High-load plant-derived fermentation oil carrier and its preparation method

Abstract

This invention relates to a high-load plant-derived fermented oil carrier and its preparation method, belonging to the technical field of fermented oil carriers. The raw materials for preparing the high-load plant-derived fermented oil carrier include: 45-55 parts rapeseed methyl oleate, 5-8 parts macadamia seed oil, 5-10 parts glucose, 35-45 parts deionized water, 2-5 parts peptone, 1.5-3 parts activated bacterial solution, and 5-8 parts porous microsphere framework. A composite oil phase of rapeseed methyl oleate and macadamia seed oil is selected, and combined with glucose, peptone, and Candida albicans for staged two-stage fermentation. The fermentation metabolism is thorough, followed by sterilization, high-speed centrifugation, ethanol purification, and negative pressure dehydration purification. The resulting fermented oil has few impurities, low moisture content, excellent component compatibility, and good compatibility with the porous microsphere framework.

Description

Technical Field

[0001] This invention belongs to the field of fermentation oil carrier technology, specifically relating to high-load plant-derived fermentation oil carriers and their preparation methods. Background Technology

[0002] The core function of fermented oil carriers is to solve problems such as easy oxidation, poor water solubility, and poor stability of vegetable oils in application, so as to achieve high-content oil loading and efficient utilization.

[0003] Traditional high-load plant-derived fermentation oil carriers are mostly simple oil mixtures, lacking the support of a dedicated porous adsorption framework with controllable pore size and high specific surface area. Moreover, they have not undergone targeted modification, resulting in an inherently insufficient capacity for carrying, dispersing, and retaining plant active substances. They easily reach dissolution saturation with a slightly higher loading, which in turn leads to the aggregation and association of active molecules, resulting in slight turbidity, flocculent matter, and bottom precipitation. With prolonged storage, they will further develop into obvious stratification and a large amount of crystal precipitation, severely damaging the homogeneity of the product. Summary of the Invention

[0004] The purpose of this invention is to provide a high-load plant-derived fermentation oil carrier and its preparation method in order to solve the above-mentioned problems.

[0005] The present invention achieves the above objectives through the following technical solutions: This invention provides a high-load plant-derived fermented oil carrier. By weight, the raw materials for preparing the high-load plant-derived fermented oil carrier include: 45-55 parts rapeseed methyl ester, 5-8 parts macadamia seed oil, 5-10 parts glucose, 35-45 parts deionized water, 2-5 parts peptone, 1.5-3 parts activated bacterial solution, and 5-8 parts porous microsphere framework. The raw materials for preparing the porous microsphere framework, by weight, include: 45-55 parts of a mixture, 3-5 parts of a crosslinking agent, 0.3-0.8 parts of an initiator, 10-20 parts of a pore-forming agent, 1-3 parts of an emulsifier, and 65-75 parts of deionized water.

[0006] As a further optimization of the present invention, the mixture is a mixture of butyl acrylate and methyl methacrylate, and the mass ratio of butyl acrylate to methyl methacrylate is (5-6):(4-5).

[0007] As a further optimization of the present invention, the crosslinking agent is ethylene glycol dimethacrylate.

[0008] As a further optimization of the present invention, the initiator is azobisisobutyronitrile; the porogen is n-pentane or ethyl acetate; and the emulsifier is a mixture of Tween-80 and Span-80, with a mass ratio of (0.6-1.2):(0.5-0.8).

[0009] As a further optimization of the present invention, the method for preparing the porous microsphere framework includes: Preparation of the aqueous phase: Deionized water and emulsifier are mixed and dissolved to form an aqueous phase; Preparation of the oil phase: Mix the mixture, crosslinking agent, initiator, and pore-forming agent evenly to form an oil phase; Emulsion preparation: Slowly drop the oil phase into the aqueous phase and stir at 500-800 r / min for 30-60 min to form a uniform and stable emulsion; Polymerization: Transfer the emulsion to a reactor, heat to 55-65℃, and polymerize at a speed of 300-500 r / min for 3-5 hours; Post-processing: After the reaction was completed, the mixture was cooled to room temperature, centrifuged, washed, and dried to obtain crude porous microspheres. Surface modification: The crude porous microspheres were dispersed in anhydrous ethanol at a solid-liquid ratio of 1g:40mL, and silane coupling agent KH-570 was added. The mixture was stirred and modified for 2-3 hours at 35-45℃ and 150-200r / min. After filtration and drying, the porous microsphere framework was obtained.

[0010] As a further optimization of the present invention, the amount of silane coupling agent is 0.8%-1.2% of the crude weight of the porous microspheres.

[0011] As a further optimization of the present invention, the centrifugation speed in the post-processing is 3000-4000 r / min and the time is 10-15 min; the washing is carried out by alternating washing with deionized water and 75% ethanol 3-4 times, centrifugation after each washing, and finally washing once with phosphate buffer at pH=7.0.

[0012] As a further optimization of the present invention, the preparation process of the activated bacterial solution is as follows: PDA liquid culture medium is selected, Candida oryzae is inoculated, and cultured in a shake flask at 30℃ and 220rpm for 18-24h to obtain the activated bacterial solution.

[0013] As a further optimization of the present invention, the viable count of *Candida nectarine* is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

[0014] This invention also provides a method for preparing a high-loading plant-derived fermentation oil carrier, comprising the following steps: S1, Culture medium preparation and sterilization: Mix glucose, deionized water and peptone according to the weight parts, adjust the pH to 5.0-5.5 with phosphoric acid, sterilize at 121℃ for 30 min, cool to 30℃ to obtain sterile aqueous culture medium; mix rapeseed oil methyl ester and macadamia seed oil evenly, filter through a 0.22μm sterile filter membrane to obtain sterile oil phase; S2, Culture medium preparation: Under aseptic conditions, add 50% of the total amount of sterile oil phase to the sterile aqueous culture medium and stir evenly to obtain the fermentation culture medium; S3, primary fermentation: Add the activated bacterial solution from step 1 to the fermentation medium obtained in step 2, seal and purge with nitrogen to remove oxygen, and ferment for 56-72 hours at 28-30℃ and 150-200 rpm. S4, secondary fermentation: Add the remaining sterile oil phase to the primary fermentation body, adjust the stirring speed to 180-220 rpm, and continue fermentation at 28-30℃ for 120-144 h. S5, Sterilization: After the secondary fermentation is completed, heat the volume to 80℃ and keep it at that temperature for 30 minutes, then cool it to room temperature; S6, Separation and Purification: Centrifuge the product obtained in step S5 at 8000-10000×g for 20 min to remove bacterial residue and collect the upper oil phase; add anhydrous ethanol at a volume ratio of 1:1 to the oil phase and stir at 200 rpm for 10-15 min. After stirring, centrifuge at 4℃ and 8000-10000×g for 15 min and collect the upper oil phase; filter through a 0.22μm sterile filter membrane and dehydrate at 60℃ and -0.07~-0.09MPa until the water content in the product is ≤0.5% to obtain refined fermented oil; S7, Carrier compounding: The refined fermented oil is mixed with the porous microsphere framework, stirred and adsorbed at 40-50℃ and 120-150r / min for 1-2 hours, and then allowed to stand and age for 30 minutes to obtain a high-load plant-derived fermented oil carrier.

[0015] The beneficial effects of this invention are as follows: a compound oil phase of rapeseed methyl ester and macadamia seed oil is selected, and glucose, peptone and Candida albicans are used for staged two-stage fermentation. The fermentation and metabolism are sufficient. After sterilization, high-speed centrifugation, ethanol refining and negative pressure dehydration refining, the fermented oil has fewer impurities, lower moisture content, and excellent component compatibility. It has good compatibility with the porous microsphere skeleton and can greatly improve the overall interface stability of the carrier. A porous microsphere framework was constructed by combining butyl acrylate and methyl methacrylate, and then crosslinked with ethylene glycol dimethacrylate, pore-forming agent, and modified with silane coupling agent KH-570 to form a porous microsphere framework with uniform pore structure and large specific surface area. This framework has strong adsorption, retention, dispersion and encapsulation capabilities for plant active ingredients, enabling high loading of active substances, effectively inhibiting the aggregation, precipitation and crystallization of active molecules, and significantly improving the uniformity of the microspheres. Detailed Implementation

[0016] The present application will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0017] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products.

[0018] In this invention, the PDA liquid culture medium formula is as follows: 200 g / L potato, 20 g / L glucose, and 1000 mL deionized water; adjust the initial pH to 5.5, autoclave at 121°C for 30 min, and cool to 30°C for later use.

[0019] Example 1 The mixture is a mixture of butyl acrylate and methyl methacrylate, with a mass ratio of butyl acrylate to methyl methacrylate of 5:4. The emulsifier is a mixture of Tween-80 and Span-80, with a mass ratio of 0.6:0.5. Methods for preparing porous microsphere frameworks include: Preparation of the aqueous phase: Mix 65 parts of deionized water with 1 part of emulsifier to form an aqueous phase; Preparation of the oil phase: Mix 45 parts of the mixture, 3 parts of ethylene glycol dimethacrylate, 0.3 parts of azobisisobutyronitrile, and 10 parts of n-pentane evenly to form the oil phase; Emulsion preparation: The oil phase is slowly added dropwise to the aqueous phase, and the mixture is stirred at 500 r / min for 30 min to form a uniform and stable emulsion; Polymerization: The emulsion was transferred to a reactor, heated to 55°C, and polymerized at 300 r / min for 3 h. Post-processing: After the reaction was completed, the mixture was cooled to room temperature and centrifuged at 3000 r / min for 10 min. The mixture was washed three times with deionized water and 75% ethanol, centrifuged after each wash, and finally washed once with phosphate buffer at pH 7.0. After drying, the crude porous microspheres were obtained. Surface modification: The crude porous microspheres were dispersed in anhydrous ethanol at a solid-liquid ratio of 1g:40mL, and silane coupling agent KH-570 (the amount of silane coupling agent was 0.8% of the crude porous microspheres) was added. The mixture was stirred and modified at 35℃ and 150r / min for 2h. After filtration and drying, the porous microsphere skeleton was obtained. The preparation process of the activated bacterial solution is as follows: PDA liquid medium is selected, Candida oryzae is inoculated, and cultured in shake flasks at 30℃ and 220rpm for 18h to obtain the activated bacterial solution (the viable count of Candida oryzae is 1×10⁻⁶).8 (CFU / mL) Culture medium preparation and sterilization: Mix 5 parts glucose, 3 parts deionized water and 2 parts peptone, adjust the pH to 5.0 with phosphoric acid, sterilize at 121℃ for 30 min, and cool to 30℃ to obtain a sterile aqueous culture medium; mix 45 parts rapeseed methyl ester and 5 parts macadamia seed oil evenly, filter through a 0.22μm sterile filter membrane to obtain a sterile oil phase; Culture medium preparation: Under aseptic conditions, add 50% of the total amount of sterile oil phase to the sterile aqueous culture medium and stir evenly to obtain the fermentation culture medium; Primary fermentation: Add 1.5 parts of activated bacterial solution to the fermentation medium obtained in step 2, seal and purge with nitrogen to remove oxygen, and ferment for 56 hours at 28°C and 150 rpm. Secondary fermentation: Add the remaining sterile oil phase to the primary fermentation body, adjust the stirring speed to 180 rpm, and continue fermentation at 28℃ for 120 h; Sterilization: After the secondary fermentation is completed, heat the mixture to 80°C and keep it at that temperature for 30 minutes, then cool it to room temperature; Separation and purification: The cell obtained in step S5 was centrifuged at 8000×g for 20 min to remove bacterial residue and the upper oil phase was collected. Anhydrous ethanol with a volume ratio of 1:1 to the oil phase was added and stirred at 200 rpm for 10 min. After stirring, the mixture was centrifuged at 4℃ and 8000×g for 15 min and the upper oil phase was collected. The mixture was filtered through a 0.22 μm sterile filter membrane and dehydrated at 60℃ and -0.07 MPa until the water content in the product was 0.5% to obtain refined fermented oil. Carrier compounding: The refined fermented oil was mixed with 5 parts of porous microsphere framework, stirred and adsorbed at 40℃ and 120r / min for 1h, and then allowed to stand and age for 30min to obtain a high-load plant-derived fermented oil carrier.

[0020] Example 2 The mixture is a mixture of butyl acrylate and methyl methacrylate, with a mass ratio of butyl acrylate to methyl methacrylate of 5:5; The emulsifier is a mixture of Tween-80 and Span-80, with a mass ratio of 0.8:0.6. Methods for preparing porous microsphere frameworks include: Preparation of the aqueous phase: Mix and dissolve 70 parts of deionized water with 2 parts of emulsifier to form an aqueous phase; Preparation of the oil phase: Mix 50 parts of the mixture, 4 parts of ethylene glycol dimethacrylate, 0.5 parts of azobisisobutyronitrile, and 15 parts of n-pentane evenly to form the oil phase; Emulsion preparation: The oil phase is slowly added dropwise to the aqueous phase and stirred at 650 r / min for 45 min to form a uniform and stable emulsion; Polymerization: The emulsion was transferred to a reactor, heated to 60°C, and polymerized at 400 r / min for 4 h. Post-processing: After the reaction was completed, the mixture was cooled to room temperature and centrifuged at 3500 r / min for 12 min. The mixture was washed three times with deionized water and 75% ethanol, centrifuged after each wash, and finally washed once with phosphate buffer at pH 7.0. After drying, the crude porous microspheres were obtained. Surface modification: The crude porous microspheres were dispersed in anhydrous ethanol at a solid-liquid ratio of 1g:40mL, and silane coupling agent KH-570 (the amount of silane coupling agent was 1% of the crude porous microspheres) was added. The mixture was stirred and modified at 40℃ and 175r / min for 2.5h. After filtration and drying, the porous microsphere skeleton was obtained. The preparation process of the activated bacterial solution is as follows: PDA liquid medium is selected, and *Candida mellea* is inoculated. The mixture is then cultured in shake flasks at 30℃ and 220 rpm for 21 hours to obtain the activated bacterial solution (with a *Candida mellea* viable count of 1×10⁻⁶). 9 (CFU / mL) Culture medium preparation and sterilization: Mix 8 parts glucose, 40 parts deionized water and 3 parts peptone, adjust the pH to 5.0 with phosphoric acid, sterilize at 121℃ for 30 min, and cool to 30℃ to obtain a sterile aqueous culture medium; mix 50 parts rapeseed methyl ester and 6 parts macadamia seed oil evenly, filter through a 0.22μm sterile filter membrane to obtain a sterile oil phase; Culture medium preparation: Under aseptic conditions, add 50% of the total amount of sterile oil phase to the sterile aqueous culture medium and stir evenly to obtain the fermentation culture medium; Primary fermentation: Add 2 parts of activated bacterial solution to the fermentation medium obtained in step 2, seal and purge with nitrogen to remove oxygen, and ferment for 65 hours at 29°C and 185 rpm. Secondary fermentation: Add the remaining sterile oil phase to the primary fermentation body, adjust the stirring speed to 200 rpm, and continue fermentation at 29°C for 130 h; Sterilization: After the secondary fermentation is completed, heat the mixture to 80°C and keep it at that temperature for 30 minutes, then cool it to room temperature; Separation and purification: The cell obtained in step S5 was centrifuged at 9000×g for 20 min to remove bacterial residue and the upper oil phase was collected. Anhydrous ethanol with a volume ratio of 1:1 to the oil phase was added and stirred at 200 rpm for 12 min. After stirring, the mixture was centrifuged at 4℃ and 9000×g for 15 min and the upper oil phase was collected. The mixture was filtered through a 0.22 μm sterile filter membrane and dehydrated at 60℃ and -0.08 MPa until the water content in the product was 0.5% to obtain refined fermented oil. Carrier compounding: The refined fermented oil was mixed with 6 parts of porous microsphere framework, stirred and adsorbed at 45℃ and 135r / min for 1.5h, and allowed to stand and age for 30min to obtain a high-load plant-derived fermented oil carrier.

[0021] Example 3 The mixture is a mixture of butyl acrylate and methyl methacrylate, with a mass ratio of butyl acrylate to methyl methacrylate of 6:5; The emulsifier is a mixture of Tween-80 and Span-80, with a mass ratio of 1.2:0.8. Methods for preparing porous microsphere frameworks include: Preparation of the aqueous phase: Mix and dissolve 75 parts of deionized water with 3 parts of emulsifier to form an aqueous phase; Preparation of the oil phase: Mix 55 parts of the mixture, 5 parts of ethylene glycol dimethacrylate, 0.8 parts of azobisisobutyronitrile, and 20 parts of n-pentane evenly to form the oil phase; Emulsion preparation: The oil phase is slowly added dropwise to the aqueous phase and stirred at 800 r / min for 60 min to form a uniform and stable emulsion; Polymerization: The emulsion was transferred to a reactor, heated to 65°C, and polymerized at 500 r / min for 5 h. Post-processing: After the reaction was completed, the mixture was cooled to room temperature and centrifuged at 4000 r / min for 15 min. The mixture was washed four times with deionized water and 75% ethanol, centrifuged after each wash, and finally washed once with phosphate buffer at pH 7.0. After drying, the crude porous microspheres were obtained. Surface modification: The crude porous microspheres were dispersed in anhydrous ethanol at a solid-liquid ratio of 1g:40mL, and silane coupling agent KH-570 (the amount of silane coupling agent was 1.2% of the crude porous microspheres) was added. The mixture was stirred and modified at 45℃ and 200r / min for 3h. After filtration and drying, the porous microsphere skeleton was obtained. The preparation process of the activated bacterial solution is as follows: PDA liquid medium is selected, and *Candida leescens* is inoculated. The mixture is then cultured in shake flasks at 30℃ and 220 rpm for 24 hours to obtain the activated bacterial solution (with a *Candida leescens* viable count of 1×10⁻⁶). 9 (CFU / mL) Culture medium preparation and sterilization: Mix 10 parts glucose, 45 parts deionized water and 5 parts peptone, adjust the pH to 5.5 with phosphoric acid, sterilize at 121℃ for 30 min, and cool to 30℃ to obtain a sterile aqueous culture medium; mix 55 parts rapeseed methyl ester and 8 parts macadamia seed oil evenly, filter through a 0.22μm sterile filter membrane to obtain a sterile oil phase; Culture medium preparation: Under aseptic conditions, add 50% of the total amount of sterile oil phase to the sterile aqueous culture medium and stir evenly to obtain the fermentation culture medium; Primary fermentation: Add 3 parts of activated bacterial solution to the fermentation medium obtained in step 2, seal and purge with nitrogen to remove oxygen, and ferment for 72 hours at 30°C and 200 rpm. Secondary fermentation: Add the remaining sterile oil phase to the primary fermentation body, adjust the stirring speed to 220 rpm, and continue fermentation at 30°C for 144 h; Sterilization: After the secondary fermentation is completed, heat the mixture to 80°C and keep it at that temperature for 30 minutes, then cool it to room temperature; Separation and purification: The product obtained in step S5 was centrifuged at 10000×g for 20 min to remove bacterial residue and the upper oil phase was collected; anhydrous ethanol with a volume ratio of 1:1 was added to the oil phase and stirred at 200 rpm for 15 min. After stirring, the product was centrifuged at 10000×g for 15 min at 4℃ and the upper oil phase was collected; the product was filtered through a 0.22 μm sterile filter membrane and dehydrated at 60℃ and -0.09 MPa until the water content in the product was 0.5% to obtain refined fermented oil; Carrier compounding: The refined fermented oil was mixed with 8 parts of porous microsphere framework, stirred and adsorbed at 50℃ and 150r / min for 2h, and then allowed to stand and age for 30min to obtain a high-load plant-derived fermented oil carrier.

[0022] Comparative Example 1 The mixture is a mixture of butyl acrylate and methyl methacrylate, with a mass ratio of butyl acrylate to methyl methacrylate of 5:5; The emulsifier is a mixture of Tween-80 and Span-80, with a mass ratio of 0.8:0.6. Methods for preparing porous microsphere frameworks include: Preparation of the aqueous phase: Mix and dissolve 70 parts of deionized water with 2 parts of emulsifier to form an aqueous phase; Preparation of the oil phase: Mix 50 parts of the mixture, 4 parts of ethylene glycol dimethacrylate, 0.5 parts of azobisisobutyronitrile, and 15 parts of n-pentane evenly to form the oil phase; Emulsion preparation: The oil phase is slowly added dropwise to the aqueous phase and stirred at 650 r / min for 45 min to form a uniform and stable emulsion; Polymerization: The emulsion was transferred to a reactor, heated to 60°C, and polymerized at 400 r / min for 4 h. Post-processing: After the reaction was completed, the mixture was cooled to room temperature and centrifuged at 3500 r / min for 12 min. The mixture was washed three times with deionized water and 75% ethanol, centrifuged after each wash, and finally washed once with phosphate buffer at pH 7.0. After drying, a porous microsphere framework was obtained. The preparation process of the activated bacterial solution is as follows: PDA liquid medium is selected, and *Candida mellea* is inoculated. The mixture is then cultured in shake flasks at 30℃ and 220 rpm for 21 hours to obtain the activated bacterial solution (with a *Candida mellea* viable count of 1×10⁻⁶). 9 (CFU / mL) Culture medium preparation and sterilization: Mix 8 parts glucose, 40 parts deionized water and 3 parts peptone, adjust the pH to 5.0 with phosphoric acid, sterilize at 121℃ for 30 min, and cool to 30℃ to obtain a sterile aqueous culture medium; mix 50 parts rapeseed methyl ester and 6 parts macadamia seed oil evenly, filter through a 0.22μm sterile filter membrane to obtain a sterile oil phase; Culture medium preparation: Under aseptic conditions, add 50% of the total amount of sterile oil phase to the sterile aqueous culture medium and stir evenly to obtain the fermentation culture medium; Primary fermentation: Add 2 parts of activated bacterial solution to the fermentation medium obtained in step 2, seal and purge with nitrogen to remove oxygen, and ferment for 65 hours at 29°C and 185 rpm. Secondary fermentation: Add the remaining sterile oil phase to the primary fermentation body, adjust the stirring speed to 200 rpm, and continue fermentation at 29°C for 130 h; Sterilization: After the secondary fermentation is completed, heat the mixture to 80°C and keep it at that temperature for 30 minutes, then cool it to room temperature; Separation and purification: The cell obtained in step S5 was centrifuged at 9000×g for 20 min to remove bacterial residue and the upper oil phase was collected. Anhydrous ethanol with a volume ratio of 1:1 to the oil phase was added and stirred at 200 rpm for 12 min. After stirring, the mixture was centrifuged at 4℃ and 9000×g for 15 min and the upper oil phase was collected. The mixture was filtered through a 0.22 μm sterile filter membrane and dehydrated at 60℃ and -0.08 MPa until the water content in the product was 0.5% to obtain refined fermented oil. Carrier compounding: The refined fermented oil was mixed with 6 parts of porous microsphere framework, stirred and adsorbed at 45℃ and 135r / min for 1.5h, and allowed to stand and age for 30min to obtain a high-load plant-derived fermented oil carrier.

[0023] Comparative Example 2 The preparation process of the activated bacterial solution is as follows: PDA liquid medium is selected, and *Candida mellea* is inoculated. The mixture is then cultured in shake flasks at 30℃ and 220 rpm for 21 hours to obtain the activated bacterial solution (with a *Candida mellea* viable count of 1×10⁻⁶). 9 (CFU / mL) Culture medium preparation and sterilization: Mix 8 parts glucose, 40 parts deionized water and 3 parts peptone, adjust the pH to 5.0 with phosphoric acid, sterilize at 121℃ for 30 min, and cool to 30℃ to obtain a sterile aqueous culture medium; mix 50 parts rapeseed methyl ester and 6 parts macadamia seed oil evenly, filter through a 0.22μm sterile filter membrane to obtain a sterile oil phase; Culture medium preparation: Under aseptic conditions, add 50% of the total amount of sterile oil phase to the sterile aqueous culture medium and stir evenly to obtain the fermentation culture medium; Primary fermentation: Add 2 parts of activated bacterial solution to the fermentation medium obtained in step 2, seal and purge with nitrogen to remove oxygen, and ferment for 65 hours at 29°C and 185 rpm. Secondary fermentation: Add the remaining sterile oil phase to the primary fermentation body, adjust the stirring speed to 200 rpm, and continue fermentation at 29°C for 130 h; Sterilization: After the secondary fermentation is completed, heat the mixture to 80°C and keep it at that temperature for 30 minutes, then cool it to room temperature; Separation and purification: The cell obtained in step S5 was centrifuged at 9000×g for 20 min to remove bacterial residue and the upper oil phase was collected. Anhydrous ethanol with a volume ratio of 1:1 to the oil phase was added and stirred at 200 rpm for 12 min. After stirring, the mixture was centrifuged at 4℃ and 9000×g for 15 min and the upper oil phase was collected. The mixture was filtered through a 0.22 μm sterile filter membrane and dehydrated at 60℃ and -0.08 MPa until the water content in the product was 0.5% to obtain refined fermented oil. Carrier compounding: The refined fermented oil was stirred and adsorbed at 45℃ and 135r / min for 1.5h, and then allowed to stand and age for 30min to obtain a high-load plant-derived fermented oil carrier.

[0024] Performance testing The high-load plant-derived fermented oil carriers prepared by the methods of Examples 1-3 and Comparative Examples 1-2 were subjected to loading performance testing. The testing conditions were as follows: 2.5%, 3%, 3.5%, 4%, 4.5%, and 5% of the total carrier amount of asiaticoside were added to the high-load plant-derived fermented oil carriers prepared by the methods of Examples 1-3 and Comparative Examples 1-2, respectively, and two groups were set up for constant temperature standing. Room temperature group: Temperature 25℃±2℃, placed indoors in a dark place; Refrigeration group: Refrigerator with constant temperature of 4℃±1℃; Observation time points: Observations and records were made uniformly at three time points: day 7, day 15, and day 30. The observation results are used as stability values ​​using a standardized scoring method: 5 points: The product is uniform and transparent / uniformly dispersed, with no layering, no precipitation, and no exudation; 4 points: Slightly opalescent, very slightly cloudy, no layering, no precipitation, no crystallization; 3 points: Slight stratification / small amount of flocculent matter / trace amount of sediment at the bottom; 2 points: Obvious layering, visible precipitation, slight crystallization; 1 point: Severe stratification, large amount of precipitation, obvious crystals, and severe turbidity.

[0025] The observation results are shown in Table 1-6: Table 1. Day 7 of the group under normal temperature

[0026] Table 2. Day 15 of the group under normal temperature

[0027] Table 3. Day 30 of the group under normal temperature

[0028] Table 4. Refrigerated group at day 7

[0029] Table 5. Refrigerated group at day 15

[0030] Table 6. Refrigerated group at day 30

[0031] As can be seen from Tables 1-6, under normal temperature conditions, the examples have a significant advantage over the comparative examples: in the comparative examples, stratification, precipitation, and crystallization occur in a short time under high load; the examples can delay the instability process and maintain a relatively homogeneous state even after being placed at room temperature for a long time. Low temperature can improve the overall stability of all groups, but the improvement and final stability of the examples are still significantly higher than those of the comparative examples: under refrigeration, the comparative examples at 4.5% and 5% are still prone to stratification and precipitation; the examples can still maintain a good dispersion state under high load in refrigeration environment, and the advantages of low temperature loading are further highlighted.

[0032] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A high-load plant-derived fermentation oil carrier, characterized in that, The raw materials for preparing the high-load plant-derived fermented oil carrier, by weight, include: 45-55 parts rapeseed methyl ester, 5-8 parts macadamia seed oil, 5-10 parts glucose, 35-45 parts deionized water, 2-5 parts peptone, 1.5-3 parts activated bacterial solution and 5-8 parts porous microsphere skeleton. The raw materials for preparing the porous microsphere framework, by weight, include: 45-55 parts of a mixture, 3-5 parts of a crosslinking agent, 0.3-0.8 parts of an initiator, 10-20 parts of a pore-forming agent, 1-3 parts of an emulsifier, and 65-75 parts of deionized water.

2. The high-loading plant-derived fermentation oil carrier according to claim 1, characterized in that, The mixture is a mixture of butyl acrylate and methyl methacrylate, with a mass ratio of butyl acrylate to methyl methacrylate of (5-6):(4-5).

3. The high-loading plant-derived fermentation oil carrier according to claim 1, characterized in that, The crosslinking agent is ethylene glycol dimethacrylate.

4. The high-loading plant-derived fermentation oil carrier according to claim 1, characterized in that, The initiator is azobisisobutyronitrile; the porogen is n-pentane or ethyl acetate; the emulsifier is a mixture of Tween-80 and Span-80, with a mass ratio of (0.6-1.2):(0.5-0.8).

5. The high-loading plant-derived fermentation oil carrier according to claim 1, characterized in that, The method for preparing the porous microsphere framework includes: Preparation of the aqueous phase: Deionized water and emulsifier are mixed and dissolved to form an aqueous phase; Preparation of the oil phase: Mix the mixture, crosslinking agent, initiator, and pore-forming agent evenly to form an oil phase; Emulsion preparation: Slowly drop the oil phase into the aqueous phase and stir at 500-800 r / min for 30-60 min to form a uniform and stable emulsion; Polymerization: Transfer the emulsion to a reactor, heat to 55-65℃, and polymerize at a speed of 300-500 r / min for 3-5 hours; Post-processing: After the reaction was completed, the mixture was cooled to room temperature, centrifuged, washed, and dried to obtain crude porous microspheres. Surface modification: The crude porous microspheres were dispersed in anhydrous ethanol at a solid-liquid ratio of 1g:40mL, and silane coupling agent KH-570 was added. The mixture was stirred and modified for 2-3 hours at 35-45℃ and 150-200r / min. After filtration and drying, the porous microsphere framework was obtained.

6. The high-loading plant-derived fermentation oil carrier according to claim 5, characterized in that, The amount of silane coupling agent used is 0.8%-1.2% of the crude weight of the porous microspheres.

7. The high-loading plant-derived fermentation oil carrier according to claim 5, characterized in that, In the post-processing, the centrifugation speed is 3000-4000 r / min and the time is 10-15 min; the washing is performed by alternating between deionized water and 75% ethanol 3-4 times, centrifuging after each washing, and finally washing once with phosphate buffer at pH=7.

0.

8. The high-loading plant-derived fermentation oil carrier according to claim 1, characterized in that, The preparation process of the activated bacterial solution is as follows: PDA liquid culture medium is selected, Candida micrantha is inoculated, and cultured in a shake flask at 30℃ and 220rpm for 18-24h to obtain the activated bacterial solution.

9. The high-loading plant-derived fermentation oil carrier according to claim 1, characterized in that, The viable count of *Candida nectarinowii* was 1×10⁻⁶. 8 -1×10 9 CFU / mL.

10. A method for preparing a high-loading plant-derived fermented oil carrier according to any one of claims 1-9, characterized in that, Includes the following steps: S1, Culture medium preparation and sterilization: Mix glucose, deionized water and peptone according to the weight parts, adjust the pH to 5.0-5.5 with phosphoric acid, sterilize at 121℃ for 30 min, cool to 30℃ to obtain sterile aqueous culture medium; mix rapeseed oil methyl ester and macadamia seed oil evenly, filter through a 0.22μm sterile filter membrane to obtain sterile oil phase; S2, Culture medium preparation: Under aseptic conditions, add 50% of the total amount of sterile oil phase to the sterile aqueous culture medium and stir evenly to obtain the fermentation culture medium; S3, primary fermentation: Add the activated bacterial solution from step 1 to the fermentation medium obtained in step 2, seal and purge with nitrogen to remove oxygen, and ferment for 56-72 hours at 28-30℃ and 150-200 rpm. S4, secondary fermentation: Add the remaining sterile oil phase to the primary fermentation body, adjust the stirring speed to 180-220 rpm, and continue fermentation at 28-30℃ for 120-144 h. S5, Sterilization: After the secondary fermentation is completed, heat the volume to 80℃ and keep it at that temperature for 30 minutes, then cool it to room temperature; S6, Separation and Purification: Centrifuge the product obtained in step S5 at 8000-10000×g for 20 min to remove bacterial residue and collect the upper oil phase; add anhydrous ethanol at a volume ratio of 1:1 to the oil phase and stir at 200 rpm for 10-15 min. After stirring, centrifuge at 4℃ and 8000-10000×g for 15 min and collect the upper oil phase; filter through a 0.22μm sterile filter membrane and dehydrate at 60℃ and -0.07~-0.09MPa until the water content in the product is ≤0.5% to obtain refined fermented oil; S7, Carrier compounding: The refined fermented oil is mixed with the porous microsphere framework, stirred and adsorbed at 40-50℃ and 120-150r / min for 1-2 hours, and then allowed to stand and age for 30 minutes to obtain a high-load plant-derived fermented oil carrier.

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