Method for extracting phlorizin with assistance of compound enzyme

Abstract

The invention discloses a compound enzyme-assisted phlorizin extraction method, and particularly relates to the technical field of phlorizin extraction.The method comprises the following steps that 1, plant raw materials rich in phlorizin are selected and pretreated; 2, weighing the raw material powder, adding the raw material powder into an enzymolysis reaction kettle, and adding a citric acid-sodium citrate buffer solution; 3, cellulase, pectinase and papain are weighed and compounded into an enzymolysis system, and the enzymolysis system is injected into an enzymolysis reaction kettle; 4, the temperature in the enzymolysis reaction kettle is adjusted in real time, and an ultrasonic generator is started to work; 5, discharging the reaction liquid into a horizontal spiral centrifuge, and collecting supernate; and 6, extracting the precipitate generated in the step 5 for multiple times by using a proper amount of ethanol, and combining the extract and the supernatant. The method breaks through the limitation of a traditional process, under the mild condition, the phlorizin yield is increased by 35%, the thermal degradation rate is smaller than 2%, the impurity removal rate is larger than 90%, meanwhile, the extraction time is shortened by 66%, and the comprehensive cost is reduced by 40%.

Description

Technical Field

[0001] The present invention relates to the technical field of phloridzin extraction, and specifically relates to a method for extracting phloridzin assisted by a composite enzyme. Background Art

[0002] Phloridzin is a dihydrochalcone natural compound widely present in apple tree bark, tea leaves and some Rosaceae plants, and has significant hypoglycemic, antioxidant and anti-inflammatory activities. In recent years, with the increasing incidence of metabolic diseases such as diabetes, the demand for phloridzin as a pharmaceutical intermediate and functional food additive has increased sharply.

[0003] Traditional methods for extracting phloridzin include hot water extraction method, organic solvent extraction method and single enzymatic hydrolysis method, but they have certain limitations. The hot water extraction method requires long-term treatment at a high temperature of 60 - 100°C, resulting in a thermal degradation rate of phloridzin ≥ 15%, and significant loss of activity; in the organic solvent extraction method, organic solvents such as ethanol and acetone are likely to remain in the final product and require additional desolvation treatment, increasing the process complexity; when only using cellulase or pectinase in the single enzymatic hydrolysis method, the enzymatic hydrolysis time needs to be more than 24 hours, and the yield is less than 70%. At the same time, the enzyme is easily inactivated during the long-term reaction (half-life ≤ 2 hours), and the enzyme solution needs to be frequently supplemented, increasing the cost by 15%. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting phloridzin assisted by a composite enzyme to solve the above deficiencies in the technology.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A method for extracting phloridzin assisted by a composite enzyme, comprising the following steps:

[0006] Step 1: Select plant raw materials rich in phloridzin and perform pretreatment;

[0007] Step 2: Weigh the pretreated raw material powder, add it to an enzymatic hydrolysis reaction kettle, and add a citric acid - sodium citrate buffer solution according to a solid - liquid ratio of 1:20 - 1:30 g / ml;

[0008] Step 3: Accurately weigh cellulase, pectinase and papain with an electronic balance, compound them into an enzymatic hydrolysis system according to a certain ratio, and inject the enzyme solution into the enzymatic hydrolysis reaction kettle through a metering pump. The total concentration of the composite enzyme is 0.5% - 1.5% w / v, and at the same time, inject a 0.1% trehalose solution into the enzymatic hydrolysis reaction kettle;

[0009] Step 4: Adjust the temperature in the enzymatic hydrolysis reaction kettle in real time to make the internal temperature 45 - 50°C and the pH value 4.5 - 5.5, and turn on the ultrasonic generator to work, and continuously perform enzymatic hydrolysis for 8 hours;

[0010] Step 5: After the enzymatic hydrolysis reaction ends, the enzymatic hydrolysis reactor discharges the reaction solution into a horizontal screw centrifuge. The reaction solution is centrifuged at 4000 - 5000 r / min for 15 - 20 minutes, and the supernatant is collected.

[0011] Step 6: The precipitate generated in Step 5 is extracted multiple times with an appropriate amount of ethanol, with each extraction time being 1 - 2 hours. The extraction solutions and the supernatant are combined.

[0012] Step 7: The combined extraction solution is filtered using a vacuum pump and a suction filtration device to remove insoluble impurities, obtaining a clear extraction solution.

[0013] Preferably, in Step 1: The plant raw material can be the root bark, stem, or young leaves of an apple tree.

[0014] The pretreatment is as follows: The plant raw material is washed and then dried to a constant weight in an oven at 60°C. Then, the dried raw material is crushed using a shearing crusher and passed through a 40 - 60 mesh sieve to obtain a uniform powder, increasing the contact area to improve the enzymatic hydrolysis efficiency.

[0015] Preferably, in Step 3: Cellulase at 200 U / g, pectinase at 150 U / g, and protease at 50 U / g are compounded in a ratio of 3:2:1. Cellulase mainly destroys the cell wall skeleton structure, pectinase decomposes the pectin in the middle lamella, and protease assists in removing protein impurities bound to the active ingredients. The three act synergistically to target the degradation of cellulose, pectin, and protein components in the plant cell wall, accelerating the release of phloridzin and increasing the enzymatic hydrolysis efficiency by 65% compared to a single - enzyme system.

[0016] Preferably, in Step 4: The ultrasonic generator is a 25 kHz ultrasonic generator, the ultrasonic power density is 30 W / L, and the ultrasonic action interval is to apply for 10 minutes every 30 minutes to avoid enzyme denaturation caused by continuous ultrasound. The ultrasonic action promotes the diffusion of enzyme molecules and accelerates the breaking of the cell wall. At the same time, it avoids enzyme inactivation caused by high temperature. Through the cavitation effect, the contact efficiency between the enzyme and the substrate is enhanced. The enzymatic hydrolysis time is shortened from 24 hours to 8 hours, and the phloridzin release rate is increased by 18%.

[0017] Preferably, in Step 4: A three - blade inclined - paddle stirrer and a jacketed water circulation system are configured in the enzymatic hydrolysis reactor. In the early stage, the reaction solution in the enzymatic hydrolysis reactor is stirred by the three - blade inclined - paddle stirrer to ensure full contact and mixing of the enzyme solution and the raw material, and the temperature in the enzymatic hydrolysis reactor is controlled through the jacketed water circulation system.

[0018] Preferably, in Step 4: The probe of the ultrasonic generator is a titanium alloy horn probe, which is evenly distributed at the bottom of the inner cavity of the enzymatic hydrolysis reactor to avoid local overheating.

[0019] Preferably, in the fifth step: the centrifuge tube used in the horizontal spiral centrifuge can be a polypropylene centrifuge tube with corrosion resistance to avoid the influence of metal ion pollution on subsequent purification. The supernatant needs to be stored in a light-proof container to prevent the degradation of phloridzin due to light or oxidation.

[0020] Preferably, in the seventh step: after suction filtration, add a 0.45 μm microfiltration membrane for filtration. The 0.45 μm microfiltration membrane can remove macromolecular impurities.

[0021] Preferably, the enzymatic hydrolysis reactor is a 500 L stainless steel reactor with three-layer sieve plates. The pore diameters of the three-layer sieve plates are 0.5 mm, 0.8 mm, and 1.0 mm respectively. The surface of the sieve plates is coated with a polytetrafluoroethylene coating to prevent raw materials from adhering.

[0022] In the above technical solutions, the technical effects and advantages provided by the present invention are as follows:

[0023] 1. By forming a synergistic enzymatic hydrolysis system through the compounding of cellulase, pectinase and protease in a ratio of 3:2:1, combining with 25 kHz ultrasonic assistance and 0.1% trehalose enzyme activity protection technology, the efficient and directional extraction of phloridzin from plant raw materials is realized. This technology breaks through the limitations of traditional processes. Under mild conditions, the yield of phloridzin is increased by 35%, the thermal degradation rate < 2%, the impurity removal rate > 90%, at the same time, the extraction time is shortened by 66%, and the comprehensive cost is reduced by 40%;

[0024] 2. Through the multi-enzyme synergistic effect, the cellulose, pectin and protein components of the plant cell wall are targeted for degradation, accelerating the release of phloridzin, and increasing the enzymatic hydrolysis efficiency by 65% compared with a single enzyme system;

[0025] 3. During the enzymatic hydrolysis reaction process, the ultrasonic cavitation effect is synchronously applied to accelerate the release of intracellular components, and the enzyme activity half-life is extended from 2 hours to 6 hours by 0.1% trehalose enzyme to ensure the stability of the enzymatic hydrolysis reaction. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings.

[0027] Figure 1 It is the operation flow chart of the present invention;

[0028] Figure 2 It is the multi-enzyme synergistic action mechanism diagram of the present invention. Detailed Embodiments

[0029] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below.

[0030] Example 1:

[0031] A method for extracting phloridzin assisted by a composite enzyme, as Figures 1 to 2 shown, includes the following steps:

[0032] Step 1: Select apple root bark, wash it, dry it to constant weight in an oven at 60°C, and then crush it with a shear crusher and pass through a 40-60 mesh sieve to obtain uniform powder;

[0033] Step 2: Weigh 10 g of the raw material powder, add it to an enzymatic hydrolysis reactor, and add 250 mL of citric acid-sodium citrate buffer solution (pH 4.8) according to a solid-liquid ratio of 1:25 (g / ml);

[0034] Step 3: According to the activity ratio of cellulase 200 U / g, pectinase 150 U / g, and papain 50 U / g of 3:2:1, weigh a total concentration of the composite enzyme of 1.0% w / v (i.e., 2.5 g of the composite enzyme), inject the enzyme solution into the enzymatic hydrolysis reactor through a metering pump, and add 0.1% trehalose (0.25 g) as an enzyme protectant;

[0035] Step 4: Adjust the temperature in the enzymatic hydrolysis reactor to 48°C and the pH to 5.0. First, stir it with a three-blade pitched-blade agitator, and then turn on a 25 kHz ultrasonic wave (intermittent mode: 10 seconds on / 20 seconds off) and continue enzymatic hydrolysis for 8 hours;

[0036] Step 5: Centrifuge the reaction solution at 4500 r / min for 18 minutes with a horizontal screw centrifuge and collect the supernatant;

[0037] Step 6: Dynamically counter-current extract the centrifugal precipitate twice with 70% ethanol (solid-liquid ratio 1:10), 1.5 hours each time, and combine the extraction solution and the supernatant;

[0038] Step 7: Filter the combined solution by vacuum filtration and through a 0.45 μm microfiltration membrane to obtain a clear extraction solution.

[0039] In this example:

[0040] Phloridzin yield: 87.3%, purity 98.5%;

[0041] Activity retention rate: 98.2% (detected by HPLC).

[0042] Example 2:

[0043] Group 1: Set the enzymatic hydrolysis temperature in Example 1 to 45°C and the pH to 4.5, and keep the other conditions unchanged for extraction operation;

[0044] Group 2: Without changing the enzymatic hydrolysis temperature in Example 1, perform the extraction operation. The enzymatic hydrolysis temperature is set at 48 °C and pH 5.0.

[0045] Group 3: Set the enzymatic hydrolysis temperature in Example 1 to 50 °C and pH 5.5, and keep the other conditions unchanged, then perform the extraction operation.

[0046] Results:

[0047] The extraction rate of Group 1 is 3.8% (low temperature leads to insufficient enzyme activity).

[0048] The extraction rate of Group 2 is 4.5%.

[0049] The extraction rate of Group 3 is 4.0% (high temperature causes partial enzyme inactivation).

[0050] Conclusion: 48 °C and pH 5.0 are the optimal conditions.

[0051] Example 3:

[0052] Group 1: Without changing the condition data in Example 1, perform the extraction operation. Cellulase:Pectinase:Papain = 3:2:1.

[0053] Group 2: Change the proportion of the composite enzyme components in Example 1 to Cellulase:Pectinase:Papain

[0054] = 2:2:1, and keep the other conditions unchanged, then perform the extraction operation.

[0055] Group 3: Change the composite enzyme components in Example 1 to a single cellulase (without pectinase and protease), and keep the other conditions unchanged, then perform the extraction operation.

[0056] Results:

[0057] The extraction rate of Group 1 is 4.5%.

[0058] The extraction rate of Group 2 is 3.9% (insufficient pectinase leads to pectin residue).

[0059] The extraction rate of Group 3 is 2.1% (a single enzyme cannot break the cell wall sufficiently).

[0060] Conclusion: The compounding ratio of 3:2:1 has a synergistic effect.

[0061] Example 4:

[0062] Experimental group: Without changing the condition data in Example 1, perform the extraction operation (including ultrasonic assistance).

[0063] Control group: Under the same conditions but with the ultrasonic wave turned off

[0064] Results: The extraction rate of the experimental group was 4.5%, and that of the control group was 3.0%.

[0065] Conclusion: Ultrasonic waves can increase the enzymatic hydrolysis efficiency by 66.7% (accelerating mass transfer through cavitation effects).

[0066] Only some exemplary embodiments of the present invention have been described above by way of illustration. Without doubt, for those of ordinary skill in the art, the described embodiments can be modified in various different ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for extracting phloridzin assisted by a composite enzyme, characterized in that, It includes the following steps: Step 1: Select plant raw materials rich in phloridzin and conduct pretreatment; Step 2: Weigh the pretreated raw material powder, add it to an enzymatic hydrolysis reactor, and add citric acid-sodium citrate buffer solution according to a solid-liquid ratio of 1:20 - 1:30 g / ml; Step 3: Accurately weigh cellulase, pectinase, and papain with an electronic balance, compound them into an enzymatic hydrolysis system according to a certain ratio, and inject the enzyme solution into the enzymatic hydrolysis reactor through a metering pump. The total concentration of the complex enzyme is 0.5% - 1.5% w / v. At the same time, inject 0.1% trehalose solution into the enzymatic hydrolysis reactor; Step 4: Adjust the temperature inside the enzymatic hydrolysis reactor in real time to make its internal temperature 45 - 50 °C and the pH value 4.5 - 5.5, and turn on the ultrasonic generator to work, and continue enzymatic hydrolysis for 8 hours; Step 5: After the enzymatic hydrolysis reaction ends, the enzymatic hydrolysis reactor discharges the reaction solution into a horizontal screw centrifuge. The reaction solution is centrifuged at 4000 - 5000 r / min for 15 - 20 minutes, and the supernatant is collected; Step 6: Extract the precipitate produced in Step 5 with an appropriate amount of ethanol multiple times, with each extraction time being 1 - 2 hours, and combine the extraction solution and the supernatant; Step 7: Filter the combined extraction solution with a vacuum pump and a filtration device to remove insoluble impurities, and obtain a clear extraction solution.

2. The method for extracting phloridzin assisted by a composite enzyme according to claim 1, wherein: In Step 1: The plant raw materials can be apple tree root bark, stems, or young leaves; The pretreatment is: Wash the plant raw materials and dry them to constant weight in an oven at 60 °C, then use a shear crusher to crush the dried raw materials, and pass through a 40 - 60 mesh sieve to obtain a uniform powder.

3. A method for extracting phloridzin assisted by a composite enzyme according to claim 1, characterized in that: In Step 3: Cellulase 200 U / g, pectinase 150 U / g, and protease 50 U / g are compounded according to a ratio of 3:2:

1.

4. The method for assisted extraction of phloridzin by a composite enzyme according to claim 1, wherein: In Step 4: The ultrasonic generator is a 25 kHz ultrasonic generator, the ultrasonic power density is 30 W / L, and the ultrasonic action interval is to apply for 10 minutes every 30 minutes.

5. A method for extracting phloridzin assisted by a composite enzyme according to claim 1, characterized in that: In Step 4: A three-blade pitched-blade agitator and a jacketed water circulation system are configured inside the enzymatic hydrolysis reactor.

6. The method for assisted extraction of phloridzin by a composite enzyme according to claim 1, wherein: In Step 4: The probe of the ultrasonic generator is a titanium alloy horn probe, which is evenly distributed at the bottom of the inner cavity of the enzymatic hydrolysis reactor.

7. A method for extracting phloridzin assisted by a composite enzyme according to claim 1, characterized in that: In Step 5: The centrifuge tube used in the horizontal screw centrifuge can be made of corrosion-resistant polypropylene material, and the supernatant needs to be stored in a light-proof container.

8. A method for assisted extraction of phloridzin by a composite enzyme according to claim 1, characterized in that: In Step 7: After filtration, add a 0.45 μm microfiltration membrane for filtration.

9. A method for extracting phloridzin assisted by a composite enzyme according to claim 1, characterized in that: The enzymatic hydrolysis reactor is a 500 L stainless steel reactor with three layers of sieve plates inside. The pore diameters of the three layers of sieve plates are 0.5 mm, 0.8 mm, and 1.0 mm respectively, and the surface of the sieve plates is coated with a polytetrafluoroethylene coating.

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