Camellia seed meal oligopeptide as well as preparation method and application thereof

Abstract

The invention discloses camellia seed meal oligopeptide as well as a preparation method and application thereof. The method comprises the following steps: S1, pretreating camellia seed meal to obtain a pretreated substance; s2, performing enzymolysis and wall breaking to obtain first enzymatic hydrolysate; wherein the enzymolysis wall breaking adopts a mixed enzyme, and the mixed enzyme comprises amylase and cellulase; s3, extracting crude protein to obtain crude protein; s4, carrying out crude protein enzymolysis on the crude protein to obtain a second enzymatic hydrolysate; wherein the crude proteolysis adopts mixed protease, and the mixed protease comprises first protease and second protease; wherein the first protease is one or more of alkaline protease, neutral protease and acid protease; the second protease is papain; and S5, purifying the second enzymatic hydrolysate to obtain the camellia seed meal oligopeptide. The average molecular weight of the obtained product is smaller than 1000 Daltons, and the product has biological activity and high application value in the fields of cosmetics and functional food.

Description

Technical Field

[0001] This invention relates to a camellia seed meal oligopeptide, its preparation method, and its application. Background Technology

[0002] Bioactive oligopeptides have low molecular weights, making them easily permeable to the skin barrier and absorbed by the body. They possess physiological regulatory functions such as immune regulation, hormone regulation, antibacterial and antiviral effects, and can lower blood pressure and blood lipids. Furthermore, they are highly safe and are among the most popular raw materials in the international cosmetics and food industries. Camellia has hemostatic, antitussive, antidiarrheal, and anti-inflammatory properties and is a plant of high economic value. Its flower and seed extracts are commonly used in cosmetics and food products. Camellia seeds, in particular, contain various natural beauty ingredients and can be used to create a variety of cosmetics that can lighten dark spots, nourish the skin, and provide moisturizing effects.

[0003] Camellia seeds contain approximately 10%–20% protein, composed of 17 amino acids, 8 of which are essential amino acids for the human body. Hydrolysis of camellia seed protein can yield various bioactive peptides with effects such as enhancing immunity, anti-aging, lowering blood pressure, and lowering blood lipids, which can be applied to the research and development of cosmetics and functional foods. Camellia seed meal, the residue left after oil extraction from camellia seeds, contains not only abundant protein, lipids, and carbohydrates, but also far more in greater quantities than the extracted tea oil. However, most camellia seed meal is used for pond cleaning or as fuel, leading to significant resource waste. Currently, there are few reports on the preparation and efficacy research of oligopeptides from camellia seed meal, both domestically and internationally. Summary of the Invention

[0004] To overcome the low-value utilization of camellia seed meal in existing technologies, this invention provides a camellia seed meal oligopeptide, its preparation method, and its applications. The camellia seed meal oligopeptide of this invention has an average molecular weight of less than 1000 Daltons, high biological activity, and significant application value in the fields of cosmetics and functional foods. Furthermore, the preparation method of this invention is simple, safe, and low-cost.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing camellia seed meal oligopeptides, which includes the following steps:

[0007] S1. Pre-treat the camellia tea residue to obtain the pre-treated product;

[0008] S2. Enzymatic hydrolysis to break the cell wall, obtaining the first enzymatic hydrolysate; wherein, the enzymatic hydrolysis to break the cell wall uses a mixed enzyme, the mixed enzyme including amylase and cellulase;

[0009] S3. Crude protein extraction to obtain crude protein;

[0010] S4. The crude protein is subjected to crude enzymatic hydrolysis to obtain a second enzymatic hydrolysate; wherein the crude enzymatic hydrolysis uses a mixed protease, the mixed protease comprising a first protease and a second protease; wherein the first protease is one or more of alkaline protease, neutral protease and acidic protease; and the second protease is papain;

[0011] S5. Purify the second enzymatic hydrolysate to obtain camellia tea meal oligopeptide.

[0012] In this invention, in step S1, the camellia seed cake can be in powder form. The powder is obtained by crushing and sieving the camellia seed cake. Preferably, the sieve aperture is 60-100 mesh, for example, 80 mesh.

[0013] In this invention, the pretreatment operation in step S1 can be carried out using conventional operations in the art, preferably defatting and removing tea saponins.

[0014] In some preferred embodiments, the defatting and desaponification process includes washing the camellia tea pomace with an ethanol solution.

[0015] The washing process preferably includes mixing camellia seed cake with an ethanol solution and then filtering.

[0016] Preferably, the ratio of the mass of the camellia seed cake to the volume of the ethanol solution is 1:(8-20), for example, 1:10.

[0017] The ethanol solution refers to an aqueous solution of ethanol, and the volume fraction of the ethanol solution is preferably 50% to 80%, for example, 70%.

[0018] The mixing temperature is preferably 50-60°C, for example, 50°C.

[0019] The mixing time is preferably 1.5 to 3 hours, for example, 2 hours.

[0020] The washing operation is preferably repeated 2 to 4 times, for example 3 times.

[0021] In this invention, in step S2, the mass ratio of amylase to cellulase in the mixed enzyme is preferably (2-5):1, for example: 25:9, 4:1 or 1:1.

[0022] In this invention, step S2 preferably includes the following steps: mixing the pretreated material from step S1 with water, adding the mixed enzyme, performing enzymatic hydrolysis, inactivating the enzyme, filtering, and obtaining a first enzymatic hydrolysate.

[0023] The preferred mass ratio of the pretreatment material, water and mixed enzyme is 100:(800-2000):(1-5), for example: 100:1000:3.4, 100:800:2.5 or 100:1500:4.

[0024] The enzymatic hydrolysis temperature is preferably 45–60°C, for example, 50°C.

[0025] The enzymatic hydrolysis time is preferably 1.5 to 2.5 hours, for example, 2 hours.

[0026] The enzymatic hydrolysis is preferably carried out under stirring conditions.

[0027] The enzyme inactivation can be carried out using conventional methods, but preferably using a water bath heating method. The water bath heating temperature is preferably 80–95°C; the water bath heating time is preferably 10–20 minutes, for example, 15 minutes.

[0028] In this invention, the crude protein extraction in step S3 can be performed using conventional methods in the art, preferably using alkaline extraction and acid precipitation.

[0029] In some preferred embodiments, the alkaline extraction and acid precipitation method includes: adjusting the first enzymatic hydrolysate to alkaline, stirring, centrifuging, filtering to obtain an extract, adjusting the extract to acidic, centrifuging, filtering, washing to obtain crude protein.

[0030] The operation of adjusting the first enzymatic hydrolysate to alkalinity is preferably performed by using sodium hydroxide to adjust the pH value of the first enzymatic hydrolysate to 9-10, for example, pH=10.

[0031] The stirring temperature is preferably 50-60°C, for example, 55°C.

[0032] The stirring time is preferably 3 to 6 hours, for example, 5 hours.

[0033] The operation of adjusting the extract to acidity is preferably performed by using hydrochloric acid to adjust the pH value of the extract to 3-5, for example: pH=3.5 or 4.

[0034] The step of adjusting the extract to acidity and before centrifugation preferably includes standing for 0.5 to 1 hour, for example, 0.5 hours.

[0035] In this invention, step S4 preferably includes the following steps: mixing the crude protein from step S3 with water to obtain a crude protein solution, adding a first protease, performing a first enzymatic hydrolysis and a first enzyme inactivation, adding a second protease, performing a second enzymatic hydrolysis and a second enzyme inactivation, filtering, and obtaining a second enzymatic hydrolysate.

[0036] The ratio of crude protein to water is preferably 1:(8-20), for example, 1:10 or 1:15.

[0037] The ratio of crude protein to the first protease is preferably 200:(1-2).

[0038] The ratio of crude protein to the second protease is preferably 400:(1-4), for example, 200:1.

[0039] The aforementioned mass ratio is calculated based on the mass of undried crude protein.

[0040] Preferably, the addition of the first protease includes adjusting the pH of the crude protein solution.

[0041] When the first protease is an acidic protease, the pH of the crude protein solution is adjusted to 3-5 before adding the first protease, for example, pH=3.5.

[0042] When the first protease is an alkaline protease, the pH of the crude protein solution is adjusted to 9-10, for example, pH=10, before adding the first protease.

[0043] When the first protease is a neutral protease, the pH of the crude protein solution is adjusted to 6-7.5, for example, pH=7, before adding the first protease.

[0044] When the first protease is a neutral protease, the pH of the crude protein solution does not need to be adjusted before adding the first protease.

[0045] Preferably, before adding the second protease, the pH of the crude protein solution is adjusted to 6.5–7.5, for example, pH = 7.

[0046] The temperature of the first enzymatic hydrolysis is preferably 40-50°C, for example, 45°C.

[0047] The time for the first enzymatic hydrolysis is preferably 5 to 12 hours, for example, 7 hours.

[0048] The temperature of the secondary enzymatic hydrolysis is preferably 45-55°C, for example, 50°C.

[0049] The secondary enzymatic hydrolysis time is preferably 2 to 4 hours, for example, 3 hours.

[0050] Preferably, both the primary and secondary enzymatic hydrolysis are carried out under stirring conditions.

[0051] The primary and secondary enzyme inactivation can be performed using conventional methods, but water bath heating is preferred. The water bath heating temperature is preferably 80–95°C, for example, 95°C; and the water bath heating time is preferably 10–20 minutes, for example, 15 minutes.

[0052] In this invention, step S5 preferably includes decolorization, desalting, and drying in sequence.

[0053] In some preferred embodiments, the decolorization operation includes: first adjusting the pH of the second enzymatic hydrolysate in step S4 to 9-10, for example, pH=9, and then using a decolorizing agent for decolorization.

[0054] The decolorizing agent is preferably one or more of activated carbon, chlorine dioxide, and hydrogen peroxide.

[0055] The volume ratio of the decolorizing agent to the second enzymatic hydrolysate is preferably (1-5):100, for example, 3:100.

[0056] The decolorization temperature is preferably 50-60°C, for example, 55°C.

[0057] The decolorization time is preferably 3 to 6 hours, for example, 4 hours.

[0058] In some preferred embodiments, the desalination operation includes filtering the decolorized second enzymatic hydrolysate through a nanofiltration membrane. Preferably, the filtration includes: first filtering through a 100 Dalton (Da) nanofiltration membrane, and then filtering through a 1000 Dalton (Da) nanofiltration membrane.

[0059] In some preferred embodiments, the drying is spray drying.

[0060] In some preferred embodiments, the drying process further includes a concentration of the desalted retentate.

[0061] In this invention, other operations not specifically described, such as crushing, sieving, filtering, stirring, centrifuging, washing, cooling, settling and drying, can all be performed in a manner conventional in the art.

[0062] The present invention also provides a camellia seed meal oligopeptide, which is prepared by the above-described method for preparing camellia seed meal oligopeptides.

[0063] In this invention, the average molecular weight of the camellia seed meal oligopeptide is preferably 400-1000 Daltons.

[0064] In this invention, the camellia seed meal oligopeptide preferably includes glutamic acid, leucine, alanine, serine, phenylalanine, tyrosine, valine, and threonine.

[0065] In this invention, the total amino acid content of the camellia seed meal oligopeptide is preferably 16% to 22%, for example: 16.7%, 18.1% or 20.6%.

[0066] In some preferred embodiments, the camellia seed meal oligopeptide comprises 4.8%–6.0% glutamic acid, 2.2%–2.8% leucine, 1.8%–2.6% alanine, 1.4%–2.5% serine, 1.2%–2.0% phenylalanine, 1.0%–1.5% tyrosine, 0.86%–1.2% valine, and 0.8%–1.1% threonine.

[0067] In one specific embodiment, the camellia seed meal oligopeptide comprises 5.76% glutamic acid, 2.63% leucine, 2.44% alanine, 2.26% serine, 1.73% phenylalanine, 1.31% tyrosine, 0.86% valine, and 0.82% threonine.

[0068] In one specific embodiment, the camellia seed meal oligopeptide comprises 5.12% glutamic acid, 2.34% leucine, 1.85% alanine, 1.46% serine, 1.28% phenylalanine, 1.04% tyrosine, 0.92% valine, and 0.87% threonine.

[0069] In one specific embodiment, the camellia seed meal oligopeptide comprises 5.04% glutamic acid, 2.53% leucine, 1.94% alanine, 1.43% serine, 1.39% phenylalanine, 1.12% tyrosine, 1.05% valine and 0.99% threonine.

[0070] The present invention also provides the application of the aforementioned camellia seed meal oligopeptide in the fields of cosmetics or functional foods.

[0071] The cosmetics described in this invention refer to chemical industrial products or fine chemical products that are applied to any part of the human body surface, such as skin, hair, nails, lips, and teeth, by means of smearing, spraying, or other similar methods, for the purpose of cleaning, maintaining, beautifying, modifying and changing appearance, or correcting body odor and maintaining a good condition.

[0072] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0073] The reagents and raw materials used in this invention are all commercially available.

[0074] The positive and progressive effects of this invention are as follows:

[0075] (1) The camellia seed meal oligopeptide prepared by the method of the present invention has an average molecular weight of less than 1000 Daltons and has good biological activity.

[0076] (2) The camellia seed oligopeptide prepared by this invention has good effects in repairing hair cuticle damage, improving hair toughness and beauty anti-aging, and has high application value in the fields of cosmetics and functional foods.

[0077] (3) The method for preparing camellia seed meal oligopeptide provided by the present invention has low raw material cost and is simple and safe to operate. Attached Figure Description

[0078] Figure 1 This is a relative molecular mass distribution diagram of the camellia seed meal oligopeptide obtained in Example 1 of the present invention.

[0079] Figure 2 This is a relative molecular mass distribution diagram of the camellia seed meal oligopeptide obtained in Example 2 of the present invention.

[0080] Figure 3 This is a relative molecular mass distribution diagram of the camellia seed meal oligopeptide obtained in Example 3 of the present invention.

[0081] Figure 4 This is a diagram showing the relative molecular mass distribution of the camellia seed meal oligopeptide obtained in Comparative Example 1 of this invention. Detailed Implementation

[0082] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0083] Example 1

[0084] S1. Crush the camellia seed cake through an 80-mesh sieve, take 200g of camellia seed cake powder, add 2L of 70% ethanol, stir at 50℃ for 2h, filter, and wash twice to obtain the pretreated material.

[0085] S2. Add 2L of water, 2.5% amylase (5g), and 0.9% cellulase (1.8g) to the pretreated material. Stir and hydrolyze at 50℃ for 2h, then heat in a water bath at 95℃ for 15min to inactivate the enzyme. Filter to obtain the first hydrolysate.

[0086] S3. Adjust the pH of the first enzymatic hydrolysate to 10 using NaOH solution, stir at 55℃ for 5 hours, centrifuge, filter to obtain the extract, then adjust the pH of the extract to 3.5 using HCl solution, let stand for 0.5 hours, centrifuge, filter, wash to obtain crude protein;

[0087] S4. Add water to the crude protein, with a crude protein to water mass ratio of 1:10, to obtain a crude protein solution. Adjust the pH of the crude protein solution to 3.5 using HCl solution. Add 1% of the crude protein mass of acidic protease. Stir at 50°C for 7 hours. Heat in a water bath at 95°C for 15 minutes to inactivate the enzyme. Adjust the pH of the crude protein solution to 7 again using NaOH solution. Add 0.5% of the crude protein mass of papain. Stir at 50°C for 2 hours. Heat in a water bath at 95°C for 15 minutes to inactivate the enzyme. Filter to obtain the second enzymatic hydrolysate.

[0088] S5. Adjust the pH of the second enzymatic hydrolysate to 9 using NaOH solution, add 3% hydrogen peroxide (by volume of the second enzymatic hydrolysate), and decolorize at 60℃ for 4 hours until the solution turns pale orange-yellow. Filter the decolorized second enzymatic hydrolysate through a nanofiltration membrane to remove salts, first through a 100 Da nanofiltration membrane, then through a 1000 Da nanofiltration membrane to obtain the retentate. Concentrate and spray dry to obtain camellia seed meal oligopeptides.

[0089] Example 2

[0090] S1. Crush the camellia seed cake through an 80-mesh sieve, take 200g of camellia seed cake powder, add 2L of 70% ethanol, stir at 50℃ for 2h, filter, and wash twice to obtain the pretreated material.

[0091] S2. Add 1.6L of water, 2% amylase (4g), and 0.5% cellulase (1g) to the pretreated material. Stir and hydrolyze at 45℃ for 2.5h, then heat in a water bath at 95℃ for 15min to inactivate the enzyme. Filter to obtain the first hydrolysate.

[0092] S3. Adjust the pH of the first enzymatic hydrolysate to 10 using NaOH solution, stir at 50℃ for 6 hours, centrifuge, filter to obtain the extract, then adjust the pH of the extract to 3 using HCl solution, let stand for 0.5 hours, centrifuge, filter, wash to obtain crude protein;

[0093] S4. Add water to the crude protein, with a crude protein to water mass ratio of 1:8, to obtain a crude protein solution. Adjust the pH of the crude protein solution to 10 using NaOH solution. Add alkaline protease at 0.5% of the crude protein mass. Stir at 40°C for 5 hours. Inactivate the enzyme by heating in a water bath at 95°C for 15 minutes. Adjust the pH of the crude protein solution to 7 again using HCl solution. Add papain at 0.25% of the crude protein mass. Stir at 45°C for 3 hours. Inactivate the enzyme by heating in a water bath at 95°C for 15 minutes. Filter to obtain the second enzymatic hydrolysate.

[0094] S5. Adjust the pH of the second enzymatic hydrolysate to 9 using NaOH solution, add 1% hydrogen peroxide (by volume of the second enzymatic hydrolysate), and decolorize at 55℃ for 3 hours until the solution turns pale orange-yellow. Filter the decolorized second enzymatic hydrolysate through a nanofiltration membrane to remove salts, first through a 100 Da nanofiltration membrane, then through a 1000 Da nanofiltration membrane to obtain the retentate. Concentrate and spray dry to obtain camellia seed meal oligopeptides.

[0095] Example 3

[0096] S1. Crush the camellia seed cake through an 80-mesh sieve, take 200g of camellia seed cake powder, add 2L of 70% ethanol, stir at 50℃ for 2h, filter, and wash twice to obtain the pretreated material.

[0097] S2. Add 3L of water, 2% amylase (4g), and 2% cellulase (4g) to the pretreated material. Stir and hydrolyze at 60℃ for 1.5h, then heat in a water bath at 95℃ for 15min to inactivate the enzyme. Filter to obtain the first hydrolysate.

[0098] S3. Adjust the pH of the first enzymatic hydrolysate to 10 using NaOH solution, stir at 60℃ for 3 hours, centrifuge, filter to obtain the extract, then adjust the pH of the extract to 4 using HCl solution, let stand for 0.5 hours, centrifuge, filter, wash to obtain crude protein.

[0099] S4. Add water to the crude protein, with a crude protein to water mass ratio of 1:15, to obtain a crude protein solution. Adjust the pH of the crude protein solution to 3.5 using HCl solution. Add 1% of the crude protein mass of acidic protease. Stir at 45°C for 12 hours. Heat in a water bath at 95°C for 15 minutes to inactivate the enzyme. Adjust the pH of the crude protein solution to 7 again using NaOH solution. Add 1% of the crude protein mass of papain. Stir at 55°C for 4 hours. Heat in a water bath at 95°C for 15 minutes to inactivate the enzyme. Filter to obtain the second enzymatic hydrolysate.

[0100] S5. Adjust the pH of the second enzymatic hydrolysate to 9 using NaOH solution, add 5% hydrogen peroxide (by volume of the second enzymatic hydrolysate), and decolorize at 60℃ for 4 hours until the solution turns pale orange-yellow. Filter the decolorized second enzymatic hydrolysate through a nanofiltration membrane to remove salts, first through a 100 Da nanofiltration membrane, then through a 1000 Da nanofiltration membrane to obtain the retentate, concentrate, and spray dry to obtain camellia seed meal oligopeptides.

[0101] Comparative Example 1

[0102] S1. Crush the camellia seed cake through an 80-mesh sieve, take 200g of camellia seed cake powder, add 2L of 70% ethanol, stir at 50℃ for 2h, filter, and wash twice to obtain the pretreated material.

[0103] S2. Add 4L of water to the pretreated material to obtain the pretreated solution;

[0104] S3. The pH of the pretreated solution was adjusted to 10 using NaOH solution, stirred at 55℃ for 5 hours, centrifuged, filtered to obtain the extract, and then the pH of the extract was adjusted to 3.5 using HCl solution. After standing for 0.5 hours, it was centrifuged, filtered, and washed to obtain crude protein.

[0105] S4. Add water to the crude protein, with a crude protein to water mass ratio of 1:20, to obtain a crude protein solution. Add 1% of the crude protein mass of neutral protease, stir at 55°C for 7 hours, and heat in a water bath at 95°C for 15 minutes to inactivate the enzyme. Add 0.5% of the crude protein mass of papain, stir at 50°C for 2 hours, and heat in a water bath at 95°C for 15 minutes to inactivate the enzyme. Filter to obtain the enzymatic hydrolysate.

[0106] S5. Adjust the pH of the enzymatic hydrolysate to 9 using NaOH solution, add 3% hydrogen peroxide (by volume of the hydrolysate), and decolorize at 60℃ for 4 hours until the solution turns pale orange-yellow. Filter the decolorized hydrolysate through a nanofiltration membrane to remove salts, first through a 100 Da nanofiltration membrane, then through a 1000 Da nanofiltration membrane to obtain the retentate. Concentrate and spray dry to obtain camellia seed meal oligopeptides.

[0107] Example 1

[0108] Average molecular weight test of camellia seed meal oligopeptides

[0109] (1) Test method

[0110] This invention uses the method of the Japan Food Analysis Center to test the relative molecular weight of camellia seed meal oligopeptides using conventional testing methods (GPC) in the field, and calculates their average molecular weight.

[0111] ① Preparation of standard solutions

[0112] The standard was dissolved in a 0.1 mol / L sodium nitrate solution to prepare a 0.05 w / v solution (i.e., 0.05 g of standard was contained in every 100 mL of 0.1 mol / L sodium nitrate solution). Specific information about the standard is shown in Table 1 below.

[0113] Table 1 Standard Product Information Table

[0114]

[0115] ② Sample solution preparation

[0116] Take about 20 mg of sample, dissolve it in 10 ml of 0.1 mol / L sodium nitrate solution, and filter it through a microporous membrane to obtain the sample solution.

[0117] ③ Testing instruments and conditions

[0118] This invention uses a Shimadzu high-performance liquid chromatograph for testing; wherein the chromatographic column model is TSKgel G3000PWXL (Ф7.8mm×300mm); the mobile phase is 0.1mol / L sodium nitrate solution; the column temperature is 50℃; the flow rate is 1mL / min; the injection volume is 100μL; and the elution time is 20min.

[0119] ④ Test content

[0120] First, inject the standard solution into the Shimadzu high-performance liquid chromatograph and plot the time-molecular weight standard curve using the GPC program; second, inject the sample solution into the Shimadzu high-performance liquid chromatograph and calculate the average molecular weight using the GPC program.

[0121] (2) Test Results

[0122] Test results are as follows Figure 1-4 As shown in the relative molecular mass distribution graph, the horizontal axis represents elution time, the right vertical axis represents the retention volume, and the left vertical axis represents the response value; specific data are shown in Table 2. The average molecular weight of Examples 1-3 is less than 1000 Daltons, while the average molecular weight of Comparative Example 1 is 1137 Daltons.

[0123] Table 2 shows the average molecular weight of the products obtained in Examples 1-3 and Comparative Example 1.

[0124]

[0125] Example 2

[0126] Amino acid content and distribution test of camellia seed meal oligopeptides

[0127] (1) Test method

[0128] This invention uses Shimadzu's amino acid analysis method and conventional high-performance liquid chromatography (HPLC) to test camellia seed meal oligopeptides.

[0129] ① Preparation of standard solutions

[0130] Amino acid standard stock solution (2.5 μmol / mL): commercially available, or accurately weigh an appropriate amount of amino acid standard, dissolve it in phase A, and mix well.

[0131] Amino acid standard mixed working solution: Pipette 0.1 mL, 0.25 mL, and 0.5 mL of amino acid standard stock solution into 5 mL volumetric flasks, add water to the mark, mix well, and set aside.

[0132] ② Sample preparation

[0133] Determination of free amino acids: Weigh 100 mg of camellia seed meal oligopeptide sample into a 10 mL volumetric flask, dissolve in phase A and dilute to the mark. Filter this solution through a 0.45 μm aqueous membrane for analysis.

[0134] Determination of amino acids after hydrolysis: Weigh 20 mg of camellia seed meal oligopeptide into a gas phase headspace vial, add 2 mL of 6 mol / L hydrochloric acid solution, purge with nitrogen and immediately seal, place in an oven at 110 ℃ and heat for more than 3 hours, remove and cool, then adjust the pH to about 2-6 with 3 mol / L LiOH solution (the volume of 3M LiOH solution is about 2.9 mL), take this solution and pass it through a 0.45 μm aqueous phase membrane for analysis.

[0135] ③ Testing instruments and conditions

[0136] This invention uses a Shimadzu high-performance liquid chromatograph for testing; wherein the chromatographic column is a lithium-type amino column, model Shimadzu SHIM-PACK AMINO-LI; the detection wavelength is 440 and 570 nm; the column temperature is 39℃; the reaction chamber temperature is 130℃; the injection volume is 10 μL; and the flow rate is 0.6 mL / min.

[0137] Mobile phase A: lithium citrate buffer, pH = 2.68; Mobile phase B: lithium citrate buffer, pH = 10.00; Mobile phase C: 0.84% ​​lithium hydroxide monohydrate, 0.2 mL / min; Derivatizing solution: ninhydrin derivatizing solution. Elution steps were 1-A, 2-A, and 3-A. Gradient elution was used.

[0138] ④ Calculation of amino acid content

[0139] Preparation of standard curve: Inject the amino acid standard mixed working solution into the Shimadzu high performance liquid chromatograph and measure the corresponding chromatographic peak height or peak area. Plot the standard curve with the concentration of the amino acid standard mixed working solution as the abscissa and the response value (peak area or peak height) as the ordinate.

[0140] Determination of sample solution: Inject the sample solution into the Shimadzu high performance liquid chromatograph and measure the corresponding chromatographic peak height or peak area. The concentration of amino acids in the test solution is obtained according to the standard curve.

[0141] The amino acid content in camellia seed meal oligopeptide samples was calculated using the following formula:

[0142]

[0143] Where A is the amino acid content (%) in the sample; c is the concentration of amino acids in the sample test solution (mg / mL); V is the final volume of the sample (mL); and m is the sample mass (mg).

[0144] (2) Test Results

[0145] The test results are shown in Table 3. The total amino acid content and the content of each amino acid in Examples 1-3 are higher than those in Comparative Example 1.

[0146] Table 3. Amino acid content and distribution of the products obtained in Examples 1-3 and Comparative Example 1

[0147]

[0148]

[0149] Example 3

[0150] Yield test of camellia seed meal oligopeptides

[0151] (1) Test method

[0152] Yield of camellia seed meal oligopeptides = Mass of camellia seed meal oligopeptides (g) / Mass of camellia seed meal powder (g)

[0153] (2) Test Results

[0154] The test results are shown in Table 4.

[0155] Table 4 Yields of the products obtained in Examples 1-3

[0156]

[0157] The camellia seed meal oligopeptides prepared by this invention have an average molecular weight of less than 1000 Daltons, exhibit biological activity, and have high application value in the fields of cosmetics and functional foods.

Claims

1. A method for preparing camellia seed meal oligopeptides, characterized in that, It includes the following steps: S1. Pre-treat the camellia tea residue to obtain the pre-treated product; S2. Enzymatic hydrolysis to break the cell wall, obtaining the first enzymatic hydrolysate; wherein, the enzymatic hydrolysis to break the cell wall uses a mixed enzyme, the mixed enzyme including amylase and cellulase; S3. Crude protein extraction to obtain crude protein; S4. The crude protein is subjected to crude enzymatic hydrolysis to obtain a second enzymatic hydrolysate; wherein the crude enzymatic hydrolysis uses a mixed protease, the mixed protease comprising a first protease and a second protease; wherein the first protease is one or more of alkaline protease, neutral protease and acidic protease; and the second protease is papain; S5. Purify the second enzymatic hydrolysate to obtain camellia tea meal oligopeptide.

2. The method for preparing camellia seed meal oligopeptides as described in claim 1, characterized in that, In step S1, the pretreatment operation involves defatting and removing tea saponins; Preferably, the defatting and desaponification process includes washing the camellia tea pomace with an ethanol solution; The washing operation preferably includes mixing camellia seed cake with an ethanol solution and then filtering it; Preferably, the ratio of the mass of the camellia seed cake to the volume of the ethanol solution is 1:(8-20), for example, 1:

10. Wherein, the ethanol solution refers to an aqueous ethanol solution, and the volume fraction of the ethanol solution is preferably 50% to 80%, for example, 70%; The mixing temperature is preferably 50–60°C, for example, 50°C; The mixing time is preferably 1.5 to 3 hours, for example, 2 hours; The washing operation is preferably repeated 2 to 4 times, for example 3 times.

3. The method for preparing camellia seed meal oligopeptides as described in claim 1, characterized in that, In step S2, the mass ratio of amylase to cellulase in the mixed enzyme is (2-5):1, for example: 25:9, 4:1 or 1:1; And / or, in step S2, the enzymatic hydrolysis and cell wall breaking operation includes: mixing the pretreated material from step S1 with water, adding the mixed enzyme, enzymatic hydrolysis, enzyme inactivation, filtration, and obtaining the first enzymatic hydrolysate; The preferred mass ratio of the pretreatment material, water and mixed enzyme is 100:(800-2000):(1-5), for example: 100:1000:3.4, 100:800:2.5 or 100:1500:4; The enzymatic hydrolysis temperature is preferably 45–60°C, for example, 50°C; The enzymatic hydrolysis time is preferably 1.5 to 2.5 hours, for example, 2 hours; The enzymatic hydrolysis is preferably carried out under stirring conditions; The enzyme inactivation is preferably performed by water bath heating; the water bath heating temperature is preferably 80-95°C; and the water bath heating time is preferably 10-20 minutes, for example, 15 minutes.

4. The method for preparing camellia seed meal oligopeptides as described in claim 1, characterized in that, In step S3, the crude protein extraction is performed using an alkaline extraction and acid precipitation method. Preferably, the alkaline extraction and acid precipitation method includes: adjusting the first enzymatic hydrolysate to alkaline, stirring, centrifuging, filtering to obtain an extract, adjusting the extract to acidic, centrifuging, filtering, washing to obtain crude protein; The operation of adjusting the first enzymatic hydrolysate to alkalinity is preferably to adjust the pH value of the first enzymatic hydrolysate to 9-10 using sodium hydroxide, for example: pH=10; The stirring temperature is preferably 50-60°C, for example, 55°C; The stirring time is preferably 3 to 6 hours, for example, 5 hours; The operation of adjusting the extract to acidity is preferably performed by using hydrochloric acid to adjust the pH value of the extract to 3-5, for example: pH=3.5 or 4; The step of adjusting the extract to acidity and before centrifugation preferably includes standing for 0.5 to 1 hour, for example, 0.5 hours.

5. The method for preparing camellia seed meal oligopeptides as described in claim 1, characterized in that, In step S4, the operation of crude protein hydrolysis includes: mixing the crude protein from step S3 with water to obtain a crude protein solution, adding a first protease, performing enzymatic hydrolysis once and enzyme inactivation once, adding a second protease, performing enzymatic hydrolysis twice and enzyme inactivation twice, filtering to obtain a second hydrolysate; The ratio of crude protein to water is preferably 1:(8-20), for example, 1:10 or 1:15; Preferably, the ratio of crude protein to the first protease is 200:(1-2); The preferred mass ratio of crude protein to the second protease is 400:(1-4), for example, 200:1; Preferably, the addition of the first protease further includes adjusting the pH of the crude protein solution; when the first protease is an acidic protease, the pH of the crude protein solution is adjusted to 3-5, for example, pH=3.5, before adding the first protease; when the first protease is an alkaline protease, the pH of the crude protein solution is adjusted to 9-10, for example, pH=10, before adding the first protease; when the first protease is a neutral protease, the pH of the crude protein solution is adjusted to 6-7.5, for example, pH=7, before adding the first protease. Preferably, when the first protease is a neutral protease, the pH of the crude protein solution is not adjusted before adding the first protease; Preferably, before adding the second protease, the pH of the crude protein solution is adjusted to 6.5–7.5, for example, pH = 7; The temperature of the first enzymatic hydrolysis is preferably 40-50°C, for example, 45°C; The time for the first enzymatic hydrolysis is preferably 5 to 12 hours, for example, 7 hours; The preferred temperature for the secondary enzymatic hydrolysis is 45–55°C, for example, 50°C. The secondary enzymatic hydrolysis time is preferably 2 to 4 hours, for example, 3 hours; Preferably, both the primary and secondary enzymatic hydrolysis are carried out under stirring conditions. Preferably, the primary enzyme inactivation and the secondary enzyme inactivation are carried out by water bath heating; the water bath heating temperature is preferably 80-95℃, for example, 95℃; and the water bath heating time is preferably 10-20 min, for example, 15 min.

6. The method for preparing camellia seed meal oligopeptides as described in claim 1, characterized in that, In step S5, the purification operation includes decolorization, desalting, and drying in sequence; Preferably, the decolorization operation includes: first adjusting the pH of the second enzymatic hydrolysate in step S4 to 9-10, for example, pH=9, and then using a decolorizing agent for decolorization; The decolorizing agent is preferably one or more of activated carbon, chlorine dioxide, and hydrogen peroxide; The volume ratio of the decolorizing agent to the second enzymatic hydrolysate is preferably (1-5):100, for example, 3:100; The decolorization temperature is preferably 50-60°C, for example, 55°C; The decolorization time is preferably 3 to 6 hours, for example, 4 hours.

7. The method for preparing camellia seed meal oligopeptides as described in claim 6, characterized in that, The desalting operation includes filtering the decolorized second enzymatic hydrolysate through a nanofiltration membrane; preferably, the filtration includes: first filtering through a 100 Dalton nanofiltration membrane, and then filtering through a 1000 Dalton nanofiltration membrane. And / or, the drying is spray drying; And / or, prior to the drying process, the process further includes a concentration of the desalted retentate.

8. A camellia seed meal oligopeptide, characterized in that, It is prepared using the method for preparing camellia seed meal oligopeptide as described in any one of claims 1-7.

9. The camellia seed meal oligopeptide as described in claim 8, characterized in that, The average molecular weight of the camellia seed oligopeptide is 400-1000 Daltons; And / or, the camellia seed meal oligopeptides include glutamic acid, leucine, alanine, serine, phenylalanine, tyrosine, valine, and threonine; And / or, the total amino acid content of the camellia seed meal oligopeptide is 16% to 22%, for example: 16.7%, 18.1% or 20.6%; Preferably, the camellia seed meal oligopeptide comprises 4.8%–6.0% glutamic acid, 2.2%–2.8% leucine, 1.8%–2.6% alanine, 1.4%–2.5% serine, 1.2%–2.0% phenylalanine, 1.0%–1.5% tyrosine, 0.86%–1.2% valine, and 0.8%–1.1% threonine; More preferably, the camellia seed meal oligopeptide comprises 5.76% glutamic acid, 2.63% leucine, 2.44% alanine, 2.26% serine, 1.73% phenylalanine, 1.31% tyrosine, 0.86% valine and 0.82% threonine. More preferably, the camellia seed meal oligopeptide comprises 5.12% glutamic acid, 2.34% leucine, 1.85% alanine, 1.46% serine, 1.28% phenylalanine, 1.04% tyrosine, 0.92% valine and 0.87% threonine. More preferably, the camellia seed meal oligopeptide comprises 5.04% glutamic acid, 2.53% leucine, 1.94% alanine, 1.43% serine, 1.39% phenylalanine, 1.12% tyrosine, 1.05% valine and 0.99% threonine.

10. The application of the camellia seed meal oligopeptide as described in claim 8 or 9 in the fields of cosmetics or functional foods.

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