Method for preparing high-quality camellia oleosa seed oil through whole enzymatic method based on enzymatic extraction and enzymatic demulsification as well as product and application of high-quality camellia oleosa seed oil

Abstract

The invention discloses a method for preparing high-quality camellia oleosa seed oil by a whole enzymatic method based on enzymatic extraction and enzymatic demulsification as well as a product and application of the high-quality camellia oleosa seed oil. The method comprises the following steps: firstly, carrying out enzymolysis on camellia seed powder slurry by adopting single acid protease, then centrifuging to obtain clear oil I, and collecting an emulsion layer; then diluting an emulsion layer with distilled water, adjusting the pH value, sequentially adding medium-temperature alpha-amylase and acid protease for enzymolysis demulsification, and centrifuging to obtain demulsified clear oil II; the clear oil I and the clear oil II are combined, high-quality camellia oleosa seed oil can be obtained without any refining, and the quality index of the camellia oleosa seed oil reaches the standard of first-grade oil specified by GB / T11765-2018 camellia oleosa seed oil. The whole enzyme method has the advantages of mild reaction conditions, no use of any organic solvent, simple operation, easy amplification and industrialization, greenness, environmental protection, high efficiency, low cost and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of deep processing of agricultural and forestry products, and particularly relates to a method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification, as well as products and applications thereof. Background Art

[0002] Camellia oleifera Abel, a species of genus Camellia in the Theaceae family, is a unique edible woody oil tree native to southern China. Along with coconut, olive, and oil palm, it is considered one of the world's four major woody oil plants. Camellia oleifera seed oil is a high-quality edible oil, free of erucic acid and cholesterol. It contains over 90% unsaturated fatty acids, 80%-83% oleic acid, and 7%-13% linoleic acid. Camellia oleifera oil also contains squalene, antioxidants, and essential trace elements such as vitamins A, B, D, and E, providing valuable health benefits. Linolenic acid, in particular, is essential for the human body but cannot be synthesized. Furthermore, camellia oil has a low freezing point and high smoke point, making it easily digestible and highly absorbable. It also produces few harmful substances during cooking and has a rich, fragrant and palatable flavor, making it a culinary delicacy with a long history of consumption in China. Data indicates that 90% of the world's camellia trees grow in the hilly regions of southern China. In terms of production volume, over 90% of global camellia oil production is concentrated in China. Because its chemical, physical properties and nutritional components are very similar to those of olive oil, camellia seed oil is known as "Oriental Olive Oil".

[0003] Currently, the main processes for preparing camellia oil include pressing, organic solvent extraction, and supercritical CO₂ extraction. Pressing methods are divided into hot pressing and cold pressing. Hot pressing offers a high oil extraction rate, but the high temperatures during pretreatment significantly destroy the active substances in the oil, resulting in low-quality, dark-colored oil that requires further refining. Cold pressing, while eliminating the need for high-temperature treatment, preserves most of the oil's natural properties. However, it suffers from a low oil yield, high energy consumption, and difficulty in achieving continuous mass production. While organic solvent extraction offers a high oil yield, it also carries the problem of residual organic solvents, which not only reduces food safety but also poses a certain environmental risk. Supercritical CO₂ extraction is a novel extraction and separation technology that offers a high oil extraction rate, eliminates residual solvents, and effectively preserves the active substances in the oil. However, this method requires expensive equipment and is currently still in the experimental and pilot stages, not yet reaching industrial production capacity. Therefore, there is a pressing need for a green, environmentally friendly, and efficient method for preparing high-quality camellia oil.

[0004] The aqueous enzymatic method is a high-quality camellia oil extraction method that has been widely studied in recent years. However, camellia seeds contain 7.28-16.24% tea saponins, and the enzymatic hydrolysis process easily produces a large amount of emulsion, which seriously reduces the yield and quality of the clear oil. Therefore, the emulsion must be effectively demulsified. Currently, in the research on demulsification methods for camellia oil extraction using the aqueous enzymatic method, the methods that have been proven to be effective mainly include freeze-thaw demulsification, organic solvent demulsification, and centrifugal demulsification. However, the organic solvent demulsification method has the problem of organic solvent residue, and the centrifugal demulsification and freeze-thaw demulsification methods are energy-intensive and costly. Therefore, there is a need to further develop greener and more efficient demulsification methods. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a method for preparing high-quality camellia oil by a fully enzymatic method based on enzymatic extraction and enzymatic demulsification. This method uses only a single acidic protease in the enzymatic extraction to achieve efficient extraction, avoiding the need to use a mixture of multiple enzymes for extraction in traditional aqueous enzymatic extraction. At the same time, in the demulsification step, medium-temperature α-amylase and acidic protease are used sequentially to achieve efficient enzymatic demulsification. The entire preparation process is simple, does not require high temperature, and does not use any organic solvents. It has the advantages of being green, environmentally friendly, efficient, and low-cost. The prepared camellia oil is of good quality and can meet the standards of first-grade oil specified in GB / T 11765-2018 without refining.

[0006] The invention also provides prepared high-quality camellia seed oil and application thereof.

[0007] Technical solution: To achieve the above-mentioned purpose, the present invention provides a method for preparing high-quality camellia oil by a fully enzymatic method based on enzymatic extraction and enzymatic demulsification, comprising the following steps:

[0008] (1) shelling and crushing camellia seeds to obtain camellia seed powder;

[0009] (2) mixing camellia seed powder and buffer solution to form a slurry;

[0010] (3) adding an enzyme preparation to the slurry for stirring and enzymatic hydrolysis and extraction, heating to inactivate the enzyme after the enzymatic hydrolysis and extraction reaction is completed, and centrifuging the enzymatic hydrolysis and extraction reaction liquid at high speed after cooling to obtain a first layer of extracted clear oil, a second layer of emulsion layer, a third layer of clear liquid, and a fourth layer of precipitated residue in sequence, absorbing the first layer of extracted clear oil to obtain clear oil I, and recovering the second layer of emulsion layer;

[0011] (4) distilling the recovered emulsion layer with distilled water, adjusting the pH, adding an enzyme preparation, stirring, and performing enzymatic demulsification. After the enzymatic demulsification reaction is completed, heating is performed to inactivate the enzyme. After cooling, the enzymatic demulsification reaction liquid is centrifuged at high speed to obtain a first layer of demulsified clear oil, a second layer of emulsion, a third layer of clear liquid, and a fourth layer of precipitated residue. The first layer of demulsified clear oil is absorbed to obtain clear oil II.

[0012] (5) The clear oil I and the clear oil II are combined and dried to obtain high-quality camellia oil; the clear liquid and the emulsion in steps (3) and (4) are combined, and the precipitated residues in steps (3) and (4) are combined for recovery of other useful components.

[0013] The oil-tea camellia seeds in step (1) include any one or more of oil-tea camellia seeds XL-210, GY-12, and L-34-22-1. The present invention is not limited to the oil-tea camellia seed variety and can be applied to any variety of oil-tea camellia seeds and can also be applied to any variety of oil-tea camellia seeds mixed in any proportion.

[0014] The slurry in step (2) is prepared by uniformly mixing camellia seed powder and buffer solution at a material-liquid ratio of 1:3 to 1:7 (g / mL).

[0015] Preferably, the most preferred solid-liquid ratio is 1:4.68 (g / mL).

[0016] Wherein, the buffer solution in step (2) is 0.1 M citric acid-sodium citrate buffer solution with a pH of 2.8 to 4.4; the most preferred pH is 3.2.

[0017] Wherein, the enzyme preparation described in step (3) is acidic protease, and the addition amount is 100-500 U / g of camellia oleifera seed powder; the most preferred enzyme addition amount is 387.47 U / g (based on the mass of camellia oleifera seed powder).

[0018] Wherein, the enzymatic hydrolysis and extraction reaction temperature in step (3) is 40-60°C, the stirring speed is 100-200 rpm, and the time is 2-6 hours.

[0019] The most preferred enzymatic extraction temperature is 55°C, the stirring speed is 150 rpm, and the time is 5 hours.

[0020] The ratio of the amount of distilled water added in step (4) to the emulsion layer is 0.7:1 to 1.1:1 (mL / g). The most preferred ratio of distilled water to the emulsion layer is 0.9:1 (mL / g).

[0021] The procedure and conditions for adding the enzyme preparation for enzymatic demulsification after adjusting the pH in step (4) are as follows: first, adjusting the pH of the system to 4.8-6.4 with NaOH, then adding medium-temperature α-amylase, stirring at 80-400 U / g of the emulsified layer at 60-80°C and 150 rpm for 10-50 min; then adjusting the pH of the system to 2.4-4.0 with HCl, adding acidic protease, stirring at 80-400 U / g (based on the mass of the emulsified layer) at 45-65°C and 150 rpm for 10-50 min.

[0022] Most preferably, the pH of the system is first adjusted to 5.6 with NaOH, the amount of medium-temperature α-amylase added is 340 U / g, and the enzymatic hydrolysis is carried out at 70°C for 20 minutes; the pH of the system is adjusted to 3.2 with HCl, the amount of acidic protease added is 240 U / g, and the enzymatic hydrolysis is carried out at 55°C for 20 minutes.

[0023] Wherein, the drying in step (5) is carried out at 50-60° C. for 10-12 hours.

[0024] Preferably, the method comprises the following steps:

[0025] (1) shelling camellia seeds and crushing them into 40 mesh to obtain camellia seed powder;

[0026] (2) mixing camellia seed powder and buffer solution to form a slurry;

[0027] (3) adding an enzyme preparation to the slurry and stirring at 150 rpm for enzymatic hydrolysis and extraction; after the enzymatic hydrolysis and extraction reaction is completed, the enzyme activity is inactivated by boiling water bath for 10 minutes; after cooling, the enzymatic hydrolysis and extraction reaction liquid is centrifuged at 10°C and 10,000 rpm for 15 minutes to obtain a first layer of extracted clear oil, a second layer of emulsified layer, a third layer of clear liquid, and a fourth layer of precipitated residue; the first layer of extracted clear oil is absorbed to obtain clear oil I, and the second layer of emulsified layer is recovered.

[0028] (4) distilled water was added to the recovered emulsion layer for dilution, and an enzyme preparation was added after adjusting the pH to perform enzymatic demulsification. After the enzymatic demulsification reaction was completed, the enzyme activity was inactivated by boiling water bath for 10 minutes. After cooling, the enzymatic demulsification reaction liquid was centrifuged at 10°C and 10,000 rpm for 5 minutes to obtain the first layer of demulsified clear oil, the second layer of emulsion, the third layer of clear liquid, and the fourth layer of precipitated residue. The first layer of demulsified clear oil was absorbed to obtain clear oil II.

[0029] More preferably, the method comprises the steps of:

[0030] (1) shelling camellia seeds and crushing them into 40 mesh to obtain camellia seed powder;

[0031] (2) Mixing camellia seed powder and buffer uniformly to prepare a slurry; wherein, adding citric acid-sodium citrate buffer (0.1 M, pH 3.2) with a solid-liquid ratio of 1:4.68 g / mL;

[0032] (3) adding an enzyme preparation to the slurry and stirring at 150 rpm for enzymatic hydrolysis and extraction; after the enzymatic hydrolysis and extraction reaction is completed, the enzyme activity is inactivated by boiling water bath for 10 minutes; after cooling, the enzymatic hydrolysis and extraction reaction liquid is centrifuged at 10° C. and 10,000 rpm for 15 minutes to obtain a first layer of extracted clear oil, a second layer of emulsified layer, a third layer of clear liquid, and a fourth layer of precipitated residue in sequence; the first layer of extracted clear oil is absorbed to obtain clear oil I, and the second layer of emulsified layer is recovered; wherein acidic protease is used, the reaction temperature is 55° C., the enzyme addition amount is 387.47 U / g, and the stirring reaction time is 5 hours at 150 rpm;

[0033] (4) Distilled water was added to the recovered emulsion layer for dilution, and an enzyme preparation was added after adjusting the pH to perform enzymatic demulsification. After the enzymatic demulsification reaction was completed, the enzyme activity was inactivated by boiling water bath for 10 minutes. After cooling, the enzymatic demulsification reaction liquid was centrifuged at 10°C and 10,000 rpm for 5 minutes to obtain the first layer of demulsified oil, the second layer of emulsion, the third layer of clear liquid, and the fourth layer of precipitated residue in sequence. The first layer of demulsified oil was absorbed to obtain clear oil II; wherein, the ratio of distilled water to the emulsion layer was 0.9:1 mL / g, and medium-temperature α-amylase was added at an enzyme amount of 340 U / g. The mixture was stirred at 150 rpm for 20 minutes at 70°C and pH 5.6 (using NaOH to adjust the system), and then acid protease was added at an enzyme amount of 240 U / g. The mixture was stirred at 150 rpm for 20 minutes at 55°C and pH 3.2 (using HCl to adjust the system).

[0034] The camellia oil prepared by the method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification described in the present invention is high-quality camellia oil that meets the GB / T 11765-2018 first-grade oil standard.

[0035] The high-quality camellia seed oil of the present invention is used as a raw material or ingredient for common edible oil, health food, pharmaceutical products and cosmetics.

[0036] The camellia oil prepared by the present invention has high quality without requiring refining. Compared with the extraction method, it avoids organic solvent residues; compared with the hot pressing method, it avoids the loss of active substances caused by high temperatures. The present invention utilizes enzymatic demulsification to prepare the camellia oil. Currently, there are no effective enzymatic demulsification methods domestically or internationally. This method avoids the organic solvent residues associated with commonly used organic solvent demulsification methods, as well as the high energy consumption and high costs of freeze-thaw and centrifugal demulsification methods.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0038] (1) The present invention uses a single acidic protease to achieve efficient extraction of camellia oil, avoiding the need to use a mixture of multiple enzymes in traditional aqueous enzymatic extraction. When multiple enzymes are mixed, the control of enzymatic extraction conditions is difficult and complicated due to their different optimal temperatures and optimal pH values. In addition, the single enzymatic extraction method of the present invention avoids the destruction of active substances in camellia oil during hot pressing and avoids the residual organic solvent during organic solvent leaching.

[0039] (2) The present invention uses a moderate temperature α-amylase and an acid protease for sequential enzymatic hydrolysis to achieve efficient enzymatic demulsification of the emulsion layer. This enzymatic demulsification method is a novel invention of the present invention. It avoids the organic solvent residues caused by the commonly used organic solvent demulsification method and also avoids the high energy consumption and high cost of freeze-thaw demulsification and centrifugal demulsification methods.

[0040] (3) The present invention not only ensures a high yield of camellia oil, but also achieves high quality of camellia oil. By adopting the new "whole enzyme method" process of the present invention, the prepared camellia oil can meet the quality standard of first-grade oil specified in GB / T11765-2018 without refining.

[0041] (4) The medium-temperature α-amylase and acid protease used in the present invention can be purchased in large quantities and at low cost. The whole enzyme method has mild reaction conditions, simple operation, and is easy to scale up and industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a relationship diagram showing the effects of nine enzymes and their complex enzymes on the clear oil yield during the initial screening;

[0043] Figure 2 The temperature stability and pH stability of acidic protease;

[0044] Figure 3 This is the relationship diagram of the influence of six enzymes and their composite enzymes on the demulsification rate during the initial screening; Note: *Mta+Acp, first add medium-temperature α-amylase, react at 70℃, pH 5.6 for 30 minutes, then add acid protease, and react at 55℃, pH 3.2 for 35 minutes; *Acp+Mta, first add acid protease, react at 55℃, pH 3.2 for 35 minutes, then add medium-temperature α-amylase, and react at 70℃, pH 5.6 for 30 minutes; Mta+Acp, add medium-temperature α-amylase and acid protease at the same time, and react at 70℃, pH 5.6 for 65 minutes; Acp+Mta, add medium-temperature α-amylase and acid protease at the same time, and react at 55℃, pH 3.2 for 65 minutes;

[0045] Figure 4 This is the relationship diagram of the effect of different ratios of distilled water to emulsion layer on demulsification rate;

[0046] Figure 5 This is the relationship diagram of the effect of reaction temperature of different medium-temperature α-amylases on demulsification rate;

[0047] Figure 6 This is the relationship diagram of the effect of reaction pH on demulsification rate of different medium-temperature α-amylases;

[0048] Figure 7 This is the relationship diagram of the effect of reaction time of different medium-temperature α-amylase on demulsification rate;

[0049] Figure 8 This is the relationship diagram of the effect of different medium-temperature α-amylase addition amounts on the demulsification rate;

[0050] Figure 9 This is the relationship diagram of the effect of reaction temperature of different acidic proteases on demulsification rate;

[0051] Figure 10 This is the relationship diagram of the effect of reaction pH on demulsification rate of different acidic proteases;

[0052] Figure 11 This is a graph showing the effect of reaction time of different acidic proteases on demulsification rate;

[0053] Figure 12 This is a relationship diagram showing the effect of different acid protease addition amounts on demulsification rates;

[0054] Figure 13 The following is a graph showing the effect of different demulsification processes on demulsification efficiency. Note: Acp+Mta: simultaneous addition of moderate-temperature α-amylase (340 U / g) and acid protease (240 U / g), reaction at 55°C, pH 3.2 for 40 minutes, with a distilled water to emulsion ratio of 0.9:1. Mta+Acp: simultaneous addition of moderate-temperature α-amylase (340 U / g) and acid protease (240 U / g), reaction at 70°C, pH 5.6 for 40 minutes, with a distilled water to emulsion ratio of 0.9:1. #Mta+Acp: moderate-temperature α-amylase (340 U / g) was added first, reaction at 70°C, pH 5.6 for 20 minutes, then the pH of the system was adjusted to 3.2, the temperature was maintained constant, and acid protease (240 U / g) was added, and the reaction continued for 20 minutes. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to the examples.

[0056] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0057] Experimental materials:

[0058] The experimental raw materials used in the examples were three tea seeds: XL-210, GY-12, and L-34-22-1, provided by the Experimental Forestry Farm of the Hunan National Tea Engineering Technology Research Center. After sun-drying and shelling, the three seeds were mixed in a mass ratio of 4:3:3 and ground to produce 40-mesh tea seed powder. The present invention can utilize any single tea seed variety or a mixture of multiple varieties in any proportion, with substantially consistent properties. Acidic protease (FDG-2237, 500,000 U / g), neutral protease (FDG-2209, 50,000 U / g), papain (FDG-2203, 100,000 U / g), mesophilic α-amylase (FDG-2231, 10,000 U / g), fungal α-amylase (FDG-0011, 8600 U / g), cellulase (FDG-2225, 11,000 U / g), hemicellulase (FDG-2255, 50,000 U / g), and pectinase (FDG-2259, 300,000 U / g) were purchased from Xiasheng Biotechnology Development Co., Ltd. Alcalase 2.4L (2.4 Au / g, approximately 240,000 U / g) was purchased from Tianjin Yinuo Biotechnology Co., Ltd. All enzymes were food grade, all reagents used were analytically pure, and all other raw materials were commercially available. The NaOH and HCl used to adjust the pH were both food grade.

[0059] The experimental instruments are shown in Table 1.

[0060] Table 1 List of experimental instruments

[0061] name model factory electronic balance AL-204210G Mettler-Toledo Instruments (Shanghai) Co., Ltd. Constant temperature stirring oil bath JY-2 Jintan Tianjing Experimental Instrument Factory Desktop high-speed centrifuge TGL-16M Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd. Pure water system ELIX 10 Nanjing Hanlong Experimental Equipment Co., Ltd.

[0062] Example 1

[0063] (1) Accurately weigh 20 g of camellia seed powder into a 250 mL conical flask, add 80 mL of citric acid-sodium citrate buffer (0.1 M, pH = 3.0), and mix well to obtain a slurry.

[0064] (2) Acidic protease is added to the slurry obtained in step (1) at an enzyme dosage of 200 U / g (based on the mass of camellia oleifera seed powder). The slurry is placed in a constant temperature stirring oil bath pot and stirred and extracted at 50°C and 150 rpm for 3 hours. After the reaction is completed, the enzyme is inactivated in a boiling water bath for 10 minutes. After cooling, the slurry is centrifuged at 10°C and 10,000 rpm for 15 minutes to obtain a first layer of extracted clear oil, a second layer of emulsion, a third layer of clear liquid, and a fourth layer of precipitated residue. The first layer of extracted clear oil is absorbed to obtain clear oil I, and the second layer of emulsion is recovered.

[0065] Example 2

[0066] (1) Accurately weigh 20 g of camellia seed powder into a 250 mL conical flask, add 100 mL of citric acid-sodium citrate buffer (0.1 M, pH = 3.2), and mix well to obtain a slurry.

[0067] (2) Acidic protease is added to the slurry obtained in step (1) at an enzyme dosage of 400 U / g (based on the mass of camellia oleifera seed powder). The slurry is placed in a constant temperature stirring oil bath and stirred and extracted at 55°C and 150 rpm for 4 hours. After the reaction is completed, the enzyme is inactivated in a boiling water bath for 10 minutes. After cooling, the slurry is centrifuged at 10°C and 10,000 rpm for 15 minutes to obtain a first layer of extracted clear oil, a second layer of emulsion, a third layer of clear liquid, and a fourth layer of precipitated residue. The first layer of extracted clear oil is absorbed to obtain clear oil I, and the second layer of emulsion is recovered.

[0068] Example 3

[0069] (1) Accurately weigh 20 g of camellia seed powder into a 250 mL conical flask, add 120 mL of citric acid-sodium citrate buffer (0.1 M, pH = 3.6), and mix well to obtain a slurry.

[0070] (2) Acidic protease is added to the slurry obtained in step (1) at an enzyme dosage of 300 U / g (based on the mass of camellia oleifera seed powder). The slurry is placed in a constant temperature stirring oil bath pot and stirred and extracted at 60°C and 150 rpm for 5 hours. After the reaction is completed, the enzyme is inactivated in a boiling water bath for 10 minutes. After cooling, the slurry is centrifuged at 10°C and 10,000 rpm for 15 minutes to obtain a first layer of extracted clear oil, a second layer of emulsion, a third layer of clear liquid, and a fourth layer of precipitated residue. The first layer of extracted clear oil is absorbed to obtain clear oil I, and the second layer of emulsion is recovered.

[0071] Example 4

[0072] (1) Accurately weigh 5 g of the emulsion layer of Example 2 and place it in a 50 mL centrifuge tube. Add 5 mL of distilled water to dilute it. Add an appropriate amount of NaOH solution to the dilution to adjust the pH to 5.2.

[0073] (2) Add medium-temperature α-amylase to the dilution solution at an enzyme dosage of 240 U / g (based on the mass of the emulsion layer), place it in a constant temperature stirring oil bath, and react at 65°C and 150 rpm for 30 minutes. Then, add an appropriate amount of HCl to the reaction solution to adjust the pH to 2.8, add acid protease at an enzyme dosage of 160 U / g (based on the mass of the emulsion layer), and react at 50°C and 150 rpm for 30 minutes. After the reaction is completed, boil the solution in a boiling water bath for 10 minutes to inactivate the enzyme. After cooling, centrifuge at 10°C and 10,000 rpm for 5 minutes to obtain the first layer of demulsified oil, the second layer of emulsion, the third layer of clear liquid, and the fourth layer of precipitated residue. The first layer of clear oil is absorbed to obtain demulsified oil (clear oil II).

[0074] Example 5

[0075] (1) Accurately weigh 5 g of the emulsion layer and place it in a 50 mL centrifuge tube. Add 4.5 mL of distilled water to dilute it. Add an appropriate amount of NaOH solution to the dilution solution to adjust the pH to 5.6.

[0076] (2) Add mesophilic α-amylase to the dilution solution at an enzyme dosage of 320 U / g (based on the mass of the emulsion layer), place it in a constant temperature stirring oil bath, and react at 70°C and 150 rpm for 20 minutes. Then, add an appropriate amount of HCl to the reaction solution to adjust the pH to 3.2, add acid protease at an enzyme dosage of 240 U / g (based on the mass of the emulsion layer), and react at 55°C and 150 rpm for 40 minutes. After the reaction is completed, boil the solution in a boiling water bath for 10 minutes to inactivate the enzyme. After cooling, centrifuge at 10°C and 10,000 rpm for 5 minutes to obtain the first layer of demulsified oil, the second layer of emulsion, the third layer of clear liquid, and the fourth layer of precipitated residue. The first layer of clear oil is absorbed to obtain demulsified oil (clear oil II).

[0077] Example 6

[0078] (1) Accurately weigh 5 g of the emulsion layer and place it in a 50 mL centrifuge tube. Add 4 mL of distilled water to dilute it. Add an appropriate amount of NaOH solution to the dilution solution to adjust the pH to 6.0.

[0079] (2) Add medium-temperature α-amylase to the dilution solution at an enzyme dosage of 400 U / g (based on the mass of the emulsion layer), place it in a constant temperature stirring oil bath, and react at 75°C and 150 rpm for 40 minutes. Then, add an appropriate amount of HCl to the reaction solution to adjust the pH to 3.6, add acid protease at an enzyme dosage of 320 U / g (based on the mass of the emulsion layer), and react at 60°C and 150 rpm for 20 minutes. After the reaction is completed, boil the solution in a boiling water bath for 10 minutes to inactivate the enzyme. After cooling, centrifuge at 10°C and 10,000 rpm for 5 minutes to obtain the first layer of demulsified oil, the second layer of emulsion, the third layer of clear liquid, and the fourth layer of precipitated residue. The first layer of clear oil is absorbed to obtain demulsified oil (clear oil II).

[0080] Example 7

[0081] Determination of total oil content in camellia seed kernel and emulsion layer:

[0082] The total oil content of camellia oleifera seed kernel and emulsion layer was determined respectively with reference to the first method “Soxhlet extraction method” specified in GB 5009.6-2016.

[0083] Calculation of clear oil yield:

[0084]

[0085] Calculation of demulsification rate:

[0086]

[0087] Calculation of whey oil yield:

[0088]

[0089] Example 8

[0090] Analysis of physical and chemical properties of camellia seed oil.

[0091] Camellia oleifera oil's color, odor, transparency, fatty acid composition, moisture and volatile matter content, insoluble impurities, acid value, and peroxide value were determined according to the evaluation standards for camellia oleifera oil (GB / T 11765-2018). Sensory evaluation included color, odor, and transparency, analyzed according to GB / T 5009.37 and GB / T 5525; fatty acid composition, analyzed according to GB 5009.168 (normalization method); moisture and volatile matter content, analyzed according to GB 5009.236; and insoluble impurities, acid value, and peroxide value, analyzed according to GB / T 15688, GB 5009.229, and GB 5009.227, respectively.

[0092] Test Example 1

[0093] Effect of different single enzymes on clear oil yield. According to the method of Example 2, the effects of nine enzymes, including acid protease (Acp, 55°C, pH 3.6), neutral protease (Nep, 50°C, pH 7), Alcalase 2.4L (Alc, 55°C, pH 8.5), papain (Pap, 50°C, pH 6.6), mesophilic α-amylase (Mta, 70°C, pH 5.6), fungal α-amylase (Fua, 50°C, pH 5), cellulase (Cel, 70°C, pH 5.4), hemicellulase (Hem, 55°C, pH 4) and pectinase (Pec, 55°C, pH 3.6) on clear oil yield under their respective temperature and pH conditions were investigated. Other steps were the same as in Example 2. These enzymes play an important role in the oil extraction process by degrading proteins, starch, cellulose, hemicellulose and pectin, destroying the cell walls of oilseeds and the network structure of proteins and starch that wrap the oil. The results are shown in Figure 2. Figure 1 As shown in Figure A, acidic protease achieved the highest clear oil yield, at 66.45 ± 0.26%. When using neutral protease, the clear oil yield was less than 20%, indicating significant differences in clear oil yields between different proteases. Similar results were observed when extracting camellia oil using amylase. In contrast, the clear oil yields when extracting camellia oil using the three carbohydrases (cellulase, hemicellulase, and pectinase) were more moderate, ranging between 35% and 50%. In summary, acidic protease is the most effective single enzyme for camellia oil extraction.

[0094] Test Example 2

[0095] Effect of different single enzyme combinations on clear oil yield.

[0096] According to the method of Example 2, based on the results of the single enzyme extraction experiment, acid protease was selected as the main enzyme and compounded with other enzymes. The complex enzyme extraction test was carried out under the conditions of the optimal temperature of 55°C and the optimal pH of 3.6 for acid protease (wherein the amount of acid protease added was 400 U / g and the amount of other enzymes added was 200 U / g). Other conditions were the same as those in Example 2. Since the single enzyme extraction efficiency of neutral protease and fungal α-amylase was poor, they were discarded when considering the enzyme compounding. The results are shown in Figure 2. Figure 1 As shown in Figure B, "Acp + Alc" was the optimal enzyme complex, achieving a clear oil yield of 66.49 ± 0.90%, which was not significantly different from extraction with acid protease alone. The other enzyme complexes showed slightly lower clear oil yields compared to single acid protease. This may be because the enzyme complex increased the protein content in the reaction system, resulting in enhanced emulsification and reduced clear oil yield. Therefore, to simplify the process, acid protease alone was selected for camellia oil extraction.

[0097] Test Example 3

[0098] Optimization of extraction conditions of acid protease from camellia seed oil.

[0099] In view of the importance of enzyme stability in enzyme-catalyzed reactions, the temperature stability and pH stability of acidic protease were first determined, which helps to select the appropriate range to optimize the extraction conditions. Figure 2 As shown. Based on the results of enzyme activity stability determination, Central Composite Design (CCD) was used to design and optimize the extraction conditions to obtain the maximum extraction efficiency. The different parameter conditions were changed according to the method of Example 2 for experiment. Other conditions were the same as those in Example 2. The coding and actual variable levels are shown in Table 2. After response surface optimization, the optimal theoretical conditions for acid protease extraction were: reaction temperature 54.67°C, reaction time 5h, pH 3.2, solid-liquid ratio 1:4.68, enzyme addition amount 387.47U / g. Under these conditions, the predicted clear oil yield was 67.06%. Afterwards, three verification experiments were carried out according to this condition. For the convenience of operation, the temperature was set to 55°C. At this time, the clear oil yield was 66.94±0.70%, which was not significantly different from the predicted value, indicating that the established model was basically objective and reliable.

[0100] Table 2 Response surface coding and actual variable levels

[0101]

[0102]

[0103] Test Example 4

[0104] Effects of different enzymes on the demulsification rate of the emulsion layer produced during the extraction of acid protease from camellia seed powder.

[0105] According to the analysis of the main components of the emulsion layer, according to the method of Example 4, the effects of six enzymes, namely acid protease (Acp, 55°C, pH 3.2), neutral protease (Nep, 50°C, pH 7), Alcalase 2.4L (Alc, 55°C, pH 8.5), papain (Pap, 50°C, pH 6.6), medium-temperature α-amylase (Mta, 70°C, pH 5.6), and fungal α-amylase (Fua, 50°C, pH 5), on the demulsification rate under their respective temperature and pH conditions were investigated. The initial enzyme addition amount was 340 U / g, the reaction time was 65 min, and the other conditions were the same as in Example 4. The results are shown in FIG. Figure 3 As shown, it can be seen that among the four proteases, the acidic protease has the highest demulsification rate of 57.91±0.62%, and among the two amylases, the medium-temperature α-amylase has the highest demulsification rate of 58.55±0.10%. Therefore, these two enzymes were compounded for further screening. Four groups of comparative experiments were conducted to investigate the effects of different enzyme addition orders and reaction conditions on the demulsification rate. In the first group, "*Mta+Acp", medium-temperature α-amylase was added first and then acidic protease, and the reactions were carried out step by step under their respective optimal conditions; in the second group, "*Acp+Mta", acidic protease was added first and then medium-temperature α-amylase, and the reactions were carried out step by step under their respective optimal conditions; in the third group, "Mta+Acp", both enzymes were added at the same time, and the reactions were carried out under the optimal conditions of medium-temperature α-amylase; in the fourth group, "Acp+Mta", both enzymes were added at the same time, and the reactions were carried out under the optimal conditions of acidic protease. The results are shown in the figure below. Figure 3 As shown, it can be seen that "*Mta+Acp" has the best effect, with an emulsion breaking rate of 67.97±0.41%.

[0106] Test Example 5

[0107] Effect of the ratio of distilled water to emulsion layer on demulsification rate.

[0108] According to the method of Example 4, the demulsification conditions are as follows: first add medium-temperature α-amylase at an enzyme dosage of 340U / g, stir at 150rpm for 30min at 70℃ and pH 5.6, then add acid protease with the same enzyme activity, stir at 150rpm for 35min at 55℃ and pH 3.2. The effect of the ratio of distilled water to emulsion layer on the demulsification rate was investigated. The ratios of distilled water to emulsion layer in step (1) were 0.7:1, 0.8:1, 0.9:1, 1:1, and 1.1:1 (mL / g), respectively, and the other conditions were the same as in Example 4. The results are as follows: Figure 4As shown, the demulsification rate initially increases and then decreases with increasing distilled water to emulsion ratio, reaching a maximum of 71.79 ± 0.24% at a ratio of 0.9:1. When the distilled water to emulsion ratio is low, the reaction system becomes viscous and has poor fluidity, reducing the chance of enzyme-substrate collisions and leading to a low demulsification rate. When the distilled water to emulsion ratio is high, both the enzyme and substrate concentrations are diluted, reducing the chance of collisions, resulting in a lower demulsification rate. Therefore, a distilled water to emulsion ratio of 0.9:1 (mL / g) was selected as the optimal ratio.

[0109] Test Example 6

[0110] Effect of reaction temperature of mesophilic α-amylase on demulsification rate.

[0111] According to the method of Example 4, the demulsification conditions are as follows: the ratio of distilled water to the emulsified layer is 0.9:1, first add medium-temperature α-amylase, the enzyme amount is 340U / g, and stir at 150rpm for 30min under pH 5.6 conditions, then add acid protease with the same enzyme activity, and stir at 150rpm for 35min under 55℃ and pH 3.2 conditions. The effect of the reaction temperature of medium-temperature α-amylase on the demulsification rate was investigated. The selected temperatures were 60℃, 65℃, 70℃, 75℃, and 80℃, respectively, and the other conditions were the same as in Example 4. The results are as follows Figure 5 As shown, within the 60-70°C range, the demulsification rate gradually increases with increasing temperature, reaching a maximum of 71.79 ± 0.24% at 70°C. However, above 70°C, the demulsification rate decreases. As the temperature increases, the viscosity of the reaction system decreases, promoting oil aggregation. Furthermore, at higher temperatures, proteins tend to detach from the emulsion interface, thinning the interface and making it more susceptible to protease action. However, it should be noted that higher temperatures may inactivate the enzyme molecules, resulting in a decrease in the demulsification rate. Therefore, 70°C is selected as the optimal reaction temperature for mesophilic α-amylase.

[0112] Test Example 7

[0113] Effect of reaction pH on demulsification rate of mesophilic α-amylase.

[0114] According to the method of Example 4, the demulsification conditions are as follows: the ratio of distilled water to emulsified layer is 0.9:1, first add medium-temperature α-amylase, the enzyme amount is 340U / g, stir at 150rpm at 70℃ for 30min, then add acid protease with the same enzyme activity, stir at 150rpm at 55℃ and pH 3.2 for 35min. The effect of the reaction pH of medium-temperature α-amylase on the demulsification rate was investigated. The selected pH values were 4.8, 5.2, 5.6, 6.0 and 6.4, respectively, and the other conditions were the same as in Example 4. The results are shown in Figure 4. Figure 6As shown in the figure, the demulsification rate shows a significant upward trend within the pH range of 4.8 to 5.6, reaching a maximum of 71.79 ± 0.24% at pH 5.6. Thereafter, when the pH exceeds 5.6, the demulsification rate gradually decreases. The optimal pH for mesophilic α-amylase is around 5.6. Both high and low pH values lead to reduced enzyme activity, thereby reducing the demulsification rate. Therefore, pH 5.6 is selected as the optimal reaction pH for mesophilic α-amylase.

[0115] Test Example 8

[0116] Effect of reaction time of mesophilic α-amylase on demulsification rate.

[0117] According to the method of Example 4, the demulsification conditions are as follows: the ratio of distilled water to the emulsified layer is 0.9:1, first add medium-temperature α-amylase, the enzyme amount is 340U / g, and stir at 150rpm at 70℃ and pH 5.6, then add acid protease with the same enzyme activity, and stir at 150rpm at 55℃ and pH 3.2 for 35min. The effect of the reaction time of medium-temperature α-amylase on the demulsification rate was investigated. The selected times were 10min, 20min, 30min, 40min, and 50min, respectively, and the other conditions were the same as in Example 4. The results are as follows Figure 7 As shown, within the 10-20 minute range, the demulsification rate significantly increases with increasing reaction time for mesophilic α-amylase, reaching a maximum of 73.73 ± 0.20% at 20 minutes. Thereafter, the demulsification rate decreases with increasing reaction time. The initial enzymatic reaction is the zero-order phase, during which the demulsification rate rapidly increases. After a period of reaction, the enzymatic rate gradually decreases due to factors such as decreased substrate concentration, product inhibition, and reduced enzyme activity. Furthermore, with extended reaction time, the reaction system may re-emulsify, leading to a decrease in the demulsification rate. Therefore, 20 minutes is selected as the optimal reaction time for mesophilic α-amylase.

[0118] Test Example 9

[0119] Effect of the amount of medium-temperature α-amylase added on the demulsification rate.

[0120] According to the method of Example 4, the demulsification conditions are as follows: the ratio of distilled water to the emulsified layer is 0.9:1, medium-temperature α-amylase is added first, and stirred at 150 rpm for 20 minutes at 70°C and pH 5.6, followed by the addition of acid protease at an enzyme dosage of 340 U / g, and stirring at 150 rpm for 35 minutes at 55°C and pH 3.2. The effect of the amount of medium-temperature α-amylase added on the demulsification rate was investigated. The selected enzyme dosages were 80 U / g, 160 U / g, 240 U / g, 320 U / g, and 400 U / g, respectively, and the other conditions were the same as in Example 4. The results are shown in FIG. Figure 8As shown, the demulsification rate shows an overall trend of initially increasing and then decreasing with increasing enzyme dosage of mesophilic α-amylase. The demulsification rate reaches its maximum at 73.58±0.10% when the enzyme dosage is 340 U / g. When the enzyme dosage is too low, the enzymatic hydrolysis reaction is insufficient, so the demulsification rate gradually increases with increasing enzyme concentration. However, the enzyme itself is a protein, and excessive enzyme addition can easily cause the system to re-emulsify, thereby reducing the demulsification rate. Therefore, 340 U / g is selected as the optimal enzyme dosage for mesophilic α-amylase.

[0121] Test Example 10

[0122] Effect of reaction temperature of acid protease on demulsification rate.

[0123] According to the method of Example 4, the demulsification conditions are as follows: the ratio of distilled water to emulsified layer is 0.9:1, first add medium-temperature α-amylase, the enzyme amount is 340U / g, stir at 150rpm for 20min at 70℃ and pH 5.6, then add acid protease with the same enzyme activity, stir at 150rpm for 35min at pH 3.2. The effect of the reaction temperature of acid protease on the demulsification rate was investigated. The selected temperatures were 45℃, 50℃, 55℃, 60℃ and 65℃, respectively, and the other conditions were the same as in Example 4. The results are as follows Figure 9 As shown, the effect of reaction temperature on demulsification efficiency is similar to that of mesophilic α-amylase. When the temperature is below 55°C, the demulsification efficiency gradually increases, reaching a maximum of 73.58±0.10% at 55°C. However, when the temperature is above 55°C, the demulsification efficiency shows a significant downward trend. Therefore, 55°C is selected as the optimal reaction temperature for acidic protease.

[0124] Test Example 11

[0125] The effect of protease reaction pH on demulsification rate.

[0126] Refer to the method of Example 4, wherein the demulsification conditions are: the ratio of distilled water to emulsified layer is 0.9:1, first add medium temperature α-amylase, the enzyme amount is 340U / g, stir at 150rpm for 20min at 70℃ and pH 5.6, then add acid protease with the same enzyme activity, stir at 150rpm for 35min at 55℃. The effect of the reaction pH of acid protease on the demulsification rate was investigated. The selected pH values were 2.4, 2.8, 3.2, 3.6, and 4.0, respectively, and the other conditions were the same as in Example 4. The results are shown in FIG. Figure 10As shown, the reaction pH of acidic proteases also has a dual effect on demulsification efficiency. When the pH is < 3.2, the demulsification efficiency increases with increasing pH, reaching a maximum of 73.58 ± 0.10% at pH 3.2. When the pH exceeds 3.2, the demulsification efficiency gradually decreases. This is consistent with the optimal pH value for acidic proteases, so pH 3.2 was selected as the optimal reaction pH for acidic proteases.

[0127] Test Example 12

[0128] Effect of reaction time of acid protease on demulsification rate.

[0129] According to the method of Example 4, the demulsification conditions are as follows: the ratio of distilled water to the emulsified layer is 0.9:1, first add medium-temperature α-amylase, the enzyme amount is 340U / g, and stir at 150rpm for 20min at 70℃ and pH 5.6, then add acid protease with the same enzyme activity, and stir at 150rpm for different times at 55℃ and pH 3.2. The effect of the reaction time of acid protease on the demulsification rate was investigated. The selected times were 10min, 20min, 30min, 40min, and 50min, respectively, and the other conditions were the same as in Example 4. The results are as follows Figure 11 As shown in the figure, it can be seen that the effect of the reaction time of acidic protease on the demulsification rate shows a trend of first increasing and then decreasing, reaching a maximum of 74.18±0.24% at 20 minutes. After that, the demulsification rate gradually decreases with the extension of the reaction time. Therefore, 20 minutes is selected as the optimal reaction time for acidic protease.

[0130] Test Example 13

[0131] Effect of the amount of acid protease added on the demulsification rate.

[0132] According to the method of Example 4, the demulsification conditions are as follows: the ratio of distilled water to the emulsified layer is 0.9:1, first add medium-temperature α-amylase, the enzyme dosage is 340U / g, stir at 150rpm for 20min at 70℃ and pH 5.6, then add acid proteases with different enzyme activities, stir at 150rpm for 20min at 55℃ and pH 3.2. The effect of the enzyme dosage of acid protease on the demulsification rate was investigated. The selected enzyme dosages were 80U / g, 160U / g, 240U / g, 320U / g and 400U / g, respectively, and the other conditions were the same as in Example 4. The results are as follows Figure 12As shown, the effect of enzyme dosage on demulsification efficiency for acidic protease differs slightly from that for mesophilic α-amylase. When the enzyme dosage is less than 240 U / g, the demulsification efficiency increases rapidly with increasing enzyme dosage. Within the enzyme dosage range of 240 to 340 U / g, the demulsification efficiency remains relatively stable, with no significant change. Subsequently, as the enzyme dosage increases further, the demulsification efficiency decreases rapidly. Therefore, 240 U / g is selected as the optimal enzyme dosage for acidic protease.

[0133] Test Example 14

[0134] The influence of different demulsification processes on demulsification rate.

[0135] Through the optimization of demulsification conditions in Experiments 5 to 13, it was found that moderate-temperature α-amylase and acid protease have different optimal reaction pH and temperature. Using them together means that the demulsification process requires two addition steps, namely, changing the temperature and pH of the system, which will increase the complexity of the demulsification process. Moreover, in large-scale industrial production, achieving rapid temperature changes is relatively difficult. Therefore, to simplify the demulsification process, three sets of demulsification experiments were conducted. The first group "Acp + Mta" was added with medium-temperature α-amylase (340U / g) and acid protease (240U / g) at the same time, and the mixture was stirred at 150rpm for 40min at 55℃ and pH 3.2, with the ratio of distilled water to the emulsion layer being 0.9:1; the second group "Mta + Acp" was added with medium-temperature α-amylase (340U / g) and acid protease (240U / g) at the same time, and the mixture was stirred at 150rpm for 40min at 70℃ and pH 5.6, with the ratio of distilled water to the emulsion layer being 0.9:1; the third group "#Mta + Acp" was added with medium-temperature α-amylase (340U / g) first, and the mixture was stirred at 150rpm for 20min at 70℃ and pH 5.6, then the pH of the system was adjusted to 3.2, the temperature was kept constant, and the acid protease (240U / g) was added, and the mixture was stirred at 150rpm for 20min. The results are as follows: Figure 13 As shown in the results, the demulsification rates of "Acp+Mta", "Mta+Acp" and "#Mta+Acp" were 65.96±0.76%, 70.98±0.19% and 71.16±0.34% respectively, which were significantly lower than the final demulsification rates optimized in Experimental Examples 5 to 13. Therefore, it is recommended that in large-scale industrial production, in order to simplify the process, the process conditions of "#Mta+Acp" can be selected. In small-scale industrial production, it is more appropriate to successively add medium-temperature α-amylase and acid protease to change the reaction temperature and pH in the demulsification process.

[0136] Test Example 15

[0137] Analysis of physical and chemical properties of camellia seed oil.

[0138] In order to evaluate the quality of the camellia oil obtained by enzymatic extraction and demulsification, the physicochemical properties of camellia oil were measured. The clear oil 1 obtained by the above-mentioned enzymatic extraction optimal process was used as oil sample 1, and the demulsified clear oil obtained by the above-mentioned enzymatic demulsification optimal process was used as oil sample 2. The clear oil yield of enzymatic extraction was 66.94%, and the enzymatic demulsified clear oil yield was 16.01%. The two clear oils were mixed in a mass ratio of 66.94:16.01 (i.e., 4.18:1) as oil sample 3. The physicochemical property analysis of the three oil samples is shown in Table 3 and Table 4. It can be seen that the three oil samples all meet the quality standards of the first-class oil specified in GB / T 11765-2018, and their fatty acid composition is also within the scope of the national standard.

[0139] Table 3 Quality analysis of camellia oil

[0140]

[0141] Note: *This quality indicator is derived from the Chinese national standard GB / T 11765-2018.

[0142] Table 4 Basic composition and main physical parameters of camellia oil

[0143]

[0144]

[0145] Note: *This quality indicator is derived from the Chinese national standard GB / T 11765-2018.

[0146] In summary, the process of the present invention for preparing camellia oil, i.e., first extracting the camellia oil using acidic protease, was optimized to yield a clear oil yield of 66.94±0.70% using the method of Example 2, acidic protease, a reaction temperature of 55°C, a solid-liquid ratio of 1:4.68 (adjusted with 0.1M citric acid-sodium citrate buffer, pH 3.2), an enzyme dosage of 387.47 U / g, and stirring at 150 rpm for 5 hours. The emulsion layer was then collected and demulsified by sequentially adding moderate-temperature α-amylase and acidic protease according to the method of Example 4. The optimized demulsification conditions are as follows: the ratio of distilled water to emulsion layer is 0.9:1 mL / g, medium-temperature α-amylase is added first, the enzyme dosage is 340 U / g, and the reaction is stirred at 150 rpm for 20 minutes at 70°C and pH 5.6. Then, acid protease is added with an enzyme dosage of 240 U / g, and the reaction is continued with stirring at 150 rpm for 20 minutes at 55°C and pH 3.2. The demulsification rate reaches 74.31±0.27%, and the demulsified clear oil yield is 16.01%. Therefore, the final total clear oil yield is 82.95%. In addition, all three oil samples meet the quality standards of first-class oil specified in GB / T 11765-2018. It can be seen that the method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification disclosed in the present invention is a green, environmentally friendly and efficient production method.

Claims

1. A method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification, characterized in that: The following steps are involved: (1) shelling and crushing camellia seeds to obtain camellia seed powder; (2) mixing camellia seed powder and buffer solution to form a slurry; (3) adding an enzyme preparation to the slurry for stirring and enzymatic hydrolysis and extraction, heating to inactivate the enzyme after the enzymatic hydrolysis and extraction reaction is completed, and centrifuging the enzymatic hydrolysis and extraction reaction liquid at high speed after cooling to obtain a first layer of extracted clear oil, a second layer of emulsion layer, a third layer of clear liquid, and a fourth layer of precipitated residue in sequence, absorbing the first layer of extracted clear oil to obtain clear oil I, and recovering the second layer of emulsion layer; (4) distilling the recovered emulsion layer with distilled water, adjusting the pH, adding an enzyme preparation, stirring, and performing enzymatic demulsification. After the enzymatic demulsification reaction is completed, heating is performed to inactivate the enzyme. After cooling, the enzymatic demulsification reaction liquid is centrifuged at high speed to obtain a first layer of demulsified clear oil, a second layer of emulsion, a third layer of clear liquid, and a fourth layer of precipitated residue. The first layer of demulsified clear oil is absorbed to obtain clear oil II. (5) The clear oil I and the clear oil II are combined and dried to obtain high-quality camellia oil; the clear liquid and the emulsion in steps (3) and (4) are combined, and the precipitated residues in steps (3) and (4) are combined for recovery of other useful components.

2. The method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification according to claim 1, characterized in that: The slurry described in step (2) is prepared by uniformly mixing camellia seed powder and buffer solution at a material-liquid ratio of 1:3 to 1:7 (g / mL).

3. The method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification according to claim 1, characterized in that: The buffer solution in step (2) is preferably a citric acid-sodium citrate buffer solution with a pH of 2.8 to 4.

4.

4. The method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification according to claim 1, characterized in that: The enzyme preparation described in step (3) is acidic protease, and the addition amount is 100-500 U / g camellia seed powder.

5. The method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification according to claim 1, characterized in that: The enzymatic extraction reaction temperature in step (3) is 40-60° C., the stirring speed is 100-200 rpm, and the time is 2-6 hours.

6. The method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification according to claim 1, characterized in that: The ratio of the amount of distilled water added in step (4) to the emulsion layer is 0.7:1 to 1.1:1 (mL / g).

7. The method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification according to claim 1, characterized in that: The procedure and conditions for adding the enzyme preparation for enzymatic demulsification after adjusting the pH in step (4) are as follows: first, adjust the pH of the system to 4.8-6.4 with NaOH, then add the medium-temperature α-amylase, according to 80-400 U / g emulsion layer, and stir the enzymatic hydrolysis at 60-80°C and 150 rpm for 10-50 minutes; then adjust the pH of the system to 2.4-4.0 with HCl, add the acidic protease, according to 80-400 U / g emulsion layer, and stir the enzymatic hydrolysis at 45-65°C and 150 rpm for 10-50 minutes.

8. The method for preparing high-quality camellia oil by a full enzymatic method based on enzymatic extraction and enzymatic demulsification according to claim 1, characterized in that: The drying in step (5) is performed at 50-60° C. for 10-12 hours.

9. High-quality camellia oil prepared by the method for preparing high-quality camellia oil by the full enzymatic method based on enzymatic extraction and enzymatic demulsification according to claim 1.

10. Use of the high-quality camellia seed oil according to claim 9 as a raw material or ingredient for common edible oil, health food, pharmaceutical products, or cosmetics.

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