Preparation method and application of macadamia nut oil

Abstract

The invention relates to the technical field of deep processing of vegetable fat, in particular to a preparation method and application of squalene-containing macadamia nut oil. Through the synergistic effect of directional enzymolysis conversion and low-temperature step-by-step extraction and the combined effect of the three enzymes (cellulase destroys cell walls, lipase releases grease and beta-glucosidase removes inhibitors, and the three enzymes form a wall breaking-oil releasing-synergistic synergistic reaction) by utilizing a compound enzyme which is fixedly matched and proportioned. The compound enzyme can be used for synchronously treating cellulose, lipid and glycoside substances, so that the release efficiency of squalene is improved; and step-by-step extraction is utilized to reduce extraction impurities, so that the content of squalene in the macadamia nut oil can be increased, and the extraction and enrichment of squalene in the macadamia nut meat can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of vegetable oil deep processing, and particularly relates to a preparation method of macadamia nut oil and application thereof. BACKGROUND

[0002] The macadamia nut oil is a rare terpenoid with antioxidant and immunomodulatory functions due to its excellent fatty acid composition (containing more than 80% of monounsaturated fatty acids) and chemical components such as phytosterols, tocopherols and squalene, and is a natural antioxidant and moisturizer, and is therefore widely used in the field of cosmetics.

[0003] At present, the preparation methods of the macadamia nut oil containing squalene mainly include cold pressing method and solvent extraction method. The cold pressing method can retain the heat-sensitive components such as squalene by mechanically pressing the macadamia nut kernels to obtain oil, but the yield of the macadamia nut oil is low. The solvent extraction method uses organic solvents such as petroleum ether and n-hexane to extract oil, and then purifies squalene through saponification and complexation reaction (such as silver nitrate), but the solvents such as petroleum ether still remain even after vacuum concentration, and need to be refined twice, thereby causing the loss of squalene and the content of squalene in the macadamia nut oil is significantly reduced.

[0004] Therefore, in view of the natural squalene resource in the macadamia nut kernels, a method for improving the content of squalene in the macadamia nut oil while ensuring the yield of the macadamia nut oil is urgently needed, so as to realize the extraction and enrichment of squalene in the macadamia nut kernels. SUMMARY

[0005] To solve the above problems, the present application provides a preparation method of macadamia nut oil, which can improve the content of squalene in the macadamia nut oil while ensuring the yield of the macadamia nut oil through the combined action of composite enzymes under the cooperation of directional enzymatic conversion and low-temperature step-by-step extraction.

[0006] In one aspect, the present application provides a preparation method of macadamia nut oil, which comprises the following steps: S1, adding macadamia nut kernels into a citric acid-disodium hydrogen phosphate buffer solution, and adding composite enzymes to perform enzymolysis to obtain an enzymolysis material; The composite enzymes are compounded by cellulase, lipase and beta-glucosidase at a mass ratio of 15:8:5; The addition amount of the composite enzymes is 1.0% of the mass of the macadamia nut kernels; S2, separating the enzymolysis material in S1 after supercritical CO2 extraction to obtain a first oil phase and a residue; S3, separating the residue in S2 after subcritical butane dynamic extraction to obtain a second oil phase; S4, mixing the first oil phase obtained in S2 and the second oil phase obtained in S3, collecting heavy components after short path molecular distillation to obtain macadamia nut oil.

[0007] Further, the temperature for the enzymatic hydrolysis in S1 is 40-50℃, and the time is 4-6 hours.

[0008] Further, the specific steps for the supercritical CO2 extraction in S2 include: extraction under the conditions of pressure 25-30 MPa, temperature 35-40℃, CO2 flow rate 20-30 L / h for 1.5-2.5 hours.

[0009] Further, the specific steps for the supercritical CO2 extraction in S2 include: extraction under the conditions of pressure 28 MPa, temperature 38℃, CO2 flow rate 25 L / h for 2 hours.

[0010] Further, the pressure for the subcritical butane dynamic extraction in S3 is 0.5-1.2 MPa, the temperature is 40-50℃, and the time is 1-3 hours.

[0011] Further, the temperature for the short path molecular distillation in S4 is 180-200℃, and the vacuum degree is ≤5 Pa.

[0012] Further, the pH of the citric acid-disodium hydrogen phosphate buffer is 5.0-6.5.

[0013] Further, the solid-liquid ratio of the macadamia nut kernel to the citric acid-disodium hydrogen phosphate buffer is 1: (2-4) (g / L).

[0014] Further, the particle size of the macadamia nut kernel is 40-80 mesh.

[0015] In another aspect, the application also provides a use of the squalene-containing macadamia nut oil obtained by the above method in cosmetics.

[0016] Beneficial effects: 1. The application provides a preparation method of macadamia nut oil, which utilizes the synergistic effect of directional enzymatic hydrolysis conversion and low-temperature step-by-step extraction, and the combined action of three enzymes (cellulase breaks down the cell wall, lipase releases oil, and β-glucosidase removes inhibitors, forming a synergistic reaction of "wall breaking-oil releasing-synergistic effect"), so that the complex enzymes can simultaneously process cellulose, lipids and glycosides, improving the release efficiency of squalene; then step-by-step extraction is used to reduce impurities and improve the content of squalene in macadamia nut oil, thereby realizing the extraction and enrichment of squalene in macadamia nut kernel; 2、The application adds the macadamia nut kernel and the complex enzyme into the citric acid-disodium hydrogen phosphate buffer solution with pH 5.0-6.5, not only maintains the optimum pH of enzyme activity, ensures that the complex enzyme maintains high activity in the respective optimum pH range, but also inhibits metal ion interference, prevents the inhibition of enzyme activity or the initiation of lipid oxidation; and the ionic strength of the buffer solution can adjust the electrostatic interaction between the enzyme and the substrate (cell wall polysaccharide, lipid protein complex), and enhance the binding efficiency of the enzyme and the substrate; 3、The application carries out step-by-step extraction on the enzyme hydrolysate, uses supercritical CO2 to preferentially extract nonpolar components (such as squalene, triglyceride), and then uses subcritical butane to subsequently extract residual polar components (such as phospholipid, pigment), thereby avoiding incomplete extraction due to solvent polarity limitation in single extraction, and improving the extraction rate of squalene. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 DPPH radical scavenging rate in Example 5.

[0018] Figure 2 Antioxidant efficacy in Example 6.

[0019] Figure 3 Antioxidant phenotype graph in Example 6.

[0020] The implementation, functional characteristics and advantages of the present application will be further described with reference to the embodiments in conjunction with the drawings. DETAILED DESCRIPTION

[0021] In order to better understand the above technical solutions, the exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to convey the scope of the present disclosure to those skilled in the art.

[0022] I. Invention principle: HPLC-MS identification shows that there are a large number of bound squalene precursors (such as cycloartanol, β-amyrin, etc.) in macadamia nut kernels. The present application uses the synergistic effect of directional enzymatic conversion and low-temperature step-by-step extraction, and the combined action of three enzymes (cellulase to break the cell wall, lipase to release oil, and β-glucosidase to remove inhibitors) to form a synergistic reaction of "wall breaking-oil releasing-synergistic effect", so that the complex enzyme can simultaneously process cellulose, lipids and glycosides, and the release efficiency of squalene is improved. Then, step-by-step extraction is used to reduce impurities and improve the content of squalene, achieving efficient release and enrichment of squalene.

[0023] II. Basic mechanism: 1. Mechanism of buffer: (1) Maintaining the optimal pH for enzyme activity; biological enzyme reactions are highly sensitive to pH, and buffers (such as phosphate, citrate buffer) can stabilize the pH of the reaction system, ensuring that the complex enzymes maintain high activity within their respective optimal pH range (e.g., cellulase pH 4.5-5.5, lipase pH 7-8); (2) Inhibition of metal ion interference; chelating components (such as citric acid) in the buffer can bind heavy metal ions (such as Fe 3+ , Cu 2+ ), preventing them from inhibiting enzyme activity or causing lipid oxidation; (3) Promoting substrate contact; the ionic strength of the buffer can adjust the electrostatic interaction between enzymes and substrates (cell wall polysaccharides, lipid-protein complexes), enhancing the binding efficiency of enzymes and substrates.

[0024] Buffer regulation process: phased reaction (sequential regulation), first step: under acidic conditions (pH 4.5-5.5), activate cellulase using citrate buffer to complete cellulose hydrolysis; second step: adjust the pH to neutral (pH 7-8), activate lipase using phosphate buffer for lipid decomposition. Advantage: through step-by-step operation, ensure that each enzyme works efficiently at its optimal pH, avoiding activity loss.

[0025] 2. Mechanism of cellulase: breaks down cellulose in macadamia nut cell walls, destroys cell structure, and releases encapsulated oil and squalene. Endo-enzyme randomly cuts cellulose chains, and exo-enzyme cuts cellobiose from exposed ends, increasing substrate accessibility.

[0026] 3. Mechanism of lipase: hydrolyzes the ester bonds of triglycerides, releasing free fatty acids and glycerol, which may promote the separation of squalene and lipids, selectively degrades complex lipids (such as phospholipids, waxes) in nut oil, reducing subsequent extraction impurities.

[0027] 4. Mechanism of β-glucosidase: degrades residual cellobiose to glucose, avoiding feedback inhibition of cellulase, and breaks down glycolipid complexes to release bound squalene.

[0028] 5. Mechanism of supercritical CO2 extraction: CO2 in supercritical state (critical point: 31.1℃, 7.38 MPa) has high diffusivity of gas and strong solubility of liquid, can penetrate into the raw material and dissolve non-polar components (such as triglycerides, squalene). The low-temperature operation (usually <60℃) of supercritical CO2 extraction avoids the degradation of heat-sensitive components; no solvent residue, high safety.

[0029] 6. Mechanism of subcritical butane extraction: butane dissolves lipids with higher polarity (such as phospholipids and sterols) in a subcritical state (critical point: 152°C, 3.8 MPa) with less damage to heat-sensitive components. Subcritical butane extraction has high selectivity for polar components and can be used as a supplement to CO2 extraction.

[0030] 7. Reason for step-by-step extraction: two extractions are performed in sequence, with supercritical CO2 preferentially extracting non-polar components (such as squalene and triglycerides), and subcritical butane subsequently extracting residual polar components (such as phospholipids and pigments). The polarity of butane is slightly higher than that of CO2, so butane can extract residual polar substances that cannot be completely extracted by supercritical CO2, thereby expanding the polarity range and avoiding incomplete extraction due to the polarity limitations of a single solvent.

[0031] 8. Disadvantages of single extraction: a single solvent cannot cover the full polarity range, resulting in low extraction efficiency of target components (squalene) or excessive impurities affecting subsequent purification. At the same time, to compensate for the polarity defect, an excessive amount of solvent (such as ethanol > 20%) is required, which inhibits diffusion efficiency and increases energy consumption by 30-40% for recovery. Furthermore, the inability to regulate parameters step by step leads to the decomposition of heat-sensitive components and the precipitation of non-polar substances, ultimately resulting in a triple loss of extraction rate, purity, and cost.

[0032] 9. Analysis of the contradiction between the mechanism of short-path molecular distillation and high-temperature conditions Mechanism: Under high vacuum (<1 Pa), the boiling points of the components of the mixture are significantly reduced, and the light components (squalene) are preferentially vaporized, which are then collected by condensation in stages. The separation is based on the difference in molecular free path, avoiding the long-term high-temperature exposure of traditional distillation.

[0033] Analysis of the contradiction between the process conditions of 190°C and heat sensitivity: Actual temperature control: Although the heating temperature of the distillation apparatus is set to 190°C, the actual temperature of the material under high vacuum is significantly lower than the set value (less than 120°C), and the heating time is extremely short (seconds). Therefore, the set temperature of 190°C in the process of short-path molecular distillation for purifying and collecting light components will not cause a large amount of degradation of heat-sensitive components (squalene).

[0034] Example 1 The present application is realized through the following test steps, which include: S1, preparation of complex enzymes: cellulase, lipase, and β-glucosidase are compounded in a certain mass ratio; S2, enzymatic pretreatment: after the macadamia nut is ground to 40-80 mesh, the material is added to a pH 5.0-6.5 citric acid-disodium hydrogen phosphate buffer solution at a solid-liquid ratio of 1:(2-4) (g / L), and the complex enzymes in S1 are added. Enzymatic hydrolysis is carried out at 40-50°C for 4-6 hours to obtain an enzymatic hydrolysate; S3, supercritical CO2 primary extraction: the enzymatic hydrolyzate was placed in a supercritical extraction device, and extracted under the conditions of a pressure of 25-30 MPa, a temperature of 35-40 °C, and a CO2 flow rate of 20-30 L / h for 1.5-2.5 hours to separate a first oil phase and a residue; S4, subcritical butane secondary extraction: the residue in S3 was subjected to subcritical butane dynamic extraction under the conditions of a pressure of 0.5-1.2 MPa, a temperature of 40-50 °C, and an extraction time of 1-3 hours to obtain a second oil phase; S5, molecular distillation purification: the first oil phase and the second oil phase were mixed, and light components were separated in a short-path molecular distillation device under the conditions of a temperature of 180-200 °C and a vacuum degree of ≤5 Pa to collect a squalene-rich phase to obtain squalene-containing macadamia nut oil; S6, determination of the squalene content, the macadamia nut oil yield, and the squalene yield of the squalene-containing macadamia nut oil in S5; Example 2 In this example, a composite enzyme optimization test was performed, and the other steps were the same as in Example 1.

[0035] Scheme 1: compared with the above test steps, other conditions were unchanged, only the components and mass ratio of the composite enzyme (cellulase: lipase = 15:8) were changed, and β-glucosidase was no longer added; Scheme 2: compared with the above test steps, other conditions were unchanged, only the components and mass ratio of the composite enzyme (cellulase: β-glucosidase = 15:5) were changed, and lipase was no longer added; Scheme 3: compared with the above test steps, other conditions were unchanged, only the components and mass ratio of the composite enzyme (lipase: β-glucosidase = 8:5) were changed, and cellulase was no longer added; Scheme 4: compared with the above test steps, other conditions were unchanged, only the matching mode of the composite enzyme was changed, and the composite enzyme (cellulase: lipase: β-glucosidase = 15:7:5); Scheme 5: compared with the above test steps, other conditions were unchanged, only the matching mode of the composite enzyme was changed, and the composite enzyme (cellulase: lipase: β-glucosidase = 15:8:5); Scheme 6: compared with the above test steps, other conditions were unchanged, only the matching mode of the composite enzyme was changed, and the composite enzyme (cellulase: lipase: β-glucosidase = 15:9:5); Scheme 7: compared with the above test steps, other conditions were unchanged, only the matching mode of the composite enzyme was changed, and the composite enzyme (cellulase: lipase: β-glucosidase = 12:8:5); Scheme 8: compared with the above test steps, other conditions were unchanged, only the matching mode of the composite enzyme was changed, and the composite enzyme (cellulase: lipase: β-glucosidase = 16:8:5); Scheme 9: Compared with the above experimental procedure, other conditions remain the same, only the combination of complex enzyme is changed, complex enzyme (cellulase: lipase: β-glucosidase = 15:8:4); Scheme 10: Compared with the above experimental procedure, other conditions remain the same, only the combination of complex enzyme is changed, complex enzyme (cellulase: lipase: β-glucosidase = 15:8:6).

[0036] Example 3 The squalene content, macadamia nut oil yield and squalene yield of the scheme in Example 2 were determined; the same batch of macadamia nut kernels was used for all example tests (the initial squalene content of the macadamia nut kernel raw material was 300 mg / kg).

[0037] The determination method is: gas chromatography (Gas Chromatography), usually combined with flame ionization detector or mass spectrometer, and includes necessary lipid extraction and saponification / unsaponifiable matter extraction and other pretreatment steps.

[0038] The mass of squalene obtained by extraction is: 300 mg.

[0039] The calculation formulas of squalene content (mg / kg), squalene content (mg / kg), macadamia nut oil yield (%) and squalene yield (%) in macadamia nut oil are respectively: The test results are shown in Table 1: Table 1: Determination results of squalene content, macadamia nut oil yield and squalene yield of samples The factors in this Example 2 were subjected to quadratic regression, and a quadratic regression equation was obtained. The experimental variable design: Independent variables: mass ratio of cellulase (X1), lipase (X2), β-glucosidase (X3); Dependent variable: response value Y (squalene content).

[0040] Table 2: Experimental results of different enzyme ratio combinations According to the quadratic regression model and coefficient analysis, the quadratic regression equation is used to fit the data in Table 2, and the model expression is: Y = B0+ B1X1 + B2X2 + B3X3 + B4X1 2 + B5X22 + B6X3 2 + B7X1X2 + B8X1X3 + B9X2X3 Table 3 Regression model parameters of the effect of three enzyme ratios on index response values Model reliability: coefficient of determination R 2 = 0.986 (very high degree of fitting), overall model P < 0.001 (significantly credible).

[0041] As can be seen from the above table, the response value Y (140-215) of the three-enzyme combination of schemes 4-10 is significantly higher than that of the two-enzyme / single-enzyme combination (81-154), proving that the synergistic effect of the three enzymes can greatly improve the efficiency.

[0042] The quadratic regression analysis shows that the quadratic term coefficients of the three enzymes are negative and significant (P < 0.05), indicating that there is an optimal threshold for the enzyme ratio; combined with the model extreme value calculation and experimental data (Y = 215 of scheme 5 is the maximum value), the optimal combination mass ratio is determined as: cellulase: lipase: β-glucosidase = 15:8:5. The response value (215) under the optimal ratio is 39.6% higher than that of the optimal two-enzyme combination (scheme 2, Y = 154) and 10.8%-53.6% higher than that of other three-enzyme ratios (such as schemes 6-10), which has significant practical value.

[0043] Scheme 5 (15:8:5) achieves the highest squalene yield (72%), which is the best scheme for comprehensive extraction of squalene. Cellulase (15 parts) fully destroys the cell wall (mainly containing cellulose), releases oil and squalene (the oil yield of scheme 3 without this enzyme is only 35%, and excessive amount reduces the purity); lipase (8 parts) moderately hydrolyzes triglycerides to release bound squalene (the content of scheme 2 without this enzyme is only 154 mg / kg, and excessive amount degrades squalene); β-glucosidase (5 parts) precisely hydrolyzes glycosidic bonds to activate squalene precursors (low dosage scheme 10 contains 195 mg / kg, high dosage reduces). Scheme 5 breaks through the squalene yield (72.00%) by using the golden enzyme ratio, and its mechanism is to precisely control the activity of lipase to release the maximum amount of squalene, while the three enzymes synergistically improve the extraction efficiency.

[0044] Example 4 After screening by the above examples 1-3, the optimal preparation method is selected, and the steps are as follows: S1, preparing a composite enzyme: cellulase, lipase, and β-glucosidase are combined in a mass ratio of 15:8:5; S2, enzymatic pretreatment: 1000 g of macadamia nut kernel was crushed to 60 mesh, then added to a citric acid-disodium hydrogen phosphate buffer solution with pH 5.5 at a solid-liquid ratio of 1:3 (g / L), and added with the compound enzyme in S1, and then enzymatically treated at 45°C for 5 hours to obtain an enzymatic hydrolysate; the moisture content of the macadamia nut kernel was ≤5%; the amount of the compound enzyme added was 1.0 wt% of the mass of the raw material; S3, supercritical CO2 primary extraction: the enzymatic hydrolysate was placed in a supercritical extraction device, and extracted at a pressure of 28 MPa, a temperature of 38°C, and a CO2 flow rate of 25 L / h for 2 hours to separate a first oil phase and a residue; S4, subcritical butane secondary extraction: the residue in S3 was subjected to subcritical butane dynamic extraction at a pressure of 0.8 MPa, a temperature of 45°C, and an extraction time of 2 hours to obtain a second oil phase; S5, molecular distillation purification: the first oil phase and the second oil phase were combined, and the light components were separated in a short-path molecular distillation device at a temperature of 190°C, a vacuum degree of 3 Pa, and a feeding rate of 1.0 mL / min, and a squalene-rich phase was collected to obtain a macadamia nut oil containing squalene.

[0045] Comparative Example 1 The same batch of macadamia nut kernels (the initial squalene content of the macadamia nut kernel raw material was 300 mg / kg) was used for comparative tests (the macadamia nut kernel raw material was not subjected to enzymatic treatment, and was treated by a traditional pressing method); Scheme 11 The macadamia nut kernel (moisture content ≤5%) was crushed to 60 mesh, and a pH 5.5 buffer solution was added at a solid-liquid ratio of 1:3 (g / L), and a traditional pressing method (without enzyme treatment) was used to prepare crude oil by a traditional screw pressing method (pressure 30 MPa, temperature 60-70°C), and refined macadamia nut oil was obtained after degumming, deacidification, and bleaching; the obtained nut oil was determined according to the determination method of Example 3.

[0046] Table 4: Determination results of squalene content, macadamia nut oil yield, and squalene yield of samples The enzymatic method (Scheme 5) is the optimal process for extracting macadamia nut squalene, which is significantly better than the traditional pressing method (Scheme 11). The enzymatic method is significantly better than other schemes with pure natural and high extraction efficiency (the squalene yield in Scheme 5 is 4.5 times higher than that in Scheme 11), and significantly improves the squalene content in the macadamia nut oil compared to the traditional pressing method.

[0047] Example 5 In this example, the antioxidant efficacy of the macadamia nut oil containing squalene obtained by different preparation processes was studied by DPPH free radical scavenging rate and IC 50 value experimental results; The sample to be tested (macadamia nut oil) is reacted with a DPPH solution, and the degree of decrease in absorbance at 517 nm is detected by spectrophotometry to quantitatively evaluate the antioxidant activity. Three groups are divided, which are a blank group, a control group and a test product group. The test product group is the macadamia nut oil with different squalene contents obtained by different extraction processes and different enzyme ratios in the above test scheme. The free radical scavenging rate results of the test product group are shown in Table 5 and Figure 1

[0048] DPPH is a stable purple free radical (maximum absorption wavelength 517 nm), and its single electron can be captured by an antioxidant, resulting in discoloration of the solution (decrease in absorbance); by measuring the change in absorbance, the free radical scavenging rate (the higher the scavenging rate, the stronger the antioxidant capacity) is calculated.

[0049] Table 5 DPPH free radical scavenging rate and IC 50 value experimental results Based on the above data analysis and Table 1, it can be seen that scheme 5 (cellulase: lipase: β-glucosidase = 15:8:5) is the most preferred: its DPPH scavenging rate (75.16%) is the highest, and its IC 50 (0.95 mg / mL) is the lowest, indicating that the antioxidant efficiency of natural ingredients is significantly improved by the synergistic effect of three enzymes (cellulase to break the wall to release active substances, lipase to hydrolyze lipid barriers, and β-glucosidase to convert high-activity free state). In cosmetics, strong antioxidant capacity can have multiple synergies: anti-photoaging (scavenging free radicals to prevent collagen degradation), skin lightening (melanin inhibition), barrier repair (lipid peroxidation prevention), synergistic sun protection, and anti-inflammatory stability, meeting the demand for high-efficiency natural anti-aging.

[0050] Example 6 This example studies the antioxidant efficacy of macadamia nut oil obtained by different preparation processes through zebrafish antioxidant experiments; Zebrafish antioxidant experiment: reactive oxygen species (ROS) are oxygen-containing chemically reactive chemicals, including peroxides, superoxides, hydroxyl radicals, etc. ROS is a natural byproduct of normal metabolism of oxygen and plays an important role in cell signaling and body balance. However, during ultraviolet exposure, ROS levels increase dramatically, causing serious damage to cell structure, which is called oxidative stress. Menaquinone can produce active oxygen free radicals, and when the amount of free radicals is greater than the body's clearance capacity, oxidative stress occurs, and superoxide dismutase (SOD) activity decreases during oxidative stress. Therefore, the antioxidant effect of the test sample can be evaluated by zebrafish yolk sac fluorescence intensity and SOD activity.

[0051] ​The experiment was divided into a normal group, a model group, and an experimental group (test sample or positive control drug). In the zebrafish antioxidant experiment, compared with the model group, the fluorescence intensity of the zebrafish yolk sac was significantly reduced ( P <0.05), possessing antioxidant effects. The test groups consisted of different extraction processes and enzyme ratios used in this experiment. Statistical analysis was performed using Dunnett's T-test, with the normal control group used as the standard, to compare the yolk sac staining intensity of each experimental group. P <0.05 indicates a significant difference.

[0052] The experimental group used the test sample or positive control (N-acetyl-L-cysteine) for antioxidant experiments at a dosage of 1 mg / mL.

[0053] The other control groups used macadamia nut oil containing squalene obtained from their own experiments, at a concentration of 1 mg / mL.

[0054] The calculation formula is as follows: Antioxidant efficacy (%) = ×100% Wherein, S (model control group) is the fluorescence intensity of the modeling agent containing menaquinone; S (sample group) represents the fluorescence intensity of the sample. S (normal group) represents the fluorescence intensity of water containing only fish. Table 6 Antioxidant efficacy of the test group The results are shown in Table 6. Figure 2 and Figure 3 As shown, based on comprehensive in vitro antioxidant data and key in vivo zebrafish model validation results (lowest yolk sac brightness), Scheme 5 was determined to be the optimal scheme. Its antioxidant components exhibit excellent bioavailability, stability, and in vivo activity, efficiently translating in vitro potential into real biological protective effects. Therefore, the application of potent antioxidants (such as Scheme 5) in cosmetics is crucial. It effectively neutralizes free radicals, thereby delaying skin aging (reducing wrinkles and sagging), mitigating photodamage, providing anti-inflammatory and soothing effects, brightening skin tone, strengthening the skin barrier, protecting other active ingredients, and ultimately improving overall skin health and appearance. Based on its superior in vitro and in vivo performance, Scheme 5 holds promise as a core ingredient in highly effective anti-aging skincare formulations.

[0055] It should be noted that although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

Claims

1. A method of preparing macadamia oil characterised in that, The method comprises the following steps: S1, adding macadamia nut kernels and complex enzymes into citric acid-disodium hydrogen phosphate buffer solution for enzymolysis to obtain an enzymolysis material; The complex enzyme is compounded by cellulase, lipase and beta-glucosidase at a mass ratio of 15:8:5; The addition amount of the complex enzyme is 1.0% of the mass of the macadamia nut kernels; S2, separating the enzymolysis material in S1 after supercritical CO2 extraction to obtain a first oil phase and a residue; S3, separating the residue in S2 after subcritical butane dynamic extraction to obtain a second oil phase; S4, mixing the first oil phase obtained in S2 and the second oil phase obtained in S3, and collecting heavy components after short-path molecular distillation to obtain macadamia nut oil.

2. The production method according to claim 1, wherein The temperature of the enzymolysis in S1 is 40-50℃, and the time is 4-6 hours.

3. The production method according to claim 1, wherein The specific steps of the supercritical CO2 extraction in S2 include: extracting under the conditions of a pressure of 25-30 MPa, a temperature of 35-40℃, a CO2 flow rate of 20-30 L / h, and for 1.5-2.5 hours.

4. The production method according to claim 3, wherein The specific steps of the supercritical CO2 extraction in S2 include: extracting under the conditions of a pressure of 28 MPa, a temperature of 38℃, a CO2 flow rate of 25 L / h, and for 2 hours.

5. The production method according to claim 1, wherein The pressure of the subcritical butane dynamic extraction in S3 is 0.5-1.2 MPa, the temperature is 40-50℃, and the time is 1-3 hours.

6. The production method according to claim 1, wherein The temperature of the short-path molecular distillation in S4 is 180-200℃, and the vacuum degree is ≤5 Pa.

7. The production method according to claim 1, wherein The pH of the citric acid-disodium hydrogen phosphate buffer solution is 5.0-6.

5.

8. The production method according to claim 1, wherein The solid-liquid ratio of the macadamia nut kernels to the citric acid-disodium hydrogen phosphate buffer solution is 1:(2-4) (g / L).

9. The production method according to claim 1, wherein The particle size of the macadamia nut kernels is 40-80 mesh.

10. The use of the macadamia nut oil prepared by the preparation method according to any one of claims 1-9 in cosmetics.

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