Method for preparing eucalyptol inclusion compound by double-enzyme method

Abstract

The invention relates to a method for preparing an eucalyptol clathrate compound by a double-enzyme method, and belongs to the field of clathrate compound preparation. On the basis that beta-CGT enzyme acts on a starch substrate to prepare cyclodextrin traditionally, dextrin debranching enzyme (SsGDE enzyme) is additionally added to act on the starch substrate synergistically, the proportion of alpha-1, 6 glucosidic bonds in the substrate is reduced, the production efficiency of beta-cyclodextrin is improved, meanwhile, the utilization degree of beta-CGT enzyme on the substrate is improved, and compared with an emulsification technology, the method has the advantages that the yield of beta-cyclodextrin is increased, and the yield of beta-cyclodextrin is increased. The stability and the effect of the eucalyptol inclusion compound are greatly improved, and a foundation is laid for application of the eucalyptol inclusion compound in the fields of medicines, daily chemicals and agriculture.

Description

Technical Field

[0001] The present invention relates to the technical field of inclusion compound preparation, and in particular to a method for preparing eucalyptol inclusion compound by a double enzyme method. Background Art

[0002] Eucalyptol, also known as 1,8-cineole, is a kind of monoterpenoid compound, which is mainly distributed in the volatile oils of plants such as eucalyptus leaves, rosemary, and galangal. Eucalyptol has anti-inflammatory and antioxidant activities, and can be used in combination with other drugs for the treatment of influenza, enteritis, and various infections; eucalyptol also has a strong smell and can be used as a fragrance for formulating products such as perfumes, detergents, and toothpastes; in addition, eucalyptol can play a role by inhibiting the synthesis of acetylcholinesterase in insects, has insecticidal properties, and has good homology with plants, and has the effects of improving the quality and increasing the yield of crops, and can be developed into a new type of biological pesticide.

[0003] However, eucalyptol is almost insoluble in water, volatile, and has poor stability, which limits its application to a certain extent. Currently, methods such as solid-based nanoencapsulation, liposome encapsulation, and nanoemulsion have been developed to solve the above problems. In the latest research process, a method for preparing essential oil emulsion by an enzyme method was disclosed, in which the combination of emulsification and an enzyme method for preparing a cyclodextrin system was used to prepare eucalyptol inclusion compound, which improved the water solubility of eucalyptol to a certain extent. However, the emulsifier content in this system is 5%, and the relatively high concentration of emulsifier content limits the biological activity of the essential oil to a certain extent.

[0004] Therefore, there is an urgent need to provide a more efficient method for embedding eucalyptol to improve the eucalyptol inclusion compound. Summary of the Invention

[0005] Regarding the method of preparing eucalyptol inclusion complex by enzymatic method and improving the stability of eucalyptol inclusion complex by adding emulsifier, the presence of emulsifier limits the biological activity of essential oil to a certain extent, thus greatly reducing the product efficacy. The present invention provides a method for preparing eucalyptol inclusion complex by double enzyme method. On the basis of the traditional preparation of cyclodextrin by using β-CGT enzyme to act on starch substrate, pullulanase (SsGDE enzyme) is additionally added to act on starch substrate synergistically, significantly improving the production efficiency of β-cyclodextrin and replacing the existing enzymatic emulsification technology. The pullulanase (Saccharolobus solfataricus STB09 glycogendebranching enzyme, SsGDE) derived from the thermophilic archaeon Saccharolobus solfataricus STB09 has high debranching efficiency and can act synergistically with β-CGT enzyme. Specifically, from the perspective of enzyme activity, SsGDE exhibits high debranching activity and thermal stability at 70 °C, so introducing SsGDE in the liquefaction stage can continuously play a promoting role throughout the cyclization reaction process. From the perspective of the structure of cyclization products and the action mode of enzymes, β-CGT enzyme tends to act from the non-reducing end of long branched chains and is easily inhibited by small molecular oligosaccharides. While SsGDE can hydrolyze the α-1,6 glycosidic bonds in the cyclization substrate, and its debranching products are mainly segments with DP 6-12, DP 13-24, DP 25-36 and DP≥37, containing only a very low proportion of segments with DP<6. This not only helps to eliminate the interference of branched chains on β-CGT enzyme, but also does not make β-CGT enzyme affected by debranching products. Most importantly, this part of the short-chain dextrin that has not been cyclized after debranching also has a certain inclusion ability and can include free essential oil molecules that have not completely entered the cyclodextrin cavity. This enables pullulanase to greatly improve the wall material concentration of the included essential oil while promoting the conversion rate of β-cyclodextrin, and significantly improve the efficacy of eucalyptol inclusion complex compared with the emulsification technology.

[0006] The present invention is achieved by the following technical solutions:

[0007] The first object of the present invention is to provide a method for preparing eucalyptol inclusion complex by double enzyme method, including the following steps:

[0008] (1) Adding CGT enzyme and pullulanase to the starch aqueous solution for enzymatic hydrolysis to obtain a cyclodextrin mixture solution;

[0009] (2) Mixing eucalyptol and co-emulsifier evenly to obtain a mixture of eucalyptol and co-emulsifier;

[0010] (3) Adding the mixture of eucalyptol and co-emulsifier obtained in step (2) to the cyclodextrin mixture solution obtained in step (1), and simultaneously carrying out cyclization and embedding to obtain an inclusion complex solution;

[0011] (4) Shear and homogenize the clathrate solution obtained in step (3) to obtain the eucalyptol clathrate.

[0012] In one embodiment of the present invention, in step (1), the starch in the aqueous starch solution is selected from corn starch;

[0013] And / or, calculated by the mass percentage of the raw materials for preparing the eucalyptol clathrate, the addition amount of the starch in the aqueous starch solution is 5 wt%.

[0014] In one embodiment of the present invention, in step (1), the aqueous starch solution is prepared by the following method:

[0015] Dissolve starch in water, and stir at 40°C to 60°C and 200 rpm to 500 rpm for 5 min to 15 min until the liquid is clear and transparent to obtain the aqueous starch solution.

[0016] In one embodiment of the present invention, in step (1), the CGTase is selected from one or more of α-CGTase, β-CGTase, and γ-CGTase. Among them, α-CGTase, β-CGTase, and γ-CGTase can all be obtained commercially; the enzyme activity of α-CGTase is 200 U / mL, the enzyme activity of β-CGTase is 30 U / mL, and the enzyme activity of γ-CGTase is 6 U / mL.

[0017] In one embodiment of the present invention, in step (1), the addition amount of the CGTase is 2 U / g starch to 8 U / g starch; more specifically, for example, 2 U / g starch, 3 U / g starch, 4 U / g starch, 5 U / g starch, 6 U / g starch, 7 U / g starch, 8 U / g starch, and the numerical range between any two points; preferably 4 U / g starch.

[0018] In one embodiment of the present invention, in step (1), the dextrin debranching enzyme is derived from the thermophilic archaea Saccharolobus solfataricus STB09; the enzyme activity is 2000 U / mL.

[0019] In one embodiment of the present invention, in step (1), the addition amount of the dextrin debranching enzyme is 750 U / g starch to 1000 U / g starch; more specifically, for example, 750 U / g starch, 800 U / g starch, 850 U / g starch, 900 U / g starch, 950 U / g starch, 1000 U / g starch, and the numerical range between any two points; preferably 750 U / g starch.

[0020] In one embodiment of the present invention, in step (1), the conditions for enzymatic hydrolysis are as follows: heating to 90°C; stirring at 200 rpm to 500 rpm for 5 min to 60 min, and then cooling to 40°C to 60°C.

[0021] In one embodiment of the present invention, in step (2), based on the mass percentage of the raw materials for preparing the eucalyptol inclusion compound, the addition amount of eucalyptol is 1 wt% to 10 wt%; more specifically, for example, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt% and the numerical range between any two points; preferably 10 wt%.

[0022] In one embodiment of the present invention, in step (2), the co-emulsifier is selected from ethanol;

[0023] And / or, based on the mass percentage of the raw materials for preparing the eucalyptol inclusion compound, the addition amount of the co-emulsifier is 2.5 wt% to 10 wt%; more specifically, for example, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, 10 wt% and the numerical range between any two points; preferably 10 wt%.

[0024] In one embodiment of the present invention, in step (2), the conditions for mixing are as follows: under the condition of 40°C to 60°C, stirring at 200 rpm to 500 rpm for 1 min to 10 min.

[0025] In one embodiment of the present invention, in step (3), the conditions for cyclization and encapsulation are as follows: under the condition of 40°C to 60°C, stirring at 200 rpm to 500 rpm for 90 min to 110 min.

[0026] In one embodiment of the present invention, in step (4), the shearing is: shearing at 5000 rpm to 10000 rpm for 2 min ；

[0027] And / or, the pressure for homogenization is 300 Bar to 500 Bar.

[0028] The second object of the present invention is to provide the eucalyptol inclusion compound prepared by the above method.

[0029] The third object of the present invention is to provide the application of the eucalyptol inclusion compound in the fields of medicine, daily chemicals or agriculture.

[0030] The fourth object of the present invention is to provide a biological pesticide comprising the eucalyptol inclusion compound.

[0031] The above technical solution of the present invention has the following advantages compared with the prior art:

[0032] (1) Based on the traditional method of using β-CGTase to act on starch substrates to prepare cyclodextrin, the present invention additionally adds dextrin debranching enzyme (SsGDE enzyme) to act on starch substrates synergistically, significantly improving the production efficiency of β-cyclodextrin.

[0033] (2) The present invention replaces the traditional emulsification technology, greatly improving both the stability and efficacy of the eucalyptol inclusion complex.

[0034] (3) The eucalyptol inclusion complex prepared by the present invention is extremely stable and does not delaminate after being placed at 20 °C, 40 °C, and 60 °C for 6 months, which is beneficial for the storage, transportation, and use of the product.

[0035] (4) The dilution stability of the eucalyptol inclusion complex prepared by the present invention is very good. It can be diluted in any proportion and remain stable without delamination, significantly improving the poor stability and insufficient water solubility of essential oils.

[0036] (5) The eucalyptol inclusion complex product prepared by the present invention has a good insecticidal effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, wherein,

[0038] Figure 1 is a physical diagram of the eucalyptol inclusion complex in Example 1;

[0039] Figure 2 is a particle size measurement diagram of the eucalyptol inclusion complex in Example 1;

[0040] Figure 3 is a physical diagram of the effect of different starch concentrations on the enzyme inclusion complex;

[0041] Figure 4 is a microscope diagram of the effect of different starch concentrations on the stability of the enzyme inclusion complex;

[0042] Figure 5 is a measurement diagram of the effect of different starch concentrations on the particle size distribution of the enzyme inclusion complex;

[0043] Figure 6 is a physical diagram of the effect of the addition time of dextrin debranching enzyme on the enzyme inclusion complex;

[0044] Figure 7 is a physical diagram of the effect of not adding dextrin debranching enzyme on the stability of the eucalyptol inclusion complex. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments shall not be construed as limiting the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0047] Testing method:

[0048] 1. The stability of the emulsion was evaluated by the direct observation method:

[0049] The emulsion was placed in a centrifuge tube and left standing at different temperatures (20 °C, 40 °C, 60 °C), and the state of the emulsion was observed regularly with the naked eye to check whether it was homogeneous and stable.

[0050] 2. Evaluation of the dilution performance of the emulsion:

[0051] After gradient dilution of the system, the direct observation method was used: a part of the liquid was taken from the prepared system and diluted by a factor of 10 in a gradient manner so that the dilution factor of the system was 10 - 10000 times. It was stored in a centrifuge tube and left standing at room temperature, and the state of the diluted solution was observed regularly with the naked eye.

[0052] 3. Determination of the particle size of the inclusion complex:

[0053] The particle size of the optimized system was measured using a laser particle size analyzer, and the particle size distribution was represented by the particle size span (Span). The calculation formula for the particle size span is as follows:

[0054] Span = (d 90 - d 10 ) / d 50

[0055] where d 90 , d 50 and d 10 are the particle size values corresponding to 90%, 50% and 10% of the cumulative particle size distribution, respectively.

[0056] 4. Evaluation of the insecticidal performance of the eucalyptol inclusion complex product:

[0057] Referring to the agricultural industry standards NYT 1154.6 - 2006 and NYT 1154.16 - 2013, the toxicity of the optimized system against Bemisia tabaci and aphids was determined by the insect - dipping method and leaf - dipping method. Specifically, for the aphid treatment group, 7 treatment concentrations of 500mg / L, 250mg / L, 125mg / L, 62.5mg / L, 31.25mg / L, 15.63mg / L, and 7.81mg / L were set, and for the Bemisia tabaci treatment group, 7 treatment concentrations of 250mg / L, 125mg / L, 62.5mg / L, 31.25mg / L, 15.63mg / L, 7.81mg / L, and 3.91mg / L were set; and a water control was set. Each treatment was repeated 4 times, and there were 20 - 40 insects in each repetition. The specific operations are as follows:

[0058] Insect - dipping method: The selected aphids together with the leaves were immersed in the liquid medicine for 5 s and then taken out and blotted dry with absorbent paper, and then placed in a disposable plastic culture cup lined with filter paper that had been pre - moisturized. There were 30 - 40 insects in each cup, and each concentration was repeated 4 times, with 120 - 160 aphids treated at each concentration. The control was treated with water. After inoculating the insects, the culture cups were placed in an incubator at (26 ± 2)°C with a photoperiod of 16:8 (L:D). The results were checked after 48 h of treatment.

[0059] Agar - moisturized leaf - dipping method: The agar was prepared into 15 - 17 g / L with distilled water. 2 mL of liquid agar was aspirated with a micropipette and added to the bottom of a flat - bottomed glass tube, taking care not to contaminate the tube wall and not to generate air bubbles. After the liquid agar cooled and solidified and the steam on the tube wall had evaporated completely, it was diluted with distilled water into 7 series of concentrations of liquid medicine; circular leaves of Brassica parachinensis with a diameter of 18 mm were immersed in each liquid medicine for 5 s, taken out and air - dried at room temperature, and then adhered to the agar surface with the back facing up; the control was treated with distilled water; adult Bemisia tabaci that had emerged for 24 h were introduced, the tube mouth was sealed with gauze, there were about 30 test insects in each treatment, and each treatment was repeated 4 times. The finger - shaped tubes with the test insects were placed upside - down in the insect - rearing room for normal rearing. The rearing conditions were L / D = 14:10, T = 26 ± 2°C, RH = 75 ± 5%, and the situation of the introduced test insects was checked 1 h later. If they had died, they were not counted in the number of introduced insects; the test results were checked again at 48 h respectively, and the mortality rate was calculated.

[0060] The experimental results were statistically analyzed using SPSS. With the mortality rate as the vertical coordinate Y and the chemical concentration as the horizontal coordinate X, the toxicity regression equation of each chemical was established, and the LC 50 value and 95% confidence limit were calculated.

[0061] Example 1

[0062] This example provides a method for preparing eucalyptol inclusion complex by a double - enzyme method, which includes the following steps:

[0063] (1) Add 5 g of corn starch to 82.5 g of distilled water, stir at 50 °C and 300 rpm for 10 min to obtain a clear and transparent starch aqueous solution;

[0064] (2) Add 4 U / g (starch) of β-CGTase to the starch aqueous solution for enzymatic hydrolysis. Immediately after adding 750 U / g (starch) of dextrin debranching enzyme at 50 °C, raise the temperature to 90 °C and stir at 300 rpm for 30 min for enzymatic hydrolysis, and then cool down to 50 °C;

[0065] (3) Preheat 10 g of eucalyptol and 2.5 g of co-emulsifier absolute ethanol at 50 °C for 5 min to obtain a mixed solution of eucalyptol and co-emulsifier;

[0066] (4) Add the mixed solution of eucalyptol and co-emulsifier in step (3) to the cyclized solution in step (2), stir at 50 °C and 300 rpm for 90 min for cyclization and encapsulation to obtain an encapsulated solution;

[0067] (5) Shear the encapsulated solution obtained in step (4) at 5000 rpm for 2 min and then homogenize it in a homogenizer under a pressure of 300 Bar for 2 min to obtain eucalyptol inclusion compound ( Figure 1 ).

[0068] The obtained eucalyptol inclusion compound was tested for stability and dilution performance, and the test results are as follows:

[0069] The eucalyptol inclusion compound obtained in Example 1 was stable and did not delaminate. It still did not delaminate after being placed at 20 °C, 40 °C, and 60 °C for 6 months, showing good stability, which is beneficial to the storage, transportation, and use of the product. At the same time, it remained stable after being diluted 10 - 10000 times without delamination, indicating that Example 1 well solved the problems of poor stability and water solubility of eucalyptol.

[0070] The obtained eucalyptol inclusion compound was analyzed for particle size, and the results are as Figure 2 shown. It can be seen from Figure 2 that the particle size span of the inclusion compound is 1.06, indicating that the particle size distribution symmetry within the inclusion compound is good and it is easy to maintain stability.

[0071] The obtained eucalyptol inclusion compound was evaluated for insecticidal performance, and the test results are shown in Table 1 below:

[0072] It can be seen from Table 1 that the inclusion compound in Example 1 has good insecticidal effects and biopesticide application performance. Specifically, LC 50 refers to the toxicant concentration at which half of the test pests die, which can better and objectively evaluate the control effect of the agent on pests. It can be seen that the inclusion compound in Example 1 has better control effects on Bemisia tabaci and aphids than the eucalyptol emulsion prepared by the single enzyme method.

[0073] Table 1 Insecticidal performance evaluation results

[0074] <![CDATA[LC 50 (mg / L)]]> Bemisia tabaci 7.89 Aphids (immersion method) 13.08

[0075] Example 2 Optimization of starch type

[0076] This example provides a method for preparing eucalyptol inclusion compound by double enzyme method. Similar to Example 1, the difference is only that: in Example 1, corn starch is replaced with wheat starch, tapioca starch or waxy corn starch to prepare eucalyptol inclusion compound.

[0077] The obtained eucalyptol inclusion compound was tested, and the test results are as follows:

[0078] Table 2

[0079]

[0080] It can be seen from Table 2 that: obvious stratification appears at the bottom of the waxy corn starch group, the upper and lower colors are uneven in the wheat starch group, and the colors of the corn starch and tapioca starch systems are uniform and relatively stable. However, the tapioca starch has a high branched-chain content, and the prepared inclusion compound has a large viscosity, which is not suitable for preparing an inclusion compound system. Finally, corn starch was selected as the reaction substrate.

[0081] Example 3 Optimization of starch concentration

[0082] This example provides a method for preparing eucalyptol inclusion compound by double enzyme method. Similar to Example 1, the difference is only that: calculated according to the mass percentage of the raw materials for preparing eucalyptol inclusion compound, the corn starch concentration in Example 1 was adjusted to 0%, 0.5%, 1%, 1.5%, 2%, 3%, 4% or 5% to prepare eucalyptol inclusion compound.

[0083] The obtained inclusion compound was tested, and the test results are as Figures 3 to 5 shown. After standing the inclusion compound for a period of time, it was found that as the substrate concentration increased, the system developed from an oil-water two-phase system into an upper oil phase, a middle water phase and a lower precipitate. As the starch concentration increased, the volume of the upper oil phase decreased, and the volume of the lower precipitate gradually increased. In order to further study the composition of each phase, the substances in different phases were observed by optical microscope. The results showed that the upper layer was mainly suspended oil droplets that had not been completely included, the middle layer was the water phase with a low content of inclusion compound inside, and the lower layer was inclusion compound particles with a higher density. As the starch concentration increased, the concentration of the inclusion compound after mixing the system also gradually increased, and the content of the inclusion compound in the middle water phase also increased significantly, and the system tended to be stable. Comparing the effects of different starch concentrations on the particle size distribution of the enzyme-prepared inclusion compound, as Figure 5As shown, with the increase of starch concentration, the particle size distribution of the enzyme - formed inclusion complex shows certain regular changes. Specifically, when the starch concentration is relatively low at 0.5%, the particle size distribution of the formed inclusion complex is relatively narrow, and there are more small - sized particles; as the starch concentration increases (1% - 2%), the proportion of large - sized inclusion complexes increases significantly. With the further increase of starch concentration (3% - 5%), the particle size distribution range of the inclusion complex gradually becomes narrower, indicating that the formation process of the inclusion complex is more stable and uniform. Therefore, the corn starch concentration of 5% was finally selected.

[0084] Example 4 Dosage of β - CGT enzyme

[0085] This example provides a method for preparing eucalyptol inclusion complex by double - enzyme method, which is similar to Example 1, with the only difference being that the dosage of β - CGT enzyme in Example 1 is adjusted to 1U / g, 2U / g or 8U / g to prepare the eucalyptol inclusion complex.

[0086] The obtained eucalyptol inclusion complex was tested, and the test results are as follows:

[0087] Table 3

[0088]

[0089]

[0090] It can be seen from Table 3 that the enzyme addition amount of 4U / g is the most suitable. That is, when the enzyme addition amount is less, the degree of amylase hydrolysis is insufficient, which will cause aging and precipitation, affecting the product properties; when the enzyme addition amount is too high, it will affect the preparation cost.

[0091] Example 5 Addition time of dextrin debranching enzyme

[0092] This example provides a method for preparing eucalyptol inclusion complex by double - enzyme method, which is similar to Example 1, with the only difference being that the addition time point of dextrin debranching enzyme in Example 1 is adjusted to add dextrin debranching enzyme when the temperature is raised to 70°C (YH70), add dextrin debranching enzyme when the temperature is raised to 90°C (YH90), or add dextrin debranching enzyme when the temperature is lowered to 50°C (HH50) after the enzymatic hydrolysis to prepare the eucalyptol inclusion complex.

[0093] The obtained eucalyptol inclusion complex was tested, and the test results are as follows:

[0094] Table 4

[0095]

[0096] As can be seen from Table 4, adding SsGDE during the enzymatic hydrolysis process can significantly enhance the stability of the inclusion complex and reduce the volume of the oil layer. However, adding SsGDE after enzymatic hydrolysis has no improvement effect on the system stability. This is because dextrin debranching enzymes have different substrate specificities, and due to the action of β-CGT enzyme in the cyclization system, its structure and composition are in a continuous changing state. Therefore, the addition time of the debranching enzyme may have a greater impact on its application effect. The dextrin debranching enzyme SsGDE has good thermal stability and can withstand high-temperature treatment at 70 °C, which enables SsGDE to be added in the liquefaction stage and cooperate with CGT enzyme throughout the cyclization process for the production of short-chain dextrins such as β-cyclodextrin. That is, adding SsGDE in the liquefaction stage has the best effect, and as the addition time of SsGDE is postponed, its promoting effect on β-cyclodextrin production gradually weakens. Therefore, the addition time of the dextrin debranching enzyme was finally selected to be before the start of enzymatic hydrolysis.

[0097] Example 6 Dosage of Dextrin Debranching Enzyme

[0098] This example provides a method for preparing eucalyptol inclusion complex by a two-enzyme method, which is similar to Example 1, except that: the dosage of dextrin debranching enzyme in Example 1 was adjusted from 750 U / g to 500 U / g, 550 U / g, 600 U / g, 650 U / g, 700 U / g, 800 U / g, 850 U / g, 900 U / g, 950 U / g or 1000 U / g to prepare the eucalyptol inclusion complex.

[0099] The obtained eucalyptol inclusion complex was tested, and the test results are as follows:

[0100] Table 5

[0101]

[0102] As can be seen from Table 5, as the enzyme addition amount increases, the stability of the eucalyptol inclusion complex gradually enhances. This is because at the same time, SsGDE modifying the substrate can increase its linear proportion. Using DE 4 maltodextrin modified by SsGDE to a certain extent as the substrate can improve the cyclization activity of β-CGT enzyme. These are also the reasons why SsGDE has a good promoting effect on β-cyclodextrin production. In addition, SsGDE has a stronger debranching ability for DE4 maltodextrin than other substrates such as starch, which is beneficial for slow debranching when the influence of branches is small at the initial stage of the reaction to prevent the formation of highly linear starch that is prone to retrogradation and affect the action of β-CGT enzyme. As the reaction progresses, the restriction of branches on β-CGT enzyme gradually increases, and at this time SsGDE shows good debranching performance, reducing the interference of branches on β-CGT enzyme. Therefore, as the cyclization reaction progresses, the promoting effect of SsGDE on β-cyclodextrin production gradually increases. Therefore, considering economic factors comprehensively, the dosage of dextrin debranching enzyme was finally selected to be 750 U / g.

[0103] Example 7 Optimization of Cyclization Time

[0104] This example provides a method for preparing eucalyptol inclusion complex by a two - enzyme method. Similar to Example 1, the only difference is that the cyclization time in Example 1 is adjusted to 70 min, 80 min, 100 min, 110 min or 120 min, and the eucalyptol inclusion complex is prepared.

[0105] The obtained eucalyptol inclusion complex was tested, and the test results are as follows:

[0106] Table 6

[0107]

[0108] It can be seen from Table 6 that when the reaction time is less than 90 min, due to insufficient cyclodextrin production, part of the eucalyptol is not embedded and precipitates, thus affecting the stability of the system. Excessive cyclization time will lead to an increase in by - products, and at the same time, due to reactions such as starch aging, precipitation occurs, which is not conducive to the formation of the system. Considering comprehensively, 90 min is the most suitable cyclization reaction time.

[0109] Example 8 Optimization of Eucalyptol Concentration

[0110] This example provides a method for preparing eucalyptol inclusion complex by a two - enzyme method. Similar to Example 1, the only difference is that the addition amount of eucalyptol in Example 1, which is 10 g, is adjusted to 1 g, 5 g, 20 g, 40 g or 60 g. At the same time, the amount of water is adjusted to ensure that the total percentage is 100% (calculated according to the mass percentage of the raw materials for preparing the eucalyptol inclusion complex, the added mass percentage of eucalyptol is replaced from 10 wt% in Example 1 to 1 wt%, 5 wt%, 20 wt%, 40 wt% and 60 wt%), and the eucalyptol inclusion complex is prepared.

[0111] The obtained eucalyptol inclusion complex was tested, and the test results are as follows:

[0112] Table 7

[0113]

[0114] It can be seen from Table 7 that when the amount of eucalyptol used is too small, although the embedding rate is relatively high, the actual essential oil loading is insufficient, and the function of the essential oil cannot be fully exerted. When the amount of eucalyptol used is too large, it will lead to an excessive loading in the embedding system, exceeding the bearing capacity of the wall material, and too much essential oil cannot be effectively embedded, resulting in part of the essential oil overflowing, thus reducing the embedding rate. Therefore, the final amount of eucalyptol used is selected as 10 g.

[0115] Example 9 Optimization of the Type of Co - emulsifier

[0116] This example provides a method for preparing eucalyptol inclusion complex by double enzyme method. Similar to the example, the difference is only that: the co-emulsifier ethanol in Example 1 is adjusted to glycerol, n-butanol, ethylene glycol, propylene glycol or polyglycerol ester, and the others are kept the same as in Example 1 to obtain the eucalyptol inclusion complex.

[0117] The obtained eucalyptol inclusion complex was tested, and the test results are as follows:

[0118] Table 8

[0119]

[0120] It can be seen from Table 8 that: the co-emulsifier can affect the particle size and polydispersity index of the inclusion complex by reducing the oil-water interfacial tension, and thus affect the formation and stability of the inclusion complex. Therefore, a suitable co-emulsifier can increase the loading amount of essential oil in the inclusion complex. It can be seen from the table that: the system prepared with ethanol as the co-emulsifier has good stability and can be diluted in any proportion. Therefore, ethanol was finally selected as the co-emulsifier.

[0121] Optimization of the co-emulsifier concentration in Example 10

[0122] This example provides a method for preparing eucalyptol inclusion complex by double enzyme method. Similar to the example, the difference is only that: the dosage of anhydrous ethanol as the co-emulsifier in Example 1 is adjusted to 0 g, 5 g, 10 g or 40 g, and at the same time, the dosage of water is adjusted to ensure that the total percentage is 100% (calculated according to the mass percentage of the raw materials for preparing the eucalyptol inclusion complex, the added mass percentage of eucalyptol is replaced by 0 wt%, 5 wt%, 10 wt% or 40 wt% of Example 1), and the eucalyptol inclusion complex is prepared.

[0123] The obtained eucalyptol inclusion complex was tested, and the test results are as follows:

[0124] Table 9

[0125]

[0126]

[0127] It can be seen from Table 9 that: with the increase of the co-emulsifier concentration, the stability of the eucalyptol inclusion complex first increases and then decreases. When the ethanol ratio is relatively high, due to the low solubility of starch and its decomposition products in alcohol, precipitation occurs, resulting in obvious instability and layering of the system. Considering that ethanol is a flammable and explosive liquid under normal temperature and pressure, reducing the ethanol content in the system is beneficial to the transportation and storage of pesticide products. Therefore, the ethanol dosage of 2.5 g was finally selected.

[0128] Optimization of the shear rate in Example 11

[0129] This example provides a method for preparing eucalyptol inclusion compound by double enzyme method. Similar to the example, the only difference is that the shear rate in Example 1 is adjusted to 2800 r / min, 5000 r / min, 8000 r / min, 10000 r / min or 15000 r / min, and the others are the same as in Example 1, to obtain the eucalyptol inclusion compound.

[0130] The obtained eucalyptol inclusion compound was tested, and the test results are as follows:

[0131] Table 10

[0132]

[0133] It can be seen from Table 10 that as the shear rate increases, the stability of the inclusion compound first increases and then decreases. When the shear rate is 5000 r / min, due to sufficient energy input and the energy input may reach the critical point, the stability of the inclusion compound is better; when the shear rate is 15000 r / min, the stability decreases, which may be due to the excess energy destroying the equilibrium state that has been formed in the inclusion compound. Therefore, 5000 r / min was finally selected as the optimal shear rate.

[0134] Optimization of the shear time in Example 12

[0135] This example provides a method for preparing eucalyptol inclusion compound by double enzyme method. Similar to the example, the only difference is that the shear time in Example 1 is adjusted to 0, 0.5 min, 1 min, 2 min or 3 min, and the others are the same as in Example 1, to obtain the eucalyptol inclusion compound.

[0136] The obtained eucalyptol inclusion compound was tested, and the test results are as follows:

[0137] Table 11

[0138]

[0139] It can be seen from Table 11 that when the shear time is 0 min and 0.5 min, the stability of the inclusion compound is poor and the particle size distribution shows three peaks; when the shear time increases to 2 min, the particle size distribution of the inclusion compound is more uniform and there is only a single peak; when the shear time increases to 3 min, the particle size distribution shows a bimodal peak, and the phenomenon of inclusion compound dispersion may occur. The reason for this result may be that the formation of the oil-water interface requires a certain time. When the shear time is short, the cyclodextrin molecules have not yet transferred to the newly formed oil-water interface, and the stability of the inclusion compound is poor; but if the shear time is too long, it will destroy the interfacial adsorption state of the cyclodextrin molecules and affect the stability of the inclusion compound. Therefore, 2 min was finally selected as the optimal shear time.

[0140] In Comparative Example 1, β-cyclodextrin was used as the inclusion wall material.

[0141] This comparative example provides a method for preparing eucalyptol inclusion compound using β-cyclodextrin as the inclusion wall material, which includes the following steps:

[0142] Dissolve 1.5 g of β-cyclodextrin in water, add 10 g of eucalyptol, then add 2.5 g of ethanol, and finally make up the water to 86 g to form a 100 g system, obtaining the eucalyptol inclusion compound;

[0143] After 20 minutes, observe the phenomenon and find that: a small amount of precipitation appears at the bottom of the inclusion compound. Observe the samples after 1 day, 10 days, and 30 days, and find that the precipitation increases. From this analysis, the β-cyclodextrin system can indeed play a certain embedding effect, but due to the low solubility of cyclodextrin, and the addition of co-emulsifier ethanol will further reduce the solubility of the embedded product, ultimately resulting in precipitation of the sample, which is not conducive to the stable preservation of the product.

[0144] Comparative example 2 uses γ-cyclodextrin alone as the inclusion wall material

[0145] This comparative example provides a method for preparing eucalyptol inclusion compound using γ-cyclodextrin as the inclusion wall material, which includes the following steps:

[0146] Dissolve 1.5 g of γ-cyclodextrin in water, add 10 g of eucalyptol, then add 2.5 g of ethanol, and finally make up the water to 86 g to form a 100 g system, obtaining the eucalyptol inclusion compound;

[0147] After 20 minutes, observe the phenomenon and find that: a small amount of oil floating appears in the inclusion compound. Observe the samples after 1 day, 10 days, and 30 days, and find that the inclusion compound shows stratification, with the upper layer being the oil layer and the lower layer being a darker liquid. From this analysis: perhaps due to the large pore size of γ-cyclodextrin, it cannot be accurately adapted to eucalyptol, and part of the eucalyptol does not enter the embedding system. At the same time, the successfully embedded eucalyptol sinks due to the increased density, resulting in liquid stratification.

[0148] At the same time, due to the too high production cost of γ-cyclodextrin, γ-cyclodextrin is generally not used for embedding in the process flow.

[0149] Comparative example 3 uses β-cyclodextrin and γ-cyclodextrin as the inclusion wall material

[0150] This comparative example provides a method for preparing eucalyptol inclusion compound using β-cyclodextrin and γ-cyclodextrin as the inclusion wall material, which includes the following steps:

[0151] Dissolve 1.5 g of cyclodextrin (the mass ratio of γ-cyclodextrin to β-cyclodextrin is 1:1) in water, add 10 g of eucalyptol, then add 2.5 g of ethanol, and finally make up the water to form a 100 g system, obtaining the eucalyptol inclusion compound;

[0152] After observing the phenomenon 20 minutes later, it was found that the system was stable and homogeneous. After observing the samples after 1 day, 10 days, and 30 days, it was found that the system did not layer. Comparing the dilution stability, it was found that the sample was stable at a 10-fold dilution, but layered occurred after being diluted 100-fold and left for 10 days. Moreover, compared with Example 1, the preparation process of pure cyclodextrin is complex and the processing cost is high. In large-scale production of the process, economic costs need to be considered. Considering comprehensively, it is more appropriate to choose the enzymatic preparation method of Example 1.

[0153] Eucalyptol inclusion complex without adding dextrin debranching enzyme in Comparative Example 4

[0154] This comparative example provides a method for preparing eucalyptol inclusion complex by a double-enzymatic method, which is similar to Example 3, except that: dextrin debranching enzyme is not added, and the eucalyptol inclusion complex is prepared.

[0155] The obtained eucalyptol inclusion complex was subjected to a stability test, and the test results are as Figure 7 shown. By comparing Figure 3 it can be further found that the oil layer content of the system with added dextrin debranching enzyme is significantly reduced, and the inclusion complex content in the lower-layer precipitate is higher, which further illustrates that dextrin debranching enzyme has a significant promoting effect on the stability of eucalyptol inclusion complex.

[0156] Obviously, the above examples are only for clearly illustrating the examples and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. A method for preparing eucalyptol inclusion compound by double enzyme method, characterized in that: The steps include: (1) adding CGTase and dextrin debranching enzyme to the starch aqueous solution for enzymolysis to obtain a cyclodextrin mixture solution; (2) uniformly mixing eucalyptol and the co-emulsifier to obtain a mixed solution of eucalyptol and the co-emulsifier; (3) adding the mixed solution of eucalyptol and the co-emulsifier obtained in step (2) to the cyclodextrin mixture solution obtained in step (1), and simultaneously performing cyclization and embedding to obtain an inclusion compound solution; (4) Shearing and homogenizing the inclusion compound solution obtained in step (3) to obtain the eucalyptol inclusion compound.

2. The method according to claim 1, characterized in that In step (1), the starch in the starch aqueous solution is selected from corn starch.

3. The method according to claim 1, characterized in that In step (1), the starch aqueous solution is prepared by the following method: Dissolve starch in water, stir for 5 min to 15 min at 40° C. to 60° C. and 200 rpm to 500 rpm, and mix until the liquid becomes clear and transparent to obtain a starch aqueous solution.

4. The method according to claim 1, characterized in that: In step (1), the CGTase is selected from one or more of α-CGTase, β-CGTase and γ-CGTase.

5. The method according to claim 1, characterized in that: In step (1), the amount of CGTase added is 2U / g starch to 8U / g starch.

6. The method according to claim 1, characterized in that In step (1), the dextrin debranching enzyme is derived from the thermophilic archaeon Saccharolobus solfataricus STB09.

7. The method according to claim 1, characterized in that In step (1), the added amount of the dextrin debranching enzyme is 750U / g starch to 1000U / g starch.

8. The method according to claim 1, characterized in that: In step (1), the conditions for enzymatic hydrolysis are: heating to 90°C; stirring at 200 rpm to 500 rpm for 5 min to 60 min, and then cooling to 40°C to 60°C.

9. The method according to claim 1, characterized in that: In step (2), the amount of eucalyptol added is 1 wt% to 10 wt% based on the mass percentage of the raw materials for preparing the eucalyptol inclusion compound.

10. The method according to claim 1, characterized in that In step (2), the co-emulsifier is selected from ethanol; And / or, based on the mass percentage of the raw materials for preparing the eucalyptol inclusion compound, the amount of the auxiliary emulsifier added is 2.5wt% to 10wt%.

11. The method according to claim 1, characterized in that: In step (2), the mixing conditions are: stirring at 40°C to 60°C and 200rpm to 500rpm for 1min to 10min.

12. The method according to claim 1, characterized in that In step (3), the conditions for the cyclization and embedding are: stirring at 40°C to 60°C and 200rpm to 500rpm for 90min to 110min.

13. The method according to claim 1, characterized in that In step (4), the shearing is: 5000 rpm to 10000 rpm for 2 min; And / or, the homogenization pressure is 300 Bar to 500 Bar.

14. The eucalyptol inclusion compound prepared by the method according to any one of claims 1 to 13.

15. Use of the eucalyptol inclusion compound according to claim 14 in the fields of medicine, daily chemicals or agriculture.

16. A biological insecticide, characterized in that: The biopesticide comprises the eucalyptol inclusion compound according to claim 14.

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