Method for enriching unsaturated fatty acid in tussah chrysalis oil through ultrasonic-assisted beta-cyclodextrin inclusion method
Through ultrasonic assisted β-cyclodextrin encapsulation, the problem of low extraction efficiency of unsaturated fatty acids in tussar pupa oil is solved, and efficient extraction and utilization is achieved. It not only improves the added value of the product, but also simplifies the process flow and reduces energy Consumption.
Patent Information
- Application Number
- CN202411516468.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art is difficult to efficiently extract unsaturated fatty acids, especially α-linolenic acid, in the tusk pupa oil, which leads to the failure to fully utilize the added value of tusk pupa oil.
Ultrasonic assisted β-cyclodextrin inclusion is used to accelerate the interaction between β-cyclodextrin and unsaturated fatty acids in tussum pupa oil through ultrasonic waves to form β-cyclodextrin inclusions, thereby improving the utilization rate of unsaturated fatty acids.
It significantly improves the extraction and utilization rate of unsaturated fatty acids in tussaurus pupa oil, increases the added value of the product, simplifies the process flow, and reduces energy consumption.
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Figure CN119432500A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food processing and relates to a method for enriching unsaturated fatty acids in tussah pupa oil by using an ultrasound-assisted beta-cyclodextrin inclusion method. Background Art
[0002] Tussah silkworms are insects of the family Macrochrysalis in the order Lepidoptera. They live on oak trees and feed on oak leaves. From ancient times to the present, tussah silkworms have been widely used. Ancient people used tussah silkworm resources as medicine. In modern society, tussah silkworms have also been proven to have a wide range of uses through various scientific studies and experiments, which has broadened the use of tussah silkworm resources, especially in the field of medical care.
[0003] Tussah silkworm pupae have extremely high edible and medicinal value and are one of China's unique insect resources with important economic value. Fat and protein are the two nutrients with the highest content in tussah silkworms, and tussah silkworm fat is mostly composed of unsaturated fatty acids, such as linolenic acid and linoleic acid. Tussah silkworm pupa oil is the oil produced by tussah silkworms through a series of operations. It is clear and transparent light yellow, but often accompanied by the unpleasant fishy smell unique to tussah silkworms. Its unsaturated fatty acid content reaches about 79%, especially the content of linolenic acid and linoleic acid, which are essential fatty acids for the human body, which account for more than half of the unsaturated fatty acid content. How to efficiently extract α-linolenic acid from tussah silkworm pupa oil and develop healthy and nutritious tussah silkworm pupa oil products is the top priority for the tussah silkworm industry to move towards high-level and high-value-added fields.
[0004] β-Cyclodextrin is also known as cycloheptanoglucan, cyclomaltoheptaose, cyclomaltodextrin, etc. Its molecular formula is C 42 H 70 O 35 It is a cyclic oligomeric non-reducing sugar with a hydrophilic surface and a hydrophobic inner cavity. It is composed of 7 glucose molecules connected by α-1,4-glycosidic bonds. The molecular weight is about 1135Da, the cavity height is about 7.9A°, the inner cavity diameter is about 6.5A°, and the cavity volume is about 262A°.
[0005] β-cyclodextrin inclusion fatty acids can be divided into solid phase preparation methods (grinding method, closed container heating method, room temperature shaking method, etc.) and liquid phase preparation methods (coprecipitation method, ultrasound-assisted method, supercritical fluid method, spray drying method, freeze drying method, etc.) according to the different operation methods and embedded materials.
[0006] Ultrasonic-assisted β-cyclodextrin inclusion method utilizes the lipophilicity of its internal cavity to provide a good binding environment for fatty acids in the aqueous phase, and utilizes ultrasonic conditions to accelerate the interaction between molecules. Fatty acid molecules with greater polarity are partially or completely embedded in the β-cyclodextrin cavity and combined to form β-cyclodextrin inclusion complexes. No covalent bonds are generated during the formation of β-cyclodextrin fatty acid inclusion complexes. Its formation mainly relies on van der Waals forces, hydrogen bonds, and hydrophobic forces between β-cyclodextrin solutions and solutions or between β-cyclodextrin and fatty acid molecules.
[0007] This method combines the physical effect of ultrasound with the inclusion properties of β-cyclodextrin, and has several advantages. First, ultrasound induces the cavitation effect of bubbles in the solution, making the mixing between β-cyclodextrin and the substance to be included more uniform, thereby improving the inclusion efficiency. In addition, the formation and rupture of bubbles increase the local shear force in the system, which helps to accelerate the inclusion process, thereby significantly shortening the reaction time. Therefore, compared with the traditional inclusion method, which usually requires a long time of stirring and reaction, the application of ultrasound can significantly improve production efficiency. On the other hand, the effect of ultrasound makes the structure of β-cyclodextrin more uniform and improves the quality of the inclusion compound. This can make the particle size distribution of the inclusion compound more consistent, the morphology more stable, and reduce the performance differences caused by uneven inclusion. Traditional inclusion methods usually require high temperatures and long-term mechanical stirring, high energy consumption and easy to induce side reactions. The ultrasound-assisted inclusion method can complete the reaction at a lower temperature and in a shorter time, thereby effectively reducing energy consumption. Ultrasound can also improve the solubility and bioavailability of the substance to be included. At the same time, the application of ultrasound can reduce the pyrolysis reaction caused by high temperature, thereby reducing the formation of by-products. Operation at room temperature can better control the reaction conditions, thereby reducing the occurrence of side reactions. Finally, the ultrasound-assisted inclusion method is simple to operate and easy to control. By adjusting the frequency and power of ultrasound, the reaction conditions can be flexibly optimized to meet different inclusion requirements. Compared with some traditional chemical methods, ultrasound-assisted methods usually involve fewer chemical additives and solvents, and therefore have better environmental protection and sustainability. Summary of the invention
[0008] The invention utilizes ultrasound-assisted beta-cyclodextrin inclusion method to extract unsaturated fatty acids in tussah pupae, thereby improving the utilization rate of unsaturated fatty acids in tussah pupae oil and further improving the added value of tussah pupae oil.
[0009] A production process for ultrasonic-assisted extraction of tussah pupa oil comprises the following steps:
[0010] (1) Pretreatment: fresh tussah pupae are sorted, cleaned, and sterilized, frozen overnight and then slow-frozen for 10 minutes, shelled, visceral removed, pulped, poured into a culture dish, refrigerated and pre-frozen, freeze-dried at -50°C for 48 hours in a freeze dryer, and sieved through 40 mesh to obtain tussah pupae powder;
[0011] (2) Supercritical CO2 extraction of oil: Extraction conditions: extraction pressure 25-35 MPa, extraction temperature 45-55°C, extraction CO2 flow rate 40 L / h, extraction time 60 min. Weigh the dried powder of tussah pupa, pour it into the barrel, put the barrel into the extraction kettle, open the gas cylinder, open the air inlet valve of the high-pressure pump, start extraction, and collect the extract;
[0012] (3) Pretreatment (saponification) of tussah pupa oil: Weigh 1 g of tussah pupa oil and mix it with 5 mL of saponification medium NaOH solution or anhydrous ethanol solution, and then perform saponification reaction. After the sample is fully saponified, add 25 mL of petroleum ether for extraction. Repeat three times until the solution is clear. Adjust the pH to 2-3 with 10% hydrochloric acid, and place it in a forced air drying oven for 1 hour to remove petroleum ether and other impurities. The remaining substance is the mixed fatty acid of tussah pupa oil.
[0013] (4) Ultrasonic-assisted β-cyclodextrin inclusion: After thawing at room temperature, vacuum filtration was performed to obtain a white solid with a slightly yellowish surface, which was washed three times with 100 mL of anhydrous ethanol solution and centrifuged (8500 r / min, 12 min), the supernatant was combined, and the solid part was placed in a forced air drying oven and dried to constant weight. The obtained white powder was the β-cyclodextrin-tussah pupa oil complex; the mixture was transferred to a beaker, petroleum ether was added, and the mixture was stirred with a magnetic stirrer for 1 h, then vacuum filtered, and the petroleum ether was recovered by a rotary evaporator (55°C). The remaining liquid was the unsaturated fatty acid extract of tussah pupa oil;
[0014] In the above-mentioned β-cyclodextrin inclusion method for enriching unsaturated fatty acids in tussah pupa oil, the amount of β-cyclodextrin input corresponding to the mixed fatty acids FFA of tussah pupa oil obtained per 1g of tussah pupa oil is 18.5g.
[0015] In the above-mentioned method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method, in step 3), the pH is adjusted to 2-3.
[0016] In the above-mentioned method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method, in step 4), the ultrasonic treatment time is 60 minutes.
[0017] In the above-mentioned method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method, in step 4), the ultrasonic temperature of the ultrasonic treatment is 65°C.
[0018] The beneficial effects of the present invention are:
[0019] (1) The UV-visible spectrum shows that when ethanol is used as the saponification medium and saponification pretreatment is performed, the characteristic peak absorbance of tussah pupa oil is the largest. This indicates that saponification pretreatment is beneficial to the further extraction of unsaturated fatty acids. Figure 1 shown.
[0020] (2) The results of Fourier transform infrared spectroscopy showed that the characteristic peaks of the oil after pretreatment still existed, and the structure could be deduced: the pretreatment method of the invention did not change the secondary structure of the pupa oil and could be applied in experiments and production.
[0021] (3) Orthogonal experiment was used to optimize the auxiliary β-cyclodextrin inclusion method. The best experimental conditions were ultrasonic time of 60 min, ultrasonic temperature of 65°C, and β-cyclodextrin input of 18.5 g. The inclusion rate of mixed fatty acids (mainly α-linolenic acid) of tussah pupa oil was the highest, reaching 39.28±2.57%. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Select UV results for the experimental conditions of tussah pupa oil pretreatment.
[0023] Figure 2 Select infrared result - absorbance value for the experimental conditions of tussah pupa oil pretreatment.
[0024] Figure 3 Select infrared result - transmittance for the experimental conditions of tussah pupa oil pretreatment.
[0025] Figure 4 This is the effect of β-cyclodextrin input on the extraction rate of unsaturated fatty acids.
[0026] Figure 5 The effect of ultrasonic time on the extraction rate of unsaturated fatty acids.
[0027] Figure 6 The effect of ultrasonic temperature on the extraction rate of unsaturated fatty acids.
[0028] Fig. 7A This is the GC-MS result of tussah pupa oil.
[0029] Figure 7B The figure is the GC-MS result of inclusion complex. DETAILED DESCRIPTION
[0030] The present invention is further described in detail below through specific examples. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.
[0031] Example 1 Ultrasound-assisted β-cyclodextrin inclusion method to extract unsaturated fatty acids from tussah pupae
[0032] (1) Raw material pretreatment: The tussah silkworms are sorted, cleaned, sterilized, freeze-dried, and crushed into tussah powder using a high-speed crusher as the raw material. The tussah silkworms used are 5-year-old tussah silkworms of the species Tussah genus produced in Dandong City, Liaoning Province;
[0033] (2) Extraction of pupa oil: Weigh the pretreated tussah powder and put it into the extraction kettle. When the temperature reaches a predetermined temperature of 45-55°C, open the CO2 cylinder to supply gas. When the extraction pressure reaches a pressure of 25-35 MPa, adjust the flow rate to 40 L / h. Use ethanol as the extractant to extract the tussah oil at a constant temperature and pressure, and calculate the time at the same time. After the set time of 50-70 minutes, collect the tussah oil from the outlet of the separation kettle. The collected oil is rotary evaporated at 60°C for 30 minutes to obtain high-purity pupa oil.
[0034] (3) Saponification of pupa oil: Weigh 1 g of tussah pupa oil and mix it with 5 mL of anhydrous ethanol solution, a saponification medium, and perform saponification reaction. After the sample is fully saponified, add 25 mL of petroleum ether for extraction. Repeat three times until the solution is clear. Adjust the pH of the extract to 2-3 with 10% hydrochloric acid and place it in a forced air drying oven for 1 hour to remove petroleum ether and other impurities. The remaining substance is the mixed fatty acid FFA of tussah pupa oil.
[0035] (4) Ultrasonic-assisted β-cyclodextrin inclusion: Weigh β-cyclodextrin and dissolve it in water to make a solution. Accurately weigh 1g of tussah pupa oil saponified to obtain mixed fatty acid FFA of tussah pupa oil, add it to the saturated solution of β-cyclodextrin, put the mixed solution into an ultrasonic cleaner, set the ultrasonic temperature and time, and after the ultrasonic treatment time is over, place it in the freezer of a refrigerator (-18°C) and freeze it for 18 hours. After thawing at room temperature, vacuum filtration is performed to obtain a white solid with a slightly yellowish surface. The solid is washed with anhydrous ethanol solution three times and centrifuged. The supernatant is combined, and the solid part is placed in a forced air drying oven to dry to constant weight. The obtained white powder is the β-cyclodextrin-tussah pupa oil complex; transfer it to a beaker, add petroleum ether, stir it with a magnetic stirrer, and then vacuum filter it. The petroleum ether is recovered by a rotary evaporator. The remaining liquid is the unsaturated fatty acid extract of tussah pupa oil.
[0036] Example 2 Ultrasound-assisted β-cyclodextrin inclusion method single factor experiment
[0037] Effect of β-cyclodextrin input on the extraction rate of unsaturated fatty acids: 12.5g, 15.5g, 18.5g, 21.5g, and 24.5g of β-cyclodextrin were accurately weighed, and 100mL of pure water was added and heated in a water bath at 60℃ for 10min to prepare β-cyclodextrin solution. 1g of tussah pupa oil was accurately weighed and saponified to obtain FFA, which was added to the β-cyclodextrin solution. The mixed solution was placed in an ultrasonic cleaner, and the ultrasonic temperature was set to 55℃ and the time was 60min. After the ultrasonic treatment time was over, it was placed in the freezer of a refrigerator (-18℃) for 18h and then de-inclusion was performed.
[0038] like Figure 4 As shown in the figure, when the amount of β-cyclodextrin input is 18.5g, the unsaturated fatty acids in the tussah pupa oil are included to the maximum extent. However, when the amount of β-cyclodextrin input continues to increase, the absorption value of the characteristic peak of tussah pupa oil shows a downward trend. It is speculated that when the unsaturated fatty acids are completely saturated, the remaining β-cyclodextrin will include other impurities in the solution, such as saturated fatty acids, and affect the content of unsaturated fatty acids after de-inclusion.
[0039] The effect of ultrasonic time on the extraction rate of unsaturated fatty acids was set to 30min, 40min, 50min, 60min, and 70min, the ultrasonic power was set to 50w, the ultrasonic temperature was set to 55℃, 18.5g of β-cyclodextrin was accurately weighed, and after adding pure water, it was heated in a water bath at 60℃ for 10min to prepare a saturated solution of β-cyclodextrin. Accurately 1g of tussah pupa oil was saponified to obtain FFA, added to the saturated solution of β-cyclodextrin, and magnetically stirred for 5min. The mixed solution was placed in an ultrasonic cleaner and de-inclusion was performed after the ultrasonic treatment time was over.
[0040] like Figure 5 As shown in the figure, when the ultrasonic time is 60 minutes, the absorption value of the characteristic peak of unsaturated fatty acids in tussah pupa oil is the largest, but then it begins to decline. It is speculated that there may be an over-inclusion phenomenon. Ultrasonic treatment can accelerate the mutual movement between molecules. The longer the ultrasonic time, the more obvious the effect, and the possibility of β-cyclodextrin including other impurities will also increase; it may also be due to the long ultrasonic time, which destroys the stable structure of the complex of β-cyclodextrin and unsaturated fatty acids in tussah pupa oil and causes the unsaturated fatty acids to detach from the cavity of β-cyclodextrin, resulting in a decrease in the inclusion rate.
[0041] The effect of ultrasonic temperature on the extraction rate of unsaturated fatty acids was set at 35℃, 45℃, 55℃, 65℃, and 75℃, with an ultrasonic power of 50w and an ultrasonic temperature of 50℃. 18.5g of β-cyclodextrin was accurately weighed, and after adding pure water, a saturated solution of β-cyclodextrin was prepared by heating in a water bath at 60℃ for 10min. 1g of tussah pupa oil was saponified to obtain FFA, which was added to the saturated solution of β-cyclodextrin and stirred magnetically for 5min. The mixed solution was placed in an ultrasonic cleaner and de-inclusion was performed after the ultrasonic treatment time was over.
[0042] like Figure 6 As shown, under ultrasonic conditions, the solubility of β-cyclodextrin in water is the highest at 65°C. At this time, the internal cavity of β-cyclodextrin is the loosest, and the unsaturated fatty acids of tussah pupa oil are most easily included, so the inclusion rate is the highest at this temperature.
[0043] The best experimental conditions were optimized, with an ultrasonic time of 60 min, an ultrasonic temperature of 65°C, and an input amount of 18.5 g of β-cyclodextrin. After the ultrasonic treatment time was over, de-inclusion was performed.
[0044] Example 3 GC-MS analysis of the composition and content of unsaturated fatty acids in tussah pupa oil before and after inclusion
[0045] Agilent GC-MS 7894A+5975C (GC-MS) was used; the chromatographic column was DB-5MS (30m×0.25mm×0.25μm); the front injection port temperature was 250℃. Heating program: start and end temperature, 40℃, hold for 1min; heating rate, 5℃ / min to 190℃, hold for 5min, 6℃ / min to 250℃, hold for 5min. Carrier gas: helium (99.999%); flow rate: 3mL / min. Split ratio: 10:1; injection volume: 1μL; transfer line temperature: 250℃; mass spectrometry reference conditions: ion source temperature: 230℃; quadrupole temperature: 150℃; ionization mode: EI; solvent delay time: 3min; data acquisition mode: full scan mode; mass-to-charge ratio (m / z): 35~8004.
[0046] The calibration curve was prepared by diluting the standard with n-hexane to form 5 standard solutions with different mass concentrations. The peak area of each standard solution was detected in turn according to the above chromatographic conditions. The peak area was used as the ordinate and the concentration was used as the abscissa to calculate various standard curves, linear ranges and correlation coefficients.
[0047] The gas chromatography-mass spectrometry results of the two samples are shown in FIG7 . As can be seen from FIG7 , the ultrasound-assisted β-cyclodextrin inclusion method successfully enriched the α-linolenic acid in the tussah pupa oil.
[0048] Through quantitative analysis by internal standard method, according to the content and conversion coefficient of each fatty acid methyl ester, the fatty acid composition and content of the two samples were obtained, as shown in the following table:
[0049] Table 1 Fatty acid composition and content of tussah pupa oil
[0050]
[0051] Table 2 Composition and content of fatty acids in inclusion complex
[0052]
[0053] The fatty acid composition and content of the two samples were analyzed as shown in the following table:
[0054] Table 3 Comparison of fatty acid contents of tussah pupa oil and inclusion complex
[0055]
[0056] According to the table above, except for α-linolenic acid, the contents of other unsaturated fatty acids also increased to varying degrees. It also proved that ultrasound-assisted β-cyclodextrin inclusion method is conducive to the enrichment of unsaturated fatty acids such as α-linolenic acid that are beneficial to the human body, which has guiding significance for the enrichment of unsaturated fatty acids in the future.
Claims
1. A method for enriching unsaturated fatty acids in tussah pupa oil by ultrasound-assisted β-cyclodextrin inclusion method, characterized in that: The steps of this method are: 1) Pretreatment: fresh tussah silkworm pupae are sorted and cleaned, frozen overnight and then slow-frozen, shelled, visceral removed, pulped, poured into a culture dish, refrigerated in a refrigerator for pre-freezing, and freeze-dried in a freeze dryer to obtain tussah silkworm pupae powder; 2) Supercritical CO2 extraction of oil: weigh the tussah pupa powder, pour it into the barrel, put the barrel into the extraction kettle, open the gas cylinder, open the air inlet valve of the high-pressure pump, start extraction, and collect the extract; 3) Saponification treatment: weigh the tussah pupa oil and mix it with the saponification medium, perform saponification reaction, and add petroleum ether to extract the sample after it is fully saponified. Repeat three times until the solution is clear, adjust the pH with hydrochloric acid, and place it in a forced air drying oven for 1 hour to remove petroleum ether and other impurities. The remaining substance is the mixed fatty acid FFA of tussah pupa oil; 4) Ultrasonic-assisted β-cyclodextrin inclusion: take the mixed fatty acids of tussah pupa oil obtained in step 3), add them to the β-cyclodextrin solution, subject the mixed solution to ultrasonic treatment, then freeze at -18°C for 18 hours, then thaw at room temperature and filter under reduced pressure to obtain a white solid with a slightly yellowish surface, wash and centrifuge, combine the supernatants, and dry the solid part to constant weight. The obtained white powder is the β-cyclodextrin-tussah pupa oil complex; add petroleum ether, stir and filter under reduced pressure, recover the petroleum ether on a rotary evaporator, and the remaining liquid is the unsaturated fatty acid extract of tussah pupa oil.
2. The method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method according to claim 1, characterized in that: In step 2), the extraction pressure is 30 MPa, the extraction temperature is 50° C., the extraction CO2 flow rate is 40 L / h, and the extraction time is 60 min.
3. The method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method according to claim 1, characterized in that: In step 3), the saponification medium is NaOH solution or anhydrous ethanol, and the mass fraction of the NaOH solution is 5%.
4. The method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method according to claim 3, characterized in that: In step 3), the solid-liquid ratio of the tussah pupa oil to the saponification medium is 1 g:5 mL.
5. The method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method according to claim 4, characterized in that: The amount of β-cyclodextrin corresponding to the mixed fatty acid FFA of tussah pupa oil obtained per 1g of tussah pupa oil is 18.5g.
6. The method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method according to claim 1, characterized in that: In step 3), the pH is adjusted to 2-3.
7. The method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method according to claim 1, characterized in that: In step 4), the ultrasonic treatment time is 60 min.
8. The method for enriching unsaturated fatty acids in tussah pupa oil by β-cyclodextrin inclusion method according to claim 1, characterized in that: In step 4), the ultrasonic temperature of the ultrasonic treatment is 65°C.