Composition of collagen peptide and elastin peptide, method of producing the same and use thereof

Abstract

Disclosed is a composition of a collagen peptide and an elastin peptide, method of producing the same and use thereof. The composition of the present disclosure consists of a collagen peptide and an elastin peptide; the collagen peptide is prepared by enzymatic hydrolysis of a collagen material with pepsin or trypsin, and the elastin peptide is prepared by enzymatic hydrolysis of an elastin material with papain and/or protease Protamex. In the present disclosure, an elastin peptide and a collagen peptide with molecular weight in a specific range are prepared by specific processes, and the composition composed of the two at a suitable ratio can simultaneously and significantly increase the amount of the elastin and collagen in a damaged skin in a small usage amount, and significantly increase the content of hyaluronic acid and hydroxyproline while decrease the content of MMP3, meanwhile, inhibit skin inflammatory factors.

Claims

1. A method of producing a composition of a collagen peptide and an elastin peptide, comprising: performing enzymatic hydrolysis of the collagen material with pepsin or trypsin, passing the enzymatic hydrolysate through a 1 to 2 m filter membrane, and subjecting the filtrate to spray drying to obtain the collagen peptide; removing fats and impurities on the surface of a material containing elastin, performing enzymatic hydrolysis of the material containing elastin with papain and/or Bacillus protease, passing the enzymatic hydrolysate through a 0.45 to 1 m filter membrane, and subjecting the filtrate to spray drying to obtain the elastin peptide; and mixing the collagen peptide and the elastin peptide to obtain the composition.

2. The method according to claim 1, wherein prior to enzymatic hydrolysis of the material containing collagen, drying and pulverizing the material containing collagen and wherein the enzymatic hydrolysis step comprises adding water and pepsin or trypsin in an amount equal to 1% of the amount of the collagen material to perform enzymatic hydrolysis, passing the enzymatic hydrolysate through a 1 to 2 m filter membrane, and subjecting the filtrate to spray drying to obtain the collagen peptide; and wherein the removing fats and impurities from the surface of a material a material containing elastin is performed by soaking the material containing elastin with a NaOH solution, wherein prior to the enzymatic hydrolysis step comprises washing and mincing the material containing elastin, and adding water and adjusting pH to 7.5 to 8.5, wherein the enzymatic hydrolysis step comprises adding papain and/or Bacillus protease at an amount equal to 0.4-0.8% of the amount of the material containing elastin to perform enzymatic hydrolysis, wherein the filtering step comprises filtering the enzymatic hydrolysate by a plate and frame filter and a 0.45 to 1 m filter membrane, and subjecting the filtrate to spray drying after concentrating to obtain the elastin peptide; and mixing the collagen peptide and the elastin peptide to obtain the composition.

3. The method according to claim 2, wherein prior to the drying step, the material containing collagen is subjected to chopping; wherein the drying step comprises drying in an 80 C. oven for 12 hours, wherein after pulverizing, the powder is reconstituted by adding water in an amount 20 to 30 times the amount of the powder, heating the resulting solution to a temperature of 60 C.5 C., adding pepsin or trypsin in an amount equal to 1% of the amount of the solution to perform enzymatic hydrolysis for 4 to 8 hours, increasing the temperature to 80 C.5 C. and maintain the temperature for 0.5 to 1 hour, inactivating the enzyme, passing the enzymatic hydrolyzate through a 1 to 2 m filter membrane, maintaining the temperature at 555 C. during filtration, and subjecting the filtrate to spray drying to obtain the collagen peptide; wherein prior to the removal step, washing the material containing elastin, wherein the soaking step comprises adding a 0.5% NaOH solution and soaking the material containing elastin at 40 C. to 50 C. for 1 to 2 hours to remove fats and impurities on the surface of the material containing elastin, wherein the washing step comprises washing the material containing elastin until a neutral pH is obtained then draining and mincing the material containing elastin to obtain a crude extract of elastin, wherein the adding water method steps requires adding water in an amount 2 to 4 times the amount of the material containing elastin and adjusting pH to 7.5 to 8.5, adding papain and/or Bacillus protease at an amount equal to 0.4-0.8% of the amount of the material containing elastin to perform enzymatic hydrolysis, maintaining the temperature at 60 C. for 6 to 8 hours, increasing the temperature to 90 C. and maintaining the temperature for 0.5 to 1 hour, inactivating the enzyme, filtering the enzymatic hydrolysate by a plate and frame filter and a 0.45 to 1 m filter membrane, collecting the filtrate to obtain a solution comprising elastin peptide, and subjecting the solution to spray drying after concentrating the solution to obtain the elastin peptide; and mixing the collagen peptide and the elastin peptide to obtain the composition.

4. The method according to claim 1, wherein the mass ratio of the collagen peptide to the elastin peptide is 1.6:0.1-2.4:0.1.

Description

DETAILED DESCRIPTION

(1) The present disclosure discloses a composition of a collagen peptide and an elastin peptide, a method of producing the same and use thereof. Those skilled in the art can implement the present disclosure by learning from the contents of this application and appropriately improving the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present disclosure. The composition of the present disclosure and the method and use thereof have been described by way of examples, and those skilled in the art obviously can change the composition of the present disclosure and the preparation method and application thereof or make appropriate modifications and combinations without departing from the contents, spirit, and scope of the present disclosure, to implement and apply the techniques of the present disclosure.

(2) For the specific examples of the present disclosure involving comparative tests, all the tests are carried out under the same test environments and using the same raw materials except for the distinguishing technical features.

(3) The composition of a collagen peptide and an elastin peptide provided by the present disclosure, method of producing the same and use thereof will be further described below.

Example 1

Preparation of the Composition of the Present Disclosure

(4) Cod skin was chopped and dried in an oven at 80 C. for 12 hours, then pulverized into powders by a pulverizer, and placed in a cool and dry place for use. The cod skin powders was weighed, and water 20-30 times of the weight of the powders was added to make a solution. The solution was heated to 60 C.5 C., and pepsin 1% amount of the cod skin was added to perform an enzymatic hydrolysis for 4-8 hours. The temperature of the solution was increased to 805 C. and then maintained for 0.5-1 hour. The enzymes were inactivated, and then the enzymatic hydrolyzate was passed through a 1 m filter membrane and the temperature was maintained at 555 C. The filtrate was collected and subjected to spray drying to give a collagen peptide. The molecular weight of the collagen was determined by high performance size exclusion chromatography to be 2000-6000 Da.

(5) Bovine ligaments were washed, and 0.5% NaOH solution was added to soak the bovine ligaments at 40-50 C. for 1-2 hours to remove the fats and impurities on the surface. The bovine ligaments were washed to neutral, drained, and minced to obtain a crude extract of elastin. Water 2-4 times of the bovine ligaments was added and the pH was adjusted to 7.5-8.5. Protease Protamex (purchased from Novozymes) 0.4-0.8% amount of the bovine ligaments was added in an amount of. The temperature of the solution was maintained at 60 C. for 6-8 hours, and then increased to 90 C. and maintained for 0.5-1 hour. The enzymes were inactivated, and the enzymatic hydrolyzate was filtered by a plate and frame filter and a 0.45 m filter membrane. The filtrate was collected to give an elastin peptide solution. After concentrating the filtrate, the filtrate was subjected to powder-sprayed to give an elastin peptide. The molecular weight of the elastin was determined by high performance gel exclusion chromatography to be 300-2000 Da.

(6) The prepared collagen peptide and elastin peptide were compounded into a composition in a weight ratio of (1.6-3.2):(0.075-0.2); the ratio may preferably be (1.6-2.4):0.1, such as 1.6:0.1 (3.2 g collagen peptide+0.2 g elastin peptide) or 2.4:0.1 (4.8 g collagen peptide+0.2 g elastin peptide).

Example 2

Animal Experiment

(7) 1. Experimental Materials

(8) D-galactose: purchased from Sigma-Aldrich; Mouse Hyaluronic acid (HA) ELISA kit, Mouse hydroxyproline (Hyp) ELISA kit, Mouse Collagen Type I (Col I) ELISA kit, MMP3 (matrix metalloproteinase 3) ELISA kit, Mouse Elastin ELISA kit, IL-1 ELISA kit and IL-4 kit: purchased from Wuhan Huamei Bioengineering Co., Ltd.; GSH-PX (Glutathione peroxidase): purchased from Nanjing Jiancheng Bioengineering Institute.

(9) 2. Experimental Equipment

(10) Electronic Balance: Mettler Toledo, Model: PL303; Skin Moisture Analyzer: Huntkey Portable Elastic Moisture Skin Tester (HUNTKEY JAPAN Co., Ltd.), Model: HKJ-SK03P; UV Sterilization Lamp: Philips Black Light Purple Tube TL-D UV Sterilization Lamp (Philips Lighting Company), Model: TL-D/BLB; Centrifuge: Kylin-Bell Lab Instruments, Model: LX-200; Microplate Reader: BIO-TEK, US, Model: ELX-800; Homogenizer: SONICS MATERIALS INC, Model: VCX 130PB; EP tube, Syringe, etc.

(11) 3. Experimental Animals

(12) BALBc-nu nude mice (SPF grade, male, 3-4 weeks old), provided by the Animal Experimental Center of Sun Yat-sen University and housed in the SPF barrier animal room.

(13) 4. Experimental Methods

(14) Experimental animal groups and treatment model groups were daily injected subcutaneously with 10% D-gal 1.0 g.Math.kg.sup.1 into the nape of the neck, and were UV irradiated with an UV irradiation wavelength of 350-400 nm for 40 min.Math.d.sup.1, wherein the light source was about 40 cm vertically from the mice. The modeling was continued for 42 days. The normal control group was daily injected subcutaneously with an equal volume of normal saline into the nape of the neck and housed under normal light. During the experiment, mice were free to food and drink, and the water was changed daily. At the same time of modeling, mice in each sample group were fed with the samples of different dosages for intervention. The samples were diluted proportionally before administration to give the same volume to each group. The administration method was oral lavage feeding once a day for 42 days. The normal group and the model control group were given the same volume of distilled water with the same type of administration method. Group setting: blank group, model group, A collagen alone groups (A1: 3.2 g; A2: 6.4 g; A3: 12.8 g), B elastin peptide alone groups (B1: 0.2 g; B2: 0.4 g; B3: 0.8 g), C collagen peptide+elastin peptide groups (C1: 1.7 g (1.6 g+0.1 g); C2: 3.4 g (3.2 g+0.2 g); C3: 6.8 g (6.4 g+0.4 g));

(15) Three control groups were set up:

(16) Group D1, referred to the process of Example 1, except that the enzymes used were alkaline protease (for collagen peptide, used in an amount 1% of the raw material) and flavor protease (for elastin peptide, used in an amount 0.8% of the raw material), and the weight ratio of the collagen peptide to the elastin peptide was 1.6:0.1, i.e., 3.2 g collagen peptide+0.2 g elastin peptide;

(17) Group D2, referred to the process of Example 1, except that the pore size of the filter membrane was changed, the collagen peptide was filtered by a filter membrane with a pore diameter of 4.0 m, and the elastin peptide was filtered by a filter membrane with a pore diameter of 2.0 m, and the weight ratio of the collagen peptide to the elastin peptide was 1.6:0.1, i.e., 3.2 g collagen peptide+0.2 g elastin peptide;

(18) Group D3, referred to the process of Example 2 of patent CN106519020, and the amount of the functional peptide used for composition was 3.4 g;

(19) Type I collagen, elastin, hyaluronic acid, hydroxyproline, MMP3, IL-1, IL-4, GSH-PX in skin tissue were detected. After blood collection, the mice were sacrificed by cervical dislocation. An area of 1.5 cm1.5 cm back skin was taken. The rest of the back skin was taken for dry skin measurement of water content and skin histopathological examination. The subcutaneous tissues were cut off and weighed, rinsed with 4 C. 0.9% NaCl solution, dried with paper towels. The skin sample was chopped, and 4 C. 0.9% NaCl solution (the total volume of 0.9% NaCl solution was 9 times the weight of the skin) and an appropriate amount of protein lysis buffer were added. The skin sample was homogenized with a tissue homogenizer (on ice, 10 s each time, 30 s interval, repeating 5-6 times). The homogenate was centrifuged at 3,000 r/min at 4 C. for 15 min. An appropriate amount of supernatant was collected and subjected to tests according to the instructions of the ELISA kits to measure the activity of type I collagen, elastin, hyaluronic acid, hydroxyproline, MMP3, IL-1, IL-4, and GSH-PX in the skin tissue.

(20) 5. Statistical Analysis

(21) The experimental data were statistically processed by GraphPad Prism 6.0 biostatistics software: the measurement data were expressed as meanstandard deviation (meanSD), analyzed by variance analysis combined with Dunnett's multiple comparison method; the data were analyzed by Kruskal-Wallis rank sum test.

(22) 6. Results

(23) (1) Collagen

(24) As shown in Table 1, type I collagen content in the skin of the nude mice of the model group was significantly lower than that in the blank group (P<0.001); in A3 group using collagen peptide alone and B3 group using elastin peptide alone, type I collagen in the skin was significantly increased as compared with the model group (P<0.001), but the use amounts were relatively high; in C2 group of the composition of the present disclosure, though the use amount was a quarter of the total amount of A3 group+B3 group, type I collagen in the skin was significantly increased as compared with the model group (P<0.01); in C3 group of the composition of the present disclosure, though used in a half of the total amount of A3 group+B3 group, type I collagen in the skin was significantly increased as compared with the model group (P<0.001). The results demonstrate that the compositions of the present application possess significantly synergistic effects.

(25) In the three control groups, by adjusting the type of enzymes and the pore size of the filter membrane, both of D1 and D2 groups did not significantly increase the collagen amount; although D3 group significantly increased the collagen amount, the amount of elastin was not significantly increased.

(26) TABLE-US-00001 TABLE 1 Groups Collagen I ( IOD ) Elastin ( IOD ) Blank 5228 146*** 5044 141*** group Model 2777 63 2585 242 group A1 2855 39 2051 39 A2 3361 60 3088 432 A3 4774 167*** 3206 75 B1 2822 163 2749 209 B2 3373 144 3530 254 B3 3808 94*** 4360 315** C1 2887 116 3144 23 C2 3653 205** 3630 282** C3 4925 47*** 4877 64*** D1 3263 174 3430 214 D2 3423 251 3390 152 D3 3723 209** 3360 174 Note: *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the model group
(2) Elastin

(27) As shown in Table 1, the elastin content in the skin of the nude mice of the model group was significantly lower than that of the blank group (P<0.001); in neither of A1-3 groups using collagen peptide alone and B1-2 groups using elastin peptide alone, the elastin content in the skin of the nude mice was significantly increased as compared with the model group, especially in the A1 group using collagen peptide alone, the elastin content decreased instead; in B3 group using elastin peptide alone, the elastin in the skin was significantly increased (P<0.0001), but the use amount was relatively high, up to 0.8 g; in C3 group of the composition of the present disclosure, although the use amount was a half of the total amount of A3 group+B3 group, which was the same as the total amount of A2 group+B2 group, the elastin in the skin was significantly increased as compared with the model group (P<0.001). The results demonstrate that the compositions of the present disclosure possess significantly synergistic effects.

(28) In the three control groups, by adjusting the type of enzymes and the pore size of the filter membrane, both of D1 and D2 groups did not significantly increase the elastin amount; D3 group did not significantly increase the elastin amount either.

(29) (3) MMP3, Hyaluronic Acid, Hydroxyproline

(30) As shown in Table 2, as compared with the blank group, the contents of MMP3, hyaluronic acid and hydroxyproline in the skin of the nude mice of the model group were significantly different (P<0.001); in A1-3 groups using collagen peptide alone and B1-3 groups using elastin peptide alone, the contents of MMP3, hyaluronic acid and hydroxyproline in the skin of the nude mice were significantly changed as compared with the model group (P<0.05; P<0.01), but the use amount was relatively high; in C2 group of the composition of the present disclosure, though the use amount was a quarter of the total amount of A3 group+B3 group, the contents of MMP3, hyaluronic acid and hydroxyproline were significantly changed in the skin of the nude mice as compared with the model group (P<0.05); in C3 group of the composition of the present disclosure, though the use amount was a half of the total amount of A3 group+B3 group, the contents of MMP3, hyaluronic acid and hydroxyproline in the skin of the nude mice were significantly changed as compared with the model group (P<0.01; P<0.001). The results demonstrate that the compositions of the present disclosure possess significantly synergistic effects.

(31) Although D1-D3 groups significantly increased the amount of hyaluronic acid, they did not achieve the intended purpose of increasing the amount of hydroxyproline and decreasing the amount of MMP3.

(32) TABLE-US-00002 TABLE 2 Results of ELISA kit detection MMP3 Hyaluronic acid Hydroxyproline Groups (ng/mg) (pg/mg) (ng/mg) Blank 121.51 21.45*** 232.09 11.31*** 28.13 7.16** group Model 265.50 36.21 121.23 19.51 15.22 7.36 group A1 251.71 32.23 132.34 11.32 14.65 1.23 A2 240.50 55.67 161.00 10.81* 14.25 4.23 A3 196.25 22.11** 189.20 13.11** 16.81 2.23* B1 271.73 15.15 129.14 16.31 15.21 0.22 B2 231.50 12.65 145.21 15.23* 15.28 4.99 B3 190.33 18.21** 179.11 12.95** 17.19 1.54* C1 241.78 11.26 121.12 19.52 16.11 1.02 C2 221.32 12.55* 169.01 11.63* 16.99 3.90* C3 161.12 21.66*** 190.94 20.13** 19.16 0.54** D1 240.50 55.67 161.00 10.81* 14.25 4.23 D2 231.50 12.65 145.21 15.23* 15.28 4.45 D3 240.50 55.67 162.00 10.81* 15.25 3.23 Note: *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the model group;
(4) Skin Inflammatory Factors

(33) As shown in Table 3, as compared with the blank group, contents of inflammatory factor IL-1 and IL-4 in the skin of the nude mice of the model group were significantly increased, while content of GSH-Px was significantly decreased; although in A1-3 groups using collagen peptide alone and B1-3 groups using elastin peptide alone, one or more of the above indicators were significantly increased, the composition of the present disclosure also has a significant improvement in its one-quarter or one-half amount. The results demonstrate that the compositions of the present disclosure possess significantly synergistic effects.

(34) D1-D3 groups can only improve the content of IL-1 inflammatory factor, but not for the two other indexes, which was obviously inferior to the effect of improving the various inflammatory factors indexes by the present disclosure.

(35) TABLE-US-00003 TABLE 3 Results of ELISA kit detection Groups IL-1 (pg/ml) IL-4 (pg/ml) GSH-Px (ng/L) Blank 94.67 2.45*** 255.02 9.31*** 265.93 2.11*** group Model 130.41 3.41 287.35 3.11 194.46 2.17 group A1 129.28 3.29* 288.60 6.14 193.99 1.98 A2 128.38 5.17* 279.71 11.21 198.88 4.12* A3 104.94 1.23*** 256.46 3.27** 198.68 2.34 B1 128.81 3.34* 284.48 6.26 195.04 1.45 B2 117.80 2.43*** 282.65 5.21 194.71 2.67 B3 96.97 2.43*** 276.29 4.11** 199.74 2.21* C1 129.98 1.18* 283.96 2.26 197.40 3.11* C2 129.67 3.15* 287.29 9.11 197.46 4.09* C3 114.15 2.23*** 279.98 7.25** 196.68 1.34* D1 129.28 3.29* 281.60 4.42 192.93 1.87 D2 129.28 3.29* 282.60 5.44 194.99 1.58 D3 128.97 2.45* 283.40 4.12 196.44 1.68 Note: *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the model group

(36) The above are merely preferred embodiments of the present disclosure. It should be noted that one of ordinary skill in the art can also make several improvements and refinements without departing from the principles of the present disclosure. These improvements and refinements should also be regarded as the scope of protection of the present disclosure.