Enzyme-assisted preparation method of rose essential oil and rose essential oil

Abstract

The present invention relates to the technical field of essential oil extraction, and in particular to an enzyme-assisted rose essential oil preparation method and rose essential oil. The rose essential oil preparation method comprises the following steps: mixing rose petals with water, homogenizing to prepare rose homogenate; adding immobilized cellulase to the rose homogenate, adjusting the pH to 4.0-5.0, performing enzymolysis treatment, adjusting the pH to 7.4-8.0, taking out the immobilized cellulase, and obtaining an enzymolysis homogenate; the immobilized cellulase is a super-hydrophilic membrane with cellulase connected to the surface through polyhistidine; steam distilling the enzymolysis homogenate, collecting the distillate, performing oil-water separation, and obtaining rose essential oil. In the preparation method of the present invention, a super-hydrophilic membrane with cellulase connected to the surface through polyhistidine is used as an immobilized cellulase, which can improve the enzymolysis efficiency and reduce the loss of enzymolysis homogenate components when the immobilized cellulase is separated, thereby achieving a higher yield of rose essential oil.

Description

Technical Field

[0001] The invention relates to the technical field of essential oil extraction, and in particular to an enzyme-assisted rose essential oil preparation method and rose essential oil. Background Art

[0002] Rose essential oil is a concentrated liquid of volatile compounds extracted from roses. It contains terpenes, oxygen-containing compounds, alkane compounds, benzene derivatives and other ingredients. It has a fragrant smell and is a high-grade, expensive fragrance. It also has moisturizing, antibacterial, antioxidant, decompression, and sleep-inducing functions. It is widely used in the fields of cosmetics, food, and medicine. At present, the amount of natural rose essential oil extracted is small. It is often hailed as "liquid gold" in the international market and is one of the most expensive essential oils. It is of great significance to study the efficient extraction method of rose essential oil.

[0003] The traditional steam distillation method is simple to operate, but the yield is not high. Combining cellulase hydrolysis with steam distillation can greatly increase the yield of rose essential oil. For example, in the paper "Study on High-efficiency Extraction of Rose Essential Oil by High-voltage Pulsed Electric Field and Its Antibacterial Properties", rose homogenate was enzymatically hydrolyzed before steam distillation, which increased the yield of rose essential oil by about 20%. However, the existing enzyme-assisted steam distillation technology has the problem that cellulase cannot be recovered and reused.

[0004] If the cellulase is immobilized by traditional immobilized enzyme technology, although the problem of enzyme reuse can be solved, on the one hand, the substrate particles in the rose homogenate are large, and the immobilized enzyme is difficult to fully contact the plant cell wall, resulting in low enzymatic efficiency, which in turn leads to a low yield of rose essential oil. On the other hand, in the process of separating the immobilized enzyme after enzymatic hydrolysis, it is easy to bring out essential oil and other components in the rose homogenate, which will also reduce the yield of rose essential oil. These two problems limit the application of immobilized enzyme technology in rose essential oil extraction. Summary of the invention

[0005] In order to solve the technical problem that the yield of rose essential oil is low when the existing immobilized enzyme technology is applied to the extraction of rose essential oil, the present invention provides an enzyme-assisted rose essential oil preparation method. In the preparation method, a super-hydrophilic membrane with cellulase connected to the surface via polyhistidine is used as immobilized cellulase, which can improve the enzymatic hydrolysis efficiency and reduce the loss of enzymatic homogenate components when the immobilized cellulase is separated, thereby achieving a higher yield of rose essential oil.

[0006] The specific technical scheme of the present invention is:

[0007] In a first aspect, the present invention provides an enzyme-assisted method for preparing rose essential oil, comprising the following steps:

[0008] (1) mixing rose petals with water and homogenizing them to prepare rose homogenate;

[0009] (2) adding immobilized cellulase to the rose homogenate, adjusting the pH to 4.0-5.0, performing enzymatic hydrolysis, adjusting the pH to 7.4-8.0, taking out the immobilized cellulase, and obtaining an enzymatic hydrolysis homogenate; the immobilized cellulase is a super-hydrophilic membrane with cellulase connected to the surface via polyhistidine;

[0010] (3) The enzymatic hydrolyzed homogenate is subjected to steam distillation, the distillate is collected, and the oil-water separation is performed to obtain rose essential oil.

[0011] In the immobilized cellulase adopted by the present invention, polyhistidine has reversible pH responsiveness, is positively charged and hydrophilic in an acidic environment, and is uncharged in an alkaline environment. Cellulase is connected to a super-hydrophilic membrane through polyhistidine, and the pH responsiveness of polyhistidine is utilized to improve enzymatic hydrolysis efficiency and the yield of rose essential oil. Specifically, at an enzymatic hydrolysis pH (4.0-5.0) suitable for cellulase, polyhistidine is positively charged and hydrophilic, wherein the hydrophilicity is conducive to better contact of the cellulase connected to the polyhistidine with a substrate in rose homogenate and catalytic reaction, and the positive charge is conducive to adsorbing plant cells with negative charges on the surface, and is also conducive to contact of the cellulase with the substrate and catalytic reaction, thereby improving the enzymatic hydrolysis efficiency and finally achieving a higher yield of rose essential oil. After the enzymatic hydrolysis is completed, in an environment of pH 7.4-8.0, polyhistidine is uncharged, which can prevent the components in the enzymatic homogenate from binding to the polyhistidine due to electrostatic attraction, thereby facilitating the separation of the immobilized cellulase, reducing the loss of components in the enzymatic homogenate caused by the separation of immobilized cellulose, and improving the yield of rose essential oil. At the same time, it can also keep the immobilized cellulase at a high activity after repeated use. In addition, the pH response characteristic of polyhistidine is reversible, so it can still play a role when the immobilized cellulase is reused.

[0012] In addition, the present invention adopts a super-hydrophilic membrane as a carrier of the immobilized cellulase, and the hydrophobic rose essential oil is not easy to adhere thereto, so that the rose essential oil brought out from the enzymatic homogenate during the separation of the immobilized cellulase can be reduced. Moreover, the super-hydrophilic membrane has good anti-adhesion property, so that the adhesion of other components in the enzymatic homogenate (including plant cells and substances released after the lysis thereof) can be reduced. Moreover, the small amount of substances brought out by the immobilized cellulase after removal can be recovered by washing with a small amount of water, so that the use of the immobilized enzyme can be prevented from having an adverse effect on the yield of the rose essential oil.

[0013] Preferably, in step (2), one end of the molecular chain of the polyhistidine is connected to the superhydrophilic membrane, and the other end is connected to the cellulase; the degree of polymerization of the polyhistidine is 20-50.

[0014] When the polymerization degree of polyhistidine is too small, its molecular chain is too short. During the enzymatic hydrolysis process, it is not conducive to utilizing the hydrophilicity of polyhistidine to promote the contact between the enzyme and the substrate in the rose homogenate, thus resulting in low enzymatic hydrolysis efficiency.

[0015] Preferably, in step (2), the method for preparing the immobilized cellulase comprises the following steps:

[0016] (2.1) Using a super-hydrophilic film with hydroxyl groups on the surface and an epoxy silane coupling agent as raw materials, the epoxy silane coupling agent is grafted onto the super-hydrophilic film with hydroxyl groups on the surface through a hydrolysis condensation reaction between hydroxyl groups and silaneoxy groups, thereby preparing a super-hydrophilic film with epoxy groups on the surface;

[0017] (2.2) Using a super-hydrophilic membrane with epoxy groups on the surface and polyhistidine as raw materials, polyhistidine is grafted onto the super-hydrophilic membrane with epoxy groups on the surface through a ring-opening reaction between amino groups and epoxy groups, thereby preparing a super-hydrophilic membrane modified by polyhistidine grafting;

[0018] (2.3) Using a polyhistidine grafted modified superhydrophilic membrane and cellulase as raw materials, cellulase is grafted onto the polyhistidine grafted modified superhydrophilic membrane through a condensation reaction between amino groups and carboxyl groups to obtain immobilized cellulase.

[0019] In the above preparation method, in step (2.1), the hydroxyl group on the surface of the super-hydrophilic membrane is reacted with the silaneoxy group (-SiOR, R is an alkyl group) in the epoxysilane coupling agent to introduce epoxy groups on the surface of the super-hydrophilic membrane; in step (2.2), the terminal amino group in polyhistidine is reacted with the epoxy group on the surface of the super-hydrophilic membrane to graft the polyhistidine onto the surface of the super-hydrophilic membrane; in step (2.3), the terminal carboxyl group in polyhistidine is reacted with the amino group in cellulase to graft the cellulase onto the free end of the polyhistidine molecular chain (the end not connected to the super-hydrophilic membrane). By using the above method, the cellulase can be connected to the super-hydrophilic membrane through polyhistidine, and the two ends of the molecular chain of polyhistidine are respectively connected to the super-hydrophilic membrane and the cellulase.

[0020] Preferably, in step (2.1), the super-hydrophilic membrane with hydroxyl groups on the surface is a polyvinylidene fluoride membrane with tannic acid and polyethylene glycol bonded to the surface.

[0021] The surface of polyvinylidene fluoride membrane is modified to be super-hydrophilic by using tannic acid and polyethylene glycol, which can introduce hydroxyl groups on the membrane surface, thereby enabling the super-hydrophilic membrane to be grafted with polyhistidine and cellulase.

[0022] Preferably, in step (2.1), the method for preparing the super-hydrophilic membrane having hydroxyl groups on the surface comprises the following steps: immersing the polyvinylidene fluoride membrane in a tannic acid solution, separating the product after immersion, and then immersing the membrane in a polyethylene glycol solution, separating the product after immersion, to obtain a super-hydrophilic membrane having hydroxyl groups on the surface.

[0023] Furthermore, the weight average molecular weight of the polyethylene glycol is 6000-8000 Da.

[0024] Furthermore, the concentration of the tannic acid solution is 1.5-2.5wt%; and the immersion time in the tannic acid solution is 0.5-1.5h.

[0025] Furthermore, the concentration of the polyethylene glycol solution is 5-10wt%; and the immersion time in the polyethylene glycol solution is 0.5-1.5h.

[0026] Preferably, the specific process of step (2.1) comprises the following steps: mixing an epoxy silane coupling agent and a solvent in a mass volume ratio of 1 g:10-20 mL, performing a hydrolysis reaction, immersing the super-hydrophilic film with hydroxyl groups on the surface therein, reacting at 30-40° C. for 2-3 hours, separating the product, and obtaining a super-hydrophilic film with epoxy groups on the surface.

[0027] Preferably, the specific process of step (2.2) includes the following steps: immersing the super-hydrophilic membrane with epoxy groups on the surface into a polyhistidine solution with a concentration of 3-6wt%, reacting at 25-35°C for 3-4h, separating the product, and obtaining a super-hydrophilic membrane grafted with polyhistidine.

[0028] Preferably, the specific process of step (2.3) includes the following steps: immersing the super-hydrophilic membrane modified by polyhistidine grafting into an activator solution, taking it out after carboxyl activation, and then immersing it in a cellulase solution, separating the product after sufficient reaction, and obtaining immobilized cellulase.

[0029] Furthermore, in step (2.3), the solutes of the activator solution are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCL) and N-hydroxysuccinimide (NHS).

[0030] Furthermore, in step (2.3), the time for the carboxyl activation is 20 to 40 minutes; and the time for the full reaction is 20 to 30 hours.

[0031] Furthermore, in step (2.3), the concentration of the cellulase solution is 0.5-2.5 wt %.

[0032] Preferably, in step (1), the mass volume ratio of the rose petals to water (solid-liquid ratio) is 1 g:4-7 mL.

[0033] Preferably, in step (1), after homogenization, sodium chloride is added and mixed so that the prepared rose homogenate contains 5-15wt% sodium chloride.

[0034] Preferably, in step (2), the mass ratio of the immobilized cellulase to the rose homogenate is 1:20-30.

[0035] Preferably, in step (2), the temperature of the enzymatic hydrolysis treatment is 40-50°C and the time is 2-4 hours.

[0036] Preferably, in step (2), after taking out the immobilized cellulase, the immobilized cellulase is rinsed in water according to a mass volume ratio of 1 g:3-5 mL and then taken out, the rinse solution obtained is mixed with the enzymatic hydrolysis slurry obtained in step (2), and then step (3) is performed.

[0037] Preferably, in step (2), after adjusting the pH to 7.4-8.0, the mixture is allowed to stand for 10-20 minutes before the immobilized cellulase is taken out.

[0038] In a second aspect, the present invention provides a rose essential oil, which is prepared by the above preparation method.

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] (1) In the preparation process of rose essential oil, the present invention uses a super-hydrophilic membrane with cellulase connected to the surface via polyhistidine as immobilized cellulase, which can utilize the reversible pH response characteristics of polyhistidine and the super-hydrophilicity of the membrane surface to improve the yield of rose essential oil and enable the immobilized cellulase to maintain a high activity after multiple uses;

[0041] (2) By controlling the degree of polymerization of polyhistidine, the present invention can better utilize its hydrophilicity in the enzymatic hydrolysis environment, promote the contact between cellulase and the substrate in the rose homogenate, and thus improve the enzymatic hydrolysis efficiency. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the embodiments.

[0043] Overall embodiment

[0044] An enzyme-assisted method for preparing rose essential oil comprises the following steps:

[0045] (1) mixing rose petals with water and homogenizing them to prepare rose homogenate;

[0046] (2) adding immobilized cellulase to the rose homogenate, adjusting the pH to 4.0-5.0, performing enzymatic hydrolysis, adjusting the pH to 7.4-8.0, taking out the immobilized cellulase, and obtaining an enzymatic hydrolysis homogenate; the immobilized cellulase is a super-hydrophilic membrane with cellulase connected to the surface via polyhistidine; one end of the molecular chain of the polyhistidine is connected to the super-hydrophilic membrane, and the other end is connected to the cellulase; the degree of polymerization of the polyhistidine is 20-50;

[0047] (3) The enzymatic hydrolyzed homogenate is subjected to steam distillation, the distillate is collected, and the oil-water separation is performed to obtain rose essential oil.

[0048] As a specific implementation method, the specific process of step (1) includes the following steps: rose petals and water are homogenized in a mass volume ratio (solid-liquid ratio) of 1 g:4-7 mL, sodium chloride is added, and the mixture is mixed to prepare rose homogenate, wherein the prepared rose homogenate contains 5-15 wt % of sodium chloride.

[0049] As a specific implementation, in step (2), the method for preparing the immobilized cellulase comprises the following steps:

[0050] (2.1) Using a super-hydrophilic film with hydroxyl groups on the surface and an epoxy silane coupling agent as raw materials, the epoxy silane coupling agent is grafted onto the super-hydrophilic film with hydroxyl groups on the surface through a hydrolysis condensation reaction between hydroxyl groups and silaneoxy groups, thereby preparing a super-hydrophilic film with epoxy groups on the surface;

[0051] (2.2) Using a super-hydrophilic membrane with epoxy groups on the surface and polyhistidine as raw materials, polyhistidine is grafted onto the super-hydrophilic membrane with epoxy groups on the surface through a ring-opening reaction between amino groups and epoxy groups, thereby preparing a super-hydrophilic membrane modified by polyhistidine grafting;

[0052] (2.3) Using a polyhistidine grafted modified superhydrophilic membrane and cellulase as raw materials, cellulase is grafted onto the polyhistidine grafted modified superhydrophilic membrane through a condensation reaction between amino groups and carboxyl groups to obtain immobilized cellulase.

[0053] As a specific implementation, in step (2.1), the super-hydrophilic membrane with hydroxyl groups on the surface is a polyvinylidene fluoride membrane with tannic acid and polyethylene glycol bound to the surface, and its preparation method comprises the following steps: immersing the polyvinylidene fluoride membrane in a tannic acid solution with a concentration of 1.5~2.5wt%, separating the product after immersing for 0.5~1.5h, and then immersing it in a polyethylene glycol (weight average molecular weight of 6000~8000Da) solution with a concentration of 5~10wt%, separating the product after immersing for 0.5~1.5h, and obtaining a super-hydrophilic membrane with hydroxyl groups on the surface.

[0054] As a specific implementation method, the specific process of step (2.1) includes the following steps: mixing an epoxy silane coupling agent and a solvent in a mass volume ratio of 1 g: 10-20 mL, performing a hydrolysis reaction, immersing the super-hydrophilic film with hydroxyl groups on the surface therein, reacting at 30-40° C. for 2-3 hours, and separating the product to obtain a super-hydrophilic film with epoxy groups on the surface.

[0055] As a specific implementation method, the specific process of step (2.2) includes the following steps: immersing the super-hydrophilic membrane with epoxy groups on the surface into a polyhistidine solution with a concentration of 3-6wt%, reacting at 25-35°C for 3-4h, separating the product, and obtaining a super-hydrophilic membrane grafted with polyhistidine.

[0056] As a specific implementation method, the specific process of step (2.3) includes the following steps: dissolving EDC.HCL and NHS in a solvent according to a mass volume ratio of 1.5~2.5g:1g:200~500mL to obtain an activator solution; immersing the super-hydrophilic membrane modified by polyhistidine grafting in the activator solution, activating the carboxyl group for 20~40min, taking it out, and then immersing it in a cellulase solution with a concentration of 0.5~2.5wt%, reacting for 20~30h, separating the product, and obtaining immobilized cellulase.

[0057] As a specific implementation, in step (2), the mass ratio of the immobilized cellulase to the rose homogenate is 1:20-30; the temperature of the enzymatic hydrolysis treatment is 40-50°C, and the time is 2-4 hours.

[0058] As a specific implementation, in step (2), after adjusting the pH to 7.4-8.0, the mixture is allowed to stand for 10-20 minutes before the immobilized cellulase is taken out.

[0059] As a specific implementation method, in step (2), after taking out the immobilized cellulase, the immobilized cellulase is placed in water and rinsed according to a mass volume ratio of 1 g:3-5 mL, and then taken out, the rinse solution obtained is mixed with the enzymatic hydrolysis slurry obtained in step (2), and then step (3) is performed.

[0060] Example 1

[0061] Immobilized cellulase was prepared by the following steps:

[0062] (A) Preparation of superhydrophilic membrane:

[0063] After ultrasonically cleaning the polyvinylidene fluoride membrane with ethanol, it was immersed in a pre-prepared 2wt% tannic acid solution (solvent: water) according to m:V=1g:20mL, taken out after soaking for 1 hour, rinsed with water to remove unbound tannic acid, and then dried. It was immersed in a pre-prepared 10wt% PEG6000 solution (solvent: water) according to m:V=1g:20mL (polyvinylidene fluoride membrane: PEG6000 solution), soaked for 1 hour, taken out, rinsed with water to remove unbound polyethylene glycol, and dried to obtain a hydroxyl-superhydrophilic membrane (i.e., a superhydrophilic membrane with hydroxy groups on the surface).

[0064] (B) Grafted epoxy group:

[0065] γ-Glycidyloxypropyltrimethoxysilane (KH-560) was added to an ethanol-water mixture (containing 83% v / v ethanol) according to m:V=1g:10mL, the pH was adjusted to 5.0 after mixing, and hydrolyzed with stirring at room temperature for 20 minutes. Then, the hydroxyl-superhydrophilic membrane was immersed therein according to m:V=1g:20mL, stirred at 30°C for 3 hours, taken out, rinsed with water and dried to obtain an epoxy-superhydrophilic membrane (i.e., a superhydrophilic membrane with epoxy groups on the surface).

[0066] (C) Grafted polyhistidine:

[0067] According to m:V=1g:20mL, the epoxy-superhydrophilic membrane was immersed in a pre-prepared 5wt% poly-L-histidine solution (the solvent was dimethyl sulfoxide, and the degree of polymerization of poly-L-histidine was 20), stirred at 30°C for 3.4 hours, then taken out, rinsed with acetone and water in turn, and then dried to obtain a polyhistidine-superhydrophilic membrane (i.e., a superhydrophilic membrane grafted with poly-L-histidine).

[0068] (D) Grafted cellulase:

[0069] EDC.HCL and NHS were dissolved in water according to m:m:V=2g:1g:250mL, and the polyhistidine-superhydrophilic membrane was immersed in it according to m:V=1g:20mL, stirred at room temperature for 30min, taken out and drained, and immersed in a pre-prepared 2wt% cellulase solution (solvent is water) according to m:V=1g:20mL, stirred at room temperature for 24h, taken out, rinsed with water and dried to obtain immobilized cellulase. It was measured that the water contact angle of the membrane surface of the immobilized cellulase was 2.0°.

[0070] The immobilized cellulase prepared in this example was used to prepare rose essential oil, and the steps were as follows:

[0071] (1) Preprocessing:

[0072] Rose petals were mixed with distilled water at a solid-liquid ratio of 1 g:5 mL, homogenized, 10 wt % sodium chloride was added (ie, the sodium chloride content in the final rose homogenate was 10 wt %), and mixed to obtain rose homogenate.

[0073] (2) Enzymatic hydrolysis:

[0074] Add immobilized cellulase to the rose homogenate at m:m=25:1, adjust the pH to 4.5 with hydrochloric acid and sodium hydroxide solution, stir at 50°C for 3h, adjust the pH to 7.4 with hydrochloric acid and sodium hydroxide solution, let stand for 20min, and use tweezers to remove the immobilized cellulase to obtain enzymatic homogenate. Rinse the removed immobilized cellulase in distilled water at m:V=1g:4mL, remove the membrane, and combine the rinse solution with the enzymatic homogenate.

[0075] (3) Steam distillation:

[0076] The mixed solution of the rinse solution and the enzymatic homogenate was subjected to steam distillation, and the distillate was condensed and collected. The time was counted from the collection of the first drop of distillate, and the distillation was stopped after 4 hours. The distillate was subjected to ether extraction to separate the oil, and the rose essential oil was obtained after evaporation and removal of the ether. The yield of rose essential oil was measured to be 0.072%.

[0077] According to the above steps (1) to (3), when the immobilized cellulase was reused 5 times, the yield of rose essential oil was 0.065%, and the calculated enzyme activity retention rate was 90.2%; when it was reused 10 times, the yield of rose essential oil was 0.058%, and the calculated enzyme activity retention rate was 80.5%.

[0078] Example 2

[0079] Immobilized cellulase was prepared by the following steps:

[0080] (A) Preparation of superhydrophilic membrane:

[0081] After ultrasonically cleaning the polyvinylidene fluoride membrane with ethanol, it was immersed in a pre-prepared 2.5wt% tannic acid solution (solvent: water) according to m:V=1g:20mL, taken out after soaking for 30 minutes, rinsed with water to remove unbound tannic acid, and then immersed in a pre-prepared 5wt% PEG8000 solution (solvent: water) according to m:V=1g:20mL (polyvinylidene fluoride membrane: PEG8000 solution) after drying. After soaking for 1.5 hours, it was taken out, rinsed with water to remove unbound polyethylene glycol, and dried to obtain a hydroxyl-superhydrophilic membrane (i.e., a superhydrophilic membrane with hydroxy groups on the surface).

[0082] (B) Grafted epoxy group:

[0083] KH-560 was added into an ethanol-water mixture (containing 83% v / v ethanol) at m:V=1g:15mL, the pH was adjusted to 4.0 after mixing, and hydrolyzed with stirring at room temperature for 15min. Then, the hydroxyl-superhydrophilic membrane was immersed therein at m:V=1g:20mL, stirred at 40°C for 2h, taken out, rinsed with water and dried to obtain an epoxy-superhydrophilic membrane (i.e., a superhydrophilic membrane with epoxy groups on the surface).

[0084] (C) Grafted polyhistidine:

[0085] According to m:V=1g:20mL, the epoxy-superhydrophilic membrane was immersed in a pre-prepared 6wt% poly-L-histidine solution (the solvent was dimethyl sulfoxide, and the degree of polymerization of poly-L-histidine was 30), stirred at 25°C for 4 hours, then taken out, rinsed with acetone and water in turn, and then dried to obtain a polyhistidine-superhydrophilic membrane (i.e., a superhydrophilic membrane grafted with poly-L-histidine).

[0086] (D) Grafted cellulase:

[0087] EDC.HCL and NHS were dissolved in water according to m:m:V=2.5g:1g:500mL, and the polyhistidine-superhydrophilic membrane was immersed in it according to m:V=1g:20mL, stirred at room temperature for 40min, taken out and drained, and immersed in a pre-prepared 2.5wt% cellulase solution (solvent is water) according to m:V=1g:20mL, stirred at room temperature for 20h, taken out, rinsed with water and dried to obtain immobilized cellulase. It was measured that the water contact angle of the membrane surface of the immobilized cellulase was 1.9°.

[0088] The immobilized cellulase prepared in this example was used to prepare rose essential oil, and the steps were as follows:

[0089] (1) Preprocessing:

[0090] Rose petals were mixed with distilled water at a solid-liquid ratio of 1 g:7 mL, homogenized, 15 wt % sodium chloride was added (ie, the sodium chloride content in the final rose homogenate was 15 wt %), and mixed to obtain rose homogenate.

[0091] (2) Enzymatic hydrolysis:

[0092] Add immobilized cellulase to the rose homogenate at m:m=30:1, adjust the pH to 5.0 with hydrochloric acid and sodium hydroxide solution, stir at 40°C for 4h, adjust the pH to 7.6 with hydrochloric acid and sodium hydroxide solution, let stand for 10min, and use tweezers to remove the immobilized cellulase to obtain enzymatic homogenate. Rinse the removed immobilized cellulase in distilled water at m:V=1g:3mL, remove the membrane, and combine the rinse solution with the enzymatic homogenate.

[0093] (3) Steam distillation:

[0094] The mixed solution of the rinse solution and the enzymatic homogenate was subjected to steam distillation, and the distillate was condensed and collected. The time was started from the collection of the first drop of distillate, and the distillation was stopped after 4 hours. The distillate was subjected to ether extraction to separate the oil, and the rose essential oil was obtained after evaporation and removal of the ether. The yield of rose essential oil was measured to be 0.078%.

[0095] According to the above steps (1) to (3), when the immobilized cellulase was reused for 5 times, the yield of rose essential oil was 0.071%, and the calculated enzyme activity retention rate was 91.0%; when it was reused for 10 times, the yield of rose essential oil was 0.065%, and the calculated enzyme activity retention rate was 83.3%.

[0096] Example 3

[0097] Immobilized cellulase was prepared by the following steps:

[0098] (A) Preparation of superhydrophilic membrane:

[0099] After ultrasonically cleaning the polyvinylidene fluoride membrane with ethanol, it was immersed in a pre-prepared 1.5wt% tannic acid solution (solvent: water) according to m:V=1g:20mL, taken out after soaking for 1.5h, rinsed with water to remove unbound tannic acid, and then dried. It was immersed in a pre-prepared 8wt% PEG6000 solution (solvent: water) according to m:V=1g:20mL (polyvinylidene fluoride membrane: PEG6000 solution), soaked for 30min, taken out, rinsed with water to remove unbound polyethylene glycol, and dried to obtain a hydroxyl-superhydrophilic membrane (i.e., a superhydrophilic membrane with hydroxyl groups on the surface).

[0100] (B) Grafted epoxy group:

[0101] KH-560 was added into an ethanol-water mixture (containing 83% v / v ethanol) at m:V=1g:20mL, the pH was adjusted to 5.5 after mixing, and hydrolyzed with stirring at room temperature for 30min. Then, the hydroxyl-superhydrophilic membrane was immersed therein at m:V=1g:20mL, stirred at 35°C for 2.5h, taken out, rinsed with water and dried to obtain an epoxy-superhydrophilic membrane (i.e., a superhydrophilic membrane with epoxy groups on the surface).

[0102] (C) Grafted polyhistidine:

[0103] According to m:V=1g:20mL, the epoxy-superhydrophilic membrane was immersed in a pre-prepared 3wt% poly-L-histidine solution (the solvent was dimethyl sulfoxide, and the degree of polymerization of poly-L-histidine was 50), stirred at 35°C for 3h, then taken out, rinsed with acetone and water in turn, and then dried to obtain a polyhistidine-superhydrophilic membrane (i.e., a superhydrophilic membrane grafted with poly-L-histidine).

[0104] (D) Grafted cellulase:

[0105] EDC.HCL and NHS were dissolved in water according to m:m:V=1.5g:1g:200mL, and the polyhistidine-superhydrophilic membrane was immersed in it according to m:V=1g:20mL, stirred at room temperature for 20min, taken out and drained, and immersed in a pre-prepared 0.5wt% cellulase solution (solvent is water) according to m:V=1g:20mL, stirred at room temperature for 30h, taken out, rinsed with water and dried to obtain immobilized cellulase. It was measured that the water contact angle of the membrane surface of the immobilized cellulase was 2.4°.

[0106] The immobilized cellulase prepared in this example was used to prepare rose essential oil, and the steps were as follows:

[0107] (1) Preprocessing:

[0108] Rose petals were mixed with distilled water at a solid-liquid ratio of 1 g:4 mL, homogenized, 5 wt % sodium chloride was added (ie, the sodium chloride content in the final rose homogenate was 5 wt %), and mixed to obtain rose homogenate.

[0109] (2) Enzymatic hydrolysis:

[0110] Add immobilized cellulase to the rose homogenate at m:m=20:1, adjust the pH to 4.0 with hydrochloric acid and sodium hydroxide solution, stir at 45°C for 2h, adjust the pH to 8.0 with hydrochloric acid and sodium hydroxide solution, let stand for 15min, and use tweezers to remove the immobilized cellulase to obtain enzymatic homogenate. Rinse the removed immobilized cellulase in distilled water at m:V=1g:5mL, remove the membrane, and combine the rinse solution with the enzymatic homogenate.

[0111] (3) Steam distillation:

[0112] The mixed solution of the rinse solution and the enzymatic homogenate was subjected to steam distillation, and the distillate was condensed and collected. The time was counted from the time the first drop of distillate was collected, and the distillation was stopped after 4 hours. The distillate was subjected to ether extraction to separate the oil component, and the rose essential oil was obtained after evaporation and removal of the ether. The yield of rose essential oil was measured to be 0.069%.

[0113] According to the above steps (1) to (3), when the immobilized cellulase was reused for 5 times, the yield of rose essential oil was 0.061%, and the calculated enzyme activity retention rate was 88.4%; when it was reused for 10 times, the yield of rose essential oil was 0.054%, and the calculated enzyme activity retention rate was 78.2%.

[0114] Example 4

[0115] In the process of preparing immobilized cellulase in this embodiment, poly-L-histidine with a degree of polymerization of 8 is used, and the rest is the same as in Example 1. The specific steps are as follows:

[0116] (A) Preparation of superhydrophilic membrane:

[0117] After ultrasonically cleaning the polyvinylidene fluoride membrane with ethanol, it was immersed in a pre-prepared 2wt% tannic acid solution (solvent: water) according to m:V=1g:20mL, taken out after soaking for 1 hour, rinsed with water to remove unbound tannic acid, and then dried. It was immersed in a pre-prepared 10wt% PEG6000 solution (solvent: water) according to m:V=1g:20mL (polyvinylidene fluoride membrane: PEG6000 solution), soaked for 1 hour, taken out, rinsed with water to remove unbound polyethylene glycol, and dried to obtain a hydroxyl-superhydrophilic membrane (i.e., a superhydrophilic membrane with hydroxy groups on the surface).

[0118] (B) Grafted epoxy group:

[0119] KH-560 was added into an ethanol-water mixture (containing 83% v / v ethanol) at m:V=1g:10mL, the pH was adjusted to 5.0 after mixing, and hydrolyzed with stirring at room temperature for 20min. Then, the hydroxyl-superhydrophilic membrane was immersed therein at m:V=1g:20mL, stirred at 30°C for 3h, taken out, rinsed with water and dried to obtain an epoxy-superhydrophilic membrane (i.e., a superhydrophilic membrane with epoxy groups on the surface).

[0120] (C) Grafted polyhistidine:

[0121] According to m:V=1g:20mL, the epoxy-superhydrophilic membrane was immersed in a pre-prepared 5wt% poly-L-histidine solution (the solvent was dimethyl sulfoxide, and the degree of polymerization of poly-L-histidine was 8), stirred at 30°C for 3.4 hours, then taken out, rinsed with acetone and water in turn, and then dried to obtain a polyhistidine-superhydrophilic membrane (i.e., a superhydrophilic membrane grafted with poly-L-histidine).

[0122] (D) Grafted cellulase:

[0123] EDC.HCL and NHS were dissolved in water according to m:m:V=2g:1g:250mL, and the polyhistidine-superhydrophilic membrane was immersed in it according to m:V=1g:20mL, stirred at room temperature for 30min, taken out and drained, and immersed in a pre-prepared 2wt% cellulase solution (solvent is water) according to m:V=1g:20mL, stirred at room temperature for 24h, taken out, rinsed with water and dried to obtain immobilized cellulase. It was measured that the water contact angle of the membrane surface of the immobilized cellulase was 1.6°.

[0124] The immobilized cellulase prepared in this example was used to prepare rose essential oil, and the steps were the same as in Example 1. After measurement, the yield of rose essential oil was 0.057%. When the immobilized cellulase was reused 5 times, the yield of rose essential oil was 0.052%, and the calculated enzyme activity retention rate was 91.2%; when it was reused 10 times, the yield of rose essential oil was 0.048%, and the calculated enzyme activity retention rate was 84.2%.

[0125] Data analysis: Compared with Example 1, the yield of rose essential oil in Example 4 was reduced by 20.8%, indicating that when the polymerization degree of polyhistidine is too small, the yield of rose essential oil will be reduced. Cause analysis: The molecular chain of polyhistidine is too short, which is not conducive to utilizing the hydrophilicity of polyhistidine to promote the contact between the enzyme and the substrate in the rose homogenate during the enzymatic hydrolysis process, thus resulting in low enzymatic hydrolysis efficiency.

[0126] Comparative Example 1

[0127] In the process of preparing rose essential oil in this comparative example, non-immobilized cellulase was used, and the rest was the same as in Example 1, and the specific steps were as follows:

[0128] (1) Preprocessing:

[0129] Rose petals were mixed with distilled water at a solid-liquid ratio of 1 g:6 mL, homogenized, 10 wt % sodium chloride was added (ie, the sodium chloride content in the final rose homogenate was 10 wt %), and mixed to obtain rose homogenate.

[0130] (2) Enzymatic hydrolysis:

[0131] Cellulase was added to the rose homogenate at a ratio of m:m=100:1, the pH was adjusted to 4.5 with hydrochloric acid and sodium hydroxide solution, and the enzymatic homogenate was obtained after stirring at 50°C for 3 hours.

[0132] (3) Steam distillation:

[0133] The enzymatic homogenate was subjected to steam distillation, and the distillate was collected by condensation. The time was counted from the time the first drop of distillate was collected, and the distillation was stopped after 4 hours. The distillate was subjected to ether extraction to separate the oil, and the rose essential oil was obtained after evaporation and removal of the ether. The yield of rose essential oil was measured to be 0.075%.

[0134] Comparative Example 2

[0135] In the process of preparing immobilized cellulase in this comparative example, the cellulase is directly grafted onto the super-hydrophilic membrane without passing through polyhistidine. The specific steps are as follows:

[0136] (A) Preparation of superhydrophilic membrane:

[0137] After ultrasonically cleaning the polyvinylidene fluoride membrane with ethanol, it was immersed in a pre-prepared 2wt% tannic acid solution (solvent: water) according to m:V=1g:20mL, taken out after soaking for 1 hour, rinsed with water to remove unbound tannic acid, and then dried. It was immersed in a pre-prepared 10wt% PEG6000 solution (solvent: water) according to m:V=1g:20mL (polyvinylidene fluoride membrane: PEG6000 solution), soaked for 1 hour, taken out, rinsed with water to remove unbound polyethylene glycol, and dried to obtain a hydroxyl-superhydrophilic membrane (i.e., a superhydrophilic membrane with hydroxy groups on the surface).

[0138] (B) Grafted amino group:

[0139] 3-Aminopropyltriethoxysilane (KH-550) was added to an ethanol-water mixture (containing 83% v / v ethanol) according to m:V=1g:10mL, the pH was adjusted to 5.0 after mixing, and hydrolyzed with stirring at room temperature for 20 minutes. Then, the hydroxyl-superhydrophilic membrane was immersed therein according to m:V=1g:20mL, stirred at 30°C for 3 hours, taken out, rinsed with water and dried to obtain an amino-superhydrophilic membrane (i.e., a superhydrophilic membrane with amino groups on the surface).

[0140] (C) Grafted Cellulase:

[0141] EDC.HCL and NHS were dissolved in water according to m:m:V=2g:1g:250mL, 2wt% of cellulase was added thereto (i.e., the content of cellulase in the mixed solution was 2wt%), and after stirring at room temperature for 30min, the amino-superhydrophilic membrane was immersed therein according to m:V=1g:20mL, and after stirring at room temperature for 24h, it was taken out, rinsed with water, and dried to obtain immobilized cellulase. It was measured that the water contact angle of the membrane surface of the immobilized cellulase was 1.3°.

[0142] The immobilized cellulase prepared in this comparative example was used to prepare rose essential oil, and the steps were the same as those in Example 1. After measurement, the yield of rose essential oil was 0.053%. When the immobilized cellulase was reused 5 times, the yield of rose essential oil was 0.048%, and the calculated enzyme activity retention rate was 90.6%; when it was reused 10 times, the yield of rose essential oil was 0.044%, and the calculated enzyme activity retention rate was 83.0%.

[0143] Data analysis: The yield of rose essential oil in comparative example 2 is significantly lower than that in comparative example 1, while the yield in example 1 is comparable to that in example 1, indicating that compared with directly grafting cellulase onto the super-hydrophilic membrane, connecting the two via polyhistidine can reduce the effect of enzyme immobilization on the yield of rose essential oil. Cause analysis: In the pH environment of enzymatic hydrolysis, polyhistidine is positively charged and hydrophilic, which is conducive to the cellulase connected to polyhistidine to better contact and catalyze the reaction with the substrate in the rose homogenate, thereby improving the efficiency of enzymatic hydrolysis and ultimately achieving a higher yield of rose essential oil; and, after the enzymatic hydrolysis is completed, by adjusting the pH to alkaline, polyhistidine can be converted into an uncharged state, thereby reducing the loss of components in the enzymatic homogenate caused by the separation of immobilized cellulose, and avoiding adverse effects on the yield of rose essential oil.

[0144] Comparative Example 3

[0145] In this comparative example, the same steps as in Example 1 were used to prepare immobilized cellulase.

[0146] In the process of preparing rose essential oil in this comparative example, the pH is not adjusted to alkaline after the enzymatic hydrolysis is completed, and the rest is the same as in Example 1, and the specific steps are as follows:

[0147] (1) Preprocessing:

[0148] Rose petals were mixed with distilled water at a solid-liquid ratio of 1 g:5 mL, homogenized, 10 wt % sodium chloride was added (ie, the sodium chloride content in the final rose homogenate was 10 wt %), and mixed to obtain rose homogenate.

[0149] (2) Enzymatic hydrolysis:

[0150] Add immobilized cellulase to the rose homogenate at m:m=25:1, adjust the pH to 4.5 with hydrochloric acid and sodium hydroxide solution, stir at 50°C for 3h, and then use tweezers to remove the immobilized cellulase to obtain enzymatic homogenate. Rinse the removed immobilized cellulase in distilled water at m:V=1g:4mL, remove the membrane, and combine the rinse solution with the enzymatic homogenate.

[0151] (3) Steam distillation:

[0152] The mixed solution of the rinse solution and the enzymatic homogenate was subjected to steam distillation, and the distillate was condensed and collected. The time was counted from the time the first drop of distillate was collected, and the distillation was stopped after 4 hours. The distillate was subjected to ether extraction to separate the oil component, and the rose essential oil was obtained after evaporation and removal of the ether. The yield of rose essential oil was measured to be 0.063%.

[0153] According to the above steps (1) to (3), when the immobilized cellulase was reused for 5 times, the yield of rose essential oil was 0.055%, and the calculated enzyme activity retention rate was 87.3%; when it was reused for 10 times, the yield of rose essential oil was 0.046%, and the calculated enzyme activity retention rate was 73.0%.

[0154] Data analysis: Compared with Example 1, the yield of rose essential oil in Comparative Example 3 is significantly lower, and the enzyme activity retention rate of immobilized cellulase after repeated use is also lower, indicating that the characteristic of polyhistidine undergoing charge conversion under pH induction can improve the yield of rose essential oil and enable the immobilized cellulase to maintain a high activity during repeated use. Cause analysis: After the enzymolysis is completed, the polyhistidine is converted into an uncharged state by adjusting the pH, which can prevent the components in the enzymolysis homogenate from binding to the polyhistidine due to electrostatic attraction, thereby facilitating the separation of the immobilized cellulase, reducing the loss of components in the enzymolysis homogenate caused by the separation of immobilized cellulose, improving the yield of rose essential oil, and at the same time enabling the immobilized cellulase to maintain a high activity after multiple uses.

[0155] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0156] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. An enzyme-assisted method for preparing rose essential oil, characterized in that: include: 1) Mix rose petals with water and homogenize to obtain rose homogenate; 2) Add immobilized cellulase to the rose homogenate, adjust the pH to 4.0-5.0, perform enzymatic hydrolysis, adjust the pH to 7.4-8.0, remove the immobilized cellulase, and obtain enzymatic hydrolysis homogenate; The immobilized cellulase is a super-hydrophilic membrane with cellulase connected to the surface via polyhistidine, and the preparation steps include: 2.1) Through the hydrolysis condensation reaction between hydroxyl groups and silaneoxy groups, the epoxysilane coupling agent is grafted onto the polyvinylidene fluoride membrane with tannic acid and polyethylene glycol on the surface to obtain a super-hydrophilic membrane with epoxy groups on the surface; 2.2) Grafting polyhistidine with a degree of polymerization of 20 to 50 onto the membrane obtained in step 2.1) through a ring-opening reaction between an amino group and an epoxy group to obtain a polyhistidine-grafted superhydrophilic membrane; 2.3) Grafting cellulase onto the membrane obtained in step 2.2) through a condensation reaction between amino groups and carboxyl groups; 3) Perform steam distillation on the enzymatic homogenate, collect the distillate, separate the oil and water, and obtain rose essential oil.

2. The preparation method according to claim 1, characterized in that In step 2), one end of the polyhistidine molecular chain is connected to the super-hydrophilic membrane, and the other end is connected to the cellulase.

3. The preparation method according to claim 1, characterized in that: The specific process of step 2.1) includes the following steps: mixing an epoxy silane coupling agent and a solvent in a mass volume ratio of 1 g: 10-20 mL, performing a hydrolysis reaction, immersing a polyvinylidene fluoride membrane having tannic acid and polyethylene glycol bound to the surface therein, reacting at 30-40° C. for 2-3 hours, separating the product, and obtaining a super hydrophilic membrane with epoxy groups on the surface.

4. The preparation method according to claim 1, characterized in that: In step 2), the mass ratio of the immobilized cellulase to the rose homogenate is 1:20-30.

5. The preparation method according to claim 1 or 4, characterized in that: In step 2), the temperature of the enzymatic hydrolysis treatment is 40-50°C.

6. The preparation method according to claim 5, characterized in that: In step 2), the enzymatic hydrolysis treatment time is 2 to 4 hours.

7. The preparation method according to claim 1, characterized in that: In step 2), after taking out the immobilized cellulase, rinse it in water at a mass volume ratio of 1 g:3-5 mL and then take it out, mix the rinse solution obtained with the enzymatic homogenate obtained in step 2), and then proceed to step 3).

8. A rose essential oil, characterized in that: The rose essential oil is prepared by the preparation method according to any one of claims 1 to 7.