CN107475221A - A kind of new lysozyme formulation and preparation method thereof - Google Patents
A kind of new lysozyme formulation and preparation method thereof Download PDFInfo
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- CN107475221A CN107475221A CN201710852051.0A CN201710852051A CN107475221A CN 107475221 A CN107475221 A CN 107475221A CN 201710852051 A CN201710852051 A CN 201710852051A CN 107475221 A CN107475221 A CN 107475221A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- 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)
- C12Y302/01017—Lysozyme (3.2.1.17)
Abstract
The invention discloses a kind of preparation method of new lysozyme formulation, comprise the following steps：(1) the egg clear solution of clarification is prepared；(2) polyanionic polysaccharide solution is prepared；(3) egg, polyanionic polysaccharide solution to same pH are adjusted respectively, egg and polyanionic polysaccharide solution are sufficiently mixed, stirring, the lysozyme in egg is set to interact to form lysozyme polyanionic polysaccharide complex precipitate by electrostatic attraction with polyanionic polysaccharide；(4) lysozyme polyanionic polysaccharide complex precipitate is collected by centrifugation, through being dried to obtain lysozyme formulation.The present invention is interacted by electrostatic attraction using polyanionic polysaccharide and lysozyme and produces the principle precipitated to reach the purpose of lysozyme separation and preparation, said preparation can both keep stability of the lysozyme during saving and processing, also with good Targeting delivery performance.
Description
Technical field
The invention belongs to protease preparation preparing technical field, and in particular to a kind of new lysozyme formulation and its preparation side
Method.
Background technology
Lysozyme is also known as muramidase, can optionally decompose microorganism wall composition, and itself is nontoxic, thus
It is a kind of good bactericide of natural security performance, preservative, can be widely applied to food antiseptic, pharmaceutical preparation, dailyization
The industries such as work.Lysozyme is present in the body fluid such as egg white, the tear of mammal, saliva, blood plasma, urine, milk and microorganism.People
Antalzyme activity in milk, tear and saliva is about 3 times of lysozyme from egg white vigor, but in egg lysozyme content
Most abundant (about 3mg/mL).
The method of separation and the purifying of lysozyme mainly has ammonium sulfate precipitation method, affinity chromatography, ion-exchange and coagulated
Glue chromatography etc..Although ammonium sulfate precipitation method purity is high, equipment is simple and convenient to operate, the lysozyme salt content obtained
Height, need to repeatedly saltouing, salt is molten could to obtain high purity product；Affinity chromatography separation lysozyme is also a kind of effective means, parent
Being separated with chromatography has higher selectivity, lysozyme can be isolated from egg white, but because affinity column cost is high, make
The shortcomings of difficult, is restricted its application；The sample that Strong acid ion-exchanger goes out, which compares concentration, sample recovery rate height, activity, to be protected
Hold that good, separation cycle is relatively short, but to be eluted out lysozyme as eluant, eluent using substantial amounts of salting liquid, then pass through
The lysozyme of higher degree can be just obtained after dialysis removal salt ion；Gel chromatography disposal ability is smaller, post material dress post
It is required that also very high, slightly structure inequality or slight crack can all have influence on the separating effect of sample, therefore gel chromatography is not general single
The purifying of lysozyme is solely used in, and often prepares a small amount of high-purity egg white lysozyme as a kind of two level means of purification.
Lin Liufeng (structure of lysozyme/pectin compound system and its application study in nutrition and drug delivery system,
Hua Zhong Agriculture University's master thesis, in June, 2015) it have studied interaction between lysozyme and pectin, and using molten
Bacterium enzyme and pectin heat 30min in pH10.7,80 DEG C of water-bath, and to obtain lysozyme/pectin nanogel more to load tea
Phenol, the stability of Tea Polyphenols can be effectively improved and improve load capacity etc..The main research material of this paper is lysozyme knot
Crystal, when near isoelectric points of proteins heating (80 DEG C) make bacteriolyze enzyme denaturation assemble to form spherical nanogel, heating process
Although the positive charge region on middle lysozyme surface can be combined with pectin negative electrical charge, mainly deposited using between lysozyme and pectin
Electrostatic repulsion prevent the lysozyme of denaturation from forming bulky grain, and heating process easily causes part lysozyme
Enzymatic activity reduces.
Xu Wei (lysozyme/polysaccharide assembly behavior and regulation and control, Hua Zhong Agriculture University Ph.D. Dissertation, in December, 2015) will
Xanthans, lysozyme soln pH are adjusted to 11.8, and after mixing 1h according to different quality ratio, 80 DEG C are heated 30min, naturally cold
But xanthans/lysozyme nanogel is obtained after removing impurity to 0.8 μm of filter membrane excessively after room temperature；Then again with xanthans/molten
Bacterium enzyme nanogel is aqueous phase, using atoleine as oil phase, is mixed respectively with different oil/water volume ratios, in 6000r/min conditions
Lower homogeneous 5min prepares Pickering emulsions.The main object of the technical research is still antalzyme crystallization body, and lysozyme exists
It is more than isoelectric point (about 11) negatively charged, exist with xanthans by electrostatic repulsion in the form of molten altogether；Pass through heating
Composite Nano gel is formed after effect promotion bacteriolyze enzyme denaturation with xanthans, therefore not by electrostatic attraction interaction shape
Into.The paper also studied carragheen/bacteriolyze enzyme interacting simultaneously, but carragheen needs the heating stirring at 70 DEG C to incubate
It could be used after educating 30min, and lysozyme mother liquor stirs 2h and could used at ambient temperature.Lysozyme mother liquor at room temperature with
Different carragheens/lysozyme proportioning (3：1、2：1、1：1、1：2、1：3) carrageenan solutions are added dropwise and are prepared after stirring 10min
Carragheen/bacteriolyze multienzyme complex, forms soluble complex under conditions of pH6.8, but the sky of the lysozyme in compound
Between conformation occur it is obvious change, molecule hydrophobic region moieties are exposed to molecular surface, the activity decrease of lysozyme in compound
For original a quarter.Preparation environment temperature in the technology is room temperature, and carragheen needs heating stirring, and need by
Lysozyme is added dropwise in carrageenan solutions, and preparation condition is complicated, causes the activity of lysozyme in compound to be greatly reduced.
(Responds Surface Methodology optimizes microbic muramidase microcapsule preparation process to Fei Guoqin etc., takes state's qin etc., food section
Learn, the 4th phase of volume 35, the 11-15 pages, 2014) microbic muramidase and sodium alginate are mixed into homogeneous solution, by compacted
The end pin hole of dynamic pump is at the uniform velocity added dropwise in the calcium chloride of certain mass concentration and the mixing acetum of chitosan, treats whole drops
Continue to be statically placed in after complete wherein to solidify, then with water washing is distilled 2 times, drained with gauze, be laid in a diameter of 10cm glass
It is freeze-dried in ware after pre-freeze 6h, that is, obtains lysozyme microballoon.The technology utilizes sodium alginate and bivalent metal ion Ca2+
The principle that reaction can form microballoon is embedded to lysozyme, while sodium alginate, chitosan can also be acted on by electrostatic attraction
Composite membrane is formed in microsphere surface, interaction whether occurs between lysozyme and sodium alginate not to be influenceed on embedding；But
The sodium alginate micro ball particle diameter of calcification is big, stability is poor, easily act in polyion or in calcium ion diffusion and potassium, sodium ion are big
Soften during amount aggregation and " leakage enzyme " phenomenon occur；Also, embedding process is carried out, it is necessary in original enzyme after the completion of enzyme separation circuit
Extra technique is added in production procedure, causes embedding techniques cost higher；Moreover, the enzyme system prepared is embedded by this technology
Agent can not realize the sustained release and controlled release of enzyme when in use, because enzyme is embedded in matrix, therefore can be only applied to have good
In good diffusible liquid food, it can not be played a role in viscosity larger liquid and solid-state food.
In recent years, bacteriolyze enzyme source deficiency, price rise steadily, and birds, beasts and eggs processing enterprise produces greatly in process of manufacture
The damaged egg of amount, useless egg white, eggshell etc., the warp of enterprise not only can be largely improved using these materials extraction lysozyme
Ji benefit, increase the added value of birds, beasts and eggs converted products, and the insufficient situation of market lysozyme can be alleviated.So such as
What safely and effectively from egg white separation and Purification of Lysozyme, and during enzyme preparation is prepared guarantee lysozyme high enzyme activity
Power, turn into urgent problem to be solved.Therefore, explore that a kind of operating procedure is simple, cost is cheap, pollution-free, stability is high and has
There is lysozyme formulation of control-release function and preparation method thereof, it is significant for the application of expansion lysozyme.
The content of the invention
For above mentioned problem existing for lysozyme formulation in the prior art, the invention provides a kind of new lysozyme formulation
And preparation method thereof.
For achieving the above object, the present invention is achieved using following technical proposals：
(1) Fresh Egg broken shell is separated into egg white, adds deionized water, be slowly stirred uniformly, filter off except insoluble matter obtains
Filtrate；Adjust filtrate pH value to centrifugation after standing 4h at 4.5,4 DEG C and remove insoluble matter, collect the egg that supernatant is clarified
Solution；
(2) 1%-2% (w/v) polyanionic polysaccharide solution is prepared, in 4 DEG C of fully hydrations overnight；
(3) egg clear solution, polyanionic polysaccharide solution to same pH are adjusted respectively, egg and polyanion is more
Sugar juice is sufficiently mixed, stirring reaction, the lysozyme in egg is interacted with polyanionic polysaccharide by electrostatic attraction
Form lysozyme-polyanionic polysaccharide complex precipitate；
(4) lysozyme-polyanionic polysaccharide complex precipitate is collected by centrifugation, through being dried to obtain lysozyme formulation.
Further, in the step (1), by egg and deionized water according to mass ratio 1 under condition of ice bath:1-2 is mixed
With glass bar is gently mixed to be advisable with non-foaming, and double-layer nylon cloth filters to take to obtain filtrate.
Further, filtrate pH value is adjusted to 4.5 with acetum in the step (1)；After standing 4h at 4 DEG C, at 4 DEG C
10000r/min centrifugations 20min removes insoluble matter, collects 33.3%-50% (w/w) egg clear solution that supernatant is clarified.
Further, the polyanionic polysaccharide in the step (2) is sodium alginate, one kind in carboxymethyl chitosan or two
Person's mixture.
Further, the moon by egg clear solution and is gathered using acetum or sodium hydroxide solution respectively in the step (3)
Nonionic polysaccharide solution ph is adjusted to 3.0-5.0；Egg and polyanionic polysaccharide solution are sufficiently mixed, keep egg with gathering
The mass ratio 25-100 of anion polysaccharide:1；The stirring reaction condition is stirring reaction 30-60min under ice bath.
Further, it is multiple to be collected using 4000r/min centrifugation 20min for lysozyme-polyanionic polysaccharide in the step (4)
Compound precipitates.
Further, the drying means of the step (4) is freeze-drying, is dried in vacuo, one kind in spray drying.
Further, the enzyme activity rate of recovery in the step (4) in lysozyme-polyanionic polysaccharide complex precipitate is
68.3%-87.5%, protein separation multiple are 1.72-3.21.
Further, lysozyme formulation is prepared according to the preparation method of the new lysozyme formulation of the present invention.
Further, in simulate the gastric juice after 2h, the lysozyme activity retention rate not embedded is 35.1%, the lysozyme system
Agent activity preservation rate is 75.2%-83.3%；Release rate of the lysozyme formulation in artificial simulation gastric juices after 2h be
37.5%-42.2%, then be placed in artificial simulation intestinal juice after 4h, total release rate is 87.6%-92.8%.
Compared with prior art, advantages of the present invention and advantageous effects are：
(1) existing patented technology is to separate chicken using the precipitation method, ion-exchange or the gel chromatography of routine
Lysozyme in egg white and egg shell.And the present invention adds appropriate polyanionic polysaccharide in egg, by adjusting egg
Insoluble compound polyelectrolyte is formed with polysaccharide ratio, mixed solution pH etc. clearly, by being drying to obtain lysozyme formulation, no
The purpose of concentrating and separating lysozyme can only be reached, but also the stability of enzyme can be improved simultaneously and obtain required controlled capability
Can, the deficiency of existing lysozyme formulation technology can be made up, therefore, there is good application in the exploitation of novel foodstuff enzyme preparation
Potentiality.
(2) existing research report is using the repercussion study between antalzyme crystallization body and polysaccharide, although few
Number has been related to electrostatic attraction interaction, but is not separated and Purification of Lysozyme from egg using the principle, also not
Prepare polysaccharide-lysozyme formulation.Because egg complicated component, disturbed containing substantial amounts of foreign protein, it is necessary to select and lysozyme
The stronger polysaccharide of binding specificity, successfully lysozyme could be separated from egg.
(3) present invention prepares lysozyme formulation by electrostatic attraction interaction between polyelectrolyte, and the method need not appoint
What organic solvent, catalyst or chemical cross-linking agent, there is safe, good biocompatibility, that technique is simple, cost is cheap etc. is aobvious
Advantage is write, therefore, the heat endurance of lysozyme can be improved to prepare lysozyme formulation by compound polyelectrolyte, extend its storage
Tibetan phase and validity period, the production efficiency of processing food can be improved, reduces cost.
(4) present invention need not heat during lysozyme formulation is prepared, it is possible to increase lysozyme-polyanionic polysaccharide is multiple
The enzyme activity rate of recovery in compound precipitation, the enzyme activity loss in enzyme preparation product is reduced, therefore, prepared and obtained by the method for the present invention
Enzyme activity is high in the enzyme preparation product obtained.
(5) the lysozyme particle diameter that prepared by the present invention is small, and high safety, enzyme stability is high, cannot be only used for liquid food, also
The liquid larger available for viscosity and solid-state food, and the preparation condition of the present invention is simple to operation, and equipment investment is few, is easy to
Industrialized production.
Summary, the present invention use polyanionic polysaccharide and lysozyme, and precipitation is produced by electrostatic adelphotaxy
Principle reach the purpose of lysozyme separation and preparation, the enzyme preparation formulation can both keep lysozyme in saving and processing process
In stability, moreover it is possible to keep lysozyme that there is good Targeting delivery performance.The preparation method of the present invention is simple to operate, safety
Property it is good, production cost is low, and selectivity is stronger, and the enzyme activity rate of recovery is high, and equipment investment is few, is easy to industrialized production.
Brief description of the drawings
After the embodiment of the present invention is read in conjunction with the figure, the other features and advantages of the invention will become more clear
Chu.
Fig. 1 is the schematic flow sheet of the present invention.
Embodiment
2% (w/v) polyanionic polysaccharide solution：2g polyanionic polysaccharides accurately are weighed, add appropriate deionized water, are used
Constant temperature blender with magnetic force is stirred to abundant dissolving, is settled to 100mL with deionized water, is obtained 2% polyanionic polysaccharide storing solution,
4 DEG C of refrigerator overnights are positioned over fully to be hydrated.Using preceding required concentration is diluted to deionized water.
For preparing the polyanionic polysaccharide of 2% (w/v) polyanionic polysaccharide solution as sodium alginate, carboxymethyl chitosan
One or both of mixture.
Embodiment 1
(1) Fresh Egg broken shell is separated into egg white, by egg white and deionized water according to mass ratio 1 under condition of ice bath:1 is mixed
With glass bar is gently mixed to be advisable with non-foaming, and double-layer nylon cloth filters to get filtrate.Filtrate is adjusted using 2mol/L acetums
PH value is to after standing 4h at 4.5,4 DEG C, and 10000r/min centrifuges 20min, 50% (w/w) for taking supernatant to be clarified at 4 DEG C
Egg clear solution.
(2) 50% (w/w) egg clear solution and 2% sodium alginate soln pH value are adjusted respectively using 2mol/L acetums
To 4.0, egg clear solution and sodium alginate soln are according to volume ratio 1:1 mixing, keep the mass ratio of egg and sodium alginate
25:1, the stirring reaction 60min under ice bath, the lysozyme in egg interact to be formed with sodium alginate by electrostatic attraction
Lysozyme-sodium alginate complex precipitate；4000r/min centrifuges 20min, collects lysozyme-sodium alginate complex precipitate, cold
Jelly is dried to obtain lysozyme formulation.The lysozyme enzyme activity rate of recovery under the conditions of this in lysozyme-sodium alginate complex precipitate is
87.5%, protein separation multiple is 3.21.
(3) in simulate the gastric juice after 2h, the lysozyme activity retention rate not embedded is 35.1%, and the lysozyme formulation is lived
Property retention rate be 83.3%, lysozyme formulation activity preservation rate than do not embed when significantly improves.The lysozyme formulation is artificial
Release rate in simulate the gastric juice after 2h is 37.5%, then the total release rate being placed in artificial simulation intestinal juice after 4h is 87.6%.
Embodiment 2
(1) Fresh Egg broken shell is separated into egg white, by egg white and deionized water according to mass ratio 1 under condition of ice bath:1 is mixed
With glass bar is gently mixed to be advisable with non-foaming, and double-layer nylon cloth filters to get filtrate.Filtrate is adjusted using 2mol/L acetums
PH value is to after standing 4h at 4.5,4 DEG C, and 10000r/min centrifuges 20min, 50% (w/w) for taking supernatant to be clarified at 4 DEG C
Egg clear solution.
(2) 50% (w/w) egg solution ph is adjusted to 5.0 using 1mol/L sodium hydroxide solutions, using 2mol/L
2% sodium alginate soln pH value is adjusted to 5.0 by acetum, and egg clear solution and sodium alginate soln are according to volume ratio 4:1 is mixed
Close, keep the mass ratio 100 of egg and sodium alginate:1, the stirring reaction 30min under ice bath, lysozyme in egg with
Sodium alginate interacts to form lysozyme-sodium alginate complex precipitate by electrostatic attraction；4000r/min centrifuges 20min,
Lysozyme-sodium alginate complex precipitate is collected, vacuum drying obtains lysozyme formulation.Lysozyme-sodium alginate under the conditions of being somebody's turn to do
The lysozyme enzyme activity rate of recovery in complex precipitate is 78.9%, and protein separation multiple is 2.26.
(3) in simulate the gastric juice after 2h, the lysozyme activity retention rate not embedded is 35.1%, and the lysozyme formulation is lived
Property retention rate be 76.3%, lysozyme formulation activity preservation rate than do not embed when significantly improves.The lysozyme formulation is artificial
Release rate in simulate the gastric juice after 2h is 39.6%, then the total release rate being placed in artificial simulation intestinal juice after 4h is 92.8%.
Embodiment 3
(1) Fresh Egg broken shell is separated into egg white, by egg white and deionized water according to mass ratio 1 under condition of ice bath:2 is mixed
With glass bar is gently mixed to be advisable with non-foaming, and double-layer nylon cloth filters to get filtrate.Filtrate is adjusted using 2mol/L acetums
PH value is to after standing 4h at 4.5,4 DEG C, and 10000r/min centrifuges 20min, the 33.3% (w/ for taking supernatant to be clarified at 4 DEG C
W) egg clear solution.
(2) using 2mol/L acetums respectively by 33.3% (w/w) egg clear solution and 1% carboxymethyl chitosan solution
PH value is adjusted to 3.0, and egg clear solution and carboxymethyl chitosan solution are according to volume ratio 2:1 mixing, keeps egg and carboxymethyl
The mass ratio 66.7 of chitosan:1, the stirring reaction 40min under ice bath, the lysozyme in egg pass through with carboxymethyl chitosan
Electrostatic attraction interacts to form lysozyme-carboxymethyl chitosan complex precipitate；4000r/min centrifuges 20min, collects bacteriolyze
Enzyme-carboxymethyl chitosan complex precipitate, spray drying obtain lysozyme formulation.Lysozyme-carboxymethyl chitosan under the conditions of being somebody's turn to do
The lysozyme enzyme activity rate of recovery in complex precipitate is 68.3%, and protein separation multiple is 2.05.
(3) in simulate the gastric juice after 2h, the lysozyme activity retention rate not embedded is 35.1%, and the lysozyme formulation is lived
Property retention rate be 75.2%, lysozyme formulation activity preservation rate than do not embed when significantly improves.The lysozyme formulation is artificial
Release rate in simulate the gastric juice after 2h is 42.2%, then the total release rate being placed in artificial simulation intestinal juice after 4h is 89.3%.
Embodiment 4
(1) Fresh Egg broken shell is separated into egg white, by egg white and deionized water according to mass ratio 1 under condition of ice bath:2 is mixed
With glass bar is gently mixed to be advisable with non-foaming, and double-layer nylon cloth filters to get filtrate.Filtrate is adjusted using 2mol/L acetums
PH value is to after standing 4h at 4.5,4 DEG C, and 10000r/min centrifuges 20min, the 33.3% (w/ for taking supernatant to be clarified at 4 DEG C
W) egg clear solution.
(2) using 2mol/L acetums respectively by 33.3% (w/w) egg clear solution and 1% carboxymethyl chitosan solution
PH value is adjusted to 4.0, and egg clear solution and carboxymethyl chitosan solution are according to volume ratio 1:1 mixing, keeps egg and carboxymethyl
The mass ratio 33.1 of chitosan:1, the stirring reaction 50min under ice bath, the lysozyme in egg pass through with carboxymethyl chitosan
Electrostatic attraction interacts to form lysozyme-carboxymethyl chitosan complex precipitate；4000r/min centrifuges 20min, collects bacteriolyze
Enzyme-carboxymethyl chitosan complex precipitate, vacuum drying obtain lysozyme formulation.Lysozyme-carboxymethyl chitosan under the conditions of being somebody's turn to do
The lysozyme enzyme activity rate of recovery in complex precipitate is 73.7%, and protein separation multiple is 1.72.
(3) in simulate the gastric juice after 2h, the lysozyme activity retention rate not embedded is 35.1%, and the lysozyme formulation is lived
Property retention rate be 80.1%, lysozyme formulation activity preservation rate than do not embed when significantly improves.The lysozyme formulation is artificial
Release rate in simulate the gastric juice after 2h is 41.3%, then the total release rate being placed in artificial simulation intestinal juice after 4h is 90.5%.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to foregoing reality
Example is applied the present invention is described in detail, for the person of ordinary skill of the art, still can be to foregoing implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these are changed or replaced
Change, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.
Claims (10)
1. a kind of preparation method of new lysozyme formulation, it is characterised in that comprise the following steps：
(1) Fresh Egg broken shell is separated into egg white, adds deionized water, be slowly stirred uniformly, filter off except insoluble matter obtains filtrate；
Adjust filtrate pH value to centrifugation after standing 4h at 4.5,4 DEG C and remove insoluble matter, collect the egg clear solution that supernatant is clarified；
(2) 1%-2% (w/v) polyanionic polysaccharide solution is prepared, in 4 DEG C of fully hydrations overnight；
(3) egg clear solution, polyanionic polysaccharide solution to same pH are adjusted respectively, egg is molten with polyanionic polysaccharide
Liquid is sufficiently mixed, stirring reaction, makes the lysozyme in egg interact to be formed by electrostatic attraction with polyanionic polysaccharide
Lysozyme-polyanionic polysaccharide complex precipitate；
(4) lysozyme-polyanionic polysaccharide complex precipitate is collected by centrifugation, through being dried to obtain lysozyme formulation.
2. the preparation method of new lysozyme formulation according to claim 1, it is characterised in that：In the step (1),
By egg white and deionized water according to mass ratio 1 under condition of ice bath:1-2 is mixed, and glass bar is gently mixed to be advisable with non-foaming, double-deck
Nylon cloth filters to take to obtain filtrate.
3. the preparation method of new lysozyme formulation according to claim 1, it is characterised in that：Used in the step (1)
Acetum adjusts filtrate pH value to 4.5；After standing 4h at 4 DEG C, 10000r/min centrifuges 20min and removes insoluble matter at 4 DEG C,
Collect 33.3%-50% (w/w) egg clear solution that supernatant is clarified.
4. the preparation method of new lysozyme formulation according to claim 1, it is characterised in that：In the step (2)
Polyanionic polysaccharide is one or both of sodium alginate, carboxymethyl chitosan mixture.
5. the preparation method of new lysozyme formulation according to claim 1, it is characterised in that：Adopted in the step (3)
Egg clear solution and polyanionic polysaccharide solution ph are adjusted to 3.0-5.0 respectively with acetum or sodium hydroxide solution；Will
Egg is sufficiently mixed with polyanionic polysaccharide solution, keeps the mass ratio 25-100 of egg and polyanionic polysaccharide:1；Institute
Stirring reaction condition is stated as stirring reaction 30-60min under ice bath.
6. the preparation method of new lysozyme formulation according to claim 1, it is characterised in that：Adopted in the step (4)
20min is centrifuged with 4000r/min, collects lysozyme-polyanionic polysaccharide complex precipitate.
7. the preparation method of new lysozyme formulation according to claim 1, it is characterised in that：The step (4) is done
Drying method is freeze-drying, is dried in vacuo, one kind in spray drying.
8. the preparation method of new lysozyme formulation according to claim 1, it is characterised in that：It is molten in the step (4)
The enzyme activity rate of recovery in bacterium enzyme-polyanionic polysaccharide complex precipitate is 68.3%-87.5%, and protein separation multiple is
1.72-3.21。
9. lysozyme formulation is prepared in the preparation method of the new lysozyme formulation according to claim any one of 1-8.
10. new lysozyme formulation according to claim 9, it is characterised in that：In simulate the gastric juice after 2h, do not embed
Lysozyme activity retention rate is 35.1%, and the lysozyme formulation activity preservation rate is 75.2%-83.3%；The lysozyme system
Release rate of the agent in artificial simulation gastric juices after 2h is 37.5%-42.2%, then is placed in artificial simulation intestinal juice after 4h, total release
Rate is 87.6%-92.8%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108771636A (en) * | 2018-08-13 | 2018-11-09 | 陈丽芬 | A kind of lysozyme formulation |
| CN112655941A (en) * | 2020-12-24 | 2021-04-16 | 河南科技学院 | Method for constructing high internal phase emulsion gel based on dihydromyricetin/hen egg lysozyme compound emulsifier |
| CN114768652A (en) * | 2022-04-06 | 2022-07-22 | 安徽荣达食品有限公司 | Preparation system and method of lysozyme-based antibacterial emulsion |
| CN116440255A (en) * | 2023-06-09 | 2023-07-18 | 枣庄健袖生物医药有限公司 | Antibacterial combined preparation containing egg white lysozyme and synthesis process thereof |
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| CN112655941A (en) * | 2020-12-24 | 2021-04-16 | 河南科技学院 | Method for constructing high internal phase emulsion gel based on dihydromyricetin/hen egg lysozyme compound emulsifier |
| CN114768652A (en) * | 2022-04-06 | 2022-07-22 | 安徽荣达食品有限公司 | Preparation system and method of lysozyme-based antibacterial emulsion |
| CN114768652B (en) * | 2022-04-06 | 2024-03-19 | 安徽荣达食品有限公司 | System and method for preparing lysozyme-based antibacterial emulsion |
| CN116440255A (en) * | 2023-06-09 | 2023-07-18 | 枣庄健袖生物医药有限公司 | Antibacterial combined preparation containing egg white lysozyme and synthesis process thereof |
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