Camellia oil cake protein and extraction method and application thereof

Abstract

The invention discloses a tea seed cake protein and an extraction method and application thereof, and belongs to the technical field of food processing. The extraction method comprises: (1) crushing tea seed cake and mixing with petroleum ether for degreasing; (2) mixing the defatted tea seed cake with water, adjusting the pH value to 10-11, extracting at a temperature of 35-45°C, and collecting the supernatant; (3) adjusting the pH value of the supernatant to 3.0±0.5 for acid precipitation to obtain a crude extract of tea seed cake protein; (4) adding anhydrous ethanol for stirring and eluting, collecting the precipitate, adding water for redissolution, and freeze-drying to obtain the tea seed cake protein. The extraction method provided by the invention can efficiently extract protein from tea seed cake, and the product has high purity. The tea seed cake protein extracted by the invention has a reasonable amino acid composition, excellent gel properties, emulsification properties, foaming properties and antioxidant properties, and can be used as a protein enhancer or a protein raw material for plant protein meat in food processing.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a method for extracting protein from camellia oleifera cake and its application. Background Art

[0002] Camellia oleifera Abel. is a woody oil crop. Tea oil made from camellia oleifera is rich in nutrients, including oleic acid, linoleic acid, etc. It has a mellow taste and is a high-quality edible oil. With the increase in production and the improvement of deep processing technology, the competitiveness of camellia oil in the vegetable edible oil consumer market continues to increase. However, the cake produced after oil extraction has not been efficiently utilized. Most of it is used as livestock feed or directly discarded. Its potential value urgently needs to be fully explored.

[0003] Camellia oleifera cake contains rich protein, sugar, saponin, etc. Related analysis and research show that camellia oleifera cake contains 10% to 25% crude protein, 35% to 40% sugar, 5% crude fat, 6% crude fiber, and 10% to 15% saponin. At present, the extraction and application research of camellia oleifera cake protein is mainly concentrated in the food field. 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, and protein fortifiers for protein drinks and instant foods. Therefore, the extraction of camellia oleifera cake protein is very important for the utilization of camellia oleifera resources.

[0004] Patent document CN 102617701 A discloses a method for extracting protein from oil-tea cake, which comprises the following steps: irradiating a first mixture with ultrasound to obtain a product after ultrasound irradiation; removing solids and organic phases from the product after ultrasound irradiation to obtain a first clear liquid; contacting the first clear liquid with an acid to precipitate protein in the first clear liquid to obtain a product after contact with the acid; and separating the protein precipitate from the product after contact with the acid. The method involves multiple steps, including ultrasound irradiation, removal of solids and organic phases, and separation of acid precipitation and protein precipitation. The complicated operation undoubtedly increases the production cost.

[0005] Patent document CN 114539346 A discloses a method for extracting oil-tea cake protein, the steps of which are to add acetone to oil-tea cake, quickly stir evenly under low temperature conditions, filter, and rinse with trimethylsilyl ether to obtain a primary pre-treated material; add fixed enzymes and protein stabilizers to the primary treated material, perform enzymatic hydrolysis, and obtain a secondary pre-treated material; add the secondary pre-treated material to a buffer solution, perform differential pressure extraction, filter, and obtain an extract; adjust the pH of the extract, let it stand until precipitation occurs, and freeze-dry the obtained precipitate to obtain oil-tea cake protein. This method uses an organic solvent as a solvent, and acetone is a volatile organic compound, and its use and handling require special attention to avoid pollution to the environment.

[0006] In recent years, the research on plant protein meat has been quite in-depth at home and abroad, and a variety of products with different raw materials and qualities have been developed. However, as a new natural plant protein raw material, the development and utilization of camellia oleifera cake is still insufficient, and the research on its physical, chemical and functional properties is relatively small. Therefore, it is an urgent problem for technicians in this field to study how to use camellia oleifera cake protein in the processing of plant protein meat to replace traditional commercial plant protein. Summary of the invention

[0007] The purpose of the present invention is to provide a method for efficiently extracting protein from camellia oleifera cake to obtain a protein extract with excellent performance for use in food processing.

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

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

[0010] (1) crushing tea oil cake and mixing it with petroleum ether to perform degreasing treatment, and removing the organic solvent after degreasing to obtain defatted tea oil cake;

[0011] (2) mixing defatted tea oil cake with water at a solid-liquid ratio of 1:25-35 g / mL, adjusting the pH value to 10-11, extracting at a temperature of 35-45° C. for 50-70 min, and then centrifuging to collect the supernatant;

[0012] (3) adjusting the pH value of the supernatant to 3.0±0.5 for acid precipitation, and collecting the precipitate by centrifugation to obtain a crude protein extract extracted from camellia seed cake;

[0013] (4) adding anhydrous ethanol to the crude protein extract of camellia oleifera seed cake at a solid-liquid ratio of 1:10-20 g / mL, stirring and eluting for 20-40 min, collecting the precipitate by centrifugation, re-dissolving with water, adjusting the pH value of the solution to 7.0±0.2, and freeze-drying to obtain the camellia oleifera cake protein.

[0014] In step (1), the camellia oleifera cake is defatted. The presence of fat will affect the efficiency and purity of subsequent protein extraction, as well as the shelf life of subsequent products.

[0015] Preferably, the tea oil cake is crushed to 80-100 mesh and then defatted.

[0016] Preferably, the oil-tea cake raw material is mixed with petroleum ether at a ratio of 1:10-20 g / mL, stirred continuously for 1-2 hours, allowed to stand until the upper organic solvent is clarified, the organic solvent is recovered, and the operation is repeated until degreasing is completed (petroleum ether is very light yellow). After degreasing, it is naturally air-dried to remove the residual petroleum ether.

[0017] In step (2), the protein is extracted from the oil-tea cake by alkaline extraction. The solubility in different acid and alkaline environments is different. Studies have shown that the protein in the oil-tea cake is easily soluble in an alkaline environment with a pH value of 10 to 11.

[0018] Preferably, the defatted tea oil cake is crushed into 60 mesh and then extracted.

[0019] Preferably, 1 mol / L NaOH solution is used to adjust the pH value.

[0020] Preferably, defatted camellia oleifera cake is mixed with water at a solid-liquid ratio of 1:30 g / mL, the pH value is adjusted to 11, and extraction is performed at 40°C for 60 min; the centrifugation condition is 5000 rpm for 10 min.

[0021] In step (3), the protein in the extract is precipitated by acid precipitation. Plant protein dissolves in an alkaline environment and precipitates under acidic isoelectric conditions. Studies have shown that the isoelectric point of tea oil cake protein is 2.98.

[0022] Preferably, 1 mol / L HCl solution is used to adjust the pH value.

[0023] Preferably, the acid precipitation conditions are: adjusting the pH value of the supernatant to 3.0, and allowing to settle at room temperature for 30 minutes; and the centrifugation conditions are centrifugation at 5000 rpm for 10 minutes at 4°C.

[0024] In step (4), the crude protein extract extracted from the camellia seed cake is purified, and anhydrous ethanol is used to remove inorganic salts, residual polyphenols, etc.

[0025] Preferably, anhydrous ethanol is added to the crude protein extract of camellia oleifera seed cake at a solid-liquid ratio of 1:10 g / mL, the mixture is stirred and eluted for 30 minutes, the precipitate is collected by centrifugation, 5 to 8 times the volume of water is added for redissolution, the pH value of the solution is adjusted, and the mixture is freeze-dried to obtain the camellia oleifera seed cake protein. Freeze-drying can remove residual organic solvents.

[0026] The present invention also provides tea oil cake protein prepared by the above extraction method. The tea oil cake protein provided by the present invention has high purity, relatively concentrated protein molecular weight distribution, contains 17 kinds of amino acids, including 7 essential amino acids, is a high-quality protein with high economic value. Through performance testing, the tea oil cake protein provided by the present invention has excellent gel properties, emulsification properties, foaming properties and antioxidant properties.

[0027] The present invention also provides the application of the camellia oleifera cake protein in food processing. Specifically, the camellia oleifera cake protein provided by the present invention has high nutritional value and can be used as a protein enhancer in food processing; the camellia oleifera cake protein provided by the present invention has excellent foaming property and foam stability, and is suitable for the production of cakes, ice cream, etc.; the camellia oleifera cake protein provided by the present invention has excellent emulsifying activity and emulsifying stability, and can be used as an emulsifier for water-in-oil emulsions; the camellia oleifera cake protein provided by the present invention has good heat-induced gel properties, water / oil holding properties, and antioxidant activity, and can be used as a raw material for plant protein meat, and has good thermal deformation stability and high thermal stability, consumes lower energy during plant protein meat processing, and has lower processing costs.

[0028] The present invention has the following beneficial effects:

[0029] (1) The extraction method provided by the present invention can efficiently extract protein from camellia oleifera cake. The method is simple and easy to operate, has no solvent residue, has high product purity, and has a relatively concentrated protein molecular weight distribution between 10 and 15 kDa.

[0030] (2) The oil-tea cake protein extracted by the present invention has a reasonable amino acid composition, is a high-quality protein, has a high economic value, and can be used as a protein raw material for producing protein beverages, instant foods, and baked foods. In addition, the oil-tea cake protein has excellent gel properties, emulsifying properties, foaming properties, and antioxidant properties, and can be used as a protein raw material for plant protein meat and applied in food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the extraction steps based on tea oil cake protein.

[0032] Figure 2 The effect of initial pH value on the extraction rate of camellia oleifera cake protein.

[0033] Figure 3 The effect of the solid-liquid ratio on the protein extraction rate of camellia oleifera cake.

[0034] Figure 4 The effect of extraction time on the extraction rate of camellia oleifera cake protein.

[0035] Figure 5 The effect of extraction temperature on the extraction rate of camellia oleifera cake protein.

[0036] Figure 6 This is a diagram of the experiment of eluting and purifying protein from tea cake.

[0037] Figure 7 To analyze the thermal properties of tea oil cake protein and other commercial proteins.

[0038] Figure 8This is the SDS-PAGE image of tea oil cake protein and other commercial proteins.

[0039] Fig. 9 It is the particle size and polydispersity index of camellia oil cake protein and other commercial proteins.

[0040] Fig.10 This is the isoelectric point diagram of tea oil cake protein.

[0041] Fig.11 It is the solubility of tea oil cake protein and other commercial proteins.

[0042] Fig.12 It is the water and oil holding capacity of tea oil cake protein and other commercial proteins.

[0043] Fig.13 It is used to improve the foaming property and foam stability of camellia oil cake protein and other commercial proteins.

[0044] Fig.14 It is the emulsifying activity and emulsifying stability of camellia oleifera cake protein and other commercial proteins.

[0045] Fig.15 The antioxidant capacity of tea oil cake protein.

[0046] Fig.16 To compare the antioxidant capacity of tea oil cake protein and other commercial proteins.

[0047] Fig.17 It is the in vitro digestibility of tea oil cake protein and other commercial proteins.

[0048] Fig.18 This is the temperature scanning heating process of the gel properties of tea oil cake protein and other commercial proteins.

[0049] Fig.19 It is the process of temperature scanning, heat preservation and cooling for the gel properties of camellia oleifera cake protein and other commercial proteins.

[0050] Fig. 20 Frequency scan of the gel properties of camellia oleifera cake protein and other commercial proteins. DETAILED DESCRIPTION

[0051] 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.

[0052] 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.

[0053] The camellia oleifera cake used in the following examples was purchased from Zhejiang Jiusheng Camellia Oil Technology Co., Ltd., which is the residue cake after the camellia oleifera seeds are squeezed for oil. Soybean protein powder (SPC) was purchased from Linyi Shansong Biological Products Co., Ltd., peanut protein powder (PP) was purchased from Anyang Tianxiang Food Technology Co., Ltd., pea protein powder (PPI) was purchased from Linyi Yuwang Plant Protein Co., Ltd., and wheat protein (WG) was purchased from Fengqiu County Huafeng Flour Co., Ltd.

[0054] The method used for performance testing in the following embodiments is as follows:

[0055] 1. Detection of amino acid composition

[0056] The amino acid analyzer was used for detection (ninhydrin post-column derivatization ion exchange chromatography), with specific reference to the national standard GB5009.124-2016.

[0057] 2. Thermal characteristics analysis

[0058] 5-10 mg of sample was placed in an aluminum box and placed in a differential scanning calorimeter (DSC). The sample was heated from 20°C to 200°C at a rate of 10°C / min at a flow rate of 50 mL / min in a high-purity N2 atmosphere. An empty pan was used as a reference during the measurement.

[0059] 3. SDS-PAGE

[0060] SDS-PAGE of protein isolates was performed to measure the molecular weight of protein fractions. Protein samples (5 mg / mL) were added to SDS-PAGE loading buffer, boiled for 10 min, and centrifuged (6000 rpm, 5 min). The loading amount was 15 μL (the loading amount of marker was 10 μL) in a precast polyacrylamide gel at a voltage of 80 V. After electrophoresis, the gel was stained with Coomassie Blue R-250 for 15 min and then decolorized overnight.

[0061] 4. Particle size distribution, polydispersity index (PDI)

[0062] Protein solutions (1 mL) of the same concentration were equilibrated at 25° C. for 2 min, and then the particle size and PDI were measured using a nanoparticle size analyzer at 25° C. The samples were diluted before analysis to avoid multiple scattering effects.

[0063] 5. Detection of isoelectric point

[0064] Prepare fresh protein suspension (2 mg / mL) in deionized water and adjust the pH of these samples to 2.0-7.0 with 0.1 M HCl or 0.1 M NaOH. Filter through a 0.45 mm membrane before analysis. Use a Malvern particle size analyzer to record Zeta potential data at different pH values. Plot a curve of Zeta potential versus pH, and the isoelectric point is usually the pH value where the curve crosses zero.

[0065] 6. Solubility detection

[0066] Dissolve 200 mg of different protein samples in 20 mL of deionized water and stir at room temperature for 30 min with a magnetic stirrer at 500 rpm. Then centrifuge at 8000 rpm (25°C) for 10 min. Determine the protein content in the supernatant using the BCA method with bovine serum albumin (BSA) as the standard. After centrifugation, take an appropriate amount of supernatant for dilution and calculate the protein content of the supernatant with bovine serum albumin (BSA) as the standard protein. Measure the absorbance of the sample at 562 nm with an enzyme marker, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0067] 7. Testing of water and oil holding capacity

[0068] Place 100 mg of accurately weighed sample (M0) in a dry centrifuge tube and weigh the sample and centrifuge tube (M1). Then add 2 mL of deionized water (or oil) to the centrifuge tube. Vortex the mixture for 1 min, let stand at room temperature for 30 min, and centrifuge at 8000 rpm for 10 min at 25°C. Carefully pour out the supernatant and weigh the tube with the precipitate again (M2). The WHC / OHC of the sample is expressed as the weight of water / oil absorbed per gram of the sample being tested. WHC or OHC (g / g) is calculated as follows:

[0069]

[0070] Among them, M1 is the weight of the protein and the centrifuge tube, M2 is the weight of the protein and the centrifuge tube after centrifugation, and M0 is the weight of the protein.

[0071] 8. Testing of foaming properties and foaming stability

[0072] Pour 20 mL of a 20 mg / mL protein solution into the measuring cylinder. Record the initial volume V0 and homogenize the solution in a homogenizer at 10,000 rpm for 2 min. Calculate the foam capacity and foam stability using the equation: Where V0 is the volume of the protein solution before homogenization, V1 is the volume of the protein solution after homogenization, and V t It is the foam volume of the protein solution after homogenization at room temperature for 30 minutes.

[0073] The foaming property is calculated as follows:

[0074]

[0075] The foaming stability is calculated as follows:

[0076]

[0077] 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.

[0078] 9. Testing of emulsification and emulsion stability

[0079] Disperse 15mL of a 1% (w / v) protein solution in 5mL of camellia oil. Homogenize at 10,000rpm for 2min, then immediately take 50μL of the emulsion from the bottom of the mixture and dissolve it in 5mL of 0.1% (w / v) SDS. The absorbance (A0) recorded immediately at 500nm is used as an estimate of the emulsification capacity. After 10min, take out a 50μL aliquot of the sample and dissolve it in 5mL of 0.1% (w / v) SDS. After mixing, record the absorbance A1 at 500nm. Emulsification and emulsion stability are calculated according to the following formula:

[0080]

[0081] 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).

[0082] 10. Detection of antioxidant capacity in vitro

[0083] DPPH free radical scavenging ability: Take 0.5mL sample solution and 4mL DPPH solution (0.1mmol / L, dissolved in methanol) and mix them evenly. The sample concentrations are set to 0.2, 0.4, 0.6, 0.8, and 1.0mg / mL. At room temperature and away from light for 30min, deionized water is used as a reference, and the absorbance at 517nm is measured as Ai; the reagent blank group is 0.5mL sample solution and 4mL methanol mixed to measure the absorbance as Aj; the sample blank tube is measured with 0.5mL deionized water and 4mL DPPH solution to measure the absorbance as A0, and each concentration is measured 3 times.

[0084]

[0085] ABTS free radical scavenging ability: Mix equal volumes of ABTS (7mM) and KS2O (2.45mM) solutions and place in the dark for 16 hours to generate ABTS stock solution. Add deionized water to dilute the solution to an absorbance of (0.70±0.02) at 734nm. Take 0.2mL of sample solution and add 4mL of ABTS solution, shake well, and react at room temperature for 6min. Distilled water is used as a reference. The absorbance value at 734nm is measured as Ai. Deionized water is used instead of polypeptide solution to measure A0, and deionized water is used instead of ABTS solution to measure Aj.

[0086]

[0087] 11. In vitro digestibility test

[0088] The protein sample was dissolved in distilled water (2% w / v) to prepare a 2% protein solution. The protein solution was kept in a 37°C water bath for 10 min, the pH was adjusted to 1.6 with 1 mol / L HCl, and then pepsin (4% w / w, based on protein) was added. Incubate at 37°C for 2 h using a benchtop constant temperature oscillator. Subsequently, 1 mol / L NaOH was added to adjust the solution pH to 7.5, and trypsin (4% w / w, based on protein) was added. The suspension was incubated continuously at 37°C for 2 h, and then incubated in a boiling water bath for 10 min to stop digestion. The gastrointestinal digestion product was obtained by centrifugation (11000 rpm, 15 min, 4°C), and the supernatant was collected. The protein content of each sample was determined by the Kjeldahl method, and the enzyme digestibility (ED) was calculated. The in vitro digestibility was expressed as the ratio of the protein content in the solution before and after digestion.

[0089] ED (%) = protein content in the supernatant after digestion / total protein content × 100.

[0090] 12. Detection of gel properties

[0091] About 1 mL of protein dispersion (15% w / v) was loaded onto the lower plate set at 25°C and the upper plate was lowered to contact the sample. The gap between the two plates was 1 mm. To prevent the sample from drying out during the experiment, light silicone oil was applied to the exposed part. The rheological properties were measured by the following tests.

[0092] Temperature scan: The temperature scan was heated from 20°C to 95°C at a heating rate of 2°C / min. The sample was kept at 95°C for 10 min and cooled from 95°C to 20°C at a cooling rate of 2°C / min. The storage modulus (G') and loss modulus (G") of the temperature scan were measured as a function of time at a constant frequency of 1 Hz and a shear strain of 1%.

[0093] Frequency sweep: The rheological behavior of the samples was studied by comparing the storage modulus (G') and loss modulus (G") as a function of frequency. The storage modulus and loss modulus were recorded as a function of dynamic frequency from 0.1 to 100 Hz at a shear strain of 1% (25 °C).

[0094] Example 1

[0095] This embodiment provides a method for extracting protein from camellia oleifera cake, which is as follows:

[0096] (1) Dry the camellia oleifera cake, grind it in a grinder at 30,000-35,000 r / min for 4-7 seconds, pass the ground material through an 80-100 mesh sieve, discard the material above the sieve, and use the material below the sieve as the raw material for preparing camellia oleifera cake protein.

[0097] (2) The camellia oleifera cake protein raw material obtained in step (1) is mixed with petroleum ether at a W / V ratio of 1:10 g / mL, and stirred continuously for 2 h. The mixture is allowed to stand until the upper organic solvent is clarified, and the organic solvent is recovered. The operation is repeated until the degreasing is complete (the petroleum ether is a very light yellow color), and the mixture is naturally air-dried for 12 h to obtain the defatted camellia oleifera cake.

[0098] (3) The defatted tea oil cake was crushed and passed through a 60-mesh sieve to obtain defatted tea oil cake powder. The defatted tea oil cake powder was mixed with water at a W / V ratio of 1:30 g / mL, and 1 mol / L NaOH was added to adjust the pH to 11.0. The temperature was 40°C, and the mixture was stirred for 60 min. The mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected.

[0099] (4) Acid precipitation: The pH of the supernatant was adjusted to 3 with 1 mol / L HCl, and the solution was allowed to stand at room temperature for 30 min. The solution was centrifuged at 5000 rpm for 10 min at 4°C, and the precipitate was collected to obtain a crude protein extract of camellia oleifera cake.

[0100] In this study, a single-factor experiment of alkaline extraction was conducted on the initial pH value, liquid-to-solid ratio, extraction temperature and extraction time of step (3). After the extraction, the mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected to obtain the relationship between each factor and the protein extraction rate.

[0101] 1. Effect of initial pH value on the extraction rate of tea cake protein

[0102] Under the conditions of solid-liquid ratio of 1:30 g / mL, temperature of 40°C and time of 60 min, the effects of different initial pH values ​​(7, 8, 9, 10, 11) on the protein extraction rate were investigated.

[0103] Effect of initial pH on protein extraction rate Figure 2 As shown, the results showed that when the initial pH value was 11, the protein extraction rate was the highest at 39.46%.

[0104] 2. Effect of liquid-to-solid ratio on protein extraction rate of camellia oleifera cake

[0105] Under the conditions of initial pH value of 11, temperature of 40℃ and time of 60min, the effects of different solid-liquid ratios (1:10, 1:20, 1:30, 1:40, 1:50 g / mL) on protein extraction rate were investigated.

[0106] Effect of liquid-to-solid ratio on protein extraction rate Figure 3 As shown, the results showed that when the liquid-to-solid ratio was 1:30 g / mL, the highest protein extraction rate was 37.70%.

[0107] 3. Effect of extraction time on the protein extraction rate of camellia oleifera cake

[0108] Under the conditions of solid-liquid ratio of 1:30 g / mL, temperature of 40°C and initial pH of 11, the effects of different times (30, 60, 90, 120, 150 min) on protein extraction rate were investigated.

[0109] Effect of extraction time on protein extraction rate Figure 4 As shown, the results showed that when the extraction time was 60 min, the protein extraction rate was the highest at 37.17%.

[0110] 4. Effect of extraction temperature on the extraction rate of camellia oil cake protein

[0111] Under the conditions of solid-liquid ratio of 1:30 g / mL, initial pH value of 11 and time of 60 min, the effects of different temperatures (30, 40, 50, 60, 70°C) on protein extraction rate were investigated.

[0112] Effect of extraction temperature on protein extraction rate Figure 5 As shown, the results showed that when the extraction temperature was 40°C, the protein extraction rate was the highest at 37.53%.

[0113] The optimal process was obtained from single factor experiments: initial pH value 11, solid-liquid ratio 1:30 g / mL, extraction temperature 40℃ and extraction time 60 min. The extraction rate of precipitated protein was 37.53%.

[0114] Example 2

[0115] This embodiment provides a method for purifying a crude protein extract of camellia oleifera cake, which is as follows:

[0116] Deionized water was added to the crude protein extract of camellia seed cake prepared in Example 1 at a solid-liquid ratio of 1 g:10 mL, and the mixture was stirred at 500 rpm for 30 min at room temperature. The mixture was centrifuged and the supernatant was discarded. The above operation was repeated to collect the precipitate, and the pH value of the solution was adjusted to 7.0±0.2 after re-dissolving with water, and freeze-dried to obtain camellia seed cake protein (SCP).

[0117] Example 3

[0118] This embodiment provides a method for purifying a crude protein extract of camellia oleifera cake, which is as follows:

[0119] Anhydrous ethanol was added to the crude protein extract of camellia seed cake prepared in Example 1 at a solid-liquid ratio of 1:10 g / mL, stirred at room temperature and 500 rpm for 30 min, centrifuged, and the supernatant was discarded. The above operation was repeated, and the precipitate was collected. After re-dissolving with water, the pH value of the solution was adjusted to 7.0±0.2, and freeze-dried to obtain camellia seed cake protein (SCP).

[0120] Example 4

[0121] This embodiment provides a method for purifying a crude protein extract of camellia oleifera cake, which is as follows:

[0122] The crude protein extract extracted from the camellia seed cake prepared in Example 1 was dialyzed using a dialysis bag with a specification of 14 kDa. The dialyzation was performed at 4°C for 3 days, with the water changed every 8 to 10 hours. The precipitate was collected, re-dissolved in water, the pH value of the solution was adjusted to 7.0±0.2, and freeze-dried to obtain camellia seed cake protein (SCP).

[0123] Example 5

[0124] This embodiment provides a method for purifying a crude protein extract of camellia oleifera cake, which is as follows:

[0125] Acetone was added to the crude protein extract of camellia seed cake prepared in Example 1 at a solid-liquid ratio of 1:10 g / mL, and the mixture was stirred at room temperature and 500 rpm for 30 min. The mixture was centrifuged and the supernatant was discarded. The above operation was repeated to collect the precipitate, and the pH value of the solution was adjusted to 7.0±0.2 after re-dissolving with water, and freeze-dried to obtain camellia seed cake protein (SCP).

[0126] Example 6

[0127] This embodiment provides a method for purifying a crude protein extract of camellia oleifera cake, which is as follows:

[0128] Anhydrous ethanol was added to the crude protein extract extracted from the camellia seed cake prepared in Example 1 at a solid-liquid ratio of 1:10 g / mL, stirred at room temperature at 500 rpm for 30 min, centrifuged, the supernatant was discarded, the above operation was repeated, and the precipitate was collected; then, at a solid-liquid ratio of 1:5 g / mL, the precipitate was eluted with acetone, stirred at room temperature at 500 rpm for 30 min, centrifuged, the supernatant was discarded, the above operation was repeated, the precipitate was collected, and the pH value of the solution was adjusted to 7.0±0.2 after redissolution with water, and freeze-dried to obtain camellia seed cake protein (SCP).

[0129] Example 7

[0130] This embodiment provides a method for purifying a crude protein extract of camellia oleifera cake, which is as follows:

[0131] Anhydrous ethanol was added to the crude protein extract of camellia seed cake prepared in Example 1 at a solid-liquid ratio of 1:10 g / mL, and the mixture was stirred at room temperature and 500 rpm for 30 min, centrifuged, and the supernatant was discarded. The above operation was repeated to collect the precipitate; the precipitate was dialyzed using a dialysis bag with a specification of 14 kDa at 4° C. for 3 days, with the water being changed every 8 to 10 hours. The precipitate was collected, and the pH value of the solution was adjusted to 7.0±0.2 after redissolution with water, and freeze-dried to obtain camellia seed cake protein (SCP).

[0132] Test Example 1:

[0133] 1. Purity detection

[0134] The protein purity of the tea oil cake prepared in Example 2-7 was tested by Kjeldahl nitrogen determination method. The results are as follows: Figure 6 As shown, the camellia oil cake protein was eluted by different methods. The results showed that the protein purity was the highest under ethanol elution, and the best elution and purification method was determined to be ethanol elution.

[0135] The following specifically conducts various performance tests on the camellia oil cake protein prepared in Example 3.

[0136] 2. Amino Acid Composition

[0137] The amino acid composition was tested according to the national standard GB5009.124-2016, and the results are shown in Table 1.

[0138] Table 1. Amino acid composition

[0139] Amino acid name Content (mg / g) Amino acid name Content (mg / g) Aspartic acid Asp 40.247 MethionineMet 10.59 Threonine 14.745 Isoleucine Ile 14.334 Ser 20.041 Leucine 33.341 Glu 132.614 Tyrosine 14.964 Glycine 19.054 Phenylalanine 18.39 Alanine Ala 23.244 His 7.179 Cystine 9.486 Lysine 14.755 Valine 15.13 Arginine 46.595 Proline Pro 14.399

[0140] Table 1 shows that 17 kinds of amino acids can be detected by the oil-tea cake protein extracted by the present invention, of which 7 kinds are essential amino acids (EAA) for human body. Glutamic acid, arginine, aspartic acid and leucine are 4 kinds of amino acids with higher content. The content of basic amino acids is lower than that of acidic amino acids, indicating that oil-tea cake protein is acidic under natural conditions, which is consistent with the experimental result that its isoelectric point is about 3.0. In terms of content and demand, essential amino acids account for 24.51% of the total amino acids, among which leucine has the highest content, accounting for 30.28% of the total essential amino acids. Among non-essential amino acids, glutamic acid has the highest content, accounting for 29.47% of the total amino acids. Glutamic acid is an amino acid that plays a role in maintaining function in brain tissue and occupies an important position in the process of sugar metabolism and protein metabolism. The essential amino acid content of oil-tea cake protein meets the WHO adult standard except histidine and lysine, indicating that oil-tea cake protein can be used as a good amino acid dietary resource.

[0141] In summary, the amino acid composition of camellia oleifera cake protein is reasonable. It is a high-quality protein with high economic value and can be used as a protein raw material for producing protein beverages, instant foods, and baked foods.

[0142] 3. Thermal characteristics analysis

[0143] DSC was used to determine the T d and DH, the thermal stability of proteins is expressed as “T d ” stands for temperature, i.e., the temperature at which denaturation occurs, and DH represents the amount of heat required to induce denaturation as well as the degree of ordered structure. Structurally ordered proteins generally have good thermal stability because more energy is required to destroy their denaturation. Proteins with different Td values ​​can meet the needs of different food industries when used as ingredients for cooking, gelling, and pasteurization processes. Therefore, it is feasible to achieve a variety of protein applications by controlling the temperature as needed, however, from a technical point of view, materials with lower denaturation temperatures tend to consume lower energy to change the protein conformation and therefore have lower processing costs.

[0144] like Figure 7 As shown in Table 2, a single endothermic peak was observed in the thermal scanning process of 20-200°C except for wheat protein. d ) was 125.28±4.7℃ and the denaturation enthalpy (DH) was 216.91±15.36J / g. Compared with other proteins, it has a lower T d , and DH is higher, indicating that tea cake protein has good thermal deformation stability and high thermal stability. When processing plant protein meat, it may consume less energy and have lower processing costs.

[0145] Table 2

[0146]

[0147] 4. Determination of protein relative molecular weight by SDS-PAGE

[0148] like Figure 8 As shown, the molecular weight distribution of the camellia oleifera cake protein extracted by the present invention is relatively concentrated, roughly distributed between 10-15 kDa, and the molecular weight distribution of the remaining proteins is relatively wide.

[0149] 5. Particle size distribution, polydispersity index (PDI)

[0150] like Fig. 9 As shown, the particle size of camellia oleifera cake protein is 345.16±24.79nm, and the polydispersity index is 0.38±0.06. Compared with other proteins, camellia oleifera cake protein has a smaller particle size and polydispersity index, indicating that camellia oleifera cake protein has better extrudate processing performance.

[0151] 6. Determination of isoelectric point

[0152] Zeta potential changes of tea oil cake protein under different pH conditions Fig.10 As shown. The maximum number of negative charges was observed at pH 5.98, at which time more anionic groups were exposed on the protein surface. When the pH dropped from 5.98 to 2.98, the absolute value of its surface charge also decreased. At pH 2.98, the charge dropped to 0, and this pH was considered to be the isoelectric point (IEP) of tea oil cake protein.

[0153] 7. Solubility determination

[0154] Solubility (S) is one of the key properties of protein, which is closely related to the foaming, emulsifying and gelling properties of protein.

[0155] like Fig.11 As shown, camellia oleifera cake protein has good solubility, with a solubility of 30.82±1.3% and an effective solubility of 51.31±2.3%. Compared with other proteins, camellia oleifera cake protein has a lower solubility, which is equivalent to soybean and peanut proteins, while its effective solubility is higher, which is equivalent to soybean protein.

[0156] 8. Analysis of water holding capacity and oil holding capacity

[0157] Water / oil holding capacity is one of the indicators used to measure the ability of protein products to absorb or absorb water / oil, which is an important functional property that affects the taste and flavor retention of protein isolates. In addition, it has an important impact on the taste and texture of meat, shelf life extension, and processing characteristics. Good water and oil holding properties play a role in increasing product yield and improving product quality. High water holding capacity may be related to the stronger swelling, dissociation and unfolding ability of protein concentrates, thereby exposing additional binding sites during extrusion.

[0158] like Fig.12 As shown in the figure, the water holding capacity of camellia oleifera cake protein is 4.53±0.21g / g, and the oil holding capacity is 4.32±0.25g / g. Compared with other proteins, the water holding capacity of camellia oleifera cake protein is comparable to that of wheat and pea proteins; while the oil holding capacity is similar to that of wheat protein and higher than that of other proteins, indicating that it has excellent water / oil holding capacity and can be used as a good raw material for extrusion of plant protein meat.

[0159] 9. Foamability and foam stability

[0160] like Fig.13 As shown in the figure, the foaming property of camellia oleifera cake protein is 82.02±3.58%, and the foam stability of protein is 80.21±1.28%. Compared with other proteins, its foaming property is much higher than other proteins, while its foam stability is comparable to that of soybean protein and peanut protein, indicating that camellia oleifera cake protein has excellent foaming property and foam stability, and is suitable for the production of cakes, ice cream, etc.

[0161] 10. Emulsification activity and emulsification stability

[0162] Emulsifying activity (EAI) indicates the ability of protein to be adsorbed at the oil-water interface, and emulsifying stability (ESI) indicates the stability of the formed emulsion adsorption layer.

[0163] like Fig.14 As shown in Figure 2, the emulsifying activity of tea cake protein was 7.17±0.24 m 2 / g, and the protein emulsification stability was 88.97±0.99%. Compared with other proteins, the emulsification activity of camellia oleifera cake protein was much higher than that of other proteins, while the emulsification stability was comparable to that of soybean protein, indicating that it had excellent emulsification activity and emulsification stability and could be used as an emulsifier for oil-in-water emulsions.

[0164] 11. In vitro antioxidant capacity

[0165] like Fig.15 and Fig.16As shown in the results, when the protein concentration was 1 mg / mL, the antioxidant activity of camellia oleifera cake protein was significantly higher than that of other proteins, with a DPPH free radical scavenging rate of 72.79±1.66% and an ABTS free radical scavenging rate of 92.32±1.15%. As the concentration of camellia oleifera cake protein increased, the in vitro antioxidant activity showed a good linear relationship with the concentration. The results showed that camellia oleifera cake protein can be added as a good antioxidant in the preparation of plant protein meat.

[0166] 12. In vitro digestibility

[0167] The different in vitro digestibility of different proteins may be due to the fact that protein composition, enzyme type and impurities affect IVD. Some high molecular weight peptides present in the sample may increase the difficulty of digestion. The digestion mode, digestion time and enzyme selection will also have a significant impact on IVD. The extraction method will also have a significant impact.

[0168] like Fig.17 As shown, the in vitro digestibility of camellia oleifera cake protein was significantly higher than that of other proteins, at 73.67±5.52%, indicating that it has good in vitro digestibility and can provide previous reference conditions for the digestibility research of finished plant protein meat.

[0169] 13. Detection of gel properties

[0170] Thermally induced gelation and changes in rheological behavior of proteins during cooking are important criteria for food applications, e.g. Figure 18-Figure 20 The G' and G" of camellia oleifera cake protein are 2110.3Pa and 625.08Pa respectively. Compared with other proteins, the G' and G" of camellia oleifera cake protein are much smaller than wheat protein, comparable to pea protein, and higher than soybean and peanut protein.

[0171] Temperature sweeps elucidate the viscoelastic behavior of samples by strain and frequency. During heating of the protein suspension, the protein gel network continues to develop through progressive denaturation. Fig.18 It can be observed that in the initial stage of heating, after the temperature rises for a period of time, G” increases sharply and exceeds G”. When G’>G”, it indicates gel formation. Fig.19It can be observed that the modulus continues to increase when the API is kept at 95 °C. During cooling, G' shows a new increase, indicating that the protein molecules aggregate to a greater extent during cooling and a gel network structure with a certain rigidity is formed. This increase is called gel enhancement. During this gelation process, the proteins are melted and denatured, resulting in differences in irreversible interactions (hydrophobic interactions and disulfide bonds) between proteins due to exposure to internal hydrophobic regions and sulfhydryl groups. By comparing the final viscoelasticity of the mixture with the set temperature profile, it is possible to gain insight into the final product properties after heating and cooling during the extrusion process. In addition, it can be used to predict the structural strength of the extrudate, alleviating the limitations of empirical-based techniques. The results show that Camellia oleifera protein has good heat-induced gel properties, which is essential for simulating typical meat texture and juiciness. Strong gels can retain a large amount of water and maintain water absorption rate. Therefore, Camellia oleifera protein is an ideal vegetarian meat extender for quality and product performance.

[0172] Frequency sweep reflects the relationship between the viscoelasticity of the sample and the applied stress change. It can reflect the effect of different processing conditions or methods on the viscoelasticity of the product. It is the most commonly used method in oscillation testing. By plotting the relationship between the logarithm of the storage modulus (G′) and the logarithm of the loss modulus (G″) and the frequency, Fig. 20 It can be seen that the G' and G" of protein samples except peanut protein increase with the increase of frequency. When the frequency is low, G' and G" show an intersecting trend. At this time, the ordered protein chains begin to entangle with each other. Then, as the frequency continues to increase, G' is significantly greater than G", and the sample exists in a stable gel state. Finally, the G' and G" of camellia oleifera cake protein are 2353.5Pa and 1362.7Pa. Compared with other proteins, the G' and G" of camellia oleifera cake protein are smaller than wheat protein, equivalent to pea protein, and higher than soybean and peanut protein. The results show that the camellia oleifera cake protein gel has higher strength in the frequency scanning experiment. Strong gels can withstand mechanical stress and heating better than weak gels, and the gel network structure is more compact and delicate. The gelling properties are closely related to the juiciness of meat. This property is considered to be the key to the taste of meat-enhancing products imitating real meat. Juiciness is beneficial because it improves the overall palatability and provides a pleasant eating experience.

[0173] 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 tea oil cake protein, characterized in that: The following steps are involved: (1) crushing tea oil cake and mixing it with petroleum ether to perform degreasing treatment, and removing the organic solvent after degreasing to obtain defatted tea oil cake; (2) mixing defatted tea oil cake with water at a solid-liquid ratio of 1:25-35 g / mL, adjusting the pH value to 10-11, extracting at a temperature of 35-45° C. for 50-70 min, and then centrifuging to collect the supernatant; (3) adjusting the pH value of the supernatant to 3.0±0.5 for acid precipitation, and collecting the precipitate by centrifugation to obtain a crude protein extract extracted from camellia seed cake; (4) adding anhydrous ethanol to the crude protein extract of camellia oleifera seed cake at a solid-liquid ratio of 1:10-20 g / mL, stirring and eluting for 20-40 min, collecting the precipitate by centrifugation, re-dissolving with water, adjusting the pH value of the solution to 7.0±0.2, and freeze-drying to obtain the camellia oleifera cake protein.

2. The method for extracting tea oil cake protein according to claim 1, characterized in that: In step (1), the camellia oleifera cake is crushed to 80-100 meshes and then defatted.

3. The method for extracting tea cake protein as claimed in claim 1 or 2, characterized in that: In step (1), the camellia oleifera cake raw material and petroleum ether are mixed at a ratio of 1:10-20 g / mL, stirred continuously for 1-2 h, allowed to stand until the upper organic solvent is clarified, the organic solvent is recovered, and the operation is repeated until degreasing is complete.

4. The method for extracting tea oil cake protein as claimed in claim 1, characterized in that: In step (2), the defatted tea oil cake is crushed into 60 meshes and then extracted.

5. The method for extracting tea cake protein according to claim 1 or 4, characterized in that: In step (2), defatted camellia oleifera cake and water are mixed at a solid-liquid ratio of 1:30 g / mL, the pH value is adjusted to 11, and extraction is performed at 40° C. for 60 min; the centrifugation condition is 5000 rpm for 10 min.

6. The method for extracting tea oil cake protein according to claim 1, characterized in that: In step (2), the pH value is adjusted using a 1 mol / L NaOH solution; in step (3), the pH value is adjusted using a 1 mol / L HCl solution.

7. The method for extracting tea oil cake protein according to claim 1 or 6, characterized in that: In step (3), the acid precipitation conditions are: adjusting the pH value of the supernatant to 3.0, and allowing the supernatant to stand for precipitation for 30 minutes at room temperature; and the centrifugation conditions are centrifugation at 5000 rpm for 10 minutes at 4°C.

8. The method for extracting tea oil cake protein according to claim 1, characterized in that: In step (4), anhydrous ethanol is added to the crude protein extract of camellia oleifera seed cake at a solid-liquid ratio of 1:10 g / mL, the mixture is stirred and eluted for 30 minutes, the precipitate is collected by centrifugation, 5 to 8 times the volume of water is added to re-dissolve, the pH value of the solution is adjusted, and the mixture is freeze-dried to obtain the camellia oleifera cake protein.

9. Camellia oleifera cake protein prepared by the extraction method according to any one of claims 1 to 8.

10. Use of the camellia oil cake protein as claimed in claim 9 in food processing.

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