Composite tenderizing enzyme preparation, preparation method and application of composite tenderizing enzyme preparation in prepared vegetables
By developing compound tenderizing enzyme preparations and high-pressure treatment technology, we work together to improve the tendering effect of meat, solving the nutritional loss and high cost problems of traditional meat pre-made vegetables tendering technology, and achieving safe, healthy, nutritious and efficient meat pre-made vegetables products.
Patent Information
- Application Number
- CN202510230049.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-06
AI Technical Summary
The tendering technology of existing meat products pre-made vegetables has the problem of nutritional loss and high physical tendering costs due to chemical tendering, and lacks safe, healthy, nutritious and efficient meat pre-made vegetables.
Develop a compound tender enzyme preparation, including papain, trypsin and Aspergillus oryzae protease, which synergizes to improve the tendering effect of meat through high-pressure treatment and enzymatic decomposition technology.
Significantly improve the tendering effect of meat, reduce the shear force of meat, improve the water holding capacity, and achieve safe, healthy, nutritious and efficient pre-made meat dishes.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food processing, and in particular relates to a composite tenderizing enzyme preparation, a preparation method and application thereof in pre-prepared dishes. Background Art
[0002] Pre-prepared dishes are finished or semi-finished dishes made from vegetables and meat through pre-processing or pre-cooking. In recent years, pre-prepared dishes have entered a period of rapid development. According to the 2022 China Pre-prepared Meal Industry Development Report, the scale of China's pre-prepared meal market has reached 419.6 billion yuan in 2022 from 300 billion yuan in 2017, with an annual compound growth rate of about 16.36%, and is expected to exceed 830 billion yuan in 2025. The demand for "pre-prepared dishes" in China's catering, food and other markets is gradually increasing, and meat ingredients account for a large proportion of pre-prepared dishes. my country's meat production has been ranked first in the world for more than 20 consecutive years, and the market size of meat products is huge. Developing safe, healthy, nutritious, delicious and efficient meat pre-prepared dishes meets people's needs for healthy diet and expectations for a better life, and is also the core and key link for pre-prepared meal companies to seize market share. Researching or guiding the research of key technologies for processing safe, reliable, nutritious and efficient pre-prepared meat ingredients is not only a problem that pre-prepared meal companies need to consider in their development, but also the social responsibility of scientific research institutes and an important part of the work of relevant government departments.
[0003] The edible quality indicators of prepared meat dishes mainly include taste, tenderness, juiciness (water retention) and smell, among which the tenderness of meat is an important indicator for evaluating the quality of meat products. It is also one of the edible qualities that consumers pay most attention to and a key factor in determining the quality of meat products. Traditional tenderizing technologies for meat ingredients in prepared dishes are generally divided into chemical tenderizing and physical tenderizing. Chemical tenderizing is a more common tenderizing technology, which mainly includes salt tenderizing and organic acid tenderizing, which can easily destroy the protein tissue structure and cause nutritional loss. Physical tenderizing includes electrical stimulation, mechanical tenderizing, etc., which mainly achieves physical destruction of muscle structure, enhances protein hydrolysis and accelerates the maturation process, and promotes the deformation and dissolution of muscle-related proteins. This method requires large investment and high cost.
[0004] Biological tenderization mainly refers to the use of proteases to tenderize meat. Proteases generally include plant proteases, animal proteases and microbial proteases. These enzymes can break down connective tissue proteins in muscles and make the meat softer. Compared with traditional tenderization methods, enzyme tenderization has the advantages of safety, easy operation and wide sources. Developing efficient tenderization enzymes to provide technical support and safety guarantees for the development of the pre-prepared food industry is an important requirement for the development of the food processing industry. Summary of the invention
[0005] The object of the present invention is to provide a composite tenderizing enzyme preparation, a preparation method and application thereof in pre-prepared dishes, so as to solve the technical problems in the above-mentioned prior art.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] A composite tenderizing enzyme preparation is prepared from the following raw materials in parts by weight: 10-30 parts of composite enzyme, 0.1-0.3 parts of auxiliary material A, 0.05-0.15 parts of auxiliary material B, and 0.2-0.5 parts of protective agent; the composite enzyme comprises papain, trypsin, and Aspergillus oryzae protease.
[0008] Furthermore, the papain, trypsin and Aspergillus oryzae protease are prepared in a mass ratio of 2-4:1-2:1-2.
[0009] Furthermore, the activity of papain is 3000U / mL; the activity of trypsin is 2000U / mL; and the activity of Aspergillus oryzae protease is 2500U / mL.
[0010] Furthermore, the auxiliary material A is cysteine.
[0011] Furthermore, the auxiliary material B is calcium chloride.
[0012] Furthermore, the protective agent is trehalose.
[0013] The present invention also provides a method for preparing a composite tenderizing enzyme preparation, comprising the following steps:
[0014] S1: stirring and mixing papain, trypsin and Aspergillus oryzae protease to obtain a mixed enzyme solution;
[0015] S2: Prepare auxiliary materials A and B into solutions respectively, first add auxiliary material A solution into the mixed enzyme solution, stir for 3-5 minutes, then add auxiliary material B solution, continue stirring for 3-5 minutes, and finally add protective agent, stir for 5-10 minutes to obtain the composite tenderizing enzyme preparation.
[0016] Furthermore, in S1, the stirring and mixing temperature is 4-10°C, the rotation speed is 100-200 rpm, and the time is 5-10 min.
[0017] The present invention also provides an application of a composite tenderizing enzyme preparation in pre-prepared pork dishes, comprising the following steps:
[0018] S1. Meat pretreatment: Select appropriate parts of pork for pretreatment according to the type of pre-prepared dishes;
[0019] S2. High-pressure treatment: The pretreated meat is placed in a high-pressure device and subjected to ultra-high pressure reaction at a temperature of 20-50°C, 300-500Mpa, and a time of 10-20 minutes;
[0020] S3. Enzymatic hydrolysis: The composite tenderizing enzyme preparation and the high-pressure treated meat are enzymatically hydrolyzed at 30-50°C and pH 6-8 for 1-2 hours.
[0021] Furthermore, in S2, the added amount of the composite tenderizing enzyme preparation is 0.1%-0.3% of the meat mass.
[0022] The principles and effects of each raw material of the present invention are:
[0023] Papain: Papain is a proteolytic enzyme that can specifically recognize and hydrolyze a variety of proteins in meat, such as myofibrillar proteins, including myosin, actin and collagen. During the enzymatic hydrolysis process, papain breaks down large molecular proteins into smaller peptide segments by cutting the peptide bonds inside the protein molecules, making the muscle fiber structure of pork loose, reducing the toughness of pork, and thus improving tenderness.
[0024] Trypsin: Trypsin has a strong ability to hydrolyze peptide bonds formed by basic amino acids, such as lysine and arginine. After papain initially hydrolyzes pork protein, trypsin can recognize and act on the newly exposed peptide bonds containing basic amino acids, further breaking down the protein into smaller peptide segments. This relay hydrolysis method can more comprehensively break down the protein in pork, further refine the degree of protein hydrolysis, and significantly improve the tenderness of pork.
[0025] Aspergillus oryzae protease: Aspergillus oryzae protease can deeply degrade the myofibrillar protein in pork. Based on the action of papain and trypsin, Aspergillus oryzae protease can act on the remaining myofibrillar protein that has not been completely decomposed, further decomposing it into smaller peptides and amino acids, making the texture of pork more evenly tender.
[0026] Cysteine: Cysteine is an activator of papain. Papain is a thiol protease, and the thiol group (-SH) in cysteine can interact with the group in the active center of papain to maintain the active conformation of the enzyme. This enables papain to more effectively recognize and bind to protein substrates in pork, enhancing its hydrolytic activity and thus improving the tenderizing effect. Cysteine also has an antioxidant effect. In an enzymatic hydrolysis environment, some substances may oxidize the thiol group in the active center of papain, causing the enzyme to become inactive. Cysteine can protect the thiol group in the active center of papain from oxidation, maintain the activity of the enzyme, and enable it to continue to hydrolyze the protein in pork.
[0027] Calcium chloride: Calcium ions (Ca 2+ ) is an activator of trypsin. Calcium ions can bind to specific amino acid residues in the active center of trypsin, stabilize the structure of the active center of trypsin, and place the amino acid residues in the active center in a suitable position, which is conducive to its specific binding with the substrate peptide bonds in pork protein. At the same time, calcium ions can promote the binding of trypsin to pork protein substrates, change the charge distribution or conformation of pork protein, and make it easier for trypsin to approach and bind to specific peptide bonds of protein. At the same time, calcium chloride can also regulate the stability of trypsin, and within a certain concentration range, prevent trypsin from autolyzing or denaturing during enzymatic hydrolysis, so that it can continue to play a tenderizing role.
[0028] Trehalose: Trehalose is a non-reducing disaccharide that can form a protective film on the surface of the enzyme. During the enzymatic hydrolysis process, this protective film can protect the three proteases in the complex enzyme preparation from adverse factors such as temperature changes and pH fluctuations. For example, in high or low temperature environments, trehalose can prevent the enzyme from denaturing and inactivating, ensuring that the enzyme can continue to play a tenderizing role. Trehalose can also interact with the moisture in pork to stabilize the moisture content of pork. During the tenderizing process, the stable moisture content is conducive to the contact and reaction between the enzyme and the pork protein, and it can also keep the pork with a good taste and texture after processing.
[0029] The preparation principle of the composite tenderizing enzyme preparation of this application is:
[0030] S1: Many enzymes have better stability in low temperature environments, and their activity can be better preserved at low temperatures. Papain, trypsin and Aspergillus oryzae protease are all proteins, and their active structures are sensitive to temperature. Higher temperatures may cause protein denaturation, change the spatial conformation of the enzyme, and then lose activity. Controlling the temperature at 4-10°C can slow down the thermal motion of the enzyme, reduce the frequency and energy of collisions between its molecules, reduce the risk of changes in the conformation of the active center caused by collisions, and ensure that the activity of the enzyme will not drop significantly due to temperature factors during the mixing process.
[0031] Low temperature can also inhibit the growth and reproduction of microorganisms, avoiding microbial contamination and degradation of enzyme preparations. Within this temperature range, the metabolism and reproduction rate of microorganisms are significantly reduced, thus ensuring the quality and purity of enzyme preparations.
[0032] The appropriate stirring speed helps the three enzymes to mix evenly in the solution. The shear force generated by stirring can overcome the viscosity of the liquid, allowing the three enzymes to be fully dispersed in the solution and avoiding local concentrations that are too high or too low. Through stirring, papain, trypsin and Aspergillus oryzae protease can be evenly distributed in the solution, providing conditions for subsequent synergistic effects.
[0033] The stirring speed should not be too high, because too high a speed will generate a large shear force, which may damage the structure of the enzyme, resulting in a decrease or loss of enzyme activity. Controlling the speed at 100-200rpm can ensure uniform mixing of the enzyme and minimize damage to the enzyme molecular structure.
[0034] S2: Cysteine and calcium chloride are more likely to interact with enzyme molecules in solution. After the solution is prepared, their ions or molecules can diffuse and move freely, and are more likely to approach and chemically react or physically interact with enzyme molecules. For cysteine, after the solution is prepared, its sulfhydryl group can better interact with the active center of papain; for calcium chloride, calcium ions can diffuse freely in the solution and better bind to trypsin.
[0035] The solution state also makes it easier to control the amount and uniformity of addition. By preparing a solution of a certain concentration of cysteine and calcium chloride, the amount added to the mixed enzyme solution can be precisely controlled as needed, and after adding the mixed enzyme solution, it can be evenly dispersed, making the activity regulation and activation of each part of the entire enzyme preparation more uniform and consistent.
[0036] Cysteine is mainly used as an activator of papain. The purpose of adding cysteine solution first is to activate and stabilize papain. Within 3-5 minutes of stirring, the cysteine molecules in the cysteine solution can fully contact with papain. The sulfhydryl group in cysteine can interact with the group in the active center of papain to maintain the active conformation of papain and enhance its activity. At the same time, stirring can promote the diffusion of cysteine in the mixed enzyme solution, ensuring that papain can be activated and stabilized by cysteine at all sites.
[0037] The calcium ions in calcium chloride are activators of trypsin. Adding calcium chloride solution after cysteine has completed the activation of papain can avoid mutual interference between the two and ensure the activation effect of each on the target enzyme. During the stirring process, calcium ions can bind to specific amino acid residues in the active center of trypsin, stabilize the active center structure of trypsin, and improve its activity.
[0038] The application principle of the composite tenderizing enzyme preparation in the meat pre-cooked dishes is:
[0039] S1: Pork from different parts has different tissue structures and meat quality characteristics. On the one hand, it is convenient for subsequent processing and makes the processing more uniform; on the other hand, it also meets the cooking and eating requirements of finished pre-prepared dishes.
[0040] S2: Under ultra-high pressure (300-500Mpa) environment, the protein structure in pork will change. The tertiary and quaternary structures of myofibrillar proteins in pork, mainly myosin and actin, are destroyed. The head and tail structures of myosin will depolymerize, and the originally tight helical structure will become loose, exposing more internal peptide bonds and hydrophobic groups. This structural change makes the protein more susceptible to subsequent enzyme preparations. At the same time, high-pressure treatment will also change the structure of collagen, partially unraveling its triple helical structure and reducing its toughness.
[0041] The temperature is controlled at 20-50℃, which is conducive to the change of pork structure during ultra-high pressure treatment, but will not cause excessive protein denaturation due to excessive temperature. In this temperature range, the pressure can be effectively transmitted to the inside of the pork, causing moderate changes in the protein structure. If the temperature is too low, the effect of pressure on the change of protein structure may not be obvious; if the temperature is too high, it will cause excessive protein denaturation, produce unpleasant flavor and texture, and may affect the effect of subsequent enzymatic hydrolysis.
[0042] The ultra-high pressure treatment time is 10-20 minutes. During this time, the protein structure of pork can be fully changed to achieve the ideal pretreatment effect. If the time is too short, the protein structure will not be fully changed, and it may not provide enough action sites for subsequent enzymatic hydrolysis; if the time is too long, it may cause excessive damage to the tissue structure of pork, resulting in loss of nutrients and deterioration of texture, while also increasing production costs and processing time.
[0043] S3: The dosage of compound tenderizing enzyme preparation is 0.1-0.3% of the meat mass. If the dosage is too little, the enzyme will not work fully and the ideal tenderizing effect cannot be achieved. If the dosage is too much, on the one hand, it will increase the cost, and on the other hand, the texture of the pork may become too soft and rotten due to excessive hydrolysis, and lose its original taste and flavor.
[0044] The enzymatic hydrolysis temperature is controlled at 30-50℃. At this temperature, the papain, trypsin and Aspergillus oryzae protease in the composite enzyme preparation have good activity within this temperature range. This temperature range can take into account the activities of the three enzymes and ensure that they can work synergistically to tenderize the pork.
[0045] The pH is controlled at 6-8. Under this pH condition, the active center of the enzyme can maintain a suitable ionization state, which is conducive to the binding of the enzyme to the substrate and the catalytic reaction.
[0046] The enzymatic hydrolysis time is 1-2 hours. During this period of time, the compound enzyme preparation can fully hydrolyze the protein in pork. At first, papain will first act on the collagen and myofibrillar protein in pork to loosen the fiber structure; over time, trypsin and Aspergillus oryzae protease will further break down the protein and degrade it into smaller peptides and amino acids, thereby achieving a tenderizing effect. If the enzymatic hydrolysis time is too short, the tenderizing effect is not obvious; if the enzymatic hydrolysis time is too long, it may lead to over-hydrolysis, making the pork texture worse.
[0047] The beneficial effects of the present invention compared to the prior art are:
[0048] (1) The composite enzyme of the present invention uses papain, trypsin and Aspergillus oryzae protease as main raw materials. Papain first opens the structure of protein, trypsin then cuts specific peptide bonds, and finally Aspergillus oryzae protease performs deep hydrolysis. This relay hydrolysis method can more comprehensively decompose the protein in pork. The three work together to synergistically enhance the effect, thereby significantly improving the tenderization effect of meat.
[0049] (2) In the composite enzyme preparation of the present invention, by adding cysteine, it can interact with the groups in the active center of papain to maintain the active conformation of the enzyme. This enables papain to more effectively recognize and bind to the protein substrate in pork, enhance its hydrolysis activity, and thus improve the tenderizing effect; by adding calcium chloride, calcium ions can bind to specific amino acid residues in the active center of trypsin, stabilize the structure of the active center of trypsin, and place the amino acid residues in the active center in a suitable position, which is conducive to its specific binding with the substrate peptide bond in pork protein. The two can jointly enhance the effect of the composite enzyme, thereby improving the tenderizing effect.
[0050] (3) The composite tenderizing enzyme preparation of the present invention and high pressure treatment jointly tenderize meat. After ultra-high pressure treatment, the structure of pork protein becomes loose, exposing more peptide bonds, so that the composite enzyme can more easily bind to these newly exposed substrates, thereby improving the binding efficiency of the enzyme and the substrate. DETAILED DESCRIPTION
[0051] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, preferred embodiments are given below to further describe the present invention in detail. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.
[0052] A composite tenderizing enzyme preparation is prepared from the following raw materials in parts by weight: 10-30 parts of composite enzyme, 0.1-0.3 parts of auxiliary material A cysteine, 0.05-0.15 parts of auxiliary material B calcium chloride, and 0.2-0.5 parts of protective agent trehalose; the composite enzyme comprises papain, trypsin, and Aspergillus oryzae protease in a mass ratio of 2-4:1-2:1-2.
[0053] The activity of the papain is 3000U / mL; the activity of the trypsin is 2000U / mL; and the activity of the Aspergillus oryzae protease is 2500U / mL.
[0054] A method for preparing a composite tenderizing enzyme preparation comprises the following steps:
[0055] S1: stirring and mixing papain, trypsin and Aspergillus oryzae protease at a temperature of 4-10° C. and a rotation speed of 100-200 rpm for 5-10 minutes to obtain a mixed enzyme solution;
[0056] S2: Prepare auxiliary materials A and B into solutions respectively, first add auxiliary material A solution into the mixed enzyme solution, stir for 3-5 minutes, then add auxiliary material B solution, continue stirring for 3-5 minutes, and finally add protective agent, stir for 5-10 minutes to obtain the composite tenderizing enzyme preparation.
[0057] The application of a composite tenderizing enzyme preparation in pork pre-cooked dishes comprises the following steps:
[0058] S1. Meat pretreatment: Select appropriate parts of pork for pretreatment according to the type of pre-prepared dishes;
[0059] S2. High-pressure treatment: The pretreated meat is placed in a high-pressure device and subjected to ultra-high pressure reaction at a temperature of 20-50°C, a pressure of 300-500 MPa, and a time of 10-20 minutes;
[0060] S3. Enzymatic hydrolysis: 0.1%-0.3% of the composite tenderizing enzyme preparation based on the weight of the meat is added to the high-pressure treated meat and enzymatically hydrolyzed for 1-2 hours at 30-50°C and pH 6-8.
[0061] The following is a more specific example.
[0062] Example 1
[0063] A composite tenderizing enzyme preparation is prepared from the following raw materials in parts by weight: 10 parts of composite enzyme, 0.2 parts of cysteine, 0.1 parts of calcium chloride and 0.5 parts of trehalose; the composite enzyme comprises papain, trypsin and Aspergillus oryzae protease in a mass ratio of 4:2:1.
[0064] A method for preparing a composite tenderizing enzyme preparation comprises the following steps:
[0065] S1: Stir and mix papain, trypsin and Aspergillus oryzae protease at 8°C and 180 rpm for 5 min to obtain a mixed enzyme solution;
[0066] S2: Prepare solutions of cysteine and calcium chloride respectively, first add the cysteine solution into the mixed enzyme solution, stir for 4 minutes, then add the calcium chloride solution, continue stirring for 3 minutes, and finally add trehalose, stir for 8 minutes to obtain the composite tenderizing enzyme preparation.
[0067] The application of a composite tenderizing enzyme preparation in pork pre-cooked dishes comprises the following steps:
[0068] S1. Meat pretreatment: According to the type of prepared dish, select a suitable part of pork for pretreatment. In this embodiment, the type of prepared dish is pork chops, and the selected part is pork tenderloin;
[0069] S2. High-pressure treatment: The pretreated meat is placed in a high-pressure device and subjected to ultra-high pressure reaction at a temperature of 50°C, a pressure of 500 MPa, and a time of 12 minutes;
[0070] S3. Enzymatic hydrolysis: 0.2% of the composite tenderizing enzyme preparation by weight of the meat and the high-pressure treated meat are enzymatically hydrolyzed at 50°C and pH 7 for 1 hour.
[0071] Example 2
[0072] A composite tenderizing enzyme preparation is prepared from the following raw materials in parts by weight: 20 parts of composite enzyme, 0.15 parts of cysteine, 0.05 parts of calcium chloride and 0.4 parts of trehalose; the composite enzyme comprises papain, trypsin and Aspergillus oryzae protease in a mass ratio of 2:1:1.
[0073] A method for preparing a composite tenderizing enzyme preparation comprises the following steps:
[0074] S1: Papain, trypsin and Aspergillus oryzae protease were stirred and mixed at a temperature of 6°C and a rotation speed of 200 rpm for 6 minutes to obtain a mixed enzyme solution;
[0075] S2: Prepare solutions of cysteine and calcium chloride respectively, first add the cysteine solution into the mixed enzyme solution, stir for 5 minutes, then add the calcium chloride solution, continue stirring for 4 minutes, and finally add trehalose, stir for 5 minutes to obtain the composite tenderizing enzyme preparation.
[0076] The application of a composite tenderizing enzyme preparation in pork pre-cooked dishes comprises the following steps:
[0077] S1. Meat pretreatment: According to the type of prepared dish, select a suitable part of pork for pretreatment. In this embodiment, the type of prepared dish is pork chops, and the selected part is pork tenderloin;
[0078] S2. High-pressure treatment: The pretreated meat is placed in a high-pressure device and subjected to ultra-high pressure reaction at a temperature of 20°C, a pressure of 400 MPa, and a time of 15 minutes;
[0079] S3. Enzymatic hydrolysis: 0.1% of the composite tenderizing enzyme preparation based on the weight of the meat and the high-pressure treated meat are enzymatically hydrolyzed at 40°C and pH 8 for 2 hours.
[0080] Example 3
[0081] A composite tenderizing enzyme preparation is prepared from the following raw materials in parts by weight: 30 parts of composite enzyme, 0.1 parts of cysteine, 0.15 parts of calcium chloride and 0.2 parts of trehalose; the composite enzyme comprises papain, trypsin and Aspergillus oryzae protease in a mass ratio of 3:2:1.
[0082] A method for preparing a composite tenderizing enzyme preparation comprises the following steps:
[0083] S1: Papain, trypsin and Aspergillus oryzae protease were stirred and mixed at a temperature of 10°C and a rotation speed of 150 rpm for 8 minutes to obtain a mixed enzyme solution;
[0084] S2: Prepare solutions of cysteine and calcium chloride respectively, first add the cysteine solution into the mixed enzyme solution, stir for 3 minutes, then add the calcium chloride solution, continue stirring for 5 minutes, and finally add trehalose, stir for 10 minutes to obtain the composite tenderizing enzyme preparation.
[0085] The application of a composite tenderizing enzyme preparation in pork pre-cooked dishes comprises the following steps:
[0086] S1. Meat pretreatment: According to the type of prepared dish, select the appropriate part of pork for pretreatment; the type of prepared dish in this embodiment is pork chops, and the selected part is pork tenderloin;
[0087] S2. High-pressure treatment: The pretreated meat is placed in a high-pressure device and subjected to ultra-high pressure reaction at a temperature of 30°C, a pressure of 300 MPa, and a time of 20 minutes;
[0088] S3. Enzymatic hydrolysis: 0.3% of the composite tenderizing enzyme preparation based on the meat mass and the high-pressure treated meat are enzymatically hydrolyzed at 50°C and pH 7 for 2 hours.
[0089] Example 4
[0090] A composite tenderizing enzyme preparation is prepared from the following raw materials in parts by weight: 15 parts of composite enzyme, 0.3 parts of cysteine, 0.12 parts of calcium chloride and 0.3 parts of trehalose; the composite enzyme comprises papain, trypsin and Aspergillus oryzae protease in a mass ratio of 4:1:2.
[0091] A method for preparing a composite tenderizing enzyme preparation comprises the following steps:
[0092] S1: Stir papain, trypsin and Aspergillus oryzae protease at 4°C and 100 rpm for 10 min to obtain a mixed enzyme solution;
[0093] S2: Prepare solutions of cysteine and calcium chloride respectively, first add the cysteine solution into the mixed enzyme solution, stir for 4 minutes, then add the calcium chloride solution, continue stirring for 3 minutes, and finally add trehalose, stir for 6 minutes to obtain the composite tenderizing enzyme preparation.
[0094] The application of a composite tenderizing enzyme preparation in pork pre-cooked dishes comprises the following steps:
[0095] S1. Meat pretreatment: According to the type of prepared dish, select a suitable part of pork for pretreatment. In this embodiment, the type of prepared dish is pork chops, and the selected part is pork tenderloin;
[0096] S2. High-pressure treatment: The pretreated meat is placed in a high-pressure device and subjected to ultra-high pressure reaction at a temperature of 40°C, a pressure of 450 MPa, and a time of 10 minutes;
[0097] S3. Enzymatic hydrolysis: 0.15% of the composite tenderizing enzyme preparation based on the meat mass and the high-pressure treated meat are enzymatically hydrolyzed at 30°C and pH 6 for 1 hour.
[0098] Comparative Example 1
[0099] The method is basically the same as Example 1, except that no composite tenderizing enzyme preparation is used for enzymolysis.
[0100] Comparative Example 2
[0101] It is basically the same as Example 1, except that in the composite tenderizing enzyme preparation, trypsin and Aspergillus oryzae protease are not added, but only papain is used.
[0102] Comparative Example 3
[0103] The method is basically the same as Example 1, except that papain and Aspergillus oryzae protease are not added to the composite tenderizing enzyme preparation, but only trypsin is used.
[0104] Comparative Example 4
[0105] It is basically the same as Example 1, except that papain and trypsin are not added to the composite enzyme preparation, but only Aspergillus oryzae protease is used.
[0106] Comparative Example 5
[0107] It is basically the same as Example 1, except that cysteine is not added to the composite tenderizing enzyme preparation.
[0108] Comparative Example 6
[0109] It is basically the same as Example 1, except that no calcium chloride is added to the composite tenderizing enzyme preparation.
[0110] Comparative Example 7
[0111] The method is basically the same as Example 1, except that the meat is not subjected to high pressure treatment.
[0112] 1. Meat tenderization experiment
[0113] 1.1 Shear force: The pork tenderloin after enzymatic hydrolysis of Examples 1-4 and Comparative Examples 1-6 was subjected to shear force measurement according to the method of NY / T 1180-2006. The shear force measurement parameters were as follows: probe HDP / BSW; downward pressure distance 25.00 mm; speed before test 1.00 mm / s; speed during test 1.00 mm / s; speed after test 10.00 mm / s; number of cycles 2 times; sample size 2 cm×1 cm×1 cm; repeated 3 times and the average value was taken.
[0114] 1.2 Water holding capacity: Samples of the same mass of the pork tenderloin after enzymatic hydrolysis in Examples 1-4 and Comparative Examples 1-6 were taken respectively, centrifuged in a centrifuge, and then weighed again to calculate the water holding capacity.
[0115] Water holding capacity = sample water content - (weight before centrifugation - weight after centrifugation) / 10 × 100%
[0116] Table 1 Shear force and water holding capacity
[0117]
[0118]
[0119] It can be seen from Table 1 that:
[0120] (1) In Examples 1-4 using the composite tenderizing enzyme preparation and tenderizing method of the present invention, the shear force of the tenderized meat was 2.14kg, 2.78kg, 3.02kg, and 2.44kg, respectively, and the water holding capacity was 56.46%, 54.21%, 52.12%, and 55.78%, respectively. It can be seen that the composite tenderizing enzyme preparation of the present application can significantly improve the tenderizing effect of meat, reduce the shear force of meat, and improve the water holding capacity.
[0121] The comparative examples 1-4 which do not use the composite tenderizing enzyme preparation of the present application and do not fully use the components of the composite tenderizing enzyme preparation of the present application have shear forces of 6.06kg, 4.51kg, 5.04kg, and 5.23kg, respectively, and water holding capacities of 31.70%, 41.25%, 38.51%, and 37.12%, respectively, which are all inferior to the examples 1-4 of the present application. This indicates that the papain, trypsin, and Aspergillus oryzae protease in the composite enzyme preparation of the present application can interact with each other, synergistically enhance the effect, and jointly improve the tenderizing effect of meat.
[0122] This is because: papain is a thiol protease that can specifically recognize peptide bonds in protein molecules in pork, especially the carboxyl side peptide bonds of arginine and lysine residues. In the initial stage of enzymatic hydrolysis, papain will come into contact with the main protein components in pork, such as myosin, actin and collagen, and hydrolyze peptide bonds, cutting large molecular proteins into smaller fragments. At the same time, the hydrolysis of pork protein by papain will cause the muscle fiber structure to become loose, while the myofibrillar protein in pork is responsible for the contraction and relaxation of muscles, and its structure is relatively tight. Papain can weaken the connection between muscle fibers and spread the fiber bundles, which initially improves the tenderness of pork. At the same time, it allows more water to be accommodated between protein molecules, increasing the water retention of pork.
[0123] In addition, the hydrolysis of papain will expose more protein structures and cleavage sites that were previously hidden. These newly exposed sites provide better working conditions for trypsin and Aspergillus oryzae protease, allowing them to bind to pork proteins more effectively, creating favorable conditions for the subsequent hydrolysis process.
[0124] Trypsin is a serine protease that has a strong hydrolysis effect on peptide bonds formed by basic amino acids, such as lysine and arginine. After papain initially hydrolyzes pork protein, trypsin can recognize and act on newly exposed peptide bonds containing basic amino acids. This specific cutting action allows protein molecules to be further broken down into smaller peptide segments.
[0125] At the same time. Trypsin and papain have different action sites. Papain first opens the protein structure, and trypsin then cuts specific peptide bonds. This relay hydrolysis method can more comprehensively decompose the protein in pork. For example, for some complex myofibrillar protein structures, the synergistic effect of the two enzymes can more effectively decompose them. The hydrolysis of trypsin further refines the degree of protein hydrolysis and significantly improves the tenderness of pork.
[0126] In addition, the hydrolysis products of trypsin, including some small peptides and amino acids, can regulate the activity of Aspergillus oryzae protease. These products may act as activators or inhibitors, affecting the binding ability of Aspergillus oryzae protease with pork protein or its catalytic activity, thereby promoting the deep hydrolysis of pork protein by Aspergillus oryzae protease under appropriate conditions.
[0127] Aspergillus oryzae protease can deeply degrade the main myofibrillar proteins such as myosin in pork. Based on the action of papain and trypsin, Aspergillus oryzae protease can act on the remaining myofibrillar proteins that have not been completely decomposed, further decomposing them into smaller peptides and amino acids, making the texture of pork more evenly tender. At the same time, some specific peptides produced by Aspergillus oryzae protease during the hydrolysis process can feedback regulate the activity of papain and trypsin. These peptides may bind to papain and trypsin, change their conformations, and thus affect the binding ability of their active centers with substrates, so that the entire complex enzyme system can self-regulate according to the degree of hydrolysis of pork protein and maintain efficient tenderization.
[0128] Therefore, in the composite tenderizing enzyme preparation of the present application, by using papain, trypsin and Aspergillus oryzae protease, they can work together, synergistically enhance the effect, and jointly improve the tenderizing effect on meat.
[0129] (2) In Comparative Examples 5-6, cysteine or calcium chloride in the composite tenderizing enzyme preparation of the present application was not used, and the shear forces were 3.65 kg and 3.87 kg, respectively, and the water holding capacity was 38.48% and 50.26%, respectively, which were all inferior to Examples 1-4 of the present application. This indicates that the addition of cysteine and calcium chloride to the composite tenderizing enzyme preparation of the present application can improve the tenderizing effect of the composite tenderizing enzyme preparation.
[0130] This is because: papain is a thiol protease, and the thiol group (-SH) in cysteine can interact with the group in the active center of papain. In the active center of the enzyme, cysteine can participate in maintaining the active conformation of the enzyme, ensuring that the active site of papain is in the correct spatial position, so that it can effectively recognize and bind to the protein substrate in pork. At the same time, cysteine also has an antioxidant effect, protecting the thiol group in the active center of papain from oxidation, maintaining the activity of the enzyme, and enabling it to continue to hydrolyze the protein in pork, thereby improving the tenderizing effect. In addition, cysteine can also regulate the enzymatic reaction process of papain. By interacting with the substrate binding site or other regulatory sites of papain, the affinity of the enzyme for the substrate is changed, so that the enzyme's hydrolysis activity for specific peptide bonds in pork protein is enhanced, which is more conducive to destroying the fiber structure of pork and improving the tenderizing efficiency.
[0131] Active center stability: calcium ions (Ca 2+ ) is an activator of trypsin. The calcium ions provided by calcium chloride can bind to specific amino acid residues in the active center of trypsin. These calcium ions can stabilize the structure of the active center of trypsin, so that the amino acid residues in the active center are in a suitable position, which is conducive to its specific binding with the substrate peptide bond in pork protein. For example, calcium ions can interact with aspartic acid residues in the active center of trypsin, maintain the three-dimensional structure of the active center, and thus improve the activity of trypsin.
[0132] Calcium ions in calcium chloride can promote the binding of trypsin to pork protein substrates. Calcium ions can change the charge distribution or conformation of pork protein, making it easier for trypsin to approach and bind to specific peptide bonds of the protein. In the presence of calcium ions, the charge environment around some basic amino acid residues in pork protein, such as lysine and arginine, may change, and the peptide bonds formed by these amino acid residues are more easily recognized and hydrolyzed by trypsin, thereby enhancing the tenderizing effect of trypsin on pork. Calcium chloride can also regulate the stability of trypsin. Within a certain concentration range, calcium ions can prevent trypsin from autolyzing or denaturing during enzymatic hydrolysis, and can interact with certain domains in the trypsin molecule to enhance the interaction force within the enzyme molecule, so that trypsin can maintain good stability under different temperature, pH and other conditions, and continue to play its tenderizing effect.
[0133] (3) In Comparative Example 7, the shear force of the meat that has not been subjected to high-pressure treatment is 4.16 kg and the water holding capacity is 49.21%, respectively. This indicates that the present application can further improve the tenderizing effect of meat by combining the composite tenderizing enzyme preparation with high-pressure treatment.
[0134] This is because: Ultra-high pressure treatment will cause significant changes in the protein structure in pork. Under high pressure, the tertiary and quaternary structures of myofibrillar proteins such as myosin and actin will be destroyed. For example, the head and tail structures of myosin will depolymerize, and the originally tight spiral structure will become loose, exposing more internal peptide bonds and hydrophobic groups. This structural change makes the protein more susceptible to enzyme action. Ultra-high pressure will also destroy the cell structure of pork. The cell membrane and cell wall will rupture under high pressure, and the components in the cell, such as various enzymes and small molecules, will be released. The texture of pork becomes softer and the contact opportunity between the compound enzyme preparation and the protein is increased. The protein that was originally confined in the cell by the cell membrane and cell wall can better contact with the enzyme after the cell structure is destroyed, thereby improving the efficiency of enzymatic hydrolysis.
[0135] Since the structure of pork protein becomes loose after ultra-high pressure treatment, exposing more peptide bonds, papain, trypsin and Aspergillus oryzae protease in the complex enzyme preparation can more easily bind to these newly exposed substrates. Papain can find and hydrolyze specific peptide bonds in collagen more quickly, while trypsin can more effectively act on peptide bonds containing basic amino acids exposed by ultra-high pressure, and Aspergillus oryzae protease can also better access the internal structure of myofibrillar protein, thereby performing deep hydrolysis. In addition, pork treated with ultra-high pressure provides more favorable conditions for enzymatic hydrolysis, which speeds up the enzymatic hydrolysis reaction. The binding efficiency of the enzyme and the substrate is improved, the activation energy of the reaction is reduced, and the complex enzyme preparation can hydrolyze more protein in the same amount of time.
[0136] The above content cannot be used to determine that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can make several simple deductions or substitutions without departing from the concept of the present invention, which should be regarded as belonging to the scope of patent protection of the present invention determined by the submitted claims.
Claims
1. A composite tenderizing enzyme preparation, characterized in that: The invention is prepared from the following raw materials in parts by weight: 10-30 parts of compound enzyme, 0.1-0.3 parts of auxiliary material A, 0.05-0.15 parts of auxiliary material B and 0.2-0.5 parts of protective agent; the compound enzyme comprises papain, trypsin and Aspergillus oryzae protease.
2. A composite tenderizing enzyme preparation according to claim 1, characterized in that: The papain, trypsin and Aspergillus oryzae protease are prepared in a mass ratio of 2-4:1-2:1-2.
3. A composite tenderizing enzyme preparation according to claim 1, characterized in that: The activity of the papain is 3000U / mL; the activity of the trypsin is 2000U / mL; and the activity of the Aspergillus oryzae protease is 2500U / mL.
4. A composite tenderizing enzyme preparation according to claim 1, characterized in that: The auxiliary material A is cysteine.
5. The composite tenderizing enzyme preparation according to claim 1, characterized in that: The auxiliary material B is calcium chloride.
6. The composite tenderizing enzyme preparation according to claim 1, characterized in that: The protective agent is trehalose.
7. A method for preparing a composite tenderizing enzyme preparation according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1: stirring and mixing papain, trypsin and Aspergillus oryzae protease to obtain a mixed enzyme solution; S2: Prepare auxiliary materials A and B into solutions respectively, first add auxiliary material A solution into the mixed enzyme solution, stir for 3-5 minutes, then add auxiliary material B solution, continue stirring for 3-5 minutes, and finally add protective agent, stir for 5-10 minutes to obtain the composite tenderizing enzyme preparation.
8. The method for preparing a composite tenderizing enzyme preparation according to claim 7, characterized in that: In S1, the stirring and mixing temperature is 4-10°C, the rotation speed is 100-200 rpm, and the time is 5-10 min.
9. Use of a composite tenderizing enzyme preparation according to claim 7 or 8 in pre-prepared pork dishes, characterized in that: The following steps are involved: S1. Meat pretreatment: Select appropriate parts of pork for pretreatment according to the type of pre-prepared dishes; S2. High-pressure treatment: The pretreated meat is placed in a high-pressure device and subjected to ultra-high pressure reaction at a temperature of 20-50°C, a pressure of 300-500 MPa, and a time of 10-20 minutes; S3. Enzymatic hydrolysis: The composite tenderizing enzyme preparation and the high-pressure treated meat are enzymatically hydrolyzed at 30-50°C and pH 6-8 for 1-2 hours.
10. The use of a composite tenderizing enzyme preparation according to claim 9 in pre-prepared pork dishes, characterized in that: In S2, the added amount of the composite tenderizing enzyme preparation is 0.1%-0.3% of the meat mass.