A pigeon meat antioxidant peptide and an anti-fatigue peptide and a preparation method thereof

Abstract

The present invention belongs to the field of functional peptides, and specifically discloses a pigeon meat antioxidant peptide and an anti-fatigue peptide and a preparation method thereof, comprising the following steps: (1) Take pigeon meat, mince it, add water and a compound enzyme of neutral protease and alkaline protease, carry out constant-temperature enzymatic hydrolysis, centrifuge to obtain a supernatant, and lyophilize to obtain the pigeon meat antioxidant peptide; (2) The supernatant in step (1) is intercepted by an ultrafiltration membrane separation method to obtain a fraction of 1-5 kDa, and lyophilized to obtain the pigeon meat anti-fatigue peptide. The antioxidant peptide of the present invention has relatively high contents of essential amino acids and antioxidant active amino acids, reaching 47.97% and 29.27% respectively. The anti-fatigue peptide of the present invention is a peptide with a molecular weight lower than 5000 Da, which is relatively easy to be absorbed by the human body, has the effect of significantly improving the enzyme activity of SOD enzyme, and has a significant anti-fatigue effect.

Description

Technical Field

[0001] The present invention belongs to the field of functional peptides, and particularly relates to an antioxidant peptide and an anti-fatigue peptide, and a preparation method and application thereof. Background Art

[0002] Bioactive peptides are usually active small peptides composed of 2 - 20 amino acid residues, which can regulate the life activities and physiological activities of biological organisms, and can be released from precursor proteins by specific cleavage in various ways. Their functions include but are not limited to antioxidant, anti-aging, blood pressure lowering, and anti-fatigue. Active peptides can not only supply essential amino acids to the human body, but also maintain the stability of the internal environment of the human body, improve the enzyme activity and antioxidant capacity of the body, etc., and have great research value and prospects.

[0003] At present, there are various preparation methods for antioxidant peptides and anti-fatigue peptides, including chemical / physical extraction, microbial fermentation, acid-base hydrolysis, and protease hydrolysis, etc. Among them, extraction and acid-base hydrolysis methods have problems such as high cost and many by-products. Therefore, when preparing a certain type of target peptide, protease enzymatic hydrolysis or microbial fermentation method with high cost performance is usually used as the preparation method. And protease enzymatic hydrolysis has many influencing factors and complex products. The specific process conditions include solid-liquid ratio, enzymatic hydrolysis time, enzyme addition amount, temperature, etc. Determining an optimal enzymatic hydrolysis process according to the product requirements is the top priority of the protease hydrolysis method.

[0004] In recent years, the breeding industry of breeding pigeons in China has developed rapidly and has a bright future, and its market demand is extremely large. Pigeon meat is rich in amino acids, has a high protein content and a low lipid content, and its fatty acid and amino acid composition very much meets the nutritional needs of humans. It is an ideal food raw material that meets the healthy nutritional needs of modern people. At present, there are few reports on the research of preparing active peptides from pigeon meat. Summary of the Invention

[0005] The purpose of the present invention is to provide a peptide with antioxidant and anti-fatigue activities. Using fresh pigeon breast meat as raw material, pigeon meat antioxidant peptide is obtained by enzymatic hydrolysis method, and further ultrafiltration separation and purification are carried out to obtain pigeon meat anti-fatigue peptide. This pigeon meat peptide has antioxidant activity and anti-fatigue activity through tests.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A preparation method for pigeon meat antioxidant peptide and anti-fatigue peptide, comprising the following steps:

[0008] (1) Take pigeon meat, mince it, add water and a compound enzyme of neutral protease and alkaline protease, carry out constant temperature enzymatic hydrolysis, centrifuge to obtain the supernatant, and freeze-dry to obtain pigeon meat antioxidant peptide;

[0009] (2) The supernatant of step (1) is intercepted by ultrafiltration membrane separation to obtain a fraction with a molecular weight of 1 - 5 kDa, and freeze-dried to obtain pigeon meat anti-fatigue peptide.

[0010] Preferably, the constant temperature enzymatic hydrolysis conditions in step (1) are as follows: the material-liquid ratio of pigeon meat to water is 1:0.5 - 1:2.5 g / mL, the mass ratio of neutral protease to alkaline protease is 0.1:1 - 1:0.1, the temperature is 40 - 60 °C, the enzyme addition amount is 400 - 1200 U / g, the pH value is 7 - 9, and the enzymatic hydrolysis time is 2 - 4 h.

[0011] Preferably, the constant temperature enzymatic hydrolysis conditions are as follows: the material-liquid ratio is 1:1.5 g / mL, the mass ratio of neutral protease to alkaline protease is 3:1, the temperature is 50 °C, the enzyme addition amount is 800 U / g, the pH is 7.5, and the enzymatic hydrolysis time is 3 h.

[0012] Preferably, before mincing the pigeon meat in step (1), the obvious fat and fascia parts need to be removed first, and the enzymatic hydrolysis is carried out at a constant temperature in a shaker.

[0013] Preferably, the centrifugation conditions in step (1) are as follows: 4 °C, 10000 ± 2000 r / min, 15 ± 5 min, and the supernatant is taken.

[0014] Preferably, the supernatant in step (2) is pre-filtered with a 0.45 μm aqueous filter membrane first. The pigeon meat anti-fatigue peptide is the fraction with a molecular weight of 1 - 5 kDa intercepted after separation by ultrafiltration membranes with molecular weights of 1 kDa, 5 kDa, and 10 kDa. After the exhaustive swimming test of mice, the fraction with a molecular weight of 1 - 5 kDa is preferably obtained as the pigeon meat anti-fatigue peptide.

[0015] Application of the pigeon meat antioxidant peptide and anti-fatigue peptide obtained by the above method as functional food additives.

[0016] The present invention uses pigeon breast meat as the raw material, and uses protease enzymatic hydrolysis method to compound enzymes to prepare a peptide with antioxidant and anti-fatigue activities. First, the better-performing ones are screened out from four proteases as the compound enzymes. After the screening is completed, the process of compound enzymatic hydrolysis of pigeon meat to prepare antioxidant peptide is optimized by single-factor experiment and response surface optimization method. Then, the amino acids of the pigeon meat peptide prepared under the optimal conditions are determined. Finally, the pigeon meat peptide is ultrafiltered, and after the exhaustive swimming test of mice on pigeon meat peptides with different molecular weights, by analyzing the exhaustive swimming time, blood urea nitrogen content, and blood SOD enzyme content of each group of mice, a fraction with anti-fatigue efficacy and a molecular weight of 1 - 5 kDa is obtained. It is expected to provide a research basis and reference for the health attributes and potential functional values of pigeon meat.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The anti-fatigue peptide of the present invention is a peptide with a molecular weight lower than 5000 Da, which is relatively easy to be absorbed by the human body, has the effect of significantly improving the enzyme activity of SOD enzyme, and has a significant anti-fatigue effect.

[0019] (2) The antioxidant peptide of the present invention has a high content of essential amino acids and antioxidant active amino acids, reaching 47.97% and 29.27% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a graph showing the effects of enzymatic hydrolysis compound enzymes on the degree of hydrolysis and ABTS scavenging rate.

[0021] Figure 2 It is a graph showing the effects of the pH value of enzymatic hydrolysis on the degree of hydrolysis and ABTS scavenging rate.

[0022] Figure 3 It is a graph showing the effects of the ratio of material to liquid in enzymatic hydrolysis on the degree of hydrolysis and ABTS scavenging rate.

[0023] Figure 4 It is a graph showing the effects of the amount of added enzyme in enzymatic hydrolysis on the degree of hydrolysis and ABTS scavenging rate.

[0024] Figure 5 It is a graph showing the effects of the enzymatic hydrolysis time on the degree of hydrolysis and ABTS scavenging rate.

[0025] Figure 6 It is a graph showing the effects of enzymatic hydrolysates with different molecular weights on the exhaustive swimming time of mice.

[0026] Figure 7 It is a graph showing the detection results of the SOD enzyme activity in mouse serum.

[0027] Figure 8 It is a graph showing the detection results of the BUN level in mouse blood.

[0028] Figure 9 It is a graph showing the antioxidant activities of enzymatic hydrolysis of pigeon meat by different proteases. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be further described in detail below with reference to specific embodiments, but the implementation manners of the present invention are not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0030] The pigeon meat was purchased from Meizhou Jinlü Modern Agricultural Development Co., Ltd.

[0031] The chicken peptide (CP) was purchased from Shaanxi Hana Biotechnology Co., Ltd.

[0032] Taurine, papain, flavor protease, neutral protease and alkaline protease were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0033] Example 1

[0034] Take pigeon breast meat (White King pigeons at 21 days old), mince it after removing obvious fat and fascia, add deionized water, add a compound enzyme of neutral protease and alkaline protease, adjust the pH value, after enzymatic hydrolysis for a certain time, inactivate the enzyme at 95 °C for 15 min, centrifuge after cooling to obtain the supernatant (4 °C, 10,000 r / min, 15 min), and perform suction filtration to further remove solids to obtain pigeon meat peptide (PP), and store it after vacuum freeze-drying.

[0035] Determine the degree of hydrolysis by OPA method and the scavenging rate of ABTS free radicals for the products with different enzymatic hydrolysis conditions. The specific results of the optimization process are shown in Figures 1 - 5 , comprehensively Figures 1 - 5 It can be concluded that the optimal enzymatic hydrolysis conditions are: solid-liquid ratio 1:1.5 (m:V), compound enzyme ratio 3:1 (neutral protease: alkaline protease), enzyme addition amount 800 U / g, pH value 7.5, and enzymatic hydrolysis time 3 h.

[0036] Example 2

[0037] Take 30 g of pigeon breast meat (White King pigeons at 21 days old), mince it after removing obvious fat and fascia, add 1:1.5 g / mL (m:V) deionized water, add a compound enzyme of neutral protease and alkaline protease with a mass ratio of 3:1, enzymatically hydrolyze under the conditions of temperature 50 °C, enzyme addition amount 800 U / g, adjust the pH value to 7.5, and enzymatic hydrolysis time 3 h. After enzymatic hydrolysis is completed, inactivate the enzyme at 95 °C for 15 min, centrifuge after cooling to obtain the supernatant (4 °C, 10,000 r / min, 15 min), and perform suction filtration to further remove solids to obtain pigeon meat peptide (PP), and store it after vacuum freeze-drying. Under these conditions, when the concentration of pigeon meat peptide is 1 mg / mL, the scavenging rate of ABTS free radicals is 36.10%, and the degree of hydrolysis is 47.51%, showing good antioxidant activity.

[0038] Determine the total amino acid composition of the product under the above optimal conditions. The conditions for determining the total amino acid composition are as follows: Weigh a certain amount of pigeon meat peptide, add 6 mol / L HCl, seal it after filling with nitrogen, hydrolyze at 110 °C for 24 h, then make up the volume to 1 mg / mL with water, filter through a 0.22 μm filter membrane, and load the sample for detection. The results are shown in Table 1 for details.

[0039] Amino acid automatic analyzer conditions: Chromatographic column model: ADVANCEBIO AAA; Column temperature: 40 °C; Detection wavelength: 338 nm; Flow rate: 1 mL / min; Reaction solution: Ninhydrin; Injection volume: 20 μL.

[0040] Table 1: Total amino acid content in pigeon meat peptide

[0041]

[0042] Note: * indicates essential amino acids for the human body, and # indicates hydrophobic amino acids

[0043] Example 3

[0044] (1) Preparation of pigeon meat anti-fatigue peptide:

[0045] Take the pigeon meat antioxidant peptide in Example 2, first pre-filter it with a 0.45 μm aqueous filter membrane, and then use the ultrafiltration membrane separation method to intercept components with different molecular weights to obtain pigeon meat anti-fatigue peptides with different molecular weights. The proportions of components <1 kDa, 1 - 5 kDa, 5 - 10 kDa, and >10 kDa are 39.51%, 42.11%, 13.74%, and 6.63% respectively.

[0046] (2) Efficacy verification of pigeon meat anti-fatigue peptide:

[0047] Grouping and feeding of mice: A total of 40 male ICR mice were selected for the experiment. The mice were adaptively fed in the SPF-class animal house of the Animal Experiment Center of Guangdong Pharmaceutical University for one week before the experiment. The room temperature was 25 ± 1°C, the humidity was 50% - 70%, and the light was alternated between light and dark for 12 hours. The number of mice in each cage was 5, and they were fed freely with standard feed. The water bottles were changed every other day, and the cages were cleaned 2 times a week.

[0048] A total of 8 groups were set up in the experiment: (1) control group, (2) taurine control group, (3) chicken peptide (CP) control group, (4) <1 kDa group, (5) 1 - 5 kDa group, (6) 5 - 10 kDa group, (7) >10 kDa group, (8) pigeon meat peptide (PP) group. After that, there were 5 mice in each group. Three mice were selected for the exhaustive swimming experiment, and the other two were sampled for measuring indicators. Dosage setting: The pigeon meat peptides and chicken peptides with different molecular weights were gavaged at a dose of 1 mg / g·bw / d; the control group was gavaged with distilled water; taurine was gavaged at a dose of 0.5 mg / g·bw / d for 28 consecutive days.

[0049] ① Determination of the exhaustive swimming time of mice:

[0050] Thirty minutes after the last administration, 8 groups of mice were loaded with lead sheets at the tail root at 5% of their body weight and forced to swim in a water tank (water temperature 25 ± 1°C, water depth not less than 35 cm). The time from the start of swimming to death was recorded as the exhaustive swimming time when they sank to the bottom and could not float for 10 s as the death standard.

[0051] ② Determination of physiological and biochemical indexes of mice:

[0052] Thirty minutes after the last administration to the mice, they were placed in a water tank (water temperature 25±1°C, water depth not less than 35 cm) to swim freely. After resting for 30 minutes, blood was collected from the orbital venous plexus, and serum was obtained by centrifugation. The contents of blood urea nitrogen (BUN) and superoxide dismutase (SOD) in the serum were measured by the Elisa method.

[0053] The results of the exhaustive swimming test are as Figure 6 shown: Among the groups with different molecular weight samples, the 1-5kda group had the best effect, and the swimming time was 49.34±16.06 min, which was significantly improved compared with the 21.08±0.90 min of the blank group, indicating that the ultrafiltration fraction of pigeon meat peptides has a certain anti-fatigue effect.

[0054] The results of the SOD enzyme activity assay are as shown in 7: The <1k and 1-5k fractions had a significant effect on increasing the SOD enzyme activity. Among them, the enzyme activity of the 1-5kDa group reached 190.13 mmol / L, which was significantly higher than that of the control group (134.53 mmol / L).

[0055] The results of the blood BUN assay are as Figure 8 shown: Among all groups, the 1-5k fraction and taurine had a better ability to reduce the blood urea nitrogen level, and the results were basically consistent with those of the exhaustive swimming experiment, indicating that the 1-5k fraction may achieve the anti-fatigue effect by clearing bad metabolites.

[0056] Example 4

[0057] (1) Preparation of pigeon meat antioxidant peptides by enzymatic hydrolysis with different proteases

[0058] Take 30 g of pigeon breast meat (white king pigeons at 21 days of age), remove obvious fat and fascia, then mince it, add 1:1.5 g / mL (m:V) deionized water, and add different proteases for enzymatic hydrolysis under the conditions shown in Table 2. After enzymatic hydrolysis, inactivate the enzyme at 95°C for 15 min, cool it, and centrifuge to obtain the supernatant (4°C, 10,000 r / min, 15 min), then perform suction filtration to further remove solids to obtain pigeon meat peptides (PP), and store them after vacuum freeze-drying. Under these conditions, the comprehensive antioxidant properties of the enzymatic hydrolysis products of alkaline protease and neutral protease are better, and the IC50 values of each index are generally lower (see Figure 9 ), proving that these two proteases can be used as enzymes for enzymatic hydrolysis of pigeon meat protein to prepare antioxidant peptides with better activity.

[0059] Table 2 Enzymatic hydrolysis process conditions of different proteases

[0060]

[0061] Example 5

[0062] Take 30 g of pigeon breast meat (White King pigeons at 21 days of age), mince it after removing obvious fat and fascia, add deionized water at a ratio of 1:1.5 g / mL (m:V), add a compound enzyme of neutral protease and alkaline protease with a mass ratio of 2:1, enzymatically hydrolyze under the conditions of a temperature of 50 °C, an enzyme addition amount of 600 U / g, adjust the pH value to 7.5, and an enzymatic hydrolysis time of 3 h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95 °C for 15 min. After cooling, centrifuge to obtain the supernatant (4 °C, 10,000 r / min, 15 min), and perform suction filtration to further remove solids to obtain pigeon meat peptide (PP), which is stored after vacuum freeze-drying. Under these conditions, when the concentration of pigeon meat peptide is 1 mg / mL, the ABTS free radical scavenging rate is 28.08%, and the degree of hydrolysis is 38.11%. Both indicators are lower than those of the optimal process.

[0063] Example 6

[0064] Take 30 g of pigeon breast meat (White King pigeons at 21 days of age), mince it after removing obvious fat and fascia, add deionized water at a ratio of 1:1.5 g / mL (m:V), add a compound enzyme of neutral protease and alkaline protease with a mass ratio of 3:1, enzymatically hydrolyze under the conditions of a temperature of 50 °C, an enzyme addition amount of 600 U / g, adjust the pH value to 7.5, and an enzymatic hydrolysis time of 4 h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95 °C for 15 min. After cooling, centrifuge to obtain the supernatant (4 °C, 10,000 r / min, 15 min), and perform suction filtration to further remove solids to obtain pigeon meat peptide (PP), which is stored after vacuum freeze-drying. Under these conditions, when the concentration of pigeon meat peptide is 1 mg / mL, the ABTS free radical scavenging rate is 28.81%, and the degree of hydrolysis is 38.42%. Both indicators are lower than those of the optimal process.

[0065] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of pigeon meat antioxidant peptide and anti-fatigue peptide, characterized in that, it comprises the following steps: Take pigeon meat and mince it, add water and a compound enzyme of neutral protease and alkaline protease for constant-temperature enzymatic hydrolysis, centrifuge to obtain a supernatant, and the supernatant is subjected to any one of the following treatments: (1) The supernatant is freeze-dried to obtain pigeon meat antioxidant peptide; (2) The supernatant is intercepted by ultrafiltration membrane separation method to obtain a fraction of 1-5 kDa, and then freeze-dried to obtain pigeon meat anti-fatigue peptide; The conditions for the constant-temperature enzymatic hydrolysis are: the material-liquid ratio of pigeon meat to water is 1:0.5-1:2.5 g / mL, the mass ratio of neutral protease to alkaline protease is 0.1:1-1:0.1, the temperature is 40-60 °C, the enzyme addition amount is 400-1200 U / g, the pH value is 7-9, and the enzymatic hydrolysis time is 2-4 h.

2. The preparation method according to claim 1, characterized in that: The conditions for the constant-temperature enzymatic hydrolysis are: the material-liquid ratio is 1:1.5 g / mL, the mass ratio of neutral protease to alkaline protease is 3:1, the temperature is 50 °C, the enzyme addition amount is 800 U / g, the pH is 7.5, and the enzymatic hydrolysis time is 3 h.

3. The preparation method according to claim 1 or 2, characterized in that: Before mincing the pigeon meat, the obvious fat and fascia parts need to be removed first, and then subjected to constant-temperature enzymatic hydrolysis in a shaker.

4. The preparation method according to claim 1 or 2, characterized in that: The conditions for the centrifugation are: 4 °C, 10000±2000 r / min, 15±5 min, and the supernatant is taken.

5. The preparation method according to claim 1 or 2, characterized in that: In (2), the supernatant is first pre-filtered with a 0.45 μm aqueous filter membrane.

6. Pigeon meat antioxidant peptide and anti-fatigue peptide obtained by the preparation method according to any one of claims 1 to 5.

7. Applications of the pigeon meat antioxidant peptide and anti-fatigue peptide according to claim 6 as antioxidant functional food additives and as anti-fatigue functional food additives respectively.

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