CN111744439B - Hypsizygus marmoreus polysaccharide chelated zinc microcapsule and preparation method thereof - Google Patents
Hypsizygus marmoreus polysaccharide chelated zinc microcapsule and preparation method thereof Download PDFInfo
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/30—Zinc; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
- B01J13/043—Drying and spraying
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention aims to provide a hypsizygus marmoreus polysaccharide chelated zinc microcapsule and a preparation method thereof, wherein the hypsizygus marmoreus polysaccharide is chelated with metal zinc ions, and the microcapsule has the advantages of high chelating rate, complete polysaccharide structure, less environmental pollution, low use cost, small polysaccharide destructiveness, small workload, short chelating time and the like; the microcapsule is prepared by utilizing a coating technology, so that the dispersibility and the solubility of the hypsizygus marmoreus polysaccharide chelated zinc microcapsule are improved, and the antioxidant activity of the hypsizygus marmoreus polysaccharide chelated zinc is protected; the prepared hypsizygus marmoreus polysaccharide chelated zinc microcapsule has obvious anti-tumor physiological activity and obtains obvious technical effect.
Description
Technical Field
The invention belongs to the technical field of polysaccharide extraction and preparation, and particularly relates to a hypsizygus marmoreus polysaccharide chelated zinc microcapsule and a preparation method thereof.
Background
Hypsizygus marmoreus (Hypsizygus marmoreus) also known as Hypsizygus marmoreus and Hypsizygus marmoreus belongs to Basidiomycota, Basidiomycetes, Agaricales, Tricholomataceae and Equisetum, and is a rare edible fungus used as both medicine and food. The seafood mushroom has delicious taste and rich nutrition, enjoys the beauty of the 'golden bough and jade leaves' in the edible mushrooms, is rich in nutrient substances such as polysaccharide, various essential amino acids, mineral substances, vitamins and the like, and has extremely high medicinal value and nutritional value.
The extraction method of the polysaccharide of the hypsizygus marmoreus mainly comprises the traditional water extraction method, the ultrasonic-assisted extraction method, the microwave-assisted extraction method and the like. Different extraction methods have the following advantages and disadvantages: the extraction efficiency can be obviously improved by using a microwave method and ultrasonic waves, but the ultrasonic waves and the microwave can also damage the structure of the polysaccharide with poor thermal stability and even possibly reduce the biological activity of the polysaccharide; compared with the two methods, the hot water extraction method has the advantages of less environmental pollution, low use cost, small polysaccharide destructiveness, high chelating rate and the like, but the method has large workload and long extraction time.
The polysaccharide of the hypsizygus marmoreus has multiple effects of resisting aging, reducing blood fat, resisting oxidation and the like, and the polysaccharide content and the structure of the polysaccharide have important influence on the antioxidant activity. The zinc element has close relationship with the growth, development, tissue repair and cell division and reproduction of organisms, and is an essential trace element for maintaining normal physiological action and metabolic function of human bodies. In organisms, zinc exists mainly in combination with macromolecular substances such as polysaccharide and the like to generate a complex substance with relatively stable properties. The zinc in the current zinc supplement product mainly exists in an inorganic form, and can generate certain toxic action on human bodies when being taken too much; compared with inorganic zinc, the zinc existing in an organic form has a mode of action in a human body closer to that of zinc per se, so that the zinc is easier to absorb by the human body, and the antioxidant activity of the zinc is higher than that of the inorganic zinc.
The microcapsule (microcapsule) is prepared by using natural or synthetic high molecular material as wall material and coating active substance (core material) by physical, chemical or physical-chemical method to form semipermeable or sealed capsule membrane. Microencapsulation, which is a microencapsulation technique, is a technique for forming fine particles by embedding a solid or liquid in a film-forming material. The microencapsulation method includes a spray drying method, an inclusion complex method, a phase separation method, an interface polymerization method, and the like. The use value of the polysaccharide chelate is greatly limited by oxidative degradation of the polysaccharide chelate, and the stability and the availability of the polysaccharide chelate in functional products can be improved by adopting a microcapsule technology for encapsulation treatment, so that the physiological function of the polysaccharide chelate is promoted to play, and the polysaccharide chelate has great potential application value.
In recent years, the experiments of chelating plant polysaccharide and edible fungus polysaccharide with metal ions have been reported, but the related researches on chelating seafood mushroom polysaccharide with zinc are reported at present. Therefore, the hypsizygus marmoreus polysaccharide is chelated with zinc and prepared into microcapsules by taking the hypsizygus marmoreus polysaccharide as an object, and the aim of providing a basis for developing a novel nutritional type zinc supplement in the market is fulfilled.
Disclosure of Invention
At present, few reports are made on the utilization of hypsizygus marmoreus polysaccharide chelated zinc microcapsules. The invention aims to provide a hypsizygus marmoreus polysaccharide chelated zinc microcapsule and a preparation method thereof, wherein the hypsizygus marmoreus polysaccharide and metal zinc ions are chelated, so that the hypsizygus marmoreus polysaccharide chelated zinc microcapsule has the advantages of high chelation rate, complete polysaccharide structure, less environmental pollution, low use cost, small polysaccharide destructiveness, small workload, short chelation time consumption and the like; and the microcapsule preparation is carried out by utilizing the coating technology to protect the antioxidant activity of the microcapsule; the prepared hypsizygus marmoreus polysaccharide chelated zinc microcapsule has obvious anti-tumor physiological activity and obtains obvious technical effect.
The invention adopts the following technical scheme to solve the technical problems:
the preparation method comprises the technical steps of drying, crushing, sieving, leaching, centrifuging, supernatant concentrating, protein removing by a sevage method, alcohol precipitation, centrifuging, freeze drying, chelating, microcapsule preparation and the like. The preparation method has remarkable chelating effect on the hypsizygus marmoreus polysaccharide and metal ion zinc, and has the advantages of high chelating rate, complete polysaccharide structure and high biological activity; the method has the advantages of less environmental pollution, low use cost, small polysaccharide destructiveness, small workload, short chelation time and the like, and the coating technology is utilized for microencapsulation preparation to protect the antioxidant activity of the polysaccharide; the prepared hypsizygus marmoreus polysaccharide chelated zinc microcapsule has obvious anti-tumor physiological activity, obtains obvious technical effect, and provides basis for market research and development of novel nutritional zinc supplement.
The invention discloses a preparation method of hypsizygus marmoreus polysaccharide chelated zinc microcapsules, which comprises the following steps:
(1) and (3) drying: naturally airing fresh hypsizygus marmoreus to enable the fresh hypsizygus marmoreus to be in a semi-dry state, placing the hypsizygus marmoreus in an electric heating constant-temperature drying box, and drying by blowing;
(2) crushing and sieving: crushing the hypsizygus marmoreus prepared in the step (1) by using a crusher, sieving by using a 60-mesh sieve to prepare hypsizygus marmoreus powder, and placing the hypsizygus marmoreus powder at 4 ℃ for later use;
(3) leaching: leaching the hypsizygus marmoreus powder obtained in the step (2) for 2 hours in a hot water bath at 90 ℃;
(4) centrifuging: centrifuging the leaching solution obtained in the step (3), and collecting supernatant;
(5) concentrating the supernatant: concentrating the supernatant obtained in step (4) to one third of the original volume in vacuum;
(6) protein removal by sevage method: mixing the concentrated solution obtained in the step (5) with a mixed solution of n-butanol and chloroform according to a ratio of 4: 1, centrifuging by a centrifuge, separating a water phase from a chloroform phase, and collecting the water phase for later use;
(7) alcohol-out polysaccharide: slowly pouring 85% ethanol solution with volume 3 times of that of the polysaccharide solution into the water phase obtained in the step (6), standing at 4 ℃ for 12h, and centrifuging at the rotating speed of 4000r/min for 20min to obtain precipitate, namely a crude polysaccharide product;
(8) and (3) freeze drying: freeze-drying the polysaccharide product obtained in the step (7) to prepare a sample for later use;
(9) chelating: dissolving the polysaccharide product prepared in the step (8) to obtain a polysaccharide solution of the hypsizygus marmoreus, and adding the polysaccharide solutionThe mass ratio is (1-6): 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 2-7mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 3-8, and chelating in a hot water bath for 2-12 h; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate;
(10) preparing microcapsules: the polysaccharide-Zn of the hypsizygus marmoreus prepared in the step (9)2+Taking the chelate as a core material, wherein the ratio of sodium octenyl succinate to modified starch is 1: 0.7-1.7 as a coating wall material, the solid content is 20%, and the core-wall ratio is 1: (6-11), carrying out ultrasonic emulsification for 15-40min under the condition of 160W, and carrying out spray drying at the air inlet temperature of 190 ℃, the air outlet temperature of 70 ℃ and the spray pressure of 21 MPa.
Preferably, the fresh mushroom polysaccharide solution and Zn in the chelation process of the invention2+The mass ratio of the solution is 4: 1.
preferably, in the present invention, the number of times of leaching is three.
Preferably, in the invention, the ratio of n-butanol to chloroform in the mixture of n-butanol and chloroform is 1: 4, mixing according to a mass ratio.
Preferably, Zn is chelated in the process of the present invention2+The initial mass concentration of the solution was 5 mg/mL.
Preferably, the chelation time in the chelation process in the present invention is 6 h.
Preferably, the pH of the solution in the chelation process of the invention is 6.
Preferably, sodium octenylsuccinate and modified starch are used as wall materials in the preparation process of the microcapsule, and the ratio of the wall materials to the modified starch is 1: 1.3.
preferably, the ratio of the core wall to the wall in the microcapsule preparation process in the invention is 1: 9.
preferably, the ultrasonic emulsification time is 30min under the condition of 160W in the microcapsule preparation process.
By implementing the technical scheme provided by the invention, the following beneficial effects can be obtained:
the invention provides a hypsizygus marmoreus polysaccharide chelated zinc microcapsule and a preparation method thereof, wherein the hypsizygus marmoreus polysaccharide is chelated with metal zinc ions, so that the microcapsule has the advantages of high chelating rate, complete polysaccharide structure, less environmental pollution, low use cost, small polysaccharide destructiveness, small workload, short chelating time consumption and the like; the microcapsule is prepared by utilizing a coating technology, so that the dispersibility and the solubility of the hypsizygus marmoreus polysaccharide chelated zinc microcapsule are improved, and the antioxidant activity of the hypsizygus marmoreus polysaccharide chelated zinc is protected; the prepared hypsizygus marmoreus polysaccharide chelated zinc microcapsule has obvious anti-tumor physiological activity and obtains obvious technical effect.
Drawings
FIG. 1 is a graph showing the effect of chelation time on chelation effect.
FIG. 2 shows polysaccharide and Zn of Hypsizygus marmoreus2+The influence of the mass ratio (mg/mg) of (A) on the chelating effect is shown.
FIG. 3 shows Zn2+Graph of the effect of the initial mass concentration (mg/mL) on the chelation effect.
FIG. 4 is a graph showing the effect of solution pH on chelating effect.
FIG. 5 shows the chelation time with Hypsizygus marmoreus polysaccharide and Zn2+And (4) an interactive response surface map of the mass ratio.
FIG. 6 shows chelation time vs. Zn2+An interactive response surface map of the initial mass concentration.
FIG. 7 is a graph showing the interaction response of chelation time with pH.
FIG. 8 shows polysaccharide and Zn of Hypsizygus marmoreus2+Mass ratio of Zn to Zn2+An interactive response surface map of the initial mass concentration.
FIG. 9 shows Hypsizygus marmoreus polysaccharide and Zn2+Mass ratio versus pH.
FIG. 10 shows Zn2+Initial mass concentration versus pH.
FIG. 11 shows Hypsizygus Marmoreus Polysaccharide (HMP) and Hypsizygus marmoreus polysaccharide-Zn2+(Zn-HMP) chelate infrared spectrogram.
FIG. 12 is a scanning electron microscope image of hypsizygus marmoreus polysaccharide at different magnifications, wherein FIG. 12a is 5000 times and FIG. 12b is 10000 times.
FIG. 13 shows polysaccharide-Zn from Hypsizygus marmoreus2+Scanning electron microscope for chelate compounds under different timesFIG. 13a is a 5000-fold magnification view, and FIG. 13b is a 10000-fold magnification view.
FIG. 14 shows the scavenging energy diagram of hypsizygus marmoreus polysaccharide chelated zinc microcapsules for hydroxyl radical (. OH).
FIG. 15 is a graph showing the scavenging ability of hypsizygus marmoreus polysaccharide chelated zinc microcapsules on DPPH free radicals.
Detailed Description
The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.
The materials of the invention are: n-butanol, chloroform, anhydrous ethanol, concentrated sulfuric acid, phenol, glucose, potassium sodium tartrate, 3, 5-dinitrosalicylic acid, sodium hydroxide, sodium selenite, hydrogen peroxide (30%), ferrous sulfate, 1-diphenyl-2-trinitrophenylhydrazine, a swing type high-speed traditional Chinese medicine pulverizer (Yuanyao chemical machinery manufacturing limited, Jiangyin), an electric heating constant-temperature air-blast drying oven (Shanghai Baidian instruments equipment Co., Ltd.), a digital display constant-temperature water bath (Guohua electric appliances Co., Ltd.), a CENTRIFUGE CENTRIFUGE (Kyowa Katsu instruments manufacturing Co., Ltd.), a rotary evaporator (Shanghai Yangrong Biochemical apparatus works), a pH meter (Switzerland Meter TLER TOLEDO Co., Ltd.), a visible spectrophotometer (Shanghai Xinmao instruments Co., Ltd.), a Fourier transform infrared spectrometer (Tiandong Hongkong science development Co., Ltd.), a water circulation type multi-purpose vacuum pump (Zhenzhou Zi instruments equipment Co., Ltd.), Thermal field emission scanning electron microscope (carl zeiss, germany), the equipment and instruments used in the preparation method are all common equipment in the field, and can be purchased through public channels.
All materials, reagents and equipment selected for use in the present invention are well known in the art, but do not limit the practice of the invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the invention.
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The first embodiment is as follows: preparation of hypsizygus marmoreus polysaccharide chelated zinc microcapsule
The invention discloses a preparation method of hypsizygus marmoreus polysaccharide chelated zinc microcapsules, which comprises the following steps:
(1) drying: naturally airing fresh hypsizygus marmoreus to enable the fresh hypsizygus marmoreus to be in a semi-dry state, placing the hypsizygus marmoreus in an electric heating constant-temperature drying box, and drying by blowing;
(2) crushing and sieving: crushing the hypsizygus marmoreus prepared in the step (1) by using a crusher, sieving by using a 60-mesh sieve to prepare hypsizygus marmoreus powder, and placing the hypsizygus marmoreus powder at 4 ℃ for later use;
(3) leaching: leaching the hypsizygus marmoreus powder obtained in the step (2) for 2 hours in a hot water bath at 90 ℃;
(4) centrifuging: centrifuging the leaching solution obtained in the step (3), and collecting supernatant;
(5) concentrating the supernatant: concentrating the supernatant obtained in step (4) to one third of the original volume in vacuum;
(6) protein removal by sevage method: mixing the concentrated solution obtained in the step (5) with a mixed solution of n-butanol and chloroform according to a ratio of 4: 1, centrifuging by a centrifuge, separating a water phase from a chloroform phase, and collecting the water phase for later use;
(7) alcohol-precipitated polysaccharide: slowly pouring 85% ethanol solution with volume 3 times of that of the polysaccharide solution into the water phase obtained in the step (6), standing at 4 ℃ for 12h, and centrifuging at the rotating speed of 4000r/min for 20min to obtain precipitate, namely a crude polysaccharide product;
(8) and (3) freeze drying: freeze-drying the polysaccharide product obtained in the step (7) to prepare a sample for later use;
(9) chelating: dissolving the polysaccharide product prepared in the step (8) to obtain a hypsizygus marmoreus polysaccharide solution, and adding the polysaccharide product into the hypsizygus marmoreus polysaccharide solution according to the mass ratio of (1-6): 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 2-7mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 3-8, and chelating in a hot water bath for 2-12 h; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate;
(10) preparing microcapsules: taking the hypsizygus marmoreus prepared in the step (9)sugar-Zn2+Taking chelate as a core material, wherein the ratio of sodium octenyl succinate to modified starch is 1: 0.7-1.7 as a coating wall material, the solid content is 20%, and the core-wall ratio is 1: (6-11), carrying out ultrasonic emulsification for 15-40min under the condition of 160W, wherein the air inlet temperature of spray drying is 190 ℃, the air outlet temperature is 70 ℃, and the spray pressure is 21 MPa.
Example two: preparation of hypsizygus marmoreus polysaccharide chelated zinc microcapsule
Based on the first embodiment, a certain amount of polysaccharide product is dissolved to obtain a hypsizygus marmoreus polysaccharide solution, and the polysaccharide solution is added in a mass ratio of 1: 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 2mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 3, and chelating in a hot water bath for 2 hours; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate; in the coating process, the ratio of sodium octenyl succinate to modified starch is 1 in terms of m/m: 0.7 as wall material, polysaccharide-Zn of Hypsizygus marmoreus2+The chelate is used as a core material, the solid content is 20%, and the core wall ratio is 1: 6,160W, performing ultrasonic emulsification for 15min, and performing spray drying at air inlet temperature of 190 ℃, air outlet temperature of 70 ℃ and spray pressure of 21MPa to obtain the hypsizygus marmoreus polysaccharide chelated zinc microcapsule.
Example three: preparation of hypsizygus marmoreus polysaccharide chelated zinc microcapsule
Based on the first embodiment, a certain amount of polysaccharide product is dissolved to obtain a hypsizygus marmoreus polysaccharide solution, and the polysaccharide product is added into the hypsizygus marmoreus polysaccharide solution according to the mass ratio of 2: 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 3mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 4, and chelating in a hot water bath for 4 hours; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate; in the coating process, the ratio of sodium octenyl succinate to modified starch is 1 in terms of m/m: 0.9 as wall material, polysaccharide-Zn of Hypsizygus marmoreus2+The chelate is used as a core material, the solid content is 20%, and the core wall ratio is 1: 7,160W, performing ultrasonic emulsification for 20min, and performing spray drying at air inlet temperature of 190 ℃, air outlet temperature of 70 ℃ and spray pressure of 21MPa to obtain the hypsizygus marmoreus polysaccharide chelated zinc microcapsule.
Example four: preparation of hypsizygus marmoreus polysaccharide chelated zinc microcapsule
Based on the first embodiment, a certain amount of polysaccharide product is dissolved to obtain a hypsizygus marmoreus polysaccharide solution, and the polysaccharide product is added into the hypsizygus marmoreus polysaccharide solution according to the mass ratio of 3: 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 4mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 5, and chelating in a hot water bath for 6 hours; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate; in the coating process, the ratio of sodium octenyl succinate to modified starch is 1 in terms of m/m: 1.1 mix as wall material, polysaccharide-Zn of Hypsizygus marmoreus2+The chelate is used as a core material, the solid content is 20%, and the core wall ratio is 1: and (3) carrying out ultrasonic emulsification for 25min under the condition of 8,160W, and carrying out spray drying at the air inlet temperature of 190 ℃, the air outlet temperature of 70 ℃ and the spray pressure of 21MPa to obtain the hypsizygus marmoreus polysaccharide chelated zinc microcapsule.
Example five: preparation of hypsizygus marmoreus polysaccharide chelated zinc microcapsule
Based on the first embodiment, a certain amount of polysaccharide product is dissolved to obtain a hypsizygus marmoreus polysaccharide solution, and the polysaccharide product is added into the hypsizygus marmoreus polysaccharide solution according to the mass ratio of 4: 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 5mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 6, and chelating in a hot water bath for 8 hours; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate; in the coating process, the ratio of sodium octenyl succinate to modified starch is 1 in terms of m/m: 1.3 mix as wall material, polysaccharide-Zn of Hypsizygus marmoreus2+The chelate is used as a core material, the solid content is 20%, and the core wall ratio is 1: 9,160W, ultrasonic emulsifying for 30min, spray drying at air inlet temperature of 190 deg.C and air outlet temperature of 70 deg.C under spray pressure of 21MPa to obtain the final product.
Example six: preparation of hypsizygus marmoreus polysaccharide chelated zinc microcapsule
Based on the first embodiment, a certain amount of polysaccharide product is dissolved to obtain a hypsizygus marmoreus polysaccharide solution, and the polysaccharide solution is added with the components in a mass ratio of 5: 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 6mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 7, and chelating in a hot water bath for 10 hours; then, the product is processedAdding 3 times of volume of absolute ethyl alcohol, and standing for 12 hours; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate compound; in the coating process, the ratio of sodium octenyl succinate to modified starch is 1 in terms of m/m: 1.5 as wall material, polysaccharide-Zn of Hypsizygus marmoreus2+The chelate is used as a core material, the solid content is 20%, and the core wall ratio is 1: performing ultrasonic emulsification for 35min under the condition of 10W to 160W, and performing spray drying at the air inlet temperature of 190 ℃, the air outlet temperature of 70 ℃ and the spray pressure of 21MPa to obtain the hypsizygus marmoreus polysaccharide chelated zinc microcapsule.
Example seven: preparation of hypsizygus marmoreus polysaccharide chelated zinc microcapsule
Based on the first embodiment, a certain amount of polysaccharide product is dissolved to obtain a hypsizygus marmoreus polysaccharide solution, and the polysaccharide product is added into the hypsizygus marmoreus polysaccharide solution according to the mass ratio of 6: 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 7mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 8, and chelating in a hot water bath for 12 hours; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate compound. In the coating process, the ratio of sodium octenyl succinate to modified starch is 1 in terms of m/m: 1.7 as wall material, the solid content is 20%, the core-wall ratio is 1: 11, ultrasonic emulsification is carried out for 40min under the condition of 160W, the air inlet temperature of spray drying is 190 ℃, the air outlet temperature is 70 ℃, and the spray pressure is 21MPa, thus obtaining the seafood mushroom polysaccharide chelated zinc microcapsule.
Example eight: optimization of preparation method of hypsizygus marmoreus polysaccharide chelated zinc
In order to determine the optimal processing process for preparing the hypsizygus marmoreus polysaccharide chelated zinc, the chelating rate is used as an index, and the hypsizygus marmoreus polysaccharide and Zn are prepared in the preparation process of the hypsizygus marmoreus polysaccharide chelated zinc2+Chelating time of, polysaccharide of, and Zn of hypsizygus marmoreus2+Mass ratio of (1), Zn2+The initial mass concentration and the solution pH of the solution were subjected to a one-factor experiment. On the basis of the Zn, Zn is added2+The chelation rate is a response value, and a response surface analysis experiment is carried out according to a Box-Behnken center combination method.
1. Inspection method
(1) Calculation of chelation Rate
Adding anhydrous ethanol with three times volume into the chelating solution, and standingAfter 12h, the supernatant was centrifuged and Zn was measured by atomic spectrophotometer2+The concentration was measured, and the chelation rate was calculated by the following formula.
In the formula: a is the chelating rate of zinc; c0Is Zn in the solution before chelation2+Mass concentration of (1) (mg/mL); c is Zn in the chelated solution2+Mass concentration of (1) (mg/mL).
2. Single factor test
(1) Effect of chelation time on chelation Rate
Taking Zn2+Solution with mass concentration of 4mg/mL, hypsizygus marmoreus polysaccharide and Zn2+The mass ratio (mg/mg) of (1) is 4: 1 adding polysaccharide, adjusting the pH of the mixed solution to 7, and carrying out chelation reaction for 2, 4, 6, 8, 10 and 12 hours respectively to investigate the influence of different chelation time on chelation rate. As shown in figure 1, the chelating time is explored for hypsizygus marmoreus polysaccharide and Zn2+When the chelating rate is influenced, the chelating time is prolonged from 2h to 12h, and the hypsizygus marmoreus polysaccharide and Zn are mixed2+The chelating ratio of (2) is likely to increase and then decrease, and it is presumed that the chelating period of time is too long, which results in unstable structure of the chelating ring, thereby decreasing the chelating ratio. Thus, a suitable chelation time was determined to be 6 h.
(2) Hypsizygus marmoreus polysaccharide and Zn2+Influence of the mass ratio of (mg/mg) on the chelation rate
Taking Zn2+The mass concentration of the solution is 4mg/mL, and the polysaccharide and the Zn of the hypsizygus marmoreus are respectively added2+The mass ratio (mg/mg) of (1): 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1 adding polysaccharide, adjusting the pH of the mixed solution to 7, carrying out chelation reaction for 6h, and discussing the influence of different mass ratios on chelation rates. As shown in figure 2, as the mass ratio is increased, the hypsizygus marmoreus polysaccharide and Zn are mixed2+The chelation rate of (a) gradually increases, and the rate is increased when the mass ratio is 4: 1, the chelation rate reaches the maximum and then gradually decreases; when the mass ratio is too low, it is not preferable to form a stable cyclic structure and the chelating rate is low, but when the mass ratio reaches a certain value, the chelating rate is decreased by increasing the mass ratio. Thus, it is possible to provideDetermining a suitable mass ratio of 4: 1.
(3)Zn2+effect of initial Mass concentration (mg/mL) on chelation Rate
Taking Zn2+Solutions with mass concentration of 2, 3, 4, 5, 6, 7mg/L, hypsizygus marmoreus polysaccharide and Zn2+The mass ratio (mg/mg) of (1) is 4: 1 adding polysaccharide, adjusting pH of the mixed solution to 7, carrying out chelation reaction for 6h, and discussing different Zn2+Influence of initial mass concentration on chelation rate, and determination of optimal Zn2+The initial mass concentration. As shown in figure 3, hypsizygus marmoreus polysaccharide and Zn2+Chelating ratio of (2) with Zn2+The initial mass concentration increases, but too high a mass concentration leads to a decrease in the chelation rate. Zn2+The initial mass concentration of the polysaccharide is too low, so that the reaction quantity with the hypsizygus marmoreus polysaccharide is too small, and the chelation rate is not high; however, the continued increase in concentration after reaching the maximum may destabilize the chelate ring formed, affecting the polysaccharide and Zn of Hypsizygus marmoreus2+Thereby decreasing the chelation rate. Thus, suitable Zn was determined2+The initial mass concentration was 4 mg/mL.
(4) Effect of solution pH on chelation Rate
Taking Zn2+A solution with the mass concentration of 4mg/mL, prepared from hypsizygus marmoreus polysaccharide and Zn2+The mass ratio (mg/mg) of (1) is 4: 1 adding polysaccharide, respectively adjusting the pH of the mixed solution to 3, 4, 5, 6, 7 and 8, carrying out chelation reaction for 6h, and discussing the influence of different pH values on chelation rate. As shown in figure 4, the pH of the reaction solution was adjusted to the pH of the polysaccharide and Zn in Hypsizygus marmoreus2+Has a remarkable influence on the chelating rate, and the hypsizygus marmoreus polysaccharide and Zn grow from 3 to 8 of the pH value of the solution2+The chelation rate of (a) shows a tendency of increasing first and then decreasing. When the pH value is too low, the solution is acidic, so that part of the hypsizygus marmoreus polysaccharide is hydrolyzed by acid to cause low chelation rate; however, when the pH is too high, the anions in the solution compete with the active groups of the hypsizygus marmoreus polysaccharide, thereby preventing the formation of chelate. Therefore, the optimum pH was determined to be 6.
3. Response surface optimization test
Selecting chelation time, hypsizygus marmoreus polysaccharide and Zn on the basis of single-factor test2+Mass ratio of (mg)/mg)、Zn2+Carrying out Box-Behnken center combination experiment on initial mass concentration (mg/mL) and pH value of solution, selecting reasonable level according to actual experiment conditions, and taking Zn as the basis2+The chelation rate (R) is used as a response value, a four-factor three-level response surface analysis experiment is designed, and the values and codes of the combined design level are shown in Table 1.
Table 1: factor level coding table
On the basis of a single-factor experiment, selecting chelation time (A), hypsizygus marmoreus polysaccharide and Zn according to a Box-Behnken test design principle2+Mass ratio of (B) and Zn2+Initial mass concentration (C), pH value of the solution (D) and Zn as response surface analysis2+The chelation rate (R) was used as a response value, and the experimental design and results are shown in Table 2. Regression analysis was performed on the data using design expert8.0 software.
Table 2: Box-Behnken experimental design and results
According to the experimental results of Table 2, in order to examine each interaction term pair Zn2+Under the condition that other factors are fixed and unchanged, the Design-Expert 8.05 software is used for operating the regression equation to make a response surface map of the interaction term, as shown in the attached drawings 5, 6, 7, 8, 9 and 10.
Table 3: regression model analysis of variance
| Source of variant | Sum of squares | Degree of freedom | Mean square | F value | P value | Significance of |
| Model (model) | 80.45 | 14 | 5.75 | 286.77 | <0.0001 | ** |
| A | 0.46 | 1 | 0.46 | 23.16 | 0.0003 | ** |
| B | 0.12 | 1 | 0.12 | 6.09 | 0.0271 | ** |
| C | 0.39 | 1 | 0.39 | 19.22 | 0.0006 | * |
| D | 34.21 | 1 | 34.21 | 1707.08 | <0.0001 | ** |
| AB | 6.25×10-4 | 1 | 6.25×10-4 | 0.031 | 0.8623 | |
| AC | 0.060 | 1 | 0.060 | 3.00 | 0.1055 | |
| AD | 0.25 | 1 | 0.25 | 12.48 | 0.0033 | ** |
| BC | 9.03×10-3 | 1 | 9.03×10-3 | 0.45 | 0.5131 | |
| BD | 2.25×10-4 | 1 | 2.25×10-4 | 0.011 | 0.9171 | |
| CD | 9.02×10-3 | 1 | 9.02×10-3 | 0.45 | 0.5131 | |
| A2 | 4.00 | 1 | 4.00 | 199.69 | <0.0001 | ** |
| B2 | 4.33 | 1 | 4.33 | 215.90 | <0.0001 | ** |
| C2 | 6.22 | 1 | 6.22 | 310.37 | <0.0001 | ** |
| D2 | 42.61 | 1 | 42.61 | 2126.35 | <0.0001 | ** |
| Residual error | 0.28 | 14 | 0.020 | |||
| Missimilitude term | 0.20 | 10 | 0.020 | 0.96 | 0.5693 | |
| Pure error | 0.083 | 4 | 0.021 | |||
| Total regression | 80.73 | 28 |
Note: p is more than 0.05, the effect is not significant; p < 0.05 was significant; p < 0.001 was very significant.
Performing regression fitting on the data in the table 3 to obtain independent variable and Zn2+The quadratic polynomial regression equation for chelation ratio (R) is:
R＝82.36-0.2A+0.1B+0.18C+1.69D+0.25AD-0.79A2-0.82B2-0.98C2- 2.56D2
by performing regression analysis on the data, the regression analysis of variance of the equation is shown in table 3. As shown in Table 3, A, B, C, D, AD and A2、B2、C2、D2Term pair Zn2+The chelating rate influence is obvious, the mismatching item P is more than 0.05 and is not obvious, and the correlation coefficient R of the model2The result shows that the model has good fitting degree and small experimental error, and can be used for analyzing and predicting polysaccharide-Zn of hypsizygus marmoreus2+A preparation method of the chelate.
4. Response surface optimization and verification
Using Design-Expert software, by Zn2+The maximum chelation rate is taken to jointly solve the simplified regression equation to obtain the polysaccharide-Zn for preparing the hypsizygus marmoreus2+Optimizing conditions of the chelate. Considering the operability and simplicity in the practical experiment process, the chelating time is set to be 6h, and the hypsizygus marmoreus polysaccharide and Zn are2+The mass ratio is set as 4: 1. zn2+The initial mass concentration was set to 5mg/mL and the pH of the solution was set to 6. To test the accuracy and validity of the results obtained, 3 parallel experiments were performed using the above-described optimized parameters, and the results are shown in table 4. As can be seen from Table 4, the experimental result value is closer to the theoretical predicted value of the response surface, and therefore, the optimized formula parameters obtained based on the response surface method are considered to be accurate and reliable.
Table 4: response surface software prediction optimal preparation method condition
Example nine: infrared spectroscopic analysis of hypsizygus marmoreus polysaccharide and zinc chelate thereof
As shown in FIG. 11, the polysaccharide (HMP) of Hypsizygus marmoreus is located at 1367cm-1The peak at is-COO-Has an antisymmetric stretching vibration peak of 3387cm-1The broad peak at (A) is the stretching vibration peak of-OH. HMP-Zn compared to the IR spectrum of HMP2+The main differences of (a) are: (1) the stretching vibration peak of-OH in Zn-HMP is shifted to 3423cm-1It shows that-OH in HMP participates in Zn2+Chelating; (2) Zn-HMP at 1384cm-1The existence of a stretching vibration peak indicates Zn2+And a coordination bond is formed between the compound and carboxylic acid or carboxylate. The above phenomena can indicate that the polysaccharide and Zn of the hypsizygus marmoreus2+Chelation occurred.
Example ten: scanning electron microscope observation of hypsizygus marmoreus polysaccharide and zinc chelate thereof
As shown in fig. 12a and 12 b: respectively are scanning electron microscope images of the hypsizygus marmoreus polysaccharide powder sample under different magnifications, and the results show that: the polysaccharide of Hypsizygus marmoreus has smooth appearance and many pore structures on the surface, and small particulate matter is embedded on the surface and in the hollow space, as shown in the attached figures 13a and 13 b: respectively are polysaccharide-Zn of hypsizygus marmoreus2+Scanning electron microscope images of the powder sample under different magnifications show that the shape of the powder sample under 10000 times of scanning is obviously different from that of the hypsizygus marmoreus polysaccharide before chelation.
Example eleven: optimization of preparation method of hypsizygus marmoreus polysaccharide chelated zinc microcapsules
And optimizing the preparation method of the hypsizygus marmoreus polysaccharide chelated zinc microcapsule based on the scheme provided in the second embodiment to the sixth embodiment.
Based on the first embodiment, a certain amount of polysaccharide product is dissolved to obtain a hypsizygus marmoreus polysaccharide solution, and the polysaccharide product is added into the hypsizygus marmoreus polysaccharide solution according to the mass ratio of 4: 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 5mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 6, and chelating in a hot water bath for 8 hours; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate; in the coating process, sodium octenyl succinate and modified starch are mixed to form a wall material, the solid content is 20 percent, ultrasonic emulsification is carried out under the condition of 160W, the air inlet temperature of spray drying is 190 ℃, the air outlet temperature is 70 ℃,spraying pressure is 21MPa, and the seafood mushroom polysaccharide chelated zinc microcapsule is prepared.
1. Preparation method for preparing hypsizygus marmoreus polysaccharide chelated zinc microcapsules through orthogonal optimization
And designing a single-factor experiment, and respectively exploring the influence of the wall material ratio, the core-wall ratio and the emulsifying time in the microcapsule preparation process on the embedding rate in the preparation method for preparing the hypsizygus marmoreus polysaccharide chelated zinc microcapsules. Three-factor three-level orthogonal experiments were performed on the basis of the single-factor experiments, see table 5.
Table 5: orthogonality test factors and levels
Microcapsule embedding rate experiment: weighing 10mg of self-made hypsizygus marmoreus polysaccharide chelated zinc microcapsules, dissolving the microcapsules in 20mL of 5% trisodium citrate solution, selecting glucose as a standard substance, precipitating with ethanol, measuring the polysaccharide content in the microcapsules by a phenol-sulfuric acid colorimetric method, and calculating the embedding rate of the hericium erinaceus polysaccharide chelated zinc microcapsules:
the results and analysis of the orthogonal optimization test are shown in Table 6.
Table 6: results and analysis of orthogonal assays
As can be seen from table 6, within the range of the experimental design, the core-wall ratio has the greatest effect on the preparation of the hypsizygus marmoreus polysaccharide chelated zinc microcapsules, followed by the wall material ratio and the emulsification time. The best preparation method for preparing the hypsizygus marmoreus polysaccharide chelated zinc microcapsule is prepared as A2B2C2The wall material proportion is 1: 1.3, core-wall ratio of 1: and 9, emulsifying for 30min, namely preparing the hypsizygus marmoreus polysaccharide chelated zinc microcapsule according to the preparation method provided in the fifth embodiment.
Example twelve: determination of antioxidant activity of hypsizygus marmoreus polysaccharide chelated zinc microcapsules
(1) Ability to scavenge hydroxyl radical (. OH)
Based on the product obtained by the best method for preparing the hypsizygus marmoreus polysaccharide chelated zinc microcapsules in the example eight, 9mmol/L FeSO is respectively added into each test tube with a plug4Adding polysaccharide/polysaccharide-Zn with different mass concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2mg/mL) into 1mL of solution and 2mL of 9mmol/L of salicylic acid ethanol solution2+Chelate microcapsule solution 2mL, 8.8mmol/L H was added2O22mL of the solution was reacted at 37 ℃ for 30 min. The absorbance of the sample was measured at a wavelength of 510nm by blank zeroing with distilled water.
The sample's clearance for OH radicals can be expressed as follows:
in the formula: a. the0The absorbance of the blank liquid; a. thexIs the absorbance of the added sample solution.
The experimental results of the hypsizygus marmoreus polysaccharide chelated zinc microcapsules on the removal of hydroxyl radicals (OH) are shown in the attached figure 14, and it can be seen that both the hypsizygus marmoreus polysaccharide and the hypsizygus marmoreus polysaccharide chelated zinc microcapsules have good removal capability on the hydroxyl radicals (OH), the removal rate of the hydroxyl radicals is continuously improved along with the increase of the concentration of the hypsizygus marmoreus polysaccharide, and the removal rate of the hypsizygus marmoreus polysaccharide chelated zinc microcapsules under the same concentration is obviously higher than that before chelation and embedding.
(2) Ability to scavenge DPPH free radicals
Based on the product obtained by the optimal preparation method of the hypsizygus marmoreus polysaccharide chelated zinc microcapsule in example eight, 25.6mg of DPPH free radical is accurately weighed and dissolved in a 100mL volumetric flask with 70% ethanol and uniformly shaken for later use. Taking 2mL of the PPH free radical, adding 1mL of hypsizygus marmoreus polysaccharide/hypsizygus marmoreus polysaccharide chelated zinc microcapsule solution with different mass concentrations (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2mg/mL), and carrying out dark reaction for 30min at room temperature. The absorbance of the sample was measured at a wavelength of 517nm with distilled water as a reference.
The sample clearance of DPPH free radicals can be expressed as follows:
in the formula: a. thei: absorbance of 2mL DPPH +1mL hypsizygus marmoreus polysaccharide/hypsizygus marmoreus polysaccharide chelated zinc microcapsule solution;
Aj: absorbance of 2mL hypsizygus marmoreus polysaccharide/hypsizygus marmoreus polysaccharide chelated zinc microcapsule solution +1mL 70% ethanol solution;
A0: absorbance of 2mL DPPH +1mL 70% ethanol solution;
as shown in fig. 15, the hypsizygus marmoreus polysaccharide/hypsizygus marmoreus polysaccharide chelated zinc microcapsules have good capacity of removing DPPH radicals, the removal rate of the hypsizygus marmoreus polysaccharide chelated zinc microcapsules under the same concentration is obviously higher than that before chelation and embedding, and the removal effect of chelates on DPPH radicals is linearly increased within a certain concentration range, so that a good dose-effect relationship is presented.
Example thirteen: inhibition of cancer cell proliferation by hypsizygus marmoreus polysaccharide chelated zinc microcapsules
An optimal preparation method of the hypsizygus marmoreus polysaccharide chelated zinc microcapsule is obtained through a series of optimization experiments, and the obtained product is subjected to anticancer effect identification.
(1) Preparation of hypsizygus marmoreus polysaccharide chelated zinc microcapsule solution
Weighing 250mg of hypsizygus marmoreus polysaccharide chelated zinc microcapsules, dissolving in 10mL of 5% glucose isotonic solution, vortexing for about 10min, adding 10mL of 1640 culture solution containing fetal calf serum and double antibodies, adding 40 mu L (0.2%, v/v) of dimethyl sulfoxide, continuously vortexing for about 10min, passing through a 0.45 mu m water film after full dissolution, and fixing the volume to 25mL by using the 1640 culture solution, thus obtaining the mother solution containing 10mg/mL of hypsizygus marmoreus polysaccharide chelated zinc microcapsules. Diluting with 1640 culture solution to get series of hypsizygus marmoreus polysaccharide chelated zinc microcapsule solutions (prepared when in use) of 0.001, 0.01, 0.1, 1, 10 mg/mL.
(2) Cancer cell culture
Cell culture human liver cancer HepG-2 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum in 5% CO2Culturing and subculturing at 37 ℃ in an incubator, and taking cells in logarithmic phase for experiment.
(3) MTT method for measuring cell proliferation inhibition rate
HepG-2 cells at the logarithmic growth phase of passage 3 to 4 were digested with trypsin, washed, centrifuged, and prepared into a cell suspension (5.0X 104 cells/mL), and 200. mu.L of the cell suspension was inoculated into a 96-well plate (1.0X 104 cells/well). The cell culture plates were placed in a 37 ℃ cell incubator at 5% CO2Incubating for 24h under the condition, observing the adherent growth of visible cells under a microscope, sucking out the culture solution, adding 200 μ L of hypsizygus marmoreus polysaccharide chelated zinc microcapsule solution with the concentrations of 0.001, 0.01, 0.1, 1 and 10mg/mL respectively, and taking the blank of cells and the blank of solvent as the reference. And after the culture is continued for 48h, replacing serum-free culture solution, adding 5mg/mL MTT30 mu L into each hole, continuing to culture for 4h, absorbing the original culture solution, adding 200 mu L DMSO into each hole to dissolve crystals, measuring the absorbance at 570nm by using an enzyme labeling instrument, and calculating the cell survival rate under different concentrations. In the above experiment, 8 duplicate wells were set for each concentration, and each set was repeated 3 times.
In the formula: a1 is the absorbance of the experimental group; a2 is cell blank absorbance; a0 is the solvent blank absorbance.
The survival rate of cancer cells cultured in hypsizygus marmoreus polysaccharide chelated zinc microcapsule solution with different concentrations and standard error are counted, and the results are shown in table 7.
Table 7: inhibition effect of hypsizygus marmoreus polysaccharide chelated zinc microcapsules on HepG-2 cells
With the increase of the concentration of the hypsizygus marmoreus polysaccharide chelated zinc microcapsule solution, the survival rate of cancer cells is rapidly reduced. When the concentration of the hypsizygus marmoreus polysaccharide chelated zinc microcapsule solution is 0.001mg/mL, the inhibition rate is 18.13 percent; when the concentration is 10mg/mL, the inhibition rate on the cancer cells reaches 72.65%. The hypsizygus marmoreus polysaccharide chelated zinc microcapsule prepared by the invention has obvious anti-tumor physiological activity and obtains obvious technical effect.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.
Claims (4)
1. A preparation method of hypsizygus marmoreus polysaccharide chelated zinc microcapsules is characterized by comprising the following steps:
(1) drying: naturally airing fresh hypsizygus marmoreus to enable the fresh hypsizygus marmoreus to be in a semi-dry state, placing the hypsizygus marmoreus in an electric heating constant-temperature drying box, and drying by blowing;
(2) crushing and sieving: crushing the hypsizygus marmoreus prepared in the step (1) by using a crusher, sieving by using a 60-mesh sieve to prepare hypsizygus marmoreus powder, and placing the hypsizygus marmoreus powder at 4 ℃ for later use;
(3) leaching: leaching the hypsizygus marmoreus powder obtained in the step (2) for 2 hours in a hot water bath at 90 ℃;
(4) centrifuging: centrifuging the leaching solution obtained in the step (3), and collecting supernatant;
(5) concentrating the supernatant: concentrating the supernatant obtained in step (4) to one third of the original volume in vacuum;
(6) deproteinizing by sevage method: mixing the concentrated solution obtained in the step (5) with a mixed solution of n-butanol and chloroform according to a ratio of 4: 1, centrifuging by a centrifuge, separating a water phase from a chloroform phase, and collecting the water phase for later use;
(7) alcohol-precipitated polysaccharide: slowly pouring 85% ethanol solution with volume 3 times of that of the polysaccharide solution into the water phase obtained in the step (6), standing at 4 ℃ for 12h, and centrifuging at the rotating speed of 4000r/min for 20min to obtain precipitate, namely a crude polysaccharide product;
(8) and (3) freeze drying: freeze-drying the polysaccharide product obtained in the step (7) to prepare a sample for later use;
(9) chelating: dissolving the polysaccharide product prepared in the step (8) to obtain a hypsizygus marmoreus polysaccharide solution, and adding the polysaccharide product into the hypsizygus marmoreus polysaccharide solution according to the mass ratio of (1-6): 1 hypsizygus marmoreus polysaccharide solution and Zn with concentration of 2-7mg/mL2+A solution; fully stirring, adjusting the pH value of the solution to 3-8, and chelating in a hot water bath for 2-12 h; adding 3 times of anhydrous ethanol, and standing for 12 h; centrifuging at 3000r/min for 5min to obtain precipitate; drying to obtain polysaccharide-Zn of Hypsizygus marmoreus2+A chelate;
(10) preparing microcapsules: taking the polysaccharide-Zn of the hypsizygus marmoreus prepared in the step (9)2+Taking the chelate as a core material, wherein the ratio of sodium octenyl succinate to modified starch is 1: 0.7-1.7 as a coating wall material, the solid content is 20%, and the core-wall ratio is 1: (6-11), carrying out ultrasonic emulsification for 15-40min under the condition of 160W, wherein the air inlet temperature of spray drying is 190 ℃, the air outlet temperature is 70 ℃, and the spray pressure is 21 MPa;
and in the mixed solution of the n-butanol and the chloroform, the n-butanol and the chloroform are mixed according to the ratio of 1: 4, mixing according to a mass ratio;
the polysaccharide solution of the seafood mushrooms and Zn are added in the chelation process2+The mass ratio of the solution is 4: 1;
said, chelating process of Zn2+The initial mass concentration of the solution is 5 mg/mL;
in the microcapsule preparation process, sodium octenylsuccinate and modified starch are used as wall materials, and the proportion of the wall materials is 1: 1.3;
the ratio of the core wall to the wall in the microcapsule preparation process is 1: 9;
in the microcapsule preparation process, the ultrasonic emulsification time is 30min under the condition of 160W.
2. The method for preparing hypsizygus marmoreus polysaccharide chelated zinc microcapsules according to claim 1, wherein the chelation time in the chelation process is 6 h.
3. The method for preparing hypsizygus marmoreus polysaccharide chelated zinc microcapsule according to claim 1, wherein the pH value of the solution during chelation is 6.
4. The hypsizygus marmoreus polysaccharide chelated zinc microcapsule obtained by the preparation method of the hypsizygus marmoreus polysaccharide chelated zinc microcapsule according to any one of claims 1 to 3.
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