Method for enzymatic decomposition of citrus peel and extraction of volatile oil by aspergillus niger and application of aspergillus niger

Abstract

The invention discloses a method for enzymatic decomposition of citrus peel and extraction of volatile oil by aspergillus niger, and relates to the field of extraction processes of citrus peel extracts.The method comprises the steps that citrus peel is pretreated to prepare citrus peel fine powder, the citrus peel fine powder is added into a citric acid-disodium hydrogen phosphate buffer solution, aspergillus niger is added, and fermentation is performed to obtain a fermentation mixture; uniformly mixing the fermentation mixture with water, adding an enzymatic decomposition system, carrying out ultrasonic microwave extraction, taking an extract liquid and a gas impact bottle collection liquid, mixing, and filtering; the enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate; the preparation method comprises the following steps: carrying out subcritical low-temperature extraction by using propane as a subcritical fluid, adding sodium carbonate into an extraction filtrate, and carrying out heating reaction, solid-phase extraction, suction filtration and freeze drying to obtain the limonene essential oil. The extraction process is simple, the working time is short, and the prepared limonene essential oil is safe, has a mosquito repelling effect, has good compatibility with the ecological environment and is biodegradable.

Description

Technical Field

[0001] The invention relates to the field of extraction technology of citrus peel extract, in particular to a method for enzymatically decomposing citrus peel by using Aspergillus niger and application of the method in extracting volatile oil. Background Art

[0002] Except for some of the citrus peels used as traditional Chinese medicine tangerine peel and a small part used as seasoning, the rest is mostly discarded. According to the volatile oil content of fresh peels of 1-3%, about 80,000-240,000 tons of volatile oil are discarded every year.

[0003] The main components of citrus peel essential oil are: monoterpenes, sesquiterpenes and oxygenated aromatic hydrocarbons. Monoterpenes are the main components of citrus peel essential oils, mainly D-limonene, γ-terpinene, β-myrcene and α-pinene. Citrus peel essential oils have antibacterial and anti-inflammatory effects and can remove free radicals. At the same time, citronellal obtained by refining and purifying D-limonene has a good mosquito repellent effect.

[0004] Citrus peel contains about 30% pectin and cellulose. There are chemical bonds between polysaccharides, hemicellulose and pectin in the peel cells. Conventional alkali liquid-pressing will result in a small amount of carbohydrates in the product. At present, the cell wall formed by the interweaving of cellulose, hemicellulose and pectin, the degradation of any one type of component is bound to be restricted by other components. Therefore, it is impossible to extract as much terpene volatile oils such as D-limonene from the peel cells as possible.

[0005] Aspergillus niger mainly includes Aspergillus niger and Aspergillus oryzae, which are important fermentation industrial strains. They can degrade the difficult-to-absorb substances such as crude fiber and phytic acid in the substrate and improve the extraction efficiency of volatile oils in the peel. Pectinase can cut the α-1,4 glycosidic bonds in the pectin molecules and degrade the pectin molecules into small molecules. Pyridoxal phosphate is a coenzyme of transaminases and decarboxylases in amino acid metabolism, which can promote the decarboxylation of glutamate and increase the production of γ-aminobutyric acid. Therefore, the enzymatic system composed of the above-mentioned pectinase and pyridoxal phosphate is extremely important for Aspergillus niger to decompose citrus peel. Summary of the invention

[0006] The present invention provides a method for enzymatically decomposing citrus peels with Aspergillus niger and extracting volatile oils, which is based on black mold-pectin enzymatic decomposition / ultrasonic microwave, combined with freeze grinding-subcritical low-temperature extraction technology, and can significantly improve the essential oil extraction rate; after cationic styrene microsphere chromatography filtration, high-purity single D-limonene essential oil can be obtained. It is specifically achieved by the following technologies.

[0007] In a first aspect of the present invention, a method for enzymatically decomposing citrus peel and extracting volatile oil using Aspergillus niger is provided, comprising the following steps: S1, pre-treating citrus peel to prepare citrus peel fine powder, adding the powder to a citric acid-disodium hydrogen phosphate buffer solution, adding Aspergillus niger, and fermenting to obtain a fermentation mixture; S2, mixing the fermentation mixture and water, adding the enzymatic decomposition system, performing ultrasonic microwave extraction, taking the extract and the liquid collected in the gas impact bottle, mixing and filtering; repeating this process 3 times, and combining the filtrate; The enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate, and the mass ratio of the three is 1:(0.1-0.15):(0.04-0.06); S3, performing subcritical low-temperature extraction using propane as a subcritical fluid to obtain an extraction filtrate; S4, adding sodium carbonate to the extraction filtrate, heating for reaction, solid phase extraction, suction filtration, freeze drying, and obtaining single limonene essential oil.

[0008] Further, in step S1, the Aspergillus niger is composed of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434, and the mass ratio is (0.32-0.45):(0.4-0.45):(0.1-0.28).

[0009] Furthermore, in step S1, the mass ratio of Aspergillus niger to citrus peel powder is (0.05-0.2):1.

[0010] Furthermore, in step S1, the fermentation temperature is 20-40°C, and the fermentation time is 12-72h.

[0011] Furthermore, in step S2, the total concentration of pectin lyase, polygalacturonase and pyridoxal phosphate in the enzymatic decomposition system is 0.3-0.8 g / L.

[0012] Furthermore, in step S2, the specific method of ultrasonic microwave extraction is: firstly ultrasonic extraction at 33-42°C and 60-180W for 45-60min, and then microwave extraction at 200-600W for 1-2min.

[0013] Furthermore, in step S4, the amount of sodium carbonate added is 0.2-0.5 g / L.

[0014] Furthermore, in step S3, the subcritical low-temperature extraction method is: temperature 40°C, extraction pressure 0.4-0.7MPa, extraction times 3-5 times; mixed oil concentration 10-40%, temperature 40-45°C, evaporation temperature of mixed oil 45-60°C, desolventizing vacuum degree of mixed oil -0.095MPa; meal desolventizing temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm.

[0015] The second aspect of the present invention provides a citrus peel volatile oil prepared by the above method.

[0016] The third aspect of the present invention provides an application method of the citrus peel volatile oil prepared by the above method, which can be used for repelling mosquitoes or preparing mosquito repellent preparations.

[0017] Those skilled in the art should be aware that mosquito repellent preparations include but are not limited to mosquito repellent water, mosquito repellent spray, etc.

[0018] Compared with the prior art, the advantages of the present invention are: the extraction process of the present invention is simple, the process time is short, the extraction efficiency is high, and the market prospect is broad; the prepared single limonene essential oil is safe and has mosquito repellent effect, has good compatibility with the ecological environment, and is biodegradable. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1-7 GC-MS test results of test example 1; Figure 8 This is the infrared spectrum detection result of test example 1. DETAILED DESCRIPTION

[0020] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The method for enzymatically decomposing citrus peels and extracting volatile oil using Aspergillus niger provided by the present invention comprises the following steps: S1, pre-treating citrus peel to prepare citrus peel fine powder, adding the powder to a citric acid-disodium hydrogen phosphate buffer solution, adding Aspergillus niger, and fermenting to obtain a fermentation mixture; S2, mixing the fermentation mixture and water, adding an enzymatic decomposition system, performing ultrasonic microwave extraction, taking the extract and the liquid collected in the gas impact bottle, mixing and filtering; repeating this process 3 times, and combining the filtrate; The enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate, and the mass ratio of the three is 1:(0.1-0.15):(0.04-0.06); S3, performing subcritical low-temperature extraction using propane as a subcritical fluid to obtain an extraction filtrate; S4, adding sodium carbonate to the extraction filtrate, heating for reaction, solid phase extraction, suction filtration, freeze drying, and obtaining single limonene essential oil.

[0022] In some implementation cases of the present invention, in step S1, the citrus peel is freeze-dried and then processed by a crusher to obtain coarse particles of 40-100 mesh; washed - naturally dried - hot-baked - ultra-fine freeze-grinded (100-200 mesh), and sterilized at 124°C for 30 minutes to obtain citrus peel fine powder.

[0023] In some implementation cases of the present invention, in step S1, ultrafine freeze-grinding is performed using a cryo-grinder, and the grinding parameters are electromagnetic frequency 5-15 Hz, liquid nitrogen filling, 500 ml agate grinding jar, orbital speed not less than 200 rpm, rotation speed 300-500 rpm, running time 5-20 min, to obtain citrus peel fine powder with a particle size of 0.5-5 um.

[0024] In some embodiments of the present invention, in step S1, the electromagnetic frequency of the cryo-grinder used for grinding is 5 Hz, 10 Hz or 15 Hz.

[0025] In some embodiments of the present invention, in step S1, the operation time of the freezing grinder for grinding is 5 min, 8 min, 10 min, 12 min, 14 min, 16 min, 18 min or 20 min.

[0026] In some embodiments of the present invention, in step S1, the Aspergillus niger is composed of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434, and the mass ratio is (0.32-0.45):(0.4-0.45):(0.1-0.28).

[0027] Further specifically, in step S1, the mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434 can be 0.32:0.4:0.1, 0.32:0.45:0.1, 0.32:0.4:0.28, 0.32:0.45:0.28, 0.45:0.4:0.1, 0.45:0.45:0.1, 0.45:0.4:0.28 or 0.45:0.45:0.28.

[0028] In some implementation cases of the present invention, in step S1, the mass ratio of Aspergillus niger to citrus peel powder is (0.05-0.2):1.

[0029] Further specifically, the mass ratio of Aspergillus niger to citrus peel powder is 0.05:1, 0.1:1, 0.15:1 or 0.2:1.

[0030] In some implementation cases of the present invention, in step S1, the fermentation temperature is 20-40° C., and the fermentation time is 12-72 hours.

[0031] More specifically, in step S1, the fermentation temperature is 20, 25, 30, 35 or 40°C.

[0032] More specifically, in step S1, the fermentation time is 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 or 72 hours.

[0033] In some embodiments of the present invention, in step S2, the mass ratio of pectin lyase, polygalacturonase and pyridoxal phosphate is 1:0.1:0.04, 1:0.1:0.06, 1:0.15:0.04, 1:0.15:0.06, 1:0.1:0.05 or 1:0.15:0.05.

[0034] In some embodiments of the present invention, in step S2, the total concentration of pectin lyase, polygalacturonase and pyridoxal phosphate in the enzymatic decomposition system is 0.3-0.8 g / L.

[0035] More specifically, in step S2, the total concentration of pectin lyase, polygalacturonase and pyridoxal phosphate is 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L or 0.8 g / L.

[0036] In some implementation cases of the present invention, in step S2, the specific method of ultrasonic microwave extraction is: first 33-42°C, 60-180W ultrasonic extraction for 45-60min, and then 200-600W microwave extraction for 60-120s.

[0037] More specifically, the ambient temperature of the ultrasonic microwave extraction is 33, 34, 36, 38, 40 or 42°C.

[0038] More specifically, the ultrasonic power of the ultrasonic extraction is 60 W, 80 W, 100 W, 120 W, 140 W, 160 W or 180 W. The ultrasonic extraction time is 45 min, 50 min, 55 min or 60 min.

[0039] More specifically, the microwave power of the microwave extraction is 200 W, 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, 550 W or 600 W. The time of the microwave extraction is 60 s, 70 s, 80 s, 90 s, 100 s, 110 s or 120 s.

[0040] In some implementation cases of the present invention, in step S4, the amount of sodium carbonate added is 0.2-0.5 g / L.

[0041] Further specifically, the amount of sodium carbonate added is 0.2 g / L, 0.3 g / L, 0.4 g / L or 0.5 g / L.

[0042] It should be known that based on the known extraction principle of subcritical low temperature extraction, the sample to be extracted cannot contain water. Therefore, it is necessary to dehydrate the product after enzymatic decomposition and ultrasonic microwave extraction, such as freeze drying.

[0043] In some implementation cases of the present invention, the sample to be extracted is freeze-dried to obtain sample powder. The freeze-drying method is: temperature -40°C to -60°C, vacuum degree 2-10 Pa, and freeze-drying time is 48 hours.

[0044] In some implementation cases of the present invention, in step S3, the subcritical low-temperature extraction method is: temperature 40°C, extraction pressure 0.4-0.7MPa, extraction times 3-5 times; mixed oil concentration is 10-48%, temperature 40-45°C, evaporation temperature of mixed oil is 45-60°C, and desolventizing vacuum degree of mixed oil is -0.095MPa; meal desolventizing temperature is 45°C, crude oil residual solvent is <30ppm, and meal residual solvent is <500ppm.

[0045] More specifically, in step S3, the extraction pressure is 0.4 MPa, 0.5 MPa, 0.6 MPa or 0.7 MPa.

[0046] Further specifically, in step S3, the concentration of the mixed oil used in the subcritical low-temperature extraction is 27-48%. Specifically, in step S3, the concentration of the mixed oil used in the subcritical low-temperature extraction is 27%, 30%, 33%, 36%, 39%, 42%, 45% or 48%.

[0047] Preferably, the mixed oil concentration used in subcritical low-temperature extraction is 45%.

[0048] The application method of the citrus peel volatile oil provided by the present invention can be used to repel mosquitoes or prepare mosquito repellent preparations. Optionally, the mosquito repellent preparations include but are not limited to mosquito repellent water, mosquito repellent spray, etc.

[0049] Example 1 The method provided in this embodiment for enzymatically decomposing citrus peels with Aspergillus niger and extracting volatile oil comprises the following steps: S1. The citrus peel is freeze-dried and then processed by a crusher to obtain coarse particles of 40-100 mesh; washed, naturally dried, hot-dried, ultra-finely frozen (100-200 mesh), and sterilized at 124°C for 30 minutes; The ultrafine freeze grinding is performed by using a cryo-grinder, and the grinding parameters are: electromagnetic frequency 10 Hz, liquid nitrogen filling, agate grinding jar 500 ml, revolution speed 200 rpm, rotation speed 500 rpm, running time 10 min, to obtain citrus peel fine powder with a particle size of 1-2 um; 20 g of citrus peel powder is added to 100 g of citric acid-disodium hydrogen phosphate buffer solution (pH value is 6.4-6.8), and Aspergillus niger (spore content 400-500 million / g) is added at 0.16 g / g based on the mass of the citrus peel powder, and fermented to obtain a fermentation mixture at a fermentation temperature of 30°C and a fermentation time of 30 hours; The mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434 is 0.4:0.45:0.2.

[0050] S2, mixing the fermentation mixture and water at a material-liquid ratio of 1:20, and adding an enzymatic decomposition system; the enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate, and the mass ratio of the three is 1:0.15:0.05, and the concentration of the three is 0.5g / L; Ultrasonic microwave extraction (first 40°C, 180W ultrasonic extraction for 60min, then 500W microwave extraction for 120s), take the extract and the liquid collected in the gas shock bottle, mix and filter; cycle this process 3 times, combine the filtrate, and freeze-dry; S3, using propane as a subcritical fluid to perform subcritical low-temperature extraction on the freeze-dried product (temperature 40°C, extraction pressure 0.6MPa, extraction times 5 times; mixed oil concentration 45%, temperature 45°C, evaporation temperature of mixed oil 60°C, desolventizing vacuum degree of mixed oil -0.095MPa; meal desolventizing temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm), to obtain an extraction filtrate; S4. After adding sodium carbonate (addition amount 0.5 g / L) to the extraction filtrate, heating the reaction at 60° C. for 30 min, extracting by 12-phase solid phase extraction, and filtering with a 0.22-μm polytetrafluoroethylene microporous filter membrane at room temperature, freeze-drying, and obtaining single limonene essential oil.

[0051] It was determined that the extraction amount of the single limonene essential oil obtained in this example was 3.62 g / kg.

[0052] Example 2 The method provided in this embodiment for enzymatically decomposing citrus peels with Aspergillus niger and extracting volatile oil comprises the following steps: S1. The citrus peel is freeze-dried and then processed by a crusher to obtain coarse particles of 40-100 mesh; washed, naturally dried, hot-dried, ultra-finely frozen (100-200 mesh), and sterilized at 124°C for 30 minutes; The ultrafine freeze grinding is performed by using a cryo-grinding machine, and the grinding parameters are electromagnetic frequency 5 Hz, liquid nitrogen filling, agate grinding jar 500 ml, revolution speed 300 rpm, rotation speed 500 rpm, running time 20 min, to obtain citrus peel fine powder with a particle size of 0.5-2.5 um; 20 g of citrus peel powder was added to 100 g of citric acid-disodium hydrogen phosphate buffer solution (pH value was 6.4-6.8), and Aspergillus niger (spore content was 300-400 million / g) was added at 0.08 g / g based on the mass of the citrus peel powder, and fermented to obtain a fermentation mixture at a fermentation temperature of 40°C and a fermentation time of 36 hours; The mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434 is 0.32:0.45:0.1.

[0053] S2, mixing the fermentation mixture and water at a material-liquid ratio of 1:20, and adding an enzymatic decomposition system; the enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate, and the mass ratio of the three is 1:0.1:0.04, and the concentration of the three is 0.8 g / L; Ultrasonic microwave extraction (first 33°C, 180W ultrasonic extraction for 45min, then 600W microwave extraction for 60s), take the extract and the liquid collected in the gas shock bottle, mix and filter; cycle this process 3 times, combine the filtrate, and freeze-dry; S3, using propane as a subcritical fluid to perform subcritical low-temperature extraction on the freeze-dried product (temperature 40°C, extraction pressure 0.5MPa, extraction times 3 times; mixed oil concentration 27%, temperature 40°C, evaporation temperature of mixed oil 45°C, desolventizing vacuum degree of mixed oil -0.095MPa; meal desolventizing temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm), to obtain an extraction filtrate; S4. After adding sodium carbonate (addition amount 0.5 g / L) to the extraction filtrate, heating the reaction at 60° C. for 30 min, extracting by 12-phase solid phase extraction, and filtering with a 0.22-μm polytetrafluoroethylene microporous filter membrane at room temperature, freeze-drying, and obtaining single limonene essential oil.

[0054] It was determined that the extraction amount of the single limonene essential oil obtained in this example was 3.16 g / kg.

[0055] Example 3 The method provided in this embodiment for enzymatically decomposing citrus peels with Aspergillus niger and extracting volatile oil comprises the following steps: S1. The citrus peel is freeze-dried and then processed by a crusher to obtain coarse particles of 40-100 mesh; washed, naturally dried, hot-dried, ultra-finely frozen (100-200 mesh), and sterilized at 124°C for 30 minutes; The ultrafine freeze grinding is performed by using a cryo-grinder, and the grinding parameters are: electromagnetic frequency 15 Hz, liquid nitrogen filling, agate grinding jar 500 ml, revolution speed 400 rpm, rotation speed 500 rpm, running time 5 min, to obtain citrus peel fine powder with a particle size of 2.5-4 um; 20 g of citrus peel powder was added to 100 g of citric acid-disodium hydrogen phosphate buffer solution (pH value was 6.4-6.8), and Aspergillus niger (spore content was 400-500 million / g) was added at 0.2 g / g based on the mass of the citrus peel powder, and fermented to obtain a fermentation mixture at a fermentation temperature of 35°C and a fermentation time of 72 hours; The mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434 is 0.45:0.4:0.28.

[0056] S2, mixing the fermentation mixture and water at a material-liquid ratio of 1:20, and adding an enzymatic decomposition system; the enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate, and the mass ratio of the three is 1:0.15:0.06, and the concentration of the three is 0.3g / L; Ultrasonic microwave extraction (first 42°C, 60W ultrasonic extraction for 60min, then 200W microwave extraction for 120s), take the extract and the liquid collected in the gas shock bottle, mix and filter; cycle this process 3 times, combine the filtrate, and freeze-dry; S3, using propane as a subcritical fluid to perform subcritical low-temperature extraction on the freeze-dried product (temperature 40°C, extraction pressure 0.7MPa, extraction times 5 times; mixed oil concentration 48%, temperature 45°C, evaporation temperature of mixed oil 60°C, desolventizing vacuum degree of mixed oil -0.095MPa; meal desolventizing temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm), to obtain an extraction filtrate; S4. After adding sodium carbonate (addition amount 0.5 g / L) to the extraction filtrate, heating the reaction at 60° C. for 30 min, extracting by 12-phase solid phase extraction, and filtering with a 0.22-μm polytetrafluoroethylene microporous filter membrane at room temperature, freeze-drying, and obtaining single limonene essential oil.

[0057] It was determined that the extraction amount of the single limonene essential oil obtained in this example was 3.35 g / kg.

[0058] Example 4 The method provided in this embodiment for enzymatically decomposing citrus peels with Aspergillus niger and extracting volatile oil comprises the following steps: S1. The citrus peel is freeze-dried and then processed by a crusher to obtain coarse particles of 40-100 mesh; washed, naturally dried, hot-dried, ultra-finely frozen (100-200 mesh), and sterilized at 124°C for 30 minutes; The ultrafine freeze grinding is performed using a cryo-grinder, and the grinding parameters are: electromagnetic frequency 10 Hz, liquid nitrogen filling, agate grinding jar 500 ml, revolution speed 350 rpm, rotation speed 500 rpm, running time 18 min, to obtain citrus peel fine powder with a particle size of 1.5-2.5 um; 20 g of citrus peel powder was added to 100 g of citric acid-disodium hydrogen phosphate buffer solution (pH value was 6.4-6.8), and Aspergillus niger (spore content was 400-500 million / g) was added at 0.15 g / g based on the mass of the citrus peel powder, and fermented to obtain a fermentation mixture at a fermentation temperature of 30°C and a fermentation time of 66 hours; The mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434 is 0.35:0.45:0.15; S2, mixing the fermentation mixture and water at a material-liquid ratio of 1:20, and adding an enzymatic decomposition system; the enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate, and the mass ratio of the three is 1:0.1:0.05, and the concentration of the three is 0.6 g / L; Ultrasonic microwave extraction (first 37°C, 120W ultrasonic extraction for 50min, then 400W microwave extraction for 100s), take the extract and the liquid collected in the gas shock bottle, mix and filter; cycle this process 3 times, combine the filtrate, and freeze-dry; S3, using propane as a subcritical fluid to perform subcritical low-temperature extraction on the freeze-dried product (temperature 40°C, extraction pressure 0.5MPa, extraction times 3 times; mixed oil concentration 40%, temperature 45°C, evaporation temperature of mixed oil 55°C, desolventizing vacuum degree of mixed oil -0.095MPa; meal desolventizing temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm), to obtain an extraction filtrate; S4. After adding sodium carbonate (addition amount 0.3 g / L) to the extraction filtrate, heating the reaction at 60° C. for 30 min, extracting by 12-phase solid phase extraction, and filtering with a 0.22-μm polytetrafluoroethylene microporous filter membrane at room temperature, freeze-drying, and obtaining single limonene essential oil.

[0059] It was determined that the extraction amount of the single limonene essential oil obtained in this example was 3.77 g / kg.

[0060] Comparative Example 1 The method for enzymatically decomposing citrus peel and extracting volatile oil using Aspergillus niger provided in this comparative example is basically the same as that in Example 1, except that the mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonarius CICC 41254 and Aspergillus niger 41434 is 0.5:0.45:0.05.

[0061] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 3.23 g / kg.

[0062] Comparative Example 2 The method for enzymatically decomposing citrus peel and extracting volatile oil using Aspergillus niger provided in this comparative example is basically the same as that in Example 1, except that the mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonarius CICC 41254 and Aspergillus niger 41434 is 0.3:0.35:0.35.

[0063] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 3.16 g / kg.

[0064] Comparative Example 3 The method for enzymatically decomposing citrus peel and extracting volatile oil using Aspergillus niger provided in this comparative example is basically the same as that in Example 2, except that the mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonarius CICC 41254 and Aspergillus niger 41434 is 0.6:0.3:0.1.

[0065] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 2.97 g / kg.

[0066] Comparative Example 4 The method for enzymatically decomposing citrus peel and extracting volatile oil using Aspergillus niger provided in this comparative example is basically the same as that in Example 3, except that the mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonarius CICC 41254 and Aspergillus niger 41434 is 0.5:0.3:0.2.

[0067] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 3.08 g / kg.

[0068] Comparative Example 5 The method for enzymatically decomposing citrus peel and extracting volatile oil by Aspergillus niger provided in this comparative example is basically the same as that in Example 4, except that: the Aspergillus niger is composed of Aspergillus niger CICC 40273 and Aspergillus carbonarius CICC 41254, and the mass ratio of Aspergillus niger CICC 40273 to Aspergillus carbonarius CICC 41254 is 0.5:0.5 (i.e., 1:1).

[0069] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 3.12 g / kg.

[0070] Comparative Example 6 The method for enzymatically decomposing citrus peel and extracting volatile oil using Aspergillus niger provided in this comparative example is basically the same as that in Example 4, except that: the Aspergillus niger is composed of carbon black Aspergillus CICC 41254 and Aspergillus niger 41434, and the mass ratio of carbon black Aspergillus CICC41254 to Aspergillus niger 41434 is 0.5:0.5 (i.e., 1:1).

[0071] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 3.21 g / kg.

[0072] Comparative Example 7 The method for enzymatically decomposing citrus peel with Aspergillus niger and extracting volatile oil provided in this comparative example is basically the same as that in Example 1, except that in step S2, the mass ratio of pectin lyase, polygalacturonase and pyridoxal phosphate is 1:0.05:0.1.

[0073] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 3.14 g / kg.

[0074] Comparative Example 8 The method for enzymatically decomposing citrus peel with Aspergillus niger and extracting volatile oil provided in this comparative example is basically the same as that in Example 1, except that in step S2, the mass ratio of pectin lyase, polygalacturonase and pyridoxal phosphate is 1:0.2:0.02.

[0075] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 2.98 g / kg.

[0076] Comparative Example 9 The method for enzymatically decomposing citrus peel with Aspergillus niger and extracting volatile oil provided in this comparative example is basically the same as that in Example 1, except that: in step S2, the enzymatic decomposition system only contains pectin lyase and polygalacturonase, and the mass ratio is 1:0.15.

[0077] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 3.05 g / kg.

[0078] Comparative Example 10 The method for enzymatically decomposing citrus peel and extracting volatile oil using Aspergillus niger provided in this comparative example is basically the same as that in Example 1, except that in step S2, the enzymatic decomposition system contains only polygalacturonase and pyridoxal phosphate, and the mass ratio is 0.15:0.05.

[0079] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 3.11 g / kg.

[0080] Comparative Example 11: The method for extracting volatile oil from citrus peel provided in this comparative example, compared with Example 1, does not perform Aspergillus niger fermentation treatment, nor does it perform enzymatic decomposition. The specific method is: S1. The citrus peel is freeze-dried and then processed by a crusher to obtain coarse particles of 40-100 mesh; washed, naturally dried, hot-dried, ultra-finely frozen (100-200 mesh), and sterilized at 124°C for 30 minutes; The ultrafine freeze grinding is performed by using a cryo-grinder, and the grinding parameters are: electromagnetic frequency 10 Hz, liquid nitrogen filling, agate grinding jar 500 ml, revolution speed 200 rpm, rotation speed 500 rpm, running time 10 min, to obtain citrus peel fine powder with a particle size of 1-2 um; S2. Subcritical low-temperature extraction of citrus peel fine powder using propane as subcritical fluid (temperature 40°C, extraction pressure 0.6MPa, extraction times 5 times; mixed oil concentration 45%, temperature 45°C, evaporation temperature of mixed oil 60°C, desolventization vacuum degree of mixed oil -0.095MPa; meal desolventization temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm), to obtain extraction filtrate; S3. After adding sodium carbonate (addition amount 0.5 g / L) to the extraction filtrate, heating the reaction at 60° C. for 30 min, extracting by 12-phase solid phase extraction, and filtering with a 0.22-μm polytetrafluoroethylene microporous filter membrane at room temperature, freeze-drying, and obtaining single limonene essential oil.

[0081] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 0.72 g / kg.

[0082] Comparative Example 12 The method for extracting volatile oil from citrus peel provided in this comparative example, compared with Example 1, only performs Aspergillus niger fermentation treatment without enzymatic decomposition, and the specific method is as follows: S1. The citrus peel is freeze-dried and then processed by a crusher to obtain coarse particles of 40-100 mesh; washed, naturally dried, hot-dried, ultra-finely frozen (100-200 mesh), and sterilized at 124°C for 30 minutes; The ultrafine freeze grinding is performed by using a cryo-grinder, and the grinding parameters are: electromagnetic frequency 10 Hz, liquid nitrogen filling, agate grinding jar 500 ml, revolution speed 200 rpm, rotation speed 500 rpm, running time 10 min, to obtain citrus peel fine powder with a particle size of 1-2 um; 20 g of citrus peel powder is added to 100 g of citric acid-disodium hydrogen phosphate buffer solution (pH value is 6.4-6.8), and Aspergillus niger (spore content 400-500 million / g) is added at 0.16 g / g based on the mass of the citrus peel powder, and fermented to obtain a fermentation mixture at a fermentation temperature of 30° C. and a fermentation time of 30 h; freeze-drying to obtain a fermentation mixture dry powder; The mass ratio of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434 is 0.4:0.45:0.2.

[0083] S2. Using propane as a subcritical fluid, subcritical low-temperature extraction is performed on the fermentation mixture dry powder (temperature 40°C, extraction pressure 0.6MPa, extraction times 5 times; mixed oil concentration 45%, temperature 45°C, evaporation temperature of mixed oil 60°C, desolventization vacuum degree of mixed oil -0.095MPa; meal desolventization temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm), to obtain an extraction filtrate; S3. After adding sodium carbonate (addition amount 0.5 g / L) to the extraction filtrate, heating the reaction at 60° C. for 30 min, extracting by 12-phase solid phase extraction, and filtering with a 0.22-μm polytetrafluoroethylene microporous filter membrane at room temperature, freeze-drying, and obtaining single limonene essential oil.

[0084] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 1.22 g / kg.

[0085] Comparative Example 13 The method for extracting volatile oil from citrus peel provided in this comparative example, compared with Example 1, does not perform Aspergillus niger fermentation treatment, but only performs enzymatic decomposition, and the specific method is: S1. The citrus peel is freeze-dried and then processed by a crusher to obtain coarse particles of 40-100 mesh; washed, naturally dried, hot-dried, ultra-finely frozen (100-200 mesh), and sterilized at 124°C for 30 minutes; The ultrafine freeze grinding is performed by using a cryo-grinder, and the grinding parameters are: electromagnetic frequency 10 Hz, liquid nitrogen filling, agate grinding jar 500 ml, revolution speed 200 rpm, rotation speed 500 rpm, running time 10 min, to obtain citrus peel fine powder with a particle size of 1-2 um; S2, mixing citrus peel powder and water at a solid-liquid ratio of 1:20, and adding an enzymatic decomposition system; the enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate, and the mass ratio of the three is 1:0.15:0.05, and the concentration of the three is 0.5g / L; Ultrasonic microwave extraction (first 40°C, 180W ultrasonic extraction for 60min, then 500W microwave extraction for 120s), take the extract and the liquid collected in the gas shock bottle, mix and filter; cycle this process 3 times, combine the filtrate, and freeze-dry; S3, using propane as a subcritical fluid to perform subcritical low-temperature extraction on the freeze-dried product (temperature 40°C, extraction pressure 0.6MPa, extraction times 5 times; mixed oil concentration 45%, temperature 45°C, evaporation temperature of mixed oil 60°C, desolventizing vacuum degree of mixed oil -0.095MPa; meal desolventizing temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm), to obtain an extraction filtrate; S4. After adding sodium carbonate (addition amount 0.5 g / L) to the extraction filtrate, heating the reaction at 60° C. for 30 min, extracting by 12-phase solid phase extraction, and filtering with a 0.22-μm polytetrafluoroethylene microporous filter membrane at room temperature, freeze-drying, and obtaining single limonene essential oil.

[0086] It was determined that the extraction amount of the single limonene essential oil obtained in this comparative example was 1.46 g / kg.

[0087] Test Example 1: Characterization and analysis of limonene essential oil.

[0088] The single limonene essential oil prepared in Example 1 was characterized and analyzed by GC-MS and infrared spectroscopy.

[0089] GC-MS analysis conditions: carrier gas is high-purity helium (99.999%), flow rate is 1mL / min, non-split injection. Injection port temperature is 250℃, injection volume is 1μL. Program temperature rise mode is adopted: column temperature is 80℃, maintained for 3min, then heated to 280℃ at 8℃ / min, maintained for 30min. MS conditions: ion source temperature is 230℃, interface temperature is 250℃, solvent delay is 2min.

[0090] The total ion chromatogram of GC-MS analysis of limonene essential oil is shown in Figure 1-7 As shown in Table 1 below. It can be seen that according to the NIST library matching results, the component with a retention time of 4 min is determined to be the target product α-pinene, the component with a retention time of 5 min is determined to be the target product β-pinene, the component with a retention time of 6 min is determined to be the target product limonene, the component with a retention time of 7 min is determined to be the target product linalool, the component with a retention time of 10 min is determined to be the target product d-carvone, and the component with a retention time of 16 min is determined to be the target product nerolidol.

[0091] Table 1 GC-MS test results

[0092] The content of limonene is 51wt%, carveol is 5wt%, linalool is 4wt%, longifolene is 15wt%, pinene is 7wt%, etc., which are the main components of limonene mixed essential oil.

[0093] from Figure 8 The infrared spectrum shown in the figure shows that 3408cm -1 The strong and broad infrared peak at 1411 cm -1 Nearby and 1182cm -1 The in-plane bending and stretching vibration of -C-OH of alcohols in the vicinity can prove that essential oils contain more alcohols. The antisymmetric stretching vibration of -CH3 is at 2924cm -1 Display, 1457cm -1 and 1373cm -1 Two positions can be attributed to the variable angle vibration of CH3 (asymmetric mode and symmetric mode); 1638cm -1 The nearby peaks can be attributed to the stretching vibration of the C=C bond.

[0094] These observations indicate the presence of olefins in the single limonene essential oil sample. We observed that the 2855 cm -1 、1738cm -1 There are two weak peaks at 1261cm -1 、1182cm -1 The absorption peak at is the COC stretching vibration region of fatty acid esters. The common peak position shows that the essential oil contains alcohols, olefins, ketone aldehydes and ester compounds, which is consistent with the results of GC-MS.

[0095] Test Example 2: Analysis of the extraction rate of limonene essential oil in each embodiment and comparative example.

[0096] The cell wall formed by the cellulose, hemicellulose and pectin in the citrus peel is interwoven with each other. The degradation of any one type of component is inevitably restricted by other components, thereby affecting the extraction rate of limonene essential oil. The Aspergillus niger CICC 40273 used in the present invention is used to produce polygalacturonase (pectinase), xylanase and cellulase; Carbonaceous Aspergillus CICC41254 is used to produce tannase, pectinase and cellulase; Aspergillus niger 41434 is used to produce tannase, pectinase and cellulase.

[0097] By comparing Example 1 with Comparative Examples 1-2, Example 2 with Comparative Example 3, Example 3 with Comparative Example 4, and Example 4 with Comparative Examples 5 and 6, it can be seen that changing the mass ratio of Aspergillus niger CICC 40273, Carbonaceous Aspergillus CICC 41254, and Aspergillus niger 41434, or lacking any one of Aspergillus niger, will have a significant effect on the extraction rate of the single limonene essential oil. Only by using the three Aspergillus niger provided by the present invention and fermenting according to a specific mass ratio can more single limonene essential oil be extracted.

[0098] By comparing Example 1 and Comparative Examples 7-10, it can be seen that changing the mass ratio of pectin lyase, polygalacturonase and pyridoxal phosphate in the enzymatic decomposition system, or lacking any one of the components, will have a significant impact on the extraction rate of the single limonene essential oil. Only by using the enzymatic decomposition system provided by the present invention for treatment can more single limonene essential oil be extracted.

[0099] By comparing Example 1 and Comparative Examples 11-13, it can be seen that Aspergillus niger fermentation, enzymatic decomposition system and ultrasonic microwave extraction have a significant synergistic effect on the extraction rate of single limonene essential oil. When any of the process steps is missing, the acquisition of single limonene essential oil will be affected.

[0100] Test Example 3: Test on the mosquito repellent effect of the single limonene essential oil of each embodiment and comparative example.

[0101] The limonene essential oil extracted in Example 1 was prepared into a mosquito repellent spray according to the formula shown in Table 2 below.

[0102] Table 2 Mosquito repellent spray formula

[0103] According to the above scheme, limonene essential oil was prepared into mosquito repellent dispersions D1-D5 at 10%, 13%, 16%, 19% and 22%, and the mosquito repellent performance of the fabric was evaluated by taking the mosquito bite prevention rate as the test index. The mosquito repellent (finishing) effect formula was based on the repellent repellent rate calculation formula of GB / T13917.9-2009 "Indoor efficacy test and evaluation of registered pesticides for hygienic insecticides", and the test method was optimized by simulating indoor environment. The test results are shown in Table 3 below.

[0104] Table 3 Mosquito repellent effect test results

[0105] The above table shows that the rate of preventing mosquito bites can be significantly improved, wherein the addition amount of limonene essential oil is higher than 16%, and the mosquito repellent rate can reach more than 90%.

[0106] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for enzymatically decomposing citrus peel and extracting volatile oil using Aspergillus niger, characterized in that: The following steps are involved: S1, pre-treating citrus peel to prepare citrus peel fine powder, adding the powder to a citric acid-disodium hydrogen phosphate buffer solution, adding Aspergillus niger, and fermenting to obtain a fermentation mixture; S2, mixing the fermentation mixture and water, adding an enzymatic decomposition system, performing ultrasonic microwave extraction, taking the extract and the liquid collected in the gas shock bottle, mixing and filtering; repeating this process 3 times, and combining the filtrate; The enzymatic decomposition system contains pectin lyase, polygalacturonase and pyridoxal phosphate, and the mass ratio of the three is 1:(0.1-0.15):(0.04-0.06); S3, performing subcritical low-temperature extraction using propane as a subcritical fluid to obtain an extraction filtrate; S4, adding sodium carbonate to the extraction filtrate, heating for reaction, solid phase extraction, suction filtration, freeze drying, and obtaining single limonene essential oil.

2. The method for enzymatically decomposing citrus peel and extracting volatile oil by Aspergillus niger according to claim 1, characterized in that: In step S1, the Aspergillus niger consists of Aspergillus niger CICC 40273, Aspergillus carbonii CICC 41254 and Aspergillus niger 41434, and the mass ratio is (0.32-0.45):(0.4-0.45):(0.1-0.28).

3. The method for enzymatically decomposing citrus peel and extracting volatile oil by Aspergillus niger according to claim 1, characterized in that: In step S1, the mass ratio of Aspergillus niger to citrus peel powder is (0.05-0.2):

1.

4. The method for enzymatically decomposing citrus peel and extracting volatile oil by Aspergillus niger according to claim 1, characterized in that: In step S1, the fermentation temperature is 20-40°C and the fermentation time is 12-72h.

5. The method for enzymatically decomposing citrus peel and extracting volatile oil by Aspergillus niger according to claim 1, characterized in that: In step S2, the total concentration of pectin lyase, polygalacturonase and pyridoxal phosphate in the enzymatic decomposition system is 0.3-0.8 g / L.

6. The method for enzymatically decomposing citrus peel and extracting volatile oil by Aspergillus niger according to claim 1, characterized in that: In step S2, the specific method of ultrasonic microwave extraction is: firstly ultrasonic extraction at 33-42°C and 60-180W for 45-60min, and then microwave extraction at 200-600W for 60-120s.

7. The method for enzymatically decomposing citrus peel and extracting volatile oil by Aspergillus niger according to claim 1, characterized in that: In step S4, the amount of sodium carbonate added is 0.2-0.5 g / L.

8. The method for enzymatically decomposing citrus peel and extracting volatile oil by Aspergillus niger according to claim 1, characterized in that: In step S3, the subcritical low-temperature extraction method is: temperature 40°C, extraction pressure 0.4-0.7MPa, extraction times 3-5 times; mixed oil concentration 10-40%, temperature 40-45°C, evaporation temperature of mixed oil 45-60°C, desolventizing vacuum degree of mixed oil -0.095MPa; meal desolventizing temperature 45°C, crude oil residual solvent <30ppm, meal residual solvent <500ppm.

9. A citrus peel volatile oil prepared by the method according to any one of claims 1 to 8.

10. An application of citrus peel volatile oil, characterized in that: The citrus peel volatile oil is prepared by the method according to any one of claims 1 to 8 and is used for repelling mosquitoes or preparing mosquito repellent preparations.

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