A method for extracting ACE inhibitory peptides from tilapia skin

Abstract

The present invention specifically relates to a method for extracting ACE inhibitory peptides from tilapia skin. Tilapia skin is a byproduct rich in collagen, and has important development significance as a raw material for screening ACE inhibitors. The present invention is designed to provide a method for extracting ACE inhibitors using tilapia skin as a raw material, comprising the following steps: defatting the tilapia skin and crushing it, adding acid to extract to obtain fish skin collagen crude protein, adding enzyme to the fish skin collagen crude protein for enzymolysis, and retaining the part of the enzymolysis product with a molecular weight less than 3kDa is the above-mentioned ACE inhibitory peptide; in particular, the above-mentioned enzyme is a product of Bacillus subtilis or Bacillus coagulans under gelatin-induced fermentation.

Description

Technical Field

[0001] The invention relates to the technical field of collagen extraction, and in particular to a method for extracting ACE inhibitory peptides from tilapia skin, the ACE inhibitory peptides extracted by the method, and applications of the ACE inhibitory peptides in clinical practice and drug development. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Tilapia is an omnivorous fish with strong reproductive capacity. It is a major farmed fish species and an important part of my country's freshwater aquaculture species. It is not only favored by diners for its delicious taste, but also its by-products have high application value. Among them, tilapia skin is rich in collagen, and most of them are type I and type III, which are highly similar to the types of collagen in human skin. Therefore, tilapia skin is expected to be used in the manufacture of medical supplies such as wound dressings and artificial skin. These products have good biocompatibility and plasticity, meeting the special needs of the medical field. In addition, the hydrolyzate of collagen in fish skin has also been reported to have good medical activity, such as antimicrobial peptides, antihypertensive peptides, etc., such as the preparation of angiotensin converting enzyme (ACE) inhibitory peptides.

[0004] Angiotensin converting enzyme inhibitors (ACEI) are substances that inhibit the activity of ACE and are also commonly used antihypertensive drugs, including captopril, enalapril, ramipril, etc. Their antihypertensive mechanism is: (1) Inhibition of ACE activity: ACEI inhibits ACE in plasma and tissues, reduces the formation of angiotensin II in the systemic circulation and in tissues such as the heart, kidneys, and blood vessels, weakens the effects of angiotensin II in contracting blood vessels, promoting myocardial and vascular hypertrophy, and promoting aldosterone secretion, causing a decrease in peripheral vascular resistance, improved arterial compliance, reduced water and sodium retention, and preventing myocardial and vascular remodeling. (2) Inhibition of bradykinin degradation: It increases the bradykinin content in the blood, thereby promoting the production of nitric oxide (NO) and prostacyclin (PGI2), enhancing their vasodilatory and antihypertensive effects.

[0005] At present, the main methods for extracting collagen from fish skin include acid extraction, alkaline extraction or enzymatic extraction. The acid / alkaline extraction method will cause certain damage to the collagen structure. The enzymatic method mainly uses animal collagenase (such as trypsin), plant collagenase (such as papain) or microbial collagenase to hydrolyze collagen macromolecules. Microbial collagenase is a protease that can degrade collagen secreted by certain microorganisms under specific conditions. It has the following advantages: thorough degradation, more degradation sites, more adaptable substrate types, and can be prepared in batches by fermentation. Summary of the invention

[0006] In view of the above situation, the present invention believes that tilapia skin, as a byproduct rich in collagen, has great economic significance for screening highly active ACE inhibitors. In order to obtain a more thorough enzymatic hydrolysis effect and improve the activity of enzymatic hydrolysis products, designing induction and cultivating a more suitable microbial collagenase is expected to achieve the above technical effects. Bacillus subtilis and Bacillus coagulans are strains reported in the prior art to have collagenase secretion activity. The present invention attempts to induce and cultivate the above-mentioned varieties with collagenase secretion activity in order to obtain a microbial collagenase product with higher activity and more thorough enzymatic hydrolysis.

[0007] Based on the above technical ideas, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for extracting ACE inhibitory peptides from tilapia skin, comprising the following steps:

[0009] Tilapia skin is defatted and crushed, acid solution is added to extract to obtain crude collagen protein from the skin, enzyme is added to the crude collagen protein for enzymolysis, and the part of the enzymolysis product with a molecular weight less than 3kDa is the above-mentioned ACE inhibitory peptide; in particular, the above-mentioned enzyme is the product of Bacillus subtilis or Bacillus coagulans under gelatin-induced fermentation.

[0010] In the above preparation method, the extraction process of the fish skin collagen crude protein is as follows:

[0011] The degreasing is performed by soaking in an isopropanol solution, the concentration of the isopropanol solution is 8-12%, and the soaking time is 20-40 hours. In order to improve the degreasing effect, the fish skin should be cleaned in advance, the fat and fascia tissue on the surface of the fish skin should be fully removed, and the fish skin should be cut into small pieces and then soaked for degreasing.

[0012] The pulverization can be carried out by high-speed grinding, and the diameter of the powder particles after pulverization is preferably 180-220 μm. A feasible grinding method is ball milling. In an embodiment with better effect provided by the present invention, the defatted fish skin is pre-freeze-dried and then ball-milled to improve the pulverization efficiency.

[0013] The acid solution is preferably an inorganic acid solution, such as formic acid, acetic acid or propionic acid, with a concentration of 500-700 mmol / L, an extraction time of 70-80 hours, and the extraction process is at room temperature; after the extraction is completed, the reaction system is centrifuged, and the retained solution is partially salted out to obtain the above-mentioned fish skin collagen crude protein; the salting out is preferably an inorganic salt, such as sodium chloride or ammonium sulfate.

[0014] In the above preparation method, the preparation method of the enzyme is as follows: fermenting Bacillus subtilis or Bacillus coagulans under gelatin-induced conditions, and salting out the fermentation product to obtain the above enzyme.

[0015] Furthermore, during the above fermentation process, the gelatin content in the culture medium is 0.4-0.6%, and the culture medium uses gelatin and peptone as nitrogen sources, and the mass ratio of the two is 0.8-1.2:1.

[0016] In existing studies, gelatin is often used as a coagulant for solid or semi-solid culture media to fix and observe the growth status of bacteria. At this time, the amount of gelatin added is 10-12%. The present invention envisions inducing the secretion of collagenase in microorganisms by adding gelatin as a fermentation substrate. It has been verified that when the amount of gelatin added to the culture medium is 0.4-0.6%, the activity of collagenase in microbial fermentation products can be effectively improved. The present invention uses Bacillus subtilis and Bacillus coagulans that can secrete collagenase as experimental subjects. It has been verified that Bacillus gellingensis has better responsiveness under the above-mentioned induced culture method, and the enzyme activity of collagenase in the fermentation product is higher. The present invention performs gelatin zymogram detection on the enzyme products induced by the fermentation of the above two strains. The results show that the enzyme generated by the fermentation of Bacillus coagulans has a higher content of matrix metalloproteinases, which has a higher decomposition efficiency and specificity on interstitial collagen.

[0017] In one embodiment of the present invention, the method of inducing fermentation culture is as follows:

[0018] Bacillus subtilis or Bacillus coagulans is inoculated into a seed liquid culture medium and fermented for a period of time, then inoculated into a fermenter for cultivation, the inoculation amount is 5-7%, the stirring speed is 220-270 r / min, the ventilation volume is 2.5-3.5 L / min, the fermentation temperature is 30-38°C, the fermentation time is 30-36 h, and the fermentation medium includes the following components: 2.0% glucose, 0.5% peptone, 0.4-0.6% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, the balance is water, and the pH is 7.5.

[0019] In the above preparation method, the reaction parameters of the enzymatic hydrolysis are as follows: adding enzyme to the fish skin collagen crude protein for enzymatic hydrolysis, the amount of enzyme added is 1200-1700 U / g crude protein, the reaction time is 1.5-3 hours, the temperature is 38° C., and the pH is 7.0-7.2. After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysis can be terminated by high temperature inactivation, or the subsequent steps can be directly performed, which can be flexibly adjusted according to the production purpose.

[0020] After the above enzymatic hydrolysis reaction is completed, the reaction system is centrifuged to retain the solution portion, which is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The retained portion is the ACE inhibitory peptide.

[0021] The second aspect of the present invention provides an ACE inhibitory peptide prepared by the method described in the first aspect.

[0022] The above-mentioned ACE inhibitory peptide is a mixture of small peptides with a molecular weight of less than 3kDa. The present invention verifies through ACE inhibitory activity that the above-mentioned ACE inhibitory peptide has good inhibitory activity, especially the ACE inhibitory peptide induced by Bacillus coagulans gelatin enzyme production, whose inhibitory activity is significantly better than other small peptides.

[0023] Based on the inhibitory activity of the above ACE inhibitory peptide, it is expected to be used as an ACE inhibitor in the clinical treatment of hypertension, congestive heart failure and myocardial infarction, diabetic nephropathy and other kidney diseases and the corresponding drug development. Especially in the field of antihypertensive drugs, ACE inhibitors have antihypertensive activity recognized in the art. Therefore, the present invention also provides the following technical solutions:

[0024] The third aspect of the present invention provides a pharmaceutical composition, wherein the composition comprises the ACE inhibitory peptide described in the first aspect.

[0025] In a fourth aspect, there is provided use of the ACE inhibitory peptide described in the second aspect and the pharmaceutical composition described in the third aspect in the preparation of an antihypertensive drug.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. One aspect of the present invention provides a method for enzymatically extracting ACE inhibitory peptides using tilapia skin as raw material. In this study, the present invention adds a small amount of gelatin to the fermentation substrate to induce microbial fermentation to produce more collagenase. It has been verified that the above-mentioned induction fermentation method can effectively increase the activity of collagenase in the fermentation product of Bacillus coagulans and produce more matrix metalloproteinases.

[0028] 2. The present invention adopts the above-mentioned induced fermentation enzyme to add to the fish skin crude protein for enzymatic hydrolysis. The small-weight (<3kDa) peptide produced by enzymatic hydrolysis has higher ACE inhibitory activity. The results show that the product enzyme of the above-mentioned induced fermentation has better collagen decomposition activity and more thorough enzymatic hydrolysis of collagen. Small peptide products with better activity and higher market value can be obtained through enzymatic hydrolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 The standard curve of tyrosine concentration-OD value in the collagenase activity detection described in Example 1;

[0031] Figure 2 This is the fermentation curve of the two strains gelatin induction group described in Example 1;

[0032] Figure 3 The following are the gelatin zymogram test results of the crude enzyme solution of the gelatin-induced group of the two strains described in Example 1. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0036] Example 1

[0037] 1. Materials and methods

[0038] 1.1 Materials and reagents

[0039] Tilapia skin, purchased from Jimo Aquatic Product Market;

[0040] Folin-phenol reagent was purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.;

[0041] Tyrosine reference substance was purchased from Chengdu Gelipu Biotechnology Co., Ltd.;

[0042] ACE inhibitory activity kit was purchased from Shanghai Chupeptide Biotechnology Co., Ltd.;

[0043] Bacillus coagulans, purchased from Ningbo Testo Biotechnology Co., Ltd., catalog number TS279762;

[0044] Bacillus subtilis was purchased from Ningbo Testo Biotechnology Co., Ltd., catalog number TS276952.

[0045] 1.2 Research Methods

[0046] 1.2.1 Collagenase activity detection method

[0047] Using tyrosine as the standard, the Folin-phenol method was used to determine the activity of collagenase. Enzyme activity was defined as the amount of enzyme that converts 1 μmol of substrate per minute, which was defined as one unit of enzyme activity. After mixing gradient concentrations of tyrosine solution, Na2CO3 solution, and Folin-phenol reagent, the mixture was incubated in a water bath for 15 min, and the OD value was detected to establish a standard curve of tyrosine concentration-OD value (see attached). Figure 1 ), the enzyme activity calculation formula is as follows:

[0048] Enzyme activity = A / t×N

[0049] Where A is the mass of tyrosine, t is the reaction time, and N is the dilution multiple of the enzyme solution.

[0050] 1.2.2 Determination of ACE inhibitory activity

[0051] Prepare 6.5mmol / L HHL solution (solvent is 0.3mol / L borate buffer, pH8.0), add 20μL of the sample to be tested to 50μL HHL solution, add ACE enzyme after incubation, terminate the reaction after 1.5h, extract the reaction product with ethyl acetate, detect the absorbance at 228nm, replace the sample solution with an equal dose of solvent in the control group, and do not add sample solution to the blank group. The ACE inhibition rate is calculated as follows:

[0052] ACE inhibition rate (%) = [1-(A 样品 -A 空白样品 ) / (A 对照 -A 空白对照 )]×100%

[0053] In the above formula, A 样品 is the OD value of the sample to be tested, A 空白样品 is the OD value of the blank sample group, A 对照 is the OD value of the control group, A 空白对照 is the OD value of the blank control group.

[0054] 2. Research Methods

[0055] (1) Fermentation of bacterial strains: The above-mentioned Bacillus subtilis and Bacillus coagulans were inoculated into seed liquid culture medium (1% beef extract, 2% peptone, 0.5% NaCl, the balance was water, pH 7.5) at a fermentation temperature of 37°C. After 24 h of shaking flask culture, the culture was transferred to a fermenter with an inoculation size of 6%, a stirring speed of 250 r / min, a ventilation volume of 3 L / min, a fermentation temperature of 30°C, and a total fermentation time of 60 h. Sampling was performed every three hours.

[0056] The culture medium of the above fermentation tank includes the following components: 2.0% glucose, 1% nitrogen source, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, and the balance is water, with a pH of 7.5.

[0057] (2) Extraction of crude enzyme: Centrifuge the fermentation broth at 6500 r / min and 4°C for 15 min, retain the supernatant, and slowly add solid ammonium sulfate to the solution while stirring until saturated. Salt out at 4°C overnight to fully precipitate the collagenase. Centrifuge and retain the precipitate, transfer to 0.2 mol / L pH 7.8 phosphate buffer to dissolve, and obtain the crude enzyme solution for later use.

[0058] (3) Extraction of ACE inhibitory peptides: Soften the fish skin in a 2% NaCl solution for 2 h, brush repeatedly with a wire brush until all scales and fascia tissue are removed, cut into 1 cm × 1 cm pieces and add to a 10% isopropanol solution, soak at 4°C for 24 h for degreasing, rinse repeatedly with distilled water until neutral, freeze-dry and grind at high speed to obtain fish skin powder with a particle diameter of about 200 μm. Weigh the fish skin powder and add it to 10 times the volume of acetic acid solution (600 mmol / L) and stir continuously for 72 h, centrifuge at 12000 r / min for 10 min, retain the supernatant, add NaCl for salting out and centrifuge to retain the precipitate to obtain fish skin collagen crude protein.

[0059] The crude enzyme solution prepared in (2) was added to the above fish skin collagen crude protein, the amount added was 1500U / g crude protein, the pH was 7.2, the enzymatic hydrolysis time was 2h, the enzymatic hydrolysis temperature was 38°C, and after the enzymatic hydrolysis reaction was completed, the reaction system was transferred to a 100°C water bath and heated for 10min to terminate the reaction, and then the enzymatic hydrolyzate was centrifuged and the precipitate was discarded. The supernatant was ultrafiltered using a 3kDa ultrafiltration membrane, and the portion with a molecular weight less than 3kDa was retained for freeze-drying, and re-added to 20mmol / L NaAC-HAC to be re-dissolved to 30mg / mL, recorded as ACE inhibitory peptide, and the ACE inhibitory activity was measured.

[0060] 3. Experimental results

[0061] (1) In the above-mentioned bacterial fermentation stage, this example studied the results of fermentation using different nitrogen sources. In order to obtain a fermentation product with higher collagenase activity, this example attempted to add part of gelatin to the fermentation broth to replace the traditional nitrogen source for induction fermentation. The induction culture scheme in the fermentation tank culture stage is shown in Table 1 below, wherein the nitrogen source is 1% peptone, that is, all nitrogen sources use traditional peptone, which is recorded as the "peptone group"; the nitrogen source is 1% gelatin, that is, all nitrogen sources are replaced by gelatin, which is recorded as the "gelatin group"; and peptone + gelatin (0.5% + 0.5%), that is, the nitrogen source is composed of peptone and gelatin, and the mass ratio of the two is 1:1, which is recorded as the "gelatin induction group". The enzyme activity at the fermentation peak and the inhibitory activity of the ACE inhibitory peptide using the above-mentioned culture scheme are shown in Table 1:

[0062] Table 1 Fermentation tank culture scheme and enzyme activity and ACE inhibitory activity

[0063]

[0064] During the fermentation process, this example found that the fermentation and reproduction speed of the peptone group of the two strains was faster, and the enzyme activity could reach a peak value in about 30 hours, while the gelatin group of the two strains had a slower reproduction speed and the corresponding enzyme activity was significantly reduced. The fermentation speed of the gelatin-induced group was slightly lower than that of the peptone group, but the enzyme activity did not decrease. The enzyme activity of the Bacillus coagulans group also increased significantly. This example plotted the fermentation curves of the gelatin-induced group of the two strains ( Figure 2 ), and gelatin zymography was used to detect the crude enzyme solution of the gelatin-induced groups of the above two strains. The results are as follows Figure 3 .

[0065] from Figure 2 It can be seen that the fermentation enzyme activity of Bacillus coagulans reached the highest level at around 36h, significantly exceeding that of other culture groups. Combined with the above ACE inhibitory activity, the crude enzyme liquid separated from the gelatin-induced group of Bacillus coagulans has higher enzymatic activity, and the ACE inhibitory peptide obtained by enzymatic hydrolysis of fish skin collagen crude protein has stronger activity.

[0066] Figure 3 The gelatin zymogram electrophoresis separation results of the crude enzyme solutions of the two induced groups are shown. It can be clearly seen from the figure that the crude enzyme solution of the gelatin induced group of Bacillus coagulans has obvious white bands at 95kD and 72kD, which means that the crude enzyme solution of the gelatin induced group of Bacillus coagulans contains more matrix metalloproteinases MMP-2 and MMP-9, which can specifically and efficiently degrade extracellular matrix components and improve the degradation efficiency of collagen.

[0067] Example 2

[0068] Combined with the research results in the above Example 1, this example provides a method for extracting ACE inhibitory peptides from tilapia skin, wherein the molecular weight of the ACE inhibitory peptides is less than 3 kDa and has good ACE inhibitory activity. The extraction method steps are as follows:

[0069] (1) Inoculate Bacillus coagulans into seed liquid culture medium (1% beef extract, 2% peptone, 0.5% NaCl, the balance is water, pH 7.5), ferment at 38°C, culture in shake flasks for 24 hours, then transfer to a fermenter (2.0% glucose, 0.5% peptone, 0.4% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, the balance is water, pH 7.5), inoculation amount of 6%, stirring speed of 250r / min, ventilation volume of 3L / min, fermentation temperature of 30°C, fermentation time of 36h. After fermentation, centrifuge the fermentation broth at 6500r / min and 4°C for 15min, and retain the supernatant. Add ammonium sulfate to the supernatant while stirring until saturated. Let it stand at 4°C overnight to allow the collagenase in the fermentation product to fully precipitate. Transfer the retained precipitate into 0.2 mol / L pH 7.8 phosphate buffer for re-dissolution. Repeat the dialyzation against water 1 to 3 times to obtain a crude enzyme solution for use.

[0070] (2) Wash the tilapia skin, cut it into small pieces, and soak it in a 10% isopropanol solution for degreasing for 20 to 40 hours. Rinse it, freeze-dry it, and grind it in a planetary ball mill to a particle diameter of 200 μm. Weigh the ground fish skin powder, add it to 10 times the volume of acetic acid solution (600 mmol / L), stir it continuously for 72 hours, centrifuge it at 12000 r / min for 10 minutes, retain the supernatant, add NaCl for salting out, and centrifuge it to retain the precipitate to obtain fish skin collagen crude protein.

[0071] (3) Add the crude enzyme solution to the crude fish skin collagen protein, the amount added is 1500U / g crude protein, the pH is 7.2, the enzymatic hydrolysis time is 2h, the enzymatic hydrolysis temperature is 38°C, after the reaction is completed, the reaction system is heated to 100°C for 10min to terminate the reaction, and then the enzymatic hydrolyzate is centrifuged and the precipitate is discarded. The supernatant after centrifugation is ultrafiltered using a 3kDa ultrafiltration membrane, and the portion with a molecular weight less than 3kDa is the ACE inhibitory peptide.

[0072] Example 3

[0073] In this embodiment, another method for extracting ACE inhibitory peptides from tilapia skin is provided. The extraction method steps are as follows:

[0074] (1) Inoculate Bacillus coagulans into seed liquid culture medium (2% beef extract, 3% peptone, 0.5% NaCl, pH7.5), ferment at 40°C, culture in shake flask for 20 hours, then transfer to fermenter (2.0% glucose, 0.5% peptone, 0.5% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, balance water, pH7.5), inoculation amount of 7%, stirring speed of 220 r / min, ventilation volume of 2.5 L / min, fermentation temperature of 35°C, fermentation time of 33 hours. After fermentation, centrifuge the fermentation broth at 6000 r / min and 4°C for 20 minutes, and retain the supernatant. Add ammonium sulfate to the supernatant while stirring until saturated. Let it stand at 4°C overnight to allow the collagenase in the fermentation product to fully precipitate. Transfer the retained precipitate by centrifugation to 0.2 mol / L pH 7.8 phosphate buffer for re-dissolution. Repeat the dialyzation against water 1 to 3 times to obtain a crude enzyme solution for use.

[0075] (2) Wash the tilapia skin, cut it into small pieces, and soak it in 8% isopropanol solution for degreasing for 20 to 40 hours. Rinse it, freeze-dry it, and grind it in a planetary ball mill to a particle diameter of 180 μm. Weigh the ground fish skin powder, add it to 12 times the volume of acetic acid solution (600 mmol / L), stir it continuously for 80 hours, centrifuge it at 13000 r / min for 8 minutes, retain the supernatant, add NaCl for salting out, and centrifuge it to retain the precipitate to obtain fish skin collagen crude protein.

[0076] (3) Add the above crude enzyme solution to the above fish skin collagen crude protein, the addition amount is 1200U / g crude protein, the pH is 7.2, the enzymatic hydrolysis time is 3h, the enzymatic hydrolysis temperature is 38°C, after the reaction is completed, the reaction system is heated to 100°C for 12min to terminate the reaction, and then the enzymatic hydrolyzate is centrifuged and the precipitate is discarded. The supernatant after centrifugation is ultrafiltered using a 3kDa ultrafiltration membrane, and the portion with a molecular weight less than 3kDa is the ACE inhibitory peptide.

[0077] Example 4

[0078] In this embodiment, another method for extracting ACE inhibitory peptides from tilapia skin is provided. The extraction method steps are as follows:

[0079] (1) Inoculate Bacillus subtilis into seed liquid culture medium (0.8% beef extract, 1.8% peptone, 0.5% NaCl, the balance is water, pH 7.5), fermentation temperature 35℃, shake flask culture for 30h, then transfer to fermentation tank (2.0% glucose, 0.5% peptone, 0.6% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, the balance is water, pH 7.5), inoculation amount 5%, stirring speed 270r / min, ventilation volume 3.5L / min, fermentation temperature 30℃, fermentation time 30h. After fermentation, centrifuge the fermentation liquid at 5500r / min and 4℃ for 18min, and retain the supernatant. Add ammonium sulfate to the supernatant while stirring until saturated. Let it stand at 4°C overnight to allow the collagenase in the fermentation product to fully precipitate. Transfer the retained precipitate by centrifugation to 0.2 mol / L pH 7.8 phosphate buffer for re-dissolution. Repeat the dialyzation against water 1 to 3 times to obtain a crude enzyme solution for use.

[0080] (2) Wash the tilapia skin, cut it into small pieces, and soak it in a 12% isopropanol solution for degreasing for 20 to 40 hours. Rinse it, freeze-dry it, and grind it in a planetary ball mill to a particle diameter of 220 μm. Weigh the ground fish skin powder, add it to 8 times the volume of acetic acid solution (600 mmol / L), stir it continuously for 70 hours, centrifuge it at 11000 r / min for 12 minutes, retain the supernatant, add NaCl for salting out, and centrifuge it to retain the precipitate to obtain fish skin collagen crude protein.

[0081] (3) Add the crude enzyme solution to the crude fish skin collagen protein in an amount of 1700 U / g crude protein, pH 7.2, enzymolysis time 1.5 h, enzymolysis temperature 38°C, terminate the reaction, then centrifuge the enzymolysis solution and discard the precipitate. Ultrafilter the supernatant after centrifugation using a 3 kDa ultrafiltration membrane, and the portion with a molecular weight less than 3 kDa is the ACE inhibitory peptide.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting ACE inhibitory peptides from tilapia skin, characterized in that: The steps include: The tilapia skin is defatted and crushed, and an acid solution is added to extract the crude collagen protein from the skin, and an enzyme is added to the crude collagen protein for enzymolysis, and the part with a molecular weight less than 3 kDa in the enzymolysis product is retained as the above-mentioned ACE inhibitory peptide; The preparation method of the enzyme is as follows: Bacillus subtilis or Bacillus coagulans is inoculated into a seed liquid culture medium, fermented and cultured for a period of time, and then inoculated into a fermenter for culture, the inoculation amount is 5-7%, the stirring speed is 220-270 r / min, the ventilation volume is 2.5-3.5 L / min, the fermentation temperature is 30-38°C, the fermentation time is 30-36 hours, the fermentation medium includes the following components: 2.0% glucose, 0.5% peptone, 0.4-0.6% gelatin, 0.1% CaCl2, 0.4% Na2HPO4, 0.03% KH2PO4, the balance is water, the pH is 7.5, and the fermentation product is salted out to obtain the enzyme.

2. The method according to claim 1, characterized in that The degreasing step is as follows: removing fat and fascia tissue on the surface of the fish skin, cutting it into small pieces and adding it into an isopropanol solution for soaking and degreasing; the concentration of the isopropanol solution is 8-12%, and the soaking time is 20-40 hours.

3. The method according to claim 1, characterized in that The crushing step is as follows: pre-freeze-drying the defatted tilapia skin, and then ball-milling the skin, so that the diameter of the powder particles after crushing is 180-220 μm.

4. The method according to claim 1, characterized in that The acid solution is an acetic acid solution with a concentration of 500-700 mmol / L, an extraction time of 70-80 hours, and the extraction process is performed at room temperature.

5. The method according to claim 1, characterized in that After the extraction is completed, the reaction system is centrifuged, and the retained solution is partially salted out to obtain the fish skin collagen crude protein; the salting out is carried out using sodium chloride or ammonium sulfate.

6. The method according to claim 1, characterized in that The reaction parameters of the enzymatic hydrolysis are as follows: adding enzyme to the fish skin collagen crude protein for enzymatic hydrolysis, the amount of enzyme added is 1200-1700 U / g crude protein, the reaction time is 1.5-3 hours, the temperature is 38° C., and the pH is 7.0-7.

2.

7. An ACE inhibitory peptide prepared by the method according to any one of claims 1 to 6.

8. A pharmaceutical composition, characterized in that The composition comprises the ACE inhibitory peptide according to claim 7.

9. Use of the ACE inhibitory peptide according to claim 7 and the pharmaceutical composition according to claim 8 in the preparation of antihypertensive drugs.

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