CN117343132B - ACE (angiotensin converting enzyme) inhibitory peptide derived from salmon skin and preparation method thereof - Google Patents

ACE (angiotensin converting enzyme) inhibitory peptide derived from salmon skin and preparation method thereof Download PDF

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CN117343132B
CN117343132B CN202311240481.9A CN202311240481A CN117343132B CN 117343132 B CN117343132 B CN 117343132B CN 202311240481 A CN202311240481 A CN 202311240481A CN 117343132 B CN117343132 B CN 117343132B
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ace
inhibitory peptide
ace inhibitory
alteromonas
enzymolysis
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CN117343132A (en
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边斐
岳寿松
赵华
陈高
马德源
张燕
于金慧
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Shandong Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Abstract

The invention belongs to the technical field of microbial enzymolysis and comprehensive utilization of aquatic product processing waste, and particularly relates to salmon skin ACE (angiotensin converting enzyme) inhibitory peptide and a preparation method thereof. The invention provides a novel marine alteromonas P-1%AteromonasProduction process of sp.P-1) production enzyme preparation, and enzymolysis process of enzyme preparation for preparing novel ACE inhibitory peptide, hexapeptide GLPGPP obtained by enzymolysis has strong ACE inhibitory activity, and IC 50 The value is 0.206mg/mL, and provides a sequence source for the artificial synthesis of antihypertensive peptides in the future. The salmon skin disclosed by the invention is an excellent protein raw material for producing ACE inhibitory peptide, and lays a research and development foundation for the development and utilization of marine new bacteria and new enzyme resources and the resource conversion and high-value utilization of aquatic product processing wastes.

Description

ACE (angiotensin converting enzyme) inhibitory peptide derived from salmon skin and preparation method thereof
Technical Field
The invention belongs to the technical field of microbial enzymolysis and comprehensive utilization of aquatic product processing waste, and particularly relates to an ACE (angiotensin converting enzyme) inhibitory peptide derived from salmon skin and a preparation method thereof.
Background
Hypertension is a common chronic disease and is also an important cause of cardiovascular and cerebrovascular diseases, long-term hypertension can lead to arteriosclerosis, angina pectoris, myocardial infarction, end-stage renal disease and other diseases, and the death rate of cardiovascular and cerebrovascular diseases caused by hypertension is also first. The etiology of hypertension is quite complex, and it is currently widely believed that the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS), in which Angiotensin Converting Enzyme (ACE) plays a vital role in regulating blood pressure, play a central role. Studies have shown that: ACE is a zinc-containing carboxypeptidase enzyme capable of converting liver secreted angiotensin i to angiotensin ii in the RAS system, promoting vasoconstriction, resulting in increased blood pressure; in the KKS system, ACE reduces bradykinin, prostaglandin synthesis, resulting in an increase in blood pressure.
Antihypertensive peptides are capable of inhibiting ACE activity to lower blood pressure, also known as ACE inhibiting peptides. In the past, most of the common antihypertensive drugs on the market are chemically synthesized ACE inhibitors, and although the antihypertensive effect of the antihypertensive drugs is remarkable, serious side effects such as headache, rapid heart rate, dizziness, cough, dysgeusia and the like are accompanied. After the polypeptide with blood pressure reducing activity is extracted from snake venom for the first time in 1965 by Ferriera and an assistant, natural blood pressure reducing peptide derived from animals, plants and microorganisms gradually becomes a hot spot for developing blood pressure reducing medicines due to the advantages of obvious blood pressure reducing effect, no toxic or side effect and the like.
The ocean is a huge natural product treasury, and a special environment with high salt, high pressure, oxygen deficiency and lack of illumination, so that the ocean organism generates and accumulates a large amount of substances with special chemical structures, special biological activities and functions in the metabolic process, and the ocean organism has unique advantages and characteristics compared with Liu Shengdong plant protein in protein properties, so that the ocean organism also becomes an important source of ACE inhibitory peptides. Enzymatic hydrolysis is currently the most common method for obtaining antihypertensive peptides, and by selecting appropriate enzymes, active fragments of ACE inhibitory peptides are released under optimized reaction conditions. Table 1 illustrates the partially disclosed sequences and activities of the marine ACE inhibitory peptides obtained by enzymatic hydrolysis, and it can be seen that the proteases commonly used for the preparation of ACE inhibitory peptides are mainly commercial enzymes, including alkaline protease, papain, neutral protease, trypsin, pepsin, etc.
In recent years, marine bacteria have become an important source for selection of new bacteria and new enzymes, and the new enzymes of the marine sources possibly have novel catalytic properties and enzyme cleavage sites, and have great application potential in the field of preparation of bioactive peptides. The enzymatic hydrolysate of protease-hydrolyzed shrimp isolated from marine yeast Aureobasidium pullulans exhibited 85.3% ACE inhibitory activity and 52.1% antioxidant activity (Mar Biotechnol (NY), 2007,9 (3): 343-351.). Metalloproteinase from marine bacteria Anoxybacillus caldiproteolyticus A02591 hydrolyzes soybean protein, and hydrolysate ACE inhibitory activity IC 50 Three novel peptides RPSYT, VLIVP and LAIPVNKP (J Sci Food agric.,2023Jun 20.doi:10.1002/jsfa.12797.) were identified at 0.135 mg/mL. Salmon was digested with proteases from marine bacteria Pseudoalteromonas sp.sqn1 (Mar Drugs,2017,15,4) and Vibrio sp.sqs2-3 (Food chem.,2018, 248:346-352.), both of which exhibited strong antioxidant activity, and novel antioxidant peptides PMRGGGGYHY were identified in the hydrolysate of the Vibrio sp.sqs2-3 protease. The development and the utilization of new bacteria and new enzymes from ocean sources are promoted, and the development and the utilization are demands of scientific research and industrialization.
Salmon occupies a large portion of the world's processed fish. The share of salmon in the world aquatic product trade has grown enormously over the last decades: since 2013, salmon already accounts for nearly 20% of world fish trade, and becomes a single commodity with highest trade value in a global aquatic product trade chain; in 2021, the sales of salmon cultivated worldwide is 165 billion dollars, 221% greater than 2012. Byproducts of salmon processing, including bones, skins, heads, and viscera, contain high levels of protein (10-23% (w/w)) and serve as candidate substrates for the production of functional peptides.
TABLE 1 sequence and Activity of Marine ACE inhibitory peptides by enzymatic hydrolysis
Disclosure of Invention
Aiming at the research current situation and industrial demand mentioned in the background art, the invention provides a novel production process for producing an enzyme preparation by using marine alteromonas P-1 (Ateromonas sp.P-1), and an enzymolysis process for preparing salmon skin protein ACE inhibitory peptide by using the enzyme preparation, wherein hexapeptide GLPGPP obtained by enzymolysis has strong ACE inhibitory activity, and provides a sequence source for the artificial synthesis of ACE inhibitory peptide in the future. The Alternomonas sp.P-1 is preserved in China general microbiological culture Collection center, and the preservation time is as follows: 2023, 08, 04, deposit address: beijing, chaoyang area, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, deposit number: CGMCC No.28095, classified naming: alteromonas sp.
The innovation and beneficial effects of the invention are as follows:
1. the enzyme preparation for preparing ACE inhibitory peptide is prepared by fermenting a new strain of Alternomonas sp.P-1, and no report of preparing ACE inhibitory peptide by bacteria and protease from the strain is found at present.
The Alternomonas sp.P-1 provided by the invention is obtained by ultraviolet mutagenesis of Alternomonas sp.21CJ28. Alternomonas sp.21CJ28 was from a sediment of the intertidal zone of the sea area of Qingdao in China, whose 16S rRNA was 97.13% sequence identity with a sequence in the Alternomonas sp.RKMC-009 whole genome sequence in the NCBI database (GenBank: CP031010.1, microbiol resource Announc, 2019,8 (25), e 00508-19). It is currently believed that 16S rRNA similarity below 98.65% is considered a new species (Stackebrandt E, ebers J. Microbiol today, 2006,4 (4): 6-9.; mincheol Kim, jongsik Chun Method Microbiol.; 2014,41: 61-74.). Thus, alternomonas sp.21CJ28 belongs to a new species of Alternomonas (Genbank No. OR 078494.1). The 16S rRNA sequence of Alternomonas sp.P-1 and Alternomonas sp.21CJ28 is 100%, and P-1 is preserved in China general microbiological culture Collection center with a strain preservation number of CGMCC No.28095.
2.Alteromonas sp.P-1 is a high-yield strain of protease, the activity of the protease reaches the maximum value 97.23U/mL when the protease is fermented for about 30 hours in a fermentation tank, the high activity of the protease reduces the production cost and the use cost of the enzyme preparation, and the foundation is laid for the subsequent market application of the enzyme preparation.
Production and extraction of Alternomonas sp.P-1 protease: 2216E fermentation medium (0.5% corn flour, 0.5% soybean meal, 0.25% bran, 0.1% CaCl) was prepared 2 ，0.4％ Na 2 HPO 4 ，0.03％ KH 2 PO 4 After seawater preparation, naOH is used for adjusting the pH to 7.5), a 7L fermentation tank and 4L of culture medium liquid loading amount are adopted. Alternomonas sp.P-1 seed solution is added into a fermentation medium according to 6 percent, and fermentation parameters are selected: initial stirring speed 300 r.min -1 Ventilation of 4L min -1 The fermentation temperature is 25 ℃, and the fermentation time is 30 hours. After fermentation, the fermentation broth is centrifuged (10000 r/min,10min,4 ℃) to obtain supernatant, the supernatant is precipitated by 40-75% ammonium sulfate, the precipitated enzyme sludge is resuspended by 50mM Tris-HCl with pH 9.0, insoluble substances are removed by centrifugation for 20min at 10000r/min, and the enzyme broth is dialyzed by 50mM Tris-HCl until the ammonium sulfate is removed.
3. The invention provides a protein sequence GLPGPP of ACE inhibitory peptide, ACE inhibitory activity IC 50 =0.206 mg/mL, providing a source of sequence for future artificial synthesis of ACE inhibitory peptides.
The ACE inhibitory peptide is obtained by hydrolyzing salmon skin with microbial protease,
(1) The preparation method of the fish skin protein comprises the following steps: cutting fish skin into small pieces, washing with water, soaking the fish skin slices in alkali liquor for at least 2h to remove fat and non-protein components in the fish skin, washing the fish skin slices with water, heating at 75deg.C for 30min, centrifuging to obtain fish skin protein solution, dialyzing the supernatant, lyophilizing the fish skin protein, and making into protein lyophilized powder.
(2) Carrying out enzymolysis on the fish skin protein in the step (1) by using the Alternomonas sp.P-1 protease prepared in the step (2): the ratio of protease to substrate was 1:100 (w/w), the enzymatic hydrolysis temperature was 45℃and the reaction time was 5h, the reaction was stopped at 95℃for 10min, and the hydrolysate was collected by centrifugation at 10000 Xg for 10min at 4 ℃.
(3) Separating, purifying and identifying the sequence of the enzymolysis product in the step (2): and centrifuging the enzymolysis product for 60min by using an ultrafiltration tube with a cut-off molecular weight of 3000Da at 4500 Xg to obtain a filtrate with a molecular weight of less than 3000Da, wherein the ACE inhibition activity of the filtrate is 85.23%. The filtrate was separated and purified by Sephadex G-15 (2.5X150 cm) chromatography, and the fractions with high ACE inhibitory activity were screened out by measuring and comparing the ACE inhibitory activities of the fractions. The peptide component with high ACE inhibition activity is desalted by a Pierce polypeptide desalting column, is concentrated in vacuum by Speedvac, is dissolved in 0.1% formic acid, and the sequence of the peptide in the component is identified by using an EASYnLC 1200 liquid phase tandem Q exact HF mass spectrum, which shows that the main protein peptide with the 80 percent of the ratio is a short peptide containing six amino acids, and the sequence is GLPGPP. Peptide sequence synthesis by solid phase synthesis, and ACE inhibition activity IC of peptide fragment verification 50 The value is 0.206mg/mL, belonging to antihypertensive peptides with higher ACE inhibitory activity.
The salmon skin disclosed by the invention is an excellent protein raw material for producing ACE inhibitory peptide, and lays a research and development foundation for the development and utilization of marine new bacteria and new enzyme resources and the resource conversion and high-value utilization of aquatic product processing wastes.
Drawings
FIG. 1 shows the enzyme activity of Alternomonas sp.P-1 in example 1 of the present invention over time during fermentation in a fermenter and shake flask;
FIG. 2 is an extracellular enzyme spectrum of Alternomonas sp.P-1 fermentation enzyme in example 1 of the present invention;
FIG. 3 is a Sephadex G-15 chromatogram of the antihypertensive peptide of example 4 of the present invention having a molecular weight of less than 3000 Da;
FIG. 4 shows ACE inhibitory activity of the components (I to VII) isolated and purified in example 4 of the present invention.
Detailed Description
In order that the invention may be better understood, a further description of the invention will be provided with reference to specific examples, which will be apparent to those skilled in the art. The embodiments are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Example 1Alteromonas sp.P-1 identification
Alternomonas sp.21CJ28 is a seashore-derived intertidal deposit with 16S rRNA sequence of 1455bp (Genbank No. OR 078494.1) and most similar to it is a sequence in Alternomonas sp.RKMC-009 whole genome sequence (GenBank: CP031010.1, microbiol Resour Announc2019,8 (25), e 00508-19), with a sequence identity of 97.13%. It is currently believed that 16S rRNA similarity below 98.65% is considered a new species (Stackebrandt E, ebers J. Microbiol today.2006,4 (4): 6-9.; mincheol Kim, jongsik Chun. Method Microbiol.2014,41: 61-74.). Thus, alternomonas sp.21CJ28 is a new species of this genus.
Alternomonas sp.P-1 is obtained by domestication of Alternomonas sp.21CJ28 through ultraviolet mutagenesis, and the sequence identity of the Alternomonas sp.P-1 and the 16SrRNA is 100%, so that Alternomonas sp.21CJ28 is a new species of Alteromonas. Alternomonas sp.P-1 is preserved in China general microbiological culture Collection center with a strain preservation number of CGMCC No.28095.
Example 2Alteromonas sp.P-1 enzyme production and enzyme extraction
2216E fermentation medium: corn flour 0.5%, soybean meal 0.5%, bran 0.25%, caCl 0.1% 2 ，0.4％Na 2 HPO 4 ，0.03％ KH 2 PO 4 After seawater is prepared, naOH is used for adjusting pH to 7.5.
Seed culture: 100. Mu.L of the bacterial liquid was taken from the glycerol tube and inoculated into a 500mL Erlenmeyer flask containing 100mL 2216E medium, and cultured at 25℃for 24 hours at 200 r/min.
Fermentation culture: 7L fermenter, liquid loading 4LThe seed liquid is added into the culture medium according to 6 percent, and the initial stirring speed is 300 r.min -1 Ventilation of 4L min -1 The fermentation temperature is 25 ℃, and the fermentation time is 30 hours.
Protease extraction and crude separation: centrifuging the fermentation liquor at 4 ℃ and 10000r/min for 10min to obtain supernatant, precipitating the supernatant by using 40-75% ammonium sulfate, re-suspending the precipitated enzyme sludge by using Tris-HCl with the pH of 9.0 and 50mM, centrifuging at 10000r/min for 20min to remove insoluble matters, dialyzing the enzyme liquor by using Tris-HCl with the pH of 9.0 and 50mM until the ammonium sulfate is removed, and freeze-drying for later use.
Protease activity assay: protease activity was measured by Folin-phenol method at 30℃for 10min. The amount of enzyme that converts 1. Mu. Mol of substrate per minute under the reaction conditions is defined as one enzyme activity unit (U) (Zhang Shuzheng, enzyme preparation industry (below) [ J ]. 1984.).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): according to Wang Guzheng, the procedure of the protein technical Manual (scientific Press, 2000) is described.
Gelatin zymogram (zymogram): according to the patent 'an active electrophoresis detection method of protease spectrum' (ZL 201310018497.5).
The fermentation result shows that after the fermentation tank ferments for 6 hours, the extracellular protease activity of Alternomonas sp.P-1 increases faster, the protease activity reaches the maximum value 97.23U/mL when the fermentation tank cultures for about 30 hours, and then the synthesis of the enzyme is basically stopped, and the enzyme activity is kept stable; after 48h, the enzyme activity began to decrease with increasing fermentation time. Compared with the shake flask fermentation enzyme production, the total enzyme activity of the fermentation tank enzyme production is higher, the fermentation period is shorter (figure 1), the production cost and the use cost of the enzyme preparation are reduced, and a foundation is laid for the subsequent enzyme preparation market application.
Gelatin zymograms showed a negative band at approximately 60kDa and 45kDa (FIG. 2), indicating that Alternomonas sp.P-1 extracellular may secrete at least one protease.
EXAMPLE 3 enzymolysis of fish skin protein
Extraction of fish skin protein: cutting fish skin into 5cm×5cm small pieces, washing with tap water, immersing the slices in 0.1% NaOH (1:10, w/v) for 2 hours, removing fat and non-protein components, washing the sample with tap water for 2 hours, heating at 75 ℃ for 30 minutes, centrifuging at 10000×g for 10 minutes to extract soluble protein, dialyzing the supernatant, intercepting molecular weight of a dialysis bag by 8-14 k Da, and freeze-drying the protein into protein solid powder after dialysis.
Enzymolysis: the enzyme to substrate ratio was 1:100 (w/w), the enzymatic hydrolysis temperature was 45℃and the reaction time was 5h, the reaction was stopped at 95℃for 10min, and the hydrolysate was collected by centrifugation at 10000 Xg for 10min at 4 ℃.
Degree of hydrolysis (hydrolysis degree, HD) determination: HD was measured by the OPA (phthalic aldehyde) method (Nielsen, P.M.J.food Sci,2001,66 (5), p.642-646),
the degree of hydrolysis under the enzymolysis condition is 24.5%.
EXAMPLE 4 separation of enzymatic hydrolysate and measurement of ACE inhibitory Activity of peptide component
The enzymatic hydrolysate was centrifuged at 4500 Xg for 60min using an ultrafiltration tube (Amicon Ultra, millipore, USA) with a molecular weight cut-off of 3000Da to obtain a cut-off with a molecular weight of more than 3000Da and a filtrate with a molecular weight of less than 3000Da, which was lyophilized to give 50mg/mL, and the ACE inhibitory activity of the filtrate was measured.
The filtrate was purified by Sephadex G-15 (2.5X150 cm) chromatography, the mobile phase was water, the flow rate was 0.5mL/min, absorbance was measured at 220nm during collection, and a tube was collected every 5 min. Each tube sample was lyophilized and formulated into a 100. Mu.g/mL peptide sample, and the ACE inhibitory activity of each component was measured.
ACE inhibition activity assay: ACE reacts with N-hippuryl-histidyl leucine (HHL) to produce Hippuric Acid (HA), and when the amount of HHL is fixed, the production of HA is linear with ACE activity; the antihypertensive peptide can inhibit the activity of ACE, so that the amount of HA generated by the reaction is reduced, and the inhibitory activity of the antihypertensive peptide on ACE can be analyzed by detecting the amount of the generated HA. HHT was prepared according to the method of Wu (Wu et al, J.chromatogrA 2002,950 (1): 125-130.), mixed with 0.1M boric acid buffer at a concentration of 6.5mM, 40. Mu.L of the enzymatic hydrolysate was mixed with 25. Mu.L of ACE solution (100U/L), incubated at 37℃for 10min, 40. Mu.L of substrate HHT was added, incubation was continued at 37℃for 30min, 85. Mu.L of 1M HCl was added to terminate the reaction, the reaction solution was filtered through a 0.22 μm membrane, and the product HA was detected by high pressure liquid chromatography (High Performance Liquid Chromatography \HPLC) 1260 agent information II and ZRBAX extension-C18 column (4.6X1250 mm, particle size 5 μm).
ACE inhibitory activity (%) = (a-B)/a×100%, a is the HA content of the control (without sample)), and B is the HA content generated by the reaction with the sample.
The ACE inhibitory activity of the filtrate with molecular weight less than 3000Da was 85.23% as measured by HPLC.
Further separating and purifying the filtrate with molecular weight less than 3000Da by Sephadex G-15, and separating to obtain 7 peaks (I-VII) (figure 3); and after the eluent corresponding to each absorption peak is collected and freeze-dried for a plurality of times, measuring ACE inhibition activity. The ACE inhibitory activities of peaks I to VII were 30%,43.15%,78.03%,92.45%,61%,45.35%,34.87%, respectively, and the ACE inhibitory activities of peaks III and IV were significantly higher than those of the other components, indicating that peptides with antihypertensive activity were mainly present in both components (FIG. 4).
Example 5 sequence identification of antihypertensive peptides
The appropriate amount of peak IV fraction was desalted using Pierce polypeptide desalting column, and Speedvac concentrated in vacuo, then dissolved in 0.1% formic acid, and the peptide sequence in the fraction was identified using EASYnLC 1200 liquid phase tandem Q exact HF mass spectrometry.
The flow rate of the column is controlled at 300nL/min, the temperature of the column is 40 ℃, the electrospray voltage is 2kV, and the phase A of the mobile phase is 0.1% formic acid solution; phase B is an acetonitrile solution containing 0.1% formic acid.
The procedure is: 0-5 min,94% A,6% B; 6-40 min, 94-80% of A and 6-20% of B; 41-50 min, 80-65% of A, 20-35% of B; 51-60 min, 65-0% of A and 35-100% of B.
LC-MS identifies that 80% of peptide in peak IV component is hexapeptide with sequence GLPGPP, and the peptide belongs to Salmo salar collagen alpha-1 (XXVII) B chain.
Research shows that the characteristics of the antihypertensive peptide such as molecular weight, peptide chain length, amino acid composition and the like directly influence the inhibition effect of the antihypertensive peptide on ACE, and the short peptide is easier to combine with an ACE active site, is easy to absorb into blood circulation and keeps the activity, so that the ACE inhibition activity of the short peptide consisting of 2-12 amino acids is stronger. Studies show that active peptides containing Gly (G) have obvious inhibition effect on N-terminal domains of ACE, and when G is at two ends of the peptide, the selective inhibition of ACE activity is more obvious (Liu Chang, food-borne bioactive peptides have inhibition mechanism study on N and C domains of ACE [ D ], jilin: jilin university, 2021); the pyrrole ring of Pro (P) readily interacts with aromatic amino acid residues to form pi-alkyl groups resulting in higher ACE inhibitory activity (Zhang et al, eur Food Res technology, 2019, 245:1743-1753.). The main protein peptide accounting for 80% of the components with the highest ACE inhibition activity is a short peptide containing six amino acids, and the peptide sequence contains 2 Gly and 3 Pro, so that the peptide meets the reported peptide chain length and amino acid composition characteristics of the high-activity ACE inhibition peptide.
GLPGPP peptide fragment was synthesized by solid phase synthesis, and IC of synthetic peptide was obtained by measuring ACE inhibitory activities of synthetic peptides (0.05-1.0 mg/mL) at different concentrations 50 Values. IC (integrated circuit) 50 The value is defined as the concentration of peptide required to reduce ACE activity by 50%, IC 50 The calculation of the values uses a linear logarithmic method toPlotting log (inhibitor concentration mg/mL) and the value corresponding to the intersection of the straight line and X-axis is M,10 M IC for inhibiting ACE activity as inhibitor 50 Values. ACE inhibition activity IC of calculated GLPGPP 50 The value was 0.206mg/ml.

Claims (1)

1. The preparation method of the ACE inhibitory peptide from salmon skin is characterized in that the amino acid sequence of the ACE inhibitory peptide is GLPGPP; the ACE inhibitory peptide is prepared from marine microorganismsAlteromonassp, P-1 protease enzymatic hydrolysis of salmon skin protein, saidAlteromonassp.P-1 is deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganismsThe number is CGMCC No. 28095;
the said processAlteromonassp, P-1 protease, the specific preparation method is as follows: will beAlteromonasInoculating sp, P-1 seed solution into a fermentation tank for fermentation culture, wherein the volume of the seed solution accounts for 6% of the volume of the culture medium, and the initial stirring speed is 300 r.min -1 Ventilation 4 L.min -1 The fermentation temperature is 25 ℃, and the fermentation time is 30 h;
by usingAlteromonasWhen the sp, P-1 protease is used for carrying out enzymolysis on salmon skin protein,Alteromonasthe ratio of sp, P-1 protease to salmon skin protein substrate is 1:20-200 (w/w), wherein the enzymolysis temperature is 35-55 ℃, the reaction time is 2-8 h, and the reaction is stopped for 10min at 95 ℃ to inactivate enzyme; and then, centrifuging the product obtained by enzymolysis for 60min by using an ultrafiltration tube with a cut-off molecular weight of 3000Da at 4500 Xg to obtain a filtrate with a molecular weight of less than 3000Da, and separating and purifying the filtrate by using Sephadex G-15 and Sephadex 2.5X106 cm to obtain the ACE inhibitory peptide.
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