CN105861478A - Nanometer chitin-lysozyme composite particles and preparation method thereof - Google Patents
Nanometer chitin-lysozyme composite particles and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of nanometer chitin-lysozyme composite particles. The method comprises the steps that 1, chitin is dissolved in 2.5-3 M HCl to prepare a solution with the mass-volume concentration being 30-35% is prepared; intense stirring is carried out for 1.5-6 h at 95-105 DEG C for acid hydrolysis; turbid liquid obtained after hydrolysis is centrifuged, washed with water and precipitated; the precipitate obtained after washing is dispersed in water to obtain turbid liquid again, and the obtained turbid liquid is placed in a dialysis bag for dialysis; a sample obtained after dialysis is subjected to ultrasonic treatment for 2-5 min and then placed under the temperature of -85-75 DEG C to be frozen, vacuum freeze drying is carried out, and nanometer chitin is obtained; 2, the nanometer chitin is dissolved in a phosphate buffer solution to prepare a solution with the concentration of 1.96*10<-5>-9.84*10<-5> M, and the solution is subjected to ultrasonic treatment for 1-2 min to be evenly dispersed; lysozyme is added till the concentration of the lysozyme in the solution is 0.3*0.8 mg/mL, stirring is carries out for 2-4 h at the speed of 200-500 rpm, and nanometer chitin-lysozyme composite particles are formed. The composite particles greatly improve activity of lysozyme, and have a good antibacterial effect on Gram-positive bacteria and Gram-negative bacteria.
Description
Technical field
The invention belongs to food medicine enzyme raising field alive, be specifically related to a kind of nano-chitosan-lysozyme composite particles and preparation thereof
Method.
Background technology
In daily life, food is often corrupt, rotten, can cause the generation of a series of disease after eating by mistake, serious threat people's
Healthy.Cause food spoilage reason more, have physical factor, chemical factor and biological factor, wherein polluted by microorganism
Caused food spoilage is mostly important and universal.In view of the toxic and side effects of conventional chemical preservative, exploitation is a kind of safe, high
The food preservative of effect has important economic worth.
Lysozyme itself is a kind of native protein, has no side effect it is considered to be the safest a kind of food additive.It is special
Gate action is in the hydrolytic enzyme of microorganism wall, because it has bacteriolysis, and can be as the bio-preservative of natural safety non-toxic.Molten
Bacterium enzyme all has certain killing and inhibitory action to antibacterial, fungus and virus, can stop or delay food to become to a certain extent
Matter, extends food shelf life.The most by a lot of countries and tissue (U.S., Japan, FAO/WHO and China) approval conduct
Food preservative and antistaling agent use.
Lysozyme, as a kind of natural albumen, also has certain alimentary health-care function, adds appropriate bacteriolyze in baby milk
Enzyme, can make Lac Bovis seu Bubali emulsifying.This strengthening model have kill corrupt coccus in intestinal, maintain intestinal flora normalization, promote double
The functions such as discrimination bacillus field planting, propagation, so the anti-infection ability of infant can be strengthened, contribute to growing up healthy and sound of infant.Simultaneously
Also can add in toothpaste, it can suppress the formation of bacterial plaque and tartar, contributes to preventing the generation of teenager dental caries.Lysozyme is also
There is pharmacological action, have been widely used for now clinical medicine, can enhancing immunity, promote hemostasis, blood coagulation, anti-inflammation etc..
The bacteriolysis specificity of egg white lysozyme is very strong, only specific microorganism is had sufficient dissolution, generally to gram
Positive bacteria action effect is notable, but the invalid or poor effect to gram negative bacteria.Simultaneously as lysozyme is conventional with other
It is higher that price compared by chemical preservative, the most domestic use as food preservative the most less.In order to change this limitation, research
A kind of new method improving lysozyme activity, it is achieved commercialization, widens its application in food service industry and has great importance.
Summary of the invention
The problem existed for existing lysozyme, it is an object of the invention to provide a kind of method improving lysozyme activity, utilizes
Nano-chitosan absorption lysozyme, prepares nano-chitosan-lysozyme composite particles, to improve lysozyme activity and to play it right
The inhibitory action of gram negative bacteria.
The technical solution used in the present invention is:
The preparation method of a kind of nano-chitosan-lysozyme composite particles, specifically includes following steps:
(1) preparation of nano-chitosan
Chitin is dissolved in the HCl of 2.5-3M, prepares the solution that mass body volume concentrations is 30-35%；At 95-105 DEG C acutely
Stirring 1.5-6h carries out acid hydrolysis；The centrifugal also water washing and precipitating of suspension after acid hydrolysis；Precipitation after washing is dispersed in water must hang again
Turbid liquid, and the suspension obtained is placed in bag filter dialysis；Sample ultrasonic 2-5min obtained after dialysis, is positioned over-85～-75 DEG C
In freeze, freeze final vacuum lyophilization and obtain nano-chitosan.
(2) preparation of nano-chitosan-lysozyme composite particles
Being dissolved in phosphate buffer by nano-chitosan, making concentration is 1.96 × 10-5-9.84×10-5The solution of M, ultrasonic 1-2min
Make it be uniformly dispersed；Adding lysozyme, the concentration making Lysozyme in Solution is that 0.3-0.8mg/mL, 200-500rpm stir 2-4h,
Form nano-chitosan-lysozyme composite particles.
Further, the centrifugal force that in step (1), suspension is centrifugal is 3000-5000g, time 2-3min.
Further, in step (1), suspension is washed 5-10 time.
Further, in step (1), bag filter is placed in deionized water and dialyses, and dialyses out by the little molecule in suspension,
Dialysis time is 1-2 days, and every 1-3h changes a water, and the molecular cut off of bag filter is 10000-12000.
Further, in step (1), ultrasonic rear sample is the most freezing, then lyophilizing, and the most freezing purpose is to maintain the original shape of sample
Looks characteristic, vacuum 5-15Pa of vacuum lyophilization, temperature-85～-75 DEG C, time 24-72h.
Further, in step (2), the pH value of phosphate buffer is 6.0-6.5, and concentration is 10-20mM.
Further, in step (1), (2), ultrasonic power is 20KHz.
Another object of the present invention is to provide nano-chitosan-lysozyme composite particles that method described above prepares.
The beneficial outcomes of the present invention is:
(1) present invention uses nano adsorption preparation method, i.e. chitin acidolysis to make nano-particle, nanometer under optimum pH
Grain absorption lysozyme, improves the activity of lysozyme.
(2) technique preparing nano-chitosan in the present invention, by using acid hydrolysis, the method for dialysis to prepare nano-chitosan,
Nano-chitosan productivity prepared by the method is high, good stability, is evenly distributed.
(3) method of preparing nano-chitosan-lysozyme composite particles in the present invention, by using good stability, being evenly distributed
Nano-chitosan absorption lysozyme, shortens the path that enzyme-to-substrate combines, increases the speed of enzyme-to-substrate reaction, thus improve bacteriolyze
The activity of enzyme.
(4) method preparing nano-chitosan-lysozyme composite particles in the present invention is simple, easily operates, and cost is relatively low, is suitable for big
Large-scale production, high volume applications is in food service industry.
(5) in the present invention, gram positive bacteria and gram negative bacteria are all had good by the nano-chitosan-lysozyme composite particles of preparation
Good fungistatic effect.
Accompanying drawing explanation
Fig. 1 is that embodiment 1-5 absorption lysozyme on nano-chitosan is relative to enzyme collection of illustrative plates alive.
Fig. 2 is the transmission electron microscope picture of embodiment 1 nano-chitosan absorption lysozyme.
Fig. 3 is the ultraviolet figure that embodiment 1-5 nano-chitosan interacts with lysozyme.
Fig. 4 is the fluorescence pattern that embodiment 1-5 nano-chitosan interacts with lysozyme.
Fig. 5 is the synchronous fluorescence collection of illustrative plates (Δ λ=15nm) that embodiment 1-5 nano-chitosan interacts with lysozyme.
Fig. 6 is the synchronous fluorescence collection of illustrative plates (Δ λ=60nm) that embodiment 1-5 nano-chitosan interacts with lysozyme.
Fig. 7 is the infrared spectrum that embodiment 1-5 nano-chitosan interacts with lysozyme.
Fig. 8 is the Stern-Volmer cancellation collection of illustrative plates that embodiment 1-5 nano-chitosan interacts with lysozyme.
Fig. 9 is the antibacterial collection of illustrative plates (escherichia coli) of lysozyme in embodiment 1-5 composite particles.
Figure 10 is the antibacterial collection of illustrative plates (golden yellow glucose coccus) of lysozyme in embodiment 1-5 composite particles.
Figure 11 is the antibacterial collection of illustrative plates (bacillus subtilis) of lysozyme in embodiment 1-5 composite particles.
Detailed description of the invention
Below in conjunction with specific embodiment and accompanying drawing, the present invention is described in further details.
Embodiment 1
(1) preparation of nano-chitosan
5g chitin is dissolved in the HCl of 150mL 3M；At 100 DEG C, it is stirred vigorously 2h carries out acid hydrolysis；After acid hydrolysis
Suspension 3000g is centrifuged 2min, water washing and precipitating 5 times；Precipitation after washing be dispersed in water again suspension, and will obtain
Suspension is placed in bag filter dialysis 2 days, and every 2h changes a water；The ultrasonic 3min of sample 20KHz obtained after dialysis, is positioned over
-80 DEG C are freezed, freezes final vacuum lyophilization (vacuum 10Pa, temperature-80 DEG C, time 48h) and obtain nano-chitosan.
(2) preparation of nano-chitosan-lysozyme composite particles
Being dissolved in the 20mM phosphate buffer of pH6.2 by 4mg nano-chitosan, making concentration is 1.96 × 10-5The solution of M,
The ultrasonic 1min of 20KHz makes it be uniformly dispersed；Adding 40mg lysozyme, 500rpm stirs 4h, formation nano-chitosan-molten
Bacterium enzyme composite particles.
Embodiment 2
(1) preparation of nano-chitosan
4.5g chitin is dissolved in the HCl of 150mL 3M；At 103 DEG C, it is stirred vigorously 3h carries out acid hydrolysis；After acid hydrolysis
Suspension 5000g be centrifuged 1min, water washing and precipitating 6 times；Precipitation after washing is dispersed in water and obtains suspension again, and will obtain
Suspension be placed in bag filter dialysis 1.5 days, every 1h changes a water；The ultrasonic 3min of sample 20KHz obtained after dialysis, puts
It is placed in-78 DEG C and freezes, freeze final vacuum lyophilization (vacuum 5Pa, temperature-78 DEG C, time 30h) and obtain nano-chitosan.
(2) preparation of nano-chitosan-lysozyme composite particles
Being dissolved in the 10mM phosphate buffer of pH6.5 by 8mg nano-chitosan, making concentration is 3.93 × 10-5The solution of M,
The ultrasonic 2min of 20KHz makes it be uniformly dispersed；Adding 50mg lysozyme, 400rpm stirs 4h, formation nano-chitosan-molten
Bacterium enzyme composite particles.
Embodiment 3
(1) preparation of nano-chitosan
5.2g chitin is dissolved in the HCl of 150mL 3M；At 98 DEG C, it is stirred vigorously 5h carries out acid hydrolysis；After acid hydrolysis
Suspension 4000g is centrifuged 2min, water washing and precipitating 8 times；Precipitation after washing be dispersed in water again suspension, and will obtain
Suspension is placed in bag filter dialysis 1 day, and every 1h changes a water；The ultrasonic 3min of sample 20KHz obtained after dialysis, is positioned over
-85 DEG C are freezed, freezes final vacuum lyophilization (vacuum 15Pa, temperature-85 DEG C, time 72h) and obtain nano-chitosan.
(2) preparation of nano-chitosan-lysozyme composite particles
Being dissolved in the 15mM phosphate buffer of pH6.3 by 12mg nano-chitosan, making concentration is 5.90 × 10-5The solution of M,
The ultrasonic 1min of 20KHz makes it be uniformly dispersed；Adding 60mg lysozyme, 500rpm stirs 2h, formation nano-chitosan-molten
Bacterium enzyme composite particles.
Embodiment 4
(1) preparation of nano-chitosan
5g chitin is dissolved in the HCl of 150mL 2.5M；At 95 DEG C, it is stirred vigorously 1.5h carries out acid hydrolysis；After acid hydrolysis
Suspension 4000g be centrifuged 2min, water washing and precipitating 10 times；Precipitation after washing is dispersed in water and obtains suspension again, and will obtain
Suspension be placed in bag filter dialysis 1.5 days, every 2h changes a water；The ultrasonic 3min of sample 20KHz obtained after dialysis, puts
It is placed in-80 DEG C and freezes, freeze final vacuum lyophilization (vacuum 10Pa, temperature-80 DEG C, time 24h) and obtain nanometer carapace
Element.
(2) preparation of nano-chitosan-lysozyme composite particles
Being dissolved in the 10mM phosphate buffer of pH6.3 by 16mg nano-chitosan, making concentration is 7.87 × 10-5The solution of M,
The ultrasonic 1min of 20KHz makes it be uniformly dispersed；Adding 80mg lysozyme, 500rpm stirs 4h, formation nano-chitosan-molten
Bacterium enzyme composite particles.
Embodiment 5
(1) preparation of nano-chitosan
4.5g chitin is dissolved in the HCl of 150mL 3M；At 100 DEG C, it is stirred vigorously 6h carries out acid hydrolysis；After acid hydrolysis
Suspension 3000g be centrifuged 3min, water washing and precipitating 5 times；Precipitation after washing is dispersed in water and obtains suspension again, and will obtain
Suspension be placed in bag filter dialysis 2 days, every 3h changes a water；The ultrasonic 3min of sample 20KHz obtained after dialysis, places
Freeze in-75 DEG C, freeze final vacuum lyophilization (vacuum 5Pa, temperature-75 DEG C, time 60h) and obtain nano-chitosan.
(2) preparation of nano-chitosan-lysozyme composite particles
Being dissolved in the 20mM phosphate buffer of pH6.4 by 20mg nano-chitosan, making concentration is 9.84 × 10-5The solution of M,
The ultrasonic 2min of 20KHz makes it be uniformly dispersed；Adding 30mg lysozyme, 400rpm stirs 3h, formation nano-chitosan-molten
Bacterium enzyme composite particles.
Nano-chitosan-lysozyme the composite particles preparing embodiment 1-5 carries out performance measurement:
1. the relative enzyme of nano-chitosan-lysozyme composite particles is lived
The mensuration that enzyme is lived is with reference to the method for (2008) such as Yang.The micrococcus lysodeikticus solution (0.25mg/mL) taking 1.5mL adds
In the cuvette of 1cm, add lysozyme or the lysozyme-nano-chitosan complex of 100 μ L, extinction when measuring 450nm
The change of degree.
Enzyme work=K relativelymix/Kfree× 100%
Wherein KfreeIt is the initial slope of pure lysozyme, KmixIt it is the slope of lysozyme-nano-chitosan complex.
As it is shown in figure 1, the lysozyme activity being not added with nano-chitosan is 100%, add the nano-chitosan lysozyme of 4mg
Enzyme work relatively is 120%, and along with nano-chitosan addition increases, the relative enzyme of lysozyme is lived to be increased, when adding of nano-chitosan
When dosage is 20mg, enzyme is lived and is reached maximum 254%.Compared with the control, the lysozyme enzyme notable increasing alive of nano-chitosan is added
Add.
2. the form of nano-chitosan-lysozyme composite particles and size
The transmission electron microscope figure for nano-chitosan-lysozyme composite particles as shown in Figure 2, it is seen then that nano-chitosan in
Fusiform, a length of 100-500nm, a width of about 5-50nm, smooth surface, size is uniform.As seen from the figure, adsorb molten
After bacterium enzyme, nano-chitosan particle diameter does not change, but occurs the circular spheroid of some 3-9nm on surface, it was demonstrated that lysozyme is
Successfully it is adsorbed onto on nano-chitosan.
3. lysozyme interacts with nano-chitosan
3.1 uv-spectrogram analyses
As it is shown on figure 3, compared with former lysozyme ultraviolet figure, the peak of the lysozyme adding nano-chitosan occurs in that movement, and intensity
Reduce.Along with the interpolation of nano-chitosan, absworption peak gradually moves to high wavelength, when adding 20mg, and absworption peak red shift 3nm,
This is because when in system, nano-chitosan concentration is gradually increased, the protein peptide chain that nano-chitosan has in lysozyme molecule is stretched
Exhibition, the hydrophobic group making the tryptophan within lysozyme and tyrosine residue is exposed out, so that lysozyme absorption peak strength strengthens,
This shows that nano-chitosan and lysozyme there occurs reaction.
3.2 fluorescence pattern analyses
As shown in Figure 4, when excitation wave is 295nm, former lysozyme has maximum absorption band at 336nm, along with nanometer in system
Chitin concentration is gradually increased, and peak is gradually lowered by force, and blue-shifted phenomenon occurs.When adding 16mg nano-chitosan, lysozyme
Maximum absorption wavelength move to 334nm from 336nm, show in system add nano-chitosan cause the endogenous glimmering of lysozyme
The cancellation of light, illustrates in the process of mixing, the two there occurs that interaction forms nano-chitosan-lysozyme complex.
3.3 synchronous fluorescence atlas analysis
As it is shown in figure 5, the tyrosine residue fluorescence of fluorescence display protein during Δ λ=λ em-λ ex=15nm, along with system is received
Rice chitin concentration is gradually increased, and peak is gradually lowered by force, but the position at peak is not moved.As shown in Figure 6, Δ λ=λ em-
Synchronous fluorescent spectrum during λ ex=60nm only shows protein trp residue fluorescence, along with in system, nano-chitosan concentration is gradually
Increasing, peak is gradually lowered by force, and the position of fluorescence peak there occurs blue shift slightly, moves to 275nm from 276nm, shows color ammonia
Microenvironment residing for acid changes, and illustrates in the process of mixing, the two there occurs that interaction formation nano-chitosan-lysozyme is multiple
Compound.
3.4 infrared spectrum analysis
As it is shown in fig. 7, lysozyme is at 1241cm-1The absworption peak at place is the vibration peak of C-O, 1551cm-1It is that N-H bond bending shakes
The absworption peak that movable property is raw.After nano-chitosan and lysozyme interact, 1241cm-1The absworption peak at place moves right, and N-H key exists
1551cm-1Bending vibration peak move to low wavelength, show that the conformation of protein there occurs change, illustrate to add after nano-chitosan,
Lysozyme there occurs interaction with it.
4. lysozyme and nano-chitosan combination and binding site
As shown in Figure 8, F0/F (y) and [Q] (x) in the cancellation Stern-Volmer curve that lysozyme and nano-chitosan interact
Between there is good linear relationship, the fluorescence lifetime of biomacromolecule is generally 10-8S, therefore quenching rate Kq is
5.78×1011Lmol-ls-l, its numerical value maximally diffuses collisional quenching speed constant more than quencher and is about biomacromolecule
2.0×1010, it is possible to speculate that nano-chitosan and lysozyme combination are static cancellation.
According to log [(F0-F)/F] and the linear relationship of log [CHNC], calculating binding site is 1.03, and binding constant is
3.29×103Lmol-1, as shown in table 1.
Table 1 is the incorporating parametric and binding site table that in the present invention, nano-chitosan interacts with lysozyme.(Ka representative is knot
Closing constant, what n represented is binding site)
The most antibacterial collection of illustrative plates
As it is shown in figure 9, lysozyme to the suppression ratio of escherichia coli (gram negative bacteria) in downward trend after first rising.Along with
In system, nano-chitosan concentration is gradually increased, and colibacillary suppression ratio is gradually increasing by lysozyme, shows that nano-chitosan is permissible
Strengthen the lysozyme suppression ratio to gram negative bacteria.
As shown in Figure 10, after the suppression of staphylococcus aureus (gram positive bacteria) is taken the lead in rising by lysozyme steadily.Along with
In system, nano-chitosan concentration is gradually increased, and the suppression ratio of bacillus subtilis is gradually increasing by lysozyme, shows nano-chitosan
The lysozyme suppression ratio to gram positive bacteria can be strengthened.
As shown in figure 11, after the suppression of bacillus subtilis (gram positive bacteria) is taken the lead in rising by lysozyme steadily.Along with body
In system, nano-chitosan concentration is gradually increased, and the suppression ratio of bacillus subtilis is gradually increasing by lysozyme, shows that nano-chitosan can
To strengthen the lysozyme suppression ratio to gram positive bacteria.
Claims (8)
1. a preparation method for nano-chitosan-lysozyme composite particles, specifically includes following steps:
(1) preparation of nano-chitosan
Chitin is dissolved in the HCl of 2.5-3M, prepares the solution that mass body volume concentrations is 30-35%；At 95-105 DEG C acutely
Stirring 1.5-6h carries out acid hydrolysis；The centrifugal also water washing and precipitating of suspension after acid hydrolysis；Precipitation after washing is dispersed in water again
Obtain suspension, and the suspension obtained is placed in bag filter dialysis；Sample ultrasonic 2-5min obtained after dialysis, is positioned over
-85～-75 DEG C in freeze, freeze final vacuum lyophilization and obtain nano-chitosan.
(2) preparation of nano-chitosan-lysozyme composite particles
Being dissolved in phosphate buffer by nano-chitosan, making concentration is 1.96 × 10-5-9.84×10-5The solution of M, ultrasonic 1-2min
Make it be uniformly dispersed；Adding lysozyme, the concentration making Lysozyme in Solution is 0.3-0.8mg/mL, 200-500rpm stirring
2-4h, forms nano-chitosan-lysozyme composite particles.
The preparation method of nano-chitosan the most according to claim 1-lysozyme composite particles, it is characterised in that step (1)
The centrifugal force that middle suspension is centrifuged is 3000-5000g, time 2-3min.
The preparation method of nano-chitosan the most according to claim 1-lysozyme composite particles, it is characterised in that step (1)
Middle suspension is washed 5-10 time.
The preparation method of nano-chitosan the most according to claim 1-lysozyme composite particles, it is characterised in that step (1)
Middle bag filter is placed in deionized water dialyses, and dialyses out by the little molecule in suspension, and dialysis time is 1-2 days,
Every 1-3h changes a water, and the molecular cut off of bag filter is 10000-12000.
The preparation method of nano-chitosan the most according to claim 1-lysozyme composite particles, it is characterised in that step (1)
In ultrasonic rear sample first freezing, then lyophilizing, the most freezing purpose is to maintain the original topographical property of sample, vacuum lyophilization
Vacuum 5-15Pa, temperature-85～-75 DEG C, time 24-72h.
The preparation method of nano-chitosan the most according to claim 1-lysozyme composite particles, it is characterised in that step (2)
The pH value of middle phosphate buffer is 6.0-6.5, and concentration is 10-20mM.
The preparation method of nano-chitosan the most according to claim 1-lysozyme composite particles, it is characterised in that step (1),
(2) in, ultrasonic power is 20KHz.
8. nano-chitosan-lysozyme the composite particles prepared according to the arbitrary described method of claim 1-7, it is characterised in that by weighing
Profit requires that the arbitrary described method of 1-7 is prepared from.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479997A (en) * | 2016-11-28 | 2017-03-08 | 陕西师范大学 | Lysozyme nanocrystalline colloidal sol and the protein polycrystalline hydrogel prepared using which and preparation method |
| CN108690305A (en) * | 2018-05-30 | 2018-10-23 | 安徽农业大学 | A kind of preparation method of high-performance organic-inorganic composite membrane |
| CN108751126A (en) * | 2018-06-08 | 2018-11-06 | 陕西师范大学 | The method for preparing three-dimensional self-supporting film based on lysozyme nano thin-film |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050457A (en) * | 2007-03-20 | 2007-10-10 | 王艳 | Method for fixing chitosan enzyme by using cross-linking adsorption of chitosan - glutaraldehyde |
-
2016
- 2016-04-19 CN CN201610242360.1A patent/CN105861478A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050457A (en) * | 2007-03-20 | 2007-10-10 | 王艳 | Method for fixing chitosan enzyme by using cross-linking adsorption of chitosan - glutaraldehyde |
Non-Patent Citations (3)
| Title |
|---|
| ASIER M. SALABERRIA 等: "Different routes to turn chitin into stunning nano-objects", 《EUROPEAN POLYMER JOURNAL》 * |
| JIANG SUISUI 等: "Enhanced antibacterial activity of lysozyme immobilized on chitin nanowhiskers", 《FOOD CHEMISTRY》 * |
| 周纪宁 等: "甲壳素/壳聚糖及其衍生物在固定化酶中的应用", 《化工新型材料》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479997A (en) * | 2016-11-28 | 2017-03-08 | 陕西师范大学 | Lysozyme nanocrystalline colloidal sol and the protein polycrystalline hydrogel prepared using which and preparation method |
| CN106479997B (en) * | 2016-11-28 | 2019-05-14 | 陕西师范大学 | Lysozyme nanocrystalline colloidal sol and the protein polycrystalline hydrogel and preparation method for using its preparation |
| CN108690305A (en) * | 2018-05-30 | 2018-10-23 | 安徽农业大学 | A kind of preparation method of high-performance organic-inorganic composite membrane |
| CN108751126A (en) * | 2018-06-08 | 2018-11-06 | 陕西师范大学 | The method for preparing three-dimensional self-supporting film based on lysozyme nano thin-film |
| CN108751126B (en) * | 2018-06-08 | 2019-10-25 | 陕西师范大学 | The method for preparing three-dimensional self-supporting film based on lysozyme nano thin-film |
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