Preparation method of tea seed oil with high content of vitamin a

Abstract

This invention belongs to the field of refining technology for diglyceride camellia seed oil, specifically relating to a method for preparing high-vitamin A content diglyceride camellia seed oil. This invention involves nano-sizing the cellulose extracted from camellia seed cake, then combining the resulting nanocellulose with graphene. The resulting camellia seed cake nanocellulose / graphene composite material adsorbs glycerol and is then mixed with freshly pressed camellia seed oil and lipase for enzymatic glycerolysis. The resulting esterification product is filtered and then molecularly distilled. Vitamin A (prepared from the deodorized distillate fermentation product obtained by yeast and *Microbacterium chrysogenum*) is added to the refined diglyceride camellia seed oil to obtain high-vitamin A content diglyceride camellia seed oil. This method not only improves the utilization rate of fresh camellia seed cake and the deodorized distillate of tea oil refining, increasing the yield of vitamin A, but also reduces vitamin A loss during esterification, shortens esterification time, and improves esterification efficiency.

Description

Technical Field

[0001] This invention belongs to the field of refining technology of diglyceride camellia seed oil, specifically relating to a method for preparing diglyceride camellia seed oil with high vitamin A content. Background Technology

[0002] Camellia seed oil, derived from the seeds of the camellia plant, is widely used as an edible vegetable oil in traditional Asian cooking. Multiple studies have shown that camellia seed oil offers numerous health benefits, including anti-inflammatory, antioxidant, antibacterial, cardiovascular disease prevention, tumor growth inhibition, and Alzheimer's disease prevention. These benefits are closely related to the abundant fatty acids, tocopherols, phytosterols, and other bioactive components in camellia seed oil, as well as trace amounts of vitamin A. Therefore, camellia seed oil has significant potential for development in functional foods.

[0003] In recent years, with the increasing demand from consumers for healthy and functional foods, the food and condiment industry has been developing rapidly. Diacylglycerol, as a potential healthy and functional oil alternative, has received widespread attention. As a natural oil component, diglycerol is present in many vegetable oils, but its content is usually no more than 10%. Currently, the main preparation processes for diglycerol oil are enzymatic hydrolysis and enzymatic glycerolysis, both of which require esterification reactions at temperatures between 55 and 65°C.

[0004] Deodorized tea oil distillate contains a small amount of vitamin A, approximately 0.05-0.3%. After refining and deodorizing, the vitamin A content in tea seed oil is approximately 0.5-1 mg / kg. During the glycerol esterification reaction, due to the high esterification temperature and long reaction time, the vitamin A content in the diglyceride oil is lost. However, vitamin A is crucial for various physiological functions, including vision, reproduction, the immune system, epithelial cell differentiation, and normal bone growth and development. Vitamin A deficiency can lead to night blindness, weakened immunity, and stunted growth; vitamin A deficiency is a global health concern.

[0005] In conclusion, how to increase the vitamin A content in diglyceride oil while simultaneously improving its extraction rate has become an urgent problem to be solved. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a method for preparing camellia seed oil with high vitamin A content diglyceride. This method is simple and can not only effectively improve the esterification rate of diglycerides, but also reduce the loss of vitamin A and increase the vitamin A content in the diglyceride oil.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing camellia seed oil with high vitamin A content diglyceride, comprising the following steps: Cellulose was extracted from camellia seed cake, and the resulting camellia seed cake cellulose was nano-processed to obtain nanocellulose. The nanocellulose and graphene are combined to obtain a nanocellulose / graphene composite material of camellia seed cake. Glycerol and the aforementioned camellia seed cake nanocellulose / graphene composite material are mixed, and glycerol is adsorbed onto the camellia seed cake nanocellulose / graphene composite material to obtain glycerol-camellia seed cake nanocellulose / graphene composite material. Freshly pressed camellia seed oil, glycerol-camellia seed cake nanocellulose / graphene composite material, and lipase are mixed and subjected to enzymatic glycerolysis to obtain esterified products; the temperature of the enzymatic glycerolysis reaction is 30~45℃. The esterification product was filtered, and the resulting mixture of diglyceride camellia seed oil was subjected to molecular distillation to obtain refined diglyceride camellia seed oil. Vitamin A was added to the refined diglyceride camellia seed oil to obtain diglyceride camellia seed oil with high vitamin A content; The method for preparing vitamin A is as follows: the deodorized distillate fermentation product, methanol and sulfuric acid solution are mixed and subjected to esterification reaction to obtain the esterified material; The esterified material, ethanol, and NaOH solution are mixed and a saponification reaction is carried out to obtain a saponified solution. Transfer the saponified liquid into a separatory funnel, add petroleum ether, shake, and let it stand to separate into layers. Take the upper clear liquid for later use. Wash the supernatant with water until the pH value is 7, then add sodium chloride, let it stand to dehydrate, filter and collect the filtrate for later use; The filtrate was concentrated under reduced pressure to obtain the distillation product; The distillation product was passed through a silica gel chromatography column for fractional elution, and the eluent was collected. The eluent was concentrated under reduced pressure until no solvent odor was detected, yielding vitamin A; The preparation method of the deodorized distillate fermentation product is as follows: a mixed bacterial suspension of yeast and flavobacterium is inoculated onto a fermentation medium containing tea oil refined deodorized distillate for fermentation to obtain the deodorized distillate fermentation product.

[0008] Preferably, the cellulose extraction operation steps are as follows: crushing camellia seed cake, defatting the obtained camellia seed cake powder with petroleum ether, drying the defatted camellia seed cake powder, sieving, mixing the obtained sieved powder with NaOH solution for cellulose extraction, and obtaining crude camellia seed cake cellulose. The crude camellia seed cake cellulose was mixed with H2O2 solution and oxidized to obtain camellia seed cake cellulose.

[0009] Preferably, the composite operation steps are as follows: mixing the nanocellulose aqueous solution and the graphene aqueous solution and sequentially performing ultrasonication, magnetic stirring, vacuum filtration and drying; the dried composite material is repeatedly subjected to freeze-thaw treatment and then freeze-dried to obtain the camellia seed cake nanocellulose / graphene composite material.

[0010] Preferably, the mass ratio of the glycerol to the camellia seed cake nanocellulose / graphene composite material is 1:1 to 2.5.

[0011] Preferably, the mass ratio of the freshly pressed camellia seed oil to the glycerol-camellia seed cake nanocellulose / graphene composite material is 4~8:1.

[0012] Preferably, the amount of vitamin A added to the refined diglyceride camellia seed oil is 4000~6000μg / kg.

[0013] Preferably, the enzymatic glycerol hydrolysis reaction takes 6-10 hours.

[0014] Preferably, the inoculation amount of the mixed bacterial suspension of yeast and microbacterium flavonoids is 2-6% of the mass of the tea oil refining and deodorizing distillate.

[0015] Preferably, the fermentation temperature is 28~32℃ and the time is 48~96h.

[0016] Preferably, the lipase is Lipozyme TL IM lipase; the mass of the lipase is 4-8% of the mass of freshly pressed camellia seed oil.

[0017] This invention provides a method for preparing camellia seed oil with high vitamin A content diglyceride, comprising the following steps: Cellulose was extracted from camellia seed cake, and the resulting camellia seed cake cellulose was nano-processed to obtain nanocellulose. The nanocellulose and graphene are combined to obtain a nanocellulose / graphene composite material of camellia seed cake. Glycerol and the aforementioned camellia seed cake nanocellulose / graphene composite material are mixed, and glycerol is adsorbed onto the camellia seed cake nanocellulose / graphene composite material to obtain glycerol-camellia seed cake nanocellulose / graphene composite material. Freshly pressed camellia seed oil, glycerol-camellia seed cake nanocellulose / graphene composite material, and lipase are mixed and subjected to enzymatic glycerolysis to obtain esterified products; the temperature of the enzymatic glycerolysis reaction is 30~45℃. The esterification product was filtered, and the resulting mixture of diglyceride camellia seed oil was subjected to molecular distillation to obtain refined diglyceride camellia seed oil. Vitamin A was added to the refined diglyceride camellia seed oil to obtain diglyceride camellia seed oil with high vitamin A content; The method for preparing vitamin A is as follows: the deodorized distillate fermentation product, methanol and sulfuric acid solution are mixed and subjected to esterification reaction to obtain the esterified material; The esterified material, ethanol, and NaOH solution are mixed and a saponification reaction is carried out to obtain a saponified solution. Transfer the saponified liquid into a separatory funnel, add petroleum ether, shake, and let it stand to separate into layers. Take the upper clear liquid for later use. Wash the supernatant with water until the pH value is 7, then add sodium chloride, let it stand to dehydrate, filter and collect the filtrate; The filtrate was concentrated under reduced pressure to obtain the distillation product; The distillation product was passed through a silica gel chromatography column for fractional elution, and the eluent was collected. The eluent was concentrated under reduced pressure until no solvent odor was detected, yielding vitamin A; The preparation method of the deodorized distillate fermentation product is as follows: a mixed bacterial suspension of yeast and flavobacterium is inoculated onto a fermentation medium containing tea oil refined deodorized distillate for fermentation to obtain the deodorized distillate fermentation product.

[0018] The beneficial effects of this invention are: (1) The refining process of diglyceride camellia seed oil in this invention is simple, convenient, and safe. In the esterification process, only enzymatic glycerolysis is required at 30-45℃. Compared with the traditional glycerolysis esterification process, it can not only reduce the esterification temperature, shorten the esterification time, and increase the esterification rate of diglycerides, but also significantly reduce the loss of vitamin A during esterification, thereby increasing the vitamin A content in the diglyceride oil and improving the quality of the diglyceride camellia seed oil.

[0019] (2) When the present invention uses the camellia seed cake nanocellulose / graphene composite material as the glycerol adsorbent, the glycerol is adsorbed onto the composite material, which significantly reduces its influence on the stability and activity of lipase. At the same time, the adsorbed glycerol participates in the glycerolysis reaction through slow release, which also reduces the coverage of the lipase active site and improves the reaction efficiency of enzymatic glycerolysis.

[0020] (3) The present invention uses a composite material of camellia seed cake nanocellulose / graphene. The two-dimensional honeycomb structure of graphene and the nano-network of nanocellulose intertwine to form a large number of mesopores, providing abundant physical adsorption sites for glycerol molecules. At the same time, the surfaces of nanocellulose and graphene are rich in hydroxyl groups, which can form intermolecular hydrogen bonds with multiple hydroxyl groups of glycerol molecules, thereby enhancing the adsorption effect.

[0021] (4) The present invention uses a fermentation medium containing deodorized distillate by mixing yeast and flavobacterium. Under the synergistic fermentation of yeast and flavobacterium, a large amount of β-carotene, a precursor for the synthesis of vitamin A, is provided, which promotes the synthesis of vitamin A. Thus, vitamin A in the deodorized distillate can be effectively enriched. This not only improves the comprehensive utilization rate of the deodorized distillate, a by-product of refining, but also effectively increases the yield of vitamin A. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the preparation method of camellia seed oil with high vitamin A content in an embodiment of the present invention; Figure 2 A graph showing the trend of changes in β-carotene content due to different fermentation times; Figure 3 A graph showing the trend of changes in vitamin A content due to different fermentation times; Figure 4 This is a graph showing the trend of the effect of different inoculation amounts on vitamin A yield. Detailed Implementation

[0023] This invention provides a method for preparing camellia seed oil with high vitamin A content diglyceride, comprising the following steps: Cellulose was extracted from camellia seed cake, and the resulting camellia seed cake cellulose was nano-processed to obtain nanocellulose. The nanocellulose and graphene are combined to obtain a nanocellulose / graphene composite material of camellia seed cake. Glycerol and the aforementioned camellia seed cake nanocellulose / graphene composite material are mixed, and glycerol is adsorbed onto the camellia seed cake nanocellulose / graphene composite material to obtain glycerol-camellia seed cake nanocellulose / graphene composite material. Freshly pressed camellia seed oil, glycerol-camellia seed cake nanocellulose / graphene composite material, and lipase are mixed and subjected to enzymatic glycerolysis to obtain esterified products; the temperature of the enzymatic glycerolysis reaction is 30~45℃. The esterification product was filtered, and the resulting mixture of diglyceride camellia seed oil was subjected to molecular distillation to obtain refined diglyceride camellia seed oil. Vitamin A was added to the refined diglyceride camellia seed oil to obtain diglyceride camellia seed oil with high vitamin A content; The method for preparing vitamin A is as follows: the deodorized distillate fermentation product, methanol and sulfuric acid solution are mixed and subjected to esterification reaction to obtain the esterified material; The esterified material, ethanol, and NaOH solution are mixed and a saponification reaction is carried out to obtain a saponified solution. Transfer the saponified liquid into a separatory funnel, add petroleum ether, shake, and let it stand to separate into layers. Take the upper clear liquid for later use. Wash the supernatant with water until the pH value is 7, then add sodium chloride, let it stand to dehydrate, filter and collect the filtrate for later use; The filtrate was concentrated under reduced pressure to obtain the distillation product; The distillation product was passed through a silica gel chromatography column for fractional elution, and the eluent was collected. The eluent was concentrated under reduced pressure until no solvent odor was detected, yielding vitamin A; The preparation method of the deodorized distillate fermentation product is as follows: a mixed bacterial suspension of yeast and flavobacterium is inoculated onto a fermentation medium containing tea oil refined deodorized distillate for fermentation to obtain the deodorized distillate fermentation product.

[0024] Unless otherwise specified, the present invention does not have special requirements on the source of raw materials used, and commercially available products well known to those skilled in the art can be used.

[0025] This invention uses camellia seed cake to extract cellulose, and then performs nano-processing on the obtained camellia seed cake cellulose to obtain nanocellulose.

[0026] As one implementation method, the cellulose extraction operation steps are as follows: crushing camellia seed cake, defatting the obtained camellia seed cake powder with petroleum ether, drying the defatted camellia seed cake powder, sieving, mixing the obtained sieved powder with NaOH solution for cellulose extraction, and obtaining crude camellia seed cake cellulose. The crude camellia seed cake cellulose was mixed with H2O2 solution and oxidized to obtain camellia seed cake cellulose.

[0027] The present invention does not have any particular limitation on the source of the camellia seed cake, and camellia seed cake known in the art can be used.

[0028] In one embodiment, the drying temperature is 60°C; the sieve used for sieving is a 300-mesh sieve; the concentration of the NaOH solution is 0.5~2 mol / L, specifically 1 mol / L in this embodiment; the powder-to-water ratio of the sieve powder to the NaOH solution is 1:10~45, specifically 1:30 in this embodiment; the powder-to-water ratio refers to the ratio of the mass (g) of the sieve powder to the volume (mL) of the sodium hydroxide solution; the cellulose extraction is carried out under heating and stirring conditions; the heating and stirring temperature is 60~80°C, specifically 70°C in this embodiment, the time is 3~7 h, specifically 5 h in this embodiment, and the stirring rate is 6000~9000 r / min, specifically 8000 r / min in this embodiment.

[0029] In one embodiment, after cellulose extraction, the process further includes: sequentially subjecting the cellulose extraction liquid to solid-liquid separation, washing, and freeze-drying to obtain crude camellia seed cake cellulose; the solid-liquid separation is centrifugal separation; the centrifugation speed is 4000~9000 r / min, specifically 8000 r / min in this embodiment, and the time is 5~20 min, specifically 10 min in this embodiment; the washing is repeated until the pH of the supernatant from centrifugation is 7; the washing reagent is pure water. This invention does not specifically limit the temperature and time of the freeze-drying; temperatures and times well-known in the art can be used.

[0030] In one embodiment, the solid-liquid ratio of the crude camellia seed cake cellulose to the H2O2 solution is 1:10~40, specifically 1:30 in this embodiment; the solid-liquid ratio refers to the mass ratio of the crude camellia seed cake cellulose to the H2O2 solution; the concentration of the H2O2 solution is 30%, specifically 30wt% in this embodiment; the oxidation treatment involves room temperature oxidation followed by cell disruption using a cell disruptor, and finally, oxidation in the dark; the room temperature oxidation time is 1.5~5h, specifically 3h in this embodiment; the disruption power is 500~700W, specifically 600W in this embodiment, and the time is 10~30min, specifically 15min in this embodiment; the oxidation in the dark is 8~24h, specifically 12h in this embodiment.

[0031] In one embodiment, after the oxidation treatment, the process further includes: sequentially performing solid-liquid separation, washing, and freeze-drying on the product of the oxidation treatment to obtain camellia seed cake cellulose; the solid-liquid separation is centrifugal separation; the centrifugation speed is 7000~9000 r / min, specifically 8000 r / min in this embodiment, and the time is 8~15 min, specifically 10 min in this embodiment; the washing is repeated until the pH of the supernatant is 7; the washing reagent is pure water. The freeze-drying temperature is -50℃, and the time is 24 h.

[0032] As one implementation method, the specific steps of the nano-processing are as follows: the cellulose from the camellia seed cake is mixed with water and stirred. The resulting cellulose suspension is poured into a micro-jet feed tank for circulation treatment. The liquid is collected, freeze-dried, and nanocellulose is obtained.

[0033] In one embodiment, the water is deionized water; the solid-liquid ratio of the camellia seed cake cellulose to water is 1:5~20, specifically 1:10 in this embodiment; the solid-liquid ratio refers to the mass ratio of camellia seed cake cellulose to water; the stirring speed is 400~1000 r / min, specifically 500 r / min in this embodiment, and the time is 15~45 min, specifically 30 min in this embodiment; the pressure of the circulation treatment is 80~120 MPa, specifically 100 MPa in this embodiment, the feed temperature is 25~35℃, specifically 30℃ in this embodiment, and the number of cycles is 3~6 times, specifically 5 times in this embodiment; the freeze-drying temperature is -10℃, and the time is 6 hours.

[0034] After obtaining the nanocellulose, the present invention combines the nanocellulose with graphene to obtain a camellia seed cake nanocellulose / graphene composite material.

[0035] In one embodiment, the composite operation steps are as follows: a nanocellulose aqueous solution and a graphene aqueous solution are mixed and sequentially subjected to ultrasonication, magnetic stirring, vacuum filtration, and drying; the dried composite material is repeatedly subjected to freeze-thaw treatment and then freeze-dried to obtain a camellia seed cake nanocellulose / graphene composite material; the concentration of the nanocellulose aqueous solution is 1.0~2.5wt%, specifically 2.0wt% in this embodiment; the concentration of the graphene aqueous solution is 1.0~3.0wt%, specifically 2.0wt% in this embodiment; the volume ratio of the nanocellulose aqueous solution to the graphene aqueous solution is 1:5~20, specifically 1:10 in this embodiment; the ultrasonic power is 100~300W, specifically 200W in this embodiment, the frequency is 20~40kHz, specifically 30kHz in this embodiment, and the time is 20~40min, specifically 30min in this embodiment; the magnetic stirring speed is 200~500rpm, specifically 300rpm in this embodiment. The drying process takes 12-36 hours, specifically 24 hours in this embodiment; the drying temperature is 50-70℃, specifically 60℃ in this embodiment, and the drying time is 12-24 hours, specifically 24 hours in this embodiment; after drying, the process further includes cooling to 25℃; the freeze-thaw treatment involves sequential freezing and thawing; the freezing temperature is -40 to -60℃, specifically -50℃ in this embodiment, and the freezing time is 4-8 hours, specifically 5 hours in this embodiment; the process is repeated 2-4 times, specifically 3 times in this embodiment; the freeze-drying process includes pre-freezing, primary drying, and secondary drying; the pre-freezing temperature is -40 to -80℃, specifically -50℃ in this embodiment, and the drying time is 2-5 hours, specifically 3 hours in this embodiment; the primary drying temperature is -20 to 10℃, specifically 0℃ in this embodiment, and the drying time is 8-20 hours, specifically 12 hours in this embodiment; the secondary drying temperature is 20-40℃, specifically 30℃ in this embodiment, and the drying time is 4-8 hours, specifically 6 hours in this embodiment.

[0036] After obtaining the camellia seed cake nanocellulose / graphene composite material, the present invention mixes glycerol with the camellia seed cake nanocellulose / graphene composite material, and the glycerol is adsorbed onto the camellia seed cake nanocellulose / graphene composite material to obtain the glycerol-camellia seed cake nanocellulose / graphene composite material.

[0037] In one embodiment, the mass ratio of the glycerol to the camellia seed cake nanocellulose / graphene composite material is 1:1 to 2.5, and in a specific embodiment it is 1:2.

[0038] After obtaining the glycerol-camellia seed cake nanocellulose / graphene composite material, the present invention mixes freshly pressed camellia seed oil, the glycerol-camellia seed cake nanocellulose / graphene composite material and lipase, and performs enzymatic glycerolysis to obtain esterified products.

[0039] As one embodiment, the method for preparing the freshly pressed camellia seed oil is as follows: a) Remove impurities from fresh fruit; After removing impurities from fresh camellia seeds, the moisture content of the fresh camellia seeds after impurity removal is 40-60 wt%, with 50 wt% in the specific embodiment. b) Knead and crush; After removing impurities, fresh camellia seeds are sent to a kneading machine for extrusion and kneading. c) Seed hull sorting; Fresh fruit processed by the rubbing machine is placed into a seed shell sorting drum screen to separate the camellia fruit shells from the camellia seeds. d) Color sorting; A color sorter was used to screen the sorted camellia seeds to remove residual shells and unqualified seeds. e) Drying; The color-sorted camellia seeds were transferred to a drum dryer and dried at 110°C for 30 minutes. The moisture content after drying was controlled at 3-5 wt%, with 4 wt% in the specific embodiment. f) Remove impurities from camellia seeds; The dried camellia seeds are fed into a magnetic separator and a flat rotary screen to remove impurities; g) Peeling; The impurity-removed camellia seeds are transported to a shelling machine for processing to obtain camellia seed shells and kernels. The roller linear speed of the shelling machine is 8.5~10.0m / s, specifically 9.0m / s in this embodiment, ensuring a shelling rate of over 98%, and a shell content of <4% in the camellia seed kernels, specifically 3wt% in this embodiment, and a kernel content of <1% in the camellia seed shells, specifically 0.8wt% in this embodiment. h) Oil extraction; The obtained camellia seed kernels are pressed at low temperature and filtered to obtain fresh camellia seed oil.

[0040] In one embodiment, the temperature of the low-temperature pressing is 40~45℃, and in a specific embodiment it is 40℃. In one embodiment, the mass ratio of freshly pressed camellia seed oil to glycerol-camellia seed cake nanocellulose / graphene composite material is 4~8:1, specifically 4:1, 6:1 or 8:1 in the embodiments; the lipase is Lipozyme TLIM lipase; the mass of the lipase is 4~8% of the mass of freshly pressed camellia seed oil, specifically 6% in the embodiments; the temperature of the enzymatic glycerol hydrolysis reaction is 30~45℃, specifically 40℃ in the embodiments, and the time is 6~10h, specifically 6h, 8h or 10h in the embodiments; after the enzymatic glycerol hydrolysis reaction, the process further includes: filtering to remove the lipase and camellia seed cake nanocellulose / graphene composite material to obtain the esterification product.

[0041] After obtaining the esterification product, the present invention filters the esterification product, and the resulting diglyceride camellia seed oil mixture is subjected to molecular distillation to obtain refined diglyceride camellia seed oil.

[0042] In one embodiment, the molecular distillation is a three-stage molecular distillation, comprising a first-stage distillation, a second-stage distillation, and a third-stage distillation performed sequentially. The temperature of the first-stage distillation is 130~160℃, specifically 150℃ in this embodiment, and the vacuum degree is 10~40Pa, specifically 30Pa in this embodiment. The temperature of the second-stage distillation is 140~170℃, specifically 150℃ in this embodiment, and the vacuum degree is 10~40Pa, specifically 30Pa in this embodiment. The temperature of the third-stage distillation is 200~220℃, specifically 210℃ in this embodiment, and the vacuum degree is 3~10Pa, specifically 5Pa in this embodiment.

[0043] After obtaining the refined diglyceride camellia seed oil, the present invention adds vitamin A to the refined diglyceride camellia seed oil to obtain a diglyceride camellia seed oil with high vitamin A content.

[0044] As one embodiment, the method for preparing vitamin A is as follows: The deodorized distillate fermentation product, methanol and sulfuric acid solution are mixed and subjected to esterification to obtain the esterified material; The esterified material, ethanol, and NaOH solution are mixed and a saponification reaction is carried out to obtain a saponified solution. Transfer the saponified liquid into a separatory funnel, add petroleum ether, shake, and let it stand to separate into layers. Take the upper clear liquid for later use. Wash the supernatant with water until the pH value is 7, then add sodium chloride, let it stand to dehydrate, filter and collect the filtrate for later use; The filtrate was concentrated under reduced pressure to obtain the distillation product; The distillation product was passed through a silica gel chromatography column for fractional elution, and the eluent was collected. The eluent was concentrated under reduced pressure until no solvent odor was detected, yielding vitamin A.

[0045] In one embodiment, the mixing of the deodorized distillate fermentation product, methanol, and sulfuric acid solution is as follows: the deodorized distillate fermentation product is heated to 50°C and stirred until homogeneous, then methanol and sulfuric acid solution are added sequentially; the mass ratio of the deodorized distillate fermentation product to methanol is 1:1~3, specifically 1:2 in this embodiment; the mass concentration of the sulfuric acid solution is 1.5~4%, specifically 3% in this embodiment; the volume of the sulfuric acid solution is 2~4% of the volume of methanol, specifically 3% in this embodiment; the temperature of the esterification reaction is 45~60°C, specifically 55°C in this embodiment, and the time is 1~3 hours, specifically 2 hours in this embodiment; the esterification reaction is carried out under constant temperature and stirring conditions; fatty acids are removed through the esterification reaction; after the esterification reaction, the mixture is further further subjected to: cooling to room temperature, adding an equal volume of distilled water, separating and discarding the aqueous phase, washing the organic phase with water until neutral, filtering, and obtaining the esterified material.

[0046] In one embodiment, the ethanol is anhydrous ethanol; the ratio of the esterified material to ethanol is 1~2:1~2, specifically 1:1 in this embodiment; the ratio refers to the ratio of the mass (g) of the esterified material to the volume (mL) of ethanol; the mass fraction of the NaOH solution is 15~25%, specifically 20% in this embodiment; the mass of the NaOH solution is 6~12% of the mass of the esterified material, specifically 8% in this embodiment; the temperature of the saponification reaction is 50~70℃, specifically 60℃ in this embodiment, and the time is 0.5~2h, specifically 1h in this embodiment; the saponification reaction is carried out under constant temperature stirring conditions; after the saponification reaction, the reaction further includes cooling to room temperature.

[0047] In one embodiment, the petroleum ether is a petroleum ether with a boiling range of 30-60°C; the petroleum ether is of equal volume to the saponification liquid; the shaking time is 0.5-3 min, specifically 1 min in this embodiment; the water used for washing the supernatant is distilled water of equal volume to the supernatant; the mass of sodium chloride is 5% of the mass of the saponification liquid; the settling time is 30-60 min, specifically 40 min in this embodiment; the temperature for vacuum concentration of the filtrate is 35-40°C, specifically 35°C in this embodiment, and the vacuum degree is 0.08-0.09 MPa, specifically 0.085 MPa in this embodiment; the fractional elution is performed sequentially with petroleum ether and petroleum ether-diethyl ether; the petroleum ether-diethyl ether is a mixed solution of petroleum ether and diethyl ether, wherein the volume ratio of petroleum ether to diethyl ether is 90-95:5-10, specifically 92:8 in this embodiment; the temperature for vacuum concentration of the eluent is 35-45°C, specifically 40°C in this embodiment.

[0048] As one embodiment, the method for preparing the deodorized distillate fermentation product is as follows: yeast and *Microbacterium flavonoids* (… microbacterium flavumA mixed bacterial suspension (abbreviated as M. flavum) was inoculated onto a fermentation medium containing tea oil refining and deodorizing distillate for fermentation to obtain the deodorizing distillate fermentation product. This invention does not specifically limit the source of the yeast and *Microbacterium flavum* strains; yeast and *Microbacterium flavum* from sources well known in the art can be used.

[0049] As one embodiment, the method for preparing the mixed bacterial suspension of yeast and *Microbacterium flavonoids* is as follows: mixing yeast suspension and *Microbacterium flavonoids* suspension to obtain the mixed bacterial suspension of yeast and *Microbacterium flavonoids*; the volume ratio of the yeast suspension to the *Microbacterium flavonoids* suspension is 1:1 to 4, specifically 1:1, 1:3, or 1:4 in the embodiments; the concentration of the yeast suspension is 8 × 10⁻⁶. 7 ~2×10 8 CFU / mL, specifically 8 × 10⁻⁶ in the example. 7 CFU / mL, 1×10 8 CFU / mL or 2×10 8 CFU / mL; the concentration of the *Microbacterium chrysogenum* suspension was 8 × 10⁻⁶ CFU / mL. 7 ~2×10 8 CFU / mL, specifically 8 × 10⁻⁶ in the example. 7 CFU / mL, 1×10 8 CFU / mL or 2×10 8 CFU / mL; the inoculation amount of the mixed bacterial suspension of yeast and flavobacterium is 2-6% of the mass of tea oil refining and deodorizing distillate, specifically 2%, 3% or 6% in the embodiments.

[0050] In one embodiment, the preparation steps for the yeast expansion culture are as follows: yeast is transferred to malt extract agar medium to obtain single colonies; then, the single colonies are inoculated into yeast extract peptone glucose medium and statically cultured for later use; the composition of the malt extract agar medium is 2 wt% malt extract, 2 wt% glucose, 0.1 wt% peptone, 1.5 wt% agar, and the remainder is sterile water; the composition of the yeast extract peptone glucose medium is 2 wt% glucose, 2 wt% peptone, 1 wt% yeast extract, and the remainder is sterile water; the static culture temperature is 28~33℃, specifically 30℃ in this embodiment, and the time is 18~30h, specifically 24h in this embodiment.

[0051] As one implementation method, the preparation steps for the expanded culture of *Xanthomonas flavovirens* are as follows: The activated *Microbacterium flavonoids* strain was inoculated into *Microbacterium flavonoids* liquid seed culture medium and cultured in a constant temperature shaker for later use. The composition of the *Microbacterium flavonoids* liquid seed culture medium was 60 g / L glucose, 25 g / L peptone, 2 g / L NaNO3, 1 g / L MgSO4·7H2O, 1 g / L KH2PO4·3H2O, and 1000 mL distilled water. The pH value of the *Microbacterium flavonoids* liquid seed culture medium was 5.0. Before inoculation, the *Microbacterium flavonoids* liquid seed culture medium was sterilized at 121℃ for 30 min.

[0052] As one implementation method, the preparation steps of the yeast suspension and the *Microbacterium chrysogenum* suspension are as follows: The yeast culture solution and the *Microbacterium chrysogenum* culture solution after expansion culture are centrifuged for the first time to remove the supernatant, physiological saline is added, and the mixture is vortexed and mixed evenly. The supernatant is removed by a second centrifugation, and physiological saline is added again, washing repeatedly four times. Finally, physiological saline is used to replenish and mix the mixture. Using physiological saline as a blank control, the absorbance of the bacterial solution is adjusted to 0.8 at a wavelength of 600 nm using physiological saline. The yeast suspension and the *Microbacterium chrysogenum* suspension are prepared. The rotation speeds of the first and second centrifugations are independently 3000~4500 r / min, specifically 4000 r / min in this embodiment. The first centrifugation time is 10~25 min, specifically 15 min in this embodiment. The first centrifugation time is 3~8 min, specifically 5 min in this embodiment. The concentration of the physiological saline is 0.9 wt%.

[0053] As one embodiment, the preparation method of the fermentation medium containing the tea oil refined and deodorized distillate is as follows: Sterilized tea oil refined and deodorized distillate is added to the sterilized fermentation medium to achieve a concentration of 50 g / L, thus obtaining the fermentation medium containing the tea oil refined and deodorized distillate; the sterilization temperature is 121℃, specifically 30 min in this embodiment. This invention does not specifically limit the source of the tea oil refined and deodorized distillate; any tea oil refined and deodorized distillate well-known in the art can be used.

[0054] In one embodiment, the fermentation medium consists of 0.5 g / L yeast extract, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, and 1000 mL distilled water; the pH value of the fermentation medium is 7.0.

[0055] In one implementation, the fermentation temperature is 28~32℃, specifically 30℃ in this embodiment, and the fermentation time is 48~96h, specifically 72h in this embodiment.

[0056] As one implementation method, the amount of vitamin A added to the refined diglyceride camellia seed oil is 4000~6000μg / kg, and in a specific embodiment it is 5000μg / kg.

[0057] The preparation method provided by this invention can not only improve the utilization rate of fresh camellia seed shells and deodorized distillate, and increase the yield of vitamin A, but also reduce the loss of vitamin A during esterification, shorten the esterification time, and improve the esterification efficiency.

[0058] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1 A method for preparing camellia seed oil with high vitamin A content diglyceride, the process flow is as follows: Figure 1 As shown, it includes the following steps: 1) Preparation of Camellia Seed Oil: a) Cleaning fresh fruit: After removing impurities from the fresh camellia seeds, the moisture content of the removed fresh camellia seeds is 50 wt%. b) Knead and crush: After removing impurities, fresh camellia seeds are sent to a kneading machine for extrusion and kneading. c) Seed hull sorting: Fresh fruit processed by the rubbing machine is placed into a seed shell sorting drum screen to separate the camellia fruit shells from the camellia seeds. d) Color sorting: A color sorter was used to screen the sorted camellia seeds to remove residual shells and unqualified seeds. e) Drying: After color sorting, the camellia seeds were transferred to a drum dryer and dried at 110℃ for 30 minutes, with the moisture content controlled at 4 wt% after drying. f) Removing impurities from camellia seeds: The dried camellia seeds are fed into a magnetic separator and a flat rotary screen to remove impurities; g) Peeling: The impurity-removed camellia seeds are transported to a shelling machine for processing, yielding camellia seed shells and kernels. The roller linear speed of the shelling machine is set to 9.0 m / s to ensure a shelling rate of over 98%, with a shell content of 3 wt% in the camellia seed kernels and a kernel content of 0.8 wt% in the camellia seed shells. h) Oil extraction: The obtained camellia seed kernels are pressed at a low temperature of 40℃ and then filtered to obtain fresh camellia seed oil.

[0060] 2) Mixed fermentation of yeast and yellow microbacterium: a) The tea oil refining and deodorizing distillate was sterilized at 121℃ for 30 minutes to obtain the sterilized deodorizing distillate.

[0061] b) Prepare a concentration of 1×10 8 CFU / mL yeast suspension and 1×10 8 CFU / mL of *Microbacterium flavonoids* suspension: The preparation steps for yeast expansion culture are as follows: transfer yeast to malt extract agar medium (the composition of malt extract agar medium is 2wt%, glucose 2wt%, peptone 0.1wt%, agar 1.5wt%, and the remainder is sterile water) to obtain single colonies; then inoculate the single colonies into yeast extract peptone glucose medium (the composition of yeast extract peptone glucose medium is 2wt%, peptone 2wt%, yeast extract 1wt%, and the remainder is sterile water), and incubate at 30℃ for 24h for later use.

[0062] The preparation steps for the expanded culture of *Microbacterium flavonoids* are as follows: The activated *Microbacterium flavonoids* strain (… microbacterium flavum The bacteria (abbreviated as M. flavum) were inoculated into liquid seed culture medium of Microbacterium flavum (60 g / L glucose, 25 g / L peptone, 2 g / L NaNO3, 1 g / L MgSO4·7H2O, 1 g / L KH2PO4·3H2O, 1000 mL distilled water, pH 5.0, sterilized at 121℃ for 30 min) and cultured on a constant temperature shaker for later use.

[0063] After the expansion culture was completed, the expanded culture solutions of yeast and flavobacterium were centrifuged at 4000 r / min for 15 min, the supernatant was removed, 10 mL of 0.9 wt% physiological saline was added, and the mixture was vortexed to mix evenly. The culture solution was centrifuged at 4000 r / min for 5 min, the supernatant was removed, and 0.9 wt% physiological saline was added. The mixture was washed repeatedly 4 times. Finally, physiological saline was added to make up the difference and mix well. Physiological saline was used as a blank control. The absorbance of the culture solution was adjusted to 0.8 with physiological saline at a wavelength of 600 nm to prepare yeast and flavobacterium suspensions. c) Mix the yeast suspension and the flavobacterium suspension at a volume ratio of 1:3 to obtain a mixed bacterial suspension; d) The sterilized deodorized distillate was added to the sterilized fermentation medium to achieve a concentration of 50 g / L, thus obtaining a fermentation medium containing the deodorized distillate. The fermentation medium used in preparing the deodorized distillate consisted of 0.5 g / L yeast extract, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, 1000 mL distilled water, pH 7.0, and was sterilized at 121℃ for 15 min. The mixed bacterial suspension was inoculated into the fermentation medium containing the deodorized distillate at an inoculation amount of 3% of the tea oil refining deodorized distillate, and then fermented at 30℃ for 72 h to obtain the deodorized distillate fermentation product.

[0064] 3) Preparation of Camellia seed cake nanocellulose / graphene composite material: Camellia seed cake was crushed to obtain Camellia seed cake powder, which was then defatted with petroleum ether. The dried powder was passed through a 300-mesh sieve. The sieved powder was then mixed with 1 mol / L NaOH solution at a powder-to-water ratio of 1:30 (powder mass (g) to sodium hydroxide solution volume (mL)) using a magnetic stirrer at 70℃ for 5 h. The mixture was then centrifuged at 8000 r / min for 10 min, and the precipitate was collected and washed repeatedly until the pH of the supernatant reached 7. After freeze-drying, crude Camellia seed cake cellulose was obtained. Crude camellia seed cake cellulose was mixed with 30wt% H2O2 solution at a solid-liquid ratio of 1:30 (mass ratio of crude camellia seed cake cellulose to H2O2 solution) and oxidized. After oxidation at room temperature for 3 hours, it was disrupted in a cell disruptor at 600W for 15 minutes, allowed to stand in the dark for 12 hours, and then centrifuged at 8000 rpm for 10 minutes. The precipitate was collected and washed repeatedly until the pH of the supernatant was 7. After freeze-drying at -50℃ for 24 hours, camellia seed cake cellulose was obtained. Camellia seed cake cellulose was weighed and added to deionized water at a solid-liquid ratio of 1:10 (mass ratio of camellia seed cake cellulose to water). The mixture was stirred at 500 rpm for 30 minutes to prepare a cellulose suspension. The cellulose suspension was poured into a microfluidic feed tank at a pressure of 100 MPa and a feed temperature of 30℃, and circulated 5 times. The liquid was collected and freeze-dried at -10℃ for 6 hours to obtain nano-cellulose powder.

[0065] A 2.0 wt% aqueous solution of nanocellulose and a 2.0 wt% aqueous solution of graphene were mixed at a mass ratio of 1:10, sonicated for 30 min, and magnetically stirred for 24 h to ensure uniform dispersion. The mixture was then vacuum filtered and dried at 60 °C. After cooling to 25 °C, the mixture was frozen at -50 °C for 5 h, thawed, and refrozen. This refrozen-refrozen process was repeated three times. The sample was then freeze-dried. The freeze-drying process involved a pre-freezing temperature of -50 °C for 3 h, a first drying temperature of 0 °C for 12 h, and a second drying temperature of 30 °C for 6 h, yielding a nanocellulose / graphene composite material from camellia seed cake.

[0066] 4) Enzymatic glycerol hydrolysis: Glycerol and camellia seed cake nanocellulose / graphene composite material were mixed evenly at a mass ratio of 1:2. Glycerol was then adsorbed onto the camellia seed cake nanocellulose / graphene composite material to form a glycerol-camellia seed cake nanocellulose / graphene composite material. Freshly pressed camellia seed oil was added to the enzyme reaction vessel. The glycerol-camellia seed cake nanocellulose / graphene composite material was added at a mass ratio of 6:1. Lipozyme TL IM lipase was added to the glycerol hydrolysis reaction system at a mass ratio of 6% by weight of camellia oil. The reaction temperature was 40℃ and the reaction time was 10h. After filtration, Lipozyme TL IM lipase and camellia seed cake nanocellulose / graphene composite material were removed to obtain the esterified product. 5) Molecular distillation: After filtration of the esterification products, the resulting mixture of diglyceride camellia seed oil is subjected to molecular distillation. The first-stage distillation conditions are: temperature 150℃, vacuum degree 30Pa; the second-stage distillation conditions are: temperature 150℃, vacuum degree 30Pa; and the third-stage distillation conditions are: temperature 210℃, vacuum degree 5Pa, to obtain refined diglyceride camellia seed oil.

[0067] 6) Extraction of Vitamin A: Heat the deodorized distillate of tea oil to 50℃ and stir well. Add methanol to the deodorized distillate at a mass ratio of 1:2 (deodorized distillate:methanol), then add 3wt% concentrated sulfuric acid (3% of methanol volume). Esterify at 55℃ for 2 hours to remove fatty acids. After the reaction, cool to room temperature, add an equal volume of distilled water, separate and discard the aqueous phase, wash the organic phase with water until neutral, and filter to obtain the esterified material. Add anhydrous ethanol (material-to-liquid ratio 1:1, ratio of esterified material mass (g) to ethanol volume (mL)) + 20wt% of the esterified material to the esterified material. NaOH solution (8% of the mass of the esterified material) was used for saponification by stirring at 60℃ for 1 hour, followed by cooling to room temperature. The saponified liquid was transferred to a separatory funnel, and an equal volume of petroleum ether with a boiling range of 30-60℃ was added. The mixture was shaken for 1 minute and allowed to stand for separation. The supernatant was collected for later use. The supernatant was washed with an equal volume of distilled water until the pH reached 7. Then, 5 wt% sodium chloride (by weight of the saponified liquid) was added, and the mixture was allowed to stand for 40 minutes to dehydrate. The filtrate was filtered and collected for later use. The filtrate was concentrated under reduced pressure at 35℃ and a vacuum of 0.085 MPa to obtain the distillation product. The distillation product was passed through a silica gel column for fractional elution using petroleum ether and petroleum ether-diethyl ether (petroleum ether volume:diethyl ether volume = 92:8) as eluents. The eluent was collected and concentrated under reduced pressure at 40℃ until no solvent odor was detected to obtain the vitamin A product.

[0068] 7) Adding Vitamin A: Add 5000 μg / kg of Vitamin A to the diglyceride camellia seed oil and mix thoroughly to obtain diglyceride camellia seed oil with high Vitamin A content.

[0069] Example 2 A method for preparing camellia seed oil with high vitamin A content diglyceride includes the following steps: 1) Preparation of Camellia Seed Oil: a) Cleaning fresh fruit: After removing impurities from the fresh camellia seeds, the moisture content of the removed fresh camellia seeds is 50 wt%. b) Knead and crush: After removing impurities, fresh camellia seeds are sent to a kneading machine for extrusion and kneading. c) Seed hull sorting; Fresh fruit processed by the rubbing machine is placed into a seed shell sorting drum screen to separate the camellia fruit shells from the camellia seeds. d) Color sorting: A color sorter was used to screen the sorted camellia seeds to remove residual shells and unqualified seeds. e) Drying: After color sorting, the camellia seeds were transferred to a drum dryer and dried at 110℃ for 30 minutes, with the moisture content controlled at 4 wt% after drying. f) Removing impurities from camellia seeds: The dried camellia seeds are fed into a magnetic separator and a flat rotary screen to remove impurities; g) Peeling: The impurity-removed camellia seeds are transported to a shelling machine for processing, yielding camellia seed shells and kernels. The roller linear speed of the shelling machine is set to 9.0 m / s to ensure a shelling rate of over 98%, with a shell content of 3 wt% in the camellia seed kernels and a kernel content of 0.8 wt% in the camellia seed shells. h) Oil extraction: The obtained camellia seed kernels are pressed at a low temperature of 40℃ and then filtered to obtain fresh camellia seed oil.

[0070] 2) Mixed fermentation of yeast and yellow microbacterium: a) The tea oil refining and deodorizing distillate was sterilized at 121℃ for 30 minutes to obtain the sterilized deodorizing distillate.

[0071] b) Prepare a concentration of 2×10 8 CFU / mL yeast suspension and 2×10 8 CFU / mL of *Microbacterium flavonoids* suspension: The preparation steps for yeast expansion culture are as follows: transfer yeast to malt extract agar medium (the composition of malt extract agar medium is 2wt%, glucose 2wt%, peptone 0.1wt%, agar 1.5wt%, and the remainder is sterile water) to obtain single colonies; then inoculate the single colonies into yeast extract peptone glucose medium (the composition of yeast extract peptone glucose medium is 2wt%, peptone 2wt%, yeast extract 1wt%, and the remainder is sterile water), and incubate at 30℃ for 24h for later use.

[0072] The preparation steps for the expanded culture of *Microbacterium flavum* are as follows: The activated *Microbacterium flavum* strain is inoculated into *Microbacterium flavum* liquid seed culture medium (60 g / L glucose, 25 g / L peptone, 2 g / L NaNO3, 1 g / L MgSO4·7H2O, 1 g / L KH2PO4·3H2O, 1000 mL distilled water, pH 5.0, sterilized at 121℃ for 30 min), and cultured in a constant temperature shaker for later use.

[0073] After the expansion culture was completed, the expanded culture solutions of yeast and flavobacterium were centrifuged at 4000 r / min for 15 min, the supernatant was removed, 10 mL of 0.9 wt% physiological saline was added, and the mixture was vortexed to mix evenly. The culture solution was centrifuged at 4000 r / min for 5 min, the supernatant was removed, and 0.9 wt% physiological saline was added. The mixture was washed repeatedly 4 times. Finally, physiological saline was added to make up the difference and mix well. Physiological saline was used as a blank control. The absorbance of the culture solution was adjusted to 0.8 with physiological saline at a wavelength of 600 nm to prepare yeast and flavobacterium suspensions. c) Mix the yeast suspension and the flavobacterium suspension at a volume ratio of 1:4 to obtain a mixed bacterial suspension; d) The sterilized deodorized distillate was added to the sterilized fermentation medium to achieve a concentration of 50 g / L, thus obtaining a fermentation medium containing the deodorized distillate. The fermentation medium used in preparing the deodorized distillate consisted of 0.5 g / L yeast extract, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, pH 7.0, and 1000 ml distilled water, and was sterilized at 121℃ for 15 min. The mixed bacterial suspension was inoculated into the fermentation medium containing the deodorized distillate at an inoculum amount of 6% of the tea oil refining deodorized distillate, and then fermented at 30℃ for 96 h to obtain the deodorized distillate fermentation product.

[0074] 3) Preparation of Camellia seed cake nanocellulose / graphene composite material: Camellia seed cake was crushed to obtain Camellia seed cake powder, which was then defatted with petroleum ether. The dried powder was passed through a 300-mesh sieve. The sieved powder was then mixed with 1 mol / L NaOH solution at a powder-to-water ratio of 1:30 (powder mass (g) to sodium hydroxide solution volume (mL)) using a magnetic stirrer at 70℃ for 5 h. The mixture was then centrifuged at 8000 r / min for 10 min, and the precipitate was collected and washed repeatedly until the pH of the supernatant reached 7. After freeze-drying, crude Camellia seed cake cellulose was obtained. Crude camellia seed cake cellulose was mixed with 30wt% H2O2 solution at a solid-liquid ratio of 1:30 (mass ratio of crude camellia seed cake cellulose to H2O2 solution) and oxidized. After oxidation at room temperature for 3 hours, it was disrupted in a cell disruptor at 600W for 15 minutes, allowed to stand in the dark for 12 hours, and then centrifuged at 8000 rpm for 10 minutes. The precipitate was collected and washed repeatedly until the pH of the supernatant was 7. After freeze-drying at -50℃ for 24 hours, camellia seed cake cellulose was obtained. Camellia seed cake cellulose was weighed and added to deionized water at a solid-liquid ratio of 1:10 (mass ratio of camellia seed cake cellulose to water). The mixture was stirred at 500 rpm for 30 minutes to prepare a cellulose suspension. The cellulose suspension was poured into a microfluidic feed tank at a pressure of 100 MPa and a feed temperature of 30℃, and circulated 5 times. The liquid was collected and freeze-dried at -10℃ for 6 hours to obtain nano-cellulose powder.

[0075] A 2.0 wt% aqueous solution of nanocellulose and a 2.0 wt% aqueous solution of graphene were mixed at a mass ratio of 1:10, sonicated for 30 min, and magnetically stirred for 24 h to ensure uniform dispersion. The mixture was then vacuum filtered and dried at 60 °C. After cooling to 25 °C, the mixture was frozen at -50 °C for 5 h, thawed, and refrozen. This refrozen-refrozen process was repeated three times before freeze-drying. The freeze-drying process consisted of a pre-freezing temperature of -50 °C for 3 h, a first drying temperature of 0 °C for 12 h, and a second drying temperature of 30 °C for 6 h, yielding a nanocellulose / graphene composite material from camellia seed cake.

[0076] 4) Enzymatic glycerol hydrolysis: Glycerol and camellia seed cake nanocellulose / graphene composite material were mixed evenly at a mass ratio of 1:2.5. Glycerol was then adsorbed onto the camellia seed cake nanocellulose / graphene composite material to form a glycerol-camellia seed cake nanocellulose / graphene composite material. Freshly pressed camellia seed oil was added to the enzyme reaction vessel. The glycerol-camellia seed cake nanocellulose / graphene composite material was added at a mass ratio of 4:1. Lipozyme TL IM lipase was added to the glycerol hydrolysis reaction system at a mass ratio of 8% of the camellia oil mass. The reaction temperature was 45℃ and the reaction time was 10h. After filtration, Lipozyme TL IM lipase and camellia seed cake nanocellulose / graphene composite material were removed to obtain the esterified product. 5) Molecular distillation: After filtration of the esterification products, the resulting mixture of diglyceride camellia seed oil is subjected to molecular distillation. The first-stage distillation conditions are: temperature 150℃, vacuum degree 30Pa; the second-stage distillation conditions are: temperature 150℃, vacuum degree 30Pa; and the third-stage distillation conditions are: temperature 210℃, vacuum degree 5Pa, to obtain refined diglyceride camellia seed oil.

[0077] 6) Extraction of Vitamin A: Heat the deodorized tea oil distillate to 50℃ and stir until homogeneous. Add methanol to the deodorized distillate at a mass ratio of 1:2 (deodorized distillate:methanol), then add 3wt% concentrated sulfuric acid (3% of methanol by volume). Esterify at 55℃ with constant stirring for 2 hours to remove fatty acids. After the reaction, cool to room temperature, add an equal volume of distilled water, separate and discard the aqueous phase, wash the organic phase with water until neutral, and filter to obtain the esterified material. Add anhydrous ethanol (material-to-liquid ratio 1:1, ratio of esterified material mass (g) to ethanol volume (mL)) + 20% NaOH solution (8% of the esterified material mass) to the esterified material. Saponify by stirring at 60℃ for 1 hour, then cool to room temperature. The saponified liquid was transferred to a separatory funnel, and an equal volume of petroleum ether with a boiling range of 30-60℃ was added. The mixture was shaken for 1 minute and allowed to stand for separation. The supernatant was collected for later use. The supernatant was washed with an equal volume of distilled water until the pH reached 7. Then, 5 wt% sodium chloride (by weight of the saponified liquid) was added, and the mixture was allowed to stand for 40 minutes to dehydrate. The filtrate was filtered and collected for later use. The filtrate was concentrated under reduced pressure at 35℃ and a vacuum of 0.085 MPa to obtain the distillation product. The distillation product was passed through a silica gel column and eluted in fractions with petroleum ether and petroleum ether-diethyl ether (petroleum ether volume:diethyl ether volume = 92:8). The eluent was collected. The eluent was concentrated under reduced pressure at 40℃ until no solvent odor was detected to obtain the vitamin A product.

[0078] 7) Adding Vitamin A: Add 6000 μg / kg of Vitamin A to the diglyceride camellia seed oil and mix thoroughly to obtain diglyceride camellia seed oil with high Vitamin A content.

[0079] Example 3 A method for preparing camellia seed oil with high vitamin A content diglyceride includes the following steps: 1) Preparation of Camellia Seed Oil: a) Cleaning fresh fruit: After removing impurities from fresh camellia seeds, the moisture content of the removed seeds is 50 wt%. b) Knead and crush: After removing impurities, fresh camellia seeds are sent to a kneading machine for extrusion and kneading. c) Seed hull sorting: Fresh fruit processed by the rubbing machine is placed into a seed shell sorting drum screen to separate the camellia fruit shells from the camellia seeds. d) Color sorting: A color sorter was used to screen the sorted camellia seeds to remove residual shells and unqualified seeds. e) Drying: After color sorting, the camellia seeds were transferred to a drum dryer and dried at 110℃ for 30 minutes, with the moisture content controlled at 4 wt% after drying. f) Removing impurities from camellia seeds: The dried camellia seeds are fed into a magnetic separator and a flat rotary screen to remove impurities; g) Peeling: The impurity-removed camellia seeds are transported to a shelling machine for processing, yielding camellia seed shells and kernels. The roller linear speed of the shelling machine is set to 9.0 m / s to ensure a shelling rate of over 98%, with a shell content of 3 wt% in the camellia seed kernels and a kernel content of 0.8 wt% in the camellia seed shells. h) Oil extraction: The obtained camellia seed kernels are pressed at a low temperature of 40℃ and then filtered to obtain fresh camellia seed oil.

[0080] 2) Mixed fermentation of yeast and yellow microbacterium: a) The tea oil refining and deodorizing distillate was sterilized at 121℃ for 30 minutes to obtain the sterilized deodorizing distillate.

[0081] b) Prepare a concentration of 8×10 7 CFU / mL yeast suspension and 8×10 7 CFU / mL of *Microbacterium flavonoids* suspension: The preparation steps for yeast expansion culture are as follows: transfer yeast to malt extract agar medium (the composition of malt extract agar medium is 2wt%, glucose 2wt%, peptone 0.1wt%, agar 1.5wt%, and the remainder is sterile water) to obtain single colonies; then inoculate the single colonies into yeast extract peptone glucose medium (the composition of yeast extract peptone glucose medium is 2wt%, peptone 2wt%, yeast extract 1wt%, and the remainder is sterile water), and incubate at 30℃ for 24h for later use.

[0082] The preparation steps for the expanded culture of *Microbacterium flavum* are as follows: The activated *Microbacterium flavum* strain is inoculated into *Microbacterium flavum* liquid seed culture medium (60 g / L glucose, 25 g / L peptone, 2 g / L NaNO3, 1 g / L MgSO4·7H2O, 1 g / L KH2PO4·3H2O, 1000 mL distilled water, pH 5.0, sterilized at 121℃ for 30 min), and cultured in a constant temperature shaker for later use.

[0083] After the expansion culture was completed, the expanded culture solutions of yeast and flavobacterium were centrifuged at 4000 r / min for 15 min, the supernatant was removed, 10 mL of 0.9 wt% physiological saline was added, and the mixture was vortexed to mix evenly. The culture solution was centrifuged at 4000 r / min for 5 min, the supernatant was removed, and 0.9 wt% physiological saline was added. The mixture was washed repeatedly 4 times. Finally, physiological saline was added to make up the difference and mix well. Physiological saline was used as a blank control. The absorbance of the culture solution was adjusted to 0.8 with physiological saline at a wavelength of 600 nm to prepare yeast and flavobacterium suspensions. c) Mix the yeast suspension and the flavobacterium suspension at a volume ratio of 1:1 to obtain a mixed bacterial suspension; d) The sterilized deodorized distillate was added to the sterilized fermentation medium to achieve a concentration of 50 g / L, thus obtaining a fermentation medium containing the deodorized distillate. The fermentation medium used in preparing the deodorized distillate consisted of 0.5 g / L yeast extract, 1.0 g / L MgSO4, 5.0 g / L K2HPO4, pH 7.0, and 1000 ml distilled water, and was sterilized at 121℃ for 15 min. The mixed bacterial suspension was inoculated into the fermentation medium containing the deodorized distillate at an inoculation amount of 2% of the tea oil refining deodorized distillate, and then fermented at 30℃ for 48 h to obtain the deodorized distillate fermentation product.

[0084] 3) Preparation of Camellia seed cake nanocellulose / graphene composite material: Camellia seed cake was crushed to obtain Camellia seed cake powder, which was then defatted with petroleum ether. The dried powder was passed through a 300-mesh sieve. The sieved powder was then mixed with 1 mol / L NaOH solution at a powder-to-water ratio of 1:30 (powder mass (g) to sodium hydroxide solution volume (mL)) using a magnetic stirrer at 70℃ for 5 h. The mixture was then centrifuged at 8000 r / min for 10 min, and the precipitate was collected and washed repeatedly until the pH of the supernatant reached 7. After freeze-drying, crude Camellia seed cake cellulose was obtained. Crude camellia seed cake cellulose was mixed with 30wt% H2O2 solution at a solid-liquid ratio of 1:30 (mass ratio of crude camellia seed cake cellulose to H2O2 solution) and oxidized. After oxidation at room temperature for 3 hours, it was disrupted in a cell disruptor at 600W for 15 minutes, allowed to stand in the dark for 12 hours, and then centrifuged at 8000 rpm for 10 minutes. The precipitate was collected and washed repeatedly until the pH of the supernatant was 7. After freeze-drying at -50℃ for 24 hours, camellia seed cake cellulose was obtained. Camellia seed cake cellulose was weighed and added to deionized water at a solid-liquid ratio of 1:10 (mass ratio of camellia seed cake cellulose to water). The mixture was stirred at 500 rpm for 30 minutes to prepare a cellulose suspension. The cellulose suspension was poured into a microfluidic feed tank at a pressure of 100 MPa and a feed temperature of 30℃, and circulated 5 times. The liquid was collected and freeze-dried at -10℃ for 6 hours to obtain nano-cellulose powder.

[0085] A 2.0 wt% aqueous solution of nanocellulose and a 2.0 wt% aqueous solution of graphene were mixed at a mass ratio of 1:10, sonicated for 30 min, and magnetically stirred for 24 h to ensure uniform dispersion. The mixture was then vacuum filtered and dried at 60 °C. After cooling to 25 °C, the mixture was frozen at -50 °C for 5 h, thawed, and refrozen. This refrozen-refrozen process was repeated three times. The sample was then freeze-dried. The freeze-drying process involved a pre-freezing temperature of -50 °C for 3 h, a first drying temperature of 0 °C for 12 h, and a second drying temperature of 30 °C for 6 h to obtain a nanocellulose / graphene composite material from camellia seed cake.

[0086] 4) Enzymatic glycerol hydrolysis: Glycerol and camellia seed cake nanocellulose / graphene composite material were mixed evenly at a mass ratio of 1:1. Glycerol was then adsorbed onto the camellia seed cake nanocellulose / graphene composite material to form a glycerol-camellia seed cake nanocellulose / graphene composite material. Freshly pressed camellia seed oil was added to the enzyme reaction vessel. The glycerol-camellia seed cake nanocellulose / graphene composite material was added at a mass ratio of 8:1. Lipozyme TL IM lipase was added to the glycerol hydrolysis reaction system at a mass ratio of 4% of the camellia oil mass. The reaction temperature was 30℃ and the reaction time was 10h. After filtration, Lipozyme TL IM lipase and camellia seed cake nanocellulose / graphene composite material were removed to obtain the esterification product. 5) Molecular distillation: After filtration of the esterification products, the resulting mixture of diglyceride camellia seed oil is subjected to molecular distillation. The first-stage distillation conditions are: temperature 150℃, vacuum degree 30Pa; the second-stage distillation conditions are: temperature 150℃, vacuum degree 30Pa; and the third-stage distillation conditions are: temperature 210℃, vacuum degree 5Pa, to obtain refined diglyceride camellia seed oil.

[0087] 6) Extraction of Vitamin A: Heat the deodorized tea oil distillate to 50°C and stir until homogeneous. Add methanol at a mass ratio of 1:2 (deodorized distillate:methanol) to the deodorized distillate, then add 3wt% concentrated sulfuric acid (3% of methanol by volume). Esterify at 55°C with constant stirring for 2 hours to remove fatty acids. After the reaction, cool to room temperature, add an equal volume of distilled water, separate the liquid and discard the aqueous phase. Wash the organic phase with water until neutral, and filter to obtain the esterified material. Add anhydrous ethanol (material-to-liquid ratio 1:1, ratio of esterified material mass (g) to ethanol volume (mL)) + 20wt% NaOH solution (8% of the esterified material mass). Saponify at 60°C with constant stirring for 1 hour, then cool to... At room temperature; transfer the saponified liquid to a separatory funnel, add an equal volume of petroleum ether with a boiling range of 30-60℃, shake for 1 min, allow to stand and separate into layers, and collect the supernatant for later use; wash the supernatant with an equal volume of distilled water until the pH reaches 7, then add 5 wt% sodium chloride (by weight of the saponified liquid), allow to stand for 40 min to dehydrate, filter and collect the filtrate for later use; concentrate the filtrate under reduced pressure at 35℃ and a vacuum of 0.085 MPa to obtain the distillation product; pass the distillation product through a silica gel chromatography column, using petroleum ether and petroleum ether-diethyl ether (petroleum ether volume:diethyl ether volume = 92:8) as eluents for fractional elution, and collect the eluent; concentrate the eluent under reduced pressure at 40℃ until no solvent odor remains to obtain the vitamin A product.

[0088] 7) Adding Vitamin A: Add 4000 μg / kg of Vitamin A to the diglyceride camellia seed oil and mix thoroughly to obtain diglyceride camellia seed oil with high Vitamin A content.

[0089] Comparative Example 1 The difference between the comparative example and Example 1 is that step 2) uses a single yeast strain for fermentation, and the specific operation steps are as follows: a) The tea oil refining and deodorizing distillate was sterilized at 121°C for 30 minutes to obtain the sterilized deodorizing distillate.

[0090] b) Prepare a concentration of 1×10 8 CFU / mL yeast suspension: The preparation steps for yeast culture expansion are as follows: transfer yeast to malt extract agar medium (the composition of malt extract agar medium is 2wt%, glucose 2wt%, peptone 0.1wt%, agar 1.5wt%, and the remainder is sterile water) to obtain single colonies; then inoculate the single colonies into yeast extract peptone glucose medium (the composition of yeast extract peptone glucose medium is 2wt%, peptone 2wt%, yeast extract 1wt%, and the remainder is sterile water), and incubate at 30℃ for 24h for later use.

[0091] After the expansion culture is completed, the yeast culture solution is centrifuged at 4000 r / min for 15 min, the supernatant is removed, 10 ml of 0.9 wt% physiological saline is added, and the mixture is vortexed to mix evenly. The culture solution is then centrifuged at 4000 r / min for 5 min, the supernatant is removed, and 0.9 wt% physiological saline is added. The mixture is washed repeatedly 4 times, and finally, physiological saline is added to make up the difference and mix well. Physiological saline is used as a blank control. The absorbance of the culture solution is adjusted to 0.8 with physiological saline at a wavelength of 600 nm to prepare a yeast culture suspension. c) Inoculate the yeast suspension into the fermentation medium containing the deodorized distillate at an inoculation amount of 3% of the tea oil refining and deodorizing distillate. The fermentation temperature is 30℃ and the fermentation time is 72h to obtain the deodorized distillate fermentation product.

[0092] Comparative Example 2 The difference between the comparative example and Example 1 is that step 2) uses a single type of *Microbacterium flavonoids* for fermentation, and the specific operation steps are as follows: a) The tea oil refining and deodorizing distillate was sterilized at 121°C for 30 minutes to obtain the sterilized deodorizing distillate.

[0093] b) Prepare a concentration of 1×10 8 CFU / mL *Microbacterium flavum* suspension: The preparation steps for the expanded culture of *Microbacterium flavum* are as follows: Inoculate the activated *Microbacterium flavum* strain into *Microbacterium flavum* liquid seed culture medium (60 g / L glucose, 25 g / L peptone, 2 g / L NaNO3, 1 g / L MgSO4·7H2O, 1 g / L KH2PO4·3H2O, 1000 mL distilled water, pH 5.0, sterilized at 121℃ for 30 min), and culture on a constant temperature shaker for later use.

[0094] After the expansion culture was completed, the bacterial culture of *Microbacterium chrysogenum* was centrifuged at 4000 r / min for 15 min, the supernatant was removed, 10 ml of 0.9 wt% physiological saline was added, and the mixture was vortexed to mix evenly. The bacterial culture was centrifuged at 4000 r / min for 5 min, the supernatant was removed, and 0.9 wt% physiological saline was added. The mixture was washed repeatedly 4 times. Finally, physiological saline was added to make up the difference and mix well. Physiological saline was used as a blank control. The absorbance of the bacterial culture was adjusted to 0.8 with physiological saline at a wavelength of 600 nm to prepare a *Microbacterium chrysogenum* suspension. c) The suspension of *Microbacterium flavonoids* was inoculated into a fermentation medium containing deodorized distillate at an inoculation amount of 3% of the mass of tea oil refining and deodorizing distillate. The fermentation temperature was 30℃ and the fermentation time was 72 h to obtain the deodorized distillate fermentation product.

[0095] Standard indicator test (1) The acid value and peroxide value of the camellia seed oil with lipid-lowering function prepared in Examples 1-3 were determined, and the results are shown in Table 1: Table 1. Results of acid value and peroxide value determination of camellia seed oil with high vitamin A content prepared in Examples 1-3

[0096] As shown in Table 1, the acid value of the camellia seed oil prepared by the method of the present invention is controlled at 0.1~1.0 mgKOH / g, and the peroxide value is 0.009~0.08 g / 100g, which meets the standard of Grade 1 pressed camellia seed oil in GB / T 11765-2018.

[0097] (2) The yield of vitamin A obtained in the preparation of finished camellia seed oil in Examples 1-3 and Comparative Examples 1-2 was determined. The yield of vitamin A was calculated based on the refined and deodorized distillate of camellia oil. The yield of vitamin A = weight of vitamin A powder / content of vitamin A in the refined and deodorized distillate of camellia oil × 100%. The results are shown in Table 2. Method for detecting vitamin A content: The fermentation product or the refined and deodorized distillate of camellia oil was freeze-dried at -55℃ for 72h in a freezer and then pulverized and freeze-dried into fermentation product powder or refined and deodorized distillate powder. 10g of fermentation product powder or refined and deodorized distillate powder of camellia oil was weighed into a 250mL conical flask, 100mL of 15g / L vitamin C-ethanol solution and 25mL of 55wt% KOH solution were added, and the mixture was magnetically stirred for 45min at 53℃. The saponified solution was transferred to a 500 mL separatory funnel and extracted three times with 100 mL of petroleum ether each time. The extracts were combined and washed with water 3-5 times until neutral. The petroleum ether layer was collected and dehydrated with anhydrous Na₂SO₄. The petroleum ether was recovered under low temperature and reduced pressure until only 1-2 mL remained. The solution was then transferred to a 10 mL amber bottle, dried under nitrogen, dissolved in methanol, and brought to a final volume of 10 mL for later use. Chromatographic conditions: High performance liquid chromatograph (HPLC), Agilent 1260; column: Athena C30, length 250 mm, inner diameter 4.6 mm, particle size 5 μm; flow rate 0.8 mL / min; injection volume 10 μL; column temperature 20℃; UV detector wavelengths 325 nm and 294 nm (dual wavelengths); elution time 30 min; mobile phase: methanol:water (96:4, v / v). Standard curve for vitamin A: y = 0.0091x + 0.0223, R 2 =0.9997.

[0098] Table 2. Yields of Vitamin A obtained in Examples 1-3 and Comparative Examples 1-2

[0099] As shown in Table 2, the vitamin A yield obtained by the method of mixed fermentation and deodorization of distillate using yeast and microbacterium flavum in Examples 1-3 reached over 90%, which was significantly higher than that in Comparative Examples 1-2. This indicates that the synergistic fermentation of the two bacteria (yeast and microbacterium flavum) can significantly improve the vitamin A yield in the deodorized distillate compared to single-strain fermentation of deodorized distillate using yeast or microbacterium flavum, thus playing a role in the efficient enrichment of vitamin A.

[0100] (3) The changes in β-carotene and vitamin A with fermentation time during the preparation of finished camellia seed oil in Examples 1-3 and Comparative Examples 1-2 were studied. The results are as follows: Figures 2-3 As shown.

[0101] Fermentation was carried out on the corresponding bacteria of Examples 1-3 and Comparative Examples 1-2 respectively. The fermentation products (containing culture medium, deodorized distillate and bacterial cells) after fermentation for 36h, 48h, 60h, 72h, 84h, 96h, 108h and 120h were tested for β-carotene and vitamin A content.

[0102] Determination of β-carotene content: The detection method referred to the study on the mechanism of squalene inhibiting β-carotene loss during silage fermentation of alfalfa silage. The specific steps were as follows: The fermentation product was freeze-dried at -55℃ for 72h in a freezer, and then pulverized and freeze-dried into fermentation product powder. 2g of fermentation product powder was accurately weighed into a 100mL Erlenmeyer flask, and 15mL of 10wt% vitamin C solution, 15mL of methanol, 35mL of anhydrous ethanol, and 10mL of KOH aqueous solution were added sequentially. The mixture was then saponified in a water bath at 80℃ in the dark for 30min. After cooling to room temperature, 35mL of the saponified solution was transferred to a 50mL centrifuge tube, 3mL of heptane and 1mL of distilled water were added, and the mixture was shaken well. The mixture was centrifuged at 3500r / min for 5min, and then another 3mL of heptane was added, and the mixture was shaken well. After centrifuging at 3500r / min for 5min, 1mL of the supernatant was used to determine the β-carotene content. β-carotene content was determined by high-performance liquid chromatography (HPLC) using an Inertsil ODS-4 column at 45℃. The mobile phase was methanol-acetonitrile solution (methanol:acetonitrile = 9:1), the flow rate was 2 mL / min, and a DAD detector was used. β-carotene was detected at a wavelength of 450 nm. The standard curve for β-carotene was: y = 57869x - 23457, R0 2 =0.999 (x: concentration μg / mL, y: peak area).

[0103] from Figures 2-3 It can be seen that during the fermentation process, the β-carotene content in the fermentation products corresponding to Examples 1-3 showed a significant increase followed by a stabilization and then a slight decrease over time. In contrast, the β-carotene content in the fermentation products corresponding to Comparative Examples 1 and 2 showed a slow increase followed by a stabilization and then a slight decrease over time. Furthermore, the β-carotene content in Examples 1-3 was higher than that in Comparative Examples 1-2 throughout the entire fermentation process. This indicates that only when yeast and *Microbacterium flavonoids* are fermented together can the synthesis of β-carotene be effectively promoted, resulting in a significantly higher increase in β-carotene content compared to fermentation by yeast or single-strain *Microbacterium flavonoids*.

[0104] from Figure 3It can be seen that the vitamin A content in the fermentation products corresponding to Examples 1-3 also showed a significant increase followed by a slight decrease. The vitamin A content in the fermentation products corresponding to Comparative Examples 1 and 2 did not increase significantly, and showed a slight decrease after 72 hours of fermentation. This phenomenon may be due to the cell death phase and the rapid consumption of nutrients, which causes vitamin A to be consumed as a substrate, resulting in a greater consumption of vitamin A than synthesis, ultimately leading to a decrease in vitamin A content.

[0105] Furthermore, throughout the fermentation process, the vitamin A content in Examples 1-3 was higher than that in Comparative Examples 1-2, indicating that the co-fermentation of yeast and flavobacterium significantly increased the vitamin A content in the fermentation product compared to single-strain fermentation. This may be related to the fact that the co-fermentation of yeast and flavobacterium increased the β-carotene content, thereby promoting the synthesis of vitamin A.

[0106] (4) Camellia seed oil was prepared according to the steps of Example 1. The effect of the inoculation amount of different yeast and flavobacterium suspensions on the vitamin A yield was studied. The inoculation amounts of yeast and flavobacterium suspensions were 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%. The results are shown in Table 3.

[0107] from Figure 4 It can be seen that as the inoculum amount of the mixed yeast and flavobacterium increases, the yield of phytosterols first increases and then plateaus, showing a significant downward trend after the inoculum amount reaches 6%. This may be because excessive inoculum amount generates a large amount of metabolic waste, which negatively affects the growth of yeast and flavobacterium and also affects the yield of phytosterols. The staining degree shows a trend of first decreasing, then plateauing, and then increasing. Considering the trends of both, the preferred inoculum amount is 2-6%, and the optimal inoculum amount is 3%.

[0108] Table 3. Effects of yeast and *Microbacterium flavonoids* suspension inoculum size on vitamin A yield.

[0109] (5) Camellia seed oil with diglycerides was prepared according to the steps of Example 1. The effects of fermentation time of different yeast and flavobacterium suspensions on vitamin A yield were studied. The fermentation times of yeast and flavobacterium suspensions were 24h, 48h, 60h, 72h, 84h, 96h and 108h. The results are shown in Table 4.

[0110] As shown in Table 4, with the extension of fermentation time of the mixed yeast and flavobacterium tumefaciens, the vitamin A yield first increased and then leveled off, showing a significant downward trend after 96 hours. This phenomenon may be due to the decline of the microbial cells, which hinders the synthesis of vitamin A in the fermentation product. In addition, the nutrients in the later stage are rapidly consumed, causing vitamin A to be consumed by the microbial strain as a substrate. This leads to a decrease in the vitamin A content in the fermentation product after 96 hours of fermentation, ultimately resulting in a significant decrease in the vitamin A yield. The preferred fermentation time is 48-96 hours, and the optimal fermentation time is 72 hours.

[0111] Table 4. Effects of fermentation time of yeast and *Microbacillus flavus* suspensions on vitamin A yield.

[0112] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing camellia seed oil with high vitamin A content diglycerides, characterized in that, Includes the following steps: Cellulose was extracted from camellia seed cake, and the resulting camellia seed cake cellulose was nano-processed to obtain nanocellulose. The nanocellulose and graphene are combined to obtain a nanocellulose / graphene composite material of camellia seed cake. Glycerol and the aforementioned camellia seed cake nanocellulose / graphene composite material are mixed, and glycerol is adsorbed onto the camellia seed cake nanocellulose / graphene composite material to obtain glycerol-camellia seed cake nanocellulose / graphene composite material. Freshly pressed camellia seed oil, glycerol-camellia seed cake nanocellulose / graphene composite material, and lipase are mixed and subjected to enzymatic glycerolysis to obtain esterified products; the temperature of the enzymatic glycerolysis reaction is 30~45℃. The esterification product was filtered, and the resulting mixture of diglyceride camellia seed oil was subjected to molecular distillation to obtain refined diglyceride camellia seed oil. Vitamin A was added to the refined diglyceride camellia seed oil to obtain diglyceride camellia seed oil with high vitamin A content; The method for preparing vitamin A is as follows: the deodorized distillate fermentation product, methanol and sulfuric acid solution are mixed and subjected to esterification reaction to obtain the esterified material; The esterified material, ethanol, and NaOH solution are mixed and a saponification reaction is carried out to obtain a saponified solution. Transfer the saponified liquid into a separatory funnel, add petroleum ether, shake, and let it stand to separate into layers. Take the upper clear liquid for later use. Wash the supernatant with water until the pH value is 7, then add sodium chloride, let it stand to dehydrate, filter and collect the filtrate for later use; The filtrate was concentrated under reduced pressure to obtain the distillation product; The distillation product was passed through a silica gel chromatography column for fractional elution, and the eluent was collected. The eluent was concentrated under reduced pressure until no solvent odor was detected, yielding vitamin A; The preparation method of the deodorized distillate fermentation product is as follows: a mixed bacterial suspension of yeast and flavobacterium is inoculated onto a fermentation medium containing tea oil refined deodorized distillate for fermentation to obtain the deodorized distillate fermentation product.

2. The preparation method according to claim 1, characterized in that, The steps for extracting cellulose are as follows: crush the camellia seed cake, defatt the obtained camellia seed cake powder with petroleum ether, dry the defatted camellia seed cake powder, sieve it, mix the sieved powder with NaOH solution for cellulose extraction, and obtain crude camellia seed cake cellulose. The crude camellia seed cake cellulose was mixed with H2O2 solution and oxidized to obtain camellia seed cake cellulose.

3. The preparation method according to claim 1, characterized in that, The composite process involves mixing an aqueous solution of nanocellulose and an aqueous solution of graphene, followed by ultrasonication, magnetic stirring, vacuum filtration, and drying. The dried composite material is then subjected to repeated freeze-thaw treatments and freeze-dried to obtain a nanocellulose / graphene composite material from camellia seed cake.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the glycerol to the camellia seed cake nanocellulose / graphene composite material is 1:1~2.

5.

5. The preparation method according to claim 1, characterized in that, The mass ratio of freshly pressed camellia seed oil to glycerol-camellia seed cake nanocellulose / graphene composite material is 4~8:

1.

6. The preparation method according to claim 1, characterized in that, The amount of vitamin A added to the refined diglyceride camellia seed oil is 4000~6000μg / kg.

7. The preparation method according to claim 1, characterized in that, The enzymatic glycerol hydrolysis reaction takes 6-10 hours.

8. The preparation method according to claim 1, characterized in that, The inoculation amount of the mixed bacterial suspension of yeast and flavobacterium is 2-6% of the mass of the tea oil refining and deodorizing distillate.

9. The preparation method according to claim 1, characterized in that, The fermentation temperature is 28~32℃ and the time is 48~96h.

10. The preparation method according to claim 1, characterized in that, The lipase is Lipozyme TL IM lipase; the mass of the lipase is 4-8% of the mass of freshly pressed camellia seed oil.

Images