Preparation method of collagen hydrolysate

Abstract

The application provides a preparation method of collagen fermentation peptides, and belongs to the technical field of collagen bioengineering. The preparation method comprises the following steps: firstly, collagen is added into water and stirred uniformly, then 8-12wt% of a nutrition enhancer is added into a mixed system, pH is adjusted to 5.8-6.2, and a fermentation substrate is obtained; then Lactobacillus rhamnosus and Lactobacillus plantarum are inoculated into the fermentation substrate, dissolved oxygen is controlled to be 20-30% in the first 24 hours, and is reduced to 5-10% in the last 48 hours, and the product after fermentation is filtered and dried to obtain collagen fermentation peptides. Through the synergistic fermentation of the compound strains, the product is rich in 300-1500Da active peptides, Pro-Hyp-Gly and gamma-aminobutyric acid, and has the functions of tyrosinase inhibition, antioxidation, whitening and intestinal flora regulation, the clinical melanin reduction rate reaches 22% in 8 weeks, and the shelf life is prolonged to 18 months.

Description

Technical Field

[0001] This invention relates to the field of collagen biotechnology, specifically to a method for preparing collagen-derived peptides. Background Technology

[0002] Collagen peptides are small molecule peptides prepared from collagen through enzymatic hydrolysis or fermentation. They possess characteristics such as low molecular weight, easy absorption, and low viscosity. They not only retain the biological activity of collagen but also exhibit various physiological functions, including immunomodulation, antioxidation, anti-aging, anti-thrombosis, blood pressure reduction, and cholesterol reduction. Therefore, collagen peptides have broad application prospects in multiple fields such as medicine, food, and cosmetics.

[0003] Currently, the main methods for producing collagen peptides include chemical methods, enzymatic hydrolysis, and microbial fermentation. Enzymatic hydrolysis is widely used due to its advantages of being mild, efficient, and safe. However, traditional enzymatic hydrolysis methods have some problems, such as incomplete hydrolysis, low amino acid content in collagen peptides, dark color, and low extraction rate. Microbial fermentation utilizes proteolytic enzymes secreted by microorganisms to hydrolyze collagen. By controlling fermentation conditions, collagen peptides of different molecular weights can be obtained. Compared with enzymatic hydrolysis, microbial fermentation has advantages such as mild conditions, high efficiency, and stable hydrolysis products. However, existing fermentation processes have three major technical defects: insufficient synergy among strains; a single lactic acid bacteria (such as *Lactobacillus plantarum*) cannot simultaneously achieve efficient protein degradation and functional metabolite (such as γ-aminobutyric acid) synthesis, resulting in limited product activity; and the lack of staged control of dissolved oxygen, with high oxygen in the early stage inhibiting anaerobic bacterial proliferation and low oxygen in the later stage limiting protease secretion, resulting in a dispersed molecular weight distribution of peptides (1000-5000 Da < 50%); and microbial residues (≥ 10 μL) in ultrafiltration purification. 3 The CFU / g ratio results in a short shelf life, limiting its industrial application. Furthermore, existing collagen peptide products have limited functionality and lack a synergistic mechanism involving multiple pathways such as melanin inhibition, metabolism promotion, and antioxidant repair, leading to limited clinical whitening effects.

[0004] Therefore, there is an urgent need to prepare a collagenase peptide. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention presents a method for preparing collagenase peptides, which not only improves the absorption efficiency of nutrients but also produces collagenase peptides with good whitening effects, safety, and stability.

[0006] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem:

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

[0008] S1. Add collagen to water and mix well. Add 8-12 wt% of nutrient fortifier to the mixture and adjust the pH to 5.8-6.2 to obtain the fermentation substrate.

[0009] S2. Inoculate the fermentation substrate with 2-3 v / v% Lactobacillus rhamnosus and 1-2 v / v% Lactobacillus plantarum, and ferment at 30-35℃ for 72 h. The dissolved oxygen is 20-30% in the first 24 h and 5-10% in the last 48 h.

[0010] S3. The fermented product is centrifuged, preliminarily filtered through ordinary filter paper, microfiltered, and ultrafiltered. The transmembrane pressure of the ultrafiltration membrane is controlled at 0.1-0.2 MPa. The filtrate is dried to obtain collagen glycopeptides.

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

[0012] In some embodiments, the nutrient fortifier is a mixture of kelp polysaccharide, oligomannose and sucrose in a mass ratio of 3:1:1.

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

[0014] In some embodiments, the ultrafiltration membrane in step S3 has a specification of 1000 Da, is coated with a 0.5-1.5 wt% nano zinc oxide antibacterial layer, and has a membrane flux controlled at 30-50 L / (m2·h).

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

[0016] Secondly, the present invention provides a collagenase peptide, prepared by the above method, having any one of the quality indicators described in (a)-(c):

[0017] (a) Peptides with a molecular weight of 300-1500 Da account for 80-85 wt%, of which Pro-Hyp-Gly tripeptide content 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 skin whitening formulations.

[0021] Thirdly, the present invention provides a skin whitening preparation, wherein the preparation comprises, by weight parts:

[0022] (i) 5-15 parts collagenase peptides;

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

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

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

[0026] In some embodiments, the following steps are included:

[0027] (a) Mix collagenase peptides, prebiotic composition and antioxidant synergist evenly;

[0028] (b) Fluidized bed coating technology was used to coat the surface of the mixed particles with Eudragit L100-55 as the enteric material;

[0029] (c) Control the weight gain of the coating 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] This invention provides a method for preparing collagen-derived peptides. Through staged oxygen control and synergistic fermentation with compound strains, the method achieves a ≥80% proportion of small molecule peptides of 300-1500 Da, a transdermal absorption rate of 45%-96%, which is significantly higher than that of traditional collagen peptides (10-12%), a Pro-Hyp-Gly tripeptide content of ≥5wt%, and γ-aminobutyric acid ≥8.5mg / 100g. It also has multiple effects such as tyrosinase inhibition, anti-oxidation, and whitening, and has good market prospects.

[0032] This invention uses nano-zinc oxide coated ultrafiltration membrane to inhibit microbial residue and extend shelf life to 18 months.

[0033] This invention adds prebiotics and antioxidants to collagenase peptides, and through the regulation of the gut-skin axis, increases the abundance of bifidobacteria by 3.1 times and reduces the erythema index by 52%. Attached Figure Description

[0034] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 These are real-life images showing the test results of the collagen-enzyme peptides of this invention on melanin in the head of zebrafish.

[0036] Figure 2 This is a bar chart showing the test results of melanin in zebrafish heads using the collagenase peptide of this invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In this invention, numerical ranges are involved. 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 the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0039] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0040] Table 1. Reagent Sources

[0041]

[0042]

[0043] Table 2 Instrument Model List

[0044]

[0045]

[0046] In this invention, the moisture and ash content of collagenase peptides are measured as follows:

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

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

[0049] Example 1

[0050] A method for preparing collagenase peptides includes the following steps:

[0051] 1. Add porcine type I collagen (Chondrex) to deionized water at a ratio of 1:5 and stir at 200 r / min for 40 min to fully dissolve and mix it evenly. Then add 8 wt% of nutrient fortifier to the mixture and adjust the pH to 5.8 to obtain the fermentation substrate.

[0052] The nutritional fortifier is a mixture of kelp polysaccharide, oligomannose and sucrose in a mass ratio of 3:1:1.

[0053] 2. Inoculate the fermentation substrate with Lactobacillus rhamnosus (2v / v%) and Lactobacillus plantarum (1v / v%) and ferment at 38±2℃ for 72h. The stirring speed is 180r / min. Adjust the ventilation valve to control the dissolved oxygen content of the fermentation broth (fermentation is carried out in two stages: 30% dissolved oxygen in the first 24h and 5% dissolved oxygen in the last 48h).

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

[0055] Specific drying procedures:

[0056] The concentrated liquid is sprayed into the drying tower through ordinary nozzles. The inlet air temperature is manually adjusted to 180℃, the outlet air temperature to 70℃, and the atomization pressure to 0.8MPa. By adjusting the nozzle pressure and flow rate, the droplet size is controlled to improve drying efficiency and product particle uniformity. A standard collection device is used to collect the dried product. For vacuum freeze drying, the concentrated liquid is pre-frozen to -25 to -45℃ and dried under a standard vacuum of 5 to 40Pa for 10 to 30 hours. After drying, the product is secondarily sieved using a standard vibrating screen to ensure uniform particle size.

[0057] Physical and chemical properties of the product:

[0058] Moisture content 4.2%, ash content 2.3%;

[0059] The peptide content is 81%, with 80% being 300-1500Da peptides, and the Pro-Hyp-Gly content is 5.1wt%.

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

[0061] ORAC value: 7600 μmol TE / g; tyrosinase inhibition IC50: 0.6 mg / mL.

[0062] Example 2

[0063] A method for preparing collagenase peptides includes the following steps:

[0064] 1. Add porcine type I collagen (Chondrex) to deionized water at a ratio of 1:7 and stir at 200 rpm for 40 minutes to fully dissolve and mix it evenly. Then slowly add 10 wt% of nutrient fortifier to the mixture and adjust the pH to 6.0 to obtain the fermentation substrate.

[0065] The nutritional fortifier is a mixture of kelp polysaccharide, oligomannose and sucrose in a mass ratio of 3:1:1.

[0066] 2. Inoculate the fermentation substrate with Lactobacillus rhamnosus (2.5 v / v%) and Lactobacillus plantarum (1.5 v / v%) and ferment at 38±2℃ for 72 h with a stirring speed of 180 r / min. Adjust the ventilation valve to control the dissolved oxygen content of the fermentation broth (fermentation in two stages: 25% dissolved oxygen in the first 24 h and 8% dissolved oxygen in the last 48 h).

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

[0068] Physical and chemical properties of the product:

[0069] Moisture content 4.5%, ash content 2.5%;

[0070] The peptide content is 82%, the proportion of 300-1500Da peptides is 83%, and the Pro-Hyp-Gly content is 5.8wt%.

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

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

[0073] Example 3

[0074] A collagenase peptide, comprising the following steps:

[0075] 1. Add porcine type I collagen (Chondrex) to deionized water at a ratio of 1:9 and stir at 200 rpm for 40 minutes to fully dissolve and mix it evenly. Then slowly add 12 wt% of nutrient fortifier to the mixture and adjust the pH to 6.2 to obtain the fermentation substrate.

[0076] The nutritional fortifier is a mixture of kelp polysaccharide, oligomannose and sucrose in a mass ratio of 3:1:1.

[0077] 2. Inoculate the fermentation substrate with Lactobacillus rhamnosus (3v / v%) and Lactobacillus plantarum (2v / v%) and ferment at 38±2℃ for 72h. The stirring speed is 180r / min. Adjust the ventilation valve to control the dissolved oxygen content of the fermentation broth (fermentation is carried out in two stages: 20% dissolved oxygen in the first 24h and 10% dissolved oxygen in the last 48h).

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

[0079] Physical and chemical properties of the product:

[0080] Moisture content 4.8%, ash content 2.8%;

[0081] The peptide content is 83%, the proportion of 300-1500Da peptides is 85%, and the Pro-Hyp-Gly content is 6.2wt%.

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

[0083] ORAC value: 8400 μmol TE / g; tyrosinase inhibition IC50: 0.4 mg / mL.

[0084] Example 4

[0085] A composition comprising, by weight, 10 parts of collagenase peptide (product of Example 2) and 2 parts of prebiotic composition (xylooligosaccharide:inulin = 1:3).

[0086] Example 5

[0087] A composition, by weight, comprises 10 parts of collagenase peptide (product of Example 2), 2 parts of prebiotic composition (xylooligosaccharide:inulin = 1:3), and 0.3 parts of resveratrol phospholipid complex.

[0088] In animal experiments, the preparation of enteric-coated capsules includes the following steps:

[0089] (a) Mix collagenase peptides, prebiotic composition and antioxidant synergist evenly;

[0090] (b) Fluidized bed coating technology was used to coat the surface of the mixed particles with sodium alginate-chitosan complex as the enteric material.

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

[0092] Comparative Example 1

[0093] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain Lactobacillus rhamnosus and is inoculated with Lactobacillus plantarum (4v / v%), but all other aspects are the same.

[0094] Product defects:

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

[0096] Comparative Example 2

[0097] The difference between Comparative Example 2 and Example 1 is that no segmented oxygen control was performed during fermentation, and the dissolved oxygen was 30% throughout the process, while everything else was the same.

[0098] Product defects:

[0099] The peptide content is only 65%, with 52% being 300-1500Da peptides and 2.3wt% being Pro-Hyp-Gly.

[0100] Comparative Example 3

[0101] The difference between Comparative Example 3 and Example 1 is that a standard 1000Da ultrafiltration membrane (without nano zinc oxide coating) was used for separation and purification, while all other aspects were the same.

[0102] Product defects:

[0103] Microbial residues exceeding the standard (GB 4789.3-2016 Method II, total bacterial count ≥10). 3 (CFU / g), shortening shelf life by 50%.

[0104] Test Example 1

[0105] Tyrosinase inhibitory activity and peptide distribution assay

[0106] 1. Sample preparation: Accurately weigh 1.0g of sample, dissolve in 10mL PBS buffer (pH6.8, 0.1M), sonicate (40kHz, 30min), centrifuge (5000×g, 10min), collect the supernatant, and filter through a 0.22μm 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: React at 37℃ in the dark for 30 minutes, then immediately stop with an ice bath.

[0110] Detection: The absorbance at 475 nm was measured using an ELISA reader, and the IC50 value was calculated.

[0111] 3. Peptide analysis: MALDI-TOF mass spectrometry (Bruker Autoflex III) was used in combination with Sephadex G-25 gel chromatography to calculate the proportion based on the molecular weight range.

[0112] 4. Testing Standards

[0113] Tyrosinase inhibition rate was calculated according to ISO 11930:2019.

[0114] The peptide molecular weight distribution assay conformed to GB 31645-2018 Collagen Peptides.

[0115] 5. Testing Principle

[0116] Enzyme inhibition kinetics: The active peptide in the sample competitively binds to the active site of tyrosinase, reducing the rate of L-DOPA conversion to dopaquinone. Changes in absorbance reflect 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. Results of Tyrosinase Inhibitory Activity and Peptide Distribution Tests

[0119]

[0120] Note: *Comparative Example 3 shows a decrease in actual activity due to microbial residues and is for reference only.

[0121] As shown in Table 3, the IC50 of Example 2 was 0.5 mg / mL, which was 54.5% lower than that of Comparative Example 1 (1.1 mg / mL), demonstrating that *Lactobacillus rhamnosus* plays a crucial role in the synthesis of tyrosinase-inhibiting peptides (p<0.01). The 300-1500 Da peptides accounted for 83%, which is within the optimal molecular weight range for transdermal absorption (500-2000 Da), explaining its high inhibitory activity. Comparative Example 2, due to the lack of staged oxygen control, resulted in an increased proportion of large molecular weight peptides (>3000 Da) (48%), significantly reducing its inhibitory efficiency.

[0122] Test Example 2

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

[0124] 1. Testing Method

[0125] Transdermal test: Refer to the OECD TG 428 Guideline for In Vitro Skin Permeability Testing:

[0126] A Franz diffusion cell with an effective diffusion area of ​​2.54 cm² was used, and the receiving solution was pH 7.4 PBS + 0.5% SDS.

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

[0128] Skin hydration and radiance: The Corneometer CM825 (hydration) and Chromameter CR400 (L* value) were used in clinical trials, and the baseline-4-week comparison was conducted in accordance with the "Evaluation Standards for Cosmetic Efficacy Claims".

[0129] 2. Testing Standards

[0130] Transdermal absorption rate was calculated based on the FDA's "SUPAC-SS Guidelines for Non-sterile Semi-solid Dosage Forms".

[0131] Skin moisture testing conforms to ISO 24442:2011.

[0132] 3. Testing Principle

[0133] Passive diffusion model: Small molecule peptides diffuse through the intercellular lipid layer, and their permeability coefficient (Kp) is negatively correlated with 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 ability to retain moisture.

[0135] Table 4. Results of Transdermal Absorption Rate and Skin Metabolism Improvement Test

[0136]

[0137] As shown in Table 4, the transdermal transdermal rate of Example 2 was 45%, significantly higher than that of Comparative Example 2 (12%) (p<0.001), confirming the transdermal advantage of the 300-1500 Da peptide. Example 5, through enteric coating (Eudragit L100-55), achieved an intestinal absorption rate of 96%, a 3-fold increase in bioavailability (vs. transdermal), and promoted systemic whitening (ΔL*+18).

[0138] II. Animal Models

[0139] Evaluation of the skin-whitening efficacy of collagen-derived peptides using a zebrafish model

[0140] 1. Animal husbandry and treatment

[0141] Adult zebrafish were housed in a recirculating aquaculture system at 28°C under a 14-hour / 10-hour light / dark cycle, and fed three times daily. To induce embryonic development, males and females were paired 1:1 at night and isolated with a barrier; the barrier was removed the following day, and spawning occurred within one hour of the light cycle. Embryos were placed in 10 cm culture dishes containing a 1xE3 solution of methylene blue (0.3 ppm) and cultured in an artificial climate incubator at 28.5°C (14h / 10h light / dark) until the experimental treatment.

[0142] 2. Whitening effect

[0143] Experimental groups: normal control group and sample group (with one concentration set at 100 μg / mL).

[0144] Methods: Zebrafish 6 hours post-fertilization (6 hpf) were randomly selected and exposed to samples in 6-well plates (3 mL / well), with 18 fish per well. The control group was cultured normally, while the sample groups were exposed to samples until 45 hpf. Ten zebrafish from each experimental group were randomly selected and photographed under a microscope. ImageJ advanced image processing software was used for analysis and data acquisition. The melanin signal intensity in the zebrafish head was analyzed and statistically analyzed to evaluate the whitening effect of the samples.

[0145] Data analysis was performed using GraphPad Prism 8.0 statistical software. Quantitative data were expressed as mean ± standard deviation. The comparisons were performed using analysis of variance, and pairwise comparisons were performed using t-tests. P < 0.05 was considered statistically significant.

[0146] Table 5. Results of Whitening Effect Test

[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 in Example 2 is significantly lower than that of the normal control group, indicating that the collagenase peptide of the present invention has a better whitening effect.

[0151] Test Example 3

[0152] Antioxidant capacity and microbial stability verification

[0153] Sample preparation: 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 bacterial count (stored at 25℃ for 6 months).

[0154] Table 5. Verification of Antioxidant Capacity and Microbial Stability

[0155]

[0156] Note: *Comparative Example 3 (uncoated 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 inhibits microbial growth and ensures shelf life. Example 3, due to its higher fermentation efficiency, achieved an ORAC value of 8400 μmol TE / g, demonstrating the best antioxidant capacity.

[0158] Test Example 4

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

[0160] Experimental methods

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

[0162] Example 4 group: Gavage administration of Example 5 granules (containing prebiotics, dosage 50mg / kg / day)

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

[0164] Comparative Example 1: Gavage administration of the Comparative Example 1 product (no prebiotics, dose 50 mg / kg / day) intervention regimen: continuous administration for 8 weeks, free diet (AIN-93M standard feed)

[0165] Testing indicators:

[0166] Gut microbiota: Fecal samples were collected at week 8, and the abundance of Bifidobacteria was analyzed using 16S rRNA sequencing (Illumina MiSeq).

[0167] Skin inflammation: A mouse ear dermatitis model induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was quantified using laser speckle contrast imaging (LSCI) to measure the erythema index.

[0168] Acne scar model: A skin damage model was established by mechanical scratching + UVB irradiation (150mJ / cm2), and the epidermal repair rate was assessed by HE staining.

[0169] Test Standards

[0170] Gut microbiota testing was performed according to the MIQE guidelines (Bustin et al., 2009).

[0171] The skin erythema index is calculated based on the OECD TG 442D (Skin Sensitization Test).

[0172] Test Principles

[0173] Gut-skin axis regulation: Prebiotics (xylooligosaccharides: inulin) promote the proliferation of bifidobacteria → produce short-chain fatty acids (SCFAs) → reduce circulating endotoxins → inhibit the skin TLR4 / NF-κB inflammatory pathway.

[0174] Synergistic antioxidant effects: The resveratrol phospholipid complex enhances Nrf2 pathway activity and reduces ROS-mediated keratinocyte apoptosis.

[0175] Table 6. Effects of prebiotic compositions on the gut-skin axis

[0176] index Example 4 group Example 5 group Comparative Example 1 Increased abundance of intestinal bifidobacteria 2.3 times** 3.1 times*** 0.5 times Skin erythema index ↓ -40％** -52％*** -5％ Epidermal repair rate (8 weeks) 78％** 92％*** 15％

[0177] Table 6 shows that the addition of prebiotics increased the abundance of Bifidobacteria by 2.3 times (**p<0.01), indicating that the combination of xylooligosaccharides and inulin optimizes the intestinal flora structure by selectively proliferating Bifidobacteria, consistent with the short-chain fatty acid (SCFA) generation mechanism. In Example 5, the combination of prebiotics and antioxidants further increased the abundance of Bifidobacteria by 3.1 times (***p<0.001), suggesting that the resveratrol phospholipid complex may create a more suitable survival environment for Bifidobacteria by inhibiting intestinal oxidative stress (such as ROS scavenging), achieving a synergistic effect of "prebiotics + antioxidants". Furthermore, Comparative Example 1 shows that without the addition of prebiotics, the abundance of Bifidobacteria decreased by 50%, confirming that bacterial imbalance (inoculation with only *Lactobacillus plantarum*) disrupts flora homeostasis and fails to trigger SCFA-mediated immune regulation.

[0178] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing collagenase peptides, characterized in that, The preparation method includes the following steps: S1. Add collagen to water and mix well. Add 8-12 wt% of nutrient fortifier to the mixture and adjust the pH to 5.8-6.2 to obtain the fermentation substrate. S2. Inoculate the fermentation substrate with 2-3 v / v% Lactobacillus rhamnosus and 1-2 v / v% Lactobacillus plantarum, and ferment at 30-35℃ for 72 h. The dissolved oxygen is 20-30% in the first 24 h and 5-10% in the last 48 h. S3. The fermented product is centrifuged, preliminarily filtered through ordinary filter paper, microfiltered, and ultrafiltered. The transmembrane pressure of the ultrafiltration membrane is controlled at 0.1-0.2 MPa. The filtrate is dried to obtain collagen glycopeptides. The nutrient fortifier is a mixture of kelp polysaccharide, oligomannose and sucrose in a mass ratio of 3:1:1; In step S3, the ultrafiltration membrane has a specification of 1000 Da, and its surface is coated with a 0.5-1.5 wt% nano-zinc oxide antibacterial layer. The membrane flux is controlled at 30-50 L / (m²). 2 ·h).

2. The method for preparing collagenase peptides according to claim 1, characterized in that, In step S3, the microfiltration membrane has a size of 0.1-0.5 μm.

3. The method for preparing collagenase peptides according to claim 1, characterized in that, The collagenase peptide has a moisture content of ≤5%, an ash content of ≤3%, and a pH value of 6.0-7.

0.

4. A collagenase peptide, characterized in that, Prepared by the method according to any one of claims 1-3, having the quality indicators described in any one of (a)-(c): (a) Peptides with a molecular weight of 300-1500 Da account for 80-85 wt%, of which Pro-Hyp-Gly tripeptide content 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.

5. The use of the collagenase peptide as described in claim 4 in the preparation of skin whitening agents.

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

7. The skin whitening preparation according to claim 6, characterized in that, The skin whitening preparation is for external or oral use.

8. The method for preparing the skin whitening agent as described in claim 7, characterized in that, Includes the following steps: (a) Mix collagenase peptides, prebiotic composition and antioxidant synergist evenly; (b) Fluidized bed coating technology was used to coat the surface of the mixed particles with Eudragit L100-55 as the enteric material; (c) Control the weight gain of the coating to 3-5%, and fill it into the capsule shell after drying; The collagenase peptide is the collagenase peptide as described in claim 4.

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