Preparation method of collagen yeast peptide

Abstract

The invention provides a preparation method of collagen yeast peptide, and belongs to the technical field of collagen biology. The preparation method comprises the following steps: adding collagen into water, uniformly stirring, then adding a nutrition enhancer accounting for 8-12wt% of the mixed system, and adjusting the pH value to 5.8-6.2 to obtain a fermentation substrate; and inoculating lactobacillus rhamnosus and lactobacillus plantarum into the fermentation substrate, controlling the dissolved oxygen to be 20-30% in the first 24 hours, reducing the dissolved oxygen to be 5-10% in the later 48 hours, and filtering and drying the fermented product to obtain the collagen fermented peptide. Through synergistic fermentation of composite strains, the product is rich in 300-1500Da bioactive peptide, Pro-Hyp-Gly and gamma-aminobutyric acid and has the functions of inhibiting tyrosinase, resisting oxidation, whitening and regulating intestinal flora, the melanin reduction rate reaches 22% in clinical 8 weeks, and the shelf life is prolonged to 18 months.

Description

Technical Field

[0001] The present invention relates to the field of collagen biotechnology, and in particular to a method for preparing collagen enzyme peptide. Background Art

[0002] Collagen peptides are small molecule peptides produced through enzymatic hydrolysis or fermentation of collagen. They possess properties such as low molecular weight, easy absorption, and low viscosity. They not only retain the biological activity of collagen but also possess a variety of physiological functions, including immunomodulation, antioxidant, anti-aging, anti-thrombotic, blood pressure, and cholesterol-lowering properties. Therefore, collagen peptides have broad application prospects in various fields, including medicine, food, and cosmetics.

[0003] At present, the production methods of collagen peptides mainly include chemical method, enzymatic method and microbial fermentation method. Among them, enzymatic method is widely used due to its advantages such as mildness, high efficiency and safety. However, there are some problems with traditional enzymatic method, such as incomplete enzymatic hydrolysis, low amino acid content in collagen peptides, dark color, low extraction rate, etc. The microbial fermentation method uses the proteolytic enzymes secreted by microorganisms to hydrolyze collagen. By controlling the fermentation conditions, collagen peptides of different molecular weights can be obtained. Compared with the enzymatic method, the microbial fermentation method has the advantages of mild conditions, high efficiency and stable hydrolysis products. However, the existing fermentation process has three major technical defects: insufficient strain synergy, a single lactic acid bacteria (such as Lactobacillus plantarum) cannot simultaneously achieve efficient protein degradation and functional metabolite synthesis (such as γ-aminobutyric acid), resulting in limited product activity; and the dissolved oxygen is not regulated in stages. High oxygen in the early stage inhibits the proliferation of anaerobic bacteria, and low oxygen in the later stage limits the secretion of proteases, resulting in a discrete molecular weight distribution of peptides (1000-5000Da accounts for <50%); at the same time, microbial residues in ultrafiltration purification (≥10 3 CFU / g) results in a short shelf life, limiting industrial applications. Furthermore, existing collagen peptide products have a single function and lack a synergistic mechanism for the multi-pathway of "inhibiting melanin production, promoting metabolism, and anti-oxidative repair," resulting in limited clinical whitening effects.

[0004] Based on this, there is an urgent need to prepare a collagen enzyme peptide. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention designs a method for preparing collagen enzyme peptides, which not only improves the absorption efficiency of nutrients, but also the prepared collagen enzyme peptides have good whitening effect, safety and stability.

[0006] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing collagenase peptide, the preparation method comprising the following steps:

[0008] S1. The collagen was added to water and mixed evenly, 8-12wt% of a nutrient enhancer was added to the mixed system, and the pH was adjusted to 5.8-6.2 to obtain a fermentation matrix;

[0009] S2. The fermentation medium was inoculated with Lactobacillus rhamnosus 2-3 v / v% and Lactobacillus plantarum 1-2 v / v%, and fermented at 30-35 ° C for 72 h, with dissolved oxygen 20-30% in the first 24 h and 5-10% after 48 h;

[0010] S3. The fermented product is centrifuged, preliminarily filtered with ordinary filter paper, filtered through a microfiltration membrane, and then filtered through an ultrafiltration membrane. The transmembrane pressure of the ultrafiltration membrane is controlled at 0.1-0.2 MPa. The filtrate is dried to obtain collagen fermentation acid peptide.

[0011] Specifically, the drying adopts a dual-fluid atomizer with an air inlet temperature of 180-200° C., an air outlet temperature of 70-90° C., and an atomization pressure of 0.8-1.2 MPa.

[0012] In some embodiments, the nutritional enhancer is a mixture of laminarin, oligomannan and sucrose in a mass ratio of 3:1:1.

[0013] In some embodiments, the microfiltration membrane in step S3 has a specification of 0.1-0.5 μm.

[0014] In some embodiments, the ultrafiltration membrane in step S3 has a specification of 1000Da, a surface coated with a 0.5-1.5wt% nano zinc oxide antibacterial layer, and a membrane flux controlled at 30-50L / (m 2 ·h).

[0015] In some embodiments, the collagen enzyme peptide has a moisture content of ≤5%, an ash content of ≤3%, and a pH value of 6.0-7.0.

[0016] In a second aspect, the present invention provides a collagen enzyme peptide prepared by the above method, having the quality indicators described in any one of (a) to (c):

[0017] (a) Peptides with molecular weights of 300-1500 Da account for 80-85 wt%, of which the content of Pro-Hyp-Gly tripeptide is ≥5 wt%;

[0018] (b) γ-aminobutyric acid 8-10 mg / 100 g, total essential amino acids 15-18 g / 100 g;

[0019] (c) ORAC value 7500-8500 μmol TE / g, tyrosinase inhibition IC50 value 0.4-0.6 mg / mL.

[0020] In some embodiments, the collagenase peptide is used in the preparation of a skin whitening preparation.

[0021] In a third aspect, the present invention provides a skin whitening preparation, comprising, by weight:

[0022] (i) 5-15 parts of the collagenase peptide according to claim 6;

[0023] (ii) 1-3 parts of a prebiotic composition, wherein the prebiotic is a mixture of xylo-oligosaccharides and inulin at a ratio of 1:2-1:4;

[0024] (iii) 0.1-0.5 parts of an antioxidant synergist selected from fullerenediol or resveratrol phospholipid complex.

[0025] In some embodiments, the skin whitening preparation is a topical or oral preparation.

[0026] In some embodiments, the steps include:

[0027] (a) mixing the collagen enzyme peptide, the prebiotic composition and the antioxidant synergist evenly;

[0028] (b) using fluidized bed coating technology, Eudragit L100-55 is used as the enteric material to coat the surface of the mixed particles;

[0029] (c) Control the coating weight gain to 3-5%, and fill it into the capsule shell after drying.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a preparation method of collagen acid peptide. Through staged oxygen control and composite bacterial strain synergistic fermentation, the proportion of 300-1500Da small molecule peptides is achieved to be ≥80%, and the transdermal absorption rate reaches 45%-96%, which is significantly higher than traditional collagen peptides (10-12%). The content of Pro-Hyp-Gly tripeptide is ≥5wt%, and the content of γ-aminobutyric acid is ≥8.5mg / 100g. It has multiple functions of tyrosinase inhibition, anti-oxidation and whitening, and has good market prospects.

[0032] The present invention adopts nano zinc oxide to coat the ultrafiltration membrane, inhibits microbial residue, and extends the shelf life to 18 months.

[0033] The present invention adds prebiotics and antioxidants to collagen enzyme peptides, and through the regulation of the intestinal-skin axis, the abundance of bifidobacteria is increased by 3.1 times and the erythema index is reduced by 52%. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings required for use in the implementation. Obviously, the drawings described below are only some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is a real picture of the zebrafish head melanin test results of the collagen enzyme peptide of the present invention.

[0036] Figure 2 This is a bar graph of the test results of melanin in the zebrafish head using the collagen enzyme peptide of the present invention. DETAILED DESCRIPTION

[0037] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] In the present invention, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Furthermore, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0039] In the present invention, the test methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0040] Table 1 Reagent sources

[0041]

[0042]

[0043] Table 2 Instrument Models

[0044]

[0045]

[0046] In the present invention, the moisture and ash detection indicators of collagen enzyme peptide are as follows:

[0047] Moisture content: GB 5009.3-2016 Method 1, standard requirement: ≤6.0;

[0048] Ash content: GB 5009.4-2016 Method 1, standard requirement: ≤7.0.

[0049] Example 1

[0050] A method for preparing collagen enzyme peptide comprises the following steps:

[0051] 1. Porcine type I collagen (Chondrex) was added to deionized water at a material-liquid ratio of 1:5 and stirred at a stirring speed of 200 r / min for 40 min to fully dissolve and mix uniformly. Then, 8 wt% of a nutrient enhancer was added to the mixture and the pH was adjusted to 5.8 to obtain a fermentation matrix.

[0052] The nutritional enhancer is prepared by mixing kelp polysaccharide, oligomannosaccharide and sucrose in a mass ratio of 3:1:1.

[0053] 2. Lactobacillus rhamnosus (2 v / v%) and Lactobacillus plantarum (1 v / v%) were inoculated into the fermentation medium and fermented at 38±2°C for 72 h with a stirring speed of 180 r / min. The dissolved oxygen content of the fermentation liquid was adjusted by adjusting the ventilation valve (fermentation was divided into two stages: 30% dissolved oxygen in the first 24 h and 5% dissolved oxygen in the last 48 h).

[0054] 3. After the fermentation is completed, the fermentation liquid is transferred to a centrifuge and centrifuged at a speed of 3000r / min for 15min to remove solid impurities and bacteria. The supernatant is first filtered with ordinary filter paper (pore size of 3-6μm), and then passed through a microfiltration membrane with a pore size of 0.1μm and an ultrafiltration membrane with a molecular weight cutoff of 1000Da (coated with 0.5wt% nano zinc oxide, transmembrane pressure of 0.1MPa) for separation and purification. The filtered liquid is then transferred to a vacuum concentration tank (vacuum degree -0.08MPa, temperature 60°C) and concentrated to a relative density of 1.18, and then spray-dried or freeze-dried to obtain a powder, which is collagen ferment acid peptide.

[0055] Drying specific operations:

[0056] The concentrate is sprayed into the drying tower through a standard nozzle. The inlet air temperature is manually adjusted at 180°C, the outlet air temperature at 70°C, and the atomization pressure at 0.8 MPa. By adjusting the nozzle pressure and flow rate, the droplet size is controlled to improve drying efficiency and product particle uniformity. The dried product is collected using a standard collection device. During vacuum freeze drying, the concentrate is pre-frozen to -25-45°C and dried at a standard vacuum of 5-40 Pa for 10-30 hours. After drying, the product is screened again using a standard vibrating screen to ensure uniform particle size.

[0057] Physical and chemical properties of the product:

[0058] Moisture 4.2%, ash 2.3%;

[0059] The peptide content is 81%, the 300-1500Da peptide segment accounts for 80%, and the Pro-Hyp-Gly content is 5.1wt%;

[0060] γ-aminobutyric acid 8.5mg / 100g, total essential amino acids 15.2g / 100g;

[0061] ORAC value is 7600 μmol TE / g, and tyrosinase inhibition IC50 is 0.6 mg / mL.

[0062] Example 2

[0063] A method for preparing collagen enzyme peptide comprises the following steps:

[0064] 1. Porcine type I collagen (Chondrex) was added to deionized water at a material-liquid ratio of 1:7 and stirred at a stirring speed of 200 r / min for 40 min to fully dissolve and mix uniformly. Then, 10 wt% of a nutritional enhancer was slowly added to the mixture and the pH was adjusted to 6.0 to obtain a fermentation matrix.

[0065] The nutritional enhancer is prepared by mixing kelp polysaccharide, oligomannosaccharide and sucrose in a mass ratio of 3:1:1.

[0066] 2. Lactobacillus rhamnosus (2.5 v / v%) and Lactobacillus plantarum (1.5 v / v%) were inoculated into the fermentation medium and fermented at 38±2°C for 72 h with a stirring speed of 180 r / min. The dissolved oxygen content of the fermentation liquid was adjusted by adjusting the ventilation valve (fermentation was divided into two stages: 25% dissolved oxygen in the first 24 h and 8% dissolved oxygen in the last 48 h).

[0067] 3. After the fermentation is completed, the fermentation liquid is transferred to a centrifuge and centrifuged at a speed of 4000r / min for 15min to remove solid impurities and bacteria. The supernatant is first filtered with ordinary filter paper (pore size of 3-6μm), and then passed through a microfiltration membrane with a pore size of 0.3μm and an ultrafiltration membrane with a molecular weight cutoff of 1000Da (coated with 1.0wt% nano zinc oxide, transmembrane pressure of 0.15MPa) for separation and purification. The filtered liquid is then transferred to a vacuum concentration tank (vacuum degree -0.08MPa, temperature 60°C) and concentrated to a relative density of 1.18, and then spray-dried or freeze-dried to obtain a powder, which is collagen ferment acid peptide.

[0068] Physical and chemical properties of the product:

[0069] Moisture 4.5%, ash 2.5%;

[0070] The peptide content is 82%, the 300-1500Da peptide segment accounts for 83%, and the Pro-Hyp-Gly content is 5.8wt%;

[0071] γ-aminobutyric acid 9.0mg / 100g, total essential amino acids 16.0g / 100g;

[0072] ORAC value is 8000 μmol TE / g, and tyrosinase inhibition IC50 is 0.5 mg / mL.

[0073] Example 3

[0074] A collagen acid peptide comprises the following steps:

[0075] 1. Porcine type I collagen (Chondrex) was added to deionized water at a material-liquid ratio of 1:9 and stirred at a stirring speed of 200 r / min for 40 min to fully dissolve and mix uniformly. Then, 12 wt% of a nutrient enhancer was slowly added to the mixture and the pH was adjusted to 6.2 to obtain a fermentation matrix.

[0076] The nutritional enhancer is prepared by mixing kelp polysaccharide, oligomannosaccharide and sucrose in a mass ratio of 3:1:1.

[0077] 2. Lactobacillus rhamnosus (3 v / v%) and Lactobacillus plantarum (2 v / v%) were inoculated into the fermentation medium and fermented at 38±2°C for 72 h with a stirring speed of 180 r / min. The dissolved oxygen content of the fermentation liquid was adjusted by adjusting the ventilation valve (fermentation was divided into two stages: 20% dissolved oxygen in the first 24 h and 10% dissolved oxygen in the last 48 h).

[0078] 3. After the fermentation is completed, the fermentation liquid is transferred to a centrifuge and centrifuged at a speed of 5000r / min for 15min to remove solid impurities and bacteria. The supernatant is first filtered with ordinary filter paper (pore size of 3-6μm), and then passed through a microfiltration membrane with a pore size of 0.5μm and an ultrafiltration membrane with a molecular weight cutoff of 1000Da (coated with 1.5wt% nano zinc oxide, transmembrane pressure of 0.2MPa) for separation and purification. The filtered liquid is then transferred to a vacuum concentration tank (vacuum degree -0.08MPa, temperature 60°C) and concentrated to a relative density of 1.18, and then spray-dried or freeze-dried to obtain a powder, which is collagen ferment acid peptide.

[0079] Physical and chemical properties of the product:

[0080] Moisture 4.8%, ash 2.8%;

[0081] The peptide content is 83%, the 300-1500Da peptide segment accounts for 85%, and the Pro-Hyp-Gly content is 6.2wt%;

[0082] γ-aminobutyric acid 10.0mg / 100g, total essential amino acids 17.8g / 100g;

[0083] ORAC value is 8400 μmol TE / g, and tyrosinase inhibition IC50 is 0.4 mg / mL.

[0084] Example 4

[0085] A composition comprises, by mass, 10 parts of collagen enzyme peptide (product of Example 2) and 2 parts of a prebiotic composition (xylo-oligosaccharide:inulin=1:3).

[0086] Example 5

[0087] A composition comprises, by mass, 10 parts of collagen enzyme peptide (product of Example 2), 2 parts of a prebiotic composition (xylo-oligosaccharide:inulin=1:3), and 0.3 parts of a resveratrol phospholipid complex.

[0088] In animal experiments, enteric-coated capsules were prepared, including the following steps:

[0089] (a) mixing the collagen enzyme peptide, the prebiotic composition and the antioxidant synergist evenly;

[0090] (b) using fluidized bed coating technology, sodium alginate-chitosan complex is used as the enteric material and coated on the surface of the mixed particles;

[0091] (c) Control the coating weight gain to 3-5%, and fill it into the capsule shell after drying.

[0092] Comparative Example 1

[0093] Comparative Example 1 differs from Example 1 in that Lactobacillus rhamnosus is not included, and Lactobacillus plantarum (4 v / v%) is inoculated, while all other conditions are the same.

[0094] Product defects:

[0095] The γ-aminobutyric acid content was reduced to 3.2 mg / 100 g, and the tyrosinase inhibition IC50 was 1.1 mg / mL (activity was significantly reduced).

[0096] Comparative Example 2

[0097] Comparative Example 2 differs from Example 1 in that no segmented oxygen control is performed during fermentation, and the dissolved oxygen content is 30% throughout the fermentation process; other aspects are the same.

[0098] Product defects:

[0099] The peptide content is only 65%, the 300-1500Da peptide segment accounts for 52%, and the Pro-Hyp-Gly content is 2.3wt%.

[0100] Comparative Example 3

[0101] The difference between Comparative Example 3 and Example 1 is that, during separation and purification, an ordinary 1000Da ultrafiltration membrane (without nano zinc oxide coating) is used, and all other conditions are the same.

[0102] Product defects:

[0103] Microbial residue exceeds the standard (GB 4789.3-2016 second method, total colony count ≥ 10 3 CFU / g), the shelf life is shortened by 50%.

[0104] Test Example 1

[0105] Tyrosinase inhibitory activity and peptide distribution test

[0106] 1. Sample treatment: Accurately weigh 1.0 g of sample, dissolve in 10 mL of PBS buffer (pH 6.8, 0.1 M), ultrasonically treat (40 kHz, 30 min), centrifuge (5000 × g, 10 min), collect the supernatant, and filter through a 0.22 μm filter membrane for later use.

[0107] 2. Tyrosinase inhibition test: refer to GB / T 34819-2017 Determination of tyrosinase activity in cosmetics:

[0108] Reaction system: 0.1 mL sample solution (0.1-5 mg / mL) + 0.1 mL tyrosinase solution (100 U / mL, dissolved in PBS) + 0.1 mL L-dopa (1 mM)

[0109] Conditions: 37℃ in the dark for 30min, then immediately terminate in ice bath

[0110] Detection: Measure the absorbance at 475 nm using a microplate reader and calculate the IC50 value

[0111] 3. Peptide analysis: MALDI-TOF mass spectrometry (Bruker Autoflex III) was used in combination with Sephadex G-25 gel chromatography, and the proportion was calculated by integrating the molecular weight interval.

[0112] 4. Test standards

[0113] Tyrosinase inhibition rate calculation refers to ISO 11930:2019

[0114] The peptide molecular weight distribution test complies with GB 31645-2018 Collagen Peptide

[0115] 5. Test Principle

[0116] Enzyme inhibition kinetics: The active peptide in the sample competitively binds to the active center of tyrosinase, reducing the rate of conversion of L-dopa to dopaquinone. The change in absorbance reflects the inhibition efficiency.

[0117] Molecular sieving effect: Gel chromatography separates peptides according to molecular weight, and mass spectrometry accurately determines the content of characteristic peptides (such as Pro-Hyp-Gly).

[0118] Table 3 Tyrosinase inhibitory activity and peptide distribution test results

[0119]

[0120] Note: *Comparative Example 3 has decreased actual activity due to residual microorganisms and is for reference only.

[0121] As can be seen from Table 3, the IC50 of Example 2 is 0.5 mg / mL, which is 54.5% lower than that of Comparative Example 1 (1.1 mg / mL), proving that Lactobacillus rhamnosus plays a key role in the synthesis of tyrosinase inhibitory peptides (p<0.01). The 300-1500 Da peptide segment accounts for 83%, which is in line with the optimal molecular weight range for transdermal absorption (500-2000 Da), explaining its high inhibitory activity. In Comparative Example 2, the proportion of large molecular peptides (>3000 Da) increased (48%) due to the lack of staged oxygen control, and the inhibition efficiency decreased significantly.

[0122] Test Example 2

[0123] 1. Transdermal absorption rate and skin metabolism improvement test

[0124] 1. Test method

[0125] Transdermal test: Refer to OECD TG 428 In vitro skin penetration test guideline:

[0126] Using Franz diffusion cell, the effective diffusion area is 2.54cm 2 The receiving solution is pH 7.4 PBS + 0.5% SDS.

[0127] Excised human skin (200 μm thickness) was pretreated and fixed, and 1 mL of sample solution (10 mg / mL) was applied and kept constant at 37°C. Samples were taken at 1 / 2 / 4 / 8 / 24 h, and the peptide content was detected by HPLC (C18 column, UV 220 nm).

[0128] Skin moisture and brightness: Corneometer CM825 (moisture) and Chromameter CR400 (L* value) were used clinically to conduct a baseline-4-week comparison in accordance with the "Standards for the Evaluation of Cosmetic Efficacy Claims".

[0129] 2. Test standards

[0130] The transdermal absorption rate is calculated according to the FDA's SUPAC-SS Non-sterile Semisolid Dosage Formulation Scale Guide.

[0131] Skin moisture testing complies with ISO 24442:2011.

[0132] 3. Test Principle

[0133] Passive diffusion model: Small molecule peptides diffuse through the intercellular lipid layer, and their permeability coefficient (Kp) is negatively correlated with the molecular weight (LogKp = -0.006×MW+0.86).

[0134] Stratum corneum hydration: Peptides promote the synthesis of natural moisturizing factor (NMF) and enhance the skin's water retention capacity.

[0135] Table 4 Transdermal absorption rate and skin metabolism improvement test results

[0136]

[0137] As shown in Table 4, Example 2 achieved a transdermal penetration rate of 45%, significantly higher than Comparative Example 2 (12%) (p < 0.001), confirming the transdermal superiority of the 300-1500 Da peptide segment. Example 5, through enteric coating (Eudragit L100-55), achieved an intestinal absorption rate of 96%, a three-fold increase in bioavailability (vs. transdermal), and promoted systemic whitening (ΔL* +18).

[0138] 2. Animal Model

[0139] Evaluation of the whitening efficacy of collagenase peptide using a zebrafish model

[0140] 1. Animal breeding and handling

[0141] Adult zebrafish were housed in a recirculating water system at 28°C with a 14-hour / 10-hour light / dark cycle and fed three times daily. To produce embryos, male and female zebrafish were paired 1:1 at night and separated by a baffle; the baffle was removed the next day, and eggs were laid within 1 hour of the light cycle. Embryos were placed in 10-cm Petri dishes containing a 1xE3 solution containing methylene blue (0.3 ppm) and incubated in an artificial climate incubator at 28.5°C (14-hour / 10-hour light / dark) until experimental treatment.

[0142] 2. Whitening effect

[0143] Experimental groups: normal control group, sample group (set to a concentration of 100 μg / mL).

[0144] Treatment Methods: Zebrafish 6 hours post-fertilization (hpf) were randomly selected and exposed to the sample in a 6-well plate (3 mL / well), with 18 fish per well. The control group was cultured normally, while the sample group was exposed to the sample until 45 hpf. Ten zebrafish were randomly selected from each experimental group and photographed under a microscope. Data were collected and analyzed using ImageJ advanced image processing software. The intensity of melanin signals in the zebrafish heads was statistically analyzed to evaluate the whitening efficacy of the sample.

[0145] Data were analyzed using GraphPad prism 8.0 statistical software, and the measured data were expressed as mean ± standard deviation. The data were compared with analysis of variance and t-test for pairwise comparison. P < 0.05 was considered statistically significant.

[0146] Table 5 Whitening effect test results

[0147]

[0148]

[0149] Compared with the normal control group, *** represents p < 0.001

[0150] From Table 5 and Figure 1 、 2 It can be seen that the melanin signal intensity of the collagenase peptide of Example 2 is significantly lower than that of the normal control group, indicating that the collagenase peptide of the present invention has a good whitening effect.

[0151] Test Example 3

[0152] Antioxidant capacity and microbial stability verification

[0153] Sample processing: Take 1.0 g of solid particles, disperse them in 10 mL of deionized water, and test the ORAC value; take another unsterilized sample to test the total colony count (stored at 25°C for 6 months).

[0154] Table 5 Antioxidant capacity and microbial stability verification table

[0155]

[0156] Note: *Comparative Example 3 (not coated with nano zinc oxide) had an ORAC value that dropped to 6000 (6 months) due to microbial proliferation.

[0157] As shown in Table 5, the nano zinc oxide coating (Examples 2-3) effectively inhibited microbial growth and ensured shelf life. Example 3 had an ORAC value of 8400 μmol TE / g due to higher fermentation efficiency, indicating the best antioxidant capacity.

[0158] Test Example 4

[0159] Effects of a prebiotic composition on the gut-skin axis (mouse model)

[0160] Experimental methods

[0161] Animal grouping: 60 BALB / c mice (female, 6 weeks old) were randomly divided into 3 groups (n=20):

[0162] Example 4 group: Oral administration of Example 5 granules (containing prebiotics, dose 50 mg / kg / d)

[0163] Example 5 group: Oral administration of Example 6 granules (containing prebiotics + antioxidants, dose 50 mg / kg / d)

[0164] Comparative Example 1 group: Oral administration of the product of Comparative Example 1 (without prebiotics, dosage 50 mg / kg / d)

[0165] Intervention plan: Continuous administration for 8 weeks, free diet (AIN-93M standard feed)

[0166] Detection indicators:

[0167] Gut flora: Feces were collected at week 8, and the abundance of Bifidobacterium was analyzed using 16S rRNA sequencing (Illumina MiSeq)

[0168] Skin inflammation: A mouse ear dermatitis model was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and the erythema index was quantified by laser speckle contrast imaging (LSCI).

[0169] Acne scar model: mechanical scratching + UVB irradiation (150mJ / cm 2 ) Establish a skin injury model and evaluate the epidermal repair rate by HE staining

[0170] Test standards

[0171] Intestinal flora testing was performed according to the MIQE Guidelines (Bustin et al., 2009).

[0172] Skin erythema index is calculated according to OECD TG 442D (Skin sensitization test)

[0173] Test Principle

[0174] Regulation of the gut-skin axis: Prebiotics (xylo-oligosaccharides, inulin) promote the proliferation of Bifidobacteria → produce short-chain fatty acids (SCFAs) → reduce circulating endotoxins → inhibit the skin TLR4 / NF-κB inflammatory pathway

[0175] Antioxidant synergy: Resveratrol phospholipid complex enhances Nrf2 pathway activity and reduces ROS-mediated keratinocyte apoptosis.

[0176] Table 6 Effects of prebiotic composition on the gut-skin axis

[0177] index Example 4 Group Example 5 group Comparative Example 1 Intestinal Bifidobacterium abundance ↑ 2.3 times** 3.1 times*** 0.5 times Skin erythema index↓ -40％** -52％*** -5％ Epidermal repair rate (8 weeks) 78％** 92％*** 15％

[0178] As shown in Table 6, after adding prebiotics, the abundance of bifidobacteria increased to 2.3 times (**p<0.01), indicating that the combination of oligoxylose and inulin optimizes the intestinal flora structure by selectively proliferating bifidobacteria, which is consistent with the short-chain fatty acid (SCFAs) production mechanism. The combination of prebiotics and antioxidants in Example 5 further increased the abundance of bifidobacteria to 3.1 times (***p<0.001), suggesting that resveratrol phospholipid complexes may create a more suitable living environment for bifidobacteria by inhibiting intestinal oxidative stress (such as ROS removal), thereby achieving "prebiotics + antioxidant" synergistic enhancement. As can be seen from Comparative Example 1, without the addition of prebiotics, the abundance of bifidobacteria decreased by 50%, confirming that strain imbalance (only inoculating Lactobacillus plantarum) destroys flora homeostasis and cannot trigger SCFAs-mediated immune regulation.

[0179] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing collagenase peptide, characterized in that: The preparation method comprises the following steps: S1. The collagen was added to water and mixed evenly, 8-12wt% of a nutrient enhancer was added to the mixed system, and the pH was adjusted to 5.8-6.2 to obtain a fermentation matrix; S2. The fermentation medium was inoculated with Lactobacillus rhamnosus 2-3 v / v% and Lactobacillus plantarum 1-2 v / v%, and fermented at 30-35 ° C for 72 h, with dissolved oxygen 20-30% in the first 24 h and 5-10% after 48 h; S3. The fermented product is centrifuged, preliminarily filtered with ordinary filter paper, filtered through a microfiltration membrane, and then filtered through an ultrafiltration membrane. The transmembrane pressure of the ultrafiltration membrane is controlled at 0.1-0.2 MPa. The filtrate is dried to obtain collagen fermentation acid peptide.

2. The method for preparing a collagenase peptide according to claim 1, wherein: The nutritional enhancer is prepared by mixing kelp polysaccharide, oligomannosaccharide and sucrose in a mass ratio of 3:1:

1.

3. The method for preparing a collagenase peptide according to claim 1, wherein: The microfiltration membrane specification in step S3 is 0.1-0.5 μm.

4. The method for preparing a collagenase peptide according to claim 1, wherein: The ultrafiltration membrane in step S3 has a specification of 1000Da, and is coated with a 0.5-1.5wt% nano zinc oxide antibacterial layer on its surface. The membrane flux is controlled at 30-50L / (m 2 ·h).

5. The method for preparing a collagenase peptide according to claim 1, wherein: The collagen enzyme peptide has a moisture content of ≤5%, an ash content of ≤3%, and a pH value of 6.0-7.

0.

6. A collagen acid peptide, characterized in that Prepared by the method according to any one of claims 1 to 5, having the quality indicators described in any one of (a) to (c): (a) Peptides with molecular weights of 300-1500 Da account for 80-85 wt%, of which the content of Pro-Hyp-Gly tripeptide is ≥5 wt%; (b) γ-aminobutyric acid 8-10 mg / 100 g, total essential amino acids 15-18 g / 100 g; (c) ORAC value 7500-8500 μmol TE / g, tyrosinase inhibition IC50 value 0.4-0.6 mg / mL.

7. Use of the collagenase peptide as claimed in claim 6 in preparing a skin whitening preparation.

8. A skin whitening preparation, characterized in that: The preparation comprises, by mass: (i) 5-15 parts of the collagenase peptide according to claim 6; (ii) 1-3 parts of a prebiotic composition, wherein the prebiotic is a mixture of xylo-oligosaccharides and inulin at a ratio of 1:2-1:4; (iii) 0.1-0.5 parts of an antioxidant synergist selected from fullerenediol or resveratrol phospholipid complex.

9. The skin whitening preparation according to claim 8, characterized in that The skin whitening preparation is an external or oral preparation.

10. The method for preparing the skin whitening preparation according to claim 9, wherein: The following steps are involved: (a) mixing the collagen enzyme peptide, the prebiotic composition and the antioxidant synergist evenly; (b) using fluidized bed coating technology, Eudragit L100-55 is used as the enteric material to coat the surface of the mixed particles; (c) Control the coating weight gain to 3-5%, and fill it into the capsule shell after drying.

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