Plant essential oil extraction using specific enzyme combinations to optimize water vapor distillation process

Abstract

The present invention relates to a plant essential oil extraction method using a specific enzyme combination to optimize a steam distillation method, which can simultaneously maximize the activity of enzymes and maximize the distillation effect by using a combination of heat-resistant pectinase and / or heat-resistant cellulase, and can significantly increase the plant essential oil extraction amount by using a combination of different enzymes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the field of plant essential oil extraction, and more specifically to using a specific enzyme combination to optimize the plant essential oil extraction by a steam distillation method. Background Art

[0002] More than 80% of existing plant essential oils are extracted by steam distillation. According to literature records, the use of enzymes such as pectinase or cellulase can enhance the extraction of plant essential oils by steam distillation. However, the prior art of using steam distillation often uses a single type of general enzyme for extraction, and therefore has the following defects. First, the limited heat resistance of general pectinase or cellulase leads to limited extraction temperature, so it is impossible to maximize the activity of the enzyme and maximize the distillation effect at the same time. Second, a single type of enzyme has limited effect on enhancing the extraction of plant essential oils. Third, the antibacterial effect of the obtained plant essential oil is not strong. Summary of the invention

[0003] The technical problem to be solved by the present invention is that, in response to at least one of the above-mentioned defects of the prior art, a method for extracting plant essential oils using a specific enzyme combination to optimize the steam distillation method is provided. By adopting a combination of heat-resistant pectinase and / or heat-resistant cellulase, the enzyme activity and the distillation effect can be maximized at the same time, and the plant essential oil extraction amount can be significantly increased by combining different enzymes.

[0004] The technical solution adopted by the present invention to solve the technical problem is: constructing a plant essential oil extraction method using a specific enzyme combination to optimize the steam distillation method, comprising:

[0005] S1. expressing a gene fragment containing an exocellulase of cellulomonas, an endocellulase of cellulomonas, and a β-glucosidase of Thermotoga maritima in Escherichia coli, and inducing culture of the Escherichia coli to obtain an exocellulase of cellulomonas, an endocellulase of cellulomonas, and a β-glucosidase of Thermotoga maritima;

[0006] S2. After mixing the obtained exocellulase of cellulomonas, endocellulase of cellulomonas and β-glucosidase of Thermotoga maritima with the plant, essential oil is extracted by steam distillation at room temperature or after preheating, wherein the mixing ratio of the exocellulase of cellulomonas, endocellulase of cellulomonas and β-glucosidase of Thermotoga maritima is 1:1:1.

[0007] Another technical solution adopted by the present invention to solve the technical problem is to construct a plant essential oil extraction method using a specific enzyme combination to optimize the steam distillation method, comprising:

[0008] S1. expressing a gene fragment containing an exocellulase of cellulomonas, an endocellulase of cellulomonas and a pectinase of Thermotoga maritima in Escherichia coli, and inducing culture of the Escherichia coli to obtain an exocellulase of cellulomonas, an endocellulase of cellulomonas and a pectinase of Thermotoga maritima;

[0009] S2. After mixing the obtained exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritima with the plant, essential oil is extracted by steam distillation at room temperature or after preheating, wherein the mixing ratio of the exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritima is 1:1:1.

[0010] In the plant essential oil extraction method using a specific enzyme combination to optimize the steam distillation method described in the present invention, the preheating temperature is 50 to 60 degrees Celsius; the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes; the ratio of the enzyme composition to the plant is in the range of 1:1000 to 1:10.

[0011] In the plant essential oil extraction method using a specific enzyme combination to optimize the steam distillation method of the present invention, it further comprises:

[0012] S3. Using Candida lipase to treat the extracted plant essential oil.

[0013] Another technical solution adopted by the present invention to solve the technical problem is to construct a plant essential oil extraction method using a specific enzyme combination to optimize the steam distillation method, comprising:

[0014] S1. expressing a gene fragment containing a heat-resistant pectinase and / or a heat-resistant cellulase in a heat-resistant enzyme biological vector, and inducing and culturing the heat-resistant enzyme biological vector to obtain the heat-resistant pectinase and / or the heat-resistant cellulase;

[0015] S2. Mixing the obtained heat-resistant pectinase and / or heat-resistant cellulase with plants and extracting essential oils by steam distillation.

[0016] In the method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation of the present invention, the heat-resistant biological carrier includes Escherichia coli, yeast and Bacillus subtilis;

[0017] The thermostable cellulase comprises β-glucosidase from Thermotoga maritima, exocellulase from Cellulomonas and / or endocellulase from Cellulomonas; and / or

[0018] The thermostable pectinase includes pectinase from Thermotoga maritima and / or pectinase from Bacillus firmus.

[0019] In the method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation according to the present invention, step S2 further comprises:

[0020] S21, after mixing the β-glucosidase of Thermotoga maritima with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50 to 60 degrees Celsius;

[0021] S22, after mixing the exocellulase of Cellulomonas with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50 to 60 degrees Celsius;

[0022] S23, after mixing the exocellulase of Cellulomonas and the endocellulase of Cellulomonas with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50-60 degrees Celsius, wherein the ratio of the exocellulase of Cellulomonas to the endocellulase of Cellulomonas is 1:1;

[0023] S24, after mixing the pectinase of Thermotoga maritima with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50 to 60 degrees Celsius;

[0024] S25, after mixing the pectinase of Bacillus firmus with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50-60 degrees Celsius;

[0025] S26. Mixing the exocellulase of Cellulomonas, the endocellulase of Cellulomonas and the β-glucosidase of Thermotoga maritima with the plant, and extracting the essential oil by steam distillation at room temperature or after preheating to 50-60 degrees Celsius; wherein the ratio of the exocellulase of Cellulomonas, the endocellulase of Cellulomonas and the β-glucosidase of Thermotoga maritima is 1:1:1; or

[0026] S27. Mix the exocellulase of cellulomonas, the endocellulase of cellulomonas and the pectinase of Thermotoga maritima with the plant, and extract the essential oil by steam distillation at room temperature or after preheating to 50-60 degrees Celsius; wherein the ratio of the exocellulase of cellulomonas, the endocellulase of cellulomonas and the pectinase of Thermotoga maritima is 1:1:1.

[0027] In the method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation as described in the present invention, in step S2, the heating temperature is 100 degrees Celsius, the heating time is more than 30 minutes, and the ratio of the enzyme composition or the enzyme composition to the plant is in the range of 1:1000 to 1:10.

[0028] The method for extracting essential oils from plants by using a specific enzyme combination to optimize the steam distillation method described in the present invention further comprises: S3, treating the extracted essential oils with lipase.

[0029] Another technical solution adopted by the present invention to solve its technical problem is: constructing a plant essential oil, which is extracted according to any of the above-mentioned plant essential oil extraction methods using a specific enzyme combination to optimize the steam distillation method.

[0030] The method for extracting essential oils by using a specific enzyme combination to optimize the steam distillation method of the present invention can simultaneously maximize the activity of the enzymes and maximize the distillation effect by using a combination of heat-resistant pectinase and / or heat-resistant cellulase, and can significantly increase the amount of essential oil extracted by using a combination of different enzymes. Furthermore, by using Candida lipase to treat the extracted essential oils, the antibacterial ability of the essential oils can be effectively enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0032] Figure 1 The results of lavender essential oil extraction using different enzyme combinations under preheating and room temperature conditions are shown;

[0033] Figure 2A-2C The graph showing the antibacterial relationship between various essential oil concentrations and time of the lavender essential oil of the present invention in the Escherichia coli MIC antibacterial experiment is shown;

[0034] Figures 3A-3C The graph shows the antibacterial effect of the lavender essential oil of the present invention in the antibacterial experiment on Escherichia coli. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] The present invention expresses a gene fragment containing a heat-resistant pectinase and / or a heat-resistant cellulase in a heat-resistant enzyme biological carrier, and induces the culture of the heat-resistant enzyme biological carrier to obtain a heat-resistant pectinase and / or a heat-resistant cellulase, and then uses the obtained heat-resistant pectinase and heat-resistant cellulase to enhance the extraction amount of plant essential oil. In a preferred embodiment of the present invention, the heat-resistant biological carrier includes Escherichia coli, yeast and Bacillus subtilis; the heat-resistant cellulase includes β-glucosidase of Thermatogamaritima, exocellulase of Cellulomonas and / or endoglucanase of Cellulomonas. The heat-resistant pectinase includes pectinase of Thermatogamaritima and / or pectinase of Pectobacterium atrosepticum. In the present invention, the plant includes any suitable part of any plant from which essential oil can be extracted, such as flowers, leaves, roots, seeds, fruits, bark, resin, wood core, etc. of any plant. In a preferred embodiment of the present invention, the plant includes plant materials such as lavender, rose, orange peel, etc. In a preferred embodiment of the present invention, the aforementioned heat-resistant pectinase and / or heat-resistant cellulase include secretion signal peptide fragments to enhance the production efficiency of the enzyme. Here, any known secretion signal peptide fragment can be added to the aforementioned heat-resistant pectinase and heat-resistant cellulase by any method known in the prior art to enhance the production efficiency of the enzyme.

[0037] The present invention further tests the effects of different thermostable pectinases, thermostable cellulases and combinations thereof on enhancing the extraction of essential oils by steam distillation. The present invention further tests the effects of a preheating step on enhancing the extraction of essential oils by steam distillation. The present invention further tests the effects of different thermostable pectinases, thermostable cellulases and combinations thereof on enhancing the extraction of essential oils by steam distillation under a preheating step.

[0038] In a preferred embodiment of the present invention, gene fragments containing exocellulase of cellulomonas, endocellulase of cellulomonas and β-glucosidase of Thermotoga maritime are expressed in Escherichia coli, and the Escherichia coli is induced and cultured to obtain exocellulase of cellulomonas, endocellulase of cellulomonas and β-glucosidase of Thermotoga maritime. Preferably, the exocellulase of cellulomonas, endocellulase of cellulomonas and β-glucosidase of Thermotoga maritime can respectively include secretion signal peptide fragments to enhance the production efficiency of the enzyme. Subsequently, the exocellulase of cellulomonas, endocellulase of cellulomonas and β-glucosidase of Thermotoga maritime obtained are mixed in a ratio of 1:1:1, and after mixing with plants, essential oils are extracted by steam distillation at room temperature or after preheating. Here, various enzymes can be mixed first and then mixed with plants, or all raw materials can be directly mixed together. Preferably, the mixture is preheated to 50 to 60 degrees Celsius before steam distillation is used for essential oil extraction, more preferably preheated to 50 degrees Celsius. Here, the ratio of the enzyme composition to the plant is preferably in the range of 1:1000 to 1:10. More preferably, 100g of plant is adapted to 10g to 15g of crude enzyme composition, and most preferably, 100g of plant is adapted to 15g of crude enzyme composition. In a further preferred embodiment of the present invention, if a purified enzyme composition is used, its amount can be less, as mentioned above, the ratio of the enzyme composition to the plant can be in the range of 1:1000 to 1:10. Here, those skilled in the art can make adjustments in actual experiments according to the purity and activity of the selected enzymes, all of which fall within the scope of protection of the present invention.

[0039] Preferably, the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes. More preferably, the extracted plant essential oil can be treated with Candida lipase to improve the antibacterial ability of the plant essential oil.

[0040] In a preferred embodiment of the present invention, gene fragments containing exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritime are expressed in Escherichia coli, and the Escherichia coli is induced and cultured to obtain exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritime. Preferably, the exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritime can respectively include secretion signal peptide fragments to enhance the production efficiency of enzymes. Subsequently, the exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritime obtained are mixed with plants, and essential oil is extracted by steam distillation at room temperature or after preheating, wherein the mixing ratio of the exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritime is 1:1:1. Here, various enzymes can be mixed first and then mixed with plants, or all raw materials can be directly mixed together. Preferably, after the mixture is preheated to 50 to 60 degrees Celsius, more preferably to 50 degrees Celsius, steam distillation is used to extract the essential oil. Here, the ratio of the enzyme composition to the plant is preferably in the range of 1:1000 to 1:10. More preferably, 100g of the plant is adapted to 10g to 15g of the crude enzyme composition, and 100g of the plant is adapted to 15g of the crude enzyme composition. Preferably, the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes. More preferably, the extracted plant essential oil can be treated with Candida lipase to improve the antibacterial ability of the plant essential oil.

[0041] In a preferred embodiment of the present invention, only thermostable pectinase or thermostable cellulase can be selected. For example, gene fragments of β-glucosidase of Thermotoga maritime, exocellulase of Cellulomonas, endocellulase of Cellulomonas, pectinase of Thermotoga maritime or pectinase of Bacillus firmus can be expressed in Escherichia coli, yeast or Bacillus subtilis. Then, the Escherichia coli, yeast or Bacillus subtilis is induced and cultivated to obtain β-glucosidase of Thermotoga maritime, exocellulase of Cellulomonas, endocellulase of Cellulomonas, pectinase of Thermotoga maritime or pectinase of Bacillus firmus. These enzymes are then used separately or in combination for plant essential oil extraction.

[0042] For example, the β-glucosidase of Thermotoga marineus or the exocellulase of Cellulomonas can be mixed with the plant, and then the essential oil can be extracted by steam distillation at room temperature or after preheating to 50 to 60 degrees Celsius (more preferably 50 degrees Celsius). Here, the ratio of β-glucosidase or exocellulase to plant is preferably in the range of 1:1000 to 1:10. More preferably, 100g of plant is adapted to 15g of crude β-glucosidase or 15g of crude exocellulase. Preferably, the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes. More preferably, the extracted plant essential oil can be treated with Candida lipase to improve the antibacterial ability of the plant essential oil.

[0043] For another example, the exocellulase of Cellulomonas and the endocellulase of Cellulomonas can be mixed with plants, and then steam distillation is used to extract essential oils at room temperature or after preheating to 50 to 60 degrees Celsius (more preferably 50 degrees Celsius), wherein the ratio of the exocellulase of Cellulomonas to the endocellulase of Cellulomonas is 1:1. Here, the ratio range of the enzyme mixture to the plant is preferably 1:1000 to 1:10. More preferably, 100g of plant is adapted to 7.5g of crude exocellulase of Cellulomonas and 7.5g of crude endocellulase of Cellulomonas. Preferably, the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes. More preferably, the extracted plant essential oil can be treated with Candida lipase to improve the antibacterial ability of the plant essential oil.

[0044] For another example, the pectinase of Thermotoga marine or the pectinase of Bacillus firmus can be mixed with the plant, and then the essential oil can be extracted by steam distillation at room temperature or after preheating to 50-60 degrees Celsius (more preferably 50 degrees Celsius). The ratio of the pectinase of Thermotoga marine or the pectinase of Bacillus firmus to the plant is preferably in the range of 1:1000 to 1:10. More preferably, 100g of the plant is adapted to 15g of crude pectinase of Thermotoga marine or 15g of crude pectinase of Bacillus firmus. Preferably, the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes. More preferably, the extracted plant essential oil can be treated with Candida lipase to improve the antibacterial ability of the plant essential oil.

[0045] For another example, the exocellulase of Cellulomonas, the endocellulase of Cellulomonas and the β-glucosidase of Thermotoga maritime can be mixed with plants, and the essential oil can be extracted by steam distillation at room temperature or preheated to 50-60 degrees Celsius (more preferably 50 degrees Celsius); wherein the ratio of the exocellulase of Cellulomonas, the endocellulase of Cellulomonas and the β-glucosidase of Thermotoga maritime is 1:1:1. Here, the ratio of the enzyme mixture to the plant is preferably in the range of 1:1000 to 1:10. More preferably, 100g of plant is adapted to 5g of crude exocellulase of Cellulomonas, 5g of crude endocellulase of Cellulomonas and 5g of crude β-glucosidase of Thermotoga maritime. Preferably, the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes. More preferably, the extracted plant essential oil can be treated with Candida lipase to improve the antibacterial ability of the plant essential oil.

[0046] For another example, the exocellulase of Cellulomonas, the endocellulase of Cellulomonas and the pectinase of Thermotoga marine can be mixed with plants, and the essential oil can be extracted by steam distillation at room temperature or after preheating to 50-60 degrees Celsius (more preferably 50 degrees Celsius); wherein the ratio of the exocellulase of Cellulomonas, the endocellulase of Cellulomonas and the pectinase of Thermotoga marine is 1:1:1. Here, the ratio of the enzyme mixture to the plant is preferably in the range of 1:1000 to 1:10. More preferably, 100g of plant is adapted to 5g of crude exocellulase of Cellulomonas, 5g of crude endocellulase of Cellulomonas and 5g of crude pectinase of Thermotoga marine. Preferably, the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes. More preferably, the extracted plant essential oil can be treated with Candida lipase to improve the antibacterial ability of the plant essential oil.

[0047] The present invention also relates to plant essential oils extracted by the plant essential oil extraction method using the aforementioned specific enzyme combination to optimize the steam distillation method. For example, the plant essential oil can be lavender essential oil, rose essential oil, orange peel essential oil, etc.

[0048] The method for extracting essential oils by using a specific enzyme combination to optimize the steam distillation method of the present invention can simultaneously maximize the activity of the enzymes and maximize the distillation effect by using a combination of heat-resistant pectinase and / or heat-resistant cellulase, and can significantly increase the amount of essential oil extracted by using a combination of different enzymes. Furthermore, by using Candida lipase to treat the extracted essential oils, the antibacterial ability of the essential oils can be effectively enhanced.

[0049] The plant essential oil extraction method using a specific enzyme combination to optimize the steam distillation method of the present invention is described below in conjunction with specific embodiments. In the present invention, various biological materials and reagents used can be purchased from the market, such as Sigma and GenScript in the United States.

[0050] 1. Enzyme extraction

[0051] First, a gene fragment containing a thermostable pectinase and / or a thermostable cellulase is expressed in Escherichia coli, yeast or Bacillus subtilis. Here, any suitable gene fragment of a thermostable pectinase and / or a thermostable cellulase known in the art can be used, and the expression of these gene fragments is also known in the art. In order not to obscure the inventive point of the present invention, it will not be described here.

[0052] Then, the Escherichia coli, yeast or Bacillus subtilis is induced and cultured to obtain the desired thermostable pectinase and / or thermostable cellulase. We use Escherichia coli for the following description.

[0053] Add the starter culture (E. coli producing pectinase and / or cellulase) to Luria-Bertani medium and shake at 250 rpm at 37°C for 2.5 hours until the optical density (OD) 600 = 0.5-0.6. Use 0.5M isopropyl-β-D-thiogalactoside (PTG induction) (1 / 1000, v / v) for 6 hours to culture the bacteria. Take 50 ml of the culture as the bacterial culture to be tested. Centrifuge at a centrifugal rate of >4000 rpm for 10 minutes. Discard the supernatant and wash the cell pellet thoroughly with PBS (45 ml for 1L culture). Clean the ultrasonic generator tip with ethanol and distilled water. Pour the mixture into a steel ultrasonic treatment cup, immerse the ultrasonic pipette tip in the liquid, and place it slightly on the bottom of the cup (do not touch the bottom or place it around the surface of the liquid). 40% sonication of the cell mixture, repeat the following operation 5 times: 5 seconds on, 5 seconds off, and then stand for 1 minute within 1 minute. The cup is then placed in ice (it must be covered around the cup to absorb heat). The sonicated solution is stored in 50 ml tubes in a -4C refrigerator. The enzyme extract can be used within the next few days.

[0054] The applicant repeated the above-mentioned induction culture process using yeast and Bacillus subtilis, and the results were the same, which will not be repeated here.

[0055] 2. Extraction of essential oils by steam distillation

[0056] The obtained heat-resistant pectinase and / or heat-resistant cellulase are mixed with plants and essential oil is extracted by steam distillation. We take different heat-resistant enzymes or enzyme combinations to extract lavender essential oil as an example to illustrate as follows.

[0057] 1. Assemble the distillation apparatus. Specifically, connect a round bottom flask (1-2L) to the condenser. Install a thermometer on the top of the flask to monitor the temperature. Make sure the condenser is connected to a cooling water source so that cold water circulates through the condenser jacket. Place a collection flask at the end of the condenser to collect the distillate.

[0058] 2. Mix the raw materials. Specifically, weigh and prepare 100 grams of lavender flowers. Dried or fresh flowers can be used, and fresh flowers are preferred because they usually contain a higher content of essential oils. Place the lavender flowers in a round-bottom flask. Add 500-1000 mL of water to the round-bottom flask to ensure that the flowers are submerged in water. Add different enzymes, enzyme combinations or controls in the proportions of Examples 1 to 9 to the round-bottom flask to test the effects of different thermostable enzymes or enzyme combinations on the extraction of essential oils by steam distillation. The experimental results are shown in FIG. Figure 1 shown.

[0059] Example 1 (P1): 15 g of crude β-glucosidase from Thermotoga maritima;

[0060] Example 2 (P6): 15 g crude exocellulase from Cellulomonas spp.;

[0061] Example 3 (P7): 7.5 g of crude cellulomonas exocellulase + 7.5 g of crude cellulomonas endocellulase;

[0062] Example 4 (P3): 15 g crude pectinase from Thermotoga maritima;

[0063] Example 5 (P5): 15 g of crude pectinase from Bacillus firmus;

[0064] Example 6 (P1+P7): 5g of crude β-glucosidase from Thermotoga maritima + 5g of crude exocellulase from Cellulomonas + 5g of crude endocellulase from Cellulomonas;

[0065] Example 7 (P3+P7): 5g crude pectinase from Thermotoga maritima + 5g crude exocellulase from Cellulomonas + 5g crude endocellulase from Cellulomonas;

[0066] Example 8 (PET11a): 15 g PET11a;

[0067] Example 9 (water): 15 g water.

[0068] 3. Heating distillation. Specifically, preheat the mixed raw materials to 50 degrees Celsius, then heat the flask with a heating jacket or Bunsen burner. Slowly heat the water to boiling (about 100°C). The steam generated will pass through the lavender flowers and take away the essential oils for more than 30 minutes. In order to verify the effect of preheating on the extraction of essential oils by steam distillation, a control experiment was conducted under the same conditions without preheating and directly heated at room temperature. The experimental results are as follows Figure 1 shown.

[0069] 4. Condensation. Specifically, the steam carries the volatile lavender essential oil through the condenser, where it cools and liquefies into a mixture of water and oil. Make sure the cold water in the condenser flows steadily to keep the system cool.

[0070] 5. Obtaining essential oil. Specifically, the distillate (including a mixture of water and oil) will be collected in a collection bottle. Continue the distillation process until no oil is visible. Transfer the distillate to a separatory funnel and let it stand for a while. The essential oil will float on the water surface due to its low density. Carefully drain the water layer (floral water) from the bottom and collect the lavender essential oil on the upper layer.

[0071] 6. Antibacterial treatment. Specifically, the extracted lavender essential oil was treated with Candida rugosa lipase, and the applicant surprisingly found that the treated essential oil had antibacterial ability. Any known treatment method in the art can be used, which will not be described here.

[0072] Figure 2A-2C The graph showing the antibacterial relationship between various essential oil concentrations and time of the lavender essential oil of the present invention in the E. coli MIC antibacterial experiment is shown. Figures 3A-3C The graph shows the antibacterial effect of the lavender essential oil of the present invention in the antibacterial experiment on Escherichia coli.

[0073] Combine the following Figure 1-3C The various essential oils of the present invention and their antibacterial effects are described below.

[0074] like Figure 1 As shown, whether it is heated distillation at room temperature or preheated to 50 degrees Celsius before heating distillation, the essential oil obtained by using PET11a and water is less than 1 ml, and preheating does not significantly enhance the extraction of essential oil. When using a thermostable enzyme or enzyme combination, no matter which thermostable enzyme or enzyme combination is used, the essential oil yield obtained by heating distillation after preheating to 50 degrees Celsius is higher than that obtained by heating distillation at room temperature, which fully demonstrates that the preheating operation can significantly increase the yield of essential oil.

[0075] See also Figure 1It can be seen that in Example 3, a heat-resistant enzyme composition of 7.5g of exocellulase from Cellulomonas + 7.5g of endocellulase from Cellulomonas was used, and when the mixture was preheated to 50 degrees Celsius and then heated and distilled, the yield was about 1.7ml, which was significantly increased compared to when the heat-resistant enzyme composition was not used. In Example 6 (P1+P7), a heat-resistant enzyme composition of 5g of β-glucosidase from Thermotoga maritima + 5g of exocellulase from Cellulomonas + 5g of endocellulase from Cellulomonas was used, and when the mixture was preheated to 50 degrees Celsius and then heated and distilled, the yield was about 1.9ml, which was nearly doubled compared to when the heat-resistant enzyme composition was not used. In Example 7 (P3+P7), a heat-resistant enzyme composition of 5g of pectinase from Thermotoga maritima + 5g of exocellulase from Cellulomonas + 5g of endocellulase from Cellulomonas was used, and when it was preheated to 50 degrees Celsius and then heated and distilled, the output was about 2ml, which was more than doubled compared with when no heat-resistant enzyme composition was used.

[0076] See also Figure 1 It can be seen that in Examples 1, 2, 4 and 5, when a single heat-resistant enzyme is used, the essential oil yield obtained by heating and distilling after preheating to 50 degrees Celsius is higher than that obtained by heating and distilling at room temperature, especially for the β-glucosidase of Thermotoga maritima in Example 1 (P1) and the pectinase of Thermotoga maritima in Example 4 (P3), the preheating operation can significantly increase the essential oil yield. However, for the exocellulase of Cellulomonas in Examples 2 (P6) and 5 (P5), the preheating operation has limited effect on increasing the essential oil yield.

[0077] comprehensive Figure 1It can be seen that by using a thermostable enzyme composition, especially a mixture of exocellulase from Cellulomonas, endocellulase from Cellulomonas and β-glucosidase from Thermotoga maritima in a ratio of 1:1:1, or exocellulase from Cellulomonas, endocellulase from Cellulomonas and pectinase from Thermotoga maritima in a ratio of 1:1:1, and extracting essential oil by steam distillation after preheating, the yield of essential oil can be significantly increased. This is an unexpected beneficial effect for essential oil extraction by steam distillation. In the conventional steam distillation method for extracting essential oils, without adding chemical reagents, 100 g of lavender flowers can basically only extract less than 1 ml of lavender essential oil, and it is difficult to improve it through general engineering technology (see Cannon, JB, Cantrell, CL, Astatkie, T. and Zheljazkov, VD, 2013. Modification of yield and composition of essential oils by distillation time. Industrial Crops and Products, 41, pp. 214-220; Chen, X., Guo, D., Gong, X., Wan, N. and Wu, Z., 2023. Optimization of Steam Distillation Process for Volatile Oils from Forsythia suspensa and Lonicera japonica according to the Concept of Quality by Design. Separations, 10 (1), p. 25.). The present invention, by adopting a heat-resistant enzyme combination, can increase the yield of essential oil by more than double without any chemical reagents, thereby achieving unexpected beneficial effects.

[0078] Figure 2A-2C The graph showing the antibacterial relationship between various essential oil concentrations and time of the lavender essential oil of the present invention in the E. coli MIC antibacterial experiment is shown. Figure 2A This is a schematic diagram of the antibacterial results of the Escherichia coli MIC antibacterial experiment with different concentrations of untreated lavender essential oil. Figure 2B The figure shows the antibacterial results of the Escherichia coli MIC antibacterial experiment using lavender essential oil of different concentrations treated with water. Figure 2C The following is a schematic diagram of the antibacterial results of the MIC antibacterial experiment of Escherichia coli treated with Candida lipase using different concentrations of lavender essential oil. Figure 2A-2C It can be clearly seen that OD600 shows the amount of bacteria. Figure 2CThe most obvious bar graph is that the bacterial growth is significantly slower (1ug / ml). Therefore, it can be seen that the antibacterial effect of lavender essential oil is significantly improved after treatment with Candida lipase.

[0079] Figures 3A-3C The graph shows the antibacterial effect of the lavender essential oil of the present invention in the antibacterial experiment on Escherichia coli. Figure 3A For the blank group, Figure 3B 1ug PET11a, Figure 3C This is 1ug of lavender essential oil treated with Candida lipase. Figures 3A-3C It can be seen that after Candida lipase treatment, the antibacterial effect of lavender essential oil was significantly improved.

[0080] It should be noted that the lavender essential oil used in this experiment was obtained by preheating to 50 degrees Celsius and then heating and distilling the thermostable enzyme combination in Example 7. Of course, the antibacterial effects obtained by the thermostable enzymes or thermostable enzyme combinations in the remaining embodiments of the present invention are the same, and will not be repeated here.

[0081] In a further preferred embodiment of the present invention, the aforementioned experiment was repeated on other plant materials such as rose and orange peel, and similar results were obtained, indicating that the aforementioned plant essential oil extraction method of the present invention using a specific enzyme combination to optimize the steam distillation method can still significantly increase the amount of plant essential oil extracted on other plant materials.

[0082] The method for extracting essential oils by using a specific enzyme combination to optimize the steam distillation method of the present invention can simultaneously maximize the activity of the enzymes and maximize the distillation effect by using a combination of heat-resistant pectinase and / or heat-resistant cellulase, and can significantly increase the amount of essential oil extracted by using a combination of different enzymes. Furthermore, by using Candida lipase to treat the extracted essential oils, the antibacterial ability of the essential oils can be effectively enhanced.

[0083] Although the present invention is described by specific embodiments, it should be understood by those skilled in the art that various changes and equivalent substitutions may be made to the present invention without departing from the scope of the present invention. In addition, various modifications may be made to the present invention for specific situations or materials without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed, but should include all implementation methods falling within the scope of the claims of the present invention.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation, characterized in that: include: S1. expressing a gene fragment containing an exocellulase of cellulomonas, an endocellulase of cellulomonas, and a β-glucosidase of Thermotoga maritima in Escherichia coli, and inducing culture of the Escherichia coli to obtain an exocellulase of cellulomonas, an endocellulase of cellulomonas, and a β-glucosidase of Thermotoga maritima; S2. After mixing the obtained exocellulase of cellulomonas, endocellulase of cellulomonas and β-glucosidase of Thermotoga maritima with the plant, essential oil is extracted by steam distillation at room temperature or after preheating, wherein the mixing ratio of the exocellulase of cellulomonas, endocellulase of cellulomonas and β-glucosidase of Thermotoga maritima is 1:1:

1.

2. A method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation, characterized in that: include: S1. expressing a gene fragment containing an exocellulase of cellulomonas, an endocellulase of cellulomonas and a pectinase of Thermotoga maritima in Escherichia coli, and inducing culture of the Escherichia coli to obtain an exocellulase of cellulomonas, an endocellulase of cellulomonas and a pectinase of Thermotoga maritima; S2. After mixing the obtained exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritima with the plant, essential oil is extracted by steam distillation at room temperature or after preheating, wherein the mixing ratio of the exocellulase of cellulomonas, endocellulase of cellulomonas and pectinase of Thermotoga maritima is 1:1:

1.

3. The method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation according to claim 1 or 2, characterized in that: The preheating temperature is 50 to 60 degrees Celsius; the heating temperature of the steam distillation method is 100 degrees Celsius, and the heating time is more than 30 minutes; the ratio of the enzyme composition to the plant is in the range of 1:1000 to 1:

10.

4. The method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation according to claim 1 or 2, characterized in that: Further including: S3. Using Candida lipase to treat the extracted plant essential oil.

5. A method for extracting essential oils from plants by using a specific enzyme combination to optimize steam distillation, characterized in that: include: S1. expressing a gene fragment containing a heat-resistant pectinase and / or a heat-resistant cellulase in a heat-resistant enzyme biological vector, and inducing and culturing the heat-resistant enzyme biological vector to obtain the heat-resistant pectinase and / or the heat-resistant cellulase; S2. Mixing the obtained heat-resistant pectinase and / or heat-resistant cellulase with plants and extracting essential oils by steam distillation.

6. The method for extracting essential oils from plants by using a specific enzyme combination to optimize steam distillation according to claim 5, characterized in that: The heat-resistant biological carriers include Escherichia coli, yeast and Bacillus subtilis; The thermostable cellulase comprises β-glucosidase from Thermotoga maritima, exocellulase from Cellulomonas and / or endocellulase from Cellulomonas; and / or The thermostable pectinase includes pectinase from Thermotoga maritima and / or pectinase from Bacillus firmus.

7. The method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation according to claim 6, characterized in that: The step S2 further comprises: S21, after mixing the β-glucosidase of Thermotoga maritima with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50 to 60 degrees Celsius; S22, after mixing the exocellulase of Cellulomonas with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50 to 60 degrees Celsius; S23, after mixing the exocellulase of Cellulomonas and the endocellulase of Cellulomonas with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50-60 degrees Celsius, wherein the ratio of the exocellulase of Cellulomonas to the endocellulase of Cellulomonas is 1:1; S24, after mixing the pectinase of Thermotoga maritima with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50 to 60 degrees Celsius; S25, after mixing the pectinase of Bacillus firmus with the plant, extracting the essential oil by steam distillation at room temperature or after preheating to 50-60 degrees Celsius; S26. Mixing the exocellulase of Cellulomonas, the endocellulase of Cellulomonas and the β-glucosidase of Thermotoga maritima with the plant, and extracting the essential oil by steam distillation at room temperature or after preheating to 50-60 degrees Celsius; wherein the ratio of the exocellulase of Cellulomonas, the endocellulase of Cellulomonas and the β-glucosidase of Thermotoga maritima is 1:1:1; or S27. Mix the exocellulase of cellulomonas, the endocellulase of cellulomonas and the pectinase of Thermotoga maritima with the plant, and extract the essential oil by steam distillation at room temperature or after preheating to 50-60 degrees Celsius; wherein the ratio of the exocellulase of cellulomonas, the endocellulase of cellulomonas and the pectinase of Thermotoga maritima is 1:1:

1.

8. The method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation according to claim 7, characterized in that: In step S2, the heating temperature is 100 degrees Celsius, the heating time is more than 30 minutes, and the ratio of the enzyme composition or the enzyme composition to the plant is in the range of 1:1000 to 1:

10.

9. The method for extracting essential oils from plants using a specific enzyme combination to optimize steam distillation according to any one of claims 5 to 8, characterized in that: Further including: S3. Using lipase to treat the extracted plant essential oil.

10. The plant essential oil extracted by the plant essential oil extraction method using a specific enzyme combination to optimize the steam distillation method according to any one of claims 1 to 9.

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