Anti-sugar and anti-aging cod collagen peptide as well as preparation method and application thereof

Abstract

The invention provides an anti-sugar and anti-aging cod collagen peptide as well as a preparation method and application thereof, according to the method, the yield and functional characteristics of active peptide are remarkably improved by combining ultrasonic wave with nanometer grinding pretreatment and combining step-by-step directional enzymolysis and a three-stage ultrafiltration membrane precise separation technology. The method specifically comprises the following steps: carrying out ultrasonic pretreatment on dry cod skin, and then soaking and activating in warm water containing natural anti-sugar components; grinding and dissociating collagenous fibers by adopting high-pressure homogenization and nanoscale zirconium oxide beads; the high-purity peptide fragment with the molecular weight of 500-3000Da is obtained through step-by-step enzymolysis of a compound enzyme I and an enzyme II in combination with gradient interception of a three-stage polyethersulfone ultrafiltration membrane. The total nitrogen content of the obtained product is larger than or equal to 14.5%, the proportion of the target peptide fragment is larger than or equal to 96.7%, the yield is larger than or equal to 18.1%, and the product has remarkable anti-saccharification and anti-aging activity and can be applied to skin care products such as essence and masks to achieve collagen repair and skin barrier function improvement.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to a cod collagen peptide with anti-glycation and anti-aging effects, and its preparation method and application. Background Art

[0002] Common extraction methods of cod collagen include five categories: hot water method, acid method, alkali method, enzymatic method, and salt method. Among them, the enzymatic extraction method is considered to be the extraction method with the highest biological activity of the product because of its good stability, mild extraction conditions, adjustable and controllable, fast reaction rate, and environmental friendliness. Combined with other research data. Research shows that cod collagen can not only effectively supplement the collagen lost by the skin, but also show significant effects in anti-aging, anti-glycation, and slowing down skin aging.

[0003] In terms of anti-aging, cod collagen can promote the metabolism of skin cells, enhance cell vitality, stimulate fibroblasts to synthesize more collagen and elastic fibers, thereby improving the elasticity and firmness of the skin and reducing the generation of wrinkles. At the same time, it also has an antioxidant effect, can scavenge free radicals in the body, reduce the damage of oxidative stress to skin cells, and delay the process of cell aging.

[0004] In terms of anti-glycation, cod collagen can inhibit the glycation reaction and reduce the generation of advanced glycation end products (AGEs). AGEs are products of non-enzymatic glycation reactions between sugars and macromolecules such as proteins and lipids. Their large accumulation in the skin will cause cross-linking and denaturation of collagen and elastic fibers, making the skin lose elasticity and dull in color. By inhibiting the generation of AGEs, cod collagen blocks the damage of the glycation reaction to the skin, thereby effectively preventing and improving the aging problems caused by skin glycation.

[0005] In terms of slowing down skin aging, cod collagen can enhance the moisturizing ability of the skin and maintain the skin moisture balance. The stability of the skin moisture content is crucial for maintaining the softness, gloss, and elasticity of the skin. In addition, it can also regulate the immune function of the skin, enhance the skin barrier function, resist the invasion of external environmental factors on the skin, and further slow down the skin aging speed.

[0006] Although there are currently many research reports on the anti-aging, anti-glycation, and slowing down skin aging of cod collagen, there are still many problems to be further explored in this field. For example, the research on the absorption mechanism, optimal supplement dosage and method, safety and effectiveness of long-term supplementation of cod collagen in the body is not yet perfect. Therefore, deeply revealing the mechanism of action of cod collagen in anti-aging, anti-glycation, and slowing down skin aging has important theoretical significance and practical application value for the development of highly efficient and safe anti-aging skin care products and improving people's skin health level. Summary of the Invention

[0007] In view of this, the present invention provides an anti-glycation and anti-aging cod collagen peptide, its preparation method and application.

[0008] The technical solution of the present invention is realized as follows: A preparation method of an anti-glycation and anti-aging cod collagen peptide, comprising the following steps: (1) Place dried cod skin in an ultrasonic cleaning tank with 40 - 60 times the mass of deionized water, and perform ultrasonic pretreatment at a temperature of 30 - 70°C, a power of 300 - 700 w, and a frequency of 20 - 40 kHz for 20 - 40 min; (2) After draining, soak it in warm water at a solid-liquid ratio of 1:15 - 25 at 60 - 80°C for 8 - 12 h; (3) Put the soaked cod skin into a high-pressure homogenizer, perform homogenization treatment at a pressure of 1200 - 1400 bar, a temperature of 50 - 70°C, and a flow rate of 8 - 15 L / h, and then perform nano-grinding treatment to form a nano-level homogenate; (4) Seal the homogenate and cook it at 100 - 110°C for 1 - 3 h; (5) Cool down to 50 ± 2°C, adjust the pH to 7 - 9, add 1 - 3% of complex enzyme I based on the mass of the substrate and enzymolyze for 0.8 - 1.2 h, then add 0.5 - 1.5% of complex enzyme II based on the mass of the substrate and enzymolyze for 2.5 ± 0.5 h. During the enzymolysis process of complex enzyme II, when one-third of the enzymolysis process remains, the pH of the system gradually decreases from the initial 7 - 9 to 5.5 - 6.2; (6) Centrifuge at a speed of 2000 - 3000 rpm for 25 - 35 min to separate the supernatant and the residue; (7) After the supernatant is clarified by ceramic membrane and membrane concentrated, it is fractionated by an ultrafiltration membrane with a molecular weight cut-off of 500 - 3000 Da; (8) Sterilize with a three-stage filter element, and perform aseptic packaging after spray drying.

[0009] Furthermore, during the soaking in step (2), 0.5 - 0.8% of the natural anti-glycation active ingredient based on the mass of the dried cod fillet is added. The natural anti-glycation active ingredient is rosmarinic acid, ferulic acid, and chlorogenic acid with a mass ratio of 3:2:1, and the preliminary loading of the anti-glycation component is realized synchronously during the warm water soaking process.

[0010] Furthermore, the nano-grinding treatment in step (3) is to grind with zirconia beads with a particle size of 0.1 - 0.3 mm at a rotation speed of 1000 - 1500 rpm for 20 - 30 min to reduce the average particle size of the material to 1 - 2 μm and form a nano-level homogenate.

[0011] Further, the complex enzyme I in step (5) is prepared by weighing bromelain, flavorzyme and papain at a mass ratio of (1-3):(0.2-1.2):(0.3-1.5), and the enzyme activity of each is greater than 1.5×10 5 u / g.

[0012] Further, the complex enzyme II in step (5) is subtilisin, trypsin and β-glucosidase at a mass ratio of 1:(1-2):(0.1-0.3), and the enzyme activity of each is greater than 1.8×10 5 u / g. β-glucosidase can degrade glycosaminoglycan impurities in cod skin and enhance the anti-glycation reaction activity of collagen peptides.

[0013] Further, in step (7), the pore size of the ceramic membrane is 0.1-0.2 μm, the membrane filtration pressure is 0.2-0.3 MPa, and the temperature is controlled at 40-50°C.

[0014] Further, a pulsed electric field is applied during the clarification process of the ceramic membrane, the electric field strength is 5-10 V / cm, the pulse frequency is 20-50 Hz, an alternating electric field is formed on the membrane surface, membrane fouling is inhibited and the impurity interception efficiency is increased by more than 30%.

[0015] Further, in step (7), the ultrafiltration fractionation uses a hollow fiber ultrafiltration membrane, and the feed liquid flow rate during ultrafiltration is 0.5-1.0 m / s.

[0016] Further, a cod collagen peptide has a total nitrogen content ≥14.5%, the proportion of target peptide segments ≥96.7%, and the yield of cod collagen peptide ≥18.1%.

[0017] Further, the application of the cod collagen peptide in the preparation of anti-aging and anti-glycation skin care products, and the skin care products include essence, mask or cream.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This application processes cod skin by ultrasonic cleaning, which makes the surface impurities cleaner. Heating can be carried out simultaneously during ultrasonic treatment, avoiding subsequent heat treatment. The cavitation effect of ultrasonic waves improves the soaking and crushing efficiency of cod skin, which is beneficial to improving the extraction rate in the later stage. Adding natural anti-glycation active ingredients to soak can synchronously achieve the preliminary loading of anti-glycation components. On the other hand, through high-pressure homogenization pretreatment, the processing efficiency of cod skin is also improved, shortening the extraction time. Nano-grinding treatment significantly improves the solubility and bioavailability of collagen peptides. The cod skin liquid after homogenization and grinding is subjected to two-step enzymatic hydrolysis. The enzymatic hydrolysis sites are unique and the degree of enzymatic hydrolysis is more thorough. Adding β-glucosidase is used to degrade glycosaminoglycan impurities in cod skin, enhancing the anti-glycation reaction activity of collagen peptides. Through the synergistic process of composite enzyme step-by-step enzymatic hydrolysis, pulsed electric field ceramic membrane clarification, and nano-grinding, functional peptide segments with a molecular weight of 500-3000 Da are directionally enriched, making the prepared cod collagen peptide have the characteristics of good anti-aging effect and easy absorption. Description of the Drawings

[0019] Figure 1 The clearance rate of cod collagen peptide in Example 3 against DPPH free radicals; Figure 2 The clearance rate of cod collagen peptide in Example 3 against superoxide anion free radicals; Figure 3 The clearance rate of cod collagen peptide in Example 3 against hydroxyl free radicals; Figure 4 The inhibition rate of cod collagen peptide in Example 3 against fructosamine. The inhibition rates of 0.1 and 1.5 mg / mL on the left side of the figure are those of the positive control group. Detailed Description of the Invention

[0020] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0021] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0022] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels unless otherwise specified.

[0023] Example 1 A preparation method of anti-glycation and anti-aging cod collagen peptide, comprising the following steps: (1) Place dry cod skin in an ultrasonic cleaning tank with 40 times the mass of deionized water, and perform ultrasonic pretreatment for 20 min at a temperature of 30 °C, a power of 300 w, and a frequency of 20 kHz; (2) After draining, soak in warm water at 60 °C for 8 h at a material-liquid ratio of 1:15. During soaking, add natural anti-glycation active ingredients accounting for 0.5% of the mass of the dried cod fish slices. The natural anti-glycation active ingredients are rosmarinic acid, ferulic acid, and chlorogenic acid with a mass ratio of 3:2:1; (3) Put the soaked cod fish skin into a high-pressure homogenizer and perform homogenization treatment at a pressure of 1200 bar, a temperature of 50 °C, and a flow rate of 8 L / h, and then perform nano-grinding treatment to form a nano-level homogeneous liquid; (4) After sealing the homogeneous liquid, cook it at 100 °C for 1 h; (5) Cool down to 50 °C, adjust the pH to 7, add complex enzyme I accounting for 1% of the substrate mass and enzymolyze for 0.8 h in sequence, then add complex enzyme II accounting for 0.5% of the substrate mass and enzymolyze for 2.5 h. During the enzymolysis process of complex enzyme II, when one-third of the enzymolysis process remains, the pH of the system gradually decreases from the initial 7 to 5.5; Complex enzyme I is weighed according to the ratio of 1:0.2:0.3 of bromelain, flavor protease, and papain, and the enzyme activity is greater than 1.5×10 5 u / g; Complex enzyme II is subtilisin, trypsin, and β-glucosidase with a mass ratio of 1:1:0.1, and the enzyme activity is greater than 1.8×10 5 u / g; (6) Centrifuge at 2000 rpm for 25 min to separate the supernatant and the residue; (7) After the supernatant is clarified by a ceramic membrane and membrane concentrated, it is fractionated by an ultrafiltration membrane with a molecular weight cut-off of 500 - 3000 Da; The pore size of the ceramic membrane is 0.1 μm, the membrane filtration pressure is 0.2 MPa, the temperature is controlled at 40 °C, and a pulsed electric field is applied during the ceramic membrane clarification process, with an electric field strength of 5 V / cm and a pulse frequency of 20 Hz. The ultrafiltration fractionation described above uses a three-stage series ultrafiltration membrane, which sequentially includes polyethersulfone hollow fiber ultrafiltration membranes with molecular weight cut-offs of 3000 Da, 1000 Da, and 500 Da. During the ultrafiltration process, the feed liquid flow rate is 0.5 m / s; (8) Sterilize with a three-stage filter element and perform aseptic packaging after spray drying.

[0024] Example 2 A preparation method of anti-glycation and anti-aging cod collagen peptide, comprising the following steps: (1) Place the dried cod fish skin in an ultrasonic cleaning tank with 60 times the mass of deionized water and perform ultrasonic pretreatment at a temperature of 70 °C, a power of 700 w, and a frequency of 40 kHz for 40 min; (2) After draining, soak in warm water at 80 °C for 12 h at a material-liquid ratio of 1:25. During soaking, add natural anti-glycation active ingredients accounting for 0.8% of the mass of the dried cod fish slices. The natural anti-glycation active ingredients are rosmarinic acid, ferulic acid, and chlorogenic acid with a mass ratio of 3:2:1; (3) Put the soaked cod skin into a high-pressure homogenizer, and perform homogenization treatment at a pressure of 1400 bar, a temperature of 70 °C and a flow rate of 15 L / h, and then perform nano-grinding treatment to form a nano-level homogeneous liquid; (4) After sealing, steam the homogeneous liquid at 110 °C for 3 h; (5) Cool down to 52 °C, adjust the pH to 9, and add complex enzyme I at 3% of the substrate mass in sequence for enzymatic hydrolysis for 1.2 h, and then add complex enzyme II at 1.5% of the substrate mass for enzymatic hydrolysis for 3 h. During the enzymatic hydrolysis of complex enzyme II, when one-third of the enzymatic hydrolysis process remains, the pH of the system gradually decreases from the initial 9 to 6.2; Complex enzyme I is composed of bromelain, flavor protease and papain weighed in a mass ratio of 3:1.2:1.5, and the enzyme activity is greater than 1.5×10 5 u / g; Complex enzyme II is composed of subtilisin, trypsin and β-glucosidase with a mass ratio of 1:2:0.3, and the enzyme activity is greater than 1.8×10 5 u / g; (6) Centrifuge at 3000 rpm for 35 min to separate the supernatant and the residue; (7) After the supernatant is clarified by a ceramic membrane and membrane concentrated, it is fractionated by an ultrafiltration membrane with a molecular weight cut-off of 500-3000 Da; the pore size of the ceramic membrane is 0.2 μm, the membrane filtration pressure is 0.3 MPa, the temperature is controlled at 50 °C, and a pulsed electric field is applied during the clarification process of the ceramic membrane, the electric field strength is 10 V / cm, and the pulse frequency is 50 Hz. The ultrafiltration fractionation uses three-stage series ultrafiltration membranes, which sequentially include polyethersulfone hollow fiber ultrafiltration membranes with molecular weight cut-offs of 3000 Da, 1000 Da and 500 Da, and the feed liquid flow rate during ultrafiltration is 1.0 m / s; (8) Sterilize with a three-stage filter element and perform aseptic packaging after spray drying.

[0025] Example 3 A method for preparing anti-glycation and anti-aging cod collagen peptides, comprising the following steps: (1) Place the dry cod skin in an ultrasonic cleaning tank with 50 times the mass of deionized water, and perform ultrasonic pretreatment at a temperature of 50 °C, a power of 500 w and a frequency of 30 kHz for 30 min; (2) After draining, soak it in warm water at 70 °C at a material-liquid ratio of 1:20 for 10 h, and add 0.7% of the natural anti-glycation active ingredient based on the mass of the dry cod fillet during soaking. The natural anti-glycation active ingredient is rosmarinic acid, ferulic acid and chlorogenic acid with a mass ratio of 3:2:1; (3) Put the soaked cod skin into a high-pressure homogenizer, and perform homogenization treatment at a pressure of 1300 bar, a temperature of 60 °C and a flow rate of 12 L / h, and then perform nano-grinding treatment to form a nano-level homogeneous liquid; (4) After sealing, steam the homogeneous liquid at 105 °C for 2 h; (5) Cool down to 50 °C, adjust the pH to 8, and successively add Complex Enzyme I at 2% of the substrate mass for enzymatic hydrolysis for 1 h, then add Complex Enzyme II at 1% of the substrate mass for enzymatic hydrolysis for 2.5 h; during the enzymatic hydrolysis with Complex Enzyme II, when one-third of the enzymatic hydrolysis process remains, the pH of the system gradually decreases from the initial 8 to 5.8. Complex Enzyme I is prepared by weighing bromelain, flavor protease, and papain in a mass ratio of 2:1:1, and the enzyme activity is greater than 1.5×10 5 u / g; Complex Enzyme II is prepared by weighing subtilisin, trypsin, and β-glucosidase in a mass ratio of 1:1.5:0.2, and the enzyme activity is greater than 1.8×10 5 u / g; (6) Centrifuge at 2500 rpm for 30 min to separate the supernatant and the residue; (7) After the supernatant is clarified by ceramic membrane and membrane concentrated, it is fractionated by an ultrafiltration membrane with a molecular weight cut-off of 500 - 3000 Da; the pore size of the ceramic membrane is 0.1 μm, the membrane filtration pressure is 0.25 MPa, the temperature is controlled at 45 °C, a pulsed electric field is applied during the ceramic membrane clarification process, the electric field strength is 8 V / cm, and the pulse frequency is 30 Hz. The ultrafiltration fractionation uses a three-stage series ultrafiltration membrane, which successively includes polyethersulfone hollow fiber ultrafiltration membranes with molecular weight cut-offs of 3000 Da, 1000 Da, and 500 Da. During the ultrafiltration process, the feed liquid flow rate is 0.8 m / s; (8) Sterilize with a three-stage filter element and perform aseptic packaging after spray drying.

[0026] Comparative Example 1 Other steps are the same as those in Example 3, except that the dosage of Complex Enzyme I in step (5) is 3%.

[0027] Comparative Example 2 Other steps are the same as those in Example 3, except that in step (5), the enzymatic hydrolysis time remains unchanged and Complex Enzyme II is not used.

[0028] Comparative Example 3 Other steps are the same as those in Example 3, except that the dosage of the second complex enzyme in step (5) is 2%.

[0029] Comparative Example 4 Other steps are the same as those in Example 3, except that in step (5), the enzymatic hydrolysis time of the second complex enzyme is extended to 4 h.

[0030] Comparative Example 5 Other steps are the same as those in Example 2, except that the ultrasonic time in step (1) is 1.5 h.

[0031] Comparative Example 6 The other steps are the same as those in Example 3, except that the ultrasonic time in step (1) is 1.5 h and the enzymatic hydrolysis time of the second complex enzyme in step (5) is 4 h.

[0032] Comparative Example 7 The other steps are the same as those in Example 3. Only the nano-grinding treatment in step (3) is omitted, and the parameters of the remaining steps remain unchanged.

[0033] Comparative Example 8 The other steps are the same as those in Example 3. No pulsed electric field is applied during the ceramic membrane clarification process in step (7).

[0034] I. Result Testing The cod fish collagen peptides obtained from the above Examples 1-3 and Comparative Examples 1-8 were tested: 1. Total nitrogen content: The Kjeldahl method was used. The sample was heated and digested together with concentrated sulfuric acid and a catalyst to convert organic nitrogen into ammonium salts. Then, through steps such as distillation, absorption, and titration, the total nitrogen content in the sample was calculated, and then converted into protein content.

[0035] 2. Peptide segment ratio: The high performance liquid chromatography (HPLC) method was used. Using the principle of molecular exclusion chromatography, according to the retention time of peptide segments with different molecular weights, the peptide segments with relative molecular weights of 500-3000 Da, 1000-2000 Da, and 500-1000 Da were separated and measured, and their ratios in the total peptide segments were calculated respectively.

[0036] 3. Yield of cod fish collagen peptide: The dry cod fish skin raw material was accurately weighed, and its original weight (m1, unit: g) was recorded. The collagen peptide powder obtained by final spray drying was completely collected and weighed (m2, unit: g).

[0037] Calculation of yield: Yield (%) = (m2 / m1) × 100%.

[0038] 4. Test Results

[0039] Result Analysis: The total nitrogen contents of Examples 1-3 were all ≥ 14.5%, and that of Example 3 reached 14.8%, which was significantly higher than that of the comparative examples (the highest was 14.2%). This indicates that through the synergistic process of stepwise enzymatic hydrolysis by complex enzymes, pulsed electric field ceramic membrane clarification, and nano-grinding, the extraction purity of protein (peptide segments) was effectively improved in the examples.

[0040] Examples 1-3 were all ≥ 96.7%, and that of Example 3 reached 97.2%, indicating that the ultrafiltration fractionation process and nano-grinding homogenization effectively enriched high-activity peptide segments.

[0041] The yield of Example 3 (18.5%) indicates that the combination of pulsed electric field-assisted membrane clarification and precise enzymatic hydrolysis parameters reduces the generation of ineffective by-products.

[0042] II. In vitro activity verification of anti-aging efficacy 1. DPPH radical scavenging rate Add 1.5 mg of the sample to 1.5 mL of 0.1 mmol / L DPPH· (95% ethanol), mix well and incubate at 25 °C for 30 min. Measure the absorbance at 517 nm. The Vc solution is used as a control. The DPPH· scavenging ability W (%) is calculated as follows: W (%) = [1 - (A1 - A2) / A0] × 100% Where A0 is the absorbance value of 1.5 mL of distilled water and 1.5 mL of 95% ethanol containing 0.1 mmol / L DPPH, A1 is the absorbance value of 1.5 mL of cod collagen oligopeptide containing 0.1 mmol / L DPPH·, and A2 is the absorbance value of 1.5 mL of hydrolysis product and 1.5 mL of 95% ethanol.

[0043] 2. Superoxide anion radical scavenging rate According to the instructions of the anti-superoxide anion radical kit, add different concentrations of cod collagen peptide samples and the reagents contained in the kit in sequence, mix well with a vortex mixer, incubate in a 37 °C constant temperature water bath for 40 min, add the color developer and mix well, and measure the absorbance at 550 nm after 10 min. The calculation formula is: Scavenging rate (%) = 〔(A2 - A1) / A2〕× 100% Where the measurement tube is A1 and the control tube is A2.

[0044] 3. Hydroxyl radical scavenging rate The hydroxyl radicals generated by the Fenton reaction react with salicylic acid and have a special absorption peak at 540 nm. The absorption ability of the hydroxyl radicals of the sample is evaluated according to the magnitude of the absorbance value.

[0045] Take 50 μL of sample solutions with different mass concentrations (5, 10, and 20 mg / mL), add the corresponding reagents according to the kit requirements, mix well, react accurately at 37 °C for 20 min. If the solution is turbid, centrifuge at 8000 r / min at room temperature for 5 min, take 200 μL of the clarified solution and place it in a 96-well plate, and immediately measure the absorbance value at 510 nm. Among them, the measurement tube is A1, the control tube is A2, and the blank tube is A0. Set 3 groups in parallel and use the following formula to calculate the scavenging rate of hydroxyl radicals.

[0046] Scavenging rate (%) = 〔1 - (A1 - A2) / A0〕× 100% The results are as Figures 1 - 3, as the concentration increases, the scavenging rate of cod collagen peptide on DPPH free radicals gradually increases, proving that it has a good linear dependence on this free radical; cod collagen peptide has a good scavenging effect on superoxide anion free radicals, and when the total concentration is 20 mg / mL, it is close to 100% scavenging; as the concentration increases, cod collagen peptide has a good scavenging effect on hydroxyl free radicals, and when the total concentration is 30 mg / mL, the scavenging rate is the highest.

[0047] III. In vitro activity verification of anti-glycation effect Comparative Example 9 Other steps are the same as those in Example 3, omitting the addition of natural anti-glycation active ingredients in step (2), and the parameters of the remaining steps remain unchanged.

[0048] Fructose model preparation: Mix 2 mL of fructose solution (300 mg / mL) with 2 mL of cod collagen peptide solutions with different mass concentrations in Example 3 and Comparative Example 9, add 2 mL of 30 mg / mL BSA (bovine serum albumin) solution. All the above reactants are dissolved in 50 mmol / L phosphate buffer solution with a pH value of 7.4 (containing 0.02% procLin300). Use a certain mass concentration of (0.1, 1.5 mg / mL) aminoguanidine (AG) solution to replace the cod collagen peptide solution as the positive control group, use phosphate buffer solution to replace the composition solution as the blank group, and use phosphate buffer solution to replace the fructose solution as the co-incubation group of BSA and cod collagen peptide solution. Each mass concentration is repeated in parallel 3 times. After incubating each sample in a water bath at 50 °C for 24 h, cool it with running water.

[0049] Determination of fructosamine content: Add 0.2 mL of glycated protein samples treated with different masses of cod collagen peptide samples and 0.8 mL of nitroblue tetrazolium chloride reagent (0.3 mmol) to 2 mL of carbonate buffer solution (100 mmol / L, pH value of 10.1). After reacting at room temperature for 30 min, measure the OD value at 530 nm with an enzyme-labeled instrument.

[0050] The calculation formula for the inhibition rate of fructosamine (%) is as follows: R1 = 〔(F0 - F1) / F0〕× 100% Where: R1 - inhibition rate of fructosamine, %; F0 - OD value of the sample in the absence of cod collagen peptide (or AG); F1 - OD value of the sample in the presence of cod collagen peptide (or AG).

[0051] Test results:

[0052] Reference Figure 4, as the concentration increases, cod collagen peptide has a good inhibitory effect on fructosamine. When the total concentration is 2.5 mg / mL, the inhibition rate is the highest, reaching 42%.

[0053] In summary, cod collagen peptide can not only resist aging but also resist glycation, which opens up new ways for the application of products.

[0054] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of anti-glycation and anti-aging cod fish collagen peptide, characterized in that: It includes the following steps: (1) Place dried cod skin in deionized water with a mass 40 - 60 times that of the dried cod skin, and perform ultrasonic pretreatment at a temperature of 30 - 70°C, a power of 300 - 700 W, and a frequency of 20 - 40 kHz for 20 - 40 min; (2) After draining, add natural anti-glycation active ingredients accounting for 0.5 - 0.8% of the mass of the dried cod fish slices to warm water at 60 - 80°C at a solid-liquid ratio of 1:15 - 25 and soak for 8 - 12 h; (3) Put the soaked cod skin into a high-pressure homogenizer, perform homogenization treatment at a pressure of 1200 - 1400 bar, a temperature of 50 - 70°C, and a flow rate of 8 - 15 L / h, and then perform nano-grinding treatment to form a nano-level homogeneous liquid; (4) Seal the homogeneous liquid and cook it at 100 - 110°C for 1 - 3 h; (5) Cool down to 50 ± 2°C, adjust the pH to 7 - 9, add complex enzyme I accounting for 1 - 3% of the substrate mass and enzymolyze for 0.8 - 1.2 h, then add complex enzyme II accounting for 0.5 - 1.5% of the substrate mass and enzymolyze for 2.5 ± 0.5 h; (6) Centrifuge at a rotational speed of 2000 - 3000 rpm for 25 - 35 min to separate the supernatant and the residue; (7) After the supernatant is clarified by a ceramic membrane and membrane concentrated, it is fractionated by an ultrafiltration membrane with a molecular weight cut-off of 500 - 3000 Da; (8) Sterilize with a three-stage filter element, and perform aseptic packaging after spray drying.

2. The preparation method of a sugar-resistant and anti-aging cod collagen peptide according to claim 1, wherein: The natural anti-glycation active ingredient in step (2) is rosmarinic acid, ferulic acid, and chlorogenic acid with a mass ratio of 3:2:

1.

3. The preparation method of an anti-glycation and anti-aging cod fish collagen peptide according to claim 1, characterized in that, The nano-grinding treatment in step (3) is to grind with zirconia beads with a particle size of 0.1 - 0.3 mm at a rotational speed of 1000 - 1500 rpm for 20 - 30 min to reduce the average particle size of the material to 1 - 2 μm and form a nano-level homogeneous liquid.

4. The preparation method of an anti-glycation and anti-aging cod fish collagen peptide according to claim 1, characterized in that, The complex enzyme I in step (5) is prepared by weighing bromelain, flavorzyme and papain in a mass ratio of (1 - 3):(0.2 - 1.2):(0.3 - 1.5), and the enzyme activity of each is greater than 1.5×10 5 u / g.

5. The preparation method of an anti-glycation and anti-aging cod collagen peptide according to claim 1, characterized in that, The complex enzyme II in the step (5) is subtilisin, trypsin and β-glucosidase with a mass ratio of 1:(1-2):(0.1-0.3), and the enzyme activity of each is greater than 1.8×10 5 u / g.

6. The preparation method of an anti-glycation and anti-aging cod collagen peptide according to claim 1, characterized in that, In step (7), the aperture of the ceramic membrane is 0.1 - 0.2 μm, the membrane filtration pressure is 0.2 - 0.3 MPa, and the temperature is controlled at 40 - 50°C.

7. The preparation method of an anti-glycation and anti-aging cod fish collagen peptide according to claim 6, characterized in that, During the clarification process of the ceramic membrane, a pulsed electric field is applied, the electric field intensity is 5 - 10 V / cm, and the pulse frequency is 20 - 50 Hz.

8. The preparation method of a sugar-resistant and anti-aging cod collagen peptide as described in claim 1, characterized in that, In step (7), the ultrafiltration fractionation uses a three-stage series ultrafiltration membrane, which sequentially includes polyethersulfone hollow fiber ultrafiltration membranes with molecular weight cut-offs of 3000 Da, 1000 Da, and 500 Da. During the ultrafiltration process, the flow rate of the feed liquid is 0.5 - 1.0 m / s, and the peptide segments in the molecular weight range of 500 - 3000 Da are collected as the target product.

9. A cod fish collagen peptide prepared by the method according to any one of claims 1-8, characterized in that, Its total nitrogen content ≥ 14.5%, the proportion of the target peptide segments ≥ 96.7%, and the yield of cod collagen peptides ≥ 18.1%.

10. Use of the cod fish collagen peptide as described in claim 9 in the preparation of anti-aging and anti-glycation skin care products, characterized in that: The skin care products include essence, facial mask, or cream.

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