Fish maw collagen peptide with low molecular weight and high antioxidant activity as well as preparation method and application of fish maw collagen peptide

Abstract

The invention discloses a fish maw collagen peptide with low molecular weight and high antioxidant activity as well as a preparation method and application thereof, and belongs to the technical field of functional product development. The fish maw collagen peptide is prepared by adopting an ultrasonic, microwave or high hydrostatic pressure assisted enzymolysis method, wherein the content of peptide with the relative molecular mass of 70-500Da is 72.47%-77.78%, the content of hydrophobic amino acid is 37.30%-38.66%, the content of GL is 0.03-0.65 g / 100g, and the content of GPA is 0.6-1.8 g / 100. The antioxidant activity is high, and the effect of enhancing immunity is achieved. In addition, the method also has the advantages of simple process flow, low cost, mild reaction conditions, environmental friendliness and the like. The method provided by the invention can provide solid theoretical support in the aspects of increasing the economic value of fish byproducts, increasing the utilization rate of fish maw collagen products, reasonably utilizing resources, developing novel health-care and skin-care products and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of functional product development, and in particular relates to a low-molecular-weight fish maw collagen peptide with high antioxidant activity, and a preparation method and application thereof. Background Art

[0002] Collagen peptides are usually composed of 2 to 10 amino acids, and due to their small molecular size, they show excellent biological activity and easy absorption. Collagen peptides have multiple functions, including anti-oxidation and improving immunity. They also contain strong activity and high safety. They can be widely used in food, cosmetics and pharmaceutical preparations, and have become a research focus. Studies have shown that different molecular weights and amino acid compositions determine the biological activity of collagen peptides. Peptides with lower molecular weights are more likely to approach free radicals, thereby inhibiting the generation of free radicals and achieving an antioxidant effect; the amino acid composition also has a greater effect on the biological activity of collagen peptides. The higher the content of hydrophobic amino acids in the sequence, the stronger the antioxidant activity. Acidic and basic amino acids can exhibit high antioxidant activity by chelating metal ions. Immunomodulatory function is an important active function of active peptides. By extracting immunomodulatory peptides from natural food raw materials, health products or nutritional products can be made to improve human immunity. In recent years, there have been a lot of studies on the immune regulation function of the body by extracting bioactive peptides from marine organisms.

[0003] Fish maw, one of the eight sea immortals in my country, is a finished product made by cutting open fresh fish maw, removing blood vessels and surface mucosa, cleaning, flattening and drying, or soaking fish maw in a certain concentration of alum water to remove mucosa, cutting into slices, and then dehydrating and drying. Fish maw contains rich collagen and a variety of essential amino acids for the human body. However, the unique triple helix structure of collagen is very stable, and it is difficult to decompose it when the heating time is short and the heating temperature is normal, so it is not easily absorbed by the human body. Fish maw collagen peptides are small molecule peptide chains that are decomposed into fish maw collagen macromolecules by enzymatic hydrolysis and other methods, making them easy for the human body to absorb and utilize. In addition, fish maw collagen immune protein peptides are usually derived from by-products of deep-sea fish, with abundant resources and far away from industrial pollution. Compared with traditional terrestrial animal collagen such as cattle and pigs, it has stronger biocompatibility, lower immunogenicity and disease transmission risks, and no religious restrictions.

[0004] Although fish maw collagen peptides have good safety and biocompatibility, they are usually too large to be used efficiently, and there are deficiencies in the research on the preparation of low molecular weight, high antioxidant activity fish maw small molecule immune protein peptides. Existing methods for preparing fish collagen peptides, for example:

[0005] CN103992385B discloses a method for preparing antioxidant collagen peptide from yellow croaker maw, comprising the following steps: (1) pretreatment: washing the yellow croaker maw with tap water, homogenizing it with a high-speed tissue crusher, and adding Na2HPO4-NaH2PO4 buffer; (2) adjusting the pH value, and keeping it warm at 40-50°C for 10-15 minutes; (3) performing a primary enzymatic hydrolysis with a neutral protease; (4) performing a secondary enzymatic hydrolysis with trypsin; (5) centrifuging; (6) collecting a portion with a molecular weight less than 3 kDa by ultrafiltration; (7) performing ion exchange and gel column chromatography to collect the component with the highest superoxide anion free radical scavenging activity; and (8) RP-HPLC purification.

[0006] Patent CN110577975B discloses a method for extracting and preparing fish maw collagen oligopeptides, comprising the following steps: (1) cleaning; (2) pretreatment with tannic acid; (3) enzymatic hydrolysis; (4) membrane filtration; (5) nanofiltration; (6) vacuum falling film concentration; (7) sterilization filtration; and (8) spray drying.

[0007] Patent CN116284341B discloses a method for preparing a low-immunogenic, blood pressure-lowering, and antioxidant deep-sea fish skin small molecule collagen peptide, comprising the following steps: (1) physical pretreatment, defatting, and removal of foreign proteins; (2) removal of terminal peptides; (3) combined enzymatic hydrolysis with neutral protease and papain; (4) ultrafiltration; (5) separation and purification of filtered components by Sephedax G-25 molecular sieve chromatography, and purification of the most active component by reverse phase chromatography C18 column; (6) 60 Co irradiation to inactivate endotoxin; (7) sterilization; (8) packaging.

[0008] However, the methods for preparing fish maw collagen peptides using the above existing methods have some simple steps, which only use the action of biological enzymes to decompose fish gelatin, but the content of small molecule peptides obtained is low; some steps are too complicated, the investment cost is too high, and it is difficult to carry out large-scale production. In addition, the existing methods for preparing fish maw collagen peptides either focus on reducing the peptide molecules or focus on preparing and separating functional peptides. There are few studies on preparing small peptides with high antioxidant or immune activity. Although some pure peptide products synthesized according to known sequences have high purity and strong activity, they can only be obtained by chemical synthesis, which is costly, polluting, and difficult to mass produce.

[0009] Therefore, the art is in urgent need of a fish maw small molecule immune protein peptide with low molecular weight, high antioxidant activity, strong immune activity, simple preparation process, low cost, mild reaction conditions, and environmental friendliness, and a preparation method thereof. Summary of the invention

[0010] In order to solve the above technical problems, the present invention provides a low molecular weight fish maw collagen peptide with high antioxidant activity and a preparation method and application thereof. The low molecular weight fish maw collagen peptide with high antioxidant activity is composed of a series of components with a relative molecular mass of less than 2000Da, wherein the content of peptides with a relative molecular mass of less than 1000Da is 91.74% to 93.67%, the content of peptides with a relative molecular mass between 70 and 500Da is 72.47% to 77.78%, and the content of hydrophobic amino acids is 37.30% to 38.66%; wherein the mass spectrometry results screen out two small peptide sequences with high matching degrees, namely GL and GPA, and the quantitative results show that the content of GL is 0.03 to 0.65g / 100g, and the content of GPA is 0.6 to 1.8g / 100g. The fish maw collagen peptide has strong antioxidant activity, with a DPPH free radical scavenging IC50 value of 4.14-6.25 mmol / L and an ABTS free radical scavenging IC50 value of 7.22-10.36 mmol / L. The fish maw collagen peptide has the effect of enhancing immunity, can enhance the function of immune organs such as the spleen and thymus, improve the ability of immune cells such as white blood cells and lymphocytes, and promote the activity of immune cytokines such as IL-3 and IL-6.

[0011] In short, the collagen peptide prepared by the method of the present invention has low molecular weight, high antioxidant activity, strong immune activity, and has the advantages of simple process flow, low cost, mild reaction, and environmental friendliness, and can be used in the preparation of antioxidant products or immune-enhancing products in the fields of food, health products, cosmetics, or medicines. The present invention can provide solid theoretical support in terms of improving the economic value of fish by-products, improving the utilization rate of fish maw collagen products, rationally utilizing resources, and developing new health care and skin care products.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] One of the purposes of the present invention is to provide a low molecular weight fish maw collagen peptide with high antioxidant activity. The low molecular weight fish maw collagen peptide with high antioxidant activity is composed of a series of components with a relative molecular mass of less than 2000Da, wherein the content of peptides with a relative molecular mass of less than 1000Da is 91.74% to 93.67%, the content of peptides with a relative molecular mass between 70 and 500Da is 72.47% to 77.78%, the content of hydrophobic amino acids is 37.30% to 38.66%, the content of small peptide sequence GL is 0.03 to 0.65g / 100g, and the content of small peptide sequence GPA is 0.6 to 1.8g / 100g.

[0014] The second object of the present invention is to provide a method for preparing the low molecular weight and high antioxidant activity fish maw collagen peptide, the preparation method comprising the following steps:

[0015] (1) Defatting and drying fish maw;

[0016] (2) Soaking, stewing and colloid milling: The fish glue is fully soaked and stewed for 15 to 30 minutes, and then the colloid mill tooth gap is set to 80 to 120 mm, and the sample is subjected to colloid milling for 20 to 40 minutes;

[0017] (3) Enzymatic hydrolysis: adjust the pH value of the fish gelatin homogenate to 6-10, add protease at an enzyme-substrate ratio of 2000-5000 U / g, and then use high hydrostatic pressure treatment, ultrasound or microwave method to assist enzymatic hydrolysis at 35-65°C for 4-7 hours;

[0018] (4) Inactivation of enzyme and centrifugation: Inactivate the enzyme in the enzymatic solution at 90-100°C for 15-30 min; then centrifuge at 4000-6000 r / min for 15-30 min, and finally collect the supernatant;

[0019] (5) Secondary alcohol precipitation and rotary evaporation concentration: anhydrous ethanol is added to the supernatant after centrifugation to make the ethanol volume fraction in the final system reach 40-60%, then the supernatant is taken after standing and centrifugation, and the ethanol is concentrated by rotary evaporation, and then anhydrous ethanol is added again for secondary alcohol precipitation and rotary evaporation concentration;

[0020] (6) Freeze-drying: The concentrated enzymatic supernatant is subjected to vacuum freeze-drying to obtain the fish maw collagen peptide, which is the low molecular weight and high antioxidant activity fish maw collagen peptide.

[0021] Furthermore, the fish maw in step (1) is derived from any one or more of sea bass, largemouth bass, Nile perch, river bass and greater leatherback.

[0022] Furthermore, the protease in step (3) is selected from any one or more of papain, flavor protease and hydrolase protease.

[0023] Furthermore, in step (3), when the protease is papain and the auxiliary enzymatic hydrolysis method is a high hydrostatic pressure treatment method, the pH value of the fish glue homogenate is first adjusted to 6-8, and then papain is added and mixed, followed by vacuum packaging and placing in a high hydrostatic pressure treatment equipment, and treating for 4-7 hours at a temperature of 40-50°C and a hydrostatic pressure of 100-300MPa.

[0024] Furthermore, in step (3), when the protease is flavor protease and the auxiliary enzymatic hydrolysis method is ultrasonic method, the pH value of the fish gelatin homogenate is first adjusted to 7-9, and then the flavor protease is added and mixed, followed by vacuum packaging and placing in an ultrasonic instrument, and treating for 4-7 hours at 35-65° C. and an ultrasonic power of 200-400 W.

[0025] Furthermore, in step (3), when the enzyme is a hydrolyzing protease and the auxiliary enzymatic hydrolysis method is a microwave method, the pH value of the fish glue homogenate is first adjusted to 7-10, and then the hydrolyzing protease is added and mixed, followed by vacuum packaging and placing in a microwave reactor, and treating for 4-7 hours at 37-50° C. and a microwave power of 50-300 W.

[0026] Furthermore, the standing time in step (5) is 10 to 30 minutes; the centrifugal speed is 3000 to 5000 r / min, and the time is 15 to 30 minutes; the rotary evaporation concentration speed is 100 to 200 r / min, and the temperature is 30 to 50°C.

[0027] Furthermore, the freeze drying in step (6) is firstly quick-frozen at -80°C for 10 to 12 hours, and then freeze-dried in a freeze dryer for 48 to 72 hours.

[0028] The third object of the present invention is to provide a method for preparing the low molecular weight and high antioxidant activity fish maw collagen peptide or the low molecular weight and high antioxidant activity fish maw collagen peptide and its application in the preparation of antioxidant products and / or immunomodulatory products, wherein the products include but are not limited to food, health products, cosmetics and medicines.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The low molecular weight fish maw collagen active peptide provided by the present invention has dual effects of anti-oxidation and immunity, can inhibit the generation of free radicals and enhance immunity. It has broad application prospects in food, health products, cosmetics or medicines, promotes the further development of fish maw products, and improves the nutritional value and application value of fish maw. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention is a flow chart of the preparation process of fish maw collagen peptide (fish maw small molecule immune protein peptide). DETAILED DESCRIPTION

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. The products, equipment, etc. used in the following examples are commercially available unless otherwise specified, and the methods used are consistent with conventional methods unless otherwise specified.

[0033] The preparation process flow chart of fish maw collagen peptide (fish maw small molecule immune protein peptide) in the present invention is as follows: Figure 1 shown.

[0034] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0035] Example 1 Preparation of a low molecular weight fish maw collagen immune protein peptide I with high antioxidant activity

[0036] (1) Fish maw degreasing: Use petroleum ether to degrease fish maw (sea bass) and dry it for later use.

[0037] (2) Soaking, stewing and colloid milling: Add 20 to 50 times the dry weight of fish glue to fully soak, stew for 15 to 30 minutes after soaking; set the colloid mill tooth gap to 120 mm, and perform colloid milling on the sample for 35 minutes.

[0038] (3) High hydrostatic pressure treatment (HPP) assisted enzymatic hydrolysis: The pH value of the homogenate obtained in step (2) was adjusted to 6.0 using HCL and NaOH, and papain (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S10011-25g, enzyme amount 3000U / mg) was added. After mixing, the mixture was vacuum packed in a polyethylene bag and immediately placed in an HPP equipment for treatment at 45°C and a hydrostatic pressure of 100 MPa for 6 h.

[0039] (4) Inactivation of enzyme and centrifugation: The enzymatic solution obtained in step (3) was inactivated at 100°C for 20 min, cooled to room temperature, centrifuged at 4°C and 4000 rpm for 20 min, and the supernatant was collected;

[0040] (5) secondary alcohol precipitation and rotary evaporation: adding anhydrous ethanol to the supernatant after centrifugation in step (4) to make the ethanol volume fraction in the final system reach 40%, letting it stand and centrifuging, taking the supernatant, and rotary evaporation to remove ethanol, adding anhydrous ethanol again to make the ethanol volume fraction in the system reach 40%, letting it stand and centrifuging, taking the supernatant, and rotary evaporation to remove ethanol;

[0041] (6) Freeze drying: The concentrated enzymatic supernatant obtained in step (4) is divided into glass plates, each plate having about 25 to 30 mL, and is quick-frozen at -80°C for 12 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain fish maw collagen peptide freeze-dried powder;

[0042] (7) Determination of molecular weight distribution of fish maw collagen peptide I: The molecular weight distribution of fish maw collagen peptide I was determined by high performance liquid chromatography. The fish maw collagen peptide I freeze-dried powder was dissolved in deionized water to prepare a 15 mg / mL solution. The sample solution was filtered through a 0.45 μm filter and then injected. The sample volume was 10 μL. The molecular weight determination results of fish maw collagen peptide I are shown in Table 1.

[0043] Table 1 Relative molecular weight distribution of small molecule immune protein peptide I in fish maw

[0044]

[0045] As shown in Table 1, the relative molecular weight distribution of fish maw small molecule protein peptide I is mainly concentrated in 70-500 Da, and the relative percentage content of this molecular weight peptide reaches 72.47%.

[0046] (8) Determination of amino acid composition of fish maw collagen peptide I: The sample was treated with acid hydrolysis to obtain a clear and transparent hydrolyzate. After the hydrolyzate was filtered through a 0.22 μm water filter membrane, 400 μL was taken and added to the injection bottle. The amino acid composition of fish maw collagen peptide I was analyzed using a high performance liquid chromatograph dedicated to hydrolysis amino acid analysis. The results of the amino acid composition determination of fish maw collagen peptide I are shown in Table 2.

[0047] Table 2 Determination of amino acid composition of fish maw collagen immune protein peptide I

[0048]

[0049] (Note: “*” indicates hydrophobic amino acids.)

[0050] The higher the content of hydrophobic amino acids in the collagen peptide sequence, the stronger the antioxidant activity. The stronger the immune activity of the peptide segment, the acidic and basic amino acids can also show high antioxidant activity by chelating metal ions. Some amino acids such as arginine and glutamic acid may participate in inflammatory responses and reduce the release of inflammatory mediators. As shown in Table 2, the relative percentage of hydrophobic amino acids in fish maw small molecule immune protein peptide I is 38.03%, the acidic amino acid content is 17.21%, and the basic amino acid content is 13.19%, of which the contents of glutamic acid and arginine are 12.23% and 8.78%, respectively.

[0051] Example 2 Preparation of a low molecular weight and high antioxidant activity fish maw small molecule protein peptide II

[0052] (1) Fish maw degreasing: Use petroleum ether to degrease fish maw (largemouth bass) and dry it for later use.

[0053] (2) Soaking, stewing and colloid milling: Add 20 to 50 times the dry weight of fish glue to fully soak, stew for 15 to 30 minutes after soaking; set the colloid mill tooth gap to 120 mm, and perform colloid milling on the sample for 35 minutes.

[0054] (3) Ultrasonic-assisted enzymatic hydrolysis: The pH value of the homogenate obtained in step (2) was adjusted to 7.5 using HCL and NaOH, and flavor protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S10153-25g, enzyme amount 3000U / mg) was added. After mixing, the mixture was vacuum-packed in a polyethylene bag and immediately placed in an ultrasonic instrument at 50°C with an ultrasonic power of 200-400W for 6 h.

[0055] (4) Inactivation of enzyme and centrifugation: The enzymatic solution obtained in step (3) was inactivated at 100°C for 20 min, cooled to room temperature, centrifuged at 4°C and 4000 rpm for 20 min, and the supernatant was collected;

[0056] (5) secondary alcohol precipitation and rotary evaporation: adding anhydrous ethanol to the supernatant after centrifugation in step (4) to make the ethanol volume fraction in the final system reach 50%, letting it stand and centrifuging, taking the supernatant, and rotary evaporation to remove ethanol, adding anhydrous ethanol again to make the ethanol volume fraction in the system reach 50%, letting it stand and centrifuging, taking the supernatant, and rotary evaporation to remove ethanol;

[0057] (6) Freeze drying: The concentrated enzymatic supernatant obtained in step (5) is divided into glass plates, each plate having about 25 to 30 mL, and is quick-frozen at -80°C for 12 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain fish maw collagen peptide freeze-dried powder;

[0058] (7) Molecular weight distribution of fish maw collagen peptide II: The molecular weight distribution of fish maw collagen peptide was determined by high performance liquid chromatography. The fish maw collagen peptide freeze-dried powder was dissolved in deionized water to prepare a 15 mg / mL solution. The sample solution was filtered through a 0.45 μm filter and then injected with a sample volume of 10 μL. The molecular weight determination results of fish maw collagen peptide II are shown in Table 3.

[0059] Table 3 Relative molecular weight distribution of low molecular weight fish maw collagen peptide II

[0060]

[0061] As shown in Table 3, the relative molecular weight distribution of fish maw small molecule protein peptide II is mainly concentrated in 70-500 Da, and the relative percentage of this molecular weight peptide reaches 75.49%.

[0062] (8) Amino acid composition of fish maw small molecule protein peptide II: The sample was treated by acid hydrolysis to obtain a clear and transparent hydrolyzate. After the hydrolyzate was filtered through a 0.22 μm water filter membrane, 400 μL was taken and added to the injection bottle. The amino acid composition of fish maw small molecule protein peptide II was analyzed using a high performance liquid chromatograph dedicated to hydrolysis amino acid analysis. The results of the amino acid composition determination of fish maw small molecule protein peptide II are shown in Table 4.

[0063] Table 4 Determination of amino acid composition of low molecular weight fish maw collagen peptide II

[0064]

[0065] (Note: “*” indicates hydrophobic amino acids.)

[0066] The higher the content of hydrophobic amino acids in the collagen peptide sequence, the stronger the antioxidant activity. The stronger the immune activity of the peptide segment, the acidic amino acids and basic amino acids can also show high antioxidant activity by chelating metal ions. Certain amino acids such as arginine and glutamic acid may participate in inflammatory responses and reduce the release of inflammatory mediators. As shown in Table 4, the relative percentage of hydrophobic amino acids in fish maw collagen immune protein peptide II is 37.3%, the acidic amino acid content is 16.59%, and the basic amino acid content is 13.22%, of which the contents of glutamic acid and arginine are 11.53% and 9.26%, respectively.

[0067] Example 3 Preparation of a low molecular weight fish maw collagen peptide III with high antioxidant activity

[0068] (1) Defatting fish maw: Defatting fish maw (perch) with petroleum ether and drying for later use.

[0069] (2) Soaking, stewing and colloid milling: Add 20 to 50 times the dry weight of fish glue to fully soak, stew for 15 to 30 minutes after soaking; set the colloid mill tooth gap to 120 mm, and perform colloid milling on the sample for 35 minutes.

[0070] (3) Microwave-assisted enzymatic hydrolysis: The pH value of the homogenate obtained in step (2) was adjusted to 9.5 using HCL and NaOH, and hydrolyzing protease (purchased from Novozymes, product number: Alcalase 2.4L, enzyme amount: 3000U / mg) was added. After mixing, the mixture was vacuum-packed in a polyethylene bag and immediately placed in a microwave reactor. The mixture was treated at 45°C with a microwave power of 250W for 6 h.

[0071] (4) Inactivation of enzyme and centrifugation: The enzymatic solution obtained in step (3) was inactivated at 100°C for 20 min, cooled to room temperature, centrifuged at 4°C and 4000 rpm for 20 min, and the supernatant was collected;

[0072] (5) secondary alcohol precipitation and rotary evaporation: adding anhydrous ethanol to the supernatant after centrifugation in step (4) to make the ethanol volume fraction in the final system reach 60%, letting it stand and centrifuging, taking the supernatant, and rotary evaporation to remove ethanol, adding anhydrous ethanol again to make the ethanol volume fraction in the system reach 60%, letting it stand and centrifuging, taking the supernatant, and rotary evaporation to remove ethanol;

[0073] (6) Freeze drying: The concentrated enzymatic supernatant obtained in step (6) is divided into glass plates, each plate having about 25 to 30 mL, and is quick-frozen at -80°C for 12 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain fish maw collagen peptide freeze-dried powder;

[0074] (7) Determination of molecular weight distribution of fish maw small molecule protein peptide III: The molecular weight distribution of fish maw small molecule protein peptide was determined by high performance liquid chromatography. The fish maw small molecule protein peptide freeze-dried powder was dissolved in deionized water to prepare a 15 mg / mL solution. The sample solution was filtered through a 0.45 μm filter and then injected with a sample volume of 10 μL. The molecular weight determination results of the fish maw small molecule protein peptide are shown in Table 5.

[0075] Table 5 Relative molecular weight distribution of low molecular weight fish maw collagen peptide III

[0076]

[0077]

[0078] As shown in Table 5, the relative molecular weight distribution of fish maw small molecule protein peptide III is mainly concentrated in 70-500 Da, and the relative percentage content of this molecular weight peptide reaches 77.78%.

[0079] (8) Determination of amino acid composition of fish maw small molecule protein peptide III: The sample was treated by acid hydrolysis to obtain a clear and transparent hydrolyzate. After the hydrolyzate was filtered through a 0.22 μm water filter membrane, 400 μL was taken and added to the injection bottle. The amino acid composition of fish maw small molecule protein peptide III was analyzed using a high performance liquid chromatograph dedicated to hydrolysis amino acid analysis. The results of the amino acid composition determination of fish maw small molecule protein peptide III are shown in Table 6.

[0080] Table 6 Determination of amino acid composition of low molecular weight fish maw small molecule protein peptide III

[0081]

[0082] (Note: “*” indicates hydrophobic amino acids.)

[0083] The higher the content of hydrophobic amino acids in the collagen peptide sequence, the stronger the antioxidant activity. The stronger the immune activity of the peptide segment, the acidic amino acids and basic amino acids can also show high antioxidant activity by chelating metal ions. Certain amino acids such as arginine and glutamic acid may participate in inflammatory responses and reduce the release of inflammatory mediators. As shown in Table 6, the relative percentage of hydrophobic amino acids in fish maw collagen immune protein peptide III is 38.66%, the acidic amino acid content is 16.95%, and the basic amino acid content is 13.28%, of which the contents of glutamic acid and arginine are 11.18% and 9.11%, respectively.

[0084] Summary of Examples 1 to 3

[0085] Generally speaking, different molecular weights and amino acid compositions determine the biological activity of collagen peptides. Peptides with smaller molecular weights are more easily absorbed and closer to free radicals, thereby inhibiting the generation of free radicals and achieving an antioxidant effect. Fish maw small molecule immune protein peptides I to III are composed of a series of components with a relative molecular weight of less than 2000Da, and their relative molecular weight distribution is mainly concentrated in 70-500Da. The relative percentage of peptides in this molecular weight range is 72.47% to 77.78%, among which the relative content of fish maw small molecule immune protein peptide III in the molecular weight range of 70-500Da is the highest, up to 77.78%.

[0086] The amino acid composition also has a great influence on the biological activity of collagen peptides. The higher the content of hydrophobic amino acids in the sequence, the stronger the antioxidant activity. The stronger the immune activity of the peptide segment, the acidic and basic amino acids can also exhibit high antioxidant activity by chelating metal ions. The hydrophobic amino acid content of fish maw small molecule immune protein peptides I to III is 37.30% to 38.66%, among which the hydrophobic amino acid content of fish maw small molecule immune protein peptide III is the highest at 38.66%.

[0087] The above description shows that the fish maw collagen peptide III obtained by microwave-assisted enzymatic hydrolysis combined with secondary alcohol precipitation has a lower molecular weight composition, a higher relative content of hydrophobic amino acids, and a stronger biological activity, and is more suitable for the development of foods, health products, cosmetics or medicines related to anti-oxidation, free radical scavenging and immunity.

[0088] Example 4 Determination of Antioxidant Activity of Small Molecule Immune Protein Peptides from Fish Maw

[0089] The antioxidant activity experiment measures the DPPH free radical scavenging ability and ABTS free radical scavenging rate of the sample, and determines its half-inhibitory concentration (IC50) by measuring the free radical scavenging ability of the sample. The specific determination method of each free radical scavenging rate is as follows:

[0090] ①DPPH free radical scavenging rate

[0091] Prepare 0.4mmol / L DPPH solution, dissolve it in 95% volume fraction ethanol, prepare it and use it immediately, and store it in the dark at 0-4℃. Prepare sample solutions of different concentrations (a series of dilution multiples) to cover the expected IC50 range. Take a 96-well plate, add 0.5mL of DPPH solution and 50μL of sample solutions of different concentrations, add 150μL of DPPH solution to the wells containing the samples, and mix thoroughly. After mixing, place the 96-well plate at room temperature, react for 30 minutes in the dark, and measure the absorbance of each well at a wavelength of 517nm. Use deionized water instead of sample solution, mix it with the DPPH reaction solution, and use it as a blank control group. The DPPH free radical scavenging rate is calculated as shown in formula (1):

[0092]

[0093] Wherein: A1 is the absorbance value of the sample well; A0 is the absorbance value of the sample control group (deionized water replaces the sample); A2 is the absorbance value of the blank control well containing only DPPH solution.

[0094] ②ABTS free radical scavenging rate

[0095] Prepare 7mmol / L ABTS solution and 2.45mmol / L potassium persulfate solution with distilled water, mix ABTS and potassium persulfate in a volume ratio of 1:1, and place in a dark place at 23℃ for 12-16h to form ABTS cation radicals. Dilute the ABTS cation radical solution appropriately with distilled water before use (about 20-25 times) so that the absorbance of the diluted solution at 734nm wavelength is 0.70±0.02 to obtain ABTS cation radical working solution. Prepare sample solutions of different concentrations (a series of dilution multiples) to cover the expected IC50 range. Take a 96-well plate, add 100μL of sample solutions of different concentrations and 100μL of ABTS cation radical working solution to each well, and mix thoroughly. After mixing, place the 96-well plate at 30℃ for 10min. After the reaction is completed, immediately measure the absorbance at 734nm. Use deionized water instead of sample solution and mix it with ABTS cation radical working solution as a blank control group. The calculation of ABTS free radical scavenging rate is shown in formula (2):

[0096]

[0097] Wherein: A1 is the absorbance value of the sample well; A0 is the absorbance value of the sample control group (deionized water replaces the sample); A2 is the absorbance value of the blank control well containing only ABTS solution.

[0098] By plotting the relationship curve between sample concentration and free radical scavenging rate, the sample concentration when the scavenging rate is 50%, that is, the IC50 value, is found. The measurement results are shown in Table 7.

[0099] Table 7 Determination of antioxidant activity of small molecule immune protein peptides from fish maw

[0100]

[0101] Free radicals containing unpaired electrons are very active and can trigger a series of harmful reactions to health by taking electrons (evolution process), including oxidative damage, inflammatory response and immune system damage. The antioxidant activity of fish maw small molecule immune protein peptides I to III was evaluated by measuring their DPPH free radical scavenging ability and ABTS free radical scavenging rate ability. The DPPH free radical IC50 value was 4.14-6.25mmol / L, and the ABTS free radical IC50 value was 7.22-10.36mmol / L. Among them, the free radical scavenging ability of fish maw small molecule immune protein peptide III was significantly higher than that of the other two groups (P < 0.05). Combined with the results of molecular weight distribution and amino acid composition determination, the present invention believes that fish maw small molecule immune protein peptide III is more suitable for the development of food, health products, cosmetics or medicines related to anti-oxidation, free radical scavenging and immunity.

[0102] Example 5 Amino Acid Sequence Analysis and Peptide Quantification of Small Molecular Immune Protein Peptides from Fish Maw

[0103] (1) Amino acid sequence analysis

[0104] The peptide amino acid sequences in the peptide mixture were sequenced and identified using liquid chromatography-mass spectrometry and proteomics methods. The fish maw small molecule immune protein peptide freeze-dried powder was dissolved in methanol to prepare a 10 mg / mL collagen peptide solution.

[0105] Specific chromatographic conditions: Acclaim PepMap C 18 Chromatographic column (75 μm×25 cm), mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile solution containing 0.1% formic acid; sample volume 5.0 μL, elution flow rate 300.0 nL / min. Mass spectrometry ionization mode is ESI+, positive charge spray voltage is 2.0 kV.

[0106] A fish maw protein database was established from the Uniprot database, and then the database was compared using PEAKS Studio software. De novo sequencing analysis was performed, and 9 peptides with high matching degrees were screened out based on their relative abundance, molecular weight, protein source, and alignment standard scores. The results are shown in Table 8.

[0107] Table 8 Peptide sequences with higher matching degree in fish maw collagen peptides

[0108]

[0109] Note: Since the sequence listing requirements indicate that the sequence must contain at least 4 specially defined amino acids unless it is intentionally skipped, only the amino acid sequences numbered 3-9 were prepared.

[0110] (2) Peptide content determination

[0111] After the small molecule peptides in the fish maw collagen peptide were dissolved and extracted with water, they were separated by reverse phase liquid chromatography column, detected by triple quadrupole mass spectrometry in series, and quantified by external standard method. Among them, the standard products: ① dipeptide GL (Gly-Leu, purity 98%, relative molecular mass 188.22g / mol), purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; ② tripeptide GPA (Gly-Pro-Ala, purity 98%. Relative molecular mass 244.26g / mol), purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0112] Chromatographic conditions: Waters Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) or other similar columns with similar performance, mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in methanol, gradient elution; sample volume 1.0 μL, elution flow rate 0.2 mL / min, column temperature 30°C. Mass spectrometry ionization mode ESI+, curtain gas flow 30 psi, spray voltage 5500 V, nebulization temperature 550°C, nebulizing gas (GS1) 55 psi, auxiliary gas (GS2) 50 psi.

[0113] Weigh 10 mg (accurate to 0.0001 g) of GL and GPA standards into a 50 mL volumetric flask, add water to dissolve and dilute to scale to obtain a solution with a concentration of 200 μg / mL as a standard stock solution. Weigh 20 mg of the sample into a 100 mL volumetric flask, add water to dissolve and dilute to scale, shake well, and filter through a 0.22 μm aqueous PTFE or PVDF filter membrane to obtain the solution.

[0114] Tandem triple quadrupole mass spectrometry detection and external standard method quantification were adopted. The results showed that the GL (Gly-Leu) content in fish maw collagen peptides I to III was 0.03 to 0.65 g / 100 g, and the GPA (Gly-Pro-Ala) content was 0.6 to 1.8 g / 100 g.

[0115] Example 6 Fish maw small molecule immune protein peptides I to III can alleviate cyclophosphamide (CTX)-induced immune damage in mice

[0116] 1. Experimental methods

[0117] (1) Animal grouping and dosing regimen

[0118] 40 healthy SPF-grade 6-week-old male C57BL / 6J mice were housed in an SPF-grade barrier environment after being settled in, with a controlled temperature of 23-27°C, a relative humidity of 40%-60%, and an artificial light / dark cycle of 12h. All mice were given free access to food and water and fed with irradiated basic feed. Adaptive feeding was performed for 7 days before the formal experiment to allow the mice to adapt to the environment. After one week of adaptation, the mice were randomly divided into 5 groups according to their weight, namely, normal control group, cyclophosphamide model group, cyclophosphamide + fish maw small molecule peptide I group (fish maw small molecule peptide I group), cyclophosphamide + fish maw small molecule peptide II group (fish maw small molecule peptide II group), and cyclophosphamide + fish maw small molecule peptide III group (fish maw small molecule peptide III group), with 8 mice in each group. All five groups were adaptively fed for the first 7 days. The intervention started on the eighth day, with 200 μL of saline orally administered to the CON and CTX groups every day; and 200 μL of fish maw small molecule peptides were orally administered to the fish maw small molecule peptide groups I to III for a total of 12 days. The intervention started on the 17th day, with 200 μL of cyclophosphamide intraperitoneally injected into the CTX and fish maw small molecule peptide groups I to III every day for a total of 3 days.

[0119] (2) Tissue sample collection

[0120] On the 20th day after arrival, the mice were fasted overnight but not water, and were anesthetized after being weighed. The mice were eyeballed and whole blood was collected. Part of the blood was stored in an enzyme-inactivated centrifuge tube, and after standing at room temperature for 3 hours, it was centrifuged at 4°C and 3000rpm for 15 minutes to separate the serum; part of the blood was transferred to a sodium heparin anticoagulation centrifuge tube, and after standing for 1 hour, it was centrifuged under the same conditions to obtain plasma; 50μL of whole blood was used for the detection of basic blood indicators. The mice were killed by cervical dislocation, and their thymus and spleen were removed by sterile dissection, and the organ samples were collected on ice, weighed and recorded, and the blood samples and organ tissues were frozen in a -80°C refrigerator.

[0121] (3) Organ index measurement

[0122] Organ index was calculated according to the following formula: Organ index (mg / g) = organ weight (mg) / body weight (g)

[0123] (4) Determination of blood indicators

[0124] Take 50 μL of whole blood and place it in an anticoagulant tube. Shake it up and down to mix the blood and anticoagulant evenly. Use an animal automatic blood cell analyzer to detect the number of white blood cells, red blood cells and lymphocytes.

[0125] (5) Serum cytokine level determination

[0126] The IL-3, IL-6, and TNF-α contents in serum were determined. The cytokine determination was performed using Elisa kits. The enzyme labeling reagent was added to the 96-well plate according to the instructions, and then the incubation and washing steps were performed. After the color reaction, the stop solution was added to stop the reaction, and the absorbance value of each well at a wavelength of 450 nm was immediately measured, and the cytokine concentration was calculated based on the obtained standard curve.

[0127] 2. Experimental results

[0128] (1) No obvious toxic side effects

[0129] During the experimental drug intervention, no abnormal behavior was observed in the mice, the mice survived normally, and no mice died during the experiment.

[0130] (2) Organ index measurement

[0131] The spleen and thymus are important immune organs for the growth, differentiation, maturation and immune response of immune cells. Their immunoregulatory effects are closely related to the increase of organ indexes. Each organ index reflects the functional status of the immune organs to a certain extent. The results of organ index determination are shown in Table 9. After the CTX modeling, the thymus index and spleen index of mice were significantly reduced (P>0.05), indicating that the immune injury mouse modeling was successful. From the three groups of fish maw small molecule peptides I to III, it can be seen that the fish maw small molecule immune protein peptide significantly increased the spleen index and thymus index of the model group mice (P<0.05), which preliminarily showed that the fish maw small molecule protein immune peptide can alleviate cyclophosphamide-induced immune damage.

[0132] Table 9 Effects of small molecule immune protein peptides from fish maw on organ indexes of mice

[0133]

[0134] (3) Measurement of blood indicators

[0135] The results of blood index measurement are shown in Table 10. After the cyclophosphamide modeling, the white blood cell count, red blood cell count and lymphocyte count of mice were significantly reduced (P < 0.05), indicating that the immune-damaged mouse modeling was successful; compared with the model group, fish maw small molecule peptide I to III groups can significantly adjust the white blood cell count, red blood cell count and lymphocyte count in the blood of immune-damaged mice (P < 0.05), among which the lymphocyte count of mice in fish maw small molecule peptide I and III groups had no significant difference with that in the normal group (P ≥ 0.05), which shows that fish maw small molecule peptide I to III can significantly alleviate cyclophosphamide-induced immune damage and has immunoregulatory function.

[0136] Table 10 Effects of small molecule immune protein peptides from fish maw on blood indicators in mice

[0137]

[0138] (4) Analysis of mouse serum immune cytokines

[0139] When the body is stimulated by immunogens or stimulants, it will release cytokines through transcription and translation. Their release can enhance the defense ability of macrophages and is a signal molecule that plays a key role in the body's immune response. Interleukins play an important role in promoting the proliferation and differentiation of B lymphocytes. IL-3 is a type of cytokine produced by lymphocytes, which promotes the generation of megakaryocytes. It stimulates the generation and differentiation of hematopoietic progenitor cells and improves the hematopoietic microenvironment of the bone marrow, thereby completing the immune response; IL-6 is a common proinflammatory factor that plays an important role in humoral immunity. It is related to the organ damage and bone marrow suppression caused by cyclophosphamide. At the same time, it will enhance the biological activity of TNF-α and aggravate the inflammatory response. Tumor necrosis factor-α (TNF-α) is mainly produced by activated macrophages or monocytes, which can promote the proliferation of T cells and the secretion of chemokines and interleukin-6 (IL-6) in other infected sites. It is an important inflammatory response mediator in the body.

[0140] The results of mouse serum immune cytokine determination are shown in Table 11. Compared with the normal group, the IL-3, IL-6, and TNF-α of the model group mice were significantly reduced (P < 0.05); compared with the model group, the immune cytokine levels of the fish maw small molecule peptide I ~ III intervention group were significantly increased (P < 0.05), which shows that fish maw small molecule protein peptides I ~ III have a promoting effect on the secretion of cellular inflammatory factors in the blood, and can effectively alleviate the immune damage of mice caused by cyclophosphamide.

[0141] Table 11 Effects of small molecule immune protein peptides from fish maw on inflammatory factors in mouse blood

[0142]

[0143] The above experiments show that the fish maw small molecule immune protein peptides I to III prepared by the method of microwave-assisted enzymatic hydrolysis combined with secondary alcohol precipitation in the present invention have a significant improvement effect on the spleen index and thymus index of cyclophosphamide-induced immunosuppressive mice (P < 0.05), can significantly adjust the number of white blood cells, red blood cells and lymphocytes in the blood of immune-damaged mice (P < 0.05), and have a significant promoting effect on the secretion of cellular inflammatory factors (IL-3, IL-6, TNF-α) in the blood (P < 0.05), indicating that the fish maw small molecule immune protein peptide III can effectively alleviate the immune damage of mice caused by cyclophosphamide, has the ability to enhance immune function, and has the potential for development as a food-borne active peptide.

[0144] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A low molecular weight fish maw collagen peptide with high antioxidant activity, characterized in that: It consists of a series of components with a relative molecular mass of less than 2000Da, among which the content of peptides with a relative molecular mass of less than 1000Da is 91.74% to 93.67%, the content of peptides with a relative molecular mass between 70 and 500Da is 72.47% to 77.78%, the content of hydrophobic amino acids is 37.30% to 38.66%, the content of small peptide sequence GL is 0.03 to 0.65g / 100g, and the content of small peptide sequence GPA is 0.6 to 1.8g / 100g.

2. The method for preparing the low molecular weight and high antioxidant activity fish maw collagen peptide according to claim 1, characterized in that: The following steps are involved: (1) Defatting and drying fish maw; (2) Soaking, stewing and colloid milling: The fish glue is fully soaked and stewed for 15 to 30 minutes, and then the colloid mill tooth gap is set to 80 to 120 mm, and the sample is subjected to colloid milling for 20 to 40 minutes; (3) Enzymatic hydrolysis: adjust the pH value of the fish gelatin homogenate to 6-10, add protease at an enzyme-substrate ratio of 2000-5000 U / g, and then use high hydrostatic pressure treatment, ultrasound or microwave method to assist enzymatic hydrolysis at 35-65°C for 4-7 hours; (4) Inactivation of enzyme and centrifugation: Inactivate the enzyme in the enzymatic solution at 90-100°C for 15-30 min; then centrifuge at 4000-6000 r / min for 15-30 min, and finally collect the supernatant; (5) Secondary alcohol precipitation and rotary evaporation concentration: anhydrous ethanol is added to the supernatant after centrifugation to make the ethanol volume fraction in the final system reach 40-60%, then the supernatant is taken after standing and centrifugation, and the ethanol is concentrated by rotary evaporation, and then anhydrous ethanol is added again for secondary alcohol precipitation and rotary evaporation concentration; (6) Freeze-drying: The concentrated enzymatic supernatant is subjected to vacuum freeze-drying to obtain the fish bladder collagen peptide, which is the low molecular weight and high antioxidant activity fish maw collagen peptide.

3. The preparation method according to claim 2, characterized in that: The fish maw in step (1) is derived from any one or more of Lateolabrax japonicus, Largemouth bass, Nile perch, River bass and Greater bream.

4. The preparation method according to claim 2, characterized in that: The protease in step (3) is selected from any one or more of papain, flavor protease and hydrolase protease.

5. The preparation method according to claim 4, characterized in that: In step (3), when the protease is papain and the auxiliary enzymatic hydrolysis method is a high hydrostatic pressure treatment method, the pH value of the fish glue homogenate is first adjusted to 6-8, and then papain is added and mixed, followed by vacuum packaging and placing in a high hydrostatic pressure treatment device, and treating for 4-7 hours at a temperature of 40-50° C. and a hydrostatic pressure of 100-300 MPa.

6. The preparation method according to claim 4, characterized in that: In step (3), when the protease is flavor protease and the auxiliary enzymatic hydrolysis method is ultrasonic method, the pH value of the fish gelatin homogenate is first adjusted to 7-9, and then the flavor protease is added and mixed, followed by vacuum packaging and placing in an ultrasonic instrument, and treating for 4-7 hours at 35-65° C. and an ultrasonic power of 200-400 W.

7. The preparation method according to claim 4, characterized in that: In step (3), when the enzyme is a hydrolyzing protease and the auxiliary enzymatic hydrolysis method is a microwave method, the pH value of the fish gelatin homogenate is first adjusted to 7-10, and then the hydrolyzing protease is added and mixed, followed by vacuum packaging and placing in a microwave reactor, and treating for 4-7 hours at 37-50° C. and a microwave power of 50-300 W.

8. The preparation method according to claim 2, characterized in that: The standing time in step (5) is 10 to 30 minutes; the centrifugal speed is 3000 to 5000 r / min, and the time is 15 to 30 minutes; the rotary evaporation concentration speed is 100 to 200 r / min, and the temperature is 30 to 50°C.

9. The preparation method according to claim 2, characterized in that: The freeze drying in step (6) is firstly quick-freezing at -80°C for 10 to 12 hours, and then freeze-drying in a freeze dryer for 48 to 72 hours.

10. Use of the low molecular weight fish maw collagen peptide with high antioxidant activity according to claim 1 or the method for preparing the low molecular weight fish maw collagen peptide with high antioxidant activity according to any one of claims 2 to 9 in the preparation of antioxidant products and / or immunomodulatory products, characterized in that: The products include but are not limited to food, health products, cosmetics and medicines.

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