CN114569582A - Enzyme preparation, preparation method and application thereof - Google Patents

Enzyme preparation, preparation method and application thereof Download PDF

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Publication number
CN114569582A
CN114569582A CN202210052928.9A CN202210052928A CN114569582A CN 114569582 A CN114569582 A CN 114569582A CN 202210052928 A CN202210052928 A CN 202210052928A CN 114569582 A CN114569582 A CN 114569582A
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solution
enzyme
layer
enzyme preparation
chitosan
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张洪才
徐建雄
冯苗苗
陈斌
白一菲
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Shanghai Jiaotong University
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Shanghai Jiaotong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/501—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5015—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A61K9/5042—Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5089—Processes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/02—Oxycellulose; Hydrocellulose; Cellulosehydrate, e.g. microcrystalline cellulose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)

Abstract

The invention discloses an enzyme preparation and a preparation method thereofA method, the enzyme preparation comprising: the chitosan/chitosan composite material comprises a core material comprising chitosan nanoparticles and enzyme, a first layer of wall material comprising sodium alginate and trehalose, and a second layer of wall material comprising nanocrystalline cellulose, wherein the first layer of wall material is coated on the surface of the core material, and the second layer of wall material is coated on the surface of the first layer of wall material. The preparation method comprises the following steps: (1) adding enzyme into chitosan solution, adding sodium tripolyphosphate, forming enzyme-loaded chitosan nanoparticles through ion crosslinking, (2) adding CaCl2Adding a mixed solution of sodium alginate and trehalose into the solution to form a first layer of wall material, and filtering to obtain an enzyme preparation in a single-layer microcapsule form; (3) adding the enzyme preparation into the nanocrystalline cellulose solution, and adsorbing the nanocrystalline cellulose on the surface of the first layer of wall material to form a second layer of wall material. The enzyme preparation has simple preparation method and high enzyme embedding efficiency, can obviously improve the activity of the enzyme preparation in gastrointestinal tracts, and can be used in the fields of medicines, agriculture, foods, feeds and the like.

Description

Enzyme preparation, preparation method and application thereof
Technical Field
The invention relates to an enzyme preparation embedding material, in particular to a novel enzyme preparation in a double-layer microcapsule form, a preparation method and application thereof.
Background
The enzyme preparation is a nontoxic and green bioactive substance, has the functions of catalyzing decomposition, improving the intestinal environment, promoting the digestion and absorption of nutrient substances, eliminating free radicals, protecting epithelial cells of the intestinal tract and the like, and is easy to be influenced by factors such as heavy metal ions, pH, surfactants, temperature and the like to reduce the activity and even inactivate the activity. How to prevent the enzyme from being inactivated in food or feed processing has important significance.
Chitosan is a biopolymer obtained by deacetylating chitin, has the advantages of no toxicity, biocompatibility, biodegradability, abundant hydroxyl and amino groups, wide sources and the like, and is widely applied to the field of enzyme preparation embedding. The patent office inquires Chinese patent publication numbers: CN101664111B discloses a preparation method of chitosan nanoparticle embedded alpha-galactosidase used in feed and formed by taking chitosan and sodium Tripolyphosphate (TPP) as raw materials; CN113388169A discloses a method for preparing a high-temperature-resistant composite preservative film for food by using chitosan, glycerol, montmorillonite, Tween-80, high-temperature-resistant lysozyme and holy ginger essential oil; CN113061595A discloses a method for immobilizing phospholipase C by using epoxy group modified magnetic chitosan nanoparticles as a carrier; CN113652415A publicThe method comprises the steps of attaching chitosan to the surface of an egg shell membrane and carrying out coprecipitation on magnetic Fe3O4Uniformly depositing and distributing on the eggshell membrane to prepare magnetic Fe3O4the/CS/ESMP is used as an enzyme carrier for immobilizing the beta-glucosidase; CN107699558B discloses a method for preparing a double-layer immobilized enzyme with chitosan and sodium alginate as base materials. However, most of the methods for embedding enzyme preparations by using chitosan as a wall material disclosed by the prior art are single-layer embedding, the release speed is high, sustained release cannot be realized in gastrointestinal tracts, and for example, the yield of the chitosan nanoparticle embedded enzyme preparation is low, which is not beneficial to industrial production.
Nanocrystalline Cellulose (CNC) is a rigid rod-like particle obtained by hydrolyzing cellulose with sulfuric acid or enzyme, has the advantages of wide source, small size, large specific surface area, low cost, no toxicity, biodegradability, biocompatibility and the like, and is widely applied to the aspects of biomedicine, agriculture, food, cosmetics and the like. The patents of embedding enzyme preparation by using CNC as wall material disclosed by the query of the national intellectual property office are less, but there is an article for stabilizing enzyme preparation by CNC, such as Deng (Food Research International 2020, 137,109380) and the like report that the stability of the enzyme is improved and the in vitro release time is prolonged by embedding beta-galactosidase by combining CNC and chitosan nanoparticles; zhang (International Journal of Biological Macromolecules,2021,191,71-78) and the like use CNC as a functional cross-linking agent, Pickering emulsifier and stabilize lysozyme embedded with quaternary ammonium salt chitosan nano particles by an ion gel method. Zhang (Carbohydrate Polymers,2020,236,115974) et al reported a preparation method of adding CNC to lysozyme-loaded beta-chitosan nanoparticles to enhance their antibacterial stability. The defects of the prior art are as follows: (1) CNC is chitosan nanoparticles loaded with stable enzyme in solution, and is difficult to produce quantitatively; (2) CNC-stabilized nanoparticle solutions have short storage times (less than 2 months).
Disclosure of Invention
In view of the above-mentioned defects in the prior art, the object of one aspect of the present invention is to provide a double-layer microcapsule embedded enzyme preparation, and to achieve the object of the present invention, the present invention adopts the following technical solutions:
an enzyme preparation, comprising:
a core material comprising chitosan nanoparticles and an enzyme,
a first layer of wall material which comprises sodium alginate and trehalose,
a second layer of wall material comprising nanocrystalline cellulose,
wherein the first layer of wall material is coated on the surface of the core material, and the second layer of wall material is coated on the surface of the first layer of wall material.
Preferably, the enzyme is selected from one or more of glucose oxidase, xylanase, phytase, protease, lipase and amylase.
More preferably, the enzyme is selected from glucose oxidase and/or xylanase and/or phytase.
Preferably, the mass ratio of the chitosan to the enzyme is 1-5: 0.2 to 0.5.
Preferably, the mass ratio of the sodium alginate to the trehalose is 10-20: 0.1 to 1.
More preferably, the mass ratio of sodium alginate to trehalose is 10-20: 1.
more preferably, the mass ratio of sodium alginate to trehalose is 15: 1.
preferably, the preparation method of the nanocrystalline cellulose comprises the following steps:
(1) dispersing microcrystalline cellulose in 50-75% sulfuric acid by mass, strongly mechanically stirring for 8-12 h at 40-60 ℃, diluting a reaction mixture with 4-12 times of water, and quenching a hydrolysis reaction;
(2) adding 20-40% by mass of H into the quenched reaction mixture in the step (1)2O2After the aqueous solution reacts for 20-40 min, centrifugally separating crystals;
(3) adding the crystal into water, washing with ultrasonic waves, centrifuging to remove excessive acid, dialyzing (molecular weight cut-off of 14000kDa), and freeze-drying to obtain the nanocrystalline cellulose.
Preferably, in the step (1) of the preparation method of the nanocrystalline cellulose, the use amount of the sulfuric acid is 1g of the microcrystalline cellulose, and 1-12 mL of the sulfuric acid is used for dispersing.
Preferably, in step (2) of the process for preparing nanocrystalline cellulose, H2O2The amount of the aqueous solution is 1g of microcrystalline cellulose 0.3-2 mLH2O2And (4) oxidizing the aqueous solution.
Another object of the present invention is to provide a process for preparing the above enzyme preparation, which comprises the steps of:
(1) preparation of a core material: adding enzyme into the chitosan solution, adding sodium tripolyphosphate, and forming enzyme-loaded chitosan nanoparticles by ionic crosslinking between chitosan and tripolyphosphate ions;
(2) coating of the first layer of wall material: adding CaCl2Adding the solution into the final solution obtained in the step (1), uniformly mixing, adding a mixed solution of sodium alginate and trehalose, adsorbing the sodium alginate and the trehalose on the surface of the chitosan nanoparticle, solidifying to form a first layer of wall material, and filtering to obtain an enzyme preparation in the form of a single-layer microcapsule;
(3) coating of the second layer of wall material: adding the enzyme preparation in the form of single-layer microcapsules into the nano-crystalline cellulose solution, adsorbing the nano-crystalline cellulose on the surface of the first layer of wall material to form a second layer of wall material, and filtering to obtain the enzyme preparation in the form of double-layer microcapsules.
Preferably, the preparation of the core material in the step (1) comprises: dissolving chitosan in 0.5-4% acetic acid solution, fully dissolving to obtain 1-10 mg/mL chitosan solution, adjusting pH to 5.0-6.0 with NaOH solution, adding enzyme, fully dissolving, centrifuging, taking supernatant, mixing 0.10-0.50 mg/mL sodium tripolyphosphate solution and the supernatant according to a volume ratio of 2-8: 1, and stirring at 100-200 rpm to obtain the enzyme-loaded chitosan nanoparticle solution. More preferably, the mass concentration of acetic acid used is 1 to 3%, most preferably 2%. More preferably, the solution of chitosan has a mass concentration of 2-3 mg/mL, most preferably 2.5 mg/mL. Preferably, the method for sufficiently dissolving chitosan in the acetic acid solution is as follows: firstly, stirring the acetic acid solution added with the chitosan for 10-30 h, and then carrying out ultrasonic treatment for 2-40 min.
Preferably, the step (2) of coating the first layer of wall material comprises:adding CaCl2Adding the solution into the chitosan nanoparticle solution loaded with the enzyme, uniformly mixing, and adding Ca2+The final molar concentration of the sodium alginate-trehalose microcapsule is 0.5-2 mol/L, a mixed solution of sodium alginate and trehalose is dripped into the microcapsule, the microcapsule is solidified for 20-40 min, and the enzyme preparation in the form of a single-layer microcapsule is obtained by filtering, wherein the mass ratio of the sodium alginate to the trehalose in the mixed solution of the sodium alginate and the trehalose is 10-20: 0.1-1 percent, the mass concentration of sodium alginate is 5-30 percent, the mass concentration of trehalose is 0.5-4 percent, and the mixed solution of sodium alginate and trehalose and Ca-containing solution2+The volume ratio of the solution (A) to (B) is 1-5: 1.
Preferably, the step (3) of coating the second-layer wall material comprises the steps of adding the enzyme preparation in the form of the single-layer microcapsule into a nanocrystalline cellulose solution with the mass concentration of 0.05-5%, slightly stirring for 0.5-2 hours, and filtering to obtain the enzyme preparation in the form of the double-layer microcapsule. More preferably, the concentration of the nanocrystalline cellulose solution is between 0.1% and 1%. Preferably, the dosage of the nano-crystalline cellulose solution is 1g of the microcapsule coated with the first layer wall prepared in the step (2) and added into 20-100 mL of the nano-crystalline cellulose solution.
The UV-Vis spectrum shows that the enzyme (xylanase and glucose oxidase 1:1 mix) was successfully embedded in the enzyme preparation in microencapsulated form according to the invention. When the concentration of the enzyme is 35mg/mL, in the enzyme preparation in the form of a single-layer microcapsule formed by only coating the core material with sodium alginate and trehalose, the embedding rates of xylanase and glucose oxidase are 83.34% and 87.16%, respectively, and CNC coating with the mass concentrations of 0.1%, 0.5% and 1% is continuously used, so that in the enzyme preparation in the form of a double-layer microcapsule, the embedding rates of xylanase are 85.92%, 88.75% and 82.83%, respectively, and the embedding rates of glucose oxidase are 86.42%, 88.34% and 86.10%, respectively. The simulated gastrointestinal tract digestion experiment shows that the enzyme preparation in the form of double-layer microcapsules formed by CNC coating can improve the activity of enzyme in the gastrointestinal tract, which is far higher than that of free enzyme. In the whole digestion process, the cumulative release rate of free enzyme is a trend of increasing firstly and then decreasing, the cumulative release rate of xylanase in the stomach stage is 49.06-51.11%, the release rate in the small intestine stage is 45-30.72%, and the cumulative release rates of glucose oxidase in the two stages are 47.61-58.67% and 47.66-32.99% respectively. The release rates of the two enzymes are always in an ascending trend in the process of simulating gastrointestinal tract digestion by the enzyme preparation in the form of the single-layer microcapsule and the enzyme preparation in the form of the double-layer microcapsule, the release rate of the double-layer microcapsule is slightly smaller than that of the enzyme preparation in the form of the single-layer microcapsule in the stomach period, and the release rates are opposite in the small intestine period.
In a further aspect, the present invention provides the use of the above enzyme preparation for the preparation of a medicament, food or feed for increasing the activity of the enzyme in the gastrointestinal tract of an animal.
The enzyme preparation is of a double-layer microcapsule structure, a core material comprises chitosan nanoparticles and enzyme, a first layer of wall material comprises sodium alginate and trehalose, and an outermost layer of wall material (a second layer of wall material) comprises CNC. Wherein the chitosan contains abundant amino and hydroxyl, the amino can generate ion crosslinking with polyanion carried by sodium tripolyphosphate through electrostatic attraction to form chitosan nanoparticles, and the COO carried by the sodium alginate-With small amounts of chitosan-NH4 +Performing electrostatic attraction on CaCl2In the presence of (a) to form a first layer of microcapsules, -OSO of the CNC surface3 -And Ca2+The CNC can be coated on the whole microsphere surface through electrostatic attraction between the CNC and the microsphere. Trehalose was used as a lyoprotectant in the above results.
According to the invention, the CNC is designed into the second layer of wall material of the microcapsule by utilizing the strong acid resistance and stability of the CNC, and the microcapsule is used for preparing the double-layer embedded enzyme preparation microcapsule, so that the enzyme can be effectively protected from being damaged and degraded by gastric acid, and the microcapsule has important application value in the fields of food, livestock raising, agriculture and the like.
Compared with the prior art, the method has the following beneficial effects:
(1) chitosan and CNC are high molecular polymers, and have the advantages of wide sources, biodegradability, biocompatibility and the like.
(2) The enzyme preparation in the form of the double-layer microcapsule enables the enzyme to realize sustained release in the gastrointestinal tract, improves the activity of the enzyme in the gastrointestinal tract, and has practical application value.
(3) Preparing double-layer microcapsules by three-time ion crosslinking, wherein the first time comprises the following steps: chitosan and TPP; and (3) for the second time: sodium alginate and CaCl 2; and thirdly: CNC and CaCl2, three-time ion cross-linked microcapsules, the structure is more compact, and the embedded enzyme can be effectively released in a targeted manner in the small intestine.
Drawings
FIG. 1 is a flow chart of a method of preparing an enzyme preparation of the present invention.
FIG. 2 is a UV-Vis spectrum of an enzyme preparation in the form of single-layer microcapsules of different enzyme concentrations. Wherein A1, A2, A3, A4, A5 are the ultraviolet-visible spectrum diagrams of the enzyme preparation containing enzyme concentrations of 20, 25, 30, 35 and 40mg/mL, respectively. The top right panel is a UV-Vis spectrum of an enzyme preparation containing the above enzyme concentrations at 200-220 nm.
FIG. 3 is a photograph of an actual enzyme preparation in the form of non-microcapsules, in which panel A shows a photograph of an enzyme preparation in the form of single-layer microcapsules prepared in example 4; panel B shows a photograph of the enzyme preparation prepared in example 1 in the form of a double-layer microcapsule coated with CNC at a mass concentration of 0.1%; panel C shows a photograph of the enzyme preparation prepared in example 2 in the form of a double-layer microcapsule coated with CNC at a mass concentration of 0.5%; panel D shows a photograph of the enzyme preparation prepared in example 3 in the form of a double-layer microcapsule coated with CNC at a mass concentration of 1%.
FIG. 4 is the encapsulation efficiency and yield of xylanase and glucose oxidase, wherein (A) is a graph showing the encapsulation efficiency of enzyme preparations in the form of single-layer microcapsules, wherein A1, A2, A3, A4, A5 represent enzyme preparations containing enzyme concentrations of 20, 25, 30, 35 and 40mg/mL, respectively; (B) the figure shows the encapsulation efficiency and yield of enzyme preparations in the form of double-layer microcapsules, in which A4, CNC0.1/A4、CNC0.5/A4、 CNC1the/A4 indicates enzyme entrapment concentrations of 35mg/mL, respectively, using CNC-coated enzyme preparations at mass concentrations of 0.1, 0.5, and 1%.
FIG. 5 is a graph showing the release effect of an enzyme preparation in a microcapsule form in PBS buffer at pH 4.5, wherein the release effects of xylanase and glucose oxidase are shown in (A) and (B), respectively, wherein A4, CNC0.1/A4、CNC0.5/A4、CNC1the/A4 indicates enzyme entrapment concentrations of 35mg/mL, respectively, using CNC-coated enzyme preparations at mass concentrations of 0.1, 0.5, and 1%.
FIG. 6 is a graph of the release effect of the microcapsules on the enzyme in PBS buffer at pH 7.4, where (A) and (B) are graphs of the release effect of xylanase and glucose oxidase, respectively, where A4, CNC0.1/A4、 CNC0.5/A4、CNC1the/A4 indicates enzyme entrapment concentrations of 35mg/mL, respectively, using CNC-coated enzyme preparations at mass concentrations of 0.1, 0.5, and 1%.
FIG. 7 is a graph showing the release effect of microcapsules on enzymes in simulated GI digestive fluid, wherein (A) and (B) are graphs showing the release effect of xylanase and glucose oxidase in simulated GI digestive fluid, respectively, wherein A4, CNC0.1/A4、CNC0.5/A4、CNC1the/A4 represents the enzyme entrapment concentration of 35mg/mL, CNC-coated enzyme preparations with mass concentrations of 0.1, 0.5 and 1%, respectively.
Detailed Description
Aiming at the condition of the prior art, the inventor of the application firstly invents the enzyme preparation in the form of a double-layer microcapsule by taking chitosan and CNC as wall materials and taking chitosan nanoparticles loaded with enzyme as core materials through deep research, and can realize the sustained release of the enzyme in the gastrointestinal tract so as to improve the enzyme activity. The preparation method of the enzyme preparation comprises the following steps of (1) preparing a core material: adding enzyme into a chitosan solution, adding sodium tripolyphosphate, and forming enzyme-loaded chitosan nanoparticles between chitosan and tripolyphosphate ions through ion crosslinking, (2) coating a first layer of wall material: adding CaCl2Adding the solution into the final solution obtained in the step (1), uniformly mixing, adding a mixed solution of sodium alginate and trehalose, adsorbing the sodium alginate and the trehalose on the surface of the chitosan nanoparticle, solidifying to form a first layer of wall material, and filtering to obtain an enzyme preparation in the form of a single-layer microcapsule; (3) coating of the second layer of wall material: adding the enzyme preparation in the form of the single-layer microcapsule into a nanocrystalline cellulose solution, adsorbing nanocrystalline cellulose on the surface of a first layer of wall material to form a second layer of wall material, and filtering to obtain the enzyme preparation in the form of the double-layer microcapsule. The present invention has been completed based on this finding.
The specific flow chart of the preparation method of the invention is shown in figure 1.
In the context of the present invention, enzymes include, but are not limited to, glucose oxidase, xylanase, phytase, protease, lipase and amylase. In one embodiment of the invention, the enzyme of the invention is xylanase and glucose oxidase, and the mass ratio of the xylanase to the glucose oxidase is 1: 1.
nanocrystalline cellulose is commercially available in the description of the invention, and may also be prepared as described herein.
In one embodiment of the invention, the preparation method of the nanocrystalline cellulose comprises the steps of dispersing 1g of microcrystalline cellulose (MCC) in 3-10 mL of sulfuric acid with the mass fraction of 50-75%, strongly mechanically stirring for 10 hours at 50 ℃, diluting a reaction mixture with 5-10 times of water, and quenching a hydrolysis reaction; then 0.5-1.6 mL of 30 wt% H is added2O2Aqueous solution, oxidized cellulose nanocrystal suspension; after reacting for 30min, centrifuging at 8000rpm for 10min to separate crystal, washing with ultrasonic wave, and centrifuging for multiple times to remove excessive acid; finally, dialyzing and freeze-drying to prepare the nanocrystalline cellulose. In this process, the sulfuric acid is preferably present in an amount of 75% by mass, and the reaction mixture is preferably diluted with 9 times the amount of water in the quenching hydrolysis reaction.
In one embodiment of the present invention, the process for preparing the enzyme preparation of the present invention comprises the steps of: (1) preparation of a core material: dissolving chitosan in 2% acetic acid solution to obtain chitosan with concentration of 2.5mg/mL, stirring overnight for 24h, then performing ultrasonic treatment for 30min to fully dissolve the chitosan, adjusting the pH value to 5.5 with NaOH solution, then adding enzyme into the solution, centrifuging, taking supernatant, mixing TPP solution with concentration of 0.25mg/mL and the supernatant according to a certain ratio (1: 5), and stirring at low speed (150rpm) to obtain chitosan nanoparticle solution loaded with enzyme; (2) preparing a first layer of wall material: the concentration of CaCl2Adding the solution into the chitosan nanoparticle solution, mixing uniformly, and adding Ca2+Adding dropwise a mixed solution of sodium alginate (1% by mass) and trehalose (15% by mass) into the solution at a final molar concentration of 1mol/L, solidifying for 30min, and filtering to obtain enzyme preparation in the form of single-layer microcapsule containing sodium alginate and trehaloseMixing the solution with Ca2+The volume ratio of the solution (a) to (b) is 1: 1; (3) preparing a second layer of wall material: adding the enzyme preparation in the form of the single-layer microcapsule into a CNC solution with the mass concentration of 0.1-1%, slightly stirring, reacting for 1h, filtering to obtain the enzyme preparation in the form of the CNC-coated double-layer microcapsule, and freeze-drying.
In the description of the present invention, "solution" generally refers to an aqueous solution unless otherwise specified.
In the description of the present invention, "plural" means two or more.
The invention will be further illustrated with reference to the following specific examples. The specific embodiment is implemented on the premise of the technical scheme of the invention, and a detailed implementation mode and an operation process are given. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions. Unless otherwise indicated, ratios and percentages are by weight. It should be understood that the auxiliary materials that can be added in the present invention are not limited to the examples, and those skilled in the art can select different auxiliary materials to add according to the needs.
In the following examples, chitosan was used having a molecular weight of 1400kDa and prepared in the laboratory according to conventional methods.
In the following examples, sodium alginate was used with CAS number 9005-38-3 from Aladdin.
In the following examples, trehalose was used having a CAS number of 99-20-7 and a molecular weight of 342.3 and was purchased from Aladdin.
In the following examples, microcrystalline cellulose, code No. A600279, size 100 μm, was used and purchased from Biotechnology engineering (Shanghai) GmbH. In the following examples, nanocrystalline cellulose was prepared by the following method:
the hydrolysis reaction was quenched by dispersing 9g of MCC in 50mL of 70% by mass sulfuric acid, vigorously stirring at 50 ℃ for 10h, and diluting the reaction mixture with 8 times water. Then 8mL of 30 wt% H was added2O2Solution, oxidized nanocrystalline cellulose suspension. After 30min of reaction, the mixture was centrifuged at 8000rpm for 10min for separationCrystallization, washing with ultrasound, and centrifuging several times to remove excess acid. Finally, the nano-crystalline cellulose is prepared by dialysis (molecular weight cut-off) with 14000kDa and freeze-drying.
Example 1
First step preparation of enzyme-loaded chitosan nanoparticles (core materials)
The chitosan was dissolved in 2% acetic acid solution at a chitosan concentration of 2.5mg/mL, and stirred overnight to dissolve the chitosan sufficiently. The pH was then adjusted to 5.5 with 5M NaOH solution and the total amount of glucose oxidase and xylanase was adjusted to 1:1 was added to the chitosan solution at a concentration of 35 mg/mL. After the solution is fully dissolved, the solution is placed in a centrifuge for centrifugation at the speed of 3500 rpm. Taking the supernatant, and mixing TPP solution with the concentration of 0.25mg/mL as 1: 5 (v: v) was dropped into the supernatant, and stirred at 150rpm for 30min to form a solution of enzyme-loaded chitosan nanoparticles.
Second step preparation of first layer wall material
Adding CaCl2Adding the solution into chitosan nanoparticle solution, Ca2+The final molar concentration was 1 mol/L. The mixed solution of sodium alginate (1%) and trehalose (15%) was then mixed with a syringe (needle diameter 0.45mm) according to a 1:1 (v: v) dropping into the chitosan nanoparticles and CaCl2The enzyme preparation was solidified for 30min to form a single-layer microcapsule.
Step three, preparing a second layer of wall material
And (3) filtering the enzyme preparation in the form of the single-layer microcapsule obtained in the step two (without washing), putting the filtered enzyme preparation in a CNC solution with the mass concentration of 0.1% (1g of filter residue is added into about 5-10 mLCNC solution), fully reacting for 1h, filtering to obtain the enzyme preparation in the form of the double-layer microcapsule coated with the CNC, and freeze-drying.
Evaluation of Effect
(1) Release of enzyme preparation in bilayer microcapsule form in PBS pH 4.5
0.1g of the enzyme preparation in the form of lyophilized bilayer microcapsules was put into 10mL of PBS having a pH of 4.5, followed by the experiment in a shaker. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2M), respectively, in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, respectively, and measuring absorbance at 540nm and 615nm, respectively. The result shows that when the CNC mass concentration is 0.1%, the xylanase reaches the maximum release rate of 90.87% in 6h, and the glucose oxidase reaches the maximum release rate of 90.84% in 6.5 h.
(2) Release of enzyme preparation in bilayer microcapsule form in PBS pH 7.4
0.1g of the enzyme preparation in the form of lyophilized bilayer microcapsules was put into 10mL of PBS buffer solution with pH 7.4, followed by experiment in a shaker. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2M), respectively, in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, respectively, and measuring absorbance at 540nm and 615nm, respectively. The result shows that when the CNC mass concentration is 0.1%, the xylanase reaches the maximum release rate of 91.42% in 5.5h, and the glucose oxidase reaches the maximum release rate of 91.11% in 6.5 h.
(3) Simulated gastrointestinal release of enzyme preparations in the form of double-layer microcapsules
0.1g of the enzyme preparation in the form of lyophilized double-layer microcapsules was placed in 10mL of PBS (pH 7), mixed with an equal volume of simulated gastric fluid (2g of NaCl,3.2g of pepsin, 7mL of HCl, pH adjusted to 2.5 with NaOH solution and finally made to volume 1000mL) and incubated in a shaker (shaker was preheated to 37 ℃). The whole stomach period is 2 h. 2 portions of 0.1mL sample solution were taken every 30min and absorbance was measured. After the sample liquid is taken, the same amount of simulated gastric juice is added. After the end of the gastric phase the pH was adjusted to 6.8 with NaOH solution. Then 20mL of simulated intestinal fluid (6.8g KH) was added2PO4Dissolved in 250mL of water, 77mL of 0.2M NaOH solutionThe solution, 500mL of water, 10g of trypsin, pH adjusted to 6.8 with NaOH solution or HCl) was sampled every 30min and the absorbance was measured. The results showed that the cumulative release rate of xylanase at the end of gastric phase was 53.13% and glucose oxidase was 47.62%, while the cumulative release rate of xylanase and glucose oxidase at the end of small intestinal phase was 73.64% and 68.95%, respectively.
Example 2
First step preparation of enzyme-loaded chitosan nanoparticles (core Material)
The chitosan was dissolved in 2% acetic acid solution at a chitosan concentration of 2.5mg/mL, and stirred overnight to dissolve the chitosan sufficiently. The pH was then adjusted to 5.5 with 5M NaOH solution and the glucose oxidase and xylanase were mixed at a 1:1 was added to the chitosan solution at a concentration of 35 mg/mL. After the solution is fully dissolved, the solution is placed in a centrifuge for centrifugation at the speed of 3500 rpm. Taking the supernatant, and mixing TPP solution with the concentration of 0.25mg/mL as 1: 5 (v: v) was dropped into the supernatant, and stirred at 150rpm for 30min to form a solution of enzyme-loaded chitosan nanoparticles.
Second step preparation of first layer wall material
Adding CaCl2Adding the solution into chitosan nanoparticle solution, Ca2+The final molar concentration was 1 mol/L. The mixed solution of sodium alginate (1%) and trehalose (15%) was then mixed with a syringe (needle diameter 0.45mm) according to a 1:1 (v: v) dropping into the chitosan nanoparticles and CaCl2The enzyme preparation was solidified for 30min to form a single-layer microcapsule.
The third step is the preparation of the second layer wall material
And (3) filtering the microcapsule obtained in the step two (without flushing), putting the microcapsule into a CNC solution with the mass concentration of 0.5%, fully reacting for 1h, filtering to obtain the enzyme preparation coated with the CNC in a double-layer microcapsule form, and freeze-drying.
Evaluation of Effect
(1) Release of enzyme preparation in bilayer microcapsule form in PBS pH 4.5
0.1g of the enzyme preparation in the form of lyophilized bilayer microcapsules was put into 10mL of PBS having a pH of 4.5, followed by the experiment in a shaker. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2M), respectively, in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, respectively, and measuring absorbance at 540nm and 615nm, respectively. The result shows that when the CNC mass concentration is 0.5%, the xylanase reaches the maximum release rate of 91.95% in 7.5h, and the glucose oxidase reaches the maximum release rate of 92.55% in 8.5 h.
(2) Release of enzyme preparation in bilayer microcapsule form in PBS pH 7.4
0.1g of the enzyme preparation in the form of lyophilized bilayer microcapsules was put into 10mL of PBS buffer solution with pH 7.4, followed by experiment in a shaker. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2M), respectively, in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, respectively, and measuring absorbance at 540nm and 615nm, respectively. The result shows that when the CNC mass concentration is 0.5%, the xylanase reaches the maximum release rate of 91.42% in 6.5h, and the glucose oxidase reaches the maximum release rate of 92% in 7 h.
(3) Simulated gastrointestinal release of enzyme preparations in the form of double-layer microcapsules
0.1g of the enzyme preparation in the form of lyophilized double-layer microcapsules was placed in 10mL of PBS (pH 7), mixed with an equal volume of simulated gastric fluid (2g of NaCl,3.2g of pepsin, 7mL of HCl, pH adjusted to 2.5 with NaOH solution and finally made to volume 1000mL) and incubated in a shaker (shaker was preheated to 37 ℃). The whole stomach period is 2 h. 2 portions of 0.1mL sample solution were taken every 30min and absorbance was measured. After the sample liquid is taken, the same amount of simulated gastric juice is added. After the gastric period, using NaOH solutionThe pH was adjusted to 6.8. Then 20mL of simulated intestinal fluid (6.8g KH) was added2PO4Dissolved in 250mL of water, 77mL of a 0.2M NaOH solution, 500mL of water, 10g of trypsin, adjusted to pH 6.8 with a NaOH solution or HCl), and samples were taken every 30min to measure the absorbance. The results showed that the cumulative release rate of xylanase at the end of gastric phase was 41.62% and glucose oxidase was 42.63%, while the cumulative release rate of xylanase and glucose oxidase at the end of small intestinal phase was 77.13% and 72.11%, respectively.
Example 3
First step preparation of enzyme-loaded chitosan nanoparticles (core materials)
The chitosan was dissolved in 2% acetic acid solution at a chitosan concentration of 2.5mg/mL, and stirred overnight to dissolve the chitosan sufficiently. The pH was then adjusted to 5.5 with 5M NaOH solution and the glucose oxidase and xylanase were mixed at a 1:1 was added to the chitosan solution at a concentration of 35 mg/mL. After the solution is fully dissolved, the solution is placed in a centrifuge for centrifugation at the speed of 3500 rpm. Taking the supernatant, and mixing TPP solution with the concentration of 0.25mg/mL as 1: 5 (v: v) was dropped into the supernatant, and stirred at 150rpm for 30min to form a solution of enzyme-loaded chitosan nanoparticles.
Second step preparation of first layer wall material
Adding CaCl2Adding the solution into chitosan nanoparticle solution, Ca2+The final molar concentration was 1 mol/L. The mixed solution of sodium alginate (1%) and trehalose (15%) was then mixed with a syringe (0.45 mm needle diameter) according to 1:1 (v: v) dropping into the chitosan nanoparticles and CaCl2The enzyme preparation was solidified for 30min to form a single-layer microcapsule.
Step three, preparing a second layer of wall material
And (3) filtering the microcapsule obtained in the step two (without flushing), putting the microcapsule into a CNC solution with the mass concentration of 1%, fully reacting for 1h, filtering to obtain the enzyme preparation coated with the CNC in a double-layer microcapsule form, and freeze-drying.
Evaluation of Effect
(1) Release of enzyme preparation in bilayer microcapsule form in PBS pH 4.5
0.1g of the enzyme preparation in the form of lyophilized bilayer microcapsules was put into 10mL of PBS having a pH of 4.5, followed by the experiment in a shaker. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2M), respectively, in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, respectively, and measuring absorbance at 540nm and 615nm, respectively. The result shows that when the CNC mass concentration is 1%, the xylanase achieves the maximum release rate of 90.61% in 10h, and the glucose oxidase achieves the maximum release rate of 91.19% in 10 h.
(2) Release of enzyme preparation in bilayer microcapsule form in PBS pH 7.4
0.1g of the enzyme preparation in the form of lyophilized bilayer microcapsules was put into 10mL of PBS buffer solution with pH 7.4, followed by experiment in a shaker. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2M), respectively, in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, respectively, and measuring absorbance at 540nm and 615nm, respectively. The result shows that when the CNC mass concentration is 1%, the xylanase reaches the maximum release rate of 94.42% in 7.5h, and the glucose oxidase reaches the maximum release rate of 91.75% in 9 h.
(3) Simulated gastrointestinal release of enzyme preparations in the form of double-layer microcapsules
0.1g of the enzyme preparation in the form of lyophilized double-layer microcapsules was placed in 10mL of PBS (pH 7), mixed with an equal volume of simulated gastric fluid (2g of NaCl,3.2g of pepsin, 7mL of HCl, pH adjusted to 2.5 with NaOH solution and finally made to volume 1000mL) and incubated in a shaker (shaker was preheated to 37 ℃). The whole stomach period is 2 h. Take 2 parts of 0 every 30 min.1mL of the sample solution was measured for absorbance. After the sample liquid is taken, the same amount of simulated gastric juice is added. After the end of the gastric phase the pH was adjusted to 6.8 with NaOH solution. Then 20mL of simulated intestinal fluid (6.8g KH) was added2PO4Dissolved in 250mL of water, 77mL of a 0.2M NaOH solution, 500mL of water, 10g of trypsin, adjusted to pH 6.8 with a NaOH solution or HCl), and samples were taken every 30min to measure the absorbance. The results showed that the cumulative release rate of xylanase at the end of gastric phase was 32.63% and glucose oxidase was 44.25%, while the cumulative release rate of xylanase and glucose oxidase at the end of small intestinal phase was 64.77% and 61.37%, respectively.
Comparative example 1
First step preparation of enzyme-loaded chitosan nanoparticles (core materials)
The chitosan was dissolved in 2% acetic acid solution at a concentration of 2.5mg/mL, and stirred overnight to dissolve the chitosan sufficiently. The pH was then adjusted to 5.5 with 5M NaOH solution and the glucose oxidase and xylanase were mixed at 1:1 was added to the chitosan solution at a concentration of 35 mg/mL. After the solution is fully dissolved, the solution is placed in a centrifuge for centrifugation at the speed of 3500 rpm. Taking the supernatant, and mixing TPP solution with the concentration of 0.25mg/mL as 1: 5 (v: v) was dropped into the supernatant, and stirred at 150rpm for 30min to form a solution of enzyme-loaded chitosan nanoparticles.
Second step preparation of first layer wall material
Adding CaCl2Adding the solution into chitosan nanoparticle solution, Ca2+The final molar concentration was 1 mol/L. The mixed solution of sodium alginate (1%) and trehalose (15%) was then mixed with a syringe (needle diameter 0.45mm) according to a 1:1 (v: v) dropping into the chitosan nanoparticles and CaCl2The enzyme preparation was solidified for 30min to form a single-layer microcapsule. The enzyme preparation in the form of single-layer microcapsules was subsequently washed three times with an acetate-sodium acetate buffer at pH 5.5 so that no more enzyme was attached to the surface of the microcapsules, and subsequently lyophilized.
Evaluation of Effect
(1) Release of enzyme preparation in single-layer microcapsule form in PBS pH 4.5
0.1g of the enzyme preparation in the form of lyophilized monolayer microcapsules was placed in 10mL of PBS having a pH of 4.5, followed by the experiment in a shaker. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2mol/L) in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, and measuring the absorbance at 540nm and 615nm, respectively. The result shows that the xylanase achieves the maximum release rate of 92.07% in 6h, and the glucose oxidase achieves the maximum release rate of 92.88% in 7 h.
(2) Release of enzyme preparation in single-layer microcapsule form in PBS pH 7.4
0.1g of the freeze-dried CNC-coated chitosan microcapsule beads was put into 10mL of PBS buffer with pH 7.4, and then put into a shaker to perform the experiment. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2mol/L) in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, and measuring the absorbance at 540nm and 615nm, respectively. The result shows that the xylanase achieves the maximum release rate of 92.52 percent at 5h and the glucose oxidase achieves the maximum release rate of 90.09 percent at 6 h.
(3) Mimicking gastrointestinal release of an enzyme preparation in the form of a single layer microcapsule
0.1g of the enzyme preparation in the form of lyophilized double-layer microcapsules was placed in 10mL of PBS (pH 7), mixed with an equal volume of simulated gastric fluid (2g of NaCl,3.2g of pepsin, 7mL of HCl, pH adjusted to 2.5 with NaOH solution and finally made to volume 1000mL) and incubated in a shaker (shaker was preheated to 37 ℃). The whole stomach period is 2 h. 2 portions of 0.1mL sample solution were taken every 30min and absorbance was measured. After the sample liquid is taken, the same amount of simulated gastric juice is added. End of stomach stageThe pH was then adjusted to 6.8 with NaOH solution. Then 20mL of simulated intestinal fluid (6.8g KH) was added2PO4Dissolved in 250mL of water, 77mL of a 0.2M NaOH solution, 500mL of water, 10g of trypsin, adjusted to pH 6.8 with a NaOH solution or HCl), and samples were taken every 30min to measure the absorbance. The results showed that the cumulative release rate of xylanase at the end of gastric phase was 58.39% and glucose oxidase was 52.93%, while the cumulative release rate of xylanase and glucose oxidase at the end of small intestinal phase was 68.08% and 61.66%, respectively.
Comparative example 2
Evaluation of Effect of free enzyme
(1) Release of free enzyme in PBS at pH 4.5
0.05g of xylanase and glucose oxidase were weighed out accurately into 10mL of PBS having a pH of 4.5, and then placed in a shaker for the experiment. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2M), respectively, in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, respectively, and measuring absorbance at 540nm and 615nm, respectively. The result shows that the maximum release rate of the free xylanase is 92.03 percent at 3.5h, and the maximum release rate of the free glucose oxidase is 90.97 percent at 3.5 h.
(2) Release of free enzyme in PBS at pH 7.4
0.05g of xylanase and glucose oxidase was accurately weighed into 10mL of PBS buffer with pH 7.4, and then placed in a shaker to perform the experiment. The shaker needs to be preheated. The whole release time is 12h, 2 portions of 0.1mL sample solution are taken every 15min in the first 1h of the release experiment, and are taken every 30min in the subsequent time, and PBS with the same volume is added after the sample solution is taken. 0.1mL of the sample solution was reacted with 0.5mL of a xylan solution (1%) and a glucose solution (0.2M), respectively, in a water bath at 37 ℃ for 30min, followed by addition of 0.5mL of a DNS reagent and an indigo carmine solution, respectively, heating in a boiling water bath for 5min and 13min, respectively, and measuring absorbance at 540nm and 615nm, respectively. The result shows that the maximum release rate of the free xylanase reaches 91.42 percent in 4 hours, and the maximum release rate of the free glucose oxidase reaches 93.7 percent in 4 hours.
(3) Free enzyme simulated in vitro release
0.05g of xylanase and glucose oxidase were weighed accurately into 10mL of PBS (pH 7), mixed with an equal volume of simulated gastric fluid (2g NaCl,3.2g pepsin, 7mL HCl, pH adjusted to 2.5 with NaOH solution, and finally made up to 1000mL) and incubated in a shaker (shaker preheated to 37 ℃ C.). The whole stomach period is 2 h. 2 portions of 0.1mL sample solution were taken every 30min and absorbance was measured. After the sample liquid is taken, the same amount of simulated gastric juice is added. After the end of the gastric phase the pH was adjusted to 6.8 with NaOH solution. Then 20mL of simulated intestinal fluid (6.8g KH) was added2PO4Dissolved in 250mL of water, 77mL of a 0.2M NaOH solution, 500mL of water, 10g of trypsin, adjusted to pH 6.8 with a NaOH solution or HCl), and samples were taken every 30min to measure the absorbance. The results showed that the cumulative release rate of free xylanase at the end of gastric phase was 59.11% and glucose oxidase was 52.93%, while the cumulative release rate of free xylanase and glucose oxidase at the end of small intestinal phase was 58.65% and 32.99%, respectively.
Comparing examples 1-3 with comparative examples 1 and 2, it can be seen that (1) the two-layer microcapsule enzyme preparation has better encapsulation efficiency, in vitro release and gastrointestinal release than the single-layer microcapsule preparation, and the two-layer and single-layer microcapsule preparation has better encapsulation efficiency, in vitro release and gastrointestinal release than the free enzyme preparation.
The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. An enzyme preparation, comprising:
a core material comprising chitosan nanoparticles and an enzyme,
a first layer of wall material which comprises sodium alginate and trehalose,
a second layer of wall material comprising nanocrystalline cellulose,
wherein the first layer of wall material is coated on the surface of the core material, and the second layer of wall material is coated on the surface of the first layer of wall material.
2. The enzyme preparation according to claim 1, wherein the enzyme is selected from one or more of glucose oxidase, xylanase, phytase, protease, lipase and amylase.
3. The enzyme preparation according to claim 1 or 2, wherein the mass ratio of the chitosan to the enzyme is 1-5: 0.2 to 0.5.
4. The enzyme preparation according to claim 1 or 2, wherein the mass ratio of the sodium alginate to the trehalose is 10-20: 0.1 to 1.
5. The method according to claim 1, wherein the method for preparing nanocrystalline cellulose comprises the following steps:
(1) dispersing microcrystalline cellulose in 50-75% sulfuric acid by mass, strongly mechanically stirring for 8-12 h at 40-60 ℃, diluting a reaction mixture with 4-12 times of water, and quenching a hydrolysis reaction;
(2) adding 20-40% by mass of H into the quenched reaction mixture in the step (1)2O2Reacting the aqueous solution for 20-40 min, and then centrifuging and separating a crystal;
(3) adding the crystal into water, washing with ultrasonic wave, centrifuging to remove excessive acid, dialyzing, and lyophilizing to obtain nanocrystalline cellulose.
6. A process for the preparation of an enzyme preparation according to any one of claims 1 to 5, characterized in that the process comprises the steps of:
(1) preparation of a core material: adding enzyme into the chitosan solution, adding sodium tripolyphosphate, and forming enzyme-loaded chitosan nanoparticles between chitosan and tripolyphosphate ions through ion crosslinking;
(2) coating of a first layer of wall material: adding CaCl2Adding the solution into the final solution obtained in the step (1), uniformly mixing, adding a mixed solution of sodium alginate and trehalose, adsorbing the sodium alginate and the trehalose on the surface of the chitosan nanoparticle, solidifying to form a first layer of wall material, and filtering to obtain an enzyme preparation in the form of a single-layer microcapsule;
(3) coating of the second layer of wall material: adding the enzyme preparation in the form of the single-layer microcapsule into a nanocrystalline cellulose solution, adsorbing nanocrystalline cellulose on the surface of a first layer of wall material to form a second layer of wall material, and filtering to obtain the enzyme preparation in the form of the double-layer microcapsule.
7. The method of preparing an enzyme preparation according to claim 6, wherein the step (1) of preparing the core material comprises: dissolving chitosan in 0.5-4% acetic acid solution, fully dissolving to obtain 1-10 mg/mL chitosan solution, adjusting the pH to 5.0-6.0 with NaOH solution, adding enzyme, fully dissolving, centrifuging, taking supernatant, mixing 0.10-0.50 mg/mL sodium tripolyphosphate solution with the supernatant according to the volume ratio of 1: 2-8, and stirring at 200-400 rpm to obtain the enzyme-loaded chitosan nanoparticle solution.
8. The process for preparing an enzyme preparation according to claim 6, wherein the step (2) of coating the first wall material comprises: adding CaCl2Adding the solution into the chitosan nanoparticle solution loaded with the enzyme, uniformly mixing, and adding Ca2+The final molar concentration of the sodium alginate solution is 0.5-2 mol/L, a mixed solution of sodium alginate and trehalose is dripped into the sodium alginate solution, the mixture is solidified for 20-40 min, and the single-layer micro-gel is obtained by filtrationAn enzyme preparation in the form of a capsule,
in the mixed solution of sodium alginate and trehalose, the mass ratio of sodium alginate to trehalose is 10-20: 0.1-1 percent, the mass concentration of sodium alginate is 5-30 percent, the mass concentration of trehalose is 0.5-4 percent,
wherein the mixed solution of sodium alginate and trehalose and Ca2+The volume ratio of the solution (A) to (B) is 1-5: 1.
9. The method for preparing an enzyme preparation according to claim 6, wherein the step (3) of coating the second wall material comprises: and adding the enzyme preparation in the form of the single-layer microcapsule into a nanocrystalline cellulose solution with the mass concentration of 0.05-5%, slightly stirring for 0.5-2 h, and filtering to obtain the enzyme preparation in the form of the double-layer microcapsule.
10. Use of an enzyme preparation according to any one of claims 1 to 5 for the preparation of a medicament, food or feed for increasing the activity of an enzyme in the gastrointestinal tract of an animal.
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