A method for extracting camphor essential oil by fermentation using a mixed enzyme preparation

Abstract

This invention provides a method for preparing borneol essential oil by fermentation extraction using a mixed enzyme preparation, belonging to the field of plant essential oil technology. The method involves using borneol-type borneol-based borneol... Cinnamomum burmannii ​ The leaves of *C. var. Borneol* were microwave-dried, mixed, and pulverized to obtain leaf powder. A compound enzyme was added for enzymatic hydrolysis using a citric acid-sodium citrate buffer solution as the solvent. After hydrolysis, the enzyme was inactivated by heating, followed by fermentation with a compound microbial agent. The fermentation product was extracted with a mixed solution of ionic liquid and acetonitrile, then extracted with petroleum ether and refluxed with anhydrous ethanol. The organic solvent was removed by vacuum drying to obtain camphor-type *C. var. Borneol* leaf essential oil. This camphor essential oil has a high yield and can significantly reduce the release of TNF-α and IL-1β inflammatory mediators, alleviate inflammatory responses, inhibit inflammatory processes, and achieve therapeutic or preventative effects against chronic pharyngitis.

Description

Technical Field

[0001] This invention relates to the field of plant essential oil technology, specifically to a method for preparing camphor essential oil by fermentation extraction using a mixed enzyme preparation. Background Technology

[0002] Chronic pharyngitis is a diffuse chronic inflammation of the pharyngeal mucosa and submucosa, often part of chronic upper respiratory tract inflammation. It is mainly caused by poor habits, occupational factors, living environment, physical factors, genetic factors, and inflammation itself. As a common disease in otolaryngology, chronic pharyngitis accounts for 10% to 12% of all throat diseases, occurring mostly in adults, with a higher incidence in urban areas, especially among teachers and singers. Patients often experience a foreign body sensation, itching, burning, dryness, or mild pain in the throat. Due to the stimulation of surrounding inflammation, the pharyngeal mucosa is often congested and thickened, appearing dark red.

[0003] Borneol-type incense ( Cinnamomum burmannii chvar. borneol ) is a chemical strain of Cinnamomum cassia. Natural dextrorotatory borneol is extracted from its leaves. Dextrorotatory borneol is also known as camphor or borneol. Camphor is a precious and rare medicinal material, a high-grade fragrance and an important chemical raw material, which is widely used in the pharmaceutical, fragrance, cosmetic and food industries. Existing technical reports include the use of papain, cellulase, hemicellulase, pectinase, xylanase, amylase, etc., to treat camphor-type osmanthus ( Cinnamomum burmannii Studies have been conducted on the treatment of leaves of Chvar. Borneol, which has effectively improved the extraction of essential oils, but research on changes in essential oil composition and functional improvements is lacking.

[0004] This invention investigates the application of enzymatic hydrolysis and fermentation technologies in the preparation of camphor oil. Based on the screening of a series of flavor hydrolytic enzymes and fermentation strains, a complex enzyme consisting of xylanase, hemicellulase, lipase, and pectinase, as well as a complex microbial agent composed of Saccharomyces cerevisiae, Bacillus licheniformis, and Lactobacillus acidophilus, was selected. For borneol-type osmanthus ( Cinnamomum burmannii The leaves of chvar. Borneol were processed to improve the yield and purity of borneol-type borneol essential oil and enhance its functional activity. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is the low extraction rate and the insignificant functional activity of the prior art.

[0006] The technical solution of this invention is achieved as follows: Borneol-type incense ( Cinnamomum burmanniiAfter microwave drying, the leaves of *C. var. Borneol* were mixed and pulverized to obtain leaf powder. A compound enzyme was added for enzymatic hydrolysis using a citric acid-sodium citrate buffer solution as a solvent. After enzymatic hydrolysis, the enzyme was heated to inactivate the enzyme. Then, the product was fermented with a compound microbial agent. The fermentation product was extracted with a mixed solution of ionic liquid and acetonitrile, then extracted with petroleum ether and refluxed with anhydrous ethanol. After vacuum drying to remove organic solvents, the essential oil of *C. var. Borneol* leaves was obtained.

[0007] As a further improvement to the present invention, the following steps are included: S1 Pretreatment: Prepare camphor-type incense ( Cinnamomum burmannii The leaves of Chvar. Borneol were microwave-dried, mixed, and pulverized. S2 Enzymatic hydrolysis: The solid obtained in step S1 is added to a citric acid-sodium citrate buffer solution, a compound enzyme is added for enzymatic hydrolysis, the enzyme is inactivated, the enzymatic hydrolysis product is obtained, filtered under reduced pressure, and dried under vacuum. S3 Fermentation: Add activated Saccharomyces cerevisiae, Bacillus licheniformis, and Lactobacillus acidophilus to the enzymatic hydrolysis product of step S2, ferment, sterilize, filter, and obtain the fermentation product; S4 Ionic liquid extraction: The fermentation product obtained in step S4 is added to the ionic liquid-acetonitrile mixed solution, CO2 is slowly introduced, the mixture is shaken, the CO2 is stopped, and a highly extractable ionic liquid-acetonitrile mixed solution is obtained. The mixture is allowed to stand and separate into layers, the water layer is removed, the solution is dried, and the solution is filtered to obtain the extract. S5 Organic solvent extraction: The extract from step S4 was refluxed with petroleum ether, filtered, the filtrate was concentrated by rotary evaporator, vacuum dried, refluxed with anhydrous ethanol, filtered, the filtrate was evaporated to dryness and then vacuum dried to remove organic solvent, yielding camphor-type Aristolochia leaf essential oil.

[0008] As a further improvement of the present invention, the complex enzyme in step S2 is composed of xylanase, hemicellulase, lipase and pectinase, with an enzyme activity ratio of 1.5:2:0.8:1. As a further improvement of the present invention, the concentration of the citric acid-sodium citrate buffer solution in step S2 is 0.8 mol / L; In another preferred embodiment of the present invention, the application of a complex enzyme composed of xylanase, hemicellulase, lipase and pectinase in improving the yield of camphor-type cinnamon essential oil is also provided. In another preferred embodiment of the present invention, the use of the borneol-type cinnamon leaf essential oil in the preparation of products for the prevention or treatment of chronic pharyngitis is also provided. The technical solution of the present invention has the following effects: 1. The compound enzyme treatment prepared by this invention can improve the yield of borneol-type borneol essential oil, while also increasing the content of the main aroma substances, reducing the content of harmful substances, and improving the quality of borneol essential oil.

[0009] 2. The borneol-type cinnamon essential oil prepared by compound enzyme treatment can reduce the release of inflammatory mediators such as TNF-α and IL-1β, thereby alleviating the inflammatory response and inhibiting the inflammatory process, thus achieving the treatment or prevention of chronic pharyngitis. Attached Figure Description

[0010] Figure 1 Effects of different compound enzyme treatments on the yield of camphor-type borneol essential oil Figure 2 Changes in the main components of camphor-type cinnamon bark essential oil after treatment with different compound enzymes Figure 3 Effects of compound enzyme-assisted extraction of camphor-type cinnamon essential oil on TNF-α in rats with chronic pharyngitis Figure 4 Effects of compound enzyme-assisted extraction of camphor-type cinnamon bark essential oil on IL-1β in rats with chronic pharyngitis Detailed Implementation

[0011] The methods and applications of the present invention have been described through preferred embodiments. Those skilled in the art will be able to make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the technology of the present invention.

[0012] Example 1: Preparation of essential oil from borneol-type Aristolochia leaves using compound enzyme-assisted extraction Group A: Pectinase + Xylanase + Lipase S1 Pretreatment: Dry borneol-type incense ( Cinnamomum burmannii The leaves of chvar. Borneol were crushed and passed through a 60-mesh sieve to obtain camphor-type cinnamon leaf powder; S2 Enzymatic hydrolysis: Weigh 25g of camphor-type Cinnamomum cassia leaf powder prepared in step S1, add 100U / g pectinase, 150U / g xylanase powder and 80U / g lipase powder, use 0.8 mol / L citrate-sodium citrate buffer solution as solvent, adjust pH to 5.5, enzymatically hydrolyze at 45℃ for 4 hours, heat to 100℃ to inactivate enzyme for 5 minutes, filter, and retain the solid part; S3 Fermentation: Activated Saccharomyces cerevisiae, Bacillus licheniformis, and Lactobacillus acidophilus were added to the enzymatic hydrolysis product of step S2, with inoculum amounts of 1.5%, 2%, and 1.5%, respectively. The product was fermented at 37°C for 24 hours, sterilized with ultraviolet light, filtered, and the fermentation product was obtained. S4 Ionic Liquid Extraction: Add 20 parts by weight of the ionic liquid-acetonitrile mixed solution to the fermentation product of step S3, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2, and obtain a highly extractable ionic liquid-acetonitrile mixed solution. Allow it to stand and separate the layers, separate the liquids, remove the water layer, wash, dry, filter, and obtain the extract. S5 Organic solvent extraction: The extract from step S4 was refluxed with petroleum ether for 1.5 h, filtered, concentrated by rotary evaporator, refluxed with anhydrous ethanol at 60 °C for 1 h, filtered, the filtrate was evaporated to dryness and then vacuum dried to remove organic solvent, thus obtaining the essential oil of Aralia elata leaf of group A (borneol type).

[0013] Group B: Pectinase + Xylanase + Hemicellulase S1 Pretreatment: Dry borneol-type incense ( Cinnamomum burmannii The leaves of chvar. Borneol were crushed and passed through a 60-mesh sieve to obtain camphor-type cinnamon leaf powder; S2 Enzymatic hydrolysis: Weigh 25g of camphor-type Cinnamomum cassia leaf powder prepared in step S1, add 100U / g pectinase, 150U / g xylanase powder and 200U / g hemicellulase powder, use 0.8 mol / L citrate-sodium citrate buffer solution as solvent, adjust pH to 5.5, enzymatically hydrolyze at 45℃ for 4 hours, heat to 100℃ to inactivate enzyme for 5 minutes, filter, and retain the solid part; S3 Fermentation: Activated Saccharomyces cerevisiae, Bacillus licheniformis, and Lactobacillus acidophilus were added to the enzymatic hydrolysis product of step S2, with inoculum amounts of 1.5%, 2%, and 1.5%, respectively. The product was fermented at 37°C for 24 hours, sterilized with ultraviolet light, filtered, and the fermentation product was obtained. S4 Ionic Liquid Extraction: Add 20 parts by weight of the ionic liquid-acetonitrile mixed solution to the fermentation product of step S3, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2, and obtain a highly extractable ionic liquid-acetonitrile mixed solution. Allow it to stand and separate the layers, separate the liquids, remove the water layer, wash, dry, filter, and obtain the extract. S5 Organic solvent extraction: The extract from step S4 was refluxed with petroleum ether for 1.5 h, filtered, concentrated by rotary evaporator, refluxed with anhydrous ethanol at 60 °C for 1 h, filtered, the filtrate was evaporated to dryness and then vacuum dried to remove organic solvent, thus obtaining the essential oil of Aralia elata leaf of group A (borneol type).

[0014] Group C: Xylanase + Hemicellulase + Lipase S1 Pretreatment: Dry borneol-type incense ( Cinnamomum burmannii The leaves of chvar. Borneol were crushed and passed through a 60-mesh sieve to obtain camphor-type cinnamon leaf powder; S2 Enzymatic hydrolysis: Weigh 25g of camphor-type Cinnamomum cassia leaf powder prepared in step S1, add xylanase 150U / g, hemicellulase 200U / g and lipase 80U / g powder, use 0.8 mol / L citrate-sodium citrate buffer solution as solvent, adjust pH to 5.5, enzymatically hydrolyze at 45℃ for 4 hours, heat to 100℃ to inactivate enzyme for 5 minutes, filter, and retain the solid part; S3 Fermentation: Activated Saccharomyces cerevisiae, Bacillus licheniformis, and Lactobacillus acidophilus were added to the enzymatic hydrolysis product of step S2, with inoculum amounts of 1.5%, 2%, and 1.5%, respectively. The product was fermented at 37°C for 24 hours, sterilized with ultraviolet light, filtered, and the fermentation product was obtained. S4 Ionic Liquid Extraction: Add 20 parts by weight of the ionic liquid-acetonitrile mixed solution to the fermentation product of step S3, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2, and obtain a highly extractable ionic liquid-acetonitrile mixed solution. Allow it to stand and separate the layers, separate the liquids, remove the water layer, wash, dry, filter, and obtain the extract. S5 Organic solvent extraction: The extract from step S4 was refluxed with petroleum ether for 1.5 h, filtered, concentrated by rotary evaporator, refluxed with anhydrous ethanol at 60 °C for 1 h, filtered, the filtrate was evaporated to dryness and then vacuum dried to remove organic solvent, thus obtaining the essential oil of Aralia elata leaf of group A (borneol type).

[0015] Group D: Xylanase + Hemicellulase + Lipase + Pectinase S1 Pretreatment: Dry borneol-type incense ( Cinnamomum burmannii The leaves of chvar. Borneol were crushed and passed through a 60-mesh sieve to obtain camphor-type cinnamon leaf powder; S2 Enzymatic hydrolysis: Weigh 25g of camphor-type Cinnamomum cassia leaf powder prepared in step S1, add xylanase 150U / g, hemicellulase 200U / g, lipase 80U / g and pectinase 100U / g powder, use 0.8 mol / L citrate-sodium citrate buffer solution as solvent, adjust pH to 5.5, enzymatically hydrolyze at 45℃ for 4 hours, heat to 100℃ to inactivate enzyme for 5 minutes, filter, and retain the solid part; S3 Fermentation: Activated Saccharomyces cerevisiae, Bacillus licheniformis, and Lactobacillus acidophilus were added to the enzymatic hydrolysis product of step S2, with inoculum amounts of 1.5%, 2%, and 1.5%, respectively. The product was fermented at 37°C for 24 hours, sterilized with ultraviolet light, filtered, and the fermentation product was obtained. S4 Ionic Liquid Extraction: Add 20 parts by weight of the ionic liquid-acetonitrile mixed solution to the fermentation product of step S3, introduce CO2 at a rate of 0.5 mL / min for 30 min, shake, stop introducing CO2, and obtain a highly extractable ionic liquid-acetonitrile mixed solution. Allow it to stand and separate the layers, separate the liquids, remove the water layer, wash, dry, filter, and obtain the extract. S5 Organic solvent extraction: The extract from step S4 was refluxed with petroleum ether for 1.5 h, filtered, concentrated by rotary evaporator, refluxed with anhydrous ethanol at 60 °C for 1 h, filtered, the filtrate was evaporated to dryness and then vacuum dried to remove organic solvent, thus obtaining the essential oil of Aralia elata leaf of group A (borneol type).

[0016] Group E: Dried camphor-type incense ( Cinnamomum burmannii The leaves of *C. chvar.* Borneol* were pulverized and passed through a 60-mesh sieve to obtain camphor-type *C. chvar.* leaf powder. 25g of this powder was weighed out, and without any enzymatic hydrolysis, it was filtered under reduced pressure. The treated camphor-type *C. chvar.* leaves were placed in a vacuum desiccator and dried at 40°C for 2 hours. The enzyme-treated and dried camphor-type *C. chvar.* sample was then extracted with petroleum ether under reflux for 1.5 hours. After filtration, the filtrate was concentrated using a rotary evaporator, dried under vacuum at 40°C, refluxed with anhydrous ethanol at 60°C for 1 hour, filtered again, and the filtrate was evaporated to dryness and then vacuum dried to remove organic solvents, thus obtaining group E camphor-type *C. chvar.* leaf essential oil.

[0017] Essential oil yield (%) = (Mass of essential oil obtained from vacuum drying / Mass of camphor-type aralia elata raw material) × 100% Table 1 Enzymatic hydrolysis parameters of various enzyme preparations pectinase 50℃ pH: 5.0 Beijing Solarbio Technology Co., Ltd. Lipase 45℃ pH: 6.0 Beijing Bio-Lab Technology Co., Ltd. hemicellulase 50℃ pH: 5.5 Beijing Bio-Lab Technology Co., Ltd. Xylanase 45℃ pH: 5.5 Shanghai Yuanye Biotechnology Co., Ltd. The chemical components of the essential oils were determined by gas chromatography-mass spectrometry (GC-MS). The instrument used was a Shimadzu GCMS-QP2020W / O system. GC conditions were: SH-RxiTM-5Sil MS column (30m × 0.25 mm, 0.25μm); temperature program: initial column temperature 70℃, increased to 160℃ at 2℃ / min, held for 2 min, then increased to 220℃ at 10℃ / min, held for 5 min, for a total run time of 51 min. The injection volume was 0.5μL, using split injection, with an injection port temperature of 230℃; nitrogen was used as the carrier gas, with a constant flow rate of 1.19 mL / min. GC conditions were: ion source temperature 200℃; scan mass range m / z 50–500. The effects of different compound enzyme treatments on the yield of camphor-type *Aralia elata* essential oil were statistically analyzed. Figure 1 Simultaneously, the main components of the essential oils in group AE were analyzed using GC-MS instruments and parameters. The results are shown in [Figure 1]. Figure 2 .

[0018] Figure 1 The results showed that the effects of different compound enzyme treatments in groups A and D on the yield of camphor-type Cinnamomum cassia essential oil were significantly different from those in group E (p < 0.05). Among them, the compound enzyme treatments of group A (pectinase + xylanase + lipase), group B (pectinase + xylanase + hemicellulase), and group C (xylanase + hemicellulase + lipase) had similar yields of camphor-type Cinnamomum cassia essential oil. However, the compound enzyme treatment of group D (xylanase + hemicellulase + lipase + pectinase) had a significantly higher effect on the yield of camphor-type Cinnamomum cassia essential oil than the other groups A, B, and C (p < 0.05). The compound enzyme treatment in group D yielded the highest yield of camphor-type Cinnamomum cassia essential oil.

[0019] Figure 2 The results showed that the combined enzyme treatment in group AD significantly increased the release of effective compounds, including borneol, camphor, linalool, and caryophyllene, compared with group E, especially for borneol and camphor. Among them, the combined enzyme treatment in group D significantly promoted the release of effective compounds, including borneol, camphor, linalool, caryophyllene, limonene, and eucalyptol, compared with group AC.

[0020] Example 2: Study on the effect of compound enzyme-assisted extraction of essential oil from borneol-type Aristolochia debilis leaf on a mouse model of chronic pharyngitis Forty-eight male SD rats were randomly divided into eight groups: a control group, a model group, group A, group B, group C, group D, group E, and a throat-clearing group, with eight rats in each group. The rats were labeled with picric acid, and the weight of each rat was recorded. All rats were fed standard feed and had free access to water. From day 1 to 15, the animals in the model group, group A, group B, group C, group D, group E, and the throat-clearing group were sprayed with 2.5% ammonia water twice a day (morning and afternoon), three puffs each time using a laryngeal sprayer. The control group received the same treatment with distilled water sprayed into their throats from day 1 to 15. Rats were observed daily for food intake, activity level, water intake, oral secretions, and any scratching of the oropharynx. The pharynx was also observed once daily. After model establishment, from day 16 to 25, mice in the throat-clearing group were administered throat-clearing granules (composed of Achyranthes bidentata, Eupatorium fortunei, Plantago asiatica, and Dioscorea opposita) at a dose of 10 g / kg daily, diluted with 55°C distilled water to a concentration of 0.1 g / ml for pharyngeal spraying. Groups A, B, C, D, and E were administered borneol-type Cinnamomum camphora leaf essential oil prepared in Example 1 (groups A, B, C, D, and E) at a dose of 1 g / kg daily, diluted with 55°C distilled water to a concentration of 0.1 g / ml for pharyngeal spraying. A solution of the same volume as the throat-clearing group was also administered using 55°C distilled water for pharyngeal spraying. The blank group and model group received the same volume of distilled water. Finally, IL-1β and TNF-α were detected using ELISA. Figure 3 .

[0021] Figure 3The results showed that, compared with the blank group, the TNF-α level in the model group was significantly increased (P<0.05), indicating that the chronic pharyngitis mouse model was successful. Compared with the model group, the TNF-α level in the positive control Qingyan group was significantly reduced. Compared with the borneol-type Yinxiang leaf essential oil in group D, the borneol-type Yinxiang leaf essential oil in group AC had a significant effect on reducing serum TNF-α secretion (P<0.05).

[0022] Figure 4 The results showed that, compared with the blank group, the IL-1β level in the model group was significantly increased (P<0.05), indicating that the chronic pharyngitis mouse model was successful. Compared with the model group, the IL-1β level in the positive control Qingyan group was significantly decreased. The essential oil of borneol-type Yinxiang leaves in group D had a significant effect on reducing serum IL-1β secretion compared with the model group (P<0.01). At the same time, the essential oil of borneol-type Yinxiang leaves in group D had a significant effect on reducing serum IL-1β secretion compared with the essential oil of borneol-type Yinxiang leaves in groups AC.

[0023] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for fermenting extraction of camphor oil using a mixed enzyme preparation, characterized by Includes the following steps: S1 Pretreatment: The leaves of borneol-type Cinnamomum burmanniich var. Borneol were microwave-dried, mixed, and pulverized. S2 Enzymatic hydrolysis: The solid obtained in step S1 is added to a citric acid-sodium citrate buffer solution, a compound enzyme is added for enzymatic hydrolysis, the enzyme is inactivated, the enzymatic hydrolysis product is obtained, filtered under reduced pressure, and dried under vacuum. S3 Fermentation: Add activated Saccharomyces cerevisiae, Bacillus licheniformis, and Lactobacillus acidophilus to the enzymatic hydrolysis product of step S2, ferment, sterilize, filter, and obtain the fermentation product; S4 Ionic liquid extraction: The fermentation product obtained in step S4 is added to the ionic liquid-acetonitrile mixed solution, CO2 is slowly introduced, the mixture is shaken, the CO2 is stopped, and a highly extractable ionic liquid-acetonitrile mixed solution is obtained. The mixture is allowed to stand and separate into layers, the water layer is removed, the solution is dried, and the solution is filtered to obtain the extract. S5 Organic solvent extraction: The extract from step S4 is extracted by reflux with petroleum ether, filtered, the filtrate is concentrated by rotary evaporator, vacuum dried, refluxed with anhydrous ethanol, filtered, the filtrate is evaporated to dryness and then vacuum dried to remove organic solvent, thus obtaining camphor-type Aristolochia debilis leaf essential oil. The complex enzyme in step S2 is composed of xylanase, hemicellulase, lipase and pectinase, with an enzyme activity ratio of 1.5:2:0.8:

1. The ratio of Saccharomyces cerevisiae, Bacillus licheniformis, and Lactobacillus acidophilus in step S2 is 1.5:2:1.

5.

2. The application of a complex enzyme composed of xylanase, hemicellulase, lipase and pectinase in improving the yield of camphor-type cinnamon essential oil.

3. The application of the borneol essential oil prepared by the method according to claim 1 in the preparation of products for the prevention or treatment of chronic pharyngitis.

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