MLCT-based oil body nanostructured lipid carriers, preparation method and application thereof

Abstract

This invention discloses an oil-based nanostructured lipid carrier based on MLCT, its preparation method, and its application. The method includes mixing long-chain oil with decanoic acid triglyceride, adding immobilized lipase, vacuum drying, stirring, filtering, deacidification, and vacuum evaporation to obtain MLCT-structured lipids. Glyceryl monostearate is mixed with the MLCT-structured lipids and heated to form the oil phase. Trichosanthes kirilowii seed oil is mixed with ultrapure water and heated to form the aqueous phase. The aqueous and oil phases are then mixed, heated, ultrasonically broken up, and subjected to an ice bath to obtain the oil-based nanostructured lipid carrier. This invention replaces traditional small-molecule emulsifiers with natural oil bodies (OB) as emulsifiers, meeting consumer health needs while exhibiting excellent stability and safety; it also reduces the risk of chemically synthesized residues. Furthermore, the introduction of MLCT revolutionizes the digestibility of NLCs at the metabolic mechanism level. In vitro simulated digestion experiments show that the free fatty acid release rate is higher than 80%, providing a new solution for the development of functional food and drug delivery systems.

Description

Technical Field

[0001] This invention belongs to the field of functional food technology, specifically relating to an oil-based nanostructured lipid carrier based on MLCT, its preparation method, and its application. Background Technology

[0002] In the fields of drug delivery and functional foods, nanoemulsion systems have become highly valuable carrier systems due to their simple preparation process, good potential for large-scale production, and efficient encapsulation of active substances. However, their thermodynamic instability, leading to problems such as particle size growth, aggregation, and phase separation, severely limits their practical applications. To address this challenge, solid lipid nanoparticles (SLNs) have emerged. This system successfully combines the controlled-release properties of solid lipids with the advantages of nanoemulsions by using solid lipids instead of liquid lipids and utilizing a crystalline lattice to encapsulate nutrients. However, SLNs face the challenge of polymorphic transformation during storage, where fatty acid side chains spontaneously transform from thermodynamically unstable α-type crystals to β-type crystals. Due to their highly ordered structure and larger grain size, β-type crystals cause the expulsion of lipid-soluble active substances from the lattice, significantly reducing drug loading. Oil bodies (OBs) are organelles within plant cells that store lipids. They consist of a triacylglycerol core encased in a membrane structure composed of a single layer of phospholipids and oleoproteins. This structure endows them with excellent environmental tolerance, maintaining structural stability under conditions of temperature changes, pH fluctuations, and mechanical stress. The phospholipid membrane and oleoproteins of oil bodies possess excellent emulsifying activity; using them as emulsifiers in non-dairy lichens (NLCs) can significantly reduce dependence on synthetic emulsifiers, promoting the greening of NLC systems. However, the dense membrane structure of oil bodies, while providing emulsifying and protective properties, can also prevent the digestion of the encapsulated lipid components, potentially leading to a decline in the nutritional performance of NLCs.

[0003] Nanostructured lipid carriers (NLCs), as an improved product of SLNs, effectively enhance drug loading and storage stability by introducing liquid lipids to regulate the crystallization behavior of solid lipids and reduce crystallinity. However, traditional NLC preparation processes rely on petroleum-based synthetic surfactants to overcome the high interfacial energy at the oil-water interface, which not only increases production costs but also poses a problem of poor environmental friendliness. With the surge in consumer demand for green and healthy products, the development of NLC systems based on natural biosurfactants has become a research hotspot. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0006] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an oil-based nanostructured lipid carrier based on MLCT, its preparation method, and its application.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing an oil-based nanostructured lipid carrier based on MLCT, comprising, Long-chain oil was mixed with capric acid triglyceride, then immobilized lipase was added, vacuum dried, stirred and filtered, deacidified, and vacuum evaporated to obtain MLCT structured lipid. Glyceryl monostearate was mixed with MLCT structured lipid and heated to form the oil phase, while Trichosanthes kirilowii seed oil was mixed with ultrapure water and heated to form the aqueous phase. The aqueous and oil phases were then mixed, heated, ultrasonically broken up, and subjected to an ice bath to obtain the oil-based nanostructured lipid carrier.

[0008] As a preferred embodiment of the preparation method described in this invention, the long-chain oil includes high-oleic rapeseed oil and camellia oil.

[0009] In a preferred embodiment of the preparation method described in this invention, the mass ratio of the long-chain oil to decanoic acid triglyceride is 6:4.

[0010] In a preferred embodiment of the preparation method described in this invention, the mass ratio of the monostearate glycerol to the MLCT structured lipid is 1:3~19.

[0011] In a preferred embodiment of the preparation method described in this invention, the mass fraction of Trichosanthes kirilowii seed oil in the aqueous phase is 2.5-5.5%.

[0012] As a preferred embodiment of the preparation method described in this invention, the method for preparing the Trichosanthes seed oil includes mixing the Trichosanthes seed oil with a sodium chloride solution, pulping, filtering, centrifuging to obtain an upper emulsion, adding a sucrose solution to disperse and adjust the pH, performing a second centrifugation, taking the upper emulsion, dispersing and washing, and then centrifuging again.

[0013] In a preferred embodiment of the preparation method described in this invention, the mass ratio of the oil phase to the water phase is 2.5~7.5:92.5~97.5.

[0014] In a preferred embodiment of the preparation method described in this invention, the pH is adjusted to 11.

[0015] Another objective of this invention is to overcome the shortcomings of the prior art and provide an oil-based nanostructured lipid carrier prepared by a specific method.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of an oil-based nanostructured lipid carrier in biosurfactants.

[0017] Beneficial effects of this invention: This invention innovatively replaces traditional small-molecule emulsifiers with natural oil bodies (OB) as emulsifiers, meeting consumer health needs while exhibiting superior stability and safety, and reducing the risk of chemically synthesized residues. Secondly, the introduction of MLCT revolutionizes the digestibility of NLCs at the metabolic mechanism level. In vitro simulated digestion experiments show that the free fatty acid release rate is higher than 80%, providing an innovative solution for the development of functional food and drug delivery systems. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is the FFA release curve of the NLC in this embodiment of the invention.

[0019] Figure 2 This invention illustrates the effect of oil concentration on NLC particle size in an embodiment of the invention.

[0020] Figure 3 This is a transmission electron microscope image of NLC from an embodiment of the present invention.

[0021] Figure 4 The TGA and DTG curves of the NLC in this embodiment of the invention are shown.

[0022] Figure 5 The storage stability results of NLC in the embodiments of the present invention are shown. Detailed Implementation

[0023] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0024] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0025] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0026] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available. See Table 1 for details.

[0027] Table 1

[0028] Example 1 This embodiment provides a method for preparing an oil-based nanostructured lipid carrier using MLCT, specifically: (1) Remove the shells from the seeds of Trichosanthes kirilowii to obtain the kernels. Soak 100g of the kernels in pure water at 4 ℃ for 24h. Then, take out 100g of the kernels, drain the water, add 7 times the mass of 0.1 mol / L NaCl solution, mix and slurry. After slurrying, filter three times to remove the residue. Then, centrifuge the filtrate at 10000 g for 20 min, take the upper emulsion, disperse it with 4 times the mass of 25% sucrose aqueous solution, adjust the pH value to 11, and then centrifuge again under the same conditions to obtain the crude oil body. Then disperse and wash it with 4 times the mass of ultrapure water, and centrifuge again under the same conditions. The upper emulsion obtained by centrifugation is the Trichosanthes kirilowii seed oil body.

[0029] (2) After weighing the oil sample with a mass ratio of high oleic rapeseed oil (long-chain oil) to decanoic acid triglyceride (medium-chain oil, MCT) of 6:4, the mixture was magnetically stirred to ensure thorough mixing. Immobilized lipase Lipozyme TLIM was then added at a concentration of 9%. Under vacuum drying conditions, the mixture was magnetically stirred at 65 °C for 6 h at a stirring speed of 200 r / min. After the reaction was completed, the lipase was removed by centrifugation at 6000 r / min for 10 min, and the mixture was filtered to obtain the crude MLCT product. Next, the centrifuged immobilized lipase Lipozyme TLIM was washed sequentially with n-hexane and anhydrous ethanol, air-dried at room temperature, and stored under refrigeration. Finally, the acid value of the crude MLCT product was determined according to the national standard GB5009.229-2016 to calculate the amount of alkali to be added. The crude MLCT structured lipid product was mixed with n-hexane and placed in a three-necked flask equipped with a reflux condenser. The mixture was heated and stirred at 60°C. After stabilization, the alkali solution calculated based on the acid value was added for alkali refining and deacidification. After deacidification, the soap was removed, the solvent was removed by vacuum rotary evaporation, and the product was centrifuged again to obtain the MLCT structured lipid. Finally, the acid value was determined to determine whether the MLCT had been completely deacidified.

[0030] (3) Solid lipid GMS and MLCT structured lipid at room temperature were mixed at a ratio of 1:3 and heated to 75°C to obtain the oil phase. Trichosanthes seed oil was mixed with ultrapure water and heated to 75°C to obtain the aqueous phase. The amount of Trichosanthes seed oil added was 4.5% (w / w). The oil phase and aqueous phase were mixed at a ratio of 5:95 and homogenized at 8000 r / min for 3 min. Then, ultrasonic cell disruptor was used to sonicate at 200 W power for 20 min, with 2 s on and 2 s off. The dispersion and ultrasonic process were kept at 75°C throughout. After ultrasonication, the mixture was placed in an ice water bath for 30 min to allow crystal formation, thus obtaining the oil nanostructured lipid carrier (NLC). After storage at 4°C for 24 h, subsequent measurements were performed.

[0031] Example 2 The difference from Example 1 is that the high-oleic rapeseed oil in step (2) is replaced with camellia oil. The remaining steps and processes are all the same as in Example 1, and the MLCT structured lipid synthesized in this example using camellia oil as raw material is obtained.

[0032] Comparative Example 1 The difference from Example 1 is that in step (3), the MLCT structure lipid is replaced with high oleic rapeseed oil.

[0033] Example 3 The difference from Example 1 is that the amount of Trichosanthes kirilowii seed oil added in step (3) is 2.5%, and the remaining steps are all the same as in Example 1, so as to obtain the MLCT structured lipid of this example.

[0034] Example 4 The difference from Example 1 is that the amount of Trichosanthes kirilowii seed oil added in step (3) is 3.5%.

[0035] Example 5 The difference from Example 1 is that the amount of Trichosanthes kirilowii seed oil added in step (3) is 5.5%.

[0036] Example 6 Comparison of free fatty acid release from NLCs with different oil concentrations in Examples 1 and 3-5 with the release level in Comparative Example 1: In vitro simulated digestion was performed using simulated gastric juice containing 3.2 mg / mL pepsin, 0.7% (v / v) HCl, and 2 mg / mL NaCl, preheated to 37°C. 0.5 g of NLCs were mixed with 20 mL of gastric digestion solution, and the pH was adjusted to 2.5 with 1 M HCl. The mixture was then incubated in a 37°C water bath with shaking for 2 h (100 rpm). After simulated gastric digestion, the pH was adjusted to 7 with 1 mol / L NaOH. 1.5 mL of preheated intestinal fluid (0.25 mol / L CaCl₂·2H₂O, 3.75 mol / L NaCl) and 3.5 mL of bile salt solution (53.57 mg / mL) were added. The pH was adjusted to 7, and 2.5 mL of preheated lipase solution (24 mg / mL) was added. The mixture was incubated at 37°C with shaking for 2 h (100 rpm). 0.1 M NaOH was added to maintain the pH at 7, and the FFA release level was calculated by measuring the amount of NaOH used in the reaction.

[0037] The final FFA release levels of NLC samples with oil concentrations ranging from 2.5% to 5.5% are as follows: Figure 1 As shown, the concentrations of lipid matrix and oil bodies in NLCs jointly affect their digestibility. The samples exhibited a two-stage characteristic: rapid FFA release in the first 20 minutes, followed by a slow release phase. The final release level was 4.5% (81.92%) > control (70.30%). The FFA release in the MLCT-treated group was higher than that in the control group (using high-oleic rapeseed oil), indicating that MLCT improved the digestibility and nutritional value of NLCs.

[0038] Example 7 Effects of different oil concentrations on NLC particle size, potential, and PDI value in Examples 1 and 3-5 Examples 1 and 3-5, with oil concentrations ranging from 2.5% to 5.5%, were diluted 800-fold with ultrapure water at pH 7.0 to avoid multiple scattering. The particle size, PDI, and zeta potential of the samples were then measured using a Zetasizer particle size analyzer at 25°C. The average particle size of NLCs is significantly affected by the oil concentration. Figure 2 As shown, the particle size initially decreases and then increases with increasing oil concentration. However, when the oil content is further increased, the electrostatic repulsion is insufficient to support system stability, some charges are shielded, leading to a decrease in negative charge and an increase in particle size. Therefore, an oil concentration of 4.5% was selected as the optimal formulation.

[0039] Example 8 Transmission electron microscopy images of NLC with different oil concentrations in Examples 1 and 3-5 Examples 1 and 3-5, with oil concentrations ranging from 2.5% to 5.5%, were observed using a TEM and an SC 1000 CCD camera at a scale of 200 kV and 1 µm. The shape and size of the samples were imaged using a TEM. Figure 3 As shown, transmission electron microscopy revealed that all NLCs exhibited a spherical and uniform shape with a clearly defined core-shell structure. No particle aggregation was observed, indicating good dispersion of the system. Oil droplets were uniformly encapsulated by oil-body proteins, and the interfacial layer formed by these proteins effectively prevented droplet aggregation. Transmission electron microscopy observation of the NLC system further demonstrates the feasibility of using the oil-body NLC system as a carrier delivery system.

[0040] Example 9 Thermogravimetric analysis (TGA) by NLC for different oil concentrations in Examples 1 and 3-5 Thermogravimetric analysis was used to measure the thermal degradation behavior of the nanocarriers. 6-7 mg of lyophilized oil concentrations from Examples 1 and 3-5, ranging from 2.5% to 5.5%, were placed in an aluminum pot and heated from 25°C to 600°C at a heating rate of 10°C / min under a nitrogen atmosphere. The results are as follows: Figure 4 As shown.

[0041] Below 100 °C, the sample mass decreased only slightly, which is related to the evaporation of residual free water in the sample. The sample began to lose mass at around 250 °C and completely decomposed at 480 °C, indicating that NLC prepared by using oil as an emulsifier has good thermal stability.

[0042] Example 10 Storage stability of NLC with different oil concentrations in Examples 1 and 3-5 Examples 1 and 3-5, with oil concentrations ranging from 2.5% to 5.5%, were stored at 4°C, and particle size and zeta potential were measured on days 0, 3, 7, 14, and 21.

[0043] Particle size and zeta potential after 21 days of storage are as follows: Figure 5 As shown, all NLC particles had a diameter in the range of 200-300 nm, a potential greater than 25 mV, and were negatively charged, maintaining good stability during 21 days of storage.

[0044] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing an oil-based nanostructured lipid carrier based on MLCT, characterized in that: include, Long-chain oil was mixed with capric acid triglyceride, then immobilized lipase was added, vacuum dried, stirred and filtered, deacidified, and vacuum evaporated to obtain MLCT structured lipids. Glyceryl monostearate was mixed with MLCT structured lipid and heated to form the oil phase, while Trichosanthes kirilowii seed oil was mixed with ultrapure water and heated to form the aqueous phase. The aqueous and oil phases were then mixed, heated, ultrasonically broken up, and subjected to an ice bath to obtain the oil-based nanostructured lipid carrier.

2. The preparation method according to claim 1, characterized in that: The long-chain oils include high-oleic rapeseed oil or camellia oil.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the long-chain oil to decanoic acid triglyceride is 6:

4.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the monostearate glycerol to the MLCT structural lipid is 1:3~19.

5. The preparation method according to claim 1, characterized in that: The mass fraction of Trichosanthes kirilowii seed oil in the aqueous phase is 2.5-5.5%.

6. The preparation method according to claim 1, characterized in that: The method for preparing Trichosanthes seed oil includes mixing Trichosanthes seed oil with sodium chloride solution, pulping, filtering, centrifuging to obtain an upper emulsion, adding sucrose solution to disperse and adjust pH, performing a second centrifugation, taking the upper emulsion, dispersing and washing, and then centrifuging again.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the oil phase to the water phase is 2.5~7.5:92.5~97.

5.

8. The preparation method according to claim 6, characterized in that: The pH was adjusted to 11.

9. The oil-based nanostructured lipid carrier prepared by any one of the preparation methods described in claims 1 to 8.

10. The application of the oil-based nanostructured lipid carrier as described in claim 9 in biosurfactants.

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