Method for extracting camellia oleifera seed meal polypeptide
The method of extracting peptides from camellia seed meal by microwave-assisted extraction and mixed enzymatic hydrolysis solves the problems of low extraction rate and weak activity in existing technologies, realizes efficient and environmentally friendly peptide production, and enhances the utilization value of camellia seed meal.
Patent Information
- Application Number
- CN202511068529.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-11-07
AI Technical Summary
Existing technologies have low peptide extraction rates and weak activity in camellia seed meal, making industrial production difficult and posing pollution risks, thus failing to meet the needs of green environmental protection and comprehensive resource utilization.
A multi-step optimization method was adopted to extract peptides from camellia seed meal, which combines microwave-assisted extraction, continuous flow centrifugation, and a mixed enzymatic hydrolysis method using neutral protease, trypsin, and alkaline protease. This method includes pH adjustment, enzymatic hydrolysis, and ultrafiltration, thereby improving peptide yield and activity.
It significantly improved the extraction rate and activity of peptides from camellia seed meal, reduced the burden on the gastrointestinal tract, achieved efficient resource utilization and environmentally friendly production, and enhanced the medicinal value of camellia seed meal.
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Figure CN120905342A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of polypeptide extraction, and particularly relates to an extraction method of oil-tea camellia seed meal polypeptide. BACKGROUND
[0002] Oil-tea camellia tree is a plant of the Camellia genus of the Theaceae family, and its planting and oil-tea camellia leaf development are extremely popular in China, with a huge annual output and rich resources. Oil-tea camellia seed, as a mature seed of the oil-tea camellia tree, can be used for oil extraction, and the produced oil-tea camellia seed oil is nutritious. However, the byproduct oil-tea camellia seed meal, which contains various nutrients, has not been reasonably utilized for a long time, resulting in a large amount of resource waste.
[0003] The protein content in the oil-tea camellia seed meal accounts for 12% to 15%, and contains 17 kinds of amino acids, of which 8 are essential amino acids for the human body, which constitute oil-tea camellia seed protein. The protein can produce various active polypeptides after enzymolysis, and the main molecular weight range is 300 to 3000 Da, and the average peptide chain length is between 3 to 10 amino acid residues, which can be easily digested and absorbed by the human body, and has high utilization value.
[0004] However, the existing technology for extracting protein polypeptides from oil-tea camellia seed meal has many deficiencies. The traditional extraction method often has low protein extraction rate, high energy consumption, and may use organic solvents in the extraction process, causing pollution. In the enzymolysis process, the use of single enzymolysis not only prolongs the enzymolysis time, but also leads to low oil-tea camellia seed meal polypeptide yield, weak polypeptide activity, and difficulty in being efficiently absorbed by the human body, increasing the burden on the gastrointestinal function. At the same time, the existing preparation method has certain limitations and cannot be applied to industrial mass production, which cannot realize the high value-added utilization of oil-tea camellia seed meal, cannot well meet the current green environmental protection and resource comprehensive utilization concept, and restricts the research and development prospect of oil-tea camellia seed meal. SUMMARY
[0005] The application aims to provide an extraction method of oil-tea camellia seed meal polypeptide, which can effectively improve the yield of oil-tea camellia seed meal polypeptide, and the polypeptide extracted by the method has high activity and is easy to be absorbed by the body, reducing the burden on the gastrointestinal function.
[0006] To solve the above technical problems, the technical scheme adopted by the application is as follows:
[0007] An extraction method of oil-tea camellia seed meal polypeptide, comprising the following steps:
[0008] S1, crushing and sieving the oil-tea camellia seed meal, mixing with pure water according to the solid-liquid ratio, adjusting the pH to alkaline, microwave-assisted leaching, continuous flow centrifugation, and obtaining supernatant;
[0009] S2, adjusting the pH of the supernatant extracted in S1 to acid, standing, continuous flow centrifugation, collecting the precipitate, washing, spray drying, and obtaining oil-tea camellia seed meal crude protein;
[0010] S3, the oil-tea camellia seed meal crude protein obtained in S2 is prepared into a water solution with a concentration of 3.0%-5.0%, heated for denaturation treatment, cooled, adjusted to an acidic pH for standing, adjusted to a weak alkaline pH, neutral protease, trypsin and alkaline protease are added synchronously, oscillated for enzymolysis, the enzyme is inactivated after the enzymolysis, centrifuged, the precipitate is discarded, filtered by an ultrafiltration membrane, desalted by tangential flow filtration, filtered, the supernatant is taken, continuously flow centrifuged, the supernatant is taken, spray dried, and oil-tea camellia seed meal polypeptide is obtained.
[0011] Preferably, in S1, the oil-tea camellia seed meal is crushed to 60-80 mesh.
[0012] Preferably, in S1, the ratio of the oil-tea camellia seed meal to pure water is 1:15-1:25.
[0013] Preferably, in S1, the pH is adjusted to 9.0-11.0 using a NaOH solution with a concentration of 4.0%.
[0014] Preferably, in S1, the microwave-assisted extraction process parameters are specifically: the temperature is 25-30°C, the microwave power is 500-600W, and the time is 1.5-2h.
[0015] Preferably, in S1, the continuous flow centrifugation process parameters are specifically: the supernatant is obtained by continuous flow centrifugation under the conditions of a centrifugal force of 400-500xg, a temperature of 0-4°C, and a feed flow rate of 170-180L / h.
[0016] Preferably, in S2, the pH is adjusted to 3.2-4.1 using a hydrochloric acid solution with a concentration of 3.0%, and the solution is allowed to stand for 2 days.
[0017] Preferably, in S2, the continuous flow centrifugation process parameters are specifically: the precipitate is collected by continuous flow centrifugation under the conditions of a centrifugal force of 400-500xg, a temperature of 0-4°C, and a feed flow rate of 170-180L / h.
[0018] Preferably, in S2, the spray drying process parameters are specifically: the inlet temperature is 100-120°C, the outlet temperature is 80-90°C, and the feed rate is 0.8-1.0L / h.
[0019] Preferably, in S3, the heating denaturation treatment temperature is 85-95°C, the time is 15-20min, and the temperature is cooled to 30-35°C.
[0020] Preferably, in S3, the pH is adjusted to 5.8-6.5 and allowed to stand for 1.7-2.3h, and then the pH is adjusted to 7.5-8.0.
[0021] Preferably, in S3, the enzymolysis temperature is 45-50 DEG C, and the enzymolysis time is 2.8-3.5 h.
[0022] Preferably, in S3, the temperature for enzyme inactivation is 85-95 DEG C, and the time is 20-30 min.
[0023] Preferably, in S3, the centrifugal speed is 11000-13000 rpm / min, and the time is 15-20 min.
[0024] Preferably, in S3, the pore size of the ultrafiltration membrane is 1-3 kDa.
[0025] Preferably, in S3, the tangential flow filtration desalination process parameters are as follows: the membrane pore size is 3-5 kDa, the transmembrane pressure is 0.7-0.9 bar, the tangential flow rate is 1.5-2.5 m / s, the feed concentration is 3.0-4.5 g / L, the pH is 6-8, and the temperature is 20-30 DEG C.
[0026] Preferably, in S3, the continuous flow centrifugation process parameters are as follows: the supernatant is obtained by continuous flow centrifugation under the conditions of 400-500 x g centrifugal force, 0-4 DEG C, and a feed flow rate of 170-180 L / h.
[0027] Preferably, in S3, the spray drying process parameters are as follows: the inlet temperature is 100-120 DEG C, the outlet temperature is 80-90 DEG C, and the feed rate is 0.8-1.0 L / h.
[0028] The application also provides the oil tea seed meal polypeptide extracted by the method.
[0029] Compared with the prior art, the application has the following advantages and technical effects:
[0030] The application discloses an extraction method of oil tea seed meal polypeptide.
[0031] The application selects neutral protease, trypsin and alkaline protease for mixed enzymolysis, the enzymolysis process not only saves time cost, but also produces polypeptides with smaller molecular weight and higher activity, and the active polypeptides are more easily absorbed by the human body, reduce the burden of gastrointestinal function, and improve the medicinal value of the oil tea seed meal.
[0032] The application adopts the extraction method of taking pure water as the extraction liquid, applying alkali extraction and acid precipitation, and multi-factor synergistic regulation of the extraction process, so that the protein extraction process of oil tea seed meal after supercritical extraction of oil tea seed is better optimized, and the method can be applied to large-scale industrial production, energy consumption is reduced, and pollution is prevented.
[0033] The technical solutions of the application are further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS
[0034] Figure 1 The process flow chart for extracting oil tea seed meal polypeptides according to the application is shown in the figure.
[0035] Figure 2 The polypeptide content statistical graph of 5 parts of enzymatic hydrolysis filtrate in Example 1 is shown in the figure.
[0036] Figure 3 The sample molecular weight distribution graph is shown in the figure.
[0037] Figure 4 The P-A4 ultraviolet absorption chromatogram is shown in the figure.
[0038] Figure 5 The P-C ultraviolet absorption chromatogram is shown in the figure.
[0039] Figure 6 The statistical graph of the tumor inhibition results of oil tea seed meal polypeptides is shown in the figure.
[0040] Figure 7 The statistical graph of the total SOD activity detection results of oil tea seed meal polypeptides is shown in the figure.
[0041] Figure 8 The statistical graph of the DPPH free radical scavenging rate of oil tea seed meal polypeptides is shown in the figure. DETAILED DESCRIPTION
[0042] The technical solutions of the application are further described below with reference to the drawings and examples.
[0043] Unless otherwise defined, the technical terms or scientific terms used in the application should be understood as the usual meanings understood by those skilled in the art.
[0044] Test material sources:
[0045] In the application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art, and can be purchased through commercial channels.
[0046] Example 1 of the application refers to the process flow chart shown in the figure. Figure 1
[0047] The raw material of the application is oil tea seed meal formed after supercritical extraction of oil tea seed, in the form of dry powder.
[0048] Example 1
[0049] The application discloses an extraction method of oil-tea camellia seed meal polypeptide, which comprises the following steps:
[0050] S1, crushing the oil-tea camellia seed meal through an 80-mesh screen, mixing the oil-tea camellia seed meal with pure water at a material-liquid ratio of 1:20, adjusting the pH to 10.0 by using a 4.0% NaOH solution, microwave-assisted extraction for 2 hours at 25 DEG C, continuous flow centrifugation at a centrifugal force of 500xg, 4 DEG C and a feeding flow rate of 175 L / h, and obtaining supernatant;
[0051] S2, adjusting the pH of the supernatant obtained in S1 to 3.5 by using a 3.0% hydrochloric acid solution, standing for 24 hours, observing that flocculent precipitate is separated out at the bottom, continuously centrifuging at a centrifugal force of 500xg, 4 DEG C and a feeding flow rate of 175 L / h, collecting the precipitate, washing, spray drying for 30 minutes at an inlet temperature of 120 DEG C, an outlet temperature of 90 DEG C and a feeding rate of 1.0 L / h, and obtaining oil-tea camellia seed meal crude protein, and the crude protein extraction rate can reach 69.8%;
[0052] S3, preparing the oil-tea camellia seed meal crude protein obtained in S2 into a 5.0% water solution, heating and denaturing at 91 DEG C for 20 minutes, cooling to 35 DEG C, adjusting the pH to 6.0 by using a 4.0% sodium hydroxide solution and a 3.0% hydrochloric acid solution, standing for 2 hours, dividing into five parts, adjusting the pH to the optimum pH of each enzyme, respectively adding 5000u of neutral protease, 5000u of trypsin, 5000u of alkaline protease, 5000u of acid protease and 5000u of pepsin into each part, carrying out enzymolysis according to the optimum enzymolysis conditions of each enzyme in Table 1, shaking and stirring during the enzymolysis, and carrying out enzymolysis for 3 hours; after the enzymolysis, the temperature is raised to 85 DEG C for 20 minutes to inactivate the enzymes, centrifuging at a speed of 13000r / min for 15 minutes, discarding the precipitate, and filtering by using a 3Kd ultrafiltration membrane. Figure 2
[0053] Table 1 Optimum enzymolysis conditions of enzymes
[0054] Enzyme name Optimum temperature / °C Optimum pH Alkaline protease 50 9.0 Acid protease 45 3.0 Neutral protease 37 7.0 Trypsin 37 8.1 Pepsin 37 2.0
[0055] As shown in Table 1, three enzymes with the highest polypeptide content after enzymolysis can be selected, which are neutral protease, trypsin and alkaline protease.
[0056] According to Figure 2 the polypeptide content after enzymolysis of each enzyme, the enzymolysis combination of compound enzymes is designed, and a total of 10 groups are designed, and the number and enzymolysis combination are shown in Table 2.
[0057] Table 2 Classification of compound enzyme enzymolysis combinations
[0058]
[0059]
[0060] The oil-tea camellia seed meal crude protein obtained in S2 was prepared into an aqueous solution with a concentration of 5.0%, heated and denatured at 91℃ for 20 min, cooled to 30℃, and then adjusted to pH 6.0 using a 4.0% sodium hydroxide solution and a 3.0% hydrochloric acid solution, and then left to stand for 2 h. The pH was then adjusted to 8.0, and each enzyme was added to each group according to Table 2. 5000 u of neutral protease, 5000 u of trypsin, and 5000 u of alkaline protease, 5000 u of acid protease, and 5000 u of pepsin were added to each gram of substrate, and the pH and temperature were changed according to the optimal conditions of each enzyme in Table 1. Each enzyme was shaken and stirred for 3 h, and then the temperature was raised to 85℃ to inactivate the enzyme for 20 min. The mixture was centrifuged at 13000 r / min for 15 min, and the precipitate was discarded. The sample was filtered through an ultrafiltration membrane with a pore size of 3Kd, and desalted by tangential flow filtration. The specific parameters were as follows: the membrane pore size was 5kDa, the transmembrane pressure was 0.9 bar, the tangential flow rate was 2.5 m / s, the feed concentration was 4.0 g / L, the pH was 7.0, and the temperature was 25℃. The filtrate was obtained, and then the supernatant was obtained by continuous flow centrifugation under the conditions of 500 x g centrifugal force, 4℃, and a feed flow rate of 175 L / h. The supernatant was spray dried under the conditions of an inlet temperature of 120℃, an outlet temperature of 90℃, and a feed rate of 1.0 L / h for 30 min, and then oil-tea camellia seed meal polypeptides were obtained.
[0061] The enzyme hydrolyzed polypeptides obtained in the above step were subjected to molecular weight distribution determination and activity verification.
[0062] The polypeptide molecular weight distribution determination was analyzed by SEC-volume exclusion chromatography, and the specific test scheme was as follows:
[0063] After mixing, 200 μL of the sample was centrifuged at 12000 rpm / min for 10 min, and the supernatant was filtered with a water-based 0.22 μm filter membrane. Then, 80 μL of the filtered sample was taken and loaded into a liquid phase vial.
[0064] The Marker information is shown in Table 3.
[0065] Table 3 Marker molecular weight information
[0066] Marker Molecular weight (Da) BSA 66430 IgG 150000 Small peptides 3246 Tyrosine 181 Cytochrome c 12365
[0067] The liquid chromatography conditions were as follows:
[0068] (a) The column temperature was 25℃;
[0069] (b) The flow rate was 0.7 mL / min;
[0070] (c) Injection volume: 20 μL;
[0071] (d) Column: SRT-C SEC-120, 5 μm, 7.8*300 mm;
[0072] (e) Mobile phase: 150 mmol / L phosphate mobile phase (8.99 g of anhydrous sodium dihydrogen phosphate and 10.65 g of anhydrous disodium hydrogen phosphate to 1 L of water);
[0073] (f) Detection wavelength: UV 214 nm.
[0074] The results are shown in Figure 3-5 .
[0075] It can be seen from Figure 3 that the macromolecular proteins with peak time before 14 min have smaller molecular weight after enzyme digestion, and the molecular weight distribution table shows that P-A4 and P-B2 groups have the best enzyme digestion effect, and the proportion of small molecular weight below 3 kDa is large.
[0076] It can be seen from Figure 4 that the small molecular polypeptides in the P-A4 group have a peak time after 14.50 min.
[0077] It can be seen from Figure 5 that the protein molecules in the P-C group have peaks at 7.54 min, 11.16 min and 13.73 min. According to the principle of SEC-volume exclusion chromatography, the molecules with large molecular weight have short peak time, so combined Figure 4 comparison, it can be seen that the large molecular weight proteins in the P-C group are digested into small molecular weight polypeptides after P-A4 group enzyme digestion.
[0078] EC109 esophageal cancer cells were used to detect the tumor inhibition effect of polypeptide molecules. BC109 cells were cultured in complete culture medium containing 10% fetal bovine serum and 90% DMEM, and placed in a 37°C constant temperature incubator with a CO2 concentration of 5%. The liquid was changed every 1-2 days, and the cells were subcultured when the cell density reached 80%-90%. The cells were plated in a 96-well plate, and the drug was given after 24 h. After 24 h of drug culture, the plate was collected and added with culture medium containing 10% CCK-8 reagent. The reaction was cultured at 37°C for 30 min. The absorbance was detected using a full-automatic enzyme marker at a wavelength of 450 nm. The results are shown in Figure 6 .
[0079] It can be seen from Figure 6 that the IC 50 of the P-A4 group is 200 mg / ml, the IC 50 of the P-B2 group is 400 mg / ml, and the IC 50800mg / ml, it is concluded that the polypeptide of P-A4 group inhibited tumor, the inhibition effect of P-A4 group is the best, the enzyme cutting effect of P-B2 group is the second, and the activity of polypeptide after enzyme cutting is far more than that of P-C group without enzyme cutting.
[0080] The antioxidant activity of the polypeptide was detected by total SOD activity detection kit (WST-8 method) and DPPH free radical scavenging rate detection kit (colorimetric method), and the results are shown in Figure 7-8 .
[0081] It can be seen from Figure 7-8 that the total SOD activity inhibition rate of P-A4 group is stronger than that of P-B2 group, and P-C group is the weakest; at the same time, the DPPH free radical scavenging ability of P-A4 group is stronger than that of P-B2 group, and stronger than that of P-C group. It can be concluded that the activity of polypeptide of P-A4 group is the strongest, and the polypeptide yield is 85.96%.
[0082] Comparative example 1
[0083] S1, crush the oil tea seed meal through a 60 mesh sieve, mix with pure water according to the solid-liquid ratio of 1:10, adjust the pH to 9.0 using a 4.0% NaOH solution, microwave assisted extraction at 20℃ for 3h, microwave power is 550W, centrifuge at 400xg, continuous flow centrifugation at 0℃ with a feed flow rate of 175L / h, and obtain the supernatant;
[0084] S2, adjust the pH of the supernatant extracted in S1 to 4.5 using a 3.0% hydrochloric acid solution, stand for 24h, observe that flocculent precipitate is precipitated at the bottom, centrifuge at 500xg, 4℃, continuous flow centrifugation with a feed flow rate of 175L / h, collect the precipitate, wash, spray dry at an inlet temperature of 120℃, an outlet temperature of 90℃, a feed rate of 1.0L / h for 30min, and obtain the crude protein of oil tea seed meal, the crude protein extraction rate can reach 60.1%.
[0085] S3, prepare the crude protein of oil tea seed meal obtained in S2 into a 5.5% aqueous solution, heat denaturation treatment at 96℃ for 25min, cool to 35℃, adjust the pH to 6.6 using a 4.0% sodium hydroxide solution and a 3.0% hydrochloric acid solution, stand for 2.5h, adjust to the optimum pH of each enzyme, add 5000u of neutral protease, 5000u of trypsin and 5000u of alkaline protease to each gram of substrate respectively, and carry out enzyme hydrolysis according to the optimum enzyme hydrolysis conditions of each enzyme in Table 1, shake and stir during enzyme hydrolysis, enzyme hydrolysis time is 4.0h, heat to 84℃ to inactivate the enzyme for 35min after enzyme hydrolysis, centrifuge at 10000r / min for 25min, discard the precipitate, and filter through a ultrafiltration membrane with a pore size of 3Kd. Determine the polypeptide content of 5 enzyme hydrolysis filtrates by BCA kit, and the polypeptide yield is 75.62%.
[0086] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit it, and even though the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.
Claims
1. A method for extracting a polypeptide from oil-tea camellia seed meal, characterized in that, The method comprises the following steps: S1, crushing and sieving the oil-tea camellia seed meal, mixing with pure water according to a material-liquid ratio, adjusting pH to alkaline, microwave-assisted extraction, continuous flow centrifugation, and obtaining supernatant; S2, adjusting the supernatant obtained in S1 to acid, standing, continuous flow centrifugation, collecting precipitate, washing, spray drying, and obtaining oil-tea camellia seed meal crude protein; S3, preparing the oil-tea camellia seed meal crude protein obtained in S2 into a water solution with a concentration of 3.0%-5.0%, heating denaturation treatment, cooling, adjusting pH to acid and standing, adjusting pH to weak alkaline, synchronously adding neutral protease, trypsin and alkaline protease, oscillating enzymolysis, heating to inactivate enzyme after enzymolysis, centrifugation, discarding precipitate, ultrafiltration membrane filtration, tangential flow filtration desalination, filtration, taking supernatant, continuous flow centrifugation, taking supernatant, spray drying, and obtaining oil-tea camellia seed meal polypeptide.
2. The method of claim 1, wherein, In S1, the material-liquid ratio of the oil-tea camellia seed meal to pure water is 1:15-1:
25.
3. The method of claim 1, wherein, In S1, the pH is adjusted to 9.0-11.0 by using a NaOH solution with a concentration of 4.0%.
4. The method of claim 1, wherein, In S1, the microwave-assisted extraction process parameters are as follows: temperature is 25-30℃, microwave power is 500-600W, and time is 1.5-2h.
5. The method of claim 1, wherein, In S2, the pH is adjusted to 3.2-4.1 by using a hydrochloric acid solution with a concentration of 3.0%, and standing for 2 days.
6. The method of claim 1, wherein, In S3, the heating denaturation treatment temperature is 85-95℃, and the time is 15-20min, and the cooling temperature is 30-35℃.
7. The method of claim 1, wherein, In S3, the pH is adjusted to 5.8-6.5 and standing for 1.7-2.3h, and then the pH is adjusted to 7.5-8.
0.
8. The method of claim 1, wherein, In S3, the enzymolysis temperature is 45-50℃, and the enzymolysis time is 2.8-3.5h.
9. The method of claim 1 wherein, In S3, the temperature for heating to inactivate enzyme is 85-95℃, and the time is 20-30min.
10. The oil-tea camellia seed meal polypeptide obtained by the method according to any one of claims 1-9.