A method for solvent-free extraction of pine pollen essential oil using pine pollen

Abstract

The present invention discloses a method for solvent-free extraction of pine pollen essential oil from pine pollen, comprising the steps of: (1) mixing broken-wall pine pollen and purified water in a weight ratio of 1:5 - 1:15, adding a composite enzyme to obtain a feed liquid, adjusting the pH value and temperature conditions of the feed liquid, and performing enzymatic hydrolysis treatment for a certain time to obtain a pine pollen enzymatic hydrolysate; (2) transferring the pine pollen enzymatic hydrolysate to an extraction kettle equipped with a microwave generating device for extraction, the extraction kettle is externally connected with a condensation reflux device, turning on the microwave generating device, adjusting the microwave power to 240W - 560W, setting the extraction temperature to 45°C - 50°C, and the extraction time to 0.5 - 1.5h; (3) the essential oil components obtained by extraction are subjected to condensation reflux, oil phase separation, and collection to obtain pine pollen essential oil. The method for preparing pine pollen essential oil provided by the present invention has the characteristics of no solvent residue, high safety, and high extraction efficiency, and is also simple to operate, low in cost and energy consumption, and is a green and environmentally friendly preparation technology.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for solvent-free extraction of pine pollen essential oil from pine pollen. Background Art

[0002] Pine pollen, also known as pine flower and pine yellow, refers to the dried pollen of pine trees such as Pinus Massoniana and Pinus tabulaeformis Carr. collected artificially, and is a traditional pollen variety for both food and medicine in China. Pine pollen has a sweet and neutral taste, and belongs to the liver and spleen meridians, with the effects of moistening the heart and lungs, replenishing qi, enhancing complexion, and improving will. Since ancient times, there have been detailed records of pine pollen in more than 30 important traditional Chinese medical works in China. As the male reproductive cells produced by the stamens of pine plants, pine pollen contains rich protein, dietary fiber, fat, active polysaccharides, minerals, nucleic acids, flavonoids, saponins, vitamins, and essential nutrients and bioactive substances such as zinc, manganese, and iron for the human body, and has the reputation of "natural micro-nutrition library". The protein content in pine pollen is 10%-25%, including 22 kinds of amino acids, and 8 kinds of essential amino acids for the human body. The lipid content in pine pollen is 5%-12%, including various unsaturated fatty acids, phospholipids, sterols, carotenoids, etc. More than 90 kinds of lipid components have been found, such as myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, stigmasterol, campesterol, β-sitosterol, β-carotene, etc., and the unsaturated fatty acids account for more than 70% of the total fatty acids. The dietary fiber content in pine pollen is as high as more than 40%, including cellulose, hemicellulose, pectin, lignin, sporopollenin, callose, raffinose, stachyose, etc. In addition, pine pollen also contains active polysaccharide components with immunomodulatory and anti-cancer effects, which are mainly composed of monosaccharide components such as fucose, arabinose, L-xylose, D-mannose, and D-glucose.

[0003] Pine pollen essential oil is an active substance extracted from pine pollen, and its main components are fatty acids, alkanes, terpenoids and their oxygen-containing derivatives and other volatile components, which can produce a pleasant and natural coordinated pine flower aroma. Pine pollen essential oil contains a variety of bioactive components, has inhibitory effects on Escherichia coli, Staphylococcus aureus, Proteus, etc., and has wide application values in industries such as perfumes and flavors, cosmetics, food, and medicine.

[0004] At present, the main methods for extracting pine pollen essential oil include steam distillation, organic solvent extraction, and supercritical carbon dioxide extraction. The steam distillation method has disadvantages such as low extraction efficiency and long time consumption, and it is often difficult to effectively obtain the target components. The disadvantage of the organic solvent extraction method is that the recovery of organic solvents takes a long time and consumes a lot of energy. Long-term high-temperature reflux is likely to damage the inherent components of pine pollen, and the residual amount of organic solvents is large, which is likely to cause potential safety hazards in use. The supercritical carbon dioxide extraction method has advantages such as non-toxicity, odorlessness, easy refinement and recovery, but its disadvantage is that the supercritical extraction equipment is expensive, the one-time investment is large, and its operating cost is much higher than other extraction methods.

[0005] The composition of pine pollen is very complex, containing a variety of macromolecular substances such as proteins, polysaccharides, and nucleic acids. The components of pine pollen essential oil are volatile small-molecular substances. In the natural state, most of them are in a state of being chemically bonded to other macromolecular substances. According to the characteristics of the composition of pine pollen itself, targeted technical means are used to release them from the bound state, effectively collect them, and then analyze and identify their components, analyze their typical component composition, which has important value for the development and application of pine pollen essential oil. Summary of the Invention

[0006] In view of the above deficiencies in the prior art, the present invention provides a method for solvent-free extraction of pine pollen essential oil using pine pollen.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] A method for solvent-free extraction of pine pollen essential oil using pine pollen includes the following steps:

[0009] (1) Mix the wall-broken pine pollen and purified water evenly according to a weight ratio of 1:5 - 1:15, add a composite enzyme to obtain a feed liquid, adjust the pH value and temperature conditions of the feed liquid, and perform enzymatic hydrolysis for a certain time to obtain a pine pollen enzymatic hydrolysate;

[0010] (2) Transfer the pine pollen enzymatic hydrolysate to an extraction kettle equipped with a microwave generating device for extraction. The extraction kettle is externally connected to a condensation reflux device. Turn on the microwave generating device, adjust the microwave power to 240W - 560W, set the extraction temperature to 45°C - 50°C, and the extraction time to 0.5 - 1.5h;

[0011] (3) The essential oil components obtained by extraction are condensed and refluxed, separated from the oil phase, and collected to obtain pine pollen essential oil.

[0012] The extracted essential oil components are continuously collected into the collector through the condensation reflux device. After the extraction is completed, use an oil phase separation device to collect the essential oil extract in the collection tube of the condensation reflux device to obtain pine pollen essential oil. Through sensory evaluation and comparison with the original pine pollen powder and wall-broken pine pollen samples, the obtained pine pollen essential oil has typical pine pollen flavor characteristics.

[0013] Furthermore, the complex enzyme is composed of pectinase, papain and porcine pancreatic lipase.

[0014] Furthermore, the mass ratio of the pectinase, papain and porcine pancreatic lipase is 1:1:2, and the addition amount of the complex enzyme is 200 U / g of pine pollen.

[0015] Furthermore, in step (1), the pH of the material liquid is adjusted to 6.0, the enzymatic hydrolysis temperature is 45°C - 60°C, and the enzymatic hydrolysis time is 30 - 120 minutes.

[0016] Furthermore, the enzymatic hydrolysis temperature is 50°C and the enzymatic hydrolysis time is 60 minutes.

[0017] Furthermore, in step (1), the weight ratio of the wall-broken pine pollen to purified water is 1:10.

[0018] Furthermore, in step (2), the microwave power is 400 W and the extraction time is 0.75 h.

[0019] Furthermore, GC-MS is used to analyze and detect the obtained pine pollen essential oil, and the characteristic component composition of the pine pollen essential oil and the proportion relationship between them are obtained. A total of 137 compounds with a matching factor greater than 80 are identified. The relative percentage content of each compound is calculated by the peak area normalization method. Among them, the proportion of acid substances is 89.68%, the proportion of alcohol substances is 0.62%, the proportion of alkane substances is 2.92%, the proportion of aldehyde substances is 1.2%, the proportion of ester substances is 0.78%, the proportion of alkene substances is 0.1%, the proportion of furan substances is 0.08%, the proportion of amide substances is 2.54%, and the proportion of other substances is 2.07%.

[0020] Furthermore, the GC-MS analysis and detection parameters are as follows:

[0021] Chromatographic column: Agilent HP-5MS (30.0 m × 0.25 mm × 0.25 μm);

[0022] Program column temperature: The initial temperature is 60°C, the temperature is raised to 140°C at a rate of 8°C / min and held for 2 min, then the temperature is raised to 240°C at a rate of 2°C / min and held for 2 min, and then the temperature is raised to 280°C at a rate of 8°C / min and held for 5 min; Carrier gas: helium (≥99.999%), rate 1.0 mL / min; Injection port temperature: 260°C; Injection mode: split injection, split ratio 10:1; Injection volume: 1.0 μL;

[0023] Mass spectrometry conditions: Ionization mode: EI, voltage 70 eV; Ion source temperature: 230 °C, quadrupole temperature: 150 °C; Transfer line temperature: 280 °C; Determination mode: full scan mode (SCAN); Collision gas: nitrogen (≥99.999%); Solvent delay: 2.5 min.

[0024] Compared with the prior art, the present invention has the following technical effects:

[0025] Based on the structural characteristics and composition features of organic compound components such as proteins, dietary fibers, active polysaccharides, and lipids contained in pine pollen itself, the present invention provides a method for solvent-free extraction of pine pollen essential oil from pine pollen. By treating the pine pollen raw material with a specific biological enzyme combination technology, the volatile essential oil components contained therein can be effectively released, greatly improving the yield of the extracted pine pollen essential oil. On this basis, by optimizing key influencing factors such as microwave extraction power, solid-liquid ratio, and extraction time, the extraction efficiency is further improved. The method for extracting pine pollen essential oil provided by the present invention has the characteristics of no solvent residue, high safety, and high extraction efficiency, and is simple to operate, low in cost, and low in energy consumption, and is a green and environmentally friendly preparation technology. Description of the Drawings

[0026] Figure 1 Shows the analysis diagrams of the effects of single factors of extraction time, microwave power, and solid-liquid ratio on the yield of pine pollen essential oil in the examples of the present invention (a: effect of extraction time on the yield of pine pollen essential oil, b: effect of microwave power on the yield of pine pollen essential oil, c: effect of solid-liquid ratio on the yield of pine pollen essential oil);

[0027] Figure 2 Shows the response surface and contour diagrams of the effects of two-factor interactions on the yield of pine pollen essential oil in the examples of the present invention (a: response surface and contour diagrams of the effect of the interaction between extraction time and microwave power on the yield of pine pollen essential oil, b: response surface and contour diagrams of the effect of the interaction between extraction time and solid-liquid ratio on the yield of pine pollen essential oil, c: response surface and contour diagrams of the effect of the interaction between solid-liquid ratio and microwave power on the yield of pine pollen essential oil);

[0028] Figure 3 Shows the total ion chromatogram of GC-MS of pine pollen essential oil in the examples of the present invention. Detailed Embodiments

[0029] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Although the description of the present invention will be introduced in conjunction with preferred embodiments, this does not mean that the features of this invention are limited to this implementation manner. On the contrary, the purpose of introducing the invention in conjunction with the implementation manner is to cover other alternatives or modifications that may be extended based on the claims of the present invention. In order to provide a deep understanding of the present invention, many specific details will be included in the following description. The present invention can also be implemented without using these details. In addition, in order to avoid confusion or obscuring the key points of the present invention, some specific details will be omitted in the description. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0030] In Examples 1, 2, and 3, the yield rate of pine pollen essential oil was calculated according to the following formula:

[0031] Yield rate of pine pollen essential oil (%) = (mass of pine pollen essential oil / mass of wall-broken pine pollen) × 100%

[0032] Example 1

[0033] A method for solvent-free extraction of pine pollen essential oil, which respectively examines the effects of different single enzymes and different composite enzymes on the yield rate of pine pollen essential oil. The steps are as follows:

[0034] 1) Take several portions of 200 g of wall-broken pine pollen, add purified water to the wall-broken pine pollen according to a ratio of 1:10 (w:w) respectively, mix evenly, and then add cellulase, hemicellulase, pectinase, papain, pepsin, porcine pancreatic lipase, β-glucanase, and xylanase respectively. The addition amount of each enzyme is 200 U / g of pine pollen. Stir to make it mix evenly, adjust the pH of the material-liquid to 6.0, and under the condition of 55 °C, carry out enzymatic hydrolysis treatment for 90 minutes to obtain pine pollen enzymatic hydrolysate;

[0035] 2) Transfer the above-mentioned pine pollen enzymatic hydrolysate to an extraction kettle for extraction. The extraction kettle is externally connected with a condensation reflux device. Set the extraction temperature at 45 °C - 50 °C and the extraction time at 1 h. The essential oil components extracted are continuously collected into a collector through the condensation reflux device;

[0036] 3) After the extraction is completed, use an oil phase separation device to collect the essential oil extract in the collection tube of the condensation reflux device to obtain pine pollen essential oil.

[0037] Under the action of different single enzymes, the yield rates of pine pollen essential oil are shown in Table 1. It can be seen from the experimental results that under the action of single enzymes such as pectinase, papain, and porcine pancreatic lipase, the yield rates of pine pollen essential oil are relatively high.

[0038] Table 1 Yield rates of pine pollen essential oil under the action of different single enzymes

[0039]

[0040] Referring to the operation steps of 1), 2) and 3) in the above-mentioned embodiment, with the yield of pine pollen essential oil as the investigation index, different combinations and ratios of three enzymes, namely pectinase, papain and porcine pancreatic lipase, were carried out to screen out a better composite enzyme combination. The experimental results are shown in Table 2.

[0041] Table 2 Yield of pine pollen essential oil under the action of different composite enzymes

[0042]

[0043] It can be seen from the experimental results in Table 2 that when any two combinations or three combinations of pectinase, papain and porcine pancreatic lipase are used, the yield of pine pollen essential oil is significantly increased, indicating that there is a synergistic effect between different enzymes. When the ratio of pectinase, papain and porcine pancreatic lipase is 1:1:2, the yield of pine pollen essential oil is the highest.

[0044] When the ratio of pectinase, papain and porcine pancreatic lipase is 1:1:2, with the yield of pine pollen essential oil as the reference index, the addition amount of the composite enzyme, the enzymolysis temperature and the enzymolysis time were investigated respectively. The experimental results are shown in Table 3, Table 4 and Table 5. It can be seen from Table 3 that when the addition amount of the composite enzyme is 200 U / g of pine pollen, the yield of pine pollen essential oil is the highest. It can be seen from Table 4 that when the enzymolysis temperature is 50 °C, the yield of pine pollen essential oil is the highest. It can be seen from Table 5 that when the enzymolysis time is 30 min, the yield of pine pollen essential oil is relatively low. When the enzymolysis time is 60 min, 90 min and 120 min, the yield of pine pollen essential oil has almost no difference. Considering the extraction efficiency comprehensively, the enzymolysis time was finally determined to be 60 min.

[0045] Table 3 Effect of the addition amount of the composite enzyme on the yield of pine pollen essential oil

[0046]

[0047] Table 4 Effect of the enzymolysis temperature on the yield of pine pollen essential oil

[0048]

[0049]

[0050] Table 5 Effect of the enzymolysis time on the yield of pine pollen essential oil

[0051]

[0052] Example 2

[0053] A method for solvent-free extraction of pine pollen essential oil was carried out with single-factor experimental designs of solid-liquid ratio, microwave power and extraction time respectively. The steps are as follows:

[0054] 1) Take 200 g of broken-wall pine pollen. After mixing the broken-wall pine pollen evenly with purified water in a certain proportion, transfer the material liquid to an extraction kettle equipped with a microwave generating device for extraction. The extraction kettle is externally connected to a condensation reflux device. Turn on the microwave generating device, adjust the microwave power, set the extraction temperature at 45°C - 50°C, set different extraction times, and the extracted essential oil components are continuously collected into the collector through the condensation reflux device. Take the yield of pine pollen essential oil as an index to investigate the effects of the material-liquid ratio, microwave power, and extraction time on the extraction effect;

[0055] 2) Fix the microwave power at 500 W and the material-liquid ratio at 1:10 unchanged. Select the times of 0.5 h, 0.75 h, and 1 h, and operate according to 1) above in this example to investigate the effects of different extraction times on the yield;

[0056] 3) Fix the extraction time at 0.75 h and the material-liquid ratio at 1:10 unchanged. Select the medium microwave powers of 240 W, 400 W, and 560 W respectively, and operate according to 1) above in this example to investigate the effects of different extraction powers on the yield;

[0057] 4) Fix the extraction time at 0.75 h and the microwave power at 500 W unchanged. On the premise of a fixed sample loading amount of adding 200 g of broken-wall pine pollen each time, change the material-liquid ratio by adding different amounts of purified water. Select the material-liquid ratios (g / g) of 1:5, 1:10, and 1:15 respectively, and operate according to 1) above in this example to investigate the effects of different material-liquid ratios on the yield.

[0058] The results of the single-factor experiment on the optimization of the solvent-free extraction process of pine pollen essential oil are as follows:

[0059] When the microwave power is fixed at 500 W and the material-liquid ratio is 1:10, investigate the influence of the extraction time factor on the yield of pine pollen essential oil. The experimental results are shown in Table 6.

[0060] Table 6 Results of the single-factor experiment on extraction time

[0061]

[0062]

[0063] From Figure 1 (a), it can be seen that when the time is 0.5 - 0.75 h, the extraction of pine pollen essential oil is incomplete, and the yield of essential oil increases with the increase of time. When the time is 0.75 h, the yield is similar to that at 1 h, and both are significantly higher than the yield at 0.5 h, indicating that when the time exceeds 0.75 h, the pollen extract is almost completely produced, and very little extract is obtained by extending the extraction time. Therefore, in order to save costs and improve efficiency, 0.75 h is selected as the optimal time condition.

[0064] When the fixed extraction time was 0.75 h and the solid-liquid ratio was 1:10, the yields of pine pollen essential oil at different extraction powers were investigated, as shown in Table 7.

[0065] Table 7 Results of single-factor experiment on microwave power

[0066]

[0067] As Figure 1 shown in (b), under the conditions of fixed time and solid-liquid ratio, when the power was in the range of 200 - 400 W, the yield increased with the increase of microwave power, and the highest yield was obtained at 400 W. When the power exceeded 400 W, increasing the power instead affected the extraction process and led to a decrease in pollen extract, that is, 400 W was the optimal power.

[0068] When the fixed extraction time was 0.75 h and the microwave power was 400 W, the test results of the yields of pine pollen essential oil at different solid-liquid ratios are shown in Table 8.

[0069] Table 8 Results of single-factor experiment on solid-liquid ratio

[0070]

[0071] As Figure 1 shown in (c), while keeping the mass of added pollen unchanged, during the process of changing the solid-liquid ratio by increasing the volume of ultrapure water, the yield showed a trend of first increasing and then decreasing. When the solid-liquid ratio was 1:5, due to incomplete dissolution or insufficient immersion of pollen, the extraction yield was relatively low; when the solid-liquid ratio reached 1:10, the yield was the highest; when the solid-liquid ratio reached 1:15, the yield decreased instead. The optimal solid-liquid ratio was determined to be 1:10.

[0072] On the basis of the above single-factor experiment results, using Design-Expert 10.0.7 for response surface experiment operation, time (A), power (B), and solid-liquid ratio (C) were selected as independent variables, and a three-factor and three-level response surface was designed to optimize the solvent-free microwave extraction process of pine pollen, as shown in Table 9.

[0073] Table 9 Results of three-factor and three-level response surface experiment

[0074] Serial number A Time (h) B Power (W) C Liquid-to-material ratio Yield (%) 1 0.5 240 10 0.31 2 1 240 10 0.33 3 0.5 560 10 0.3 4 1 560 10 0.37 5 0.5 400 5 0.43 6 1 400 5 0.475 7 0.5 400 15 0.42 8 1 400 15 0.52 9 0.75 240 5 0.38 10 0.75 560 5 0.395 11 0.75 240 15 0.37 12 0.75 560 15 0.39 13 0.75 400 10 0.555 14 0.75 400 10 0.48 15 0.75 400 10 0.47 16 0.75 400 10 0.51 17 0.75 400 10 0.465

[0075] Performing regression analysis on the data in the above table, the quadratic polynomial regression model equation of the extraction yield (Y) of broken-wall pine pollen with respect to time (A), power (B), and solid-liquid ratio (C) was obtained:

[0076] Y = 0.50 + 0.029A + 0.0081B + 0.0025C + 0.013AB + 0.014AC + 0.0013BC - 0.046A 2 - 0.12B2 +0.011C 2

[0077] Analysis table 10 of the regression coefficients and significance test results of the experimental model.

[0078] Table 10 Model regression coefficients and significance test

[0079]

[0080]

[0081] Analyzing the data in the above table, the P value of the equation model used in the experiment is 0.0027 (P < 0.01), and the model is extremely significant. The P value of the lack-of-fit term = 0.91397 > 0.05, indicating that the lack-of-fit term is not significant and the model error is small. Among the three factors affecting the yield, the time factor has the most significant influence. The primary and secondary order of the factors affecting the yield of solvent-free microwave extraction of pine pollen is: A > B > C, that is, extraction time > microwave power > solid-liquid ratio.

[0082] As can be seen from the above table, the optimal process conditions are time 0.87 h, microwave power 409.98 W, and solid-liquid ratio 1:10. Under these conditions, the extraction yield of pine pollen essential oil is the highest, and the yield is 0.555%.

[0083] Analysis of the interaction of extraction influencing factors:

[0084] Figure 2 (a) shows the influence of extraction time and microwave power on the yield. When the time is fixed, the yield first increases and then decreases with the increase of power; under the condition of fixed power, it can be seen that with the extension of time, the yield gradually increases. It can also be seen from the contour plot that fixing any one of the two factors of time and power will have a greater impact on the yield. However, the contour lines tend to be circular, indicating that the interaction between time and power is not significant.

[0085] Figure 2 (b) shows the interaction effect of extraction time and solid-liquid ratio. As can be seen from the figure, when the time is fixed, the change of solid-liquid ratio has little effect on the yield; when the solid-liquid ratio is fixed, the yield increases with the increase of extraction time. The interaction between time and solid-liquid ratio is not significant.

[0086] Figure 2 (c) reflects the interaction effect of microwave power and solid-liquid ratio. After fixing the power, different solid-liquid ratios have little effect on the yield; when the solid-liquid ratio remains unchanged, the influence of the power factor on the yield is that as the power gradually increases, the yield first increases and then decreases. It can be seen from the contour plot that the interaction between power and solid-liquid ratio is not significant.

[0087] Example 3

[0088] A method for solvent-free extraction of pine pollen essential oil, the steps are as follows

[0089] (1) Take 200 g of broken-wall pine pollen, add purified water to the broken-wall pine pollen in a ratio of 1:10 (w:w), mix evenly, then add a composite enzyme composed of pectinase, papain, and porcine pancreatic lipase, mix well, adjust the pH of the material-liquid to 6.0, and carry out enzymatic hydrolysis treatment at 50 °C for 60 minutes to obtain a pine pollen enzymatic hydrolysis solution. The addition amount of the composite enzyme is 200 U / g of pine pollen, and the composition of the composite enzyme: the weight ratio of pectinase, papain, and porcine pancreatic lipase is 1:1:2;

[0090] (2) Transfer the above pine pollen enzymatic hydrolysis solution to an extraction kettle equipped with a microwave generating device for extraction. The extraction kettle is externally connected to a condensation reflux device. Turn on the microwave generating device, adjust the microwave power to 400 W, set the extraction temperature at 45 °C - 50 °C, and the extraction time at 0.75 h. The extracted essential oil components are continuously collected into the collector through the condensation reflux device.

[0091] (3) After the extraction is completed, use an oil phase separation device to collect the essential oil extract in the collecting tube of the condensation reflux device to obtain pine pollen essential oil. Through sensory evaluation and comparison with the original pine pollen powder and broken-wall pine pollen samples, the obtained pine pollen essential oil has a typical pine pollen flavor.

[0092] Perform GC-MS analysis on the pine pollen essential oil sample obtained in Example 3. The experimental parameter conditions are as follows:

[0093] Chromatographic column: Agilent HP-5MS (30.0 m × 0.25 mm × 0.25 μm).

[0094] Program column temperature: The initial temperature is 60 °C, heated to 140 °C at a rate of 8 °C / min and held for 2 min, then heated to 240 °C at a rate of 2 °C / min and held for 2 min, and then heated to 280 °C at a rate of 8 °C / min and held for 5 min; Carrier gas: Helium (≥99.999%), rate 1.0 mL / min; Injection port temperature: 260 °C; Injection mode: Split injection, split ratio 10:1; Injection volume: 1.0 μL.

[0095] Mass spectrometry conditions: Ionization mode: EI, voltage 70 eV; Ion source temperature: 230 °C, quadrupole temperature: 150 °C; Transfer line temperature: 280 °C; Determination mode: Full scan mode (SCAN); Collision gas: Nitrogen (≥99.999%); Solvent delay: 2.5 min.

[0096] The pine pollen essential oil is detected by GC-MS, and a variety of chemical components are identified. Its total ion current chromatogram is as Figure 3As shown, there are 137 chemical components with a matching factor greater than 80. The relative percentage content of each compound is calculated by the peak area normalization method. The results of the components with a matching factor greater than 80 are shown in Table 11.

[0097] Table 11 Chemical Components of Pine Pollen Extract

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] The present invention uses broken-wall pine pollen as the raw material. According to the structural characteristics and composition features of the components contained in pine pollen itself, a composite enzyme composed of pectinase, papain, and porcine pancreatic lipase is used for enzymatic hydrolysis treatment, so that the volatile essential oil components in pine pollen can be effectively released, greatly improving the extraction yield of pine pollen essential oil. On this basis, by optimizing the influencing factors such as microwave extraction power, solid-liquid ratio, and extraction time, the extraction efficiency is further improved. The method for preparing pine pollen essential oil provided by the present invention has the characteristics of no solvent residue, high safety, and high extraction efficiency, and is also simple to operate, low in cost, and low in energy consumption, which is a green and environmentally friendly preparation technology.

[0106] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for solvent-free extraction of pine pollen essential oil using pine pollen, characterized in that , including the following steps: (1) Mix the broken-wall pine pollen and purified water evenly at a weight ratio of 1:5 - 1:15, add a complex enzyme to obtain a feed liquid, adjust the pH value and temperature conditions of the feed liquid, and perform enzymatic hydrolysis for a certain period of time to obtain a pine pollen enzymatic hydrolysate; (2) Transfer the pine pollen enzymatic hydrolysate to an extraction kettle equipped with a microwave generating device for extraction. The extraction kettle is externally connected to a condensation reflux device. Turn on the microwave generating device, adjust the microwave power to 240W - 560W, set the extraction temperature to 45°C - 50°C, and the extraction time to 0.5 - 1.5h; (3) The essential oil components obtained by extraction are subjected to condensation reflux, oil phase separation, and collection to obtain pine pollen essential oil; The complex enzyme is composed of pectinase, papain, and porcine pancreatic lipase. The mass ratio of pectinase, papain, and porcine pancreatic lipase is 1:1:2, and the addition amount of the complex enzyme is 200 U / g of pine pollen; In step (1), adjust the pH of the feed liquid to 6.0, the enzymatic hydrolysis temperature is 50°C, and the enzymatic hydrolysis time is 60 minutes.

2. The method for solvent-free extraction of pine pollen essential oil using pine pollen as claimed in claim 1, wherein In step (1), the weight ratio of the broken-wall pine pollen to purified water is 1:

10.

3. The method for solvent-free extraction of pine pollen essential oil using pine pollen as claimed in claim 1, wherein In step (2), the microwave power is 400W, and the extraction time is 0.75h.

4. The method for solvent-free extraction of pine pollen essential oil using pine pollen as claimed in claim 1, wherein Analyze and detect the obtained pine pollen essential oil by GC-MS to obtain the characteristic component composition of the pine pollen essential oil and the proportional relationship between them. A total of 137 compounds with a matching factor greater than 80 are identified. Among them, acids account for 89.68%, alcohols account for 0.62%, alkanes account for 2.92%, aldehydes account for 1.2%, esters account for 0.78%, alkenes account for 0.1%, furans account for 0.08%, amides account for 2.54%, and other substances account for 2.07%.

5. The method for solvent-free extraction of pine pollen essential oil using pine pollen as claimed in claim 4, wherein The GC-MS analysis and detection parameters are as follows: Chromatographic column: Agilent HP-5MS; Program column temperature: The initial temperature is 60 °C, the temperature is raised to 140°C at a rate of 8 °C / min and held for 2 min, then the temperature is raised to 240°C at a rate of 2 °C / min and held for 2 min, and then the temperature is raised to 280 °C at a rate of 8°C / min and held for 5 min; Carrier gas: helium, rate 1.0 mL / min; Injection port temperature: 260 °C; Injection mode: split injection, split ratio 10:1; Injection volume: 1.0 μL; Mass spectrometry conditions: Ionization mode: EI, voltage 70 eV; Ion source temperature: 230 °C, quadrupole temperature: 150 °C; Transfer line temperature: 280 °C; Determination mode: full scan mode; Collision gas: nitrogen; Solvent delay: 2.5 min.

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