CN115232852A - Preparation method of sea buckthorn oligopeptide with ACE (angiotensin converting enzyme) inhibitory activity - Google Patents

Preparation method of sea buckthorn oligopeptide with ACE (angiotensin converting enzyme) inhibitory activity Download PDF

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CN115232852A
CN115232852A CN202211087975.3A CN202211087975A CN115232852A CN 115232852 A CN115232852 A CN 115232852A CN 202211087975 A CN202211087975 A CN 202211087975A CN 115232852 A CN115232852 A CN 115232852A
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sea buckthorn
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彭强
袁木荣
袁芳廷
李文霞
袁海佳
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Puredia Ltd
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Abstract

The invention discloses a preparation method of sea buckthorn oligopeptide with ACE inhibitory activity, which comprises the following steps: pretreating raw materials, grinding into thick liquid, carrying out enzymolysis and decoloration, inactivating enzyme, carrying out ultrafiltration, drying and the like. The invention directly takes the seabuckthorn seed meal as the raw material for enzymolysis, combines the steps of protein extraction and enzymolysis, has simpler process and improves the yield of the peptide. The invention adopts the ACE inhibition directional enzymolysis technology, adopts the enzyme preparation of food additive grade, and the process conditions can be applied to the production of functional food ingredients. The decolorization adopts laccase and activated carbon fiber composite treatment, avoids the damage of the traditional chemical decolorization method to the peptide product, and has better decolorization effect. And the preparation condition is mild, the method is simple, and the peptide inhibition activity is high. The obtained ultrafiltrate or powder can be optionally added into other food, and can be used as functional food additive or health product, with wide application.

Description

Preparation method of sea buckthorn oligopeptide with ACE (angiotensin converting enzyme) inhibitory activity
Technical Field
The invention relates to the technical field of biomass extraction and preparation, in particular to a preparation method of oligopeptide with ACE inhibitory activity.
Background
Sea buckthorn, latin name: hippophae rhamnoides linn. Hippophae rhamnoides is a generic term for plants and their fruits. According to the determination, the sea buckthorn fruit contains active substances such as various vitamins, fatty acids, trace elements, linoleic acid, sea buckthorn flavone, superoxide and the like and various amino acids required by a human body, and the vitamin is called king of vitamin C and life energy. The fructus Hippophae has effects of relieving cough, eliminating phlegm, invigorating stomach, resolving food stagnation, promoting blood circulation and removing blood stasis. Modern medical research shows that seabuckthorn can reduce cholesterol, relieve angina attack and prevent and treat coronary atherosclerotic heart disease. The seed meal of seabuckthorn is the waste of seabuckthorn after extracting seabuckthorn oil, and the seed meal contains various nutrient components such as abundant protein, sugar and edible cellulose, but is only used as feed at present.
ACE Angiotensin converting enzyme (Angiotensin converting enzyme) plays an important role in a human blood pressure regulating system, is widely distributed in each tissue of the whole body, and can convert Angiotensin I into Angiotensin II, so that blood vessel is contracted, and blood pressure is increased. Can also be combined with bradykinin in the blood pressure reducing system to make it inactive, resulting in imbalance of the blood pressure reducing system of human body. Active peptides can bind to the active site of ACE, inhibiting its action.
The prior art lacks of researches on preparation of ACE inhibitory peptide from seabuckthorn seed meal, the preparation process of the active peptide of seabuckthorn seed meal at present is that seabuckthorn protein is extracted firstly, and then polypeptide is prepared by enzymolysis with protein as a raw material, the product process is complex, and the product yield is not high. In addition, the existing process does not solve the problem of pigment residue of peptide products, so that the purity of the products is not high. Therefore, how to improve the yield and purity of the active peptide of the seabuckthorn seed meal and enable the active peptide to have ACE inhibitory activity has important significance for the production and application of the seabuckthorn peptide.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art and provides a preparation method of sea buckthorn oligopeptide with ACE inhibitory activity, which can prepare ACE inhibitory peptide powder, has mild preparation conditions, simple method and high peptide inhibitory activity.
In order to solve the technical problems, the invention adopts the following technical scheme: a preparation method of sea buckthorn oligopeptide with ACE inhibitory activity is characterized by comprising the following steps: the method for obtaining the sea buckthorn ACE inhibitory peptide by taking sea buckthorn seed meal as a raw material through enzymolysis and membrane separation specifically comprises the following steps:
step 1, raw material pretreatment: micronizing the seabuckthorn seed meal to form seabuckthorn seed powder, and mixing the seabuckthorn seed powder with water to obtain seabuckthorn seed powder suspension;
step 2, proteolysis: heating the sea buckthorn seed powder suspension to 50-60 ℃, adjusting the pH to 9.5-10.5, and adding alkaline protease accounting for 1% of the mass of the sea buckthorn seed powder for hydrolysis; after the hydrolysis is finished, respectively adding neutral protease accounting for 1 percent of the mass of the sea buckthorn seed powder and laccase accounting for 0.05 percent of the mass of the sea buckthorn seed powder for continuous hydrolysis; inactivating enzyme after hydrolysis, and then performing centrifugal operation, wherein the supernatant is a seabuckthorn protein hydrolysis solution;
step 3, membrane separation and decoloration: the sea-buckthorn protein hydrolysate is filtered through an ultrafiltration membrane to obtain components below 2000Da, the salt and small molecular weight impurities are removed through a nanofiltration membrane, the separated liquid is decolorized through activated carbon fibers, and the concentrated and spray-dried sea-buckthorn ACE inhibitory peptide is obtained.
In the step 1, the sea buckthorn seed meal is subjected to superfine grinding and is sieved by a 100-mesh sieve to obtain sea buckthorn seed powder, and then the sea buckthorn seed powder is mixed with water according to the weight ratio of 1:10 in the ratio of 10.
In step 2, hydrolyzing with alkaline protease (model number AP 200) for 5h, adding neutral protease (model number EF 108) and laccase, and continuing hydrolyzing for 3h.
In the step 3, the activated carbon fiber is prepared by matching 500L of distilled water with 10 inches of activated carbon fiber, and the decoloring time is 1h.
The invention has the advantages that: 1. in the prior art, protein is extracted from seabuckthorn seed meal by an alkali extraction and acid precipitation process, and then the protein is used as a raw material for enzymolysis, so that partial protein is not precipitated due to different isoelectric points of different proteins, and the waste of protein resources is caused. The invention directly takes the seabuckthorn seed meal as the raw material for enzymolysis, combines the steps of protein extraction and enzymolysis, has simpler process and improves the yield of peptide.
2. The invention adopts an ACE inhibitory directional enzymolysis technology, in the enzymolysis condition, the ACE inhibitory activity is taken as an index, and the active peptide obtained under the enzymolysis condition has better ACE inhibitory activity. Meanwhile, the enzymes adopted in the prior art are mainly biochemical-level enzymes, so that the disclosed preparation process cannot be applied to actual production. The invention is close to the actual production, adopts the enzyme preparation of food additive grade, and the process conditions can be applied to the production of functional food ingredients.
3. The decolorization adopts laccase and activated carbon fiber composite treatment, avoids the damage of the traditional chemical decolorization method to the peptide product, and has better decolorization effect.
In addition, the obtained ultrafiltrate or powder can be optionally added into other foods, can be used as additive of functional foods or health products, and has wide application.
Drawings
FIG. 1 is a graph showing the relationship between the concentration of ACE inhibitory peptide of sea buckthorn and inhibitory activity;
FIG. 2 is a graph of inhibition of ACE kinetics by sea buckthorn oligopeptide with double inversions;
FIG. 3 is a diagram showing the ACE inhibitory rate change state of sea buckthorn oligopeptide treated at different temperatures;
FIG. 4 is a diagram showing the change of ACE inhibition rate of sea buckthorn oligopeptide treated at different pH values;
fig. 5 is a total ion flow diagram of the hippophae rhamnoides oligopeptide component.
Detailed Description
The invention is further described in the following by the embodiments in conjunction with the drawings:
in this example, the preparation of the hippophae rhamnoides seed meal ACE inhibitory peptide:
step 1, raw material pretreatment: carrying out superfine grinding on the sea buckthorn seed meal, sieving the ground sea buckthorn seed meal with a 100-mesh sieve to obtain sea buckthorn seed powder, and mixing the sea buckthorn seed powder with water according to a ratio of 1:10 in proportion.
Step 2, proteolysis: heating the suspension of the seabuckthorn seed powder to 55 ℃, adjusting the pH value to 10, adding alkaline protease (AP 200 of Angel corporation) accounting for 1 percent of the mass of the seabuckthorn seed powder for hydrolysis for 5 hours, adding neutral protease (model number is EF 108) accounting for 1 percent of the mass of the seabuckthorn seed powder and laccase accounting for 0.05 percent of the mass of the seabuckthorn seed powder for continuous hydrolysis for 3 hours, inactivating the enzyme and then centrifuging, wherein the supernatant is obtained by hydrolysis and dissolution of seabuckthorn protein.
Step 3, membrane separation and decoloration: the sea-buckthorn protein hydrolysate is passed through ultrafiltration membrane to obtain components below 2000Da, and the components are desalted and low molecular weight impurity is removed by nanofiltration membrane, the separated liquid is decolorized by active carbon fiber (500L distilled water is mixed with 10 inch active carbon fiber) for 1h, and then concentrated and spray dried to obtain sea-buckthorn ACE inhibitory peptide.
Characteristic study:
1. determination of ACE inhibition and IC50
Three test tubes were prepared with borate buffer solution having a pH of 8.3, HHL solution of 8mM and ACE solution of 0.1U/mL, and the reagents were added to the test tubes according to the parameters shown in Table 1 to react with each other. After the reaction was completed, 1.7mL of ethyl acetate was added to each tube, followed by shaking for 20s and allowing to stand for liquid separation. The ethyl acetate layer (1 mL) was taken out and placed in a drying oven at a constant temperature of 120 ℃ to dry for 30min. After drying, taking out the test tube, adding 3mL of ultrapure water for redissolving, adjusting the ultraviolet spectrophotometer at 228nm, and measuring the light absorption value of the redissolved solution, wherein the calculation formula is as follows:
Figure BDA0003835946710000041
in the formula: a. The B : the light absorption value of the control tube; a. The A : the light absorption value of the sample tube; a. The C : absorbance of blank tube
TABLE 1 sample addition for the test for determining ACE inhibition
Figure BDA0003835946710000042
The ACE inhibition rate of the sea buckthorn oligopeptide is calculated to be 84.98%, the relationship between the concentration of the ACE inhibition peptide of the sea buckthorn seed meal and the inhibition rate is shown in figure 1, the concentration of the ACE inhibition peptide and the inhibition activity show a positive correlation trend, and the IC50 of 4.358mg/mL can be calculated according to a relationship curve.
2. Calculation of inhibition constants Km and Vmax
The sea-buckthorn polypeptide prepared by the invention is diluted to 19.5, 9.75 and 0mg/mL, and the three concentrations of enzymolysis liquid react with HHL of 8.0, 4.0, 2.0 and 0.8mmol/L respectively. Measuring the reaction rate, taking 1/V as an ordinate and 1/[ S ] as an abscissa, making a Lineweaver-Burk double reciprocal diagram, analyzing an inhibition mode, and calculating inhibition constants Km and Vmax through the intercept of a straight line and the abscissa.
The kinetic pattern of ACE inhibition by the polypeptides was analyzed according to figure 2. The straight line of reciprocal double of sea buckthorn oligopeptide is crossed with the 1/[ S ] axis, which shows that the inhibition type of sea buckthorn oligopeptide belongs to non-competitive inhibition. The result proves that the sea buckthorn oligopeptide has no competitive relationship with a substrate, can be combined with an ACE-HHL complex or a site except an ACE active site, changes the spatial conformation of ACE and reduces the activity of the ACE. The intercept of the horizontal and vertical coordinates shows that Vmax of the sea-buckthorn oligopeptides of 19.5, 9.75 and 0 mmol/L are respectively 0.015, 0.008 and 0.006, km is 4.29 x 10 < -3 > mol/L, and as can be seen from FIG. 2, the ACE inhibition rate and the concentration of the sea-buckthorn oligopeptides have positive correlation change, and the higher the concentration of the sea-buckthorn oligopeptides is, the higher the inhibition rate is, and the stronger the inhibition capacity is.
3. Studies on the stability of sea-buckthorn oligopeptide
The sea-buckthorn oligopeptide prepared by the method is subjected to stability research, (1) the thermal stability research: and (3) carrying out water bath on the peptide liquid in a water bath kettle at the temperature of 4, 20, 40, 60, 80 and 100 ℃ for 2h, cooling to room temperature, and determining the ACE inhibition rate of the peptide. (2) pH stability study: the pH of the peptide solution is 2, 4, 6, 8, 10 and 12, the peptide solution is kept at 37 ℃ for 2 hours, the pH is adjusted to 8.3, and the ACE inhibition rate of the peptide is measured. (3) stability of digestive tract: weighing 0.1g of peptide freeze-dried powder, dissolving in 0.1M HCL solution to prepare 1% (w/v) solution, adjusting the pH to 2, adding 1% (w/w) pepsin, hydrolyzing for 3h at 37 ℃, inactivating the enzyme for 15min in boiling water bath, adjusting the pH of an enzymolysis system to 8 by using 1M NaOH, adding 1% (w/w) trypsin, hydrolyzing for 3h at 37 ℃, inactivating the enzyme in boiling water bath, and determining the ACE inhibition rate of the final enzymolysis system.
As can be seen from the results of measuring the inhibition rates of the sea buckthorn oligopeptides treated for 2 hours at different temperatures and at different pH values in the graphs of FIG. 3 and FIG. 4, the inhibition rates are not greatly changed, and the sea buckthorn oligopeptides have good thermal stability and pH stability. If the small peptide is applied to industrial production, the small peptide is completely feasible, and the structure of the small peptide is not easily damaged by acid, alkali and high temperature conditions.
The peptide was digested with pepsin and trypsin, and it was found that the ACE inhibition of 1% (w/v) peptide solution after digestion was 67.46 ± 3.83%, and that the ACE inhibition of undigested peptide solution of the same concentration was 70.24 ± 2.06%, which was slightly higher than the ACE inhibition of the digested peptide. This is because the polypeptide is enzymatically digested by pepsin and trypsin, the structure is destroyed, and it becomes an inactive fragment. Therefore, the sea buckthorn seed meal ACE inhibitory peptide still has good inhibitory capacity after digestion, which is related to the fact that the inhibitory peptide contains a large number of hydrophobic residues, and the more the content of the hydrophobic residues, the stronger the inhibitory activity of the peptide can be kept.
4. Sea-buckthorn oligopeptide amino acid composition research and peptide segment characteristic detection
Taking a certain amount of sea buckthorn oligopeptide sample, hydrolyzing the sample for 22 hours at 110 ℃ by using 6M HCL, taking out the sample, neutralizing the sample by using NaOH, filtering and centrifuging the sample, and taking supernatant for determination. High performance liquid chromatograph parameters, chromatographic conditions: the column temperature is 40 ℃; the flow rate is stabilized at 1.0mL/min; ultraviolet detector conditions: wavelength 338nm; the wavelength is changed to 262nm at 22.5 min; fluorescence detector conditions: excitation wavelength is 340nm, and emission wavelength is 450nm;22.5 The excitation wavelength was changed at min to 266nm and the emission wavelength was 305nm.
According to the analysis of the table 2, the sea buckthorn seed meal polypeptide is rich in amino acid types, comprises 17 amino acids, the total amount of the amino acids is 53.61g/100g, wherein 27.88% of 8 essential amino acids are contained, the content of the amino acids is more glutamic acid, arginine and aspartic acid, the content of the three amino acids reaches 55.47% of the total amount, and methionine and cystine are limited amino acids. According to the amino acid properties, the classification and comparison show that the acidic amino acid has 39.33 percent with the largest percentage, the hydrophobic amino acid is used next, the content is 29.24 percent, the basic amino acid and the neutral amino acid have low contents, respectively accounting for 20.92 percent and 11.27 percent, the aromatic amino acid has the smallest content, only accounting for 5.88 percent, and the positively charged amino acids such as lysine, tyrosine, phenylalanine and proline also account for 2 to 4 percent of the total amount.
TABLE 2 amino acid composition analysis table for sea buckthorn oligopeptide
Figure BDA0003835946710000061
Figure BDA0003835946710000071
Adopting a three-in-one ultrahigh resolution liquid chromatography-mass spectrometer to identify peptide fragments, wherein the capillary liquid chromatography conditions are as follows: and (3) analyzing the column: mobile phase A:0.1% formic acid; mobile phase B:0.1% formic acid and 80% acetonitrile; flow rate: 300nL/min. Mass spectrum conditions: primary mass spectrum parameters: resolution:60000; AGC target:4.0e5; maximum IT: 100ms; scanning range:375 to 1500; secondary mass spectrum parameters: resolution:15000; AGC target: 5.0e4; maximum IT:256ms; scan range Mode Auto: m/z Normal; HCD Collision Energy (%): 30. the peptide characterization calculation website (https:// pepcalc. Com) was used to calculate the isoelectric point (pI), net charge, and solubility of the polypeptide.
The results of the assay using the NCBI of ACE inhibitory peptides, uniprot database alignment were shown in fig. 5 and table 3 for 10 ACE inhibitory peptides. The molecular weight distribution of the peptide fragments measured at this time is between 1000 and 3000Da, wherein the molecular weight of the peptide fragments AGGGGGGGGGGSRRL and DDEARINQLFL is smaller and slightly larger than 1kDa, and the rest detected peptide fragments are larger than 1.5kDa, because most of the peptide fragments obtained by enzymolysis are not recorded in a database, so that the result is not detected. From these results, it can be seen that most of the 10 peptides contained hydrophobic amino acids at the C-terminus or N-terminus, or positively charged amino acid residues, and also contained a large amount of hydrophobic and aromatic amino acids. In addition, the existing research shows that arginine in the peptide can also improve the ACE inhibitory activity of the peptide, and most of peptide fragments screened at this time contain arginine, which has similar results with the amino acid sequences of ACE inhibitory peptides screened in other researches.
TABLE 3 detection results and physicochemical characteristics of peptide fragments
Serial number Amino acid sequence Mass to charge ratio Isoelectric point Static charge Solubility in water
1 FRVAWTEKNDGQRAPLANN 2187.105 pH9.86 1 Good
2 LIISVAYARVAKKLWLCNMIGDVTTEQY 3198.705 pH8.7 0.9 Poor
3 VIRSRASDGCLEVKEFEDIPP 2360.191 pH4.16 -2.1 Good
4 AGGGGGGGGGGSRRL 1172.588 pH12.1 2 Good
5 LQPREGPAGGTTALREELSLGPEAALDTPPAGP 3268.681 pH3.88 -3 Good
6 DDEARINQLFL 1333.675 pH3.54 -2 Good
7 FAVSTLTSYDWSDRDDATQGRKL 2632.264 pH4.19 -1 Good
8 RQLSLEGSGLGVEDLKDN 1929.988 pH3.93 -2 Good
9 GGGGGGGGGGGGGGGIGGGGGGGGGGGAR 1841.798 pH10.84 1 Good
10 KEALGEGCFGNRIDRIGDVSGMGCNRRTPAP 3276.578 pH8.07 0.9 Good
Through a peptide characteristic calculation website, the 10 peptides are shown to have wide isoelectric point distribution range, the isoelectric points are distributed between pH 3-12, the electrostatic charge is distributed between-2.1 and 2, the peptide segment 2 has poor solubility, and the solubility of most of the peptides is good, which shows that the peptides can be used as the auxiliary materials or raw materials of powder or infusion type health care products for processing and production due to the good solubility of the peptides.
The present invention has been described in detail, and it should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Claims (4)

1. A preparation method of sea buckthorn oligopeptide with ACE inhibitory activity is characterized by comprising the following steps: the method for obtaining the sea buckthorn ACE inhibitory peptide by taking sea buckthorn seed meal as a raw material through enzymolysis and membrane separation specifically comprises the following steps:
step 1, raw material pretreatment: micronizing fructus Hippophae seed meal to obtain fructus Hippophae seed powder, and mixing with water to obtain semen Hippophae powder suspension;
step 2, proteolysis: heating the sea-buckthorn seed powder suspension to 50-60 ℃, adjusting the pH to 9.5-10.5, and adding alkaline protease accounting for 1% of the mass of the sea-buckthorn seed powder for hydrolysis; after the hydrolysis is finished, respectively adding neutral protease accounting for 1 percent of the mass of the sea buckthorn seed powder and laccase accounting for 0.05 percent of the mass of the sea buckthorn seed powder for continuous hydrolysis; inactivating enzyme after hydrolysis, and centrifuging to obtain supernatant as fructus Hippophae protein hydrolysis solution;
step 3, membrane separation and decoloration: the method comprises the steps of enabling a sea-buckthorn protein hydrolysis solution to pass through an ultrafiltration membrane to obtain components below 2000Da, removing salt and small molecular weight impurities through a nanofiltration membrane, decoloring separated liquid through activated carbon fibers, concentrating, and performing spray drying to obtain sea-buckthorn ACE inhibitory peptide.
2. The method of preparing hippophae rhamnoides oligopeptide with ACE inhibitory activity according to claim 1, wherein the method comprises the following steps: in the step 1, the sea buckthorn seed meal is subjected to superfine grinding and is sieved by a 100-mesh sieve to obtain sea buckthorn seed powder, and then the sea buckthorn seed powder is mixed with water according to the weight ratio of 1:10 in proportion.
3. The method of preparing hippophae rhamnoides oligopeptide with ACE inhibitory activity according to claim 1, wherein the method comprises the following steps: in step 2, alkaline protease with model number AP200 is used for hydrolysis for 5h, and neutral protease with model number EF108 and laccase are added for continuous hydrolysis for 3h.
4. The method of preparing hippophae rhamnoides oligopeptide with ACE inhibitory activity according to claim 1, wherein the method comprises the following steps: in the step 3, the activated carbon fiber is prepared by matching 500L of distilled water with 10 inches of activated carbon fiber, and the decoloring time is 1h.
CN202211087975.3A 2022-09-07 2022-09-07 Preparation method of sea buckthorn oligopeptide with ACE (angiotensin converting enzyme) inhibitory activity Pending CN115232852A (en)

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Publication number Priority date Publication date Assignee Title
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CN108277248A (en) * 2017-12-29 2018-07-13 磐安县派普特生物科技有限公司 A kind of method of ultrasonic wave extraction active seabuckthorn polypeptide
CN109652271A (en) * 2019-02-18 2019-04-19 林道新 A kind of production method of health care Spondias axillaris fruit wine
CN114517218A (en) * 2020-11-19 2022-05-20 巫世贵 Sea-buckthorn oligopeptide powder and preparation method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009125071A2 (en) * 2008-04-11 2009-10-15 Aromtech Ltd Extract with high amount of bioactive components, and its use
CN108277248A (en) * 2017-12-29 2018-07-13 磐安县派普特生物科技有限公司 A kind of method of ultrasonic wave extraction active seabuckthorn polypeptide
CN109652271A (en) * 2019-02-18 2019-04-19 林道新 A kind of production method of health care Spondias axillaris fruit wine
CN114517218A (en) * 2020-11-19 2022-05-20 巫世贵 Sea-buckthorn oligopeptide powder and preparation method and application thereof

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Title
张程慧等: "沙棘的综合价值研究进展", 食品工业科技, vol. 38, no. 22, 1 August 2017 (2017-08-01), pages 331 - 335 *
黄鹏等: "沙棘生物活性肽的制备及功效研究", 食品与机械, vol. 26, no. 6, 18 November 2010 (2010-11-18), pages 67 - 69 *

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