Method for preparing hydroxytyrosol by combining compound enzyme with molecular imprinting adsorption

Abstract

The invention is applicable to the technical field of hydroxytyrosol preparation, and provides a method for preparing hydroxytyrosol by combining compound enzyme with molecular imprinting adsorption, which comprises the following steps: crushing fresh olive fruits, and collecting olive fruit pulp; putting the olive pulp into an enzymolysis tank, adding cellulose-pectin compound enzyme and citric acid, and performing enzymolysis; transferring the olive pulp subjected to enzymolysis into a microwave extraction tank, adding purified water, carrying out intermittent irradiation extraction, centrifuging after the extraction is completed, collecting supernate, and carrying out vacuum concentration; and adsorbing the concentrated supernate by using a chromatographic column filled with a hydroxytyrosol imprinted polymer, eluting with water and an ethanol solution in sequence, collecting ethanol eluent, and concentrating to obtain extract, namely the high-purity hydroxytyrosol. The problems that an existing fermentation method is low in yield and high in cost are solved. Generation of byproducts is reduced, and the separation and purification process is simplified; green and sustainable hydroxytyrosol industrial production is realized; and the purification period is shortened to be within 5 hours.

Description

Technical Field

[0001] This invention belongs to the field of hydroxytyrosol preparation technology, and particularly relates to a method for preparing hydroxytyrosol using a combination of complex enzymes and molecular imprinted adsorption. Background Technology

[0002] Hydroxytyrosol, chemically known as 3,4-dihydroxyphenylethanol, has the molecular formula C8H10H2O. 10 O3, a natural polyphenol compound, possesses strong antioxidant activity and is widely used in the food, pharmaceutical, and cosmetic industries. Hydroxytyrosol mainly exists in the form of esters in olive fruits and leaves, but traditional methods for extracting hydroxytyrosol from olive fruits have the following drawbacks:

[0003] Organic solvents (such as methanol) have low extraction efficiency (yield <2%), and solvent residues affect product safety.

[0004] Conventional resins have poor adsorption selectivity, require multiple purification processes, and have complex processes.

[0005] High-temperature extraction leads to the oxidative degradation of hydroxytyrosol, resulting in a loss of more than 30% of its activity.

[0006] Existing technologies also include ultrasound-assisted ethanol extraction, which can increase the yield by 3%, but consumes a large amount of solvent; macroporous resin adsorption purification, which can achieve a purity of 90%, but requires 5 elutions and takes more than 8 hours; and supercritical CO2 extraction, which leaves no solvent residue, but has high equipment costs and is difficult to industrialize. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing hydroxytyrosol using a combination of complex enzymes and molecularly imprinted adsorption, thereby addressing the problems mentioned in the background section.

[0008] The present invention is implemented as follows: a method for preparing hydroxytyrosol using a complex enzyme combined with molecular imprinting adsorption includes the following steps:

[0009] S1. Crush fresh olives and collect olive pulp;

[0010] S2. Place the collected olive pulp in an enzymatic hydrolysis tank, add cellulose pectin complex enzyme and citric acid, and carry out enzymatic hydrolysis.

[0011] S3. Transfer the enzymatically hydrolyzed olive pulp into a microwave extraction vessel, add purified water, and perform intermittent irradiation extraction. After extraction, centrifuge and collect the supernatant, and then concentrate it under vacuum to one-third of the original volume.

[0012] S4. The concentrated supernatant is subjected to adsorption on a chromatography column packed with hydroxytyrosol imprinted polymer, and eluted sequentially with water and ethanol solutions. The ethanol eluent is collected and concentrated to a paste-like consistency, which is high-purity hydroxytyrosol.

[0013] This invention provides a method for preparing hydroxytyrosol using a combination of complex enzymes and molecularly imprinted adsorption. For the first time, it systematically integrates three technologies—complex enzymatic hydrolysis, microwave-assisted extraction, and molecularly imprinted adsorption—to form a synergistic and complete process chain, rather than simply adding them together. By using highly selective molecularly imprinted polymers (MIPs) to replace traditional macroporous resins, the purity of the product is increased from approximately 90% to over 98%, while the elution steps are reduced from 5 to 1. Attached Figure Description

[0014] Figure 1 This is a three-dimensional structural diagram of a method for preparing hydroxytyrosol using a composite enzyme combined with molecular imprinting adsorption, provided in an embodiment of the present invention.

[0015] Figure 2 This is a front view of a method for preparing hydroxytyrosol using a combination of complex enzymes and molecular imprinting adsorption, provided as an embodiment of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] A method for preparing hydroxytyrosol using a complex enzyme-linked molecularly imprinted adsorption method includes the following steps:

[0018] S1. Wash the freshly grown olives, crush them using a hammer mill, and collect the olive pulp.

[0019] S2. Place the collected olive pulp in an enzymatic hydrolysis tank, and add cellulosic pectin complex enzyme (the ratio of cellulosic enzyme to pectinase is 3:1 (W / W), and the total enzyme activity is ≥200U / g substrate) and citric acid (add until the pH of the mixed solution is about 5.0, then stop adding), and carry out enzymatic hydrolysis.

[0020] S3. Transfer the enzymatically hydrolyzed olive pulp into a microwave extraction tank, add purified water (the amount added is 10 times the mass of the olive pulp), and perform intermittent irradiation extraction (microwave power is 300W, time is 3 minutes on and 1 minute off, for a total duration of 20 minutes). After extraction, centrifuge (using a screw discharge centrifuge) and collect the supernatant. Vacuum concentrate to one-third of the original volume at a temperature of 65℃.

[0021] S4. The concentrated supernatant was subjected to adsorption on a chromatography column packed with hydroxytyrosol imprinted polymer (adsorption capacity 350 mg / g, adsorption time 30 min). The supernatant was eluted sequentially with water (volume 1-5 BV) and ethanol solution (volume concentration 40%). The ethanol eluent was collected and concentrated to a paste state (water content ≤0.5%), which is high-purity hydroxytyrosol.

[0022] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0023] Example 1: A method for preparing hydroxytyrosol using a composite enzyme pretreatment combined with molecular imprinting adsorption, comprising the following steps:

[0024] S1. Wash and crush the artificially cultivated olives to obtain 200 kg of olive pulp for later use;

[0025] S2. Place olive pulp in an enzymatic hydrolysis tank, add 2kg of cellulose pectin complex enzyme and 200g of citric acid in a ratio of 3:1 (W / W), and stir for 2 hours for enzymatic hydrolysis.

[0026] S3. Take out the enzymatically hydrolyzed olive pulp and place it in a microwave extraction tank. Add 2000L of purified water and microwave extract intermittently for 20 minutes. Centrifuge to obtain 1700L of supernatant, which is then concentrated to obtain 670L.

[0027] S4: Load the 670L concentrate onto a hydroxytyrosol-imprinted polymer chromatography column with a column diameter-to-height ratio of 1:10. After adsorption for 30 min, elute with 4 BV of purified water and then with 3 BV of 40% ethanol. Collect the ethanol eluent and concentrate it to a paste with a water content of less than 0.5% to obtain 8.2 kg of hydroxytyrosol.

[0028] The test results of the sample prepared in Example 1 were as follows: Figure 1 As shown, the purity is 98.23% and the yield is 4.1%.

[0029] Comparative Analysis: A parallel comparative experiment was conducted using the closest existing technical solution (resin adsorption purification method). The same batch of enzymatic hydrolysis concentrate was used, and purification was performed using conventional AB-8 macroporous resin. The specific procedure was as follows: sample loading → water washing → elution with 10% ethanol → elution with 30% ethanol → elution with 50% ethanol → elution with 70% ethanol. The 70% ethanol eluent was collected and concentrated. Liquid chromatography analysis showed the following results: Figure 2 As shown, the purity is 90.15% and the yield is 3.01%.

[0030] In summary, the method proposed in this embodiment of the invention improves cell wall disruption efficiency through enzymatic hydrolysis-microwave synergy, exhibits strong MIP adsorption specificity, achieves one-step purification, is environmentally friendly and beneficial, and complies with FDA / EMA standards; the final yield of hydroxytyrosol is 4.1%, with a purity of 98.23%, and the solvent consumption is reduced by 60% (only ethanol elution is required); the total time is 5.5 hours, and energy consumption is reduced by 30%.

[0031] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements 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 preparing hydroxytyrosol using a complex enzyme combined with molecular imprinting adsorption, characterized in that, Includes the following steps: S1. Crush fresh olives and collect olive pulp; S2. Place the collected olive pulp in an enzymatic hydrolysis tank, add cellulose pectin complex enzyme and citric acid, and carry out enzymatic hydrolysis. S3. Transfer the enzymatically hydrolyzed olive pulp into a microwave extraction vessel, add purified water, and perform intermittent irradiation extraction. After extraction, centrifuge and collect the supernatant, and then concentrate it under vacuum to one-third of the original volume. S4. The concentrated supernatant is subjected to adsorption on a chromatography column packed with hydroxytyrosol imprinted polymer, and eluted sequentially with water and ethanol solutions. The ethanol eluent is collected and concentrated to a paste-like consistency, which is high-purity hydroxytyrosol.

2. The method for preparing hydroxytyrosol using a composite enzyme combined with molecular imprinting adsorption according to claim 1, characterized in that, In step S1, the crushing process uses a hammer mill.

3. The method for preparing hydroxytyrosol using a composite enzyme combined with molecular imprinting adsorption according to claim 1, characterized in that, In step S2, the weight ratio of cellulase to pectinase in the cellulose pectin complex enzyme is 3:1-1.2, and the total enzyme activity is ≥200U / g substrate; the enzymatic hydrolysis time is 2-2.5 hours.

4. The method for preparing hydroxytyrosol using a complex enzyme combined with molecular imprinting adsorption according to claim 1, characterized in that, In step S2, the amount of citric acid added is sufficient to adjust the pH to 5.

0.

5. The method for preparing hydroxytyrosol using a composite enzyme combined with molecular imprinting adsorption according to claim 1, characterized in that, In step S3, the amount of purified water added is 8-12 times the mass of olive pulp.

6. The method for preparing hydroxytyrosol using a complex enzyme combined with molecular imprinting adsorption according to claim 1, characterized in that, In step S3, the microwave power extracted by intermittent irradiation is 280-320W, and the time is 3 minutes on and 1 minute off, for a total duration of 16-24 minutes.

7. The method for preparing hydroxytyrosol using a complex enzyme combined with molecular imprinting adsorption according to claim 1, characterized in that, In step S4, the adsorption capacity of the chromatography column filled with hydroxytyrosol imprinted polymer is 350 mg / g, and the adsorption time is 30 min.

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