Preparation method of camellia oleosa seed oil with function of reducing blood fat

Abstract

The invention discloses a preparation method of camellia oleosa seed oil with a function of reducing blood fat. The preparation method comprises the following steps: 1) sterilizing decolorized spent bleaching clay; 2) performing mixed fermentation of aspergillus niger and microbacterium flavum: firstly preparing a fermentation culture medium of the decolorized spent bleaching clay, then inoculating the fermentation culture medium containing the decolorized spent bleaching clay with the mixed bacterial suspension of the aspergillus niger and the microbacterium flavum, and performing fermentation to obtain a decolorized spent bleaching clay fermentation product; (3) extracting the phytosterol; (4) growing grains by using ethanol, and freeze-drying to obtain phytosterol powder; and 5) adding the phytosterol powder: adding the phytosterol powder into the camellia oleosa seed oil, and fully and uniformly mixing to obtain the finished camellia oleosa seed oil with the function of reducing blood fat. The method not only can effectively improve the yield of the phytosterol, but also can improve the blood fat reducing effect of the camellia oleosa seed oil.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of functional camellia oil, and in particular to a method for preparing camellia oil with blood lipid lowering function. Background Art

[0002] Vegetable oil is mainly obtained by squeezing the fruits, seeds, germs and other parts of plants. According to the raw materials, it can be divided into peanut oil, corn oil, camellia oil and other types. Camellia oil is the first health care plant edible oil recommended by the Food and Agriculture Organization of the United Nations. In order to obtain fragrant and transparent camellia oil, it is usually necessary to refine the oil obtained by squeezing or leaching. During the refining process, it needs to go through processes such as degumming, decolorization and deodorization.

[0003] However, when activated clay is used for decolorization, it absorbs pigments, phospholipids and other substances in oils and fats, and also absorbs bioactive substances in camellia oil that have lipid-lowering effects, such as phytosterols, so that the decolorized waste clay (also called decolorized waste clay) contains phytosterols. Due to the low content of phytosterols in decolorized waste clay and the difficulty in extraction, companies generally sell them directly. In order to improve the comprehensive utilization of such by-products and reduce the waste of waste clay resources, how to enrich phytosterols in decolorized waste clay and make functional foods with auxiliary lipid-lowering effects has become a research hotspot.

[0004] CN202111131385.1 discloses a method for recovering bioactive components from waste activated clay treated with vegetable oil decolorization, through ultrasonic extraction, organic solvent extraction and centrifugation to obtain supernatant, gradient elution to collect eluate, and detection of active components to obtain tocopherol, squalene, phytosterols, flax cyclic peptide, polyphenols and many other active components. The waste clay after centrifugation is acidified, washed with water, filtered, dried and crushed to obtain regenerated clay. The present invention is simple, convenient, fast and can recover a series of bioactive components adsorbed in vegetable oil from waste clay in large quantities and realize secondary recycling of waste clay.

[0005] The above recovery method uses ultrasound-assisted and organic solvent extraction to recover phytosterols from waste activated clay, but has the following defects:

[0006] 1) When the method is used to recover the phytosterols in the waste activated clay, a large amount of organic solvent is required, which not only reduces the yield of phytosterols but also reduces the safety performance;

[0007] 2) There is no research to verify the lipid-lowering effect of camellia oil produced by extracting phytosterols using this method.

[0008] In summary, how to provide a method for preparing camellia oil that can both improve the comprehensive utilization rate of by-products (bleaching waste clay) and increase the yield of phytosterols, and give camellia oil better lipid-lowering effect, has become a technical problem that needs to be solved urgently. Summary of the invention

[0009] 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 which has a simple process, can effectively improve the yield of phytosterols and has the function of lowering blood lipids.

[0010] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing camellia oil with blood lipid lowering function, comprising the following steps:

[0011] 1) Sterilization of decolorized waste clay;

[0012] 2) Mixed fermentation of Aspergillus niger and Microbacterium flavum: firstly prepare a fermentation medium containing decolorized waste clay, then inoculate a mixed bacterial suspension of Aspergillus niger and Microbacterium flavum onto the fermentation medium containing decolorized waste clay for fermentation to obtain a decolorized waste clay fermentation product;

[0013] 3) Extraction of phytosterols: cooling the fermentation product of decolorized waste clay, adding ethanol and then performing ultrasonic treatment to obtain a crude phytosterol extract;

[0014] 4) growing crystals in ethanol and freeze-drying to obtain phytosterol powder;

[0015] 5) Adding phytosterol powder: adding phytosterol powder to camellia oil, mixing thoroughly and evenly, to obtain finished camellia oil with lipid-lowering function.

[0016] Furthermore, in the step 1), the sterilization temperature of the decolorized waste clay is 121° C., and the sterilization time is 15 minutes.

[0017] Further, the specific operation steps of step 2) mixed fermentation of Aspergillus niger and Microbacterium flavum are:

[0018] a) adding the sterilized bleaching waste clay to the sterilized fermentation medium to make the concentration of the bleaching waste clay 60 g / L, to obtain a fermentation medium containing the bleaching waste clay;

[0019] b) Prepare spores at a concentration of 2×10 7 -2×10 8 CFU / ml of Aspergillus niger suspension and 2×10 7 -2×10 8 CFU / ml suspension of Microbacterium flavum;

[0020] c) mixing the Aspergillus niger suspension and the Microbacterium flavum suspension in a mixing volume ratio of 1:1-3 to obtain a mixed bacterial suspension;

[0021] d) inoculating the mixed bacterial suspension into a fermentation medium containing decolorized waste clay at an inoculation rate of 4-10%, and then fermenting the medium at a temperature of 28-35° C. for 48-96 hours to obtain a decolorized waste clay fermentation product.

[0022] Furthermore, the composition of the fermentation medium used in preparing the fermentation medium containing decolorized waste clay in step a) is 0.5 g / L yeast powder, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, 1000 mL distilled water, pH 7.0, and sterilized at 121°C for 15 min.

[0023] Preferably, the spore concentration of the Aspergillus niger suspension in step b) is 1×10 8 CFU / ml, the spore concentration of the yellow microbacterium suspension was 1.5×10 8 CFU / ml.

[0024] Preferably, in step c), the mixing volume ratio of the Aspergillus niger suspension to the Microbacterium flavum suspension is 1:2.

[0025] Preferably, the inoculation amount of the mixed bacterial suspension in step d) is 6%.

[0026] Furthermore, the specific operation steps of step 3) are as follows: after the fermentation is completed, the decolorized waste clay fermentation product is cooled to 4°C, and 80% ethanol is added to the decolorized waste clay fermentation product at a volume ratio of 1:10-1:30, and mixed; then, ultrasonic treatment is performed at a temperature of 40-50°C and a power of 400-600W for 90 minutes, the ultrasonication is performed twice, the supernatant is extracted, and concentrated to obtain a crude plant sterol extract.

[0027] Furthermore, the specific operation steps of step 4) are: adding 95% ethanol to the crude plant sterol extract at a solid-liquid ratio of 1:10-20g / mL for crystal growth, the crystal growth temperature is 30°C, the crystal growth time is 4h, and after the crystal growth is completed, centrifugation is performed, the bottom precipitate is collected, and freeze-dried to obtain purified plant sterol powder.

[0028] Furthermore, in the step 5), the amount of phytosterol powder added is 16000 mg / kg-20000 mg / kg.

[0029] The beneficial effects of the method for preparing camellia oil with blood lipid lowering function of the present invention are as follows:

[0030] (1) The preparation process of the camellia oil of the present invention is simple. By using Aspergillus niger and Microbacterium flavum to mix and ferment a fermentation medium containing bleaching waste clay, under the synergistic fermentation of Aspergillus niger and Microbacterium flavum, a large amount of key precursor acetyl coenzyme A and a large amount of energy substance ATP are provided for the synthesis of phytosterols, thereby promoting the large-scale synthesis of phytosterols, thereby effectively enriching the phytosterols in the bleaching waste clay, which can not only improve the comprehensive utilization rate of the refining by-product bleaching waste clay, but also effectively improve the yield of phytosterols;

[0031] (2) The present invention adopts the mixed fermentation method of Aspergillus niger and Microbacterium flavum, which can also improve the solubility of phytosterols in camellia oil, thereby ensuring product quality and stability;

[0032] (3) The present invention adds phytosterol powder obtained by mixed fermentation of Aspergillus niger and Microbacterium flavum to camellia oil, so that the prepared camellia oil can effectively reduce the content of triglycerides and has a better lipid-lowering effect;

[0033] (4) The acid value of the camellia oil prepared by the method of the present invention is controlled at 0.1-1.0 mgKOH / g, the peroxide value is controlled at 0.010-0.020 g / 100 g, and the phytosterol content is controlled at 16000 mg / kg-20000 mg / kg, which meets the first-level pressing standard of camellia oil GB / T11765-2018. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 — is a process flow chart of preparing camellia oil with blood lipid lowering function according to the present invention;

[0035] Figure 2 - A trend diagram showing the change of the content of acetyl-CoA in the fermentation product with the fermentation time when preparing camellia oil in Example 1-3 and Comparative Example 1-2;

[0036] Figure 3 - A trend diagram showing the change of ATP content in the fermentation product with fermentation time when preparing camellia oil in Example 1-3 and Comparative Example 1-2;

[0037] Figure 4 —A trend diagram showing the change of the content of phytosterols in the fermentation product over the fermentation time when preparing camellia oil in Example 1-3 and Comparative Example 1-2;

[0038] Figure 5 —A typical image of the blood vessel staining intensity of the tail of zebrafish after being treated with the camellia oil with blood lipid lowering function prepared by the present invention;

[0039] Figure 6— is a trend diagram showing the effect of the mixed volume ratio of Aspergillus niger suspension and Microbacterium flavum suspension on the yield of phytosterols;

[0040] Figure 7 — is a trend diagram showing the effect of the inoculation amount of Aspergillus niger and Microbacterium flavum suspension on the yield of phytosterols;

[0041] Figure 8 — is a trend chart showing the effect of mixed fermentation time of Aspergillus niger suspension and Microbacterium flavum suspension on the yield of phytosterols. DETAILED DESCRIPTION

[0042] 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.

[0043] The strain number of Aspergillus niger used in the present invention is ATCC16404, and the English name of Microbacterium flavum used is microbacterium flavum, abbreviated as M. flavum, and its strain number is ATCC11063.

[0044] Example 1

[0045] A method for preparing camellia oil with blood lipid lowering function, the process flow chart is as follows Figure 1 As shown, the following steps are included:

[0046] 1) Sterilization of decolorized waste clay: sterilize the decolorized waste clay at 121°C for 15 minutes; the decolorized waste clay is obtained by treating pre-decolorized oil with activated clay in the refining process of raw tea oil;

[0047] 2) Mixed fermentation of Aspergillus niger and Microbacterium flavum:

[0048] a) adding the sterilized bleaching waste clay to the sterilized fermentation medium (the fermentation medium is composed of: 0.5 g / L yeast powder, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, 1000 mL distilled water, pH 7.0, sterilized at 121° C. for 15 min) to make the concentration of the bleaching waste clay 60 g / L, to obtain a fermentation medium containing the bleaching waste clay;

[0049] b) Prepare spores at a concentration of 1×10 8 CFU / ml of Aspergillus niger suspension and 1.5×10 8 CFU / ml of yellow microbacterium suspension:

[0050] The specific operation steps of the Aspergillus niger expansion culture are as follows: inoculate the activated Aspergillus niger strain into the Aspergillus niger liquid seed culture medium (glucose 20g / L, peptone 20g / L, yeast powder 10g / L, K2HPO4 2.0g / L, distilled water 1000mL, adjust pH7.0, sterilize at 121°C for 30min), culture on a constant temperature shaker, and set aside;

[0051] The specific operation steps of expanding the culture of Microbacterium flavum are as follows: inoculate the activated Microbacterium flavum strain into a liquid seed culture medium of Microbacterium flavum (glucose 60 g / L, peptone 25 g / L, NaNO3 2 g / L, MgSO4·7H2O 1 g / L, KH2PO4·3H2O1 g / L, distilled water 1000 mL, pH 5.0, sterilized at 121°C for 30 min), culture on a constant temperature shaker, and set aside;

[0052] After the expansion culture is completed, the bacterial liquid after the expansion culture of Aspergillus niger and the bacterial liquid after the expansion culture of Microbacterium flavum are respectively centrifuged at 4000r / min for 15min, the supernatant is removed, 10ml of 0.9% physiological saline is added, and the mixture is evenly mixed by vortex vibration, the bacterial liquid is centrifuged at 4000r / min for 5min, the supernatant is removed, 0.9% physiological saline is added, and the mixture is repeatedly washed 4 times, and finally the physiological saline is used to supplement and mix, the physiological saline is used as a blank control, and the absorbance value of the bacterial liquid concentration is adjusted to 0.8 at a wavelength of 600nm with the physiological saline to prepare the Aspergillus niger suspension and the Microbacterium flavum suspension;

[0053] c) mixing the Aspergillus niger suspension and the Microbacterium flavum suspension in a mixing volume ratio of 1:2 to obtain a mixed bacterial suspension;

[0054] d) inoculating the mixed bacterial suspension into a fermentation medium containing decolorized waste clay at an inoculation rate of 6%, and then fermenting at a temperature of 35° C. for 72 hours to obtain a decolorized waste clay fermentation product;

[0055] 3) Extraction of phytosterols: After the fermentation is completed, the decolorized waste clay fermentation product is cooled to 4°C, and 80% ethanol is added to the decolorized waste clay fermentation product at a volume ratio of 1:20, and mixed; then, ultrasonic treatment is performed at a temperature of 15°C and a power of 500W for 90 minutes, and the ultrasonic treatment is performed twice, and the supernatant is extracted and concentrated to obtain a crude extract of phytosterols extracted from the decolorized waste clay;

[0056] 4) Ethanol crystallization and freeze drying: 95% ethanol was added to the crude phytosterol extract extracted from the decolorized waste clay at a solid-liquid ratio of 1:15 g / mL for crystallization. The crystallization temperature was 30° C. and the crystallization time was 4 hours. After the crystallization was completed, the precipitate was collected and freeze-dried to obtain purified phytosterol powder (the purity of the phytosterol powder was 99.26% after testing);

[0057] 5) Adding phytosterol powder: Add phytosterol powder to camellia oil at an addition amount of 20,000 mg / kg, mix thoroughly and evenly, and obtain finished camellia oil with lipid-lowering function.

[0058] Example 2

[0059] A method for preparing camellia oil with blood lipid lowering function comprises the following steps:

[0060] 1) Sterilization of decolorized waste clay: sterilize the decolorized waste clay at 121°C for 15 minutes; the decolorized waste clay is obtained by treating pre-decolorized oil with activated clay in the refining process of raw tea oil;

[0061] 2) Mixed fermentation of Aspergillus niger and Microbacterium flavum:

[0062] a) adding the sterilized bleaching waste clay to the sterilized fermentation medium (the fermentation medium is composed of: 0.5 g / L yeast powder, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, 1000 mL distilled water, pH 7.0, sterilized at 121° C. for 15 min) to make the concentration of the bleaching waste clay 60 g / L, to obtain a fermentation medium containing the bleaching waste clay;

[0063] b) Prepare spores at a concentration of 1×10 8 CFU / ml of Aspergillus niger suspension and 1.5×10 8 CFU / ml of yellow microbacterium suspension:

[0064] The specific operation steps of the Aspergillus niger expansion culture are as follows: inoculate the activated Aspergillus niger strain into the Aspergillus niger liquid seed culture medium (glucose 20g / L, peptone 20g / L, yeast powder 10g / L, K2HPO4 2.0g / L, distilled water 1000mL, adjust pH7.0, sterilize at 121°C for 30min), culture on a constant temperature shaker, and set aside;

[0065] The specific operation steps of expanding the culture of Microbacterium flavum are as follows: inoculate the activated Microbacterium flavum strain into a liquid seed culture medium of Microbacterium flavum (glucose 60 g / L, peptone 25 g / L, NaNO3 2 g / L, MgSO4·7H2O 1 g / L, KH2PO4·3H2O1 g / L, distilled water 1000 mL, pH 5.0, sterilized at 121°C for 30 min), culture on a constant temperature shaker, and set aside;

[0066] After the expansion culture is completed, the bacterial liquid after the expansion culture of Aspergillus niger and the bacterial liquid after the expansion culture of Microbacterium flavum are respectively centrifuged at 4000r / min for 15min, the supernatant is removed, 10ml of 0.9% physiological saline is added, and the mixture is evenly mixed by vortex vibration, the bacterial liquid is centrifuged at 4000r / min for 5min, the supernatant is removed, 0.9% physiological saline is added, and the mixture is repeatedly washed 4 times, and finally the physiological saline is used to supplement and mix, the physiological saline is used as a blank control, and the absorbance value of the bacterial liquid concentration is adjusted to 0.8 at a wavelength of 600nm with the physiological saline to prepare the Aspergillus niger suspension and the Microbacterium flavum suspension;

[0067] c) mixing the Aspergillus niger suspension and the Microbacterium flavum suspension in a mixing volume ratio of 1:1 to obtain a mixed bacterial suspension;

[0068] d) inoculating the mixed bacterial suspension into a fermentation medium containing decolorized waste clay at an inoculation rate of 4%, and then fermenting at a temperature of 28° C. for 48 hours to obtain a decolorized waste clay fermentation product;

[0069] 3) Extraction of phytosterols: After the fermentation is completed, the decolorized waste clay fermentation product is cooled to 4°C, and 80% ethanol is added to the decolorized waste clay fermentation product at a volume ratio of 1:20, and mixed; then, ultrasonic treatment is performed at a temperature of 15°C and a power of 500W for 90 minutes, and the ultrasonic treatment is performed twice, and the supernatant is extracted and concentrated to obtain a crude extract of phytosterols extracted from the decolorized waste clay;

[0070] 4) Ethanol crystallization and freeze drying: 95% ethanol was added to the crude phytosterol extract extracted from the decolorized waste clay at a solid-liquid ratio of 1:15 g / mL for crystallization. The crystallization temperature was 30°C and the crystallization time was 4 hours. After the crystallization was completed, the precipitate was collected and freeze-dried to obtain purified phytosterol powder (the purity of the phytosterol powder was 99.19% after testing);

[0071] 5) Adding phytosterol powder: Add phytosterol powder to camellia oil at an addition amount of 20,000 mg / kg, mix thoroughly and evenly, and obtain finished camellia oil with lipid-lowering function.

[0072] Example 3

[0073] A method for preparing camellia oil with blood lipid lowering function comprises the following steps:

[0074] 1) Sterilization of decolorized waste clay: sterilize the decolorized waste clay at 121°C for 15 minutes; the decolorized waste clay is obtained by treating pre-decolorized oil with activated clay in the refining process of raw tea oil;

[0075] 2) Mixed fermentation of Aspergillus niger and Microbacterium flavum:

[0076] a) adding the sterilized bleaching waste clay to the sterilized fermentation medium (the fermentation medium is composed of: 0.5 g / L yeast powder, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, 1000 mL distilled water, pH 7.0, sterilized at 121° C. for 15 min) to make the concentration of the bleaching waste clay 60 g / L, to obtain a fermentation medium containing the bleaching waste clay;

[0077] b) Prepare spores at a concentration of 1×10 8 CFU / ml of Aspergillus niger suspension and 1.5×10 8 CFU / ml of yellow microbacterium suspension:

[0078] The specific operation steps of the Aspergillus niger expansion culture are as follows: inoculate the activated Aspergillus niger strain into the Aspergillus niger liquid seed culture medium (glucose 20g / L, peptone 20g / L, yeast powder 10g / L, K2HPO4 2.0g / L, distilled water 1000mL, adjust pH7.0, sterilize at 121°C for 30min), culture on a constant temperature shaker, and set aside;

[0079] The specific operation steps of expanding the culture of Microbacterium flavum are as follows: inoculate the activated Microbacterium flavum strain into a liquid seed culture medium of Microbacterium flavum (glucose 60 g / L, peptone 25 g / L, NaNO3 2 g / L, MgSO4·7H2O 1 g / L, KH2PO4·3H2O1 g / L, distilled water 1000 mL, pH 5.0, sterilized at 121°C for 30 min), culture on a constant temperature shaker, and set aside;

[0080] After the expansion culture is completed, the bacterial liquid after the expansion culture of Aspergillus niger and the bacterial liquid after the expansion culture of Microbacterium flavum are respectively centrifuged at 4000r / min for 15min, the supernatant is removed, 10ml of 0.9% physiological saline is added, and the mixture is evenly mixed by vortex vibration, the bacterial liquid is centrifuged at 4000r / min for 5min, the supernatant is removed, 0.9% physiological saline is added, and the mixture is repeatedly washed 4 times, and finally the physiological saline is used to supplement and mix, the physiological saline is used as a blank control, and the absorbance value of the bacterial liquid concentration is adjusted to 0.8 at a wavelength of 600nm with the physiological saline to prepare the Aspergillus niger suspension and the Microbacterium flavum suspension;

[0081] c) mixing the Aspergillus niger suspension and the Microbacterium flavum suspension at a mixing volume ratio of 1:3 to obtain a mixed bacterial suspension;

[0082] d) inoculating the mixed bacterial suspension into a fermentation medium containing decolorized waste clay at an inoculation rate of 10%, and then fermenting at a temperature of 35° C. for 96 hours to obtain a decolorized waste clay fermentation product;

[0083] 3) Extraction of phytosterols: After the fermentation is completed, the decolorized waste clay fermentation product is cooled to 4°C, and 80% ethanol is added to the decolorized waste clay fermentation product at a volume ratio of 1:20, and mixed; then, ultrasonic treatment is performed at a temperature of 15°C and a power of 500W for 90 minutes, and the ultrasonic treatment is performed twice, and the supernatant is extracted and concentrated to obtain a crude extract of phytosterols extracted from the decolorized waste clay;

[0084] 4) Ethanol crystallization and freeze drying: 95% ethanol was added to the crude phytosterol extract extracted from the decolorized waste clay at a solid-liquid ratio of 1:15 g / mL for crystallization. The crystallization temperature was 30° C. and the crystallization time was 4 hours. After the crystallization was completed, the mixture was centrifuged at 4000 rpm for 5 minutes, and the bottom precipitate was collected and freeze-dried to obtain purified phytosterol powder (the purity of the phytosterol powder was 99.21% after testing);

[0085] 5) Adding phytosterol powder: Add phytosterol powder to camellia oil at an addition amount of 16000 mg / kg, mix thoroughly and evenly, and obtain finished camellia oil with lipid-lowering function.

[0086] Comparative Example 1

[0087] The difference between this comparative example and Example 1 is that: Step 2) uses a single Aspergillus niger for fermentation, and the specific operation steps are:

[0088] a) adding the sterilized bleaching waste clay to the sterilized fermentation medium (the fermentation medium is composed of: 0.5 g / L yeast powder, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, 1000 mL distilled water, pH 7.0, sterilized at 121° C. for 15 min) to make the concentration of the bleaching waste clay 60 g / L, to obtain a fermentation medium containing the bleaching waste clay;

[0089] b) Prepare spores at a concentration of 1×10 8 CFU / ml of Aspergillus niger suspension:

[0090] The specific operation steps of the Aspergillus niger expansion culture are as follows: inoculate the activated Aspergillus niger strain into the Aspergillus niger liquid seed culture medium (glucose 20g / L, peptone 20g / L, yeast powder 10g / L, K2HPO4 2.0g / L, distilled water 1000mL, adjust pH7.0, sterilize at 121°C for 30min), culture on a constant temperature shaker, and set aside;

[0091] After the expansion culture is completed, the bacterial liquid after the expansion culture of Aspergillus niger is centrifuged at 4000r / min for 15min, the supernatant is removed, 10ml of 0.9% physiological saline is added, and the mixture is evenly mixed by vortex vibration, the bacterial liquid is centrifuged at 4000r / min for 5min, the supernatant is removed, 0.9% physiological saline is added, and the mixture is repeatedly washed 4 times, and finally the physiological saline is used to supplement and mix, and the physiological saline is used as a blank control, and the absorbance value of the bacterial liquid concentration is adjusted to 0.8 at a wavelength of 600nm with the physiological saline to prepare an Aspergillus niger suspension;

[0092] c) inoculating the Aspergillus niger suspension into the fermentation medium containing the decolorized waste clay at an inoculation rate of 6%, and then fermenting the medium at a temperature of 35° C. for 72 hours to obtain a decolorized waste clay fermentation product.

[0093] Comparative Example 2

[0094] The difference between this comparative example and Example 1 is that: Step 2) uses a single yellow microbacterium for fermentation, and the specific operation steps are:

[0095] a) adding the sterilized bleaching waste clay to the sterilized fermentation medium (the fermentation medium is composed of: 0.5 g / L yeast powder, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, 1000 mL distilled water, pH 7.0, sterilized at 121° C. for 15 min) to make the concentration of the bleaching waste clay 60 g / L, to obtain a fermentation medium containing the bleaching waste clay;

[0096] b) Prepare spores at a concentration of 1.5×10 8 CFU / ml of yellow microbacterium suspension:

[0097] The specific operation steps of expanding the culture of Microbacterium flavum are as follows: inoculate the activated Microbacterium flavum strain into a liquid seed culture medium of Microbacterium flavum (glucose 60 g / L, peptone 25 g / L, NaNO3 2 g / L, MgSO4·7H2O 1 g / L, KH2PO4·3H2O1 g / L, distilled water 1000 mL, pH 5.0, sterilized at 121°C for 30 min), culture on a constant temperature shaker, and set aside;

[0098] After the expansion culture is completed, the bacterial solution after the expansion culture of the yellow microbacterium is centrifuged at 4000r / min for 15min, the supernatant is removed, 10ml of 0.9% physiological saline is added, and the mixture is evenly mixed by vortexing, the bacterial solution is centrifuged at 4000r / min for 5min, the supernatant is removed, 0.9% physiological saline is added, and the mixture is repeatedly washed 4 times, and finally the physiological saline is added to mix evenly, and the physiological saline is used as a blank control, and the absorbance value of the bacterial solution concentration is adjusted to 0.8 at a wavelength of 600nm with the physiological saline to prepare a yellow microbacterium bacterial suspension;

[0099] c) inoculating a suspension of Microbacterium flavum into a fermentation medium containing decolorized waste clay at an inoculation rate of 6%, and then fermenting the medium at a temperature of 35° C. for 72 hours to obtain a decolorized waste clay fermentation product.

[0100] Experimental Example 1

[0101] In this experimental example, the acid value and peroxide value of the camellia oil with blood lipid lowering function prepared in Examples 1-3 were measured, and the results are shown in Table 1:

[0102] Table 1 Determination results of acid value and peroxide value of camellia oil with lipid-lowering function prepared in Examples 1-3

[0103] Acid value (mgKOH / g) Peroxide value (g / 100g) Example 1 0.11 0.012 Example 2 0.12 0.017 Example 3 0.12 0.014

[0104] It can be seen from Table 1 that the acid value of the camellia oil prepared by the method of the present invention is controlled at 0.1-1.0 mgKOH / g, which is less than 2.0 mgKOH / g, and the peroxide value is controlled at 0.010-0.020 g / 100 g, which is less than 0.25 g / 100 g, which meets the first-level pressing standard of camellia oil GB / T11765-2018.

[0105] Experimental Example 2

[0106] In this experimental example, the solubility of the phytosterols prepared in Examples 1-3 and Comparative Examples 1-2 and purchased phytosterols (named as the purchased group, the manufacturer is Baoji Fangsheng Biological Development Co., Ltd.) in camellia oil was measured.

[0107] The solubility test method of phytosterol in camellia oil is as follows: weigh 100.0g of camellia oil of each treatment group into a 200ml beaker, place in a glycerol bath, start heating from 20°C, adjust the temperature every 5°C until 100°C, stay at each temperature for 5 minutes, add phytosterol, observe the dissolution of phytosterol at each temperature, and add until insoluble matter appears, record the amount of phytosterol added, repeat the operation once, and take the average value. The results are shown in Table 2:

[0108] Table 2 Determination results of the solubility of phytosterols in camellia oil of Examples 1-3, Comparative Examples 1-2 and the purchased group

[0109] Solubility (g / 100g oil) Example 1 15.5 Example 2 15.1 Example 3 15.3 Comparative Example 1 7.4 Comparative Example 2 8.9 Purchase Group 6.8

[0110] It can be seen from the above table that the solubility of phytosterols prepared by the methods of Examples 1-3 in camellia oil reached 15g / 100g oil, which is significantly higher than the solubility of comparative example 1, comparative example 2 and commercially purchased phytosterols; comparative example 1 and comparative example 2 respectively use Aspergillus niger and Microbacterium flavum for single-strain fermentation, which can slightly improve the solubility of phytosterols in oil to a certain extent, but there is no significant difference between them and the purchased group, indicating that only under the synergistic effect of Aspergillus niger and Microbacterium flavum can the solubility of phytosterols in camellia oil be effectively improved, and the camellia oil can further have a better lipid-lowering effect.

[0111] Experimental Example 3

[0112] This experimental example measured the yield of phytosterols obtained when preparing finished tea seed oil in Example 1-3 and Comparative Example 1-2. The yield of phytosterols was calculated based on the decolorized waste clay. The yield of phytosterols = weight of phytosterol powder / content of phytosterols in decolorized waste clay * 100%. The results are shown in Table 3. Among them, the method for determining the content of phytosterols is: the dried decolorized waste clay is made into a uniform powder, 1.0g of the powdered decolorized waste clay is accurately weighed, 30ml of n-hexane is added, and ultrasonic-assisted (normal temperature 30°C) extraction is performed for 1h, and then the extract is filtered to obtain the extract. The filter residue is repeatedly extracted 4 times, and 30ml of n-hexane is added each time. The extracts are combined, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to recover the solvent. Before the solvent is almost evaporated, it is transferred to a porcelain evaporating dish, evaporated in a water bath, and finally the phytosterol extract is obtained, which is accurately weighed. The obtained plant sterol extract was fully dissolved with chloroform, placed in a 10ml brown volumetric flask, diluted to the scale, 1ml of the sample solution to be tested was removed, 2nl of acetic acid was added, 1 drop of concentrated sulfuric acid was added, and after oscillation for 1min, it was allowed to stand for 24min, and then its absorbance value was detected at a wavelength of 610nm, and the content was calculated according to the standard curve. When the plant sterol standard curve was prepared, the plant sterol concentrations were 0mg / ml, 0.1mg / ml, 0.2mg / ml, 0.4mg / ml, 0.6mg / ml, 0.8mg / ml, and 1.0mg / ml, respectively. The standard curve is: y=1.7672x+0.0208, R 2 =0.9966, where y represents the absorbance value and x represents different phytosterol concentrations.

[0113] Table 3 Yield of phytosterols obtained in Examples 1-3 and Comparative Examples 1-2

[0114] Treatment Group Yield (%) Example 1 95.47 Example 2 94.45 Example 3 94.78 Comparative Example 1 80.11 Comparative Example 2 82.42

[0115] As can be seen from Table 3, the yield of phytosterols obtained by the mixed fermentation of Aspergillus niger and Microbacterium flavum decolorizing waste clay in Example 1-3 reached more than 94%, which is significantly higher than that in Comparative Example 1-2, indicating that the synergistic fermentation of two bacteria (Aspergillus niger and Microbacterium flavum) can further significantly improve the yield of phytosterols in the decolorized waste clay compared with the fermentation of Aspergillus niger and Microbacterium flavum with single strains, thereby playing a role in efficiently enriching phytosterols.

[0116] Experimental Example 4

[0117] This experimental example studies the variation trend of acetyl coenzyme A, ATP and phytosterol content during the fermentation process when preparing finished tea seed oil in Examples 1-3 and Comparative Examples 1-2. Figure 2-4 shown.

[0118] The fermentation products (including culture medium, bleaching waste clay, and bacteria) in the corresponding strain fermentation steps of Examples 1-3 and Comparative Examples 1-2 were respectively taken after 12h, 24h, 48h, 72h, 96h, and 120h of fermentation to detect the contents of acetyl CoA and ATP.

[0119] 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).

[0120] Determination of ATP content: Sterile water was added to the fermentation products (including culture medium, decolorized waste clay, and bacteria) cultured for 12h, 24h, 48h, 72h, 96h, and 120h, placed in a 37°C incubator, shaken at 200r / min for 5min, and the liquid was collected. After filtration through 2 layers of filter paper, a pure bacterial solution was obtained. After centrifugation at 4000r / min for 5min, the bacterial solution was resuspended with sterile water to obtain a bacterial solution. The bacterial solution was centrifuged at a centrifugal temperature of 4°C and 10000r / min for 15s, and the supernatant was discarded, and the lower bacterial cells were collected. Then, a methanol solution precooled at -30°C was quickly added, and placed at -30°C for 1h to stop the metabolic activity of the bacteria. The bacterial sample treated with methanol solution was dried in a vacuum centrifugal dryer, and a white solid substance containing ATP was obtained after removing methanol. Then, an equal volume of water was added to the centrifuge tube to dissolve ATP to obtain an ATP solution, which was then detected using an ATP detection kit. ATP concentration standard curve: y = -2.2209x 2 +3.8976x+0.0701, R 2 =0.9915, (ATP concentrations in the curve are: 0.0625umol, 0.15umol, 0.25umol, 0.5umol, 1.0umol).

[0121] The method for determining the phytosterol content in the fermentation products corresponding to different fermentation times is the same as the determination of the phytosterol content in Experimental Example 3, with the only difference being that the sample pretreatment is different. The fermentation product in this experiment needs to be freeze-dried first and then made into a uniform powder, and then 1 g of baking powder is weighed for subsequent determination steps.

[0122] from Figure 2-3 It can be seen that during the fermentation process, the contents of acetyl-CoA and ATP in the fermentation products corresponding to Examples 1-3 showed a trend of significantly increasing and then tending to a stable state as time went on, while the contents of acetyl-CoA and ATP in the fermentation products corresponding to Comparative Examples 1 and 2 showed a trend of slowly increasing and then tending to a stable state as time went on, and during the entire fermentation process, the contents of acetyl-CoA and ATP corresponding to Examples 1-3 were higher than those in Comparative Examples 1-2, indicating that only when Aspergillus niger and Microbacterium flavum were fermented in coordination could the synthesis of acetyl-CoA and ATP be effectively promoted and accelerated, so that the contents of acetyl-CoA and ATP and their increasing ranges were significantly higher than those in the fermentation of Aspergillus niger or Microbacterium flavum single strains;

[0123] from Figure 4It can be seen that the content of phytosterols in the fermentation products corresponding to Examples 1-3 also showed a trend of significantly increasing and then slightly decreasing. The content of phytosterols in the fermentation products corresponding to Comparative Examples 1 and 2 did not increase significantly, and at 96 hours of fermentation, a downward trend appeared. The emergence of this phenomenon may be related to the fact that the bacteria are in a period of decline, and the nutrients are rapidly consumed, so that phytosterols are also consumed as substrates, resulting in a greater consumption of phytosterols than the synthesis, which ultimately leads to a decrease in the content of phytosterols.

[0124] Moreover, during the entire fermentation process, the content of phytosterols corresponding to Examples 1-3 was higher than that of Comparative Examples 1-2, which also shows that the synergistic fermentation of Aspergillus niger and Microbacterium flavum can significantly increase the content of phytosterols in the fermentation product compared with single-species fermentation. This may be related to the fact that the synergistic fermentation of Aspergillus niger and Microbacterium flavum increases the content of acetyl-CoA and ATP, thereby promoting the synthesis of phytosterols.

[0125] Experimental Example 5

[0126] In this experimental example, the degree of zebrafish tail blood vessel staining by the finished camellia oil prepared in Examples 1-3, Comparative Examples 1-2, Control Group 1, and the camellia oil in the blank control group was measured to evaluate the lipid-lowering effect of each finished camellia oil. Among them, the finished camellia oil in Control Group 1 was obtained by directly adding the phytosterols of the purchased group to the camellia oil in the same amount as in Example 1; the camellia oil in the blank control group was the camellia oil used in step 5) of Example 1.

[0127] The method for determining the degree of blood vessel staining in the tail of zebrafish is as follows: 5dpf wild-type AB strain zebrafish are randomly selected in a beaker, and 30 zebrafish are treated in each beaker (experimental group). The finished camellia oil sample is given 2000ug / ml in water, the positive control group is atorvastatin 11.6ug / ml, and the normal control group (zebrafish fed with non-high-sugar and high-fat feed, not fed with camellia oil) and the model control group (zebrafish fed with high-sugar and high-fat feed, and not fed with camellia oil) are set up at the same time, and the capacity of each cup is 25ml. Except for the normal control group, the remaining experimental groups are given high-sugar and high-fat feed in water to establish a high-sugar and high-fat model of zebrafish. After 2 days of treatment at 28℃, oil red o is given for overall fat staining (oil red o is a fat-soluble azo fuel with strong lipid solvent and lipid dye properties, which specifically stains triglycerides in cells. The higher the staining degree, the higher the pixel, the higher the triglyceride content, and the worse the lipid-lowering function). After decolorization and bleaching, 10 zebrafish were randomly selected from each beaker and photographed under a dissection microscope. The data were analyzed and collected using NIS-Elenments D 3.20 advanced image processing software to analyze the degree of blood vessel staining in the zebrafish tail. The results are shown in Table 4:

[0128] Table 4 Determination results of the degree of staining of zebrafish tail blood vessels by finished tea seed oil of Examples 1-3, Comparative Examples 1-2, Control Group 1 and Blank Control Group

[0129]

[0130]

[0131] As shown in Table 4, the staining degree of zebrafish tail blood vessels corresponding to Examples 1-3 was significantly lower than that of the blank control group; and compared with the control group 1, the staining degree of Examples 1-3 was significantly lower than that of the control group 1, indicating that when preparing camellia oil with lipid-lowering function, adding phytosterols prepared by the method of the present invention to camellia oil has a better lipid-lowering effect than directly purchased phytosterols;

[0132] Compared with Comparative Examples 1 and 2, the dyeing degrees of Examples 1-3 are significantly lower than those of Comparative Examples 1 and 2, indicating that the mixed fermentation of Aspergillus niger and Microbacterium flavum in the present invention can achieve a better lipid-lowering effect than fermentation of any one of Aspergillus niger and Microbacterium flavum alone. This may be because the solubility of phytosterols in camellia oil is increased under the synergistic fermentation of Aspergillus niger and Microbacterium flavum (as confirmed in Table 2), which effectively enhances the lipid-lowering effect of phytosterols, thereby making the prepared finished camellia oil have a better lipid-lowering effect.

[0133] Experimental Example 6

[0134] This experimental example further verifies the lipid-lowering effect of the finished camellia oil prepared in Example 1, and the verification method is as follows:

[0135] This experimental example studies the maximum tolerable concentration (MTC) of camellia oil prepared in Example 1. The specific steps are as follows: wild-type AB strain zebrafish 5 days after fertilization were randomly selected in a beaker, and 30 zebrafish were treated in each beaker (experimental group). Different concentrations of finished camellia oil (125ug / ml, 250ug / ml, 500ug / ml, 1000ug / ml, 2000ug / ml, 2250ug / ml) were given in water-soluble form, and a normal control group (normal zebrafish, zebrafish not fed with high-sugar and high-fat feed, not fed with camellia oil) and a model control group (model control group: zebrafish fed with high-sugar and high-fat feed, and not fed with camellia oil) were set up at the same time, and the capacity of each cup was 25ml. Except for the normal control group, the remaining experimental groups were all given high-sugar and high-fat feed in water-soluble form to establish a high-sugar and high-fat model of zebrafish. The samples were treated at 28°C for 2 days, and the MTC of the model zebrafish was determined. The results are shown in Table 5.

[0136] Table 5 Effects of different addition amounts on the MTC of model zebrafish

[0137]

[0138]

[0139] As can be seen from Table 5, the MTC of camellia oil in assisting in lowering blood lipids is 2000ug / ml.

[0140] Using the optimal MTC concentration, 5dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Finished camellia oil 2000ug / ml was given in water, and the positive control group was atorvastatin 11.6ug / ml. A normal control group and a model control group were also set up, with a capacity of 25ml per cup. Except for the normal control group, the other experimental groups were given high-sugar and high-fat feed in water to establish a high-sugar and high-fat model of zebrafish. After 2 days of treatment at 28°C, oil red o was given for overall fat staining. After decolorization and bleaching, 10 zebrafish were randomly selected from each beaker and photographed under a dissection microscope. The NIS-Elenments D 3.20 advanced image processing software was used to analyze and collect data to analyze the degree of vascular staining in the zebrafish tail. The results are shown in Tables 6 and Figure 5 shown.

[0141] Table 6 Effect of the optimal sample addition amount on lipid-lowering in zebrafish model

[0142]

[0143] from Figure 5 As can be seen from Table 6, when the finished camellia oil was added at an amount of 2000ug / ml, the staining intensity of the blood vessels in the zebrafish tail was significantly different from that in the model control group, indicating that the finished camellia oil prepared in Example 1 can reduce the content of triglycerides, thereby verifying that the camellia oil has a good auxiliary lipid-lowering effect.

[0144] Experimental Example 7

[0145] In this experimental example, tea seed oil was prepared by referring to the steps of Example 1, and the effects of different mixing volume ratios of Aspergillus niger suspension and Microbacterium flavum suspension on the yield of phytosterols and the lipid-lowering effect of tea seed oil were studied. The mixing volume ratios of Aspergillus niger suspension and Microbacterium flavum suspension were 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4. The results are shown in Tables 7 and 8. Figure 6 shown.

[0146] From Table 7 and Figure 6It can be seen that with the increase in the proportion of yellow microbacterium, the yield of phytosterols and the lipid-lowering effect in the bacterial tissue showed a trend of first significantly increasing and then decreasing. When Aspergillus niger: yellow microbacterium = 1:4, the yield of phytosterols and the lipid-lowering effect decreased significantly. When Aspergillus niger: yellow microbacterium = 1:1-3, the yield of phytosterols reached more than 95%, and there was no obvious difference in the degree of staining (pixels), so the preferred ratio was Aspergillus niger: yellow microbacterium = 1:1-3, and the best ratio was Aspergillus niger: yellow microbacterium = 1:2.

[0147] Table 7 Effect of the mixed volume ratio of Aspergillus niger suspension and Microbacterium flavum suspension on the degree of dyeing of camellia oil

[0148]

[0149] Experimental Example 8

[0150] In this experimental example, tea seed oil was prepared by referring to the steps of Example 1, and the effects of different inoculation amounts of Aspergillus niger and Microbacterium flavum suspension on the yield of phytosterols and the lipid-lowering effect of tea seed oil were studied. The inoculation amounts of Aspergillus niger and Microbacterium flavum suspension were 2%, 4%, 6%, 8%, 10%, and 12%. The results are shown in Tables 8 and 9. Figure 7 shown.

[0151] From Table 8 and Figure 7 It can be seen that with the increase of the inoculation amount of the mixed bacterial liquid of Aspergillus niger and Microbacterium flavum, the phytosterol yield first increased and then leveled off, and showed a significant downward trend after the inoculation amount reached 10%. This may be due to the excessive inoculation amount, which produced a large amount of metabolic waste, thus having a negative impact on the growth of Aspergillus niger and Microbacterium flavum, and at the same time affecting the yield of phytosterols. The dyeing degree showed a trend of first decreasing, then leveling off, and then increasing. Combining the changing trends of the two, the preferred inoculation amount is 4-10%, and the optimal inoculation amount is 6%.

[0152] Table 8 Effect of inoculation amount of Aspergillus niger and Microbacterium flavum suspension on phytosterol yield and staining degree

[0153]

[0154] Experimental Example 9

[0155] In this experimental example, tea seed oil was prepared by referring to the steps of Example 1, and the effects of different fermentation times of Aspergillus niger and Microbacterium flavum suspensions on the yield of phytosterols and the lipid-lowering effect of tea seed oil were studied.

[0156] From Table 9 and Figure 8It can be seen that with the extension of the fermentation time of the mixed culture of Aspergillus niger and Microbacterium flavum, the phytosterol yield first increased and then leveled off, and showed a significant downward trend after 96 hours. The appearance of this phenomenon may be that the bacteria are in a period of death, which hinders the synthesis of phytosterol content in the fermentation product, and the nutrients in the later period are rapidly consumed, so that phytosterol is consumed by the bacteria as a substrate, resulting in a decrease in the content of phytosterol in the fermentation product after 96 days of fermentation, and ultimately a significant decrease in the yield of phytosterol; while the dyeing degree shows a trend of first decreasing and then leveling off, and then a significant increase. Combining the changing trends of the two, the preferred fermentation time is 48-96 hours, and the optimal fermentation time is 72 hours.

[0157] Table 9 Effect of fermentation time of Aspergillus niger and Microbacterium flavum suspension on phytosterol yield and staining degree (pixels)

[0158]

[0159] 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 having the function of lowering blood lipids, characterized in that: The following steps are involved: 1) Sterilization of decolorized waste clay; 2) Mixed fermentation of Aspergillus niger and Microbacterium flavum: firstly prepare a fermentation medium containing decolorized waste clay, then inoculate a mixed bacterial suspension of Aspergillus niger and Microbacterium flavum onto the fermentation medium containing decolorized waste clay for fermentation to obtain a decolorized waste clay fermentation product; 3) Extraction of phytosterols: cooling the fermentation product of decolorized waste clay, adding ethanol and then performing ultrasonic treatment to obtain a crude phytosterol extract; 4) growing crystals in ethanol and freeze-drying to obtain phytosterol powder; 5) Adding phytosterol powder: adding phytosterol powder to camellia oil, mixing thoroughly and evenly, to obtain finished camellia oil with lipid-lowering function.

2. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 1, characterized in that: The sterilization temperature of the decolorized waste clay in step 1) is 121° C. and the sterilization time is 15 minutes.

3. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 1, characterized in that: The specific operation steps of the step 2) mixed fermentation of Aspergillus niger and Microbacterium flavum are as follows: a) adding the sterilized bleaching waste clay to the sterilized fermentation medium to make the concentration of the bleaching waste clay 60 g / L, to obtain a fermentation medium containing the bleaching waste clay; b) Prepare spores at a concentration of 2×10 7 -2×10 8 CFU / ml of Aspergillus niger suspension and 2×10 7 -2×10 8 CFU / ml suspension of Microbacterium flavum; c) mixing the Aspergillus niger suspension and the Microbacterium flavum suspension in a mixing volume ratio of 1:1-3 to obtain a mixed bacterial suspension; d) inoculating the mixed bacterial suspension into a fermentation medium containing decolorized waste clay at an inoculation rate of 4-10%, and then fermenting the medium at a temperature of 28-35° C. for 48-96 hours to obtain a decolorized waste clay fermentation product.

4. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 3, characterized in that: The composition of the fermentation medium used in preparing the fermentation medium containing decolorized waste clay in step a) is 0.5 g / L yeast powder, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, 1000 mL distilled water, pH 7.0, and sterilized at 121° C. for 15 min.

5. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 3, characterized in that: The spore concentration of the Aspergillus niger suspension in step b) is 1×10 8 CFU / ml, the spore concentration of the yellow microbacterium suspension is 1.5×10 8 CFU / ml.

6. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 3, characterized in that: In the step c), the mixing volume ratio of the Aspergillus niger suspension and the Microbacterium flavum suspension is 1:

2.

7. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 3, characterized in that: The inoculation amount of the mixed bacterial suspension in step d) is 6%.

8. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 1, characterized in that: The specific operation steps of step 3) are as follows: after the fermentation is completed, the decolorized waste clay fermentation product is cooled to 4°C, and 80% ethanol is added to the decolorized waste clay fermentation product at a volume ratio of 1:10-1:30, and mixed; then, ultrasonic treatment is performed at a temperature of 40-50°C and a power of 400-600W for 90 minutes, the ultrasonic treatment is performed twice, the supernatant is extracted, and concentrated to obtain a crude plant sterol extract.

9. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 1, characterized in that: The specific operation steps of step 4) are: adding 95% ethanol to the crude plant sterol extract at a solid-liquid ratio of 1:10-20g / mL for crystal growth, the crystal growth temperature is 30°C, the crystal growth time is 4h, and after the crystal growth is completed, centrifugation is performed, the bottom precipitate is collected, and freeze-dried to obtain purified plant sterol powder.

10. The method for preparing camellia oil having the function of lowering blood lipids as claimed in claim 1, characterized in that: In the step 5), the amount of phytosterol powder added is 16000 mg / kg-20000 mg / kg.

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