Method for enriching 1, 8-cineole in eucalyptus oil refined by short-path molecular distillation

Abstract

The invention belongs to the technical field of separation and purification of natural products, and discloses a process of a 1, 8-cineole enrichment method for refining eucalyptus oil through short-path molecular distillation. The method is characterized by comprising the following steps: carrying out selective hydroxylation or epoxidation reaction on crude eucalyptus oil and a buffer system containing F87A / A328G mutant CYP102A1 enzyme under temperature and oxygen control conditions to convert impurities into high-boiling-point derivatives, and collecting light components through short-path molecular distillation. By adopting the technical scheme, an enzymatic selective conversion-short-path molecular distillation coupling process is constructed, the key separation problem in the preparation of the high-purity 1, 8-cineole is successfully solved, an efficient, green and large-scale technical path is provided, and the method is suitable for industrial production. The urgent requirements on high-purity natural monoterpene compounds in the fields of medicines, high-end daily chemicals and the like are met.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product separation and purification, and relates to a method for enriching 1,8-cineole in eucalyptus oil by short path molecular distillation. BACKGROUND

[0002] As an important natural plant essential oil, eucalyptus oil is widely used in the fields of medicine, daily chemical industry and food additives, etc. The content of its core active ingredient 1,8-cineole directly determines the product quality grade and market value. In order to meet the demand for high-purity 1,8-cineole for high-end applications, short path molecular distillation technology is often used in industry to effectively avoid the decomposition of heat-sensitive components. This technology realizes the selective migration of light and heavy components within the range of molecular free path by controlling the extremely short distance between the evaporation surface and the condensation surface, thereby completing the preliminary separation of components with similar boiling points at a lower operating temperature, which is significantly better than traditional atmospheric or reduced pressure distillation.

[0003] However, the existing short path molecular distillation process has difficulty in effectively separating 1,8-cineole from α-pinene, β-pinene, limonene and some oxygen-containing monoterpene impurities due to their similar molecular weight, boiling point range (mostly between 150-185℃ under atmospheric pressure) and polarity parameters. It is difficult to achieve effective separation by volatility difference during molecular distillation.

[0004] Under high vacuum conditions, the saturation vapor pressure curves of these components overlap, resulting in similar escape rates of the components on the evaporation surface, and thus the co-distillation of 1,8-cineole and impurities in the distillate. Even through multi-stage series or fine adjustment of operating parameters (such as wiper speed, feed rate, evaporation temperature gradient, etc.), only limited separation gain can be obtained, accompanied by a sharp increase in energy consumption and a decrease in processing flux. Meanwhile, excessive strengthening of distillation intensity in pursuit of higher purity may induce local overheating, causing trace oxidation or polymerization side reactions, which damage the color and odor stability of the product. SUMMARY

[0005] The present application provides a method for enriching 1,8-cineole in eucalyptus oil by short path molecular distillation. The method introduces an enzymatic pretreatment step before short path molecular distillation, selectively catalyzing the conversion of competitive impurities in eucalyptus oil with similar boiling points and polarity to 1,8-cineole to generate high-boiling or high-polarity derivatives that are easy to separate in the subsequent distillation process, thereby significantly improving the purity and yield of 1,8-cineole in the distillate. This method solves the separation efficiency bottleneck problem caused by the high similarity in physical properties between the target component and the impurities in the existing short path molecular distillation process, and realizes efficient, mild and highly selective enrichment of 1,8-cineole in crude eucalyptus oil.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: a method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation, comprising the following steps: (1) mixing crude eucalyptus oil with an aqueous buffer system containing a specific redox enzyme to form an oil-water two-phase reaction system; (2) carrying out an enzyme-catalyzed reaction under controlled temperature and oxygen conditions to selectively hydroxylate or epoxidize competitive impurities such as α-pinene, β-pinene, and limonene in the crude eucalyptus oil to generate corresponding alcohols or epoxide derivatives; (3) after the reaction, separating the aqueous phase to obtain eucalyptus oil pretreated by enzyme method; (4) sending the eucalyptus oil pretreated by enzyme method into a short-path molecular distillation apparatus, and distilling under set vacuum, evaporation temperature, and scraper rotation speed conditions to collect the light component distillate, thereby obtaining a high-purity 1,8-cineole product.

[0007] In step (1), the initial content of 1,8-cineole in the crude eucalyptus oil is higher than 65%, and it contains a total of more than 15% α-pinene, β-pinene, and limonene. The aqueous buffer system is a phosphate buffer or Tris-HCl buffer with a pH of 7.0–8.5 and an ionic strength of 0.05–0.2 mol / L. The oxidoreductase is a cytochrome P450 monooxygenase system, specifically composed of a CYP102A1 mutant derived from Bacillus subtilis, whose amino acid sequence is modified by site-directed mutagenesis, replacing phenylalanine at position 87 with alanine (F87A) and alanine at position 328 with glycine (A328G) to enhance the recognition ability and catalytic activity of monoterpene substrates. The amount of enzyme added is 0.5%–2.0% of the mass of the crude eucalyptus oil, on a dry weight basis.

[0008] In the oil-water two-phase reaction system, the volume ratio of crude eucalyptus oil to the aqueous buffer system is 1:0.8 to 1:1.5. To promote mass transfer at the two-phase interface, a nonionic surfactant, polyoxyethylene sorbitan monooleate, is added to the system at a concentration of 0.1%–0.5% (w / v). The surfactant can be completely removed by washing with water after the reaction and does not remain in the organic phase.

[0009] In step (2), the enzyme-catalyzed reaction is carried out under an inert gas atmosphere of nitrogen or argon, with the oxygen partial pressure controlled within the range of 0.5–2.0 kPa to maintain the limited oxygen supply required for the P450 enzyme catalytic cycle and to prevent non-enzymatic oxidation of eucalyptus oil components. The reaction temperature is maintained at 25–35 °C, and the reaction time is 2–6 hours. Under these conditions, α-pinene is converted to α-terpineol, β-pinene is converted to β-terpineol, and limonene is converted to 1,2-epoxylimonene or 8-hydroxylimonene. The boiling points of the above conversion products are all above 220 °C (at atmospheric pressure), and their polarity is significantly greater than that of 1,8-cineole. They are almost non-volatile in subsequent molecular distillation and remain in the distillation residue.

[0010] In step (3), the reaction is terminated by heating to 50°C and maintaining the temperature for 10 minutes, causing irreversible inactivation of the enzyme protein. The mixture is then allowed to stand and separate into layers, with the lower aqueous phase being separated. The organic phase is washed 2–3 times with deionized water, each wash using 30% of the organic phase volume, to thoroughly remove residual buffer salts, surfactants, and water-soluble byproducts. The washed organic phase is dried with anhydrous sodium sulfate and filtered to obtain enzymatically pretreated eucalyptus oil. Its 1,8-cineole content is slightly lower than that of the raw material (approximately 1%–2%), but the total amount of competing impurities is reduced by more than 80%.

[0011] In step (4), the operating conditions for short-path molecular distillation are as follows: system absolute pressure ≤ 1 Pa, evaporation surface temperature 80–110 °C, condensation surface temperature 20–40 °C, scraper rotor speed 200–400 rpm, and feed rate 50–150 g / h. Under these conditions, the mean free path of 1,8-cineole is much greater than the distance between the evaporation and condensation surfaces (usually 20–50 mm), allowing it to migrate efficiently to the condensation surface and be captured. In contrast, the mean free path of the high-boiling-point derivative molecules generated by enzymatic conversion is extremely short, preventing them from effectively reaching the condensation surface. They remain at the bottom of the evaporator and are discharged as heavy components.

[0012] In a preferred embodiment of the present invention, the oxidoreductase system further includes a coenzyme regeneration module. Glucose dehydrogenase and D-glucose are added to an aqueous buffer system, wherein the glucose dehydrogenase is derived from *Bacillus megaterium* and is added at 10%–20% of the P450 enzyme mass; the D-glucose concentration is 5–20 mmol / L. This coenzyme regeneration system catalyzes the oxidation of glucose to gluconic acid via glucose dehydrogenase, while simultaneously regenerating NADP... + It is reduced to NADPH, continuously providing reducing equivalents for the P450 enzyme, enabling the catalytic cycle to continue sustainably, avoiding the need for the exogenous addition of expensive coenzyme NADPH, and significantly reducing process costs.

[0013] In another preferred embodiment of the present invention, the short-range molecular distillation employs a two-stage cascade operation. The first-stage distillation is conducted at an evaporation temperature of 90°C and a feed rate of 120 g / h, primarily removing residual low-boiling-point components (such as small amounts of unreacted monoterpenes and water). The second-stage distillation is conducted at an evaporation temperature of 105°C and a feed rate of 80 g / h, specifically enriching 1,8-cineole. The condensates from both stages are collected separately, with only the second-stage distillate serving as the final product.

[0014] In the method described in this invention, all material contact surfaces are made of 316L stainless steel or high borosilicate glass to avoid side reactions catalyzed by metal ions.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] 1. By introducing highly selective P450 enzyme catalytic pretreatment, competitive impurities that are difficult to separate by physical means are directionally converted into high-boiling-point, high-polarity derivatives, which fundamentally changes the component property distribution of the distillation system and significantly expands the difference in behavior between 1,8-cineole and impurities in the molecular distillation process, breaking through the thermodynamic limit of traditional pure physical separation methods.

[0017] 2. The enzyme reaction is carried out under mild conditions. The molecular structure of 1,8-cineole is stable and has not undergone any chemical modification or degradation, ensuring the integrity and natural properties of the main components and meeting the strict requirements of pharmaceutical and food-grade products.

[0018] 3. The integration of the coenzyme regeneration system eliminates the need for exogenous NADPH addition to the entire biocatalytic process, significantly reducing raw material costs. It also simplifies the post-processing steps, enhancing the economic feasibility and industrialization potential of the process.

[0019] 4. After processing by the method of the present invention, the operating window of short-path molecular distillation is significantly widened: it can operate at higher feed rates and lower evaporation temperatures while still obtaining high-purity products, thereby reducing energy consumption, increasing equipment throughput, and avoiding color deterioration and odor loss caused by local overheating.

[0020] 5. The distillation residue contains high-value conversion products with well-defined structures, which have the potential for secondary development and improve the overall resource utilization efficiency and economic benefits of the process. Detailed Implementation

[0021] This invention provides a method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation. This method involves introducing an enzymatic pretreatment step before short-path molecular distillation to selectively catalytically convert competing impurities in eucalyptus oil with similar boiling points and polarities to 1,8-cineole, generating high-boiling-point or highly polar derivatives that are easily separated during subsequent distillation. This significantly improves the purity and yield of 1,8-cineole in the distillate.

[0022] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0023] Example 1: Crude eucalyptus oil with an initial 1,8-cineole content of 72%; enzyme addition of 1.2%; oil-water volume ratio of 1:1.2; reaction at 30°C and oxygen partial pressure of 1.2 kPa for 4 hours; single-stage molecular distillation (evaporation temperature 95°C, condensation temperature 30°C, rotation speed 300 rpm, pressure 1 Pa).

[0024] Preparation process: Crude eucalyptus leaf oil is mixed with an enzyme buffer system → impurities are converted by enzyme catalysis → layer washing and drying → short-path molecular distillation → light components are collected.

[0025] Example 2: Enzyme addition amount 0.5%, other formulations and processes are the same as in Example 1;

[0026] Preparation process: Same as in Example 1.

[0027] Example 3: Enzyme addition amount 2.0%, other formulations and processes are the same as in Example 1;

[0028] Preparation process: Same as in Example 1.

[0029] Example 4: Two-stage distillation was used (first-stage evaporation at 90°C and second-stage evaporation at 105°C), with the remaining formula and process the same as in Example 1;

[0030] Preparation process: Same as in Example 1 (the distillation step is changed to two stages in series).

[0031] Example 5: The enzyme-catalyzed reaction temperature was 25°C, and the remaining formulation and process were the same as in Example 1;

[0032] Preparation process: Same as in Example 1.

[0033] Example 6: The enzyme-catalyzed reaction temperature was 35°C, and the remaining formulation and process were the same as in Example 1;

[0034] Preparation process: Same as in Example 1.

[0035] Example 7: Enzyme immobilization on porous silica microspheres, with the remaining formulation and process the same as in Example 1;

[0036] Preparation process: Same as in Example 1 (the enzyme is in immobilized form).

[0037] Example 8: Add glucose dehydrogenase (15% by enzyme mass) and 10 mmol / L LD-glucose to the buffer system; the rest of the formulation and process are the same as in Example 1.

[0038] Preparation process: Same as in Example 1 (with the addition of coenzyme regeneration components).

[0039] Comparative Example 1: Crude eucalyptus leaf oil was directly subjected to two-stage short-path molecular distillation without enzyme-catalyzed pretreatment; other distillation parameters were the same as in Example 4.

[0040] Preparation process: Same as in Example 4 (without enzyme catalysis step).

[0041] Comparative Example 2: No molecular distillation was performed after enzyme-catalyzed pretreatment; the product was only washed and dried. The rest of the formulation and process were the same as in Example 1.

[0042] Preparation process: Same as in Example 1 (without distillation step).

[0043] Test method:

[0044] Enrichment performance test: The purity and yield of 1,8-cineole were determined by gas chromatography; the content of impurity derivatives in the distillation residue was detected.

[0045] Product quality testing: assess product color and odor; test peroxide value to determine the degree of oxidation; verify product stability.

[0046] Process efficiency testing: Calculate the throughput per unit time; evaluate the efficiency of enzyme reuse; calculate energy consumption and cost.

[0047] The test data comparisons are shown in Table 1 and Table 2.

[0048] Table 1. Comparison of purity and yield of 1,8-cineole

[0049] Test item 1,8-cineole purity (%) Yield (%) Example 1 97.8 89.5 Example 2 96.5 88.2 Example 3 98.2 90.3 Example 4 98.7 93.2 Example 5 96.8 87.5 Example 6 97.5 89 Example 7 97.6 88.8 Example 8 98 91 Comparative Example 1 92.3 79.5 Comparative Example 2 82.5 98

[0050] Table 2 Comparison of Residue Impurity Derivative Content and Peroxide Value

[0051] Test item Residual impurities derivative content (%) Peroxide value (meq / kg) Example 1 15 1.2 Example 2 12 1.3 Example 3 18 1.1 Example 4 16 1 Example 5 13 1.4 Example 6 17 1.2 Example 7 15 1.1 Example 8 16 1 Comparative Example 1 - 3.5 Comparative Example 2 - 2.8

[0052] The purity of 1,8-cineole in Examples 1-8 was ≥96.5% and the yield was ≥87.5%, which was far superior to the comparative example. Comparative example 1 had no enzyme pretreatment, and the impurities were difficult to separate, resulting in low purity and yield, which confirms that enzymatic conversion is the key to enrichment.

[0053] With increased enzyme addition (Examples 2→1→3), impurities are converted more thoroughly, and purity is gradually improved; two-stage distillation (Example 4) further purifies the product, achieving optimal purity and yield; the coenzyme regeneration module (Example 8) optimizes catalytic efficiency and improves yield.

[0054] The product in the example had a peroxide value ≤1.4 meq / kg and no oxidative odor; the product in the comparative example had a significantly higher peroxide value due to increased oxidation caused by the presence of impurities.

[0055] The process described in this example is mild, avoiding the decomposition of heat-sensitive components; the immobilized enzyme (Example 7) is reusable, reducing costs; and impurity derivatives in the residue are recyclable, improving resource utilization.

[0056] The method described in this invention solves the separation problem caused by similar physical properties in traditional distillation by pre-treating impurities with enzymes and separating them with short-range molecular distillation. Different parameter combinations can achieve efficient enrichment of 1,8-cineole, making it suitable for the preparation of high-purity raw materials in pharmaceutical, high-end daily chemical and other scenarios.

[0057] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for refining eucalyptus oil by short path molecular distillation to enrich 1,8-cineole, characterized in that, The method comprises the following steps: (1) mixing crude eucalyptus leaf oil with a water-phase buffer system containing specific oxidoreductase to form an oil-water two-phase reaction system; (2) performing enzyme catalysis under temperature and oxygen control to make selective hydroxylation or epoxidation of α-pinene, β-pinene and limonene in the crude eucalyptus leaf oil to generate alcohol or epoxide derivatives with boiling point higher than 220℃ and polarity greater than 1,8-eucalyptol; (3) separating the water phase after the reaction to obtain eucalyptus leaf oil pretreated by enzyme method; (4) feeding the eucalyptus leaf oil pretreated by enzyme method into a short-path molecular distillation device to perform distillation under suitable process conditions, and collecting light component distillate to obtain high-purity 1,8-eucalyptol product.

2. The process for the short path molecular distillation refining of eucalyptus leaf oil, 1,8-cineole enrichment according to claim 1, characterized in that, The oxidoreductase is a CYP102A1 mutant derived from Bacillus subtilis, and the amino acid sequence of the mutant is substituted with alanine at position 87 phenylalanine and substituted with glycine at position 328 alanine.

3. The process for the short path molecular distillation refining of eucalyptus leaf oil, 1,8-cineole enrichment according to claim 1, characterized in that, The initial content of 1,8-eucalyptol in the crude eucalyptus leaf oil is higher than 65%, and the total content of α-pinene, β-pinene and limonene is higher than 15%; the water-phase buffer system is a phosphate buffer or Tris-HCl buffer with pH 7.0-8.5 and ionic strength 0.05-0.2 mol / L; and the addition amount of the oxidoreductase is 0.5%-2.0% of the mass of the crude eucalyptus leaf oil.

4. The process for the short path molecular distillation refining of eucalyptus leaf oil for 1,8-cineole enrichment according to claim 2, characterized in that, In the oil-water two-phase reaction system, the volume ratio of the crude eucalyptus leaf oil to the water-phase buffer system is 1:0.8 to 1:1.5; and polyoxyethylene sorbitan monooleate is added to the system as a non-ionic surfactant, and the concentration of the polyoxyethylene sorbitan monooleate is 0.1%-0.5%.

5. The process for the enrichment of 1,8-cineole from eucalyptus leaf oil by short path molecular distillation as claimed in claim 1, wherein, The enzyme catalysis is performed under protection of inert gas, the reaction atmosphere is nitrogen or argon, the oxygen partial pressure of the system is controlled to be 0.5-2.0 kPa, the reaction temperature is 25-35℃, and the reaction time is 2-6 hours; during the reaction, α-pinene is converted into α-terpineol, β-pinene is converted into β-terpineol, and limonene is converted into 1,2-epoxylimonene or 8-hydroxylimonene.

6. The process for the short path molecular distillation refining of eucalyptus leaf oil, 1,8-cineole enrichment according to claim 1, characterized in that, The water-phase buffer system further comprises a coenzyme regeneration module, and the coenzyme regeneration module comprises glucose dehydrogenase derived from Bacillus megaterium and D-glucose; wherein the addition amount of the glucose dehydrogenase is 10%-20% of the mass of the oxidoreductase, and the final concentration of the D-glucose is 5-20 mmol / L.

7. The process for the short path molecular distillation refining of eucalyptus oil enriched in 1,8-cineole according to claim 1, characterized in that, The oxidoreductase is immobilized on porous silica microspheres, and the immobilization efficiency of the porous silica microspheres is higher than 90%.

8. The process for the enrichment of 1,8-cineole from eucalyptus leaf oil by short path molecular distillation as claimed in claim 1, wherein, In step (3), the enzyme is inactivated by heating to 50℃ and maintaining for 10 minutes; then the system is left to stand and stratify, and the lower water phase is separated; the organic phase is washed with deionized water for 2-3 times, and the amount of washing water is 30% of the volume of the organic phase each time; after washing, the organic phase is dried with anhydrous sodium sulfate and filtered to obtain eucalyptus leaf oil pretreated by enzyme method.

9. The process for the enrichment of 1,8-cineole from eucalyptus leaf oil by short path molecular distillation as claimed in claim 1, wherein, The short-path molecular distillation is operated in two stages in series to remove low-boiling-point components and high-boiling-point derivatives in sequence and specifically enrich 1,8-eucalyptol.

10. The process for the enrichment of 1,8-cineole from eucalyptus leaf oil by short path molecular distillation as claimed in claim 1, wherein, The distance between the evaporation surface and the condensation surface of the short path molecular distillation apparatus is 20-50 mm; the average free path of the 1,8-cineole molecules under the operating conditions is greater than the distance, while the average free path of the derivative molecules generated by enzymatic conversion is less than the distance.