CN117264690A

CN117264690A - Extraction method of camellia oil rich in polyphenols

Info

Publication number: CN117264690A
Application number: CN202311437306.9A
Authority: CN
Inventors: 孟祥河; 叶沁; 陆元超
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2023-11-01
Filing date: 2023-11-01
Publication date: 2023-12-22

Abstract

The invention discloses an extraction method of camellia oil rich in polyphenol, which comprises the steps of firstly extracting camellia oil from camellia seeds by adopting a water enzymatic method, and then extracting polyphenol in camellia seed shells by using the camellia oil, or directly extracting the camellia oil rich in polyphenol by adopting a water enzymatic method and ultrasonic assistance, so that the camellia oil rich in polyphenol is obtained under the condition of ensuring the extraction rate of the camellia oil. The camellia seed oil preparation condition is mild, the oil yield is high (up to 41.8 g of oil per 100g of camellia seed, the camellia seed oil is close to an organic solvent storage method, the polyphenol content reaches 48.32 mg/kg), the trace nutrients (vitamin E, sterol, squalene and the like) and the natural characteristic flavor of the camellia seed are reserved to the greatest extent, and particularly the high-concentration and high-activity polyphenol functional factors are enriched, so that the obtained grease is more nutritional, safer and healthier.

Description

Extraction method of camellia oil rich in polyphenols

Field of the art

The invention relates to an extraction method of camellia oil rich in polyphenol.

(II) background art

Camellia oil is rich in oleic acid and linoleic acid, with about 90% unsaturated fatty acids. Research shows that long-term intake of unsaturated fatty acid can reduce accumulation of low density lipoprotein, and helps to reduce risk of cardiovascular disease. Camellia oil is known as "Oriental olive oil" because of its similar physicochemical properties and fatty acid composition. Besides the high-quality fatty acid composition, trace active polyphenol, vitamin and plant sterol also endow the camellia oil with various health characteristics. At present, camellia oil mainly takes oil preparation by pressing, and residual oil in cake is mostly extracted by a solvent extraction method.

The aqueous enzymatic method is gaining attention because of mild extraction conditions and better retention of the flavor of the oil and trace active substances such as VE and sterol. However, the aqueous enzymatic process has low oil yield, high cost, and limited dissolution of polar polyphenols, thus resulting in inefficient extraction of important active substances. The camellia oil polyphenol has a plurality of biological activities such as good antioxidation, anti-tumor, blood vessel regulation and the like, and 27 phenolic substances including phenolic acid, flavone and the like are identified in the camellia oil at present. However, researches on camellia oil production and polyphenol enrichment have been recently reported.

(III) summary of the invention

The invention aims to provide an extraction method of camellia oil rich in polyphenol, which comprises the steps of firstly extracting camellia oil from camellia seeds by adopting a water-enzyme method, and then extracting polyphenol in camellia seed shells by using the camellia oil, or directly extracting the camellia oil rich in polyphenol by adopting a water-enzyme method and ultrasonic assistance, so that the camellia oil rich in polyphenol is obtained under the condition of ensuring the extraction rate of the camellia oil.

The technical scheme adopted by the invention is as follows:

the invention provides an extraction method of camellia oil rich in polyphenol, which comprises the following steps: adding camellia seed kernel powder and camellia seed shell powder together into a solvent, and heating to deactivate enzyme (preferably pre-treating at 90 ℃ for 10min to deactivate enzyme); adjusting pH to 8-10 (preferably with 1M NaOH aqueous solution to 9) when cooling to 50-60deg.C (preferably 55deg.C), adding alkaline protease, hydrolyzing the mixture in 45-60deg.C water bath under continuous stirring at 100-300rpm for 3-6 hr (preferably 55deg.C, 200rpm, 4 hr); stirring at 80-100deg.C for 5-15min (preferably 90deg.C for 10 min), cooling to room temperature, centrifuging (preferably 8000rpm for 30 min), and collecting supernatant to obtain camellia oil rich in polyphenols; the solvent is one of deionized water, an ethanol water solution with the volume concentration of 20%, an isopropanol water solution with the volume concentration of 25%, a glucose water solution with the volume concentration of 0.15M, a KCl water solution with the volume concentration of 0.1M, an isopropanol water solution with the volume concentration of 25% containing 0.15M glucose and an isopropanol water solution with the volume concentration of 25% containing 0.1M KCl.

Further, the mass ratio of camellia seed kernel powder to solvent is 1:5-10, preferably 1:7, preparing a base material; the mass ratio of the camellia seed kernel powder to the camellia seed shell powder is 4-8:1, preferably 5:1, a step of; the enzyme activity of the alkaline protease is 20000U/mg, and the mass ratio of the alkaline protease to camellia seed kernel powder is 0.001-0.1:1, preferably 0.07:1.

Further, the solvent was an aqueous solution of 25% isopropyl alcohol containing 0.15M glucose by volume.

Further, camellia seed kernel powder is prepared according to the following steps: manually removing the shells of fresh camellia seeds, and drying camellia seeds at 50 ℃ for 4 hours until the mass water content is below 5% (preferably 3.6%); cooling, grinding, sieving with 40 mesh sieve, and storing at-18deg.C to obtain camellia seed kernel powder.

Further, camellia seed shell powder is prepared according to the following steps: fresh camellia seed is manually shelled, camellia seed shells are dried at 50 ℃ for 4 hours until the mass water content is below 5% (preferably 4.5%), cooled, ground and sieved by a 40-mesh sieve, and stored at-18 ℃ to obtain camellia seed shell powder.

The invention also provides an extraction method of the camellia oil rich in polyphenol, which comprises the following steps of adding fresh camellia seeds with shells into distilled water, crushing by a wet method, adding cellulase and alkaline protease, kneading and extracting for 60-120min under the assistance of 100-300W ultrasound at the temperature of 40-60 ℃, centrifuging (preferably centrifuging for 30min at 8000 rpm), and taking supernatant to obtain the camellia oil rich in polyphenol.

Further, the mass water content of the fresh camellia seed with the shell is 60%, and the mass ratio of the fresh camellia seed with the shell to distilled water is 1:1-5, preferably 1:1, a step of; the enzyme activity of the cellulase is preferably 10000U/g, and the mass ratio of the added amount to the fresh camellia seed is 0.005-0.1:1, preferably 0.01:1, a step of; the enzyme activity of the alkaline protease is preferably 20000U/g, and the mass ratio of the added mass to the fresh camellia seed is 0.001-0.01:1, preferably 0.007:1.

further, the ultrasonic-assisted extraction conditions were 150W, 50℃and 90min.

Compared with the prior art, the invention has the beneficial effects that: the camellia oil rich in polyphenol is obtained by taking camellia seed kernels and camellia seed shell powder as raw materials, taking a solvent containing glucose or salt as an extracting agent and extracting under the action of enzyme; or extracting fresh camellia seed with shell with water as extractant and ultrasound assisted enzyme to obtain camellia oil rich in polyphenols. The method not only improves the utilization rate of camellia seed shells, but also selects isopropanol or distilled water added with glucose as an extracting agent, reduces the solvent pollution problem of the traditional organic solvent extraction, and simultaneously solves the emulsification problem. The invention does not adopt organic solvent, has less emulsification, high yield, mild oil preparation condition, good quality, more nutrition, safer and healthier.

The camellia seed oil preparation condition is mild, the oil yield is high (up to 41.8 g of oil per 100g of camellia seed, the oil is close to an organic solvent extraction method, and the polyphenol content reaches 48.32 mg/kg), the trace nutrients (vitamin E, sterol, squalene and the like) and natural characteristic flavor of the camellia seed are reserved to the greatest extent, and particularly, the high-concentration and high-activity polyphenol functional factors are enriched, so that the obtained grease is more nutritious, safer and healthier.

(IV) description of the drawings

Fig. 1, and the liquid chromatograms of the compositions of camellia oil polyphenols obtained in examples 12, 13 and 14; curve a represents example 12, curve B represents example 13, and curve C represents example 14.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto: the Camellia seed (Camellia seed), which is an alias Camellia seed, is the fruit of Camellia oleifera, and belongs to the Camellia genus of Camellia.

Example 1 extraction of camellia oil by conventional organic solvent extraction

10Kg of fresh camellia seed is dried for 4 hours at 50 ℃, cooled, shelled, milled and sieved by a 40-mesh sieve, and stored at-18 ℃ to obtain 2.8Kg of camellia seed powder.

10g of camellia seed kernel powder is wrapped in filter paper and placed in a Soxhlet extractor with 180mL of n-hexane. After refluxing at 80℃for 8 hours, the solvent was removed by rotary evaporation at 50℃and the concentrate was collected as camellia oil, and the oil yield was calculated and the Total Phenol Content (TPC) in the oil was measured, with the results shown in Table 1.

Oil yield (%) = mass of oil/mass of camellia seed kernel powder x 100

Determination method of total phenol content (Folin-Ciocaltea method):

2g of camellia oil was dissolved in 4mL of n-hexane and then mixed with 2mL of ethanol-water (80:20, v/v). After vortex extraction for 3min, the lower supernatant was collected. Repeating the extraction for 2 times, combining the lower clear liquid, washing with normal hexane, and removing the normal hexane to obtain polyphenol extract. 1mL of the polyphenol extract was mixed with 1mL of a dilution of Folin-Ciocalteau reagent (dilution of Folin-Ciocalteau reagent with water in a volume ratio of 1:1). After 2min at room temperature, the mixture was admixed with 4mL of 10% Na by mass ₂ CO ₃ The aqueous solutions were mixed and reacted in the dark at 50℃for 1h. Finally, absorbance was measured at 725nm, and the result was calculated as gallic acid equivalent.

Polyphenol composition analysis method (UPLC-Triple-TOF/MS):

5g of camellia seed oil was extracted as above to polyphenol and concentrated to 1.5mL, and filtered through a 0.22 μm pore size filter, and the filtrate was analyzed by HPLC.

Conditions are as follows: HPLC analysis was performed by Agilent 1260 (Agilent, santa Clara, calif.) equipped with a reverse phase ZORBAX SB-C18 column (250 mm. Times.4 mm. Times.5 μm). The mobile phase is a combination of methanol (solvent a) and water (solvent B). Gradient elution procedure: 10% A for 5min; then raising the temperature to 20% A for 5min, and keeping the temperature for 5min; then raising the temperature to 30% A for 5min, and keeping the temperature for 5min; the gradient was repeated up to 100% a concentration. The flow rate of the mobile phase was 1.0mL/min. The phenolic compounds and the content were detected with a UV detector at 280 nm. Qualitative identification analysis of phenols in oil was performed by a UPLC Triple TOF/MS 5600plus system (AB Sciex Co., framingham, USA).

Examples 2 to 4 influence of the conventional organic solvent species on the extraction yield of camellia oil

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

Example 5 aqueous enzymatic extraction of camellia oil

10g of the camellia seed kernel powder prepared by the method of example 1 was added to 70g of deionized water and pre-treated at 90℃for 10min to inactivate enzymes. When the temperature was cooled to 55℃the pH was adjusted to 9 with a 1M aqueous NaOH solution, and 0.07g of alkaline protease (enzyme activity 20000U/g) was added. Then, the mixture was hydrolyzed in a 55℃water bath under continuous stirring at 200rpm for 4 hours. After stirring sterilization at 90℃for 10min and cooling to room temperature, the mixture was centrifuged at 8000rpm for 30min. The supernatant, namely 0.258g of camellia oil, was collected, the oil yield was calculated by the method of example 1, and the total phenol content in the oil was measured, and the results are shown in Table 1.

Examples 6-11 influence of different solvents on aqueous enzymatic extraction of camellia oil

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

Example 12 Simultaneous aqueous enzymatic method of seed husks

10Kg of fresh camellia seeds are manually shelled, camellia seed kernels and camellia seed shells are dried at 50 ℃ for 4 hours until the mass water content of the camellia seed kernels is 3.6 percent and the mass water content of the camellia seed shells is 4.5 percent respectively, cooled, ground into powder, screened by a 40-mesh sieve and stored at-18 ℃ to obtain 2.8Kg of camellia seed kernel powder and 1.9Kg of camellia seed shell powder.

10g of camellia seed kernel powder and 2g of camellia seed shell powder are added into 70g of 25% isopropyl alcohol aqueous solution containing 0.15M glucose by volume concentration, and the mixture is pretreated for 10min at 90 ℃ to inactivate enzymes. When the temperature was cooled to 55 ℃,1M NaOH aqueous solution was adjusted to pH 9, 0.07g alkaline protease (0.7% based on camellia seed mass) was added. Then, the mixture was hydrolyzed in a 55℃water bath under continuous stirring at 150rpm for 4 hours. After treatment at 90℃for 10min and cooling to room temperature, the mixture was centrifuged at 8000rpm for 30min. The supernatant, namely 0.403g of camellia oil, was collected, the oil yield was calculated by the method of example 1, the total phenol content in the oil was measured, the results are shown in Table 1, and the polyphenol content was analyzed.

Example 13 extraction of Total phenols from camellia oil by shearing

20g of refined camellia oil (refined camellia oil of Qiandao Hu Yao Ji) was weighed and added with 0.5g of camellia husk powder prepared by the method of example 12. Firstly, shearing and mixing for 1min by adopting a high-speed shearing machine under the condition of 10000rpm, and repeating for 3 times; then ultrasonic mixing is carried out for 30min under 200W; finally, the mixture was centrifuged at 8000rpm for 30min, and the supernatant was collected, and the total phenol content in camellia oil was measured by the method of example 1, and the results are shown in Table 1.

Example 14 extraction of Total phenols from Camellia oil by phase transfer

5g of camellia seed shell powder prepared by the method of example 12 was weighed, 10mL of ethanol was added, ultrasonic extraction was performed at 300W for 30 minutes, the mixed solution was centrifuged at 8000rpm for 30 minutes, the supernatant was mixed with 4g of refined camellia oil (Qianlia Yao note refined camellia oil), ultrasonic mixing was performed at 150W for 30 minutes, transferred to a 25mL round bottom flask, and the solvent was removed by rotary evaporation at 45℃to determine the polyphenol content in camellia oil by the method of example 1, and the results are shown in Table 1.

Example 15 fresh camellia seed oil production

500g of fresh camellia seed with shell (water content 60%) is weighed, 500g of distilled water is added, the camellia seed with shell is crushed by a wet method, 5g of cellulase (enzyme activity 10000U/g) and 3.5g of alkaline protease (enzyme activity 20000U/g) are added, kneading, mixing and extracting are carried out for 90 minutes at 50 ℃ under the assistance of 150W ultrasonic waves, centrifugation is carried out for 30 minutes at 8000rpm, supernatant fluid is taken, namely camellia oil rich in polyphenol, the oil yield and polyphenol content are measured by the method of example 1, and the results are shown in table 1.

TABLE 1 yields and Total phenol content of camellia oil with different extraction methods

Note that: -represents no addition and E represents addition of enzyme.

Table 1 data shows:

1. example 1 conventional solvent n-hexane extraction of clear oil yields were up to 42.27%, but the active polyphenol was not high, about 13.76mg/Kg. Example 2 pure ethanol extract oil yield is not high, only 32.3%, polyphenol content is up to 48.92mg/Kg; example 3 isopropyl alcohol clear oil yield 38.2% with a polyphenol content of 143.74 mg/Kg; example 4 acetone oil extraction rate and polyphenol content were both high. However, in examples 1-4, the conventional organic solvent extraction of camellia seed kernel (dehulling) to produce oil, although the oil yield is high and the polyphenol content is high, the oil produced by the organic solvent method needs high-strength refining, and most of the polyphenol is removed by the removal refining, so that the polyphenol concentration in the refined oil is lower than 10 mg/Kg. In addition, the organic solvent method has large scale and expensive equipment, and is mainly suitable for preparing large amount of oil materials such as soybean, rapeseed oil and the like. Inflammable and explosive, high safety risk, incapacity of effectively extracting phospholipid, environment friendliness, low quality of camellia oil extracted by an organic solvent, potential safety hazard of food brought by residual solvent in the oil and low consumer acceptance, so that the method is rarely applied to extracting camellia oil from original camellia seeds in practice.

2. Example 5 used basic aqueous enzymatic method, water as solvent, hydrolyzed camellia seed kernel (dehulled) to make oil, safe, but severely emulsified, low oil yield, and low polyphenol.

Example 5 the aqueous enzymatic process using dehulled camellia seed kernel as the starting material and water as the solvent resulted in severe emulsification with a clean oil yield of only 25.8%.

3. Examples 6-11 used an improved aqueous enzymatic process with the addition of a portion of an alcoholic solvent and salt or sugar to hydrolyze camellia seed kernel (dehulling) to make oil, the problem of emulsification was solved, the oil yield was improved, and the polyphenol content was also improved, but the requirement for high polyphenols was not met.

In examples 6-10, low concentration isopropanol, ethanol, KCl and glucose are introduced, so that emulsification can be effectively reduced, and the yield of the clear oil is improved to 33.3-41.8%. Importantly, the aqueous enzymatic method has mild conditions and avoids high-temperature exposure, so that the aqueous enzymatic method is green and safe, has good oil quality and enriches more trace nutrient substances such as vitamin E, squalene, sterol and the like. The disadvantage is that the content of active polyphenol in the oil is lower than 10 mg/Kg.

Example 11 camellia seed kernel dehulled as raw material, combined isopropanol and glucose solution as solvent, and aqueous enzymatic extraction of camellia oil was performed, camellia oil yield increased to 41.8%, and polyphenol content increased by 1-fold up to 13.82mg/Kg, which may be that the addition of sugar molecules reduced the polarity of the solution, allowing the saturated polyphenol fraction to transfer to the oil phase.

4. In example 12, the improved aqueous enzymatic method is adopted to hydrolyze camellia seed kernel and shell to prepare oil, so that the oil yield is improved, the polyphenol content is improved to 23 mg/kg, and the prepared oil is safe, does not need high-strength refining and has high nutrient retention.

In the embodiment 12, camellia seed kernel and partial shells are hydrolyzed together, the camellia oil yield is 40.3%, the polyphenol content is obviously improved to 23.1mg/Kg, the balance between the oil yield and nutrition is realized, meanwhile, the emulsification problem is solved, the utilization rate of camellia seed shells is improved, and the cost is reduced.

5. Example 13 oil extraction of polyphenols method, with commercially available refined camellia oil (almost no polyphenols) and camellia shell (high polyphenols, no oil) shear mixing extraction of polyphenols, the polyphenols content in the oil is raised to 19.67mg/Kg, the characteristic is that the process is simple and convenient. The disadvantage is that the commercially available camellia oil is mainly prepared by a squeezing method, wherein the content of vitamin E, sterol and squalene is not high.

In the example 14 phase transfer method, the ethanol extraction solution of camellia peel and camellia oil are mixed and dissolved according to a certain proportion, then the ethanol solvent is removed by evaporation, polyphenol in the ethanol is transferred into the oil phase, and the polyphenol content of the camellia oil is improved to 48.32mg/Kg. The disadvantage is that the process is slightly complicated, polyphenol and grease are required to be extracted respectively, and then the polyphenol and the grease are mixed and distilled to remove solvent.

Example 15 adopts fresh fruit (shell + kernel) enzyme method to prepare oil, the whole camellia fruit is not required to be dried, and is directly crushed and added with partial enzyme to prepare the camellia fruit, so that the camellia fruit is green, safe and nutritional.

Example 15 a freshly extracted camellia oil is developed from fresh fruits, and simultaneously, bio-enzyme is added to assist in enhancing mass transfer by ultrasonic physical field, the camellia oil yield is 36.9%, the polyphenol content is as high as 22.85mg/Kg, on one hand, protein bio-dissociation promotes precipitation of oil and grease, and meanwhile, dissolution of polyphenol substances into an oil phase is promoted, and in addition, cellulase possibly promotes decomposition and conversion of glycosylated polyphenol into nonpolar enhanced free phenol, so that transfer of the glycosylated polyphenol into the oil is facilitated. The polyphenol level in the oil has fully met the phenolic rich demand.

Comprehensive analysis of the polyphenol composition in the camellia oil of example 12 shows that the polyphenol composition of example 12 has high free phenol and glycosylated phenol content (see curve A in FIG. 1), good activity and more natural process. Whereas examples 13,14 have high polyphenol content but high flavonoid content, less active glycosylated phenol (see curve B, C in FIG. 1).

Claims

1. The extraction method of the camellia oil rich in polyphenol is characterized by comprising the following steps of: adding camellia seed kernel powder and camellia seed shell powder into a solvent together, heating and inactivating enzyme; when the temperature is cooled to 50-60 ℃, regulating the pH value to 8-10, adding alkaline protease, and hydrolyzing the mixed solution in a water bath at 45-60 ℃ under continuous stirring at 100-300rpm for 3-6 hours; stirring at 80-100deg.C for 5-15min, cooling to room temperature, centrifuging, and collecting supernatant to obtain camellia oil rich in polyphenols; the solvent is one of deionized water, an ethanol water solution with the volume concentration of 20%, an isopropanol water solution with the volume concentration of 25%, a glucose water solution with the volume concentration of 0.15M, a KCl water solution with the volume concentration of 0.1M, an isopropanol water solution with the volume concentration of 25% containing 0.15M glucose and an isopropanol water solution with the volume concentration of 25% containing 0.1M KCl.

2. The extraction method of the camellia oil rich in polyphenols as claimed in claim 1, wherein the mass ratio of camellia seed kernel powder to solvent is 1:5-10; the mass ratio of the camellia seed kernel powder to the camellia seed shell powder is 4-8:1, a step of; the enzyme activity of the alkaline protease is 20000U/mg, and the mass ratio of the alkaline protease to camellia seed kernel powder is 0.001-0.1:1.

3. the process for extracting a polyphenol-enriched camellia oil as claimed in claim 1, wherein the solvent is an aqueous solution of 25% isopropyl alcohol containing 0.15M glucose by volume.

4. The process for extracting a polyphenol-enriched camellia oil as claimed in claim 1, wherein the camellia seed kernel powder is prepared by the steps of: manually removing the shells of fresh camellia seeds, and drying camellia seeds at 50 ℃ until the mass water content is below 5%; cooling, grinding, and sieving with 40 mesh sieve to obtain camellia seed kernel powder.

5. The process for extracting a polyphenol-enriched camellia oil as claimed in claim 1, wherein the camellia seed shell powder is prepared by the steps of: fresh camellia seeds are manually shelled, camellia seed shells are dried at 50 ℃ until the mass water content is below 5%, cooled, ground and sieved by a 40-mesh sieve, and camellia seed shell powder is obtained.

6. A method for extracting camellia oil rich in polyphenol is characterized by comprising the following steps of adding fresh camellia seeds with shells into distilled water, crushing by a wet method, adding cellulase and alkaline protease, kneading and extracting for 60-120min under the assistance of 100-300W ultrasound at the temperature of 40-60 ℃, centrifuging, and taking supernatant to obtain camellia oil rich in polyphenol.

7. The method for extracting camellia oil rich in polyphenols as claimed in claim 6, wherein the mass water content of the fresh camellia seed with shell is 60%, and the mass ratio of the fresh camellia seed with shell to distilled water is 1:1-5; the enzyme activity of the cellulase is 10000U/g, and the mass ratio of the added amount to the fresh camellia seed is 0.005-0.1:1, a step of; the enzyme activity of the alkaline protease is 20000U/g, and the mass ratio of the added mass to the fresh camellia seed is 0.001-0.01:1.

8. the process for extracting a polyphenol-enriched camellia oil as claimed in claim 7, wherein the ultrasound-assisted extraction conditions are 150W, 50 ℃ for 90min.