Active peptide based on chicken processing byproducts and application thereof

Abstract

The invention discloses an active peptide based on chicken processing byproducts and application of the active peptide, and belongs to the technical field of active short peptides. Chicken by-products are subjected to enzymolysis through a compound enzyme to obtain chicken-derived peptide, the influence of enzymolysis of the chicken-derived peptide on intestinal absorption and health is systematically analyzed, and the nonapeptide GPEGPPGFP with excellent oxidation resistance and the function of promoting kidney and intestinal health is prepared from the chicken-derived peptide. The nonapeptide GPEGPPGFP prepared by the invention has extremely high practical value and economic value, provides a new direction for healthy food development, and can provide a new direction for healthy food development.

Description

Technical Field

[0001] The invention relates to an active peptide based on chicken processing byproducts and application thereof, belonging to the technical field of active short peptides. Background Art

[0002] The extensive development of the poultry industry inevitably produces a large amount of chicken by-products, including chicken heads, chicken legs, chicken bones, viscera and feathers. These by-products are usually used as animal feed, plant fertilizer or condiments, but their added value is low, which limits the efficient use of resources. In addition, improper handling of these by-products may cause environmental pollution and pose a potential threat to human health. To meet these challenges, researchers have tried to improve the utilization value of chicken by-products through enzymatic hydrolysis technology and explore their potential biological activities. Studies have shown that biotransformation technology can transform food by-products into a series of high value-added products.

[0003] As bioactive molecules after protein decomposition, peptides are easier to absorb and utilize than intact proteins due to their smaller molecular weight and good structural characteristics. In recent years, peptides prepared by enzymatic hydrolysis of proteins (such as chicken) have gradually attracted attention due to their potential benefits in promoting nutrient absorption and intestinal health.

[0004] In the process of preparing polypeptides based on chicken and its processing by-products, there are still many problems that need to be solved. The document "Research on the conditions for enzymatic hydrolysis of chicken polypeptides" discloses the use of papain and trypsin chicken to prepare polypeptides; the document "Research on the preparation of functional protein peptide powder by enzymatic hydrolysis of chicken protein" discloses the preparation of functional peptides by enzymatic hydrolysis of chicken protein with papain, composite protease, alkaline protease, trypsin and flavor protease; the document "Research on the process conditions for the preparation of chicken small peptides by enzymatic hydrolysis of different proteases" discloses the use of neutral protease and acidic protease to hydrolyze chicken to prepare chicken small peptides composed of 2 to 3 amino acids. The above-mentioned technologies have the problem of high raw material costs, and the functionality of the prepared peptides only stays at antioxidant activity, and there is a lack of functional evaluation of peptides in intestinal and kidney safety.

[0005] Therefore, systematically exploring the potential functional peptides in chicken by-products and developing a highly active peptide has extremely high practical and economic value, providing a new direction for the development of healthy foods. Summary of the invention

[0006] To solve the above problems, the present invention uses a composite enzyme to hydrolyze chicken byproducts, and obtains 10 small molecule peptides through further hydrolysis and purification. On this basis, the present invention screens out a small molecule peptide GPEGPPGFP from the 10 small molecule peptides that has excellent antioxidant properties and promotes kidney and intestinal health.

[0007] The first object of the present invention is to provide an active peptide whose amino acid sequence is Gly-Pro-Glu-Gly-Pro-Pro-Gly-Phe-Pro (GPEGPPGFP).

[0008] In one embodiment, the amino acid sequence of the active peptide is shown as SEQ ID NO.1.

[0009] The present invention also provides a nucleic acid molecule, which is used to encode the above active peptide.

[0010] The present invention also provides a biological material, which contains the above-mentioned nucleic acid molecule and is an expression cassette, a transposon, a plasmid vector, a virus vector, an engineering bacterium or a transgenic cell line.

[0011] The second object of the present invention is to provide a method for preparing the above-mentioned active peptide, which comprises: preparing by enzymatic hydrolysis of chicken processing byproducts or synthesizing by genetically engineered bacteria or by artificial synthesis.

[0012] In one embodiment, the method for preparing chicken processing by-products by enzymatic hydrolysis is:

[0013] (1) Grinding chicken processing byproducts, adding alkaline protease, bromelain and flavor protease, enzymolysis, inactivation, filtering, and obtaining enzymolysis products;

[0014] (2) centrifuging the enzymatic hydrolysis product to obtain the supernatant, ultrafiltration, freeze-drying, dialysis, and freeze-drying again to obtain a chicken-derived mixed peptide sample;

[0015] (3) The chicken-derived mixed peptide sample was re-dissolved, the pH was adjusted, pepsin was added, enzymatically hydrolyzed, inactivated, cooled, the pH was adjusted to neutral, and dried to obtain active peptides with a purity of 96.57%.

[0016] In one embodiment, in step (1), the added amounts of alkaline protease, bromelain and flavor protease are 1000-2000 U / g, 1000-2000 U / g and 5000-6000 U / g, respectively.

[0017] In one embodiment, the addition amounts of alkaline protease, bromelain and flavor protease are 1000-1500 U / g, 1000-1500 U / g, and 5000-5500 U / g, respectively;

[0018] Preferably, the added amounts of alkaline protease, bromelain and flavor protease are 1000-1200 U / g, 1000-1200 U / g and 5000-5500 U / g, respectively.

[0019] In one embodiment, in step (1), the enzymatic hydrolysis is carried out at 38-42° C., pH 7.0-7.5, and for 1-2 hours.

[0020] In one embodiment, in step (2), the molecular cut-off of ultrafiltration is 1000 Da.

[0021] In one embodiment, the centrifugation in step (2) is performed at 4000-6000 rpm and 4° C. for 10-20 minutes.

[0022] In one embodiment, the dialysis time in step (2) is 20 to 30 hours.

[0023] The third object of the present invention is to provide the use of the above-mentioned active peptide or the above-mentioned nucleic acid molecule or the above-mentioned biological material or any of the above-mentioned preparation methods in the preparation of antioxidant products or products promoting kidney and intestinal health, and the products include food, medicine, health care products or cosmetics.

[0024] The fourth object of the present invention is to provide an active peptide product, characterized in that the product contains the above-mentioned active peptide; the active peptide product includes food, medicine, health care product or cosmetics.

[0025] In one embodiment, the product includes antioxidant foods, antioxidant medicines, medicines or health products that promote kidney and intestinal health, and antioxidant cosmetics.

[0026] The present invention also provides a class of active peptides, the amino acid sequence of the active peptides is as follows:

[0027] VPPPFNP, EPGEPGLP, IPGEPGPK, LPGEPGPK, ISWSNAP, DDNWDLR, DDNWDIR, STAPPPAP, PAASDPRP.

[0028] In one embodiment, a class of active peptides has certain properties; the properties include one or more of biological activity, lipophilicity, absorbability, MDCK permeability, anti-inflammatory effect, antioxidant and chelation.

[0029] Beneficial Effects of the Invention

[0030] The present invention uses a composite enzyme to hydrolyze chicken processing byproducts to obtain an enzymatic product, and further enzymatic hydrolysis and purification are performed to obtain 10 small molecule peptides. On this basis, the present invention screens out a small molecule peptide GPEGPPGFP from the 10 small molecule peptides that has excellent antioxidant properties and promotes kidney and intestinal health.

[0031] Specifically:

[0032] (1) The DPPH free radical scavenging rate of the small molecule peptide GPEGPPGFP prepared by the present invention reaches 48.09±1.77% at 1 mg / mL and 73.45±0.98% at 5 mg / mL;

[0033] (2) The clearance rate of ABTS of the small molecule peptide GPEGPPGFP prepared by the present invention reached 33.55±1.31% at 1 mg / mL and 52.29±1.31% at 5 mg / mL;

[0034] (3) The small molecule peptide GPEGPPGFP prepared by the present invention can promote kidney immune balance and maintain intestinal tissue health; including: increasing the expression of ZO-1 in the intestine (increased by 37.05±0.74% compared with conventional feed); reducing the expression of IL-6 in the kidney (reduced by 28.82±1.28% compared with conventional feed), and promoting the expression of IL-10 (increased by 27.44±1.39% compared with conventional feed). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the relative content of the top ten peptides purified in Example 1;

[0036] Figure 2 The liquid phase diagram of the mixed peptide and the 1000Da separated peptide in Example 2;

[0037] Figure 3 This is the liquid phase diagram of the chemically synthesized nonapeptide GPEGPPGFP in Example 2;

[0038] Figure 4 The effect of each component in Example 3 on the scavenging rate of DPPH free radicals;

[0039] Figure 5 The effect of each component in Example 3 on the total antioxidant activity of ABTS;

[0040] Figure 6 The histochemical image of the intestinal structure and tight junction protein ZO-1 in Example 3;

[0041] Figure 7 This is the immunohistochemistry diagram of kidney IL-6 and IL-10 in Example 3. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0043] The raw materials used in the embodiment:

[0044] Chicken processing by-products: Zhejiang Jichong Trading Co., Ltd.;

[0045] Bromelain (40,000 U / g), Shandong Weilan Biotechnology Co., Ltd.;

[0046] Alkaline protease (360,000 U / g) and flavor protease (200,000 U / g) were purchased from Wuxi Youpu Biotechnology Co., Ltd.;

[0047] Pepsin (>3000 U / mg) was purchased from Macklin.

[0048] Example 1: Preparation of active peptides from chicken

[0049] 1. A method for preparing chicken-derived active peptides, comprising the steps of:

[0050] (1) The chicken processing byproduct is uniformly crushed into minced meat by pressure extrusion in a low-temperature sterile environment at 4°C, and 0.3% alkaline protease (1080U / g), 2.5% bromelain (1000U / g) and 2.5% flavor protease (5000U / g) are added; enzymolysis is performed at 40°C for 1.5 hours; after enzymolysis, it is heated to 90°C for 15 minutes to inactivate the enzyme, and cooled; centrifuged at 5000rpm for 15 minutes at 4°C, and filtered to obtain an enzymolysis solution;

[0051] (2) The enzymatic hydrolysate was centrifuged at 5000×g for 15 minutes using a PALL ultrafiltration tube with a molecular weight cutoff (MWCO) of 1000 Da to obtain a filtrate, which was freeze-dried and dissolved in 5 mL of ultrapure water. The filtrate was dialyzed in 100 mL of ultrapure water using a dialysis membrane (MWCO 1000 Da, Spectrum Labs, USA) for 24 h. After dialysis, the hydrolysate was collected and freeze-dried to obtain a chicken peptide sample.

[0052] The chicken peptide sample was desalted using a C18 solid phase extraction column; after desalting, the sample was vacuum dried and then dissolved in a 0.1% formic acid solution to adjust the peptide concentration to 0.5 μg / μL; 2 μL of the peptide solution was taken for peptidomic analysis of the specific sequence.

[0053] The results are as follows Figure 1 As shown, 10 small molecule peptides were obtained, namely GPEGPPGFP (SEQ ID NO.1), VPPPFNP (SEQ ID NO.2), EPGEPGLP (SEQ ID NO.3), IPGEPGPK (SEQ ID NO.4), LPGEPGPK (SEQ ID NO.5), ISWSNAP (SEQ ID NO.6), DDNWDLR (SEQ ID NO.7), DDNWDIR (SEQ ID NO.8), STAPPPAP (SEQ ID NO.9), and PAASDPRP (SEQ ID NO.10).

[0054] 2. Functional activity detection

[0055] Take the small molecule peptide prepared in step 1 and perform functional activity prediction.

[0056] Enzymatic digestion and computational bioactivity analysis of chicken-derived short peptides An overall bioactivity score was calculated using a formula designed to provide a balanced assessment of peptide activity. Each factor was assigned a different weight, with bioactivity receiving the highest emphasis (30%), while lipophilicity, absorption, permeability, and other biological functions were also considered (10% each).

[0057] This approach ensures a comprehensive assessment of the functional properties of the peptides, with the comprehensive scoring formula being:

[0058] Comprehensive score = 0.3×biological activity + 0.1×lipophilicity + 0.1×absorbability + 0.1×MDCK permeability + 0.1×anti-inflammatory effect + 0.1×antioxidation + 0.1×chelation.

[0059] The activity results of the small molecule peptide prepared in step 1 are shown in Table 1.

[0060] Table 1 Sequences and biologically active short peptides

[0061]

[0062]

[0063] The results showed that the overall bioactivity scores of peptides GPEGPPGFP and VPPPFNP were 0.77 and 0.72, respectively. GPEGPPGFP had strong absorption and antioxidant capacity, while VPPPFNP had strong lipophilicity and chelating ability. Both peptides showed significant permeability and bioavailability, indicating their potential role in intestinal absorption.

[0064] In contrast, IPGEPGPK and LPGEPGPK had lower bioactivity scores of 0.44 and 0.43, respectively, and although they performed well in specific parameters such as anti-inflammatory and chelating properties, their overall bioactivity was less optimistic.

[0065] The high MDCK permeability scores of GPEGPPGFP and VPPPFNP indicated that they had strong intestinal absorption capacity, and these peptides exhibited effective membrane permeability, suggesting potential application prospects in enteric nutrition.

[0066] Peptides such as DDNWDLR and STAPPPAP showed some activity in anti-inflammatory and antioxidant tests, but the absorption scores were low.

[0067] The above results show that GPEGPPGFP and VPPPFNP have the best biological activity.

[0068] Example 2: Preparation of nonapeptide GPEGPPGFP

[0069] 1. Separation and preparation of nonapeptide GPEGPPGFP from chicken mixed peptides

[0070] The chicken peptide sample prepared in Example 1 was subjected to secondary enzymatic hydrolysis in the following steps:

[0071] After the chicken peptide sample was redissolved to a concentration of about 1.5 mg / mL, the pH was adjusted to 2.0, and pepsin with a mass fraction of 2% of the chicken peptide solution was added. The mixture was reacted in a water bath shaker at 120 r / min and 40°C for 2 hours. After the reaction, the enzyme was inactivated at 100°C for 15 minutes. After cooling, the pH was adjusted to 7.0, and the mixture was centrifuged at 6000 r / min for 5 minutes to obtain a secondary enzymatic hydrolyzate, which was spray-dried to obtain peptide powder.

[0072] The composition of the peptide powder was analyzed by high performance liquid chromatography and ion exchange column, and the peptide was GPEGPPGFP with a purity of 96.57%.

[0073] 2. Preparation of nonapeptide GPEGPPGFP by chemical synthesis

[0074] The nine-peptide GPEGPPGFP was synthesized by chemical method. The synthesized peptide was detected as a single peak by liquid chromatography. The results are as follows Figure 3 As shown, the peak time is consistent with the peptide separated in step 1.

[0075] Example 3: Performance test of nonapeptide GPEGPPGFP

[0076] The performance of nonapeptide GPEGPPGFP was tested and the results are as follows.

[0077] 1. Scavenging rate of DPPH free radical by nonapeptide GPEGPPGFP

[0078] The DPPH kit purchased from Nanjing Jiancheng Bioengineering Institute was used to prepare different concentrations of unpurified mixed peptides and pure nonapeptides (1 mg / mL, 2 mg / mL, and 5 mg / mL). Vc was used as a positive control. According to the instructions of the kit, the absorbance of each sample was measured at 517 nm. The formula DPPH clearance rate (%) = [1-(A 样品 -A 对照 ) / A 空白 ]×100%, and the DPPH scavenging rate of chicken peptide was calculated.

[0079] The results are as follows Figure 4As shown, the DPPH scavenging rate of the mixed peptide (1 mg / mL) was 26.31±1.43%; the DPPH free radical scavenging rate of the nonapeptide (1 mg / mL) reached 48.09±1.77%, and the pure nonapeptide was about 82.78% higher than the mixed peptide; the DPPH free radical scavenging rate of the nonapeptide (5 mg / mL) reached 73.45±0.98%.

[0080] 2. Scavenging rate of ABTS free radicals by nonapeptide GPEGPPGFP

[0081] The ABTS kit purchased from Nanjing Jiancheng Bioengineering Institute was used to prepare the mixed peptide and nonapeptide into different concentrations (1 mg / mL, 2 mg / mL, 5 mg / mL), and Vc was used as a positive control. According to the instructions of the kit, the absorbance of each sample was measured at 734 nm. The formula ABTS clearance rate % = [1-(A 测定 -A 对照 ) / A 空白 )]×100%, and the ABTS clearance rate of chicken peptide was calculated.

[0082] The results are as follows Figure 5 As shown, the ABTS scavenging rate of the mixed peptide (1 mg / mL) was 18.32±1.78%; the ABTS scavenging rate of the nonapeptide (1 mg / mL) reached 33.55±1.31%, which was 83.13% higher than that of the mixed peptide; the ABTS free radical scavenging rate of the nonapeptide (5 mg / mL) reached 52.29±1.31%.

[0083] 3. Effect of Nonapeptide GPEGPPGFP on Maintaining Intestinal Barrier

[0084] The nonapeptide GPEGPPGFP prepared in Example 2 was used to detect its application in promoting the intestinal barrier and kidney immune balance in mice.

[0085] Animal experiment: 40 male C57BL / 6J mice, 9 weeks old, were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The mice were placed in a specific pathogen-free (SPF) environment in the Experimental Animal Center of Jiangnan University. After a 7-day adaptation period, the mice were randomly divided into 5 groups: normal diet group, mixed peptide group, and nonapeptide group.

[0086] The normal group's feed was based on casein, L-tyrosine, corn starch, maltodextrin, sucrose, cellulose, soybean oil, mixed minerals S10022M, mixed vitamins V10037, and choline bitartrate;

[0087] The mixed peptide group and the nonapeptide group were respectively: casein and L-tyrosine in the normal group feed were replaced with mixed peptide and nonapeptide GPEGPPGFP (the addition amount of the mixed peptide and nonapeptide was 4%; the total amount of crude protein in each feed was the same).

[0088] Each group of mice had free access to water and food and were fed for 4 weeks. The mice were killed after 4 weeks, and the small intestinal structure, ZO-1 expression level, and inflammatory factor (IL-6, IL-10) expression level in the kidney were detected.

[0089] The test results of mouse small intestine and kidney are as follows:

[0090] (1) Intestinal test results

[0091] The results of small intestine tests in mice in different diet groups are as follows Figure 6 shown.

[0092] The results showed that the intestinal structures of the three groups of mice were intact, the villi were tightly arranged, and the intestinal morphology was healthy. This indicates that the peptide-containing feed will not damage the intestinal barrier; compared with the other two groups, the villi and microvilli of the small intestine of the nonapeptide group were more tightly arranged, which increased the absorption area and facilitated the absorption of nutrients.

[0093] On this basis, compared with the normal diet group and the mixed peptide group, the expression of ZO-1 in the small intestine of the nonapeptide group was significantly increased by 37.05±0.74%. It can be seen that the nonapeptide GPEGPPGFP can increase the expression level of tight junction proteins in small intestinal tissue and promote intestinal tissue health.

[0094] (2) Kidney test results

[0095] Immunohistochemistry of mice in different diet groups Figure 7 shown.

[0096] IL-6 (interleukin 6) and IL-10 (interleukin 10) are important indicators for detecting inflammation. Normally, high expression of IL-6 promotes inflammation and plays a pro-inflammatory role, while high expression of IL-10 inhibits inflammation and plays an anti-inflammatory role.

[0097] The results showed that compared with the normal diet group, the expression of IL-6 in the nonapeptide group was significantly reduced by 28.82±1.28%, and the expression of IL-10 was significantly increased by 27.44±1.39%. It can be seen that nonapeptide GPEGPPGFP can promote renal immune balance and maintain renal health.

[0098] The above results show that the nonapeptide GPEGPPGFP prepared in the example can promote kidney immune balance and maintain intestinal tissue health.

[0099] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An active peptide, characterized in that The amino acid sequence of the peptide is Gly-Pro-Glu-Gly-Pro-Pro-Gly-Phe-Pro.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule is used to encode the active peptide according to claim 1.

3. A biomaterial, characterized in that: The biological material contains the nucleic acid molecule according to claim 2, and the biological material is an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

4. The method for preparing the active peptide according to claim 1, characterized in that: The method comprises: preparing by enzymatic hydrolysis of chicken processing byproducts or synthesizing by genetic engineering bacteria or by artificial synthesis.

5. The preparation method according to claim 4, characterized in that: The method for preparing chicken processing by-products by enzymatic hydrolysis is as follows: (1) Grinding chicken processing byproducts, adding alkaline protease, bromelain and flavor protease, enzymolysis, inactivation, filtering, and obtaining enzymolysis products; (2) centrifuging the enzymatic hydrolysis product to obtain the supernatant, ultrafiltering, dialyzing, and drying to obtain a chicken-derived mixed peptide sample; (3) The chicken-derived mixed peptide sample was re-dissolved, the pH was adjusted, pepsin was added, enzymatically hydrolyzed, inactivated, cooled, the pH was adjusted to neutral, and dried to obtain active peptides with a purity of 96.57%.

6. The method according to claim 5, characterized in that In step (1), the added amounts of alkaline protease, bromelain and flavor protease are 1000-2000 U / g, 1000-2000 U / g and 5000-6000 U / g respectively.

7. The method according to claim 5, characterized in that In step (1), the enzymatic hydrolysis is carried out at 38-42° C., pH 7.0-7.5, and the enzymatic hydrolysis lasts for 1-2 hours.

8. The method according to claim 5, characterized in that In step (2), the molecular cut-off of ultrafiltration is 1000Da.

9. Use of the active peptide according to claim 1, the nucleic acid molecule according to claim 2, the biomaterial according to claim 3, or the preparation method according to any one of claims 4 to 8 in the preparation of antioxidant products or products for kidney and intestinal health, characterized in that: The products include food, medicine, health products or cosmetics.

10. An active peptide product, characterized in that: The product contains the active peptide according to claim 1; the active peptide product includes food, medicine, health care product or cosmetics.

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