A method for extracting polyphenol-rich camellia oil

Abstract

The present invention discloses a method for extracting polyphenol-rich camellia oil. First, camellia seeds are used to extract camellia oil by the aqueous enzymatic method, and then the camellia oil is used to extract polyphenols from camellia seed shells, or the camellia seeds are directly extracted with polyphenol-rich camellia oil by the aqueous enzymatic method assisted by ultrasound. Under the condition of ensuring the extraction rate of camellia oil, polyphenol-rich camellia oil is obtained. This method maximally retains the natural flavor and nutrition of camellia seeds and has the advantages of being green and environmentally friendly. The oil-making conditions of the camellia seeds in the present invention are mild, and the oil yield is high (up to 41.8 grams of oil / 100 g of camellia seeds, close to the organic solvent extraction method, and the polyphenol content reaches 48.32 mg / kg). It maximally retains the micronutrients (vitamin E, sterols, squalene, etc.) and natural characteristic flavors of camellia seeds, especially enriches high-concentration and high-activity polyphenol functional factors, and the obtained oil is more nutritious, safer, and healthier.

Description

(1) Technical Field

[0001] The present invention relates to a method for extracting camellia oil rich in polyphenols. (2) Background Art

[0002] Camellia oil is rich in oleic acid and linoleic acid, and its unsaturated fatty acids account for about 90%. Research shows that long-term intake of unsaturated fatty acids by the human body can reduce the accumulation of low-density lipoprotein, which helps to reduce the risk of cardiovascular diseases. Since camellia oil has similar physicochemical properties and fatty acid composition to olive oil, it is known as the "Oriental olive oil". In addition to its high-quality fatty acid composition, trace active polyphenols, vitamins and phytosterols in camellia oil also endow camellia oil with various health characteristics. Currently, camellia oil is mainly produced by pressing, and the residual oil in the cake is mostly extracted by solvent extraction.

[0003] The aqueous enzymatic method has attracted more and more attention because of its mild extraction conditions and better retention of the natural flavor of oilseeds and active substances such as trace VE and sterols. However, the aqueous enzymatic method has low oil yield, high cost, and limited solubility in polar polyphenols, resulting in ineffective extraction of important active substances. Camellia oil polyphenols have good antioxidant, anti-tumor, blood vessel regulation and other biological activities. At present, 27 phenolic substances, including phenolic acids and flavonoids, have been identified in camellia oil. However, there are few reports on the simultaneous enrichment of polyphenols during the production of camellia oil. (3) Summary of the Invention

[0004] The object of the present invention is to provide a method for extracting camellia oil rich in polyphenols. First, camellia seeds are used to extract camellia oil by the aqueous enzymatic method, and then the polyphenols in the camellia seed shells are extracted with camellia oil, or camellia seeds are directly used to ultrasonically assist in extracting camellia oil rich in polyphenols by the aqueous enzymatic method. Under the condition of ensuring the extraction rate of camellia oil, camellia oil rich in polyphenols is obtained. This method maximally retains the natural flavor and nutrition of camellia seeds and has the advantages of being green and environmentally friendly.

[0005] The technical solution adopted by the present invention is as follows:

[0006] The present invention provides a method for extracting polyphenol-rich camellia oil, and the method is carried out according to the following steps: adding camellia seed kernel powder and camellia seed shell powder into a solvent, heating to inactivate enzymes (preferably pretreating at 90 °C for 10 min to inactivate enzymes); when the temperature cools to 50-60 °C (preferably 55 °C), adjusting the pH to 8-10 (preferably adjusting to 9 with 1M NaOH aqueous solution), adding alkaline protease, and hydrolyzing the mixture in a water bath at 45-60 °C with continuous stirring at 100-300 rpm for 3-6 h (preferably at 55 °C, 200 rpm, for 4 h); then stirring at 80-100 °C for 5-15 min (preferably at 90 °C, for 10 min) and cooling to room temperature, centrifuging the mixture (preferably centrifuging at 8000 rpm for 30 min), and collecting the supernatant, which is polyphenol-rich camellia oil; the solvent is one of deionized water, an aqueous ethanol solution with a volume concentration of 20%, an aqueous isopropanol solution with a volume concentration of 25%, a 0.15M glucose aqueous solution, a 0.1M KCl aqueous solution, an aqueous isopropanol solution with a volume concentration of 25% containing 0.15M glucose, and an aqueous isopropanol solution with a volume concentration of 25% containing 0.1M KCl.

[0007] Furthermore, the mass ratio of camellia seed kernel powder to the solvent is 1:5-10, preferably 1:7; the mass ratio of camellia seed kernel powder to camellia seed shell powder is 4-8:1, preferably 5:1; the enzyme activity of the alkaline protease is 20000 U / mg, and the mass ratio of the alkaline protease to camellia seed kernel powder is 0.001-0.1:1, preferably 0.07:1.

[0008] Furthermore, the solvent is an aqueous isopropanol solution with a volume concentration of 25% containing 0.15M glucose.

[0009] Furthermore, the camellia seed kernel powder is prepared according to the following steps: manually removing the shells of fresh camellia seeds, drying the camellia seed kernels at 50 °C for 4 h until the mass water content is below 5% (preferably 3.6%); after cooling, grinding into powder and passing through a 40-mesh sieve, and storing at -18 °C to obtain camellia seed kernel powder.

[0010] Furthermore, the camellia seed shell powder is prepared according to the following steps: manually removing the shells of fresh camellia seeds, drying the camellia seed shells at 50 °C for 4 h until the mass water content is below 5% (preferably 4.5%), after cooling, grinding into powder and passing through a 40-mesh sieve, and storing at -18 °C to obtain camellia seed shell powder.

[0011] The present invention also provides a method for extracting polyphenol-rich camellia oil, and the method is carried out according to the following steps: adding fresh camellia seeds with shells into distilled water, wet crushing, adding cellulase and alkaline protease, kneading and extracting under ultrasonic assistance at 40-60 °C and 100-300 W for 60-120 min, centrifuging (preferably centrifuging at 8000 rpm for 30 min), and taking the supernatant, which is polyphenol-rich camellia oil.

[0012] Furthermore, the moisture content of the fresh shelled camellia seeds is 60%, and the mass ratio of the fresh shelled camellia seeds to distilled water is 1:1 - 5, preferably 1:1; the enzyme activity of the cellulase is preferably 10,000 U / g, and the mass addition amount is in a ratio of 0.005 - 0.1:1 to the mass of the fresh camellia seeds, preferably 0.01:1; the enzyme activity of the alkaline protease is preferably 20,000 U / g, and the mass addition amount is in a ratio of 0.001 - 0.01:1 to the mass of the fresh camellia seeds, preferably 0.007:1.

[0013] Furthermore, the conditions for ultrasonic-assisted extraction are 150 W, 50 °C, and 90 min.

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: The present invention uses camellia seed kernels and camellia seed shell powders as raw materials, and a solvent containing glucose or salt as an extractant to extract camellia oil rich in polyphenols under the action of enzymes; or uses fresh shelled camellia seeds as raw materials, water as an extractant, and ultrasonic-assisted enzyme extraction to obtain camellia oil rich in polyphenols. The method of the present invention not only improves the utilization rate of camellia seed shells, but also selects isopropanol or distilled water added with glucose as an extractant, reducing the solvent pollution problem of traditional organic solvent extraction and solving the emulsification problem at the same time. The present invention does not use organic solvents, has less emulsification, high yield, mild oil-making conditions, good quality, and is more nutritious, safer, and healthier.

[0015] The oil-making conditions of camellia seeds are mild, with a high oil yield (up to 41.8 g of oil / 100 g of camellia seeds, close to the organic solvent extraction method, and the polyphenol content reaches 48.32 mg / kg). It maximally retains the micronutrients (vitamin E, sterols, squalene, etc.) and natural characteristic flavors of camellia seeds, especially enriches high-concentration and high-activity polyphenol functional factors, and the obtained oil is more nutritious, safer, and healthier. (IV) Description of the Drawings

[0016] Figure 1 The liquid chromatograms of the polyphenol compositions of the camellia oils obtained in Examples 12, 13, and 14; Curve A represents Example 12, Curve B represents Example 13, and Curve C represents Example 14. (V) Specific Embodiments

[0017] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto: The camellia seeds (Camellia seed) of the present invention, also known as oil-tea camellia seeds, are the fruits of the oil-tea camellia tree and belong to the Theaceae family and the Camellia genus.

[0018] Example 1: Extracting camellia oil by traditional organic solvent extraction method

[0019] 10 Kg of fresh camellia seeds were dried at 50 °C for 4 h. After cooling, the seeds were shelled, and the camellia seed kernels were ground into powder and passed through a 40-mesh sieve, then stored at -18 °C to obtain 2.8 Kg of camellia seed kernel powder.

[0020] 10 g of camellia seed kernel powder was wrapped in filter paper and placed in a Soxhlet extractor containing 180 mL of n-hexane. After refluxing at 80 °C for 8 h, the solvent was removed by rotary evaporation at 50 °C, and the concentrated solution collected was camellia oil. The oil yield was calculated and the total phenolic content (TPC) in the oil was determined. The results are shown in Table 1.

[0021] Oil yield (%) = mass of oil / mass of camellia seed kernel powder × 100

[0022] Method for determining total phenolic content (Folin-Ciocaltea method):

[0023] 2 g of camellia oil was dissolved in 4 mL of n-hexane, and then mixed with 2 mL of ethanol-water (80:20, v / v). After vortex extraction for 3 min, the lower layer of the supernatant was collected. The extraction was repeated 2 times, and the combined lower layer of the supernatant was washed with n-hexane to remove the n-hexane, obtaining a polyphenol extract. 1 mL of the polyphenol extract was mixed with 1 mL of Folin-Ciocalteau reagent dilution (a dilution of Folin-Ciocalteau reagent and water in a volume ratio of 1:1). After standing at room temperature for 2 min, the mixture was mixed with 4 mL of a 10% (w / v) Na2CO3 aqueous solution and reacted in the dark at 50 °C for 1 h. Finally, the absorbance was measured at 725 nm, and the results were expressed in terms of gallic acid equivalents.

[0024] Method for polyphenol composition analysis (UPLC-Triple-TOF / MS):

[0025] 5 g of camellia seed oil was extracted for polyphenols as above and concentrated to 1.5 mL, then filtered through a 0.22-μm pore size filter membrane, and the filtrate was analyzed by HPLC.

[0026] Conditions: HPLC analysis was performed by Agilent 1260 (Agilent, Santa Clara, CA), which was equipped with a reversed-phase ZORBAX SB-C18 column (250 mm × 4 mm × 5 μm). The mobile phase was a combination of methanol (solvent A) and water (solvent B). Gradient elution program: 10% A was maintained for 5 min; then increased to 20% A in 5 min and maintained for 5 min; then increased to 30% A in 5 min and maintained for 5 min; this gradient was repeated until a concentration of 100% A. The flow rate of the mobile phase was 1.0 mL / min. Phenolic compounds and their contents were detected at 280 nm using a UV detector. Qualitative identification and analysis of the phenols in the oil were performed by a UPLC Triple TOF / MS 5600plus system (AB Sciex Co., Framingham, USA).

[0027] Examples 2 - 4. Influence of Traditional Organic Solvent Types on the Extraction Rate of Camellia Oil

[0028] In Example 1, n - hexane was replaced with ethanol, isopropanol, and acetone respectively, and other operations were the same. The results are shown in Table 1.

[0029] Example 5. Extraction of Camellia Oil by Aqueous Enzymatic Method

[0030] 10 g of the camellia seed kernel powder prepared by the method of Example 1 was added to 70 g of deionized water, and pretreated at 90 °C for 10 min to inactivate enzymes. When the temperature cooled to 55 °C, the pH was adjusted to 9 with 1 M NaOH aqueous solution, and 0.07 g of alkaline protease (enzyme activity 20000 U / g) was added. Then, the mixture was hydrolyzed in a water bath at 55 °C with continuous stirring at 200 rpm for 4 h. After stirring and sterilizing at 90 °C for 10 min and cooling to room temperature, the mixture was centrifuged at 8000 rpm for 30 min. The supernatant was collected, and 0.258 g of camellia oil was obtained. The oil yield was calculated by the method of Example 1, and the total phenol content in the oil was determined. The results are shown in Table 1.

[0031] Examples 6 - 11. Influence of Different Solvents on the Extraction of Camellia Oil by Aqueous Enzymatic Method

[0032] In Example 5, deionized water was replaced with an equal mass of 20% ethanol aqueous solution by volume, 25% isopropanol aqueous solution by volume, 0.15 M glucose aqueous solution, 0.1 M KCl aqueous solution, 25% isopropanol aqueous solution containing 0.15 M glucose, and 25% isopropanol aqueous solution containing 0.1 M KCl, and other operations were the same. The results are shown in Table 1.

[0033] Example 12. Simultaneous Aqueous Enzymatic Method for Seed Hulls

[0034] 10 Kg of fresh camellia seeds were manually shelled. The camellia seed kernels and camellia seed hulls were dried at 50 °C for 4 h until the moisture content of the camellia seed kernels was 3.6% and the moisture content of the camellia seed hulls was 4.5%. After cooling, they were ground and passed through a 40 - mesh sieve, and stored at - 18 °C, obtaining 2.8 Kg of camellia seed kernel powder and 1.9 Kg of camellia seed hull powder.

[0035] Add 10 g of camellia seed kernel powder and 2 g of camellia seed shell powder to 70 g of an aqueous solution of 25% isopropanol containing 0.15 M glucose, and pretreat at 90 °C for 10 min to inactivate enzymes. When the temperature cools to 55 °C, adjust the pH to 9 with 1 M aqueous NaOH solution, and add 0.07 g of alkaline protease (0.7% based on the mass of camellia seed kernel). Then, the mixture is hydrolyzed in a water bath at 55 °C with continuous stirring at 150 rpm for 4 h. After treatment at 90 °C for 10 min and cooling to room temperature, the mixture is centrifuged at 8000 rpm for 30 min. Collect the supernatant, which is 0.403 g of camellia oil. Calculate the oil yield using the method of Example 1, determine the total phenol content in the oil, and the results are shown in Table 1. Analyze the polyphenol content.

[0036] Example 13. Extraction of Total Phenols in Camellia Oil by Shearing Method

[0037] Weigh 20 g of refined camellia oil (Qiandaohu Yaoji Refined Camellia Oil) and add 0.5 g of camellia shell powder prepared by the method of Example 12. First, shear and mix at 10000 rpm for 1 min using a high-speed shearer, and repeat 3 times; then ultrasonically mix at 200 W for 30 min; finally, centrifuge the mixture at 8000 rpm for 30 min, take the supernatant, and determine the total phenol content in the camellia oil using the method of Example 1. The results are shown in Table 1.

[0038] Example 14. Extraction of Total Phenols in Camellia Oil by Phase Transfer Method

[0039] Weigh 5 g of camellia seed shell powder prepared by the method of Example 12, add 10 ml of ethanol, ultrasonically extract at 300 W for 30 minutes, centrifuge the mixture at 8000 rpm for 30 min, take the supernatant and mix it with 4 g of refined camellia oil (Qiandaohu Yaoji Refined Camellia Oil). After ultrasonically mixing at 150 W for 30 min, transfer it to a 25 mL round-bottom flask and remove the solvent by rotary evaporation at 45 °C. Determine the polyphenol content in the camellia oil using the method of Example 1. The results are shown in Table 1.

[0040] Example 15. Oil Production from Fresh Camellia Seeds

[0041] Weigh 500 g of fresh camellia seeds with shells (water content 60%), add 500 g of distilled water, crush them wet, add 5 g of cellulase (enzyme activity 10000 U / g) and 3.5 g of alkaline protease (enzyme activity 20000 U / g), knead and mix for extraction at 50 °C with ultrasonic assistance at 150 W for 90 minutes, centrifuge at 8000 rpm for 30 min, take the supernatant, which is polyphenol-rich camellia oil. Calculate the oil yield and polyphenol content using the method of Example 1. The results are shown in Table 1.

[0042] Table 1. Oil Yield and Total Phenol Content of Camellia Oil under Different Extraction Methods

[0043]

[0044]

[0045] Note: - represents no addition, and E represents the addition of enzymes.

[0046] The data in Table 1 show that:

[0047] 1. In Example 1, the highest yield of refined oil extracted by the traditional solvent n - hexane was 42.27%, but the content of active polyphenols was not high, about 13.76 mg / Kg. In Example 2, the oil yield extracted by pure ethanol was not high, only 32.3%, and the highest polyphenol content was 48.92 mg / Kg; in Example 3, the refined oil yield of isopropanol was 38.2%, and the polyphenol content in the oil was 143.74 mg / Kg; in Example 4, both the oil extraction rate and the polyphenol content of acetone were relatively high. However, for the extraction of oil from shelled camellia seeds by traditional organic solvents in Examples 1 - 4, although the oil yield was high and the polyphenol content in some cases was relatively high, the oil obtained by the organic solvent method required high - intensity refining, and most of the polyphenols would be removed during refining. Therefore, the polyphenol concentration in the refined oil was less than 10 mg / Kg. In addition, the organic solvent method has a large scale and expensive equipment, and is mainly suitable for the oil production of bulk oilseeds such as soybeans and rapeseeds. It is flammable and explosive, with a high safety risk, phospholipids cannot be effectively extracted, and it is not environmentally friendly. The quality of camellia oil extracted by organic solvents is not high, and the residual solvents in the oil pose a food safety hazard, resulting in low consumer acceptance. Therefore, it is rarely used in the actual extraction of camellia oil from original camellia seeds.

[0048] 2. In Example 5, the basic aqueous enzymatic method was adopted, using water as a solvent to hydrolyze oil from shelled camellia seeds. It is safe, but the emulsification is serious, the oil yield is low, and the polyphenol content is not high.

[0049] In Example 5, using shelled camellia seeds as raw materials, due to serious emulsification in the aqueous enzymatic method with water as a solvent, the refined oil yield was only 25.8%.

[0050] 3. In Examples 6 - 11, the improved aqueous enzymatic method was adopted, adding some alcohol solvents and salts or sugars to hydrolyze oil from shelled camellia seeds. The emulsification problem was solved, the oil yield was increased, and the polyphenol content was also improved, but it still did not meet the requirements of high polyphenols.

[0051] In Examples 6 - 10, introducing low - concentration isopropanol, ethanol, KCl, and glucose could effectively reduce emulsification, and the refined oil yield was increased to between 33.3% and 41.8%. Importantly, the aqueous enzymatic method has mild conditions and avoids high - temperature exposure, so it is green and safe, the quality of the oil is good, and it is enriched with more trace nutrients such as vitamin E, squalene, and sterols. The disadvantage is that the content of active polyphenols in the oil is relatively low, less than 10 mg / Kg.

[0052] Example 11 uses shelled camellia seed kernels as raw materials, combines isopropanol and glucose solution as solvents, and extracts camellia oil by the aqueous enzymatic method. The yield of camellia oil is increased to 41.8%, and the polyphenol content is increased by more than one time to 13.82 mg / Kg. This may be because the addition of sugar molecules reduces the polarity of the solution, causing part of the saturated polyphenols to transfer to the oil phase.

[0053] 4. Example 12 uses an improved aqueous enzymatic method to hydrolyze camellia seed kernels + shells together to produce oil. The oil yield is increased, and the polyphenol content is also improved to 23 mg / kg. The prepared oil is safe, does not require high-intensity refining, and has a high nutrient retention rate.

[0054] Example 12 uses the co-hydrolysis of camellia seed kernels and part of the fruit shells. The yield of camellia oil is 40.3%, and the polyphenol content is significantly increased to 23.1 mg / Kg, achieving a balance between oil yield and nutrition. At the same time, the emulsification problem is solved, the utilization rate of camellia seed shells is improved, and the cost is reduced.

[0055] 5. Example 13: Oil extraction polyphenol method. Commercially available refined camellia oil (almost polyphenol-free) and camellia shells (high in polyphenols and oil-free) are sheared and mixed to extract polyphenols. The polyphenol content in the oil is increased to 19.67 mg / Kg. The characteristic is that the process is simple and convenient. The disadvantage is that commercially available camellia oil is mainly produced by the pressing method, and the contents of vitamin E, sterols, and squalene are not high.

[0056] Example 14: Phase transfer method. The ethanol extraction solution of camellia skin is mixed and dissolved with camellia oil in a certain proportion, and then the ethanol solvent is evaporated and removed. The polyphenols in the ethanol are transferred to the oil phase. The polyphenol content of the obtained camellia oil is increased to 48.32 mg / Kg. The disadvantage is that the process is slightly complicated and requires the separate extraction of polyphenols and oil, and then the two are mixed and the solvent is distilled off.

[0057] Example 15 uses fresh fruits (shells + kernels) to produce oil by the enzymatic method. The whole camellia fruit does not need to be dried, and is directly crushed and added with part of the enzyme for preparation, which is green, safe, and nutritious.

[0058] Example 15 develops fresh-pressed camellia oil from fresh fruits, and at the same time adds biological enzymes and ultrasonic physical fields to enhance mass transfer. The yield of camellia oil is 36.9%, and its polyphenol content is also as high as 22.85 mg / Kg. On the one hand, the biological dissociation of proteins promotes the precipitation of oil, and at the same time promotes the dissolution of polyphenol substances into the oil phase. In addition, cellulase may also promote the decomposition of glycosylated polyphenols into free phenols with enhanced non-polarity, so it is beneficial for their transfer into the oil. The polyphenol level in the oil has fully met the demand for rich polyphenols.

[0059] Comprehensive analysis of the polyphenol composition in the camellia oil of Example 12 shows that the free phenol and glycosylated phenol contents in the polyphenol composition of Example 12 are high (see Figure 1Among them, curve A) has good activity and a more natural process. Although the polyphenol content in Examples 13 and 14 is high, the flavonoid content is high and the active glycosylated phenols are less (see Figure 1 curves B and C in

Claims

1. A method for extracting polyphenol-rich camellia oil, characterized in that, The method is carried out according to the following steps: Camellia seed kernel powder and Camellia seed shell powder are jointly added to a solvent, and heated to inactivate enzymes; when the temperature is cooled to 50-60 °C, the pH is adjusted to 8-10, alkaline protease is added, and the mixture is hydrolyzed in a water bath at 45-60 °C and continuously stirred at 100-300 rpm for 3-6 h; then, after stirring at 80-100 °C for 5-15 min and cooling to room temperature, the mixture is centrifuged, and the supernatant is collected, which is polyphenol-rich camellia oil; the solvent is an aqueous solution of 25% isopropanol containing 0.15 M glucose; the mass ratio of Camellia seed kernel powder to the solvent is 1:5-10; the mass ratio of the Camellia seed kernel powder to the Camellia seed shell powder is 4-8:

1.

2. The extraction method of polyphenol-rich camellia oil according to claim 1, characterized in that, The enzyme activity of the alkaline protease is 20000 U / mg, and the mass ratio of the alkaline protease to the Camellia seed kernel powder is 0.001-0.1:

1.

3. The extraction method of polyphenol-rich camellia oil according to claim 1, characterized in that, The Camellia seed kernel powder is prepared according to the following steps: Fresh Camellia seeds are manually shelled, and the Camellia seed kernels are dried at 50 °C until the mass water content is below 5%; after cooling, they are ground and passed through a 40-mesh sieve to obtain the Camellia seed kernel powder.

4. The extraction method of polyphenol-rich camellia oil according to claim 1, characterized in that, The Camellia seed shell powder is prepared according to the following steps: Fresh Camellia seeds are manually shelled, and the Camellia seed shells are dried at 50 °C until the mass water content is below 5%; after cooling, they are ground and passed through a 40-mesh sieve to obtain the Camellia seed shell powder.

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