Preparation method of diacylglycerol nanostructured lipid carrier loaded with fat-soluble nutrients

Abstract

The present invention relates to a preparation method of a diacylglycerol nanostructure lipid carrier for a load of fat-soluble nutrients, comprising the following steps: (S1) heating a mixture of diacylglycerol and glyceryl monostearate to a molten state to form a uniform oil phase according to a mass ratio of 5-10:1-2, adding fat-soluble nutrients to obtain an oil phase; (S2) preparing an aqueous solution of a nonionic surfactant as an aqueous phase; (S3) adding the aqueous phase to the oil phase under stirring, and homogenizing under the stirring action of a high-speed shearing machine to obtain colostrum, ultrasonication, and cooling to room temperature to obtain a diacylglycerol nanostructure lipid carrier for a load of fat-soluble nutrients. The present invention prepares a lipid carrier for a load of fat-soluble nutrients with high loading capacity, good stability, and high bioavailability using diacylglycerol, showing excellent storage and thermal stability. The potential benefits of enhancing LYC conversion and bioaccessibility are shown during LYC-NLC digestion.

Description

Technical Field

[0001] The present invention belongs to the technical field, and in particular relates to a method for preparing a diacylglycerol nanostructured lipid carrier loaded with fat-soluble nutrients. Background Art

[0002] Fat-soluble nutrients have become the most widely used nutritional supplements in food and health products. However, in actual application, there are the following main problems: (1) Fat-soluble nutrients have poor water solubility, and daily intake cannot meet their efficacy; (2) Some fat-soluble nutrients are easily oxidized during production, processing and storage due to their special structure; (3) Some fat-soluble nutrients are affected by extreme environments (such as low pH gastric acid and other digestive fluids), resulting in nutrient destruction and low bioavailability. A specific example is lycopene (LYC), a fat-soluble carotenoid with functions such as immunomodulation, anti-cancer, antioxidant and prevention of various diseases. The structure of LYC contains unsaturated double bonds and is extremely sensitive to environmental conditions. It is reported that the bioavailability of LYC is only 0.1-1.6%. Poor water solubility and gastrointestinal instability seriously limit its application in functional foods and health products.

[0003] Currently, there are various delivery systems, such as emulsions, microemulsions, new nanoemulsions, lipid carriers, hydrogels, and nanostructured lipid carriers, which have been shown to improve their stability and bioavailability. Due to the influence of carrier oil, most drug delivery systems have problems such as low content of bioactive substances. For example, the LYC loading of the Pickering emulsion system prepared with grapefruit peel stabilizer is about 0.1% (Reference 1). The alginate gel beads containing soy protein isolate coated oil droplets prepared by Lin et al. (Reference 2) contain about 0.177 mg / g of LYC. The new bigels composed of monoglyceride-beeswax oil gel and high acyl gellan gum hydrogel showed a LYC loading of about 0.1% (Reference 3). The LYC content of the emulsion prepared by Guo et al. (Reference 4) using α-lactalbumin is about 1 mg / mL. The surface whey protein is easily degraded by pepsin in the stomach, which may lead to the destruction of the system in the stomach, premature release of LYC, and inability to accurately deliver it to the intestine for effective intestinal absorption. Among them, nanostructured lipid carriers have improved loading capacity, encapsulation rate and stability due to the reduction of crystal arrangement caused by the combination of solid and liquid lipids.

[0004] Diacylglycerol (DAG) has attracted widespread attention due to its oligomeric, amphiphilic nature, and excellent emulsifying and water-retention properties. Because DAG oil has a different metabolic pathway than triacylglycerol (TAG) and does not cause fat deposition, many studies have used DAG oil as a replacement for traditional TAG oil in emulsions for applications in the food, pharmaceutical, and cosmetic industries. However, research on nutrient-loaded NLC delivery systems prepared using DAG oil remains lacking.

[0005] Document 1: Gao J, Qiu Y, Chen F, et al. Pomelo peel derived nanocellulose as Pickering stabilizers: Fabrication of Pickering emulsions and their potential as sustained-release delivery systems for lycopene[J]. Food Chemistry, 2023,415:135742.

[0006] Document 2: Lin D, Kelly AL, Miao S. The impact of pH on mechanicalproperties, storage stability and digestion of alginate-based and soy proteinisolate-stabilized emulsion gel beads with encapsulated lycopene[J]. FoodChemistry, 2022,372:131262.

[0007] Document 3: Zhu Q, Gao J, Han L, et al. Development and characterization of novelbigels based on monoglyceride-beeswax oleogel and high acyl gellan gumhydrogel for lycopene delivery[J]. Food Chemistry, 2021,365:130419.

[0008] Document 4: Guo S, Guo Q, Zhang Y, et al. Preparation of enzymatically cross-linkedα-lactalbumin nanoparticles and their application for encapsulatinglycopene[J]. Food Chemistry, 2023:136394. Summary of the Invention

[0009] To address the existing shortcomings of fat-soluble nutrients, such as difficulty in human absorption and utilization, poor stability, and low bioavailability in delivery systems, the present invention utilizes plant oil-based diacylglycerol as a liquid lipid and glyceryl monostearate as a solid lipid to prepare a nanostructured lipid carrier loaded with fat-soluble nutrients, thereby improving the bioavailability and system stability of fat-soluble nutrients. In a system embedded in the diacylglycerol-monostearylglycerol nanostructured lipid carrier (LYC-NLC), not only is its heating and storage stability improved, but the bioavailability of lycopene is also enhanced.

[0010] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0011] A method for preparing a diacylglycerol nanostructured lipid carrier loaded with fat-soluble nutrients comprises the following steps:

[0012] (S1) heating a mixture of diacylglycerol and glyceryl monostearate in a mass ratio of 5-10:1 until it is molten to form a uniform oil phase, and adding a fat-soluble nutrient to obtain an oil phase;

[0013] (S2) preparing an aqueous solution of a nonionic surfactant as the aqueous phase;

[0014] (S3) adding the aqueous phase to the oil phase under stirring, and homogenizing under stirring by a high-speed shearing machine to obtain colostrum, ultrasonically crushing the colostrum, and cooling the colostrum to room temperature to obtain a diacylglycerol nanostructured lipid carrier loaded with fat-soluble nutrients.

[0015] Preferably, the diacylglycerol is derived from at least one of soybean oil, peanut oil, tea oil, sunflower oil, olive oil, and rapeseed oil.

[0016] Furthermore, in step (S1), the mass ratio of diacylglycerol (DAG) to glyceryl monostearate (GMS) is 6-9:1.

[0017] Furthermore, in step (S1), heating to a molten state is heating to 70-80°C.

[0018] Furthermore, in step (S1), the fat-soluble nutrient is selected from at least one of lycopene, curcumin, lutein, and β-carotene.

[0019] Furthermore, in step (S1), the amount of fat-soluble nutrients added is 2-5 wt% of the total raw materials.

[0020] Furthermore, in step (S2), the nonionic surfactant is selected from at least one of Tween, Span, and polyethylene glycol fatty acid ester; the Tween is selected from at least one of Tween 20, Tween 21, Tween 40, Tween 60, and Tween 80, and the Span is selected from at least one of Span 20, Span 40, Span 60, and Span 80; the concentration of the aqueous solution of the nonionic surfactant is 1-5wt%, such as 2wt%, 3wt%, and 4wt%.

[0021] Furthermore, in step (S2), the nonionic surfactant is a compound of surfactant I and surfactant II according to a mass ratio of 1-2:1-2, 8≤the HLB value of surfactant I<12, 12≤the HLB value of surfactant I<18, and the HLB difference between surfactant I and surfactant II is between 4-7, preferably the HLB difference between surfactant I and surfactant II is between 5.3-6.4. For example, the surfactant is a compound of Span 20 (HLB=8.6) and Tween 80 (HLB=15) according to a mass ratio of 1-2:1-2, or the surfactant is a compound of Tween 61 (HLB=9.6) and Tween 60 (HLB=14.9) according to a mass ratio of 1-2:1-2. The inventors unexpectedly found that the surfactants compounded with the above-mentioned different HLBs can further improve the retention rate and bioaccessibility of the lipid carrier.

[0022] Furthermore, in step (S3), the speed of the high-speed shearing machine is 8000-15000 rpm, such as 10000 rpm; and the stirring time is 1-5 min, such as 2 min, 3 min, or 4 min.

[0023] Furthermore, in step (S3), the ultrasonic disruption treatment is carried out at a power of 100-150 W for 10-30 min, such as 20 min.

[0024] The present invention also provides the use of diacylglycerol in preparing a lipid carrier of fat-soluble nutrients.

[0025] The present invention also provides a diacylglycerol nanostructured lipid carrier loaded with fat-soluble nutrients, comprising the following raw materials in parts by mass: 5-10 parts of diacylglycerol, 1-2 parts of glyceryl monostearate, 2-5 parts of fat-soluble nutrients, 1-5 parts of nonionic surfactant, and water supplemented to 100 parts.

[0026] The present invention has achieved the following technical effects:

[0027] First, the present invention uses diacylglycerol (DAG) of varying purity to prepare lipid carriers for fat-soluble nutrients with high loading capacity, excellent stability, and high bioavailability, exhibiting excellent storage and thermal stability. Furthermore, DAG oil, due to its high lipid digestibility and encapsulation capacity, has the potential to enhance LYC conversion and bioaccessibility during LYC-NLC digestion.

[0028] 2. The preparation process of the present invention is simple and practical, does not require complex large-scale equipment and complicated operations, and the product has storage stability and thermal stability, and can effectively encapsulate fat-soluble nutrients to meet the absorption and utilization of the human body.

[0029] 3. The present invention uses a compounded surfactant to improve both retention rate and bioaccessibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the infrared spectra of the nanostructured lipid carrier loaded with lycopene prepared in the examples and comparative examples.

[0031] Figure 2 1 is the XRD pattern of the lycopene-loaded nanostructured lipid carrier prepared in Examples and Comparative Examples.

[0032] Figure 3 The bioaccessibility index and bioaccessibility of the lycopene-loaded nanostructured lipid carriers prepared in Examples and Comparative Examples are shown.

[0033] Figure 4 1 is a graph showing the relationship between the FFA release rate and time of the lycopene-loaded nanostructured lipid carriers of the examples and comparative examples.

[0034] Figure 5 is the loading rate of the lycopene-loaded nanostructured lipid carriers of Examples and Comparative Examples after storage for 21 days.

[0035] Figure 6 is the retention rate of the lycopene-loaded nanostructured lipid carriers of Examples and Comparative Examples.

[0036] Figure 7 3 is a graph showing the change in retention rate of the lycopene-loaded nanostructured lipid carriers of Examples 1-3 and Comparative Example 1 after treatment at 37° C. for 8 hours.

[0037] Figure 8 3 is a graph showing the change in retention rate of the lycopene-loaded nanostructured lipid carriers of Examples 1-3 and Comparative Example 1 treated at 63° C. for 8 h. DETAILED DESCRIPTION

[0038] The following is a further explanation of the technical solution of the present invention with reference to specific embodiments. Unless otherwise specified, the "parts" in the embodiments of the present invention are parts by mass, and the "%" are percentages by mass.

[0039] Diacylglycerol (DAG) and triacylglycerol (TAG) were purchased from Guangdong Yueshan Special Nutrition Technology Co., Ltd. Glyceryl monostearate (analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd., and lycopene was purchased from Zhongda Hengyuan Co., Ltd. (Luohe, China). Ultrapure water was prepared using a Milli-Q water purification system.

[0040] Example 1

[0041] Ingredients: 2.5 parts of lycopene, 9 parts of diacylglycerol, 1 part of glyceryl monostearate, 2 parts of Tween 80, and ultrapure water to make up to 100 parts.

[0042] (S1) mixing 99% diacylglycerol (DAG99%) and glyceryl monostearate (GMS) in a mass ratio of 9:1 and heating to 80° C. until the mixture is melted to form a uniform oil phase, adding lycopene (LYC), and stirring to obtain a uniform oil phase;

[0043] (S2) Tween 80 dissolved in water at 80°C as the aqueous phase;

[0044] (S3) The aqueous phase was added to the oil phase under mechanical stirring conditions and stirred at 800 rpm for 2 minutes. The colostrum was then homogenized using a high-speed shear at 10,000 rpm for 3 minutes. The colostrum was then sonicated at 120 W and 80 kHz for 10 minutes. The colostrum was then cooled in an ice-water bath to recrystallize the lipids, thereby obtaining a lycopene-loaded nanostructured lipid carrier, referred to as 99% DAG-NLC.

[0045] Example 2

[0046] Other conditions were the same as those in Example 1, except that 99% DAG was replaced with 80% DAG of equal mass, which was referred to as 80% DAG-NLC.

[0047] Example 3

[0048] Other conditions were the same as those in Example 1, except that 99% DAG was replaced with 60% DAG of equal mass, referred to as 60% DAG-NLC.

[0049] Comparative Example 1

[0050] Other conditions were the same as those in Example 1, except that 99% of DAG was replaced with TAG of equal mass, which was referred to as TAG-NLC.

[0051] Comparative Example 2

[0052] Other conditions were the same as those in Example 1, except that lycopene (LYC) was not added, and the mixture was referred to as K-NLC.

[0053] The components of 99% DAG, 80% DAG, and 60% DAG were analyzed, and the content of the components is shown in Table 1 below.

[0054] Table 1 Analysis of ingredient content

[0055] Element(%) TAG 60% DAG 80% DAG 99% DAG TAG 95.37 38.09 19.66 0.46 1,3-DAG 2.86 41.96 53.67 75.1 1,2-DAG 1.78 19.95 26.67 24.44

[0056] DAG exists in two isomers, 1,2-DAG and 1,3-DAG. 1,3-DAG is the main component of DAG, with a higher content than 1,2-DAG and stronger biological activity.

[0057] Figure 1 The infrared spectra of the nanostructured lipid carrier loaded with lycopene prepared in the above examples and comparative examples are shown in Figure 1. Lycopene is at 957 cm -1 A characteristic absorption peak is shown at , which is attributed to the R1HC=CR2H oscillation vibration absorption peak common in lycopene (LYC). In the infrared spectra of the lycopene-loaded nanostructured lipid carriers prepared in the Examples and Comparative Examples, this characteristic peak indicates that lycopene has been successfully loaded into the lipid carriers.

[0058] Figure 2 Figure 2 shows the XRD patterns of the lycopene-loaded nanostructured lipid carriers prepared in Examples and Comparative Examples. The characteristic peak at 2θ near 24° is a characteristic diffraction peak of LYC. The XRD patterns of the lycopene-loaded nanostructured lipid carriers prepared by K-NLC and the present invention are essentially identical. The characteristic LYC peak at 24° disappears in Comparative Example 2, further supporting the successful loading of LYC into the lipid carrier.

[0059] Application Example 1

[0060] The lycopene-loaded lipid carriers of the examples and comparative examples were tested for lycopene encapsulation efficiency, loading amount and retention rate using an indirect method with slight modifications.

[0061] LYC-NLC sample (0.1 mL) was dissolved in acetone (1 mL), and then vortexed for 1 minute before the volume was adjusted to 10 mL. The absorbance of total LYC was measured at 472 nm using a UV-visible spectrophotometer. Acetone was used as a blank control to measure the total lycopene content. A standard curve of total LYC (y = 0.2776x + 0.1067, R) was prepared over the concentration range of LYC acetone solution (0-15 μg / mL). 2=0.99445). To measure the unloaded LYC, LYC-NLC (1 mL) was dissolved in n-hexane (1 mL), then vortexed for 1 minute and centrifuged at 10,000 rpm for 10 minutes at 4°C to obtain the n-hexane phase. This process was repeated twice, and the resulting n-hexane phases were combined and diluted to a final volume of 10 mL. The absorbance of free LYC was measured at 472 nm using spectrophotometry. The absorbance of LYC n-hexane solutions (0-15 ug / mL) within the LYC concentration range was obtained as a standard curve for free LYC (y=0.265x+0.0932, R 2 =0.9949).

[0062] The encapsulation efficiency (%), loading capacity (%) and retention rate (%) of LYC were calculated according to the following formulas.

[0063]

[0064] Where C1 is the total LYC content (mg), C2 is the free LYC content (mg), M is the total lipid content (mg), x1 is the LYC content after 21 days of storage (mg), and x2 is the initial LYC content (mg). The results are shown in Table 2 below.

[0065] Table 2 Encapsulation efficiency, loading capacity and retention rate test

[0066]

[0067] It can be seen that DAG-NLC has higher encapsulation efficiency and loading capacity than TAG-NLC, especially 99% DAG-NLC has a significantly higher 21-day retention rate.

[0068] Application Example 2: Bioaccessibility Testing

[0069] In vitro lipid digestion was performed according to INFOGEST (Brodkorb A, Egger L, Alminger M, et al. INFOGEST static in vitro simulation of gastrointestinal food digestion [J]. Nature Protocols, Nature Publishing Group, 2019, 14(4): 991–1014.). 1.25X simulated oral fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared according to the protocol and diluted to 1X for use. The entire digestion process should be maintained at a constant temperature of 37°C, and all solutions and components should be preheated before use to avoid temperature fluctuations.

[0070] To simulate oral digestion, the test sample was mixed with SSF containing 0.00375 g / mL mucin and 1.5 mM CaCl2(H2O)2 at a 1:1 (v / v) ratio to form an oral solution. The resulting sample was then incubated at 37°C with mechanical shaking at 100 rpm for 5 minutes.

[0071] Simulated gastric digestion: The oral digest was then diluted 1:1 (v / v) with SGF containing pepsin (2000 U / mL in the final digestion mixture) and 0.15 mM CaCl2(H2O)2 and incubated at pH 3.0 for 1 hour under shaking conditions. Samples were collected every 30 minutes to determine LYC content.

[0072] Simulated intestinal digestion: The gastric digest was then diluted 1:1 (v / v) with SIF containing 0.6 mM CaCl2(H2O)2 and 10 mM bile salts. After thorough mixing, the sample was adjusted to pH 7.0. Trypsin was then added to initiate digestion, and the mixture was incubated at 37°C for 2 hours. Trypsin activity reached 100 U / mL and trypsin lipase activity reached 2000 U / mL. Samples were collected every 30 minutes to determine LYC content.

[0073] After the simulated gastric and intestinal digestion, 10 ml of sample was taken, 1 ml of ethanol:hexane (1:2, v / v) was added, and vortexed for 1 min to break the emulsion. Then, the supernatant was obtained by centrifugation at 25°C and 14000 rpm for 60 min. The supernatant was extracted three times and the volume was adjusted to 10 ml with n-hexane. After that, the LYC concentration in the total chyme and before small intestinal digestion in the sample was quantitatively determined by ultraviolet spectroscopy at 472 nm.

[0074] After the digestion process, the sample was boiled in a water bath for 5 minutes to inactivate the enzyme. Ten milliliters of chyme was centrifuged at 10,000 g for 30 minutes at 4°C. The supernatant was filtered through a 0.45 μm membrane filter to obtain a clear micellar phase. The extract was then demulsified with ethanol, collected with n-hexane, and the absorbance was measured at 472 nm. The concentration of LYC in the micelles was calculated.

[0075] The lycopene-loaded nanostructured lipid carriers prepared in the Examples and Comparative Examples were tested for bioaccessibility. The LYC conversion rate, bioaccessibility index, and bioaccessibility were calculated according to the following formulas:

[0076] Conversion rate = A d / A i ×100%;

[0077] Bioaccessibility index = A m / A d ×100%;

[0078] Bioaccessibility = conversion rate × bioaccessibility index × 100%;

[0079] Among them A d A represents the concentration of LYC in the total digesta. i Indicates the concentration of LYC at the end of the simulated gastric stage and before small intestinal digestion (0 min), A m represents the micellar phase LYC concentration.

[0080] See the results Figure 3 and Table 3.

[0081] Table 3 Bioaccessibility test results

[0082] project TAG-LYC 60% DAG-NLC 80% DAG-NLC 99% DAG-NLC Conversion rate (%) 61.3±4.0 79.6±0.9 85.4±2.7 89.0±5.7 Bioaccessibility index (%) 29.9±1.7 36.4±3.1 36.1±1.5 35.3±0.1 Bioaccessibility (%) 18.3±0.3 29.0±2.2 30.8±1.0 31.4±2.1

[0083] The conversion rate of LYC refers to the proportion of undegraded LYC at the end of simulated digestion. Compared with other DAG-NLCs, TAG-NLC had the lowest conversion rate (61.3 ± 4.0%) and increased with increasing DAG oil content. The bioaccessibility index represents the ratio of the LYC content in the mixed micelles to the LYC content in the chyme. Bioaccessibility is determined by both the conversion rate and the bioaccessibility index. The emulsifier and lipid layer can be hydrolyzed by enzymes and bile extracts into free fatty acids (FFA) and monoacylglycerols (MAG), ultimately leading to micelle formation. LYC bioaccessibility increased proportionally with the DAG oil content in the NLC, reaching a peak of 31.4 ± 2.1% at the highest DAG oil concentration (99%) in the lipid carrier. The increased bioaccessibility of LYC may be due to the fact that 1,3-DAG is the main component of DAG lipids. 1,3-DAG has a special structure different from TAG and is easily digested by pancreatic 1,3-specific lipase into 1(3)-monoacylglycerol and free fatty acids (FFA). In addition, the surface active MAG and FFA produced during digestion can regulate fat digestion.

[0084] Application Example 2 Study on the Release of FFA and LYC during In Vitro Digestion

[0085] The pH-stat titration method was used to measure the FFA release rate to evaluate the digestion behavior of the lycopene-loaded nanostructured lipid carriers of the present invention. Specifically, the FFA release was monitored by titrating the sample with 0.25 M NaOH at pH 7.0 for 2 hours using a pH-stat titrator (MT-V6, HOGON, China).

[0086] The FFA release was calculated according to the following formula:

[0087]

[0088] Where V NaOH represents the titration volume of NaOH (L), C NaOH is the concentration of NaOH (M), and M lipid W is the average molecular weight of the lipid mixture (g / mol). lipid is the initial lipid mass (g).

[0089] Figure 4 3 is a graph showing the relationship between the FFA release rate and time of the lycopene-loaded nanostructured lipid carriers of Example and Comparative Example 1.

[0090] During the digestion experiment, the release rate of FFAs accelerated within 5-10 minutes after adding trypsin, then gradually increased slowly until reaching a relatively constant final value. The order of FFA release was 99% DAG-NLC > 80% DAG-NLC > 60% DAG-NLC > TAG-NLC. After 120 minutes of digestion, the digestibility of TAG-NLC was the lowest (66.45%), while the digestibility of DAG-NLCs (60% DAG, 80% DAG, and 99% DAG) exceeded 75%. Compared with the TAG-NLC in Comparative Example 1, the DAG-NLC in Example 1 demonstrated a higher lipolytic effect.

[0091] Different lipid types also affect the bioaccessibility of LYC. DAG can form larger micelles to better load lipophilic bioactive compounds, resulting in a sustained increase in LYC release over time. DAG content plays a crucial role in promoting efficient release. Therefore, this precise delivery mechanism enables targeted delivery of LYC-NLC in the intestine and offers promising applications.

[0092] Application Example 3 Storage Stability Study

[0093] After the lycopene-loaded nanostructured lipid carriers of the examples and comparative examples were stored in a refrigerator at 4° C. in the dark for 21 days, the loading rate and retention rate of the lipid carriers were tested. Figure 5 is the loading rate of the lycopene-loaded nanostructured lipid carriers of Example and Comparative Example 1 after storage for 21 days; Figure 6is the retention rate of the lycopene-loaded nanostructured lipid carriers of Example and Comparative Example 1. Among them, 99% DAG-NLC has the best stability (retention rate 76.1%), while 60% DAG-NLC and 80% DAG-NLC have retention rates of 38.3% and 51.2%, respectively. It can be seen that the storage stability of DAG-NLC in Example is significantly higher than that of TAG-NLC (retention rate 34.5%). This may be because the NLC carrier can stabilize the zeta potential of the nanoparticles through electrostatic and steric hindrance. The DAG molecule is amphiphilic and adsorbs at the oil-water interface. Its polar groups are inserted into the aqueous phase, resulting in an increase in the absolute value of the zeta potential. Moreover, DAG can inhibit lipid oxidation in oil-in-water emulsions. A small amount of DAG can significantly improve the stability of the emulsion by promoting the interfacial uneven crystallization of hydrogenated lipids.

[0094] Application Example 4 Thermal Stability Study

[0095] Nanostructured lipid carriers loaded with fat-soluble nutrients are affected by the in vivo digestive environment and high ambient temperature conditions during sterilization. Using different temperatures (37°C and 63°C) as indicators, the effects of heat treatment of different oil phase LYC-NLCs for 8 hours on stability were evaluated. Tables 4 and 5 show the particle size changes of the nanostructured lipid carriers loaded with lycopene in Example 1 and Comparative Example 1 after heating for 8 hours under different conditions. It can be seen that the hydrodynamic diameters of all four NLCs (Examples 1-3, Comparative Example 1) remained within an average range of 160-190 nm during storage at 37°C, with no significant differences observed (p>0.05). However, under 63°C heat treatment conditions, the hydrodynamic diameter of the TAG-NLC of Comparative Example 1 increased significantly from (166.7±2.33 nm) to (383.93±19 nm), showing a statistically significant difference (p<0.05), while the particle size changes of the DAG-NLC of Examples 1-3 were negligible. We believe that in addition to the high melting temperature of DAG, which can maintain the stability of DAG-NLC at high temperatures, the presence of the interfacial crystalline DAG shell may also reduce the expansion and contraction of the emulsion in response to osmotic pressure and thermal treatment, which helps to improve the stability of the emulsion.

[0096] Table 4 Particle size changes after heating at 37℃ for 8h

[0097] 0h(nm) 2h(nm) 4h(nm) 6h(nm) 8h(nm) TAG-NLC 163.9±3.2 169.6±0.9 170.7±3.2 168.6±4.3 169.1±3.6 60% DAG-NLC 163.1±0.6 179.2±8.8 173.4±3.4 179.5±3.9 172.7±3.9 80% DAG-NLC 192.0±1.8 174.7±3.4 187.9±5.1 203.2±17.5 179.4±4.1 99% DAG-NLC 186.9±5.9 191.7±8.0 198.5±20.1 198.6±9.9 186.6±3.6

[0098] Table 5 Particle size changes after heating at 63℃ for 8h

[0099] 0h(nm) 2h(nm) 4h(nm) 6h(nm) 8h(nm) TAG-NLC 166.7±2.3 230.8±4.7 282.1±5.7 343.0±6.8 383.9±19 60% DAG-NLC 165.3±6.5 160.3±2.9 164.0±3.8 162.1±4.9 166.2±1.2 80% DAG-NLC 164.5±3.1 149.6±3.2 158.3±2.5 157.5±4.6 161.2±3.4 99% DAG-NLC 174.1±5.1 165.2±6.5 162.6±3.5 160.5±5.4 164.3±2.7

[0100] Figure 7is a graph showing the change in retention rate of the lycopene-loaded nanostructured lipid carriers of Examples 1-3 and Comparative Example 1 treated at 37° C. for 8 hours; Figure 8 This graph shows the retention rate of the lycopene-loaded nanostructured lipid carriers of Examples 1-3 and Comparative Example 1 after treatment at 63°C for 8 hours. After treatment at 37°C for 8 hours, the retention rates of the DAG-NLCs of the Examples and the TAG-NLC of the Comparative Example remained relatively unchanged. However, after treatment at 63°C for 8 hours, the retention rate of the TAG-NLC decreased significantly, with the retention rate of LYC on the TAG-NLC dropping to 51.9%. While the retention rates of the DAG-NLCs of the Examples were all above 80%, demonstrating that the nanostructured lipid carriers of the present invention using DAG exhibited improved thermal stability.

[0101] Example 4

[0102] Other conditions were the same as those in Example 1, except that 2 parts of Tween 80 as the surfactant were replaced by 1 part of Tween 80 and 1 part of Span 20.

[0103] Example 5

[0104] Other conditions were the same as those in Example 1, except that 2 parts of Tween 80, a surfactant, were replaced by 2 parts of Span 20.

[0105] Example 6

[0106] Other conditions were the same as those in Example 1, except that 2 parts of Tween 80 surfactant were replaced by 1 part of Span 20 and 1 part of Tween 61.

[0107] The loading amount, retention rate after 21 days of storage and bioaccessibility of Example 1 and Examples 4-6 are shown in Table 6.

[0108] Table 6 Effects of different surfactants

[0109] project Example 1 Example 4 Example 5 Example 6 Loading capacity (%) 1.48±0.02 1.47±0.03 1.26±0.04 1.31±0.02 Retention rate after 21 days of storage (%) 76.13±2.99 80.52±3.17 66.32±2.43 77.26±2.83 Bioaccessibility (%) 31.4±2.1 33.46±2.0 27.8±1.9 30.9±2.1

[0110] It can be seen that the combination of low-HLB and high-HLB surfactants can simultaneously improve the retention rate and bioaccessibility of lipid carriers, and further enhance the application performance of such products.

Claims

1. A method for preparing a diacylglycerol nanostructured lipid carrier loaded with fat-soluble nutrients, characterized in that: The raw materials are 2.5 parts lycopene, 9 parts diacylglycerol, 1 part glyceryl monostearate, 2 parts Tween 80, and ultrapure water to make up to 100 parts. The parts are by mass. The preparation method comprises the following steps: (S1) mixing 99% diacylglycerol and glyceryl monostearate in a mass ratio of 9:1, heating to 80° C. until the mixture is melted to form a uniform oil phase, adding lycopene, and stirring to obtain a uniform oil phase; diacylglycerol is a liquid lipid; (S2) Tween 80 dissolved in water at 80°C as the aqueous phase; (S3) The aqueous phase was added to the oil phase under mechanical stirring conditions and stirred at 800 rpm for 2 minutes. The colostrum was then homogenized using a high-speed shear at 10,000 rpm for 3 minutes. The colostrum was then sonicated at 120 W and 80 kHz for 10 minutes. The colostrum was then cooled in an ice-water bath to recrystallize the lipids, thereby obtaining a lycopene-loaded nanostructured lipid carrier.

2. A method for preparing a diacylglycerol nanostructured lipid carrier loaded with fat-soluble nutrients, characterized in that: The raw materials are 2.5 parts lycopene, 9 parts diacylglycerol, 1 part glyceryl monostearate, 1 part Tween 80, and 1 part Span 20, and ultrapure water is used to make up to 100 parts. The parts are by mass. The preparation method comprises the following steps: (S1) mixing 99% diacylglycerol and glyceryl monostearate in a mass ratio of 9:1, heating to 80° C. until the mixture is melted to form a uniform oil phase, adding lycopene, and stirring to obtain a uniform oil phase; diacylglycerol is a liquid lipid; (S2) Tween 80 and Span 20 dissolved in water at 80°C as the aqueous phase; (S3) The aqueous phase was added to the oil phase under mechanical stirring conditions and stirred at 800 rpm for 2 minutes. The colostrum was then homogenized using a high-speed shear at 10,000 rpm for 3 minutes. The colostrum was then sonicated at 120 W and 80 kHz for 10 minutes. The colostrum was then cooled in an ice-water bath to recrystallize the lipids, thereby obtaining a lycopene-loaded nanostructured lipid carrier.

3. The preparation method according to claim 1 or 2, wherein the diacylglycerol is derived from at least one of soybean oil, peanut oil, tea oil, sunflower oil, olive oil, and rapeseed oil.

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