Preparation method of camellia oleosa seed oil with high oxidation resistance

Abstract

The invention discloses a preparation method of camellia oleifera seed oil with high oxidation resistance, which comprises the following steps: 1) preparing a fermentation substrate: pretreating camellia oleifera seed shells and camellia oleifera fruits to obtain the fermentation substrate; (2) preparing a fermentation culture medium: preparing the fermentation culture medium by adopting a fermentation substrate, distilled water and a Basal culture medium as raw materials; (3) chlorella fermentation: inoculating the activated chlorella into a fermentation culture medium for high-temperature fermentation to obtain an intermediate product; 4) fermentation of the Antarctic micrococcus: firstly cooling the intermediate product, and then inoculating the activated Antarctic micrococcus into the cooled intermediate product for low-temperature fermentation to obtain a fermentation product; 5) coenzyme Q0 treatment: adding coenzyme Q0 into the fermentation product for enzymolysis and inactivation to obtain a fermentation enzymatic hydrolysate; and 6) squeezing: drying and steaming the fermented enzymatic hydrolysate, and squeezing to obtain the camellia oleosa seed oil with high oxidation resistance. According to the method, the EPA content and the oxidation resistance in the camellia oleosa seed oil are improved.

Description

Technical Field

[0001] The invention relates to the technical field of camellia oil preparation, and in particular to a method for preparing camellia oil with high antioxidant properties. Background Art

[0002] Camellia oil is a high-quality edible oil and a natural green food. Camellia oil has the effects of anti-oxidation, anti-inflammatory, improving immunity, and helping to lower blood lipids. Its effects are due to the trace components in camellia oil, such as sterols, squalene, tea polyphenols, etc. At the same time, camellia oil is rich in polyunsaturated fatty acids, especially oleic acid, and the relative content of eicosapentaenoic acid (EPA) is very low.

[0003] EPA is a ω-3 polyunsaturated fatty acid (PUFA) that is mainly derived from marine animals such as shellfish and fish, while the content of EPA in camellia oil produced by pressing is extremely low. However, EPA has strong antioxidant properties and multiple functional activities such as improving immunity, anti-thrombosis, anti-tumor, reducing cardiovascular and cerebrovascular diseases, and preventing neurodegenerative diseases, and is known as an important bioactive nutrient.

[0004] In order to make camellia oil have the above functional activity conferred by EPA and to increase the content of EPA in camellia oil, the commonly used method at present is to mix camellia oil and fish oil to prepare blended oil, such as:

[0005] CN201010173183.9 discloses a health oil, which is mixed with fish oil and vegetable oil, and the amount of fish oil is 3-60% of the weight of the vegetable oil, and the vegetable oil is one or more of sunflower seed oil, olive oil, sesame oil, cottonseed oil, coconut oil, tea seed oil, rice bran oil, linseed oil, safflower oil, walnut oil, grape seed oil and pumpkin seed oil. In this health oil, fish oil and vegetable oil are mixed together, and fish oil is attached to food during cooking, so that fish oil can be easily taken, and the elderly and children are also easy to accept, and the problem of forgetting to eat is avoided at the same time. For children, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) contained in fish oil can improve brain capacity, enhance memory and comprehension. For the elderly, it can slow down atherosclerosis, reduce hypercholesterolemia, plasma cholesterol and triglycerides, and reduce plasma lipoprotein concentration, blood viscosity and blood pressure.

[0006] CN201910098024.8 discloses a human nutrition functional edible oil composition rich in DHA and EPA, which is configured with the following raw materials in mass percentage: 70-99% vegetable oil, 1-30% strong-flavor vegetable oil, 0.003-15% fish oil, and 0.05%-0.3% trace antioxidant; each 100g blended oil composition contains a product with a total amount of DHA and EPA of 1mg-199mg and 601mg-3000mg, which is suitable for nutritional strengthening of the general population and special populations, respectively. The vegetable oil is a mixture of one or more of cottonseed oil, soybean oil, rapeseed oil, peanut oil, rice bran oil, tea seed oil, sunflower seed oil, sesame oil, corn oil, linseed oil, walnut oil, safflower seed oil, grape seed oil, olive oil and safflower seed oil.

[0007] The above methods are all prepared in the form of blended oil by exogenously adding EPA (i.e., adding fish oil) to camellia oil to increase the EPA content in the oil. However, the EPA in the blended oil has poor stability during high-temperature cooking, which increases the loss of EPA. In addition, fish oil has a certain fishy smell, and most consumers are disgusted by the fishy smell of fish oil, which will greatly affect the flavor of camellia oil. The content of polyunsaturated fatty acids in fish oil is very high and is easily oxidized, which significantly reduces the antioxidant capacity of camellia oil. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for preparing camellia oil with high antioxidant properties, which has a simple process, a short fermentation time and can effectively increase the content and stability of EPA.

[0009] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing camellia oil with high antioxidant properties, comprising the following steps:

[0010] 1) Preparing a fermentation substrate: pretreating camellia oleifera seed shells and camellia oleifera fruits to obtain a fermentation substrate;

[0011] 2) preparing a fermentation medium: using a fermentation substrate, distilled water and Basal medium as raw materials to prepare a fermentation medium;

[0012] 3) Chlorella fermentation: inoculating the activated Chlorella into the fermentation medium for high temperature fermentation to obtain an intermediate product;

[0013] 4) Antarctic micrococcus fermentation: firstly cooling the intermediate product, then inoculating the activated Antarctic micrococcus into the cooled intermediate product for low-temperature fermentation to obtain a fermentation product;

[0014] 5) Coenzyme Q0 treatment: adding coenzyme Q0 to the fermentation product for enzymolysis and inactivation to obtain a fermentation enzymolysis solution;

[0015] 6) Pressing: The fermented enzymatic hydrolysate is dried, steamed and then pressed to obtain camellia seed oil with high antioxidant properties.

[0016] Furthermore, the specific operation steps of step 1) are: placing the camellia seed shells and camellia fruits in an oven at a temperature of 35°C to 50°C for 3h to 5h respectively, taking them out, mixing the dried camellia seed shells and camellia fruits according to a shell content of 6% to 8%, and then crushing them to 80 mesh to 120 mesh to obtain a fermentation substrate.

[0017] Furthermore, the specific operation steps of step 2) are: adding distilled water to the fermentation substrate at a solid-liquid ratio of 1:8 to 1:12, heating in a boiling water bath for 40 to 60 minutes, and then cooling to room temperature and mixing with Basal medium at a volume ratio of 1:4 to 1:6, and high-pressure sterilization at 121°C for 15 minutes, with a pH of 5 to 7.

[0018] Preferably, the solid-liquid ratio of the fermentation substrate to distilled water is 1:10.

[0019] Preferably, the fermentation substrate after adding distilled water is cooled to room temperature and mixed with Basal medium in a volume ratio of 1:6.

[0020] Furthermore, the composition of the Basal medium is: NaNO3 1250mg / L, KHPO4 1250mg / L, MgSO4·7H2O1000mg / L, EDTA 500mg / L, H3BO3 114.2mg / L, CaCl2·2H2O 111mg / L, FeSO4·7H2O49.8mg / L, ZnSO4·7H2O 88.2mg / L, MoO3 7.1mg / L, MnCl2·4H2O 14.2mg / L, CuSO4·5H2O15.7mg / L, CoNO3·6H2O 6.1mg / L, glucose 10g / L, urea 3g / L, the solvent is deionized water, and the pH is 6.14±0.05.

[0021] Furthermore, the specific operation steps of step 3) are as follows: first, the activated Chlorella is prepared into a cell density of 2.5×10 7 cells / mL of seed liquid, and then inoculating the seed liquid into a fermentation medium at a volume percentage of 4%-10% for high-temperature fermentation to obtain an intermediate product.

[0022] Preferably, the volume percentage of the Chlorella seed liquid to the fermentation medium is 4%.

[0023] Furthermore, the temperature of the high temperature fermentation is 30° C. and the fermentation time is 1 day.

[0024] Furthermore, the specific operation steps of step 4) are: firstly cool the intermediate product to 16°C, and then prepare the activated Antarctic micrococci to a cell density of 1×10 8 cells / mL of seed liquid, and then inoculating the seed liquid into the cooled intermediate product at a volume percentage of 1%-2% to perform low-temperature fermentation to obtain a fermentation product.

[0025] Preferably, the volume percentage of the Antarctic Micrococcus seed solution and the intermediate product is 1.5%.

[0026] Furthermore, the temperature of the low-temperature fermentation is 16° C., and the fermentation time is 1 day.

[0027] Furthermore, the specific operation steps of step 5) are: first dissolve coenzyme Q0 with acetone, then add the dissolved coenzyme Q0 to the fermentation product to make the concentration of coenzyme Q0 in the fermentation product 0.25 mg / mL to 0.35 mg / mL, and inactivate it after enzymatic hydrolysis at 16°C for 24 hours to obtain a fermentation hydrolyzate.

[0028] Furthermore, the specific operation steps of the inactivation are: bathing the enzymatically hydrolyzed solution at 60° C. for 45 minutes, and then cooling it in 4° C. ice water for 10 minutes.

[0029] Furthermore, in step 6), the drying temperature is 60° C. and the time is 1 hour; the steaming temperature is 70° C. and the time is 30 minutes; the pressing temperature is 65° C. and the pressing time is 23 minutes.

[0030] The beneficial effects of the method for preparing camellia oil with high antioxidant properties of the present invention are as follows:

[0031] (1) The preparation process of the present invention is simple, using camellia oleifera seed shells and camellia oleifera fruit as raw materials, firstly performing high temperature fermentation using Chlorella, then performing low temperature fermentation using Antarctic Micrococcus, and then performing enzymatic hydrolysis using coenzyme Q0 and then pressing to prepare camellia oleifera oil. The entire fermentation process after inoculation is controlled within 2 days, which can greatly shorten the fermentation time; and in the preparation process, fish oil with a fishy smell is not added, and the content of EPA in the camellia oleifera oil can be effectively increased, which can greatly improve the flavor and antioxidant properties of the camellia oleifera oil.

[0032] (2) When preparing camellia oil by fermentation, the present invention first uses Chlorella to ferment the fermentation medium prepared from camellia seed shells and camellia fruits, and then uses Antarctic Micrococcus to ferment. Under the synergistic effect of Chlorella and Antarctic Micrococcus, the content of acetyl coenzyme A for synthesizing EPA can be increased, thereby effectively promoting the synthesis of EPA, so that the content of EPA in the camellia oil can be significantly increased. In addition, during the growth process of Chlorella and Antarctic Micrococcus, the cells themselves will also generate and accumulate EPA, thereby greatly increasing the content of EPA in the camellia oil.

[0033] (3) The camellia oil prepared by the present invention meets the standard of camellia crude oil "GB / T 11765-2018 Camellia Oil", and the loss rate of EPA in the prepared camellia oil under high temperature conditions (140-180°C) is less than 1%, which effectively improves the stability of EPA in camellia oil at high temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 —is a process flow chart of a method for preparing camellia oil with high antioxidant properties according to the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with the accompanying drawings and examples, but these specific implementation schemes do not limit the protection scope of the present invention in any way.

[0036] The strains used in the present invention are:

[0037] Chlorella kessleri is a common single-cell green algae of the genus Chlorella in the Chlorophyta. It is a spherical single-cell freshwater algae. It is a strain existing in the prior art. The strain number is ATCC11468. The purchase website is http: / / www.biofeng.com / ATCC / junzhong / ATCC11468.html. Micrococcus antarcticus is an Antarctic microorganism that can grow and reproduce in extremely cold environments. The number is D70591. The purchase website is https: / / www.biomart.cn / infosupply / 113313682.htm.

[0038] Example 1

[0039] A method for preparing camellia oil with high antioxidant properties, the process flow chart of which is as follows Figure 1 As shown, the following steps are included:

[0040] 1) preparing a fermentation substrate: drying camellia oleifera seed shells and camellia oleifera fruits in an oven at 45° C. for 4 h, taking them out, mixing the dried camellia oleifera seed shells and camellia oleifera fruits at a shell content of 7%, and then crushing them into 100 meshes to obtain a fermentation substrate;

[0041] 2) preparing fermentation medium: adding distilled water to the fermentation substrate at a solid-liquid ratio of 1:10, heating in a boiling water bath for 50 min, then cooling to room temperature and mixing with Basal medium at a volume ratio of 1:6, and autoclaving at 121° C. for 15 min, with a pH of 5 to 7;

[0042] The composition of the Basal medium is: NaNO3 1250mg / L, KHPO4 1250mg / L, MgSO4·7H2O1000mg / L, EDTA 500mg / L, H3BO3 114.2mg / L, CaCl2·2H2O 111mg / L, FeSO4·7H2O 49.8mg / L, ZnSO4·7H2O88.2mg / L, MoO3 7.1mg / L, MnCl2·4H2O 14.2mg / L, CuSO4·5H2O 15.7mg / L, CoNO3·6H2O 6.1mg / L, glucose 10g / L, urea 3g / L, the solvent is deionized water, and the pH is 6.14±0.05; if solid Basal medium is required, 2% agar needs to be added;

[0043] 3) Chlorella fermentation: The activated Chlorella is inoculated into the fermentation medium for high temperature fermentation to obtain an intermediate product; the specific operation steps are as follows:

[0044] a) Activation of Chlorella: Take a piece of Chlorella moss on Basal slant medium and inoculate it on Basal solid plate medium sterilized at 121℃ for 15min. 2 / s light incubator for 7 days to obtain an activated plate;

[0045] b) Preparation of seed solution of Chlorella: A single algae colony was picked from the above-mentioned activated plate and inoculated into a 50 mL conical flask with 20 mL of Basal medium. The flask was placed in a constant temperature shaker at 30°C and 160 r / min for 4 days to prepare seed solution. The solution was then diluted with Basal medium to a cell density of 2.5×10 7 cells / mL of seed solution;

[0046] c) Inoculation of Chlorella: The cell density is 2.5×10 7 The seed liquid of Chlorella vulgaris cells / mL was inoculated into the fermentation medium at a volume percentage of 4%, and high-temperature fermentation was carried out at a temperature of 30°C for 1 day to obtain an intermediate product;

[0047] 4) Antarctic micrococcus fermentation: firstly cooling the intermediate product, and then inoculating the activated Antarctic micrococcus into the cooled intermediate product for low-temperature fermentation to obtain a fermentation product; the specific steps are as follows:

[0048] d) Cooling: Cooling the intermediate product to 16°C;

[0049] e) Activation of Antarctic Micrococcus: Take a strain of 2216E slant medium from an environment and inoculate it into 5 mL of 2216E solid plate medium precooled to 16°C, and culture it for 7 days to obtain an activated plate; the preparation composition of 2216E medium is: yeast extract powder 1.0 g / L, peptone 5.0 g / L, FePO4·2H2O 0.01 g / L, deionized water to volume, pH 7.5, and wet heat sterilization for 20 minutes; if solid 2216E medium is required, 2% agar needs to be added;

[0050] f) Preparation of seed solution of Antarctic Micrococcus: A single colony was picked from the above-mentioned activated plate and inoculated into a 50 mL conical flask with 20 mL of 2216E medium. The flask was placed in a constant temperature shaker at a temperature of 16°C and a rotation speed of 160 r / min for 3 days to prepare seed solution. The bacterial suspension was collected by centrifugation at 5000 r / min for 5 minutes, and washed and diluted with sterile water to prepare a cell density of 1×10 8 cells / mL of seed solution;

[0051] g) Inoculation of Antarctic Micrococcus: The cell density was 1×10 8 cells / mL of Antarctic Micrococcus seed solution was inoculated into the intermediate product at a volume percentage of 1.5%, and low-temperature fermentation was performed at a temperature of 16°C for 1 day to obtain a fermentation product;

[0052] 5) Coenzyme Q0 treatment: first dissolve the coenzyme Q0 with acetone (dissolved in the form of a coenzyme Q0 to acetone solid-liquid ratio of 1:100), then add the dissolved coenzyme Q0 to the fermentation product to make the concentration of coenzyme Q0 in the fermentation product 0.3 mg / mL, enzymolyze at 16°C for 24 hours, then place the enzymolyzed solution in a water bath at 60°C for 45 minutes, and cool in 4°C ice water for 10 minutes to inactivate, to obtain a fermentation enzymolyzate;

[0053] 6) Pressing: The fermentation hydrolysate is dried at 60°C, then steamed at 70°C for 30 minutes, and finally pressed at 65°C for 23 minutes to obtain camellia oil with high antioxidant properties.

[0054] Example 2

[0055] A method for preparing camellia oil with high antioxidant properties comprises the following steps:

[0056] 1) preparing a fermentation substrate: drying camellia oleifera seed shells and camellia oleifera fruits in an oven at 35° C. for 3 h, taking them out, mixing the dried camellia oleifera seed shells and camellia oleifera fruits at a shell content of 6%, and then crushing them into 80 meshes to obtain a fermentation substrate;

[0057] 2) preparing fermentation medium: adding distilled water to the fermentation substrate at a solid-liquid ratio of 1:8, heating in a boiling water bath for 40 min, then cooling to room temperature and mixing with Basal medium at a volume ratio of 1:4, and sterilizing by high pressure at 121° C. for 15 min, with a pH of 5 to 7;

[0058] The composition of the Basal medium is: NaNO3 1250mg / L, KHPO4 1250mg / L, MgSO4·7H2O1000mg / L, EDTA 500mg / L, H3BO3 114.2mg / L, CaCl2·2H2O 111mg / L, FeSO4·7H2O 49.8mg / L, ZnSO4·7H2O88.2mg / L, MoO3 7.1mg / L, MnCl2·4H2O 14.2mg / L, CuSO4·5H2O 15.7mg / L, CoNO3·6H2O 6.1mg / L, glucose 10g / L, urea 3g / L, the solvent is deionized water, and the pH is 6.14±0.05; if solid Basal medium is required, 2% agar needs to be added;

[0059] 3) Chlorella fermentation: The activated Chlorella is inoculated into the fermentation medium for high temperature fermentation to obtain an intermediate product; the specific operation steps are as follows:

[0060] a) Activation of Chlorella: Take a piece of Chlorella moss on Basal slant medium and inoculate it on Basal solid plate medium sterilized at 121℃ for 15min. 2 / s light incubator for 7 days to obtain an activated plate;

[0061] b) Preparation of seed solution of Chlorella: A single algae colony was picked from the above-mentioned activated plate and inoculated into a 50 mL conical flask with 20 mL of Basal medium. The flask was placed in a constant temperature shaker at 30°C and 160 r / min for 4 days to prepare seed solution. The solution was then diluted with Basal medium to a cell density of 2.5×10 7 cells / mL of seed solution;

[0062] c) Inoculation of Chlorella: The cell density is 2.5×10 7 The seed liquid of Chlorella vulgaris cells / mL was inoculated into the fermentation medium at a volume percentage of 6%, and high-temperature fermentation was carried out at a temperature of 30°C for 1 day to obtain an intermediate product;

[0063] 4) Antarctic micrococcus fermentation: firstly cooling the intermediate product, and then inoculating the activated Antarctic micrococcus into the cooled intermediate product for low-temperature fermentation to obtain a fermentation product; the specific steps are as follows:

[0064] d) Cooling: Cooling the intermediate product to 16°C;

[0065] e) Activation of Antarctic Micrococcus: Take a strain of 2216E slant medium from an environment and inoculate it into 5 mL of 2216E solid plate medium precooled to 16°C, and culture it for 7 days to obtain an activated plate; the preparation composition of 2216E medium is: yeast extract powder 1.0 g / L, peptone 5.0 g / L, FePO4·2H2O 0.01 g / L, deionized water to volume, pH 7.5, and wet heat sterilization for 20 minutes; if solid 2216E medium is required, 2% agar needs to be added;

[0066] f) Preparation of seed solution of Antarctic Micrococcus: A single colony was picked from the above-mentioned activated plate and inoculated into a 50 mL conical flask with 20 mL of 2216E medium. The flask was placed in a constant temperature shaker at a temperature of 16°C and a rotation speed of 160 r / min for 3 days to prepare seed solution. The bacterial suspension was collected by centrifugation at 5000 r / min for 5 minutes, and washed and diluted with sterile water to prepare a cell density of 1×10 8 cells / mL of seed solution;

[0067] g) Inoculation of Antarctic Micrococcus: The cell density was 1×10 8 cells / mL of Antarctic Micrococcus seed solution was inoculated into the intermediate product at a volume percentage of 1%, and low-temperature fermentation was carried out at a temperature of 16°C for 1 day to obtain a fermentation product;

[0068] 5) Coenzyme Q0 treatment: first dissolve the coenzyme Q0 with acetone (dissolved in the form of a coenzyme Q0 to acetone solid-liquid ratio of 1:100), then add the dissolved coenzyme Q0 to the fermentation product to make the concentration of coenzyme Q0 in the fermentation product 0.25 mg / mL, enzymolyze at 16°C for 24 hours, then place the enzymolyzed solution in a water bath at 60°C for 45 minutes, and cool in 4°C ice water for 10 minutes to inactivate, to obtain a fermentation enzymolyzate;

[0069] 6) Pressing: The fermentation hydrolysate is dried at 60°C, then steamed at 70°C for 30 minutes, and finally pressed at 65°C for 23 minutes to obtain camellia oil with high antioxidant properties.

[0070] Example 3

[0071] A method for preparing camellia oil with high antioxidant properties comprises the following steps:

[0072] 1) preparing a fermentation substrate: drying camellia oleifera seed shells and camellia oleifera fruits in an oven at 50° C. for 5 h, taking them out, mixing the dried camellia oleifera seed shells and camellia oleifera fruits at a shell content of 8%, and then crushing them into 120 meshes to obtain a fermentation substrate;

[0073] 2) preparing fermentation medium: adding distilled water to the fermentation substrate at a solid-liquid ratio of 1:12, heating in a boiling water bath for 60 min, then cooling to room temperature and mixing with Basal medium at a volume ratio of 1:5, and sterilizing by high pressure at 121° C. for 15 min, with a pH of 5 to 7;

[0074] The composition of the Basal medium is: NaNO3 1250mg / L, KHPO4 1250mg / L, MgSO4·7H2O1000mg / L, EDTA 500mg / L, H3BO3 114.2mg / L, CaCl2·2H2O 111mg / L, FeSO4·7H2O 49.8mg / L, ZnSO4·7H2O88.2mg / L, MoO3 7.1mg / L, MnCl2·4H2O 14.2mg / L, CuSO4·5H2O 15.7mg / L, CoNO3·6H2O 6.1mg / L, glucose 10g / L, urea 3g / L, the solvent is deionized water, and the pH is 6.14±0.05; if solid Basal medium is required, 2% agar needs to be added;

[0075] 3) Chlorella fermentation: The activated Chlorella is inoculated into the fermentation medium for high temperature fermentation to obtain an intermediate product; the specific operation steps are as follows:

[0076] a) Activation of Chlorella: Take a piece of Chlorella moss on Basal slant medium and inoculate it on Basal solid plate medium sterilized at 121℃ for 15min. 2 / s light incubator for 7 days to obtain an activated plate;

[0077] b) Preparation of seed solution of Chlorella: A single algae colony was picked from the above-mentioned activated plate and inoculated into a 50 mL conical flask with 20 mL of Basal medium. The flask was placed in a constant temperature shaker at 30°C and 160 r / min for 4 days to prepare seed solution. The solution was then diluted with Basal medium to a cell density of 2.5×10 7 cells / mL of seed solution;

[0078] c) Inoculation of Chlorella: The cell density is 2.5×10 7The seed liquid of Chlorella cells / mL was inoculated into the fermentation medium at a volume percentage of 10%, and high-temperature fermentation was carried out at a temperature of 30°C for 1 day to obtain an intermediate product;

[0079] 4) Antarctic micrococcus fermentation: firstly cooling the intermediate product, and then inoculating the activated Antarctic micrococcus into the cooled intermediate product for low-temperature fermentation to obtain a fermentation product; the specific steps are as follows:

[0080] The culture was carried out at 15°C and 120 rpm for 3 days, and the suspension was collected by centrifugation at 5000 r / min for 5 min. The suspension was washed with sterile water.

[0081] d) Cooling: Cooling the intermediate product to 16°C;

[0082] e) Activation of Antarctic Micrococcus: Take a strain of 2216E slant medium from an environment and inoculate it into 5 mL of 2216E solid plate medium precooled to 16°C, and culture it for 7 days to obtain an activated plate; the preparation composition of 2216E medium is: yeast extract powder 1.0 g / L, peptone 5.0 g / L, FePO4·2H2O 0.01 g / L, deionized water to volume, pH 7.5, and wet heat sterilization for 20 minutes; if solid 2216E medium is required, 2% agar needs to be added;

[0083] f) Preparation of seed solution of Antarctic Micrococcus: A single colony was picked from the above-mentioned activated plate and inoculated into a 50 mL conical flask with 20 mL of 2216E medium. The flask was placed in a constant temperature shaker at a temperature of 16°C and a rotation speed of 160 r / min for 3 days to prepare seed solution. The bacterial suspension was collected by centrifugation at 5000 r / min for 5 minutes, and washed and diluted with sterile water to prepare a cell density of 1×10 8 cells / mL of seed solution;

[0084] g) Inoculation of Antarctic Micrococcus: The cell density was 1×10 8 cells / mL of Antarctic Micrococcus seed solution was inoculated into the intermediate product at a volume percentage of 2%, and low-temperature fermentation was carried out at a temperature of 16°C for 1 day to obtain a fermentation product;

[0085] 5) Coenzyme Q0 treatment: first dissolve the coenzyme Q0 with acetone (dissolved in the form of a coenzyme Q0 to acetone solid-liquid ratio of 1:100), then add the dissolved coenzyme Q0 to the fermentation product to make the concentration of coenzyme Q0 in the fermentation product 0.35 mg / mL, enzymolyze at 16°C for 24 hours, then place the enzymolyzed solution in a water bath at 60°C for 45 minutes, and cool in 4°C ice water for 10 minutes to inactivate, to obtain a fermentation enzymolyzate;

[0086] 6) Pressing: The fermentation hydrolysate is dried at 60°C, then steamed at 70°C for 30 minutes, and finally pressed at 65°C for 23 minutes to obtain camellia oil with high antioxidant properties.

[0087] The present invention measured the EPA content and antioxidant activity of the camellia oil prepared in Examples 1-3, and tested the oil according to the crude oil quality index requirements of GB / T 11765-2018 Camellia Oil. The results are shown in Table 1. The corresponding detection method is as follows:

[0088] The detection method of EPA relative content refers to GB 5009.168-2016.

[0089] The antioxidant test steps are:

[0090] DPPH scavenging rate: Weigh 19.7 mg DPPH accurately, dissolve it, transfer it to a 50 mL brown volumetric flask, dilute it to the mark with isopropanol, and place it in the refrigerator for later use. When ready to use, transfer 8 mL of DPPH solution and dilute it to 100 mL with isopropanol to prepare a DPPH (0.8 mmol / L) free radical working solution for later use.

[0091] Take 2 mL of tea oil sample and react with 2 mL of DPPH free radical working solution, protect from light for 0.5 h, and measure its absorbance (A) at a wavelength of 517 nm. i ). At the same time, 2 mL of isopropanol was reacted with 2 mL of DPPH, and the reaction was carried out in the dark for 0.5 h. The absorbance value (A0) at a wavelength of 517 nm was measured, as well as the absorbance value (A0) of 2 mL of tea oil sample mixed with 2 mL of isopropanol. j , the free radical scavenging rate was calculated according to formula 1.

[0092] ABTS scavenging rate: Weigh 19.21 mg ABTS and 33.10 mg potassium persulfate, dissolve them in methanol and distilled water respectively, mix them in equal volumes and transfer them to a brown volumetric flask, and react for more than 12 hours in the dark. When ready for use, dilute with n-butanol to an absorbance of 0.70±0.04 at 734 nm to prepare an ABTS free radical working solution.

[0093] Take 0.4 mL of tea oil sample and react with 4 mL of ABTS free radical working solution, protect from light and react for 10 min. Use n-butanol solvent as blank control and measure its absorbance (A) at a wavelength of 734 nm. i ). Take 0.4 mL of methanol and 4 mL of ABTS to react, and react for 10 min in the dark. Measure the absorbance (A0) at a wavelength of 734 nm, and calculate the free radical scavenging rate according to formula (2).

[0094] Formula 1: Formula 2:

[0095] Among the crude oil quality indicators, the acid value determination shall refer to the first method of GB 5009.229-2016, the peroxide value determination shall refer to the first method of GB5009.227-2023, the moisture and volatile matter content determination shall refer to the second method of GB 5009.236-2016, the insoluble impurity content determination shall refer to GB / T15688-2008, and the solvent residue determination shall refer to GB 5009.262-2016.

[0096] Table 1 EPA content, antioxidant activity and quality indexes of camellia oil prepared in Examples 1-3

[0097]

[0098] It can be seen from the above table that the EPA content in the camellia oil prepared in Examples 1-3 reached more than 0.55%, and the corresponding DPPH and ABTS removal rates reached more than 85%; and the acid value (in terms of KOH) in the prepared camellia oil was less than 4 mg / g, the peroxide value was less than 0.25, the moisture and volatile content were less than 0.2%, the insoluble impurities were less than 0.2%, and no solvent residue was detected, indicating that the camellia oil with high antioxidant properties prepared by the method of Examples 1-3 all met the standards of camellia crude oil in GB / T11765-2018 Camellia Oil, indicating that the double fermentation of Chlorella and Antarctic Micrococcus used in the present invention will not produce substances that have adverse effects on camellia oil, and the camellia oil prepared by using camellia shells and camellia fruits as substrates and combining Chlorella and Antarctic Micrococcus for fermentation meets the standards of camellia crude oil in GB / T 11765-2018 Camellia Oil.

[0099] Comparative Example 1

[0100] The difference between this comparative example and Example 1 is that the fermentation of Chlorella and Antarctic Micrococcus and the treatment of coenzyme Q0 are not used, and the specific operation steps are as follows:

[0101] The camellia seed shells and camellia fruits were dried in an oven at 45°C for 4 hours respectively, the dried camellia seed shells and camellia fruits were mixed according to the shell content of 7%, and crushed into 100 mesh with a grinder; then steamed at 70°C for 30 minutes, and finally pressed at 65°C for 23 minutes to obtain camellia seed oil.

[0102] Comparative Example 2

[0103] The difference between this comparative example and Example 1 is that Antarctic Micrococcus fermentation is not used, that is, step 4) is not included, and the high temperature fermentation time in step 3) is 2 days, and the intermediate product obtained by fermentation of Chlorella is subsequently directly treated with coenzyme Q0.

[0104] Comparative Example 3

[0105] The difference between this comparative example and Example 1 is that: Chlorella fermentation is not used, that is, step 3) is not included, and step 2) of preparing the fermentation medium is modified as follows: distilled water is added to the fermentation substrate at a solid-liquid ratio of 1:10, heated in a boiling water bath for 50 minutes, and then cooled to room temperature and mixed with 2216E medium at a volume ratio of 1:6, and sterilized at high pressure at 121°C for 15 minutes, with a pH of 7.5; step 4) Antarctic micrococcus fermentation is modified as follows: first the fermentation medium is cooled, and then the activated Antarctic micrococcus is inoculated into the cooled fermentation medium for low-temperature fermentation for 2 days to obtain a fermentation product, and the specific related operating steps (step d and step g) are also modified accordingly, which will not be repeated here.

[0106] Comparative Example 4

[0107] The difference between this comparative example and comparative example 3 is that: no coenzyme Q0 treatment is used; that is, step 5) is not included, and the fermentation product obtained by fermentation of Antarctic Micrococcus is directly subjected to pressing treatment.

[0108] Comparative Example 5

[0109] The difference between this comparative example and comparative example 2 is that: no coenzyme Q0 treatment is used; that is, the intermediate product obtained by fermenting Chlorella is directly squeezed.

[0110] Comparative Example 6

[0111] The difference between this comparative example and Example 1 is that no coenzyme Q0 treatment is used, that is, step 5) is not included, and the obtained fermentation product is directly subjected to pressing treatment.

[0112] Comparative Example 7

[0113] The difference between this comparative example and Example 1 is that Antarctic Micrococcus fermentation is first used and then Chlorella fermentation is used. The specific operation steps are different in that:

[0114] 1) preparing a fermentation substrate;

[0115] 2) Preparation of fermentation medium: adding distilled water to the fermentation substrate at a solid-liquid ratio of 1:10, heating in a boiling water bath for 50 min, then cooling to room temperature and mixing with 2216E medium at a volume ratio of 1:6, and autoclaving at 121° C. for 15 min, pH 7.5;

[0116] 3) Antarctic micrococcus fermentation: firstly cooling the fermentation medium, and then inoculating the activated Antarctic micrococcus into the cooled fermentation medium for low-temperature fermentation to obtain an intermediate product; the specific steps are as follows:

[0117] a) Cooling: Cooling the fermentation medium to 16°C;

[0118] b) Activation of Antarctic Micrococcus: Take a strain of bacteria from 2216E slant medium and inoculate it into 5 mL of 2216E solid plate medium precooled to 16°C, and culture for 7 days to obtain an activated plate;

[0119] c) Preparation of seed solution of Antarctic Micrococcus: A single colony was picked from the above-mentioned activated plate and inoculated into a 50 mL conical flask with 20 mL of 2216E medium. The flask was placed in a constant temperature shaker at 16°C and 160 r / min for 3 days to prepare seed solution. The bacterial suspension was collected by centrifugation at 5000 r / min for 5 min, and washed and diluted with sterile water to prepare a cell density of 1×10 8 cells / mL of seed solution;

[0120] d) Inoculation of Antarctic Micrococcus: The cell density is 1×10 8 cells / mL of Antarctic Micrococcus seed solution was inoculated into the fermentation medium at a volume percentage of 1.5%, and low-temperature fermentation was carried out at a temperature of 16°C for 1 day to obtain an intermediate product;

[0121] 4) Chlorella fermentation: inoculating the activated Chlorella into the intermediate product for high temperature fermentation to obtain a fermentation product; the specific operation steps are as follows:

[0122] e) Activation of Chlorella: Take a piece of Chlorella moss on Basal slant medium and inoculate it on Basal solid plate medium that has been autoclaved at 121°C for 15 min. 2 / s light incubator for 7 days to obtain an activated plate;

[0123] f) Preparation of seed solution of Chlorella: A single colony was picked from the above-mentioned activated plate and inoculated into a 50 mL conical flask with 20 mL of Basal medium. The flask was placed in a constant temperature shaker at 30°C and 160 r / min for 4 days to prepare seed solution. The solution was then diluted with Basal medium to a cell density of 2.5×10 7 cells / mL of seed solution;

[0124] g) Inoculation of Chlorella: The cell density is 2.5×10 7 cells / mL of Chlorella vulgaris seed solution was inoculated into the intermediate product at a volume percentage of 4%, and high-temperature fermentation was carried out at a temperature of 30°C for 1 day to obtain a fermentation product;

[0125] 5) Coenzyme Q0 treatment: first dissolve the coenzyme Q0 with acetone (dissolved in the form of a coenzyme Q0 to acetone solid-liquid ratio of 1:100), then add the dissolved coenzyme Q0 to the fermentation product to make the concentration of coenzyme Q0 in the fermentation product 0.3 mg / mL, enzymolyze at 16°C for 24 hours, then place the enzymolyzed solution in a water bath at 60°C for 45 minutes, and cool in 4°C ice water for 10 minutes to inactivate, to obtain a fermentation enzymolyzate;

[0126] 6) Pressing: the fermentation hydrolysate is dried at 60°C, then steamed at 70°C for 30 min, and finally pressed at 65°C for 23 min to obtain camellia seed oil.

[0127] Table 2 Similarities and differences in the steps of preparing camellia seeds for use in Example 1 and Comparative Examples 1-7

[0128]

[0129]

[0130] Control group 1

[0131] The control group directly adopts the method of adding fish oil to the camellia oil prepared in Comparative Example 1 to prepare the blended oil. By adding fish oil, the EPA content in the blended oil is made the same as the EPA content in the camellia oil prepared in Example 1.

[0132] Control group 2

[0133] The control group directly adopted the method of adding a common antioxidant (tocopherol) to the camellia oil prepared in Comparative Example 1, so that the content of the antioxidant was the same as the EPA content in the camellia oil prepared in Example 1.

[0134] The present invention measured the EPA content in the camellia oil prepared in Example 1, Comparative Examples 1-7 and Control Groups 1-2, and the antioxidant activity of the camellia oil. The results are shown in Table 3.

[0135] Table 3 EPA content and antioxidant activity of camellia oil prepared in Example 1, Comparative Examples 1-7 and Control Groups 1-2

[0136] EPA relative content (%) DPPH clearance rate (%) ABTS clearance rate (%) Example 1 0.62 93 89 Comparative Example 1 ND 28 22 Comparative Example 2 0.19 56 46 Comparative Example 3 0.16 49 41 Comparative Example 4 0.11 34 24 Comparative Example 5 0.13 42 32 Comparative Example 6 0.32 64 58 Comparative Example 7 0.49 79 70 Control group 1 0.62 80 72 Control group 2 ND 82 82

[0137] Note: ND means not detected

[0138] It can be seen from the above table that by comparing Example 1 with Comparative Example 1, the relative content of EPA in the camellia oil prepared in Example 1 is 0.62%, while it is not detected in Comparative Example 1, and the antioxidant activity (DPPH removal rate and ABTS removal rate) is also significantly lower than that in Example 1, indicating that under the co-fermentation of Chlorella and Antarctic Micrococcus and with the assistance of coenzyme Q0, EPA in the camellia oil can be significantly enriched, the content of EPA in the camellia oil can be increased, and thus the antioxidant capacity can be significantly improved. The reason may be that: camellia seed shells and camellia fruits contain a large amount of sugar substances. Chlorella promotes the process of glycolysis during the fermentation process by using fermentation substrates containing camellia seed shells and camellia fruits for high-temperature fermentation, thereby increasing the content of acetyl-CoA, an intermediate for the synthesis of EPA, and further promoting the synthesis of EPA, so that EPA can be effectively enriched and the content of EPA in camellia seed oil is increased; and during the fermentation process, Chlorella also regulates the fatty acid composition in its cells, so that the relative content of EPA will also increase relatively; after Chlorella fermentation, Antarctic Micrococcus is used to continue low-temperature fermentation, which can further promote the generation of acetyl-CoA and further effectively increase the content of EPA; finally, through coenzyme Q0 treatment, the integrity of the cell membrane can be further destroyed, so that EPA can be fully released and dissolved in the fermentation substrate, thereby effectively increasing the content of EPA in camellia seed oil.

[0139] Comparative Examples 2 and 3 respectively use only Chlorella and Antarctic Micrococcus for single-strain fermentation. Compared with Example 1, the relative content of EPA is significantly reduced, and the antioxidant activity is also significantly lower than that of Example 1, indicating that when Chlorella or Antarctic Micrococcus is fermented alone, the content of EPA will be significantly reduced, which may be related to the content of acetyl-CoA generated; the reduction of EPA will further lead to a significant decrease in antioxidant activity.

[0140] Comparative Examples 4 and 5 respectively use Chlorella and Antarctic Micrococcus for single-strain fermentation and do not add coenzyme Q0 for treatment. Compared with Comparative Examples 3 and 2, the EPA content and antioxidant capacity of camellia oil are reduced; and Comparative Example 6, compared with Example 1, only does not add coenzyme Q0 for treatment, and the EPA content and antioxidant capacity are also lower than those of Example 1, indicating that the addition of coenzyme Q0 can effectively promote the dissolution of EPA in the cell membrane, and plays an important role in increasing the EPA content.

[0141] As can be seen from Comparative Example 7, the Antarctic Micrococcus fermentation is first performed, and then the Chlorella fermentation is used. The final EPA content is significantly reduced compared to Example 1, and the antioxidant activity is also significantly weakened, indicating that the order of Chlorella and Antarctic Micrococcus fermentation will also have a significant impact on the content of EPA in Camellia oleifera oil. It may be because, in the early stage of fermentation, due to the high content of sugars such as glucose in the fermentation substrate, and the degree of effect of Chlorella fermentation in promoting the generation of acetyl-CoA is greater than that of Antarctic Micrococcus, therefore, when the strain (Chlorella) with a higher content of fermentation substrate and more effective promotion of acetyl-CoA is used for fermentation first, it will be more conducive to the generation of acetyl-CoA, and then the EPA content synthesized by Chlorella fermentation first will be more than that of Antarctic Micrococcus fermentation first, and then the EPA content in Camellia oleifera oil of Example 1 is greater than that of Comparative Example 7.

[0142] Compared with Example 1, although the EPA content in the camellia oil (also blended oil) prepared by the control group 1 by adding fish oil is the same as that in Example 1, its corresponding antioxidant activity is lower than that of Example 1. In the preparation process, it is also found that the blended oil prepared by the control group 1 has a fishy smell and its flavor is worse than that of Example 1. This shows that the method of the present invention can prepare camellia oil with better flavor and stronger antioxidant activity than the method of adding fish oil.

[0143] Compared with Example 1, although the control group 2 adopts the method of directly adding antioxidants (tocopherol) to the pressed oil, its corresponding antioxidant activity is also lower than that of Example 1, indicating that the antioxidant activity of camellia seed oil prepared by the method of the present invention is better than that of directly adding antioxidants.

[0144] Experimental Example 1 Changes of Acetyl Coenzyme A during Chlorella Fermentation

[0145] This experimental example investigates the changing trend of acetyl-CoA with fermentation time during the fermentation of Chlorella. The fermentation products corresponding to 0h, 4h, 8h, 12h, 16h, 20h and 24h of fermentation were taken in step 3) of Example 1, and the content of acetyl-CoA in the fermentation products corresponding to each fermentation time point was measured. The results are shown in Table 4:

[0146] The acetyl CoA content was determined by referring to the method in "The Effect of Cofactors on 7-dehydrocholesterol Synthesis in Engineered Saccharomyces cerevisiae, Su Wan, Tianjin University". The specific steps are as follows: use BioVision's PiconProbe Acetyl CoA AssayKit was used to quantitatively test acetyl CoA. The determination method was as follows: sterile water was added to the fermentation product, placed in a 37°C incubator, and shaken at 200r / min for 5min. The liquid was collected and filtered through 2 layers of filter paper to obtain a pure bacterial solution. The cells were fully ground into powder in liquid nitrogen and weighed. 2uL of perchloric acid was added for every 1mg of bacterial powder, vortexed and mixed, and kept at low temperature. Perchloric acid was neutralized with 3M potassium bicarbonate solution, and 1uL of potassium bicarbonate solution was added for every 10uL of supernatant. The solution was vortexed continuously until no bubbles were generated, and the temperature was kept low. The solution was placed in an ice bath for 5min, and the pH of the solution was measured to ensure that it was between 6-8. The potassium perchlorate was removed by centrifugation at 5000rpm for 5min. 10uL of bacterial sample with perchloric acid removed was added to a blank 96-well plate, and 10uL of Buffer was added to each well. 2uL of CoASH Quencher was added to each well and reacted at room temperature for 5min to quench free CoA. 2uL of QuenchRemover, react at room temperature for 5 minutes; add 2uL Substrate Mix, 1uL Conversion Enzyme, 5uL Enzyme Mix and 2uL PicoProbe to each well in turn, react at 37°C for 10 minutes; use Plate Reader to measure RFU (fluorescence intensity) at Ex / Em=535 / 589nm, where y=-0.0023x 2 +4.9046x+10.2, R 2 =0.9963 (the value of y in the formula represents RFU, the value of x represents the amount of acetyl-CoA in pmol, and the concentrations of the acetyl-CoA standard samples are 150 pmol, 300 pmol, 450 pmol, 600 pmol, and 750 pmol).

[0147] Table 4 Changes in acetyl-CoA content during Chlorella fermentation

[0148]

[0149] From the table above, we can see that during the growth and metabolism of Chlorella, the content of acetyl-CoA shows a trend of substantial increase from 0h to 8h. The reason may be that Chlorella is in the logarithmic growth period during this period, and the nitrogen and carbon sources available to Chlorella are relatively sufficient. It tends to be stable from 12h to 24h. The reason may be that the content of nitrogen and carbon sources for the growth of Chlorella has been partially consumed, and the growth and metabolism rate of Chlorella will slow down. At the same time, while producing acetyl-CoA, the rate of consuming acetyl-CoA to synthesize EPA also increases due to the increase in the degree of glycolysis, so that the growth rate of acetyl-CoA begins to slow down.

[0150] Experimental Example 2 Changes of Acetyl-CoA during Fermentation of Antarctic Micrococcus

[0151] This experimental example investigates the changing trend of acetyl-CoA with fermentation time during the fermentation of Antarctic micrococcus. The fermentation products corresponding to 0h, 4h, 8h, 12h, 16h, 20h and 24h of fermentation were taken in step 4) of Example 1, and the content of acetyl-CoA in the fermentation products corresponding to each fermentation time point was measured. The results are shown in the following table:

[0152] Table 5 Changes in acetyl-CoA content during the fermentation of Antarctic Micrococcus

[0153]

[0154] From the above table, we can see that the production rate of acetyl-CoA by Antarctic Micrococcus is relatively high in 0h-4h, and it also increases significantly in 4h-8h. In 12h-24h, the growth rate decreases significantly. The reason may be similar to the change of acetyl-CoA during the fermentation of Chlorella.

[0155] Based on the results of Experimental Examples 4 and 5, it was found that during the fermentation process using Chlorella, acetyl-CoA showed a trend of first increasing rapidly and then slowly increasing. The inoculation of Antarctic Micrococcus could make the content of acetyl-CoA increase rapidly during the fermentation process and then increase slowly, thereby significantly improving the content of acetyl-CoA during the entire fermentation process, thereby providing more intermediates for the synthesis of EPA, and ultimately making the camellia oil prepared by the dual fermentation of Micrococcus antarctica contain more EPA, so that the camellia oil has high antioxidant properties.

[0156] Experimental Example 3 Effect of Cell Density of Chlorella on EPA Content and Antioxidant Activity

[0157] Camellia oleifera oil was prepared by referring to Example 1. The Chlorella vulgaris in step 3) was cultured at different cell densities (0.1×10 7 cells / mL、0.5×10 7 cells / mL、1.5×10 7 cells / mL、2.5×107 cells / mL、3×10 7 cells / mL) were inoculated into the fermentation medium, and the other steps were the same as in Example 1. The EPA content and antioxidant activity of the camellia oil prepared at each cell density were measured. The results are shown in Table 6:

[0158] Table 6 Effect of cell density of Chlorella on EPA and antioxidant activity

[0159] Chlorella algae solution concentration (cells / mL) EPA relative content (%) DPPH clearance rate (%) ABTS clearance rate (%) <![CDATA[0.1×10 7 ]]> 0.36 65 60 <![CDATA[0.5×10 7 ]]> 0.52 81 80 <![CDATA[1.5×10 7 ]]> 0.55 84 82 <![CDATA[2.5×10 7 ]]> 0.62 93 89 <![CDATA[3.0×10 7 ]]> 0.49 78 70

[0160] As shown in Table 6, with the increase of cell density of Chlorella, the relative content of EPA also increased, and the antioxidant activity was enhanced. 7 cells / mL, and then as the cell density increases, the EPA content gradually decreases and the antioxidant activity weakens. The reason may be that during the growth of Chlorella, under the condition that the supply of nutrients for cell growth and metabolism is constant, as the cell density increases, the competition between cells increases, and the nutrients that each cell can use are relatively reduced, resulting in a decrease in EPA content. The results show that the optimal cell density range of Chlorella is 0.5×10 7 cells / mL~2.5×10 7 cells / mL, and the most preferred value is 2.5×10 7 cells / mL.

[0161] Experimental Example 4 Effect of the Volume Percentage of Chlorella Inoculated into Fermentation Medium on EPA Content and Antioxidant Activity

[0162] Camellia oleifera oil was prepared with reference to Example 1, and the chlorella in step 3) was inoculated into the fermentation medium at different volume percentages (2%, 4%, 6%, 10%, 12%) of the fermentation medium, and the other steps were the same as in Example 1. The EPA content and antioxidant activity of the camellia oleifera oil prepared at each volume percentage were measured. The results are shown in Table 7:

[0163] Table 7 Effect of volume percentage of Chlorella on EPA and antioxidant activity

[0164]

[0165] From the results in Table 7, it can be seen that as the volume percentage of Chlorella inoculated with the fermentation medium increases, the EPA content and antioxidant activity both show a trend of first increasing and then decreasing, and the preferred volume percentage range is 4% to 10%, and the most preferred is 4%.

[0166] Experimental Example 5 Effect of Cell Density of Antarctic Micrococcus on EPA Content and Antioxidant Activity

[0167] Camellia oleifera oil was prepared by referring to Example 1. The Antarctic Micrococcus in step 4) was cultured at different cell densities (0.6×10 8 cells / mL、0.8×10 8 cells / mL、1×10 8 cells / mL、1.2×10 8 cells / mL、1.4×10 8 cells / mL) were inoculated into the intermediate product, and the other steps were consistent with Example 1. The EPA content and antioxidant activity of the camellia oil prepared at each cell density were measured. The results are shown in Table 8:

[0168] Table 8 Effect of cell density of Antarctic Micrococcus on EPA and antioxidant activity

[0169] Bacterial concentration (cells / mL) EPA relative content (%) DPPH clearance rate (%) ABTS clearance rate (%) <![CDATA[0.6×10 8 ]]> 0.37 65 60 <![CDATA[0.8×10 8 ]]> 0.56 84 82 <![CDATA[1.0×10 8 ]]> 0.62 93 89 <![CDATA[1.2×10 8 ]]> 0.55 84 81 <![CDATA[1.4×10 8 ]]> 0.32 64 59

[0170] As shown in Table 8, with the increase of the cell density of Antarctic Micrococcus, the EPA content and antioxidant activity in camellia oil showed a trend of first increasing and then decreasing. 8 cells / mL. The reason may be due to the intensified competition between cells. The results showed that the optimal cell density range of Antarctic Micrococcus was 0.8×10 8 cells / mL~1.2×10 8 cells / mL. The most preferred value is 1.0×10 8 cells / mL.

[0171] Experimental Example 6 Effect of the Volume Percentage of Antarctic Micrococcus Inoculated into the Intermediate Product on the EPA Content and Antioxidant Activity

[0172] Camellia oil was prepared with reference to Example 1, and the Antarctic micrococci in step 4) were inoculated into the intermediate product at different volume percentages (0.5%, 1%, 1.5%, 2%, 2.5%) of the intermediate product, and the other steps were consistent with Example 1. The EPA content and antioxidant activity of the camellia oil prepared at each volume percentage were determined. The results are shown in Table 9:

[0173] Table 9 Effect of volume percentage of Antarctic Micrococcus on EPA and antioxidant activity

[0174]

[0175] As can be seen from Table 9, as the volume percentage of Antarctic Micrococcus increases, the EPA content and antioxidant activity both show a trend of first increasing and then decreasing, and the preferred volume percentage range is 1.0% to 2.0%, and the most preferred is 1%.

[0176] Experimental Example 7 Orthogonal test of tea seed oil preparation process

[0177] According to Experimental Examples 3-6, a four-factor three-level orthogonal experiment was designed for the tea seed oil preparation process, namely L9(3 4 ) experiment. 7 cells / mL、1.5×10 7 cells / mL、2.5×10 7 cells / mL), Chlorella addition amount (4%, 6%, 10%), Antarctic Micrococcus bacterial solution concentration (0.8×10 8 cells / mL, 1.0×10 8 cells / mL、1.2×10 8 cells / mL), and the amount of Antarctic Micrococcus added (1.0%, 1.5%, 2.0%) were used for the experimental design. The test was carried out according to the steps in Example 1 according to Table 10, and the EPA content and antioxidant activity of the prepared camellia seed oil were shown in Table 11.

[0178] Table 10 Orthogonal test table of antioxidant camellia oil preparation process

[0179]

[0180] Table 11 EPA content and antioxidant test results of camellia oil

[0181] serial number EPA relative content (%) DPPH clearance rate (%) ABTS clearance rate (%) 1 0.44 75 69 2 0.35 65 60 3 0.54 83 82 4 0.30 60 57 5 0.50 80 72 6 0.39 70 65 7 0.62 93 89 8 0.49 79 72 9 0.28 59 53

[0182] It can be seen from Table 11 that under the condition No. 7, the EPA relative content, DPPH removal rate, and ABTS removal rate of camellia oil are the highest, indicating that the optimal process conditions for preparing camellia oil are: the cell density of Chlorella is 2.5×10 7 cells / mL, volume percentage is 4%; the cell density of Antarctic Micrococcus is 1.0×10 8 cells / mL, and the volume percentage is 1.5%. Under the process conditions, the prepared camellia seed oil can contain a higher content of EPA and has good antioxidant properties.

[0183] Experimental Example 8 Stability test of EPA in camellia oil under high temperature conditions

[0184] The camellia oil prepared in Example 1, Comparative Example 1 and Control Group 1 were heated at 140°C, 150°C, 160°C, 170°C and 180°C for 5 min, respectively, and then cooled. The EPA content in the camellia oil was measured, and the EPA loss rate was calculated. EPA loss rate = (EPA content before heating treatment - EPA content after heating treatment) / EPA content before heating treatment. The results are shown in Table 12.

[0185] Table 12 Effect of different heating temperatures on EPA loss rate in camellia oil (%)

[0186]

[0187] As can be seen from the above table, after the camellia oil is heated, as the temperature increases (from 140°C to 180°C), the loss rate of EPA content in the camellia oil corresponding to Example 1 increases by 0.7%, while the corresponding loss rate in the control group 1 increases by 3.3%. This indicates that the stability of EPA in the camellia oil prepared by the method of the present invention at high temperature is significantly higher than that of the camellia oil containing EPA prepared by directly adding fish oil.

[0188] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing camellia oil with high antioxidant properties, characterized in that: The following steps are involved: 1) Preparing a fermentation substrate: pretreating camellia oleifera seed shells and camellia oleifera fruits to obtain a fermentation substrate; 2) preparing a fermentation medium: using a fermentation substrate, distilled water and Basal medium as raw materials to prepare a fermentation medium; 3) Chlorella fermentation: inoculating the activated Chlorella into the fermentation medium for high temperature fermentation to obtain an intermediate product; 4) Antarctic micrococcus fermentation: firstly cooling the intermediate product, then inoculating the activated Antarctic micrococcus into the cooled intermediate product for low-temperature fermentation to obtain a fermentation product; 5) Coenzyme Q0 treatment: adding coenzyme Q0 to the fermentation product for enzymolysis and inactivation to obtain a fermentation enzymolysis solution; 6) Pressing: The fermented enzymatic hydrolysate is dried, steamed and then pressed to obtain camellia seed oil with high antioxidant properties.

2. The method for preparing camellia oil with high antioxidant properties as claimed in claim 1, characterized in that: The specific operation steps of step 1) are: putting the camellia oleifera seed shells and camellia oleifera fruits in an oven at a temperature of 35°C to 50°C for drying for 3h to 5h respectively, taking them out, mixing the dried camellia oleifera seed shells and camellia oleifera fruits according to a shell content of 6% to 8%, and then crushing them into 80 mesh to 120 mesh to obtain a fermentation substrate.

3. The method for preparing camellia oil with high antioxidant properties as claimed in claim 1, characterized in that: The specific operation steps of step 2) are: adding distilled water to the fermentation substrate at a material-liquid ratio of 1:8 to 1:12, heating in a boiling water bath for 40 to 60 minutes, then cooling to room temperature and mixing with Basal medium at a volume ratio of 1:4 to 1:6, and high-pressure sterilization at 121°C for 15 minutes, with a pH of 5 to 7.

4. The method for preparing camellia oil with high antioxidant properties as claimed in claim 1, characterized in that: The composition of the Basal culture medium is: NaNO3 1250mg / L, KHPO4 1250mg / L, MgSO4·7H2O 1000mg / L, EDTA 500mg / L, H3BO3 114.2mg / L, CaCl2·2H2O 111mg / L, FeSO4·7H2O 49.8mg / L, ZnSO4·7H2O 88.2mg / L, MoO3 7.1mg / L, MnCl2·4H2O 14.2mg / L, CuSO4·5H2O 15.7mg / L, CoNO3·6H2O 6.1mg / L, glucose 10g / L, urea 3g / L, the solvent is deionized water, and the pH is 6.14±0.

05.

5. The method for preparing camellia oil with high antioxidant properties as claimed in claim 1, characterized in that: The specific operation steps of step 3) are as follows: first, the activated Chlorella is prepared into a cell density of 2.5×10 7 cells / mL of seed liquid, and then inoculating the seed liquid into a fermentation medium at a volume percentage of 4%-10% for high-temperature fermentation to obtain an intermediate product.

6. The method for preparing camellia oil with high antioxidant properties as claimed in claim 5, characterized in that: The temperature of the high temperature fermentation is 30° C. and the fermentation time is 1 day.

7. The method for preparing camellia oil with high antioxidant properties as claimed in claim 1, characterized in that: The specific operation steps of step 4) are: firstly cool the intermediate product to 16°C, and then prepare the activated Antarctic micrococci into a cell density of 1×10 8 cells / mL of seed liquid, and then inoculating the seed liquid into the cooled intermediate product at a volume percentage of 1%-2% to perform low-temperature fermentation to obtain a fermentation product.

8. The method for preparing camellia oil with high antioxidant properties as claimed in claim 7, characterized in that: The temperature of the low-temperature fermentation is 16° C., and the fermentation time is 1 day.

9. The method for preparing camellia oil with high antioxidant properties as claimed in claim 1, characterized in that: The specific operation steps of step 5) are: first dissolve coenzyme Q0 with acetone, then add the dissolved coenzyme Q0 to the fermentation product to make the concentration of coenzyme Q0 in the fermentation product 0.25 mg / mL to 0.35 mg / mL, and inactivate after enzymolysis at 16°C for 24 hours to obtain a fermentation hydrolyzate.

10. The method for preparing camellia oil with high antioxidant properties as claimed in claim 1, characterized in that: In step 6), the drying temperature is 60° C. and the time is 1 hour; the steaming temperature is 70° C. and the time is 30 minutes; the pressing temperature is 65° C. and the pressing time is 23 minutes.

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