A method for reducing the activity of tyrosine oxidase in fermented bean curd sauce
By using sulfite, ascorbate, phosphate, vitamin C and green tea extract in fermented bean curd sauce, the problem of fermented bean curd sauce turns black, achieving color maintenance and quality improvement, and meeting food safety standards.
Patent Information
- Application Number
- CN202510023622.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-04-04
AI Technical Summary
The excessive activity of tyrosine oxidase in fermented bean curd sauce causes blackening of fermented bean curd sauce, affecting its appearance and shelf life.
The combination of sulfite and ascorbate is used as the antioxidant, the combination of sodium hexametaphosphate and sodium tripolyphosphate is used as the water retention agent, and the combination of vitamin C and green tea extract is used as the auxiliary inhibitor, and the tyrosine oxidase is extracted and purified through specific process steps to form a biological inhibitor to reduce the activity of tyrosine oxidase.
Effectively reduce the activity of tyrosine oxidase in bean curd sauce by more than 80%, keep the color of bean curd sauce unblack, maintain the best appearance and quality within the 18-month shelf life, and meets food safety standards.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of food science and food processing, in particular to a method for reducing the activity of tyrosine oxidase in fermented bean curd sauce. Background Art
[0002] Fermented bean curd is a traditional Chinese food. Fermented bean curd sauce is a sauce made with fermented bean curd as the main ingredient, combined with sesame paste, sesame oil, fine sugar and hot water. The production process includes mixing the fermented bean curd, sesame paste, sesame oil, fine sugar and hot water evenly, and then seasoning and mixing them to finally form a fermented bean curd sauce with a unique flavor, which is very popular.
[0003] Tyrosine oxidase is produced in fermented bean curd due to microbial reaction, and tyrosine oxidase will oxidize tyrosine in the fermented bean curd, causing the fermented bean curd to turn black, affecting the appearance of the fermented bean curd and shortening the shelf life of the fermented bean curd. Based on this, in order to improve the quality of the fermented bean curd and prevent it from turning black, it is urgent to find a method that can inhibit the activity of tyrosine oxidase in the fermented bean curd to prevent the fermented bean curd from turning black and extend the shelf life of the product. Summary of the invention
[0004] The technical problem to be solved by the present invention is:
[0005] Inhibit the activity of tyrosine oxidase in fermented bean curd sauce.
[0006] In order to solve the problem, one of the technical solutions adopted by the present invention is:
[0007] The production formula of fermented bean curd sauce includes the following components:
[0008]
[0009]
[0010] Specifically, the antioxidant includes a combination of sulfite and ascorbate,
[0011] Sulfites include sodium sulfite and sodium bisulfite.
[0012] Ascorbate includes sodium ascorbate, calcium ascorbate,
[0013] The ratio of sulfite to ascorbate is 1:10.
[0014] Specifically, the water retaining agent includes a combination of sodium hexametaphosphate and sodium tripolyphosphate.
[0015] The ratio of sodium hexametaphosphate to sodium tripolyphosphate is 1:2.
[0016] Specifically, the specific preparation process of the bioinhibitor is as follows:
[0017] S1. Extracting crude tyrosine oxidase enzyme solution from fermented bean curd sauce after long-term storage. First, pour out the fermented bean curd sauce and its liquid after long-term storage, mix them into PBS for thorough mixing and then centrifuge. The temperature of PBS is 10° C., the pH value is 7.0, and the centrifugation time is 10 min. After centrifugation, extract and separate the supernatant to obtain crude enzyme solution. DEAE-Sepharose is selected as the ion exchange resin according to the charge property of tyrosine oxidase. The ion exchange resin is loaded into a chromatography column. The ion exchange column is balanced with Tris-HCl buffer until the pH and ionic strength of the effluent are stable. The crude tyrosine oxidase enzyme solution is loaded onto the balanced ion exchange column at an appropriate flow rate for sample loading. The linear gradient elution is performed with buffer containing different concentrations of NaCl. The eluate is collected, and the protein concentration and enzyme activity are determined tube by tube using a spectrophotometer. After the determination, the target protein eluate is the preliminary purified enzyme solution.
[0018] S2, dialyzing the collected target protein eluate to remove excess salt and small molecule impurities, concentrating the dialyzed target protein solution to a desired volume using, for example, a freeze dryer, and determining the enzyme activity of the purified tyrosine oxidase using a dopachrome generation method to ensure that its biological activity is maintained;
[0019] S3. The purified tyrosinase was fully diluted with Shengli saline, and healthy, disease-free rabbits with well-developed immune systems were selected as immunized animals. The treated tyrosinase dilution was used as an antigen and inoculated into the rabbits by intramuscular injection. The initial inoculation dose was 3 ml per rabbit. After 20 days and 40 days, the rabbits with normal physiological status after the initial injection were given booster immunizations, and the booster immunization dose was 6 ml per rabbit.
[0020] S4. After the immunization program is completed 60 days after the initial vaccination, rabbit serum is extracted by venous blood collection. The collected serum is centrifuged at 15,000 r / min for 10 minutes to remove red blood cells and other impurities. The supernatant is collected after centrifugation, and the purified tyrosinase is used as a ligand to be combined with the fixed part of the chromatography column to form an affinity chromatography column. The serum sample containing the antibody is passed through the chromatography column to allow the antibody to specifically bind to the ligand. Finally, the antibody is eluted from the chromatography column using an appropriate eluent. After the elution is completed, the inhibitor dry powder is obtained by freeze-drying;
[0021] S5. When preparing the fermented bean curd sauce for production, the inhibitor dry powder is mixed with deionized water at a ratio of 1:200 to obtain a biological inhibitor.
[0022] Specifically, the auxiliary inhibitor includes a combination of vitamin C and green tea extract,
[0023] The ratio of vitamin C and green tea extract is 2:1.
[0024] Specifically, the processing technology of the fermented bean curd sauce is as follows: white fermented bean curd, water and thickener are ground by a colloid mill; soybean oil, starch, salt, sesame oil, chili powder and water retaining agent are added to the ground mixture and stirred evenly; the mixture is heated to 98°C in a cooking pot, white sugar is added, stirred evenly, heated to 100°C, kept warm for 20 minutes, and the heating is stopped; the temperature is lowered to 70°C, an acidity regulator is added; the temperature is further lowered to 60°C, an antioxidant is added, stirred evenly, an alcohol content regulator is added, stirred evenly, and finally a biological inhibitor and an auxiliary inhibitor are added and stirred evenly.
[0025] Specifically, the acidity regulator includes any one of white rice vinegar and citric acid. After adding the acidity regulator, the final acidity of the fermented bean curd sauce is required to be controlled at 1-1.5% (calculated as acetic acid).
[0026] Specifically, the alcohol content regulator includes any one of rice wine and alcohol. After adding the alcohol content regulator, the final alcohol content of the fermented bean curd sauce is required to be controlled at 8-10%.
[0027] Specifically, after adding salt, the final salinity of the fermented bean curd sauce is required to be controlled at 9-12%.
[0028] Beneficial effects of the present invention:
[0029] The invention provides a method for reducing the activity of tyrosine oxidase in fermented bean curd sauce, so that the activity of tyrosine oxidase in the fermented bean curd sauce is reduced by more than 80%, and the color of the fermented bean curd does not change to black or color within a shelf life of 18 months. Production comparison experiments show that under the synergistic effect of sulfite, ascorbate, phosphate, rice wine, biological inhibitor and auxiliary inhibitor, the activity of tyrosine oxidase is the lowest and the color of the fermented bean curd sauce is kept at the best. The method for reducing the activity of tyrosine oxidase in the fermented bean curd sauce is scientific and reasonable, and can maintain the quality of the fermented bean curd sauce and meet the national food safety standards. DETAILED DESCRIPTION
[0030] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.
[0031] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.
[0032] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.
[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of the present invention.
[0034] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0035] Example 1
[0036] A fermented bean curd sauce comprising the following components in parts by weight:
[0037] The production formula of fermented bean curd sauce includes the following components:
[0038]
[0039] The preparation of the fermented bean curd sauce comprises the following steps:
[0040] 1. Grind the white fermented bean curd, water and thickener through a colloid mill.
[0041] 2. Add soybean oil, starch, salt, sesame oil, chili powder and water retaining agent to the above-ground mixture and stir well.
[0042] 3. Heat the above mixture to 98°C in a cooking pot, add white sugar, stir evenly, heat to 100°C, keep warm for 20 minutes, and stop heating.
[0043] 4. Cool down to 70℃ and add acidity regulator.
[0044] 5. Continue to cool down to 60℃, add antioxidant, stir evenly, then add alcohol regulator, stir evenly, finally add biological inhibitor and auxiliary inhibitor, stir evenly.
[0045] 6. Filling to obtain the fermented bean curd sauce product.
[0046] Comparative Example 1
[0047] The difference between Comparative Example 1 and Example 1 is that the components of Comparative Example 1 do not include sulfite.
[0048] A fermented bean curd sauce comprising the following components in parts by weight:
[0049] The production formula of fermented bean curd sauce includes the following components:
[0050]
[0051]
[0052] The preparation of the fermented bean curd sauce comprises the following steps:
[0053] 1. Grind the white fermented bean curd, water and thickener through a colloid mill.
[0054] 2. Add soybean oil, starch, salt, sesame oil, chili powder and water retaining agent to the above-ground mixture and stir well.
[0055] 3. Heat the above mixture to 98°C in a cooking pot, add white sugar, stir evenly, heat to 100°C, keep warm for 20 minutes, and stop heating.
[0056] 4. Cool down to 70℃ and add acidity regulator.
[0057] 5. Continue to cool down to 60°C, add sodium ascorbate, stir evenly, then add alcohol regulator, stir evenly, and finally add biological inhibitor and auxiliary inhibitor, stir evenly.
[0058] 6. Filling to obtain the fermented bean curd sauce product.
[0059] Comparative Example 2
[0060] The difference between Comparative Example 2 and Example 1 is that the components of Comparative Example 2 do not include ascorbate.
[0061] A fermented bean curd sauce comprising the following components in parts by weight:
[0062] The production formula of fermented bean curd sauce includes the following components:
[0063]
[0064]
[0065] The preparation of the fermented bean curd sauce comprises the following steps:
[0066] 1. Grind the white fermented bean curd, water and thickener through a colloid mill.
[0067] 2. Add soybean oil, starch, salt, sesame oil, chili powder and water retaining agent to the above-ground mixture and stir well.
[0068] 3. Heat the above mixture to 98°C in a cooking pot, add white sugar, stir evenly, heat to 100°C, keep warm for 20 minutes, and stop heating.
[0069] 4. Cool down to 70℃ and add acidity regulator.
[0070] 5. Continue to cool down to 60℃, add the antioxidant sodium sulfite, stir evenly, then add the alcohol regulator, stir evenly, and finally add the biological inhibitor and auxiliary inhibitor, stir evenly.
[0071] 6. Filling to obtain the fermented bean curd sauce product.
[0072] Comparative Example 3
[0073] The difference between Comparative Example 3 and Example 1 is that the components of Comparative Example 3 do not include phosphate.
[0074] A fermented bean curd sauce comprising the following components in parts by weight:
[0075] The production formula of fermented bean curd sauce includes the following components:
[0076]
[0077]
[0078] The preparation of the fermented bean curd sauce comprises the following steps:
[0079] 1. Grind the white fermented bean curd, water and thickener through a colloid mill.
[0080] 2. Add soybean oil, starch, salt, sesame oil and chili powder to the above ground mixture and stir well.
[0081] 3. Heat the above mixture to 98°C in a cooking pot, add white sugar, stir evenly, heat to 100°C, keep warm for 20 minutes, and stop heating.
[0082] 4. Cool down to 70℃ and add acidity regulator.
[0083] 5. Continue to cool down to 60℃, add antioxidant, stir evenly, then add alcohol regulator, stir evenly, finally add biological inhibitor and auxiliary inhibitor, stir evenly.
[0084] 6. Filling to obtain the fermented bean curd sauce product.
[0085] Comparative Example 4
[0086] The difference between Comparative Example 4 and Example 1 is that no rice wine is added to the ingredients of Comparative Example 4.
[0087] The production formula of fermented bean curd sauce includes the following components:
[0088]
[0089] The preparation of the fermented bean curd sauce comprises the following steps:
[0090] 1. Grind the white fermented bean curd, water and thickener through a colloid mill.
[0091] 2. Add soybean oil, starch, salt, sesame oil, chili powder and water retaining agent to the above-ground mixture and stir well.
[0092] 3. Heat the above mixture to 98°C in a cooking pot, add white sugar, stir evenly, heat to 100°C, keep warm for 20 minutes, and stop heating.
[0093] 4. Cool down to 70℃ and add acidity regulator.
[0094] 5. Continue to cool down to 60℃, add antioxidant, stir evenly, and finally add biological inhibitor and auxiliary inhibitor, stir evenly.
[0095] 6. Filling to obtain the fermented bean curd sauce product.
[0096] Comparative Example 5
[0097] The difference between Comparative Example 5 and Example 1 is that the components of Comparative Example 5 do not include a bio-inhibitor.
[0098] A fermented bean curd sauce comprising the following components in parts by weight:
[0099] The recipe for fermented bean curd sauce includes the following ingredients:
[0100]
[0101] The preparation of the fermented bean curd sauce comprises the following steps:
[0102] 1. Grind the white fermented bean curd, water and thickener through a colloid mill.
[0103] 2. Add soybean oil, starch, salt, sesame oil and chili powder to the above ground mixture and stir well.
[0104] 3. Heat the above mixture to 98°C in a cooking pot, add white sugar, stir evenly, heat to 100°C, keep warm for 20 minutes, and stop heating.
[0105] 4. Cool down to 70℃ and add acidity regulator.
[0106] 5. Continue to cool down to 60℃, add antioxidant, stir evenly, then add alcohol regulator, stir evenly, and finally add auxiliary inhibitor, stir evenly.
[0107] 6. Filling to obtain the fermented bean curd sauce product.
[0108] Comparative Example 6
[0109] The difference between Comparative Example 6 and Example 1 is that the components of Comparative Example 5 do not include an auxiliary inhibitor.
[0110] A fermented bean curd sauce comprising the following components in parts by weight:
[0111] The recipe for fermented bean curd sauce includes the following ingredients:
[0112]
[0113] The preparation of the fermented bean curd sauce comprises the following steps:
[0114] 1. Grind the white fermented bean curd, water and thickener through a colloid mill.
[0115] 2. Add soybean oil, starch, salt, sesame oil and chili powder to the above ground mixture and stir well.
[0116] 3. Heat the above mixture to 98°C in a cooking pot, add white sugar, stir evenly, heat to 100°C, keep warm for 20 minutes, and stop heating.
[0117] 4. Cool down to 70℃ and add acidity regulator.
[0118] 5. Continue to cool down to 60℃, add antioxidant, stir evenly, then add alcohol regulator, stir evenly, and finally add biological inhibitor, stir evenly.
[0119] 6. Filling to obtain the fermented bean curd sauce product.
[0120] Performance Test:
[0121] 1. Sensory and physical and chemical analysis
[0122] The products prepared in the examples and comparative examples were stored at room temperature for 18 months and then subjected to sensory evaluation and physicochemical analysis. The sensory evaluation included evaluating the color, aroma, taste, and body of the extract. The physicochemical indicators measured the salt content, total acid (TA), alcohol content, and tyrosine oxidase activity. The results are shown in Table 1.
[0123] Table 1 Sensory evaluation and physicochemical analysis results
[0124]
[0125]
[0126] The results show that the color, aroma, taste, body and physicochemical indicators of the fermented bean curd sauce prepared in Example 1 of the present invention are the best. Since no sulfite is added to Comparative Example 1, its tyrosine oxidase activity is high, and the color of the fermented bean curd sauce becomes black and dark. Since no ascorbate is added to Comparative Example 2, its tyrosine oxidase activity is slightly high, and the color of the fermented bean curd sauce becomes slightly black and dark, and the color is poor. Since no phosphate is added to Comparative Example 3, the tyrosine oxidase activity is slightly high, the color of the fermented bean curd sauce is uneven, the surface is slightly darkened, and the color is slightly poor. Since no rice wine is added to Comparative Example 4, the tyrosine oxidase activity is slightly high, and the color of the fermented bean curd sauce is slightly darkened, and the color is slightly poor. Since no biological inhibitor was added to Comparative Example 5, the tyrosine oxidase activity was high, and the color of the fermented bean curd sauce became black and dark. Since no auxiliary inhibitor was added to Comparative Example 6, the tyrosine oxidase activity was slightly high, and the color of the fermented bean curd sauce was slightly uneven. It can be seen that under the synergistic effect of sulfite, ascorbate, phosphate, rice wine, biological inhibitor and auxiliary inhibitor, the tyrosine oxidase activity was the lowest and the color of the fermented bean curd sauce was kept best.
[0127] In summary, the preparation method of the embodiment of the present invention has the following obvious advantages:
[0128] The invention provides a method for reducing the activity of tyrosine oxidase in fermented bean curd sauce, so that the activity of tyrosine oxidase in the fermented bean curd sauce is reduced by more than 80%, and the color of the fermented bean curd does not change to black or color within a shelf life of 18 months. Production comparison experiments show that under the synergistic effect of sulfite, ascorbate, phosphate, rice wine, biological inhibitor and auxiliary inhibitor, the activity of tyrosine oxidase is the lowest and the color of the fermented bean curd sauce is kept at the best. The method for reducing the activity of tyrosine oxidase in the fermented bean curd sauce is scientific and reasonable, and can maintain the quality of the fermented bean curd sauce and meet the national food safety standards.
[0129] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A method for reducing the activity of tyrosine oxidase in fermented bean curd sauce, characterized in that The fermented bean curd sauce recipe is as follows:
2. The method for reducing the activity of tyrosine oxidase in fermented bean curd sauce according to claim 1, characterized in that: The antioxidant comprises a combination of sulfite and ascorbate, Sulfites include sodium sulfite and sodium bisulfite. Ascorbate includes sodium ascorbate, calcium ascorbate, The ratio of sulfite to ascorbate is 1:
10.
3. The method for reducing the activity of tyrosine oxidase in fermented bean curd sauce according to claim 2, characterized in that: The water retaining agent comprises a combination of sodium hexametaphosphate and sodium tripolyphosphate, The ratio of sodium hexametaphosphate to sodium tripolyphosphate is 1:
2.
4. The method for reducing the activity of tyrosine oxidase in fermented bean curd sauce according to claim 1, characterized in that: The specific preparation process of the bioinhibitor is as follows: S1. Extracting crude tyrosine oxidase enzyme solution from fermented bean curd sauce after long-term storage. First, pour out the fermented bean curd sauce and its liquid after long-term storage, mix them into PBS for thorough mixing and then centrifuge. The temperature of PBS is 10° C., the pH value is 7.0, and the centrifugation time is 10 min. After centrifugation, extract and separate the supernatant to obtain crude enzyme solution. DEAE-Sepharose is selected as the ion exchange resin according to the charge property of tyrosine oxidase. The ion exchange resin is loaded into a chromatography column. The ion exchange column is balanced with Tris-HCl buffer until the pH and ionic strength of the effluent are stable. The crude tyrosine oxidase enzyme solution is loaded onto the balanced ion exchange column at an appropriate flow rate for sample loading. A linear gradient elution is performed with a buffer containing different concentrations of NaCl. The eluate is collected, and the protein concentration and enzyme activity are determined tube by tube using a spectrophotometer. After the determination, the target protein eluate is the preliminary purified enzyme solution. S2, dialyzing the collected target protein eluate to remove excess salt and small molecule impurities, concentrating the dialyzed target protein solution to a desired volume using, for example, a freeze dryer, and determining the enzyme activity of the purified tyrosine oxidase using a dopachrome generation method to ensure that its biological activity is maintained; S3. The purified tyrosinase was fully diluted with Shengli saline, and healthy, disease-free rabbits with well-developed immune systems were selected as immunized animals. The treated tyrosinase dilution was used as an antigen and inoculated into the rabbits by intramuscular injection. The initial vaccination dose was 3 ml per rabbit. After 20 days and 40 days, the rabbits with normal physiological status after the initial injection were boosted with 6 ml per rabbit. S4. After the immunization program is completed 60 days after the initial vaccination, rabbit serum is extracted by venous blood collection. The collected serum is centrifuged at 15000r / min for 10 minutes to remove red blood cells and other impurities. The supernatant is collected after centrifugation, and the purified tyrosinase is used as a ligand to be combined with the fixed part of the chromatography column to form an affinity chromatography column. The serum sample containing the antibody is passed through the chromatography column to allow the antibody to specifically bind to the ligand. Finally, the antibody is eluted from the chromatography column using an appropriate eluent. After the elution is completed, the antibody is freeze-dried to obtain the inhibitor dry powder; S5. When preparing the fermented bean curd sauce for production, the inhibitor dry powder is mixed with deionized water at a ratio of 1:200 to obtain a biological inhibitor.
5. The method for reducing the activity of tyrosine oxidase in fermented bean curd sauce according to claim 1, characterized in that: The co-inhibitor comprises a combination of vitamin C and green tea extract, The ratio of vitamin C and green tea extract is 2:
1.
6. The method for reducing the activity of tyrosine oxidase in fermented bean curd sauce according to claim 1, characterized in that: The processing technology of the fermented bean curd sauce is as follows: white fermented bean curd, water and a thickener are ground by a colloid mill; soybean oil, starch, salt, sesame oil, chili powder and a water retaining agent are added to the ground mixture and stirred evenly; the mixture is heated to 98°C in a cooking pot, white sugar is added, stirred evenly, heated to 100°C, kept warm for 20 minutes, and the heating is stopped; the temperature is lowered to 70°C, an acidity regulator is added; the temperature is further lowered to 60°C, an antioxidant is added, stirred evenly, an alcohol content regulator is added, stirred evenly, and finally a biological inhibitor and an auxiliary inhibitor are added and stirred evenly.
7. A method for reducing tyrosine oxidase activity in fermented bean curd sauce according to claim 6, wherein the acidity regulator comprises any one of white rice vinegar and citric acid, and after adding the acidity regulator, the final acidity of the fermented bean curd sauce is required to be controlled at 1-1.5% (calculated as acetic acid).
8. A method for reducing tyrosine oxidase activity in fermented bean curd sauce according to claim 6, wherein the alcohol content regulator comprises any one of rice wine and alcohol. After adding the alcohol content regulator, the final alcohol content of the fermented bean curd sauce is required to be controlled at 8-10%.
9. The method for reducing the activity of tyrosine oxidase in fermented bean curd sauce according to claim 6, wherein after adding salt, the final salinity of the fermented bean curd sauce is required to be controlled at 9-12%.