produced using only Aspergillus oryzae. The highly efficient enzyme system produced by Aspergillus versicolor SC007 preemptively captures nutrients from the koji, thereby inhibiting the growth of other bacteria with similar needs. This reduction in crude oil bacterial count avoids high sterilization process parameters, reduces flavor loss in the finished product, and also saves costs and extends the shelf life of the product.

[0045] Example 3: Adding crude enzyme solution to koji fermentation for soy sauce Aspergillus oryzae and strain SC007 were inoculated into moist bran respectively, cultured at 30°C for 2-3 days, and seed koji was obtained, which was taken out for later use.

[0046] Experimental Group: SC007 seed koji was added with an equal amount of grade tertiary water and extracted at room temperature for 1-2 hours. The supernatant was filtered and concentrated to 100% by membrane concentration to obtain SC007 crude enzyme solution. Koji making and fermentation were carried out using Aspergillus oryzae according to the koji making process described in Example 2. After 24 hours of koji making, SC007 crude enzyme solution was added at 0.5% of the dry weight of the raw material. The crude enzyme solution and the raw material were thoroughly mixed by koji turning. After fermentation, the juice was filtered.

[0047] Control group: The control group was only inoculated with Aspergillus oryzae to make koji.

[0048] The moisture content, enzyme activity index and FN (amino acid nitrogen) index of the finished koji of the experimental group and the control group were measured respectively. Among them, the protease activity test referred to the "GB / T23527-2009 National Standard Protease Preparation" and the amino acid nitrogen content test referred to the "GB 5009.235-2016 National Food Safety Standard Determination of Amino Acid Nitrogen in Food". The specific measurement data are shown in Table 4: Table 4 Related indicators of experimental group and control group

[0049] The soy sauce crude oil obtained from the above experimental group was prepared into a finished soy sauce product. After being tasted by 15 professional tasters, they believed that it had a bright red color, sufficient salty and fresh flavor, rich sauce aroma, and mellow ester aroma.

[0050] According to the test results, the activity of the koji enzyme in the experimental group was significantly improved compared with the control group, and FN was increased by 6.6%, and the raw material utilization rate was greatly improved. In this embodiment, crude enzyme liquid was used to directly participate in the koji making process. Compared with the inoculated strains, the mechanism of action was clearer and the metabolic pathway was clearer, making the relationship between the activity of the koji enzyme and the increase in amino acid nitrogen more accurate.

[0051] Example 4: Bean paste mixed koji making and fermentation In this example, the Aspergillus oryzae and strain SC007 were added to the experimental group in the same manner as in Example 3 for soy sauce koji making, and the soybean paste koji making and fermentation were prepared according to existing techniques. The soybean paste fermentation period was 90 days, and the fermentation temperature was maintained at 25-28° C. The control group was inoculated with only Aspergillus oryzae for koji making.

[0052] The moisture content, enzyme activity index and FN (amino acid nitrogen) index of the finished koji of the experimental group and the control group were measured respectively. Among them, the protease activity test referred to the "GB / T23527-2009 National Standard Protease Preparation", the amino acid nitrogen content test referred to the "GB 5009.235-2016 National Food Safety Standard Determination of Amino Acid Nitrogen in Food", and the sauce mash colony count test referred to the "GB 4789.2-2022 National Food Safety Standard Food Microbiological Examination Determination of Total Colony Count". The specific measurement data are shown in Table 5: Table 5 Related indicators of the experimental group and the control group

[0053] Test results showed that the experimental group showed significantly higher enzyme activity in the finished koji compared to the control group, and a 9.4% increase in FN, significantly improving raw material utilization. Similarly, the total bacterial count in the mash of the experimental group was approximately 1 log lower than that of the control group, which has a beneficial impact on extending the shelf life of the product and improving its quality during shelf life. The fermented sauce obtained from the experimental group was prepared into finished bean paste. After being evaluated by 15 professional tasters, they found it to have a rich savory and savory flavor, a strong sauce aroma, and a mellow ester aroma.

[0054] From the above embodiments, it can be seen that the Aspergillus versicolor SC007 and Aspergillus oryzae provided by the present invention are used in combination for making koji for bean paste or soy sauce, which can significantly improve the protease activity of the finished koji. The increase in the enzyme activity of the finished koji makes the protein in the raw materials more fully decomposed, thereby increasing the amino acid nitrogen content in the sauce mash, and thus greatly improving the raw material utilization rate of soy sauce / sauce. At the same time, it can also reduce the total colony count of the finished product, reduce the flavor loss of the finished product, and improve the quality and flavor of the product. In addition, Aspergillus versicolor SC007 will not inhibit the growth and enzyme production of Aspergillus oryzae itself.

[0055] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements fall within the scope of protection claimed in the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Aspergillus versicolor, characterized in that The Aspergillus versicolor was deposited in the General Microbiology Center of China Culture Collection Administration Committee with the deposit number CGMCC No. 41829, the deposit date was March 12, 2025, the deposit address was Beijing, China, and the classification name was Aspergillus versicolor. Aspergillus versicolor .

2. A composite bacterial agent, characterized in that: The method comprises Aspergillus oryzae and the Aspergillus versicolor described in claim 1.

3. Use of the Aspergillus versicolor according to claim 1 or the composite bacterial agent according to claim 2 in the preparation of condiments.

4. The use according to claim 3, characterized in that The seasoning is a fermented seasoning.

5. The use according to claim 4, characterized in that The fermented condiments include soybean paste and soy sauce.

6. A method for preparing a condiment, characterized in that: The method comprises the step of preparing a seasoning using the Aspergillus versicolor described in claim 1 or the composite bacterial agent described in claim 2.

7. The method for preparing a seasoning according to claim 6, wherein: The following steps are involved: 1) koji making: inoculating the Aspergillus versicolor and Aspergillus oryzae into the koji making raw materials to make koji to obtain finished koji; 2) Fermentation: mixing the koji and salt water to prepare a mash, and fermenting the mash to obtain the seasoning.

8. The method for preparing a seasoning according to claim 7, wherein: The inoculation ratio of Aspergillus versicolor is 0.01%-0.5% of the weight of the koji-making raw materials.

9. The method for preparing a seasoning according to claim 6, wherein: The seasoning is a fermented seasoning.

10. The method for preparing a seasoning according to claim 9, characterized in that: The fermented condiments include soybean paste and soy sauce.

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