Active pinolenic acid-rich pine nut oil unsaturated fatty acids and methods of making same

Abstract

This invention belongs to the field of plant oil extraction technology, specifically relating to a method for preparing pine nut oil rich in pinolenic acid and its preparation. The method for preparing pine nut oil rich in pinolenic acid provided by this invention mainly includes the following steps: gas spraying, centrifugal oil separation, high-solids enzymatic hydrolysis, freeze-thaw reaction, saponification reaction, and urea inclusion. The beneficial effects of this invention are that by using a mixed gas for rapid slurry modification of pine nuts, combined with stepwise enzymatic hydrolysis and freeze-thaw processes, not only is the extraction rate of pine nut oil improved, but it also helps to protect components such as pinolenic acid in the pine nut oil from damage. Results show that the extraction rate of highly unsaturated pine nut oil obtained by the method of this invention is >85%, and the pinolenic acid content in the unsaturated fatty acids of the pine nut oil rich in pinolenic acid is >70%. Furthermore, the obtained pine nut oil unsaturated fatty acids exhibit good lipid-lowering effects.

Description

Technical Field

[0001] This invention belongs to the field of plant oil extraction technology, specifically relating to an active pine nut oil rich in pinolenic acid and its preparation method. Background Technology

[0002] Pine nuts are the kernels of mature seeds from pine trees such as Korean pine, after the hard shell has been removed. They are rich in nutrients, including 60-69% fat, 13-20% protein, about 10% dietary fiber, about 2% carbohydrates, and various vitamins and bioactive components. Pine nuts are rich in lipids, containing approximately 50% polyunsaturated fatty acids, 40% monounsaturated fatty acids, and about 10% saturated fatty acids. Pinolenic acid, a unique octadecyltriene fatty acid found only in pine nuts, accounts for 14-19% of the total fatty acids and is considered a key component in pine nut oil for its lipid-regulating properties.

[0003] Currently, the main method for producing products containing pinolenic acid using pine nuts as raw material is to first obtain pine nut oil through pressing / extraction, and then saponification, acidification, washing, dehydration, and molecular distillation to finally obtain products containing pinolenic acid.

[0004] However, pine nuts contain over 60% oil. In the oil extraction process, traditional pressing / leaching methods yield low oil yields, only around 50%. Hot pressing, on the other hand, easily destroys pinolenic acid due to the high temperatures, reducing the content of unsaturated fatty acids. Furthermore, extraction methods often require organic solvents, resulting in high pinolenic acid solvent residue and difficulties in high-temperature desolventizing. Currently, to avoid these problems, steam explosion technology is increasingly used in vegetable oil extraction. For example, patent CN102154055 A discloses a steam explosion-water extraction process for vegetable oils. However, the types and effects of unsaturated fatty acids in vegetable oils from different plant sources vary significantly. While steam explosion can indeed promote the dissolution of active ingredients from plant sources to some extent, improper operating conditions may actually destroy some of these active ingredients, leading to the loss of some functional unsaturated fatty acids.

[0005] Pine nuts are expensive and have a low oil yield. If the active ingredients are further damaged during the oil extraction process, the activity of pine nut oil will be significantly reduced. Therefore, to obtain pine nut oil with high activity and high unsaturated fatty acid content, the preparation method of pine nut oil must be improved. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an active pine nut oil rich in pinolenic acid and its preparation method.

[0007] The present invention provides a method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid, comprising the following steps:

[0008] (1) Gas spraying: Pine nuts are crushed, water is added to make them swell, and then 0.4-0.6 MPa of clean air is first introduced into the swelled material, followed by saturated water vapor to make the final pressure reach 1.2-1.8 MPa. Under these conditions, the material is left to stand for 10-20 minutes, and then the pressure is released within 1 second. The material is sprayed out by the rapid release of gas, and the pine nut slurry is collected.

[0009] (2) Centrifugation for oil separation: The pine nut pulp obtained in (1) is centrifuged to separate oil phase I, emulsion phase I, aqueous phase I and solid phase I;

[0010] (3) High-solidity enzymatic hydrolysis: Disperse the emulsion phase I obtained in (2) in water to obtain a mixture. First, add cellulase to the mixture for the first enzymatic hydrolysis, and then add a mixture of neutral protease and α-amylase for the second enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme, centrifuge, and obtain oil phase II, emulsion phase II, aqueous phase II and solid phase II.

[0011] (4) Freezing and thawing: After freezing and thawing, the emulsion phase II obtained in (3) is centrifuged to obtain oil phase III and water phase III. Then, the oil phase I obtained in (2), the oil phase II obtained in (3) and the oil phase III obtained in (4) are thoroughly stirred and mixed to obtain highly unsaturated pine nut oil.

[0012] (5) Saponification reaction: The highly unsaturated pine nut oil obtained in (4) is dispersed in a methanol aqueous solution, and sodium hydroxide is added to carry out the saponification reaction. After the reaction is completed, the mixture is centrifuged and extracted with hexane 2-3 times. The resulting mixture is combined to obtain the unsaturated fatty acids of pine nut oil.

[0013] (6) Urea inclusion: The pine nut oil unsaturated fatty acids obtained in (5) are added to the methanol solution of urea, heated until the urea is completely dissolved, and then an inclusion reaction is carried out. After purification, active pine nut oil unsaturated fatty acids rich in pinolenic acid are obtained.

[0014] In the above-mentioned method for preparing active pine nut oil unsaturated fatty acids provided by the present invention, the pine nut slurry obtained after (1) gas spraying treatment has an oil weight ratio of >70%, and the oil phase I obtained after (2) centrifugation oil separation accounts for >30% of the total oil weight of the pine nut raw material.

[0015] In this invention, pine nuts are first crushed and then moistened with water. Then, compressed air coupled with saturated steam is used for a short-term spraying process. The saturated steam rapidly penetrates the softened pine nuts, causing the proteins to denature and degrade under heat and pressure, and the amorphous cellulose to partially degrade. Furthermore, the rapid release of the mixed gas dissolves and disperses the pine nut oil in the slurry. This process not only improves the extraction rate of pine nut oil and its active ingredients, but also ensures that the compressed air maintains effective spraying pressure while lowering the processing temperature. The short spraying time also prevents excessive oxidation of unsaturated fatty acids and other components in the pine nut oil, thus avoiding the loss of active ingredients.

[0016] In the above steps, preferably, in steps (2), (3) and (4), the centrifugation is carried out at a centrifugation rate of 8000 to 12000 r / min and for a centrifugation time of 20 to 30 min.

[0017] Preferably, in (3), the volume ratio of emulsion phase I to water is 1:3-5, the weight of cellulase added accounts for 0.3-0.6% of the total weight of the mixture, the pH of the first enzymatic hydrolysis using cellulase is 5-6, and the hydrolysis temperature is 45-55℃; the weight of neutral protease added accounts for 1.0-2.0% of the total weight of the mixture, the weight of α-amylase added accounts for 0.1-0.2% of the total weight of the mixture, the pH of the second enzymatic hydrolysis using a mixture of neutral protease and α-amylase is 6-7, and the hydrolysis time is 4-8h.

[0018] Preferably, in (4), the freezing and thawing process is specifically operated as follows: first, quick-freeze at -20 to -18°C for 45 to 50 hours, and then thaw completely at room temperature.

[0019] More preferably, the freezing and thawing process described in (4) is specifically operated as follows: first freeze at -20 to -18°C for 48 hours, and then thaw completely at room temperature.

[0020] After the above treatments, after treatments (1) to (4), the extraction rate of the highly unsaturated pine nut oil obtained in (4) is >85%. The highly unsaturated pine nut oil contains the following components: linoleic acid 50-60%, oleic acid 14-18%, pinolenic acid 10-20%, palmitic acid 5-8%, octadecadienoic acid 2.5-4%, stearic acid 2-3%, cis-11-eicosenoic acid 1-2%, eicosanoic acid 0.6-1.2%, and eicosadienoic acid 0.2-0.8%. Among them, the content of unsaturated fatty acids is >90%, and the content of ω-6 fatty acids is >60%.

[0021] Preferably, in (5), the volume ratio of the highly unsaturated pine nut oil to the methanol aqueous solution is 1:3 to 5, the volume ratio of methanol to water is 2 to 5:1, and the mass of sodium hydroxide is 20 to 60% of the mass of the highly unsaturated pine nut oil.

[0022] More preferably, in (5), the volume ratio of the highly unsaturated pine nut oil to the methanol aqueous solution is 1:4, the volume ratio of methanol to water is 3:1, and the mass of sodium hydroxide is 40% of the mass of the highly unsaturated pine nut oil.

[0023] Preferably, in (5), the saponification reaction is carried out at 40-70°C for 0.5-3 hours, and after the reaction is completed, the pH value of the system is adjusted to 1-2 with hydrochloric acid solution.

[0024] Preferably, in (5), the saponification reaction is carried out at 60°C for 1 hour.

[0025] Preferably, in (6), the weight-to-volume ratio of urea to methanol is 0.1-0.5 g: 1 mL, the weight ratio of pine nut oil unsaturated fatty acids to urea is 1: 4-6, the inclusion reaction temperature is -20 to -10 °C, and the reaction time is 8-24 h.

[0026] More preferably, in (6), the weight-to-volume ratio of urea to methanol is 0.3g:1mL.

[0027] Preferably, in (6), the purification process is specifically performed as follows: after the reaction is completed, the mixed solution is taken, methanol is recovered, the remaining solution is acidified with 0.1-0.5 mol / L hot hydrochloric acid solution, and then the acidified solution is extracted with n-hexane 2-3 times to recover n-hexane. The weight-volume ratio of urea to hydrochloric acid solution is (0.2-1.0) g: 1 mL, and the weight-volume ratio of urea to n-hexane is (0.1-0.5) g: 1 mL.

[0028] More preferably, in (6), the purification process is specifically performed as follows: take the mixed solution after the reaction is completed, recover methanol, acidify the remaining liquid with 0.1 mol / L hot hydrochloric acid solution, then extract the acidified solution with n-hexane 3 times and recover n-hexane, wherein the weight-volume ratio of urea to hydrochloric acid solution is 0.5 g: 1 mL, and the weight-volume ratio of urea to n-hexane is 0.25 g: 1 mL.

[0029] Furthermore, the highly unsaturated pine nut oil prepared by the above-mentioned method provided by this invention, as well as the pine nut oil unsaturated fatty acids rich in pinolenic acid obtained in the final product, are the key technical contents protected by this invention.

[0030] Similarly, the application of pine nut oil unsaturated fatty acids rich in pinolenic acid in the preparation of lipid-lowering products also falls within the scope of protection of this invention. Specifically, the application involves using pine nut oil unsaturated fatty acids rich in pinolenic acid in the preparation of lipid-lowering products, which are selected from any of the following: food, health powder, and functional beverages.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) Pine nuts were rapidly modified by compressed air coupled with saturated steam through slurrying. Saturated steam could quickly penetrate and swell the pine nuts, causing the protein to denature and degrade rapidly under heat and pressure, and the amorphous region of cellulose to degrade partially. After the mixed gas was rapidly released, the pine nuts became an emulsion, and the particle size was reduced to the millimeter level and dispersed in the slurry. This facilitated the dissolution of various active ingredients in the pine nuts and improved the extraction rate of pine nut oil. The results showed that more than 30% of pine nut oil could be collected after slurrying and centrifugation once.

[0033] (2) The stepwise enzymatic hydrolysis combined with freeze-thaw operation further improved the extraction rate and unsaturated fatty acid content of pine nut oil, while also protecting the pinolenic acid component from damage. The extraction rate of the high unsaturated pine nut oil obtained by the above method is >85%, and the pinolenic acid content in the unsaturated fatty acids of the final pine nut oil rich in pinolenic acid is >70%, and the recovery rate of pinolenic acid is >60%.

[0034] (3) The present invention provides a highly unsaturated pine nut oil, which is rich in a variety of unsaturated fatty acid components, consisting of 50-60% linoleic acid, 14-18% oleic acid, 10-20% pinolenic acid, 5-8% palmitic acid, 2.5-4% octadecadienoic acid, 2-3% stearic acid, 1-2% cis-11-eicosenoic acid, 0.6-1.2% eicosanoic acid, and 0.2-0.8% eicosanoic acid. The content of unsaturated fatty acids is >90%, and the content of ω-6 fatty acids is >60%.

[0035] (4) The pine nut oil rich in pinolenic acid obtained by the method of the present invention also shows good auxiliary effect in lowering blood lipids. Attached Figure Description

[0036] Figure 1 This is a gas chromatogram of the unsaturated fatty acids in the active pine nut oil obtained in Example 1 of the present invention.

[0037] Figure 2 This is Experiment Example 1 of the present invention, showing the proportion of oil content in oil phase I to the total oil content under different saturated vapor pressures and reaction times. Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0039] Example 1

[0040] A method for preparing active pine nut oil with pinolenic acid-rich unsaturated fatty acids includes the following steps:

[0041] (1) Gas spraying: The mature pine nuts after shelling and peeling are crushed and soaked in distilled water. Then, the soaked material is first passed through 0.4MPa clean air and then saturated water vapor to make the final pressure reach 1.5MPa. Under this condition, it is left to stand for 10 minutes and then depressurized within 1 second. The material is sprayed out by gas rapid release and pine nut slurry is collected.

[0042] (2) Centrifugation for oil separation: The pine nut slurry obtained in (1) was centrifuged at 10000 r / min for 30 min to separate oil phase I, emulsion phase I, aqueous phase I and solid phase I.

[0043] (3) High-solidity enzymatic hydrolysis: The emulsion phase I obtained in (2) was dispersed in water to obtain a mixture. The volume ratio of emulsion phase I to water was 1:4. Cellulase was added to the mixture for the first enzymatic hydrolysis. The weight of cellulase added was 0.4% of the total weight of the mixture. The pH was adjusted to 5.5 and the first enzymatic hydrolysis was carried out at 50°C for 2 hours. Then the pH was adjusted to 6.8 and a mixture of neutral protease and α-amylase was added for the second enzymatic hydrolysis. The weight of neutral protease added was 1.5% of the total weight of the mixture and the weight of α-amylase added was 0.15% of the total weight of the mixture. The second enzymatic hydrolysis was carried out at 50°C for 6 hours. After the enzymatic hydrolysis was completed, the enzyme was inactivated by boiling water bath and centrifuged at 10000 r / min for 30 min to obtain oil phase II, emulsion phase II, aqueous phase II and solid phase II.

[0044] (4) Freezing and thawing: The emulsion phase II obtained in (3) was frozen at -18℃ for 48h, and then completely thawed at room temperature. After thawing, it was centrifuged at 10000r / min for 30min to obtain oil phase III and water phase III. Then, the oil phase I obtained in (2), the oil phase II obtained in (3) and the oil phase III obtained in (4) were thoroughly stirred and mixed to obtain highly unsaturated pine nut oil.

[0045] (5) Saponification reaction: The highly unsaturated pine nut oil obtained in (4) was dispersed in a methanol aqueous solution. The volume ratio of highly unsaturated red pine nut oil to methanol aqueous solution was 1:4, and the volume ratio of methanol to water was 3:1. Then, sodium hydroxide accounting for 40% of the mass of highly unsaturated pine nut oil was added, and the reaction was carried out in a water bath at 60°C for 1 hour. After the reaction was completed, the pH value of the system was adjusted to 1.5 with hydrochloric acid solution. After centrifugation, the upper layer solution was extracted twice with n-hexane, and n-hexane was recovered under reduced pressure to obtain unsaturated fatty acids of pine nut oil.

[0046] (6) Urea inclusion: The pine nut oil unsaturated fatty acids obtained in (5) were added to a methanol solution of urea. After heating until the urea was completely dissolved, the inclusion reaction was carried out at -15℃ for 12h. The mass ratio of pine nut oil unsaturated fatty acids to urea was 1:5, and the weight-volume ratio of urea to methanol was 0.3g:1mL. After the reaction was completed, methanol was recovered. The remaining liquid was acidified with dilute hot hydrochloric acid solution. The acidified solution was extracted twice with n-hexane. After recovering n-hexane, pine nut oil unsaturated fatty acids rich in pinolenic acid were obtained. The weight-volume ratio of urea to hydrochloric acid solution was 0.5g:1mL, and the weight-volume ratio of urea to n-hexane was 0.25g:1mL.

[0047] The unsaturated fatty acids of the pine nut oil rich in pinolenic acid obtained in this example were characterized by gas chromatography according to the national standard GB 5009.168-2016. The composition and content of unsaturated fatty acids were analyzed. The gas chromatogram is attached. Figure 1 .

[0048] Appendix Figure 1 The results showed that the pinolenic acid-rich pine nut oil unsaturated fatty acids prepared according to the method of this embodiment mainly contained pinolenic acid and linoleic acid, of which the content of pinolenic acid was >70%.

[0049] Example 2

[0050] A method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid, the steps are the same as in Example 1, the difference from Example 1 is that: (1) clean air at 0.4 MPa is introduced first, and then saturated water vapor is introduced to make the final pressure reach 1.2 MPa; (3) the weight of cellulase added accounts for 0.3% of the weight of the mixture, the weight of neutral protease added accounts for 1.0% of the weight of the mixture, and the weight of α-amylase added accounts for 0.1% of the weight of the mixture; (6) the mass ratio of pine nut oil unsaturated fatty acids to urea is 1:4, and the temperature of the inclusion reaction is -10℃.

[0051] Example 3

[0052] A method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid, the steps are the same as in Example 1, the difference from Example 1 is that: (1) clean air at 0.4 MPa is introduced first, and then saturated water vapor is introduced to make the final pressure reach 1.8 MPa; (3) the weight of cellulase added accounts for 0.6% of the weight of the mixture, the weight of neutral protease added accounts for 2.0% of the weight of the mixture, and the weight of α-amylase added accounts for 0.2% of the weight of the mixture; (6) the mass ratio of pine nut oil unsaturated fatty acids to urea is 1:6, and the temperature of the inclusion reaction is -20℃.

[0053] Comparative Example 1

[0054] This comparative study focuses on investigating the effect of gas spraying operation on the quality of unsaturated fatty acids in pine nut oil.

[0055] The only difference between this comparative example and Example 1 is that the mature, shelled and peeled pine nuts are crushed and then subjected to steam spraying treatment, i.e., the treatments (1) and (2) are not performed. Instead, enzymatic hydrolysis is carried out directly. All other operations and conditions are the same as in Example 1.

[0056] Comparative Example 2

[0057] A method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid, the steps are the same as in Example 1, the difference from Example 1 is that: (1) the final pressure of the mixed gas is 1.1 MPa, the treatment time is 10 min and 20 min respectively, and the pine nut oil unsaturated fatty acids rich in pinolenic acid are labeled as TL1 and TL2 respectively.

[0058] In addition, the final pressure of the mixed gas was set to 1.2 MPa and 1.8 MPa respectively, and the processing time was 8 min. The unsaturated fatty acids of the pine nut oil rich in pinolenic acid were labeled as PL1 and PL2 respectively. The remaining operations and conditions were the same as in Example 1.

[0059] Comparative Example 3

[0060] A method for preparing pine nut oil unsaturated fatty acids rich in pinolenic acid, the steps are the same as in Example 1, the difference from Example 1 is that: (1) the final pressure of the mixed gas is 1.9 MPa, the treatment time is 10 min and 20 min respectively, and the pine nut oil unsaturated fatty acids rich in pinolenic acid are labeled as TH1 and TH2 respectively.

[0061] In addition, the final pressure of the mixed gas was set to 1.2 MPa and 1.8 MPa respectively, and the processing time was 22 min. The unsaturated fatty acids of the pine nut oil rich in pinolenic acid were labeled as PH1 and PH2 respectively. All other operations and conditions were the same as in Example 1.

[0062] Comparative Example 4

[0063] A method for preparing pine nut oil unsaturated fatty acids rich in pinolenic acid, the steps are the same as in Example 1, the difference from Example 1 is that: (3) the pH of the system is adjusted to 6.0, and cellulase, neutral protease and α-amylase are added for enzymatic hydrolysis, the enzyme amounts are 0.4%, 1.5% and 0.15% (w / w) respectively, and the reaction time is 6h.

[0064] Comparative Example 5

[0065] A method for preparing pine nut oil unsaturated fatty acids rich in pinolenic acid, the steps are the same as in Example 1, the difference being that: (3) only a single type of enzyme preparation is added for one enzymatic hydrolysis, specifically:

[0066] Cellulase was added to adjust the pH to 5.5, the reaction temperature was 50℃, the reaction time was 2h, and the enzyme dosage was 0.4% (w / w) for enzymatic hydrolysis. The resulting pine nut oil rich in pinolenic acid was labeled as E1.

[0067] Neutral protease was added, the pH was adjusted to 7.0, the reaction temperature was 50℃, the reaction time was 6 hours, and the enzyme dosage was 1.5%. Enzymatic hydrolysis was carried out, and the resulting pine nut oil rich in pinolenic acid was labeled as E2.

[0068] α-Amylase was added, the pH was adjusted to 6.0, the reaction temperature was 50℃, the reaction time was 6 hours, and the enzyme dosage was 0.15%. The resulting pine nut oil rich in pinolenic acid was labeled as E3.

[0069] Comparative Example 6

[0070] A method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid, the steps are the same as in Example 1, the difference from Example 1 is that: (6) the mass ratio of pine nut oil unsaturated fatty acids to urea is adjusted to 1:3 and 1:7 respectively, heated until the urea is completely dissolved, and then the mixture is encapsulated at -15℃ for 12h. The pine nut oil unsaturated fatty acids rich in pinolenic acid are labeled as M1 and M2 respectively.

[0071] Comparative Example 7

[0072] A method for preparing pine nut oil unsaturated fatty acids rich in pinolenic acid, the steps are the same as in Example 1, the difference from Example 1 is that: (6) the mass ratio of fatty acid to urea is 1:5, the temperature during inclusion is set to -9℃ and -21℃ respectively, the inclusion reaction is 12h, the rest are the same as in Example 1, and the pine nut oil unsaturated fatty acids rich in pinolenic acid are labeled as I1 and I2 respectively.

[0073] The extraction rates of highly unsaturated pine nut oil obtained from the above embodiments and comparative examples, as well as the content of pinolenic acid in the unsaturated fatty acids of pine nut oil, are shown in Table 1 below.

[0074] Table 1. Extraction rate of highly unsaturated pine nut oil and the content of pinolenic acid in unsaturated fatty acids.

[0075]

[0076]

[0077] The data in Table 1 show that the content of pinolenic acid in the unsaturated fatty acids of pine nut oil prepared by the method of the present invention is significantly higher than that of the unsaturated fatty acids of pine nut oil prepared under the comparative conditions. In particular, under the conditions of Example 1, the extraction rate of highly unsaturated pine nut oil is as high as 92.31%, and the content of pinolenic acid in the unsaturated fatty acids of pine nut oil is as high as 77.86%.

[0078] In addition, in Comparative Example 1, no steam spraying treatment was performed on the pine nuts, and the extraction rate of highly unsaturated pine nut oil was only 70.23%, while the content of pinolenic acid in the unsaturated fatty acids of pine nut oil was only 55.75%, which was significantly lower than that in Example 1. This is because the reaction effect of directly enzymatic hydrolysis after crushing the pine nuts was poor, and the kernel structure was not fully broken down to release pine nut oil and its unsaturated fatty acids.

[0079] In Comparative Examples 2 and 3, the final pressure and treatment time of the mixed gas in the gas spraying were adjusted respectively. The results showed that the pressure of the gas spraying and the length of the gas spraying treatment time had a significant impact on the extraction rate of highly unsaturated pine nut oil, especially on the content of pinolenic acid in the unsaturated fatty acids of pine nut oil. Excessive spraying pressure and excessive time may cause the destruction of the pinolenic acid component and reduce the content of pinolenic acid. The results in the table show that the extraction rate of highly unsaturated pine nut oil obtained by using the conditions in Comparative Examples 2 and 3 was <85% and the content of pinolenic acid in the unsaturated fatty acids of pine nut oil was <70%.

[0080] In Comparative Example 4, the order of enzyme addition was adjusted. Various enzyme preparations were directly mixed and added to the substrate for enzymatic hydrolysis. The extraction rate of highly unsaturated pine nut oil was 83.88%, and the pinolenic acid content in the unsaturated fatty acids of pine nut oil was 63.47%. This may be due to the mutual influence of the three enzyme preparations during the catalytic reaction, which in turn affected the final enzymatic hydrolysis effect.

[0081] In Comparative Example 5, only a single type of enzyme preparation was added. Under the action of each single enzyme preparation, the extraction rate of highly unsaturated pine nut oil was less than 85%, and the content of pinolenic acid in the unsaturated fatty acids of pine nut oil was less than 70%. It can be seen that the enzymatic hydrolysis effect of a single enzyme preparation is relatively limited.

[0082] In Comparative Examples 6 and 7, although the extraction rate of highly unsaturated pine nut oil was relatively high, the pinolenic acid content in the final pine nut oil was significantly reduced due to adjustments in the mass ratio of unsaturated fatty acids to urea and the temperature of the inclusion reaction when using urea to encapsulate the unsaturated fatty acids. This is because if the weight ratio of unsaturated fatty acids to urea is too low, there will be less saturated fatty acids that are stable with urea, resulting in a lower pinolenic acid content in the pine nut oil. However, if the weight ratio of unsaturated fatty acids to urea is too high, it will lead to excessive urea consumption and increase operating costs.

[0083] In addition, if the inclusion reaction temperature is too high or the time is too short, it will affect the amount and stability of urea and saturated fatty acids, resulting in a decrease in the pinolenic acid content in pine nut oil. Conversely, if the inclusion reaction temperature is too low or the time is too long, the molecular mobility will be reduced, thus failing to achieve a good effect.

[0084] Experimental Example 1

[0085] The weight percentage of oil in the oil phase I of pine nut pulp.

[0086] The proportion of oil content in oil phase I of pine nut pulp after gas spraying and centrifugal oil separation was determined by gravimetric method. The results are shown in Table 2 and Appendix. Figure 2 .

[0087] Table 2. Percentage of oil weight in oil phase I relative to total oil weight (%)

[0088] 5min 10min 15min 20min 1.0MPa 12.4 19.1 23.7 28.1 1.2MPa 17.4 30.7 35.5 37.0 1.5MPa 18.7 31.1 39.8 36.3 1.8MPa 24.3 32.9 31.3 30.0 2.0MPa 25.8 28.7 27.9 23.8

[0089] The results in Table 2 show that after gas spraying, pine nuts can yield more than 30% oil, which helps to reduce the amount of bio-enzymes added in the subsequent oil extraction process and further improves the total extraction rate of highly unsaturated pine nut oil.

[0090] Experimental Example 2

[0091] The composition, physicochemical properties, and component content of fatty acids in highly unsaturated pine nut oils obtained by different preparation methods.

[0092] This experiment compares the composition, physicochemical properties, and component content of fatty acids in highly unsaturated pine nut oil obtained under three different preparation methods: Example 1, Comparative Example 1, and traditional solvent extraction.

[0093] The traditional solvent extraction method involves the following steps: pine nuts are crushed, and petroleum ether is used as the extraction solvent. The mixture is refluxed at 60℃ for 10 hours, and the petroleum ether is removed by rotary evaporation to obtain pine nut oil. Gas chromatography was used to analyze the fatty acid composition and content of the pine nut oil according to the national standard GB 5009.168-2016. The results are shown in Table 3 below.

[0094] Table 3. Composition and content (%) of fatty acids in highly unsaturated pine nut oil prepared by different methods

[0095] fatty acid Solvent-extracted oil Comparative Example 1 Example 1 Palmitic acid 6.19 5.83 5.85 stearic acid 2.86 2.87 2.50 Oleic acid 13.89 15.86 16.10 Octadectopadienoic acid 3.10 3.11 3.39 Linoleic acid 55.87 56.55 57.10 Pinolenic acid 11.74 11.94 13.14 Arachidonic acid 0.29 0.31 0.36 Octadecapentaenoic acid 0.25 - - cis-11-eicosenoic acid 1.42 1.59 1.65 diatocarbonate 0.81 0.87 0.28 Eicosadienoic acid 0.50 0.53 0.46 Monounsaturated fatty acids 15.31 17.45 17.75 Polyunsaturated fatty acids 71.75 72.44 74.45 Saturated fatty acids 9.86 9.57 8.63 ω-6 fatty acids 58.97 60.06 61.89

[0096] The results in Table 3 show that the pine nut oils prepared by the three different methods are mainly composed of linoleic acid, oleic acid, pinolenic acid, palmitic acid, octadecadienoic acid, stearic acid, and other components.

[0097] The high-unsaturated pine nut oil prepared using the method in Example 1 contains more than 90% unsaturated fatty acids, with 61.89% being ω-6 fatty acids and 13.14% being pinolenic acid. In contrast, the high-unsaturated pine nut oil obtained using the traditional solvent extraction method contains only 87.06% unsaturated fatty acids, and the high-unsaturated pine nut oil obtained in Comparative Example 1 contains only 89.89% unsaturated fatty acids.

[0098] The reason for the above phenomenon may be that in Example 1, the rapid slurry modification treatment of pine kernels by gas spraying can effectively destroy the structure of pine kernels, release some of the highly unsaturated oils, and promote subsequent enzymatic oil extraction. In addition, the gas treatment process of the present invention is short in time and low in temperature, which will not cause oxidation of olefin bonds in unsaturated fatty acids, thus helping to avoid damage to the pinolenic acid component.

[0099] To further verify the quality of pine nut oil obtained by different methods, the physicochemical properties of the extracted pine nut oil were further characterized.

[0100] The acid value of oils and fats was determined by titration with cold solvent indicator according to GB 5009.229-2016; the peroxide value of oils and fats was determined by titration according to GB 5009.227-2016; the iodine value of oils and fats was determined by titration according to GB / T 5532-2008; and the saponification value of oils and fats was determined by titration according to GB / T 5534-2008. The total flavonoid content in oils and fats was determined by spectrophotometer colorimetry according to GB / T 20574-2006; the vitamin E content was determined by reversed-phase high-performance liquid chromatography according to GB 5009.82-2016; the sterol content was determined by gas chromatography according to GB / T 25223-2010; and the total polyphenol content was determined according to grain standard LS / T6119-2017. The results are shown in Tables 4 and 5 below.

[0101] Table 4 Physicochemical properties of highly unsaturated pine nut oil obtained by different preparation methods

[0102]

[0103] Acid value and peroxide value are important indicators for evaluating the quality of oils and fats. Acid value determines the amount of free fatty acids in oils and fats; the lower the acid value, the lower the degree of rancidity, and the better it is for storage. Peroxide value measures the degree of oxidation of oils and fats; the higher the peroxide value, the more severe the rancidity of the oils and fats, which means that the quality of the oils and fats has declined.

[0104] The data in Table 4 show that the highly unsaturated pine nut oil prepared according to the method in Example 1 has the lowest acid value, peroxide value, saponification value, and iodine value. It is speculated that this is because the pulping modification treatment followed by compound enzymatic hydrolysis can dissolve a large number of natural antioxidants in the pine nuts into the oil, thereby reducing the oxidative rancidity of the oil.

[0105] Table 5. Content of active ingredients in highly unsaturated pine nut oil and unsaturated fatty acids prepared by different methods.

[0106]

[0107] Polyphenols and vitamin E in oils have strong antioxidant properties, phytosterols have the effects of lowering blood cholesterol and antibacterial properties, and flavonoids have a strong ability to scavenge free radicals in the body and improve blood circulation and lower cholesterol.

[0108] The data in Table 5 show that the contents of total flavonoids, total polyphenols, vitamin E, and phytosterols in the high unsaturated pine nut oil and pine nut oil unsaturated fatty acids prepared according to the method of Example 1 of this invention are significantly higher than those in solvent-extracted pine nut oil and pine nut oil and pine nut oil unsaturated fatty acids in Comparative Example 1. This indicates that the structure of pine nuts is destroyed after pulping treatment, which increases the dissolution of substances during the oil extraction process.

[0109] Experimental Example 3

[0110] Evaluation of the lipid-lowering function of pine nut oil rich in pinolenic acid and unsaturated fatty acids.

[0111] Mice were randomly divided into 5 groups of 10 each. The control group was fed a normal diet, while the other groups were fed a high-fat diet. The low, medium, and high dose groups were administered pine nut oil rich in pinolenic acid prepared in Example 1 of this invention by gavage, at doses of 0.5, 1.0, and 2.0 g / (kg·d), respectively. Feed intake was recorded daily during the feeding period. After 6 weeks of feeding, the mice were sacrificed and their final body weight and blood lipid indicators such as triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein were measured. The arteriosclerosis index was calculated, and the results are shown in Table 6 below.

[0112] Table 6 Evaluation of the lipid-lowering function of pine nut oil rich in pinolenic acid and unsaturated fatty acids.

[0113] Functional evaluation indicators control group Model group High-dose group medium dose group low-dose group Feed intake (g) 49.63 48.32 47.52 47.65 48.01 Final body weight (g) 28.43 31.65 28.80 28.97 29.42 Triglycerides (mmol / L) 0.55 1.25 0.62 0.75 0.81 Total cholesterol (mmol / L) 4.02 4.63 4.17 4.23 4.40 High-density lipoprotein (mmol / L) 2.37 1.55 2.00 1.95 1.88 Low-density lipoprotein (mmol / L) 1.60 2.32 1.74 1.88 1.90 Arteriosclerosis Index 0.70 1.99 1.09 1.17 1.34

[0114] The results showed that there was no significant difference in food intake among the groups during the feeding period; after 6 weeks of feeding, the final weight of mice in the model group was significantly higher than that in the control group, while the weight of mice in the low, medium and high dose groups of pine nut oil rich in pinolenic acid unsaturated fatty acids was significantly lower than that in the model group.

[0115] Mice in the low, medium, and high dose groups of pine nut oil rich in pinolenic acid had significantly lower levels of triglycerides, total cholesterol, and low-density lipoprotein than the model group, and significantly higher levels of high-density lipoprotein. The calculated arteriosclerosis index was also significantly lower in mice than in the model group.

[0116] This indicates that pine nut oil, rich in pinolenic acid and unsaturated fatty acids, has an auxiliary effect in lowering blood lipids, such as reducing serum triglycerides, low-density lipoprotein, and total cholesterol in mice, while increasing high-density lipoprotein, and can reduce the risk of arteriosclerosis.

[0117] In addition, in order to better compare the lipid-lowering effects of pine nut oil unsaturated fatty acids obtained by different preparation methods, this invention also measured the arteriosclerosis index of pine nut oil unsaturated fatty acids rich in pinolenic acid prepared in the above-mentioned experimental groups in a high-fat model mouse. The results are shown in Tables 7-8 below.

[0118] Table 7 Arteriosclerosis Index in High-Fat Model Mice Using Pine Nut Oil Unsaturated Fatty Acids from Each Example

[0119] experimental group Example 1 Example 2 Example 3 Arteriosclerosis Index 1.02 1.13 1.25

[0120] Table 8 Arteriosclerosis index of high-fat model mice with unsaturated fatty acids from pine nut oil in each comparative proportion.

[0121]

[0122] The arteriosclerosis index is one of the indicators for assessing the risk of developing arteriosclerosis. An elevated arteriosclerosis index indicates an increased risk of developing arteriosclerosis.

[0123] The results in Tables 7 and 8 above show that after administering the pine nut oil unsaturated fatty acids prepared in each example to high-fat model mice, the atherosclerosis index of the mice was significantly lower than that of mice given the pine nut oil unsaturated fatty acids prepared under the control conditions. This indicates that the pine nut oil unsaturated fatty acids prepared in this invention have a good auxiliary function in lowering blood lipids.

Claims

1. A method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid, characterized in that, Includes the following steps: (1) Gas spraying: Pine nuts are crushed, water is added to make them swell, and then 0.4~0.6 MPa of clean air is first introduced into the swelled material, followed by saturated water vapor to make the final pressure reach 1.2~1.8 MPa. Under these conditions, the material is left to stand for 10~20 min, and then the pressure is released within 1 second. The material is sprayed out by the rapid release of gas, and the pine nut slurry is collected. (2) Centrifugation for oil separation: The pine nut pulp obtained in (1) is centrifuged to separate oil phase I, emulsion phase I, aqueous phase I and solid phase I; (3) High-solidity enzymatic hydrolysis: Disperse the emulsion phase I obtained in (2) in water to obtain a mixture. First, add cellulase to the mixture for the first enzymatic hydrolysis, and then add a mixture of neutral protease and α-amylase for the second enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme, centrifuge, and obtain oil phase II, emulsion phase II, aqueous phase II and solid phase II. The volume ratio of emulsion phase I to water is 1:3~5, the weight of cellulase added accounts for 0.3~0.6% of the total weight of the mixture, the pH of the first enzymatic hydrolysis using cellulase is 5~6, and the enzymatic hydrolysis temperature is 45~55℃. The weight of neutral protease added accounts for 1.0-2.0% of the total weight of the mixture, and the weight of α-amylase added accounts for 0.1-0.2% of the total weight of the mixture. When using a mixture of neutral protease and α-amylase for the second enzymatic hydrolysis, the pH is 6-7, and the hydrolysis time is 4-8 h. (4) Freezing and thawing: The emulsion phase II obtained in (3) is first frozen at -20~-18℃ for 45~50 h, and then thawed completely at room temperature. After centrifugation, oil phase III and aqueous phase III are obtained. Then, oil phase I obtained in (2), oil phase II obtained in (3) and oil phase III obtained in (4) are thoroughly stirred and mixed to obtain highly unsaturated pine nut oil. (5) Saponification reaction: The highly unsaturated pine nut oil obtained in (4) is dispersed in a methanol aqueous solution, and sodium hydroxide is added to carry out the saponification reaction. After the reaction is completed, the mixture is centrifuged and extracted with hexane 2-3 times. The mixture is then combined to obtain the unsaturated fatty acids of pine nut oil. The volume ratio of the highly unsaturated pine nut oil to the methanol aqueous solution is 1:3~5, the volume ratio of methanol to water is 2~5:1, the mass of sodium hydroxide is 20~60% of the mass of the highly unsaturated pine nut oil, the saponification reaction is carried out at 40~70℃ for 0.5~3 h, and after the reaction is completed, the pH value of the system is adjusted to 1~2 with hydrochloric acid solution. (6) Urea inclusion: The pine nut oil unsaturated fatty acids obtained in (5) are added to a methanol solution of urea and heated until the urea is completely dissolved before inclusion reaction. The weight-volume ratio of urea to methanol is 0.1~0.5 g:1 mL, the weight ratio of pine nut oil unsaturated fatty acids to urea is 1:4~6, the temperature of inclusion reaction is -20~-10℃, the reaction time is 8~24 h, and after purification, active pine nut oil unsaturated fatty acids rich in pinolenic acid are obtained.

2. The method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid as described in claim 1, characterized in that, The pine nut slurry obtained after (1) gas spraying treatment has an oil weight ratio of >70%, and the oil phase I obtained after (2) centrifugation oil separation accounts for >30% of the total oil weight of the pine nut raw material.

3. The method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid as described in claim 1, characterized in that, In (2), (3) and (4), the centrifugation is carried out at a centrifugation rate of 8000~12000 r / min and a centrifugation time of 20~30 min.

4. The method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid as described in claim 1, characterized in that, The extraction rate of the highly unsaturated pine nut oil mentioned in (4) is >85%. The highly unsaturated pine nut oil contains the following components: linoleic acid 50~60%, oleic acid 14~18%, pinolenic acid 10~20%, palmitic acid 5~8%, octadecadienoic acid 2.5~4%, stearic acid 2~3%, cis-11-eicosenoic acid 1~2%, eicosanoic acid 0.6~1.2%, eicosanoic acid 0.2~0.8%, wherein the weight percentage of unsaturated fatty acids is >90%, and the weight percentage of ω-6 fatty acids is >60%.

5. The method for preparing active pine nut oil unsaturated fatty acids rich in pinolenic acid as described in claim 1, characterized in that, (6) The purification process is specifically performed as follows: take the mixed solution after the reaction is completed, recover the methanol, acidify the remaining liquid with dilute hot hydrochloric acid solution, and then extract the acidified solution with n-hexane containing urea 2 to 3 times, wherein the weight-volume ratio of urea to hydrochloric acid solution is (0.2~1.0) g: 1 mL, and the weight-volume ratio of urea to n-hexane is (0.1~0.5) g: 1 mL.

6. The application of the active pine nut oil unsaturated fatty acids rich in pinolenic acid prepared by the method described in claim 1 in the preparation of lipid-lowering products, characterized in that, The active pine nut oil rich in pinolenic acid and its unsaturated fatty acids are used in the preparation of a lipid-lowering product, which is a health powder.

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