A method for enriching 1,8-cineole from eucalyptus oil by short path molecular distillation

Abstract

The application belongs to the technical field of natural product separation and purification, and discloses a process of a method for enriching 1,8-eucalyptol from eucalyptus oil through short-path molecular distillation. The method is characterized in that: first, the crude eucalyptus oil is subjected to selective hydroxylation or epoxidation reaction with a buffer system containing F87A / A328G mutant CYP102A1 enzyme under temperature and oxygen control conditions, so that the impurities are converted into high-boiling-point derivatives, and then the light components are collected through short-path molecular distillation. By adopting the above technical scheme, the application constructs an enzyme method selective conversion-short-path molecular distillation coupling process, successfully solves the key separation problem in the preparation of high-purity 1,8-eucalyptol, provides an efficient, green and scalable technical path, and meets the urgent needs of the medical, high-end daily chemical and other fields for high-purity natural monoterpene compounds.

Description

Technical Field

[0001] This invention belongs to the field of natural product separation and purification technology, and relates to a method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation. Background Technology

[0002] Eucalyptus oil, as an important natural plant essential oil, is widely used in pharmaceuticals, daily chemicals, and food additives. The content of its core active ingredient, 1,8-cineole, directly determines the product's quality grade and market value. To meet the demand for high-purity 1,8-cineole in high-end applications, short-path molecular distillation technology, which effectively avoids the decomposition of heat-sensitive components, is commonly used in industry. This technology, by controlling the extremely short distance between the evaporation and condensation surfaces, achieves selective migration of light and heavy components within the molecular path of freedom, thus completing the preliminary separation of components with similar boiling points at lower operating temperatures, significantly superior to traditional atmospheric or vacuum distillation.

[0003] However, existing short-path molecular distillation processes are difficult to effectively separate from 1,8-cineole (boiling point about 176°C) by molecular distillation when faced with specific competitive impurities in eucalyptus oil, such as α-pinene, β-pinene, limonene, and certain oxygen-containing monoterpenes, which have molecular weights, boiling point ranges (mostly between 150–185°C at normal pressure), and polar parameters.

[0004] Under high vacuum conditions, the saturated vapor pressure curves of these components overlap, resulting in extremely similar escape rates on the evaporation surface. This leads to significant co-distillation of 1,8-cineole and impurities in the distillate. Even with multi-stage series distillation or precise control of operating parameters (such as scraper rotation speed, feed rate, and evaporation temperature gradient), only limited separation gains can be achieved, accompanied by a sharp increase in energy consumption and a decrease in throughput. Furthermore, excessively intensifying distillation in pursuit of higher purity may induce localized overheating, triggering trace oxidation or polymerization side reactions, and impairing the product's color and odor stability. Summary of the Invention

[0005] This invention provides a method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation. The method involves introducing an enzymatic pretreatment step before short-path molecular distillation to selectively catalyze the conversion of 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. This method solves the bottleneck problem of separation efficiency in existing short-path molecular distillation processes caused by the high similarity in properties between the target component and impurities, achieving 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 megaterium. This mutant is obtained by site-directed mutagenesis of wild-type CYP102A1, in which the 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] 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] 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 coexistence 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 the 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 enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation, characterized in that, Includes the following steps: (1) Crude eucalyptus oil is mixed with an aqueous buffer system containing a specific oxidoreductase to form an oil-water two-phase reaction system; the oxidoreductase is a CYP102A1 mutant derived from Bacillus megaterium, which is obtained by site-directed mutagenesis of wild-type CYP102A1, wherein the phenylalanine at position 87 of the amino acid sequence of wild-type CYP102A1 is replaced with alanine and the alanine at position 328 is replaced with glycine; (2) Under controlled temperature and oxygen conditions, enzyme catalysis is carried out to selectively hydroxylate or epoxidize α-pinene, β-pinene and limonene in crude eucalyptus oil to generate alcohols or epoxide derivatives with boiling points higher than 220℃ and polarity greater than 1,8-cineole. (3) After the reaction is completed, the aqueous phase is separated to obtain eucalyptus oil pretreated by enzyme method; the enzyme catalytic reaction is carried out under inert gas protection, the reaction atmosphere is nitrogen or argon, and the oxygen partial pressure of the system is controlled at 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 to α-terpineol, β-pinene is converted to β-terpineol, and limonene is converted to 1,2-epoxylimonene or 8-hydroxylimonene; (4) The eucalyptus oil pretreated by enzyme method is fed into a short-path molecular distillation apparatus and distilled under suitable process conditions. The light component distillate is collected to obtain a high-purity 1,8-cineole product. The distance between the evaporation surface and the condensation surface of the short-path molecular distillation apparatus is 20–50 mm. The molecular mean free path of 1,8-cineole under the operating conditions is greater than this distance, while the molecular mean free path of the derivative generated by enzyme conversion is less than this distance.

2. The method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation according to claim 1, characterized in that, The crude eucalyptus oil contains an initial content of 1,8-cineole higher than 65%, and a total content of α-pinene, β-pinene, and limonene higher than 15%; 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 amount of oxidoreductase added is 0.5%–2.0% of the mass of the crude eucalyptus oil.

3. The method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation according to claim 1, characterized in that, 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; and polyoxyethylene sorbitan monooleate is added to the system as a nonionic surfactant at a concentration of 0.1%–0.5%.

4. The method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation according to claim 1, characterized in that, The aqueous buffer system also includes a coenzyme regeneration module, which includes glucose dehydrogenase and D-glucose derived from Bacillus megaterium; wherein the amount of glucose dehydrogenase added is 10%–20% of the mass of the oxidoreductase, and the final concentration of D-glucose is 5–20 mmol / L.

5. The method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation 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%.

6. The method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation according to claim 1, characterized in that, In step (3), the enzyme is deactivated by heating to 50°C and maintaining it for 10 minutes; then the mixture is allowed to stand and separate into layers, and the lower aqueous phase is separated; the organic phase is washed with deionized water 2–3 times, with each wash water volume being 30% of the organic phase volume; after washing, the mixture is dried with anhydrous sodium sulfate and filtered to obtain eucalyptus oil pretreated by enzyme method.

7. The method for enriching 1,8-cineole in eucalyptus oil by short-path molecular distillation according to claim 1, characterized in that, The short-path molecular distillation employs a two-stage tandem operation to sequentially remove low-boiling-point components and high-boiling-point derivatives, specifically enriching 1,8-cineole.