WO2023206830A1 - 一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备及其应用 - Google Patents

一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备及其应用 Download PDF

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WO2023206830A1
WO2023206830A1 PCT/CN2022/106735 CN2022106735W WO2023206830A1 WO 2023206830 A1 WO2023206830 A1 WO 2023206830A1 CN 2022106735 W CN2022106735 W CN 2022106735W WO 2023206830 A1 WO2023206830 A1 WO 2023206830A1
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medium
long
essential oil
litsea cubeba
fatty acids
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French (fr)
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仇超颖
余雅思
汪勇
李颖
张震
何佳静
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Qingyuan Yaokang Biotechnology Co Ltd
Jinan University
University of Jinan
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Qingyuan Yaokang Biotechnology Co Ltd
Jinan University
University of Jinan
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
    • B65D19/00—Pallets or like platforms, with or without side walls, for supporting loads to be lifted or lowered
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
    • B65D81/00—Containers, packaging elements, or packages, for contents presenting particular transport or storage problems, or adapted to be used for non-packaging purposes after removal of contents
    • B65D81/24—Adaptations for preventing deterioration or decay of contents; Applications to the container or packaging material of food preservatives, fungicides, pesticides or animal repellants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
    • B65D85/00—Containers, packaging elements or packages, specially adapted for particular articles or materials
    • B65D85/30—Containers, packaging elements or packages, specially adapted for particular articles or materials for articles particularly sensitive to damage by shock or pressure
    • B65D85/34—Containers, packaging elements or packages, specially adapted for particular articles or materials for articles particularly sensitive to damage by shock or pressure for fruit, e.g. apples, oranges or tomatoes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of food, and specifically relates to the preparation and application of a nanostructured lipid carrier based on medium- and long-chain diglyceride-embedded Litsea cubeba essential oil.
  • Litsea cubeba essential oil is a lipophilic and highly volatile yellowish natural aromatic compound extracted from the fresh fruit of Litsea cubeba. It is rich in bioactive ingredients such as citral, limonene and linalool. It has Significant antifungal, antibacterial, anti-insect and antioxidant biological activities. Litsea cubeba essential oil can destroy bacterial cell membranes and cause protein denaturation. It has obvious inhibitory effects on Escherichia coli, Staphylococcus aureus, Cryptococcus neoformans, Aspergillus niger, Rhizopus oryzae, Bacillus subtilis, Candida albicans, etc. Has broad-spectrum antibacterial and antifungal effects.
  • Citral the main component of Litsea cubeba essential oil, can effectively scavenge DPPH free radicals, hydroxyl free radicals and superoxide free radicals, showing good antioxidant properties.
  • Litsea cubeba essential oil has been partially studied in the field of food preservation and preservation. It can significantly inhibit the activities of peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase in loquat and kumquat, and delay the aging of fruits; and VE , phytic acid prepared compound preservatives can extend the shelf life of shrimp and pork.
  • Litsea cubeba essential oil has significant in vitro activity and has been partially studied in the field of food preservation and preservation, due to its instability to light, heat, and oxygen during storage, the antioxidant activity decreases rapidly with time, and its own vulnerability Disadvantages such as volatility, low water solubility, and strong special flavor of Litsea cubeba essential oil cannot fully realize its value in practical applications.
  • Chinese invention patent publication (CN108464998A) discloses a highly stable peppermint essential oil emulsion.
  • the peppermint essential oil and resveratrol embedded in this method show a synergistic antibacterial effect, but this method uses the organic solvent anhydrous ethanol, and the preparation operation using the anti-solvent method is more complicated, and the encapsulation of peppermint essential oil and resveratrol is The burial rates are all low, only about 80%.
  • Chinese invention patent publication (CN113440479A) discloses a nanoemulsion of plant essential oils and its preparation method and application.
  • the nanoemulsion prepared by this method has high stability, low volatility, and has obvious inhibitory effect on Escherichia coli and Staphylococcus aureus.
  • the particle size of the emulsion prepared by this method is larger.
  • Cid110720A discloses a rosemary essential oil nanoliposome and its preparation method and application. This method overcomes the shortcomings of rosemary essential oil during use, has a better sustained-release effect, can increase the retention amount and retention time of the drug in the skin, better exerts the local effect of the drug, and has good applications. prospect. However, the emulsion prepared by this method has a low encapsulation rate of rosemary essential oil.
  • the primary purpose of the present invention is to provide a nanometer-based medium and long chain diglyceride-embedded Litsea cubeba essential oil with high efficiency, simple process, green safety, high stability and high encapsulation rate.
  • Methods for preparing structured lipid carriers includes an oil phase and a water phase.
  • the oil phase is composed of solid lipids (medium and long chain diglycerides) and liquid lipids (vegetable oil, litsea cubeba essential oil), and the water phase includes surfactants. and phosphate buffer.
  • Litsea cubeba essential oil acts as part of the liquid lipid in this system and has the dual functions of antioxidant and antibacterial and functional liquid oil. It can be better wrapped in the fat crystal lattice. Vegetable oil can reduce the order of the crystal lattice and reduce the In order to solve problems such as essential oil leakage caused by crystal form transformation, the solubility and stability of nano-lipid carriers in water are improved, and the sustained release of essential oils can be well achieved.
  • Another object of the present invention is to provide a nanostructured lipid carrier emulsion based on medium and long-chain diglyceride-embedded Litsea cubeba essential oil prepared by the above method.
  • the emulsion particle size is small and uniform, and the storage process stability is high. It also demonstrates high encapsulation efficiency and sustained release effect that traditional nanoemulsions and solid lipid nanoparticles do not have.
  • Another object of the present invention is to provide the application of the above-mentioned nanostructured lipid carrier emulsion based on medium and long chain diglyceride-embedded Litsea cubeba essential oil in the preservation of fruits and vegetables, especially in the preservation of blueberries.
  • the controllable release performance of the nanostructured lipid carrier obtained by the present invention as an encapsulating material can fully unleash the antiseptic potential of Litsea cubeba essential oil in fruit and vegetable preservation, extend the shelf life of fruits and vegetables, and increase the economic value.
  • a method for preparing a nanostructured lipid carrier based on medium- and long-chain diglyceride-embedded Litsea cubeba essential oil including the following steps:
  • Dissolve Litsea cubeba essential oil in vegetable oil then add the resulting mixed liquid oil to the melted medium and long chain diglycerides, heat and stir to obtain the oil phase; add the emulsifier to the phosphate buffer, heat and stir to obtain water phase; add the water phase to the oil phase and homogenize with high-speed shearing to obtain a coarse emulsion; immediately subject the coarse emulsion to ultrasonic treatment to obtain a dispersion;
  • the medium- and long-chain diglycerides in step (1) can be prepared by esterification of monoglycerides containing medium-chain fatty acids and long-chain fatty acids under the catalysis of lipase, or monoglycerides containing medium-chain fatty acids and long-chain fatty acids.
  • Triglycerides are prepared by transesterification under the catalysis of lipase; they can also be prepared by esterification of monoglycerides containing long-chain fatty acids and medium-chain fatty acids under the catalysis of lipase, or monoglycerides containing long-chain fatty acids and medium-chain fatty acids.
  • the triglyceride is prepared by transesterification under the catalysis of lipase; the melting point of medium and long chain diglyceride ranges from 40 to 70°C, wherein the medium chain fatty acid is selected from capric acid and lauric acid, and the long chain fatty acid is selected from meat.
  • the medium chain fatty acid is selected from capric acid and lauric acid
  • the long chain fatty acid is selected from meat.
  • myristic acid, palmitic acid, or stearic acid One of myristic acid, palmitic acid, or stearic acid.
  • the crude product is purified by molecular distillation or solvent method to obtain medium and long chain diglycerides with a purity of not less than 50%.
  • the selected distillation temperature for the molecular distillation method is 140-200°C.
  • Medium and long-chain diglycerides are produced by the esterification of monoglycerides and fatty acids (monoglycerides and long-chain fatty acids containing medium-chain fatty acids, or monoglycerides and medium-chain fatty acids containing long-chain fatty acids) under the catalysis of lipase
  • the lipase is at least one of Novozymes 435 immobilized lipase, LipozymeCALB lipase, and TLIM lipase
  • the molar ratio of monoglycerides and fatty acids is 1:3 to 3:1, and the amount of lipase is 3-5wt% of the total mass of monoglycerides and fatty acids
  • the temperature of the esterification reaction is 60-70°C
  • the reaction time is 30min-3h;
  • Triglycerides When medium and long-chain diglycerides are passed through monoglycerides and triglycerides (monoglycerides containing medium-chain fatty acids and triglycerides composed of long-chain fatty acids, or monoglycerides containing long-chain fatty acids and composed of medium-chain fatty acids) Triglycerides) are prepared by transesterification under the catalysis of lipase, wherein the lipase is at least one of Novozymes 435 immobilized lipase, LipozymeCALB lipase, and TLIM lipase; monoglycerides and triglycerides
  • the molar ratio is 1:3 ⁇ 3:1, the amount of lipase is 3-5wt% of the total mass of monoglycerides and triglycerides, the temperature of the transesterification reaction is 60-70°C, and the reaction time is 30min-3h .
  • the vegetable oil in step (1) is at least one of olive oil, soybean oil, corn oil, sunflower oil, tea seed oil, cottonseed oil, rice bran oil, peanut oil, and linseed oil.
  • Nanostructured lipid carriers as a new type of nanodelivery system, can protect loaded bioactive substances from environmental factors, improve water solubility, stability and bioavailability, and enhance controlled release and target tropism.
  • the lipid matrix of NLCs is composed of a mixture of solid lipids and liquid lipids, which can overcome the shortcomings of other colloidal carriers such as nanoemulsions and solid lipid nanoparticles.
  • bioactive substances are in solid
  • the fluidity in the matrix is weak, which can better achieve sustained release; compared with solid lipid nanoparticles (SLNs) with only a solid matrix, the presence of liquid oil slows down the formation of multiple lipid crystals of a single type.
  • the mass ratio of the oil phase and the water phase in step (1) is (5:95) ⁇ (20:80), and the amount of emulsifier is 2.0 ⁇ 8.0% of the water phase (that is, the amount of emulsifier added accounts for the amount of the oil phase and the water phase).
  • the amount of medium and long chain diglycerides is 1.0-5.0% of the total mass of the emulsion
  • the amount of vegetable oil is 1.0-5.0% of the total mass of the emulsion
  • the amount of litsea cubeba essential oil is 3.0-5.0% of the total mass of the emulsion. 10.0%.
  • the sum of the mass percentages of phosphate buffer, diglycerides, vegetable oil, Litsea cubeba essential oil and emulsifier is 100%.
  • step (1) the water phase and the oil phase are heated and the temperature is maintained at 55-75°C to ensure the melting of medium and long chain diglycerides while reducing the volatilization of Litsea Cubeba essential oil.
  • the high-speed shearing and homogenizing speed in step (1) is 8000-15000rpm
  • the homogenizing time is 2-5 minutes
  • the water bath temperature during the shearing and homogenizing process is 55-75°C.
  • the power of the ultrasonic probe in step (1) is 300-900W
  • the ultrasonic time is 4-10 minutes
  • the water bath temperature during the ultrasonic process is 55-75°C.
  • the ultrasonic-treated dispersion is placed in an ice-water bath and stirred to cool to room temperature at a stirring rate of 100 to 1000 rpm.
  • the invention discloses a method for preparing a nanostructured lipid carrier using medium and long-chain diglyceride as solid lipid to embed Litsea cubeba essential oil.
  • the diglyceride has both a hydrophilic group and a lipophilic group, which can better Reduce the interfacial tension of oil-water, and the nanostructured lipid carrier has high encapsulation rate and high stability, improves the solubility and stability of Litsea cubeba essential oil in water, slows down the volatilization rate of essential oil, and achieves long-lasting antibacterial and antioxidant effects. .
  • the nanostructured lipid carrier has a light white appearance, high encapsulation rate, good fluidity, and has the refreshing lemon scent of litsea cubeba essential oil.
  • the stability and biological activity of the essential oil can be protected during processing and storage, and ideal slow release can be achieved. This solves the problem that Litsea cubeba essential oil is difficult to use because it is oily, difficult to dissolve in water, highly volatile, and gives full play to its broad spectrum. Antibacterial and antioxidant properties.
  • the properties of lipid carriers embedding different bioactive compounds can be controlled by changing the preparation conditions such as the ratio of medium and long chain diglycerides/vegetable oil, the ratio of vegetable oils/essential oils, the type and content of emulsifiers in the nanostructured lipid carrier.
  • the particle size of the nanostructured lipid carrier based on medium- and long-chain diglyceride-embedded Litsea cubeba essential oil is 100-300 nm, and the encapsulation rate is 92-98%.
  • nanostructured lipid carrier based on medium- and long-chain diacylglycerol-embedded Litsea cubeba essential oil in fruit and vegetable preservation includes the following steps:
  • the present invention mainly has the following advantages and effects:
  • the nanostructured lipid carrier prepared by embedding Litsea cubeba essential oil with medium and long chain diacylglycerols prepared by the present invention solves the problem that Litsea cubeba essential oil is not easily soluble in water and is highly volatile and difficult to utilize, and achieves Efficient encapsulation and sustained release of Litsea cubeba essential oil, giving full play to its broad-spectrum antibacterial and antioxidant properties.
  • Litsea cubeba essential oil is encapsulated in an aqueous nanostructured lipid carrier and can be used to preserve fruits and vegetables through soaking and spraying.
  • the high stability of the nanolipid carrier also contributes to the application of fruits and vegetables in storage and cold chain transportation. Therefore, It has broad application prospects in the field of fruit and vegetable preservation and preservation.
  • the process of the present invention is simple, does not involve the application of organic reagents, the ultrasonic treatment operation is simple, environmentally friendly, and can be rapidly and continuously prepared.
  • the ratio of medium and long chain diglycerides/vegetable oil, the ratio of vegetable oil/essential oil, the type and content of emulsifiers in the lipid carrier can be used to produce emulsions embedding different bioactive compounds, which has good industrial application value.
  • the medium and long chain diglyceride used in the present invention has excellent molecular amphiphilicity, high melting point, good emulsification, and is not prone to crystalline conversion during storage.
  • the prepared nanostructured lipid carrier based on medium and long chain diacylglycerol-embedded Litsea cubeba essential oil has a light white appearance, good fluidity, moderate viscosity, high encapsulation rate and high stability, and can be used with water or phosphate buffer. Infinitely diluted and stable at low temperatures, it has outstanding advantages over traditional emulsions and solid lipid nanoparticles that are prone to crystalline transformation during storage and have low encapsulation rates.
  • the size of the carton tray of the present invention can be made according to the needs, and has strong flexibility. Compared with the traditional ordinary cartons treated with chemical preservatives, it is specially used for coated corrugated cartons, cardboard, etc. for food packaging. More excellent. By adding green and safe natural plant essential oils with broad-spectrum antibacterial effects into food packaging, it can not only alleviate the spoilage of fruits, but also reduce the residue of chemical preservatives, making packaging materials healthier and more environmentally friendly.
  • Figure 1 is a schematic flow diagram of the present invention.
  • Figure 2 is the particle size distribution diagram, microscope picture and real picture of the nanostructured lipid carrier based on medium and long chain diglyceride-embedded Litsea cubeba essential oil in Example 1.
  • Figure 3 shows the encapsulation rates of Example 1, Example 2, Example 3, Comparative Example 1 and Comparative Example 2.
  • Figure 4 is a diagram showing the effects of Example 1, Example 2, Example 3, Comparative Example 1 and Comparative Example 2 on the active oxygen scavenging ability.
  • Figure 5 shows the morphological changes of blueberries in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and the control group during storage.
  • Figure 6 shows the changes in blueberry rot rate in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and the control group.
  • Figure 7 shows the changes in weight loss rate of blueberries in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and the control group.
  • Figure 8 shows the changes in hardness of blueberries in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and the control group.
  • the reagents used in the examples can all be purchased from the market unless otherwise specified.
  • a method for preparing a nanostructured lipid carrier based on medium and long chain diacylglycerol-embedded Litsea cubeba essential oil is composed of a water phase and an oil phase containing Litsea cubeba essential oil.
  • the quality of the water phase The proportion is 90%, and the oil phase mass proportion is 10%; the water phase contains 5.4% Tween 80 and 84.6% 5mM phosphate buffer; the oil phase contains 2% medium and long chain diglycerides, 3% Olive oil and 5% litsea cubeba essential oil, the preparation steps are as follows:
  • (1) Preparation and purification of medium and long chain diglyceride raw materials Mix lauric acid and stearic acid monoglyceride at a molar ratio of 3:1, and catalyze the reaction under vacuum at 60°C for 30 minutes.
  • the catalyst is Novozymes 435 fixed lipase, the added amount is 5% of the total mass of the substrate, and the crude product after the reaction is purified by molecular distillation at 180°C to obtain the purified diglyceride, which is detected by gas chromatography.
  • the medium and long chain diglyceride is The ester ratio is greater than 60%.
  • step (2) Immediately cool the dispersion obtained in step (1) with a magnetic stirrer at 300 rpm and stir to obtain a nanostructured lipid carrier with medium and long chain diacylglycerol embedded Litsea cubeba essential oil.
  • the size of the antibacterial carton tray can be designed according to actual conditions; the preservation temperature of the refrigerator is 4 to 8°C.
  • a method for preparing a nanostructured lipid carrier based on medium- and long-chain diglycerides embedded with litsea cubeba essential oil The preparation and purification of medium- and long-chain diglycerides are the same as in Example 1.
  • the nanostructured lipid carrier is composed of a water phase and a The oil phase of Litsea cubeba essential oil is compounded. The water phase mass ratio is 90% and the oil phase mass ratio is 10%. The water phase is 7.2% Tween 20 and 82.8% 5mM phosphate buffer. ;
  • the oil phase is 2.5% medium and long chain diglyceride containing lauric acid and stearic acid, 2.5% soybean oil, and 5% Litsea cubeba essential oil.
  • the preparation steps are as in Example 1.
  • a method for preparing a nanostructured lipid carrier based on medium- and long-chain diglycerides embedded with litsea cubeba essential oil The preparation and purification of medium- and long-chain diglycerides are the same as in Example 1.
  • the nanostructured lipid carrier is composed of a water phase and a The oil phase of Litsea cubeba essential oil is compounded, the water phase mass proportion is 85%, and the oil phase mass proportion is 15%; the water phase is 7.2% Tween 20 and 77.8% 5mM phosphate buffer;
  • the oil phase includes 5% lauric acid, stearic acid medium and long chain diglyceride, 5% soybean oil and 5% Litsea cubeba essential oil.
  • the preparation steps are the same as in Example 1.
  • the particle size distribution and micromorphology of nanostructured lipid carriers were analyzed using dynamic light scattering analyzer and transmission electron microscope, respectively.
  • Determination of DPPH free radical scavenging rate Prepare sample methanol solutions with concentrations of 0.1 to 1.0 ⁇ L/mL, mix 500 ⁇ L with 3.0 mL, 0.06 mM DPPH methanol solution, and measure the absorbance at a wavelength of 517 nm. Use the methanol solution without sample Make a blank control. There are 3 parallel samples, and the free radical scavenging rate is calculated according to the following formula:
  • IC 50 is the concentration of nanostructured lipid carrier dispersion required to scavenge 50% of free radicals.
  • a sample is the absorbance value of the sample at 517nm;
  • a blank is the absorbance value of the blank control at 517nm.
  • Blueberry rot rate evaluation The rot rate is evaluated based on the degree of softening of the fruit, juice leakage caused by rot, and the degree of mold infection. It is divided into 3 levels according to the degree of corruption, namely level 0: no obvious changes on the surface; level 1: obvious softening; level 2: severe softening and juice leakage; level 3: mildew infection on the surface.
  • the decay rate is calculated according to the following formula:
  • Blueberry hardness measurement Use a texture analyzer with a 2mm probe aimed at the equatorial area of the blueberry for puncture measurement.
  • the puncture depth is 4mm
  • the speed is 1.5mm/s
  • the triggering force is 7.0g
  • each treatment is measured 15 times
  • the unit is g.
  • Example 4 Effects of the type and concentration of emulsifiers on the properties of nanostructured lipid carriers based on medium- and long-chain diglyceride-embedded Litsea cubeba essential oil.
  • 5mM phosphate buffer was used as the water phase
  • the oil phase composition was : Medium and long chain diglyceride (MLCD) containing lauric acid and stearic acid (the preparation and purification of the medium and long chain diglyceride are the same as in Example 1), and a mixed liquid oil with a mass ratio of olive oil and essential oil of 1:1 .
  • MLCD Medium and long chain diglyceride
  • lauric acid and stearic acid the preparation and purification of the medium and long chain diglyceride are the same as in Example 1
  • a mixed liquid oil with a mass ratio of olive oil and essential oil of 1:1 a mass ratio of olive oil and essential oil of 1:1 .
  • Tween 20 or Tween 80 emulsifiers
  • PDI dispersion index
  • Tween series emulsifiers can all assist in forming smaller-sized and uniformly dispersed nanostructured lipid carriers based on medium- and long-chain diglycerides. In the following examples, 5.4% Tween 80 is used for preparation.
  • Example 5 Effect of the ratio of olive oil to essential oil on the properties of nanostructured lipid carriers
  • 0.5mM phosphate buffer is used as the water phase
  • Tween 80 is used as the emulsifier
  • the components of the oil phase are: medium and long chain diacylglycerol (MLCD) (the preparation and purification of the medium and long chain diacylglycerol are the same as in the embodiment) 1), olive oil, litsea cubeba essential oil.
  • MLCD medium and long chain diacylglycerol
  • olive oil litsea cubeba essential oil.
  • the total content of essential oils and olive oil accounts for 5% of the total mass of the water phase and oil phase
  • the MLCD content accounts for 5% of the total mass of the water phase and oil phase
  • the added amount of Tween 80 accounts for the total mass of the water phase and oil phase. of 5.4%.
  • the difference in the average particle size of the emulsion is obvious when the ratio of olive oil to essential oil is different (Table 2).
  • the nanoemulsion has the smallest particle size and good dispersion.
  • Example 6 Effect of the ratio of medium and long chain diglycerides to olive oil on the properties of nanostructured lipid carriers based on medium and long chain diglycerides entrapped Litsea cubeba essential oil
  • MLCD medium and long chain diacylglycerol
  • the particle sizes of the emulsions formed at different ratios of diglyceride and olive oil are different (Table 3).
  • the mass ratio of medium and long chain diglycerides to olive oil is 2:3, the nanoemulsion formed has the smallest particle size and is evenly dispersed.
  • the formula of the nanoemulsion embedding Litsea cubeba essential oil is 5% olive oil, 5% Litsea cubeba essential oil, 5.4% Tween 80 and 84.6% phosphate buffer.
  • step (2) Immediately cool the dispersion obtained in step (1) with a magnetic stirrer at a rotation speed of 300 rpm and stir to cool down, to obtain solid lipid nanoparticles embedded with Litsea cubeba essential oil.
  • the formula of the solid lipid nanoparticles embedded with Litsea cubeba essential oil is 5% medium and long chain diglyceride, 5% Litsea cubeba essential oil, 5.4% Tween-80 and 84.6% phosphate buffer.
  • the NLC prepared in Example 1 has a light white appearance, uniform dispersion, good fluidity, and a fresh lemon fragrance.
  • the particle size is 181.21nm
  • the PDI is 0.153
  • DPPH clearance IC 50 is 0.76 ⁇ L/mL
  • the encapsulation rate dropped slightly to 93.84%, compared to Comparative Examples 1 and 2. Produces higher embedding rate, better stability and stronger free radical scavenging ability.
  • the NLC prepared in Example 1 has better antioxidant and antibacterial capabilities, and has the best preservation effect on blueberry fruits. .

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Abstract

一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法及其在果蔬保鲜中的应用。该制备方法包括以下步骤：将山苍子精油、植物油和熔化状态的中长链甘油二酯作为油相；随后将水相混入油相中，高速剪切均质，超声处理，冰浴中搅拌降温冷却，得到基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体。该包埋山苍子精油的纳米结构脂质载体具有粒径小、包封率高和稳定性好的优势，解决了山苍子精油作为油性不易溶于水和高挥发性较难利用的难题，实现了对山苍子精油的高效包埋和延缓释放，充分发挥其广谱抑菌性和抗氧化性，能够有效抑制蓝莓腐败，延长蓝莓的货架期，提高经济价值。

Description

一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备及其应用 技术领域
本发明属于食品领域，具体涉及一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备及其应用。
背景技术
山苍子精油(Litsea cubeba essential oil)是从山苍子鲜果中萃取得到的具有亲脂性和高挥发性的微黄色天然芳香化合物，富含柠檬醛、柠檬烯及芳樟醇等生物活性成分，具有显著的抗真菌、抗菌、抗虫及抗氧化等生物活性。山苍子精油能够破坏细菌的细胞膜，造成蛋白质变性，对大肠杆菌、金黄色葡萄球菌、新生隐球菌、黑曲霉、米根霉、枯草杆菌、白假丝酵母菌等均有明显的抑制作用，具有广谱抗细菌及抗真菌的作用。山苍子精油主成分柠檬醛能够有效清除DPPH自由基、羟自由基和超氧自由基，展示出较好的抗氧化性能。目前，山苍子精油在食品防腐保鲜领域已有部分研究，能显著抑制枇杷和金桔过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶的活性，延缓水果的衰老；与VE、植酸制备的复合保鲜剂均使虾肉和猪肉货架期延长。
虽然山苍子精油体外活性显著且在食品防腐保鲜领域已有部分研究，但由于其在贮藏过程中对光、热、氧的不稳定，随时间增加抗氧化活性下降较快，以及自身的易挥发、低水溶性、强烈的山苍子精油特殊风味等缺点，在实际应用中未能充分发挥其价值。
中国发明专利公开文本(CN108464998A)公开了一种具有高稳定性的薄荷精油乳状液。该方法包埋的薄荷精油和白藜芦醇显现出协同抑菌效果，但该法使用有机溶剂无水乙醇，且用反溶剂法制备操作较为复杂，且对薄荷精油、白藜芦醇的包埋率均较低，仅在80％左右。
中国发明专利公开文本(CN113440479A)公开了一种植物精油的纳米乳液及其制备方法和应用。该方法制备的纳米乳液稳定性高、挥发性低，对大肠杆菌和金黄色葡萄球菌具有明显抑制作用。但该方法制备的乳液粒径较大。
中国发明专利公开文本(CN111110720A)公开了一种迷迭香精油纳米脂质体及其制备方法与应用。此方法克服了迷迭香精油在使用过程中存在的缺陷，具有较好的缓释作用，能够增加药物在皮肤的滞留量和滞留时间，更好地发挥药物的局部作用，具有较好的应用前景。但该方法制得的乳液对迷迭香精油的包封率较低。
发明内容
为了克服上述现有技术的缺点，本发明的首要目的在于提供一种高效、工艺简单、绿色安全、高稳定性和高包封率的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法。本发明所提供纳米结构脂质载体包括油相和水相，油相由固体脂质(中长链甘油二酯)和液体脂质(植物油、山苍子精油)组成、水相包括表面活性剂和磷酸盐缓冲液。山苍子精油在该体系中充当部分的液体脂质具有抗氧化抑菌及功能性液油的双重作用，能够更好地包裹在脂肪晶体晶格中，植物油可减少晶格有序性，降低晶型转化导致的精油渗出等问题，纳米脂质载体在水中的溶解性和稳定性提升，并能很好地实现精油的缓释。
本发明的另一目的在于提供一种通过上述方法制备得到的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体乳液，乳液粒径小且均匀，贮藏过程稳定性高，并展示出传统纳米乳液和固体脂质纳米粒不具有的高包封率及缓释效果。
本发明的再一目的在于提供上述基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的乳液在果蔬保鲜中的应用，尤其是在蓝莓保鲜中的应用。本发明所得纳米结构脂质载体作为包封材料的可控释放性能可以充分发挥山苍子精油在果蔬保鲜中的防腐潜力，延长果蔬的货架期，提高经济价值。
本发明的目的通过下述技术方案实现：
一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，包括以下步骤：
(1)基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体制备：
将山苍子精油溶解在植物油中，然后将所得混合液油添加到熔化的中长链甘油二酯中，加热搅拌，获得油相；将乳化剂加入磷酸盐缓冲液中，加热搅拌，获得水相；将水相添加到油相中，高速剪切均质，得粗乳液；立即将粗乳液进行超声处理，得分散液；
(2)降温冷却：超声处理后，将分散液立即于冰浴中搅拌降温冷却，使脂质结晶，得到基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体，即包埋山苍子精油的乳液。
步骤(1)所述中长链甘油二酯可由含中链脂肪酸的单甘酯和长链脂肪酸在脂肪酶的催化下酯化制备，或含中链脂肪酸的单甘酯与含长链脂肪酸的甘油三酯在脂肪酶催化下酯交换制备；也可以通过含长链脂肪酸的单甘酯和中链脂肪酸在脂肪酶催化下酯化制备，或含长链脂肪酸的单甘酯与含中链脂肪酸的甘油三酯在脂肪酶催化下酯交换制备；中长链甘油二酯熔点范围为40～70℃，其中，中链脂肪酸选自癸酸、月桂酸中的一种，长链脂肪酸选自肉豆蔻酸、棕榈酸或硬脂酸中的一种。
通过分子蒸馏或溶剂法纯化粗产物，得到纯度不小于50％的中长链甘油二酯。所述的分子蒸馏法所选的蒸馏温度为140～200℃。
当中长链甘油二酯通过单甘酯和脂肪酸(含中链脂肪酸的单甘酯和长链脂肪酸，或者含长链脂肪酸的单甘酯和中链脂肪酸)在脂肪酶的催化作用下酯化制备时，其中脂肪酶为诺维信435固定化脂肪酶、LipozymeCALB脂肪酶、TLIM脂肪酶中的至少一种；单甘酯和脂肪酸的摩尔比为1:3～3:1，脂肪酶的用量为单甘油酯和脂肪酸的总质量的3-5wt％，酯化反应的温度为60-70℃，反应的时间为30min-3h；
当中长链甘油二酯通过单甘酯和甘油三酯(含中链脂肪酸的单甘酯和由长链脂肪酸组成的甘油三酯，或者含长链脂肪酸的单甘酯和由中链脂肪酸组成的甘油三酯)在脂肪酶的催化作用下酯交换制备时，其中脂肪酶为诺维信435固定化脂肪酶、LipozymeCALB脂肪酶、TLIM脂肪酶中的至少一种；单甘酯和甘油三酯的摩尔比为1:3～3:1，脂肪酶的用量为单甘酯和甘油三酯的总质量的3-5wt％，酯交换反应的温度为60-70℃，反应的时间为30min-3h。
优选的，步骤(1)所述植物油为橄榄油、大豆油、玉米油、葵花籽油、茶籽油、棉籽油、米糠油、花生油、亚麻籽油中的至少一种。
纳米结构脂质载体(nanostructured lipid carriers，NLCs)作为一种新型纳米递送体系，可以保护负载的生物活性物质免受环境因素的影响，提高水溶性、稳定性和生物利用度，增强控释和靶向性。NLCs的脂质基质由固体脂质和液体脂质混合而成，可克服其他胶体载体如纳米乳液、固体脂质纳米颗粒的不足，与纳米乳液(nanoemulsion,NE)相比，生物活性物质在固体基质中的流动性弱，可更好地实现缓释；而与仅有固体基质的固体脂质纳米粒(solid lipid nanoparticles，SLNs)相比，液体油的存在减缓了单一类型脂质晶体的多态性转变，并提供更多的无序晶格容纳生物活性物质，增加了包封率；其他包埋体系例如聚合物纳米颗粒通常用到有机溶剂；脂质体则制备过程复杂；微乳则存在包封率低、稳定性差等缺陷。
优选的，步骤(1)所述乳化剂为吐温80、吐温60和吐温20的一种；所述磷酸盐缓冲液(PBS)中磷酸根离子浓度为0.5-10mM、pH＝6～8。
优选的，步骤(1)中油相和水相的质量比为(5：95)～(20：80)，乳化剂的量为水相的2.0～8.0％(即乳化剂添加量占油相和水相总质量的1.6～7.6％)，中长链甘油二酯的量为乳液总质量的1.0～5.0％，植物油为乳液总质量的1.0～5.0％，山苍子精油为总质量的3.0～10.0％。磷酸盐缓冲液、甘油二酯、植物油、山苍子精油和乳化剂的质量百分比之和为100％。
优选的，步骤(1)中水相和油相加热，温度保持在55～75℃，保证中长链甘油二酯熔化的同时减少山苍子精油的挥发。
优选的，步骤(1)中所述高速剪切均质速度为8000～15000rpm，均质时间为2～5分钟，剪切均质过程中水浴温度为55～75℃。
优选的，步骤(1)中所述超声探头功率为300～900W，超声时间为4～10分钟，超声过程水浴温度为55～75℃。
优选的，步骤(2)中，将超声处理后的分散液置于冰水浴中搅拌冷却至室温，搅拌速率100～1000rpm。
本发明公开了一种以中长链甘油二酯作为固体脂质包埋山苍子精油的纳米结构脂质载体制备方法，甘油二酯同时拥有亲水基团和亲脂基团，能够更好的降低油-水的界面张力，纳米结构脂质载体具有高包封率和高稳定性，提高山苍子精油在水中的溶解性和稳定性，减缓精油挥发速度，达到持久抑菌抗氧化的效果。
一种由上述方法制备得到的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体。纳米结构脂质载体具有浅白色外观，高包封率、流动性好，有山苍子精油的柠檬清香味。加工和存储期间可保护精油的稳定性和生物活性，并能够实现理想的缓慢释放，解决了山苍子精油作为油性不易溶于水和高挥发性、较难利用的难题，充分发挥其广谱抑菌性和抗氧化能力。可通过改变纳米结构脂质载体中的中长链甘油二酯/植物油比例、植物油/精油比例、乳化剂种类和含量等制备条件调控包埋不同生物活性化合物的脂质载体性质。
所述的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的粒径为100-300nm，包封率为92-98％。
一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体在果蔬保鲜中的应用保鲜，包括以下步骤：
(1)制备抑菌纸盒：将包埋山苍子精油的乳液用喷涂方式，或涂布棒均匀涂布在瓦楞纸或白色卡纸上，自然风干或置于电热恒温鼓风干燥箱中干燥，之后折叠成抑菌纸盒；
(2)将果蔬放置于抑菌纸盒中，置于冰箱中贮藏。
相比于现有技术，本发明主要具有以下优点和效果：
(1)本发明所制备的中长链甘油二酯包埋山苍子精油制备的纳米结构脂质载体，解决了山苍子精油不易溶于水和高挥发性较难利用的难题，实现了对山苍子精油的高效包埋和缓释，充分发挥其广谱抑菌性和抗氧化性。将山苍子精油包裹在水性纳米结构脂质载体中，可以通过浸泡、喷涂的方式用于果蔬保鲜，纳米脂质载体的高稳定性也有助于果蔬在储存和冷链运输中的应用，因此在果蔬保鲜防腐领域具有广阔的应用前景。
(2)本发明工艺简单，不涉及有机试剂的应用，超声波处理操作简单，环境友好，且能够进行快速连续化制备，通过简单调控基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体中的中长链甘油二酯/植物油比例、植物油/精油比例、乳化剂种类和含量等即可制得包埋不同生物活性化合物的乳液，具有较好的工业化应用价值。
(3)本发明所用的中长链甘油二酯具有优异的分子两亲性，熔点高，乳化性好，储存过程不易发生晶型转换。制得的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体具有浅白色外观，流动性好，粘度适中，具有高包封率和高稳定性，可以用水或磷酸缓冲液无限稀释，且低温稳定，与传统乳液和容易在储存过程中发生晶型转变且包封率较低的固体脂质纳米粒相比具有突出优势。
(4)本发明纸盒托盘大小可以根据需求打板制作，具有较强的灵活性，与传统的化学防腐剂处理的普通纸盒相比，专门用于食品包装的涂布瓦楞纸盒、纸板等更具有优异性。通过在食品包装中加入绿色安全的具有广谱抑菌作用的天然植物精油，不但可以用于缓解水果的腐败，还可减少化学防腐剂的残留，使得包装材料更加健康和环保。
附图说明
图1是本发明的流程示意图。
图2为实施例1基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的粒径分布图、显微镜图和实图。
图3为实施例1、实施例2、实施例3、对比例1和对比例2的包封率。
图4为实施例1、实施例2、实施例3、对比例1和对比例2对活性氧清除能力的效果图。
图5为实施例1、实施例2、实施例3、对比例1、对比例2和对照组蓝莓贮藏期间形貌变化。
图6为实施例1、实施例2、实施例3、对比例1、对比例2和对照组蓝莓腐烂率的变化。
图7为实施例1、实施例2、实施例3、对比例1、对比例2和对照组蓝莓失重率的变化。
图8为为实施例1、实施例2、实施例3、对比例1、对比例2和对照组蓝莓硬度的变化。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例中所用试剂如无特殊说明均可从市场常规购得。
实施例1：
一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，所述乳液由水相和包含山苍子精油的油相复配而成，所述水相质量占比为90％，油相质量占比为10％；其中水相包含5.4％的吐温80和84.6％的5mM磷酸盐缓冲液；油相包含2％的中长链甘油二酯、3％的橄榄油、5％的山苍子精油，制备步骤如下：
(1)中长链甘油二酯原料制备与纯化：将月桂酸与硬脂酸单甘酯按照摩尔比3:1混合，在60℃条件下抽真空催化反应30min，催化剂为诺维信435固定化脂肪酶，添加量为底物总质量的5％，将反应后的粗产物用分子蒸馏在180℃条件下纯化，得到纯化后的甘油二酯，采用气相色谱检测，其中中长链甘油二酯比例大于60％。
(2)将上述含有月桂酸、硬脂酸的中长链甘油二酯70℃加热搅拌使其完全融化，将山苍子精油溶解在橄榄油中，并将混合物添加到熔化的中长链甘油二酯中，获得油相；将吐温80添加到5mM磷酸盐缓冲液中70℃预热，搅拌均匀，获得水相；将水相添加到油相中，混合液在12000rpm下高速剪切均质，水浴温度为70℃，制得粗乳液；再在540W功率下超声6分钟，得到包埋精油的甘油二酯分散液；
(2)将步骤(1)所得的分散液立即在300rpm转速下用磁力搅拌器并与搅拌降温冷却，得到中长链甘油二酯包埋山苍子精油的纳米结构脂质载体。
使用上述制备方法得到的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体在抑菌纸盒中的应用，保鲜蓝莓步骤如下：
(1)制备抑菌纸盒：将包埋山苍子精油的乳液以80g/m 2均匀涂布在瓦楞纸上，自然风干，折叠成抑菌纸盒。
(2)将蓝莓放置于抑菌纸盒中，置于冰箱中贮藏。
作为优选，所述抑菌纸盒托盘可根据实际情况设计大小；冰箱的保鲜温度为4～8℃。
实施例2：
一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，中长链甘油二酯的制备和纯化同实施例1，纳米结构脂质载体由水相和包含山苍子精油的油相复配而成，所述水相质量占比为90％，油相质量占比为10％；其中水相为7.2％的吐温20和82.8％5mM磷酸盐缓冲液；油相为2.5％含有月桂酸、硬脂酸的中长链甘油二酯、2.5％大豆油、5％山苍子精油，制备步骤如实施例1。
实施例3：
一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，中长链甘油二酯的制备和纯化同实施例1，纳米结构脂质载体由水相和包含山苍子精油的油相复配而成，所述水相质量占比为85％，油相质量占比为15％；其中水相为7.2％吐温20和77.8％5mM磷酸盐缓冲液；油相包括5％月桂酸、硬脂酸中长链甘油二酯、5％大豆油和5％山苍子精油，制备步骤同实施例1。
试验方法：
纳米结构脂质载体的粒径分布和微观形态分别采用动态光散射分析仪和透射电子显微镜进行分析。
包封率测量：将乳液与50％乙醇按照体积比1：7比例均匀混合，于超滤管中在4000rpm下离心5分钟，使游离精油从超滤膜中分离出来，收集滤液，用50％乙醇适当稀释。向1mL乳液中添加1mL氯仿并充分摇晃，以破坏载体并释放封装的精油，在10000下离心10分钟，取含有游离和包埋的精油的氯仿层，用50％乙醇适当稀释，使用紫外-可见分光光度计在λ max＝238nm处测量。使用浓度范围0.00001～0.00005mg/mL的山苍子精油绘制 标准曲线y＝0.00002881674x-0.00000056298，计算精油含量，其中相关系数R 2＝0.987，y为山苍子精油的浓度(mg/mL)，x为238nm下溶液的吸光度(A)值，包封率使用下列公式计算：
Figure PCTCN2022106735-appb-000001
DPPH自由基清除率测定：配制浓度分别为0.1～1.0μL/mL样品甲醇液，分别取500μL与3.0mL，0.06mM的DPPH甲醇液混合，于波长517nm处测定吸光度，以不加样品的甲醇液作空白对照。平行样为3个，自由基清除率按照下式进行：
Figure PCTCN2022106735-appb-000002
作样品浓度-清除率的线性关系图，计算IC 50，即清除50％自由基所需纳米结构脂质载体分散液的浓度。
其中A 样品为样品在517nm的吸光值；A 空白为空白对照在517nm的吸光值。
蓝莓腐烂率评价：腐烂率根据果实的软化程度、腐烂导致的果汁渗漏以及霉菌感染程度评估。根据腐败情况分为3个等级，分别为0级：表面无明显变化；1级：出现明显软化现象；2级：软化严重，出现果汁渗漏现象；3级：表面出现霉斑感染。
腐烂率按下式计算：
Figure PCTCN2022106735-appb-000003
蓝莓失重率评价：
Figure PCTCN2022106735-appb-000004
蓝莓硬度测定：采用质构仪，2mm探头对准蓝莓赤道区进行穿刺测定。穿刺深度为4mm，速度均为1.5mm/s，触发力为7.0g，每个处理测定15次，单位为g。
实施例4：乳化剂的种类和浓度对基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体性质的影响本实施例中，水相选用5mM磷酸缓冲液，油相成分为：含有月桂酸、硬脂酸的中长链甘油二酯(MLCD)(该中长链甘油二酯的制备和纯化同实施例1)，以及橄榄油与精油质量比1:1的混合液油。不同浓度的乳化剂(吐温20或吐温80)对纳米乳液的粒径、电位和分散性指数(PDI)的影响效果采用动态光散射分析仪进行测量，具体参数见表1所示，其中，精油与橄榄油总含量共占水相和油相总质量的5％，MLCD含量共占水相和油相总质量的5％，吐温80添加量为占水相和油相的总质量的比值。
表1.不同乳化剂种类及添加量时基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体性质
Figure PCTCN2022106735-appb-000005
不同乳化剂种类和浓度可形成不同粒径的乳液(表1)。吐温系列乳化剂均能辅助形成较小尺寸和均匀分散的基于中长链甘油二酯的纳米结构脂质载体，在接下来的实施例均采用5.4％的吐温80配制。
实施例5：橄榄油与精油比例对纳米结构脂质载体性质的影响
本实施例中，水相选用0.5mM磷酸缓冲液、乳化剂选用吐温80，油相成分有：中长链甘油二酯(MLCD)(该中长链甘油二酯的制备和纯化同实施例1)、橄榄油、山苍子精油。选用不同橄榄油与精油比例对纳米乳液的粒径、电位和PDI的影响，具体参见表2所示。其中，精油与橄榄油总含量共占水相和油相总质量的5％，MLCD含量共占水相和油相总质量的5％，吐温80添加量共占水相和油相总质量的5.4％。
表2.橄榄油与精油比例对纳米结构脂质载体性质的影响
Figure PCTCN2022106735-appb-000006
不同橄榄油与精油比例时乳液平均粒径差异较明显(表2)。当橄榄油与精油质量比为1：1时，纳米乳液具有最小的粒径且分散性好。
实施例6：中长链甘油二酯与橄榄油比例对基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体性质的影响
本实施例中，水相选用5mM磷酸盐缓冲液、乳化剂选用吐温80，油相成分有：含月桂酸、硬脂酸的中长链甘油二酯(MLCD)(该中长链甘油二酯的制备和纯化同实施例1)、橄榄油、山苍子精油。分析不同中长链甘油二酯与橄榄油比例对纳米乳液的粒径、电位和PDI的影响，具体参见表2所示，其中，MLCD与橄榄油总含量共占水相和油相的总质量的5％，精油和吐温80添加量均指其质量占水相和油相总质量的比值。
表3.中长链甘油二酯与橄榄油的比例对基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体性质的影响
Figure PCTCN2022106735-appb-000007
不同甘油二酯与橄榄油比例时形成的乳液粒径不同(表3)。中长链甘油二酯与橄榄油质量比为2:3时，形成的纳米乳液具有最小的粒径且分散均匀。
对比例1
(1)将山苍子精油溶解在橄榄油中搅拌，橄榄油与山苍子精油质量比为1：1，获得油相；将吐温80添加到5mM磷酸盐缓冲液中搅拌均匀，获得水相；将水相添加到油相中，混合液在15000rpm下高速剪切均质，再在540W功率下超声6分钟，得到包埋精油的传统纳米乳液。
所述包埋山苍子精油的纳米乳液的配方为5％橄榄油、5％山苍子精油、5.4％吐温80和84.6％磷酸盐缓冲液。
对比例2
(1)将含有月桂酸、硬脂酸的中长链甘油二酯(该中长链甘油二酯的制备和纯化同实施例1)60℃加 热搅拌使其完全融化，将山苍子精油添加到中长链甘油二酯中(质量比为1：1)，60℃加热搅拌，获得油相；将吐温80添加到5mM磷酸盐缓冲液中于60℃预热，搅拌均匀，获得水相；将水相添加到油相中，混合液在15000rpm下高速剪切均质，得粗乳液；再在540W功率下超声6分钟，得到包埋精油的甘油二酯分散液；
(2)将步骤(1)所得的分散液立即在300rpm转速下用磁力搅拌器并与搅拌降温冷却，得到包埋山苍子精油的固体脂质纳米粒。
所述包埋山苍子精油的固体脂质纳米粒的配方为5％中长链甘油二酯、5％山苍子精油、5.4％吐温-80和84.6％磷酸盐缓冲液。
乳液性质及蓝莓贮藏过程性质测定结果如表4和5所示：
表4乳液性质
Figure PCTCN2022106735-appb-000008
分析：从上表4可以看出，实施例1中制得的NLC具有浅白色外观、均匀分散、流动性好，具有清新的柠檬香味，粒径为181.21nm，PDI为0.153，包封率为97.69％，DPPH清除率IC 50为0.76μL/mL，且在4℃下储存60天后粒径和PDI均无显著变化，包封率略有下降至93.84％，相比于对比例1、2展示出更高的包埋率、更好的稳定性和更强的自由基清除能力。
表5蓝莓贮藏过程性质测定结果
Figure PCTCN2022106735-appb-000009
分析：从上表5可以看出，所有实施例和对比例相比于未经过任何处理的对照组对蓝莓的腐败均有一定的延缓效果，实施例1处理下的蓝莓在第1天、4天、8天和12天的腐烂率分别为0％、0.83％、3.33％和5.83％，失重率为0.29％、2.12％、6.05％和10.69％，硬度为279g、267g、255g和241g。在贮藏4天时，蓝莓无明显变化，8天时小部分蓝莓果实开始出现软化现象。
在贮藏12天后，未处理的蓝莓腐烂率是NLC处理组的2.79倍，失重百分比为1.49倍，且NLC保鲜的蓝莓果实软化程度最小。相比于实施例2、3和对比例1纳米乳液、对比例2固体脂质纳米粒，实施例1制得的NLC具有更好的抗氧化和抑菌能力，对蓝莓果实的保鲜效果最佳。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，其特征在于，包括以下步骤：
    (1)基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体制备：
    将山苍子精油溶解在植物油中，然后将所得混合液油添加到熔化的中长链甘油二酯中，加热搅拌，获得油相；将乳化剂加入磷酸盐缓冲液中，加热搅拌，获得水相；将水相添加到油相中，高速剪切均质，得粗乳液；立即将粗乳液进行超声处理，得分散液；
    (2)降温冷却：超声处理后，将分散液立即于冰浴中搅拌降温冷却，使脂质结晶，得到基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体，即包埋山苍子精油的乳液。
  2. 根据权利要求1所述的一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，其特征在于，步骤(1)所述中长链甘油二酯完全水解后对应的两种脂肪酸为中链脂肪酸和长链脂肪酸，其中，中链脂肪酸选自癸酸、月桂酸中的一种，长链脂肪酸选自肉豆蔻酸、棕榈酸或硬脂酸中的一种；步骤(1)所述中长链甘油二酯熔点范围为40～70℃，纯度不低于50％；
    优选的，步骤(1)所述中长链甘油二酯由含中链脂肪酸的单甘酯和长链脂肪酸在脂肪酶的催化下酯化制备，或含中链脂肪酸的单甘酯与含长链脂肪酸的甘油三酯在脂肪酶催化下酯交换制备；或通过含长链脂肪酸的单甘酯和中链脂肪酸在脂肪酶催化下酯化制备，或含长链脂肪酸的单甘酯与含中链脂肪酸的甘油三酯在脂肪酶催化下酯交换制备；其中，中链脂肪酸选自癸酸、月桂酸中的一种，长链脂肪酸选自肉豆蔻酸、棕榈酸或硬脂酸中的一种。
  3. 根据权利要求2所述的一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，其特征在于，当中长链甘油二酯通过单甘酯和脂肪酸在脂肪酶的催化作用下酯化制备时，其中单甘酯和脂肪酸指含中链脂肪酸的单甘酯和长链脂肪酸，或者含长链脂肪酸的单甘酯和中链脂肪酸；所述的脂肪酶为诺维信435固定化脂肪酶、LipozymeCALB脂肪酶、TLIM脂肪酶中的至少一种；单甘酯和脂肪酸的摩尔比为1:3～3:1，脂肪酶的用量为单甘油酯和脂肪酸的总质量的3-5wt％，酯化反应的温度为60-70℃，反应的时间为30min-3h；
    当中长链甘油二酯通过单甘酯和甘油三酯在脂肪酶的催化作用下酯交换制备时，其中单甘酯和甘油三酯指含中链脂肪酸的单甘酯和由长链脂肪酸组成的甘油三酯，或者含长链脂肪酸的单甘酯和由中链脂肪酸组成的甘油三酯；所述的脂肪酶为诺维信435固定化脂肪酶、LipozymeCALB脂肪酶、TLIM脂肪酶中的至少一种；单甘酯和甘油三酯的摩尔比为1:3～3:1，脂肪酶的用量为单甘酯和甘油三酯的总质量的3-5wt％，酯交换反应的温度为60-70℃，反应的时间为30min-3h。
  4. 根据权利要求1所述的一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，其特征在于，步骤(1)中所述植物油为橄榄油、大豆油、玉米油、葵花籽油、茶籽油、棉籽油、米糠油、花生油、亚麻籽油中的至少一种；
    步骤(1)中所述乳化剂为吐温80、吐温20和吐温60的一种；所述磷酸盐缓冲液浓度为0.5-10mM、pH＝6～8。
  5. 根据权利要求1所述的一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，其特征在于，
    步骤(1)中油相和水相的质量比为(5：95)～(20：80)，乳化剂的量为水相的2.0～8.0％，中长链甘油二酯的量为乳液总质量的1.0～5.0％，植物油为乳液总质量的1.0～5.0％，山苍子精油为总质量的3.0～10.0％，水相、甘油二酯、植物油、山苍子精油和乳化剂的质量百分比之和为100％。
  6. 根据权利要求1所述的一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备方法，其特征在于，步骤(1)中水相和油相加热温度保持在55～75℃，保证中长链甘油二酯熔化的同时减少山苍子精油的挥发；
    步骤(1)中所述高速剪切均质速度为8000～15000rpm，均质时间为2～5分钟，剪切均质过程中水浴温度为55～75℃；
    步骤(1)中所述超声探头功率为300～900W，超声时间为4～10分钟，超声过程水浴温度为55～75℃；将 超声处理后的分散液置于冰水浴中搅拌冷却至室温，搅拌速率100～1000rpm。
  7. 一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体，其特征在于，其由权利要求1-6任一项所述方法制得。
  8. 根据权利要求7所述的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体，其特征在于，所述的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的粒径为100-300nm，包封率为92-98％。
  9. 根据权利要求7或8所述的基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体在果蔬保鲜中的应用，尤其是在蓝莓保鲜中的应用。
  10. 根据权利要求9所述的一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的应用，其特征在于包括以下步骤：
    (1)制备抑菌纸盒：将包埋山苍子精油的乳液用喷涂方式，或涂布棒均匀涂布在瓦楞纸或白色卡纸上，自然风干或置于电热恒温鼓风干燥箱中干燥，之后折叠成抑菌纸盒；
    (2)将果蔬放置于抑菌纸盒中，置于冰箱中贮藏。
PCT/CN2022/106735 2022-04-24 2022-07-20 一种基于中长链甘油二酯包埋山苍子精油的纳米结构脂质载体的制备及其应用 Ceased WO2023206830A1 (zh)

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