Nano-fiber membrane immobilized enzyme of epsilon-polylysine combined with chitosan as well as preparation and application of nano-fiber membrane immobilized enzyme
Nanofiber membranes were prepared by the esterification reaction of ε-polylysine and chitosan, and dialdehyde chitosan immobilized naringidase was solved, and the problems of enzyme instability and toxicity of crosslinking agents in the prior art were solved, thus achieving efficient and environmentally friendly juice treatment effects.
Patent Information
- Application Number
- CN202411861258.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-03-25
AI Technical Summary
The prior art has problems such as instability, high cost, difficult to recover and recycle when removing bitterness and clarifying in citrus juices, and traditional crosslinking agents such as glutaraldehyde are toxic and affecting enzyme activity.
Nanofiber membranes were prepared by the esterification reaction of ε-polylysine and chitosan, and bisaldehyde chitosan was used as a green crosslinking agent to immobilize naringidase on the nanofiber membrane to form nanofiber membrane immobilization enzymes that bind ε-polylysine to chitosan.
It achieves efficient bitterness, clarification and antibacteriality of citrus juice, improves the stability and reuse of enzymes, reduces production costs, and avoids the toxicity problem of traditional crosslinking agents.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of food processing and provides an ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme and a preparation method and application thereof. Background Art
[0002] Citrus fruits have a unique flavor and high nutritional value. They are rich in nutrients such as vitamin A, vitamin C, vitamin B1, B2, etc. The peel contains pectin and dietary fiber, and the pulp is rich in organic acids. It also contains rich bioactive substances such as flavonoids, which have antioxidant capacity and are beneficial to health. Citrus fruits are suitable for processing into juice products because of their plump and juicy flesh and high water content. However, the content of naringin and limonin will increase during the juicing and sterilization process. These two substances are the main reasons for the bitterness of citrus juice, which seriously affects the taste and flavor of the juice.
[0003] Naringinase is often used to debitter and degrade juice. It is a hydrolytic multi-enzyme complex with β-D-glucosidase and α-L-rhamnosidase activities. The debittering and degradation reaction process of naringinase is divided into two steps, namely, α-L-rhamnosidase hydrolyzes naringin to produce prunin and rhamnose, and β-D-glucosidase hydrolyzes prunin to produce naringenin and glucose. Naringenin is a non-bitter substance, thus achieving the effect of debittering. In production and use, free naringinase has the disadvantages of instability, high cost, and difficulty in recovery and recycling. The immobilized enzyme method can improve the stability and rigidity of the enzyme, increase the number of reuses, facilitate the separation of juice and enzyme, and greatly reduce the production cost.
[0004] With the development of nanotechnology, nanoscale immobilized carriers have been widely used. Nanofibers prepared by electrospinning technology have a very large surface area to volume ratio, excellent flexibility, small pore size and high porosity. Chitosan is a natural, non-toxic, biodegradable polysaccharide with multiple active groups such as hydroxyl and amino groups. The positively charged chitosan molecules interact with the negatively charged microbial surface through electrostatic interaction, changing the permeability of the microbial membrane, thereby achieving an antibacterial effect. However, chitosan is insoluble in aqueous solution, which limits its use. ε-Polylysine is a natural antibacterial agent that is easily biodegradable, non-toxic, edible, sustainable, and has good water solubility. The hydroxyl groups of chitosan and the carboxyl groups of ε-Polylysine are combined through an esterification reaction, in which the carboxyl groups are activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), which can improve the reaction efficiency. Natural chitosan has a large number of amino groups on its molecular chain, which is positively charged in an acidic environment. It can neutralize the charge and bridge the particles in the juice, causing the particles to flocculate into agglomerates, thus achieving the effect of clarifying the juice. Modification of chitosan can improve its water solubility, enhance its antibacterial properties, and enhance the clarification effect. Modified chitosan has broad application prospects in the field of juice clarification.
[0005] At present, the combination of immobilized carrier and naringinase usually uses a cross-linking agent. Glutaraldehyde is widely used as one of the cross-linking agents. It can form strong covalent bonds with carriers and enzymes, is easy to obtain and has low cost. However, glutaraldehyde is toxic, which limits its application concentration, resulting in a low binding rate between enzyme and carrier, and it is easy to form agglomerates during the cross-linking process and bind to the enzyme active site, reducing or losing the enzyme activity. Dialdehyde chitosan is a green, non-toxic polysaccharide. The aldehyde group reacts with the amino group of the immobilized carrier and the amino group of the enzyme to form a strong covalent bond, which can fix the enzyme on the carrier. Summary of the invention
[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a green cross-linked, high-stability, good flocculation effect, synergistic antibacterial ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme and its preparation method and application in juice processing. The immobilized enzyme also has debittering, clarifying and antibacterial effects.
[0007] In order to achieve the above object, the present invention adopts the following technical solution:
[0008] In a first aspect, the present invention provides an ε-polylysine-bound chitosan nanofiber membrane immobilized enzyme, which is prepared by the following method: using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to activate the carboxyl group of ε-polylysine, and then performing an esterification reaction with the hydroxyl group of chitosan, and using the obtained reaction solution as an electrospinning solution for electrospinning to obtain an ε-polylysine-bound chitosan nanofiber membrane; in an acidic environment, the amino group on the ε-polylysine-bound chitosan nanofiber membrane and the amino group of naringinase are respectively combined with two aldehyde groups on the cross-linking agent dialdehyde chitosan to form strong covalent bonds, so that naringinase is fixed on the ε-polylysine-bound chitosan nanofiber membrane, and the ε-polylysine-bound chitosan nanofiber membrane immobilized enzyme is obtained.
[0009] In a second aspect, the present invention provides a method for preparing an enzyme immobilized on a nanofiber membrane of ε-polylysine combined with chitosan according to the first aspect, comprising the following steps:
[0010] (1) Preparation of ε-polylysine combined with chitosan nanofiber membrane by electrospinning: ε-polylysine was dissolved in distilled water to prepare a 6-8 wt % ε-polylysine solution; chitosan powder was dissolved in 1-4 vol % acetic acid aqueous solution to obtain a 2-4wt% chitosan solution; mixing the chitosan solution and the ε-polylysine solution in a volume ratio of 20:60-100, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to the mixed solution, stirring at 75-85°C for 1-2h to obtain an electrospinning solution, and obtaining a nanofiber membrane of ε-polylysine combined with chitosan through an electrospinning process;
[0011] (2) adding 1-4 g of ε-polylysine combined with chitosan nanofiber membrane to 80-140 mL of naringinase solution to obtain a mixture, wherein the naringinase solution is obtained by dissolving naringinase in acetate buffer, wherein the naringinase concentration is 2-3 mg / mL and the pH value of the acetate buffer is 4.5-5.0; further mixing the mixture with a cross-linking agent, dialdehyde chitosan, and shaking in a water bath at 25-35° C. for 90-180 min at a shaking speed of 80-100 r / min, and then washing with acetate buffer to obtain an ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme.
[0012] Preferably, in step (1), the amount of EDC added is 1-15 mg / mL, more preferably 4-7 mg / mL.
[0013] Preferably, in step (1), the volume concentration of acetic acid in the acetic acid aqueous solution is 1-3%, more preferably 2%.
[0014] Preferably, in step (1), a plastic syringe equipped with a metal needle is filled with the electrospinning solution, the needle of the syringe is connected to a high voltage power supply, and the parameters of the electrospinning process are specifically set as follows: the voltage is 3-30KV, the flow rate of the electrospinning solution is 1-5mL / h, and the needle is 5-30cm away from the collector.
[0015] Preferably, in step (2), the feed ratio of the ε-polylysine combined with chitosan nanofiber membrane and the naringinase solution is (1-2) g: (80-100) mL; the added mass of the cross-linking agent dialdehyde chitosan accounts for 2-4% of the mass of the mixture.
[0016] In the present invention, the dialdehyde chitosan is prepared by the following method: sodium metaperiodate and chitosan are mixed, the pH value of the mixture is adjusted to 3.5 using sodium bicarbonate and hydrochloric acid (diluted), reacting at 40° C. in the dark for 90 minutes, and terminating the reaction by adding ethylene glycol; then, isopropyl alcohol (IPA) is used to precipitate to form a cross-linking agent, and vacuum filtration is used to separate.
[0017] In a third aspect, the present invention provides the use of the ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme in the first aspect in juice processing. In this application, the ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme can simultaneously play the role of clarification, debittering and antibacterial.
[0018] Preferably, in the juice treatment, the pH of the juice is adjusted to 4.0-6.0, the treatment temperature is 40-45° C., and the treatment time is 1-1.5 h.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] 1. The nanofiber membrane prepared by combining ε-polylysine with chitosan provided by the present invention has the advantages of high surface area ratio, excellent toughness, small pore size and high porosity. ε-polylysine is a natural antibacterial agent with the characteristics of broad antibacterial spectrum, good water solubility, high safety and good thermal stability. The combination of the two has a bactericidal effect. At the same time, it contains a large number of amino groups. In an acidic environment, the surface of the nanofiber membrane is positively charged, and charge neutralization and bridging adsorption occur with the particles in the juice, and flocculation precipitation occurs to achieve a clarifying effect.
[0021] 2. The ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme provided by the present invention can clarify and debitter juice at the same time, and has an antibacterial effect. Since the surface of the nanofiber membrane is positively charged under acidic conditions, naringinase is also positively charged, and the same charges repel each other, they are connected by using green and non-toxic dialdehyde chitosan as a cross-linking agent. The dialdehyde group of the cross-linking agent reacts with the amino group of the nanofiber membrane and the amino group of the naringinase to produce a strong covalent bond. The loading amount and activity of the enzyme are increased. While naringinase decomposes naringin in the juice, the ε-polylysine combined with the chitosan nanofiber membrane clarifies and sterilizes the juice. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The present invention is a schematic diagram of the preparation and application of the nanofiber membrane immobilized enzyme using ε-polylysine combined with chitosan.
[0023] Figure 2 This is a comparison chart of the debittering performance of the enzyme immobilized on the nanofiber membrane of ε-polylysine combined with chitosan prepared in Example 1-2.
[0024] Figure 3 This is a comparison chart of the antibacterial properties of the nanofiber membrane-immobilized enzymes prepared in Examples 1 and 3 and Comparative Example 1. DETAILED DESCRIPTION
[0026] The technical solution of the present invention is further described below through specific embodiments, but the protection scope of the present invention is not limited thereto.
[0027] If no specific conditions are specified in the examples of the present invention, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained by conventional technical means or purchased commercially.
[0028] Example 1
[0029] (1) Preparation of ε-polylysine combined with chitosan nanofiber membrane by electrospinning:
[0030] ε-polylysine was dissolved in distilled water to prepare a 6wt% ε-polylysine solution. Chitosan powder was dissolved in a 2% acetic acid aqueous solution by stirring for 2h at room temperature to prepare a 3wt% chitosan solution. The chitosan solution and the ε-polylysine solution were mixed at a volume ratio of 20:80, 5mg / mL EDC was added to the mixed solution, and stirred at 80°C for 1h to obtain an electrospinning solution.
[0031] A plastic syringe equipped with a metal needle was filled with an electrospinning solution, and the needle of the syringe was connected to a high-voltage power supply for electrospinning to obtain a nanofiber membrane of ε-polylysine combined with chitosan. The electrospinning process parameters were: voltage of 20KV, electrospinning solution flow rate of 2mL / h, and the needle was 20cm away from the collector.
[0032] (2) Preparation of ε-polylysine-chitosan nanofiber membranes for enzyme immobilization
[0033] A sodium metaperiodate (0.1 M) solution was prepared using a 0.1 M sodium acetate buffer solution (pH = 5.0), 25 mg of chitosan was added to 10 mL of the sodium metaperiodate solution and stirred, the pH value of the mixture was adjusted to 3.5 using 0.5 mol / L sodium bicarbonate and hydrochloric acid, the mixture was reacted at 40° C. in the dark for 90 min, and the reaction was terminated by adding 0.3 mL of ethylene glycol; then, isopropyl alcohol (IPA) was used to precipitate a cross-linking agent, and vacuum filtration was used to separate to obtain dialdehyde chitosan.
[0034] 2 g of ε-polylysine combined with chitosan nanofiber membrane was added to 100 mL of naringinase solution to obtain a mixture, wherein the naringinase concentration was 2 mg / mL and the pH value of the acetate buffer was 4.5; 2% by mass of dialdehyde chitosan was added to the mixture for further mixing, and the mixture was shaken in a 30°C water bath for 150 min at a shaking speed of 100 r / min, and then washed with acetate buffer to obtain ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme.
[0035] Example 2
[0036] Refer to Example 1, except that: the shaking time of the mixture in step (2) is adjusted to 90 min, 120 min, and 180 min, and the rest is exactly the same as Example 1 to obtain ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme.
[0037] Example 3
[0038] Referring to Example 1, the difference is that in step (1), the concentration of ε-polylysine is adjusted to 6.5%, 7.0%, and 7.5%, and the rest is exactly the same as Example 1 to obtain ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme.
[0039] Comparative Example 1
[0040] (1) Preparation of chitosan nanofiber membrane by electrospinning:
[0041] At room temperature, chitosan powder was dissolved in 2% acetic acid aqueous solution by stirring for 2 h to prepare a 3 wt% chitosan solution. The chitosan solution and distilled water were mixed at a volume ratio of 20:80, 5 mg / mL EDC was added to the mixed solution, and stirred at 80°C for 1 h to obtain an electrospinning solution.
[0042] A plastic syringe equipped with a metal needle was filled with an electrospinning solution, and the needle of the syringe was connected to a high-voltage power supply for electrospinning to obtain a chitosan nanofiber membrane. The electrospinning process parameters were: voltage of 20KV, electrospinning solution flow rate of 2mL / h, and the needle was 20cm away from the collector.
[0043] (2) Preparation of chitosan nanofiber membranes for enzyme immobilization
[0044] 2 g of chitosan nanofiber membrane was added to 100 mL of naringinase solution to obtain a mixture, wherein the naringinase concentration was 2 mg / mL and the pH value of the acetate buffer was 4.5; 2% by mass of dialdehyde chitosan was added to the mixture for further mixing, and the mixture was shaken in a 30°C water bath for 150 min at a shaking speed of 100 r / min, and then washed with acetate buffer to obtain chitosan nanofiber membrane-immobilized enzyme.
[0045] The debittering performance of the ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme prepared in Examples 1-3 was tested, and the specific testing method was as follows: the pomelo purchased from the local market was washed, seeded and flattened, and then the obtained crude liquid was centrifuged to remove suspended particles, and the pH of the juice was adjusted to 4-6 by adding hydrochloric acid or ascorbic acid. The ε-polylysine combined with chitosan nanofiber membrane immobilized enzymes prepared with different shaking times prepared in Examples 1 and 2 were placed in conical flasks containing known amounts of juice. The mixture was then incubated at 45-50°C and stirred continuously for 45 minutes. After incubation, the obtained grapefruit juice was further tested.
[0046] Naringin in grapefruit juice was determined by high pressure liquid chromatography (HPLC). A Poroshell 120EC-C18 column (4.6×100 mm, 2.7 μm, Agilent Technologies) was used with acetonitrile-water as the mobile phase. The sample was introduced into the column by an injector equipped with a 20 μL sample loop. A Perkin Elmer UV-visible detector (Perkin Elmer mod. LC 295) was used to determine naringin at 283 nm. The column temperature was 25 °C and the flow rate was fixed at 1.0 mL / min. The grapefruit juice was diluted with acetonitrile in a ratio of 1:2, filtered through a 0.45 μm filter, and then 10 μL aliquots of the filtrate were injected. The amount of naringin in grapefruit juice was obtained by calibrating their corresponding standard curves.
[0047] The chitosan nanofiber membrane immobilized enzyme prepared in Comparative Example 1 was used as a blank example, and the antibacterial properties of the ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme prepared in Examples 1 and 3 were tested. The specific testing method was as follows: ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme was added to pomelo juice, and the chitosan nanofiber membrane immobilized enzyme without ε-polylysine was used as a control group. The determination was carried out according to the method given in the "National Food Safety Standard Food Microbiology Test Colony Count Determination" (GB 4789.2-2016).
Claims
1. An ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme, characterized in that: The ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme is prepared by the following method: using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to activate the carboxyl group of ε-polylysine, and then carrying out an esterification reaction with the hydroxyl group of chitosan, and using the obtained reaction solution as an electrospinning solution for electrospinning to obtain the ε-polylysine combined with chitosan nanofiber membrane; in an acidic environment, the amino groups on the ε-polylysine combined with chitosan nanofiber membrane and the amino groups of naringinase are respectively combined with two aldehyde groups on the cross-linking agent dialdehyde chitosan to form strong covalent bonds, so that the naringinase is fixed on the ε-polylysine combined with chitosan nanofiber membrane, and the ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme is obtained.
2. A method for preparing the ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme as claimed in claim 1, comprising the following steps: (1) Preparing ε-polylysine combined with chitosan nanofiber membrane by electrospinning: dissolving ε-polylysine in distilled water to prepare a 6-8wt% ε-polylysine solution; dissolving chitosan powder in a 1-4vol% acetic acid aqueous solution to obtain a 2-4wt% chitosan solution; mixing the chitosan solution and the ε-polylysine solution in a volume ratio of 20:60-100, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the mixed solution, stirring at 75-85°C for 1-2h to obtain an electrospinning solution, and obtaining an ε-polylysine combined with chitosan nanofiber membrane by an electrospinning process; (2) adding 1-4 g of ε-polylysine combined with chitosan nanofiber membrane to 80-140 mL of naringinase solution to obtain a mixture, wherein the naringinase solution is obtained by dissolving naringinase in acetate buffer, wherein the naringinase concentration is 2-3 mg / mL and the pH value of the acetate buffer is 4.5-5.0; further mixing the mixture with a cross-linking agent, dialdehyde chitosan, and shaking in a water bath at 25-35° C. for 90-180 min at a shaking speed of 80-100 r / min, and then washing with acetate buffer to obtain an ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme.
3. The preparation method according to claim 2, characterized in that: In step (1), the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride added is 1-15 mg / mL.
4. The preparation method according to claim 3, characterized in that: In step (1), the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride added is 4-7 mg / mL.
5. The preparation method according to claim 2, characterized in that: In step (1), a plastic syringe equipped with a metal needle is filled with an electrospinning solution, the needle of the syringe is connected to a high voltage power supply, and the parameters of the electrospinning process are specifically set as follows: the voltage is 3-30KV, the flow rate of the electrospinning solution is 1-5mL / h, and the needle is 5-30cm away from the collector.
6. The preparation method according to claim 2, characterized in that: In step (2), the feed ratio of the ε-polylysine combined with chitosan nanofiber membrane and the naringinase solution is (1-2) g: (80-100) mL; the added mass of the cross-linking agent dialdehyde chitosan accounts for 2-4% of the mass of the mixture.
7. Use of the ε-polylysine combined with chitosan nanofiber membrane immobilized enzyme as claimed in claim 1 in fruit juice processing.
8. The use according to claim 7, characterized in that: In the juice treatment, the pH of the juice is adjusted to 4.0-6.0, the treatment temperature is 40-45° C., and the treatment time is 1-1.5 hours.