A Camellia Protein Hydrolysate with Anti-aging Properties, Its Preparation Method and Application

Abstract

This invention relates to a camellia peptide, its preparation method, and its applications, belonging to the field of biotechnology. The invention provides a camellia peptide containing characteristic peptides with amino acid sequences of at least one selected from Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro. The invention obtains the camellia peptide by processing camellia flowers, and HPLC-MS / MS identification shows that the camellia peptide contains characteristic peptides with amino acid sequences of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro. Recent studies have demonstrated that small molecule bioactive peptides composed of amino acids can be directly absorbed and utilized by the body, providing nutrients necessary for human growth and development while also exhibiting effective physiological functions.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a camellia protein hydrolysate with anti-aging properties, its preparation method, and its application. Background Technology

[0002] Camellia japonica Linn. is an evergreen broad-leaved tree belonging to the genus Camellia (Camellia L.) of the family Theaceae. It is a tropical and subtropical species. Camellias prefer warm and humid environments, with an optimal growth temperature of 18–24℃ and an optimal relative humidity of 60%–80%. They primarily inhabit hilly areas at altitudes of 300–1100 meters. Native to China, the vast majority of camellias in my country are distributed in regions south of the Yangtze River, including Zhejiang, Jiangxi, Guangxi, Yunnan, Guizhou, Hunan, and Guangdong. Currently, camellia cultivation has spread throughout Asia, Europe, North America, Oceania, Africa, and South America. The three main cultivated camellia species worldwide are East China Camellia, Yunnan Camellia, and Camellia sasanqua, along with the unique Chinese species Camellia chrysantha, forming the four major camellia systems. Camellias, with their graceful shape, evergreen foliage, vibrant colors, diverse flower shapes, and long blooming period, are among the world's most famous ornamental trees, possessing high ornamental value. Furthermore, camellias also have significant medicinal value. For example, camellias are included in the "Drug Standards of the Ministry of Health of the People's Republic of China: Mongolian Medicine Volume" and are clinically used for clearing heat and relieving pain. Recent studies have shown that camellias are rich in flavonoids, polyphenols, triterpenes, saponins, tannins, and organic acids, as well as vitamins, proteins, fats, amino acids, and various minerals. Bioactivity studies have shown that camellias can inhibit thrombus formation, alleviate myocardial ischemia damage, improve cerebral ischemia damage, and protect the gastric mucosa. Therefore, research on the chemical and biological activities of camellias is attracting increasing attention, showing broad application prospects in the food and pharmaceutical fields.

[0003] Although camellia extract has been included in the 2021 edition of the "Catalogue of Used Cosmetic Ingredients" and is used in product development, current fragrance extraction or extract preparation processes cannot effectively dissolve it due to the characteristics of large protein molecules. A large amount of protein remains in the residue after camellia extraction, which is not fully utilized. Therefore, there is an urgent need to develop a processing technology that can hydrolyze the proteins in camellia flowers or camellia residue into small molecule peptides to broaden the application range of camellia. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a camellia protein hydrolysate with anti-aging effects, its preparation method and application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a camellia protein hydrolysate with anti-aging effects, comprising a characteristic peptide having an amino acid sequence of at least one of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro.

[0007] This invention, through cell experiments, has found that camellia peptides and camellia protein hydrolysates containing camellia peptides can reduce the positive rate of SA-β-Gal in cells, indicating that they both have anti-aging activity, can delay the aging of skin cells and skin tissues, and have advantages such as safety, long-term use, and no side effects. They can be applied to products for anti-skin aging and related symptoms.

[0008] As a preferred embodiment of the camellia protein hydrolysate of the present invention, the mass ratio of the characteristic peptides Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro in the camellia protein hydrolysate is Leu-Pro-Phe:Leu-Leu-Leu-Leu-Gly-His:Ala-Pro=(93.5-94):(0.4-0.5):(5.4-6).

[0009] Secondly, the present invention provides a method for preparing the above-mentioned camellia protein hydrolysate, comprising the following steps:

[0010] (1) Mix camellia pollen with alkaline solution, extract twice, combine the extracts, and centrifuge to obtain the supernatant;

[0011] (2) Mix the supernatant obtained in step (1) with the acid solution, let it stand, and centrifuge to obtain the precipitate;

[0012] (3) Dissolve the precipitate obtained in step (2) in water, adjust the pH to 6.0-8.0, add protease, incubate in a water bath at 45-55℃ for 4-8 hours, centrifuge to obtain the enzymatic hydrolysate;

[0013] (4) The enzymatic hydrolysate obtained in step (3) is subjected to ultrafiltration and dried to obtain camellia protein hydrolysate, which contains camellia peptides.

[0014] This invention, through amino acid analysis of the prepared camellia protein hydrolysate, found that the main amino acids in the camellia protein hydrolysate are acidic amino acids. Therefore, camellia protein was extracted from camellia flowers using an alkali dissolution and acid precipitation method, and then subjected to proteolytic hydrolysis, ultrafiltration and drying to prepare camellia protein hydrolysate containing camellia peptides. In vivo and in vitro experiments verified that the camellia protein hydrolysate has excellent anti-aging effects.

[0015] As a preferred embodiment of the preparation method of the present invention, it includes at least one of the following (Ⅰ) to (Ⅱ):

[0016] (I) In step (1), the alkaline solution is a 0.3M NaOH solution, and the ratio of camellia pollen to alkaline solution is camellia pollen:alkaline solution = 1g:20mL;

[0017] (II) In step (1), the extraction is carried out by water bath extraction at 55°C for 1 hour.

[0018] As a preferred embodiment of the preparation method of the present invention, it includes at least one of the following (III) to (IV):

[0019] (III) In step (2), the acid solution is a 0.5M HCl solution;

[0020] (Ⅳ) In step (2), after the supernatant is mixed with the acid solution, the pH is adjusted to 3.0.

[0021] As a preferred embodiment of the preparation method of the present invention, it includes at least one of the following (V) to (VI):

[0022] (V) In step (3), the protease includes at least one of papain, trypsin, flavor protease and bromelain;

[0023] (VI) In step (3), the amount of protease added is 6000 U / g.

[0024] The enzyme used in the enzymatic hydrolysis process of the preparation method described in this invention can be a combination of various enzymes, such as a combination of papain and trypsin, a combination of papain and flavor protease, or a combination of papain and bromelain.

[0025] As a preferred embodiment of the preparation method of the present invention, in step (4), the ultrafiltration is performed by placing the enzymatic hydrolysate obtained in step (3) into an ultrafiltration membrane for ultrafiltration separation, wherein the molecular weight cutoff of the ultrafiltration membrane is 2500-3000 Da.

[0026] In a preferred embodiment of the preparation method of the present invention, in step (1), the method for preparing camellia pollen includes any one of the following methods:

[0027] Method 1: Air dry the camellia flowers naturally, crush them, and pass them through a 40-mesh sieve to obtain camellia flower powder;

[0028] Method 2: Place the camellia flowers at 100℃ for 15 minutes to kill the green, dry them at 55℃ until the moisture content is below 13%, pulverize them and pass them through a 40-mesh sieve to obtain camellia flower powder.

[0029] In a preferred embodiment of the preparation method described in this invention, in step (1), the raw materials for the camellia pollen include whole camellia flowers and residues generated from the preparation of camellia flowers for fragrances, essential oils, or extracts. This invention can hydrolyze the proteins in camellia flowers or camellia flower residues, enabling the reuse of camellia flower residues, further expanding the application scope of camellia flowers, and increasing their economic benefits.

[0030] Thirdly, the present invention provides a camellia peptide, wherein the camellia peptide comprises a characteristic peptide having an amino acid sequence of at least one of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro.

[0031] This invention obtains camellia peptides by processing camellia flowers. HPLC-MS / MS analysis revealed that the camellia peptides contain characteristic peptides with amino acid sequences of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro. Recent studies have demonstrated that small-molecule bioactive peptides composed of amino acids can be directly absorbed and utilized by the body, providing not only the nutrients needed for human growth and development but also exhibiting effective physiological functions.

[0032] As a preferred embodiment of the camellia peptide of the present invention, the amino acid sequences of the characteristic peptides in the camellia peptide are Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro.

[0033] Fourthly, the present invention provides the application of the above-mentioned camellia peptides or camellia protein hydrolysates with anti-aging effects in the preparation of anti-aging products.

[0034] As a preferred embodiment of the application described in this invention, the articles include pharmaceuticals, food, health products, cosmetics, and biological products.

[0035] As a preferred embodiment of the application described in this invention, the amount of camellia protein hydrolysate used is 250-1000 mg per 1 kg body weight of the organism.

[0036] As a preferred embodiment of the application described in this invention, the anti-aging product is an anti-skin aging product.

[0037] As a preferred embodiment of the present invention, the anti-skin aging includes internal aging caused by aging or gene changes and premature aging caused by external environment. The external stimuli include, but are not limited to, sunlight exposure, pollutant stimulation, drug side effects, metabolic diseases, oxidant stimulation, emotional factors, malnutrition, and bad lifestyle habits such as smoking, drinking, lack of exercise, and staying up late.

[0038] Fourthly, the present invention provides an anti-aging cosmetic, comprising the above-mentioned camellia protein hydrolysate or camellia peptide.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] 1. This invention obtains camellia peptides by processing camellia flowers. The camellia peptides are identified by HPLC-MS / MS as containing characteristic peptides with amino acid sequences of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His and Ala-Pro.

[0041] 2. Through cell experiments, this invention has found that camellia peptides and camellia protein hydrolysates containing camellia peptides can reduce the positive rate of SA-β-Gal in cells, indicating that they have anti-aging activity, can delay the aging of skin cells and skin tissues, and have the advantages of being safe, suitable for long-term use, and without side effects. They can be applied to products for anti-skin aging and related symptoms.

[0042] 3. Through amino acid analysis of the prepared camellia protein hydrolysate, this invention found that the main amino acids in the camellia protein hydrolysate are acidic amino acids. Therefore, camellia protein was extracted from camellia flowers using the alkali dissolution and acid precipitation method, and then subjected to proteolytic hydrolysis, ultrafiltration and drying to prepare camellia protein hydrolysate containing camellia peptides. In vivo and in vitro experiments have verified that the camellia protein hydrolysate has excellent anti-aging effects. Attached Figure Description

[0043] Figure 1 This is a liquid chromatography-mass spectrometry (LC-MS) diagram of the camellia protein hydrolysate from Example 1 of the present invention.

[0044] Figure 2 This is the secondary mass spectrum of the LPF peptide in Example 1 of the present invention;

[0045] Figure 3 This is the secondary mass spectrum of the LLLLGH peptide in Example 1 of the present invention;

[0046] Figure 4 This is a secondary mass spectrum of the AP peptide in Example 1 of the present invention. Detailed Implementation

[0047] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0048] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0049] Human fibroblasts were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu49.

[0050] The solid-phase synthesis of LPF, LLLLGH, and AP characteristic peptides was commissioned to a biological company for completion.

[0051] The formulation of the staining working solution in the SA-β-Gal cell staining experiment: 1 mg / mL X-gal, 5 mM potassium ferrocyanide, 5 mM magnesium chloride, 5 mM citric acid-sodium citrate buffer, pH adjusted to 6.0.

[0052] SPF-grade female C57BL / 6J mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center (SCXK(Guangdong)2022-0002).

[0053] SPF-grade BALB / c female mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center (SCXK(Guangdong)2022-0002).

[0054] The techniques not described in detail in the following examples and effect examples are common techniques in this field. References can be made to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Press), "Molecular Biology Experiments (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiments" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0055] In the following examples, comparative examples, and effect examples, "V" refers to valine (Val), "P" refers to proline (Pro), "G" refers to glycine (Gly), "H" refers to histidine (His), "R" refers to arginine (Arg), "K" refers to lysine (Lys), "I" refers to isoleucine (Ile), "F" refers to phenylalanine (Phe), "L" refers to leucine (Leu), "W" refers to tryptophan (Trp), "A" refers to alanine (Ala), "M" refers to methionine (Met), "C" refers to cysteine (Cys), "N" refers to asparagine (Asn), "S" refers to serine (Ser), "Q" refers to glutamine (Gln), "Y" refers to tyrosine (Tyr), "D" refers to aspartic acid (Asp), "E" refers to glutamic acid (Glu), and "T" refers to threonine (Thr).

[0056] In the following examples, comparative examples, and effect examples, the LPF characteristic peptide refers to the Leu-Pro-Phe characteristic peptide, LLLLGH refers to the Leu-Leu-Leu-Leu-Gly-His characteristic peptide, and the AP characteristic peptide refers to the Ala-Pro characteristic peptide.

[0057] Example 1

[0058] This example provides a camellia protein hydrolyzate and its preparation method. The preparation method includes the following steps:

[0059] S1. Camellia flowers are naturally air-dried and pulverized through a 40-mesh sieve, or they are blanched at 100℃ for 15 minutes and then dried at 55℃ until the moisture content is <13%, pulverized through a 40-mesh sieve to obtain camellia flower powder.

[0060] S2. Take the camellia pollen obtained in step S1 and 0.3M NaOH solution. The ratio of camellia pollen to NaOH solution is 1g:20mL. Soak in a water bath at 55℃ for 1h. Repeat the soaking twice. Combine the two extracts and centrifuge at 10000rpm for 10min to obtain the supernatant.

[0061] S3. Mix the supernatant obtained in step S2 with 0.5M HCl solution, adjust the pH to 3.0, let it stand to precipitate, and centrifuge at 10000rpm for 10min to obtain the precipitate, which is crude protein.

[0062] S4. Add water to the precipitate obtained in step S3, adding 10 mL of water for every 1 g of precipitate, stir well, adjust the pH to 7.0 with 2 M NaOH solution, place in a water bath at 50℃, add 6000 U / g papain, stir at 300 rpm for 4 h for enzymatic hydrolysis, place in a water bath at 90℃ for 15 min after enzymatic hydrolysis, cool to room temperature, centrifuge at 10000 rpm for 10 min, collect the supernatant, and the obtained supernatant is the enzymatic hydrolysate;

[0063] S5. The enzymatic hydrolysate obtained in step S4 is subjected to ultrafiltration using a laboratory membrane separation device. The ultrafiltration membrane used has a molecular weight cutoff of 3000 Da, resulting in an ultrafiltration solution with a molecular weight lower than 3000 Da. The ultrafiltration solution is then spray-dried in a high-speed centrifugal spray dryer to obtain camellia protein hydrolysate, which contains camellia peptides.

[0064] Example 2-3

[0065] Examples 2-3 respectively provide a camellia protein hydrolysate and its preparation method. The preparation method is similar to that of Example 1, with the following differences:

[0066] Example 2: In step S4, the phrase "placed in a water bath at 50°C" is changed to "placed in a water bath at 45°C", while the rest of the operation remains unchanged;

[0067] Example 3: In step S4, the phrase "placed in a water bath at 50°C" is changed to "placed in a water bath at 55°C", while the rest of the operation remains unchanged.

[0068] Examples 4-5

[0069] Examples 4-5 respectively provide a camellia protein hydrolysate and its preparation method. The preparation method is similar to that of Example 1, with the following differences:

[0070] Example 4: In step S4, the phrase "adjust pH = 7.0 with 2M NaOH solution" is changed to "adjust pH = 6.0 with 2M NaOH solution", while the rest of the operation remains unchanged;

[0071] Example 5: In step S4, the phrase "adjust pH = 7.0 with 2M NaOH solution" is changed to "adjust pH = 8.0 with 2M NaOH solution".

[0072] Examples 6-9

[0073] Examples 6-9 provide a camellia protein hydrolysate and its preparation method, which are similar to those in Example 1, except as follows:

[0074] Example 6: In step S4, the "300 rpm stirring enzymatic hydrolysis for 4 hours" is adjusted to "300 rpm stirring enzymatic hydrolysis for 5 hours", and the rest of the operation remains unchanged;

[0075] Example 7: In step S4, the "300 rpm stirring enzymatic hydrolysis for 4 hours" is adjusted to "300 rpm stirring enzymatic hydrolysis for 6 hours", and the rest of the operation remains unchanged;

[0076] Example 8: In step S4, the "300 rpm stirring enzymatic hydrolysis for 4 hours" is adjusted to "300 rpm stirring enzymatic hydrolysis for 7 hours", while the rest of the operation remains unchanged;

[0077] Example 9: In step S4, the "300rpm stirring enzymatic hydrolysis for 4 hours" is adjusted to "300rpm stirring enzymatic hydrolysis for 8 hours", and the rest of the operation remains unchanged.

[0078] Examples 10-12

[0079] Examples 10-12 respectively provide a camellia protein hydrolysate and its preparation method. The preparation method is similar to that of Example 1, with the following differences:

[0080] Example 10: In step S4, the phrase "adding 6000 U / g of papain" is changed to "adding 6000 U / g of trypsin", while the rest of the operation remains unchanged;

[0081] Example 11: In step S4, the phrase "adding 6000 U / g of papain" is changed to "adding 6000 U / g of flavor protease", while the rest of the operation remains unchanged;

[0082] Example 12: In step S4, the phrase "add 6000 U / g of papain" is changed to "add 6000 U / g of bromelain", while the rest of the operation remains unchanged.

[0083] Comparative Examples 1-3

[0084] Comparative Examples 1-3 each provide a camellia protein hydrolysate and its preparation method. The preparation methods are similar to those in Example 1, with the following differences:

[0085] Comparative Example 1: In step S4, the phrase "adding 6000 U / g of papain" was changed to "adding 6000 U / g of alkaline protease", while the rest of the operation remained unchanged;

[0086] Comparative Example 2: In step S4, the phrase "adding 6000 U / g of papain" was changed to "adding 6000 U / g of neutral protease", while the rest of the operation remained unchanged;

[0087] Comparative Example 3: In step S4, the phrase "adding 6000 U / g of papain" was changed to "adding 6000 U / g of complex protease", while the rest of the operation remained unchanged.

[0088] Comparative Examples 4-6

[0089] Comparative Examples 4-6 each provide a camellia protein hydrolysate and its preparation method. The preparation methods are similar to those in Example 1, with the following differences:

[0090] Comparative Example 4: In step S4, the phrase "placed in a water bath at 50°C" is changed to "placed in a water bath at 35°C", while the rest of the operation remains unchanged;

[0091] Comparative Example 5: In step S4, the phrase "placed in a water bath at 50°C" is changed to "placed in a water bath at 40°C", while the rest of the operation remains unchanged;

[0092] Comparative Example 6: In step S4, the phrase "placed in a water bath at 50°C" is changed to "placed in a water bath at 60°C", while the rest of the operation remains unchanged.

[0093] Comparative Examples 7-8

[0094] Comparative Examples 7-8 respectively provide a camellia protein hydrolysate and its preparation method. The preparation methods are similar to those in Example 1, with the following differences:

[0095] Comparative Example 7: In step S4, the phrase "adjust pH = 7.0 with 2M NaOH solution" is changed to "adjust pH = 5.0 with 2M NaOH solution", while the rest of the operation remains unchanged;

[0096] Comparative Example 8: In step S4, the phrase "adjust pH = 7.0 with 2M NaOH solution" is changed to "adjust pH = 9.0 with 2M NaOH solution".

[0097] Comparative Examples 9-10

[0098] Comparative Examples 9-10 respectively provide a camellia protein hydrolysate and its preparation method. The preparation method is similar to that of Example 1, except as follows:

[0099] Comparative Example 9: In step S4, the "300 rpm stirring enzymatic hydrolysis for 4 hours" was adjusted to "300 rpm stirring enzymatic hydrolysis for 2 hours", while the rest of the operation remained unchanged;

[0100] Comparative Example 10: In step S4, the "300 rpm stirring and enzymatic hydrolysis for 4 hours" was changed to "300 rpm stirring and enzymatic hydrolysis for 3 hours", while the rest of the operation remained unchanged.

[0101] Example of effect 1

[0102] The camellia protein hydrolysate obtained in Example 1 was characterized using the following specific method:

[0103] 1. Determination of amino acid composition in camellia protein hydrolysate.

[0104] The amino acid content of the camellia protein hydrolysate prepared in Example 1 was analyzed according to the national safety standard GB 5009.124-2016, "Determination of Amino Acids in Food". The analysis was performed using an S7130 fully automated amino acid analyzer (SYKAM, Germany), with a Cation Separation Column LCA K06 / Na, 150 mm × 4.6 mm, and an Ammonia Filtration Column LCA K04 / Na, 100 mm × 4.6 mm. The results are shown in Table 1.

[0105] Table 1. Amino acid content of camellia protein hydrolysate

[0106] amino acids percentage(%) amino acids percentage(%) Aspartic acid 10.92 Leucine 10.08 threonine 4.25 Tyrosine 3.35 Serine 10.09 Phenylalanine 4.58 glutamic acid 17.51 Histidine 2.34 glycine 4.98 Lysine 4.67 alanine 5.80 Arginine 4.90 Valine 5.35 proline 6.59 Isoleucine 4.59 total 100

[0107] As shown in Table 1, camellia protein hydrolysate is rich in various amino acids, with a relatively balanced composition of essential amino acids, indicating high nutritional value and worthy of vigorous development and utilization. Glutamic acid and aspartic acid are the main amino acids in camellia protein hydrolysate and even camellia peptides, followed by serine and leucine. Therefore, the relatively high content of acidic amino acids makes camellia protein acidic under natural conditions, and alkali dissolution and acid precipitation can effectively extract protein from camellia.

[0108] 2. The peptide sequences of the camellia protein hydrolysate obtained in Example 1 were identified.

[0109] The amino acid sequence of the camellia protein hydrolysate prepared in Example 1 was identified using HPLC-MS / MS. The specific detection method is as follows:

[0110] The samples were separated and detected using an X500LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry (LC-MS) system (AB SCIEX, USA). The LC and MS control software was SCIEX OS 2.0 (AB SCIEX, USA); the chromatographic column was a 1×100mm HSS T3 (1.8μm) column. (American Waters).

[0111] The mobile phase consisted of a 0.1% (v / v) aqueous solution of formic acid (A) and acetonitrile (B). The elution method was as follows: 0-4.00 min 5.0% B, 4.00-6.00 min 5.0-10.0% B, 6.00-30.00 min 10.0-40.0% B, 30.00-34.00 min 40.0-90.0% B, 34.00-40.00 min 90% B, 40.00-42.00 min 90.0-5.0% B, 42.00-52.00 min, 5.0% B, flow rate 0.05 mL / min, injection volume 1 μL, column temperature 40℃; Mass spectrometry detection method: scan period 0.642 s, ESI ion source temperature 500℃, positive ion mode, spray voltage 5500 V, TOF primary scan range 100-1200 Da, secondary scan range 50-1200 Da, operating mode IDA, maximum candidate ion number 4, dynamic exclusion enabled, other parameters use the default values ​​for proteomics methods. Note that the dead volume should be minimized before using the instrument. Identification results are shown in [link to results]. Figure 1-4 And Table 2.

[0112] Table 2. Identification results of camellia peptide sequences in camellia protein hydrolysate.

[0113]

[0114] like Figure 1-4 As shown in Table 2, the present invention used HPLC-MS / MS technology to identify three characteristic amino acid sequences from camellia protein hydrolysate, including one dipeptide (AP), one tripeptide (LPF), and one hexapeptide (LLLLGH).

[0115] Example 2

[0116] The anti-aging activity of the camellia protein hydrolysate obtained in Example 1 was evaluated using the following specific method:

[0117] Cellular senescence is assessed by detecting the activity of SA-β-Gal (senescence-associated β-galactosidase) in cells. At pH 6.0, SA-β-Gal hydrolyzes X-gal substrates, producing a blue precipitate. Senescent cells exhibit increased SA-β-Gal activity, thus displaying a distinct blue stain after staining. This method is commonly used in aging-related studies and drug screening. In this example, this method was used to detect the anti-skin cell aging activity of the camellia protein hydrolysate and its characteristic amino acid sequence of camellia peptides obtained in Example 1 on human fibroblasts.

[0118] This example uses the crude protein obtained in step S3 of Example 1, the enzymatic hydrolysate obtained in step S4 of Example 1, the components with >3000Da and <3000Da obtained in step S5 of Example 1, and the solid-phase synthesized characteristic peptides LPF, LLLLGH, and AP for experiments.

[0119] Human fibroblasts were seeded in 6-well plates and allowed to adhere overnight. After washing the cells twice with PBS, 0.5 mL of PBS was added to each well to cover the cells. A UV irradiation device was placed 25 cm above the cells, with the wavelength set to 365 nm (UVA band), and real-time monitoring was performed. The UVA-induced cell senescence dose was set at 5 J / cm², with irradiation for 15 min per session. Cells not subjected to UV irradiation served as the normal control group. After the irradiation, the PBS was replaced with complete culture medium containing the drug (DMEM + 10% FBS). The amounts of crude protein, enzymatic hydrolysate, components less than 3000 Da, and components greater than 3000 Da obtained in Example 1 were added at 20 μg / mL, and the amounts of the three active peptides LPF, LLLLGH, and AP were added at 5 μM. The drug-containing culture medium was replaced again after the second day. After the fourth day, the number of senescent cells was determined using the SA-β-Gal staining method.

[0120] The procedure for SA-β-Gal cell staining is as follows: Cells were washed twice with PBS, and 1 mL of fixative was added and fixed at room temperature for 5 minutes. The fixative was discarded, and the cells were washed twice with PBS. Freshly prepared staining working solution was added, and the cells were incubated at 37°C for 16 hours. The chromogenic solution was removed, and the cells were washed twice with PBS. Finally, 2 mL of PBS was added to each well, and SA-β-Gal-positive senescent cells (appearing blue) were observed under a microscope. The positive rate was calculated, and the results are shown in Table 3.

[0121] Table 3. Positive rate of SA-β-Gal in human skin fibroblasts

[0122]

[0123]

[0124] As shown in Table 3, the crude protein in camellia protein hydrolysate exhibits extremely weak anti-aging activity, only reducing the SA-β-Gal positivity rate of human skin fibroblasts from 58.6% to 52.6%. However, the enzymatically prepared hydrolysate significantly enhances anti-aging activity, further reducing the SA-β-Gal positivity rate to 32.1%. This indicates that enzymatic hydrolysis technology can significantly enhance the anti-aging properties of camellia protein in camellia protein hydrolysate. Comparison of the activity of ultrafiltration fractions of camellia protein hydrolysate revealed that the SA-β-Gal positivity rate of the ultrafiltration fraction group with a molecular weight less than 3 kDa was only 11.4%, while the SA-β-Gal positivity rate of the ultrafiltration fraction group with a molecular weight greater than 3 kDa was 45.6%. This suggests that the anti-aging activity of camellia protein hydrolysate is mainly caused by fractions with a molecular weight less than 3 kDa. Three characteristic peptides (LPF, LLLLGH, and AP) were evaluated and found to have potent anti-aging activity, reducing the SA-β-Gal positivity rate to below 10%. Therefore, these three active peptide fragments can serve as an important basis for quality control of camellia protein hydrolysate and the camellia peptides it contains.

[0125] Example 3

[0126] The anti-aging activity of the camellia protein hydrolysates obtained in Examples 1-12 and Comparative Examples 1-10 was tested. The positive cell rate of SA-β-Gal was tested according to the method of Effect Example 2. The results are shown in Table 4.

[0127] Table 4. Effects of different enzymatic hydrolysis processes on the anti-aging activity of camellia protein hydrolysates

[0128]

[0129]

[0130] As shown in Table 4, the anti-aging activities of camellia protein hydrolysates prepared by different processes varied. The anti-aging activity of the product increased with increasing temperature, but temperatures above 50℃ actually impaired the product's activity. This may be related to the destruction of enzyme activity or increased side reactions caused by high temperatures. Therefore, it is best to control the enzymatic hydrolysis temperature within the range of 45-55℃, with 50℃ being optimal.

[0131] The pH of the enzymatic hydrolysis reaction also affects the activity of the product. The anti-aging activity of the product is better in the pH range of 6.0-8.0, with pH=7.0 being the optimal value. This may be related to the optimal pH required for the enzymatic reaction.

[0132] Furthermore, the pH of the reaction system is affected by the release of amino acid sequences. Therefore, selecting the optimal starting pH is beneficial for ensuring the activity of the product.

[0133] Finally, the inventors discovered that the anti-aging activity of camellia protein hydrolysate requires a certain enzymatic reaction time; at least 4 hours are needed for the camellia protein hydrolysate to reduce the SA-β-Gal positivity rate of human skin fibroblasts to below 20%. However, further extending the reaction time did not significantly improve the activity of the product. Therefore, considering energy consumption, a hydrolysis time of 4 hours is most suitable.

[0134] Example of effect 4

[0135] The effect of the camellia protein hydrolysate obtained in Example 1 on a photo-aged mouse model was detected using the following specific method:

[0136] Six-week-old SPF-grade female C57BL / 6J mice were acclimatized for one week in an environment with a temperature of 20-25℃, humidity of 40-60%, and 12 hours of light per day (7:00-19:00) before the experiment was conducted. All mice had their dorsal hair removed and were randomly divided into four groups: a normal control group, a UV model group, a low-dose (25 mg / mL) camellia protein hydrolysate group, a medium-dose (50 mg / mL) camellia protein hydrolysate group, and a high-dose (100 mg / mL) camellia protein hydrolysate group, with eight mice in each group. Except for the normal control group, the other groups of mice were exposed to UVA (25 mJ / cm²) once daily. 2 For eight consecutive weeks, starting from the model initiation date, mice in the treatment group received daily light exposure followed by application of camellia protein hydrolysate solution to the bald areas, with 0.3 mL applied to each mouse. The normal control group and the model group received an equal volume of distilled water. After the experiment, the mice were euthanized by cervical dislocation, the back skin was collected, subcutaneous tissue was removed, and the skin was stored in liquid nitrogen.

[0137] The level of the aging marker SA-β-Gal in skin tissue was observed through histopathological sections. The specific experimental steps are as follows:

[0138] (1) Remove mouse skin tissue from liquid nitrogen, cool and embed the skin using OCT frozen section embedding medium, slice the skin tissue longitudinally using a cryostat with a thickness of 8 μm, and attach the tissue to a glass slide.

[0139] (2) Wash away the embedding agent with double-distilled water, then immerse the tissue in 1% formaldehyde solution for 1 minute to fix it, and wash the sections with PBS several times to remove the fixative.

[0140] (3) Add an appropriate amount of SA-β-Gal staining solution to the tissue and place it in a constant temperature incubator at 37°C overnight.

[0141] (4) Eosin counterstaining: clean the SA-β-Gal staining solution, immerse the slide in 95% ethanol for 5 seconds to remove water, then immerse the slide in eosin staining solution for 2 minutes, quickly wash the slide twice with 70% ethanol, and place it in a fume hood to air dry.

[0142] (5) The tissue was mounted with neutral resin and observed and photographed under an optical microscope after solidification. The positive spots of SA-β-Gal were blue. The results are shown in Table 5.

[0143] Table 5. Levels of SA-β-Gal in skin tissue of photoaged mice

[0144]

[0145] As shown in Table 5, compared with normal control mice, the number of SA-β-Gal positive spots in the skin tissue of UV-induced photoaging mice was significantly increased (from 6.80±3.74 spots / mm). 2 Increased to 37.4±7.77 per mm 2 Different doses of camellia protein hydrolysate can reduce SA-β-Gal positive spots. A high dose (100 mg / mL) of camellia protein hydrolysate can reduce SA-β-Gal positive spots to 11.5 ± 2.56 spots / mm. 2 The above experimental results demonstrate that topical application of camellia protein hydrolysate can reduce the levels of aging markers in tissues and slow down the skin aging process. In fact, the release of small peptides formed by enzymatic hydrolysis helps enhance skin absorption, thus achieving its anti-aging effect in vivo.

[0146] Example 5

[0147] The effect of the camellia protein hydrolysate obtained in Example 1 on a D-galactose-induced aging mouse model was detected using the following specific method:

[0148] Six-week-old SPF-grade female BALB / c mice (18–22 g) were acclimatized for one week in an environment with a temperature of 20–25°C, humidity of 40–60%, and 12 hours of light per day (7:00–19:00) before the experiment was conducted. Mice were randomly divided into four groups: a normal control group, a D-galactose model group, a low-dose (250 mg / kg) camellia protein hydrolysate group, a medium-dose (500 mg / kg) camellia protein hydrolysate group, and a high-dose (1000 mg / kg) camellia protein hydrolysate group, with eight mice in each group. Except for the blank control group, the other groups of mice underwent subcutaneous injection of D-galactose (500 mg / kg) into the nape of the neck to establish a subacute skin aging model, once daily for eight consecutive weeks. From the start of model establishment, camellia protein hydrolysate was administered daily by gavage, while the normal control group and the model group received an equal volume of physiological saline. After the experiment, the mice were euthanized by cervical dislocation, the hair on their backs was cut off, the skin from their backs was collected, the subcutaneous tissue was removed, and the skin was stored in liquid nitrogen. The level of SA-β-Gal in the mouse skin was detected according to the method described in Example 4, and the results are shown in Table 6.

[0149] Table 6. Levels of SA-β-Gal in skin tissue of D-galactose-induced aging mice.

[0150]

[0151] As shown in Table 6, compared with normal control mice, the number of SA-β-Gal positive spots in the skin tissue of D-galactose-induced aging mice was significantly increased, while different doses of camellia protein hydrolysate could reduce the number of SA-β-Gal positive spots. These experimental results indicate that oral ingestion of camellia protein hydrolysate can also achieve the accumulation of aging markers in skin tissue and alleviate the skin aging process.

[0152] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A camellia protein hydrolysate with anti-aging properties, characterized in that, It contains characteristic peptides with amino acid sequences of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro; The camellia protein hydrolysate with anti-aging properties is mainly prepared by the following steps: (1) Mix camellia pollen with alkaline solution, extract twice, combine the extracts, and centrifuge to obtain the supernatant; (2) Mix the supernatant obtained in step (1) with the acid solution, let it stand, and centrifuge to obtain the precipitate; (3) Dissolve the precipitate obtained in step (2) in water, adjust the pH to 6.0-8.0, add protease, incubate in a water bath at 45-55℃ for 4-8 hours, centrifuge to obtain the enzymatic hydrolysate; (4) The enzymatic hydrolysate obtained in step (3) is subjected to ultrafiltration and dried to obtain camellia protein hydrolysate, which contains camellia peptides; In step (1), the alkaline solution is a 0.3-0.5M NaOH solution, and the ratio of camellia pollen to alkaline solution is camellia pollen:alkaline solution = 1g:(15-20)mL; In step (2), after the supernatant is mixed with the acid solution, the pH is adjusted to 3.0-4.0; In step (3), the protease is papain.

2. The camellia protein hydrolysate as described in claim 1, characterized in that, The mass ratio of the characteristic peptides Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro in the camellia protein hydrolysate is Leu-Pro-Phe:Leu-Leu-Leu-Leu-Gly-His:Ala-Pro = (93.5-94):(0.4-0.5):(5.4-6).

3. The method for preparing camellia protein hydrolysate as described in claim 1, characterized in that, Includes the following steps: (1) Mix camellia pollen with alkaline solution, extract twice, combine the extracts, and centrifuge to obtain the supernatant; (2) Mix the supernatant obtained in step (1) with the acid solution, let it stand, and centrifuge to obtain the precipitate; (3) Dissolve the precipitate obtained in step (2) in water, adjust the pH to 6.0-8.0, add protease, incubate in a water bath at 45-55℃ for 4-8 hours, centrifuge to obtain the enzymatic hydrolysate; (4) The enzymatic hydrolysate obtained in step (3) is subjected to ultrafiltration and dried to obtain camellia protein hydrolysate, which contains camellia peptides; In step (1), the alkaline solution is a 0.3-0.5M NaOH solution, and the ratio of camellia pollen to alkaline solution is camellia pollen:alkaline solution = 1g:(15-20)mL; In step (2), after the supernatant is mixed with the acid solution, the pH is adjusted to 3.0-4.0; In step (3), the protease is papain.

4. The preparation method according to claim 3, characterized in that, In step (1), the extraction is carried out in a water bath at 55°C for 1-1.5 hours.

5. The preparation method according to claim 3, characterized in that, In step (2), the acid solution is a 0.5-0.7M HCl solution.

6. The preparation method according to claim 3, characterized in that, In step (3), the amount of protease added is 5500-6000 U / g.

7. The preparation method according to claim 3, characterized in that, In step (4), the ultrafiltration is performed by placing the enzymatic hydrolysate obtained in step (3) into an ultrafiltration membrane for ultrafiltration separation, wherein the molecular weight cutoff of the ultrafiltration membrane is 2500-3000 Da.

8. A camellia peptide composition, characterized in that, The camellia peptide composition is composed of characteristic peptides with the amino acid sequences Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro.

9. The use of the camellia protein hydrolysate of claim 1 or the camellia peptide composition of claim 8 in the preparation of anti-skin aging products; wherein the products are pharmaceuticals and cosmetics.

10. An anti-aging cosmetic, characterized in that, Includes the camellia protein hydrolysate as described in claim 1 or the camellia peptide composition as described in claim 8.

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