CN108977489B - Enzymolysis peptide and medical application thereof - Google Patents
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Abstract
The invention discloses an enzymolysis peptide and medical application thereof. Prostate cancer is one of the most common malignancies of the male reproductive system, with increasing incidence with age. In recent years, the incidence of prostate cancer in China has increased, and the development of corresponding anti-cancer drugs is urgently needed. The sandalwood seed compound protease enzymolysis peptide with the molecular weight range of 1000-3000 u or 5000-7000 u provided by the invention has excellent activity of resisting prostate cancer, and the sandalwood seed papain enzymolysis peptide with the molecular weight range of 1000-3000, 3000-5000 or 7000-10000 has excellent activity of resisting prostate cancer.
Description
Technical Field
The invention belongs to the field of chemical medicine, and relates to an enzymolysis peptide and medical application thereof.
Background
Prostate cancer is the most common malignancy of the male genitourinary system, accounting for the 5 th cancer incidence worldwide. According to WHO global tumor epidemiological data (GLOBOCAN 2008), the incidence rate of the prostate cancer in 2008 is 2 nd (second to lung cancer) of the malignant tumor of the male globally, and accounts for 14 percent of all cases of the cancer of the male. The incidence of prostate cancer varies significantly around the world, with the highest incidence being about 25 times that of the lowest. In developing countries, the age-normalized incidence rate of prostate cancer is 12.0/10 ten thousand, the incidence accumulation rate of 0-74-year-old men is 1.4%, and the number 6 of malignant tumors of men is in place; in developed countries, the age-normalized incidence rate of prostate cancer in men is over 62.0/10 ten thousand, the incidence accumulation rate of 0-74 years old men is 7.8%, and the number of prostate cancer in men is 1.
In the united states, prostate cancer occurs at the 1 st of all male malignancies, accounting for about 29% of all male malignancies. Between 2004 and 2008, the incidence of prostate cancer in men, adjusted for age in the united states, was 152.9/10 ten thousand. The incidence of prostate cancer in american men increased by 2% every year between 1995 and 2001, and thereafter, the incidence declined year by year, with an annual 1.9% decrease between 2001 and 2008. China is one of the countries with low incidence and death of prostate cancer, but the incidence of prostate cancer in China shows a continuous and rapid growth trend in recent years. Estimated by the annual increase proportion (12.07%) of the last 10 years, the incidence rate of prostate cancer of Chinese men in 2012 is estimated to reach 17.35/10 ten thousand, which exceeds that of bladder cancer, and becomes the tumor with the highest incidence rate of urogenital systems of Chinese men. Prostate cancer is becoming a malignant tumor of the urinary system which seriously affects the health of men in China, and sufficient attention should be paid. The rapid increase of the incidence of prostate cancer in China may be related to the following factors: (1) with the improvement of living standard and medical standard of people in China, the aging of population is intensified, so that more patients can be detected to obtain prostate cancer; (2) the lifestyle and dietary structure are gradually westernized, resulting in a substantial increase in the incidence of prostate cancer; (3) with the improvement of the diagnosis and treatment level of the prostate cancer in China, particularly the wide application of screening of the prostate specific antigen, a plurality of prostate cancers which cannot be found before can be diagnosed at an early stage.
Therefore, the development of corresponding anti-prostate cancer drugs is urgently needed.
Disclosure of Invention
The invention aims to provide an enzymolysis peptide and medical application thereof.
The above purpose of the invention is realized by the following technical scheme:
an enzymolysis peptide, which is prepared by the following steps:
s1, extraction of sandalwood seed protein
Pulverizing peeled lignum Santali albi seed, weighing appropriate amount of powder, adding double distilled water, adjusting pH to 8.5 with NaOH solution, stirring in 40 deg.C water bath for 4 hr, filtering with gauze, and filtering to remove insoluble impurities to obtain clear solution; adjusting the pH value to an isoelectric point by using an HCl solution, standing at room temperature, centrifuging, removing supernatant to obtain milky protein precipitate, and slightly rinsing the precipitate by using distilled water for later use;
s2, enzymolysis of sandalwood seed protein
Suspending the opalescent protein precipitate in distilled water until the opalescent protein precipitate is dissolved, adding Protamex compound protease for enzymolysis, wherein the addition amount of the Protamex compound protease is 450U/g of substrate, and the enzymolysis conditions are as follows: pH value, 7.0; the enzymolysis temperature is 45 ℃; enzymolysis time is 6 hours; after enzymolysis, heating to inactivate the compound protease, and cooling to normal temperature; cooling, centrifuging, collecting supernatant, and freeze drying to obtain lyophilized powder;
s3, separation and purification of enzymolysis peptide
Dissolving the freeze-dried powder with distilled water, then sequentially separating the obtained enzymolysis peptide by using ultrafiltration membranes with the molecular weight cutoff of 7000u and 5000u, and freeze-drying 5000 u-7000 u components obtained by ultrafiltration to obtain the finished product.
Preferably, the powder is crushed into 50-60 meshes in the step S1.
Preferably, step S1 adds 600ml of double distilled water per 40g of powder.
Preferably, the isoelectric point of step S1 is 4.2.
Preferably, the centrifugation conditions of step S1 are 4000r/min 15 min.
Preferably, step S2 places the opalescent protein precipitate in distilled water at a concentration of 0.05mg/mL until dissolved.
Preferably, step S2 is performed by water bath at 85 deg.C for 10 minutes to inactivate the protease.
Preferably, the centrifugation condition of step S2 is 9000r/min × 25 min.
Preferably, 25mL of distilled water is added to 1g of lyophilized powder in step S3.
The enzymolysis peptide is used for preparing the anti-prostatic cancer medicine.
An enzymolysis peptide, which is prepared by the following steps:
s1, extraction of sandalwood seed protein
Pulverizing peeled lignum Santali albi seed, weighing appropriate amount of powder, adding double distilled water, adjusting pH to 8.5 with NaOH solution, stirring in 40 deg.C water bath for 4 hr, filtering with gauze, and filtering to remove insoluble impurities to obtain clear solution; adjusting the pH value to an isoelectric point by using an HCl solution, standing at room temperature, centrifuging, removing supernatant to obtain milky protein precipitate, and slightly rinsing the precipitate by using distilled water for later use;
s2, enzymolysis of sandalwood seed protein
Suspending the opalescent protein precipitate in distilled water until the opalescent protein precipitate is dissolved, adding Protamex compound protease for enzymolysis, wherein the addition amount of the Protamex compound protease is 450U/g of substrate, and the enzymolysis conditions are as follows: pH value, 7.0; the enzymolysis temperature is 45 ℃; enzymolysis time is 6 hours; after enzymolysis, heating to inactivate the compound protease, and cooling to normal temperature; cooling, centrifuging, collecting supernatant, and freeze drying to obtain lyophilized powder;
s3, separation and purification of enzymolysis peptide
Dissolving the freeze-dried powder with distilled water, then sequentially separating the obtained enzymolysis peptide by using ultrafiltration membranes with the molecular weight cutoff of 3000u and 1000u, and freeze-drying the components of 1000 u-3000 u obtained by ultrafiltration.
Preferably, the powder is crushed into 50-60 meshes in the step S1.
Preferably, step S1 adds 600ml of double distilled water per 40g of powder.
Preferably, the isoelectric point of step S1 is 4.2.
Preferably, the centrifugation conditions of step S1 are 4000r/min 15 min.
Preferably, step S2 places the opalescent protein precipitate in distilled water at a concentration of 0.05mg/mL until dissolved.
Preferably, step S2 is performed by water bath at 85 deg.C for 10 minutes to inactivate the protease.
Preferably, the centrifugation condition of step S2 is 9000r/min × 25 min.
Preferably, 25mL of distilled water is added to 1g of lyophilized powder in step S3.
The enzymolysis peptide is used for preparing the anti-prostatic cancer medicine.
An enzymolysis peptide, which is prepared by the following steps:
s1, extraction of sandalwood seed protein
Pulverizing peeled lignum Santali albi seed, weighing appropriate amount of powder, adding double distilled water, adjusting pH to 8.5 with NaOH solution, stirring in 40 deg.C water bath for 4 hr, filtering with gauze, and filtering to remove insoluble impurities to obtain clear solution; adjusting the pH value to an isoelectric point by using an HCl solution, standing at room temperature, centrifuging, removing supernatant to obtain milky protein precipitate, and slightly rinsing the precipitate by using distilled water for later use;
s2, enzymolysis of sandalwood seed protein
Placing the opalescent protein precipitate in distilled water for suspending until the opalescent protein precipitate is dissolved, adding papain for enzymolysis, wherein the addition amount of the papain is 420U/g of substrate, and the enzymolysis conditions are as follows: pH value, 7.0; the enzymolysis temperature is 42 ℃; the enzymolysis time is 8 hours; after enzymolysis, heating to inactivate protease, and cooling to normal temperature; cooling, centrifuging, collecting supernatant, and freeze drying to obtain lyophilized powder;
s3, separation and purification of enzymolysis peptide
Dissolving the freeze-dried powder with distilled water, then sequentially separating the obtained enzymolysis peptide by using ultrafiltration membranes with the molecular weight cutoff of 10000u and 7000u, and freeze-drying the 7000 u-10000 u components obtained by ultrafiltration to obtain the freeze-dried powder.
Preferably, the powder is crushed into 50-60 meshes in the step S1.
Preferably, step S1 adds 600ml of double distilled water per 40g of powder.
Preferably, the isoelectric point of step S1 is 4.2.
Preferably, the centrifugation conditions of step S1 are 4000r/min 15 min.
Preferably, step S2 places the opalescent protein precipitate in distilled water at a concentration of 0.05mg/mL until dissolved.
Preferably, step S2 is performed by water bath at 85 deg.C for 10 minutes to inactivate the protease.
Preferably, the centrifugation condition of step S2 is 9000r/min × 25 min.
Preferably, 25mL of distilled water is added to 1g of lyophilized powder in step S3.
The enzymolysis peptide is used for preparing the anti-prostatic cancer medicine.
An enzymolysis peptide, which is prepared by the following steps:
s1, extraction of sandalwood seed protein
Pulverizing peeled lignum Santali albi seed, weighing appropriate amount of powder, adding double distilled water, adjusting pH to 8.5 with NaOH solution, stirring in 40 deg.C water bath for 4 hr, filtering with gauze, and filtering to remove insoluble impurities to obtain clear solution; adjusting the pH value to an isoelectric point by using an HCl solution, standing at room temperature, centrifuging, removing supernatant to obtain milky protein precipitate, and slightly rinsing the precipitate by using distilled water for later use;
s2, enzymolysis of sandalwood seed protein
Placing the opalescent protein precipitate in distilled water for suspending until the opalescent protein precipitate is dissolved, adding papain for enzymolysis, wherein the addition amount of the papain is 420U/g of substrate, and the enzymolysis conditions are as follows: pH value, 7.0; the enzymolysis temperature is 42 ℃; the enzymolysis time is 8 hours; after enzymolysis, heating to inactivate protease, and cooling to normal temperature; cooling, centrifuging, collecting supernatant, and freeze drying to obtain lyophilized powder;
s3, separation and purification of enzymolysis peptide
Dissolving the freeze-dried powder with distilled water, then sequentially separating the obtained enzymolysis peptide by using ultrafiltration membranes with the molecular weight cutoff of 5000u and 3000u, and freeze-drying 3000 u-5000 u components obtained by ultrafiltration to obtain the freeze-dried powder.
Preferably, the powder is crushed into 50-60 meshes in the step S1.
Preferably, step S1 adds 600ml of double distilled water per 40g of powder.
Preferably, the isoelectric point of step S1 is 4.2.
Preferably, the centrifugation conditions of step S1 are 4000r/min 15 min.
Preferably, step S2 places the opalescent protein precipitate in distilled water at a concentration of 0.05mg/mL until dissolved.
Preferably, step S2 is performed by water bath at 85 deg.C for 10 minutes to inactivate the protease.
Preferably, the centrifugation condition of step S2 is 9000r/min × 25 min.
Preferably, 25mL of distilled water is added to 1g of lyophilized powder in step S3.
The enzymolysis peptide is used for preparing the anti-prostatic cancer medicine.
An enzymolysis peptide, which is prepared by the following steps:
s1, extraction of sandalwood seed protein
Pulverizing peeled lignum Santali albi seed, weighing appropriate amount of powder, adding double distilled water, adjusting pH to 8.5 with NaOH solution, stirring in 40 deg.C water bath for 4 hr, filtering with gauze, and filtering to remove insoluble impurities to obtain clear solution; adjusting the pH value to an isoelectric point by using an HCl solution, standing at room temperature, centrifuging, removing supernatant to obtain milky protein precipitate, and slightly rinsing the precipitate by using distilled water for later use;
s2, enzymolysis of sandalwood seed protein
Placing the opalescent protein precipitate in distilled water for suspending until the opalescent protein precipitate is dissolved, adding papain for enzymolysis, wherein the addition amount of the papain is 420U/g of substrate, and the enzymolysis conditions are as follows: pH value, 7.0; the enzymolysis temperature is 42 ℃; the enzymolysis time is 8 hours; after enzymolysis, heating to inactivate protease, and cooling to normal temperature; cooling, centrifuging, collecting supernatant, and freeze drying to obtain lyophilized powder;
s3, separation and purification of enzymolysis peptide
Dissolving the freeze-dried powder with distilled water, then sequentially separating the obtained enzymolysis peptide by using ultrafiltration membranes with the molecular weight cutoff of 3000u and 1000u, and freeze-drying the components of 1000 u-3000 u obtained by ultrafiltration.
Preferably, the powder is crushed into 50-60 meshes in the step S1.
Preferably, step S1 adds 600ml of double distilled water per 40g of powder.
Preferably, the isoelectric point of step S1 is 4.2.
Preferably, the centrifugation conditions of step S1 are 4000r/min 15 min.
Preferably, step S2 places the opalescent protein precipitate in distilled water at a concentration of 0.05mg/mL until dissolved.
Preferably, step S2 is performed by water bath at 85 deg.C for 10 minutes to inactivate the protease.
Preferably, the centrifugation condition of step S2 is 9000r/min × 25 min.
Preferably, 25mL of distilled water is added to 1g of lyophilized powder in step S3.
The enzymolysis peptide is used for preparing the anti-prostatic cancer medicine.
Has the advantages that:
the sandalwood seed compound protease enzymolysis peptide with the molecular weight range of 1000-3000 u or 5000-7000 u provided by the invention has excellent activity of resisting prostate cancer, and the sandalwood seed papain enzymolysis peptide with the molecular weight range of 1000-3000, 3000-5000 or 7000-10000 provided by the invention has excellent activity of resisting prostate cancer.
Drawings
FIG. 1 shows the IC50 values for the anti-prostate cancer activity of each zymolytic peptide.
Detailed Description
The following detailed description of the present invention is provided in connection with the accompanying drawings and examples, but not intended to limit the scope of the invention.
Example 1: protamex compound protease enzymolysis peptide
First, experimental material
Sandalwood (Santalbum L.) seeds are collected from a 5-year-old sandalwood forest, the seed coats of the sandalwood are manually removed after collection, and then the sandalwood seeds are dried in the shade in the environment of normal temperature, ventilation and no direct sunlight.
Protamex complex protease (food grade) was purchased from Novovin, Denmark (NOVO).
Second, Experimental methods and results
1. Extraction of sandalwood seed protein
Crushing peeled sandalwood seeds into 50-60 meshes of powder, weighing 40g, adding 600ml of double distilled water, adjusting the pH value to 8.5 by using 1mol/LNaOH solution, stirring in a water bath at 40 ℃ for 4 hours, filtering by using gauze, and filtering out insoluble impurities to obtain a clear solution; adjusting pH value to an isoelectric point (pI is 4.2) by using 1mol/LHCl, standing at room temperature for 30min, centrifuging (4000r/min multiplied by 15min), discarding supernatant to obtain milky protein precipitate, and slightly rinsing the protein precipitate with distilled water for three times for later use.
2. Enzymolysis of sandalwood seed protein
Placing the opalescent protein precipitate in distilled water according to the concentration of 0.05mg/mL until the opalescent protein precipitate is suspended until the opalescent protein precipitate is dissolved, adding Protamex compound protease for enzymolysis, wherein the addition amount of the Protamex compound protease is 450U/g of substrate, and the enzymolysis conditions are as follows: pH value, 7.0; the enzymolysis temperature is 45 ℃; enzymolysis time is 6 hours; after enzymolysis, inactivating the compound protease in water bath at 85 ℃ for 10 minutes, and cooling to normal temperature; after cooling, centrifuging at 9000rpm for 25 minutes, taking the supernatant, and freeze-drying to obtain the freeze-dried powder.
3. Separation and purification of enzymolysis peptide
Dissolving the freeze-dried powder with distilled water, adding 25mL of distilled water into 1g of freeze-dried powder, then sequentially separating the obtained enzymolysis peptides by ultrafiltration membranes with the molecular weight cut-off of 10000u, 7000u, 5000u, 3000u and 1000u, and freeze-drying the components obtained by ultrafiltration (the water content is less than or equal to 5%) to obtain the enzymolysis peptides with different molecular weights, which specifically comprises the following steps:
1000 u-3000 u of sandalwood seed compound protease enzymolysis peptide;
3000 u-5000 u of compound protease of sandalwood seeds is used for enzymolysis of peptide;
the sandalwood seeds are compounded with 5000 u-7000 u protease for enzymolysis of peptide;
the sandalwood seed is compounded with 7000 u-10000 u of protease for enzymolysis of peptide.
Example 2: papain enzymolysis peptide
First, experimental material
Sandalwood (Santalbum L.) seeds are collected from a 5-year-old sandalwood forest, the seed coats of the sandalwood are manually removed after collection, and then the sandalwood seeds are dried in the shade in the environment of normal temperature, ventilation and no direct sunlight.
Papain (50X 10)4U/g) was purchased from Sigma, USA.
Second, Experimental methods and results
1. Extraction of sandalwood seed protein
Crushing peeled sandalwood seeds into 50-60 meshes of powder, weighing 40g, adding 600ml of double distilled water, adjusting the pH value to 8.5 by using 1mol/LNaOH solution, stirring in a water bath at 40 ℃ for 4 hours, filtering by using gauze, and filtering out insoluble impurities to obtain a clear solution; adjusting pH value to an isoelectric point (pI is 4.2) by using 1mol/LHCl, standing at room temperature for 30min, centrifuging (4000r/min multiplied by 15min), discarding supernatant to obtain milky protein precipitate, and slightly rinsing the protein precipitate with distilled water for three times for later use.
2. Enzymolysis of sandalwood seed protein
Placing the opalescent protein precipitate in distilled water according to the concentration of 0.05mg/mL until the opalescent protein precipitate is suspended until dissolved, adding papain for enzymolysis, wherein the adding amount of the papain is 420U/g of substrate, and the enzymolysis conditions are as follows: pH value, 7.0; the enzymolysis temperature is 42 ℃; the enzymolysis time is 8 hours; after enzymolysis, inactivating papain in water bath at 85 ℃ for 10 minutes, and cooling to normal temperature; after cooling, centrifuging at 9000rpm for 25 minutes, taking the supernatant, and freeze-drying to obtain the freeze-dried powder.
3. Separation and purification of enzymolysis peptide
Dissolving the freeze-dried powder with distilled water, adding 25mL of distilled water into 1g of freeze-dried powder, then sequentially separating the obtained enzymolysis peptides by ultrafiltration membranes with the molecular weight cut-off of 10000u, 7000u, 5000u, 3000u and 1000u, and freeze-drying the components obtained by ultrafiltration (the water content is less than or equal to 5%) to obtain the enzymolysis peptides with different molecular weights, which specifically comprises the following steps:
1000 u-3000 u of papain of sandalwood seeds is used for enzymolysis of peptide;
3000 u-5000 u of papain of sandalwood seeds;
5000 u-7000 u of papain of sandalwood seeds is used for enzymolysis of peptides;
the papain of sandalwood seeds is 7000 u-10000 u for enzymolysis of peptide.
Example 3: anti-prostate cancer effect
First, experimental material
Prostate cancer PC-3 cells were purchased from ATCC. Reagents required for MTT experiments were purchased from Nanjing as a built organism.
1000 u-3000 u of sandalwood seed compound protease, 3000 u-5000 u of sandalwood seed compound protease, 5000 u-7000 u of sandalwood seed compound protease, and 7000 u-10000 u of sandalwood seed compound protease are prepared according to the method of example 1, and stored at 4 ℃ for later use.
1000 u-3000 u of sandalwood seed papain, 3000 u-5000 u of sandalwood seed papain, 5000 u-7000 u of sandalwood seed papain, and 7000 u-10000 u of sandalwood seed papain are prepared according to the method of example 2 and stored at 4 ℃ for later use.
Second, Experimental methods
The MTT method is used for detecting the inhibition effect of the medicine on the proliferation of the prostatic cancer PC-3 cells:
PC-3 cells in log phase were cultured in 96-well plates at a seeding density of 104And (4) adding enzymolysis peptides with different final concentrations into each well after the cells grow completely adherent to the wall, simultaneously setting control groups, setting 6 multiple wells in each group, and culturing for 24 h. Discarding the old culture solution, adding 5 μ L of 5mg/mL MTT solution into each well, incubating at 37 deg.C for 4h, discarding the supernatant, adding 100 μ L of LDMSO into each well, shaking thoroughly, measuring absorbance at 570nm, and calculating the inhibition rate according to the following formula：
Inhibition (%) [ 1- (absorbance value of drug/absorbance value of control) ] × 100%.
IC50 was calculated using the LOGIT method.
Third, experimental results
The results of the experiment are shown in table 1 and fig. 1. The enzymolysis peptide with different molecular weight ranges, which is prepared by adopting different enzymes, of the sandalwood seeds has different activities of resisting the prostatic cancer, wherein: the effect of the sandalwood seed compound protease 3000 u-5000 u enzymolysis peptide, the sandalwood seed compound protease 7000 u-10000 u enzymolysis peptide and the sandalwood seed papain 5000 u-7000 u enzymolysis peptide on resisting prostate cancer is not obvious, and other 5 enzymolysis peptides have stronger activity on resisting prostate cancer.
TABLE 1 IC50 values for the anti-prostate cancer activity of each zymolytic peptide
| Enzymolysis peptide | IC50 value (μ g/mL) |
| Enzymolysis peptide of compound protease 1000 u-3000 u (compound 1000-3000) | 9.52 |
| Enzymolysis peptide of compound protease 3000 u-5000 u (compound 3000-5000) | ＞100 |
| 5000 u-7000 u composite protease enzymolysis peptide (5000-7000 composite) | 8.78 |
| Composite protease 7000 u-10000 u enzymolysis peptide (composite 7000-10000) | ＞100 |
| Papain 1000 u-3000 u enzymolysis peptide (pawpaw 1000-3000) | 11.45 |
| Papain 3000 u-5000 u enzymolysis peptide (pawpaw 3000-5000) | 8.63 |
| Papain 5000 u-7000 u enzymolysis peptide (pawpaw 5000-7000) | ＞100 |
| Papain 7000u ~ 10000u enzymolysis peptide (pawpaw 7000 ~ 10000) | 10.17 |
In conclusion, the sandalwood seed compound protease enzymolysis peptide with the molecular weight range of 1000-3000 u or 5000-7000 u provided by the invention has excellent activity of resisting prostate cancer, and the sandalwood seed papain enzymolysis peptide with the molecular weight range of 1000-3000, 3000-5000 or 7000-10000 provided by the invention has excellent activity of resisting prostate cancer.
The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.
Claims (1)
1. The application of an enzymolysis peptide in preparing an anti-prostate cancer medicament is prepared by the following steps:
s1, extraction of sandalwood seed protein
Pulverizing peeled lignum Santali albi seed, weighing appropriate amount of powder, adding double distilled water, adjusting pH to 8.5 with NaOH solution, stirring in 40 deg.C water bath for 4 hr, filtering with gauze, and filtering to remove insoluble impurities to obtain clear solution; adjusting the pH value to an isoelectric point by using an HCl solution, standing at room temperature, centrifuging, removing supernatant to obtain milky protein precipitate, and slightly rinsing the precipitate by using distilled water for later use;
s2, enzymolysis of sandalwood seed protein
Placing the opalescent protein precipitate in distilled water for suspending until the opalescent protein precipitate is dissolved, adding papain for enzymolysis, wherein the addition amount of the papain is 420U/g of substrate, and the enzymolysis conditions are as follows: pH value, 7.0; the enzymolysis temperature is 42 ℃; the enzymolysis time is 8 hours; after enzymolysis, heating to inactivate protease, and cooling to normal temperature; cooling, centrifuging, collecting supernatant, and freeze drying to obtain lyophilized powder;
s3, separation and purification of enzymolysis peptide
Dissolving the freeze-dried powder with distilled water, then sequentially separating the obtained enzymolysis peptide with ultrafiltration membranes with the molecular weight cutoff of 3000u and 1000u, and freeze-drying the components of 1000 u-3000 u obtained by ultrafiltration to obtain the freeze-dried powder;
wherein: in the step S1, the raw materials are crushed into 50-60 meshes of powder, 600ml of double distilled water is added into every 40g of the powder, the isoelectric point is 4.2, and the centrifugation condition is 4000r/min multiplied by 15 min; in step S2, the opalescent protein precipitate is placed in distilled water according to the concentration of 0.05mg/mL until being dissolved, and is subjected to water bath at the temperature of 85 ℃ for 10 minutes to inactivate protease, and the centrifugation condition is 9000r/min multiplied by 25 min; in step S3, 25mL of distilled water was added to 1g of lyophilized powder.
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| CN1093600A (en) * | 1993-04-10 | 1994-10-19 | 河南省三门峡市九九三神力保健品厂 | Prostate health-care article and manufacture method thereof |
| CN100376684C (en) * | 2006-03-28 | 2008-03-26 | 华南理工大学 | Process for preparing bacteriostatic peptide by discarded tobacco leaf protein |
| CN104480177B (en) * | 2014-12-31 | 2018-05-04 | 广西壮族自治区农业科学院农产品加工研究所 | A kind of preparation method of the numb protein ACE inhibitory peptide of fire |
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