CN118813332A - A method for preparing camellia oil by biological method and simultaneously enriching polyphenols in situ - Google Patents

Abstract

The invention discloses a method for preparing camellia oil by biological method and simultaneously enriching polyphenols in situ. The invention uses camellia seed kernel powder as raw material, improves the utilization rate of camellia seeds, uses isopropanol or ethanol aqueous solution containing glucose or salt as zero emulsification extractant, reduces the solvent pollution problem of traditional organic solvent extraction, solves the emulsification problem, and adopts one-step method or two-step method to add enzyme, and the mild conversion reaction catalyzed by glycosidase promotes the transfer of free polyphenols to oil phase, effectively improves the polyphenol content in camellia oil. The method of the invention adopts a small amount of organic solvent, realizes the purpose of zero emulsification, high oil yield and good polyphenol enrichment effect, and the oil preparation conditions are mild at the same time, the camellia oil yield is as high as 41.7 grams of oil/100g camellia seeds, the extraction rate is 92.5%, and the polyphenol content is nearly 95.72mg/kg, the micronutrients and natural characteristic flavor of camellia seeds are retained to the greatest extent, and the obtained oil is more nutritious, safer and healthier.

Description

A method for preparing camellia oil by biological method and simultaneously enriching polyphenols in situ

(I) Technical field

The invention relates to a method for preparing camellia oil by biological method and simultaneously enriching polyphenols in situ.

(II) Background technology

Camellia oil has similar physical and chemical properties and fatty acid composition to olive oil, so it is known as "Oriental olive oil". Unsaturated fatty acids account for about 90% of it. Long-term consumption can reduce the accumulation of low-density lipoprotein and help reduce the risk of cardiovascular disease. The health benefits of camellia oil are not only related to its high-quality fatty acids, but also closely related to trace active polyphenols, vitamin E, phytosterols, and squalene. Phenolic substances have attracted widespread attention due to their wide range of antioxidant activities and various biological activities such as anti-inflammatory, anti-tumor, and blood lipid regulation. However, polyphenols have strong polarity. At present, camellia oil is mainly produced by pressing, and the residual oil in the cake is mainly extracted by solvent. Therefore, the polyphenol content in the obtained camellia oil is extremely low, generally less than 10 mg/kg. At present, there are very few studies on enriching and retaining polyphenols in camellia oil. Patent 201810357004.3 discloses a method of obtaining camellia oil rich in tea polyphenols by additionally strengthening and adding tea polyphenols to camellia oil, with a content of up to 2.2%. However, the polyphenols are not from the camellia itself, but from tea extracts. Patent CN202111351707.3 discloses a process for the simultaneous extraction of polyphenol tea oil, tea oil polypeptides and tea oil polysaccharides. The process uses fresh camellia seeds as raw materials, which are crushed by colloid milling, colloid milling and homogenizer, and then hydrolyzed by cellulase and protease in turn, and finally ultrasonic combined with citric acid demulsification, and the final oil yield is about 35%. On the one hand, the process is cumbersome, and multiple colloid mill treatments cause serious emulsification, and the demulsification yield in the later stage is still not high. In addition, citric acid demulsification may cause oil hydrolysis and increased acid value, and the product is easy to oxidize and spoil, which leads to high subsequent refining consumption and low product yield. The aqueous enzyme method is increasingly valued because of its mild extraction conditions and better retention of the flavor of the oil itself and trace amounts of VE, sterols and other active substances. However, the aqueous enzyme method has a low oil yield and high cost, and its solubility in polar polyphenols is limited, resulting in the failure of effective extraction of important active substances. However, there are few reports on the green and efficient zero-emulsification oil production of camellia oil and the enrichment of polyphenols.

(III) Summary of the invention

The purpose of the present invention is to provide a method for preparing camellia oil by biological method and simultaneously enriching polyphenols in situ, firstly, dispersing camellia seed powder in isopropanol or ethanol containing sucrose, using cellulase and protease to hydrolyze and release camellia oil, and adding glycosidase or pectinase to continue hydrolysis synchronously or stepwise, converting glycosylated phenol with strong polarity in aqueous solution into glycosylated phenol or free phenol with weakened polarity, the latter of which has enhanced hydrophobicity and then transferred into camellia oil phase; on the other hand, because polyphenols in water phase are transferred into oil phase, the balance between polyphenols in water phase and residue is broken, and polyphenols in residue are further dissolved. The above process is carried out dynamically and continuously, and the enrichment of polyphenols in oil phase is continuously realized until the distribution balance of polyphenols in residue, water phase and oil phase is reached. This method has mild conditions, does not emulsify, has a high camellia oil yield (92-95%), and good quality. At the same time, polyphenols are enriched in situ (polyphenol content 46.4-95.7ppm), obtaining camellia oil with natural flavor, enhanced nutrition, improved safety, and improved quality. The process is green and environmentally friendly and has great application potential.

The technical solution adopted by the present invention is:

The present invention provides a method for preparing camellia oil and simultaneously enriching polyphenols in situ by a two-step biological method. The method is carried out according to the following steps: adding camellia kernel powder to an isopropanol or ethanol aqueous solution containing glucose, heating to inactivate enzymes (preferably stirring at 90° C. for 10 minutes for pretreatment to inactivate enzymes), when the temperature is cooled to 50-60° C. (preferably 55° C.), adjusting the pH to 7-9 (preferably using a 1M NaOH aqueous solution), adding cellulase or protease to release the camellia oil, and hydrolyzing the mixed solution in a 50-60° C. water bath at 100-200 rpm for 1-4 hours; (2) adjusting the pH of the reaction solution in step (1) to 5.0 (preferably using a 1M NaOH aqueous solution); HCl adjustment), glycosidase or pectinase is added to further hydrolyze and enrich polyphenols, the mixed solution is hydrolyzed for 1-4 hours in a water bath at 45-55°C and continuously stirred at 100-300 rpm, the enzyme is inactivated (preferably at 90°C for 10 minutes by stirring) and after cooling to room temperature, the mixture is centrifuged at 8000 rpm for 30 minutes, the supernatant is collected, and camellia oil rich in polyphenols is obtained.

Furthermore, the volume concentration of the isopropanol or ethanol aqueous solution is 10-30%, preferably 20-25%; the concentration of glucose in the isopropanol or ethanol aqueous solution is 0.1-0.5M, preferably 0.15M; the mass ratio of the camellia seed kernel powder to the isopropanol or ethanol aqueous solution containing glucose is 1:5-10, preferably 1:7; the mass ratio of the camellia seed kernel powder to protease or cellulase is 100:0.1-1, preferably 100:0.7; the mass ratio of the camellia seed kernel powder to glycosidase or pectinase is 100:0.1-1, preferably 100:0.7.

Furthermore, the hydrolysis conditions in step (1) are 55° C., 150 rpm, and 4 h. The hydrolysis conditions in step (2) are 50° C., 200 rpm, and 1 h.

Furthermore, the protease includes alkaline protease or acidic protease; the glycosidase includes α-glucosidase and β-glucosidase; and the pectinase includes acidic pectinase.

Further, the glycosidase or pectinase in step (2) is one of α-glucosidase, β-glucosidase or acid pectinase, or a mixture of α-glucosidase and β-glucosidase of equal quality. More preferably, alkaline protease is selected in step (1), and β-glucosidase or acid pectinase is selected in step (2).

The present invention also provides a one-step biological method for preparing camellia oil and simultaneously enriching polyphenols in situ, the method is carried out according to the following steps: adding camellia kernel powder to an isopropanol or ethanol aqueous solution containing glucose, inactivating enzymes (preferably stirring at 90°C for 10 minutes for pretreatment and inactivating enzymes), when the temperature is cooled to 50-60°C (preferably 55°C), adjusting the pH to 7-9 (adjusting with a 1M NaOH aqueous solution), adding cellulase or protease, and then adding glycosidase or pectinase, the mixed solution is hydrolyzed for 1-4 hours in a 45-55°C water bath and continuously stirred at 100-300rpm, inactivating enzymes (preferably stirring at 90°C for 10 minutes for sterilization) and cooling to room temperature, centrifuging the mixture at 8000rpm for 30 minutes, collecting the supernatant, and obtaining camellia oil rich in polyphenols.

Further, the volume concentration of the isopropanol or ethanol aqueous solution is 10-30%, preferably 25% isopropanol; 20% isopropanol; the concentration of glucose in the isopropanol or ethanol aqueous solution is 0.1-0.5M, preferably 0.15M; the mass ratio of the camellia kernel powder to the isopropanol or ethanol aqueous solution containing glucose is 1:5-10, preferably 1:7; the mass ratio of the camellia kernel powder to the protease or cellulase is 100:0.1-1, preferably 100:0.7; the mass ratio of the camellia kernel powder to the glycosidase or pectinase is 100:0.1-1, preferably 100:0.7. The protease includes alkaline protease or acidic protease; the glycosidase includes α-glucosidase and β-glucosidase; the pectinase includes acidic pectinase. More preferably, the cellulase or protease selects alkaline protease, and the glycosidase or pectinase selects alkaline pectinase or β-glucosidase.

Furthermore, the hydrolysis conditions were 50° C., 200 rpm, and 1 h.

Polyphenols usually include glycosylated polyphenols with strong polarity and free phenols with slightly weaker polarity. After normal hydrolysis, the residual polyphenols in the camellia residues and the dissolved polyphenols (in the solution) reach equilibrium, while the polyphenol distribution of the aqueous phase and the oil phase of the solution is balanced. The addition of glycosidase or pectinase converts the highly polar glycosylated phenols into partially glycosylated phenols and free phenols with enhanced hydrophobicity, and the latter can be transferred into the oil phase to promote the enrichment of polyphenols in the oil phase. On the other hand, the bound phenols in the aqueous phase migrate into the oil phase due to enzyme conversion, causing the phenol concentration in the aqueous phase to decrease, the phenol balance in the solid and liquid is broken, and the bound phenols in the camellia residues are released into the aqueous phase under the action of the enzyme (glycosidase). The above process continues until the phenol distribution is balanced, and finally a significant enrichment of phenols in the oil phase is achieved.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

The invention uses camellia seed kernel powder as a raw material, thereby improving the utilization rate of camellia seeds; uses isopropanol or ethanol aqueous solution containing glucose or salt as a zero-emulsification extractant, thereby reducing the solvent pollution problem of traditional organic solvent extraction and solving the emulsification problem; adopts a one-step method or a two-step method to add enzymes; a mild conversion reaction catalyzed by glycosidase promotes the transfer of free polyphenols to the oil phase, thereby effectively increasing the polyphenol content in camellia oil.

The method of the invention adopts a small amount of organic solvent, realizes the purpose of zero emulsification, high oil yield and good polyphenol enrichment effect, and at the same time, the oil preparation conditions are mild, the camellia oil yield is as high as 41.7 grams of oil/100 grams of camellia seeds, the extraction rate is 92.5%, and the polyphenol content is nearly 95.72 mg/kg. Compared with the conventional aqueous enzyme method (water is used as solvent) for preparing camellia oil, the emulsification is serious, the clear oil yield is higher, that is, 25.8 grams/100 grams of camellia seeds, and the oil extraction rate is 57.22%. The method of the invention retains the micronutrients (vitamin E, sterols, squalene, etc.) and natural characteristic flavor of camellia seeds to the greatest extent, 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

Figure 1. HPLC chromatogram of polyphenol-rich camellia oil.

(V) Specific implementation methods

The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

The camellia seed of the present invention, also known as oil tea seed, is the fruit of the oil tea tree, belonging to the family Theaceae and the genus Camellia. The camellia seed kernel powder used is prepared according to the following steps: 10 kg of fresh camellia seeds are dried at 50° C. for 4 hours, after cooling, shelled and ground to powder, passed through a 40-mesh sieve, and stored at -18° C. to obtain 2.8 kg of camellia seed kernel powder.

Alkaline protease (200000U/g), neutral protease (100000U/g), acidic protease (96000U/g), α-glucosidase (700000U/mL), β-glucosidase (100000U/g) and acidic pectinase (50000U/g) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; neutral cellulase (10000U/g) was purchased from Aladdin Biochemical Technology Co., Ltd.; alkaline pectinase (30000U/g) was purchased from Heshibi Biotechnology Co., Ltd.

Comparative Example 1: Hydrolysis of alkaline protease in aqueous solution

100g of camellia kernel powder was added to 700g of distilled water and stirred at 90℃ for 10min to pre-treat the enzyme. When the temperature was cooled to 55℃, 1M NaOH aqueous solution was used to adjust the pH to 9, 0.7g of alkaline protease (0.7% based on the mass of camellia kernel) was added, and the mixture was hydrolyzed for 4h in a 55℃ water bath and continuously stirred at 150rpm; after stirring and sterilizing at 90℃ for 10min and cooling to room temperature, the mixture was centrifuged at 8000rpm for 30min. The supernatant was collected to obtain 25.8g of camellia oil, and the oil yield and oil extraction rate were calculated. The total phenol content (TPC) in the oil was determined. Three parallel experiments were performed for each experiment, and the results were expressed as mean ± standard deviation, see Table 1.

Clear oil yield (%) = oil mass/camellia seed kernel powder mass × 100%

Oil extraction rate = mass of clear oil obtained by water enzymatic method from 100g tea seed kernel (g) / mass of oil content from 100g tea seed kernel (g) × 100%

Determination of total phenolic 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 clear liquid was collected. The extraction was repeated twice, and the lower clear liquid was combined and washed with n-hexane to remove the n-hexane to obtain a polyphenol extract. 1mL of the polyphenol extract was mixed with 1mL 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 2min, the mixture was mixed with 4mL of 10% Na ₂ CO ₃ aqueous solution and reacted in the dark at 50℃ for 1h. Finally, the absorbance was measured at 725nm, and the result was calculated as gallic acid equivalent.

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

5 g of camellia seed oil was dissolved in 10 mL of n-hexane and then mixed with 5 mL of ethanol-water (80:20, v/v). After vortex extraction for 3 min, the lower clear liquid was collected. The extraction was repeated twice, and the lower clear liquid was combined and washed with n-hexane. The n-hexane was removed to obtain a polyphenol extract, which was concentrated to 1.5 mL and filtered through a 0.22 μm pore size filter membrane. The filtrate was analyzed by HPLC.

Conditions: HPLC analysis was performed by Agilent 1260 (Agilent, Santa Clara, CA), which was equipped with a reverse 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 for 5 min; then 5 min to 20% A, maintained for 5 min; then 5 min to 30% A, maintained for 5 min; repeat the gradient until 100% A concentration. The flow rate of the mobile phase was 1.0 mL/min. Phenolic compounds and content were detected at 280 nm using a UV detector. Qualitative identification of phenols in the oil was performed by a UPLC Triple TOF/MS 5600plus system (AB Sciex Co., Framingham, USA).

Comparative Example 2: Two-step hydrolysis by alkaline protease/β-glucosidase in aqueous solution

(1) 100 g of camellia kernel powder was added to 700 g of distilled water and stirred at 90°C for 10 min to pre-treat the enzyme. When the temperature was cooled to 55°C, the pH was adjusted to 9 with 1 M NaOH aqueous solution, 0.7 g of alkaline protease (0.7% based on the mass of camellia kernel) was added, and the mixed solution was hydrolyzed in a 55°C water bath and continuously stirred at 150 rpm for 4 h; (2) The reaction solution of step (1) was adjusted to pH 5.0 with 1 M HCl, 0.7 mL of β-glucosidase was added, and the mixed solution was hydrolyzed in a 50°C water bath and continuously stirred 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 to obtain 26.0 g of camellia oil, and the clear oil yield and oil extraction rate were calculated. The total phenol content (TPC) in the oil was determined. The results are shown in Table 1.

Example 1: Hydrolysis of alkaline protease in a 25% isopropanol aqueous solution containing 0.15 M glucose

100g of camellia kernel powder was added to 700g of 25% isopropanol aqueous solution containing 0.15M glucose, and stirred at 90℃ for 10min to pre-treat and inactivate enzymes. When the temperature was cooled to 55℃, 1M NaOH aqueous solution was used to adjust the pH to 9, 0.7g of alkaline protease (0.7% based on the mass of camellia kernel) was added, and the mixture was hydrolyzed for 4h in a 55℃ water bath and continuously stirred at 150rpm; after stirring and sterilizing at 90℃ for 10min and cooling to room temperature, the mixture was centrifuged at 8000rpm for 30min. The supernatant was collected to obtain 25.8g of camellia oil, and the oil yield and oil extraction rate were calculated, and the total phenol content (TPC) in the oil was determined. The results are shown in Table 1.

Example 2: Hydrolysis of alkaline protease in 25% ethanol aqueous solution containing 0.15M glucose

The 25% isopropanol aqueous solution with a volume concentration of 0.15M glucose in Example 1 was replaced with 700g of 25% ethanol aqueous solution with a volume concentration of 0.15M glucose. Other operations were the same, and 36.14g of camellia oil was obtained. The oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 3: Hydrolysis of alkaline protease in 20% ethanol aqueous solution containing 0.15M glucose

The 25% isopropanol aqueous solution containing 0.15M glucose by volume in Example 1 was replaced with 700g of 20% ethanol aqueous solution containing 0.15M glucose by volume, and other operations were the same to obtain 39.68g of camellia oil. The oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1. The HPLC chromatogram is shown in the control group in Figure 1.

Example 4: Alkaline protease/α-glucosidase two-step hydrolysis—25% isopropanol system

(1) 100 g of camellia kernel powder was added to 700 g of 25% isopropanol aqueous solution containing 0.15 M glucose and pretreated at 90° C. for 10 min to inactivate the enzyme. When the temperature was cooled to 55° C., the pH was adjusted to 9 with 1 M NaOH aqueous solution, 0.7 g of alkaline protease (0.7% based on the mass of camellia kernel) was added, and the mixture was hydrolyzed in a 55° C. water bath with continuous stirring at 150 rpm for 4 h; (2) The reaction solution of step (1) was adjusted to pH 5.0 with 1 M HCl, 0.7 mL of α-glucosidase was added, and the mixture was hydrolyzed in a 50° C. water bath with continuous stirring at 200 rpm for 1 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, i.e. 42.1 g of polyphenol-rich camellia oil, and the clear oil yield and oil extraction rate were calculated. The total phenol content (TPC) in the oil was determined. The results are shown in Table 1. The HPLC analysis of the polyphenol composition is shown in Figure 1. α-glucosidase 1h.

Example 5: Alkaline protease/α-glucosidase two-step hydrolysis—25% isopropanol system

The hydrolysis time in step (2) of Example 4 was changed from 1 h to 4 h, and the other operations were the same to obtain 41.5 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1. The HPLC analysis of polyphenol composition is shown in Figure 1 for α-glucosidase 4 h.

Example 6: Alkaline protease/α-glucosidase two-step hydrolysis—20% ethanol system

The hydrolysis solution in step (1) of Example 4 was changed to a 20% ethanol aqueous solution containing 0.15 M glucose, and the hydrolysis time in step (2) was changed from 1 h to 4 h. Other operations were the same, and 40.6 g of polyphenol-rich camellia oil was obtained. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 7: Alkaline protease/β-glucosidase two-step hydrolysis—25% isopropanol system

The α-glucosidase in step (2) of Example 4 was replaced by 0.7 g of β-glucosidase. The other operations were the same to obtain 41.5 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 8: Alkaline protease/β-glucosidase two-step hydrolysis—25% isopropanol system

The α-glucosidase in step (2) of Example 4 was replaced by 0.7 g of β-glucosidase, and the hydrolysis time was changed from 1 h to 4 h. The other operations were the same to obtain 41.6 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 9: Alkaline protease/β-glucosidase two-step hydrolysis—20% ethanol system

The hydrolysis solution in step (1) of Example 4 was changed to a 20% ethanol aqueous solution containing 0.15 M glucose, the α-glucosidase in step (2) was replaced by 0.7 g β-glucosidase, and the hydrolysis time of 1 h was changed to 4 h. The other operations were the same to obtain 40.5 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 10: Alkaline protease/acidic pectinase two-step hydrolysis—25% isopropanol system

The α-glucosidase in step (2) of Example 4 was replaced by 0.7 g of acid pectinase, and the other operations were the same to obtain 40.8 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 11: Alkaline protease/acidic pectinase two-step hydrolysis—25% isopropanol system

The α-glucosidase in step (2) of Example 4 was replaced by 0.7 g of acid pectinase, and the hydrolysis time was changed from 1 h to 4 h. The other operations were the same to obtain 41.2 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 12: Alkaline protease/α-glucosidase two-step hydrolysis—20% ethanol system

The hydrolysis solution in step (1) of Example 4 was changed to a 20% ethanol aqueous solution containing 0.15 M glucose, the α-glucosidase in step (2) was replaced by 0.7 g of acid pectinase, and the hydrolysis time of 1 h was changed to 4 h. The other operations were the same to obtain 40.7 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 13, alkaline protease/α-glucosidase+β-glucosidase-25% isopropanol system

The α-glucosidase in step (2) of Example 4 was replaced by 0.35 g α-glucosidase + 0.35 g β-glucosidase. The other operations were the same to obtain 40.9 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 14: Alkaline protease/α-glucosidase+β-glucosidase-25% isopropanol system

The α-glucosidase in step (2) of Example 1 was replaced with 0.35 g α-glucosidase + 0.35 g β-glucosidase, and the hydrolysis time was changed from 1 h to 4 h. The other operations were the same to obtain 43.5 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 15: Alkaline protease/α-glucosidase+β-glucosidase-20% ethanol system

The hydrolysis solution in step (1) of Example 4 was changed to a 20% ethanol aqueous solution containing 0.15 M glucose, the α-glucosidase in (2) was replaced with 0.35 g α-glucosidase + 0.35 g β-glucosidase, and the hydrolysis time of 1 h was changed to 4 h. The other operations were the same to obtain 41.8 g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 16: Acid protease-β-glucosidase one-step simultaneous hydrolysis - 25% isopropanol system

100g of camellia kernel powder was added to 700g of 25% isopropanol aqueous solution containing 0.15M glucose, and stirred at 90°C for 10min to pre-treat and inactivate enzymes. When the temperature was cooled to 55°C, the pH was adjusted to 7 with 1M NaOH aqueous solution, 0.7g of acid protease (0.7% based on the mass of camellia kernel) and 0.7g of β-glucosidase were added, and the mixture was hydrolyzed for 4h in a 50°C water bath and continuously stirred at 200rpm. After stirring and sterilizing at 90°C for 10min and cooling to room temperature, the mixture was centrifuged at 8000rpm for 30min. The supernatant was collected to obtain 39.9g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Example 17: Acid protease-β-glucosidase one-step simultaneous hydrolysis - 20% ethanol system

The 25% isopropanol aqueous solution containing 0.15M glucose in Example 16 was replaced with a 20% ethanol aqueous solution containing 0.15M glucose. Other operations were the same to obtain 40.2g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate, determination and total phenol content in the oil are shown in Table 1.

Example 18: Alkaline protease-alkaline pectinase one-step simultaneous hydrolysis - 20% ethanol system

100g of camellia kernel powder was added to 700g of 25% isopropanol aqueous solution containing 0.15M glucose, and stirred at 90°C for 10min to pre-treat the enzyme. When the temperature was cooled to 55°C, the pH was adjusted to 9 with 1M NaOH aqueous solution, 0.7g of alkaline protease (0.7% based on the mass of camellia kernel) and 0.7g of alkaline pectinase were added, and the mixture was hydrolyzed for 4h in a 55°C water bath and continuously stirred at 200rpm. After stirring and sterilizing at 90°C for 10min and cooling to room temperature, the mixture was centrifuged at 8000rpm for 30min. The supernatant was collected to obtain 41.9g of polyphenol-rich camellia oil. The clear oil yield, oil extraction rate and total phenol content in the oil are shown in Table 1.

Table 1. Yield and total phenol content of camellia oil under different extraction methods (theoretical oil content 45.09%)

Note: The solvent is 0.15M glucose volume concentration 25% isopropanol solution, and the mass ratio of camellia seed powder to solvent is 1:7.

Comparative Example 1 shows that the conventional aqueous enzyme method (water as solvent) produces camellia oil with serious emulsification, and the higher oil yield is 25.8g/100g camellia seeds, and the oil extraction rate is 57.22%. Comparative Example 2 uses pure water as solvent, has serious emulsification, low oil yield, and even if glycosidase is added, the polyphenol enrichment effect is not obvious. Example 1 uses 25% isopropanol aqueous solution containing 0.15M glucose as solvent, emulsification is basically eliminated, and the oil yield is increased to 41.8g/100g camellia seeds (oil extraction rate 92.7%), which is significantly improved, and the functional polyphenol content in the oil is increased from 4.35 to 13.82mg/kg. The solvents of Examples 2 and 3 are replaced with safer ethanol solutions containing 0.15M glucose 20% or 25%, and the oil extraction rate is still between 80-88%, and the polyphenol content is 8-10mg/kg. Although the new water enzyme system solves the emulsification problem and increases the polyphenol content in the oil, it is close to the oil yield (42.27g/100g camellia seeds) and polyphenol content (13.76mg/Kg) of the solvent extraction method, but it is still far lower than the 60-100mg/kg of olive oil. The highest clear oil yield of the traditional solvent n-hexane extraction is 42.27%, but the active polyphenols are not high, about 13.76mg/Kg (patent 2023114373069).

In Example 4-18, ethanol or isopropanol aqueous solution containing glucose was used as the extractant. The addition of hydrolase that hydrolyzes glycosidic bonds during protein hydrolysis to produce oil can effectively achieve the enrichment of polyphenols in camellia oil. Regardless of simultaneous hydrolysis or step-by-step hydrolysis, the content of polyphenols in the oil increased by about 2.5 to 4 times, reaching 46.35 to 95.72 mg/kg. This shows that ethanol and isopropanol glucose solutions not only inhibit emulsification, but also cooperate with glycosidase and pectinase to promote the migration of polyphenols into the oil phase.

In summary, β-glucosidase has the best effect on polyphenol enrichment, and the highest polyphenol concentration in camellia oil is nearly 100ppm, which may be related to the high abundance of β-glycosides in glycosylated phenols. Secondly, α-glucosidase and glycoside isozyme pectinase have similar effects, and the concentration of phenols in camellia oil is about 50ppm. The combination of α-glycosidase and β-glycosidase has a better effect, and the phenol concentration is increased to nearly 90%, but it does not exceed that of β-glycosidase alone. The simultaneous hydrolysis effect of protease and glycosidase is slightly lower than that of step-by-step hydrolysis. The enrichment effect of polyphenols improves with the increase of glycosidase hydrolysis time, but the difference between 1h and 4h is not very significant. For the hydrolysis solution system, the 25% isopropanol aqueous solution system containing 0.15M glucose has the highest free oil yield of 90-97%, and the best polyphenol enrichment effect of 46.35-95.72 mg/kg. The second is 20% ethanol aqueous solution containing 0.15M glucose, with a clear oil extraction rate of 87.80-90.26% and a polyphenol concentration of 49.98-77.59 mg/kg. When the ethanol concentration increased to 25%, the clear oil recovery rate and the polyphenol concentration in the oil decreased, which may be related to the inhibition of enzyme activity.

Claims (10)

1. The method for preparing camellia oil by a two-step biological method and simultaneously enriching polyphenol in situ is characterized by comprising the following steps of: adding camellia seed kernel powder into isopropanol or ethanol water solution containing glucose, heating to deactivate enzyme, cooling to 50-60deg.C, adjusting pH to 7-9, adding cellulase or protease, and hydrolyzing the mixed solution in water bath at 50-60deg.C under continuous stirring at 100-200rpm for 1-4 hr; (2) Regulating the pH value of the reaction liquid in the step (1) to 5.0, adding glycosidase or pectase, hydrolyzing the mixed liquid for 1-4 hours in a water bath at 45-55 ℃ under continuous stirring at 100-300rpm, inactivating enzyme, cooling to room temperature, centrifuging the mixture at 8000rpm for 30min, and collecting the supernatant to obtain the camellia oil rich in polyphenol.

2. The method of claim 1, wherein the volume concentration of the aqueous isopropanol or ethanol solution is 10-30%, the concentration of the glucose in the aqueous isopropanol or ethanol solution is 0.1-0.5M, the mass ratio of the camellia seed kernel powder to the aqueous isopropanol or ethanol solution containing glucose is 1:5-10, the mass ratio of the camellia seed kernel powder to the protease or cellulase is 100:0.1-1, and the mass ratio of the camellia seed kernel powder to the glycosidase or pectase is 100:0.1-1.

3. The process of claim 1, wherein the hydrolysis conditions of step (1) are 55 ℃, 150rpm, 4 hours; the hydrolysis condition in the step (2) is 50 ℃, 200rpm and 1h.

4. The method of claim 1, wherein the protease comprises an alkaline protease or an acidic protease; the glycosidase comprises alpha-glucosidase and beta-glucosidase; the pectinase comprises an acid pectinase.

5. The method of claim 1, wherein the glycosidase or pectase of step (2) is one of an alpha-glucosidase, a beta-glucosidase or an acid pectase or a mixture of alpha-glucosidase and beta-glucosidase of equal mass.

6. The method of claim 1, wherein step (1) selects an alkaline protease and step (2) selects a β -glucosidase or an acid pectinase.

7. A method for preparing camellia oil by a one-step biological method and simultaneously enriching polyphenol in situ is characterized by comprising the following steps: adding camellia seed kernel powder into isopropanol or ethanol water solution containing glucose, inactivating enzyme, cooling to 50-60deg.C, adjusting pH to 7-9, adding cellulase or protease, adding glycosidase or pectase, hydrolyzing the mixed solution in 45-55deg.C water bath under continuous stirring at 100-300rpm for 1-4 hr, inactivating enzyme, cooling to room temperature, centrifuging the mixture at 8000rpm for 30min, and collecting supernatant to obtain camellia oil rich in polyphenols.

8. The method of claim 7, wherein the aqueous isopropanol or ethanol solution has a volume concentration of 10-30%, the concentration of glucose in the aqueous isopropanol or ethanol solution is 0.1-0.5M, the mass ratio of camellia seed kernel powder to aqueous isopropanol or ethanol solution containing glucose is 1:5-10, the mass ratio of camellia seed kernel powder to protease or cellulase is 100:0.1-1, and the mass ratio of camellia seed kernel powder to glycosidase or pectase is 100:0.1-1.

9. The method of claim 7, wherein the protease comprises an alkaline protease or an acidic protease; the glycosidase comprises alpha-glucosidase and beta-glucosidase; the pectinase comprises an acid pectinase.

10. The method of claim 7, wherein the cellulase or protease selects an alkaline protease and the glycosidase or pectinase selects an alkaline pectinase or β -glucosidase.