Preparation method and application of fresh collagen peptide with anti-aging and anti-oxidation properties

Abstract

The present invention provides a preparation method and application of fresh collagen peptides with anti-aging and antioxidant properties, which relates to the field of bioengineering technology, and includes the steps of: firstly performing swelling pretreatment on the raw material, and then performing the first enzymolysis of the pretreated raw material using an acidic protease; and then performing the second enzymolysis using a composite protease to obtain fresh collagen peptides; the composite protease is a mixture of papain, aminopeptidase and trypsin. The preparation method of the present invention not only efficiently removes the fishy substances in the raw material, but also performs enzymolysis by specific enzymes and under specific enzymolysis conditions, and the acidic protease and the composite protease interact with each other, showing a synergistic enzymolysis effect, shortening the preparation time of the collagen peptide, and the fresh collagen peptide has high activity, and the antioxidant and anti-aging effects are significantly improved, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering technology, and specifically relates to a preparation method and application of anti-aging and anti-oxidation fresh collagen peptide. Background Art

[0002] Collagen peptides are enzymatic hydrolysis products of collagen and are absorbed in the form of small peptides and free amino acids. Collagen peptides have the characteristics of low molecular weight, easy absorption, low viscosity, etc., and also have the effects of immune regulation, anti-oxidation, anti-aging, anti-thrombosis, lowering blood pressure, and lowering cholesterol. Therefore, collagen peptides have been widely used in the fields of medicine, food, daily chemical industry, etc. At present, the main production methods of collagen peptides are chemical method, enzymatic method and microbial fermentation method.

[0003] Enzymatic hydrolysis can hydrolyze collagen into many small fragments of low molecular weight collagen peptides, making collagen peptides have a higher probability of passing through the intestinal barrier, increasing biological activity and reducing antigenicity. Enzymatic hydrolysis has the advantages of mild action, high efficiency and safety, and is widely used in the production and processing of collagen peptides. However, the current enzymatic hydrolysis methods still have problems such as incomplete enzymatic hydrolysis, low content of amino acids such as glycine and proline in collagen peptides, dark color, and low extraction rate; and because collagen peptide products extracted from aquatic products are very likely to produce unpleasant fishy smells, the application of collagen peptides is limited.

[0004] Chinese invention patent CN102533913A discloses a method for preparing antioxidant active peptides by co-immobilizing dual enzymes to hydrolyze fish scale collagen, including the steps of: cross-linking co-immobilizing dual enzymes, fish scale pretreatment, co-immobilized enzyme directional hydrolysis of fish scale protein, decolorization and deodorization, ultrafiltration separation of fish scale polypeptides, and finally vacuum concentration and freeze drying. The enzyme used is a composite protease composed of any two enzymes of papain, trypsin, neutral protease, Flavourzyme flavor protease, and Alcalase alkaline protease according to an activity ratio of 1:1, the enzymolysis time is 2-6h, and the buffer is a phosphate buffer. The prepared antioxidant active peptides can be used as natural antioxidants in food antioxidant preservation, and the enzyme can be used multiple times; the method provides a new way for the effective utilization of freshwater fish waste; the prepared collagen peptide products can be used in medicines, food and cosmetics. The method only uses activated carbon for decolorization and deodorization to remove odors, but the adsorption of activated carbon to odors is limited, and the loss rate of collagen peptides is high.

[0005] In addition, Chinese invention patent CN113151387A discloses a cod skin collagen peptide with antioxidant and immune-enhancing functions and a preparation method thereof. The method comprises the following steps: using cod skin as raw material, subjecting the pretreated raw material to two-step enzymolysis to obtain an enzymolysate; in the two-step enzymolysis, the enzyme used in the first step of enzymolysis is a composite protease, the composite protease is trypsin and neutral protease, and the enzyme used in the second step of enzymolysis is flavor protease. The collagen peptide prepared by this method has a high content of small molecule peptides and has excellent antioxidant and immune-enhancing functions. However, it still does not solve the problem of bad odor.

[0006] Gao Jianhua et al. (Amino acid analysis of fish scale collagen hydrolysis products by different proteases. Food Science and Technology, 2009, 34(05):117-119.) used different enzymes to hydrolyze fish scales and analyzed the obtained protein peptides and amino acid products. The yields of peptides and amino acids in the hydrolysis products of different enzymes were different, thus showing different properties. However, they did not conduct a comparative study on the relevant properties of the products prepared by the relevant single enzyme activity complex enzymes.

[0007] Although current research has documented a large number of methods for enzymatic hydrolysis of aquatic products to produce collagen peptides, it has not conducted in-depth research into the effects of enzyme types, ratios, enzymatic hydrolysis processes, buffers, etc. on the odor, antioxidant and anti-aging effects of collagen peptides during the enzymatic hydrolysis process. Moreover, the current enzymatic hydrolysis method takes a long time from raw materials to the production of collagen peptides, greatly reducing economic efficiency. Therefore, providing a method for preparing small molecule collagen peptides that is time-saving and can convert aquatic product collagen into more active functions and better odor is of great significance to broaden the application of fresh collagen peptides and improve the antioxidant and anti-aging effects of fresh collagen peptides. Summary of the invention

[0008] The present invention aims at the problems existing in the prior art, and provides a method for preparing and applying fresh collagen peptides with anti-aging and anti-oxidation properties, which solves the problems in the prior art of preparing collagen peptides from aquatic products, such as heavy fishy and bitter taste of the product, low collagen peptide yield and amino acid content, and low antioxidant and anti-aging activities. The present invention firstly performs a soaking and swelling treatment on the aquatic product raw materials to efficiently remove the fishy substances; then, an acidic protease and a composite protease are used for two-step enzymolysis, wherein the composite protease is a mixture of papain, aminopeptidase and trypsin; finally, fresh collagen peptides with a high amino acid content are obtained, and the product has no fishy smell and has high antioxidant and anti-aging effects. The prepared fresh collagen peptides are widely used in food, health products, daily necessities, and cosmetics.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] First, the present invention provides a method for preparing fresh collagen peptides with anti-aging and anti-oxidation properties, comprising the steps of: firstly performing swelling pretreatment on the raw materials, then performing a first enzymatic hydrolysis on the pretreated raw materials using an acidic protease; and then performing a second enzymatic hydrolysis using a composite protease to obtain fresh collagen peptides;

[0011] The composite protease is a mixture of papain, aminopeptidase and trypsin.

[0012] Preferably, the preparation method comprises the steps of:

[0013] (1) Swelling pretreatment: The raw material is mixed with a sodium hydroxide solution and soaked for degreasing; then mixed with a sodium chloride solution containing an acid, heated and stirred to obtain a pretreatment solution;

[0014] (2) Centrifugation: The pretreated liquid is centrifuged to obtain a pretreated raw material;

[0015] (3) First enzymatic hydrolysis: the pretreated raw material is mixed with phosphate buffer, and then the pH is adjusted to 2.0-4.0 with acetic acid, and acidic protease is added, and enzymatic hydrolysis is carried out at 36-45° C. for 1-3 hours to obtain a first enzymatic hydrolysis mixture;

[0016] (4) Second enzymatic hydrolysis: The pH of the first enzymatic hydrolysis mixture is adjusted to 7.0-8.2, and then the composite protease is added and enzymatic hydrolysis is carried out at 45-52° C. for 2-4 hours to obtain a second enzymatic hydrolysis mixture;

[0017] (5) Inactivation of enzymes: After heating the second enzymatic hydrolysis mixture to inactivate the enzymes, centrifuge and retain the supernatant; after filtering the supernatant, fresh collagen peptides are obtained.

[0018] Further preferably, in step (1), the soaking degreasing is specifically: the raw material is mixed with a sodium hydroxide solution with a mass concentration of 0.5-2% at a material-liquid ratio of 1:12-20 g / mL, and the mixture is soaked for 10-30 minutes for degreasing.

[0019] More preferably, the soaking degreasing is specifically as follows: the raw material is mixed with a sodium hydroxide solution having a mass concentration of 1% at a material-liquid ratio of 1:15 g / mL, soaked for 15 minutes for degreasing, and fully washed after degreasing.

[0020] Further preferably, in step (1), the acid in the sodium chloride solution containing acid is selected from at least one of citric acid, acetic acid, tartaric acid and hydrochloric acid; in the sodium chloride solution containing acid, the mass concentration of the acid is 1-3%, and the mass concentration of sodium chloride is 10-15%.

[0021] More preferably, in the sodium chloride solution containing acid, the mass concentration of the acid is 2% and the mass concentration of the sodium chloride is 12%.

[0022] Further preferably, in step (1), the heating and stirring is specifically heating to 65-80° C. and stirring for 15-45 min.

[0023] More preferably, the heating and stirring is specifically heating to 70-75° C. and stirring for 20-40 minutes.

[0024] More preferably, the heating and stirring is specifically heating to 70° C. and stirring for 30 minutes.

[0025] Further preferably, in step (2), the centrifugation is high-speed centrifugation, 6000-12000 rpm for 8-15 min; further preferably, the centrifugation is 8000 rpm for 10 min.

[0026] Further preferably, in step (3), the phosphate buffer comprises 0.2-0.3 M potassium dihydrogen phosphate and 0.4-0.6 M anhydrous disodium hydrogen phosphate, and the liquid-to-solid ratio of the pretreated raw material to the phosphate buffer is 1:15-25 g / mL.

[0027] More preferably, the phosphate buffer comprises 0.25 M potassium dihydrogen phosphate and 0.5 M anhydrous disodium hydrogen phosphate, and the liquid ratio of the pretreated raw material to the phosphate buffer is 1:20 g / mL.

[0028] More preferably, in step (3), the pH is 2.6-3.3; and the pH is even more preferably 3.0.

[0029] Further preferably, in step (3), the acidic protease is 537 acidic protease, and the amount added is 3-5% of the mass of the raw material; further preferably, the amount of the acidic protease added is 4% of the mass of the raw material.

[0030] Further preferably, in step (3), the enzymatic hydrolysis is specifically performed at 40-42° C. for 1.5-2.5 h, with shaking at 30-50 rpm during the enzymatic hydrolysis.

[0031] More preferably, the enzymatic hydrolysis is carried out at 42° C. for 2 h, with shaking at 30-50 rpm during the enzymatic hydrolysis.

[0032] Further preferably, in step (4), the pH is adjusted by using a potassium hydroxide solution with a mass concentration of 0.5-2% to adjust the pH to 7.5-7.9; further preferably, a potassium hydroxide solution with a mass concentration of 4% is used to adjust the pH to 7.8.

[0033] Further preferably, in step (4), in the composite protease, the mass ratio of papain, aminopeptidase and trypsin is 1.5-3:1.5-3:0.5-1.2.

[0034] More preferably, the mass ratio of papain, aminopeptidase and trypsin is 1.8-2.5:2-2.5:1.

[0035] Most preferably, the mass ratio of papain, aminopeptidase and trypsin is 2:2:1.

[0036] Further preferably, in step (4), the amount of the composite protease added is 4-6% of the mass of the raw material, and further preferably 5%.

[0037] Further preferably, in step (4), the enzymatic hydrolysis is carried out at 50° C. for 160 min, with shaking at 30-50 rpm during the enzymatic hydrolysis.

[0038] Further preferably, step (5) is specifically as follows: heating the second enzymatic hydrolysis mixture to 95-121°C, keeping it warm for 10-30 minutes to inactivate the enzyme, centrifuging it at 6000-12000 rpm for 5-15 minutes, retaining the supernatant; filtering the supernatant to obtain fresh collagen peptides.

[0039] More preferably, step (5) is specifically as follows: heating the second enzymatic hydrolysis mixture to 100°C, keeping the mixture warm for 15 minutes to inactivate the enzyme, centrifuging at 8000 rpm for 10 minutes, retaining the supernatant; filtering the supernatant to obtain fresh collagen peptides.

[0040] More preferably, the supernatant is filtered by using ceramic membrane filtration and nanofiltration membrane for nanofiltration.

[0041] More preferably, the ceramic membrane filtration adopts a ceramic membrane filtration equipment with a pressure of 0.3-0.5MPa, and the nanofiltration membrane adopts a nanofiltration membrane equipment with a pore size of 100-300D; most preferably, the ceramic membrane filtration adopts a ceramic membrane filtration equipment with a pressure of 0.4MPa, and the nanofiltration membrane adopts a nanofiltration membrane equipment with a pore size of 200D.

[0042] Then, the fresh collagen peptide prepared by the above preparation method of the present invention.

[0043] Finally, the present invention provides the use of the fresh collagen peptide in the preparation of food, health products, medical products and cosmetics.

[0044] In the present invention, the raw material is aquatic product; the aquatic product used in the present invention is not limited to tilapia and snakehead fish, and can be fish skin, fish scales, and fish bones.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The preparation method of the present invention efficiently removes the fishy substances in the raw materials by soaking and swelling the raw materials with sodium chloride and acid, and is more conducive to the subsequent protein enzymatic hydrolysis process.

[0047] 2. The preparation method of the present invention increases the content of small molecule amino acids such as glycine and proline in collagen peptides by using specific enzymes and performing enzymolysis under specific enzymolysis conditions. The acidic protease and the composite protease (papain, aminopeptidase, trypsin) interact with each other to show a synergistic enzymolysis effect, shortening the preparation time of collagen peptides. The prepared fresh collagen peptides have high activity, significantly improved antioxidant and anti-aging effects, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 1 is a graph showing the nematode survival rate test results; A is a graph showing the nematode survival rate results of the embodiment, the blank group and the control group, and B is a graph showing the nematode survival rate results of the embodiment 1 and the comparative example group. DETAILED DESCRIPTION

[0049] The following non-limiting examples can make those of ordinary skill in the art understand the present invention more comprehensively, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention according to the disclosed content, and it should also belong to the scope of the present invention. When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those of ordinary skill in the technical field to which the present invention belongs.

[0050] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Products from different manufacturers have no significant effect on the effects.

[0051] In the following examples, 537 acid protease was purchased from Shandong Longkote Enzyme Preparation Co., Ltd., with a CAS number of 9025-49-4, and the unit specific activity was determined to be 50000 U / g;

[0052] Papain was purchased from Nanjing Pangbo Bioengineering Co., Ltd., CAS No. 9001-73-4, and the unit specific activity was determined to be 100000U / g;

[0053] Aminopeptidase is a biological fermentation enzyme fermented by Aspergillus oryzae strains, purchased from Nanning Dongheng Huadao Biotechnology, and the unit specific activity was 60000U / g.

[0054] Trypsin was purchased from Henan Anrui Biotechnology Co., Ltd. with a CAS number of 8049-47-6. The unit specific activity was determined to be 4000 U / g.

[0055] Example 1

[0056] (1) Swelling pretreatment: After cleaning 20 g of tilapia fish skin, add a sodium hydroxide solution with a mass concentration of 1% according to a solid-liquid ratio of 1:15 g / mL, and soak for 15 minutes for degreasing; the defatted fish skin is fully washed, and then mixed with a sodium chloride solution with a mass concentration of 12% according to a solid-liquid ratio of 1:24 g / mL, and the sodium chloride solution also contains citric acid with a mass concentration of 2%; after stirring evenly, heat to 70° C. and keep stirring for 30 minutes to obtain a pretreatment solution;

[0057] (2) Centrifugation: The pretreated solution was centrifuged at 8000 rpm for 10 min to obtain pretreated fish skin;

[0058] (3) Primary enzymolysis: the pretreated fish skin was mixed with a phosphate buffer solution (0.25 M potassium dihydrogen phosphate, 0.5 M anhydrous disodium hydrogen phosphate) at a solid-liquid ratio of 1:20 g / mL, and then the pH was adjusted to 3.0 using acetic acid; 537 acid protease (50000 U / g) of 4% of the mass of tilapia skin was added, and enzymolysis was performed at 42° C. for 2 h. During the enzymolysis process, the mixture was continuously shaken at 30 rpm to obtain a first enzymolysis mixture;

[0059] (4) Secondary enzymolysis: The pH of the first enzymolysis mixture was adjusted to 7.8 using a 1% potassium hydroxide aqueous solution, and then a composite protease equivalent to 5% of the mass of tilapia skin (mass ratio: papain, aminopeptidase, trypsin = 2:2:1) was added, and enzymolysis was performed at 50° C. for 160 min. During the enzymolysis process, the mixture was continuously shaken at 30 rpm to obtain a second enzymolysis mixture;

[0060] (5) Inactivation of enzymes: The temperature of the second enzymatic hydrolysis mixture was raised to 100°C, and the enzyme inactivation treatment was maintained for 15 minutes. The mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was retained. The supernatant was filtered using a ceramic membrane filtration device with a pressure of 0.4 MPa and a nanofiltration membrane device with a pore size of 200D in sequence to obtain fresh collagen peptides.

[0061] Example 2

[0062] The difference from Example 1 is that the heating temperature in step (1) is different, the temperature is 80° C., specifically:

[0063] (1) Swelling pretreatment: After 20 g of tilapia skin is cleaned, a sodium hydroxide solution with a mass concentration of 1% is added according to a solid-liquid ratio of 1:15 g / mL, and the skin is soaked for 15 minutes for degreasing; the defatted skin is fully washed, and then mixed with a sodium chloride solution with a mass concentration of 12% according to a solid-liquid ratio of 1:24 g / mL, wherein the sodium chloride solution also contains citric acid with a mass concentration of 2%; after stirring evenly, the mixture is heated to 80° C. and kept stirred for 30 minutes to obtain a pretreatment solution.

[0064] The rest is the same as in Example 1.

[0065] Example 3

[0066] The difference from Example 1 is that the phosphate buffer in step (3) is different, specifically:

[0067] (3) Primary enzymolysis: The pretreated fish skin was mixed with a phosphate buffer solution (0.3 M potassium dihydrogen phosphate, 0.4 M anhydrous disodium hydrogen phosphate) at a solid-liquid ratio of 1:20 g / mL, and then the pH was adjusted to 3.3 using acetic acid; 537 acid protease (50000 U / g) of 4% of the mass of tilapia skin was added, and enzymolysis was performed at 42° C. for 2 h. During the enzymolysis process, the mixture was continuously shaken at 30 rpm to obtain a first enzymolysis mixture.

[0068] The rest is the same as in Example 1.

[0069] Example 4

[0070] The difference from Example 1 is that the enzymolysis temperature and time in step (3) are different, specifically:

[0071] (3) Primary enzymolysis: The pretreated fish skin was mixed with a phosphate buffer solution (0.25 M potassium dihydrogen phosphate, 0.5 M anhydrous disodium hydrogen phosphate) at a solid-liquid ratio of 1:20 g / mL, and then the pH was adjusted to 3.0 using acetic acid; 537 acid protease (50000 U / g) of 4% of the mass of tilapia skin was added, and enzymolysis was performed at 40° C. for 2.5 h. During the enzymolysis process, the mixture was continuously shaken at 30 rpm to obtain a first enzymolysis mixture.

[0072] The rest is the same as in Example 1.

[0073] Example 5

[0074] Different from Example 1, the mass ratio of the composite protease in step (4) is different, that is, the mass ratio is 2.5:2:1.

[0075] (4) Secondary enzymolysis: The pH of the first enzymolysis mixture was adjusted to 7.8 using a 1% potassium hydroxide aqueous solution, and then a composite protease equivalent to 5% of the weight of tilapia skin (mass ratio: papain, aminopeptidase, trypsin = 2.5:2:1) was added, and enzymolysis was performed at 50° C. for 160 min. During the enzymolysis process, the mixture was continuously shaken at 30 rpm to obtain a second enzymolysis mixture.

[0076] The rest is the same as in Example 1.

[0077] Example 6

[0078] Different from Example 1, the enzymolysis temperature and enzymolysis time in step (4) are different.

[0079] (4) Secondary enzymolysis: The pH of the first enzymolysis mixture was adjusted to 7.8 using a 1% potassium hydroxide aqueous solution, and then a composite protease equivalent to 5% of the mass of tilapia skin (mass ratio: papain, aminopeptidase, trypsin = 2:2:1) was added, and enzymolysis was performed at 45° C. for 240 min. During the enzymolysis process, the mixture was continuously shaken at 30 rpm to obtain a second enzymolysis mixture.

[0080] The rest is the same as in Example 1.

[0081] Comparative Example 1

[0082] The difference from Example 1 is that the citric acid in step (1) is replaced by acetic acid. The rest is the same as Example 1.

[0083] Comparative Example 2

[0084] The difference from Example 1 is that the enzymatic hydrolysis temperature in step (4) is replaced with 40° C. The rest is the same as Example 1.

[0085] Comparative Example 3

[0086] The difference from Example 1 is that the heating in step (1) is replaced by room temperature. That is, the defatted fish skin is fully washed, and then mixed with a sodium chloride solution with a mass concentration of 12% according to a material-liquid ratio of 1:24 g / mL, and the sodium chloride solution also contains citric acid with a mass concentration of 2%; stirred at room temperature for 30 minutes to obtain a pretreatment solution. The rest is the same as Example 1.

[0087] Comparative Example 4

[0088] The difference from Example 1 is that the centrifugation in step (2) is replaced by ordinary filtration. The rest is the same as Example 1.

[0089] Comparative Example 5-Comparative Example 10

[0090] The difference from Example 1 is that the types of enzymes used in the enzymolysis process and the ratio of the enzymes are different:

[0091] Step (3) primary enzymatic hydrolysis Step (4) Secondary enzymatic hydrolysis Comparative Example 5 537 Acid Protease Papain, 5% Comparative Example 6 537 Acid Protease Aminopeptidase, 5% Comparative Example 7 537 Acid Protease Trypsin, 5% Comparative Example 8 537 Acid Protease Papain: aminopeptidase: trypsin = 0.5:0.5:4, 5% Comparative Example 9 Pepsin Same as Example 1 Comparative Example 10 - Same as Example 1

[0092] The difference between Comparative Example 5 and Example 1 is that the composite protease in step (4) is replaced by papain.

[0093] The difference between Comparative Example 6 and Example 1 is that the composite protease in step (4) is replaced by aminopeptidase.

[0094] The difference between Comparative Example 7 and Example 1 is that the composite protease in step (4) is replaced by trypsin.

[0095] The difference between Comparative Example 8 and Example 1 is that the mass ratio of each enzyme in the composite protease in step (4) is different, and the mass ratio of papain, aminopeptidase and trypsin is 0.5:0.5:4.

[0096] The difference between Comparative Example 9 and Example 1 is that the 537 acid protease in step (3) is replaced by pepsin purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and the enzyme activity concentration during enzymolysis is kept consistent with that of Example 1.

[0097] The difference between Comparative Example 10 and Example 1 is that the enzymolysis process of step (3) is not carried out: after centrifugation in step (2), the pretreated fish skin is mixed with phosphate buffer (0.25M potassium dihydrogen phosphate, 0.5M anhydrous disodium hydrogen phosphate) at a solid-liquid ratio of 1:20 g / mL, and the pH of the first enzymolysis mixed solution is adjusted to 7.8 using a 1% potassium hydroxide aqueous solution, and then a composite protease equivalent to 5% of the mass of tilapia fish skin (mass ratio: papain, aminopeptidase, trypsin = 2:2:1) is added, and enzymolysis is carried out at 50°C for 160 min. During the enzymolysis process, the heat preservation and shaking at 30 rpm are continuously performed to obtain a second enzymolysis mixed solution.

[0098] The remaining steps are the same as those in Example 1.

[0099] Comparative Example 11

[0100] The difference from Example 1 is that the buffer used in step (3) is different, that is, potassium dihydrogen phosphate is replaced by sodium dihydrogen phosphate, that is, a phosphate buffer of 0.25 M sodium dihydrogen phosphate and 0.5 M anhydrous disodium hydrogen phosphate.

[0101] Comparative test 1 collagen peptide content

[0102] The peptide content of the fresh collagen peptides obtained in Examples 1-6 and Comparative Examples 1-11 was tested. The content was tested using the method in Appendix A of GB31645-2018. The test results of the proportion of collagen peptides with a relative molecular mass of less than 10,000 are shown in Table 1:

[0103] Table 1

[0104]

[0105] Comparative experiment 2 Anti-aging test Test index: Test the effect of the fresh collagen peptides prepared in the examples and comparative examples on acute heat stress of nematodes.

[0106] 1. Cultivation of Caenorhabditis elegans

[0107] Prepare LB (Luria-Bertani) medium, NGM medium (nematode growth medium), nematode lysis solution (sodium hypochlorite lysis solution) and M9 buffer solution (HEPENGBIO nematode M9 buffer).

[0108] A single colony of E. coli OP50 (Escherichia coli) was picked and inoculated into 5 mL of LB liquid culture medium. After shaking culture at 37°C and 200 r / min for 24 h, it was evenly spread on an NGM plate and nematodes (wild-type Caenorhabditis elegans N2, Institute of Plant Protection and Environmental Protection, Beijing Academy of Agricultural and Forestry Sciences) were inoculated in a space at the edge, which was recorded as the blank group.

[0109] Repeat the above operation, pick E. coli OP50 colonies and add fresh collagen peptides (fresh collagen peptides prepared in the examples and comparative examples) when smearing on the culture medium, so that the final mass concentration of collagen peptides (collagen peptides with a relative molecular mass of less than 10,000) in OP50 is 0.1 mg / mL. The above is the sample group.

[0110] Repeat the above operation, pick E. coli OP50 colonies and add collagen peptide powder (batch number J231019) from Hainan Huayan when smearing on the culture medium, so that the final mass concentration of collagen peptide (collagen peptide with a relative molecular mass of less than 10,000) in OP50 is 0.1 mg / mL. The above is the control group.

[0111] The plates after inoculation were placed in a 37°C incubator for overnight culture.

[0112] 2. Synchronization of Nematodes

[0113] The nematodes were processed by breaking and synchronization to ensure that they were in the same developmental stage. The well-growing nematodes were transferred to a test tube, lysate was added, and the tube was shaken 5 times and centrifuged at 3500r / min for 2min, and the supernatant was discarded; the above operation was repeated 3 times, and the bottom nematodes were spread on the sterile area of ​​NGM medium coated with E.coli OP50 and blown dry, and cultured at 20℃ for 48h to obtain L4 stage nematodes for use.

[0114] 3. Impact on the average heat resistance time and the longest heat resistance time of nematodes

[0115] The experimental materials were preheated to 35°C, and L4 nematodes were selected from the sample group and the control group and placed on the corresponding plates, 20 nematodes / plate, 10 parallels in each group, and cultured at 35°C, and this time was recorded as 0h; the plates were quickly taken out from the incubator for observation every 4h to reduce the experimental error caused by temperature changes, and the plates were discarded after observation and the survival rate and survival time data were recorded. The average heat resistance time and the longest heat resistance time of the nematodes at 35°C were recorded, and the test results are shown in Table 2.

[0116] The results of the nematode survival rate test are shown in Figure 1 .

[0117] Table 2

[0118]

[0119]

[0120] In Table 2, compared with the blank group, *P<0.05, **P<0.01; compared with Example 1, # P<0.05, ## P<0.01.

[0121] The acute heat stress resistance of nematodes is an indicator of the aging process of nematodes. As can be seen from Table 2, the average heat resistance time and the longest heat resistance time of nematodes in Example 1-6 groups are higher than those in the control group and the comparative example group. The fresh collagen peptide prepared by the present invention significantly improves the heat stress resistance of nematodes. The prepared fresh protein peptide can improve the stress resistance of nematodes to delay the aging of the body.

[0122] Comparative test 3 Antioxidant test

[0123] 1. Detect the effects of fresh collagen peptides prepared in different examples and comparative proportions and collagen peptide powder in the control group on the expression of TGF-βRⅡ and Smad7 protein in human skin fibroblasts induced by UVA. Human skin fibroblast cell lines (HSFs) were purchased from Shanghai Fusheng Industrial Co., Ltd.

[0124] 2. Sample group: Weigh a certain amount of fresh collagen peptides prepared in the examples and comparative examples of the present invention, mix them with a dimethyl sulfoxide solution, and prepare a test solution with a collagen peptide (collagen peptide with a relative molecular mass of less than 10,000) concentration of 25 mg / mL, set aside, and perform ultraviolet irradiation experiment detection.

[0125] Control group: Hainan Huayan's collagen peptide powder (batch number J231019) was mixed with dimethyl sulfoxide solution to prepare a test solution with a collagen peptide (collagen peptide with a relative molecular mass of less than 10,000) concentration of 25 mg / mL, which was set aside for ultraviolet irradiation experiment detection.

[0126] The non-illumination group only used dimethyl sulfoxide solution without ultraviolet irradiation.

[0127] UVA irradiation group: only dimethyl sulfoxide solution was used for ultraviolet irradiation.

[0128] Each group of experiments was repeated 3 times.

[0129] 3. Ultraviolet irradiation: Use SS-03UVA ultraviolet phototherapy device (Sigma, Shanghai), wavelength 320-400nm UVA irradiation, UVA dose = UVA radiation × time (s) (single irradiation), irradiation distance 15cm. UVA irradiation dose was set to 30J / cm after screening. 2 .

[0130] Immunocytochemistry was used to qualitatively determine the expression of TGF-βRⅡ and Smad7 in fibroblasts, referring to the instructions of the color development kit. The results of the positive expression rate test are shown in Table 3.

[0131] Table 3

[0132]

[0133]

[0134] In Table 3, compared with the UVA irradiation group, *P<0.05, **P<0.01; compared with Example 1, the comparative example has P<0.05, ## P<0.01.

[0135] As can be seen from Table 3, the fresh collagen peptides prepared by the present invention can promote the expression of TGF-βRⅡ and can correspondingly downregulate the expression of Smad7; after the action of the fresh collagen peptides, the expression levels of TGF-βRⅡ and Smad7 proteins in fibroblasts are close to normal levels, thereby promoting the synthesis of collagen by fibroblasts, reducing the degradation of extracellular matrix, and delaying the process of skin photoaging.

[0136] Comparative test 4 sensory evaluation

[0137] The fresh collagen peptides obtained in Example 1, Comparative Example 1, Comparative Example 3 and Comparative Example 6 were evaluated by oral sensation using commercially available collagen peptide powder.

[0138] 1. Assessment method

[0139] The evaluation team consists of 10 people, 5 boys and 5 girls, aged between 25 and 35, all of whom are in good health, have no bad habits, have strong discrimination and high sensitivity to color, aroma and taste, and evaluate at room temperature. Before each sensory evaluation, rinse your mouth with warm water to keep your mouth fresh. This experiment consists of three factors: smell (fishy smell), taste, and bitterness. The evaluation of each factor is based on the corresponding scores of excellent, medium, and poor.

[0140] 2. Sample Preparation

[0141] The samples produced in the embodiments and comparative examples, and samples of commercially available collagen peptide powder (collagen peptide powder produced by Hainan Huayan, batch number J231019) were mixed with water to form a solution with a collagen peptide concentration of 0.1 g / mL (the concentration of collagen peptides with a relative molecular mass of less than 10,000 is the same).

[0142] 3. Sensory evaluation reference standards, see Table 4 for details.

[0143] Table 4

[0144] odor taste bitterness excellent No unpleasant fishy smell Light collagen peptide unique flavor Not obvious middle A slight fishy smell More obvious collagen peptide unique taste Can taste bitterness, but the bitterness is weak Difference Fishy smell The distinctly unpleasant taste of collagen peptides Bitter taste

[0145] 4. Sensory evaluation results

[0146] The specific sensory evaluation results are shown in Table 5, and the number of people at different sensory evaluation standards is counted:

[0147] Table 5

[0148]

[0149] As can be seen from Table 5, the fresh collagen peptide prepared by the present invention has an excellent taste and a qualified sensory organoleptic property after evaluation.

[0150] Comparative experiment 5 Heterocyclic amine detection experiment

[0151] The liquid collagen peptide obtained in Example 1 and a commercially available collagen peptide powder product (collagen peptide powder produced by Hainan Huayan, batch number J231019) were tested for heterocyclic amines, and the heterocyclic amines were tested according to the standard GB5009.243-2016.

[0152] The test results are shown in Table 6:

[0153] Table 6

[0154] Test items Example 4 Commercially available collagen peptide powder 2-Amino-3,4-dimethylimidazo[4.5-f]quinoline(MeIQ) Not detected (<0.1 μg / kg) 0.65 μg / kg 2-Amino-3,8-dimethylimidazo[4,5-f]quinoline(MeIQx) Undetectable (<0.3 μg / kg) Undetectable (<0.3pg / kg) 2-Amino-3,4,8-trimethylimidazo[45-f](4,8-DiMeIQx) Undetectable (<0.2 μg / kg) Undetectable (<0.2pg / kg) 2-Amino-3,7,8-trimethylimidazo[4,5-f]quinoline(7,8-DiMeIQx) Undetectable (<0.2pg / kg) Undetectable (<0.2pg / kg) 2-Amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) Not detected (<0.1 μg / kg) 0.30 μg / kg

[0155] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing anti-aging and antioxidant fresh collagen peptides, characterized in that: Includes steps: (1) Swelling pretreatment: fish skin is mixed with sodium hydroxide solution and soaked for degreasing; then mixed with sodium chloride solution containing acid, heated and stirred to obtain a pretreatment solution; (2) Centrifugation: the pretreated liquid is centrifuged to obtain pretreated fish skin; (3) First enzymatic hydrolysis: the pretreated fish skin is mixed with a phosphate buffer, and then the pH is adjusted to 2.0-4.0 with acetic acid, 3%-5% of the weight of the fish skin 537 acid protease is added, and enzymatic hydrolysis is carried out at 42° C. for 2 hours to obtain a first enzymatic hydrolysis mixture; the phosphate buffer comprises 0.2-0.3M potassium dihydrogen phosphate and 0.4-0.6M anhydrous disodium hydrogen phosphate, and the material-liquid ratio of the pretreated fish skin to the phosphate buffer is 1:15-25 g / mL; (4) Second enzymatic hydrolysis: the pH of the first enzymatic hydrolysis mixture is adjusted to 7.0-8.2, and then a composite protease in an amount of 4%-6% by weight of the fish skin is added, and enzymatic hydrolysis is carried out at 50° C. for 160 min to obtain a second enzymatic hydrolysis mixture; the composite protease is composed of papain, aminopeptidase, and trypsin in a mass ratio of 2:2:1; (5) Inactivation of enzymes: After heating the second enzymatic hydrolysis mixture to inactivate the enzymes, centrifuge and retain the supernatant; after filtering the supernatant, fresh collagen peptides are obtained.

2. The preparation method according to claim 1, characterized in that: In step (1), the soaking and degreasing is specifically: the fish skin is mixed with a sodium hydroxide solution with a mass concentration of 0.5-2% at a solid-liquid ratio of 1:12-20 g / mL, and soaked for 10-30 minutes for degreasing; In the acid-containing sodium chloride solution, the acid is selected from at least one of citric acid and acetic acid; in the acid-containing sodium chloride solution, the mass concentration of the acid is 1-3%, and the mass concentration of the sodium chloride is 10-15%.

3. The preparation method according to claim 1, characterized in that: In step (1), the heating and stirring is specifically heating to 65-80°C and stirring for 15-45 minutes; in step (2), the centrifugation is high-speed centrifugation, centrifugation at 6000-12000 rpm for 8-15 minutes.

4. The preparation method according to claim 1, characterized in that: Step (3) specifically includes: adjusting the pH to 2.6-3.3, adding 537 acid protease, and performing enzymatic hydrolysis.

5. The preparation method according to claim 1, characterized in that: In step (3), the enzymatic hydrolysis needs to be carried out under shaking conditions at 30-50 rpm.

6. The preparation method according to claim 1, characterized in that: In step (4), the pH is adjusted by using a potassium hydroxide solution with a mass concentration of 0.5-2% to adjust the pH to 7.5-7.9; the enzymatic hydrolysis needs to be carried out under shaking conditions at 30-50 rpm; Step (5) is specifically as follows: heating the second enzymatic hydrolysis mixture to 95-121°C, keeping the mixture warm for 10-30 minutes to inactivate the enzyme, centrifuging at 6000-12000 rpm for 5-15 minutes, retaining the supernatant; filtering the supernatant to obtain fresh collagen peptides.

7. Fresh collagen peptide prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the fresh collagen peptide according to claim 7 in the preparation of foods, health products, medical products and cosmetics.

Images