Anti-sugar and anti-aging pollock collagen peptide, and preparation method and application thereof

Abstract

The application provides an anti-sugar and anti-aging cod collagen peptide as well as a preparation method and application thereof. The method significantly improves the yield and functional characteristics of active peptides by ultrasonic wave and nanometer grinding pretreatment, combined with stepwise directional enzymolysis and three-stage ultrafiltration membrane precise separation technology. Specifically, after the dry cod skin is pretreated by ultrasonic wave, it is soaked and activated in warm water containing natural anti-sugar ingredients; the collagen fibers are dissociated by high-pressure homogenization and nanoscale zirconium oxide bead grinding; high-purity peptide segments with a molecular weight of 500-3000 Da are obtained by stepwise enzymolysis of complex enzyme I and enzyme II combined with three-stage polyether sulfone ultrafiltration membrane gradient interception. The total nitrogen content of the obtained product is greater than or equal to 14.5%, the proportion of target peptide segments is greater than or equal to 96.7%, and the yield is greater than or equal to 18.1%. The product has significant anti-sugar and anti-aging activities, and can be applied to skin care products such as essence and mask to realize collagen repair and skin barrier function improvement.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular to an anti-sugar and anti-aging cod collagen peptide, a preparation method and application thereof. BACKGROUND

[0002] The common extraction methods of cod collagen protein include hot water method, acid method, alkali method, enzyme method and salt method. Among them, enzyme method is considered to be the highest in biological activity of the product due to its good stability, mild extraction conditions, adjustable and controllable reaction rate, environmental friendliness and other advantages combined with other research data. Studies have shown that cod collagen protein not only can effectively supplement the lost collagen protein of the skin, but also shows significant effects in anti-aging, anti-sugar and slowing down skin aging.

[0003] In terms of anti-aging, cod collagen protein 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 production of wrinkles. At the same time, it also has antioxidant effect, can remove free radicals in the body, reduce the damage of oxidative stress to skin cells, and delay the aging process of cells.

[0004] In terms of anti-sugar, cod collagen protein can inhibit glycosylation reaction and reduce the generation of advanced glycosylation end products (AGEs). AGEs are the products of non-enzymatic glycosylation reaction of sugars with macromolecular substances such as proteins and lipids. The accumulation of AGEs in the skin can cause cross-linking and denaturation of collagen and elastic fibers, resulting in loss of skin elasticity and dark color. By inhibiting the generation of AGEs, cod collagen protein blocks the damage of glycosylation 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 protein can enhance the moisturizing ability of the skin and maintain the water balance of the skin. The stability of skin moisture content is crucial for maintaining the softness, luster and elasticity of the skin. In addition, it can also regulate the immune function of the skin and enhance the skin barrier function to resist the invasion of external environmental factors on the skin, further slowing down the aging speed of the skin.

[0006] Although there have been many reports on the anti-aging, anti-sugar and slowing down of skin aging of cod collagen protein, there are still many problems in this field to be further explored. For example, the absorption mechanism of cod collagen protein in the body, the optimal supplement dosage and method, the safety and effectiveness of long-term supplementation, etc. are not perfect enough. Therefore, further revealing the mechanism of cod collagen protein in anti-aging, anti-sugar and slowing down of skin aging has important theoretical significance and practical application value for developing efficient and safe anti-aging skin care products and improving people's skin health level. SUMMARY

[0007] Therefore, the application provides a sugar-resistant and anti-aging cod collagen peptide as well as a preparation method and application thereof.

[0008] The technical scheme of the application is as follows: a preparation method of a sugar-resistant and anti-aging cod collagen peptide, comprising the following steps:

[0009] (1) placing dry cod skin in an ultrasonic cleaning tank filled with deionized water with a mass ratio of 40-60, and performing ultrasonic pretreatment at a temperature of 30-70 DEG C, a power of 300-700 W and a frequency of 20-40 kHz for 20-40 min;

[0010] (2) after draining, soaking the cod skin in warm water at a solid-liquid ratio of 1:15-25 for 8-12 h;

[0011] (3) putting the soaked cod skin into a high-pressure homogenizer, and performing homogenization treatment at a pressure of 1200-1400 bar, a temperature of 50-70 DEG C and a flow rate of 8-15 L / h, and then performing nano-grinding treatment to form a nano-sized homogenate;

[0012] (4) sealing the homogenate and cooking it at a temperature of 100-110 DEG C for 1-3 h;

[0013] (5) cooling the homogenate to a temperature of 50±2 DEG C, adjusting the pH to 7-9, adding a compound enzyme I with a mass ratio of 1-3% to the homogenate, and performing enzyme hydrolysis for 0.8-1.2 h, then adding a compound enzyme II with a mass ratio of 0.5-1.5% to the homogenate, and performing enzyme hydrolysis for 2.5±0.5 h, wherein during the enzyme hydrolysis of the compound enzyme II, the pH of the system is gradually reduced from 7-9 to 5.5-6.2 when the remaining one-third of the enzyme hydrolysis process is completed;

[0014] (6) centrifuging the homogenate at a speed of 2000-3000 rpm for 25-35 min to separate the supernatant from the residue;

[0015] (7) clarifying the supernatant by ceramic membrane, concentrating the supernatant by membrane, and then grading the supernatant by an ultrafiltration membrane with a molecular weight cutoff of 500-3000 Da;

[0016] (8) sterilizing the supernatant by a three-stage filter cartridge, and then performing spray drying and aseptic packaging.

[0017] Further, the natural sugar-resistant active ingredient is added to the cod skin during the soaking process in step (2), and the natural sugar-resistant active ingredient is a mixture of rosemary acid, ferulic acid and chlorogenic acid with a mass ratio of 3:2:1.

[0018] Further, the nano-milling treatment of step (3) is to use zirconium oxide beads with a particle size of 0.1-0.3mm to mill at a speed of 1000-1500rpm for 20-30min, so that the average particle size of the material is reduced to 1-2um, and a nano-level homogeneous liquid is formed.

[0019] Further, the complex enzyme I of step (5) is bromelain, flavor protease and papain with a mass ratio of (1-3):(0.2-1.2):(0.3-1.5), and the enzyme activity is greater than 1.5x10 5 u / g.

[0020] Further, the complex enzyme II of step (5) is bacillus subtilis protease, trypsin and beta-glucosidase with a mass ratio of 1:(1-2):(0.1-0.3), and the enzyme activity is greater than 1.8x10 5 u / g. The beta-glucosidase can degrade the glycosaminoglycan impurities in the cod skin, and improve the anti-glycosylation activity of the collagen peptide.

[0021] Further, the pore size of the ceramic membrane in step (7) is 0.1-0.2um, the membrane filtration pressure is 0.2-0.3MPa, and the temperature is controlled at 40-50℃.

[0022] Further, a pulse electric field is applied during the ceramic membrane clarification process, the electric field strength is 5-10V / cm, the pulse frequency is 20-50Hz, an alternating electric field is formed on the membrane surface, membrane pollution is inhibited, and the impurity retention efficiency is improved by more than 30%.

[0023] Further, the hollow fiber ultrafiltration membrane is used for the ultrafiltration classification in step (7), and the liquid flow rate during the ultrafiltration process is 0.5-1.0m / s.

[0024] Further, a kind of cod collagen peptide, total nitrogen content is greater than or equal to 14.5%, target peptide segment ratio is greater than or equal to 96.7%, and cod collagen peptide yield is greater than or equal to 18.1%.

[0025] Further, the cod collagen peptide is applied to the preparation of anti-aging, anti-sugar skin care products, and the skin care products include essence, mask or cream.

[0026] Compared with the prior art, the beneficial effects of the present application are:

[0027] The cod skin is treated by ultrasonic cleaning in the application, which makes the surface impurities be cleaned more thoroughly. The ultrasonic can be heated at the same time, which avoids the subsequent heat treatment. The cavitation effect of ultrasonic improves the soaking and crushing efficiency of cod skin, which is beneficial to improve the extraction rate in the later stage. The addition of natural anti-sugar active ingredients realizes the preliminary loading of anti-sugar components during the soaking process. On the other hand, the treatment efficiency of cod skin is also improved by the high-pressure homogenization pretreatment method, which shortens the extraction time. The nano-grinding treatment significantly improves the solubility and bioavailability of collagen peptides. The cod skin liquid after homogenization and grinding treatment is subjected to two-step enzymolysis. The enzyme hydrolysis site is unique, and the enzyme hydrolysis degree is more thorough. The addition of β-glucosidase is used to degrade the glycosaminoglycan impurities in the cod skin, which improves the anti-glycosylation activity of collagen peptides. Through the synergistic process of compound enzyme step-by-step enzymolysis, pulse electric field ceramic membrane clarification and nano-grinding, the functional peptide segment of 500-3000 Da is directionally enriched, so that the prepared cod collagen peptide has the characteristics of good anti-aging effect and easy absorption. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The DPPH free radical scavenging rate of the cod collagen peptide of Example 3;

[0029] Figure 2 The superoxide anion free radical scavenging rate of the cod collagen peptide of Example 3;

[0030] Figure 3 The hydroxyl radical scavenging rate of the cod collagen peptide of Example 3;

[0031] Figure 4 The fructosamine inhibition rate of the cod collagen peptide of Example 3, and the left side of the figure is the inhibition rate of the positive control group at 0.1 and 1.5 mg / mL. DETAILED DESCRIPTION

[0032] In order to better understand the technical content of the application, the following specific examples are provided to further illustrate the application.

[0033] The experimental methods used in the embodiments of the application are conventional methods unless otherwise specified.

[0034] The materials, reagents, etc. used in the embodiments of the application can be obtained from commercial channels unless otherwise specified.

[0035] Example 1

[0036] A preparation method of an anti-sugar and anti-aging cod collagen peptide, comprising the following steps:

[0037] (1) Dry cod skin is placed in an ultrasonic cleaning tank with 40 times the mass of deionized water, and is ultrasonically pretreated at a temperature of 30℃, a power of 300w, and a frequency of 20kHz for 20min;

[0038] (2) After draining, soak in warm water at 60℃ for 8h at a solid-liquid ratio of 1:15, and add 0.5% of a natural anti-sugar active ingredient based on the mass of the dry cod fillets during soaking, the natural anti-sugar active ingredient being rosemary acid, ferulic acid, and chlorogenic acid at a mass ratio of 3:2:1;

[0039] (3) The soaked cod skin is placed in a high-pressure homogenizer, and is homogenized at a pressure of 1200bar, a temperature of 50℃, and a flow rate of 8L / h, and is then nano-ground to form a nano-sized homogenate;

[0040] (4) The homogenate is sealed and cooked at 100℃ for 1h;

[0041] (5) Cool to 50℃, adjust the pH to 7, add 1% of a complex enzyme I based on the mass of the substrate, and enzymatically hydrolyze for 0.8h, then add 0.5% of a complex enzyme II based on the mass of the substrate, and enzymatically hydrolyze for 2.5h, during the complex enzyme II enzymatic hydrolysis process, the pH of the system is gradually reduced from the initial 7 to 5.5 when one-third of the enzymatic hydrolysis process is complete; the complex enzyme I is composed of bromelain, flavor protease, and papain at a mass ratio of 1:0.2:0.3, and the enzyme activity of each is greater than 1.5×10 5 u / g; the complex enzyme II is composed of bacillus subtilis protease, trypsin, and β-glucosidase at a mass ratio of 1:1:0.1, and the enzyme activity of each is greater than 1.8×10 5 u / g;

[0042] (6) Centrifuge at 2000rpm for 25min to separate the supernatant from the residue;

[0043] (7) The supernatant is clarified by ceramic membrane, concentrated by membrane, and then fractionated by ultrafiltration membrane with a molecular weight cutoff of 500-3000Da; the pore size of the ceramic membrane is 0.1μm, the membrane filtration pressure is 0.2MPa, the temperature is controlled at 40℃, a pulse electric field is applied during the ceramic membrane clarification process, the electric field strength is 5V / cm, and the pulse frequency is 20Hz; the ultrafiltration fractionation uses three-stage series-connected ultrafiltration membranes, including polyether sulfone hollow fiber ultrafiltration membranes with molecular weight cutoffs of 3000Da, 1000Da, and 500Da, respectively, and the flow rate of the feed liquid during the ultrafiltration process is 0.5m / s;

[0044] (8) Sterilize by three-stage filter cartridges, spray dry, and then aseptically package.

[0045] Example 2

[0046] A method for preparing an anti-sugar, anti-aging cod collagen peptide, comprising the following steps:

[0047] (1) Dry cod skin is placed in an ultrasonic cleaning tank with 60 times the mass of deionized water, and is ultrasonically pretreated at a temperature of 70℃, a power of 700w, and a frequency of 40kHz for 40min;

[0048] (2) After draining, soak in 80℃ warm water at a solid-liquid ratio of 1:25 for 12h, and add 0.8% of the mass of the dry cod skin of natural anti-sugar active ingredients during soaking, the natural anti-sugar active ingredients being rosemary acid, ferulic acid, and chlorogenic acid at a mass ratio of 3:2:1;

[0049] (3) The soaked cod skin is placed into a high-pressure homogenizer, and is homogenized at a pressure of 1400bar, a temperature of 70℃, and a flow rate of 15L / h, and is then nano-grinded to form a nano-sized homogenate;

[0050] (4) The homogenate is sealed and cooked at 110℃ for 3h;

[0051] (5) Cool to 52℃, adjust the pH to 9, and sequentially add 3% of the mass of the substrate of compound enzyme I for enzymolysis for 1.2h, and then add 1.5% of the mass of the substrate of compound enzyme II for enzymolysis for 3h, and during the compound enzyme II enzymolysis process, the pH of the system is gradually reduced from the initial 9 to 6.2 when one-third of the enzymolysis process is completed; compound enzyme I is composed of bromelain, flavor protease, and papain at a mass ratio of 3:1.2:1.5, and the enzyme activity of each is greater than 1.5×10 5 u / g; compound enzyme II is composed of bacillus subtilis protease, trypsin, and β-glucosidase at a mass ratio of 1:2:0.3, and the enzyme activity of each is greater than 1.8×10 5 u / g;

[0052] (6) Centrifuge at 3000rpm for 35min to separate the supernatant from the residue;

[0053] (7) The supernatant is clarified by ceramic membrane, concentrated by membrane, and then fractionated by ultrafiltration membrane with a molecular weight cutoff of 500-3000Da; the pore size of the ceramic membrane is 0.2μm, the membrane filtration pressure is 0.3MPa, the temperature is controlled at 50℃, a pulse electric field is applied during the ceramic membrane clarification process, the electric field strength is 10V / cm, the pulse frequency is 50Hz, three-stage series ultrafiltration membranes are used for ultrafiltration fractionation, including polyether sulfone hollow fiber ultrafiltration membranes with molecular weight cutoffs of 3000Da, 1000Da, and 500Da, respectively, and the flow rate of the feed liquid during the ultrafiltration process is 1.0m / s;

[0054] (8) Three-stage filter cartridges are used for sterilization, and then the product is packaged after spray drying.

[0055] Example 3

[0056] A method for preparing an anti-sugar, anti-aging cod collagen peptide, comprising the following steps:

[0057] (1) Dry cod skin is placed in an ultrasonic cleaning tank with 50 times the mass of deionized water, and is ultrasonically pretreated at a temperature of 50°C, a power of 500w, and a frequency of 30 kHz for 30 min;

[0058] (2) After draining, the cod skin is soaked in warm water at 70°C for 10 h at a solid-liquid ratio of 1:20, and 0.7% of a natural anti-sugar active ingredient by mass of the dry cod skin is added during the soaking, the natural anti-sugar active ingredient being rosemary acid, ferulic acid, and chlorogenic acid at a mass ratio of 3:2:1;

[0059] (3) The soaked cod skin is placed in a high-pressure homogenizer, and is homogenized at a pressure of 1300 bar, a temperature of 60°C, and a flow rate of 12 L / h, and is then subjected to nanometer grinding to form a nanoscale homogenate;

[0060] (4) The homogenate is sealed and cooked at 105°C for 2 h;

[0061] (5) The temperature is lowered to 50°C, the pH is adjusted to 8, 2% of a complex enzyme I by mass of the substrate is added, and the mixture is enzymatically hydrolyzed for 1 h, then 1% of a complex enzyme II by mass of the substrate is added, and the mixture is enzymatically hydrolyzed for 2.5 h; during the enzymatic hydrolysis of the complex enzyme II, the pH of the system is gradually lowered from the initial 8 to 5.8 when one-third of the enzymatic hydrolysis process is completed, the complex enzyme I is prepared by weighing bromelain, flavourzyme, and papain at a mass ratio of 2:1:1, and the enzyme activity of each is greater than 1.5×10 5 u / g; the complex enzyme II is prepared by weighing bacillus subtilis protease, trypsin, and β-glucosidase at a mass ratio of 1:1.5:0.2, and the enzyme activity of each is greater than 1.8×10 5 u / g;

[0062] (6) The mixture is centrifuged at a speed of 2500 rpm for 30 min, and the supernatant is separated from the residue;

[0063] (7) The supernatant is clarified by ceramic membrane, concentrated by membrane, and then fractionated by ultrafiltration membrane with a molecular weight cutoff 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 pulse electric field is applied during the ceramic membrane clarification process, the electric field strength is 8 V / cm, the pulse frequency is 30 Hz, the ultrafiltration fractionation adopts three-stage series connection of ultrafiltration membranes, including polyether sulfone hollow fiber ultrafiltration membranes with a molecular weight cutoff of 3000 Da, 1000 Da, and 500 Da, respectively, and the flow rate of the feed liquid during the ultrafiltration process is 0.8 m / s;

[0064] (8) The three-stage filter cartridges are sterilized, and the product is packaged after spray drying.

[0065] Comparative Example 1

[0066] The other steps are the same as in Example 3, except that the amount of the complex enzyme I used in step (5) is 3%.

[0067] Comparative Example 2

[0068] Other steps are the same as Example 3, except that the enzyme hydrolysis time in step (5) is unchanged, and no complex enzyme II is used.

[0069] Comparative Example 3

[0070] Other steps are the same as Example 3, except that the amount of the second complex enzyme in step (5) is 2%.

[0071] Comparative Example 4

[0072] Other steps are the same as Example 3, except that the enzyme hydrolysis time of the second complex enzyme in step (5) is extended to 4h.

[0073] Comparative Example 5

[0074] Other steps are the same as Example 2, except that the ultrasonic time in step (1) is 1.5h.

[0075] Comparative Example 6

[0076] Other steps are the same as Example 3, except that the ultrasonic time in step (1) is 1.5h, and the enzyme hydrolysis time of the second complex enzyme in step (5) is 4h.

[0077] Comparative Example 7

[0078] Other steps are the same as Example 3, except that the nanomilling treatment in step (3) is omitted, and the remaining step parameters are unchanged.

[0079] Comparative Example 8

[0080] Other steps are the same as Example 3, except that no pulse electric field is applied during the ceramic membrane clarification process in step (7).

[0081] I. Results Test

[0082] The cod collagen peptides obtained in Examples 1-3 and Comparative Examples 1-8 above were tested:

[0083] 1. Total nitrogen content: Kjeldahl nitrogen determination method was used. The sample was heated and digested with concentrated sulfuric acid and catalyst to convert organic nitrogen into ammonium salt. Then, through distillation, absorption, titration and other steps, the total nitrogen content in the sample was calculated, and then converted into protein content.

[0084] 2. Peptide segment ratio: High performance liquid chromatography (HPLC) method was used. According to the principle of molecular weight exclusion chromatography, the retention time of different molecular weight peptide segments was used to separate and determine the peptide segments with relative molecular mass of 500-3000Da, 1000-2000Da and 500-1000Da, and their ratios in the total peptide segments were calculated respectively.

[0085] 3. Cod collagen peptide yield: accurately weigh the dry cod skin raw material, record its original weight (m1, unit: g), and completely collect the collagen peptide powder obtained by final spray drying, weigh (m2, unit: g).

[0086] Calculate the yield: yield (%) = (m2 / m1) x 100%.

[0087] 4. Test results

[0088]

[0089] Result analysis:

[0090] The total nitrogen content of examples 1-3 is all ≥14.5%, among which example 3 reaches 14.8%, which is significantly higher than the comparative example (the highest is 14.2%). This shows that the example effectively improves the purity of protein (peptide segment) by the synergistic process of composite enzyme step-by-step enzymolysis, pulse electric field ceramic membrane clarification, and nano-grinding.

[0091] Examples 1-3 are all ≥96.7%, and example 3 reaches 97.2%, indicating that the ultrafiltration fractionation process and nano-grinding homogenization effectively enrich high-activity peptide segments.

[0092] The yield of example 3 (18.5%) shows that the combination of pulse electric field assisted membrane clarification and precise enzymolysis parameters reduces the generation of ineffective by-products.

[0093] II. In vitro activity verification of anti-aging efficacy

[0094] 1. DPPH free radical scavenging rate

[0095] 1.5 mg of sample was added to 1.5 mL of 0.1 mmol / L DPPH· (95% ethanol), mixed and incubated at 25°C for 30 min. The absorbance was measured at 517 nm. Vc solution was used as a control. The DPPH· scavenging capacity W (%) was calculated as follows:

[0096] W (%) = [1-(A1-A2) / A0] x 100%

[0097] Where A0 is the absorbance value of 1.5 mL distilled water and 1.5 mL 0.1 mmol / L DPPH-containing 95% ethanol, A1 is the absorbance value of 1.5 mL cod collagen oligopeptide containing 0.1 mmol / L DPPH·, and A2 is the absorbance value of 1.5 mL hydrolyzate and 1.5 mL 95% ethanol.

[0098] 2. Superoxide anion radical scavenging rate

[0099] According to the anti-superoxide anion radical kit instructions, different concentrations of cod collagen peptide samples were added to the reagents contained in the kit in turn, mixed well with a vortex mixer, placed in a 37℃ constant temperature water bath for 40min, then mixed with the chromogenic reagent, measured the absorbance at 550nm after 10min. The calculation formula is:

[0100] Clearance rate (%) = 〔(A2-A1) / A2〕x100%

[0101] Where A1 is the determination tube, and A2 is the control tube.

[0102] 3. Hydroxyl radical clearance rate

[0103] The hydroxyl radical generated by Fenton reaction reacts with salicylic acid, which has a special absorption peak at 540nm. The absorption capacity of hydroxyl radical of the sample is evaluated according to the size of the absorbance value.

[0104] Take 50μL of different mass concentrations (5, 10 and 20mg / mL) of sample solution, add the corresponding reagents according to the kit requirements, mix well, and react at 37℃ for 20min. If the solution is turbid, centrifuge at room temperature at 8000r / min for 5min, take 200μL of clear liquid in a 96-well plate, and immediately measure the absorbance value at 510nm. Where A1 is the determination tube, A2 is the control tube, and A0 is the blank tube. Set 3 groups in parallel, and calculate the clearance rate of hydroxyl radical using the following formula.

[0105] Clearance rate (%) = 〔1-(A1-A2) / A0〕x100%

[0106] The results are as follows Figures 1-3 As the concentration increases, the clearance rate of cod collagen peptide on DPPH free radical gradually increases, which proves that it has a good linear dependence on the free radical. Cod collagen peptide has good clearance effect on superoxide anion radical, and the total concentration of 20mg / mL is close to 100% clearance. As the concentration increases, cod collagen peptide has good clearance effect on hydroxyl radical, and the total concentration of 30mg / mL has the highest clearance rate.

[0107] III. In vitro activity verification of anti-sugar efficacy

[0108] Comparative Example 9

[0109] The other steps are the same as those in Example 3, and the addition of natural anti-sugar active ingredients in step (2) is omitted, and the remaining step parameters remain unchanged.

[0110] Fructose model preparation: 2 mL of fructose solution (300 mg / mL) was mixed with 2 mL of different mass concentrations of cod collagen peptide solution of Example 3 and Comparative Example 9, 2 mL of 30 mg / mL BSA (bovine serum albumin) solution was added, and the above reactants were dissolved with 50 mmol / L phosphate buffer solution (containing 0.02% procLin300) having a pH value of 7.4, a certain mass concentration (0.1, 1.5 mg / mL) of aminoguanidine (AG) solution was used instead of the cod collagen peptide solution as a positive control group, the phosphate buffer solution was used instead of the composition solution as a blank group, and the phosphate buffer solution was used instead of the fructose solution as a BSA and cod collagen peptide solution co-incubation group, each mass concentration was repeated 3 times, and each sample was incubated in a 50℃ water bath for 24 h, and then cooled with running water.

[0111] Fructose amine content determination: 0.2 mL of glycosylated protein sample treated with different mass cod collagen peptides and 0.8 mL of nitro tetrazolium chloride blue reagent (0.3 mmol) were added to 2 mL of carbonate buffer (100 mmol / L, pH 10.1), and after reaction at room temperature for 30 min, the OD value was determined at 530 nm with an enzyme-labeled instrument.

[0112] The formula for calculating the inhibition rate (%) of fructose amine is as follows:

[0113] R1= 〔(F0-F1) / F0〕×100%

[0114] In the formula, R1 is the inhibition rate of fructose amine, %, F0 is the OD value of the sample in the absence of cod collagen peptide (or AG), and F1 is the OD value of the sample in the presence of cod collagen peptide (or AG).

[0115] Test results:

[0116]

[0117] Reference Figure 4 As the concentration increases, the cod collagen peptide has a good inhibitory effect on fructose amine, and when the total concentration is 2.5 mg / mL, the inhibition rate is the highest, reaching 42%.

[0118] In summary, the cod collagen peptide not only has anti-aging effect, but also has anti-sugar effect, which opens up a new way for the application of the product.

[0119] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing an anti-sugar, anti-aging pollock collagen peptide, characterized by: The method comprises the following steps: (1) placing dry cod skin in 40-60 times mass of deionized water, and performing ultrasonic pretreatment at a temperature of 30-70 DEG C, a power of 300-700 W, and a frequency of 20-40 kHz for 20-40 min; (2) after draining, soaking the cod skin in warm water at 60-80 DEG C with the addition of 0.5-0.8% of natural anti-sugar active ingredients by mass of the cod skin, at a solid-liquid ratio of 1:15-25, for 8-12 h; (3) placing the soaked cod skin into a high-pressure homogenizer, and performing homogenization treatment at a pressure of 1200-1400 bar, a temperature of 50-70 DEG C, and a flow rate of 8-15 L / h, and then performing nano-grinding treatment, wherein the nano-grinding treatment is performed by using zirconium oxide beads with a particle size of 0.1-0.3 mm at a rotating speed of 1000-1500 rpm for 20-30 min, so that the average particle size of the material is reduced to 1-2 mu m, and a nano-level homogenate is formed; (4) sealing the homogenate and cooking it at 100-110 DEG C for 1-3 h; (5) cooling the homogenate to 50±2 DEG C, adjusting the pH to 7-9, adding 1-3% of a composite enzyme I by mass of the substrate, and performing enzymolysis for 0.8-1.2 h, and then adding 0.5-1.5% of a composite enzyme II by mass of the substrate, and performing enzymolysis for 2.5±0.5 h; (6) centrifuging the homogenate at a rotating speed of 2000-3000 rpm for 25-35 min, and separating the supernatant from the residue; (7) clarifying the supernatant by ceramic membrane, concentrating the supernatant by membrane, and grading the supernatant by ultrafiltration membrane with a molecular weight cutoff of 500-3000 Da; (8) sterilizing the supernatant by a three-stage filter cartridge, and packaging the sterilized supernatant by spray drying; the natural anti-sugar active ingredients in step (2) are rosmarinic acid, ferulic acid, and chlorogenic acid at a mass ratio of 3:2:1; The step (5) compound enzyme I is weighed with bromelain, flavour protease and papain in a mass ratio of (1-3):(0.2-1.2):(0.3-1.5), and the enzyme activity is greater than 1.5×10 5 u / g. The step (5) compound enzyme II is Bacillus subtilis protease, trypsin and beta-glucosidase in a mass ratio of 1:(1-2):(0.1-0.3), and the enzyme activity is greater than 1.8×10 5 u / g. in the ceramic membrane clarification process in step (7), a pulse electric field is applied, the electric field strength is 5-10 V / cm, and the pulse frequency is 20-50 Hz; the ultrafiltration grading is performed by using three-stage series-connected ultrafiltration membranes, which sequentially include polyether sulfone hollow fiber ultrafiltration membranes with molecular weight cutoffs 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 with a molecular weight of 500-3000 Da are collected as target products, i.e., cod collagen peptides; the total nitrogen content of the cod collagen peptides is greater than or equal to 14.5%, the proportion of the target peptide segments is greater than or equal to 96.7%, and the yield of the cod collagen peptides is greater than or equal to 18.1%.

2. The method for preparing cod collagen peptides with anti-glycation and anti-aging properties as described in claim 1, characterized in that, in step (7), the pore size of the ceramic membrane is 0.1-0.2 mu m, the membrane filtration pressure is 0.2-0.3 MPa, and the temperature is controlled at 40-50 DEG C.

3. The use of the cod collagen peptide prepared by the method of claim 1 in the preparation of anti-aging, anti-sugar skin care products, characterized by: the skin care product includes an essence, a mask, or a cream.

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