Whitening compound based on cattle spleen peptide as well as preparation method and application of whitening compound

Abstract

The invention belongs to the field of cosmetics and health care products, and provides a whitening compound based on cattle spleen peptide and a preparation method and application thereof, and the whitening compound comprises cattle spleen peptide and one or more components selected from glycyrrhiza glabra root extract, nicotinamide, ferulic acid and vitamin C. The whitening compound realizes whitening through the synergistic effect of three paths of synthesis inhibition, transport blocking and oxidation reduction, can be prepared into freeze-dried powder, hydrogel films or oral capsules, realizes high-value utilization of cattle spleen resources, and breaks through the synergistic problem of'safety-efficacy-stability 'of whitening components.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cosmetics and health products, and specifically provides a whitening compound based on bovine spleen peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Bovine spleen, as the main by-product of meat processing, has an annual waste of more than 2 million tons. For a long time, these bovine spleens have been used as feedstock or directly discarded, which not only causes a serious waste of high-quality protein resources, but also constitutes a significant pollution pressure on the environment. However, modern research shows that bovine spleen is rich in various bioactive ingredients, and bovine spleen peptide is one of them with great development value. Bovine spleen peptide is a small molecule peptide extracted from bovine spleen, which has the characteristics of low molecular weight, high bioavailability, and good safety. Recent research shows that bovine spleen peptide has shown significant potential in regulating melanin synthesis and has gradually attracted attention in the field of whitening skin care.

[0003] Skin pigmentation is a common skin problem affecting more than 60% of adults worldwide. The existing whitening technology has three major defects: 1. Safety hazard: the mainstream ingredient hydroquinone has strong mutagenicity, and the skin sensitization rate of kojic acid is as high as 15%-22%; 2. Limited efficacy: single component mechanism is insufficient (e.g., nicotinamide only blocks melanin transport, and the tyrosinase inhibition rate is <20%); 3. Stability defects: vitamin C derivatives lose more than 30% of their activity after 7 days of storage at 40°C, and are easily reacted with metal ions to cause discoloration of the product. According to statistics, the global whitening market size is $21.5 billion, and the demand gap for "natural sources + multi-target synergy + zero irritation" products continues to expand.

[0004] Although bovine spleen peptide has significant whitening potential, its industrialization still faces key technical bottlenecks: 1. Extraction process: traditional single enzymatic method results in a functional amino acid (e.g., cysteine) retention rate of <50%; 2. Separation and purification: ion exchange resin desalination is not thorough, and Na + residues cause the separation of the compound system; 3. Compound technology: simple physical mixing results in a transdermal rate of <5% and a particle size of >500 nm; 4. Drying process: conventional spray drying at a temperature of >180°C causes denaturation of the peptide chain, with an activity loss of ≥40%.

[0005] In view of the huge stock of bovine spleen processing waste and the urgent demand of the whitening market, a multi-target compound system centered on bovine spleen peptide has dual value: 1. Realizing high-value utilization of waste resources and solving environmental pollution problems; 2. Breaking through the limitations of existing whitening ingredients and improving efficacy and safety through a synergistic mechanism (inhibiting tyrosinase + blocking melanin transport + antioxidant). SUMMARY

[0006] The present application aims to provide a high-efficiency and safe, non-irritating, multi-pathway melanin production inhibition, pigment transport blocking, antioxidant defense enhancement, and stable whitening effect based on a bovine spleen peptide whitening compound and a preparation method thereof.

[0007] The present application aims to provide a high-efficiency and safe, non-irritating, multi-pathway melanin production inhibition, pigment transport blocking, antioxidant defense enhancement, and stable whitening effect based on a bovine spleen peptide whitening compound and a preparation method thereof.

[0008] In one aspect, the present application provides a bovine spleen peptide-based whitening compound, which comprises bovine spleen peptide and one or more ingredients selected from the group consisting of Glycyrrhiza glabra root extract, nicotinamide, ferulic acid, and vitamin C.

[0009] Further, the whitening compound comprises 75-95 parts of bovine spleen peptide, 5-15 parts of Glycyrrhiza glabra root extract, 2-5 parts of nicotinamide, 1-3 parts of ferulic acid, and 1-3 parts of AA2G.

[0010] Further, the whitening compound comprises 85 parts of bovine spleen peptide, 10 parts of Glycyrrhiza glabra root extract, 3.5 parts of nicotinamide, 1.5 parts of ferulic acid, and 2 parts of AA2G.

[0011] Further, the average molecular weight of the bovine spleen peptide is 1200±200 Da, the proline content is ≥12%, the cysteine content is ≥8%, the glycine content is ≥10%, and the arginine content is ≥5%.

[0012] Further, the preparation method of the bovine spleen peptide comprises:

[0013] 1) Homogenize the bovine spleen, add Tris-HCl buffer with pH 7.0-8.0, and mix uniformly;

[0014] 2) Apply pulse electric field treatment to the mixture obtained in step 1); preheat by increasing temperature; add pepsin for enzymolysis; add trypsin and glutamine enzyme for enzymolysis; and apply low-frequency ultrasound synchronously during the enzymolysis process;

[0015] 3) Inactivate the enzyme of the enzymolysis liquid obtained in step 2), centrifuge, filter, ultrafilter, desalt, concentrate, and freeze-dry to obtain bovine spleen peptide.

[0016] Further, the preparation method of the bovine spleen peptide comprises:

[0017] 1) Take fresh bovine spleen, remove fat and fascia, rinse, cut into pieces, and homogenize; add 0.1 mol / L Tris-HCl buffer with pH 7.5±0.1 at a ratio of 1 g of tissue to 18 mL, and mix until homogeneous;

[0018] 2) Apply a high-voltage pulsed electric field to the mixture obtained in step 1) at 25±1℃ for 5 min. The high-voltage pulsed electric field strength is 20 kV / cm, the pulse width is 10 μs, and the frequency is 200 Hz. Raise the temperature to 40±0.5℃ and adjust the pH to 2.0±0.1. Add pepsin with an enzyme activity ≥3500 U / mg and enzymatically hydrolyze at 40±0.5℃ for 3 h. Adjust the pH to 7.8±0.1 and add a complex enzyme system consisting of trypsin with an enzyme activity ≥2500 U / mg and glutaminase with an enzyme activity ≥1000 U / g. Enzymatically hydrolyze at 48±0.5℃ for 2.5 h.

[0019] The mass ratio of trypsin to glutaminase in the complex enzyme system is 1:0.4; the mass ratio of pepsin to the complex enzyme system is 3:2; and the total enzyme content accounts for 2.0% of the raw material mass.

[0020] Low-frequency ultrasound was applied simultaneously during the enzymatic hydrolysis process. The low-frequency ultrasound frequency was 28kHz, the power was 150W, and the intermittent mode was 3s on / 5s off. 3) The enzyme hydrolysate from step 2) was inactivated, the supernatant was collected by centrifugation, the supernatant was pre-filtered with a filter membrane, ultrafiltered with a tangential flow ultrafiltration membrane, the filtrate was collected, desalted by chromatography, concentrated, and freeze-dried to obtain bovine spleen peptide.

[0021] Furthermore, the preparation method of the whitening compound includes: taking the prescribed amounts of bovine spleen peptide, licorice root extract, nicotinamide, ferulic acid, and AA2G, dissolving them in a 5% hydroxypropyl-β-cyclodextrin solution, homogenizing under high pressure, concentrating under vacuum, and then spray drying to obtain the whitening compound powder.

[0022] On the other hand, this application provides the use of whitening compound in the preparation of health products that help improve melasma.

[0023] Furthermore, the health product is in oral dosage form.

[0024] Furthermore, the health product is in capsule form.

[0025] On the other hand, this application provides the application of whitening compound in the preparation of cosmetics with whitening function.

[0026] Furthermore, the cosmetic product is a hydrogel film.

[0027] Furthermore, the whitening compound content in the hydrogel film is 2% w / v.

[0028] On the other hand, this application provides a method for preparing the above-mentioned bovine spleen peptide.

[0029] On the other hand, this application provides a method for preparing the above-mentioned whitening compound.

[0030] In the above compound: 1. Bovine spleen peptide, as the core component, has an average molecular weight of 1200±200 Da, with proline content ≥12%, cysteine ​​content ≥8%, glycine content ≥10%, and arginine content ≥5%. Its small molecule characteristics (<1.5kDa) ensure high transdermal penetration (>18%), inhibiting tyrosinase synthesis at its source by downregulating MITF transcription factor; 2. Licorice root extract synergistically enhances the effect of bovine spleen peptide, directly inhibiting tyrosinase activity (IC50). 50 =0.2μg / mL), blocking key steps in melanin production; 3. Niacinamide blocks the transport of melanosomes to keratinocytes, reducing epidermal pigmentation; 4. Ferulic acid and AA2G form an antioxidant matrix: ferulic acid scavenge free radicals (ORAC value ≥1500μmol TE / g), inhibiting inflammatory pigmentation caused by the NF-κB pathway; AA2G provides long-lasting reducing power, reducing oxidized melanin to a colorless state while protecting peptide activity. The five components work synergistically through the "synthesis inhibition-transport blockade-redox" three pathways to reduce melanin production by 68.9% without skin irritation (100% negative rate in patch test). This compound is encapsulated with hydroxypropyl-β-cyclodextrin and processed by low-temperature spray drying (inlet air 160℃ / outlet air 85℃) to ensure that the active ingredients retain >95% after 30 days of accelerated testing at 40℃, solving the industry pain points of easy inactivation and instability of traditional whitening ingredients.

[0031] Compared with existing technologies, the advantages of this invention are: 1) Highly efficient, safe, and non-irritating: Bovine spleen peptide has a small molecular weight (1200±200Da) and a transdermal penetration rate of >18%, blocking melanin production at its source by downregulating MITF; the compound patch test has a 100% negative rate, eliminating the risk of sensitization from hydroquinone / kojic acid; 2) Multi-target synergistic effect: The five components constitute a three-pathway system of "synthesis inhibition-transport blockade-redox" (tyrosinase inhibition rate of 89.7%, melanin reduction of 68.9%); Glycyrrhiza glabra (IC) 50 =0.2μg / mL) Synergistically inhibits tyrosinase with bovine spleen peptides, nicotinamide blocks transport, and ferulic acid + AA2G scavenge ROS; 3) Innovative active protection technology: pulsed electric field-ultrasound synergistic pretreatment and step-by-step double enzymatic hydrolysis protect functional amino acids (cysteine ​​retention rate ≥95%); microwave-water bath synergistic inactivation and hydroxypropyl-β-cyclodextrin embedding + low temperature spray drying (peptide denaturation rate <3%), accelerated test 30-day activity retention >97%; 4) Flexible dosage form with strong adaptability: lyophilized powder / hydrogel film meets precise skin care needs, oral capsules (0.3g / capsule) achieve synergistic internal and external conditioning; high pressure homogenization controls particle size D90 <150nm, transdermal efficiency reaches 4.2 times that of traditional processes. This invention realizes the high-value utilization of bovine spleen resources, breaks through the impossible triangle of "safety-efficacy-stability" for whitening ingredients, and provides a revolutionary solution for the industry. Detailed Implementation

[0032] The following embodiments are provided to better understand the present invention, but are not intended to limit it. These embodiments are for illustrative purposes only and do not limit the scope of protection of the invention.

[0033] Example 1

[0034] A whitening compound based on bovine spleen peptides, the ingredients and their weight parts are as follows: 80-90 parts bovine spleen peptides, 80-90 parts bovine spleen peptides, 8-12 parts licorice root extract, 3.0-4.0 parts niacinamide, 1.0-2.0 parts ferulic acid, and 1.5-2.5 parts fat-soluble vitamin C (AA2G);

[0035] The average molecular weight of the bovine spleen peptide is 1000-1400 Da, and the content of proline is ≥11%, cysteine ​​is ≥7%, glycine is ≥9%, and arginine is ≥4.5%.

[0036] The preparation method steps are as follows:

[0037] 1) Raw material pretreatment: Take fresh beef spleen, remove fat and fascia, rinse with physiological saline until no blood is found, and cut into 5mm pieces. 3 The tissue fragments were homogenized at 20,000 rpm for 3 times × 2 min (intermittent cooling) at 4℃; 0.08-0.12 mol / L Tris-HCl buffer (pH 7.2-7.8) was added at a ratio of 1g tissue to 16-20mL, and the mixture was stirred at 100-150 rpm for 10-20 min in a dynamic mixer until homogenized.

[0038] 2) Stepwise enzymatic hydrolysis: The mixture obtained in step 1) is treated using a stepped dual-enzyme hydrolysis technique coupled with pulsed electric field-low-frequency ultrasound pretreatment: First, a high-voltage pulsed electric field (field strength 18-22kV / cm, pulse width 8-12μs, frequency 180-220Hz) is applied to the mixture at 24-26℃ for 4-6 min; then, the mixture is heated to 39-41℃, the pH is adjusted to 1.9-2.1, pepsin (enzyme activity ≥3500U / mg) is added, and low-frequency ultrasound (frequency 26-30kHz) is applied simultaneously during the enzymatic hydrolysis process. With a power of 130-170W and an intermittent mode (3s on / 5s off), enzymatically hydrolyze at 39-41℃ for 2.5-3.5h; then adjust the pH to 7.7-7.9, add a complex enzyme system of trypsin and glutaminase (mass ratio 1:0.3-0.5) (the ratio of pepsin to the complex enzyme system is 1:1-7:3), where the trypsin activity is ≥2500U / mg, the glutaminase activity is ≥1000U / g, and the total enzyme amount (including pepsin) accounts for 1.8-2.2% (w / w) of the raw material mass, and enzymatically hydrolyze at 47-49℃ for 2.0-3.0h;

[0039] 3) Inactivation and purification: The enzyme hydrolysate was inactivated using a microwave-water bath co-inactivation method (microwave power 550-650W, 58-62℃ for 4-6 min), and immediately cooled to below 4℃ in an ice bath; then centrifuged at 7500-8500 rpm for 18-22 min (4℃), and the supernatant was collected. After pre-filtration through a 0.45 μm filter membrane, it was passed through a 2.5-3.5 kDa tangential flow ultrafiltration membrane (transmembrane pressure 0.3 MPa); the filtrate was collected and desalted using a Sephadex G-25 chromatography column (flow rate 2 mL / cm). 2 ·h, conductivity ≤50μS / cm), concentrated at ≤40℃ under low pressure to a solid content of 20-30%, and finally freeze-dried at -48 to -52℃ for 20-28h to obtain bovine spleen peptide;

[0040] 4) Preparation of the compound: The prescribed amounts of bovine spleen peptide, licorice root extract, nicotinamide, ferulic acid, and AA2G were dissolved in a 4–6% hydroxypropyl-β-cyclodextrin solution. The solution was homogenized under high pressure (50–70 MPa) 2–4 times, concentrated under vacuum at ≤45℃, and then spray-dried (inlet air 150–170℃, outlet air 80–90℃) to obtain a compound powder (moisture ≤5%). The above compound can be prepared as a lyophilized powder or hydrogel film. The effective ingredient content in the topical formulation is 1.8–2.2% w / w; the oral capsule specification is 0.28–0.32 g / capsule (HPMC capsule shell). The above experimental data are non-linear, and its mechanism of action needs further verification.

[0041] Example 2

[0042] A whitening compound based on bovine spleen peptides, the ingredients and their weight parts are as follows: 85 parts bovine spleen peptides, 10 parts licorice root extract, 3.5 parts niacinamide, 1.5 parts ferulic acid, and 2.0 parts fat-soluble vitamin C (AA2G).

[0043] The preparation method steps are as follows:

[0044] 1) Raw material pretreatment: Take fresh beef spleen, remove fat and fascia, rinse with physiological saline until no blood is found, and cut into 5mm pieces. 3 The tissue fragments were homogenized at 20,000 rpm for 3 times × 2 min at 4℃ (with intermittent cooling); 0.1 mol / L Tris-HCl buffer (pH 7.5±0.1) was added at a ratio of 1 g tissue: 18 mL, and the mixture was stirred at 120 rpm for 15 min in a dynamic mixer until homogenized.

[0045] 2) Stepwise enzymatic hydrolysis: The mixture obtained in step 1 was treated using a stepped dual-enzyme hydrolysis technique coupled with pulsed electric field-low-frequency ultrasound pretreatment: First, a high-voltage pulsed electric field (field strength 20kV / cm, pulse width 10μs, frequency 200Hz) was applied to the mixture at 25±1℃ for 5min; then, the mixture was heated to 40±0.5℃, the pH was adjusted to 2.0±0.1, pepsin (enzyme activity ≥3500U / mg) was added, and low-frequency ultrasound (frequency 28kHz) was applied simultaneously during the enzymatic hydrolysis process. The enzyme was hydrolyzed at 40±0.5℃ for 3 hours using a 150W power setting (intermittent mode, 3s on / 5s off). The pH was then adjusted to 7.8±0.1, and a complex enzyme system of trypsin and glutaminase (mass ratio 1:0.4) was added (the ratio of pepsin to the complex enzyme system was 3:2). The trypsin activity was ≥2500U / mg, the glutaminase activity was ≥1000U / g, and the total enzyme amount (including pepsin) accounted for 2.0% (w / w) of the raw material (fresh bovine spleen). The enzyme was hydrolyzed at 48±0.5℃ for 2.5 hours.

[0046] 3) Inactivation and purification: The enzyme hydrolysate was inactivated using a microwave-water bath co-inactivation method (microwave power 600W, 60℃ for 5 min), and immediately cooled to below 4℃ in an ice bath; then centrifuged at 8000 rpm for 20 min (4℃), and the supernatant was collected. After pre-filtration through a 0.45 μm filter membrane, it was passed through a 3 kDa tangential flow ultrafiltration membrane (transmembrane pressure 0.3 MPa); the filtrate was collected and desalted using a Sephadex G-25 chromatography column (flow rate 2 mL / cm). 2 ·h, conductivity ≤50μS / cm), concentrated at ≤40℃ under low pressure to a solid content of 25%, and finally freeze-dried at -50℃ for 24h to obtain bovine spleen peptide;

[0047] 4) Preparation of compound: Take 85 parts of bovine spleen peptide, 10 parts of licorice root extract (glycyrrhizic acid ≥10%), 3.5 parts of nicotinamide, 1.5 parts of ferulic acid (purity ≥98%), and 2 parts of AA2G, and dissolve them in 5% hydroxypropyl-β-cyclodextrin solution; homogenize three times under high pressure at 60MPa (inlet temperature ≤25℃), concentrate under vacuum at ≤45℃, and then spray dry (inlet air 160±2℃, outlet air 85±1℃, atomizing disc 18,000rpm) to obtain a free-flowing powder (moisture ≤5%).

[0048] The above-mentioned compound can be formulated into lyophilized powder (redissolved in 5% sodium hyaluronate solution) or hydrogel film (containing 1.5% carbomer 940). The active ingredient content of the topical formulation is 2.0±0.1% w / w; the oral capsule specification is 0.300±0.005g / capsule (HPMC capsule shell). The obtained bovine spleen peptide was analyzed by SEC-HPLC: average molecular weight 1215 Da, proline content 12.7%, cysteine ​​content 8.9%, glycine content 10.5%, and arginine content 5.2%.

[0049] Example 3

[0050] Test samples: Bovine spleen peptide compound oral capsules (lyophilized powder capsules) (0.3g / capsule) and compound external gel (containing 2% active ingredient) prepared in Example 2;

[0051] Experimental animals: SPF-grade female C57BL / 6 mice (n=48, 8 weeks old, with UVB-induced skin pigmentation after dorsal hair removal: after dorsal hair removal, mice were exposed to a narrow-band UVB light source (irradiance 1.2 mW / cm²). 2 Irradiation every other day, initial dose 50 mJ / cm² 2 ×3 times, then increase to 80mJ / cm². 2 Each session lasts for 4 weeks, with a total of 12 sessions. The irradiation area is limited to 2cm. 2 Lead plates are used for shielding in non-irradiated areas.

[0052] Test environment: SPF barrier system;

[0053] Grouping (n=8 / group): Blank control group: normal saline gavage + blank gel application; Model control group: UVB irradiation (no intervention); Positive control group: 5% nicotinamide solution gavage (0.1mL / 10g·bw) + commercially available whitening gel (containing 2% arbutin) compound oral administration group: bovine spleen peptide capsules (1.2g / kg·bw) + blank gel; Compound topical application group: normal saline gavage + 2% compound gel (w / w); Compound combination group: oral + topical combined intervention.

[0054] Cycle: Intervention once daily for 6 weeks, conducted concurrently with irradiation;

[0055] The experimental results (Table 1) show that compared with the model control group, the oral, topical, and combined groups all exhibited significantly reduced melanin content, tyrosinase activity, pigmentation area, transdermal water loss (TEWL) value, and MITF protein expression, while dermal collagen density was significantly increased. The combined group showed the best improvement in most of the above indicators (such as melanin content, tyrosinase activity, pigmentation area, TEWL value, MITF protein expression, and dermal collagen density), significantly outperforming either the oral or topical groups alone. The topical and combined groups also showed significantly improved melanosome transport inhibition rate, cysteine ​​bioavailability, peptide chain integrity, and transdermal depth distribution (especially in the dermis), with the combined group demonstrating superior performance in both transdermal depth and dermal distribution.

[0056] Table 1 Effects of bovine spleen peptide compound on mouse parameters

[0057]

[0058]

[0059] Note: ▲ indicates P < 0.01 compared to the blank control group.

[0060] * indicates P < 0.05 compared to the model control group.

[0061] ** indicates that P < 0.01 compared to the positive control group.

[0062] # indicates that the combination group is significantly different from the single-use group (oral / topical), with P < 0.05.

[0063] § indicates a comparison with all groups, P < 0.01

[0064] Example 4

[0065] Control group 1 (bovine spleen peptide group): prepared according to the formula and process of Example 2 (85 parts of bovine spleen peptide).

[0066] Control group 2 (soybean peptide group): Bovine spleen peptide was replaced with an equal amount of soybean peptide (molecular weight 1200±200Da, cysteine ​​content ≤2%).

[0067] Control group 3 (whey peptide group): Bovine spleen peptide was replaced with an equal amount of whey peptide (molecular weight 1300±150Da, cysteine ​​content ≤3%).

[0068] Other components and process parameters: The proportions and preparation steps of Glycyrrhiza glabra root extract, nicotinamide, ferulic acid, and AA2G are the same as in Example 2.

[0069] Functional testing:

[0070] Test samples: Three compound topical gels (containing 2% active ingredient (w / w)).

[0071] Experimental animals: SPF-grade C57BL / 6 mice (n=24, grouping as in Example 2); experimental protocol as in Example 3; UVB-induced skin pigmentation: after hair removal on the backs of mice, a narrow-band UVB light source (irradiance 1.2 mW / cm²) was used. 2 Irradiation every other day, initial dose 50 mJ / cm² 2 ×3 times, then increase to 80mJ / cm². 2 Each session lasts for 4 weeks, with a total of 12 sessions. The irradiation area is limited to 2cm. 2 Lead plates are used to shield the non-irradiated areas.

[0072] Test environment: SPF barrier system;

[0073] Cycle: Apply the corresponding gel topically once daily for 6 weeks, in conjunction with irradiation;

[0074] The experimental results (Table 2) showed that the melanin content in the bovine spleen peptide group (18.8 μg / mg) was significantly lower than that in the soybean peptide group (28.7 μg / mg) and the whey peptide group (31.4 μg / mg) (P<0.01); the inhibition rate of tyrosinase activity was: bovine spleen peptide group (52.9%) > soybean peptide group (21.8%) > whey peptide group (17.2%).

[0075] Table 2. Effects of bovine spleen peptide complex, soybean peptide complex, and whey peptide complex on mouse indicators.

[0076] Note: ▲ indicates P<0.01 compared to the bovine spleen peptide group;

[0077] * indicates P < 0.05 compared to the model group.

[0078] The applicant further conducted an accelerated test at 40°C:

[0079] Test dosage forms: Topical gel formulations. The accelerated testing focused on three compound topical gels containing 2% (w / w) of active ingredient: bovine spleen peptide compound gel, soybean peptide compound gel, and whey peptide compound gel. Preparation process: The excipients (licorice root extract, nicotinamide, etc.) and preparation steps for the three gels were consistent with Example 2, differing only in the active peptide components; commercially available packaging or similar packaging to that used in actual applications was employed.

[0080] Test conditions: Temperature and humidity: 40℃ (±2℃), relative humidity 75% ± 5% RH (simulating high temperature and high humidity environment);

[0081] Duration: 30 days

[0082] After 30 days of experimentation, the activity retention rate of the bovine spleen peptide group (97.5%) was higher than that of the soybean peptide group (67.5%) and the whey peptide group (61.8%), which was attributed to the synergistic pretreatment of pulsed electric field and ultrasound and the protective effect of hydroxypropyl-β-cyclodextrin on the functional amino acids in the bovine spleen peptide. (Activity retention rate calculation formula: Activity retention rate = (Activity value after acceleration / Initial activity value) × 100%. Numerator: Activity value measured after 30 days; Denominator: Initial activity value measured before the start of the experiment (day 0); expressed as antioxidant activity)

[0083] The bovine spleen peptide compound of this invention has a scientifically designed formulation and clearly defined process steps, making it feasible for large-scale production. This compound can be applied in the field of skin health, targeting and repairing problems such as UV damage and pigmentation, and can also serve as an auxiliary functional ingredient for post-medical aesthetic repair. The conventional operations involved in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

Claims

1. A whitening compound based on bovine spleen peptides, characterized in that, The whitening compound contains bovine spleen peptide and one or more ingredients selected from licorice root extract, nicotinamide, ferulic acid, and vitamin C.

2. The whitening compound according to claim 1, wherein the whitening compound comprises 75-95 parts of bovine spleen peptide, 5-15 parts of licorice root extract, 2-5 parts of nicotinamide, 1-3 parts of ferulic acid, and 1-3 parts of AA2G.

3. The whitening compound according to claim 2, wherein the whitening compound comprises 85 parts of bovine spleen peptide, 10 parts of licorice root extract, 3.5 parts of nicotinamide, 1.5 parts of ferulic acid, and 2 parts of AA2G.

4. The whitening compound according to any one of claims 1-3, wherein the bovine spleen peptide has an average molecular weight of 1200±200 Da, a proline content of ≥12%, a cysteine ​​content of ≥8%, a glycine content of ≥10%, and an arginine content of ≥5%.

5. The whitening compound according to any one of claims 1-4, wherein the method for preparing the bovine spleen peptide comprises: 1) Homogenize the bovine spleen, add Tris-HCl buffer solution with pH 7.0-8.0, and mix well; 2) Apply a pulsed electric field to the mixture obtained in step 1); Preheat the mixture; add pepsin for enzymatic hydrolysis; add a complex enzyme system consisting of trypsin and glutaminase for enzymatic hydrolysis; apply low-frequency ultrasound simultaneously during enzymatic hydrolysis; the mass ratio of trypsin to glutaminase in the complex enzyme system is 1:0.3-0.5; the mass ratio of pepsin to the complex enzyme system is 1:1-7:3; the total enzyme content accounts for 1.8-2.2% of the raw material mass. 3) The enzyme hydrolysate from step 2) is inactivated, centrifuged, filtered, ultrafiltered, desalted, concentrated, and freeze-dried to obtain bovine spleen peptides.

6. The whitening compound according to claim 5, wherein the method for preparing the bovine spleen peptide comprises: 1) Take fresh beef spleen, remove fat and fascia, rinse, cut into small pieces, and homogenize; Add 0.1 mol / L Tris-HCl buffer (pH 7.5 ± 0.1) at a ratio of 1 g tissue to 18 mL, and mix until homogenized; 2) Apply a high-voltage pulsed electric field to the mixture obtained in step 1) at 25±1℃ for 5 min. The high-voltage pulsed electric field strength is 20 kV / cm, the pulse width is 10 μs, and the frequency is 200 Hz. Raise the temperature to 40±0.5℃ and adjust the pH to 2.0±0.

1. Add pepsin with an enzyme activity ≥3500 U / mg and enzymatically hydrolyze at 40±0.5℃ for 3 h. Adjust the pH to 7.8±0.1 and add a complex enzyme system consisting of trypsin with an enzyme activity ≥2500 U / mg and glutaminase with an enzyme activity ≥1000 U / g. Enzymatically hydrolyze at 48±0.5℃ for 2.5 h. The mass ratio of trypsin to glutaminase in the complex enzyme system is 1:0.4; the mass ratio of pepsin to the complex enzyme system is 3:2; and the total enzyme content accounts for 2.0% of the raw material mass. Low-frequency ultrasound was applied simultaneously during the enzymatic hydrolysis process. The low-frequency ultrasound frequency was 28kHz, the power was 150W, and the intermittent mode was 3s on / 5s off. 3) Inactivate the enzyme in the enzymatic hydrolysate from step 2), centrifuge to obtain the supernatant, pre-filter with a filter membrane, ultrafilter with a tangential flow ultrafiltration membrane, collect the filtrate, desalt by chromatography, concentrate, and freeze-dry to obtain bovine spleen peptide.

7. The whitening compound according to any one of claims 1-6, wherein the preparation method of the whitening compound comprises: Take the prescribed amounts of bovine spleen peptide, licorice root extract, nicotinamide, ferulic acid, and AA2G, dissolve them in a 5% hydroxypropyl-β-cyclodextrin solution, homogenize under high pressure, concentrate under vacuum, and then spray dry to obtain a whitening compound powder.

8. The use of the whitening compound according to any one of claims 1-7 in the preparation of health products that help improve melasma.

9. The use of the whitening compound according to any one of claims 1-7 in the preparation of cosmetics with whitening function.

10. The application according to claim 9, wherein the cosmetic is a hydrogel film.

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