Camphor essential oil and application thereof in feed additive

Abstract

The invention relates to the technical field of aquaculture, and discloses camphor essential oil and application thereof in a feed additive. The preparation method of the camphor essential oil comprises the following steps: freezing and drying camphor tree leaves, crushing, and sieving with a 40-mesh sieve to obtain camphor tree leaf powder; placing the camphor tree leaf powder in a choline chloride-lactic acid eutectic solvent, and carrying out dipping treatment to obtain dipped camphor tree leaf powder; placing the dipped camphor tree leaf powder and an enzyme preparation in water, adjusting the pH value of the system to 5.5, and performing enzymolysis to obtain enzymolysis camphor tree leaf powder; performing fractional distillation on the enzymolyzed camphor tree leaf powder to obtain a camphor essential oil precursor; placing a camphor essential oil precursor in the wall material solution, and performing ultrasonic dispersion to obtain camphor essential oil emulsion; and carrying out spray drying on the camphor essential oil emulsion to obtain the camphor essential oil. According to the camphor essential oil provided by the invention, the yield can reach 10.1%, the camphor content can reach 95.8%, the retention rate of camphor in 4 months can reach 93.5%, and the camphor essential oil has relatively high quality and stability.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and more particularly, to a camphor essential oil and its application in feed additives. Background Art

[0002] Tilapia has the advantages of fast growth, high yield, strong adaptability, etc., and is widely cultured globally. However, tilapia belongs to tropical and subtropical fish and has poor tolerance to low temperatures. When the water temperature drops to a certain extent, its growth, physiological functions and survival rate will be severely affected.

[0003] In order to improve the low-temperature tolerance of tilapia, those skilled in the art have adopted various methods. For example, improving the low-temperature tolerance of tilapia by breeding cold-tolerant varieties, but this method has a long cycle, high cost and limited effect. In addition, improving the aquaculture environment is also a common method, but this method will increase the aquaculture cost and is difficult to be widely applied in large-scale aquaculture.

[0004] As a convenient and effective means, feed additives have great potential in improving the stress resistance of aquatic animals. Therefore, developing a feed additive that can effectively enhance the low-temperature tolerance of tilapia has important practical significance and market value. Summary of the Invention

[0005] The purpose of the present invention is to provide a camphor essential oil and its application in feed additives, and this camphor essential oil feed additive can effectively improve the low-temperature tolerance of tilapia.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention:

[0008] A preparation method of camphor essential oil, comprising the following steps:

[0009] 1) Freeze-dry camphor leaves, then crush them and pass through a 40-mesh sieve to obtain camphor leaf powder;

[0010] 2) Place the camphor leaf powder obtained in step 1) in a choline chloride-lactic acid eutectic solvent for impregnation treatment to obtain impregnated camphor leaf powder;

[0011] 3) Place the impregnated camphor leaf powder obtained in step 2) and an enzyme preparation in water, adjust the pH of the system to 5.5, and carry out enzymatic hydrolysis to obtain enzymatically hydrolyzed camphor leaf powder;

[0012] 4) Carry out fractional distillation on the enzymatically hydrolyzed camphor leaf powder obtained in step 3) to obtain a camphor essential oil precursor;

[0013] 5) Place the camphor essential oil precursor obtained in step 4) into the wall material solution and disperse it by ultrasonic treatment to obtain a camphor essential oil emulsion;

[0014] 6) Spray-dry the camphor essential oil emulsion obtained in step 5) to obtain the camphor essential oil.

[0015] Further, in step 1), the freeze-drying is specifically: freeze-dry at -40 °C for 24 to 48 h.

[0016] Further, in step 2), the preparation method of the choline chloride-lactic acid eutectic solvent includes the following steps: Mix choline chloride and lactic acid according to a molar ratio of 1:(1.5 - 2), control the stirring speed at 400 rpm, and stir at 60 °C for 2 to 3 h to obtain the choline chloride-lactic acid eutectic solvent.

[0017] Further, in step 2), the impregnation treatment is specifically: impregnate at 60 °C for 3 to 5 h.

[0018] Further, in step 3), the enzyme preparation is composed of cellulase, galacturonase and β-glucanase mixed according to a mass ratio of 2:1:0.5.

[0019] Further, in step 3), the mass-volume ratio of the impregnated camphor tree leaf powder, enzyme preparation and water is 10:(2 - 3):(4 - 6).

[0020] Further, in step 3), the enzymatic hydrolysis is specifically: carry out enzymatic hydrolysis at 40 °C for 4 to 6 h.

[0021] Further, in step 4), the fractional distillation is: 1.5 KPa, 75 °C, 1 h; 40 KPa, 80 °C, 2 h; 60 KPa, 100 °C, 1 h.

[0022] Further, in step 5), the preparation method of the wall material solution includes the following steps: According to a mass ratio of arabic gum, maltodextrin and sodium alginate of 2:1:0.3, place arabic gum, maltodextrin and sodium alginate into deionized water at 60 °C, adjust the pH = 5, and add palmitic acid ascorbate with a mass fraction of 0.2 - 0.4% to obtain a wall material solution with a concentration of 12% (w / v).

[0023] Further, in step 5), the power of the ultrasonic dispersion is 200 W and the time is 2 min.

[0024] Further, in step 6), the inlet air temperature of the spray drying is 180 °C and the outlet air temperature is 85 °C.

[0025] The second technical solution of the present invention:

[0026] The camphor essential oil prepared by the above-mentioned preparation method of a camphor essential oil.

[0027] The third technical solution of the present invention:

[0028] The application of the above-mentioned camphor essential oil in a feed additive, comprising the following steps:

[0029] 1) Mix oregano essential oil, astragalus polysaccharide and capsinoid to obtain a synergistic component;

[0030] 2) Mix coconut oil, rosmarinic acid and N-acetylcysteine to obtain a supplementary component;

[0031] 3) Place soybean meal, microbial inoculum and glucose in water and ferment to obtain fermented soybean meal;

[0032] 4) First mix the camphor essential oil, the synergistic component and the supplementary component, stir to obtain an active ingredient mixture, and then add the fermented soybean meal to the active ingredient mixture, stir, and freeze-dry to obtain the camphor essential oil feed additive.

[0033] Further, in step 1), the mass ratio of the oregano essential oil, the astragalus polysaccharide and the capsinoid is (4-6):(2-3):1.

[0034] Further, in step 2), the mass ratio of the coconut oil, the rosmarinic acid and the N-acetylcysteine is (4-6):(1-3):(0.5-1.5).

[0035] Further, in step 3), the microbial inoculum is composed of Bacillus subtilis, Lactobacillus plantarum and Candida utilis mixed according to a mass ratio of 3:(0.5-1.5):(0.2-0.5).

[0036] Further, in step 3), the mass ratio of the soybean meal, the microbial inoculum, the glucose and the water is 10:(0.2-0.5):(0.5-1):(4-6).

[0037] Further, in step 3), the fermentation is specifically: fermenting at 40°C for 6-10 h.

[0038] Further, in step 4), the mass ratio of the camphor essential oil, the synergistic component, the supplementary component and the fermented soybean meal is (10-20):(5-15):(3-5):(20-30).

[0039] Further, in step 4), the freeze-drying is specifically: freeze-drying at -40°C for 12-36 h.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] The camphor essential oil provided by the present invention has a yield of up to 10.1%, a camphor content of up to 95.8%, and a camphor retention rate of up to 93.5% after 4 months, with high quality and stability.

[0042] A camphor essential oil feed additive provided by the present invention can significantly improve the survival rate of tilapia in a low-temperature environment, and the low-temperature survival rate can reach 88.2%.

[0043] A camphor essential oil feed additive provided by the present invention can reduce the stress level of tilapia, improve the antioxidant capacity of tilapia, reduce the degree of lipid peroxidation of tilapia, and reduce cell damage of tilapia. Detailed implementation manners

[0044] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention. It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention.

[0045] In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0047] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and embodiments are only exemplary.

[0048] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, they are intended to include but not limited to.

[0049] In the following examples, all raw materials used are commercially available. To ensure the comparability of the technical effects of the following examples and comparative examples, all raw materials used in the present invention are from the same manufacturer and the same batch.

[0050] In the following examples, a method for preparing camphor essential oil comprises the following steps:

[0051] 1) Freeze-dry the camphor leaves, then pulverize them and pass through a 40-mesh sieve to obtain camphor leaf powder;

[0052] Among them, the freeze-drying is: freeze-drying at -40°C for 24 - 48 h;

[0053] 2) Mix choline chloride and lactic acid according to a molar ratio of 1:(1.5 - 2), control the stirring speed at 400 rpm, and stir at 60°C for 2 - 3 h to obtain a choline chloride-lactic acid eutectic solvent;

[0054] 3) Place the camphor leaf powder obtained in step 1) into the choline chloride-lactic acid eutectic solvent obtained in step 2), and perform impregnation treatment at 60°C for 3 - 5 h to obtain impregnated camphor leaf powder;

[0055] 4) Mix cellulase, galacturonase and β-glucanase according to a mass ratio of 2:1:0.5 to obtain an enzyme preparation;

[0056] 5) According to a mass-volume ratio of impregnated camphor leaf powder, enzyme preparation and water of 10:(2 - 3):(4 - 6), place the impregnated camphor leaf powder and enzyme preparation obtained in step 3) into water, adjust the system pH = 5.5, and perform enzymatic hydrolysis at 40°C for 4 - 6 h to obtain enzymatically hydrolyzed camphor leaf powder;

[0057] 6) Perform fractional distillation on the enzymatically hydrolyzed camphor leaf powder obtained in step 5) to obtain a camphor essential oil precursor;

[0058] Among them, the fractional distillation is: 1.5 KPa, 75°C, 1 h; 40 KPa, 80°C, 2 h; 60 KPa, 100°C, 1 h;

[0059] 7) According to a mass ratio of arabic gum, maltodextrin and sodium alginate of 2:1:0.3, place arabic gum, maltodextrin and sodium alginate into deionized water at 60°C, adjust the pH = 5, and add ascorbyl palmitate with a mass fraction of 0.2 - 0.4% to obtain a wall material solution with a concentration of 12% (w / v);

[0060] 8) Place the camphor essential oil precursor obtained in step 6) into the wall material solution obtained in step 7), control the power of ultrasonic treatment at 200 W and the time at 2 min, and disperse the camphor essential oil precursor to obtain a camphor essential oil emulsion;

[0061] 9) Spray-dry the camphor essential oil emulsion obtained in step 8) to obtain the camphor essential oil;

[0062] Among them, the inlet air temperature of the spray drying is 180 °C, and the outlet air temperature is 85 °C.

[0063] Example 1

[0064] A kind of camphor essential oil

[0065] 1) Freeze-dry the camphor leaves, then crush them and pass through a 40-mesh sieve to obtain camphor leaf powder;

[0066] Among them, the freeze-drying is: freeze-dry at -40 °C for 24 h;

[0067] 2) Mix choline chloride and lactic acid according to the molar ratio of choline chloride to lactic acid of 1:1.5, control the stirring speed at 400 rpm, and stir at 60 °C for 2 h to obtain a choline chloride-lactic acid eutectic solvent;

[0068] 3) Place the camphor leaf powder obtained in step 1) into the choline chloride-lactic acid eutectic solvent obtained in step 2), and carry out impregnation treatment at 60 °C for 3 h to obtain impregnated camphor leaf powder;

[0069] 4) Mix cellulase, galacturonase and β-glucanase according to the mass ratio of cellulase, galacturonase and β-glucanase of 2:1:0.5 to obtain an enzyme preparation;

[0070] 5) According to the mass-volume ratio of impregnated camphor leaf powder, enzyme preparation and water of 10:2:4, place the impregnated camphor leaf powder and enzyme preparation obtained in step 3) into water, adjust the system pH = 5.5, and carry out enzymatic hydrolysis at 40 °C for 4 h to obtain enzymatically hydrolyzed camphor leaf powder;

[0071] 6) Carry out fractional distillation on the enzymatically hydrolyzed camphor leaf powder obtained in step 5) to obtain a camphor essential oil precursor;

[0072] Among them, the fractional distillation is: 1.5 KPa, 75 °C, 1 h; 40 KPa, 80 °C, 2 h; 60 KPa, 100 °C, 1 h;

[0073] 7) According to the mass ratio of arabic gum, maltodextrin and sodium alginate of 2:1:0.3, place arabic gum, maltodextrin and sodium alginate into deionized water at 60 °C, adjust the pH = 5, and add 0.2% ascorbyl palmitate by mass to obtain a wall material solution with a concentration of 12% (w / v);

[0074] 8) Place the camphor essential oil precursor obtained in step 6) into the wall material solution obtained in step 7), control the power of ultrasonic treatment to be 200 W and the time to be 2 min, disperse the camphor essential oil precursor, and obtain a camphor essential oil emulsion;

[0075] 9) Spray-dry the camphor essential oil emulsion obtained in step 8) to obtain the camphor essential oil;

[0076] Among them, the inlet air temperature of the spray drying is 180 °C and the outlet air temperature is 85 °C.

[0077] Example 2

[0078] A kind of camphor essential oil

[0079] 1) Freeze-dry the camphor leaves, then crush them and pass through a 40-mesh sieve to obtain camphor leaf powder;

[0080] Among them, the freeze-drying is: freeze-dry at -40 °C for 36 h;

[0081] 2) Mix choline chloride and lactic acid according to the molar ratio of choline chloride to lactic acid of 1:1.9, control the stirring speed to be 400 rpm, and stir at 60 °C for 2.5 h to obtain a choline chloride-lactic acid eutectic solvent;

[0082] 3) Place the camphor leaf powder obtained in step 1) into the choline chloride-lactic acid eutectic solvent obtained in step 2), and carry out impregnation treatment at 60 °C for 4 h to obtain impregnated camphor leaf powder;

[0083] 4) Mix cellulase, galacturonase and β-glucanase according to the mass ratio of cellulase, galacturonase and β-glucanase of 2:1:0.5 to obtain an enzyme preparation;

[0084] 5) According to the mass-volume ratio of impregnated camphor leaf powder, enzyme preparation and water of 10:2.5:5, place the impregnated camphor leaf powder and enzyme preparation obtained in step 3) into water, adjust the system pH = 5.5, and carry out enzymatic hydrolysis at 40 °C for 5 h to obtain enzymatically hydrolyzed camphor leaf powder;

[0085] 6) Carry out fractional distillation on the enzymatically hydrolyzed camphor leaf powder obtained in step 5) to obtain a camphor essential oil precursor;

[0086] Among them, the fractional distillation is: 1.5 KPa, 75 °C, 1 h; 40 KPa, 80 °C, 2 h; 60 KPa, 100 °C, 1 h;

[0087] 7) According to the mass ratio of gum arabic, maltodextrin and sodium alginate being 2∶1∶0.3, place gum arabic, maltodextrin and sodium alginate in deionized water at 60°C, adjust the pH to 5, and add palmitic acid ascorbate with a mass fraction of 0.3% to obtain a wall material solution with a concentration of 12% (w / v).

[0088] 8) Place the camphor essential oil precursor obtained in step 6) into the wall material solution obtained in step 7), control the power of ultrasonic treatment to be 200 W and the time to be 2 min, and disperse the camphor essential oil precursor to obtain a camphor essential oil emulsion.

[0089] 9) Spray-dry the camphor essential oil emulsion obtained in step 8) to obtain the camphor essential oil.

[0090] Among them, the inlet air temperature of the spray drying is 180°C and the outlet air temperature is 85°C.

[0091] Example 3

[0092] A kind of camphor essential oil

[0093] 1) Freeze-dry the camphor leaves, then crush them and pass through a 40-mesh sieve to obtain camphor leaf powder.

[0094] Among them, the freeze-drying is: freeze-dry at -40°C for 48 h.

[0095] 2) According to the molar ratio of choline chloride and lactic acid being 1∶2, mix choline chloride and lactic acid, control the stirring speed to be 400 rpm, and stir at 60°C for 3 h to obtain a choline chloride-lactic acid eutectic solvent.

[0096] 3) Place the camphor leaf powder obtained in step 1) into the choline chloride-lactic acid eutectic solvent obtained in step 2), and carry out impregnation treatment at 60°C for 5 h to obtain impregnated camphor leaf powder.

[0097] 4) According to the mass ratio of cellulase, galacturonase and β-glucanase being 2∶1∶0.5, mix cellulase, galacturonase and β-glucanase to obtain an enzyme preparation.

[0098] 5) According to the mass-volume ratio of impregnated camphor leaf powder, enzyme preparation and water being 10∶3∶6, place the impregnated camphor leaf powder and enzyme preparation obtained in step 3) into water, adjust the system pH to 5.5, and carry out enzymatic hydrolysis at 40°C for 6 h to obtain enzymatically hydrolyzed camphor leaf powder.

[0099] 6) Carry out fractional distillation on the enzymatically hydrolyzed camphor leaf powder obtained in step 5) to obtain a camphor essential oil precursor.

[0100] Among them, the fractional distillation is as follows: 1.5 KPa, 75 °C, 1 h; 40 KPa, 80 °C, 2 h; 60 KPa, 100 °C, 1 h;

[0101] 7) According to the mass ratio of gum arabic, maltodextrin and sodium alginate of 2:1:0.3, place gum arabic, maltodextrin and sodium alginate in deionized water at 60 °C, adjust the pH = 5, and add palmitic acid ester of ascorbic acid with a mass fraction of 0.4% to obtain a wall material solution with a concentration of 12% (w / v);

[0102] 8) Place the camphor essential oil precursor obtained in step 6) into the wall material solution obtained in step 7), control the power of ultrasonic treatment to be 200 W, and the time to be 2 min, and disperse the camphor essential oil precursor to obtain a camphor essential oil emulsion;

[0103] 9) Spray-dry the camphor essential oil emulsion obtained in step 8) to obtain the camphor essential oil;

[0104] Among them, the inlet air temperature of the spray drying is 180 °C, and the outlet air temperature is 85 °C.

[0105] Comparative Example 1

[0106] A kind of camphor essential oil

[0107] 1) Crush the camphor leaves and pass through a 40-mesh sieve to obtain camphor leaf powder;

[0108] 2) According to the molar ratio of choline chloride and lactic acid of 1:1.9, mix choline chloride and lactic acid, control the stirring speed to be 400 rpm, and stir at 60 °C for 2.5 h to obtain a choline chloride-lactic acid eutectic solvent;

[0109] 3) Place the camphor leaf powder obtained in step 1) into the choline chloride-lactic acid eutectic solvent obtained in step 2), and carry out impregnation treatment at 60 °C for 4 to obtain impregnated camphor leaf powder;

[0110] 4) According to the mass ratio of cellulase, galacturonase and β-glucanase of 2:1:0.5, mix cellulase, galacturonase and β-glucanase to obtain an enzyme preparation;

[0111] 5) According to the mass-volume ratio of impregnated camphor leaf powder, enzyme preparation and water of 10:2.5:5, place the impregnated camphor leaf powder and enzyme preparation obtained in step 3) in water, adjust the system pH = 5.5, and carry out enzymatic hydrolysis at 40 °C for 5 h to obtain enzymatically hydrolyzed camphor leaf powder;

[0112] 6) Carry out fractional distillation on the enzymatically hydrolyzed camphor leaf powder obtained in step 5) to obtain a camphor essential oil precursor;

[0113] Among them, the fractional distillation is as follows: 1.5 KPa, 75 °C, 1 h; 40 KPa, 80 °C, 2 h; 60 KPa, 100 °C, 1 h;

[0114] 7) According to the mass ratio of gum arabic, maltodextrin and sodium alginate being 2:1:0.3, place gum arabic, maltodextrin and sodium alginate in deionized water at 60 °C, adjust the pH = 5, and add palmitic acid ascorbate with a mass fraction of 0.3% to obtain a wall material solution with a concentration of 12% (w / v);

[0115] 8) Place the camphor essential oil precursor obtained in step 6) in the wall material solution obtained in step 7), control the power of ultrasonic treatment to be 200 W and the time to be 2 min, and disperse the camphor essential oil precursor to obtain a camphor essential oil emulsion;

[0116] 9) Spray-dry the camphor essential oil emulsion obtained in step 8) to obtain the camphor essential oil;

[0117] Among them, the inlet air temperature of the spray drying is 180 °C, and the outlet air temperature is 85 °C.

[0118] Comparative Example 2

[0119] A kind of camphor essential oil

[0120] 1) Freeze-dry the camphor leaves, then crush them and pass through a 40-mesh sieve to obtain camphor leaf powder;

[0121] Among them, the freeze-drying is carried out at -40 °C for 36 h;

[0122] 2) According to the mass ratio of cellulase, galacturonase and β-glucanase being 2:1:0.5, mix cellulase, galacturonase and β-glucanase to obtain an enzyme preparation;

[0123] 3) According to the mass-volume ratio of camphor leaf powder, enzyme preparation and water being 10:2.5:5, place the camphor leaf powder and enzyme preparation obtained in step 1) in water, adjust the system pH = 5.5, and enzymatically hydrolyze at 40 °C for 5 h to obtain enzymatically hydrolyzed camphor leaf powder;

[0124] 4) Carry out fractional distillation on the enzymatically hydrolyzed camphor leaf powder obtained in step 3) to obtain a camphor essential oil precursor;

[0125] Among them, the fractional distillation is as follows: 1.5 KPa, 75 °C, 1 h; 40 KPa, 80 °C, 2 h; 60 KPa, 100 °C, 1 h;

[0126] 5) According to the mass ratio of gum arabic, maltodextrin and sodium alginate being 2∶1∶0.3, place gum arabic, maltodextrin and sodium alginate in deionized water at 60°C, adjust the pH to 5, and add palmitic acid ester of ascorbic acid with a mass fraction of 0.3% to obtain a wall material solution with a concentration of 12% (w / v);

[0127] 6) Place the camphor essential oil precursor obtained in step 4) into the wall material solution obtained in step 5), control the power of ultrasonic treatment to be 200 W and the time to be 2 min, and disperse the camphor essential oil precursor to obtain a camphor essential oil emulsion;

[0128] 7) Spray-dry the camphor essential oil emulsion obtained in step 6) to obtain the camphor essential oil;

[0129] Among them, the inlet air temperature of the spray drying is 180°C and the outlet air temperature is 85°C.

[0130] Comparative Example 3

[0131] A kind of camphor essential oil

[0132] 1) Freeze-dry the camphor leaves, then crush them and pass through a 40-mesh sieve to obtain camphor leaf powder;

[0133] Among them, the freeze-drying is: freeze-dry at -40°C for 36 h;

[0134] 2) According to the molar ratio of choline chloride and lactic acid being 1∶1.9, mix choline chloride and lactic acid, control the stirring speed to be 400 rpm, and stir at 60°C for 2.5 h to obtain a choline chloride-lactic acid eutectic solvent;

[0135] 3) Place the camphor leaf powder obtained in step 1) into the choline chloride-lactic acid eutectic solvent obtained in step 2), and carry out impregnation treatment at 60°C for 4 to obtain impregnated camphor leaf powder;

[0136] 4) Carry out fractional distillation on the impregnated camphor leaf powder obtained in step 3) to obtain a camphor essential oil precursor;

[0137] Among them, the fractional distillation is: 1.5 KPa, 75°C, 1 h; 40 KPa, 80°C, 2 h; 60 KPa, 100°C, 1 h;

[0138] 5) According to the mass ratio of gum arabic, maltodextrin and sodium alginate being 2∶1∶0.3, place gum arabic, maltodextrin and sodium alginate in deionized water at 60°C, adjust the pH to 5, and add palmitic acid ester of ascorbic acid with a mass fraction of 0.3% to obtain a wall material solution with a concentration of 12% (w / v);

[0139] 6) Place the camphor essential oil precursor obtained in step 4) into the wall material solution obtained in step 5), control the power of ultrasonic treatment to be 200 W and the time to be 2 min, and disperse the camphor essential oil precursor to obtain a camphor essential oil emulsion;

[0140] 7) Spray-dry the camphor essential oil emulsion obtained in step 6) to obtain the camphor essential oil;

[0141] Among them, the inlet air temperature of the spray drying is 180 °C and the outlet air temperature is 85 °C.

[0142] Comparative Example 4

[0143] A kind of camphor essential oil

[0144] 1) Freeze-dry the camphor leaves, then crush them and pass through a 40-mesh sieve to obtain camphor leaf powder;

[0145] Among them, the freeze-drying is: freeze-dry at -40 °C for 36 h;

[0146] 2) Mix choline chloride and lactic acid according to the molar ratio of choline chloride to lactic acid of 1:1.9, control the stirring speed to be 400 rpm, and stir at 60 °C for 2.5 h to obtain a choline chloride-lactic acid eutectic solvent;

[0147] 3) Place the camphor leaf powder obtained in step 1) into the choline chloride-lactic acid eutectic solvent obtained in step 2), and carry out impregnation treatment at 60 °C for 4 to obtain impregnated camphor leaf powder;

[0148] 4) Mix cellulase, galacturonase and β-glucanase according to the mass ratio of 2:1:0.5 to obtain an enzyme preparation;

[0149] 5) According to the mass-volume ratio of impregnated camphor leaf powder, enzyme preparation and water of 10:2.5:5, place the impregnated camphor leaf powder and enzyme preparation obtained in step 3) into water, adjust the system pH = 5.5, and carry out enzymatic hydrolysis at 40 °C for 5 h to obtain enzymatically hydrolyzed camphor leaf powder;

[0150] 6) Carry out fractional distillation on the enzymatically hydrolyzed camphor leaf powder obtained in step 5) to obtain the camphor essential oil;

[0151] Among them, the fractional distillation is: 1.5 KPa, 75 °C, 1 h; 40 KPa, 80 °C, 2 h; 60 KPa, 100 °C, 1 h.

[0152] Detect the quality and stability of the camphor essential oils prepared in Examples 1-3 and Comparative Examples 1-4, and the detection results of the quality and stability of the camphor essential oils are shown in Table 1;

[0153] Essential oil yield (%) = mass of extracted essential oil / dry weight of raw camphor tree leaves × 100%

[0154] Table 1 Detection results of the quality and stability of camphor essential oil

[0155]

[0156]

[0157] From the data in Table 1, it can be seen that the camphor essential oil provided by the present invention has a yield of up to 10.1%, a camphor content of up to 95.8%, and a camphor retention rate of up to 93.5% after 4 months, with high quality and stability.

[0158] After verification, the camphor essential oil prepared in Example 2 has the best quality. Therefore, in the following application examples, only the camphor essential oil prepared in Example 2 is used as the raw material to prepare the camphor essential oil feed additive.

[0159] In the following application examples, the Bacillus subtilis is purchased from the China General Microbiological Culture Collection Center, with the preservation number CGMCC.No.1.8715; the Lactobacillus plantarum is purchased from the China General Microbiological Culture Collection Center, with the preservation number CGMCC.No.1.124; the Candida utilis is purchased from the China General Microbiological Culture Collection Center, with the preservation number CGMCC.No.2.2878.

[0160] In the following application examples, a preparation method of a camphor essential oil feed additive includes the following steps:

[0161] 1) Mix oregano essential oil, astragalus polysaccharide and capsinoid in a mass ratio of (4 - 6)∶(2 - 3)∶1 to obtain a synergistic component;

[0162] 2) Mix coconut oil, rosmarinic acid and N-acetylcysteine in a mass ratio of (4 - 6)∶(1 -

[0163] 3)∶(0.5 - 1.5) to obtain a supplementary component;

[0164] 3) Mix Bacillus subtilis, Lactobacillus plantarum and Candida utilis in a mass ratio of 3∶(0.5 - 1.5)∶(0.2 - 0.5) to obtain a microbial agent;

[0165] 4) According to the mass ratio of soybean meal, microbial inoculum, glucose and water being 10∶(0.2 - 0.5)∶(0.5 - 1)∶(4 - 6), place the soybean meal, the microbial inoculum obtained in step 3) and glucose in water, and ferment at 40°C for 6 - 10 h to obtain fermented soybean meal;

[0166] 5) According to the mass ratio of camphor essential oil, synergistic component, supplementary component and fermented soybean meal being (10 - 20)∶(5 - 15)∶(3 - 5)∶(20 - 30), first mix the camphor essential oil, synergistic component and supplementary component, stir at 60°C for 20 - 40 min to obtain an active ingredient mixture, then add the fermented soybean meal to the active ingredient mixture, stir at room temperature for 20 - 40 min, and finally freeze-dry at -40°C for 12 - 36 h to obtain the camphor essential oil feed additive.

[0167] Application Example 1

[0168] A camphor essential oil feed additive

[0169] 1) According to the mass ratio of oregano essential oil, astragalus polysaccharide and capsinoid being 4∶2∶1, mix the oregano essential oil, astragalus polysaccharide and capsinoid to obtain a synergistic component;

[0170] 2) According to the mass ratio of coconut oil, rosmarinic acid and N-acetylcysteine being 4∶1∶0.5, mix the coconut oil, rosmarinic acid and N-acetylcysteine to obtain a supplementary component;

[0171] 3) According to the mass ratio of Bacillus subtilis, Lactobacillus plantarum and Candida utilis being 3∶0.5∶0.2, mix Bacillus subtilis, Lactobacillus plantarum and Candida utilis to obtain a microbial inoculum;

[0172] 4) According to the mass ratio of soybean meal, microbial inoculum, glucose and water being 10∶0.2∶0.5∶4, place the soybean meal, the microbial inoculum obtained in step 3) and glucose in water, and ferment at 40°C for 6 h to obtain fermented soybean meal;

[0173] 5) According to the mass ratio of camphor essential oil, synergistic component, supplementary component and fermented soybean meal being 10∶5∶3∶20, first mix the camphor essential oil, synergistic component and supplementary component, stir at 60°C for 20 min to obtain an active ingredient mixture, then add the fermented soybean meal to the active ingredient mixture, stir at room temperature for 20 min, and finally freeze-dry at -40°C for 12 h to obtain the camphor essential oil feed additive.

[0174] Application Example 2

[0175] A camphor essential oil feed additive

[0176] 1) Mix oregano essential oil, astragalus polysaccharide and capsinoid in a mass ratio of 5:2.5:1 to obtain a synergistic component;

[0177] 2) Mix coconut oil, rosmarinic acid and N-acetylcysteine in a mass ratio of 5:2:1 to obtain a supplementary component;

[0178] 3) Mix Bacillus subtilis, Lactobacillus plantarum and Candida utilis in a mass ratio of 3:1:0.3 to obtain a microbial inoculant;

[0179] 4) Place soybean meal, the microbial inoculant obtained in step 3) and glucose in water in a mass ratio of 10:0.4:0.8:5, and ferment at 40 °C for 8 h to obtain fermented soybean meal;

[0180] 5) Mix camphor essential oil, the synergistic component, the supplementary component and the fermented soybean meal in a mass ratio of 15:10:4:25. First, mix camphor essential oil, the synergistic component and the supplementary component, and stir at 60 °C for 30 min to obtain an active ingredient mixture. Then, add the fermented soybean meal to the active ingredient mixture, stir at room temperature for 30 min, and finally freeze-dry at -40 °C for 24 h to obtain the camphor essential oil feed additive.

[0181] Application Example 3

[0182] A camphor essential oil feed additive

[0183] 1) Mix oregano essential oil, astragalus polysaccharide and capsinoid in a mass ratio of 6:3:1 to obtain a synergistic component;

[0184] 2) Mix coconut oil, rosmarinic acid and N-acetylcysteine in a mass ratio of 6:3:1.5 to obtain a supplementary component;

[0185] 3) Mix Bacillus subtilis, Lactobacillus plantarum and Candida utilis in a mass ratio of 3:1.5:0.5 to obtain a microbial inoculant;

[0186] 4) Place soybean meal, the microbial inoculant obtained in step 3) and glucose in water in a mass ratio of 10:0.5:1:6, and ferment at 40 °C for 10 h to obtain fermented soybean meal;

[0187] 5) According to the mass ratio of camphor essential oil, synergistic component, supplementary component and fermented soybean meal being 20∶15∶5∶30, first mix the camphor essential oil, synergistic component and supplementary component, stir at 60 °C for 40 min to obtain an active ingredient mixture, then add the fermented soybean meal to the active ingredient mixture, stir at room temperature for 40 min, and finally freeze-dry at -40 °C for 36 h to obtain the camphor essential oil feed additive.

[0188] Comparative Application Example 1

[0189] A camphor essential oil feed additive

[0190] Same as Application Example 2, the difference is only that step 1) is:

[0191] Mix astragalus polysaccharide and capsaicin ester according to the mass ratio of 2.5∶1 to obtain a synergistic component.

[0192] Comparative Application Example 2

[0193] A camphor essential oil feed additive

[0194] Same as Application Example 2, the difference is only that step 1) is:

[0195] Mix oregano essential oil and capsaicin ester according to the mass ratio of 5∶1 to obtain a synergistic component.

[0196] Comparative Application Example 3

[0197] A camphor essential oil feed additive

[0198] Same as Application Example 2, the difference is only that step 1) is:

[0199] Mix oregano essential oil and astragalus polysaccharide according to the mass ratio of 5∶2.5 to obtain a synergistic component.

[0200] Comparative Application Example 4

[0201] A camphor essential oil feed additive

[0202] Same as Application Example 2, the difference is only that step 2) is:

[0203] Mix rosmarinic acid and N-acetylcysteine according to the mass ratio of 2∶1 to obtain a supplementary component.

[0204] Comparative Application Example 5

[0205] A camphor essential oil feed additive

[0206] Same as Application Example 2, the difference is only that step 2) is:

[0207] Mix coconut oil and N-acetylcysteine according to the mass ratio of 5:1 to obtain a supplementary component.

[0208] Comparative Application Example 6

[0209] A camphor essential oil feed additive

[0210] Same as Application Example 2, except that step 2) is:

[0211] Mix coconut oil and rosmarinic acid according to the mass ratio of 5:2 to obtain a supplementary component.

[0212] Comparative Application Example 6

[0213] A camphor essential oil feed additive

[0214] 1) Mix oregano essential oil, astragalus polysaccharide and capsinoid according to the mass ratio of 5:2.5:1 to obtain a synergistic component;

[0215] 2) Mix coconut oil, rosmarinic acid and N-acetylcysteine according to the mass ratio of 5:2:1 to obtain a supplementary component;

[0216] 3) Mix camphor essential oil, synergistic component, supplementary component and soybean meal according to the mass ratio of 15:10:4:25. First, mix camphor essential oil, synergistic component and supplementary component, and stir at 60 °C for 30 min to obtain an active ingredient mixture. Then, add soybean meal to the active ingredient mixture, stir at room temperature for 30 min, and finally freeze-dry at -40 °C for 24 h to obtain the camphor essential oil feed additive.

[0217] Comparative Application Example 7

[0218] A camphor essential oil feed additive

[0219] Same as Application Example 2, except that step 5) is:

[0220] Mix camphor essential oil, synergistic component, supplementary component and fermented soybean meal according to the mass ratio of 15:10:4:25. First, mix camphor essential oil, synergistic component and supplementary component, and stir at 60 °C for 30 min to obtain an active ingredient mixture. Then, add fermented soybean meal to the active ingredient mixture, stir at room temperature for 30 min to obtain the camphor essential oil feed additive.

[0221] Effect verification:

[0222] Performance verification of the camphor essential oil feed additive in enhancing low-temperature tolerance

[0223] 1. Experimental grouping

[0224] 550 tilapia with consistent body weight (50±5 g) and good health status were selected and randomly divided into 11 groups, with 50 fish in each group. They were respectively the control group (fed with basal diet) and the experimental groups (fed with basal diet + camphor essential oil feed additives prepared according to Application Examples 1-3 and Comparative Application Examples 1-7).

[0225] 2. Feeding environment

[0226] The experiment was carried out in a temperature-controlled aquaculture system. The initial water temperature was maintained at 28±1℃, with an adaptation period of 7 days. Then, the temperature was gradually decreased to 10℃ at a rate of 2℃ per day and maintained at this temperature for the experiment. The experimental period was 4 weeks;

[0227] 3. Feed feeding

[0228] Feed was fed regularly 2 times a day (08:00 and 17:00), and the feeding amount was 5% of the fish body weight;

[0229] 4. Survival rate test

[0230] During the experiment, the death situation of tilapia in each group was recorded every day, and the survival rate was calculated;

[0231] Survival rate (%) = (initial number of fish - number of dead fish) / initial number of fish × 100%.

[0232] The calculation results of the survival rate are shown in Table 2:

[0233] Table 2 Calculation results of the survival rate

[0234]

[0235] As can be seen from the data in Table 2, a camphor essential oil feed additive provided by the present invention can significantly improve the survival rate of tilapia in a low-temperature environment, and the low-temperature survival rate can reach 88.2%.

[0236] 5. Serum physiological index test

[0237] After the experiment, 10 fish were randomly selected from each group, and blood was collected from the caudal vein. After centrifuging at 3000 rpm for 10 min, the serum was separated; kits were used to detect the contents of cortisol (COR), superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH) in the serum;

[0238] Among them, the COR content reflects the stress level of the fish body, the SOD activity reflects the antioxidant ability, the MDA content can measure the degree of lipid peroxidation, and the LDH activity is related to the degree of cell damage;

[0239] The test results of the serum physiological indexes are shown in Table 3;

[0240] Table 3 Test results of serum physiological indexes

[0241]

[0242] As can be seen from the data in Table 3, a camphor essential oil feed additive provided by the present invention can reduce the stress level (COR content) of tilapia, improve the antioxidant capacity (SOD activity) of tilapia, reduce the degree of lipid peroxidation (MDA content) of tilapia, and reduce cell damage (LDH activity) of tilapia.

[0243] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: the specific implementation manners of the present invention can still be modified or equivalently replaced, and any modification or equivalent replacement that does not depart from the spirit and scope of the present invention shall be covered by the protection scope of the claims of the present invention.

Claims

1. A method for preparing camphor essential oil, characterized in that, It includes the following steps: 1) Freeze-dry the camphor tree leaves, then crush them and sieve through a 40-mesh sieve to obtain camphor tree leaf powder; 2) Place the camphor tree leaf powder obtained in step 1) in a choline chloride-lactic acid eutectic solvent for impregnation treatment to obtain impregnated camphor tree leaf powder; 3) Place the impregnated camphor tree leaf powder obtained in step 2) and an enzyme preparation in water, adjust the pH of the system to 5.5, and carry out enzymatic hydrolysis to obtain enzymatically hydrolyzed camphor tree leaf powder; 4) Carry out fractional distillation on the enzymatically hydrolyzed camphor tree leaf powder obtained in step 3) to obtain a precursor of camphor essential oil; 5) Place the precursor of camphor essential oil obtained in step 4) in a wall material solution and carry out ultrasonic dispersion to obtain a camphor essential oil emulsion; 6) Carry out spray drying on the camphor essential oil emulsion obtained in step 5) to obtain the said camphor essential oil.

2. The method for preparing camphor essential oil according to claim 1, wherein in step 1), the freeze-drying is specifically: freeze-dry at -40°C for 24 - 48 h.

3. The method for preparing camphor essential oil according to claim 1, wherein in step 2), the preparation method of the choline chloride-lactic acid eutectic solvent includes the following steps: mix choline chloride and lactic acid according to a molar ratio of 1:(1.5 - 2), control the stirring speed at 400 rpm, and stir at 60°C for 2 - 3 h to obtain the choline chloride-lactic acid eutectic solvent; in step 2), the impregnation treatment is specifically: carry out impregnation treatment at 60°C for 3 - 5 h.

4. The method for preparing camphor essential oil according to claim 1, wherein in step 3), the enzyme preparation is composed of cellulase, galacturonase and β-glucanase mixed according to a mass ratio of 2:1:0.5; in step 3), the mass-volume ratio of the impregnated camphor tree leaf powder, the enzyme preparation and water is 10:(2 - 3):(4 - 6); in step 3), the enzymatic hydrolysis is specifically: carry out enzymatic hydrolysis at 40°C for 4 - 6 h.

5. The method for preparing camphor essential oil according to claim 1, wherein in step 4), the fractional distillation is: 1.5 KPa, 75°C, 1 h; 40 KPa, 80°C, 2 h; 60 KPa, 100°C, 1 h.

6. The method for preparing camphor essential oil according to claim 1, wherein in step 5), the preparation method of the wall material solution includes the following steps: place arabic gum, maltodextrin and sodium alginate according to a mass ratio of 2:1:0.3 in deionized water at 60°C, adjust the pH to 5, and add palmitic acid ester of ascorbic acid with a mass fraction of 0.2 - 0.4% to obtain a wall material solution with a concentration of 12% (w / v); in step 5), the power of the ultrasonic dispersion is 200 W and the time is 2 min.

7. The method for preparing camphor essential oil according to claim 1, wherein in step 6), the inlet air temperature of the spray drying is 180°C and the outlet air temperature is 85°C.

8. A camphor essential oil prepared by the method for preparing camphor essential oil according to any one of claims 1 - 7.

9. Use of the camphor essential oil as described in claim 8 in a feed additive, characterized in that, It includes the following steps: 1) Mix oregano essential oil, astragalus polysaccharide and capsinoid to obtain a synergistic component; 2) Mix coconut oil, rosmarinic acid and N-acetylcysteine to obtain a supplementary component; 3) Place soybean meal, microbial inoculum and glucose in water and ferment to obtain fermented soybean meal; 4) First, mix camphor essential oil, the synergistic component and the supplementary component, and stir to obtain an active ingredient mixture. Then, add the fermented soybean meal to the active ingredient mixture, stir, and freeze-dry to obtain the camphor essential oil feed additive.

10. The application according to claim 9, characterized in that In step 1), the mass ratio of the oregano essential oil, astragalus polysaccharide and capsinoid is (4-6):(2-3):1; In step 2), the mass ratio of the coconut oil, rosmarinic acid and N-acetylcysteine is (4-6):(1-3):(0.5-1.5); In step 3), the microbial inoculum is composed of Bacillus subtilis, Lactobacillus plantarum and Candida utilis mixed according to a mass ratio of 3:(0.5-1.5):(0.2-0.5); In step 3), the mass ratio of the soybean meal, microbial inoculum, glucose and water is 10:(0.2-0.5):(0.5-1):(4-6); In step 3), the fermentation is specifically: ferment at 40 °C for 6-10 h; In step 4), the mass ratio of the camphor essential oil, synergistic component, supplementary component and fermented soybean meal is (10-20):(5-15):(3-5):(20-30); In step 4), the freeze-drying is specifically: freeze-dry at -40 °C for 12-36 h.

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