Preparation method of cobra meat enzymolysis active polypeptide
Through enzymatic lysis reaction and multi-step treatment, the enzymatic lysis active polypeptide of cobra meat was successfully prepared, solving the problem of lack of such polypeptide preparation methods in the prior art, achieving an efficient and simple preparation process, and having good antioxidant properties.
Patent Information
- Application Number
- CN202510268423.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-07
- Publication Date
- 2025-06-06
AI Technical Summary
The existing technology lacks an effective preparation method for cobra enzymatic active polypeptide, which limits its application in the fields of antioxidant and other fields.
By stirring the cobra meat into minced meat and mixing it with water, adjusting the pH to 7.0, combining bromelain and neutral protease for enzymatic reaction, followed by centrifugation and dialysis to obtain the polypeptide liquid and obtain the active polypeptide powder by lyophilization.
The prepared cobra meat active polypeptide has good antioxidant activity, is simple in process, relatively easy to operate, and is suitable for large-scale production.
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Figure CN120099124A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of animal polypeptide preparation, in particular to a method for preparing cobra meat enzymatically hydrolyzed active polypeptide. Background Art
[0002] Snake meat contains 16% protein and more than 30 kinds of amino acids, including 8 essential amino acids for the human body, such as glutamic acid, aspartic acid, lysine and isoleucine. Glutamic acid can enhance the vitality of brain cells, and aspartic acid can help muscles eliminate fatigue. In addition, snake meat also contains thiamine, riboflavin, cobalamin, vitamin A, tocopherol and other vitamins, as well as zinc, copper, manganese, selenium, cobalt and other trace elements. Iron and cobalt have important physiological effects in blood tonic and hematopoiesis. It is worth mentioning that snake meat is rich in natural taurine, which plays an important role in promoting the development of brain tissue and intelligence in infants and young children. The unique effects of snake meat in enhancing immunity and anti-fatigue make its tonic effect comparable to beef and ginseng, and it is a healthy food for prolonging life.
[0003] Bioactive peptides have a variety of biological effects, including anti-oxidation, immunomodulation, anti-hypertension, anti-thrombosis, cholesterol-lowering, antibacterial, anti-viral, and anti-tumor effects. They are a natural resource treasure trove for drug screening. It is reported that python active peptides can effectively remove free radicals in the body, thereby protecting cells from oxidative damage. This characteristic makes snake meat active peptides have potential application value in preventing cardiovascular diseases and delaying aging. And low-peptide molecules are easily digested and absorbed by the human body, which can provide the human body with more nutrients such as protein, vitamins and minerals, thereby enhancing physical health. At present, there have been reports on the preparation methods of Qi snake peptides, pit viper peptides and python peptides, but there have been no reports on the preparation methods of cobra meat enzymatic active peptides. Summary of the invention
[0004] In order to solve the above problems, the present invention provides a method for preparing enzymatically hydrolyzed cobra meat active polypeptides. The cobra meat active polypeptides prepared by the preparation method provided by the present invention have good antioxidant activity and are simple in preparation.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] The present invention provides a method for preparing a cobra meat active polypeptide, comprising the following steps:
[0007] The cobra meat is minced and mixed with water, and the pH value of the mixture is adjusted to 7.0 to obtain an enzymatic substrate;
[0008] Mix bromelain, neutral protease and the enzymatic substrate, perform enzymatic reaction, and obtain enzymatic solution; the mass ratio of bromelain, neutral protease and the enzymatic substrate is 1g: 1g: 100mL; the enzymatic activity of papain is 800,000 U / g; the enzymatic activity of bromelain is 600U / mg; the enzymatic activity of neutral protease is 200,000 U / g; the enzymatic reaction time is 8h;
[0009] The enzymatic hydrolysate is centrifuged to collect the supernatant; the supernatant is dialyzed to obtain a polypeptide solution.
[0010] Preferably, the temperature of the enzymatic hydrolysis reaction is 50-60°C.
[0011] Preferably, the dialysis bag used for the dialysis is a dialysis bag with a molecular weight cutoff of 100-500D.
[0012] Preferably, the mass volume ratio of the minced meat to water is 1 g: 10-30 mL.
[0013] Preferably, the method further comprises: freeze-drying the polypeptide liquid to obtain cobra meat enzymatically hydrolyzed active polypeptide powder.
[0014] Preferably, the cobra meat is fresh cobra meat.
[0015] Preferably, the enzymatic hydrolysate is subjected to an enzyme inactivation treatment before centrifugation; the temperature of the enzyme inactivation treatment is 95-100° C., and the time is 20-30 min.
[0016] Preferably, the centrifugal speed is 10000-12000 rpm, the temperature is 4° C., and the time is 15-20 min.
[0017] The present invention provides the use of the cobra meat active polypeptide prepared by the preparation method described in the above technical scheme in the preparation of antioxidant products.
[0018] Beneficial effects:
[0019] The invention hydrolyzes the cobra meat under suitable enzymatic hydrolysis conditions, and the prepared cobra meat active polypeptide has good antioxidant activity, and the neutral protease and bromelain are subjected to enzymatic hydrolysis reaction in the same enzymatic hydrolysis system, which simplifies the enzymatic hydrolysis steps and has the advantage of simple operation. The invention provides key technical support for the efficient preparation and application of the cobra meat enzymatic hydrolysis active peptide, and has important significance in the extraction and application fields of relevant animal medicine active peptides. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0021] Figure 1 The changes of IC50 values of hydroxyl radical scavenging rate of active peptide powder obtained by enzymatic hydrolysis with different enzyme addition amounts;
[0022] Figure 2 The changes of IC50 values of hydroxyl radical scavenging rate of active peptide powder obtained by enzymatic hydrolysis at different enzymatic hydrolysis times;
[0023] Figure 3 The IC50 value of scavenging rate of hydroxyl free radicals of active peptide powder obtained by enzymatic hydrolysis under different pH values of protease;
[0024] Figure 4 The changes in IC50 values of hydroxyl radical scavenging rate of active peptide powder obtained by enzymatic hydrolysis with different enzyme combinations. DETAILED DESCRIPTION
[0025] The present invention provides a method for preparing a cobra meat active polypeptide, comprising the following steps:
[0026] The cobra meat is minced and mixed with water, and the pH value of the mixture is adjusted to 7.0 to obtain an enzymatic substrate;
[0027] Mix bromelain, neutral protease and the enzymatic substrate, perform enzymatic reaction, and obtain enzymatic solution; the mass ratio of bromelain, neutral protease and the enzymatic substrate is 1g: 1g: 100mL; the enzymatic activity of papain is 800,000 U / g; the enzymatic activity of bromelain is 600U / mg; the enzymatic activity of neutral protease is 200,000 U / g; the enzymatic reaction time is 8h;
[0028] The enzymatic hydrolysate is centrifuged to collect the supernatant; the supernatant is dialyzed to obtain a polypeptide solution.
[0029] The present invention stirs cobra meat into minced meat and mixes it with water, adjusts the pH value of the mixture to 7.0, and obtains an enzymatic hydrolysis substrate. As an embodiment, the cobra meat is fresh cobra meat. As an embodiment, the mass volume ratio of the minced meat to water is 1g:10-30mL; as another embodiment, the mass volume ratio of the minced meat to water is 1g:10-20mL; as another embodiment, the mass volume ratio of the minced meat to water is 1g:10-15mL.
[0030] After obtaining the enzymatic substrate, the present invention mixes bromelain, neutral protease and the enzymatic substrate, performs enzymatic reaction, and obtains an enzymatic solution; the mass volume ratio of the bromelain, neutral protease and the enzymatic substrate is 1g: 1g: 100mL; the enzymatic activity of the papain is 800,000 U / g; the enzymatic activity of the bromelain is 600U / mg; the enzymatic activity of the neutral protease is 200,000 U / g; the time of the enzymatic reaction is 8h. As an embodiment, the temperature of the enzymatic reaction is 50-60°C; as another embodiment, the temperature of the enzymatic reaction is 55°C. As an embodiment, the enzymatic reaction is reacted in an air oscillator. The present invention cuts the rich active protein fragments contained in cobra into small molecule polypeptides with stronger activity and easier absorption through suitable enzymatic conditions, has high antioxidant activity, and has extremely broad application prospects in the fields of medicine, health care, food preservation, etc., which target hydroxyl free radical-induced oxidative damage.
[0031] After obtaining the enzymatic hydrolysate, the present invention centrifuges the enzymatic hydrolysate and collects the supernatant. As an embodiment, the enzymatic hydrolysate is further subjected to enzyme inactivation treatment before centrifugation; the temperature of the enzyme inactivation treatment is 95-100°C and the time is 20-30 minutes; as another embodiment, the temperature of the enzyme inactivation treatment is 100°C and the time is 20-25 minutes. As an embodiment, the centrifugal speed is 10000rpm, the temperature is 4°C, and the time is 15 minutes.
[0032] After obtaining the supernatant, the present invention dialyzes the supernatant to obtain a polypeptide solution. As an embodiment, the dialysis bag used for the dialysis is a dialysis bag with a molecular weight cutoff of 100-500D.
[0033] As an embodiment, after obtaining the polypeptide liquid, the polypeptide liquid is freeze-dried to obtain cobra meat enzymatically hydrolyzed active polypeptide powder.
[0034] The active peptide obtained by the preparation method provided by the present invention has the strongest antioxidant capacity, and the entire extraction process has the characteristics of simple equipment requirements, easy control of reaction conditions, low energy consumption, high extraction efficiency, and can maintain the natural characteristics of the extract, reduce impurities, and thus obtain a higher purity product.
[0035] Based on the above advantages, the present invention provides the use of the cobra meat active polypeptide prepared by the preparation method described in the above technical solution in the preparation of antioxidant products. As an embodiment, the product can be a medicine, a health product or a food preservative.
[0036] In order to further illustrate the present invention, a method for preparing a cobra meat enzymatically hydrolyzed active polypeptide provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0037] Preparation Example
[0038] The materials, reagents, instruments and equipment used in the examples or comparative examples of the present invention are as follows:
[0039] Cobras were sourced from Leicun Snake Farm, and the experimental individuals were healthy adult snakes weighing 1.8 kg. Papain was purchased from Solebold, with an enzyme activity of 800,000 U / g, Cat: G8430; bromelain was purchased from Solebold, with an enzyme activity of 600 U / mg, Cat: B8290; neutral protease was purchased from Meilun Biotechnology, with an enzyme activity of 200,000 U / g, item number: MB2929-1; hydrochloric acid was purchased from Chengdu Kelong Chemical Co., Ltd.; sodium hydroxide was purchased from Chengdu Jinshan Chemical Reagent Co., Ltd.; salicylic acid was purchased from Solebold; anhydrous ethanol was purchased from Chengdu Kelong Chemical Co., Ltd.; dialysis bags (ready-to-use 100-500D) were purchased from Solebold; 30% hydrogen peroxide was purchased from Chengdu Jinshan Chemical Reagent Co., Ltd.; ferrous sulfate heptahydrate was purchased from Meilun Biotechnology; and pure water (15.0 MΩ·cm) was sourced from the Zhuang and Yao Medicine Laboratory of Guangxi International Zhuang Medicine Hospital.
[0040] Meat grinder, AUX; desktop high-speed refrigerated centrifuge, Hunan Hexi Instrument Equipment Co., Ltd.; 85-2A magnetic stirrer, Shanghai Huxi Industrial Co., Ltd.; electronic balance XS205DU, Mettler-Toledo Instrument (Shanghai) Co., Ltd.; THZ-92A gas bath constant temperature oscillator, Shanghai Boxun Medical Biological Instrument Co., Ltd.; SCIENTZ-30YD / A freeze dryer, Ningbo Xinzhi Freeze Drying Equipment Co., Ltd.; pHS-3C pH meter, Leici; electric constant temperature water bath, Shanghai Qixin Scientific Instrument Co., Ltd.; multi-function microplate reader, Nanning Precision Instrument Co., Ltd.
[0041] Example 1
[0042] A method for preparing cobra meat active polypeptide is as follows:
[0043] 1) Peel and remove the internal organs of the freshly slaughtered cobra meat, wash and drain, and grind it into minced meat in a meat grinder;
[0044] 2) placing the minced meat in a beaker, adding pure water to the beaker, and stirring with a magnetic stirrer to obtain a mixed solution, wherein the mass volume ratio of the minced meat to the pure water is 1 g:10 mL;
[0045] 3) placing the mixed solution in step 2) in a 55° C. water bath for heating, and adjusting the pH value to 7.0 with hydrochloric acid and sodium hydroxide;
[0046] 4) adding bromelain and neutral protease at a final concentration of 1% (10 g / L) to the mixed solution in step 3) for enzymatic hydrolysis, and oscillating the mixture at 55° C. for 8 h in an air oscillator to obtain an enzymatic hydrolyzate;
[0047] 5) The enzymatic hydrolysis solution was placed in a water bath at 100° C. for 20 min to terminate the enzymatic hydrolysis reaction, and after returning to room temperature, high-speed low-temperature centrifugation was performed using a desktop high-speed refrigerated centrifuge at 10,000 rpm, 4° C., for 15 min to obtain a supernatant; the supernatant was dialyzed using a dialysis bag with a molecular weight cutoff of 100-500D (purchased from solarbio, product number YA1069) to obtain a polypeptide solution;
[0048] 6) placing the polypeptide liquid in a freeze dryer for freeze drying to obtain cobra meat enzymatically hydrolyzed active peptide powder.
[0049] Comparative Example
[0050] The preparation method is similar to that of Example 1, except that the pH value in step 3) is different or the type of enzyme added in step 4), the amount of enzyme added (ie, the final concentration) or the enzymolysis time is different. The specific differences are shown in Table 1.
[0051] Table 1 Preparation conditions of different comparative examples and Example 1
[0052]
[0053]
[0054] Note: The papaya in the table is papain, the pineapple is bromelain, the neutral is neutral protease, and the % in the enzyme amount is the final concentration of different enzymes added to the mixed solution, i.e. g / 100mL. The same applies to the following table.
[0055] The antioxidant capacity of the polypeptides prepared in Comparative Examples 1 to 53 and Example 1 was analyzed experimentally, and the steps were as follows:
[0056] 1. Determination of hydroxyl radical (·OH) scavenging ability
[0057] 1) Experimental principle
[0058] In this experiment, the Fenton reaction was used to generate hydroxyl radicals: H 2 O 2 +Fe 2+ = OH + OH - +Fe 3+ Adding salicylic acid to the reaction system can capture hydroxyl radicals (·OH) to generate 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and 2,5-dihydroxybenzoic acid with special absorption at a wavelength of 510nm.
[0059] 2) Solution preparation
[0060] ①FeSO 4 Solution (9mmo / L): weigh FeSO 4 7H 2 O crystal 0.250208g is diluted to 100mL of pure water.
[0061] ②H 2 O 2 Solution (4.4 mmol / L): Take 30% H 2 O 2 13.4 μL of the solution was diluted to 100 mL of pure water.
[0062] ③ Salicylic acid-ethanol solution (9 mmol / L): Weigh 0.124308 g of salicylic acid solid and dilute to 100 mL of anhydrous ethanol solution.
[0063] 3) Sample processing
[0064] Take the cobra meat enzymatic active peptide powder and add pure water to mix thoroughly to prepare a 4 mg / mL solution, and dilute the concentration to 4000, 2000, 1000, 500, 250, and 125 μg / mL in sequence. Determine the absorbance value at each concentration, calculate the clearance rate at each concentration, and use GrapHpadPrism10.1.2 software to calculate the IC50 value. The smaller the IC50 value, the stronger the antioxidant activity of the peptide.
[0065] 4) Operation steps
[0066] Ax: Add 1 mL of sample solution and 0.5 mL of FeSO 4 Solution, 1 mL H 2 O 2 The solution and 1 mL of salicylic acid-ethanol solution were added to start the reaction, and the mixture was shaken and mixed. The mixture was incubated in a water bath at 37°C for 30 min, and the absorbance was measured at 510 nm.
[0067] Ax 0 : Add 1 mL of sample solution and 0.5 mL of FeSO 4 Solution, 1mL pure water and 1mL salicylic acid-ethanol solution, start the reaction, oscillate to mix, in a water bath at 37℃ for 30min, and measure the absorbance at 510nm.
[0068] A 0 : Add 1 mL of pure water and 0.5 mL of FeSO 4 Solution, 1 mL H 2 O 2The solution and 1 mL of salicylic acid-ethanol solution were added to start the reaction, and the mixture was shaken and mixed. The mixture was incubated in a water bath at 37°C for 30 min, and the absorbance was measured at 510 nm.
[0069] The calculation formula is: Clearance = [1-(Ax-Ax 0 ) / A 0 ]×100%.
[0070] 2. Results
[0071] 2.1 Effect of enzyme addition on enzymatic hydrolysis (i.e. comparative examples 1 to 15) Figure 1 and Table 2.
[0072] Table 2 IC50 values of active peptide powders from cobra meat hydrolyzed with different amounts of protease (μg / mL)
[0073] Group 1％ 2％ 3％ 4％ 5％ Neutral protease 1976±525.16 2121.67±262.88 2576.67±319.08 2879.33±666.64 2393±306.41 Bromelain 2095.67±585.29 2319.33±128.14 2226±208.73 2275.33±302.38 2491±194.92 Papain 2521.33±774.45 2627.67±969.56 2081±109.03 <![CDATA[2961±436.95 * ]]> 2566.67±434.79
[0074] Note: Figure 1 As shown in Table 2, compared with 3% papaya enzyme, * indicates P < 0.05.
[0075] The results showed that when neutral protease was used to extract active peptide powder, the best antioxidant polypeptide could be obtained when the enzyme dosage was 1%, and there was no statistically significant difference compared with other enzyme dosages (P>0.05); for bromelain, the extracted active peptide powder could obtain the strongest antioxidant polypeptide when the enzyme dosage was 1%, and there was no statistically significant difference compared with other enzyme dosages (P>0.05); and in the process of papain extracting active peptide powder, when the enzyme dosage was 3%, the best antioxidant polypeptide could be obtained, and there was a statistically significant difference compared with 4% enzyme dosage (P<0.05), and there was no statistically significant difference compared with other enzyme dosages (P>0.05).
[0076] Under different enzyme addition conditions, enzymatic hydrolysis reactions will produce peptides with different structures and compositions. As for the active peptide powder extracted by bromelain and neutral protease, as the amount of enzyme added increases, the protein is over-hydrolyzed, which will destroy the structure of the active peptide, making the structure and composition of the produced peptide unfavorable for the antioxidant effect. In addition, a large number of active peptides may aggregate to form macromolecular complexes, changing their original active conformation; or different active peptides may produce competitive effects and compete for free radical binding sites. Taking the above factors into consideration, the optimal enzyme addition amount for enzymatic hydrolysis of cobra meat active peptides was determined to be 1% for neutral protease, 1% for bromelain, and 3% for papain, so as to ensure effective enzymatic hydrolysis and antioxidant activity of proteins in cobra meat.
[0077] 2.2 Effect of enzymolysis time on enzymolysis results (i.e. comparative examples 31 to 45) Figure 2 and Table 3.
[0078] Table 3 IC50 values of cobra meat enzymatic active peptide powder prepared at different enzymatic hydrolysis times (μg / mL)
[0079] Group 5h 6h 7h 8h 9h Neutral protease <![CDATA[4208.67±758.26 ** ]]> 1976±525.16 4145.67±1336.40 1790±206.55 1806.33±31.02 Bromelain 2072±132.57 2095.67±585.29 4407±1546.34 2066±708.81 3121.33±542.59 Papain 2444.33±324.32 2081±109.03 <![CDATA[3101.33±149.51 ### ]]> 2496.67±565.76 <![CDATA[3850.67±298.66 ### ]]>
[0080] Note: Figure 2 As shown in Table 3, compared with the neutral enzymatic hydrolysis time of 8 h, ** indicates P < 0.01; compared with the papaya enzymatic hydrolysis time of 6 h, ### indicates P < 0.001.
[0081] The results showed that the active peptide powder extracted by neutral protease had the strongest antioxidant capacity at 8 hours, and the difference was statistically significant compared with 5 hours (P < 0.01); the IC50 value of the active peptide powder extracted by bromelain was the lowest at 8 hours, and its antioxidant capacity was the strongest, and there was no statistically significant difference compared with other enzymatic hydrolysis times (P > 0.05); papain was able to obtain peptides with better antioxidant properties at 6 hours of enzymatic hydrolysis, and the difference was statistically significant compared with 7 hours and 9 hours (P < 0.001), but there was no statistically significant difference compared with 8 hours (P > 0.05).
[0082] At 5 to 8 hours, as the enzymatic hydrolysis time increases, new polypeptide structures with antioxidant properties may be generated during the enzymatic hydrolysis reaction, thereby enhancing the overall antioxidant capacity. At 9 hours, the antioxidant capacity decreased. This may be because as the enzymatic hydrolysis time continues to increase, the degree of protein hydrolysis by the protease gradually deepens, causing the original antioxidant polypeptide to be further hydrolyzed into amino acids, thereby destroying its antioxidant activity structure, and ultimately causing the antioxidant capacity to decrease. Taking the above factors into consideration, 8 hours was determined as the optimal enzymatic hydrolysis time for cobra meat enzymatic active peptides. This time node can balance the relationship between polypeptide generation, structural integrity and antioxidant properties to the greatest extent under the action of multiple proteases, providing a key process parameter basis for the subsequent efficient preparation of cobra meat enzymatic active peptides and related application research.
[0083] 2.3 Results of the effect of pH value of protease on enzymolysis (i.e., comparative examples 16 to 30) are shown in Figure 3 and Table 4.
[0084] Table 4 IC50 values of cobra meat enzymatic active peptide powder prepared at different pH values (μg / mL)
[0085] Group neutral Pineapple pawpaw 6 / / 2648±169.56 6.2 / / 2758±1258.40 6.5 3657.33±2033.02 <![CDATA[3185±492.45 # ]]> <![CDATA[3761±530.86 △ ]]> 6.8 <![CDATA[4194.33±927.08 * ]]> <![CDATA[3512±260.2 3## ]]> 2308.33±138.00 7 1790±206.55 2066±708.81 2496.67±565.76 7.2 <![CDATA[3814.33±745.33 * ]]> 2001.67±357.73 / 7.5 <![CDATA[3883.33±76.49 *** ]]> <![CDATA[3331±231.18 ## ]]> /
[0086] Note: Figure 3As shown in Table 4, compared with neutral 7.0, * indicates P < 0.05, *** indicates P < 0.001; compared with pineapple 7.2, # indicates P < 0.05, ## indicates P < 0.01; compared with papaya 6.8, △ indicates P < 0.05.
[0087] The results showed that when the pH value of the neutral protease system was 7.0, a relatively lower IC50 value could be obtained, which was statistically significant compared with the pH values of 6.8, 7.2 and 7.5 (P < 0.05, P < 0.001); in the bromelain system, when the pH value was set to 7.2, a relatively lower IC50 value could be obtained, which was statistically significant compared with the pH values of 6.5, 6.8 and 7.5 of bromelain (P < 0.05, P < 0.01); in the papain system, when the pH value was set to 6.8, a relatively lower IC 50 The difference was statistically significant when compared with the pH value of 6.5 (P < 0.05).
[0088] 2.4 Effect of enzyme type combination on enzymolysis results (i.e., Comparative Example 19, Comparative Example 24, Comparative Example 28, Comparative Examples 46-53 and Example 1) Figure 4 and Table 5.
[0089] Table 5 IC50 values of cobra meat enzymatic active peptide powder prepared by different enzyme combinations (μg / mL)
[0090]
[0091]
[0092] Note: See Figure 4 In Table 5, compared with pineapple, neutral 1%, 7.0, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.
[0093] The results showed that in the pineapple neutral protease system, when the pH value was 7.0, the IC50 value of the hydroxyl radical scavenging rate was at the lowest level, and the active peptide powder with the strongest antioxidant capacity could be obtained at this time. Compared with papain at pH 6.8, bromelain at pH 7.2, neutral protease at pH 7.0, papaya bromelain in pairs, papaya neutral protease in pairs, and papaya pineapple neutral protease in pairs at pH 6.8 and pH 7.2, the differences were statistically significant (P < 0.05, P < 0.01, P < 0.001), while compared with other combinations, the differences were not statistically significant.
[0094] In summary, under the specific conditions of pH 7.0, enzymolysis time of 8h, enzymolysis temperature maintained at 55°C, and using a combination of 1% neutral protease and 1% bromelain, cobra meat enzymolysis active peptide powder with the strongest antioxidant capacity can be obtained, and the preparation method is simple, and cobra meat enzymolysis active peptide powder with the strongest antioxidant capacity can be obtained by enzymolysis in one enzymolysis system. The present invention provides key technical support for the efficient preparation and application of cobra meat enzymolysis active peptides, and is of great significance in the field of extraction and application of related animal drug active peptides.
[0095] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for preparing active polypeptide from cobra meat, characterized in that: The following steps are involved: The cobra meat is minced and mixed with water, and the pH value of the mixture is adjusted to 7.0 to obtain an enzymatic substrate; Mix bromelain, neutral protease and the enzymatic substrate, perform enzymatic reaction, and obtain enzymatic solution; the mass ratio of bromelain, neutral protease and the enzymatic substrate is 1g: 1g: 100mL; the enzymatic activity of papain is 800,000 U / g; the enzymatic activity of bromelain is 600U / mg; the enzymatic activity of neutral protease is 200,000 U / g; the enzymatic reaction time is 8h; The enzymatic hydrolysate is centrifuged to collect the supernatant; the supernatant is dialyzed to obtain a polypeptide solution.
2. The preparation method according to claim 1, characterized in that: The temperature of the enzymatic hydrolysis reaction is 50-60°C.
3. The preparation method according to claim 1, characterized in that: The dialysis bag used in the dialysis is a dialysis bag with a molecular weight cutoff of 100-500D.
4. The preparation method according to claim 1, characterized in that: The mass volume ratio of the minced meat and water is 1g:10-30mL.
5. The preparation method according to claim 1, characterized in that: The method further comprises: freeze-drying the polypeptide liquid to obtain cobra meat enzymatically hydrolyzed active polypeptide powder.
6. The preparation method according to claim 1, characterized in that: The cobra meat is fresh cobra meat.
7. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysate is subjected to enzyme inactivation treatment before centrifugation; the temperature of the enzyme inactivation treatment is 95-100° C. and the time is 20-30 minutes.
8. The preparation method according to claim 1 or 7, characterized in that: The centrifugal speed is 10000-12000 rpm, the temperature is 4° C., and the time is 15-20 min.
9. Use of the cobra meat active polypeptide prepared by the preparation method according to any one of claims 1 to 8 in the preparation of antioxidant products.