Tenderness agent for improving tenderness of stewed meat and use method of tenderness agent

Abstract

The invention discloses a tenderness agent for improving the tenderness of stewed meat and a use method of the tenderness agent. The tenderness agent comprises Na < + > and Histatin5. The use method of the tenderness agent for improving the tenderness of the stewed meat comprises the following steps: unfreezing raw meat, putting the unfrozen raw meat into a pickling solution containing the tenderness agent, and pickling at 10 DEG C for 12 hours, the pickling solution contains 200 mM of Na < + > and 1 [mu] M of Histatin 5, and the pH value is 7.5, so that the improvement of the calpain activity and the fragmentation of myofibril in the unfrozen raw meat of the stewed meat can be realized, and the tenderness of the stewed meat can be improved. The collagen can be greatly degraded in the pickling and heating processes of the raw material meat, so that the tenderizing of the meat is realized. Ca < 2 + > can be further added into the pickling liquid, that is, the pickling liquid contains 200 mM of Na < + >, 40 mM of Ca < 2 + > and 1 mu M of Histatin 5, and the pH value is 7.5, so that the calpain activity can be improved to a greater extent, myofibril fragmentation and collagen degradation are realized to a greater extent, and meat tenderization is realized. According to the use method of the tenderness agent for improving the tenderness of the stewed meat, the raw meat is unfrozen and then placed in the pickling liquid containing the tenderness agent for low-temperature pickling, and the method is simple and easy to operate and has good market popularization value.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and more particularly to a tenderizer for improving the tenderness of stewed meat and a method for using the same. Background Art

[0002] Tenderness is a key edible quality indicator that affects consumers' acceptance of beef cooking products. The industry needs post-slaughter tenderizing technology to meet consumers' strong demand for tender beef.

[0003] Summarizing previous literature, the tenderness of meat products after heating is mainly contributed by myofibrils and connective tissue in meat. When raw meat is marinated, endogenous calpain has the ability to degrade myofibril Z-line proteins and fragment myofibrils, which can reduce the force required for consumers to bite off meat. Collagen in connective tissue begins to gelatinize at temperatures above 60°C, and the gelatinization process is affected by endogenous enzymes and polysaccharide degradation during the maturation or marinating of raw meat. At present, the main methods for tenderizing meat after slaughter include electrical stimulation of carcasses after slaughter, low-temperature maturation after slaughter, ultrasonic tumbling and marinating, static marinating, high-pressure cooking, and low-temperature long-term slow cooking. Among them, electrical stimulation of carcasses after slaughter, ultrasonic tumbling and marinating, and high-pressure cooking require the purchase of processing equipment; low-temperature maturation and static marinating after slaughter require long processing time; low-temperature long-term slow cooking requires long processing time and high energy consumption. In the low-temperature maturation process after slaughter, calpain activity is activated, but beef at 4°C needs at least 5 days for the tenderness to change significantly. Static pickling is to add polyphosphates, calcium ions, sodium chloride, enzymes, etc. to the meat, and the purpose of reducing the force required to bite off muscle fibers by increasing the product pH, increasing the activity of calpain, increasing the moisture content in the meat, and achieving meat protein hydrolysis is to achieve the purpose of reducing the force required to bite off muscle fibers. The pickling time needs 12-24 hours. However, the large-scale addition of polyphosphates has caused consumers to worry about the health of meat products. During the pickling process, exogenous addition of proteases hydrolyzes the macromolecular proteins in the connective tissue and myofibrils in the muscle, thereby reducing the muscle bite resistance and improving the tenderness quality, but there is a problem of uneven distribution of enzymes in large pieces of meat, resulting in serious problems of local tenderization of meat products. Therefore, finding a new, low-cost, high-efficiency and green meat tenderization method is still a topic that needs to be studied in the meat field. At present, there is no report on the research of regulating the tenderness of meat by adding peptides to meat, especially by regulating the activity of endogenous enzymes in meat. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a tenderizer for improving the tenderness of stewed meat, which can effectively improve the tenderness quality of stewed meat.

[0006] Another object of the present invention is to provide a method for using a tenderizer for improving the tenderness of stewed meat. The method comprises thawing the frozen meat and placing the meat in a marinating liquid containing the tenderizer and marinating the meat at low temperature. The method is simple and easy to operate and has good market promotion value.

[0007] In order to achieve these purposes and other advantages according to the present invention, a tenderizer for improving the tenderness of stewed meat is provided, which includes Na + 、Histatin 5.

[0008] Preferably, the tenderizing agent for improving the tenderness of stewed meat comprises 200 mM Na + , 0~40mM Ca 2+ , 0.5~2.5μM Histatin 5.

[0009] Preferably, the tenderizing agent for improving the tenderness of stewed meat comprises 200 mM Na + , 1 μM Histatin 5, pH of the tenderizer is 7.5. Includes 200 mM Na + , without adding Ca 2+ When the concentration of Histatin 5 is 1 μM, it can significantly enhance the degradation of Desmin protein in beef myofibrils and achieve meat tenderization.

[0010] Preferably, the tenderizing agent for improving the tenderness of stewed meat comprises 200 mM Na + , 40 mM Ca 2+ , 1 μM Histatin 5; pH of the tenderizer was 7.5. When 200 mM Na + , 40 mM Ca 2+ , 1μM Histatin 5, when the pH of the tenderizer was 7.5, the degradation degree of Desmin protein in beef myofibrils was greater than that of 200mM Na + , without adding Ca 2+ , 1μM Histatin 5, the degradation degree of Desmin protein when the tenderizer pH is 7.5.

[0011] Preferably, the tenderizing agent for improving the tenderness of stewed meat is Na + is NaCl.

[0012] Preferably, the tenderizing agent for improving the tenderness of stewed meat, Ca 2+ It is CaCl2.

[0013] The present invention also provides a method for using the tenderizer for improving the tenderness of stewed meat, which comprises thawing the raw meat, putting it into a pickling liquid containing the tenderizer, and pickling it at 10° C. for 12 hours.

[0014] Preferably, in the method for using the tenderizing agent for improving the tenderness of stewed meat, the production process of the raw meat is: after the freshly slaughtered carcass is matured at 4°C for 24 hours, the fascia and visible fat are removed, and then the meat is divided into pieces, wrapped in tin foil and quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator for later use.

[0015] Preferably, in the method of using a tenderizer to improve the tenderness of stewed meat, the ratio of the weight of the raw meat to the volume of the marinade containing the tenderizer is 1g:1.5mL.

[0016] The present invention has at least the following beneficial effects:

[0017] 1. The present invention provides a tenderizer for stewing meat, which combines the bioactive peptide human histatin 5 (Histatin 5) with divalent calcium salt to form a tenderizer with simple ingredients, which has a significant and substantial promoting effect on calpain activity, can promote the endogenous calpain activity of raw meat, increase the fragmentation of myofibrils, and thus produce positive regulation on the tenderness of fresh meat; by measuring the calpain activity, the degradation of the characteristic substrate of calpain Desmin and the change of collagen content, the optimal addition amount of Histatin 5, the applicable temperature, the optimal Ca 2+ concentration, thereby maximizing the tenderness of stewed meat;

[0018] 2. When the tenderizer provided by the present invention is used to improve the tenderness of stewed meat, it is only necessary to put the raw meat into the marinating liquid made of the tenderizer and marinate it at a low temperature for a shorter time. The operation method is simple and the high efficiency of tenderness improvement is achieved.

[0019] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the present invention, a different Ca 2+ Schematic diagram of the effect of concentration on beef tenderness;

[0021] Figure 2 This is a schematic diagram of the effect of different amounts of Histatin 5 added to the Ca0 group in Experiment 2 of the present invention on the tenderness of beef;

[0022] Figure 3 This is a schematic diagram of the effect of different amounts of Histatin 5 added to the Ca5 group in Experiment 2 of the present invention on the tenderness of beef;

[0023] Figure 4This is a schematic diagram of the effect of different amounts of Histatin 5 added to the Ca10 group in Experiment 2 of the present invention on the tenderness of beef;

[0024] Figure 5 This is a schematic diagram of the effect of different amounts of Histatin 5 added to the Ca20 group in Experiment 2 of the present invention on the tenderness of beef;

[0025] Figure 6 This is a schematic diagram of the effect of different amounts of Histatin 5 added to the Ca40 group in Experiment 2 of the present invention on the tenderness of beef;

[0026] Figure 7 This is a schematic diagram of the effects of different marinating temperatures on the regulation of beef tenderness by Histatin 5 in Experiment 3 of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments so that those skilled in the art can implement the invention with reference to the description.

[0028] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0029] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0030] In the description of the present invention, the terms "lateral", "longitudinal", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" etc. to indicate directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings and are only for the convenience of describing the present invention and simplifying the description. They do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as a limitation on the present invention.

[0031] Example 1

[0032] The present invention provides a tenderizer for improving the tenderness of stewed meat, which comprises 200mM Na + , 0~40mM Ca 2+ , 0.5-2.5 μM Histatin 5; the preferred formula of the tenderizer is 200 mM Na + , 1 μM Histatin 5; or 200 mM Na + , 40 mM Ca 2+ , 1μM Histatin 5; among them, Na+ for NaCl, Ca 2+ The pH value of the tenderizer is 7.5.

[0033] Example 2

[0034] The present invention provides a method for using a tenderizer for improving the tenderness of stewed meat (a meat pickling method). Specifically, a freshly slaughtered carcass is placed at 4°C for maturation for 24 hours, and then the fascia and visible fat are removed, and then the meat is divided into pieces, wrapped with tin foil, and then quickly frozen with liquid nitrogen, and then placed in a -80°C refrigerator for storage to obtain raw meat; the raw meat is taken out and thawed naturally at 4°C for 12 hours, and then the raw meat is placed in a pickling liquid at a ratio of 1g:1.5mL of the raw meat weight to the pickling liquid containing the tenderizer, and pickled at 10°C for 12 hours. (Water is used as a solvent to prepare the pickling liquid according to the tenderizer formula)

[0035] The present invention is described below using beef as an example, but this is not intended to limit the scope of the present invention.

[0036] 1. Test materials and equipment

[0037] 1.1 Test materials

[0038] The milon of four yellow cattle bulls aged 12-18 months with the same feeding conditions were collected at Baoding Meilianhao Supermarket, and then quickly transported back to the laboratory and matured at 4°C. When the time after slaughter was 24 hours, the surface fascia and visible fat were removed, and the meat was divided into pieces weighing about 50g. After being wrapped in tin foil and quickly frozen with liquid nitrogen, it was stored in a -80°C refrigerator for subsequent experiments.

[0039] 1.2 Test reagents

[0040] Table 1 Test reagents

[0041]

[0042]

[0043] 1.3 Test equipment

[0044] Table 2 Test instruments

[0045]

[0046] 2. Determination Method

[0047] 2.1 Determination of calpain activity

[0048] (i) Take the marinated sample (the meat is marinated according to the method for using the tenderizer to improve the tenderness of stewed meat as mentioned above), add 4°C pre-cooled active protein buffer (100 μM Tris, 10 μM EDTA, 1 mL β-mercaptoethanol; pH 8.3) to the meat at a ratio of 1 g:5 mL (1 g meat corresponds to 5 mL buffer); homogenize on ice three times, each time for 20 s, with an interval of 10 s, and centrifuge the obtained homogenate at 6000 g for 10 min at 4°C. The obtained supernatant is the crude enzyme solution containing calpain, thereby obtaining a first-processed protein sample.

[0049] (ii) The protein concentration was determined using a BCA kit. The concentration of the primary treated protein sample was adjusted to 10 μg / μL with active protein buffer and mixed with an equal volume of active protein loading solution (150 μM Tris, 20% glycerol, 0.75 mL β-mercaptoethanol, 0.02% bromophenol blue; pH 6.8) to obtain a secondary treated protein sample. At this time, the concentration of the secondary treated protein sample was 5 μg / μL.

[0050] (iii) Electrophoresis was performed using 4% concentrated gel and 12% casein-containing separation gel, with casein added in an amount of 2.1 mg / mL, and the amount of the secondary treated protein sample loaded was 10 μL. The gel was electrophoresed in an ice-water bath at a constant voltage of 100 V for 5 h. After the electrophoresis, the gel was removed and washed 3 times with incubation solution for 20 min each time, and then incubated at room temperature for 16 h. After the incubation, the gel was removed, stained, destained, and placed in a gel imaging system to obtain a casein protein electrophoresis map, and the grayscale values ​​of intact calpain 2 and autolytic protease 2 were calculated using Image J. The greater the brightness of the band in the imaging photo, the greater the grayscale value, and the higher the activity of calpain.

[0051] 2.2 Determination of Desmin Content (WB Method)

[0052] After the marinated meat samples were ground with liquid nitrogen, 0.1 g was accurately weighed and added to a 1 mL centrifuge tube, and lysis buffer was added to the centrifuge tube at a ratio of 1 g:10 mL to extract the whole muscle protein. The samples were ground at 60 Hz for 1 min using a tissue grinder, and then lysed at 4 °C for 50 min. After lysis, the samples were centrifuged at 7000 r / min for 6 min, and the supernatant was aspirated to determine its protein concentration using the BCA method. The protein concentration of the supernatant was adjusted to 3.125 μg / μL with lysis buffer, and then the reducing loading buffer was added at a ratio of 4:1 (volume ratio). At this time, the sample concentration was 2.5 μg / μL, and then boiled at 100 °C for 5 min to obtain the treated sample solution;

[0053] Refer to (iii) in 2.1 for the electrophoresis test, wherein the electrophoresis conditions are as follows: use 10% separation gel instead of 12% casein-containing separation gel, and load 10 μL; transfer the sample after electrophoresis (protein already present in the electrophoresis gel) to a membrane (transfer conditions: 200 mA, 2 h, 15 min), transfer the protein to a PVDF membrane, and then place the membrane in a fully automatic chemiluminescence / fluorescence image analysis system to obtain a Desmin degradation electrophoresis diagram, and use Image J to calculate the gray value (gray value corresponding to the Desmin content).

[0054] 2.3 Determination of heat soluble and heat insoluble collagen content in beef

[0055] Hydroxyproline standard: Accurately weigh 12.5 mg (0.0125 g) of L-hydroxyproline standard, dissolve it in water and make up to 25 mL to prepare a standard stock solution with a mass concentration of 0.5 mg / mL (500 μg / mL).

[0056] Pipette 5mL of the above standard stock solution into a 500mL volumetric flask and dilute to 5μg / mL with water. Pipette 2.5mL, 5mL, 7.5mL, 12.5mL, and 15mL of the solution into a 25mL volumetric flask, dilute to 0.5μg / mL, 1.0μg / mL, 1.5μg / mL, 2.0μg / mL, and 2.5μg / mL with the solution, respectively; measure the absorbance of each concentration of the standard solution at 558nm, and draw a standard curve of hydroxyproline concentration-absorbance.

[0057] The specific steps of the collagen content determination method are as follows:

[0058] a. Take 2g of marinated beef, add 8mL of Ren's solution, homogenize with a high-speed homogenizer and swell for 1h, heat in a 77℃ water bath for 1h, cool to room temperature and centrifuge at 6000g for 15min. The supernatant contains heat-soluble collagen and the precipitate contains heat-insoluble collagen. Add 8mL and 6M concentrated hydrochloric acid to the supernatant and precipitate respectively, and nitrate at 110℃ for 16h; determine the contents of heat-soluble collagen and heat-insoluble collagen according to steps b and c respectively;

[0059] b. Filter the nitration and hydrolysis product while hot, collect the filtrate into a volumetric flask, rinse the filter paper twice with 6M hydrochloric acid, collect the rinse solution and add it to a volumetric flask, cool it down and dilute it to 100mL with distilled water, mix it well, take the dilute solution into a flask, neutralize and remove acid with 6M / 2M NaOH, adjust the pH to 8±0.2, dilute it to 50mL, shake it well for later use;

[0060] c. Take 2 mL of the final standby solution obtained in b and place it in a test tube, add 1 mL of chloramine T (50 μM chloramine T, 135 mM citric acid monohydrate, 350 mM NaOH, 0.95 M anhydrous sodium acetate, 25% n-propanol), mix well and place at room temperature for 20 min, then add 1 mL of color developer (10% p-diaminotolualdehyde, 35% perchloric acid, 65% isopropanol), seal with a sealing film, heat at 60°C for 20 min, take out the test tube after the time is up, rinse with running water and cool for at least 3 min, place at room temperature for 30 min, measure its absorbance at 558 nm, and obtain the corresponding hydroxyproline content through the standard curve. According to the corresponding relationship that the collagen content is 7.25 times that of hydroxyproline, calculate the collagen content in the sample.

[0061] 2.4 Determination of soluble collagen content in pickling liquid after pickling

[0062] When determining the collagen content in the pickling solution, directly take 2 mL of the pickling solution and add an equal volume of 6 M concentrated hydrochloric acid to nitrate at 110° C. for 16 h. The subsequent treatment is the same as the method in steps b and c in 2.3.

[0063] Test-Ca 2+ Effect of concentration on beef tenderness

[0064] 4 cows, 18 raw meat blocks (beef) from each cow were taken and thawed naturally at 4℃ for 12h, and divided into 6 treatment groups, and the corresponding tenderizers were:

[0065] Group A (Ca0): 50 mM Tris, 200 mM NaCl, 1.5 μM Histatin 5, pH 7.5;

[0066] Group B (Ca5): 50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 1.5 μM Histatin 5, pH 7.5;

[0067] Group C (Ca10): 50 mM Tris, 200 mM NaCl, 10 mM CaCl2, 1.5 μM Histatin 5, pH 7.5;

[0068] Group D (Ca20): 50 mM Tris, 200 mM NaCl, 20 mM CaCl2, 1.5 μM Histatin 5, pH 7.5;

[0069] Group E (Ca40): 50 mM Tris, 200 mM NaCl, 40 mM CaCl2, 1.5 μM Histatin 5, pH 7.5;

[0070] Group F (control): 50 mM Tris, 200 mM NaCl, pH 7.5;

[0071] The thawed beef was placed in the corresponding marinade containing different tenderizers, and marinated at 10°C for 12 hours according to the method described in Example 2. Then, the test was performed according to the determination methods of 2.1 to 2.4. The average value of the detection of 12 fresh meat samples of 4 cows in each treatment group was taken, and the gray value corresponding to the change of the intact calpain 2 activity of the 6 treatment groups was calculated ( Figure 1 (A)) and grayscale values ​​corresponding to the activity of calpain 2 ( Figure 1 (B)), grayscale value corresponding to Desmin content ( Figure 1 (C)), casein zymogram electrophoresis ( Figure 1 (D), the bands in the figure include calpain and its degradation bands), Desmin degradation immunoblot ( Figure 1 (E)), and the correlation scatter plot of calpain band and desmin degradation band ( Figure 1 (F)), changes in collagen content in beef and marinade (marinade after marinating) (Table 3); Figure 1 Medium, control: no Ca added 2+ and Histatin 5, Ca0: No Ca added 2+ , Ca5: add 5 mM Ca 2+ , Ca10: add 10 mM Ca 2+ , Ca20: add 20 mM Ca 2 + , Ca40: add 40 mM Ca 2+ Except for the control group, all other groups were supplemented with 1.5 μM Histatin 5; different letters in each picture indicate significant differences (P<0.05).

[0072] Table 3Ca 2+ Effect of concentration on thermal solubility of beef collagen and collagen content in pickling solution

[0073]

[0074]

[0075] Note: Ca0: no Ca added 2+ , Ca5: add 5 mM Ca 2+ , Ca10: add 10 mM Ca 2+ , Ca20: add 20 mM Ca 2 + , Ca40: add 40 mM Ca 2+ , Control: No Ca added2+ and Histatin 5. Except for the control group, all other groups were supplemented with 1.5 μ MHistatin 5; Different lowercase letters in the same row indicate significant differences among the groups (P<0.05)

[0076] Desmin is one of the intermediate filament proteins that connects muscle fibers to the cell membrane and connects adjacent myofibrils. It is easily degraded by the calpain system. Therefore, the degree of desmin degradation is used to indicate the activity of calpain in muscle. The results of this experiment showed that the addition of histatin 5 had an effect on the activity of both calpain 1 and 2. Figure 1 It can be seen that without adding Ca 2+ When only 1.5 μM Histatin 5 was added, calpain activity was not promoted, and the degradation of Desmin, a substrate of calpain, was significantly slowed down. The degree of Desmin degradation in the group treated with Histatin 5 was significantly lower than that in the group without 1.5 μM Histatin 5, indicating that when calpain was not activated by Ca 2+ When activated, the addition of 1.5 μM Histatin 5 did not promote calpain activity. 2+ With the increase of Ca concentration, the activity of intact calpain gradually decreased. 2+ The activity of autolytic calpain showed a trend of gradual increase. 2+ The concentration reached the highest level at 10 mM. 2+ The degradation degree of Desmin in the treatment groups with concentrations of 10, 20, and 40 mM was significantly greater than that in the Ca 2+ The treatment groups were 0mM and 5mM. 2+ The addition of will promote the autolysis of calpain, making it more active and beneficial to improving the tenderness of beef.

[0077] Collagen is the main component of connective tissue, accounting for about 2 / 3 of connective tissue. The degradation of collagen after slaughter will destroy the integrity of connective tissue, and then destroy the integrity of muscle structure, affecting meat quality. As shown in Table 3, whether Ca is added or not, 2+ After adding Histatin 5, the total collagen (P<0.001), soluble collagen (P<0.001) and insoluble collagen (P<0.001) contents in beef were significantly reduced. On the contrary, the collagen content in each marinade was significantly increased, indicating that the addition of Histatin 5 would degrade the collagen in beef and promote the transfer of degraded collagen to the marinade during the marinade process. The specific mechanism of the effect needs to be further studied. 2+With the increase of Ca concentration, the content of collagen in beef gradually decreased. 2+ The lowest concentration is 40mM, and the collagen in beef is degraded to the greatest extent. Studies have shown that collagen content is negatively correlated with the tenderness of meat, while soluble collagen content is positively correlated with the tenderness of meat. Studies have established statistical models through meta-analysis to determine the effects of collagen content and solubility on tenderness. The study found that the collagen content and solubility in beef are significantly correlated with the tenderness of beef. This experiment explored the relationship between Ca 2+ The effect of Ca concentration on the regulation of beef tenderness by Histatin 5 was found 2+ concentration has an effect on calpain activity, but Ca 2+ It is still unclear whether there is an interactive effect between concentration and histatin 5 on tenderness.

[0078] Experiment 2: Different Ca 2+ Interactive effects of concentration and histatin 5 addition on beef tenderness

[0079] 90 pieces of meat from each cow were naturally thawed at 4°C for 12 hours and divided into five groups. The tenderizer corresponding to each group included a basic solution (50mM Tris, 200mM NaCl, pH 7.5). The five groups were added with 0 (Ca0), 5 (Ca5), 10 (Ca10), 20 (Ca20), and 40mM CaCl2 (Ca40) on the basis of the basic solution. Each group was further divided into six treatment groups. The six treatment groups were added with 0.0, 0.5, 1.0, 1.5, 2.0, and 2.5μM Histatin on the basis of the basic solution. 5. Three pieces of meat were prepared in parallel for each treatment group and marinated at 10°C for 12 hours according to the method described in Example 2. Then, the test was performed according to the determination method described in 2.1 to 2.4. The average value of the test values ​​of the 12 pieces of meat samples in each treatment group was taken, and the gray value corresponding to the activity of intact calpain 2, the gray value corresponding to the desmin content, the correlation scatter plot of the calpain band and the desmin degradation band, the change of the collagen content in beef, and the change of the collagen content in the beef marinade were calculated.

[0080] in, Figure 2 (A), (B), (C), (D), and (E) are the complete calpain 2 activity change graph, Desmin content change graph, casein zymogram electrophoresis graph, Desmin degradation immunoblot graph, and correlation scatter plot of calpain band and Desmin degradation band in the six treatment groups corresponding to Ca0 (basal solution); Note: Figure 2H0: no Histatin 5 added, H0.5: 0.5 μM Histatin 5 added, H1: 1 μM Histatin 5 added, H1.5: 1.5 μM Histatin 5 added, H2: 2 μM Histatin 5 added, H2.5: 2.5 μM Histatin 5 added, no Ca was added to this treatment group 2+ , Different letters in the pictures indicate significant differences (P<0.05);

[0081] Figure 3 (A), (B), (C), (D), (E), and (F) are the complete calpain 2 activity change graph, autolytic calpain 2 (b) activity change graph, Desmin content change graph, casein zymogram electrophoresis graph, Desmin degradation immunoblot graph, and correlation scatter plot of calpain bands and Desmin degradation bands in the six treatment groups corresponding to Ca5 (addition of 5 mM CaCl2); Note: Figure 3 H0: no Histatin 5 added, H0.5: 0.5 μM Histatin 5 added, H1: 1 μM Histatin 5 added, H1.5: 1.5 μM Histatin 5 added, H2: 2 μM Histatin 5 added, H2.5: 2.5 μM Histatin 5 added, this treatment group, 5 mM Ca added 2+ (CaCl2), different letters in the picture indicate significant differences (P<0.05);

[0082] Figure 4 (A), (B), (C), (D), (E), and (F) are the complete calpain 2 activity change graph, autolytic calpain 2 (b) activity change graph, Desmin content change graph, casein zymogram electrophoresis graph, Desmin degradation immunoblot graph, and scatter plot of the correlation between calpain bands and Desmin degradation bands in the six treatment groups corresponding to Ca10 (addition of 10 mM CaCl2); Note: Figure 4 H0: no Histatin 5 added, H0.5: 0.5 μM Histatin 5 added, H1: 1 μM Histatin 5 added, H1.5: 1.5 μM Histatin 5 added, H2: 2 μM Histatin 5 added, H2.5: 2.5 μM Histatin 5 added, this treatment group, 10 mM Ca added 2+ (CaCl2), different letters in the picture indicate significant differences (P<0.05);

[0083] Figure 5(A), (B), (C), (D), (E), and (F) are the complete calpain 2 graph, autolytic calpain 2 (b) activity change graph, Desmin content change graph, casein zymogram electrophoresis graph, Desmin degradation immunoblot graph, and scatter plot of the correlation between calpain bands and Desmin degradation bands of the six treatment groups corresponding to Ca20 (addition of 20 mM CaCl2); Note: Figure 5 H0: no Histatin 5 added, H0.5: 0.5 μM Histatin 5 added, H1: 1 μM Histatin 5 added, H1.5: 1.5 μM Histatin 5 added, H2: 2 μM Histatin 5 added, H2.5: 2.5 μM Histatin 5 added, this treatment group, 20 mM Ca added 2+ (CaCl2), different letters in the picture indicate significant differences (P<0.05);

[0084] Figure 6 (A), (B), (C), (D), (E), and (F) are the complete calpain 2 graph, autolytic calpain 2 (b) activity change graph, Desmin content change graph, casein zymogram electrophoresis graph, Desmin degradation immunoblot graph, and scatter plot of the correlation between calpain bands and Desmin degradation bands of the six treatment groups corresponding to Ca40 (addition of 40 mM CaCl2); Note: Figure 6 H0: no Histatin 5 added, H0.5: 0.5 μM Histatin 5 added, H1: 1 μM Histatin 5 added, H1.5: 1.5 μM Histatin 5 added, H2: 2 μM Histatin 5 added, H2.5: 2.5 μM Histatin 5 added, this treatment group, 40 mM Ca added 2+ (CaCl2), different letters in the picture indicate significant differences (P<0.05);

[0085] Table 4 P value table

[0086]

[0087] Tables 5 and 6 show the thermal solubility of beef collagen and the content of collagen in the marinade corresponding to Ca0, Ca5, Ca10, Ca20, and Ca40, respectively;

[0088] Table 5Ca 2+ Effects of concentration and amount of histatin 5 added on thermal solubility of beef collagen

[0089]

[0090]

[0091]

[0092] In each group of indicators, different capital letters represent the same Ca 2+ There are significant differences in the concentrations. Different lowercase letters indicate significant differences at the same Histatin 5 addition amount (P<0.05)

[0093] Table 6Ca 2+ Effects of concentration and amount of histatin 5 added on collagen content in beef marinade

[0094]

[0095] Figures 2 to 6 The results showed that in each treatment group, the different addition amounts of histatin 5 led to changes in calpain activity and the degradation of calpain-degrading substrate Desmin also changed significantly. As shown in Table 4, the Ca 2+ The interaction between the concentration and the amount of histatin 5 added had a significant effect on the activity of calpain and the degradation of its characteristic substrate desmin (P<0.05). Figure 2 It can be seen that when Ca is not added to the tenderizer 2+ When the concentration of histatin 5 reached 1 μM, the activity of intact calpain was the lowest, but no calpain autolysis band appeared in the electrophoresis experiment, which is probably because no Ca was added to the tenderizer. 2+ , resulting in the inhibition of calpain activation. At this time, the degradation of Desmin also reached its maximum, indicating that without the addition of Ca 2+ In the treatment group, the concentration of Histatin 5 at 1 μM was most beneficial to improving the tenderness of beef. Figure 3 It can be seen that when Ca in the tenderizer 2+ When the concentration was 5 mM, the degradation of Desmin reached the maximum when the concentration of Histatin 5 reached 0.5 μM, indicating that in the presence of Ca 2+ In the treatment group with 5 mM, the addition of Histatin 5 at 0.5 μM was most beneficial to improving the tenderness of beef. Figure 4 It can be seen that when Ca in the tenderizer 2+ When the concentration was 10 mM and the concentration of Histatin 5 reached 0.5 μM, the activity of intact calpain was the lowest, the activity of autolytic calpain was the highest, and the degradation degree of Desmin was also the greatest at this time, indicating that in the presence of Ca 2+ In the treatment group with 10 mM, the addition of Histatin 5 at 0.5 μM was most beneficial to improving the tenderness of beef. Figure 5 It can be seen that when Ca 2+ When the concentration was 20 mM and the concentration of Histatin 5 was 2 μM, the activity of intact calpain was the lowest, the activity of autolytic calpain was the highest, and the degradation degree of Desmin was also the greatest at this time, indicating that in the presence of Ca 2+ In the treatment group with 20 mM, the addition of Histatin 5 at 2 μM was most beneficial to improving the tenderness of beef. Figure 6 It can be seen that when Ca 2+ When the concentration was 40 mM and the concentration of Histatin 5 was 1 μM, the activity of intact calpain was the lowest, the activity of autolytic calpain was the highest, and the degradation degree of Desmin was also the greatest at this time, indicating that in the presence of Ca 2+ In the treatment group with a concentration of 40 mM, the addition of Histatin 5 at a concentration of 1 μM was most beneficial to improving the tenderness of beef.

[0096] It can be seen from Table 5 and Table 6 that Ca 2+ concentration, amount of histatin 5 added, and Ca 2+ The interaction between Ca concentration and Histatin 5 addition amount had a significant effect on the collagen content in beef and its marinade (P<0.001). 2+ With the increase of Ca concentration, the total collagen (P<0.001), soluble collagen (P<0.001) and insoluble collagen (P<0.001) contents in beef were significantly reduced; the collagen content in the marinade showed a gradual increase. 2+ , the addition of Histatin 5 will cause the collagen in the beef to degrade and migrate into the marinade.

[0097] Experiment 3 Effect of marinating temperature on the regulation of beef tenderness by Histatin5

[0098] Based on the further optimized tenderizer formula and marinating conditions obtained in Experiment 2 (50mM Tris, 200mM NaCl, 20mM CaCl2, 2μM Histatin 5, pH 7.5), the marinating temperature was studied. The thawed fresh meat blocks were marinated at different marinating temperatures. Three fresh meat blocks were set for each cow in each treatment group in parallel. The specific design is as follows:

[0099] Marinate solution A: 50 mM Tris, 200 mM NaCl, 20 mM CaCl2, 2 μM Histatin 5, pH 7.5, marinate at 4 °C for 12 h;

[0100] Marinate solution B: 50 mM Tris, 200 mM NaCl, 20 mM CaCl2, 2 μM Histatin 5, pH 7.5, 10 °C for 12 h;

[0101] Marinate solution C: 50 mM Tris, 200 mM NaCl, 20 mM CaCl2, 2 μM Histatin 5, pH 7.5, marinate at 37 °C for 12 h;

[0102] Then, the test was performed according to the determination methods in 2.1 to 2.4, and the average value of the test values ​​of the three fresh meat samples in each treatment group was taken to calculate the change of the activity of autolytic calpain 2(b) ( Figure 7 (A)) and changes in Desmin content ( Figure 7 (B)), casein zymogram electrophoresis (7(C)), desmin degradation immunoblot ( Figure 7 (D) Changes in collagen content in beef and marinade (Table 7); Different letters in each group of pictures indicate significant differences (P<0.05);

[0103] This experiment investigated the effect of storage temperature on the regulation of beef tenderness by Histatin 5. The results showed that ( Figure 7 ), the activity of complete calpain was low at these three pickling temperatures, so only the autolytic calpain activity was analyzed. When the pickling temperature was 4℃, the autolytic calpain activity was lower than that of the 10℃ pickling treatment group. In the experiment, the calpain 2 band of the 37℃ pickling treatment group could not be detected, which means that calpain 2 may be degraded into smaller molecules. The results of Desmin content showed that the degree of degradation of the 10℃ and 37℃ pickling treatment groups was significantly higher than that of the 4℃ pickling treatment group. This shows that calpain is a temperature-sensitive enzyme that can be rapidly activated under higher pickling temperatures, undergoing autolytic degradation, and subsequently increasing the degradation of Desmin. It can be found from Table 7 that the degree of collagen degradation of the samples in the 4℃ pickling treatment group was significantly lower than that of the 10℃ and 37℃ pickling treatment groups. Therefore, combining production practice helps to improve the optimal storage temperature of calpain activity is 10℃.

[0104] Table 7 Effect of storage temperature on thermal solubility of beef collagen and collagen content in pickling solution

[0105]

[0106] From Experiments 1 to 3, we can see that by treating beef under different treatment conditions (different Ca 2+ The pickled pork was marinated at different concentrations, different Histatin5 addition amounts, and different storage temperatures to study the changes in calpain activity, the degradation of calpain-degrading substrate Desmin, and collagen content. The following results were obtained:

[0107] (1) High concentration of Ca 2+ The addition of will promote the autolysis of calpain, making it show higher activity, increase the degradation rate of Desmin, the characteristic substrate of calpain, and promote the degradation of collagen, which is beneficial to improve the tenderness of beef.

[0108] (2) Regardless of whether Ca is added 2+ Adding appropriate concentrations of Histatin 5 will increase the activity of calpain, increase the degradation of Desmin in meat, and promote the degradation of collagen in beef during marinating and heating.

[0109] (3) Ca 2+ , Ca 2+ The interaction with Histatin 5 has a significant effect on the activity of calpain and the degradation of its characteristic substrate Desmin, and a significant effect on the degradation of collagen during pickling and heating; the amount of Histatin 5 added has a significant effect on the degradation of calpain's characteristic substrate Desmin, and a significant effect on the degradation of collagen during pickling and heating.

[0110] (4) The degree of desmin degradation at storage temperatures of 10℃ and 37℃ was significantly higher than that at 4℃.

[0111] In summary, the optimized use conditions of the tenderizer provided by the present invention are 200mM Na + , 40 mM Ca 2+ , 1 μM Histatin 5, pH 7.5, temperature 10 °C.

[0112] Experiment 4 Histatin 5 and Ca 2+ Validation of improving the tenderness of stewed beef by combining

[0113] The above results show that Histatin 5 and Ca 2+ The addition of Histatin 5 will promote the autolysis of calpain in raw beef after slaughter, making it more active, and will also promote the degradation of collagen, which is beneficial to the improvement of beef tenderness. Therefore, based on the above test results, a group of Histatin 5 addition amounts and suitable conditions for use that can improve the activity of calpain were selected, and the texture and shear force of the indicators that characterize the tenderness of beef after Histatin 5 treatment were detected, and the reasons for the change in tenderness were further explained by using indicators such as cooking loss, shrinkage, myofibril fragmentation index (MFI), and collagen content. Considering the interaction between the components of the tenderizer and the cost issue, in this test example, the tenderizer use conditions were selected as 50mM Tris, 200mM NaCl, 40mM CaCl2, 1μM Histatin 5, pH 7.5, and temperature of 10℃.

[0114] 4.1 Experimental materials: Semimembranous muscles of 4 24-month-old Simmental bulls with the same feeding conditions were collected from Hebei Yancheng Food Co., Ltd. After low-temperature maturation at 4°C for 24 hours, they were divided into 300g pieces of meat and vacuum-packed, and quickly transported back to the laboratory within 6 hours. The surface fascia and visible fat were removed, and the pieces were divided into 90g pieces of meat, wrapped in tin foil and quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator for subsequent experiments. The reagents used in this test example are shown in Table 8, and the equipment used is shown in Table 9.

[0115] Table 8 Test reagents

[0116]

[0117]

[0118] Table 9 Test equipment

[0119] Test instrument name and model Company Ultra-low temperature storage box (DW-86L338J) Haier Group Corporation Texture Analyzer (TMS-PILOT) US FTC Company Electronic balance (AR423CN) Ohaus Instruments (Shanghai) Co., Ltd. Electric constant temperature water bath (DZKW-D-2) Beijing Yongguangming Medical Instrument Co., Ltd. ELISA reader (SYNEGRY2) Gene Company Limited Desktop high-speed refrigerated centrifuge (XZ21K) Changsha Xiangzhi Centrifuge Instrument Co., Ltd. High speed disperser (XHF-DY) Ningbo Xinzhi Biotechnology Co., Ltd.

[0120] 4.2 Test methods:

[0121] Beef marination treatment: Frozen beef was thawed at -0.5℃ for 12h. 220g frozen samples were taken from each cow for thawing, subcutaneous fat and connective tissue were removed, and cut into 2cm×2cm×2cm pieces. Marination liquid was added at a ratio of 1:1.5 (m / v). The meat pieces from each cow were evenly distributed to each treatment group. The meat samples of each marinated treatment group contained semimembranosus muscles from 4 cows. The marinade liquid was configured as follows:

[0122] Pickling solution A (Tris group): 50 mM Tris, 200 mM NaCl, pH 7.5;

[0123] Pickling solution B (Histatin 5 group): 50 mM Tris, 200 mM NaCl, 1 μM Histatin 5, pH 7.5;

[0124] Pickling liquid C (Ca 2+ Group): 50 mM Tris, 200 mM NaCl, 40 mM CaCl2, pH 7.5;

[0125] Pickling liquid D (Histatin 5+Ca 2+ Group): 50 mM Tris, 200 mM NaCl, 40 mM CaCl2, 1 μM Histatin5, pH 7.5;

[0126] The meat was marinated at 10°C for 6, 12, and 24 h. After marinating, the meat pieces were taken out for subsequent experiments.

[0127] Post-curing treatment of beef (stewing): Take the cured beef chunks from each cow, weigh them and place them in a beaker with a solid-liquid ratio of (1:2), and heat the beaker in a 95°C water bath. Insert the thermometer probe into the center of the two chunks of meat, and start timing when the center temperature reaches 90°C. The heating ends after 1 hour. Cool the cooked meat at room temperature, dry the surface moisture with filter paper, and weigh it.

[0128] 4.3 Index determination method:

[0129] 4.3.1 Calculation of juice loss after beef cooking

[0130] Weigh the weight of the beef before and after cooking. The weight of the beef before cooking is M1. After cooking, cool it to room temperature and use filter paper to absorb the moisture on the surface of the beef to accurately weigh it as M2. Calculate the cooking loss according to the following formula:

[0131] Cooking loss (%) = (M1-M2) / M1*100

[0132] 4.3.2 Determination of transverse, longitudinal and volume shrinkage of beef

[0133] Use a vernier caliper to measure the length, width and height of the meat before and after cooking. The length is along the direction of the muscle fiber. The shrinkage rate is calculated as follows: Shrinkage rate (%) = (L1-L2) / L1*100

[0134] Wherein L1 is the length, height, width or volume of the meat piece before cooking, and L2 is the length, height, width or volume of the meat piece after cooking.

[0135] 4.3.3 Determination of beef texture

[0136] The cooked beef blocks were cut into 1cm×1cm×1cm cubes along the muscle fiber direction and measured using the texture analyzer TMSACC36 / 21mm diameter aluminum alloy cylindrical probe at a test speed of 1mm / s and a compression degree of 50% using the TPA program. When measuring the texture, the muscle fiber direction of the meat block was parallel to the ground. The hardness (N), elasticity (mm), cohesion (Ratio), adhesion (mj), stickiness (mJ), chewiness (mj) and other indicators were recorded.

[0137] 4.3.4 Determination of beef shear force (WBFS)

[0138] The cooked beef blocks were cut into 1cm×1cm×1cm cubes along the muscle fiber direction, and the shear force was measured using a single blade shear procedure. The probe model used was TMS Light Weight Blade Set, and the test speed was 1mm / s. Ten samples were measured in each group, and the results were averaged.

[0139] 4.3.5 Determination of myofibril MFI value

[0140] The myofibril fragmentation index of beef after marinating was determined according to the method in the literature.

[0141] 4.3.6 Determination of heat soluble and heat insoluble collagen content in beef

[0142] The method for determining the content of heat-soluble and heat-insoluble collagen in beef is the same as that in 2.3.

[0143] 4.3.7 Data processing

[0144] SPSS26 software was used to analyze the pickling time (6, 12, 24 h) and pickling treatment (control group, Histatin 5 group, Ca 2+ group, Histatin 5+Ca 2+ Two-way ANOVA was performed with group as fixed factor to analyze the effects of pickling time, pickling treatment and the interaction between pickling time and treatment on various indicators of beef tenderness. LSD method was used for significance analysis (P<0.05), and the results were expressed as "mean ± standard deviation".

[0145] 4.4 Results and Analysis

[0146] 4.4.1 Changes in beef cooking losses

[0147] The cooking loss rate can effectively reflect the water retention capacity of meat. During the cooking process, meat will lose water and some sarcoplasmic protein and soluble protein components. The larger the cooking loss rate, the worse the water retention of the meat. The tenderness of beef depends to a large extent on its water retention capacity. During the cooking process, excessive water loss will cause the meat to become dry and rough, and the tenderness will deteriorate.

[0148] The effects of different pickling treatments on the cooking loss of beef are shown in Table 10. The experimental results show that pickling time (P = 0.005), pickling treatment (P = 0.017) and their interaction (P = 0.002) have significant effects on the cooking loss of beef. 2+ The cooking loss of beef was not significantly affected, but Histatin 5+Ca 2+ The cooking loss of the group was significantly reduced. As the marinating time increased, the cooking loss of all treatment groups showed an upward trend. The reason for the difference in results may be that the addition of marinating liquid caused the beef to absorb more water, resulting in an increase in cooking loss.

[0149] Table 10 Changes in beef cooking loss under different marinating treatments (%)

[0150]

[0151] 4.4.2 Changes in transverse, longitudinal and volume shrinkage of beef during cooking

[0152] During the cooking process, the covalent chemical bonds of collagen in the perimysium are destroyed, presenting a regular granular structure. The contraction and fragmentation of connective tissue increase significantly, the solubility of collagen fibers increases, and the contraction of the endomysium intensifies, resulting in a decrease in the gaps between muscle fibers, loss of juice in the meat, an increase in the muscle fiber density per unit area of ​​meat, and a hardening of the meat.

[0153] The effects of different pickling treatments on the volume, transverse and longitudinal shrinkage of cooked beef are shown in Table 11. It can be seen from Table 11 that pickling time (P = 0.010) and the interaction between pickling time and pickling treatment (P = 0.002) have a significant effect on the transverse shrinkage rate of beef. With the increase of pickling time, the transverse shrinkage rate of each treatment group showed an overall increasing trend. When the pickling time was 24h, the transverse shrinkage rate of the pickling treatment group with Histatin 5 added was smaller than that of the pickling treatment group without Histatin 5, but when Histatin 5 was added in the later stage of pickling, whether Ca was added or not was different. 2+ There was no significant effect on the transverse shrinkage rate. The pickling treatment (P = 0.026) had a significant effect on the longitudinal shrinkage rate of beef. When the pickling time was 24h, adding Ca 2+ The longitudinal shrinkage rate of the pickled group was significantly lower than that of the group without Ca addition. 2+ In the pickled group, Ca 2+ The addition of Histatin 5 inhibited the longitudinal shrinkage. The pickling treatment (P<0.001) and the interaction between pickling time and pickling treatment (P<0.001) had a significant effect on the volume shrinkage of beef. When the pickling time was 12h, Histatin 5+Ca 2+ The volume shrinkage rate of the group was significantly lower than that of the Histatin 5 group, indicating that Ca 2+ The addition of Histatin 5 can inhibit the volume shrinkage. In general, Histatin 5 treatment for 12 hours significantly increased the transverse, longitudinal and volume shrinkage of cooked beef.

[0154] Table 11 Effects of different pickling treatments on the volume, transverse and longitudinal shrinkage of cooked beef (%)

[0155]

[0156] 4.4.3 Changes in beef tenderness

[0157] TPA can comprehensively measure the texture characteristics of meat by imitating the chewing process, while shear force is the most commonly used test method to quantify the tenderness of meat. The smaller the shear force, the better the tenderness. The beef chunks treated with different pickling treatments were boiled, and then TPA and shear force were measured. The test results are shown in Table 12. The results showed that pickling time and pickling treatment significantly affected the shear force (P<0.01), hardness (P<0.01), adhesion (P<0.01), cohesion (P<0.01), elasticity (P<0.01), adhesiveness (P<0.01) and chewiness (P<0.01) of beef. Except for shear force, the interaction between pickling time and pickling treatment (P<0.001) had a significant effect on the other indicators. Compared with the Tris group, the shear force of the other treatment groups was significantly reduced and Histatin 5+Ca 2+ The shear force of the group was the smallest. In the later stage of pickling, the hardness of the other treatment groups was also significantly lower than that of the Tris group. As the pickling time increased, the shear force and hardness of each pickling treatment group showed a decreasing trend, the adhesion first decreased and then increased, the cohesion first decreased and then increased, and the elasticity first increased and then decreased. The results also found that the Tris group, Histatin 5 group, Ca 2+ group, Histatin 5+Ca 2+ The shear force of the groups decreased and the hardness decreased. 2+ The shear force and hardness of the group were the smallest, indicating that its tenderness was the best.

[0158] Table 12 Changes in texture and shear force of beef under different marinating treatments

[0159]

[0160] 4.4.4 Myofibril fragmentation

[0161] MFI is an important parameter for evaluating the integrity of myofibril ultrastructure and can directly reflect the degree of myofibril degradation. It is closely related to the tenderness of meat products. Generally speaking, the higher the MFI, the higher the degree of myofibril structural damage and the better the tenderness of the meat. The changes in MFI of beef under different pickling treatments are shown in Table 13. Pickling time (P=0.006) and pickling treatment (P<0.001) significantly affected the changes in MFI values ​​of beef, while the interaction between pickling treatment and pickling time (P=0.571) had no significant effect on beef MFI. With the increase of pickling time, the MFI index of beef increased significantly, indicating that its Z-line protein was degraded, myofibrils were fragmented, and tenderness increased. Compared with the Tris group, Histatin 5+Ca 2+ The MFI index of the Histatin 5 group and the Ca 2+The MFI index of the group was significantly lower than that of Histatin 5+Ca 2+ Group, Description Ca 2+ The combination of Histatin5 and Ca showed a promoting effect on the MFI index of beef. 2+ The MFI index of the group with the highest tenderness is the highest, which is consistent with the test results in 4.3.3. This phenomenon may be due to the fact that in the post-slaughter maturation process, endogenous calpain in the meat degrades myofibrils, causing myofibrils to fragment, which results in decreased shear force and improved tenderness.

[0162] Table 13 Changes in beef MFI under different pickling treatments

[0163]

[0164] 4.4.5 Changes in the content of heat-soluble and heat-insoluble collagen

[0165] The results of the interactive effects of pickling time and pickling treatment on the thermal solubility of beef collagen are shown in Table 14. With the increase of pickling time, the total collagen (P<0.001), soluble collagen (P<0.001) and insoluble collagen (P<0.001) contents were significantly reduced. This phenomenon is consistent with the research results published in the existing literature, indicating that collagen will be degraded during the pickling process. Compared with the Tris group, the total collagen, soluble collagen and insoluble collagen contents of all pickling treatment groups were significantly reduced. Histatin 5 group, Ca 2+ group, and Ca 6+ group had a significant decrease in the thermal solubility of beef collagen. 2+ group, Histatin 5+Ca 2+ The contents of total collagen, soluble collagen and insoluble collagen in the groups decreased in turn. 2+ The collagen content of the group was the lowest, indicating that it had the best tenderness. Collagen is an important component of muscle tissue and plays an important role in maintaining muscle structure, flexibility, strength, and muscle texture. Studies have shown that collagen content is negatively correlated with meat tenderness, while soluble collagen content is positively correlated with meat tenderness.

[0166] Table 14 Interactive effects of pickling time and pickling treatment on the thermal solubility of beef collagen (ug / g)

[0167]

[0168] The above experiments further confirmed that the tenderizer provided by the present invention effectively improves the tenderness of stewed meat by adding Histatin 5.

[0169] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0170] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A tenderizer for improving the tenderness of stewed meat, characterized in that: Including Na + 、Histatin 5.

2. The tenderizer for improving the tenderness of stewed meat according to claim 1, characterized in that: Includes 200 mM Na + , 0~40mM Ca 2+ , 0.5~2.5μM Histatin 5.

3. The tenderizer for improving the tenderness of stewed meat according to claim 2, characterized in that: Includes 200 mM Na + , 1 μM Histatin 5; the pH of the tenderizer is 7.

5.

4. The tenderizer for improving the tenderness of stewed meat according to claim 2, characterized in that: Includes 200 mM Na + , 40 mM Ca 2+ , 1 μM Histatin 5, tenderizer pH 7.

5.

5. The tenderizer for improving the tenderness of stewed meat according to claim 3 or 4, characterized in that: Na + is NaCl.

6. The tenderizer for improving the tenderness of stewed meat according to claim 3 or 4, characterized in that: Ca 2+ It is CaCl2.

7. The method for using the tenderizer for improving the tenderness of stewed meat according to claims 1 to 4, characterized in that: The raw meat was thawed and placed in a marinating solution containing the tenderizer and marinated at 10° C. for 12 hours.

8. The method for using the tenderizer for improving the tenderness of stewed meat according to claim 7, characterized in that: The production process of the raw meat is as follows: a freshly slaughtered carcass is placed at 4°C for maturation for 24 hours, the fascia and visible fat are removed, the meat is then divided into pieces, the pieces are wrapped in tin foil, and then quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator for later use.

9. The method for using the tenderizer for improving the tenderness of stewed meat according to claim 8, characterized in that: The ratio of the weight of the raw meat to the volume of the marinade containing the tenderizer is 1g:1.5mL.

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