Active peptides based on chicken processing by-products and uses thereof

Abstract

The application discloses an active peptide based on chicken processing by-products and application thereof, and belongs to the technical field of active short peptides. The chicken by-products are subjected to enzymolysis by using a composite enzyme to obtain chicken-derived peptides, the influence of the chicken-derived peptides on intestinal absorption and health is analyzed in a system, and a nonapeptide GPEGPPGFP with excellent antioxidant property and capable of promoting kidney and intestinal health functions is prepared from the chicken-derived peptides. The prepared nonapeptide GPEGPPGFP has extremely high practical value and economic value, and can provide a new direction for the development of healthy food.

Description

Technical Field

[0001] This invention relates to an active peptide based on chicken processing by-products and its application, belonging to the field of active short peptide technology. Background Technology

[0002] The widespread development of the poultry industry inevitably generates a large amount of chicken by-products, including chicken heads, legs, bones, internal organs, and feathers. These by-products are typically used as animal feed, plant fertilizer, or seasonings, but their added value is low, limiting the efficient use of resources. Furthermore, improper handling of these by-products can lead to environmental pollution and pose a potential threat to human health. To address these challenges, researchers have attempted to improve the utilization value of chicken by-products through enzymatic hydrolysis technology and explore their potential bioactivity. Studies have shown that biotransformation technology can convert food by-products into a range of high-value-added products.

[0003] Peptides, as bioactive molecules resulting from the breakdown of proteins, are more easily absorbed and utilized than intact proteins due to their smaller molecular weight and favorable structural properties. In recent years, peptides prepared from the enzymatic hydrolysis of proteins (such as chicken meat) have gradually attracted attention due to their potential benefits in promoting nutrient absorption and gut health.

[0004] Many problems remain to be solved in the preparation of peptides from chicken meat and its processing by-products. The literature "Study on Enzymatic Hydrolysis Conditions of Chicken Peptides" discloses the use of papain and trypsin in chicken meat to prepare peptides; the literature "Study on Enzymatic Hydrolysis of Chicken Protein to Prepare Functional Protein Peptide Powder" discloses the use of papain, complex protease, alkaline protease, trypsin, and flavor protease to prepare functional peptides from chicken protein; the literature "Study on Process Conditions for Enzymatic Hydrolysis of Chicken Small Peptides by Different Proteases" discloses the use of neutral and acidic proteases to prepare chicken small peptides composed of 2-3 amino acids from chicken meat. These technologies suffer from high raw material costs, and the functionality of the prepared peptides is limited to antioxidant activity, lacking functional evaluation of the peptides' intestinal and renal safety.

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

[0006] To address the aforementioned issues, this invention utilizes a complex enzyme to enzymatically hydrolyze chicken by-products, and through further enzymatic hydrolysis and purification, yields 10 small molecule peptides. Based on this, this invention screens from these 10 small molecule peptides to obtain the small molecule peptide GPEGPPGFP, which possesses excellent antioxidant properties and promotes kidney and intestinal health.

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

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

[0009] The present invention also provides a nucleic acid molecule for encoding the above-mentioned active peptide.

[0010] The present invention also provides a biomaterial containing the above-mentioned nucleic acid molecules, wherein the biomaterial is an expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0011] A second objective of this invention is to provide a method for preparing the above-mentioned active peptide, the method comprising: preparing it by enzymatic hydrolysis of chicken processing byproducts, synthesizing it by genetically engineered bacteria, or synthesizing it artificially.

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

[0013] (1) Chicken processing by-products are crushed, and alkaline protease, bromelain and flavor protease are added. The mixture is then hydrolyzed, inactivated and filtered to obtain the hydrolysate.

[0014] (2) Centrifuge the enzymatic hydrolysis product to collect the supernatant, ultrafilter, freeze dry, dialyze, and freeze dry again to obtain chicken-derived mixed peptide sample;

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

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

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

[0018] Preferably, the amounts of alkaline protease, bromelain, and flavor protease added 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 performed at 38–42°C, pH 7.0–7.5, for 1–2 hours.

[0020] In one implementation, in step (2), the molecular cutoff 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 implementation, the dialysis time in step (2) is 20 to 30 hours.

[0023] A third objective of this invention is to provide the application of the above-mentioned active peptides, nucleic acid molecules, biomaterials, or any of the above-mentioned preparation methods in the preparation of antioxidant products or products that promote kidney and intestinal health, said products including food, pharmaceuticals, health products, or cosmetics.

[0024] A fourth objective of this invention is to provide an active peptide product, characterized in that the product contains the aforementioned active peptides; the active peptide product includes food, pharmaceuticals, health products, or cosmetics.

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

[0026] The present invention also provides a class of bioactive peptides, the amino acid sequences of which are as follows:

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

[0028] In one embodiment, a class of bioactive peptides possesses certain properties; said properties include one or more of the following: bioactivity, lipophilicity, absorbability, MDCK permeability, anti-inflammatory effect, antioxidant effect, and chelating effect.

[0029] Beneficial effects of the present invention

[0030] This invention uses a complex enzyme to hydrolyze chicken processing byproducts to obtain enzymatic hydrolysates, and through further enzymatic hydrolysis and purification, yields 10 small molecule peptides. Based on this, this invention screens from these 10 small molecule peptides to obtain the small molecule peptide GPEGPPGFP, which exhibits excellent antioxidant properties and promotes kidney and intestinal health.

[0031] Specifically:

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

[0033] (2) The small molecule peptide GPEGPPGFP prepared in this invention achieved an ABTS scavenging rate of 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 renal immune balance and maintain intestinal tissue health; including: increasing the expression level of ZO-1 in the intestine (37.05±0.74% higher than that of conventional feed); decreasing the expression level of IL-6 in the kidney (28.82±1.28% lower than that of conventional feed); and promoting the expression level of IL-10 (27.44±1.39% higher than that of conventional feed). Attached Figure Description

[0035] Figure 1 The relative contents of the top ten peptides purified in Example 1;

[0036] Figure 2 This is a liquid phase diagram of the mixed peptides and the peptides separated at 1000 Da in Example 2;

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

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

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

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

[0041] Figure 7 Immunohistochemical images of IL-6 and IL-10 in the kidney from Example 3. Detailed Implementation

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

[0043] Raw materials used in the examples:

[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 Youpuke Biotechnology Co., Ltd.

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

[0048] Example 1: Preparation of bioactive peptides derived from chicken meat

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

[0050] (1) Chicken processing by-products were uniformly crushed into minced meat by pressure extrusion in a low-temperature sterile environment at 4°C. 0.3% alkaline protease (1080U / g), 2.5% bromelain (1000U / g) and 2.5% flavor protease (5000U / g) were added. The mixture was enzymatically hydrolyzed at 40°C for 1.5h. After hydrolysis, the mixture was heated to 90°C for 15min to inactivate the enzymes and then cooled. The mixture was centrifuged at 5000rpm at 4°C for 15min and filtered to obtain the hydrolysate.

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

[0052] Chicken-derived peptide samples were desalted using a C18 solid-phase extraction column. After desalting, the samples were vacuum dried and then dissolved in 0.1% formic acid solution to adjust the peptide concentration to 0.5 μg / μL. 2 μL of the peptide solution was taken for specific sequence analysis using peptidomics.

[0053] The results are as follows Figure 1 As shown, 10 small peptides were obtained, namely GPEGPPGFP (SEQ ID NO.1), VPPPFNP (SEQ ID NO.2), EPGEPGLP (SEQ ID NO.3), IPGEPGPK (SEQ ID NO.4), LGPEGPGPK (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 testing

[0055] The small molecule peptides prepared in step 1 were used to predict their functional activity.

[0056] Enzymatic hydrolysis and computational bioactivity analysis of chicken-derived short peptides were performed using a formula designed to provide an assessment of peptide activity balance, resulting in an overall bioactivity score. Each factor was assigned a different weight, with bioactivity receiving the highest weight (30%), while lipophilicity, absorbability, permeability, and other biological functions were also considered (each accounting for 10%).

[0057] This method ensures a comprehensive assessment of the functional properties of peptides, and the overall scoring formula is as follows:

[0058] Overall score = 0.3 × bioactivity + 0.1 × lipophilicity + 0.1 × absorption + 0.1 × MDCK permeability + 0.1 × anti-inflammatory effect + 0.1 × antioxidant effect + 0.1 × chelation effect.

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

[0060] Table 1. Sequences and bioactive 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 exhibited strong absorption and antioxidant capacity, while VPPPFNP demonstrated strong lipophilicity and chelating ability. Both peptides showed significant permeability and bioavailability, indicating their potential role in intestinal absorption.

[0064] In contrast, IPGEPGPK and LGPEGPGPK had lower bioactivity scores of 0.44 and 0.43, respectively. Although they performed well in specific parameters such as anti-inflammatory and chelating properties, their overall bioactivity was not very promising.

[0065] The high MDCK permeability scores of GPEGPPGFP and VPPPFNP indicate their strong intestinal absorption capacity. These peptides demonstrate effective membrane permeability, suggesting potential applications in enteral nutrition.

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

[0067] The results in summary indicate that GPEGPPGFP and VPPPFNP exhibit the best biological activity.

[0068] Example 2: Preparation of nonapeptide GPEGPPGFP

[0069] 1. Preparation of nonapeptide GPEGPPGFP from chicken-derived mixed peptides

[0070] The chicken peptide sample prepared in Example 1 was subjected to a second enzymatic hydrolysis, as follows:

[0071] After redissolving the chicken peptide sample to a concentration of approximately 1.5 mg / mL, the pH was adjusted to 2.0, and 2% pepsin (by mass) of the chicken peptide solution was added. The mixture was reacted in a water bath shaker at 120 rpm and 40°C for 2 h. After the reaction, the enzyme was inactivated at 100°C for 15 min. After cooling, the pH was adjusted to 7.0, and the mixture was centrifuged at 6000 rpm for 5 min to obtain a secondary enzymatic hydrolysate. The hydrolysate was then spray-dried to obtain peptide powder.

[0072] The peptide composition was analyzed using high performance liquid chromatography and ion exchange column chromatography. The peptide was identified as GPEGPPGFP with a purity of 96.57%.

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

[0074] The nonapeptide GPEGPPGFP was synthesized using a chemical method. The synthesized peptide fragment showed a single peak upon liquid chromatography detection, as shown in the following results. Figure 3 As shown, the elution time is consistent with that of the peptides separated in step 1.

[0075] Example 3: Performance Detection of Nonapeptide GPEGPPGFP

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

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

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

[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 that of 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 Biotechnology Institute, was used to prepare mixed peptides and nonapeptides at different concentrations (1 mg / mL, 2 mg / mL, 5 mg / mL). Vitamin C was used as a positive control. The absorbance of each sample was measured at 734 nm according to the kit instructions. The ABTS clearance rate % was calculated using the formula: ABTS clearance rate % = [1 - (A...]. 测定 -A 对照 ) / A 空白 )]×100%, calculate the ABTS clearance rate of chicken-derived peptides.

[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; and the ABTS free radical scavenging rate of the nonapeptide (5 mg / mL) reached 52.29 ± 1.31%.

[0083] 3. The role of nonapeptide GPEGPPGFP in maintaining the intestinal barrier

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

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

[0086] The normal group's diet consisted of casein, L-tyrosine, corn starch, maltodextrin, sucrose, cellulose, soybean oil, mixed minerals S10022M, mixed vitamins V10037, and choline tartrate as the base.

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

[0088] Mice in each group had free access to water and food and were fed for 4 weeks. After 4 weeks, the mice were sacrificed, and the structure of the small intestine, the expression level of ZO-1, and the expression levels of inflammatory factors (IL-6 and IL-10) in the kidneys were detected.

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

[0090] (1) Intestinal test results

[0091] Results of small intestine tests in mice from different diet groups are as follows: Figure 6 As shown.

[0092] The results showed that the intestinal structure of mice in all three groups was intact, the villi were tightly packed, and the intestinal morphology was healthy. This indicates that the peptide-containing diet does not damage the intestinal barrier; and compared with the other two groups, the small intestinal villi and microvilli in the nonapeptide group were more tightly packed, increasing the absorption surface area and facilitating the absorption of nutrients.

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

[0094] (2) Kidney test results

[0095] Immunohistochemistry of mice in different diet groups, such as Figure 7 As shown.

[0096] IL-6 (interleukin-6) and IL-10 (interleukin-10) are important indicators for detecting inflammation. Under normal circumstances, 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 level of IL-6 in the nonapeptide group was significantly reduced by 28.82±1.28%, while the expression level of IL-10 was significantly increased by 27.44±1.39%. This indicates that the nonapeptide GPEGPPGFP can promote renal immune balance and maintain renal health.

[0098] The results in summary indicate that the nonapeptide GPEGPPGFP prepared in the examples can promote renal immune balance and maintain intestinal tissue health.

[0099] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by 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 of claim 1.

3. A biomaterial, characterized in that, The biomaterial contains the nucleic acid molecule as described in claim 2, and the biomaterial is an expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or transgenic cell line.

4. The method for preparing the active peptide according to claim 1, characterized in that, The method includes synthesis via genetically engineered bacteria or artificial synthesis.

5. The application of the active peptide of claim 1, the nucleic acid molecule of claim 2, the biomaterial of claim 3, or the preparation method of claim 4 in the preparation of antioxidant products, characterized in that, The product in question is a health supplement.

6. An active peptide product, characterized in that, The product contains the active peptide as described in claim 1; the active peptide product includes food or health products.

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