Camellia oleifera cake protein-polysaccharide compound as well as extraction method and application thereof

Abstract

The invention discloses an oil tea cake protein-polysaccharide compound as well as an extraction method and application thereof, and belongs to the technical field of food processing. The extraction method comprises the following steps: (1) preparing an alcohol insoluble substance; (2) adding alcohol-insoluble substances into water, and carrying out ultrasonic dissolution to obtain oil-tea cake supernate; (3) adding absolute ethyl alcohol, standing and precipitating; and (4) dialyzing the precipitate, and freeze-drying. According to the method provided by the invention, the oil-tea cake protein can be more efficiently, greenly and moderately enriched, the polysaccharide is co-extracted, the oil-tea cake protein-polysaccharide compound is formed, and the compound has good stability when being used as an emulsifier to prepare an emulsion under neutral and alkaline conditions. The camellia seed meal protein extracted by the method is high-quality protein, can be used as a protein enhancer or a protein raw material to be applied to food processing, and has relatively high economic value.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a protein-polysaccharide complex based on oil-tea camellia cake, and an extraction method and application thereof. Background Art

[0002] Camellia oleifera Abel. is a small tree or shrub belonging to the genus Camellia of the family Theaceae. It is a woody oil-bearing tree species. With the development of the camellia oil industry, a large number of camellia oil by-products have been produced, among which camellia oil seed meal is one of its main by-products. Camellia oil seed meal contains 30% to 60% polysaccharides, 10% to 20% protein, 0.5% to 7.0% crude fat, 12% to 18% tea saponin and 15% to 25% crude fiber, etc., and has a high utilization value. However, it is currently mainly used as fuel, organic fertilizer or animal feed, and its utilization is still in its primary stage.

[0003] Camellia oleifera cake contains a high content of crude protein, and its amino acid composition is 16 kinds, of which 7 kinds are essential amino acids for human body, which is a kind of high-quality protein. At present, the extraction and application research of camellia oleifera cake protein is mainly concentrated in the field of food. The extracted camellia oleifera cake protein has strong emulsification and oil absorption, and is used in ice cream, baked products, meat products, imitation meat products, protein drinks, and protein fortifiers of instant foods.

[0004] At present, the main methods for extracting camellia cake protein are: organic solvent extraction, alkali dissolution and acid precipitation, enzyme method, ultrasonic assisted extraction method, and two-phase aqueous extraction method. For example, patent document CN 102363626A discloses a method for extracting camellia cake protein by alkali extraction and acid precipitation. Although the extraction process of this method is simple and the processing cost is low, it can be industrialized. However, too high or too low pH value can easily denature the protein, and the extraction rate is low. Patent document CN 103626833 B discloses a method for extracting camellia seed meal protein by ultrasonic assisted enzymatic hydrolysis. This method uses composite plant hydrolase to hydrolyze tea meal, and extracts tea meal protein by ultrasonic assisted alkali extraction and acid precipitation. Although this method is simple to handle, the extraction process conditions are mild, and the obtained camellia seed meal protein has good morphology and color. However, impurities such as oil and saponin in tea meal are not removed, and the enzyme has high requirements for time and temperature, and the actual extraction rate and purity are not high.

[0005] The polysaccharides in camellia oleifera seed meal are water-soluble, and the main components are six monosaccharides, namely rhamnose, trehalose, arabinose, mannose, galactose and glucose. Camellia seed polysaccharides have a variety of biological activities, such as anti-tumor, anti-oxidation, and hypoglycemic. The main methods for extracting plant polysaccharides are solvent extraction, enzyme extraction, ultrasonic extraction, and microwave extraction. Patent document CN116589605 A discloses a method for extracting polysaccharides from camellia oleifera cake, which uses a response surface method to optimize the removal of impurities such as protein in camellia oleifera seed meal polysaccharides to increase the content of polysaccharides. Although this method can increase the content of polysaccharides, when the Sevag method is used to remove protein, the Sevag method can only remove a small amount of protein at a time, and it is usually necessary to repeat the treatment multiple times to achieve the ideal deproteinization effect, which may lead to a long experimental operation time, and the polysaccharides may have a certain degree of loss, which will affect the yield of the final product. The reagents used, such as chloroform and n-butanol, are hazardous chemicals, and special safety measures may be required for proper handling.

[0006] Most of the extraction methods reported so far separate proteins and polysaccharides, which not only increases the number of steps but also causes certain losses to both substances. Studies have shown that compounding the protein and polysaccharides in flaxseed cake can improve its functional properties, such as emulsification. At present, there are no research reports on the extraction process of camellia oleifera cake protein-polysaccharide complexes. Therefore, how to achieve efficient extraction and multi-scenario utilization of natural blends of camellia oleifera cake protein-polysaccharide is an urgent problem that technicians in this field need to solve. Summary of the invention

[0007] The object of the present invention is to provide a method for efficiently extracting protein-polysaccharide complex from camellia oleifera cake, so as to fully utilize the effective components of camellia oleifera cake.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides a method for extracting a camellia oil cake protein-polysaccharide complex, comprising the following steps:

[0010] (1) After the oil-tea cake is crushed, anhydrous ethanol is added and stirred, and the supernatant is discarded by centrifugation. The above operation is repeated several times until the supernatant is clear; the precipitate is collected and then washed with acetone and anhydrous ethanol in turn, and dried at room temperature to obtain an alcohol-insoluble substance;

[0011] (2) adding the alcohol-insoluble substances into water for ultrasonic dissolution, centrifugation, repeating the operation 2-3 times, and combining the supernatants to obtain the camellia oil cake supernatant;

[0012] (3) adding anhydrous ethanol to the supernatant of the camellia oleifera cake, stirring, allowing to stand for stratification, and collecting the precipitate by centrifugation;

[0013] (4) The precipitate is dialyzed and then freeze-dried to obtain the tea oil cake protein-polysaccharide complex.

[0014] In step (1), anhydrous ethanol is used to dissolve and remove pigments and small molecules, and organic solvents of different polarities are used to remove soluble impurities.

[0015] Preferably, the camellia oleifera cake is crushed and then sieved to obtain camellia oleifera cake powder, and the mesh number of the sieve is 60-100 meshes.

[0016] Preferably, the material-liquid ratio of camellia oleifera cake powder and anhydrous ethanol is 1:1-3; the stirring speed is 1000-1200 rpm, the temperature is 20-25°C, and the time is 20-30 min; centrifugation is carried out at 4°C and 10000 rpm for 10-15 min.

[0017] This step was repeated 2-3 times, during which the color of the supernatant gradually became clear, indicating that most of the pigments and small molecules were removed.

[0018] The present invention uses acetone to dissolve and remove impurities after ethanol removal, and utilizes the difference in solubility of ethanol and acetone to different substances to fully remove soluble impurities. Preferably, the material-liquid ratio of precipitate to acetone or anhydrous ethanol is 1:1-3; the stirring speed is 1000-1200rpm, the temperature is 20-25℃, and the time is 20-30min; and centrifugation is carried out at 4℃ and 10000rpm for 10-15min.

[0019] In step (2), the water solubility of proteins and polysaccharides is utilized to add alcohol-insoluble substances into pure water, and ultrasonic treatment is combined to dissolve the proteins and polysaccharides in water.

[0020] Preferably, the liquid-to-solid ratio of alcohol-insoluble substance to pure water is 1:1-3; the ultrasonic power is 400-450W, the temperature is 25°C-40°C, and the time is 30-60min; centrifugation is performed at 4°C and 3000-3500rpm for 10-15min.

[0021] Repeat the above operation for the precipitation after centrifugation to fully extract the protein and polysaccharide substances in the precipitation, which is conducive to improving the extraction rate. The supernatants extracted multiple times are combined to obtain the oil-tea cake supernatant.

[0022] In step (3), the protein and polysaccharide substances in the supernatant of the camellia oleifera cake are precipitated by alcohol precipitation for further separation and purification.

[0023] Preferably, the volume ratio of the supernatant of the camellia oleifera cake to anhydrous ethanol is 1:1-3, and the mixture is allowed to stand for stratification at 4°C for 12-15 hours; and centrifuged at 4°C and 3000-3500 rpm for 10-15 minutes.

[0024] In step (4), the ratio of protein to polysaccharide is optimized by dialysis interception of target substances to obtain the camellia oleifera cake protein-polysaccharide complex.

[0025] Preferably, the dialysis cut-off molecular weight of the extract is greater than 14 kDa. The complexes with molecular weight distribution of 14-15 kDa in the extract have good antioxidant properties.

[0026] Preferably, the dialysis is performed using a dialysis bag with a specification of 14 kDa, at 4° C. for 3 to 4 days, with the water being changed every 8 to 10 hours.

[0027] The present invention provides a tea oil cake protein-polysaccharide complex prepared by the above extraction method. After testing, the protein content in the tea oil cake protein-polysaccharide complex provided by the present invention is about 51%, and the distribution of the molecular weight of the protein is relatively wide. Among the 17 amino acids that can be detected, 7 essential amino acids (EAA) for the human body account for about 27% of the total amount, which has a very high economic value. The total sugar content is about 11%, and the monosaccharides are composed of rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid, and mannuronic acid. Emulsification performance analysis shows that the tea oil cake protein-polysaccharide complex of the present invention has excellent emulsification.

[0028] The invention provides the application of the camellia oil cake protein-polysaccharide complex as an emulsifier and a protein enhancer in food processing.

[0029] The present invention has the following excellent effects:

[0030] (1) The present invention utilizes the natural advantages of the ingredients in camellia oleifera cake and uses a gentle method to extract the camellia oleifera cake protein-polysaccharide complex. Compared with the existing reported methods of extracting protein using alkali acid precipitation or extracting polysaccharides using water extraction, the method provided by the present invention can more efficiently, greenly, and moderately enrich camellia oleifera cake protein, and co-extract polysaccharides to form a camellia oleifera cake protein-polysaccharide complex. The combination of protein and polysaccharide can form a more stable emulsion. The interaction between the water absorption and oil absorption of protein and polysaccharide can enhance the stability of the interfacial layer and reduce the flocculation and aggregation of the emulsion.

[0031] (2) The tea seed cake protein-polysaccharide complex extracted by the present invention has excellent emulsifying properties and good stability when used as an emulsifier to prepare an emulsion under neutral and alkaline conditions. The tea seed cake protein extracted by the present invention is a high-quality protein that can be used as a protein enhancer or protein raw material in food processing and has high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the extraction steps of protein-polysaccharide complex based on camellia oleifera cake.

[0033] Figure 2 It is the composition of monosaccharides in the tea oil cake protein-polysaccharide complex.

[0034] Figure 3 This is the SDS-PAGE image of the tea cake protein-polysaccharide complex.

[0035] Figure 4 It is the isoelectric point of the tea cake protein-polysaccharide complex.

[0036] Figure 5 The solubility of tea oil cake protein-polysaccharide complex at different pH.

[0037] Figure 6 To test the water and oil holding capacity of tea oil cake protein-polysaccharide complex at different pH values.

[0038] Figure 7 The foaming property and bubble stability of tea oil cake protein-polysaccharide complex at different pH values.

[0039] Figure 8 The interfacial tension of the tea cake protein-polysaccharide complex at different pH values.

[0040] Fig. 9 The emulsifying activity and emulsifying stability of tea oil cake protein-polysaccharide complex at different pH values. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] The camellia oleifera cake used in the following embodiments is from Zhejiang Jiusheng Camellia oleifera Technology Co., Ltd., and is the residue cake after oil is pressed from camellia oleifera seeds.

[0044] The method used for performance testing in the following embodiments is as follows:

[0045] 1. Protein content detection: Use Kjeldahl nitrogen determination method, refer to national standard GB 5009.5-2016 for details.

[0046] 2. Detection of total sugar content: Use phenol-sulfuric acid method, refer to national standard GB / T15672-2009 for details.

[0047] 3. Detection of amino acid composition: Use an amino acid analyzer (ninhydrin post-column derivatization ion exchange chromatograph), referring to the national standard GB5009.124-2016 for specific details.

[0048] 4. Composition of monosaccharides: Preparation of standard: Take a clean 15mL EP tube, add 8mL sterile water, add 100mg each of fucose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, and glucuronic acid in turn, and dilute to 10mL with a volumetric flask after dissolution to prepare a 10mg / mL standard stock solution. Dilute the above mixed standard stock solution 100 times to prepare a 100μg / mL working solution, and dilute the above solution according to the following gradient and put it into a 1.5mL EP tube.

[0049] Sample extraction: Take a clean chromatographic bottle, accurately weigh 5 mg (± 0.05 mg) of the sample, add the prepared TFA acid solution, and heat at 121°C for 2 hours. Pass nitrogen and blow dry. Add methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add sterile water to dissolve, and transfer to the chromatographic bottle for testing.

[0050] Chromatographic parameters: The chromatographic system used was the Thermo ICS5000+ ion chromatography system (ICS5000+, (ThermoFisher Scientific, USA), using Dionex TM CarboPac TM PA10 (250×4.0 mm, 10 μm) liquid chromatography column, injection volume of 5 μL, mobile phase A (H2O), mobile phase B (100 mM NaOH), column temperature of 30°C, electrochemical detector for analysis and detection of monosaccharide components.

[0051] 5. SDS-PAGE. Perform SDS-PAGE of the protein fractions to measure the molecular weight of the protein fractions.

[0052] The protein-polysaccharide complex sample (5 mg / mL) was added to the SDS-PAGE loading buffer, boiled for 10 min, and centrifuged (7000 rpm, 5 min). The sample volume was 15 μL (the marker sample volume was 10 μL) in the precast polyacrylamide gel and the voltage was 120 V for 30 minutes. After the electrophoresis was completed, the gel was stained with Coomassie Blue R-250 for 2 hours and then destained overnight.

[0053] 6. Detection of isoelectric point:

[0054] Prepare 0.3% (w / v) tea cake protein-polysaccharide complex with ultrapure water, stir magnetically for 2 hours until completely dissolved, adjust pH to 2-4 (each 0.25 is an interval), filter through a 0.45mm membrane before testing, use a Malvern particle size analyzer, and record the Zeta potential data at different pH values. Draw a curve of the relationship between Zeta potential and pH value, and the isoelectric point is usually the pH value where the curve passes through zero.

[0055] 7. Solubility detection:

[0056] Prepare 20mL 0.01% (w / v) tea oil cake protein-polysaccharide complex with ultrapure water, stir magnetically for 2h until completely dissolved, and adjust pH to 1, 3, 5, 7, 9, 11 respectively. Then centrifuge (8000rpm, 10min, 4°C), take an appropriate amount of supernatant for dilution after centrifugation, and calculate the protein content of the supernatant with bovine serum albumin (BSA) as the standard protein. Measure the absorbance of the sample at 562nm with an enzyme marker, and calculate the protein concentration of the sample according to the standard curve and the sample volume used. The solubility of the protein is the ratio of the protein content in the supernatant to the total protein content.

[0057] 8. Testing of water and oil holding capacity:

[0058] Prepare 20mL of 0.1% (w / v) camellia oil cake protein-polysaccharide complex with ultrapure water, stir magnetically for 2h until completely dissolved, and adjust the pH to 1, 3, 5, 7, 9, and 11 respectively. Freeze-dry the solution to obtain a protein-polysaccharide complex sample. Weigh 0.1g of the protein-polysaccharide complex in a centrifuge tube and record the weight of the protein and the centrifuge tube. Vortex 0.1g of the sample with 2mL of camellia oil for 3min to mix thoroughly and let stand for 30min. Centrifuge at 4°C (8000rpm, 15min). After centrifugation, carefully decant the supernatant and weigh the remaining precipitate. WHC or OHC (g / g) is calculated as follows:

[0059]

[0060] Wherein, M1 is the weight of the protein-polysaccharide complex and the centrifuge tube, M2 is the weight of the protein-polysaccharide complex and the centrifuge tube after centrifugation, and M0 is the weight of the protein-polysaccharide complex.

[0061] 9. Testing of foaming property and foaming stability:

[0062] Prepare 20mL of 0.5% (w / v) tea cake protein-polysaccharide complex with ultrapure water, stir magnetically for 2h until completely dissolved, and adjust the pH to 1, 3, 5, 7, 9, and 11 respectively. Then use a homogenizer at a speed of 12000r / min for 3min. Record the initial volume V0 and V1 of the solution, and calculate the foaming property. The formula is as follows:

[0063]

[0064] After the uniformly formed bubbles are left to stand for 30 minutes, the volume of the bubbles is read and recorded, and the stability of the bubbles for 30 minutes is calculated using the following formula:

[0065]

[0066] Among them, V0 and V1 represent the volume of the solution before and after homogenization, V t They respectively represent the bubble volume of the solution after standing for 30 minutes.

[0067] 10. Detection of interfacial tension:

[0068] Prepare 20mL of 0.1% (w / v) camellia oleifera cake protein-polysaccharide complex with ultrapure water, stir magnetically for 2h until completely dissolved, and adjust the pH to 3, 5, 7, 9, and 11, respectively. Use the pendant drop method to evaluate the static interfacial tension at the water / oil interface. The Young-Laplace formula is used to fit the data of the drop shape curve. The 0.1% (w / v) extract was tested at 120 minutes and 25±1°C. The volume of the droplet was around 19-21μL. The experiment was repeated at least three times.

[0069] 11. Testing of emulsification and emulsion stability:

[0070] Prepare 20mL of 1% (w / v) camellia oil cake protein-polysaccharide complex with ultrapure water, stir magnetically for 2h until completely dissolved, and adjust the pH to 1, 3, 5, 7, 9, and 11, respectively. Take 15mL of the solution and add 5mL of camellia oil. Prepare the emulsion by mixing the extract solution with camellia oil at 12000rpm for 3 minutes using a homogenizer. Quickly draw 100μL of the emulsion at the bottom, add 10mL of 0.1% SDS to dilute, use the SDS solution as a blank reference, measure the initial absorbance value (A0) of the diluted emulsion at 500nm, and measure the absorbance (A0) again under the same conditions after standing for 15min. 15 ). Emulsification and emulsion stability were calculated according to the following formula:

[0071]

[0072] where C is the concentration of the sample (0.01 g / mL), L is the path length of the cuvette (1 cm), θ is the oil phase ratio (0.25), and N is the dilution factor (100).

[0073] Example 1

[0074] This embodiment provides a method for extracting a protein-polysaccharide complex from tea oil cake, which is as follows:

[0075] (1) Raw material pretreatment: First, the saponin-containing camellia oil cake was crushed and passed through an 80-mesh sieve to obtain camellia oil cake powder. Then, the camellia oil cake powder was dissolved in petroleum ether at a volume ratio of 1:10, stirred at 700 rpm in a 40°C water bath for 2 h, and the above operation was repeated again. The defatted camellia oil cake was placed in a fume hood to finally obtain dry defatted camellia oil cake.

[0076] (2) Preparation of alcohol-insoluble substances: Anhydrous ethanol was added to the defatted tea oil cake powder at a solid-liquid ratio of 1:3, and the mixture was centrifuged at 25°C and 1000 rpm for 30 min. The supernatant was discarded and the precipitate was repeatedly subjected to the above operation until the supernatant was clear. The operation was repeated 3 times, and the color of the supernatant gradually became clear during the repetition process, indicating that most of the pigments and small molecules were removed during the repetition process.

[0077] Then, the precipitate was washed with acetone and anhydrous ethanol in the same manner as above at a material-liquid ratio of 1:3. Finally, the precipitate was dried at room temperature in a fume hood to obtain a dry alcohol-insoluble substance.

[0078] In this operation, pigments and small molecules are removed and soluble impurities are removed by reagents of different polarity.

[0079] (3) Ultrasound: The alcohol-insoluble substances were added to ultrapure water at a material-liquid ratio of 1:3 and ultrasonicated (power of 450w) for 30min at a temperature of 25°C. Centrifuged at 4°C and 3500rpm for 15min to obtain the supernatant of the oil-tea cake.

[0080] (4) After adding anhydrous ethanol to the supernatant at a volume ratio of 1:3 and stirring, the mixture was allowed to stand at 4°C for stratification. The mixture was then centrifuged at 4°C and 3500 rpm for 15 min, and the lower precipitate obtained was the water-soluble camellia oleifera cake material.

[0081] (5) The lower precipitate is dialyzed with a dialysis bag of 14 kDa at 4°C for 3 days, with ultrapure water replaced every 8 hours. The liquid in the dialysis bag is collected and freeze-dried to obtain a camellia oil cake protein-polysaccharide complex.

[0082] Example 2

[0083] This embodiment provides a method for extracting a protein-polysaccharide complex from oil-tea cake. The difference from Example 1 is that in the pretreatment of the raw materials in step (1), the powder of the cake is only crushed and passed through an 80-mesh sieve, and petroleum ether degreasing is not performed. In the process of petroleum ether degreasing in Example 1, it was found that too high a temperature or too long a treatment time caused a certain degree of damage to the structure of the protein, so in Example 2, the degreasing step was removed, because in the subsequent operation process of washing with ethanol, most of the oil can be removed and the protein content is not greatly affected. Compared with the protein-polysaccharide complex extracted from the oil-tea cake in Example 1, there is no significant difference in protein content.

[0084] Example 3

[0085] This embodiment provides a method for extracting a protein-polysaccharide complex from tea oil cake. The difference from Embodiment 1 is that the operating conditions of ultrasound in step (3) are changed to 40°C.

[0086] The protein content in the extract was determined by the Kjeldahl method. Compared with the protein-polysaccharide complex extracted from the camellia oleifera cake in Example 1, the protein content was not significantly different.

[0087] Example 4

[0088] This embodiment provides a method for extracting a protein-polysaccharide complex from camellia oleifera cake. The difference from Embodiment 1 is that the ultrasonic operation conditions in step (3) are changed to 60 minutes.

[0089] The protein content in the extract was determined by Kjeldahl nitrogen determination. Compared with the protein-polysaccharide complex extracted from the camellia oleifera cake in Example 1, the protein content was only increased by about 2%.

[0090] Example 5

[0091] This embodiment provides a method for extracting a protein-polysaccharide complex from tea oil cake. The difference from Embodiment 1 is that the ultrasonic treatment in step (3) is repeated twice according to the conditions of Embodiment 1, and step (4) is performed after combining the supernatants of the two times.

[0092] The total amount of protein and polysaccharide in the extract was determined using the Kjeldahl nitrogen method and the phenol-sulfuric acid method. Compared with the protein-polysaccharide complex extracted from the camellia oleifera cake in Example 1, the extraction rate of the protein-polysaccharide complex was increased from the previous 0.8% to 2.5%.

[0093] Example 6

[0094] This example provides a method for extracting a protein-polysaccharide complex from camellia oleifera cake. The difference from Example 1 is that after step (4), an acetone-ethanol reagent exchange is performed again, and protein dialysis is not performed.

[0095] The total amount of protein in the extract was determined using the Kjeldahl method. Compared with the protein-polysaccharide complex extracted from the camellia oleifera cake in Example 1, the protein content differed by only about 1% from the result of only one reagent exchange, and there was no significant difference.

[0096] Example 7

[0097] This embodiment provides a method for extracting a protein-polysaccharide complex from camellia oleifera cake. The difference from Embodiment 1 is that in step (5), dialysis is not performed and freeze-drying is performed directly.

[0098] The total amount of protein and polysaccharide in the extract was determined by Kjeldahl nitrogen determination and phenol-sulfuric acid method. Compared with the protein-polysaccharide complex extracted from the camellia oleifera cake in Example 1, the protein content decreased from about 49% to about 32%, and the polysaccharide content increased from about 12% to about 23%. Because protein is an excellent emulsifier, the post-dialysis operation was selected to extract the protein-polysaccharide complex from the camellia oleifera cake.

[0099] Example 8

[0100] 1. This embodiment provides a method for extracting protein-polysaccharide complexes from camellia oleifera cakes. The schematic flow chart is as follows: Figure 1 As shown, the specific steps are as follows:

[0101] (1) Raw material pretreatment: First, the saponin-containing camellia oleifera cake is crushed and then passed through an 80-mesh sieve to obtain camellia oleifera cake powder.

[0102] (2) Preparation of alcohol-insoluble substances: Anhydrous ethanol was added to the oil-tea cake powder at a solid-liquid ratio of 1:3, and the mixture was centrifuged at 4°C, 10,000 rpm, and 10 min after magnetic stirring at 25°C. The supernatant was discarded and the precipitate was repeated until the supernatant was clear. Acetone was added to the precipitate at a solid-liquid ratio of 1:3, and the mixture was centrifuged at 4°C, 10,000 rpm, and 10 min after magnetic stirring at 25°C. The supernatant was discarded and the precipitate was finally dried at room temperature to obtain a dry alcohol-insoluble substance.

[0103] (3) Ultrasound: Add the alcohol-insoluble substances to ultrapure water at a material-liquid ratio of 1:3 and perform ultrasound (power of 450 W) for 30 min at a temperature of 25°C. Centrifuge at 4°C and 3500 rpm for 15 min to obtain the supernatant of the oil-tea cake. Repeat the above operation and combine the supernatants twice.

[0104] (4) After adding anhydrous ethanol to the supernatant at a volume ratio of 1:3 and stirring, the mixture was allowed to stand at 4°C for 12 hours to separate into layers. The mixture was then centrifuged at 4°C and 3500 rpm for 15 minutes to obtain the lower precipitate, which was the water-soluble camellia oleifera cake material.

[0105] (5) The lower precipitate is dialyzed with a dialysis bag of 14 kDa at 4°C for 3 days, with ultrapure water replaced every 8 hours. The liquid in the dialysis bag is collected and freeze-dried to obtain a camellia oil cake protein-polysaccharide complex.

[0106] 2. Performance Testing

[0107] 2.1 The protein, total sugar content and amino acid composition were determined according to the national standards GB 5009.5-2016, GB / T 15672-2009 and GB5009.124-2016, respectively. The results are shown in Tables 1 and 2.

[0108] Table 1. Protein and total sugar content

[0109] Element content protein 51.56％ Total Sugar 11.75％

[0110] Table 2. Amino acid composition

[0111] Amino acid name Content (mg / g) Amino acid name Content (mg / g) Aspartic acid Asp 31.519 MethionineMet 9.435 Threonine 12.73 Isoleucine Ile 13.023 Ser 17.237 Leucine 30.254 Glu 113.149 Tyrosine 12.651 Glycine 15.304 Phenylalanine 15.835 Alanine Ala 21.605 His 5.639 Cysteine ​​Cys 11.382 Lysine 12.209 Valine 12.936 Arginine 38.59 Proline Pro 13.093

[0112] Tables 1 and 2 show that the protein content in the protein-polysaccharide complex extracted by the method of the present invention is 51%, and the total sugar content is 11.75%. Among the 17 amino acids that can be detected, 7 essential amino acids (EAA) account for 27.53% of the total, of which leucine has the highest content of 30.254 mg / g. Among the hydrophobic amino acids, glutamic acid and aspartic acid account for a relatively high proportion, which can improve the antioxidant properties of protein and remove excess free radicals. Among them, the contents of two semi-essential amino acids, histidine and arginine, are also relatively high. Therefore, the extracted camellia oleifera meal protein is a high-quality protein with high economic value. It can be used as a protein enhancer for the production of protein beverages, instant foods, and baked foods, as well as a protein raw material for fermented products such as soy sauce.

[0113] 2.2 Monosaccharide composition

[0114] like Figure 2As shown, the monosaccharides in the extract are composed of rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid and mannuronic acid.

[0115] 2.3 SDS-PAGE to measure the molecular weight of protein fractions

[0116] like Figure 3 As shown, the molecular weight distribution of the protein in the extract is relatively wide, and a portion of the molecular weight is roughly distributed in the range of 14-15 kDa, and this portion of the molecular weight has good antioxidant properties.

[0117] 2.4 Determination of isoelectric point and solubility

[0118] like Figure 4 , 5 As shown, the isoelectric point of the complex is around 2.75, so its solubility is the worst around pH 3. pH also affects the solubility of protein.

[0119] In all samples with U-shaped solubility curves, there is a minimum solubility at pH 3.0 (close to pI). Near pI, the charge of the protein is less, so the interaction between the proteins is weaker, causing the proteins to aggregate more easily and thus reducing solubility. As the pH moves away from pI, the proteins have more negative or positive charges, resulting in stronger electrostatic repulsion, preventing protein aggregation and increasing their solubility. Therefore, when the pH level is from 7-11, the solubility of the complex increases, so it is better soluble under neutral and alkaline conditions like other plant and animal proteins.

[0120] 2.5 Analysis of water and oil holding capacity

[0121] like Figure 6 As shown, pH also affects water holding capacity. When pH is far away from pI, WHC gradually increases. This may be because protein molecules carry more charges, which may enhance their interaction with water molecules, thereby improving water holding capacity. Both water holding capacity and oil holding capacity have better performance under neutral or alkaline conditions. Therefore, better water absorption and oil absorption under neutral or alkaline conditions can enable the tea oil cake protein-polysaccharide complex to form a stable layer at the oil-water interface and improve the stability of the emulsion.

[0122] 2.6 Foamability and interfacial tension analysis

[0123] like Figure 7 , Figure 8As shown, the foam stability (FS) has better performance when it is close to pI; the foaming property (FA) has better performance when it is far away from pI. FS is larger when it is close to pI because the intermolecular electrostatic repulsion is small, and it is easy to form a stable structure at the gas-liquid interface to maintain the foam stability; FA is smaller because the solubility is poor and it is not easy to form foam. If the protein-polysaccharide complex of tea oil cake is used as an emulsifier, it can form a stable layer at the oil-water interface, thereby reducing the interfacial tension, preventing the aggregation of oil droplets, and improving the stability of the emulsion. The stability and foaming property of the foam are related to the behavior of protein at the gas-liquid interface. The reduction of surface tension is conducive to the formation of foam. The smaller the interfacial tension, the better the foam stability.

[0124] 2.7 Emulsification analysis

[0125] like Fig. 9 As shown, close to pI, EAI is low because of its lowest solubility near the isoelectric point, which leads to a slower movement of the protein to the oil / water interface; away from pI, EAI is larger, and the solubility and surface charge under neutral and alkaline conditions increase. In addition, alkaline conditions have more negative charges, which change the electrostatic interaction and allow the complex to be evenly dispersed in the oil-water emulsion. Therefore, under neutral and alkaline conditions, the tea cake protein-polysaccharide complex is more stable when used as an emulsifier to prepare emulsions.

[0126] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for extracting a protein-polysaccharide complex from oil-tea cake, characterized in that: The following steps are involved: (1) After the oil-tea cake is crushed, anhydrous ethanol is added and stirred, and the supernatant is discarded by centrifugation. The above operation is repeated several times until the supernatant is clear; the precipitate is collected and then washed with acetone and anhydrous ethanol in turn, and dried at room temperature to obtain an alcohol-insoluble substance; (2) adding the alcohol-insoluble substances into water for ultrasonic dissolution, centrifugation, repeating the operation 2-3 times, and combining the supernatants to obtain the camellia oil cake supernatant; (3) adding anhydrous ethanol to the supernatant of the camellia oleifera cake, stirring, allowing to stand for stratification, and collecting the precipitate by centrifugation; (4) The precipitate is dialyzed and then freeze-dried to obtain the tea oil cake protein-polysaccharide complex.

2. The method for extracting the tea oil cake protein-polysaccharide complex according to claim 1, characterized in that: In step (1), the camellia oleifera cake is crushed and then sieved to obtain camellia oleifera cake powder, and the mesh number of the sieve is 60-100 meshes.

3. The method for extracting the tea oil cake protein-polysaccharide complex according to claim 1, characterized in that: In step (1), the material-liquid ratio of camellia oleifera cake powder to anhydrous ethanol is 1:1-3; the stirring speed is 1000-1200 rpm, the temperature is 20-25° C., and the time is 20-30 min; and the centrifugation is carried out at 4° C. and 10,000 rpm for 10-15 min.

4. The method for extracting the tea oil cake protein-polysaccharide complex according to claim 1, characterized in that: In step (1), the solid-liquid ratio of the precipitate to acetone or anhydrous ethanol is 1:1-3; the stirring speed is 1000-1200 rpm, the temperature is 20-25° C., and the time is 20-30 min; and the centrifugation is performed at 4° C. and 10,000 rpm for 10-15 min.

5. The method for extracting the tea oil cake protein-polysaccharide complex according to claim 3 or 4, characterized in that: In step (2), the liquid ratio of alcohol-insoluble substance to pure water is 1:1-3; the ultrasonic power is 400-450W, the temperature is 25°C-40°C, and the time is 30-60min; and the centrifugation is carried out at 4°C and 3000-3500rpm for 10-15min.

6. The method for extracting the tea oil cake protein-polysaccharide complex according to claim 1, characterized in that: In step (3), the volume ratio of the supernatant of the camellia oleifera cake to anhydrous ethanol is 1:1-3, and the mixture is allowed to stand at 4°C for 12-15 hours to separate into layers; and centrifuged at 4°C and 3000-3500 rpm for 10-15 minutes.

7. The method for extracting the tea oil cake protein-polysaccharide complex according to claim 1, characterized in that: In step (4), dialysis is used to retain extracts with a molecular weight cutoff greater than 14 kDa.

8. The method for extracting the tea oil cake protein-polysaccharide complex according to claim 7, characterized in that: Dialysis was performed using a 14 kDa dialysis bag at 4°C for 3 to 4 days, with the water changed every 8 to 10 hours.

9. The tea oil cake protein-polysaccharide complex prepared by the extraction method according to any one of claims 1 to 8.

10. Use of the camellia oleifera cake protein-polysaccharide complex as claimed in claim 9 as an emulsifier or protein enhancer in food processing.

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