Giant salamander mucus hydrolysate as well as preparation method and application thereof

Abstract

The invention provides a giant salamander mucus hydrolysate as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a giant salamander mucus hydrolysate. The giant salamander mucus hydrolysate comprises at least one of the following polypeptides: peptide 1, peptide 2, peptide 3, peptide 4 and peptide 5, the amino acid sequences of the peptide 1 to the peptide 5 are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 5. The giant salamander mucus hydrolysate has good antioxidant activity and hyaluronidase inhibition activity, has certain effects on skin allergy resistance, inflammation resistance and the like, also has anti-aging related effects, and has wide application prospects in preparation of food, cosmetics and drugs with antioxidant, anti-aging and anti-allergy functions.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a giant salamander mucus hydrolyzate and a preparation method and application thereof. Background Art

[0002] The dermis of the giant salamander's skin contains glands, the main two types of glands are mucus glands and granular gland cells. The skin mucus of the giant salamander is the secretion product of the mucus glands and granular gland cells. The mucus glands secrete mucoglycoproteins that combine with water to form mucus, which covers the surface of the skin and forms the first natural barrier. When stimulated, the granular glands secrete a white liquid with a special smell, which is rich in glycoproteins and other biologically active substances.

[0003] In the prior art, the patent with the publication number CN101812116B and the invention name of giant salamander oligosaccharide peptide, preparation method and its application in cosmetics makes use of giant salamander skin mucus, and prepares giant salamander oligosaccharide peptide by the method of giant salamander mucus hydrolyzate. The giant salamander oligosaccharide peptide prepared by this method has the effects of antioxidant, antibacterial activity, anti-fatigue, anti-photoaging, immunomodulation, and wound healing, is non-toxic and more easily absorbed by the skin. However, the giant salamander oligosaccharide peptide prepared by this method still has the problem of low antioxidant biological activity and anti-allergic activity. Summary of the invention

[0004] In view of this, the present invention provides a giant salamander mucus hydrolyzate having high antioxidant activity and anti-allergic activity.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a giant salamander mucus hydrolyzate, comprising at least one of the following polypeptides: peptide 1, peptide 2, peptide 3, peptide 4 and peptide 5;

[0007] The amino acid sequence of peptide 1 is shown in SEQ ID NO: 1; the amino acid sequence of peptide 2 is shown in SEQ ID NO: 2; the amino acid sequence of peptide 3 is shown in SEQ ID NO: 3; the amino acid sequence of peptide 4 is shown in SEQ ID NO: 4; and the amino acid sequence of peptide 5 is shown in SEQ ID NO: 5.

[0008] Preferably, the molecular weight of the giant salamander mucus hydrolyzate is less than 3800Da;

[0009] The giant salamander mucus hydrolysate also includes a sugar chain component; the mass percentage of the sugar chain component is 2.8% to 3.2% of the total mass of the giant salamander mucus hydrolysate, and the sugar chain component is connected to the polypeptide via an O-linked structure.

[0010] Preferably, the volatile components of the giant salamander mucus hydrolyzate also include at least one of the following volatile components in percentage by mass: benzaldehyde 8.044%, 2-ethylhexanol 1.418%, 2-nonanone 0.296%, nonanal 1.308%, 3,5-diethyl-2-methyl-pyrazine 1.762%, 2-ethyl-3,5,6-trimethylpyrazine 1.176%, 1-nonanol 0.359%, ethyl benzoate 0.307%, ethyl octanoate 0.308%, decanal 0.686%, 3,4-dimethylbenzaldehyde 5.726%, benzothiazole 1.130%, 2,2,4-trimethyl-1,2-dihydroquinoline 0.385 %, geranylacetone 3.892%, 2,6-di(tert-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadiene-1-one 1.225%, B-serene 0.274%, Δ-cadinene 0.316%, ethyl laurate 0.370%, diethyl phthalate 0.249%, 3,4-dihydro-3,3,6,8-tetramethylnaphthalene-1(2H)-one 0.308%, 2-ethylhexyl benzoate 0.256%, octyl salicylate 0.640%, isopropyl myristate 0.606%, diisobutyl phthalate 15.747%, dibutyl phthalate 2.507% and ethyl palmitate 0.400%.

[0011] The present invention provides a method for preparing the giant salamander mucus hydrolyzate, comprising the following steps:

[0012] The giant salamander skin mucus is liquefied under the action of pepsin, and the obtained liquefied product is subjected to an enzyme-catalyzed reaction under the action of pepsin and cysteine ​​to obtain a giant salamander mucus hydrolyzate.

[0013] Preferably, during the liquefaction, the added mass of pepsin accounts for 0.2% to 0.5% of the mass of the giant salamander skin mucus;

[0014] The liquefaction temperature is 32-40° C., the liquefaction time is 4-6 hours, and the liquefaction pH is 1.5-2.

[0015] Preferably, the giant salamander skin mucus further includes a pretreatment before liquefaction, wherein the pretreatment is to mix the giant salamander skin mucus with water to form a homogenate; the volume ratio of the giant salamander skin mucus to water is 1:(3-7);

[0016] After the liquefaction, the supernatant is separated to obtain the liquefied product, and the pH value of the separated supernatant is adjusted to 6.5-7.5.

[0017] Preferably, during the enzyme catalytic reaction, the water content of the liquefied product is 40wt% to 80wt%; the added mass of pepsin accounts for 1% to 3% of the mass of the liquefied product; the added mass of cysteine ​​accounts for 1% to 5% of the mass of the liquefied product;

[0018] The temperature of the enzyme catalyzed reaction is 32-40° C., the time of the enzyme catalyzed reaction is 4-6 hours, and the pH value of the enzyme catalyzed reaction is 4-6.

[0019] The present invention provides the use of the giant salamander mucus hydrolyzate or the giant salamander mucus hydrolyzate prepared by the preparation method in preparing antioxidant and / or anti-allergic products.

[0020] Preferably, the anti-oxidation includes improving the DPPH free radical scavenging rate; the anti-allergy includes improving the hyaluronidase inhibition rate.

[0021] Preferably, the product comprises at least one of the following: food, cosmetics and medicine.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention provides a giant salamander mucus hydrolysate, comprising at least one of the following polypeptides: peptide 1, peptide 2, peptide 3, peptide 4 and peptide 5; the amino acid sequences of peptides 1 to 5 are shown in SEQ ID NO: 1 to SEQ ID NO: 5. The giant salamander mucus hydrolysate of the present invention has good antioxidant and anti-allergic activities, can relieve skin allergies and inflammation, and also has anti-aging related effects, and has broad application prospects in the preparation of foods, cosmetics and medicines with antioxidant, anti-aging and anti-allergic functions.

[0024] The present invention provides a method for preparing the giant salamander mucus hydrolysate, comprising the following steps: after the giant salamander skin mucus is liquefied under the action of pepsin, the obtained liquefied product is subjected to an enzyme-catalyzed reaction under the action of pepsin and cysteine ​​to obtain the giant salamander mucus hydrolysate. The present invention uses pepsin to first perform liquefaction treatment, and then uses pepsin and cysteine ​​to hydrolyze. The obtained giant salamander mucus hydrolysate improves the DPPH free radical scavenging rate and the hyaluronidase inhibition rate compared with the raw material, showing good antioxidant and anti-allergic properties, and reducing the content of fishy substances. In the embodiments of the present invention, the effects of pepsin, enzymatic changes and cysteine ​​changes on the giant salamander mucus hydrolysate are compared, and the results show that the antioxidant activity of the changed products is significantly reduced. In addition, the examples of the present invention show that the preparation method not only significantly enhances the antioxidant and hyaluronidase inhibition activity of the giant salamander oligosaccharide peptides prepared by the patent with publication number CN101812116B and patent name "Giant salamander oligosaccharide peptides, preparation method and application thereof in cosmetics", but also removes some fishy substances, is more easily accepted by consumers, has a small molecular weight, and is easily absorbed by the human body. The preparation method of the present invention provides a new technical path for giant salamander mucus hydrolyzate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the infrared spectrum of the giant salamander mucus hydrolyzate obtained in Example 1;

[0026] Figure 2 The mass spectrum of the molecular weight distribution of the giant salamander mucus hydrolysate in Example 1;

[0027] Figure 3 This is the MS / MS structural identification diagram of peptide 1 in Example 1;

[0028] Figure 4 This is the MS / MS structural identification diagram of peptide 2 in Example 1;

[0029] Figure 5 This is the MS / MS structural identification diagram of peptide 3 in Example 1;

[0030] Figure 6 This is the MS / MS structural identification diagram of peptide 4 in Example 1;

[0031] Figure 7 This is the MS / MS structural identification diagram of peptide 5 in Example 1. DETAILED DESCRIPTION

[0032] The invention provides a giant salamander mucus hydrolyzate, comprising at least one of the following polypeptides: peptide 1, peptide 2, peptide 3, peptide 4 and peptide 5; the amino acid sequence of the peptide 1 is shown in SEQ ID NO: 1 (LGVSSYMK); the amino acid sequence of the peptide 2 is shown in SEQ ID NO: 2 (TKSKDLNN); the amino acid sequence of the peptide 3 is shown in SEQ ID NO: 3 (PASKLTQA); the amino acid sequence of the peptide 4 is shown in SEQ ID NO: 4 (ITGTLYFT); and the amino acid sequence of the peptide 5 is shown in SEQ ID NO: 5 (LSSDRSQG).

[0033] In the present invention, the hydrolyzate of giant salamander mucus preferably contains amino acids in the following molar percentages: aspartic acid and asparagine 6.08±0.12%, glutamic acid and glutamine 7.63±0.08%, serine 5.93±0.05%, glycine 17.00±1.54%, histidine 2.30±0.09%, arginine 3.16±0.03%, threonine 7.26±0.11%, alanine 9.27±0.34%, proline 6.65±0.32%, tyrosine 3.28±0.12%, valine 6.84±0.14%, methionine 0.96±0.09%, isoleucine 6.36±0.45%, leucine 6.34±0.22%, phenylalanine 3.61±0.07%, and lysine 7.32±0.16%.

[0034] In the present invention, the giant salamander mucus hydrolysate preferably has a molecular weight of less than 3800Da, more preferably less than 3800m / z. The giant salamander mucus hydrolysate preferably also includes a sugar chain component; the mass percentage of the sugar chain component is preferably 2.8% to 3.2% of the total mass of the giant salamander mucus hydrolysate, and the sugar chain component is connected to the polypeptide through an O-linked structure. The structure of the giant salamander mucus hydrolysate contains at least one of the following structures: a peptide bond, an imidazole ring, and a hydroxyl group on the sugar chain. In one embodiment of the present invention, the results of Fourier transform infrared spectroscopy analysis show that the giant salamander mucus hydrolysate has characteristic peaks of amide functional groups, imidazole functional groups, and hydroxyl groups, indicating that the peptide structure of the giant salamander mucus hydrolysate contains peptide bonds, imidazole rings, and hydroxyl groups on the sugar chain. In the present invention, the relative humidity of the giant salamander mucus hydrolysate is preferably not more than 10%, and the solubility is preferably 1g / 30mL water.

[0035] In the present invention, the volatile components of the giant salamander mucus hydrolyzate also include at least one of the following volatile components in percentage by mass: benzaldehyde 8.044%, 2-ethylhexanol 1.418%, 2-nonanone 0.296%, nonanal 1.308%, 3,5-diethyl-2-methyl-pyrazine 1.762%, 2-ethyl-3,5,6-trimethylpyrazine 1.176%, 1-nonanol 0.359%, ethyl benzoate 0.307%, ethyl octanoate 0.308%, decanal 0.686%, 3,4-dimethylbenzaldehyde 5.726%, benzothiazole 1.130%, 2,2,4-trimethyl-1,2-dihydroquinoline 0.38 5%, geranylacetone 3.892%, 2,6-di(tert-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadiene-1-one 1.225%, B-serene 0.274%, Δ-cadinene 0.316%, ethyl laurate 0.370%, diethyl phthalate 0.249%, 3,4-dihydro-3,3,6,8-tetramethylnaphthalene-1(2H)-one 0.308%, 2-ethylhexyl benzoate 0.256%, octyl salicylate 0.640%, isopropyl myristate 0.606%, diisobutyl phthalate 15.747%, dibutyl phthalate 2.507%, ethyl palmitate 0.400%.

[0036] The antioxidative activity of the giant salamander mucus hydrolysate of the present invention can reach 94.69%, and the inhibition of hyaluronidase activity can reach 77.24%. It has certain effects on skin soothing, anti-allergy and anti-inflammatory, and has anti-aging related effects. At the same time, the molecular weight is small, it is easily absorbed by the human body, and it has a wide range of application prospects in the preparation of foods, cosmetics and medicines with antioxidant, anti-aging and anti-allergic functions. In addition, the content of fishy substances in the giant salamander mucus hydrolysate of the present invention is low. Compared with the giant salamander oligosaccharide peptide in the prior art, the composition structure of the volatile components of the giant salamander mucus hydrolysate of the present invention is changed, especially the content of volatile substances with strong irritating odors such as nonanal and decanal is significantly reduced, thereby reducing its fishy smell and making it more acceptable.

[0037] The present invention provides a method for preparing the giant salamander mucus hydrolyzate, comprising the following steps:

[0038] The giant salamander skin mucus is liquefied under the action of pepsin, and the obtained liquefied product is subjected to an enzyme-catalyzed reaction under the action of pepsin and cysteine ​​to obtain a giant salamander mucus hydrolyzate.

[0039] In the present invention, the skin mucus of giant salamander further includes pretreatment before liquefaction, and the pretreatment is to mix the skin mucus of giant salamander with water to form a homogenate; the volume ratio of the skin mucus of giant salamander to water is 1:(3-7), more preferably 1:(4-6), and most preferably 1:5; during the liquefaction, the mass of pepsin preferably accounts for 0.2%-0.5% of the mass of the skin mucus of giant salamander, more preferably 0.3%-0.4%, and most preferably 0.35%. The enzyme activity of the pepsin is preferably 1.2×10 6 U / g~1.8×10 6 U / g, more preferably 1.4×10 6 U / g~1.6×10 6 U / g, the most preferred is 1.5×10 6 U / g. In an embodiment of the present invention, pepsin CAS No.: 9001-75-6, purchased from Shanghai McLean Biochemical Technology Co., Ltd. The liquefaction temperature is preferably 32-40°C, more preferably 35-38°C, and most preferably 37°C. The liquefaction time is preferably 4-6h, more preferably 4.5-5.5h, and most preferably 5h. The liquefaction pH is preferably 1.5-2, more preferably 1.6-1.8, and most preferably 1.75. The liquefaction can increase the yield of antioxidant substances in the skin mucus of giant salamander and reduce the fishy smell of the hydrolyzate of giant salamander mucus.

[0040] In the present invention, after the liquefaction, the supernatant is preferably separated to obtain the liquefied product, and the pH value of the separated supernatant is preferably adjusted to 6.5-7.5, more preferably 7. The method for separating the supernatant includes centrifugation; the speed of the centrifugation is preferably 8000-10000 rpm; the time of the centrifugation is preferably 15-40 min, more preferably 20-35 min, further preferably 25-30 min, and most preferably 28 min. The method for separating the supernatant can increase the content of the effective ingredient in the liquefied product.

[0041] In the present invention, during the enzyme catalytic reaction, the water content of the liquefied product is preferably 40 wt% to 80 wt%, more preferably 50% to 70%, and most preferably 60%. The added mass of pepsin preferably accounts for 1% to 3% of the mass of the liquefied product, more preferably 1.5% to 2.5%, and most preferably 2%. The enzymatic activity of pepsin is preferably 1.2×10 6 U / g~1.8×10 6 U / g, more preferably 1.4×10 6 U / g~1.6×10 6 U / g, the most preferred is 1.5×10 6 U / g. The added mass of cysteine ​​accounts for 1% to 5% of the mass of the liquefied product, more preferably 2% to 4%, and most preferably 3%. The cysteine ​​can adjust the redox state of the reaction system, change the characteristics of the reaction product, improve the antioxidant activity and anti-allergic activity of the giant salamander mucus hydrolysate, and reduce the fishy smell. The temperature of the enzyme-catalyzed reaction is 32 to 40°C, more preferably 35 to 38°C, and most preferably 37°C. The time of the enzyme-catalyzed reaction is preferably 4 to 6h, more preferably 4.5 to 5.5h, and most preferably 5h. The pH of the enzyme-catalyzed reaction is 4 to 6, more preferably 4.5 to 5.5, and most preferably 5. The enzyme-catalyzed reaction can increase the yield of antioxidant substances in the giant salamander skin mucus and reduce the fishy smell of the giant salamander mucus hydrolysate.

[0042] The present invention adopts pepsin to liquefy first and then enzymatically react and add cysteine ​​during the enzyme catalytic reaction to improve the antioxidant and hyaluronidase inhibition rate of giant salamander mucus hydrolysate, and reduce the content of fishy substances. The embodiment in the present invention compares the effects of pepsin, enzymatic hydrolysis times and cysteine ​​on giant salamander mucus hydrolysate respectively. The results show that when pepsin is replaced with alkaline protease, trypsin, papain or neutral protease during the enzyme catalytic reaction, the antioxidant activity of the product is significantly reduced; when the number of enzymatic hydrolysis is once, the antioxidant activity of the product is significantly reduced; when cysteine ​​is not added or cysteine ​​is replaced with other types of amino acids, the antioxidant activity of the product will also be significantly reduced. It can be seen that the preparation method of the present invention is conducive to improving the antioxidant and hyaluronidase inhibition rate of the enzyme catalytic product. In addition, the preparation method of the present invention has significantly improved antioxidant properties compared to giant salamander oligosaccharide peptide (publication number CN101812116B, patent name giant salamander oligosaccharide peptide, preparation method and its application in cosmetics), low content of fishy substances, more easily accepted by consumers, and small molecular weight, easy to be absorbed by the human body. The composition structure of the volatile components of the giant salamander mucus hydrolyzate of the present invention has changed, especially the content of volatile substances with strong irritating odors such as nonanal and decanal is significantly reduced compared to the prior art, making its fishy smell smaller and more acceptable.

[0043] In the present invention, the enzymatic reaction preferably includes separating a supernatant from the enzymatic product, and the method for separating the supernatant is preferably the same as the method for separating the supernatant after liquefaction, which will not be described in detail. After obtaining the supernatant, it is preferably subjected to vacuum freeze drying to obtain a solid giant salamander mucus hydrolyzate.

[0044] The present invention provides the use of the giant salamander mucus hydrolyzate or the giant salamander mucus hydrolyzate prepared by the preparation method in preparing antioxidant and / or anti-allergic products.

[0045] In the present invention, the antioxidant effect preferably includes improving the DPPH free radical scavenging rate; the anti-allergic effect includes improving the hyaluronidase inhibition rate in vitro.

[0046] In the present invention, the working concentration of the giant salamander mucus hydrolyzate is preferably above 0.1 mg / mL, more preferably above 0.2 mg / mL, further preferably above 0.4 mg / mL, further preferably 0.6-10 mg / mL, and most preferably 0.8-1 mg / mL.

[0047] In the present invention, the product preferably includes at least one of the following: food, cosmetics and medicine.

[0048] To further illustrate the present invention, a giant salamander mucus hydrolyzate and its preparation method and application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] A method for preparing a giant salamander mucus hydrolyzate comprises the following steps:

[0051] a. The collected giant salamander skin mucus is mixed with water in a homogenizer at a material-liquid volume ratio of 1:5 to prepare a homogenized giant salamander skin mucus homogenate;

[0052] b. Adjust the pH value of the giant salamander skin mucus homogenate to 2.0, add 0.5% pepsin (1.5×10 6 U / g), and enzymatically liquefied in a 37°C water bath for 5.5 hours. The pH value of the liquefied giant salamander skin mucus was adjusted to 7.0, and centrifuged at 4°C and 9000 rpm for 30 minutes. The supernatant was taken and placed in a refrigerator for 48 hours, and then vacuum-freeze-dried to obtain a solid liquefied product;

[0053] c. Prepare a 40% mass fraction liquefied product solution, adjust the pH to 5.0, add 1.5% mass fraction pepsin (1.5×10 6 U / g) and 3% cysteine ​​by mass, and the reaction was catalyzed by enzyme in a water bath at 37°C. After 6 hours of reaction, the enzyme was inactivated at 100°C for 10 minutes, cooled to room temperature in a water bath, and centrifuged at 9000 rpm for 20 minutes; the supernatant was collected and vacuum freeze-dried to obtain the hydrolyzate of giant salamander mucus.

[0054] Example 2

[0055] Structural and Compositional Analysis of the Hydrolyzate of Giant Salamander Mucus

[0056] 1. Determination of amino acid composition

[0057] The amino acid content of the hydrolyzate of giant salamander mucus prepared in Example 1 is as follows: aspartic acid and asparagine 6.08±0.12%, glutamic acid and glutamine 7.63±0.08%, serine 5.93±0.05%, glycine 17.00±1.54%, histidine 2.30±0.09%, arginine 3.16±0.03%, threonine 7.26±0.11%, alanine 9.27±0.34%, proline The amino acid content is 6.65±0.32%, tyrosine 3.28±0.12%, valine 6.84±0.14%, methionine 0.96±0.09%, isoleucine 6.36±0.45%, leucine 6.34±0.22%, phenylalanine 3.61±0.07%, and lysine 7.32±0.16%; the sugar content is 3±0.2% of the total mass, the relative humidity is not more than 10%, and the solubility is 1g / 30mL water.

[0058] 2. Fourier Transform Infrared Spectroscopy Analysis

[0059] The results of Fourier transform infrared spectroscopy analysis of the giant salamander mucus hydrolyzate prepared in Example 1 are as follows: Figure 1 .from Figure 1 It can be observed that 3300cm -1 、1658cm -1 and 1546cm -1 The characteristic peaks of 3075cm show the presence of amide functional groups in the structure; -1 The characteristic peaks of 2964cm show that there are imidazole functional groups in the structure; -1 、1402cm -1 The characteristic peaks of show that there are hydroxyl groups in the structure. The Fourier transform infrared spectrum reflects the peptide bonds, imidazole rings and hydroxyl groups on the sugar chain in the peptide structure of the giant salamander mucus hydrolysate prepared in Example 1.

[0060] 3. Mass Spectrometry Analysis

[0061] The structure of the hydrolyzate of the giant salamander mucus prepared in Example 1 was identified by mass spectrometry. Figure 3 to Figure 7 As shown, the amino acid sequence obtained contains the following 5 polypeptides: Figure 3 to Figure 7 It was found that the amino acid sequences of peptides 1 to 5 are as follows, respectively.

[0062] Peptide 1: LGVSSYMK (SEQ ID NO: 1);

[0063] Peptide 2: TKSKDLNN (SEQ ID NO: 2);

[0064] Peptide 3: PASKLTQA (SEQ ID NO: 3);

[0065] Peptide 4: ITGTLYFT (SEQ ID NO: 4);

[0066] Peptide 5: LSSDRSQG (SEQ ID NO: 5).

[0067] The molecular weight distribution of the giant salamander mucus hydrolyzate of the present invention was measured by mass spectrometry, and the measurement results are as follows: Figure 2 As shown. Figure 2 It can be obtained that the molecular weight of the giant salamander mucus hydrolyzate prepared in Example 1 is mainly concentrated below 3800 m / z, and is easily absorbed and utilized by the human body.

[0068] Comparative Example 1

[0069] The method of Example 1 of the patent publication number CN101812116B, titled Giant Salamander Oligosaccharide Peptide, Preparation Method and Application in Cosmetics, was used to prepare Giant Salamander Oligosaccharide Peptide. The details are as follows:

[0070] a. Collect the giant salamander mucus and homogenize it with a homogenizer at 4°C;

[0071] b. Add 2 volumes of 0.05M phosphate buffer at pH 7 to 8 and 0.5% of the mass ratio of the homogenate to the resulting homogenate, and perform enzymatic hydrolysis for 24 hours; the complex enzyme is a mixture of marine alkaline protease, papain, pepsin and trypsin in an activity unit ratio of 3:3:2:2;

[0072] c. Centrifuge and take the supernatant, separate it using an ultrafiltration membrane separator with a molecular weight cutoff of less than 4000Da, and take the liquid that passes through the ultrafiltration membrane;

[0073] d. Pass through Sephadex LH-20 molecular sieve chromatography column (2.5×100cm) and collect OD 280 nm absorption peak;

[0074] e. The collected liquid is desalted by macroporous adsorption resin (HPD series) and decolorized by activated carbon, and then freeze-dried to obtain giant salamander oligosaccharide peptides.

[0075] Comparative Example 2

[0076] The method of Example 1 was used to prepare a solid liquefied product.

[0077] Comparative Example 3

[0078] The preparation method is the same as that of Example 1, except that cysteine ​​is not added.

[0079] Example 3

[0080] Volatile component analysis

[0081] The volatile substances in the giant salamander mucus hydrolyzate of Example 1 and the giant salamander oligosaccharide peptide of Comparative Example 1 were determined by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results are shown in Table 1.

[0082] Table 1 Determination results of relative content of volatile components (%)

[0083]

[0084]

[0085]

[0086] As shown in Table 1, the contents of benzaldehyde, 2-ethylhexanol, 2-nonanone, 3,5-diethyl-2-methyl-pyrazine, 2-ethyl-3,5,6-trimethylpyrazine, 1-nonanol, ethyl benzoate, decanal, 3,4-dimethylbenzaldehyde, benzothiazole, 2,2,4-trimethyl-1,2-dihydroquinoline, geranylacetone, 2,6-di(tert-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadiene-1-one, B-serene, Δ-cadinene, ethyl laurate, diethyl phthalate, 3,4-dihydro-3,3,6,8-tetramethylnaphthalene-1(2H)-one, 2-ethylhexyl benzoate, octyl salicylate, isopropyl myristate, diisobutyl phthalate, dibutyl phthalate, ethyl palmitate and other substances have changed. The volatile components in the giant salamander mucus hydrolyzate of the invention are changed, so that the fishy smell is reduced and the product is more easily accepted by consumers.

[0087] Example 4

[0088] Determination of antioxidant capacity

[0089] The DPPH radical scavenging rates of the products prepared in Example 1 and Comparative Examples 1 to 3 were measured respectively, and the specific methods are as follows.

[0090] Take 1g of the sample to be tested and fully dissolve it in 30mL of 95% ethanol, centrifuge it at 4000rpm for 15min, take a certain volume of supernatant, and add 95% edible ethanol to 2mL to prepare sample solutions of different concentrations.

[0091] Take 2 mL of sample solution with different concentrations and mix with 2 mL of 0.2 mmol / LDPPH-ethanol, keep it at room temperature and away from light for 30 min, adjust to zero with deionized water, measure the absorbance at 517 nm, and record it as A. i ; The absorbance measured by replacing DPPH-ethanol solution with 2 mL of 95% edible ethanol is taken as the sample background absorbance, recorded as A i ’; The absorbance measured by replacing the sample solution with 2 mL of 95% edible ethanol is taken as the blank control absorbance, recorded as A 0 . Calculate the DPPH free radical scavenging rate according to formula I:

[0092]

[0093] In formula I: A i - absorbance of the sample after scavenging free radicals; A i ’ -absorbance of sample background; A 0 -Blank control absorbance.

[0094] The results of the determination of the scavenging DPPH free radical of the giant salamander mucus hydrolysate are shown in Table 2. As can be seen from Table 2, the ability of the giant salamander mucus hydrolysate in Example 1 to scavenging DPPH free radical is much higher than that of other groups.

[0095] Table 2 DPPH free radical scavenging rate determination results (%)

[0096] Sample concentration (mg / mL) 0.2 0.4 0.6 0.8 1.0 Comparative Example 1 43.57±3.86 45.49±3.43 46.07±1.11 50.29±3.76 55.09±1.50 Example 1 74.08±1.80 84.69±4.26 92.45±0.91 94.49±0.46 94.69±0.58 Comparative Example 2 33.69±2.89 37.42±5.47 38.57±2.74 41.23±5.37 46.45±2.71 Comparative Example 3 40.13±5.61 41.43±2.85 44.35±4.68 47.83±2.76 49.56±3.27

[0097] Example 5

[0098] Determination of the inhibition rate of hyaluronidase in vitro

[0099] The in vitro inhibition rates of hyaluronidase of the giant salamander mucus hydrolyzate prepared in Example 1 and the giant salamander oligosaccharide peptide prepared in Comparative Example 1 were measured respectively, and the specific steps were as follows.

[0100] Take 50 μL of sample and 50 μL of hyaluronidase solution (500 U / mL), shake thoroughly and mix well, then place in a 37°C water bath for 20 min; after water bath, add 10 μL of 2.5 mol / L CaCl 2 The solution was shaken in a 37°C water bath for 20 minutes, and then 50 μL of potassium hyaluronate (0.5 mg / mL) was added, and the mixture was again shaken at 37°C for 40 minutes, and then at room temperature for 10 minutes; after the water bath again, 50 μL of distilled water, 10 μL of NaOH solution (5 mol / L), and 50 μL of acetylacetone solution were added, and the mixture was boiled in a water bath for 15 minutes, cooled for 5 minutes, cooled in a cold water bath for 10 minutes, and then at room temperature for 10 minutes; after the cold water bath, 100 μL of P-DAB color developer was added, shaken thoroughly, and 350 μL of anhydrous ethanol was added and shaken thoroughly to develop the color at room temperature for 30 minutes, and the OD was measured. 530 , as the absorbance of tube A;

[0101] Acetate buffer (pH 5.40.1 mol / L) was used instead of potassium hyaluronate to measure OD 530 , as the absorbance of tube B;

[0102] The sample was replaced with water and the hyaluronidase solution was replaced with acetate buffer. 530 , as the absorbance of tube C;

[0103] The sample was replaced with water, and the hyaluronidase solution and potassium hyaluronate were replaced with acetate buffer, and the absorbance of tube D was taken as the value.

[0104] The in vitro inhibition rate of hyaluronidase was determined according to Formula II. The results are shown in Table 3.

[0105] Hyaluronidase inhibition rate = [1-(CD) ÷ (AB)] × 100% Formula II

[0106] In formula II: A is the absorbance of tube A; B is the absorbance of tube B; C is the absorbance of tube C; D is the absorbance of tube D.

[0107] Table 3 Hyaluronidase in vitro inhibition rate test results

[0108]

[0109] As can be seen from Table 3, the giant salamander mucus hydrolyzate prepared in Example 1 has a strong inhibitory effect on hyaluronidase, and the hyaluronidase in vitro inhibition rate is significantly higher than that of Comparative Example 1, which indicates that the giant salamander mucus hydrolyzate of the present invention has anti-allergic and anti-inflammatory effects and has a very broad application prospect in cosmetic applications.

[0110] Comparative Examples 4 to 22

[0111] Comparative Examples 4 to 22 respectively use other types of amino acids to replace cysteine ​​in Example 1 to prepare giant salamander mucus hydrolysate, and other preparation steps are the same as Example 1. The amino acids used in Comparative Examples 4 to 22 are shown in Table 4.

[0112] The method of Example 4 was used to determine the antioxidant capacity of the giant salamander mucus hydrolysates prepared in Comparative Examples 4 to 22, wherein the sample concentration of the prepared giant salamander mucus hydrolysates was 0.2 mg / mL.

[0113] The test results are shown in Table 4. It can be seen from Table 4 that the ability of the giant salamander mucus hydrolyzate in Example 1 to scavenge DPPH free radicals is much higher than that of Comparative Examples 4-22.

[0114] Table 4 Comparative Examples 4 to 22 DPPH free radical scavenging rate determination results of giant salamander mucus hydrolyzate

[0115]

[0116]

[0117] Comparative Examples 23 to 26

[0118] Comparative Examples 23 to 26 respectively used other different types of proteases to replace the pepsin in the enzyme catalytic reaction in Example 1 to prepare the giant salamander mucus hydrolysate. The other preparation steps were the same as in Example 1. The enzyme activities of the different types of proteases were all 1.5×10 6 The proteases used in Comparative Examples 23 to 26 are shown in Table 5.

[0119] Among them, alkaline protease has a CAS number of 9014-01-1, which was purchased from Xiya Reagent, and the product website is: https: / / www.xiyashiji.com / goods-5624.html; trypsin was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., and the product website is http: / / www.macklin.cn / products / T819003; papain was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., and the product website is https: / / www.macklin.cn / products / P6321; neutral protease was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., and the product website is https: / / www.macklin.cn / products / D915910.

[0120] The method of Example 5 was used to determine the antioxidant capacity of the giant salamander mucus hydrolyzates prepared in Comparative Examples 20 to 26, wherein the sample concentration of the prepared giant salamander mucus hydrolyzates was 0.2 mg / mL.

[0121] The test results are shown in Table 5. It can be seen from Table 5 that the ability of the giant salamander mucus hydrolyzate in Example 1 to scavenge DPPH free radicals is much higher than that of Comparative Examples 23-26.

[0122] Table 5 DPPH free radical scavenging rate determination results of giant salamander mucus hydrolyzate of comparative examples 23 to 26

[0123] Group Protease DPPH free radical scavenging rate (%) Comparative Example 23 Alkaline protease 30.56 Comparative Example 24 Trypsin 34.69 Comparative Example 25 Papain 34.27 Comparative Example 26 Neutral protease 22.51

[0124] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A giant salamander mucus hydrolyzate, characterized in that: comprising at least one of the following polypeptides: peptide 1, peptide 2, peptide 3, peptide 4 and peptide 5; The amino acid sequence of peptide 1 is shown in SEQ ID NO: 1; the amino acid sequence of peptide 2 is shown in SEQ ID NO: 2; the amino acid sequence of peptide 3 is shown in SEQ ID NO: 3; the amino acid sequence of peptide 4 is shown in SEQ ID NO: 4; and the amino acid sequence of peptide 5 is shown in SEQ ID NO:

5.

2. The giant salamander mucus hydrolyzate according to claim 1, characterized in that: The molecular weight of the giant salamander mucus hydrolyzate is less than 3800Da; The giant salamander mucus hydrolysate also includes a sugar chain component; the mass percentage of the sugar chain component is 2.8% to 3.2% of the total mass of the giant salamander mucus hydrolysate, and the sugar chain component is connected to the polypeptide via an O-linked structure.

3. The giant salamander mucus hydrolyzate according to claim 1 or 2, characterized in that: The volatile components of the giant salamander mucus hydrolyzate also include at least one of the following volatile components in percentage by mass: benzaldehyde 8.044%, 2-ethylhexanol 1.418%, 2-nonanone 0.296%, nonanal 1.308%, 3,5-diethyl-2-methyl-pyrazine 1.762%, 2-ethyl-3,5,6-trimethylpyrazine 1.176%, 1-nonanol 0.359%, ethyl benzoate 0.307%, ethyl octanoate 0.308%, decanal 0.686%, 3,4-dimethylbenzaldehyde 5.726%, benzothiazole 1.130%, 2,2,4-trimethyl-1,2-dihydroquinoline 0.385%, Geranyl acetone 3.892%, 2,6-di(tert-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadiene-1-one 1.225%, B-serene 0.274%, Δ-cadinene 0.316%, ethyl laurate 0.370%, diethyl phthalate 0.249%, 3,4-dihydro-3,3,6,8-tetramethylnaphthalene-1(2H)-one 0.308%, 2-ethylhexyl benzoate 0.256%, octyl salicylate 0.640%, isopropyl myristate 0.606%, diisobutyl phthalate 15.747%, dibutyl phthalate 2.507% and ethyl palmitate 0.400%.

4. A method for preparing the giant salamander mucus hydrolyzate according to any one of claims 1 to 3, characterized in that: The steps include: The giant salamander skin mucus is liquefied under the action of pepsin, and the obtained liquefied product is subjected to an enzyme-catalyzed reaction under the action of pepsin and cysteine ​​to obtain a giant salamander mucus hydrolyzate.

5. The preparation method according to claim 4, characterized in that: During the liquefaction, the added mass of pepsin accounts for 0.2% to 0.5% of the mass of the giant salamander skin mucus; The liquefaction temperature is 32-40° C., the liquefaction time is 4-6 hours, and the liquefaction pH is 1.5-2.

6. The preparation method according to claim 4, characterized in that: The giant salamander skin mucus further includes pretreatment before liquefaction, wherein the pretreatment is mixing the giant salamander skin mucus with water to form a homogenate; The volume ratio of the giant salamander skin mucus to water is 1:(3-7); After the liquefaction, the supernatant is separated to obtain the liquefied product, and the pH value of the separated supernatant is adjusted to 6.5-7.

5.

7. The preparation method according to any one of claims 4 to 6, characterized in that: During the enzyme catalytic reaction, the water content of the liquefied product is 40wt% to 80wt%; the added mass of pepsin accounts for 1% to 3% of the mass of the liquefied product; the added mass of cysteine ​​accounts for 1% to 5% of the mass of the liquefied product; The temperature of the enzyme catalyzed reaction is 32-40° C., the time of the enzyme catalyzed reaction is 4-6 hours, and the pH value of the enzyme catalyzed reaction is 4-6.

8. Use of the giant salamander mucus hydrolyzate according to any one of claims 1 to 3 or the giant salamander mucus hydrolyzate prepared by the preparation method according to any one of claims 4 to 7 in the preparation of antioxidant and / or anti-allergic products.

9. The use according to claim 8, characterized in that: The anti-oxidation includes improving the DPPH free radical scavenging rate; the anti-allergy includes improving the hyaluronidase inhibition rate.

10. The use according to claim 8 or 9, characterized in that: The product includes at least one of the following: food, cosmetics and medicine.

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