A porcine liver by-product polypeptide, and a preparation method and application thereof

Abstract

The present application belongs to the technical field of bioactive peptides, and particularly relates to a pig liver by-product polypeptide, a preparation method thereof and application. The preparation method of the pig liver by-product polypeptide comprises the following specific steps: S1. Pig liver is added into a phosphate buffer solution, and supernatant is collected by crushing and centrifugation. After dialysis, pig liver protein is obtained by freeze-drying; S2. The pig liver protein is dissolved in deionized water, and after incubation and pH adjustment, protease is added for enzymolysis. After enzyme inactivation and cooling, the supernatant is obtained by centrifugation and freeze-drying to obtain the pig liver by-product polypeptide. Compared with the prior art, the present application is simple in operation, high in practicability, and the prepared pig liver by-product polypeptide has strong anti-inflammatory activity and can regulate the secretion of inflammatory factors.

Description

Technical Field

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

[0002] Inflammation is a crucial immune defense response in the body and plays a crucial role in the development and progression of metabolic syndrome. Inflammatory responses can be acute or chronic. Acute inflammatory responses produce only transient effects that heal quickly, such as localized tissue damage; chronic inflammatory responses, on the other hand, have more persistent effects. When the body is injured, related cells release inflammatory mediators; however, excessive or uncontrolled production of these mediators can 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 health threat. However, most current pharmacological interventions are insufficient to control chronic inflammatory responses and are often associated with adverse side effects due to long-term use. Therefore, the development of safe, natural functional ingredients that can treat or assist in the treatment of inflammation is crucial.

[0003] Bioactive peptides are physiologically functional peptides that can exert beneficial biological activities beyond the nutritional value of their parent proteins. They offer advantages such as widespread availability, ease of absorption and utilization by the body, and minimal side effects. Studies have reported that many food-derived bioactive peptides demonstrate significant effects in improving inflammation and regulating metabolism. These peptides can interact with various cellular signaling pathways, inhibiting the secretion of inflammatory factors, thereby exhibiting potent anti-inflammatory activity and potentially playing a role in controlling inflammatory diseases. Therefore, the development of food-derived anti-inflammatory peptides is of great significance.

[0004] Pork liver is a by-product of pork slaughter, accounting for approximately 2.5% of the pig's total weight. Pork liver has a high protein content (19%-22%), and its amino acid composition is relatively close to human nutritional requirements, making it 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 significant impact on the yield and properties of the final hydrolyzate. Therefore, improving enzymatic hydrolysis efficiency and increasing the yield and activity of peptides after enzymatic hydrolysis are important for 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 address the deficiencies of the existing technology and provide a method for preparing a pig liver by-product polypeptide optimized by response surface methodology. 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 by-product polypeptide, which specifically comprises the following steps:

[0008] S1. Add pig liver to phosphate buffer, crush and centrifuge, collect the supernatant, dialyze the supernatant, and freeze-dry to obtain pig liver protein;

[0009] S2. Dissolve the pig liver protein in deionized water, incubate and adjust the pH, then add protease for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme and cool, centrifuge, and freeze-dry the supernatant 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 said S1, the specific parameters of the crushing process are 10000 rpm / min dispersion for 10 seconds, interval of 10 seconds, and separation for 6 times; the specific parameters of the centrifugation process are 4°C, 8000g, and 20 minutes; the specific parameters of the dialysis process are 4°C and 24 hours;

[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; and the protease hydrolysis time is 2-4 hours;

[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 said 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, the inflammatory factor IL-8 was detected and the expression level of the inflammatory factor IL-8 mRNA was measured, which 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, thereby proving the possible application of the pig liver by-product polypeptide in the preparation of inflammatory factor IL-8 inhibitors; and also proving the possible application of the pig liver by-product polypeptide in the preparation of foods, health products or medicines for preventing and / or treating inflammation-related diseases.

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

[0019] 2) The present invention screened for the optimal protease for preparing pig liver polypeptides. Based on the response surface Box-Behnken experimental design principle, a prediction model for the quadratic polynomial regression equation between the NO release inhibition rate and the enzymatic hydrolysis parameters was simulated, thereby optimizing the process conditions for preparing pig liver byproduct polypeptides. Using the optimized process, enzymatic hydrolysis yielded more polypeptides containing hydrophobic and positively charged amino acids in their sequences, which better conform to the sequence characteristics of anti-inflammatory active peptides. Furthermore, the prepared pig liver byproduct polypeptides exhibited a strong inhibitory effect on the secretion of inflammatory factors.

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

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

[0022] Figure 1 The test results of peptide content and anti-inflammatory activity of pig liver protease hydrolysates hydrolyzed with different proteases in Example 1 are as follows;

[0023] Figure 2 The results of the single factor experiment in Example 2 are as follows;

[0024] Figure 3 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 5Test results of IL-8 content and IL-8 mRNA level of HepG-2 cells treated with the porcine liver by-product polypeptide prepared in Example 3;

[0027] Figure 6 Test results of different components of the porcine liver by-product polypeptide prepared in Example 3 after being separated and purified, and the effects on IL-8 secretion in HepG-2 cells. DETAILED DESCRIPTION

[0028] In the following examples, the experimental methods are conventional unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

[0029] In the following examples, the determination method of peptide content and the determination method of anti-inflammatory activity are as follows:

[0030] (1) Determination of peptide content:

[0031] The peptide content is determined by o-phthaldehyde (OPA) method. 40 mg of o-phthaldehyde is dissolved in 1 mL of methanol, 25 mL of 0.1 M sodium tetraborate solution and 2.5 mL of 20% SDS solution are added, and then 100 μL of β-mercaptoethanol is added, and the volume is made up to 50 mL with deionized water. 100 μL of sample is mixed with 2 mL of OPA solution, and incubated in the dark for 2 min, and the absorbance value is determined at 340 nm. The standard curve is drawn with trypsin solution, and the peptide content in the sample is calculated.

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

[0033] NO production inhibition ability: 40 μL of enzyme solution is placed in a 2 mL centrifuge tube, 260 μL of PBS (0.1 M, pH 7.4) and 100 mL of nitroprusside solution (100 mM) are added, and incubated under 100% light intensity for 2 h. 100 μL of incubation solution is mixed with 100 μL of Griess reagent, and the absorbance value at 540 nm is determined. The absorbance value of the reaction solution with distilled water instead of the enzyme solution is recorded as A0, and 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 = (A0-A) / A0*100%.

[0035] Albumin denaturation inhibition ability: Bovine serum albumin (BSA) was dissolved in phosphate-buffered saline (PBS, 0.1 M, pH 6.5) and diluted to 1% (w / v). A mixture of 50 μL of enzymatic hydrolysate and 450 μL of BSA solution was incubated at 90°C for 20 minutes to induce protein denaturation. After cooling to room temperature, turbidity was measured at a wavelength of 660 nm. The absorbance of the reaction solution in which distilled water replaced the enzymatic hydrolysate was recorded as A0, and the absorbance of the sample reaction solution was recorded as A. The albumin denaturation inhibition rate was calculated according to the following formula:

[0036] Albumin denaturation inhibition rate = (A0-A) / A0*100%

[0037] Example 1: Protease screening

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

[0039] (1) Purchase fresh pig liver, clean the liver, remove fat and connective tissue, add phosphate buffer (0.05M, pH 7.4) at a volume ratio of 1:3, use a high-speed tissue disruptor at 10000 rpm / min for 10 seconds, and separate 6 times with an interval of 10 seconds to fully dissolve the protein. Centrifuge (4℃, 8000g, 20min) to collect the supernatant. 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℃ for 24 hours, freeze-dry for 60 hours, and obtain pig liver protein, which is stored at -20℃ for use.

[0040] (2) Pork liver protein was dissolved in deionized water at 30 mg / mL and incubated at the optimal temperature of six proteases for 10 min. The solution was adjusted to the optimal pH and then enzymatically hydrolyzed with 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). The enzymatic hydrolysis time was 1 h, 3 h and 5 h, respectively. After the enzymatic hydrolysis, the enzyme was inactivated at 95°C for 10 min, cooled to room temperature, and centrifuged (4°C, 12000 rpm, 10 min). The supernatant was collected and its NO release inhibition rate, albumin denaturation inhibition rate and peptide content were determined to screen out the optimal protease.

[0041] Figure 1Effect of protease type on porcine liver proteolysis. The type of protease plays a crucial role in the process of enzymatic preparation of bioactive peptides. Six common proteases were used to hydrolyze porcine liver, and the hydrolysates were evaluated for peptide content and anti-inflammatory activity. The results showed that the alkaline protease hydrolysate had the highest anti-inflammatory activity and peptide content. In contrast, the pepsin hydrolysate was the least effective. Therefore, alkaline protease is most suitable for the preparation of porcine liver polypeptides with anti-inflammatory activity.

[0042] Example 2: Enzymolysis parameter screening

[0043] The selected alkaline protease was used for porcine liver proteolysis. Single-factor experiments were designed with three factors: enzyme addition amount, substrate concentration, and enzyme hydrolysis time, and the NO release inhibition rate and peptide content were used as indicators, Figure 2 Based on the single-factor test results, the effect of each factor on the NO release inhibition activity of the enzyme hydrolysate showed an increasing trend followed by a decreasing trend. The optimal range of enzyme addition amount was 3000-6000 (U / g); the optimal range of enzyme hydrolysis time was 2-4 h; and the optimal range of substrate concentration was 20-40 (mg / mL). Within this range, the peptide content of the enzyme hydrolysate was also relatively high.

[0044] Using Box-Behnken, the Design Expert 12.0 software was used to optimize the three-factor, three-level response surface method test, and the NO release inhibition rate (Y) and the three factors of enzyme hydrolysis time (A), enzyme addition amount (B), and substrate concentration (C) were polynomial fitted regression, and the regression equation was obtained: 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 enzyme hydrolysis parameters for porcine liver protein anti-inflammatory peptides. Figure 3Response surface plot of the interaction between the factors affecting the NO release inhibition rate of the enzymatic hydrolysate. The smoother the slope of the response surface, the less the impact of the factor change on the response value, while the steeper the slope, the greater the impact of the factor. In addition, there was a strong interaction between enzyme addition amount and enzymolysis time. The optimal conditions determined by analysis were enzyme addition amount of 4910 U / g, enzymolysis time of 3.2 h, and substrate concentration of 29.90 mg / mL. The verification experiment performed under these parameters showed that the NO release inhibition rate of the enzymatic hydrolysate was 24.81%, which was very close to the predicted value (25.061%). The P value (<0.0001) and F value (38.52) of the variance analysis result of the model indicated that it had statistical significance. In addition, the lack-of-fit term (p = 0.4152 > 0.05) was not significant, indicating that the model fitted well and was suitable for describing the relationship between the three factors and the NO release inhibition rate. Therefore, the equation can be used to analyze and optimize the enzymolysis conditions for preparing porcine liver polypeptides.

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

[0046] Preparation of porcine liver by-product polypeptides:

[0047] (1) The porcine liver protein was prepared according to step (1) in Example 1;

[0048] (2) The porcine 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 then added with alkaline protease for enzymolysis (4910 U / g, 3.2 h). After the enzymolysis was completed, the enzyme was inactivated at 95°C for 10 min, cooled to room temperature, centrifuged (12000 rpm) at 4°C for 10 min, and the supernatant of the enzymolysis was freeze-dried to obtain the porcine liver by-product polypeptides.

[0049] Performance characterization of porcine liver by-product polypeptides:

[0050] 1. Determination of the molecular weight distribution of porcine liver by-product polypeptides:

[0051] The BioCore SEC-300 chromatographic column (5 μm, 7.8 x 300 mm) and high performance liquid chromatography (Waters, USA) were used to determine the molecular weight distribution of the PLPHs. The porcine liver polypeptides and the standard were both prepared into a 0.5 mg / mL solution. Elution was performed with phosphate buffer at a flow rate of 0.7 mL / min, and the ultraviolet detector was used to detect the ultraviolet absorbance at a wavelength of 280 nm.

[0052] Figure 4 The molecular weight distribution of the porcine liver polypeptides after elution by high performance liquid chromatography is shown in the figure. The results show that the molecular weight of the prepared porcine liver by-product polypeptides is approximately distributed within 5 kD, and polypeptides with small molecular weight are more likely to exhibit 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 in 6-well plates at a seeding density of 10 6 Cells were cultured for 24 hours. Cells were treated with palmitic acid (PA) (final concentration 0.4 mM) and incubated with porcine liver peptide solutions (5 μg / mL, 10 μg / mL, and 20 μg / mL) for 24 hours. Cells treated with palmitic acid alone served as a positive control (PC), and cells without palmitic acid or porcine liver peptide served as a negative control (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 in the dark at 37°C for 90 min. The plate was then washed 5 times with washing buffer. 100 μL of biotinylated human IL-8 antibody was then added to each well, incubated at 37°C for 60 min, and the plate was washed 5 times. 100 μL of horseradish peroxidase-conjugated streptavidin was then added to each well, incubated at 37°C for 30 min, and washed 5 times. 100 μL of colorimetric 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 a standard curve.

[0056] (2) Determination of IL-8 mRNA expression level: 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 was 60°C (30s). 2 ΔΔCt The relative expression of mRNA was calculated by the method.

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

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

[0059] Gel filtration chromatography was used to separate 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 10 mL fractions per tube. Finally, the polypeptide solution was freeze-dried to obtain a sample powder. The optimally active component was screened for peptide sequence identification by measuring the inhibitory rate of IL-8 secretion by HepG-2 cells.

[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, Separation was performed on a Thermo Scientific (Thermo Scientific) elution buffer (80% acetonitrile, 0.1% formic acid) with a linear gradient from 3% to 35%. The eluate was directly fed into the MS / MS system for comprehensive detection. High-resolution MS1 ​​scanning mode was used to select the most intense precursor ions. MS / MS spectra were generated by high-energy collisional dissociation (HCD) with a resolution of 15,000 (m / z 200) within a 1 s period. 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 seven components, of which component 5 had the strongest inhibitory activity on IL-8 secretion. Therefore, the peptide sequence of component 5 was identified. A total of 17 highly reliable peptide sequences (ALC%>95%) were identified, and their bioactivity values ​​were all greater than 0.85. The amino acid sequences of the 17 highly reliable peptide sequences are shown in SEQ ID NOs. 1 to 17, respectively. The number of amino acid residues in the peptide segments and the molecular weight of the peptide segments were visually analyzed, and the results are shown in Table 1:

[0062] Table 1 Composition of pig liver by-product polypeptides (P5 component)

[0063]

[0064] As can be seen, the majority of the peptide sequences contained in pig liver peptides 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 more likely to bind to protein receptors, thereby exerting biological activity.

[0065] The present invention provides a porcine liver byproduct polypeptide, its preparation method, and its application. There are many methods and approaches for implementing this technical solution. The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A composition of pig liver by-product polypeptides, characterized in that: The composition includes the following 17 polypeptides: LFWFR, FFVFPR, PLFFLR, DSFFPR, NSFFPR, DWLFK, FYYPLPK, DFFRH, DNWRWH, SSWWAH, YDYWWVR, EPFWRH, DWRYYP, SDDHWFK, RDPRGF, DWRYPY and NPLLFR.

2. Use of the pig liver by-product polypeptide composition according to claim 1 in the preparation of anti-inflammatory drugs.

Images