A method for preparing protein polypeptide using bullwhip and its application

Abstract

The invention belongs to the technical field of cowhide collagen polypeptide products, and specifically relates to a method for preparing protein polypeptides from bullwhips and an application thereof. The preparation method comprises primary processing of raw materials, secondary processing of raw materials, fat homogenization, enzymolysis, separation and filtration, concentration and drying. The protease selected in the method is serine protease, the active concentration of serine protease is 3000U / g, the enzymolysis time is 2.5h, the yield of bullwhip protein polypeptide obtained by the enzymolysis is high, and the method is suitable for large-scale application, and the production efficiency is effectively improved, and the cost of waste treatment and the like is reduced. The bullwhip raw material selected in the method is the bullwhip of a specific pedigree Simmental cattle. Compared with bullwhip raw materials of other pedigrees, the bullwhip protein polypeptide prepared from the bullwhip of the Simmental cattle has the effect of resisting oxidative stress of the reproductive system, and the improvement effect of the bullwhip protein polypeptide on renal function and hypothalamus-pituitary-adrenal axis function proves that the bullwhip protein polypeptide has the effect of regulating and controlling kidney yang deficiency syndrome.

Description

Technical Field

[0001] The present invention relates to the technical field of cowhide collagen polypeptide products, and specifically relates to a method for preparing protein polypeptides using bull whip and its application. Background Art

[0002] Bullwhip, as a kind of animal whip, has been considered to have a controversial effect on improving sexual dysfunction and low immunity since ancient times. There is little research evidence on its substantial effect in modern medicine. The effects of bullwhip application vary greatly among different populations and in different processing methods. The fat, protein, hormones and other components contained in bullwhip may have certain biological activity. The protein contained in bullwhip is its main nutrient component, with a molecular weight of more than 300,000. The bioavailability of bullwhip absorbed by the human body through the digestive tract is low, and a large amount of protein is eventually decomposed into single amino acid molecules during digestion, thus losing its activity and reducing its application value. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing protein polypeptides using bullwhip and its application, thereby improving the bioavailability of bullwhip to a large extent and solving the technical problems mentioned in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: a method for preparing protein polypeptides using bullwhip, the preparation method comprising the following steps:

[0005] Step 1: Primary processing of raw materials

[0006] Take fresh bull whip, remove surrounding fat, tendons, skin, and blood vessels, open the urethra to remove impurities, break into small pieces, rinse with water, and drain;

[0007] Step 2: Secondary processing of raw materials

[0008] Soak the treated bull whip pieces in ethanol, then in sodium hydroxide solution, and then remove the remaining alkali with acid solution, drain and rinse with clean water;

[0009] Step 3: Fat homogenization

[0010] The washed bull whip pieces were drained, purified water was added at a liquid-to-solid ratio of 6:1, stirred, and passed through a high-pressure homogenizer to break the cells and disperse the substances;

[0011] Step 4: Enzymatic hydrolysis

[0012] Adding protease to the homogenized fat solution for enzymolysis;

[0013] Step 5: Separation and Filtration

[0014] The enzymatic hydrolysate is separated and filtered by a secondary ultrafiltration process to obtain a polypeptide filtrate;

[0015] Step 6: Concentration and Drying

[0016] The polypeptide filtrate is concentrated and dried to obtain powdered bullwhip peptide.

[0017] Preferably, in step 1, the source of the bull whip raw material is the fresh external genitalia of Simmental bulls of the Canadian line, and the provenance numbers include 22115033, 22117059, 22118035, 22119039, 22121191, and 22120137.

[0018] Preferably, in step 2, the concentration of the sodium hydroxide solution used is 0.5-1.5 mol / L, and the soaking time is not less than 2 h.

[0019] Preferably, in step 4, the enzyme used is serine protease.

[0020] Preferably, in step 4, the protease activity concentration is at least 3000 U / g, and the enzymatic hydrolysis time is at least 2.5 h.

[0021] Preferably, in step 5, the equipment used in the secondary ultrafiltration process is a secondary ultrafiltration equipment unit, and the obtained polypeptide filtrate is a bullwhip protein polypeptide component with a molecular weight cutoff of 1000Da-5000Da.

[0022] Preferably, the secondary ultrafiltration equipment unit includes a first ultrafiltration purifier and a second ultrafiltration purifier, the first ultrafiltration purifier and the second ultrafiltration purifier are connected, the input end of the first ultrafiltration purifier is connected to a solution storage tank, and the output end of the second ultrafiltration purifier is connected to a low-temperature storage tank. A liquid pump is provided between the solution storage tank, the first ultrafiltration purifier, the second ultrafiltration purifier and the low-temperature storage tank. Enzyme hydrolysate is stored in the solution storage tank. Under the action of the pump force, the enzyme hydrolysate enters the first ultrafiltration purifier, in the first ultrafiltration purifier, components greater than 5000Da are retained, and the remaining part of the polypeptide filtrate enters the second ultrafiltration purifier under the action of the pump force, in the second ultrafiltration purifier, components less than 1000Da are retained, thereby the bullwhip protein polypeptide component with a molecular weight of 1000Da-5000Da enters the low-temperature storage tank, waiting for further concentration and drying.

[0023] Preferably, the protein polypeptide prepared by this method contains no less than 90% protein in terms of nitrogen content and no less than 80% polypeptide.

[0024] Preferably, in step 6, the drying method includes liquid spray drying or freeze drying.

[0025] Preferably, the protein polypeptide prepared by this method is used to combat oxidative stress in the reproductive system and improve yang deficiency syndrome.

[0026] The beneficial effects of the present invention are:

[0027] (1) The bullwhip raw material selected in this method is the bullwhip of a specific lineage of Simmental cattle. Compared with bullwhip raw materials of other lineages, the bullwhip protein polypeptide prepared from the bullwhip of Simmental cattle has the effect of resisting oxidative stress of the reproductive system, and the improvement effect of bullwhip protein polypeptide on renal function and hypothalamus-pituitary-adrenal axis function proves that it has the effect of regulating kidney yang deficiency syndrome.

[0028] (2) In terms of raw material selection and processing, the bullwhip is cleaned with alcohol and alkali after processing. The bullwhip is treated with alkali solution of a specific concentration and cleaning time, which has a better cleaning effect and a relatively short cleaning time, resulting in less fat residue in the bullwhip.

[0029] (3) The protease selected in this method is serine protease with an active concentration of 3000 U / g and an enzymatic hydrolysis time of 2.5 h. Compared with other proteases, the yield of bullwhip protein polypeptide obtained by enzymatic hydrolysis is high, which is suitable for large-scale application, effectively improving production efficiency and reducing costs such as waste treatment. The bullwhip protein polypeptide obtained by serine protease enzymatic hydrolysis is also more advantageous in reducing ability and DPPH free radical scavenging rate than the bullwhip protein polypeptide obtained by other protease enzymatic hydrolysis.

[0030] (4) Compared with other molecular weight components not in this range, the bullwhip protein polypeptide molecules with a molecular weight of 1000Da-5000Da selected by this method have a reducing ability (equivalent to FeSO4•7H2O concentration) higher than 20.00μmol / L and a DPPH free radical scavenging rate higher than 40.00%, showing obvious advantages in both reducing ability and DPPH free radical scavenging rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a trend diagram of fat residue changes under different sodium hydroxide solution concentrations and different time conditions in an embodiment of the present invention;

[0032] Figure 2 This is a comparison chart of the hydrolysis degree of different types of proteases in the embodiments of the present invention;

[0033] Figure 3 This is a comparison chart of the yields of different types of protease polypeptides in the examples of the present invention;

[0034] Figure 4 The variation trend of the effect of protease dosage and enzymolysis time on protein hydrolysis rate in the examples of the present invention;

[0035] Figure 5 The standard curve for concentration-absorbance determination in the embodiment of the present invention;

[0036] Figure 6This is a comparison chart of the reducing abilities of bullwhip protein polypeptides prepared by different proteases in the examples of the present invention;

[0037] Figure 7 This is a comparison chart of the DPPH free radical scavenging ability of bullwhip protein polypeptides prepared by different proteases in the examples of the present invention;

[0038] Figure 8 This is a distribution diagram of different molecular weight components of bullwhip protein polypeptide in the embodiment of the present invention;

[0039] Fig. 9 This is a comparison chart of the reducing abilities of bullwhip protein polypeptides of different molecular weight components in the examples of the present invention;

[0040] Fig.10 This is a comparison chart of DPPH free radical scavenging rates of bullwhip protein polypeptides of different molecular weight components in the embodiments of the present invention;

[0041] Fig.11 It is a schematic diagram of the secondary ultrafiltration equipment unit of the present invention.

[0042] In the figure: 1-first ultrafiltration purification machine, 2-second ultrafiltration purification machine, 3-solution storage tank, 4-low temperature storage tank. DETAILED DESCRIPTION

[0043] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Take fresh Simmental bull whip, remove the surrounding fat, fat, tendon skin, blood line, open the urethra to remove impurities, break into small pieces, rinse with water, drain, soak in ethanol for 2h to remove the surface grease, take about 100g sample of the above treatment, a total of 7 parts, respectively, in 0.5mol / L, 0.75mol / L, 1mol / L, 1.25mol / L, 1.5mol / L, 1.75mol / L, 2mol / L hydroxide Soak in sodium solution, take samples after soaking for 0, 0.5, 1, 1.5, 2, 2.5 and 3h, respectively, take samples of about 10g each time, rinse the samples with pure water and drain, dry them in an oven at 50℃ for 1h, accurately weigh the samples (about 5-10g, accurate to 0.001g), and then put them in an evaporating dish, add about 20g quartz sand, evaporate them on a boiling water bath, and then dry them at 100℃±5℃ for 30 minutes, take them out, grind them into powder, and transfer them into a filter paper tube. The evaporating dish and the glass rod with the sample are wiped clean with cotton wool soaked in ether and placed in the filter paper tube; the filter paper tube is placed in the extraction bucket of the Soxhlet extractor, connected to the receiving bottle, and anhydrous ether is used as the solvent. It is heated in a water bath and continuously refluxed for extraction (6 times / h) for 8 hours; the receiving bottle is removed and the anhydrous ether is recovered. When there is 1-2 ml of solvent remaining in the receiving bottle, it is evaporated on a water bath, dried at 100℃±5℃ for 1 hour, cooled in a dryer for 0.5 hours, and then weighed; the fat content in the sample is calculated according to the following formula

[0045] X= ( m 1 -m 0 ) / m 2 ×100%

[0046] Where X is the percentage of fat in the sample (%), m1 is the mass of the constant-weight receiving bottle and fat (g), m0 is the mass of the receiving bottle (g), and m2 is the weighed mass of the sample (g). The test results are as follows:

[0047] Fat residue data of bull whip samples under different concentrations of alkali solution and different soaking time conditions (%)

[0048] Sodium hydroxide concentration 0h 0.5h 1h 1.5h 2h 2.5h 3h 0.5mol / L 1.036 0.993 0.926 0.860 0.788 0.811 0.804 0.75mol / L 1.012 0.954 0.787 0.672 0.552 0.460 0.464 1mol / L 1.006 0.701 0.582 0.364 0.097 0.075 0.071 1.25mol / L 1.036 0.581 0.493 0.365 0.082 0.083 0.065 1.5mol / L 1.066 0.522 0.416 0.295 0.046 0.050 0.054 1.75mol / L 1.036 0.405 0.302 0.253 0.057 0.061 0.040 2mol / L 1.020 0.355 0.274 0.194 0.060 0.072 0.053

[0049] According to the calculation results of the data in the above table and the trend of the content curve, it can be seen that the fat residue of bullwhip alkali solution immersion in the group with sodium hydroxide solution immersion concentration less than 1 mol / L is higher, and the change is no longer obvious after 2.5 hours (higher than 0.45%), while the fat residue of bullwhip alkali solution immersion in the group with sodium hydroxide solution immersion concentration greater than or equal to 1 mol / L is less (all lower than 0.10%), which is beneficial to the subsequent enzymatic hydrolysis process, and the residue tends to change stably after 2 hours; it can be seen that the sodium hydroxide immersion concentration and time used in this method have obvious advantages over other concentration and time parameters.

[0050] Take fresh bull whip, remove the surrounding fat, fat, tendons, skin, and blood vessels, break the urethra to remove impurities, break it into small pieces, rinse with clean water, drain, and then soak in ethanol for not less than 2h, soak in 1mol / L sodium hydroxide solution for 2h, then remove the remaining alkali with acid, drain and rinse with clean water; according to the "Automatic Kjeldahl Nitrogen Analyzer Method" in the "Determination of Protein in Food" (GB / T 5009.5-2016), the protein content is determined, and the coefficient of nitrogen conversion to protein is 6.25. The total nitrogen content in the washed bull whip is 8.74%, and the protein content is 54.62%.

[0051] The bullwhip samples were sampled 9 times, with about 10 g (accurate to 0.01 g) of each sample, and then 60 g of purified water was added to each sample, stirred evenly, and then the cells were broken and the material was dispersed by a high-pressure homogenizer; 8 of the samples were added with 8 kinds of proteases for comparison of enzymatic hydrolysis conditions, and the homogenized samples were fully enzymatically hydrolyzed (enzyme addition amount was 6000 U / g, and enzymatic hydrolysis time was 6 h) under the optimal enzymatic hydrolysis conditions of each protease (pH value and temperature are shown in the table below); samples without protease addition were used as blank controls.

[0052]

[0053] The above 8 enzymatic hydrolysates were filtered using ultrafiltration membrane (pore size 0.01μm) to retain macromolecular substances with molecular weight > 10000Da. 5ml of the polypeptide solution was taken from each filter, 50ml of purified water was added, the pH value was adjusted to 8.20 with alkali solution, and then 10ml of neutral formaldehyde solution was added. Then the pH value was titrated to 9.20 with 0.1mol / LNaOH standard solution. The volume of NaOH standard solution consumed was calculated as Vx (ml). 5ml of blank control stock solution was collected and blank test was performed according to the above method. The volume of NaOH standard solution consumed was V0 (ml). The degree of protein hydrolysis was calculated.

[0054] Degree of protein hydrolysis = 0.1 mol / L·(V x -V0)·(100ml / 5ml)·Nitrogen equivalent / sampling volume / total nitrogen content of substrate

[0055] In addition, 10 ml of 8 portions of enzymatic hydrolysate filtrate (counted as 10% of the sample volume) were accurately measured and the content was determined using the "automatic Kjeldahl nitrogen determination method". The test result was X (g / 100g), and the yield was then calculated.

[0056] Yield = X (g / 100g)·(100g+X) / 10g·100% / 54.62%

[0057]

[0058] The results shown in the above table combined with the attached Figure 2 , 3 It can be seen that the serine protease selected by this preparation method has a higher yield than other proteases and has more advantages. The protein polypeptide prepared by this method has a relatively high yield, which is extremely important for large-scale application, effectively improving production efficiency and reducing costs such as waste treatment.

[0059] Take fresh bullwhip, remove surrounding fat, fat, tendons, skin, and blood vessels, break the urethra to remove impurities, break it into small pieces, and then rinse and drain with clean water; soak the processed bullwhip small pieces in ethanol for not less than 2 hours, then soak in 1 mol / L sodium hydroxide solution for 2 hours, and then remove the remaining alkali with acid, drain and rinse with clean water; drain the washed bullwhip small pieces, and then add purified water at a liquid-to-solid ratio of 6:1, stir and use a high-pressure homogenizer to break cells and disperse the material. Take 6 portions of homogenate, add serine protease at 1000U / g, 2000U / g, 3000U / g, 4000U / g, 5000U / g and 6000U / g respectively, and use ultrafiltration membrane (pore size 0.01μm) to retain macromolecules with molecular weight >10000Da at 1h, 1.5h, 2h, 2.5h, 3h, 3.5h and 4h of enzymatic hydrolysis to measure the degree of protein hydrolysis. The results are shown in the following table and the changing trend is shown in the following figure.

[0060]

[0061] From the above test results, it can be seen that the active concentration of serine protease selected by this method is 3000U / g and the enzymatic hydrolysis time is 2.5h, which can achieve a protein hydrolysis rate higher than 65%. The enzymatic hydrolysis effect is significantly better than other groups with a hydrolysis time lower than 3000U / g and less than 2.5h. And with the increase of enzyme addition and enzymatic hydrolysis time, the protein hydrolysis rate no longer changes significantly. This further proves the advantages of the enzyme activity concentration and enzymatic hydrolysis time selected by this method.

[0062] Peptide biological activity test method:

[0063] ① Reducing power test. Take 12.0mL of FRAP (ferricion reducing antioxidant power) reagent, raise the temperature to 38°C, then add 0.5ml of sample solution, let it react for 10-15min, and measure it at 593nm using a UV spectrophotometer. Draw a standard curve with 0, 0.02, 0.04, 0.06, 0.08, and 0.1mmol / L FeSO4·7H2O solutions, and the reducing power of the sample is measured by the concentration of FeSO4·7H2O (μmol / L) that achieves the same absorbance.

[0064] ② Determination of DPPH free radical scavenging ability. Take 8mL of 0.1mmol / L DPPH methanol solution, mix with 2mL of the sample to be tested in a test tube and shake well, react at room temperature in the dark for 30min; adjust the spectrophotometer to 517nm wavelength to measure the absorbance against methanol, and calculate the scavenging rate of the sample for DPHH free radicals according to the following formula: scavenging rate = [1-(X1-X2) / X3] × 100% (X1 is the absorbance of DPPH-sample mixture, X2 is the absorbance of sample solution, and X3 is the absorbance of DPPH solution).

[0065] (1) Comparison of the biological activities of bullwhip protein peptides prepared by enzymatic hydrolysis using different types of proteases

[0066] Take fresh bullwhip, remove surrounding fat, fat, tendons, blood lines, break the urethra to remove impurities, break into small pieces, and then rinse with water and drain; soak the treated bullwhip small pieces in ethanol for no less than 2h, then soak in 1mol / L sodium hydroxide solution for 2h, then remove the residual alkali with acid, drain and rinse with water; drain the washed bullwhip small pieces, then add purified water at a liquid-to-solid ratio of 6:1, stir and use a high-pressure homogenizer to break cells and disperse the material; take 8 portions of homogenized fat solution, add 8 different proteases respectively, the addition amount is 3000U / g, the enzymolysis time is 2.5h, filter, concentrate and dry to obtain bullwhip protein polypeptide powder; 8 samples are weighed about 400mg powder, placed in 100ml volumetric bottles, dissolved and fixed to volume with purified water. According to the two tests of "peptide biological activity test detection method", the determination and comparison are carried out, and the results are shown in the table below.

[0067]

[0068]

[0069] From the results shown in the above charts, it can be seen that the bullwhip protein polypeptides prepared by different types of proteases have differences in reducing ability and DPPH free radical scavenging rate. The serine protease selected by this method has more advantages than other proteases in the comparison of the above two indicators.

[0070] Take about 100g of fresh bullwhip, remove the surrounding fat, fat, tendons, skin, and blood lines, break the urethra to remove impurities, break it into small pieces, and then rinse with water and drain; soak the treated bullwhip small pieces in ethanol for not less than 2h, then soak them in 1mol / L sodium hydroxide solution for 2h, then remove the residual alkali with acid solution, drain and rinse with water; drain the washed bullwhip small pieces, then add 600g of purified water, stir and use a high-pressure homogenizer to break cells and disperse substances; take 8 parts of homogenized fat solution, add 3000U / g of serine protease, and enzymolysis time is 2.5h, and the enzymolysis supernatant is passed through different holes The ultrafiltration membrane with diameter of 100 mm / s was used to collect 7 different molecular weight bullwhip protein polypeptide components, including <500Da, 500-1000Da, 1000-2000Da, 2000-3000Da, 3000-4000Da, 4000-5000Da, and >5000Da, respectively. The components were concentrated to 1 / 10 volume by reduced pressure, and then freeze-dried. The vacuum degree was set to 0.2kPa, the temperature was gradually raised from -25℃ to 30℃, and the time was 12h. The 7 samples were accurately weighed; about 400mg of powder was taken from the above 7 groups of samples, respectively placed in 100ml volumetric bottles, and purified water was added to dissolve and fix the volume. The two tests of "peptide biological activity test method" were measured and compared, and the results are shown in the table below (n=3).

[0071]

[0072]

[0073]

[0074] From the experimental results, it can be seen that the bullwhip protein polypeptide molecules with a molecular weight of 1000Da-5000Da selected by this method have a selective reducing ability (equivalent FeSO4•7H2O concentration) higher than 20.00μmol / L and a DPPH free radical scavenging rate higher than 40.00% compared with other molecular weight components not in this range. The selective reducing ability and DPPH free radical scavenging rate have obvious advantages.

[0075] Randomly obtain 5 kg of fresh bull whips from Simmental bulls of different provenance numbers (source numbers include but are not limited to 22115033, 22117059, 22118035, 22119039, 22121191, 22120137), remove surrounding fat, fat, tendons, skin, and blood vessels, open the urethra to remove impurities, break into small pieces, and then rinse with clean water and drain; soak the processed bull whip pieces in ethanol for 2 hours, then soak in 1 mol / L sodium hydroxide solution for 2 hours, and then remove the remaining alkali with acid, drain and rinse with clean water ; Drain the washed bullwhip pieces, then add 30kg purified water, stir and use a high-pressure homogenizer to break the cells and disperse the substances, add 3000U / g of serine protease, then enzymolyze for 2.5h, take the enzymolysis supernatant, collect the bullwhip protein polypeptide component filtrate with a molecular weight of 1000Da-5000Da through a secondary ultrafiltration device, and concentrate the obtained polypeptide filtrate to 1 / 10 volume by reduced pressure, then set the vacuum degree to 0.2kPa, gradually heat the temperature from -25℃ to 30℃ for 12h of freeze drying, and obtain bullwhip protein polypeptide powder. Prepare 15 consecutive batches of bullwhip protein polypeptides by the above method.

[0076] Reactive oxygen species (ROS) is a general term for highly active oxygen derivatives with one or more unbound electrons derived from oxygen. It is an important active factor involved in human metabolism. As it continues to accumulate in the body, oxidative stress occurs when the body's ROS and antioxidant system are out of balance. Oxidative stress in the reproductive system is the main cause of reproductive function and reproductive system diseases. The following experiment mainly verifies the effect of bullwhip protein peptides in improving reproductive diseases induced by oxidative stress, and at the same time shows the differences between bullwhip materials from different sources.

[0077] According to the method of Example 6, bullwhip protein polypeptides were prepared from fresh bullwhips of Luxi Yellow Cattle, Charolais Cattle, Limousin Cattle, Angus Cattle, and Japanese Wagyu Cattle; 8-week-old male SD rats were randomly divided into 9 groups (n=15), the blank control group was gavaged with 10 ml / kg·d purified water, and the other groups were gavaged with 40 mg / kg·d of triptolide for 30 consecutive days; then the blank control group and the model group were gavaged with 10 ml / kg·d purified water, the positive control group was gavaged with 30 mg / kg·d of clomiphene citrate, and the 6 groups of bullwhip protein polypeptides from different sources were gavaged with 2000 mg / kg·d for 30 consecutive days; the rats fed for 60 days were fasted for 8 hours, accurately weighed after anesthesia, blood was collected from the abdominal aorta, and then the bilateral testicles, epididymis, and seminal vesicles were removed by dissection and accurately weighed.

[0078]

[0079] At the same time, the left epididymis was taken and cut into pieces in 5 ml of normal saline, and then kept in a 37°C water bath for shaking and mixing for 5 minutes. 0.05 ml of sample solution was taken and 0.35 ml of sperm diluent (1.0% formaldehyde solution containing 5% sodium bicarbonate) was added. The sperm density was determined with a sperm density counting plate, and the number of straight-line advances of 100 sperm within a fixed range was recorded and the sperm activity rate was obtained. The results are shown in the following table.

[0080]

[0081] The results show that the three organ coefficients of the positive control group and the bullwhip protein polypeptide of Canadian Simmental cattle were significantly different from those of the model control group (p < 0.01), and the three organ coefficients of the bullwhip protein polypeptides of other sources only showed significant differences in some indicators when compared with the model group (p < 0.01); similarly, in the detection of sperm count and sperm motility, there were still significant differences between the positive control group and the bullwhip protein polypeptide of Canadian Simmental cattle and the model control group (p < 0.01), while the bullwhip protein polypeptides of other sources only showed significant differences in sperm count between the bullwhip protein polypeptides of Limousin cattle and Angus cattle and the model control group (p < 0.01); therefore, it is confirmed that bullwhip protein polypeptide has the effect of resisting oxidative stress (such as induced by triptolide) in the reproductive system, and in terms of source, the bullwhip of the Canadian Simmental cattle selected by this method as the raw material has more advantages.

[0082] Randomly obtain 10 kg of fresh bull whips from Simmental bulls of different provenance numbers (source numbers include but are not limited to 22115033, 22117059, 22118035, 22119039, 22121191, 22120137), remove the surrounding fat, fat, tendons, skin, and blood vessels, open the urethra to remove impurities, break into small pieces, and then rinse with water and drain; soak the processed bull whip pieces in ethanol for 3 hours, then soak in 1 mol / L sodium hydroxide solution for 2.5 hours, then remove the residual alkali with acid, drain and rinse with water Wash clean; drain the washed bullwhip pieces, then add 60kg purified water, stir and use a high-pressure homogenizer to break cells and disperse materials, add 3000U / g serine protease, then enzymolyze for 2.5 hours, take the enzymolysis supernatant, collect the bullwhip protein polypeptide component filtrate with a molecular weight of 2000Da-4000Da through a secondary filtration device, and concentrate the obtained polypeptide filtrate to 1 / 10 volume by reduced pressure, then set the vacuum degree to 0.2kPa, gradually heat up from -25℃ to 30℃ for 12 hours of freeze drying, and obtain bullwhip protein polypeptide powder. Prepare 10 consecutive batches of bullwhip protein polypeptides by the above method.

[0083] Kidney Yang Deficiency in Traditional Chinese Medicine refers to the deficiency of kidney Yang Qi in Traditional Chinese Medicine, which is manifested by the inability of the kidney's essence to evaporate and gasify, followed by water metabolism disorders such as soreness of waist and knees, frequent urination at night, clear and long urination, diarrhea and loose stools. The following experiments mainly verify the effects of bullwhip protein peptides on renal function and hypothalamus-pituitary-adrenal axis function, and at the same time examine the differences between bullwhip materials from different sources.

[0084] According to the method of Example 7, bullwhip protein polypeptides were prepared from fresh bullwhips of Luxi yellow cattle, Charolais cattle, Limousin cattle, Angus cattle, and Japanese Wagyu cattle; 8-week-old male SD rats were randomly divided into 9 groups (n=10), the blank control group was gavaged with 10 ml / kg·d purified water, and the other groups were gavaged with 150 mg / kg·d of adenine suspension for 30 consecutive days; then the blank control group and the model group were gavaged with 10 ml / kg·d purified water, the positive control group was gavaged with Jinkui Shenqi Pills (small honey pills, dissolved by water) 1008 mg / kg·d, and the 6 groups of bullwhip protein polypeptides from different sources were gavaged with 2000 mg / kg·d for 30 consecutive days; the rats that had been fed for 60 days were fasted but not watered on the 61st day and urine was collected on the same day, the eyeballs were removed for blood collection on the 62nd day, and the hypothalamus and kidney tissues were removed after being killed.

[0085] Renal function test: Blood was collected from rats' eyes and centrifuged at 3000 r / min for 15 minutes at 4°C to separate serum. Urine was collected from rats and centrifuged at 3000 r / min for 5 minutes to obtain the supernatant. Serum urea nitrogen, serum creatinine, urine protein, and urine creatinine content were detected using an automatic biochemical detector.

[0086]

[0087] Hypothalamus-pituitary-adrenal axis function test: 9 volumes of phosphate buffered saline were added to the hypothalamus in a centrifuge tube, and the mixture was homogenized by centrifugation at 2000 r / min for 15 minutes. The supernatant was then taken and the content of adrenocorticotropin-releasing hormone (CRH) was detected by enzyme-linked immunosorbent assay. The content of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) was detected by enzyme-linked immunosorbent assay in the serum separated by the above method. The content of urine 17-hydroxycorticosteroid (17-OHCS) was detected by enzyme-linked immunosorbent assay in the separated urine supernatant.

[0088]

[0089] The results showed that the positive control group and the bullwhip protein polypeptide of Simmental cattle showed significant differences in renal function (serum urea nitrogen, serum creatinine, urine protein, urine creatinine), hypothalamus-pituitary-adrenal axis function (corticotropin-releasing hormone, adrenocorticotropic hormone, corticosterone, urine 17-hydroxy-corticosteroid) compared with the model control group (p < 0.01). When the bullwhip protein polypeptides of other sources were compared with the model group, only some of the above 8 indicators showed significant differences. There was no significant difference (p<0.01), especially in the three indicators of urine protein, urine creatinine and urine 17-hydroxy-corticosteroid. Except for the protein polypeptide from Simmental cattle, the bullwhip protein polypeptides from other sources did not show significant differences compared with the model control group. From the above analysis, it can be seen that the improvement effect of bullwhip protein polypeptide on renal function and hypothalamus-pituitary-adrenal axis function proves that it has the effect of regulating kidney yang deficiency in traditional Chinese medicine, and in terms of seed source, the bullwhip of Simmental cattle selected by this method as raw material has more advantages.

[0090] Randomly obtain 5 kg of fresh bull whips from Simmental bulls of different provenance numbers (source numbers include but are not limited to 22115033, 22117059, 22118035, 22119039, 22121191, 22120137), remove surrounding fat, fat, tendons, skin, and blood vessels, open the urethra to remove impurities, break into small pieces, and then rinse with water and drain; soak the processed bull whip pieces in ethanol for 3 hours, then soak in 1 mol / L sodium hydroxide solution for 2.5 hours, then remove the remaining alkali with acid, drain and rinse with water clean; drain the washed bullwhip pieces, then add 30kg purified water, stir and use a high-pressure homogenizer to break the cells and disperse the substances, add 3000U / g of serine protease, and then enzymolyze for 2.5 hours, take the enzymolysis supernatant, collect the bullwhip protein polypeptide component filtrate with a molecular weight of 1000Da~5000Da through a secondary filtration device, and concentrate the obtained polypeptide filtrate to 1 / 10 volume by reduced pressure, and then set the vacuum degree to 0.2kPa, and gradually heat the temperature from -25℃ to 30℃ for 12 hours of freeze drying to obtain bullwhip protein polypeptide powder. Prepare 5 consecutive batches of bullwhip protein polypeptides by the above method.

[0091] The 30 batches of samples (batch numbers 1-30) prepared in Example 6, Example 7 and Example 8 were tested for protein content and 1000Da-5000Da polypeptide content, respectively. The specific methods are as follows: ① Protein content detection. Weigh about 2g of sample (accurate to 0.001g), and determine the protein content in the sample according to the "Automatic Kjeldahl Nitrogen Determination Method" in "Determination of Protein in Food" (GB / T 5009.5-2016); ② Determination of 1000Da-5000Da polypeptide content: weigh about 2g of sample (accurate to 0.001g) and place it in a 50ml volumetric flask, add purified water to dissolve and make up to volume, then accurately measure 25ml of polypeptide solution, filter it with a 5000Da ultrafiltration membrane, and then filter the filtrate with a 1000Da ultrafiltration membrane, discard the filtrate, retain the unfiltered part and wash the 1000Da filter membrane and make up to 25ml, and then determine the protein content in the sample according to the "Automatic Kjeldahl Nitrogen Determination Method" in "Determination of Protein in Food" (GB / T 5009.5-2016). According to the above test method, 30 sealed packaged samples were subjected to stability tests at a temperature of 25°C±2°C and a relative humidity of 60%±10%. The tests were carried out at 0, 1, 2, 3, 6 and 12 months respectively. The results are as follows.

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] The stability test results show that, in the long-term stability test of 30 batches of bullwhip protein polypeptide samples in the three embodiments within 12 months (shelf life of raw materials), the protein content of bullwhip protein polypeptide is ≥90%, and the polypeptide content is ≥80% according to the minimum extreme value determined as a reference.

[0100] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing protein polypeptides using bullwhip, characterized in that: The method comprises the following steps: Step 1: Primary processing of raw materials Take fresh bull whip, the source of bull whip raw materials is fresh external genitalia of Canadian Simmental bulls, the source numbers include 22115033, 22117059, 22118035, 22119039, 22121191, 22120137, remove surrounding fat, fat, tendons, skin, blood lines, open the urethra to remove impurities, break into small pieces, rinse with clean water, and drain; Step 2: Secondary processing of raw materials Soak the treated bull whip pieces in ethanol, then in sodium hydroxide solution, and then remove the remaining alkali with acid solution, drain and rinse with clean water; Step 3: Fat homogenization The washed bull whip pieces were drained, purified water was added at a liquid-to-solid ratio of 6:1, stirred, and passed through a high-pressure homogenizer to break the cells and disperse the substances; Step 4: Enzymatic hydrolysis Add protease to the homogenate for enzymolysis, the enzyme used is serine protease, the protease activity concentration is 3000U / g, and the enzymolysis time is 2.5h; Step 5: Separation and Filtration The enzymatic hydrolysate is separated and filtered by a secondary ultrafiltration process to obtain a polypeptide filtrate; Step 6: Concentration and drying The polypeptide filtrate is concentrated and dried to obtain powdered bullwhip peptide.

2. The method for preparing protein polypeptides using bullwhip according to claim 1, characterized in that: In step 2, the concentration of the sodium hydroxide solution used is 1 mol / L, and the soaking time is 2 h.

3. The method for preparing protein polypeptides using bullwhip according to claim 1, characterized in that: In step 5, the equipment used in the secondary ultrafiltration process is a secondary ultrafiltration equipment unit, and the obtained polypeptide filtrate is a bullwhip protein polypeptide component with a molecular weight cutoff of 1000Da-5000Da.

4. The method for preparing protein polypeptides using bullwhip according to claim 3, characterized in that: The secondary ultrafiltration equipment unit comprises a first ultrafiltration purifier (1) and a second ultrafiltration purifier (2); the first ultrafiltration purifier (1) and the second ultrafiltration purifier (2) are connected to each other; the input end of the first ultrafiltration purifier (1) is connected to a solution storage tank (3); the output end of the second ultrafiltration purifier (2) is connected to a low-temperature storage tank (4); and liquid pumps are provided between the solution storage tank (3), the first ultrafiltration purifier (1), the second ultrafiltration purifier (2) and the low-temperature storage tank (4).

5. The method for preparing protein polypeptides using bullwhip according to claim 4, characterized in that: The protein polypeptide prepared by the method contains no less than 90% protein in terms of nitrogen content and no less than 80% polypeptide.

6. The method for preparing protein polypeptides using bullwhip according to claim 1, characterized in that: In step 6, the drying method includes liquid spray drying or freeze drying.

Images