CN111961125B - Immune active peptide and preparation method thereof - Google Patents

Abstract

The invention discloses a plukenetia volubilis linneo immune active peptide and a preparation method thereof, wherein the preparation method of the plukenetia volubilis linneo immune active peptide comprises the following steps: extracting albumin of the plukenetia volubilis linneo; hydrolyzing the albumin to obtain albumin hydrolysate; carrying out ultrafiltration treatment and separation and purification treatment on the albumin hydrolysate; and collecting the polypeptide solution. According to the preparation method of the plukenetia volubilis linneo immunoactive peptide, albumin of plukenetia volubilis linneo is hydrolyzed, and the required peptide segments are separated and enriched in a targeted manner by using ultrafiltration treatment and separation and purification treatment, so that the obtained immunoactive peptide product has stronger functions and higher purity.

Description

Immune active peptide and preparation method thereof

technical field

The present invention relates to the technical field of immunoactive peptides, in particular, to an immunoactive peptide of Fructus chinensis and a preparation method thereof.

Background technique

Immune active peptides refer to a class of active polypeptides with biological functions such as enhancing immune function, transferring immune information, and promoting lymphocyte differentiation and maturation. Medicine and other fields have good application prospects. So far, researchers have isolated a variety of peptides with immunological activity from proteolysis of milk protein, soy protein, rice protein, fish and shellfish protein, collagen, etc. and applied them to clinical research, and achieved remarkable results. Effect. However, in the existing research, there is no report on the preparation of immunocompetent peptides from Metonia fruit protein.

Meiteng fruit is an emerging oil variety in tropical regions. As its fruit oil and its protein are listed as national new resource foods, the processing and comprehensive utilization of Meiteng fruit itself has gradually attracted the attention of the food processing industry. The remaining Meiteng fruit meal after oil extraction from Meiteng fruit is rich in protein, but the development and utilization of this part of the protein resources are still relatively limited. Therefore, the separation and preparation of immune active peptides from Meadowscarpus meal as raw material is of great significance for the related research on immune active peptides derived from plant proteins. realistic meaning.

SUMMARY OF THE INVENTION

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an immunoactive peptide of Fructus chinensis, which can quickly pass through the oral cavity and stomach of the human body, directly enter the small intestine, and be better digested and absorbed by the human body, and has the functions of scavenging free radicals, improving Human immunity and other functions.

The present invention also proposes a preparation method of the Fructus chinensis immunoactive peptide used for preparing the above-mentioned Fructus chinensis immune active peptide.

According to the embodiment of the first aspect of the present invention, the immunoactive peptides of Fructus Tunisiae include: TGGWSPLK, WKPW, FLTMEPR, VVLDVK, KVVL, MVVKK, LTGLNKL, RLLVWELER, KLSLEWWLK, FVKLL, LGDLGTKL, LTGLDKL, LFAEMDK, EADGTLR, VVLFK, TLLNPR, 24 peptides including AYLTGLK, WLPDVK, VLWLPR, RWQVWEDR, TVLLPR, LVRFPK, TLLFGDK, WSELVK, etc.

According to the embodiment of the present invention, the immunoactive peptide of Fructus chinensis has strong immune function, and the in vitro immune activity evaluation of the polypeptide was carried out by using the mouse macrophage model RAW 264.7. Phagocytosis has a promoting effect, and also has a certain positive effect in protecting the body and strengthening the body's immunity.

According to the preparation method of the immune active peptide of the second aspect of the present invention, comprising:

According to some embodiments of the present invention, the albumin extracted from the vine fruit comprises:

Pulverize the meteng pulp to obtain meteng pulp powder;

Take the Meadowscarpus pomegranate powder and place it in the 24L extraction kettle of the supercritical extraction equipment, and extract it under the conditions of 20MPa and 32°C for 120min, wherein the flow rate of CO2 is 60mL/min;

Discard the extracted Meteng fruit oil, take out Meteng fruit cake powder, add a certain amount of first-class water, ultrasonically treat for 15 minutes, stir at a constant temperature to extract for a certain period of time, and centrifuge at a speed of 4000 rpm for 10 minutes to separate the supernatant;

The supernatant was placed in distilled water and dialyzed for 72 hours at a temperature of 4°C, and rotary evaporated in a rotary evaporator for a certain period of time, and the lyophilized albumin was obtained by vacuum freeze drying;

hydrolyzing the albumin to obtain an albumin hydrolysate;

Ultrafiltration and separation and purification are carried out to the albumin hydrolyzate;

Collect the peptide solution.

According to the method for preparing the immunoactive peptides of the fruit of the present invention, after the albumin of the fruit of the fruit is hydrolyzed, ultrafiltration and separation and purification are used to separate and enrich the desired peptide segments in a targeted manner, and the obtained Immunoactive peptide products are more functional and have higher purity.

In addition, according to the preparation method of Fructus Fructus Immune Active Peptide according to the embodiment of the present invention, it also has the following additional technical features:

According to some embodiments of the present invention, the extraction conditions of the albumin are as follows: the ratio of solid to liquid is 1:25 (g/mL), the extraction temperature is 40° C., and the extraction time is 5 h.

According to some embodiments of the invention, the albumin is hydrolyzed using a protease.

In some embodiments of the present invention, the protease is one or more of neutral protease, papain, alkaline protease, trypsin and pepsin.

In some specific embodiments of the present invention, the hydrolysis conditions when using trypsin for hydrolysis are: the amount of enzyme added is 7000U/g, the reaction pH is 7.0, the enzymolysis temperature is 50°C, and the enzymolysis time is 4h.

According to some embodiments of the present invention, an ultrafiltration membrane with a molecular weight of 3 kDa is used to carry out ultrafiltration treatment on the albumin hydrolyzate;

The albumin hydrolyzate with a molecular weight of less than 3kDa after ultrafiltration treatment was subjected to ultrasonic treatment for 15min and then separated and purified, eluted with distilled water with a flow rate of 3mL/min, and the polypeptide solution eluted for 13min-29min was collected;

The collected polypeptide solution is concentrated.

According to some embodiments of the present invention, a Sephadex G-25 Sephadex column is used to separate and purify the albumin hydrolyzate.

According to some embodiments of the present invention, during the ultrafiltration process, nitrogen gas is used to pressurize, and the pressure is kept at 0.2 MPa, and the obtained polypeptide solution is placed on ice to ensure its activity.

According to the embodiment of the present invention, the immunoactive peptides from the fruit of the present invention have stronger functions and higher purity.

Additional aspects and advantages of the present invention will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the invention.

Description of drawings

Fig. 1 is the flow chart of the preparation method of the Fructus cannabis immune active peptide according to the embodiment of the present invention;

Fig. 2 is a schematic diagram of the effect of different proteases on the degree of albumin hydrolysis of Fructus chinensis;

Fig. 3 is the HPGPC spectrogram of the clear protease hydrolyzate of Fructus Fructus;

Fig. 4 is the molecular weight distribution chromatogram of the clear protease hydrolyzate of Fructus Fructus;

Figure 5 is a schematic diagram of the results of the effect of each ultrafiltration component of the proteolytic hydrolysate of Metenglia on the proliferation of RAW264.7 cells;

Figure 6 is a schematic diagram of the results of the effect of each ultrafiltration component of the clear protease hydrolyzate of Mettenago on the phagocytosis of RAW264.7 cells;

Fig. 7 is the elution curve diagram of the clear protease hydrolyzate of Fructus chinensis less than 3kDa fraction;

Figure 8 is a schematic diagram of the effect of Sephadex G-25 fractions on the proliferation of RAW264.7 cells;

Figure 9 is a schematic diagram of the effect of Sephadex G-25 fractions on the phagocytosis of RAW264.7 cells;

Fig. 10 is the chromatographic total ion chromatogram of Sephadex G-25 separation component P1;

Figures 11-34 correspond to the secondary mass spectra of different immunoactive peptides analyzed by liquid chromatography-tandem mass spectrometry combined with De novo sequencing, respectively.

Detailed ways

The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary, only used to explain the present invention, and should not be construed as a limitation of the present invention.

The following describes the method for preparing the immunologically active peptide of Fructus chinensis according to the embodiments of the present invention with reference to the accompanying drawings.

As shown in Figure 1, the albumin was extracted; the obtained albumin was hydrolyzed with protease; the albumin hydrolyzed solution was subjected to ultrafiltration; further separation and purification were carried out using Sephedax G-25 Sephedax Glucan gel column; the polypeptide fractions were collected, The immunoactive peptide is obtained.

Among them, the optimal extraction conditions for the extraction of albumin in Meadowscarpus pulp were as follows: the ratio of solid to liquid was 1:25 (g/mL), the extraction temperature was 40°C, and the extraction time was 5h.

The protease may be one or more of neutral protease, papain, alkaline protease, trypsin and pepsin, preferably trypsin. Among them, the optimal hydrolysis conditions when using trypsin for hydrolysis are: the amount of enzyme added is 7000U/g, the reaction pH is 7.0, the enzymolysis temperature is 50℃, and the enzymolysis time is 4h.

Preferably, the albumin hydrolyzate is ultrafiltered with an ultrafiltration membrane having a molecular weight of 3 kDa. During the ultrafiltration process, nitrogen was used to pressurize, and the pressure was kept at 0.2 MPa. The obtained polypeptide solution was placed on ice to ensure its activity.

Preferably, the above enzymatic hydrolyzate is further separated and purified by Sephadex G-25 Sephadex column. Sephadex G-25 packing was fully foamed and then packed into the column. The enzymatic hydrolyzate with molecular weight less than 3 kDa after ultrafiltration separation was ultrasonicated for 15 min and eluted with distilled water at a flow rate of 3 mL/min. Preferably, the polypeptide solution eluted in 13min-29min is collected.

In the embodiment of the present invention, after proteolysis of Meteng Guoqing, ultrafiltration membrane and Sephadex G-25 glucan gel column are used to separate and enrich the desired peptide segment, and the obtained immunoactive peptide product has better functions. Stronger and more pure. The in vitro immune activity of the peptide was evaluated by the mouse macrophage model RAW264.7. The experiment showed that the immune active peptide can promote the phagocytic ability of RAW264.7 macrophages, and can protect the body and strengthen the body's immunity. There are also some positive effects.

According to the preparation method of the embodiment of the present invention, the amino acid sequence of the immunoactive peptide obtained by the preparation method of the embodiment of the present invention is analyzed by LC/MS-MS liquid mass spectrometry, and the collected high-resolution mass spectrometry data set is subjected to de novo analysis, and TGGWSPLK, WKPW, FLTMEPR are identified. , VVLDVK, KVVL, MVVKK, LTGLNKL, RLLVWELER, KLSLEWWLK, FVKLL, LGDLGTKL, LTGLDKL, LFAEMDK, EADGTLR, VVLFK, TLLNPR, AYLTGLK, WLPDVK, VLWLPR, RWQVWEDR, TVLLPR, LVRFPK, TLLFGDK, WSELVK and other 24 peptides. Through the comparison of the protein database BLAST and the polypeptide database PIOPEP, no polypeptides identical to those obtained by the present invention were found. Therefore, the 24 peptide segments obtained by the present invention are all novel polypeptides.

The present invention will be described in further detail below in conjunction with specific examples, but these examples are intended to be used to explain the present invention and should not be construed as limiting the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The materials and reagents used in the following examples are as follows:

Table 1 Main materials for the preparation of immunoactive peptides

Table 2 Main reagents for the preparation of immunoactive peptides

Example 1 Selection of the optimal process conditions for Meteng albumin

Pulverize the pomegranate pulp, take the pomegranate pulp and place it in the 24L extraction kettle of the supercritical extraction equipment, extract under the conditions of 20MPa and 32°C for 120min, and the flow rate of _CO2 is 60mL/min. Discard the extracted Meiteng fruit oil, take out the Meiteng fruit cake powder and add a certain amount of first-grade water, ultrasonically treat it for 15 minutes, stir at a constant temperature to extract for a certain period of time, and centrifuge at a speed of 4000 rpm for 10 minutes to separate the supernatant. , the supernatant was placed in distilled water and dialyzed at a temperature of 4°C for 72 hours, then rotary evaporated in a rotary evaporator for a certain period of time, and then vacuum freeze-dried to obtain lyophilized albumin. According to the simulation experiment, taking into account the mutual influence of the ratio of solid to liquid, extraction temperature, extraction time and other factors, it is proposed to use an orthogonal experiment to determine the optimal extraction conditions for albumin. This experiment is completed by L ₉ (3 ⁴ ) orthogonal experiment. , see Table 3 for the scheme.

Table 3. Factor level table of albumin extraction test

The experimental results are shown in Table 4. According to the range analysis in Table 4, the factors that affect the extraction rate of Fructus Fructus albumin are in order: liquid-solid ratio > extraction time > extraction temperature. The size of the mean value reflects the influence level of this condition, and the mean value is the largest. is the optimal parameter for this condition. It can be seen from Table 4 that the three conditions with the largest mean value are: the ratio of solid to liquid is 1:25 (g/mL), the extraction temperature is 40°C, and the extraction time is 5h. After three verification experiments, the average extraction rate under this condition is 52.36%, which is comparable to the results of the orthogonal test, which proves that the optimized results are reliable.

Table 4 The results of the orthogonal test of albumin extraction

Example 2 Screening of the types of Meteng albumen proteolytic enzymes and selection of optimal hydrolysis conditions

The albumin was prepared at a ratio of 1:15, and the optimum temperature and pH were adjusted. Neutral protease, papain, composite protease, trypsin, gastric protease were added according to the enzyme activity ratio of 6000U/g. Protease and alkaline protease, enzymatically hydrolyzed for 3 hours, and screened the enzyme species with the degree of hydrolysis as an index. According to the simulation experiment, taking into account the interaction of factors such as enzymatic hydrolysis time, enzymatic hydrolysis temperature, amount of enzymatic addition and pH, an orthogonal experiment is proposed to determine the optimal enzymatic hydrolysis conditions for albumin. In this experiment, L ₉ (3 ⁴ ) positive The trial was completed, and the scheme is shown in Table 5.

The experimental results are shown in Figure 2. There is a significant difference in the degree of hydrolysis of the protease hydrolysate of Meteng Guo albumin with different proteases (P < 0.05). In terms of production and economic benefits, trypsin was selected to be used in the production of albumin polypeptides from Meteng. The results of the orthogonal test of proteolytic hydrolysis are shown in Table 5. From the range analysis in Table 5, it can be seen that the factors affecting the proteolysis of Meteng Guoqing are enzymatic hydrolysis temperature > enzymatic hydrolysis pH > enzymatic hydrolysis time > enzymatic hydrolysis amount, enzymatic hydrolysis The optimal process conditions are: the amount of enzyme added 7000U/g, pH 7.0, enzymolysis temperature 50℃, enzymolysis time 4h. According to the obtained optimal combination to do the verification experiment, the obtained hydrolysis degree is 28.04%±0.07, which proves that the optimized result is more reliable.

Table 5 Orthogonal test results of proteolytic hydrolysis

Example 3 Isolation, purification, identification and evaluation of immune activity of the immune active peptides

1. Ultrafiltration separation

In order to purify and enrich the active components, ultrafiltration was performed on the proteolytic hydrolysate of Metengguava to obtain a mixed solution of polypeptides with different molecular weights. The enzymatic hydrolysate was placed in an ultrafiltration cup, followed by ultrafiltration with 10kDa and 3kDa ultrafiltration membrane modules. After the ultrafiltration cup was closed, pressurized with nitrogen to keep the pressure at 0.2MPa. The obtained polypeptide solution was placed on ice to ensure its activity. Subsequently, the in vitro immune activity of each ultrafiltration fraction at a concentration of 100 μg/mL was evaluated by mouse macrophage model RAW264.7, and compared with the blank control group, 1 μg/mL lipopolysaccharide (LPS) and the same concentration without ultrafiltration Clear protein hydrolysate (SIP) was used for comparison, and the remaining samples were placed under vacuum freeze-drying at -80°C, and stored at -20°C for later use.

The in vitro immune activity evaluation of each ultrafiltration component is shown in Figure 5-Figure 6, and the ultrafiltration component less than 3kDa has the best effect on the promotion of RAW264.7 cell proliferation ability and phagocytosis of neutral red. After ultrafiltration separation, the proliferation ability of ultrafiltration fractions less than 3kDa to RAW264.7 cells increased by 72.34%, 18.09% and 85.11% compared with blank group, SIP group and LPS group, respectively; ultrafiltration fractions less than 3kDa increased RAW264.7 cells The ability to phagocytose neutral red increased by 85.39%, 30.45% and 19.95% respectively compared with the blank group, SIP group and LPS group. It shows that the ultrafiltration fraction less than 3kDa can significantly promote the proliferation and activation of RAW264.7 cells and improve immunity. As shown in Table 6, the ultrafiltration fraction less than 3kDa has a relatively good amino acid composition and can have relatively good immune activity to the body, and this judgment result is in line with the results of the in vitro immune activity test.

Table 6 Amino acid composition of each ultrafiltration fraction of the proteolytic hydrolysate of Meiteng Guoqing

2. Sephadex column purification

After the Sephadex G-25 packing was sufficiently foamed, it was packed into the column. The enzymatic hydrolysate was dissolved in distilled water and then sonicated for sample loading. Elution was performed with distilled water, the flow rate was 3 mL/min, 3 mL was collected in each tube, and the detection wavelength was 280 nm. The peak fractions after elution and separation were collected separately, and after freeze-drying, the in vitro immune activity of each fraction was evaluated by using the mouse macrophage model RAW264.7.

The experimental results are shown in Figures 7-9. After the Sephadex G-25 Sephadex column, ultrafiltration fractions less than 3 kDa were separated into three main fractions, which were fractions P1, P2 and P3 respectively. The components separated by Sephadex G-25 are non-toxic to RAW264.7 macrophages, can promote the activation of RAW264.7 macrophages and the phagocytic activity of cells, and improve immunity, and the effect of the P1 component is significantly better than that of P2. , P3 components. It can be seen from Table 7 and Table 8 that compared with the LPS group, different concentrations of P1 components have extremely significant inhibitory effects on the levels of TNF-α and IL-6 secreted by LPS-induced RAW264.7 macrophages, and further Regulates the immune function of the mechanism, and the P1 component shows better immune activity compared with the P2 and P3 components.

Table 7 Effects of Sephadex G-25 fractions on cytokine TNF-α in RAW264.7 cells

Table 8 Effects of Sephadex G-25 fractions on RAW264.7 cytokine IL-6

Note: ##. There is a very significant difference compared with the blank group (P < 0.01); **. There is a very significant difference compared with the LPS group (P < 0.01); *. There is a significant difference compared with the LPS group (P <0.05).

3. Identification of Immunoactive Peptide Sequences

1) Reductive alkylation

Add DTT solution to the sample to make the final concentration 10mmol/L, and reduce in 37℃ water bath for 4h. Subsequently, IAA solution was added to make the final concentration 50 mmol/L, and the reaction was performed in the dark for 40 min. Then use a self-packing desalting column to desalt, and evaporate the solvent in a vacuum centrifugal concentrator at 45°C.

2) LC-MS/MS detection

Capillary LC conditions are as follows:

a. Pre-column: 300μm id×5mm, packed with Acclaim PepMap RPLC C18, 5μm,

b. Analytical column: 150μm id×150mm, packed with Acclaim PepMap RPLC C18, 1.9μm,

c. Mobile phase A: 0.1% formic acid, 2% ACN;

d. Mobile phase B: 0.1% formic acid, 80% ACN;

e. Flow rate: 600nL/min;

f. Analysis time of each component: 78min;

The mass spectrometry conditions are as follows:

A. Primary mass spectrometry parameters:

Resolution: 70,000

AGCtarget: 3e6

MaximumIT: 40ms

Scanrange: 350to1800m/z

B. Secondary mass spectrometry parameters:

Resolution: 75,000

AGCtarget: 1e5

MaximumIT: 60ms

TopN: 20

NCE/stepped NCE: 27

The test results are shown in Table 9. A total of 24 novel peptides were identified in the P1 component. Through the comparison of the protein database BLAST and the polypeptide database PIOPEP, no peptides identical to the present invention were found.

Table 9 Immune active peptide sequences identified by liquid chromatography-tandem mass spectrometry analysis by De novo sequencing

In the description of the present invention, it should be understood that the terms "first" and "second" are only used for description purposes, and cannot be interpreted as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature defined as "first", "second" may expressly or implicitly include one or more of that feature. In the description of the present invention, unless otherwise specified, "plurality" means two or more.

In the description of this specification, references to the terms "one embodiment," "some embodiments," "specific embodiments," "examples," or "some examples" and the like are intended to mean specific features described in connection with the embodiment or example. , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, The scope of the invention is defined by the claims and their equivalents.

Claims (1)

1. The embelia laeta immunoactive peptide is characterized by comprising: TGGWSLK, WKPW, FLTMEPR, VWLVK, KVVL, MVVKK, LTGLNKL, RLLVWELER, KLSLEWLK, FVKLL, LGDLGTKL, LTGLDKL, LFAEMDK, EADGTLR, VVFK, TLLNPR, AYLTGLK, WLPDVK, VLWLPR, RQVWEDR, TVLLPR, LVRFPK, TLLFGDK, WSELVK24 peptide segments;

the preparation method of the plukenetia volubilis linneo immune active peptide comprises the following steps:

crushing the wisteria sinensis pulp to obtain wisteria sinensis pulp powder;

placing the Plukenetia volubilis linneo pulp powder in a 24L extraction kettle of supercritical extraction equipment, and extracting at 20MPa and 32 deg.C for 120min, wherein the extraction time is CO₂The flow rate of (2) is 60 mL/min;

discarding the extracted mei teng fruit oil, taking out mei teng fruit cake meal, adding a certain amount of first-grade water, performing ultrasonic treatment for 15min, stirring and extracting at constant temperature for a certain time, and centrifuging at the speed of 4000 revolutions per minute for 10min to obtain a supernatant;

putting the supernatant into distilled water, dialyzing for 72 hours at the temperature of 4 ℃, performing rotary evaporation in a rotary evaporator for a certain time, and performing vacuum freeze drying to obtain freeze-dried albumin;

hydrolyzing the albumin to obtain albumin hydrolysate;

carrying out ultrafiltration treatment and separation and purification treatment on the albumin hydrolysate;

collecting the polypeptide solution;

the extraction conditions of the albumin are as follows: the material-liquid ratio is 1:25g/mL, the extraction temperature is 40 ℃, and the extraction time is 5 h;

(ii) hydrolyzing the albumin with trypsin;

the hydrolysis conditions when trypsin is used for hydrolysis are as follows: the enzyme adding amount is 7000U/g, the reaction pH is 7.0, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 4 h;

carrying out ultrafiltration treatment on the albumin hydrolysate by adopting an ultrafiltration membrane with the molecular weight of 3 kDa;

performing ultrasonic treatment on albumin hydrolysate with molecular weight less than 3kDa after ultrafiltration for 15min, separating and purifying, eluting with distilled water with flow rate of 3mL/min, and collecting polypeptide solution eluted for 13-29 min;

concentrating the collected polypeptide solution;

separating and purifying the albumin hydrolysate by using a Sephadex G-25 Sephadex column;

during the ultrafiltration treatment, the pressure was increased with nitrogen gas to maintain the pressure at 0.2MPa, and the obtained polypeptide solution was placed on ice to secure its activity.

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