Pork liver by-product polypeptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioactive peptides, and particularly relates to a pork liver by-product polypeptide as well as a preparation method and application thereof. The preparation method of the pork liver by-product polypeptide comprises the following specific steps: S1, adding pork liver into a phosphate buffer solution, crushing, centrifuging, collecting supernate, dialyzing the supernate, and freeze-drying to obtain pork liver protein; s2, dissolving the pork liver protein in deionized water, incubating, adjusting the pH value, adding protease for enzymolysis, deactivating enzyme, cooling, centrifuging, taking supernate, and freeze-drying, so as to obtain the pork liver by-product polypeptide. Compared with an existing method, the method is easy to operate and high in practicability, and the prepared pork liver by-product polypeptide has high anti-inflammatory activity and can regulate secretion of inflammatory factors.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and specifically relates to a pig liver byproduct polypeptide and a preparation method and application thereof. Background Art

[0002] Inflammation is an important immune defense response of the body and plays an important role in the occurrence and development of metabolic syndrome. Inflammatory responses can be acute or chronic. Acute inflammatory responses only have short-term effects and can heal quickly, such as local tissue damage; while chronic inflammatory responses have more lasting effects. When the body is damaged, the relevant cells will release inflammatory mediators; however, when the body produces these mediators excessively or uncontrollably, it will lead to tissue damage and loss of immune function. Studies have shown that chronic inflammation is often associated with the pathogenesis of type 2 diabetes, inflammatory bowel disease, fatty liver disease and other cardiovascular diseases, and has become a public threat. However, most current pharmacological interventions are not sufficient to control chronic inflammatory responses, and long-term use is often accompanied by adverse side effects. Therefore, it is crucial to develop safe, natural functional ingredients that can treat or assist in the treatment of inflammation.

[0003] Bioactive peptides are peptide substances with physiological functions. They can exert beneficial biological activities beyond the nutritional value of their parent proteins. They have the advantages of wide sources, easy absorption and utilization by the body, and few side effects. Studies have found that many food-derived active peptides have shown significant effects in improving inflammation and regulating body metabolism. These peptides can interact with various cell signaling pathways and inhibit the secretion of inflammatory factors, thereby showing strong anti-inflammatory activity and having a certain potential role in controlling diseases related to inflammation. Therefore, the development of food-derived anti-inflammatory peptides is of great significance.

[0004] Pork liver is a byproduct of pork slaughter, accounting for about 2.5% of the total weight of the pig. Pork liver has a high protein content (19%-22%), and its amino acid composition is relatively close to human nutritional needs. It is a potential source of high-quality protein and bioactive peptides. Enzymatic hydrolysis is the main method for preparing bioactive peptides, but differences in enzymatic hydrolysis conditions have a great influence on the yield and properties of the final enzymatic hydrolysate. Therefore, improving the efficiency of enzymatic hydrolysis and increasing the yield and activity of peptides after enzymatic hydrolysis are of great significance to improving the utilization rate of liver by-products. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of a pig liver byproduct polypeptide optimized by response surface methodology in view of the deficiencies in the prior art. The prepared polypeptide has high yield and has the activity of regulating the secretion of inflammatory factors.

[0006] In order to solve the above technical problems, the present invention discloses a pig liver byproduct polypeptide, including one or more of LFWFR, FFVFPR, PLFFLR, DSFFPR, NSFFPR, DWLFK, FYYPLPK, DFFRH, DNWRWH, SSWWAH, YDYWWVR, EPFWRH, DWRYYP, SDDHWFK, RDPRGF, DWRYPY and NPLLFR peptides.

[0007] The present invention also discloses a method for preparing the above-mentioned pig liver byproduct polypeptide, which specifically comprises the following steps:

[0008] S1. Adding pig liver into phosphate buffer, crushing and centrifuging to collect the supernatant, dialyzing and freeze-drying the supernatant to obtain pig liver protein;

[0009] S2. Dissolve the pig liver protein in deionized water, incubate and adjust the pH, then add protease for enzymolysis. After the enzymolysis is completed, inactivate the enzyme and cool, centrifuge, take the supernatant and freeze-dry to obtain the pig liver by-product polypeptide.

[0010] Wherein, in S1, the volume ratio of the pig liver to the phosphate buffer is 1:(2-4); the concentration of the phosphate buffer is 0.05M, and the pH is 7.4;

[0011] Preferably, the volume ratio of the pig liver to the phosphate buffer is 1:3.

[0012] Among them, in S1, the specific parameters of the crushing process are 10000rpm / min dispersion for 10s, interval for 10s, and separation for 6 times; the specific parameters of the centrifugation process are 4°C, 8000g, and 20min; the specific parameters of the dialysis process are 4°C and 24h;

[0013] Wherein, in S2, the concentration of the pig liver protein in deionized water is 20-40 mg / mL; the amount of the protease added is 3000-6000 U / g; the protease hydrolysis time is 2-4 h;

[0014] Preferably, in S2, the concentration of the pig liver protein in deionized water is 29.9 mg / mL; the added amount of the protease is 4910 U / g; and the enzymatic hydrolysis time of the protease is 3.2 h.

[0015] Wherein, the protease is alkaline protease.

[0016] Among them, in S2, the incubation process parameters are incubation at 50°C for 10 minutes, adjusting the pH to 9, the enzyme inactivation process parameters are incubation at 95°C for 10 minutes, and the centrifugation process parameters are 4°C, 12000 rpm, and 10 minutes.

[0017] Specifically, in some embodiments of the present invention, by incubating the above-prepared pig liver by-product polypeptide with HepG-2 cells, detecting the inflammatory factor IL-8 and measuring the expression level of the inflammatory factor IL-8 mRNA, it is proved that the pig liver by-product polypeptide can significantly inhibit the secretion of the cellular inflammatory factor IL-8 and can significantly reduce the level of IL-8 mRNA, which proves the possible application of the pig liver by-product polypeptide in the preparation of the inflammatory factor IL-8 inhibitor; it also proves the possible application of the pig liver by-product polypeptide in the preparation of food, health products or drugs for preventing and / or treating inflammation-related diseases.

[0018] Beneficial effects: 1) The present invention uses pig liver as a raw material to prepare pig liver byproduct polypeptides that have the function of regulating the secretion of inflammatory factors, which makes full use of pig liver resources, increases the added value of pig liver as a byproduct, and provides a new development idea for pig slaughtering, production and processing factories.

[0019] 2) The present invention screened out the most suitable protease for preparing pig liver polypeptides, and based on the response surface Box-Behnken experimental design principle, simulated the prediction model of the quadratic polynomial regression equation between the NO release inhibition rate and the enzymatic hydrolysis parameters, and optimized the process conditions for the preparation of pig liver by-product polypeptides. After enzymatic hydrolysis using the optimized process, more polypeptides containing hydrophobic amino acids and positively charged amino acids in the sequence are obtained, which are more in line with the sequence characteristics of anti-inflammatory active peptides, and the prepared pig liver by-product polypeptides have a strong effect of inhibiting the secretion of inflammatory factors.

[0020] 3) The method of the present invention is simple to operate, highly practical, and easy to mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0022] Figure 1 The test results of peptide content and anti-inflammatory activity of pig liver protease hydrolysate hydrolyzed by different proteases in Example 1;

[0023] Figure 2 The single factor test results in Example 2;

[0024] Figure 3 is a response surface diagram of the interaction between various factors affecting the inhibition rate of NO release of enzymatic hydrolysate in Example 2;

[0025] Figure 4 The molecular weight distribution test results of the pig liver by-product polypeptide prepared in Example 3;

[0026] Figure 5The test results of IL-8 content and IL-8 mRNA level in HepG-2 cells after being treated with the pig liver byproduct polypeptide prepared in Example 3;

[0027] Figure 6 The different components of the pig liver byproduct polypeptide prepared in Example 3 after separation and purification, and the test results of their effects on IL-8 secretion in HepG-2 cells. DETAILED DESCRIPTION

[0028] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0029] Among them, in the following examples, the determination methods of peptide content and anti-inflammatory activity are as follows:

[0030] (1) Determination of peptide content:

[0031] The peptide content was determined by the o-phthalaldehyde (OPA) method. Dissolve 40 mg of o-phthalaldehyde in 1 mL of methanol, add 25 mL of 0.1 M sodium tetraborate solution and 2.5 mL of 20% SDS solution, then add 100 μL of β-mercaptoethanol and dilute to 50 mL with deionized water. Mix 100 μL of sample with 2 mL of OPA solution and incubate in the dark for 2 minutes, and measure its absorbance at 340 nm. Draw a standard curve with tryptone solution to calculate the peptide content in the sample.

[0032] (2) Determination of anti-inflammatory activity:

[0033] NO production inhibition ability: 40μL of enzymatic solution was placed in a 2mL centrifuge tube, 260μL of PBS (0.1M, pH7.4) and 100mL of sodium nitroprusside solution (100mM) were added, and incubated for 2h under 100% light intensity. 100μL of incubation solution was taken and mixed with 100μL Griess reagent, and the absorbance value at 540nm was measured. The absorbance value of the reaction solution with distilled water replacing the enzymatic solution was recorded as A 0 , the absorbance value of the sample reaction solution is recorded as A, and the NO release inhibition rate is calculated according to the following formula:

[0034] NO release inhibition rate = (A 0 -A) / A 0 *100%.

[0035] Albumin denaturation inhibition ability: Bovine serum albumin (BSA) was dissolved in phosphate buffered saline (PBS, 0.1M, pH 6.5) and diluted to 1% (w / v). A mixture of 50 μL of enzymatic solution and 450 μL of BSA solution was incubated at 90°C for 20 min to induce protein denaturation. After cooling to room temperature, the turbidity was measured at a wavelength of 660 nm. The absorbance value of the reaction solution in which distilled water replaced the enzymatic solution was recorded as A 0 , the absorbance of the sample reaction solution is recorded as A. The albumin denaturation inhibition rate is calculated according to the following formula:

[0036] Albumin denaturation inhibition rate = (A 0 -A) / A 0 *100%

[0037] Example 1: Protease screening

[0038] A method for preparing a pig liver byproduct polypeptide comprises the following steps:

[0039] (1) Purchase fresh pig liver, clean the liver, remove fat and connective tissue, add phosphate buffer (0.05M, pH7.4) at a volume ratio of 1:3, use a high-speed tissue disruptor to disperse at 10000 rpm / min for 10 seconds, and separate 6 times at intervals of 10 seconds to fully dissolve the protein, and collect the supernatant by centrifugation (4°C, 8000g, 20min). Add 3 times the volume of phosphate buffer to the remaining precipitate, repeat the above operation 3 times, combine the collected supernatants, dialyze at 4°C for 24 hours, and freeze-dry for 60 hours to obtain pig liver protein, which is stored at -20°C for future use.

[0040] (2) Pork liver protein was dissolved in deionized water at 30 mg / mL, incubated at the optimal temperature of six proteases for 10 min, adjusted to the optimal pH, and enzymatic hydrolysis was performed by adding alkaline protease (50°C, pH = 9), neutral protease (40°C, pH = 7.5), composite protease (50°C, pH = 7.5), papain (37°C, pH = 7.0), trypsin (37°C, pH = 8) and pepsin (37°C, pH = 3) for 1 h, 3 h and 5 h, respectively. After the enzymatic hydrolysis, the enzymes were inactivated at 95°C for 10 min, cooled to room temperature, and centrifuged (4°C, 12000 rpm, 10 min) to obtain the supernatant, and its NO release inhibition rate, albumin denaturation inhibition rate and peptide content were determined to screen out the most suitable protease.

[0041] Figure 1The effect of protease type on porcine liver protein hydrolysates. The type of protease plays a crucial role in the enzymatic preparation of bioactive peptides. Porcine liver protein was hydrolyzed with six common proteases, and the peptide content and anti-inflammatory activity of the hydrolysates were evaluated. The results showed that alkaline protease hydrolysates had the highest anti-inflammatory activity and peptide content. In contrast, pepsin hydrolysates had the worst effect. Therefore, alkaline protease is most suitable for the preparation of porcine liver peptides with anti-inflammatory activity.

[0042] Example 2: Enzyme hydrolysis parameter screening

[0043] The screened alkaline protease was used for enzymatic hydrolysis of pig liver protease. A single factor experiment was designed with three factors: enzymatic hydrolysis time, enzyme addition amount and substrate concentration, and NO release inhibition rate and peptide content were used as indicators. Figure 2 The results of the single factor experiment show that the influence of each factor on the NO release inhibition activity of the enzymatic hydrolysis product shows a trend of first increasing and then decreasing. The optimal range of enzyme addition is 3000-6000 (U / g); the optimal range of enzymatic hydrolysis time is 2-4h; the optimal range of substrate concentration is 20-40 (mg / mL). Within this range, the peptide content of the enzymatic hydrolysis product is also high.

[0044] The experiment was optimized by three-factor three-level response surface method using Box-Behnken software through Design Expert 12.0. The NO release inhibition rate (Y) was regressed with the three factors of enzymatic hydrolysis time (A), enzyme addition amount (B) and substrate concentration (C) by polynomial fitting. The regression equation was obtained as follows: Y = 24.77 + 1.18A + 1.70B - 0.18C - 2.16AB - 0.81AC + 0.43BC - 1.78A 2 -2.55B 2 -4.39C 2 The regression equation was calculated to obtain the optimal enzymatic hydrolysis parameters of pig liver protein anti-inflammatory peptide. Figure 3The response surface diagram is a graph of the interaction between the factors that affect the NO release inhibition rate of the hydrolyzate. The smoother the slope of the response surface, the smaller the effect of the factor change on the response value, while the steeper the slope, the greater the influence of the factor. In addition, there is a strong interaction between the amount of enzyme added and the hydrolysis time. The optimal conditions determined by analysis are that the amount of enzyme added is 4910U / g, the hydrolysis time is 3.2h, and the substrate concentration is 29.90mg / mL. The verification experiment carried out under these parameters showed that the NO release inhibition rate of the hydrolyzate was 24.81%, which is very close to the predicted value (25.061%). The results of variance analysis are the P value (<0.0001) and F value (38.52) of the model, indicating that it is statistically significant. In addition, the lack of fit term (p=0.4152>0.05) is not significant, indicating that the model fits well and is suitable for describing the relationship between the three factors and the NO release inhibition rate. Therefore, this equation can be used to analyze and optimize the hydrolysis conditions for preparing pig liver polypeptides.

[0045] Example 3: Preparation and performance characterization of pig liver by-product polypeptides

[0046] Preparation of Pork Liver Byproduct Peptides:

[0047] (1) preparing porcine liver protein according to step (1) in Example 1;

[0048] (2) Pork liver protein was dissolved in deionized water at 29.9 mg / mL, incubated at 50°C for 10 min, adjusted to pH 9.0, and alkaline protease (4910 U / g, 3.2 h) was added for enzymatic hydrolysis. After the enzymatic hydrolysis, the enzyme was inactivated at 95°C for 10 min, cooled to room temperature, centrifuged at 4°C (12000 rpm) for 10 min, and the enzymatic supernatant was freeze-dried to obtain the porcine liver byproduct polypeptide.

[0049] Performance characterization of pig liver by-product peptides:

[0050] 1. Determination of molecular weight distribution of polypeptides from pig liver byproducts:

[0051] The molecular weight distribution of PLPHs was determined using a BioCore SEC-300 column (5 μm, 7.8×300 mm) and high performance liquid chromatography (Waters, USA). The porcine liver polypeptide and the standard were prepared into a 0.5 mg / mL solution. Phosphate buffer was used for elution at a flow rate of 0.7 mL / min, and the UV detector was used to detect the UV absorbance at a wavelength of 280 nm.

[0052] Figure 4 The molecular weight distribution of the pig liver polypeptide after elution by high performance liquid chromatography. The results show that the molecular weight of the prepared pig liver byproduct polypeptide is roughly distributed within 5kD, and small molecular weight polypeptides are more likely to show anti-inflammatory activity and are easily absorbed by the human body.

[0053] 2. Determination of anti-inflammatory activity of pig liver by-product peptides:

[0054] Cell culture: HepG-2 cells (purchased from Hangzhou Meisen Cell Biotechnology Co., Ltd.) were seeded on 6-well plates at a seeding density of 10 6 Cells / well were cultured for 24 hours. Cells were treated with palmitic acid PA (final concentration was 0.4 mM) and pig liver polypeptide solution (5 μg / mL, 10 μg / mL, 20 μg / mL) was added to incubate the cells for 24 hours. Cells treated with palmitic acid alone were used as positive controls (PC), and cells without palmitic acid and pig liver polypeptide were used as negative controls (NC).

[0055] (1) Determination of inflammatory factor IL-8: IL-8 was detected using an ELISA cytokine kit (Xinbosheng Biotechnology Co., Ltd.). 100 μL of cell culture supernatant was added to a 96-well plate pre-coated with the antibody and incubated at 37°C in the dark for 90 min. The plate was then washed 5 times with washing buffer. Then 100 μL of biotinylated human IL-8 antibody was added to each well, incubated at 37°C for 60 min, and the plate was washed 5 times. Then 100 μL of horseradish peroxidase-conjugated streptavidin was added to each well, incubated at 37°C for 30 min, and washed 5 times. 100 μL of chromogenic substrate was added and incubated at 37°C in the dark for 15 min. Finally, the reaction was terminated with 100 μL of stop solution, measured at a wavelength of 450 nm, and the IL-8 content in the supernatant was calculated using the standard curve;

[0056] (2) Determination of the expression level of inflammatory factor IL-8 mRNA: Total cell RNA was extracted with Trizol, and then reverse transcribed to obtain cDNA. The cDNA sample was then mixed with the qPCR master solution. RT-qPCR was performed using the Quantstudio 6Flex system (Applied Biosystems, Foster City, USA). GAPDH was used as the internal reference gene. The operating conditions of the system were set as follows: the first stage was pre-denaturation: 95°C (30s); the second stage was cyclic reaction: 95°C (3s), 60°C (30s), 40 cycles; the third stage: 60°C (30s). 2 ΔΔCt The relative expression of mRNA was calculated by the method.

[0057] Figure 5The test results of IL-8 content and mRNA level of HepG-2 cells after being treated with the pig liver byproduct polypeptide prepared in Example 3. It can be seen that compared with the PC group, the pig liver byproduct polypeptide treatment significantly inhibited the secretion of IL-8, a cellular inflammatory factor (p < 0.05). Among them, 5, 10, and 20 μg / mL of pig liver polypeptide treatment inhibited the secretion of IL-8 by 21.60%, 34.91%, and 54.48%, respectively. In addition, RT-qPCR results showed that pig liver polypeptide significantly reduced the level of IL-8 mRNA (p < 0.05).

[0058] 3. Separation, purification and identification of pig liver protein peptides

[0059] Gel filtration chromatography was used to separate the polypeptides from pig liver byproducts. The chromatographic column was filled with G-15 gel and purified using the AKTApure purifier UPC10 protein purification system. A crude polypeptide solution (20 mg / mL) was prepared with an elution rate of 0.5 mL / min. The polypeptide eluate was automatically collected in fractions of 10 mL per tube. Finally, the polypeptide solution was freeze-dried to obtain a sample powder. By determining the inhibition rate of IL-8 secretion by HepG-2 cells, the component with the best activity was screened for peptide sequence identification.

[0060] The peptide samples were desalted by C18 solid phase extraction (SPE) column and dissolved in 0.1% formic acid aqueous solution at a concentration of 0.5 μg / μL. They were then analyzed and identified using a Q-Exactive HF-X mass spectrometer equipped with a nano-liquid chromatography system (EASY-nLC 1200, Thermo Scientific). RSLC,C18,75μm×15cm,3μm, The peptides were separated on a 400 μl (Thermo Scientific) HPLC system with a linear gradient of 3% to 35% elution buffer (80% acetonitrile, 0.1% formic acid). The eluent was directly fed into the MS / MS system for comprehensive detection. High-resolution MS1 ​​scanning mode was used to select the strongest precursor ions. MS / MS spectra were generated by high-energy collision dissociation (HCD) within a 1 s period with a resolution of 15,000 (m / z 200). The acquired data were imported into Peaks Studio (Xpro, Bioinformatics Solutions, Waterloo, Canada) for peptide sequence visualization.

[0061] Figure 6The results showed that the pig liver protein peptides were divided into 7 components, among which component 5 had the strongest inhibitory activity on IL-8 secretion, so the peptide sequence of component 5 was identified. A total of 17 highly credible peptide sequences (ALC%>95%) were identified, and their biological activity values ​​were all greater than 0.85. The amino acid sequences of the 17 highly credible peptide sequences are shown in SEQ ID NO.1 to 17. The number of amino acid residues in the polypeptide peptide segment and the molecular weight of the peptide segment were visualized and analyzed, and the results are shown in Table 1:

[0062] Table 1 Composition of pig liver byproduct polypeptides (P5 component)

[0063]

[0064] It can be seen that most of the polypeptide sequences contained in pig liver polypeptides have 5-7 amino acid residues, and nearly 94% of the peptides have a molecular weight of less than 1kDa. Low molecular weight peptides are easier for the human body to absorb completely, and are also easier to bind to protein receptors to exert biological activity.

[0065] The present invention provides a pig liver byproduct polypeptide and its preparation method and application ideas. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A pig liver byproduct polypeptide, characterized in that: Including one or more of LFWFR, FFVFPR, PLFFLR, DSFFPR, NSFFPR, DWLFK, FYYPLPK, DFFRH, DNWRWH, SSWWAH, YDYWWVR, EPFWRH, DWRYYP, SDDHWFK, RDPRGF, DWRYPY and NPLLFR peptides.

2. A method for preparing a polypeptide from a pig liver byproduct, characterized in that: The specific steps are: S1. Adding pig liver into phosphate buffer, crushing and centrifuging to collect the supernatant, dialyzing and freeze-drying the supernatant to obtain pig liver protein; S2. Dissolve the pig liver protein in deionized water, incubate and adjust the pH, then add protease for enzymolysis. After the enzymolysis is completed, inactivate the enzyme and cool, centrifuge, take the supernatant and freeze-dry to obtain the pig liver by-product polypeptide.

3. The preparation method according to claim 2, characterized in that: In S1, the volume ratio of the pig liver to the phosphate buffer is 1:(2-4); the concentration of the phosphate buffer is 0.05M, and the pH is 7.

4.

4. The preparation method according to claim 2, characterized in that: In S1, the specific parameters of the crushing process are 10000 rpm / min dispersion for 10 s, interval of 10 s, and separation for 6 times; the specific parameters of the centrifugation process are 4° C., 8000 g, and 20 min; and the specific parameters of the dialysis process are 4° C. and 24 h.

5. The preparation method according to claim 2, characterized in that: In S2, the concentration of the pig liver protein in deionized water is 20-40 mg / mL; the added amount of the protease is 3000-6000 U / g; and the enzymatic hydrolysis time of the protease is 2-4 h.

6. The preparation method according to claim 5, characterized in that: In S2, the concentration of the pig liver protein in deionized water is 29.9 mg / mL; the added amount of the protease is 4910 U / g; and the enzymatic hydrolysis time of the protease is 3.2 h.

7. The preparation method according to claim 6, characterized in that: The protease is alkaline protease.

8. The preparation method according to claim 2, characterized in that: In S2, the incubation process parameters are incubation at 50° C. for 10 min, adjusting the pH to 9, the enzyme inactivation process parameters are incubation at 95° C. for 10 min, and the centrifugation process parameters are 4° C., 12,000 rpm, and 10 min.

9. Use of the pig liver byproduct polypeptide according to claim 1 in the preparation of an inhibitor of inflammatory factor IL-8.

10. Use of the pig liver by-product polypeptide according to claim 1 in the preparation of a drug for preventing and / or treating inflammation-related diseases.

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