CN111500371A - Method for extracting orange peel essential oil by using microbial fermentation method - Google Patents
Method for extracting orange peel essential oil by using microbial fermentation method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/027—Recovery of volatiles by distillation or stripping
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Abstract
The invention discloses a method for extracting orange peel essential oil by using a microbial fermentation method, which comprises the following steps: inoculating mould spores into crushed fresh orange peel, and fermenting at 28-30 ℃ for 3-4 days to obtain orange peel fermentation product; extracting the orange peel fermentation product by a steam distillation method to obtain orange peel essential oil; the mould is Trichoderma reesei, Trichoderma longibrachiatum or Aspergillus niger. Before extracting essential oil from fresh citrus peel, fermentation pretreatment of trichoderma reesei GIM 3.553 is added. The trichoderma reesei GIM 3.553 moderately grows in the crushed orange peel to generate polysaccharide hydrolases such as cellulase, pectinase and xylanase, etc., destroy the cell wall structure, and help to release essential oil from cells, thereby improving the extraction yield of the essential oil. Compared with the conventional method without fermentation pretreatment, the extraction yield of the orange peel essential oil can be improved by more than 50%.
Description
(I) technical field
The invention belongs to the technical field of bioengineering, and particularly relates to application of a microbial fermentation technology in orange peel essential oil extraction.
(II) background of the invention
Citrus essential oil is a general name of oily liquid with aromatic odor, which is extracted from pericarp of plant of Citrus of Rutaceae (including orange, grapefruit, mandarin orange, ponkan, etc.). The citrus essential oil has unique flavor, and its main components are terpenes, sesquiterpenes, and oxygen-containing compounds composed of higher alcohols, aldehydes, ketones, esters, etc. Research shows that the citrus essential oil has the activity effects of bacteriostasis, inflammation diminishing, virus resisting, oxidation resisting and the like, and has higher application value. The application of the citrus essential oil in the fields of medicine, food and daily use chemicals is quite wide, and the citrus essential oil is a raw material for preparing various essences.
China is the third major citrus producing country which is second to Brazil and the United states, and the citrus produces a large amount of peel dregs after fresh eating or processing production and is mostly treated as garbage at present. The citrus peel residues contain a plurality of useful components including essential oil and the like, and the essential oil is extracted from the citrus peel residues, so that the comprehensive utilization of citrus can be realized, the utilization rate of citrus resources is improved, and the citrus peel residues play a positive role in the economic development and the environmental protection of citrus production areas.
At present, methods for extracting citrus essential oil include a direct squeezing method, a steam distillation method, an organic solvent extraction method, a supercritical fluid extraction method and the like. The direct pressing method can avoid the component change and volatilization loss which are possibly caused by heating the essential oil, but the extraction yield is usually lower; the steam distillation method is most widely applied, has low equipment requirement, but has the problem of thermal decomposition; organic solvent extraction methods tend to result in loss of volatile components when the solvent is removed; although the supercritical fluid extraction method is short in time consumption, the equipment cost is high, the productivity is limited, and the application in production practice has certain limitation. In contrast, steam distillation has relative advantages and is the main method for extracting citrus essential oil at present. Before extracting essential oil from orange peel, the cell wall is broken by an enzymolysis method to release essential oil in cells, so that the extraction rate of the essential oil is improved. If the orange peel essential oil is extracted by the assistance of cellulase enzymolysis, the extraction rate of the essential oil can reach 3.14 percent (Wangjian Mei, Luolinhua, Wumenguixia, and the like), the process research of extracting the orange peel essential oil by an enzymolysis-steam distillation method, the proceedings of Jinhua occupational technology academy, 2013,13(6): 86-89) is carried out, and the yield is greatly improved by about 2 percent compared with the yield extracted by a simple steam distillation method. Although the extraction yield of the citrus essential oil can be improved by carrying out enzymolysis-assisted steam distillation extraction, pure enzyme is required, the use amount is high, and the production cost is undoubtedly increased. Some moulds can produce various polysaccharide hydrolases, including cellulase, hemicellulase, pectinase and the like, for example, the citrus peel is directly fermented and pretreated by the moulds, the moulds grow moderately, and the produced polysaccharide hydrolases hydrolyze the cell wall of the citrus peel, so that essential oil in cells is favorably released, and the extraction yield of the essential oil is improved. Therefore, the invention improves the process for extracting the orange peel essential oil by the steam distillation method, and adds the step of microbial fermentation pretreatment, thereby improving the extraction yield of the orange peel essential oil.
Disclosure of the invention
The invention aims to apply the microbial fermentation technology to the extraction of orange peel essential oil, and provides a method for extracting orange peel essential oil by using a microbial fermentation method, so that the extraction yield of the orange peel essential oil is obviously improved.
The technical scheme adopted by the invention is as follows:
the invention provides a method for extracting orange peel essential oil by using a microbial fermentation method, which comprises the following steps: inoculating mould spores into crushed fresh orange peel, and fermenting at 28-30 ℃ for 3-4 days to obtain orange peel fermentation product; extracting the orange peel fermentation product by a steam distillation method to obtain orange peel essential oil; the mould is Trichoderma reesei, Trichoderma longibrachiatum or Aspergillus niger.
Further, the fresh orange peel is obtained by pulverizing fresh orange (Citrus reticulate blanco) peel into particles with a particle size of 2-3 mm with a pulverizer.
Further, the mold is trichoderma reesei (trichoderma reesei) GIM 3.553, trichoderma longibrachiatum (trichoderma longibrachiatum) GIM 3.140 or aspergillus niger (aspergillus niger) GIM 3.546, preferably trichoderma reesei GIM 3.553.
Further, the inoculation amount of the mold spores is 3-5 × 10 in terms of the weight of fresh orange peel6Per gram.
Further, the Trichoderma reesei GIM 3.553The spore is added in the form of spore solution, and the spore solution is prepared by inoculating low-temperature preserved Trichoderma reesei GIM 3.553 spore in PDA plate culture medium (culture dish with diameter of 9 cm), culturing at 28-30 deg.C for 2-3 days, adding sterile water into the culture dish, suspending the spore with inoculating loop under stirring to obtain spore solution, preferably transferring the spore solution into sterile test tube, and adjusting spore concentration with sterile water to 3-5 × 108The final concentration of the PDA plate culture medium (potato sucrose agar culture medium) is that potato 200 g/L (boiling for 30min, filtering and remaining juice), sucrose 20 g/L, agar 20 g/L, the solvent is tap water, and the pH is natural (6.5 is actually measured).
The method for extracting essential oil from orange peel fermented products comprises the steps of adding deionized water, NaCl and a little glass beads into the orange peel fermented products, oscillating and shaking uniformly, then carrying out condensation reflux extraction by adopting a steam distillation method to obtain orange peel essential oil, specifically, placing the orange peel fermented products into a round-bottomed flask, adding deionized water, NaCl and a little glass beads, oscillating and shaking uniformly, placing the round-bottomed flask into an electric heating jacket, connecting a volatile oil extractor and a reflux condenser pipe, adding water from the upper end of the condenser pipe to the scale part of the volatile oil extractor, overflowing the volatile oil into the flask, connecting the condenser pipe to cool water, turning on the electric heating jacket to heat the round-bottomed flask until the water is boiled, reducing the power of the electric heating jacket to keep the micro-boiling state in the flask, distilling until the oil amount in the volatile oil extractor is not increased any more (3-5 h), stopping heating, placing for more than 1h, turning on a piston at the lower end of the volatile oil extractor, slowly discharging all the water, discharging the essential oil, discharging the added amount of the deionized water calculated by the weight of fresh orange peel, 2m L/g of NaCl, adding the added amount of the deionized water, and measuring the heated volatile oil by the weight of the glass beads, wherein the added amount is 1-3 percent and the volatile oil extractor is measured.
The trichoderma reesei GIM 3.553, trichoderma longibrachiatum GIM 3.140 and aspergillus niger GIM 3.546 are purchased from Guangdong province microorganism strain preservation center, and the addresses are as follows: building No. 59, building No. 5 of Jie No. 100 of the first Lianzhou city, Guangdong province; a zip code 510070.
Compared with the prior art, the invention has the following beneficial effects: before extracting essential oil from fresh citrus peel, fermentation pretreatment of mould, especially Trichoderma reesei GIM 3.553, is added. The trichoderma reesei GIM 3.553 moderately grows in the crushed orange peel to generate polysaccharide hydrolases such as cellulase, pectinase and xylanase, etc., destroy the cell wall structure, and help to release essential oil from cells, thereby improving the extraction yield of the essential oil. Compared with the conventional method without fermentation pretreatment, the extraction yield of the orange peel essential oil can be improved by more than 50%.
(IV) description of the drawings
FIG. 1 is a schematic view of a volatile oil extractor.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the citrus in the embodiment of the invention is citrus (citrus reticulate Banco) which is a Rutaceae (Rutaceae) plant, and the citrus peel is the outer skin of mature citrus fruits.
The method for extracting the orange peel essential oil is operated according to a 2204 volatile oil measuring method in the four parts (2015 edition) of Chinese pharmacopoeia.
EXAMPLE 1 screening of fermentation Strain
In order to screen a microbial strain suitable for fermenting orange peel to improve the extraction yield of essential oil, the invention adopts the following method to screen a fermentation strain.
(1) Aspergillus niger GIM 3.546, Trichoderma reesei (Trichoderma reesei) GIM 3.553, Aspergillus tubingensis GIM 3.577, Trichoderma longibrachiatum GIM 3.140 and Trichoderma koningi (Trichoderma koningi) GIM3.518 strain plated spores preserved at 4 ℃ were inoculated into fresh PDA plate medium, 3 d-incubated at 28 ℃ with 5m L sterile water in the plate culture, spores were suspended with an inoculating loop agitation, spore liquid was transferred into a sterile test tube, spore concentration was adjusted to 3 × 10 using sterile water8Counts/m L (count on hemocytometer).
(2)100g of fresh orange peel is crushed into particles with the particle size of 2-3 mm, the particles are added into a triangular flask with the particle size of 250-m L and subjected to dry heat sterilization at 160 ℃ for 2h, then 1m L of spore liquid of each mould prepared in the step (1) is inoculated, the mixture is stirred uniformly, the triangular flask is tied with eight layers of gauze, and the mixture is cultured for 3d at 28 ℃ to obtain the orange peel fermented product.
(3) Transferring all the orange peel fermentation products obtained in the step (2) into a 500-m L round-bottom flask, adding 200m L of deionized water, 4g of NaCl and 20 glass beads, shaking uniformly, placing the round-bottom flask into an electric heating jacket, connecting a volatile oil extractor and a reflux condenser (shown in figure 1), adding water from the upper end of the condenser to the scale part of the volatile oil extractor until the water overflows into the flask, switching on the condenser to cool the water, starting the electric heating jacket to heat until the water in the round-bottom flask boils, reducing the power of the electric heating jacket to keep the interior of the flask slightly boiling, distilling until the oil amount in the volatile oil extractor does not increase any more (3h), stopping heating, placing for more than 1h, starting a piston at the lower end of the volatile oil extractor, slowly discharging all the water, then discharging the orange peel essential oil, and weighing.
Under the same conditions, the same procedure was carried out with the non-inoculated strain as a control.
The yields of essential oils from orange peels fermented with different mold strains, and from the unfermented orange peels (control) by steam distillation are shown in table 1.
TABLE 1 orange peel essential oil extraction yield by fermentation with different strains
As can be seen from the data in Table 1, after the orange peel is fermented by Trichoderma reesei GIM 3.553, the extraction yield of the essential oil is 2.40%, compared with the unfermented control, the yield is improved to the maximum extent and reaches 37.1%, and the strain is selected as the fermentation strain for extracting the orange peel essential oil.
The plate culture medium is potato sucrose agar culture medium (PDA), and is prepared by cleaning potato, peeling, cutting into small pieces, weighing 200g, adding 1000m L tap water, boiling for 30min, filtering with 4 layers of gauze to remove residue, adding 1000m L filtrate, adding 20g sucrose and 20g agar, measuring pH naturally (actually measuring 6.5), heating until the agar is dissolved, placing in a triangular flask, sterilizing with high pressure steam at 121 deg.C for 15min, pouring into a sterile culture dish with diameter of 9cm, and cooling.
The Aspergillus niger GIM 3.546, the Trichoderma reesei GIM 3.553, the Aspergillus tubingensis GIM 3.577, the Trichoderma longibrachiatum GIM 3.140 and the Trichoderma koningii GIM3.518 strains are all from the microorganism strain preservation center of Guangdong province and are social-oriented shared strains. Guangdong province microbial strain preservation center address: building No. 59, building No. 5 of Jie No. 100 of the first Lianzhou city, Guangdong province; a zip code 510070.
The extraction yield of the orange peel essential oil is calculated according to the following formula:
the dried mass of 100g of fresh orange peel is 31.02g, so the dry weight yield of fresh orange peel in the following examples is 31.02%.
Example 2
The trichoderma reesei GIM 3.553 is applied to the extraction of orange peel essential oil and can be operated according to the following steps.
(1) Inoculating spore obtained by plate culture of Trichoderma reesei GIM 3.553 strain preserved at 4 deg.C into fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5m L sterile water into the plate culture, suspending the spore with inoculating loop under stirring, transferring the spore solution into sterile test tube, adjusting spore concentration to 4 × 10 with sterile water8And each m L.
(2)100g of fresh orange peel is crushed to the particle size of 2-3 mm, added into a triangular flask with the particle size of 250-m L and subjected to dry heat sterilization at 160 ℃ for 2h, then inoculated with 1m L of the spore liquid prepared in the step (1), stirred uniformly, sealed by eight layers of gauze in the triangular flask, and cultured at 28 ℃ for 4d to obtain the orange peel fermented product.
(3) Transferring all the orange peel fermentation products obtained in the step (2) into a 500-m L round-bottom flask, adding 200m L of deionized water, 6g of NaCl and 20 glass beads, shaking uniformly, placing the round-bottom flask into an electric heating jacket, connecting a volatile oil extractor and a reflux condenser tube, adding water from the upper end of the condenser tube to the scale part of the volatile oil extractor, overflowing the volatile oil extractor into the flask, switching on the condenser tube to cool water, starting the electric heating jacket to heat until the water in the round-bottom flask is boiled, reducing the power of the electric heating jacket to keep the flask slightly boiling, distilling until the volatile oil in the volatile oil extractor is not increased any more (3h), stopping heating, placing for more than 1h, starting a piston at the lower end of the volatile oil extractor to slowly discharge all the water, then discharging the orange peel essential oil, and weighing.
According to the steps, 0.754g of orange peel essential oil is extracted from 100g of fresh orange peel, the extraction yield is 2.53 percent (based on the dry weight of the orange peel), and the yield is improved by 44.6 percent compared with the yield (1.75 percent) of the conventional extraction without microbial fermentation in example 1.
Example 3
The trichoderma reesei GIM 3.553 is applied to the extraction of orange peel essential oil and can be operated according to the following steps.
(1) Inoculating spore obtained by plate culture of Trichoderma reesei GIM 3.553 strain preserved at 4 deg.C into fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5m L sterile water into the plate culture, suspending the spore with inoculating loop under stirring, transferring the spore solution into sterile test tube, adjusting spore concentration to 5 × 10 with sterile water8And each m L.
(2)200g of fresh orange peel, crushing to the particle size of about 2-3 mm, adding the crushed orange peel into a 500-m L triangular flask which is subjected to dry heat sterilization at 160 ℃ for 2h, inoculating 2m L of the spore liquid prepared in the step (1), uniformly stirring, tying the triangular flask with eight layers of gauze, and culturing at 28 ℃ for 4d to obtain the orange peel fermented product.
(3) Transferring all the orange peel fermentation products obtained in the step (2) into a round-bottom flask of 1-L, adding 400m of L of deionized water, 12g of NaCl and 30 glass beads, shaking uniformly, placing the round-bottom flask into an electric heating jacket, connecting a volatile oil extractor and a reflux condenser tube, adding water from the upper end of the condenser tube to the scale part of the volatile oil extractor until the water overflows into the flask, switching on the condenser tube to cool the water, starting the electric heating jacket to heat until the water in the round-bottom flask boils, reducing the power of the electric heating jacket to keep the flask slightly boiling, distilling until the oil amount in the volatile oil extractor is not increased any more (5h), stopping heating, placing for more than 1h, starting a piston at the lower end of the volatile oil extractor, slowly discharging all the water, then discharging the orange peel essential oil, and weighing.
According to the steps, 1.58g of orange peel essential oil is extracted from 200g of fresh orange peel, the extraction yield is 2.65% (based on the dry weight of the orange peel), and the yield is increased by 51.5% compared with the yield (1.75%) of the conventional extraction without microbial fermentation in example 1.
Example 4 application of Trichoderma reesei GIM 3.553 to extraction of essential oil of orange peel
The trichoderma reesei GIM 3.553 is applied to the extraction of orange peel essential oil and can be operated according to the following steps.
(1) Inoculating spore obtained by plate culture of Trichoderma reesei GIM 3.553 strain preserved at 4 deg.C into fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5m L sterile water into the plate culture, suspending the spore with inoculating loop under stirring, transferring the spore solution into sterile test tube, adjusting spore concentration to 5 × 10 with sterile water8And each m L.
(2)500g of fresh orange peel is crushed to the particle size of 2-3 mm, added into a triangular flask of 1-L and subjected to dry heat sterilization at 160 ℃ for 2h, then inoculated with 5m L of spore liquid prepared in the step (1), stirred uniformly, the triangular flask is tied with eight layers of gauze, and cultured at 28 ℃ for 4d to obtain the orange peel fermentation product.
(3) Transferring all orange peel fermentation products obtained in the step (2) into a round-bottom flask of 2-L, adding 1000m of L of deionized water, 30g of NaCl and 50 glass beads, shaking uniformly, placing the round-bottom flask into an electric heating jacket, connecting a volatile oil extractor and a reflux condenser, adding water from the upper end of the condenser to the scale part of the volatile oil extractor, and overflowing the volatile oil into the flask, switching on the condenser to cool water, starting the electric heating jacket to heat until the water in the round-bottom flask is boiled, reducing the power of the electric heating jacket to keep the flask slightly boiling, distilling until the oil amount in the volatile oil extractor is not increased any more (5h), stopping heating, placing for more than 1h, starting a piston at the lower end of the volatile oil extractor, slowly discharging all the water, then discharging the orange peel essential oil, and weighing.
According to the steps, 3.89g of orange peel essential oil is extracted from 200g of fresh orange peel, the extraction yield is 2.61% (based on the dry weight of the orange peel), and the yield is improved by 49.2% compared with the yield (1.75%) of the conventional extraction without microbial fermentation in example 1.
Claims (7)
1. A method for extracting orange peel essential oil by using a microbial fermentation method is characterized by comprising the following steps: inoculating mould spores into crushed fresh orange peel, and fermenting at 28-30 ℃ for 3-4 days to obtain orange peel fermentation product; extracting the orange peel fermentation product by a steam distillation method to obtain orange peel essential oil; the mould is Trichoderma reesei, Trichoderma longibrachiatum or Aspergillus niger.
2. A process according to claim 1, wherein the fresh citrus peel is prepared by comminuting fresh citrus (citrus reticulate Blanco) peel to produce particles having a size of from 2 to 3 mm.
3. The method according to claim 1, wherein the mould is Trichoderma reesei (Trichoderma reesei) GIM 3.553, Trichoderma longibrachiatum (Trichoderma longibrachiatum) GIM 3.140 or Aspergillus niger (Aspergillus niger) GIM 3.546.
4. A process according to claim 1, wherein the mould spores are inoculated in an amount of from 3 to 5 × 10 parts by weight of fresh orange peel6Per gram.
5. The method according to claim 3, wherein the Trichoderma reesei GIM 3.553 is added in the form of a spore fluid prepared by: inoculating the low-temperature preserved Trichoderma reesei GIM 3.553 spores to a PDA plate culture medium, culturing at the constant temperature of 28-30 ℃ for 2-3 d, adding sterile water into a culture dish, and suspending the spores by using an inoculating ring to obtain a spore solution.
6. The method of claim 1, wherein the essential oil is extracted from the orange peel fermentation by: adding deionized water, NaCl and a little glass beads into the orange peel fermentation product, shaking uniformly, and performing condensation reflux extraction by adopting a steam distillation method to obtain the orange peel essential oil.
7. The method according to claim 6, wherein the deionized water is added in an amount of 2m L/g by volume based on the weight of fresh orange peel, and the NaCl is added in an amount of 2-3% by mass based on the mass of the deionized water.
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