Pine nut oil nano microcapsule and preparation method thereof

Abstract

The invention provides pine nut oil nano microcapsules and a preparation method thereof. The preparation method comprises the following steps: step 1, drying pine nuts to constant weight, and crushing to obtain pine nut powder; step 2 and step 3, carrying out supercritical CO2 extraction on the pine nut powder at the temperature of 47-55 DEG C, the pressure of 25-35 Mpa, the CO2 flow of 19-22 L / h and the extraction time of 120-180 min to obtain pine nut oil, and step 4, carrying out urea inclusion on the pine nut oil to obtain unsaturated fatty acid; 5, unsaturated fatty acid is subjected to microencapsulation to form solid powder, the oxidation stability of the solid powder is improved, the fluidity is good, the solid powder can be conveniently added into food, and the flavor and nutrition of the food are increased. The cold pressing-supercritical CO2 extraction technology combination yield reaches 59.5%-61%, the oil content of the meal obtained after oil extraction is low, and the method can be used for development of high-value products of polysaccharide, protein and peptide of the pine meal in the later period. Moreover, the method is green and environment-friendly, not only solves various problems in the traditional extraction method, but also has important economic value for fine and deep development and utilization of pine nuts.

Description

Technical Field

[0001] The invention relates to the technical field of pine nut oil nano-microcapsules, and in particular to pine nut oil nano-microcapsules and a preparation method thereof. Background Art

[0002] In recent years, my country's development of Pinus pumila resources has mainly focused on Pinus pumila kernels. Pinus pumila kernels are rich in nutrients, high in oil content, and rich in unsaturated fatty acids. The oil composition is similar to olive oil and apricot oil. Due to the poor oxidative stability and low bioavailability of oils, nano-microcapsule encapsulation technology is used to block the invasion of exogenous oxidants such as reactive oxygen species, so as to achieve the purpose of improving processing oxidative stability and use oxidative stability. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a pine nut oil nano-microcapsule and a preparation method thereof, comprising the following steps:

[0004] Step 1: drying the pine nuts to a constant weight, then crushing and sieving to obtain pine nut powder;

[0005] Preferably, the pine seeds in step one are Pinus tabebuia seeds.

[0006] Step 2: Pressing pine nut powder at 35-60 MPa and 25-65° C. for 20-50 minutes to obtain pine nut meal and primary oil, centrifuging the primary oil at 8000 rpm for 15 minutes, and obtaining the supernatant as pine nut oil;

[0007] Preferably, pressing is performed at 55 MPa and 65° C. for 40 min.

[0008] Step 3: Extract the pine nut powder with supercritical CO2 at a temperature of 47-55°C, a pressure of 25-35 MPa, a CO2 flow rate of 19-22 L / h, and an extraction time of 120-180 min to obtain pine nut oil. Preferably, the temperature is 55°C, the pressure is 32.6 MPa, and the CO2 flow rate is 21 L / h.

[0009] Pine nut oil can also be extracted using an ultrasound-assisted solvent extraction method, wherein the extractants are petroleum ether and ethyl acetate, the ultrasonic time is 40-60 minutes, the ultrasonic temperature is 45-55°C, the ultrasonic power is 300-500W, and the ratio of the extractant to the pine nuts (liquid-to-material ratio) is (30-80) mL:1g. Preferably, the ratio of petroleum ether to ethyl acetate is 40:60, the ultrasonic time is 40 minutes, the ultrasonic power is 350W, the liquid-to-material ratio is 40 mL:1g, and the ultrasonic temperature is 55°C.

[0010] Step 4: Mix and stir pine seed oil, urea, and ethanol solution. Throughout the process, introduce nitrogen to isolate the air. After cooling to room temperature, stir in a constant temperature water bath at 78 °C until it becomes clear. Throughout the process, introduce nitrogen to isolate the air. After naturally cooling to room temperature, conduct an inclusion reaction at -20 to 10 °C for 6 to 26 hours. Filter, and wash the filtrate with distilled water until the lower layer liquid is clear and transparent. After combined use of rotary evaporation and nitrogen purging, unsaturated fatty acids are obtained.

[0011] Preferably, the mass ratio of the pine seed oil to urea is 1:(1 - 6), and the mass ratio of the urea to the ethanol solution is 1:(3 - 8). Most preferably, the volume fraction of the ethanol solution is 95%.

[0012] Step 5: Mix whey protein and gum arabic with a mass ratio of 2.5:1 evenly. Add unsaturated fatty acids according to a wall-core ratio of 1:1, and conduct high-speed dispersion, homogenization, and emulsification at 10000 rpm for 3 minutes. Keep the system temperature at 45 °C. After adjusting the pH to 3.8 with 10% acetic acid, conduct complex coacervation for 60 minutes. Cool down to below 10 °C in an ice-water bath to end the reaction. Adjust the pH of the system to 6 with NaOH and stir and solidify at 10 °C for 30 minutes. Place it in a -80 refrigerator, and finally conduct freeze-drying.

[0013] The present invention has the following advantages:

[0014] The oil of Pinus pumila nuts forms solid powder after microencapsulation, which increases its oxidation stability, has good fluidity, is convenient to be added to food, and adds flavor and nutrition to food. This also expands the application range of the oil of Pinus pumila nuts, and provides a theoretical basis and practical experience for the development of functional foods and functional oils of the oil of Pinus pumila nuts in the future, having certain social value and economic value. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0016] Figure 1 It is a comparison chart of the oil content, acid value, and fatty acids of four pine seeds.

[0017] Figure 2 It is an optimization chart of low-temperature pressing parameters.

[0018] Figure 3 It is an optimization chart of ultrasonic-assisted solvent extraction parameters.

[0019] Figure 4 It is an optimization chart of supercritical CO2 extraction parameters.

[0020] Figure 5 It is the response surface of supercritical CO2 extraction.

[0021] Figure 6 It is the kinetic curve of supercritical CO2 extraction.

[0022] Figure 7 It is the comparison of the yield of Pinus pumila seed oil and the oil content in the meal.

[0023] Figure 8 It is the sensory evaluation chart of Pinus pumila seed oil.

[0024] Figure 9 It is the scavenging ability of Pinus pumila seed oil extracted by different methods on DPPH free radicals.

[0025] Figure 10 It is the diagram of the single-factor experiment of urea inclusion.

[0026] Figure 11 It is the diagram of the response surface optimization experiment of urea inclusion.

[0027] Figure 12 It is the diagram of wall material screening.

[0028] Figure 13 It is the diagram of the effect of pH on the absorbance of whey protein emulsion.

[0029] Figure 14 It is the diagram of the effect of each factor on the flocculation rate.

[0030] Figure 15 It is the determination of the maximum absorption wavelength of urea-included Pinus pumila oil.

[0031] Figure 16 It is the diagram of the effect of each factor on the microcapsule embedding rate.

[0032] Figure 17 It is the diagram of the effect of wall material concentration on the microcapsule embedding rate.

[0033] Figure 18 It is the zeta potential distribution diagram of the microcapsule.

[0034] Figure 19 It is the scanning electron microscope image of the microcapsule.

[0035] Figure 20 It is the transmission electron microscope image of the microcapsule.

[0036] Figure 21 It is the infrared spectrum analysis diagram.

[0037] Figure 22 It is the TG analysis diagram of the microcapsule.

[0038] Figure 23 It is the diagram of the study on the in vitro digestion characteristics of the microcapsule. Specific Embodiments

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0040] Test Example 1

[0041] Four kinds of nuts (pecans, hazelnuts, Korean pine nuts, and dwarf pine nuts) were placed in a drying oven (50 ± 1 °C) and dried to a constant weight. The oil content and acid value of pecans and hazelnuts were measured, and the oil content, acid value, and fatty acid content of Korean pine nuts and dwarf pine nuts were measured. The method referred to GB5009.6-2016 "National Food Safety Standard - Determination of Fat in Foods" for the determination of the four kinds of pine nuts, and the results are shown in Table 1 and Figure 1 .

[0042] Table 1

[0043]

[0044]

[0045] From Table 1 and Figure 1 it can be seen that the dwarf pine nuts, Korean pine nuts, and pecans have relatively high oil contents, reaching as high as 65.54%, 63.41%, and 63.87% respectively. The acid values of several oil samples are 0.42 mg / g, 3.11 mg / g, 0.33 mg / g, and 0.24 mg / g respectively, all of which meet the national food safety standards (≤ 4 mg / g). The acid value is one of the important indicators for evaluating the degree of deterioration of oils and fats. The level of the acid value represents the amount of free fatty acids produced by the hydrolysis of triglycerides in the oil. The oxidation and hydrolysis of oils and fats will both cause the acid value of the oil to rise. Generally speaking, the dwarf pine nut oil and Korean pine nut oil are the freshest. The content of unsaturated fatty acids in the dwarf pine nuts is slightly higher than that in the Korean pine nuts, which is 91.03%, and the dwarf pine nut oil contains 59.12% of polyunsaturated fatty acids. Therefore, the dwarf pine nuts are high-quality oils and fats. Finally, the dwarf pine nuts were selected as the experimental object among the four kinds of pine nuts.

[0046] Test Example 2 (Cold Press Extraction)

[0047] Clean the oil press, connect the power supply, preheat it to 45°C for 10 minutes, screen and clean the broken shells and mildewed parts in the Korean pine seeds, select 500 g of samples with uniform and plump particles, and then pour them into the oil press. Apply a pressure of 40 MPa and a pressing time of 30 minutes, collect the freshly cold-pressed Korean pine oil, centrifuge it at 8000 r / min for 15 minutes, weigh the oil layer as m, after cooling, fill it with nitrogen, and store it at -20°C for standby.

[0048] Change the pressure to 30, 35, 40, 45, 50, 55, 60 MPa respectively; change the pressing time to 10, 20, 30, 40, 50 minutes respectively, and change the pressing temperature to 25, 35, 45, 55, 65°C respectively, and detect the yield of Korean pine oil. The results are as Figure 2 .

[0049] It can be Figure 2 seen that the yield of cold-pressed Korean pine oil increases gradually with the increase of pressing pressure. When the pressing pressure increases, the mechanical external force increases and the oil is separated out. The yield reaches the maximum (52.53%) at a pressing pressure of 55 MPa. Also, for the safety of the experiment, 55 MPa is selected as the optimal pressing pressure. The yield of cold-pressed Korean pine oil increases slightly with the increase of pressing temperature. The yield is the largest (51.63%) when the pressing temperature is 65°C. It may be because the cells of Korean pine seeds are broken by heat at a certain temperature and it is easier to separate out the oil. However, when the pressing temperature reaches above 65°C, the high temperature will affect the oil quality. To reduce energy consumption and improve the oil quality, 55°C is selected as the optimal pressing temperature. The yield of Korean pine seeds increases gradually with the extension of pressing time. When the pressing time is 40 minutes, the yield reaches the maximum value (50.36%). It may be because a large amount of oil in Korean pine seeds is separated out at the beginning and the oil extraction rate is relatively high. After reaching the peak, most of the oil in Korean pine seeds has been extracted and the yield change is not significant. Therefore, 40 minutes is selected as the optimal pressing time.

[0050] Press with the optimal parameters of temperature, pressure and time respectively, and press with all the optimal parameters of temperature, pressure and time, and measure the yield of Korean pine oil. The results are shown in Table 2.

[0051] Table 2

[0052] Pressing Pressure (MPa) Pressing Temperature (°C) Pressing Time (min) Yield Rate (%) Group 1 55 45 30 52.53 Group 2 40 55 30 50.88 Group 3 40 45 40 50.36 Group 4 55 55 40 52.69

[0053] It can be seen from Table 2 that the pressing temperature is 55°C, the pressing pressure is 55 MPa, and the pressing time is 40 minutes. Under this process condition, the highest oil yield reaches 52.69%, which is significantly higher than the yields of other groups. Therefore, the optimal cold-pressing conditions are obtained.

[0054] Experimental Example 3 (Ultrasound-Assisted Solvent Extraction)

[0055] Weigh 3.00 g of pre-treated Korean pine nuts, control the ultrasonic time to 30 min, the ultrasonic temperature to 40 °C, the ultrasonic power to 350 W, and the ratio of the extractant to Korean pine nuts (liquid-to-solid ratio) to 10 mL:1 g.

[0056] The extractant is petroleum ether and ethyl acetate. The volume ratios of petroleum ether to ethyl acetate are set to 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0 respectively to investigate the effect of different ratios on the yield of Korean pine oil. The results are shown in Figure 3 .

[0057] Control the ratio of petroleum ether to ethyl acetate to 60:40, the liquid-to-solid ratio to 10 mL:1 g, the ultrasonic temperature to 40 °C, and the ultrasonic power to 350 W. Investigate the effect of different ultrasonic times (10, 20, 30, 40, 50, 60 min) on the yield of Korean pine oil. The results are shown in Figure 3 .

[0058] Control the ratio of petroleum ether to ethyl acetate to 60:40, the liquid-to-solid ratio to 10 mL:1 g, the ultrasonic time to 30 min, and the ultrasonic temperature to 40 °C. Investigate the effect of different ultrasonic powers (250, 300, 350, 400, 450, 500 W) on the yield of Korean pine oil. The results are shown in Figure 3 .

[0059] Control the ratio of petroleum ether to ethyl acetate to 60:40, the ultrasonic power to 350 W, the ultrasonic time to 30 min, and the ultrasonic temperature to 40 °C. Investigate the effect of different liquid-to-solid ratios (5 mL:1 g, 10 mL:1 g, 20 mL:1 g, 40 mL:1 g, 80 mL:1 g) on the yield of Korean pine oil. The results are shown in Figure 3 .

[0060] Control the ratio of petroleum ether to ethyl acetate to 60:40, the liquid-to-solid ratio to 10:1 mL / g, the ultrasonic time to 30 min, and the ultrasonic power to 350 W. Investigate the effect of different ultrasonic temperatures (30, 35, 40, 45, 50, 55 °C) on the yield of Korean pine oil. The results are shown in Figure 3 .

[0061] From Figure 3It can be seen that the influence of ultrasonic time on the yield of Pinus pumila oil shows that with the extension of extraction time, the extraction rate of Pinus pumila oil first increases and then levels off, reaching the maximum value of 59.83% at 40 min. When the ultrasonic power increases from 250 W to 350 W, the oil yield increases and reaches the maximum value of 60.45% at 350 W. After that, the yield decreases with the increase of ultrasonic power. This may be because with the extension of time, impurities such as insoluble substances in pine nuts are suspended in the extract or re-adsorbed onto the broken tissue particles, reducing the permeability of the solvent to the cell structure. The influence of the liquid-to-solid ratio on the yield of Pinus pumila oil shows that with the increase of the solvent dosage, the yield of Pinus pumila oil gradually increases and finally levels off. Since the dielectric constant of organic solvents is approximately equal to that of triglycerides, it is easy to immerse into the interior of cells using organic solvents such as petroleum ether and ethyl acetate, thereby effectively dissolving lipids. The gradual increase between (5 - 40) mL:1 g is mainly due to the concentration gradient between the solid and liquid phases giving the driving force in the mass transfer process. However, when the solvent is too large, for the particles deposited at the bottom, the intensity of cavitation and mechanical effects generated by ultrasound weakens, resulting in waste of the solvent and increasing the difficulty of subsequent work. Therefore, it is appropriate to select a liquid-to-solid ratio of 40 mL:1 g. The influence of ultrasonic temperature on the yield of Pinus pumila oil shows that the yield of Pinus pumila oil continuously increases with the increase of extraction temperature. The increase in temperature increases the solubility of the oil in the solvent, reduces the solvent density and viscosity, and accelerates the diffusion of the oil, resulting in an increase in the yield. However, due to too high temperature affecting the quality of the oil. Finally, the process for extracting Pinus pumila oil by ultrasonic-assisted mixed solvent is selected as follows: petroleum ether:ethyl acetate is 40:60, ultrasonic time is 40 min, ultrasonic power is 350 W, liquid-to-solid ratio is 40 mL:1 g, and ultrasonic temperature is 55 °C.

[0062] Experimental Example 4 (Cold Pressing - Supercritical CO2 Extraction)

[0063] The meal after low-temperature pressing was crushed and passed through a 20-mesh sieve to obtain Pinus pumila kernel meal. 150.0 g of Pinus pumila kernel meal and 150.0 g of filler were accurately weighed and loaded into a 1-L extraction kettle. Supercritical CO2 gas was introduced according to an extraction temperature of 51 °C, an extraction pressure of 25 MPa, and a CO2 flow rate of 16.8 L / h to continuously extract Pinus pumila kernel oil for 3 h. After the CO2 gasified, it was recycled. The extract entered the separation kettle for separation and was discharged from the bottom to obtain Pinus pumila kernel oil.

[0064] The extraction temperature (39 °C, 43 °C, 47 °C, 51 °C, 55 °C, 59 °C), extraction pressure (15 MPa, 20 MPa, 25 MPa, 30 MPa, 35 MPa), and CO2 flow rate (12.0 L / h, 14.4 L / h, 16.8 L / h, 19.2 L / h, 21.6 L / h, 24 L / h) were changed respectively.

[0065] Yield percentage of supercritical CO2 extraction = (m1 - m0) / m × 100%

[0066] Where: m1 is the mass of the grease receiving bottle and the oil / g, m0 is the mass of the grease receiving bottle / g, and m is the mass of the sample (Pinus pumila kernel meal) / g.

[0067] It can be seen from Figure 4 that under constant pressure and constant CO2 flow rate, the yield of Pinus pumila oil first gradually increases with the increase of extraction temperature. However, when the temperature exceeds 55 °C, the yield rapidly decreases. This may be because the increase in temperature also increases the vapor pressure of the compound and makes the process easier for extraction; the increase in temperature also increases the molecular motion and the kinetic energy, so the mass transfer rate and yield of the oil increase. Therefore, the possibility of the compound diffusing from the solid matrix to the SC-CO2 fluid increases. However, with the increase of the extraction temperature, the decrease in yield may be because with the increase of temperature, the density of SC-CO2 decreases, and the influence of the solvent density is slightly greater than that of the vapor pressure, resulting in a decrease in the dissolution ability of SC-CO2.

[0068] Under constant temperature and constant CO2 flow rate, the yield of Pinus pumila oil first gradually increases with the increase of extraction pressure and finally levels off with the increase of pressure. When the extraction pressure increases from 15.0 MPa to 30 MPa, the yield increases from 11.30% to 44.72%. This trend is due to the increase in the density of SC-CO2, which increases the diffusion rate of the oil, resulting in an increase in the oil yield. This may be because the distance between the solute and solvent molecules decreases with the increase of pressure, and the density significantly affects the solubility of the solute in the SC-CO2 fluid, resulting in an enhanced interaction between the oil and SC-CO2, and the solubility of the oil in SC-CO2 increases with the increase of the solvent density. However, with the increase of the extraction pressure, the yield of Pinus pumila oil levels off. This may be due to the low diffusion rate of Pinus pumila oil from the plant matrix to SC-CO2 at high working pressures.

[0069] Under constant pressure and constant temperature, the yield of Pinus pumila oil first gradually increases with the increase of CO2 flow rate and finally levels off with the increase of pressure. Generally speaking, the increase in flow rate is beneficial to the yield. The higher the flow rate, the stronger the contact between SC-CO2 and the oil in the solid matrix can be enhanced, and the mass transfer rate can be improved. The subsequent leveling-off trend may be due to the decrease in the residence time of SC-CO2 caused by the increase in flow rate, so a channeling effect may be triggered in the extraction kettle and affect the output.

[0070] Table 3

[0071]

[0072] Table 4

[0073] Experiment Number A Temperature / °C B Pressure / MPa <![CDATA[CCO2 flow rate / L / h]]> Yield Rate / % 1 47 30 19.2 43.12 2 47 35 21.6 47.1 3 51 35 19.2 46.9 4 55 25 21.6 44.95 5 51 25 24 44.56 6 55 30 19.2 46.89 7 51 30 21.6 46.97 8 55 30 24 47.11 9 47 30 24 46.28 10 55 35 21.6 47.61 11 47 25 21.6 39.73 12 51 30 21.6 46.76 13 51 30 21.6 47.12 14 51 30 21.6 45.36 15 51 35 24 47.73 16 51 30 21.6 46.88 17 51 25 19.2 40.56

[0074] Table 5

[0075] Source of Variance Sum of Squares Degree of Freedom Mean Square F Value P Value Significance Model 89.15 9 9.91 27.70 0.0001 Significant A 13.34 1 13.34 37.30 0.0005 B 47.73 1 47.73 133.45 ＜0.0001 C 8.43 1 8.43 23.56 0.0018 AB 5.55 1 5.55 15.51 0.0056 AC 2.16 1 2.16 6.04 0.0436 BC 2.51 1 2.51 7.02 0.0329 A2 0.77 1 0.77 2.17 0.1845 B2 7.58 1 7.58 21.19 0.0025 C2 0.48 1 0.48 1.35 0.2829 Residual 2.50 7 0.36 Lack of Fit 0.46 3 0.15 0.2971 0.8268 Not Significant Pure Error 2.05 4 0.51 Total Deviation 91.65 16

[0076] It can be seen from the model variance analysis in Table 3-5 that the regression equation model of this experiment has significance with P < 0.05, and the lack-of-fit term is not significant (P = 0.828 > 0.05), indicating that the model fitting is successful. The quadratic regression model equation for the supercritical CO2 yield is: Y = -356.44 + 6.48A + 8.14B + 8.86C - 0.06AB - 0.08C - 0.07BC - 0.03A 2 - 0.05B 2 - 0.06C 2 。

[0077] The optimal process parameters of cold pressing combined with supercritical CO2 determined by single factor and response surface experiments are: temperature 55°C, pressure 32.6 MPa, and CO2 flow rate 21 L / h.

[0078] The time required for extracting Korean pine nut oil with SFE-CO2 was determined through experimental kinetics. First, a series of amber bottles were weighed. Then, 150.0 g of dry and ground sample and 150.0 g of filler were weighed and loaded into a 1 L extraction kettle for kinetic analysis. The system was set at 32.6 MPa and 55°C. The oil samples were collected in amber bottles at preset time intervals: once every 5 min within the first 120 min of extraction; and finally once every 30 min from 120 min to 180 min. The CO2 flow rate was set at 21 L / h. Finally, the amber bottles were weighed again, the relationship between the cumulative mass (g) and the extraction time (min) was obtained, and the extraction kinetic curve was constructed.

[0079] The time required for extracting Korean pine kernel oil with supercritical CO2 was determined through experimental kinetics. The relationship between the cumulative mass (g) and the extraction time (min) was obtained, and the extraction kinetic curve was constructed. It can be seen that from 0 to 60 min, the yield increased rapidly with time; from 60 to 120 min, the speed gradually slowed down; and from 120 to 180 min, the yield of Korean pine kernel oil tended to be flat. Therefore, it is appropriate to select 150 min for the extraction time.

[0080] According to Figure 7 it can be known that the yields of cold pressing - supercritical CO2 and cold pressing - ultrasonic assisted mixed solvent method are similar. Among them, the oil content in the meal of the cold pressing - ultrasonic assisted mixed solvent method is the lowest at 9.69%, and that of cold pressing - supercritical CO2 is 11.51%. Although the leaching method has a high yield, it usually uses high temperature, the solvent is toxic, and it often requires an evaporation step, and the produced oil inevitably has solvent residues. Therefore, the cold pressing - supercritical CO2 method has a high yield, a relatively low oil content in the meal, and is environmentally friendly, making it a potential method for extracting oils.

[0081] Test Example 5 (Sensory Evaluation)

[0082] Improve according to the evaluation criteria of GB 15196—2015 "National Food Safety Standard Edible Oil Products" (see Table 6 for details). Select 10 professional sensory evaluators to form an evaluation group. When evaluating, adopt a random method to comprehensively evaluate the color, taste, smell, viscosity, and transparency of Korean pine nut oil prepared by cold pressing-supercritical CO2 extraction, cold-pressed Korean pine nut oil, and Korean pine nut oil extracted by ultrasonic-assisted mixed solvent method. The geometric mean value is taken as the result.

[0083] Table 6

[0084]

[0085]

[0086] According to Figure 8 It can be seen that the Korean pine nut oil prepared by the cold pressing-supercritical CO2 method has the best scores in the five sensory evaluation indexes of color, smell, taste, viscosity, and transparency, and all indexes are significantly better than those of Korean pine nut oil prepared by the cold pressing-ultrasonic-assisted mixed solvent method and the cold pressing method. It shows that the Korean pine nut oil prepared by the cold pressing-supercritical CO2 method not only has good sensory quality but also has good physicochemical properties. It can be used as a potential oil extraction method and can also be used in the fields of food and cosmetics.

[0087] Test Example 6 (Free Radical Removal)

[0088] Preparation of Korean pine nut kernel oil samples: Take 5 g of low-temperature pressed Korean pine nut kernel oil, Korean pine nut kernel oil extracted by ultrasonic-assisted mixed solvent method, and supercritical CO2 Korean pine nut kernel oil respectively and place them in two 50 mL volumetric flasks. Dilute to 50 mL with ethyl acetate:ethanol mixture (1:2) to obtain a 100 mg / mL sample solution, and dilute it step by step to 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 mg / mL for standby.

[0089] Preparation of DPPH mixture: Accurately weigh 0.0058 g of DPPH into a beaker, add 100 mL of ethanol to dissolve it fully, store it in the dark, and prepare it for use immediately.

[0090] Take 1.5 mL of the sample (Korean pine nut kernel oil) and add 1.5 mL of DPPH detection solution, mix well. Let it stand in the dark at room temperature for 30 min. Measure the absorbance value at a wavelength of 517 nm with a UV spectrophotometer. The calculation formula is as follows:

[0091]

[0092] In the formula: A0 is the absorbance value of 1.5 mL of ethyl acetate-ethanol (1:2) and 1.5 mL of DPPH mixture;

[0093] A1 is the absorbance value of the mixture of 1.5 mL of the sample and 1.5 mL of the DPPH mixture;

[0094] A2 is the absorbance value of the mixture of 1.5 mL of the sample and 1.5 mL of the ethyl acetate - ethanol (1:2) mixture.

[0095] According to Figure 9 It can be seen that the free radical scavenging ability of the Pinus pumila oil extracted by several methods increases with the increase of concentration. In the range of 5 - 70 mg / mL, its scavenging rate is positively correlated with the mass concentration, showing a good logarithmic linear relationship. When the mass concentration continues to increase, the scavenging rate changes little and gradually flattens out. The DPPH free radical scavenging rates of the Pinus pumila oil extracted by several methods are not very different.

[0096] Through the comparison of the yield of Pinus pumila kernel oil and the oil content in the meal, the sensory evaluation of Pinus pumila kernel oil, and the DPPH free radical scavenging ability, cold pressing combined with supercritical CO2 is determined as the optimal process. It shows that cold pressing combined with supercritical CO2 is green and environmentally friendly. It not only solves various problems in the above traditional extraction methods, but also has a low oil content in the meal after oil extraction, which can be used for the development of high - value products such as polysaccharides, proteins, and peptides in Pinus pumila meal in the later stage. It also has important economic value for the intensive development and utilization of Pinus pumila.

[0097] Experimental Example 7 (Single - factor experiment on urea inclusion)

[0098] Accurately weigh 5 g of Pinus pumila kernel oil and add a certain amount of urea and 95% ethanol solution respectively. Stir in a constant - temperature water bath at 78 °C until it becomes clear, and the reaction is protected by nitrogen. Then take it out and cool to room temperature, and place it in a low - temperature environment at a certain temperature for urea inclusion reaction. Filter the sample after urea inclusion, collect the filtrate, wash it with distilled water until the lower layer liquid is clear, and perform rotary evaporation and nitrogen blowing on the upper layer liquid to finally enrich and obtain ethyl esters of fatty acids in Pinus pumila kernel oil. Respectively change the inclusion time to 6, 10, 14, 18, 22, 26 h, the inclusion temperature to - 20, - 15, - 10, - 5, 0, 5, 10 °C, the ratio of ethyl esters of fatty acids in Pinus pumila kernel oil to urea (mass ratio) to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and the ratio of urea and 95% ethanol solution (mass ratio) to 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, and investigate the effects of these indexes on the iodine value respectively.

[0099] According to Figure 10 It can be seen that when the inclusion temperature is 0 °C, the iodine value reaches the maximum value. When the temperature rises above 0 °C, the iodine value of Pinus pumila oil drops sharply. This phenomenon can be explained as follows: the urea inclusion reaction is an exothermic process. When the temperature rises, the urea inclusion compound gradually decomposes, and the urea inclusion effect is poor; when the temperature is low, the urea inclusion compound gradually forms. However, when the temperature is too low, the filtration becomes more difficult and the loss of fatty acids is also greater, which will also lead to a poor urea inclusion effect. Therefore, the optimal inclusion temperature is 0 °C.

[0100] Sufficient reaction time also plays an important role during the reaction. It reaches the maximum value at an inclusion time of 22 h. When the inclusion time is decreased or increased, the iodine value decreases. This is because the inclusion reaction reaches equilibrium at 22 h. As the inclusion time is prolonged, the reaction proceeds in the opposite direction. Therefore, the optimal reaction time is 22 h.

[0101] In the urea inclusion method, the dosage of urea is a key factor affecting the inclusion effect of fatty acids. When the dosage of urea is too small, there are few hexagonal prism empty channels formed by urea inclusion compounds, and saturated or monounsaturated fatty acid ethyl esters cannot be included to the maximum extent, resulting in poor enrichment effect; while when the dosage of urea increases, the hexagonal prism empty channels of urea inclusion compounds increase, and more saturated or monounsaturated fatty acid ethyl esters are included by urea, making the content of polyunsaturated fatty acid ethyl esters increase accordingly; however, when the dosage of urea continues to increase after reaching a certain level, it will cause an excess of urea and a relative decrease in the content of polyunsaturated fatty acid ethyl esters. Therefore, it can be known from experiments that the mass ratio of fatty acid ethyl ester to urea in the subsequent experiments is 1:4.

[0102] The solvent is the place where urea includes fatty acids, and at the same time, it also plays the functions of heat transfer and mass transfer. In the experiment, we found that when the amount of absolute ethanol is small, it is difficult to dissolve urea sufficiently, the resistance between solution molecules increases, which is not conducive to the reaction; when there is too much ethanol, part of the urea dissolves in ethanol and does not precipitate in the form of crystals. During the subsequent rotary evaporation treatment, as the ethanol content decreases, urea reaches saturation again and finally precipitates in the form of including linoleic acid, which will also cause the iodine value to decrease and make it difficult to recover the solvent. Therefore, in the subsequent experiments, the ratio of ethanol to urea should be preferably 4:1.

[0103] According to Figure 11 it can be known that the optimal temperature is 0 °C, the time is 22 h, the mass ratio of fatty acid ethyl ester to urea is 1:4, and the ratio of urea to ethanol is 1 g:4 mL.

[0104] Test Example 8 (Preparation of Unsaturated Fatty Acid Nanomicrocapsules from Korean Pine Seed Oil)

[0105] After several wall materials are respectively mixed evenly with gum arabic, the pH value is adjusted with 10% acetic acid under stirring conditions, and complex coacervation is continued for 30 min. Subsequently, the supernatant after the coacervation reaction is collected, and its absorbance is measured at a wavelength of 280 nm to evaluate the coacervation effect of Korean pine seed oil, whey protein, gelatin and gum arabic. A higher absorbance value means a higher protein content in the supernatant, indicating a poor coacervation effect. At the same time, the coacervate is centrifuged at 8000 rpm for 10 minutes, and then the yield of the coacervate is determined by the constant weight method. (Screening of Wall Materials)

[0106] Take 2 g each of Korean pine seed oil, whey protein, and gelatin, dissolve them separately in 100 mL of hot water at 100 °C, stir with a magnetic stirrer for 10 min. Take 20 mL of each solution in a beaker, add 0.5 mL of Korean pine seed kernel oil to each solution, and stir for 30 min to form an emulsion. Place the emulsion in a 50 mL graduated centrifuge tube, record the initial total height of the emulsion, denoted as H. Let it stand in a 50 °C constant temperature water bath for 60 min, and then cool it to room temperature with tap water. After cooling, centrifuge at 1500 r / min for 10 min and record the height of the emulsion layer. (Emulsifying property)

[0107] Prepared a whey protein solution with a mass-volume ratio of 2%, as well as solutions of acetic acid (HAc) and sodium acetate (NaAc) with a concentration of 0.1 mol / L. Using these solutions, a series of buffer solutions with pH values between 4.0 and 5.0 were prepared. Subsequently, in each experimental test tube, 1 mL of the whey protein solution and 3 mL of the pre-adjusted buffer solution were added respectively to ensure thorough mixing. Then, an equal amount of absolute ethanol was added dropwise to each test tube using a dropper, and the test tubes were continuously shaken until the solution became turbid. To quantify this turbidity, a spectrophotometer was used to measure the percentage of light transmittance of each turbid solution at 600 nm. (Isoelectric point)

[0108] A certain amount of whey protein was mixed evenly with the arabic gum solution, and after adjusting the pH with 10% acetic acid under stirring conditions, complex coacervation was carried out for 30 min. The pH range was changed respectively, the ratio of whey protein to arabic gum was changed, and the concentration range of different wall materials was changed, and the effects of these indexes on coacervation were investigated respectively.

[0109] Bind Korean pine seed oil, whey protein, and gelatin with arabic gum respectively, and measure the absorbance of the supernatant after coacervation. The greater the absorbance, the higher the protein content in the supernatant and the worse the coacervation effect. According to Figure 12 It can be seen that the coacervation effect: Korean pine seed oil > whey protein > gelatin; among them, the emulsifying property: whey protein > Korean pine seed oil > gelatin; the coacervation rate: gelatin > whey protein > Korean pine seed oil. However, gelatin gels at room temperature to form a jelly. In summary, whey protein is selected as the protein-based wall material.

[0110] According to Figure 13 It can be seen that the absorbance is the largest at pH 4.4, that is, the light transmittance of the turbid solution is the lowest, indicating that the whey protein carries the least charge at this pH and polymerization precipitation occurs. When the pH is adjusted below the isoelectric point of whey protein and above the isoelectric point of arabic gum, it will lead to the formation of the coacervation phase.

[0111] According to Figure 14It can be seen that by changing the pH value to an appropriate size, the number of positive charges in the whey protein solution reaches the maximum, while the gum arabic maintains the original number of negative charges. At this time, coacervate products are most easily formed in the system. The absorbance reaches the highest point at pH 3.8. The system pH is lower than the isoelectric point of whey protein. At this time, the reaction between the positively charged whey protein and the negatively charged gum arabic is basically complete, so the coacervate yield reaches the highest. The interaction between protein and polysaccharide is the interaction of positive and negative charges. Only when the charges of the two reach equilibrium can the best coagulation effect be achieved. It first increases and then decreases with the increase of the wall material concentration. The absorbance reaches the highest value at a wall material concentration of 0.5%. The interaction between the two wall materials reaches the maximum.

[0112] The best coacervation effect is achieved when the ratio of WPI:GA is 2.5:1. At this time, the absorbance of the emulsion is the largest and the coagulation rate is the highest. Too low or too high a ratio will lead to waste of polysaccharide or protein. At the appropriate ratio, the charges between the two reach equilibrium, the electrostatic interaction is the strongest, and the coacervation effect is the best.

[0113] Experimental Example 9 (Determination of the microcapsule preparation process)

[0114] Determination of the maximum absorption wavelength of urea-included Pinus pumila oil

[0115] The urea-included Pinus pumila kernel oil is diluted with n-hexane at a certain concentration to prepare a Pinus pumila kernel oil - n-hexane solution at a certain concentration. Then, using n-hexane as a blank control group, the maximum absorption wavelength of the Pinus pumila kernel oil - n-hexane solution is measured by scanning in the wavelength range of 300 - 1000 nm.

[0116] Standard curve of urea-included Pinus pumila oil

[0117] The standard curve of the urea-included Pinus pumila kernel oil is determined by ultraviolet spectrophotometry. First, accurately weigh 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 g of Pinus pumila kernel oil and dissolve it in 10 mL of n-hexane, and then dilute it 1000 times with n-hexane respectively. Using n-hexane as a blank control, the absorbance of each diluted Pinus pumila kernel oil - n-hexane solution is measured at the maximum absorption wavelength of Pinus pumila kernel oil, and the corresponding relationship between the absorbance and the concentration of Pinus pumila kernel oil is obtained to draw the standard curve of Pinus pumila kernel oil.

[0118] Determination of the embedding rate

[0119] Accurately weigh 0.5 g of wet microcapsules and place them in a brown container. Add 20 mL of n-hexane and place it in a cool and dark place for 24 h. At the same time, assist with ultrasonic treatment to rupture the structure of the microcapsules, so that all the embedded Korean pine kernel oil is released. Then centrifuge at 8000 rpm for 15 min to ensure that the core material is completely separated from the wall material and dissolved in n-hexane. Collect the supernatant and dilute it, then measure its absorbance under ultraviolet light. Finally, according to the measured standard curve and dilution factor, calculate the total content of Korean pine kernel oil in the microcapsules.

[0120] Accurately weigh 0.5 g of wet microcapsules, wash and filter them three times with 20 mL of n-hexane, and combine the filtrates. Take the filtrate and dilute it, then measure its absorbance under ultraviolet light. Finally, according to the measured standard curve and dilution factor, calculate the content of surface Korean pine kernel oil, and then calculate the embedding rate of the microcapsules according to the following formula. Do three parallel groups and calculate the average value. Substitute this value into the above standard curve to obtain the surface oil and total oil of the microcapsules, and calculate the embedding rate accordingly.

[0121]

[0122] Add the Korean pine kernel oil obtained from the above experiment, disperse and homogenize it at high speed at 10000 rpm for 3 min, then dropwise add the well-homogenized gum arabic solution to the mixture, stir at 45 °C for 15 min, keep the system temperature at 45 °C, adjust the pH to 3.8 with 10% acetic acid, fix the complex coacervation time at 60 min, adjust the pH of the system to 6 with NaOH, stir and solidify at below 10 °C for 30 min to end the reaction, place it in a -80 °C refrigerator, and finally freeze-dry. Respectively change the core-wall ratio, emulsification rate, emulsification time, complex coacervation time, WPI and GA concentration and calculate the embedding rate of the microcapsules, and investigate the influence of these indexes on the embedding rate respectively.

[0123] Use a UV-visible spectrophotometer to perform a full-wavelength scan on the urea-included Korean pine seed oil-n-hexane solution, and find that the maximum absorption peak of the pine seed oil is at 450 nm. The results are shown in Figure 15 .

[0124] According to Figure 16 It can be seen that the embedding rate is the best when the core-wall ratio is 1. The effect is better in the range of 0.25 - 1. When the core-wall ratio is relatively large, the surface oil content is relatively high. It may be that as the core-wall ratio increases, the wall material adsorbed on the surface of the small oil droplets is less than the complex coacervate, and the wall material cannot form a good embedding and encapsulation effect on the core material, and the release rate of the core material will increase.

[0125] With the increase of the emulsification speed, the encapsulation rate of the microcapsules first increases and then decreases. When the emulsification speed is 10,000 rpm, the encapsulation rate reaches the maximum value of about 78%. If the emulsification speed is too low, the oil droplets cannot be well dispersed, resulting in the floating and aggregation of oil droplets. If the emulsification speed is too high, the hydrophobic interaction between droplets is enhanced, the aggregation degree between particles increases, and the microcapsules collide with each other, resulting in adhesion and a decrease in the encapsulation rate.

[0126] If the emulsification time is too short, the emulsion cannot be well dispersed, resulting in too large emulsion droplets, floating of oil droplets, and a decrease in the encapsulation rate. If the emulsification time is too long, during the process of some large oil droplets being dispersed, more medium-sized oil droplets will be dispersed into smaller oil droplets. The smaller oil droplets freely disperse in the water phase and re-aggregate, so that they cannot participate in the complex coacervation process, thus reducing the microencapsulation efficiency.

[0127] The encapsulation rate is the best when the complex coacervation time is 60 min. If the coacervation time is too long, due to a certain stirring speed, the formed microcapsules will be redispersed, resulting in a decrease in the encapsulation rate of the microcapsules.

[0128] According to Figure 17 it can be seen that when the wall material concentration is 0.75% and 1%, the encapsulation rates are both above 80%. Among them, the encapsulation rate reaches the highest at the wall material concentration of 1%. When the wall material concentration is low, the viscosity of the system is low, the encapsulation effect on the internal core material is poor, the core material is easy to overflow, the oxidation stability of the formed microcapsules is poor, and the encapsulation rate is low; when the wall material concentration increases, the viscosity of the system increases correspondingly, and the encapsulation effect becomes better; but when the viscosity of the system is too high, the wall materials are easy to form adhesions, which is not conducive to the formation of microcapsules.

[0129] Experimental Example 10 (Study on the properties of nanometer microcapsules of Korean pine seed oil)

[0130] Use a Malvern Zetasizer Nano ZS instrument (Malvern Instruments, UK) to measure the particle size and distribution width (represented by the polydispersity index PDI) of the emulsion droplets. Use the same instrument to measure the ζ potential of the emulsion.

[0131] Scanning electron microscope

[0132] Stick a layer of double-sided conductive adhesive on the sample stage. Use a toothpick to pick up a small amount of microcapsule powder and apply it on the conductive adhesive, and then use an ear bulb to blow away the excess powder. Then, after spraying gold on the sample, perform SEM observation. The acceleration voltage is 5 kV, and the time for electron microscope observation should be as short as possible to avoid electron damage caused by long-term irradiation of the electron beam.

[0133] Transmission electron microscope

[0134] Perform a more careful observation of the microstructure through a transmission electron microscope with an acceleration voltage of 75 kV. Prepare the sample by allowing the droplets to evaporate on top of a carbon-coated copper grid.

[0135] Fourier transform infrared spectroscopy

[0136] The Fourier transform infrared spectrometer was used to test the infrared spectra of gelatin, gum arabic, and pine seed oil microcapsules, respectively. The test spectral range was 400 - 4000 cm-1, and infrared spectral analysis was carried out within this range (400 times).

[0137] TG analysis

[0138] The thermal properties of the prepared microcapsules were tested using a differential thermal gravimetric analyzer in the temperature range from room temperature to 600 °C at a heating rate of 10 °C / min.

[0139] The particle size directly affects the appearance, fluidity, and dispensability of the powder. The polydispersity index is a key indicator of the oxidative stability of the emulsion because it is directly related to the uniformity of the droplet size. A lower PDI value indicates a more uniform distribution of droplet sizes and higher oxidative stability of the emulsion, while an increase in PDI reflects the instability of the emulsion, resulting in a high degree of variation in droplet size. In emulsions where coalescence and flocculation occur, the droplet sizes vary greatly, resulting in a higher PDI. Generally, a PDI value > 0.5 indicates a wide particle size distribution. The PDI of the microcapsules is 0.276 < 0.5, indicating a more uniform particle size distribution and higher oxidative stability. Moreover, a high negative or positive zeta potential (±30 mV) indicates sample stability. The zeta potential of the microcapsules is -36.3 mV, thus also indicating the oxidative stability of the sample ( Figure 18 ).

[0140] According to Figure 19 it can be seen that the microcapsules are spherical, with a relatively smooth surface, slightly adhered, and the particle size is about 200 nm, which is consistent with the particle size measurement results.

[0141] According to Figure 20 it can be seen that the microcapsules prepared under the optimal conditions are round or oval, and have a double-layer structure, with a complete appearance, appropriate size, and a smooth surface. And the particle size is about 200 nm, which is consistent with the particle size measurement results.

[0142] The Fourier transform infrared spectrometer was used to test the infrared spectra of whey protein, gum arabic, pine seed oil microcapsules, and wall materials, respectively. The test spectral range was 400 - 4000 cm-1.

[0143] From Figure 21 it can be seen that in the infrared spectrum of WPI, characteristic peaks were observed at 1654.38 cm -1 and 1549.45 cm -1 corresponding to the amide I band (C=O and C-N) and amide II band (N-H and C-N) of the protein. In addition, the infrared spectrum of WPI was at 3288.25 cm -1A relatively broad absorption peak appears on both the left and right, which is generated by the -OH stretching vibration. The infrared spectrum of GA shows a characteristic absorption peak at 3400.91 cm -1 , which is generated by the -OH and NH- stretching vibrations. The peak at 1605.14 cm -1 and the peak at 1417.13 cm -1 represent the stretching and asymmetric vibrations of -COO in the infrared spectrum. The infrared spectrum of the WPI-GA complex condensate seems to be a superposition of the infrared spectra of the two polymers (WPI and GA), and some peak changes are also observed. More specifically, the amide I band and amide II band of WPI shift from 1654.38 cm-1 to 1647.66 cm -1 and from 1549.45 cm -1 to 1555.90 cm -1 . The peak representing the carboxyl group in GA disappears (1417.13 cm -1 and 1605.14 cm -1 ), indicating that electrostatic interaction has occurred between WPI and GA. On the other hand, the -OH stretching vibration of WPI is observed to shift from 3288.25 cm -1 to 3417.32 cm -1 after condensation, indicating that hydrogen bonds are involved in the self-condensation of WPI-GA. Among them, the electrostatic attraction and hydrogen bonds between polymers lead to the formation of polymers, rather than chemical bonds. Compared with the WPI-GA complex condensate, the -OH stretching vibration of the embedded microcapsules shows a significant red shift. The -OH stretching vibration of WPI shifts from the initial 3417.328 cm -1 to 3300.198 cm -1 , probably due to the formation of hydrogen bonds between the core material and the phospholipids in WPI. The occurrence of hydrogen bonds helps to form denser condensates because WPI may capture phospholipids through hydrogen bonds, thereby reducing the porosity of the microcapsule wall.

[0144] by Figure 22It can be seen that for the differential curve corresponding to the weight loss intensity during the pyrolysis process, there are four weight loss levels of the microcapsules. For the first thermal event, there is approximately 5% mass loss of the microcapsules within the temperature range of 25°C to 100°C. The first type of mass loss is related to the moisture content of the material and is mainly attributed to the evaporation of water when the temperature is below 100°C. The second type of mass loss occurs at approximately 150°C to 250°C, with a mass loss of about 30%. This may be due to the loss of bound water, surface oil, or surface wall material in the microcapsules. The third type of mass loss occurs at 250°C to 400°C, with a mass loss of about 35%. The decomposition temperature, i.e., the peak temperature shown in the DTG curve, is observed here. This part of the mass loss is mainly caused by the rapid release of the core material after the complete decomposition of the wall material. The fourth type of mass loss is mainly the decomposition of the residual wall material. The last two steps may be related to a complex process, including the degradation of sugar rings and the decomposition of macromolecular chains of gums, indicating that microencapsulation plays a certain protective role for the core material.

[0145] Test Example 10 (Determination of the Basic Physical and Chemical Properties of Nano-Microcapsules)

[0146] Determination of Bulk Density

[0147] Transfer 2 g of microcapsule powder into a 10 mL graduated cylinder, and calculate the filled bulk density from the height of the powder in the cylinder. Here, ρ is the density of the microcapsules (g / cm 3 ), W is the mass of the microcapsules (g), V is the volume of the microcapsules (mL), and the results are shown in Table 7.

[0148] ρ = W / V

[0149] Determination of Water Content

[0150] The water content is detected according to the first method in the GB5009.3 - 2016 standard, and the results are shown in Table 7.

[0151] Determination of Angle of Repose

[0152] Vertically fix a funnel on a support, and place a receiving dish directly below it. Take 2 g of microcapsule powder and let it fall freely through the funnel. After the material forms a stable cone on the tray surface, calculate the angle of repose, and the results are shown in Table 7.

[0153] A = arctan(H / R)

[0154] where H is the distance from the lower opening of the funnel to the flat dish (cm), and R is the bottom radius of the microcapsules after scattering (cm).

[0155] Determination of Solubility

[0156] Weigh 1.00 g of Pinus pumila nut oil nano-microcapsules, quantitatively transfer them to a beaker containing 100 mL of deionized water. Stir at 25 °C, centrifuge, and then measure 25 mL of the supernatant and transfer it to a glass petri dish. Dry it at 105 °C. Use the equation to calculate the solubility of Pinus pumila nut oil nano-microcapsules, and the results are shown in Table 7.

[0157] Solubility = A / B × 100%

[0158] Where A is the weight of the powder (g) in the supernatant, and B is the weight of the powder (g) in the solution.

[0159] Table 7 Analysis of the powder characteristics of Pinus pumila nut oil nano-microcapsules

[0160] Basic Index Result Water Content (%) 2.36±0.09 Density (g / cm3) 0.46±0.03 Angle of Repose (°) 41.35±0.46 Solubility (%) 98.34±1.03

[0161] Water content is a key factor affecting the oxidative stability of products. High water content often leads to caking of dry microcapsule powder and the growth of microorganisms, resulting in lipid oxidation. Low water content will limit the molecular mobility and hinder the dispersion of microcapsules in food applications. A water content below 6% helps to extend the shelf life of powder samples. The water content of the microcapsules is 2.36 ± 0.09%, which is relatively low and not prone to deliquescence. In addition, as a key parameter affecting the processing, storage, and transportation characteristics of materials, bulk density has a significant impact on the packaging design and mixing process of products. The measured bulk density of the microcapsule product is 0.46 ± 0.03 g / cm 3 , and the higher density can effectively reduce the air penetration in the powder gap, thus inhibiting the occurrence of lipid oxidation reactions. In terms of fluidity characterization, the angle of repose is used as an evaluation index in this study. The experimental data show that the angle of repose value of the sample is 41.35 ± 0.46°, which is in the appropriate range of 30 - 45°, indicating that it has good powder flow characteristics. The dissolution rate directly reflects the release performance of the core material, is an important basis for evaluating the quality of microcapsule products, and is also a key quality index for the realization of the functions of microcapsule products. When microcapsules are used as food ingredients, they may need to be rehydrated. Therefore, microcapsules with low solubility may cause processing difficulties and economic losses in the food industry. High solubility leads to rapid adsorption, swelling, and particle rupture of the solvent, thus facilitating the release of compounds. The solubility of Pinus pumila nut oil nano-microcapsules is 98.34 ± 1.03%, indicating good redissolution performance.

[0162] Test Example 11 (Study on the in vitro digestion characteristics of Pinus pumila nut oil nano-microcapsules)

[0163] The preparation of gastric juice and intestinal juice refers to "Study on the Effects of Different Antioxidants on the Oxidative Stability of Protein Emulsions and the in vitro Digestion Characteristics of Oils", Chen Xianxin.

[0164] Take 2 g of the microcapsule sample and dissolve it in 20 mL of freshly prepared simulated gastric juice. After adjusting the pH of the system to 1.2, transfer it to a 37 °C constant temperature water bath environment and carry out digestion for 90 min under continuous mechanical stirring at 100 r / min (simulating gastrointestinal motility conditions). Throughout the experiment, the pH oxidation stability of the digestive system was maintained by dynamically adding 0.1 mol / L NaOH solution, and the amount of alkali solution used was recorded in real time.

[0165] Intestinal juice: After completing gastric digestion, cool the mixture to room temperature, adjust the pH to 6.8 with 0.1 mol / L NaOH, add an equal volume of simulated intestinal juice and adjust the pH to 6.8 in portions. Subsequently, carry out a 2-h intestinal digestion experiment under the same temperature control and stirring parameters, and simultaneously maintain the system pH by NaOH titration and record the reagent consumption data.

[0166] The content of free fatty acids (degree of digestion) during the digestion of microcapsules can be calculated by the following formula:

[0167] L = (V NaOH × C NaOH ) / (2 × M) × 100%

[0168] where L is the degree of digestion of the oil in the microcapsules, V NaOH is the content of NaOH consumed during digestion, and M is the content of oil in 2 g of microcapsules.

[0169] To explore the release kinetics of microcapsules during digestion, pepsin (simulating the gastric digestion stage) and trypsin (simulating the intestinal digestion stage) were used as characteristic enzymes to construct a digestion model, and the results are shown in Figure 23 .

[0170] In the gastric digestive juice within 0 - 120 min, the microcapsules slowly release under the action of strong acidic environment and pepsin. Although the wall material components face the risk of degradation, the high viscosity property of gum arabic enables it to form a dense and highly elastic protective layer at the wall material interface, which not only significantly enhances the mechanical stability of the microcapsules but also prevents the release behavior of the core material. This physical barrier effectively inhibits the rapid diffusion of the core material, resulting in a limited release rate. As a natural polysaccharide, the hydrogen bonds and hydrophobic interactions between the molecular chains of gum arabic may form a dynamic cross - linked network under acidic conditions, further stabilizing the microcapsule structure and delaying the erosion of the wall material by gastric juice. When the pH of the intestinal digestive juice increases, the electrostatic attraction between WPI and GA is significantly disrupted, and the loose aggregated structure makes WPI highly sensitive to the degradation by trypsin. At the same time, trypsin accelerates the deep degradation of the protein backbone by specifically cleaving peptide bonds. The synergistic effect of the two significantly destroys the structural integrity of the microcapsules, promoting the rapid diffusion of the core material and the continuous destruction of the microcapsule structure during intestinal digestion. The release rate tends to be stable (67.5%) after 140 min in intestinal fluid, indicating that the residual core material diffuses through the diffusion channels formed by the residual pores of the microcapsules or degradation products, which may result from the dynamic balance between the disintegration of the wall material and the solubility of the core material. Eventually, a sustained - release mode in which most of the core material is released in the intestine is formed, and the release rate in intestinal fluid is greater than that in gastric juice.

[0171] The results show that the microcapsule wall material exhibits a sustained - release effect in gastric digestive juice and a rapid - release property in intestinal digestive juice. It demonstrates differentiated release behaviors in the gastrointestinal digestive environment, avoiding the destruction of sensitive components by gastric acid and ensuring effective release in the intestine. Similar observations have also been reported in the studies of other complex coacervation delivery systems. Based on the results of the entire digestive process, it ensures the efficient physiological utilization of dwarf pine oil and improves the bioavailability.

[0172] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of pine seed oil nano - microcapsules, characterized in that, It includes the following steps: Step 1: Dry the pine nuts until constant weight, then crush and sieve them to obtain pine nut powder; Step 2: Press the pine nut powder at 35 - 60 Mpa and 25 - 65 °C for 20 - 50 min to obtain pine nut meal; Step 3: Perform supercritical CO2 extraction on the pine nut meal at a temperature of 47 - 55 °C, a pressure of 25 - 35 Mpa, a CO2 flow rate of 19 - 22 L / h, and an extraction time of 120 - 180 min to obtain pine seed oil; Step 4: Mix and stir the pine seed oil, urea, and ethanol solution, isolate air throughout the process. After cooling to room temperature, conduct a clathration reaction at -20 to 10 °C for 6 - 26 h, filter, wash the filtrate with distilled water until the lower layer liquid is clear and transparent. After combined use of rotary evaporation and nitrogen purging, obtain unsaturated fatty acids; Step 5: Mix and disperse whey protein, arabic gum, and unsaturated fatty acids, adjust the pH, coagulate at 40 - 50 °C for 1 - 1.5 h, end the reaction at 5 - 10 °C, then adjust the pH again, react at 8 - 12 °C for 30 - 40 min, and freeze-dry to obtain nano microcapsules.

2. The preparation method of a nano microcapsule of pine seed oil according to claim 1, characterized in that, The pine nuts described in Step 1 are Korean pine nuts.

3. The preparation method of a pine seed oil nano-microcapsule according to claim 1, characterized in that, In Step 2, press at 55 Mpa and 65 °C for 40 min.

4. The preparation method of a pine seed oil nano-microcapsule according to claim 1, characterized in that, In Step 4, the mass ratio of the pine seed oil to urea is 1:(1 - 6), and the mass ratio of the urea to the ethanol solution is 1:(3 - 8).

5. The preparation method of a pine seed oil nano-microcapsule according to claim 4, characterized in that, The volume fraction of the ethanol solution is 95%.

6. The preparation method of a nano-microcapsule of pine seed oil according to claim 1, characterized in that, In Step 5, the mass ratio of the whey protein, arabic gum, and unsaturated fatty acids is 2.5:1:3.

5.

7. A method for preparing nano-microcapsules of pine seed oil according to claim 1, characterized in that, In Step 5, the pH is adjusted to 3.8 with acetic acid solution for the first time and adjusted to 6 with NaOH solution for the second time.

8. The nano microcapsules prepared by the method according to any one of claims 1 - 7.

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