A low-molecular-weight fish maw collagen peptide with high antioxidant activity, its preparation method and application

Abstract

The present invention discloses a low-molecular-weight, high-antioxidant-activity fish maw collagen peptide, its preparation method, and application, belonging to the technical field of functional product development. The present invention prepares a fish maw collagen peptide by using ultrasound, microwave, or high hydrostatic pressure-assisted enzymatic hydrolysis, wherein the peptide content with a relative molecular mass between 70 and 500 Da is 72.47% to 77.78%, the hydrophobic amino acid content is 37.30% to 38.66%, the GL content is 0.03 to 0.65 g / 100 g, and the GPA content is 0.6 to 1.8 g / 100. It has strong antioxidant activity and has the effect of enhancing immunity. In addition, it has the advantages of a simple process flow, low cost, mild reaction conditions, and environmental friendliness. The present invention can provide solid theoretical support for improving the economic value of fish by-products, increasing the utilization rate of fish maw collagen products, rationally utilizing resources, and developing new health care and skin care products.

Description

Technical Field

[0001] The present 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 typically composed of 2 to 10 amino acids and exhibit excellent bioactivity and absorbability due to their small molecular size. Collagen peptides have multiple functions, including antioxidant and immune-enhancing properties. They possess strong activity and high safety, making them widely applicable in food, cosmetics, and pharmaceutical preparations, and have become a research focus. Studies have shown that the bioactivity of collagen peptides is determined by their molecular weight and amino acid composition. Lower molecular weight peptides are more likely to approach free radicals, thereby inhibiting their formation and achieving an antioxidant effect. Amino acid composition also significantly influences the bioactivity of collagen peptides. A high content of hydrophobic amino acids in the sequence indicates strong antioxidant activity, while acidic and basic amino acids can exhibit high antioxidant activity by chelating metal ions. Immunomodulatory function is a key active function of active peptides. By extracting immunomodulatory peptides from natural food ingredients, they can be made into health supplements or nutritional products to enhance human immunity. In recent years, there has been extensive research on the immunomodulatory effects of bioactive peptides extracted from marine organisms.

[0003] Fish maw, one of my country's eight immortals of the sea, is made by splitting fresh fish maw, removing blood vessels and the mucous membrane, cleaning it, flattening it, and then drying it. Alternatively, the maw is soaked in a certain concentration of alum water to remove the mucous membrane, cut into slices, and then dehydrated. Fish maw is rich in collagen and several essential amino acids. However, collagen's unique triple helix structure is very stable, making it difficult to break down even with short heating times or at normal temperatures, making it difficult for the body to absorb. Fish maw collagen peptides are made by enzymatically breaking down large fish maw collagen molecules into smaller peptide chains, making them easier for the body to absorb and utilize. Furthermore, fish maw collagen peptides are typically derived from byproducts of deep-sea fish. They are abundant and free from industrial pollution. Compared to collagen from traditional terrestrial animals such as cattle and pigs, they offer greater biocompatibility, lower immunogenicity, and lower disease transmission risk, and are not subject to religious restrictions.

[0004] Although fish maw collagen peptides have good safety and biocompatibility, they are usually too large to be used efficiently. In addition, 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 include:

[0005] CN103992385B discloses a method for preparing antioxidant collagen peptides 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 a Na2HPO4-NaH2PO4 buffer solution; (2) adjusting the pH value, and keeping it 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 the fraction with a molecular weight less than 3 kDa by ultrafiltration; (7) performing ion exchange and gel column chromatography to collect the fraction with the highest superoxide anion free radical scavenging activity; and (8) performing RP-HPLC purification.

[0006] Patent CN110577975B discloses a method for extracting and preparing fish maw collagen oligopeptides, which includes the following steps: (1) cleaning; (2) pretreatment with tannic acid; (3) enzymatic hydrolysis; (4) membrane filtration; (5) nanofiltration; (6) vacuum falling film concentration; (7) sterilizing 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 the filtered components using Sephedax G-25 molecular sieve chromatography, and purification of the most active component using a reverse phase chromatography C18 column; (6) 60 Co irradiation to inactivate endotoxins; (7) sterilization; (8) packaging.

[0008] However, the methods for preparing fish maw collagen peptides using the above-mentioned existing methods have some simple steps, which only rely on 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, this field is in urgent need of a small molecule immune protein peptide from fish maw with low molecular weight, high antioxidant activity, strong immune activity, simple preparation process, low cost, mild reaction conditions, and environmental friendliness, as well as a preparation method thereof. Summary of the Invention

[0010] To address the above technical problems, the present invention provides a low-molecular-weight, high-antioxidant-activity fish maw collagen peptide, its preparation method, and application. The low-molecular-weight, high-antioxidant-activity fish maw collagen peptide is composed of a series of components with a relative molecular mass of less than 2000 Da, of which the content of peptides with a relative molecular mass of less than 1000 Da is 91.74% to 93.67%, the content of peptides with a relative molecular mass between 70 and 500 Da is 72.47% to 77.78%, and the content of hydrophobic amino acids is 37.30% to 38.66%. Mass spectrometry analysis identified two small peptide sequences with high matching degrees, namely GL and GPA. Quantitative results showed that the content of GL was 0.03 to 0.65 g / 100 g, and the content of GPA was 0.6 to 1.8 g / 100 g. This fish maw collagen peptide has strong antioxidant activity, with IC50 values for scavenging DPPH free radicals ranging from 4.14 to 6.25 mmol / L and ABTS free radicals ranging from 7.22 to 10.36 mmol / L. This fish maw collagen peptide also has immune-enhancing properties, enhancing the function of immune organs such as the spleen and thymus, improving the capacity of immune cells such as white blood cells and lymphocytes, and promoting the activity of immune cytokines such as IL-3 and IL-6.

[0011] In summary, the collagen peptides prepared using the method of the present invention have low molecular weight, high antioxidant activity, and strong immune activity. They also have the advantages of a simple process, low cost, mild reaction, and environmental friendliness. They can be used in the preparation of antioxidant or immune-enhancing products in the fields of food, health products, cosmetics, or pharmaceuticals. The present invention provides solid theoretical support for increasing the economic value of fish by-products, improving the utilization rate of fish maw collagen products, rationally utilizing resources, and developing new health 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] A second object of the present invention is to provide a method for preparing the low-molecular-weight fish maw collagen peptide with high antioxidant activity, the method comprising the following steps:

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

[0016] (2) Soaking, stewing and colloid milling: The fish glue was fully soaked and stewed for 15 to 30 minutes, and then the colloid milling teeth gap was set to 80 to 120 mm, and the sample was 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 enzymes and centrifugation: Inactivate the enzymes in the enzymatic solution at 90-100°C for 15-30 min; then centrifuge at 4000-6000 rpm for 15-30 min, and collect the supernatant.

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

[0020] (6) Freeze-drying: The fish maw collagen peptide obtained by vacuum freeze-drying the concentrated enzymatic hydrolysis supernatant is the low-molecular-weight fish maw collagen peptide with high antioxidant activity.

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

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

[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 100-300 MPa.

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

[0025] Furthermore, in step (3), when the enzyme is a hydrolytic 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 hydrolytic protease is added and mixed, followed by vacuum packaging and placing in a microwave reactor, and treating at 37-50° C. and a microwave power of 50-300 W for 4-7 hours.

[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 first 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 fish maw collagen peptide with high antioxidant activity or the method for preparing the low molecular weight fish maw collagen peptide with high antioxidant activity and use it in the preparation of antioxidant products and / or immunomodulatory products, including but 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 peptide provided by the present invention has dual antioxidant and immune benefits, inhibiting the production of free radicals and enhancing immunity. It has broad application prospects in food, health products, cosmetics, or pharmaceuticals, promoting the further development of fish maw products and improving their nutritional and application value. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are intended to be within the scope of the present invention. Products, equipment, and the like 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 elaborated in detail below with reference to the embodiments.

[0035] Example 1 Preparation of a low molecular weight fish bladder 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 it, stew it 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. The mixture was treated at 45°C and a hydrostatic pressure of 100 MPa for 6 h.

[0039] (4) Inactivation of enzyme and centrifugation: The enzymatic hydrolysate obtained in step (3) was inactivated at 100°C for 20 min, cooled to room temperature, and 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) was divided into glass plates, with each plate containing about 25 to 30 mL. After quick freezing at -80°C for 12 hours, the plate was freeze-dried in a freeze dryer for 48 hours to obtain fish maw collagen peptide freeze-dried powder;

[0042] (7) Determination of the molecular weight distribution of fish maw collagen peptide I: The molecular weight distribution of fish maw collagen peptide I was determined using high performance liquid chromatography. The freeze-dried fish maw collagen peptide I 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 injected into the sample. 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 fish maw small molecule immune protein peptide I

[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 of this molecular weight peptide reaches 72.47%.

[0046] (8) Amino acid composition determination of fish maw collagen peptide I: The sample was treated with acid hydrolysis to obtain a clear and transparent hydrolyzate. The hydrolyzate was filtered through a 0.22 μm water filter membrane and 400 μL was added to a sample injection bottle. The amino acid composition of the fish maw collagen peptide was analyzed using a high-performance liquid chromatography (HPLC) instrument dedicated to hydrolysis amino acid analysis. The amino acid composition determination results 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] A high content of hydrophobic amino acids in collagen peptide sequences indicates strong antioxidant activity. The stronger the peptide's immune activity, the higher the content of acidic and basic amino acids, which can also exhibit high antioxidant activity by chelating metal ions. Certain amino acids, such as arginine and glutamate, may participate in inflammatory responses and reduce the release of inflammatory mediators. Table 2 shows that 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%. The glutamate and arginine content are 12.23% and 8.78%, respectively.

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

[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 it, stew it 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 dosage 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 hydrolysate obtained in step (3) was inactivated at 100°C for 20 min, cooled to room temperature, and 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) was divided into glass plates, with each plate containing about 25 to 30 mL. After quick freezing at -80°C for 12 hours, the plate was 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 II was determined using high performance liquid chromatography. The freeze-dried fish maw collagen peptide 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 injected into the sample at a 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 with acid hydrolysis to obtain a clear and transparent hydrolyzate. After the hydrolyzate was filtered through a 0.22 μm water filter, 400 μL was taken and added to a sample injection bottle. The amino acid composition of the fish maw small molecule protein peptide was analyzed using a high performance liquid chromatography (HPLC) specifically for 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] A high content of hydrophobic amino acids in collagen peptide sequences indicates strong antioxidant activity. The stronger the peptide's immune activity, the higher the content of acidic and basic amino acids, which can also exhibit high antioxidant activity by chelating metal ions. Certain amino acids, such as arginine and glutamate, may participate in inflammatory responses and reduce the release of inflammatory mediators. Table 4 shows that 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%. The glutamic acid and arginine content 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: Use petroleum ether to defatt the fish maw (perch) and dry it for later use.

[0069] (2) Soaking, stewing and colloid milling: Add 20 to 50 times the dry weight of fish glue to fully soak it, stew it 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 hydrolytic protease (purchased from Novozymes, product number: Alcalase 2.4 L, enzyme dosage: 3000 U / mg) was added. After mixing, the mixture was vacuum-packed in a polyethylene bag and immediately placed in a microwave reactor. The microwave power was set to 250 W at 45°C and the treatment was performed for 6 h.

[0071] (4) Inactivation of enzyme and centrifugation: The enzymatic hydrolysate obtained in step (3) was inactivated at 100°C for 20 min, cooled to room temperature, and 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) was divided into glass plates, with each plate containing about 25 to 30 mL. After quick freezing at -80°C for 12 hours, the plate was 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 using 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 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 bladder 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 of this molecular weight peptide reaches 77.78%.

[0079] (8) Amino acid composition determination of fish maw small molecule protein peptide III: 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, 400 μL was taken and added to a sample injection bottle. The amino acid composition of the fish maw small molecule protein peptide was analyzed using a high performance liquid chromatography (HPLC) specifically for 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] A high content of hydrophobic amino acids in collagen peptide sequences indicates strong antioxidant activity. The stronger the peptide's immune activity, the higher the content of acidic and basic amino acids, which can also exhibit high antioxidant activity by chelating metal ions. Certain amino acids, such as arginine and glutamate, may participate in inflammatory responses and reduce the release of inflammatory mediators. Table 6 shows that the relative percentage of hydrophobic amino acids in fish maw collagen immunoprotein peptide III is 38.66%, the acidic amino acid content is 16.95%, and the basic amino acid content is 13.28%. The glutamate and arginine content are 11.18% and 9.11%, respectively.

[0084] Summary of Examples 1 to 3

[0085] Generally speaking, the biological activity of collagen peptides is determined by their molecular weight and amino acid composition. Peptides with smaller molecular weights are more easily absorbed and more readily approach free radicals, thereby inhibiting their generation and achieving an antioxidant effect. Fish maw small molecule immune protein peptides I to III are composed of a series of components with relative molecular weights less than 2000 Da, with their relative molecular weight distribution primarily concentrated in the 70-500 Da range. The relative percentage of peptides within this molecular weight range is 72.47% to 77.78%. Among them, fish maw small molecule immune protein peptide III has the highest relative content in the 70-500 Da range, reaching 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 test measures the DPPH free radical scavenging ability and ABTS free radical scavenging ability of the sample. The half inhibitory concentration (IC50) is determined 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 for use 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 in the dark for 30 minutes, 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 instead of sample); A2 is the absorbance value of the blank control well containing only DPPH solution.

[0094] ②ABTS free radical scavenging rate

[0095] Prepare a 7 mmol / L ABTS solution and a 2.45 mmol / L potassium persulfate solution in distilled water. Mix ABTS and potassium persulfate in a 1:1 volume ratio and incubate in a dark place at 23°C for 12-16 hours to form ABTS cation radicals. Dilute the ABTS cation radical solution approximately 20-25 times with distilled water before use, so that the absorbance of the diluted solution at 734 nm is 0.70 ± 0.02. This is the ABTS cation radical working solution. Prepare sample solutions of varying concentrations (a series of dilutions) to cover the expected IC50 range. Add 100 μL of sample solution of varying concentrations and 100 μL of the ABTS cation radical working solution to each well of a 96-well plate and mix thoroughly. After mixing, incubate the 96-well plate at 30°C for 10 minutes. After completion of the reaction, immediately measure the absorbance at 734 nm. Replace the sample solution with deionized water and mix thoroughly with the ABTS cation radical working solution to serve as a blank control. The ABTS free radical scavenging rate is calculated as 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 instead of 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 at which the scavenging rate is 50%, i.e., 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 reactions that are harmful to health by taking away electrons (evolutionary process), including oxidative damage, inflammatory response and damage to the immune system. 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 antioxidant, free radical scavenging and immunity.

[0102] Example 5 Amino acid sequence analysis and peptide quantification of fish maw small molecule immune protein peptides

[0103] (1) Amino acid sequence analysis

[0104] The peptide chain 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 The chromatographic column was 75 μm × 25 cm. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The sample volume was 5.0 μL, and the elution flow rate was 300.0 nL / min. The mass spectrometer ionization mode was ESI+, and the positive spray voltage was 2.0 kV.

[0106] A fish maw protein database was established from the Uniprot database, and then database alignment was performed 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 origin, 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: As the sequence listing requirements indicate that a sequence must contain at least 4 specially defined amino acids unless it is intentionally skipped, only the amino acid sequences numbered 3-9 are listed in the sequence listing.

[0110] (2) Peptide content determination

[0111] The small peptides in fish maw collagen peptides were extracted and dissolved in water, then separated by reversed-phase liquid chromatography, detected by triple quadrupole mass spectrometry, and quantified by external standard method. The standards included: ① dipeptide GL (Gly-Leu, 98% purity, relative molecular mass 188.22 g / mol), purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; ② tripeptide GPA (Gly-Pro-Ala, 98% purity, relative molecular mass 244.26 g / mol), purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0112] Chromatographic conditions: A Waters Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) or similar column with comparable performance was used. The mobile phases were: A: 0.1% formic acid in water; B: 0.1% formic acid in methanol, gradient elution. The sample load was 1.0 μL, the elution flow rate was 0.2 mL / min, and the column temperature was 30°C. The mass spectrometer ionization mode was ESI+, the curtain gas flow was 30 psi, the spray voltage was 5500 V, the nebulizer temperature was 550°C, the nebulizer gas (GS1) was 55 psi, and the auxiliary gas (GS2) was 50 psi.

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

[0114] Tandem triple quadrupole mass spectrometry detection and external standard 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 with a controlled temperature of 23-27°C, a relative humidity of 40%-60%, and an artificial light / dark cycle of 12 hours. All mice had free access to food and water and were fed an irradiated basic feed. They were adaptively fed 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 body 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 began on the eighth day, with the CON and CTX groups receiving 200 μL of normal saline orally orally daily for 12 days; the fish maw small molecule peptide groups I to III received 200 μL of fish maw small molecule peptide orally orally. The intervention began on the 17th day, with the CTX and fish maw small molecule peptide groups receiving 200 μL of cyclophosphamide intraperitoneally daily for 3 days.

[0119] (2) Tissue sample collection

[0120] On the 20th day after arrival, mice were fasted overnight with or without water. After weighing, the mice were anesthetized. Eyeballs were removed and whole blood was collected. Serum was separated from the blood in an enzyme-inactivated centrifuge tube, allowed to stand at room temperature for 3 hours, and then centrifuged at 3000 rpm for 15 minutes at 4°C. Serum was obtained from the blood in a tube containing sodium heparin and allowed to stand for 1 hour before centrifugation under the same conditions. Plasma was obtained from the blood in 50 μL. Mice were sacrificed by cervical dislocation, and the thymus and spleen were removed by aseptic dissection. Organ samples were collected on ice, weighed, and stored frozen at -80°C.

[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 the tube 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) Determination of serum cytokine levels

[0126] Serum levels of IL-3, IL-6, and TNF-α were measured. Cytokine assays were performed using Elisa kits. Enzyme-labeled reagents were added to 96-well plates according to the manufacturer's instructions, followed by incubation and washing. After color development, stop solution was added to terminate the reaction. The absorbance of each well at 450 nm was immediately measured, and cytokine concentrations were calculated based on the resulting standard curve.

[0127] 2. Experimental results

[0128] (1) No obvious toxic or 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 generation of immune responses of immune cells. Their immunoregulatory effects are closely related to the increase in 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 model was established, the thymus index and spleen index of the mice were significantly reduced (P>0.05), indicating that the immune damage mouse model was successfully established. 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), preliminarily indicating 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) Determination of blood indicators

[0135] The results of blood index determination 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 damage 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 damage 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 of 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 immune regulation function.

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

[0137]

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

[0139] When the body is stimulated by immunogens or stimuli, it releases cytokines through transcription and translation. Their release enhances the defense capabilities of macrophages and serves as a key signaling molecule in the immune response. Interleukins play a crucial role in promoting the proliferation and differentiation of B lymphocytes. IL-3, a cytokine produced by lymphocytes, promotes megakaryocyte production by stimulating the production and differentiation of hematopoietic progenitor cells and improving the bone marrow microenvironment, thereby completing the immune response. IL-6, a common proinflammatory cytokine, plays a crucial role in humoral immunity and is associated with organ damage and bone marrow suppression caused by cyclophosphamide. It also enhances the biological activity of TNF-α, exacerbating the inflammatory response. Tumor necrosis factor-α (TNF-α), primarily produced by activated macrophages or monocytes, promotes T cell proliferation and the secretion of chemokines and interleukin-6 (IL-6) at other sites of infection, making it a key mediator of inflammatory responses 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 levels 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 to III intervention group were significantly increased (P < 0.05), which shows that the fish maw small molecule protein peptide I to III has 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 fish maw small molecule immune protein peptides 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 immunocompromised mice induced by cyclophosphamide (P < 0.05), can significantly reduce the number of white blood cells, red blood cells and lymphocytes in the blood of immunocompromised 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A low molecular weight fish maw collagen peptide with high antioxidant activity, characterized in that: The invention is composed of a series of components with a relative molecular mass of less than 2000 Da, wherein the content of peptides with a relative molecular mass of less than 1000 Da is 91.74% to 93.67%, the content of peptides with a relative molecular mass between 70 and 500 Da 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 0.65 g / 100 g, and the content of small peptide sequence GPA is 0.6 to 1.8 g / 100 g. The preparation method thereof comprises the following steps: (1) defatting and drying fish maw; the fish maw is derived from sea bass, largemouth bass or river perch; (2) Soaking, stewing and colloid milling: The fish glue was fully soaked and stewed for 15-30 min, and then the colloid mill tooth gap was set to 80-120 mm, and the sample was colloid milled for 20-40 min; (3) Enzymatic hydrolysis: the pH value of the fish gelatin homogenate is adjusted to 6-10, a protease is added at an enzyme-substrate ratio of 2000-5000 U / g, and then high hydrostatic pressure treatment, ultrasound or microwave method are used to assist enzymatic hydrolysis at 35-65°C for 4-7 h; the protease is selected from papain, flavor protease or hydrolytic protease; (4) Inactivation of enzymes and centrifugation: Inactivate the enzymes in the hydrolyzate at 90-100°C for 15-30 min; then centrifuge at 4000-6000 r / min for 15-30 min, and collect the supernatant. (5) Secondary alcohol precipitation and rotary evaporation: 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. Then anhydrous ethanol is added again for secondary alcohol precipitation and rotary evaporation; (6) Freeze-drying: The fish bladder collagen peptide obtained by vacuum freeze-drying the concentrated enzymatic hydrolysis supernatant is the low molecular weight and high antioxidant activity fish maw collagen peptide.

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; the fish maw is derived from sea bass, largemouth bass or river perch; (2) Soaking, stewing and colloid milling: The fish glue was fully soaked and stewed for 15-30 min, and then the colloid mill tooth gap was set to 80-120 mm, and the sample was colloid milled for 20-40 min; (3) Enzymatic hydrolysis: the pH value of the fish gelatin homogenate is adjusted to 6-10, a protease is added at an enzyme-substrate ratio of 2000-5000 U / g, and then high hydrostatic pressure treatment, ultrasound or microwave method are used to assist enzymatic hydrolysis at 35-65°C for 4-7 h; the protease is selected from papain, flavor protease or hydrolytic protease; (4) Inactivation of enzymes and centrifugation: Inactivate the enzymes in the hydrolyzate at 90-100°C for 15-30 min; then centrifuge at 4000-6000 r / min for 15-30 min, and collect the supernatant. (5) Secondary alcohol precipitation and rotary evaporation: 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. Then anhydrous ethanol is added again for secondary alcohol precipitation and rotary evaporation; (6) Freeze-drying: The fish bladder collagen peptide obtained by vacuum freeze-drying the concentrated enzymatic hydrolysis supernatant is the low molecular weight and high antioxidant activity fish maw collagen peptide.

3. The preparation method according to claim 2, characterized in that In step (3), when the protease is papain and the auxiliary enzymatic hydrolysis method is high hydrostatic pressure treatment, the pH value of the fish glue homogenate is first adjusted to 6-8, and then papain is added and mixed. Then, the mixture is vacuum-packed and placed in a high hydrostatic pressure treatment equipment, and treated at a temperature of 40-50°C and a hydrostatic pressure of 100-300 MPa for 4-7 hours.

4. The preparation method according to claim 2, characterized in that In step (3), when the protease is flavor protease and the auxiliary enzymatic hydrolysis method is ultrasonication, the pH value of the fish gelatin homogenate is first adjusted to 7-9, and then the flavor protease is added and mixed. Then, the mixture is vacuum-packed and placed in an ultrasonic instrument for treatment at 35-65°C and an ultrasonic power of 200-400 W for 4-7 hours.

5. The preparation method according to claim 2, characterized in that In step (3), when the enzyme is a hydrolytic 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 hydrolytic protease is added and mixed, followed by vacuum packaging and placing in a microwave reactor, and treating at 37-50°C and a microwave power of 50-300 W for 4-7 h.

6. 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 speed is 100 to 200 r / min, and the temperature is 30 to 50°C.

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

8. 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 7 in the preparation of antioxidant products and / or immunomodulatory products, characterized in that: The product includes any one of food, health care products, cosmetics and medicines.

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