CN108192000A - A kind of chitosan oligosaccharide graft copolymer G1.0 and its preparation method and application - Google Patents
A kind of chitosan oligosaccharide graft copolymer G1.0 and its preparation method and application Download PDFInfo
- Publication number
- CN108192000A CN108192000A CN201810033904.2A CN201810033904A CN108192000A CN 108192000 A CN108192000 A CN 108192000A CN 201810033904 A CN201810033904 A CN 201810033904A CN 108192000 A CN108192000 A CN 108192000A
- Authority
- CN
- China
- Prior art keywords
- chitosan oligosaccharide
- reaction
- chitosan
- graft copolymer
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
- C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3562—Sugars; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a kind of chitosan oligosaccharide graft copolymer G1.0 and its preparation method and application.The present invention uses free radical initiation grafting method, chitosan oligosaccharide is prepared using the method degradation chitosan that three kinds of physics, chemistry, enzymolysis influencing factors are combined, then using chitosan oligosaccharide as raw material, radical reaction grafted propylene acid methyl esters occurs for the active group for first causing chitosan oligosaccharide molecule by ammonium ceric nitrate, it is branched modification, divergent method is recycled to carry out Michael addition reactions to prepared chitosan oligosaccharide, ethylenediamine is grafted, prepares graft copolymer G1.0.The present invention provides a kind of good water solubility, the stronger chitosan oligosaccharide graft copolymers of bacteriostasis.The present invention provides a kind of preparation methods of chitosan oligosaccharide graft copolymer of the present invention.The present invention chitose graft copolymer have good water solubility, bacteriostasis is strong, can be used as food antiseptic bacteriostatic agent the advantages of.
Description
Technical field
The invention belongs to Food Chemistry field, a kind of a kind of bacteriostatic agent being related in Food Chemistry, and in particular to chitosan oligosaccharide
Graft copolymer and its preparation method and application.
Background technology
Chitosan (Chitosan) is the polysaccharide product that chitin (Chitin) deacetylation reaches more than 55%, be by β-
Natural alkaline polysaccharide [1] existing for the nature minority of (Isosorbide-5-Nitrae) -2-amino-2-deoxy-D-Glucose composition.Chitin is certainly
The second largest natural polysaccharide resource [2 of annual biosynthesis amount (up to 1010 tons/year) dimension element in right boundary；3], chemical constitution
Unit is N- acetyl group -2-amino-2-deoxy-D-Glucose binary linearity macromolecular polymeric by β-(Isosorbide-5-Nitrae) glucosides key connection
Object [4], be primarily present in the shell and endocuticle of oceanic invertebrate such as shrimp, crab and shellfish, fungi (yeast,
Mould, mushroom etc.), in the cell wall of algae and higher plant [5].Although chitin derive from a wealth of sources it is nexhaustible, due to point
In sub or between intermolecular two neighboring sugar unit structure, oxygen atom and another saccharide ring hydroxyl on one of saccharide ring base
In hydrogen atom be easily bonded to each other to form hydrogen bond, and there is also the close-connected hydrogen bonds of numerous CO-NH for intramolecular
[6-8], the effect of a large amount of hydrogen bonds hinder rotating freely for glycosidic bond so that chitin molecule forms high-sequential arrangement
Crystal region.The existence form of chitin has tri- kinds of crystal forms [9] of α, β, γ in nature, wherein most commonly seen with β types.Rule
Crystal structure increases the rigidity of chitin molecule, and the dissolubility for making it is very poor, not soluble in water, olefin(e) acid, diluted alkaline and general organic
Reagent significantly limits its application and development.Compared with chitin, chitosan makes moderate progress in terms of dissolubility, but molecule
In there are still more hydrogen bond, pK values be about 6.5 [10], be only dissolve in dilute acid soln, it is not soluble in water and common organic
Solvent, this weakness also limit the application of chitosan to a certain extent.Although after chitin deacetylation generation chitosan
Dissolubility is barely satisfactory, but due in the structural unit of chitosan in addition on C-3 and C-6 there are active hydroxyl group other than, C-
2 also there is active amine groups, when it is dispersed in the olefin(e) acid solution of below 10wt% ,-NH2 meetings and H+ on C-2
Protonation reaction occurs, forms the cationic polyelectrolyte of positively charged-NH3+, makes it that electropositive [11] be presented.This is great to change
Change makes chitosan possess many unique chemical property：Good biocompatibility, has no toxic side effect and prevents at degradability
The bioactivity [3] such as rotten, antibacterial, promotion wound healing, anticancer.
Chitosan and its derivative has become current hot spot in the research of antibacterial aspect with application, but due to chitosan
Poorly water-soluble, bacteriostasis are limited, significantly limit its application in the actual production process.
Chitosan be only second at present cellulose second largest natural polysaccharide resource and nature there is only a kind of day
Right alkaline polysaccharide.Because of its rich reserves, derive from a wealth of sources, cheap, and with good biocompatibility, degradability,
It has no toxic side effect and the biochemical activities such as biocidal property, is widely used in the various fields such as medicine, food, environmental protection, agricultural.From
The suppression of chitosan
Invention content
The problem of for background technology, the present invention provides a kind of good water solubility, the stronger shell of bacteriostasis are few
Sugared graft copolymer.
It is a further object to provide a kind of preparation methods of chitosan oligosaccharide graft copolymer of the present invention.
Another object of the present invention is the chitosan oligosaccharide graft copolymer using the present invention as food antiseptic bacteriostatic agent
Using.
The technical solution adopted by the present invention is as follows：A kind of chitosan oligosaccharide graft copolymer G1.0, molecular formula are as follows：
Wherein n is any integer value.
A kind of preparation method of chitosan oligosaccharide graft copolymer G1.0, the preparation method are as follows：
1) a certain amount of chitosan oligosaccharide is weighed, adds in the acetic acid of 1% (v/v) as reaction dissolvent, acetic acid addition and shell widow
The aggregate relation of sugar is as follows：Above-mentioned acetic acid solution 25ml is added in per 1g chitosan oligosaccharides；Fully after dissolving, nitrogen is passed through into bottle
Remove oxygen；A certain amount of ammonium ceric nitrate is added under nitrogen protection as initiator, the addition of initiator and chitosan oligosaccharide
Mass ratio is 0.5:1-2.5:1；Then appropriate methyl acrylate is slowly added dropwise；Above-mentioned chitosan oligosaccharide and methyl acrylate
Reaction molar ratio be 1：4—1：10；Setting mixing speed is 200rpm/min, successive reaction 2 at a temperature of 40 DEG C -55 DEG C -
10h；It waits after the completion of reacting, reaction gains G0.5 is filtered out, the reaction gains G0.5 filtered out is first then used into reaction dissolvent
1% (v/v) acetic acid filters 3 times, then acetone, ether, absolute ethyl alcohol rinse 3 times repeatedly successively, remove unreacted acrylic acid first
Ester is finally putting into 50 DEG C of vacuum drying chambers and is dried for standby；
2) G0.5 that above-mentioned steps are made spare is weighed, the methanol solvate for adding in chromatographic grade impregnates 12h, controlling reaction temperature
It is 20 DEG C, the pure ethylenediamine of analysis, the lower reaction 8h of N2 protections is added dropwise；The addition of methanol and the aggregate relation of G0.5 are as follows：
0.5ml methanol is added in per 1mgG0.5；The addition of ethylenediamine and the aggregate relation of G0.5 are as follows：It is added in per 1mgG0.5
0.08ml ethylenediamines；It waits after the completion of reacting, reaction gains G1.0 is filtered out, is rinsed 3 times with reaction dissolvent methanol, Ran Houzai
It is washed repeatedly 3 times with absolute ethyl alcohol, distilled water successively, is placed in 50 DEG C and is dried in vacuo to obtain the final product.
Preferably, contain in the methyl acrylate added in the preparation method step 1) of chitosan oligosaccharide graft copolymer G1.0
The polymerization inhibitor of self-polymerization is prevented to methoxyl group phenol MEHQ.
Preferably, in the preparation method step 1) of chitosan oligosaccharide graft copolymer G1.0 chitosan oligosaccharide and methyl acrylate it is anti-
It is 1 to answer molar ratio:8—1:8.5.
Preferably, setting mixing speed in the preparation method step 1) of chitosan oligosaccharide graft copolymer G1.0 and being
200rpm/min, continuous reaction time is 8h at a temperature of 40 DEG C -55 DEG C.
Preferably, the preparation process of reaction raw materials chitosan oligosaccharide described in the preparation method of chitosan oligosaccharide graft copolymer G1.0
It is as follows：1), weigh a certain amount of chitosan makes it be completely dissolved in a certain amount of 1% (v/v) acetic acid solution by high-speed stirred
In, then solution is transferred in quartzy three-necked bottle, adds a certain amount of 30% (v/v) H2O2, is correctly installed on microwave synthesis
In instrument cavity, ultrasonic probe is made to be less than below the interface of solution；Microwave power 300w is set, reacts 30min, is opened ultraviolet
Light shines into row assistant degradation, and to the end of experiment, then water-bath concentrated by rotary evaporation under solution is spent at 40 DEG C is freeze-dried
For 24 hours, you can obtain chitooligosaccharide- samples of the viscosity average molecular weigh M η 25000 or so；Above-mentioned acetic acid addition and chitosan it is total
Magnitude relation is as follows：The above-mentioned acetic acid solutions of 100mL, H are added in per 3g chitosans2O2The aggregate relation of addition and chitosan is as follows：
0.1mL30% (v/v) H is added in per 1g chitosans2O2；
2) it is, 1 according to mass ratio：1：1 ratio weighs a certain amount of papain, pectase and cellulase and is dissolved in
In the buffer solution of the HAC-NaAC of pH=5.5, the enzyme solution of 1mg/mL is configured to, for use；By the above-mentioned shell after microwave degradation
The pH of oligosaccharide solution is adjusted to 5.5, is 1 according to the ratio between concentration of enzyme-to-substrate：10, above-mentioned for use enzyme solution is added in, stirring is equal
It is even, it is placed in digesting 4h in 45 DEG C of constant temperature water bath；Boiling heating 10min after the completion makes enzyme-deactivating, is evaporated under reduced pressure at 40 DEG C
It is concentrated to give the reaction raw materials chitosan oligosaccharide sample solution needed for preparing.
Preferably, the preparation process of reaction raw materials chitosan oligosaccharide described in the preparation method of chitosan oligosaccharide graft copolymer G1.0
It further includes and molecular-weight gradation is carried out to the chitosan oligosaccharide sample solution for preparing gained, choose the chitosan oligosaccharide that molecular weight is 3500-7000
As raw material；Specific stage division is as follows：A diameter of 25mm molecular cut offs 7000 and diameter 34mm retention molecules are chosen respectively
The bag filter measured as 3500 carries out pre-treatment, removes metal ion；The smaller bag filter of diameter is inserted in the dialysis being relatively large in diameter
In bag, wherein one end bag filter is clamping fixed, and distilled water flushing is clean, and then pipetting claim 6 with the liquid-transfering gun of 5mL makes
Standby chitosan oligosaccharide sample solution is packed into inner layer bag filter, the gap between the double-deck bag filter of distilled water filling, bag filter clamp
Mouthful, the 5d that dialyses is then placed in the large beaker equipped with distilled water；The liquid of double-deck intermembrane space is taken out after the completion of dialysis, freezing
It is dry, you can to obtain the reaction raw materials chitosan oligosaccharide of 3500-7000 molecular weight.
Applications of the chitosan oligosaccharide graft copolymer G1.0 of the present invention as food antiseptic bacteriostatic agent.
The invention has the advantages that：
1st, noval chemical compound chitosan oligosaccharide graft copolymer G1.0 provided by the invention has good water solubility, and bacteriostasis is strong, and
It can be used as food antiseptic bacteriostatic agent.
2nd, the present invention is using free radical initiation grafting method, the side being combined using three kinds of physics, chemistry, enzymolysis influencing factors
Method degradation chitosan prepares chitosan oligosaccharide, then carries out molecular-weight gradation by double-deck dialysis to chitosan oligosaccharide again, chooses specific quantity
Molecular weight be used as raw material, radical reaction grafting third occurs for the active group for first causing chitosan oligosaccharide molecule by ammonium ceric nitrate
E pioic acid methyl ester is branched modification, and divergent method is recycled to carry out Michael addition reactions to prepared chitosan oligosaccharide, is grafted second
Diamines prepares graft copolymer G1.0.It is totally different from quaternization reaction of the prior art.
3rd, the present invention carries out microwave/ultrasonic wave/UV to chitosan and assists H2O2At two steps of quick viscosity reduction and non-specific enzymolysis
Reason, by the way that the condition of microwave and enzymolysis is controlled to realize to its fast and effective degradation treatment, the method can under low concentration into
Row, generates the condensation side reaction of carbonyl ammonia amino activity is caused to reduce this and lack when effectively avoiding hydrogen peroxide degradation chitosan
Point, and experiment condition is mild, and degradation speed is fast, and efficiently.
4th, chitosan oligosaccharide graft copolymer G1.0 bacteriostasis properties provided by the invention protrude：The present invention uses MIC assay methods,
By to Escherichia coli, two kinds of Gram-negative bacterias of pseudomonas aeruginosa and staphylococcus aureus and Bacillus subtillis two
The MIC value of kind gram-positive bacteria measures, it is found that G1.0 is respectively provided with significant fungistatic effect to these four tested bacterium.
Description of the drawings
Fig. 1 is the schematic diagram of the chitosan oligosaccharide of duplicature dialysis separation specified molecular weight.
Fig. 2 is the influence for reacting molar ratio to G0.5 grafting rates.
Fig. 3 is influence of the reaction temperature to G0.5 grafting rates.
Fig. 4 is influence of the initiator additive amount to G0.5 grafting rates.
Fig. 5 be it is seasonable between influence to G0.5 grafting rates.
Fig. 6 is the response surface 3D figures and contour map of AB, AC, BC cross action.
Fig. 7 is that the carbon nitrogen percentage composition of different algebraically molecules compares block diagram.
Fig. 8 is the thermogravimetric curve figure of G0.5-3.0.
Fig. 9 is chitosan oligosaccharide (COS), methyl acrylate (MA) and G0.5 infrared spectrograms.
Figure 10 is G0.5, ethylenediamine (EDA) and G1.0 infrared spectrograms.
Figure 11 is G0.5, G1.0 infrared spectrogram.
Figure 12 is the Zeta potential values of the COS/PAMAM of different algebraically in aqueous solution.
Specific embodiment
With reference to specific embodiment, the invention will be further described, but the present invention is not limited to following embodiments.
1 prepares chitosan oligosaccharide
1.1.1 experiment material and reagent
Table 2-1 main materials and reagent
2.1.2 laboratory apparatus and equipment
Table 2-2 key instruments and equipment
1.2.1 stepwise degradation chitosan prepares chitosan oligosaccharide
1.2.1.1 microwave/ultrasonic wave/UV auxiliary quick viscosity reductions of H2O2
It is accurate to weigh 3g chitosans it to be made to be completely dissolved in (v/v) acetic acid of 100mL certain concentrations by high-speed stirred molten
In liquid, then solution is transferred in the quartzy three-necked bottle of 250mL, adds a certain amount of 30%H2O2, correctly it is installed on microwave
In synthesizer cavity, pay attention to having to that ultrasonic probe is made not have below the interface of solution.The power of mounting ultrasonic is
200W sets microwave power and reaction time, opens ultraviolet lamp (70uw/cm2) switch progress assistant degradation, to the end of experiment,
Water-bath concentrated by rotary evaporation under solution is spent at 40 DEG C, is then freeze-dried for 24 hours, obtains chitooligosaccharide- sample.At this step
The molecular weight of chitosan is reduced to tens of thousands of orders of magnitude by reason.
L will be passed through in this experiment9(34) orthogonal test is to reaction time (min), H2O2Additive amount (mL), microwave power
(W), four essential conditions of acetic acid concentration (V/V, %) are optimized and are screened, and finally obtain best quick viscosity reduction condition.It is real
Test the factor level table such as table 2-3 of design：
Table 2-3 L9(34) orthogonal experiment factor level table
1.2.1.2 non-specific enzymolysis
It is 1 according to mass ratio：1：1 ratio weighs a certain amount of papain, pectase and cellulase and is dissolved in pH
In the buffer solution of=5.5 HAc-NaAc, the enzyme solution of 1mg/mL is configured to, for use.The above-mentioned shell after microwave degradation is low
The pH of glycan solution is adjusted to 5.5, is 1 according to the ratio between concentration of enzyme-to-substrate：10,3mg enzyme solutions are added in, are stirred evenly, juxtaposition
4h is digested in 45 DEG C of constant temperature water bath.Boiling heating 10min after the completion makes enzyme-deactivating, is evaporated under reduced pressure and is concentrated to give at 40 DEG C
Chitosan oligosaccharide sample solution.
The separation product 1.2.1.3 duplicature is dialysed
Molecular weight distribution to solve the problems, such as product in chitosan oligosaccharide preparation process is inhomogenous, herein creatively using double
The method of tunic dialysis detaches above-mentioned products therefrom.Concrete operations：A diameter of 25mm molecular cut offs are chosen respectively
The bag filter that 7000 and diameter 34mm molecular cut offs are 3500 carries out pre-treatment, removes metal ion.Diameter is smaller
Analysis bag is inserted in the bag filter being relatively large in diameter, and wherein one end bag filter is clamping fixed, and distilled water flushing is clean, then with 5mL's
The chitosan oligosaccharide sample solution that liquid-transfering gun pipettes appropriate above-mentioned preparation is packed into inner layer bag filter, the double-deck bag filter of distilled water filling
Between gap, bag filter clamp opening, dialyse 5d in distilled water under room temperature.By double-deck intermembrane space after the completion of dialysis
Liquid takes out, freeze-drying, you can obtain the pale yellow powder shape chitosan oligosaccharide sample of specified molecular weight.Duplicature dialysis principle
As shown in Figure 1.
1.2.1.4 microwave/ultrasonic wave/UV auxiliary H2O2Quick viscosity reduction orthogonal experiments
Table 2-4 Orthogonal experiment results
In summary the result of orthogonal experiment can obtain：Influencing the empirical factor of degradation chitosan is successively：During microwave
Between>H2O2Additive amount>Microwave power>Acetic acid concentration；The optimum condition of experiment is：H2O2Additive amount is 0.3mL, microwave power
300W, reaction time 30min, acetic acid concentration 1%.It repeats to test under above-mentioned optimum experimental condition, can obtain gluing equal molecule
Measure chitooligosaccharide- samples of the M η 25000 or so.
2 prepare G1.0
2.1.1 experiment material and reagent
Table 3-1 main agents
2.1.2 laboratory apparatus and equipment
Table 3-2 key instrument equipment
2.2.1 the preparation of G0.5
The accurate chitosan oligosaccharide sample for weighing the upper chapter preparations of 1g is placed in 100mL three-necked bottles, adds in 25mL1% (v/v) acetic acid
It as reaction dissolvent, fully dissolves, nitrogen 30min is passed through into bottle and removes oxygen.A certain amount of nitre is added under nitrogen protection
Then appropriate methyl acrylate is slowly added dropwise (containing preventing the polymerization inhibitor of self-polymerization to first as initiator in sour cerium ammonium
Oxygroup phenol MEHQ), here be derived from Aladdin reagent net to methoxyl group phenol MEHQ, product identification M100033,>99.0%
(GC), the MEHQ stabilizers containing≤100ppm.Setting mixing speed is 200rpm/min, successive reaction 8h under certain temperature.It treats
After the completion of reaction, G0.5 is filtered out, is first filtered 3 times with reaction dissolvent, then rinsed repeatedly with acetone, ether, absolute ethyl alcohol successively
3 times, unreacted methyl acrylate is removed, 50 DEG C of vacuum drying chambers is finally putting into and is dried for standby.
To four important factor in order in above-mentioned experiment：Initiator additive amount (g), methyl acrylate are reacted with chitosan oligosaccharide
Molar ratio (mL/g), reaction temperature (DEG C), reaction time carry out experiment of single factor conditional FP tree.Fixation response time, Ran Hougen
According to the principle of Box-Benhnken Centers by response phase method to other cross influences of three principal elements between any two
Effect is discussed, and is carried out factor level analysis using Design-Expert8.0.4, is screened the optimum synthesis condition of G0.5.
The factor level table of response phase method design is as shown in table 3-3：
The factor level table of table 3-3 response phase method experimental designs
The principle that free radical grafting reacts occurs between the ligand molecular and chitosan oligosaccharide that cause about ammonium ceric nitrate to illustrate such as
Under[83]：Ce4+The first C with saccharide residue2-NH2And C3- OH reacts, and forms a kind of compound.Subsequent C2And C3It is disproportionated, formed-
CH=NH and two kinds of free radicals of-CH (OH).Free radical may be in C2And C3Atom is centrally formed, it is also possible to it is formed on-NH, because
This ammonium ceric nitrate may cause chitosan oligosaccharide molecule and generate three kinds of different free radicals, and then graft reaction occurs, and ultimately generate
Chitosan oligosaccharide derivative.
2.2.2 the preparation of G1.0
It weighs the G0.550mg synthesized under optimum condition and is placed in 100mL three-necked bottles, and add in the immersion of 25mL methanol solvates
12h, controlling reaction temperature are 20 DEG C, and 4ml ethylenediamines, N is added dropwise2The lower reaction 8h of protection.It waits after the completion of reacting, by G1.0
It filters out, is rinsed 3 times with reaction dissolvent, then washed repeatedly for several times with absolute ethyl alcohol, distilled water successively again, be placed in 50 DEG C of vacuum
It is dry.
3.2.5.1 the grafting rate in half generation of determination of elemental analysis
1.000mg-2.000mg chitosan oligosaccharides and the per graft product in generation are accurately weighed by millionth balance, used
Sample disc is sequentially placed into after tin capsule package using elemental analyser progress C, N, the measure of H element content, and passes through the following formula
3-1 calculates half grafting rate (functional group conversion, %) for product.Using grafting rate as dependent variable into
Row experiment of single factor is explored and response surface condition optimizing.
Wherein：X-- product graft rates (%)；
Fo-- G1.5 and G2.5 is amino content in upper half generation, and G0.5 is chitosan oligosaccharide deacetylation (%)；
The relative atomic mass of Mc, Mn--C, N atom；
Contained C atoms number in Nc-- grafting molecules；
C, N percentage composition ratio in R-- products.
3.2.5.2 double jumping current potentials survey the amino content in whole generation
In the synthesis process, it is all the beginning compound using amino as end group to be grafted the whole generation molecule generated after ethylenediamine,
The amino content of whole generation molecule can be measured using double jumping constant-current titrations, its grafting efficiency can be evaluated.Accurately weigh
0.1gG1.0, G2.0, G3.0 sample are dissolved in the hydrochloric acid standard solution of 0.1mol/L, under permanent pH/mV automatical potentiometric titrimeters
It is titrated with the NaOH solution of the 0.1mol/L demarcated.The amino content of sample is calculated by formula 3-2：
Wherein, V1-- the NaOH volumes (mL) of first time jumping consumption；
V2-- NaOH (mL) volume of second of jumping consumption；
The concentration (mol/L) of C--NaOH standard solution；
0.016-- and a concentration of comparable amine amounts (g) of 1mol/LNaOH solution of 1mL；
M-- sample qualities (g).
3.2.5.3 infrared structure characterizes
By sample respectively with KBr (spectroscopic pure) with 1：50-1：100 ratio mixing, is fully ground and transparent sheet is made, lead to
It crosses NICOLET-380 types Fourier infrared spectrograph and FTIR spectrogram scannings, scanning range 4000cm is carried out to its structure-1-
400cm-1, resolution ratio 4cm-1, scanning times are 32 times.
3.2.5.4 thermogravimetric analysis (TGA)
Thermogravimetric analysis is to measure the important characterization method of substance thermal stability, is become as the temperature of program setting is raised
Gesture, degradation can occur under corresponding temperature for determinand leads to mass change, can be obtained according to weight-loss ratio variation with temperature curve
The substance further carries out qualitative analysis to the sensitivity of heat to its structure.Concrete operations are：The pact that will be dried to constant weight
The sample of 20.000mg is put into alumina crucible, is then slowly positioned on the sample cell of Instrument furnace body chamber, closes chamber
Door, at this moment program can show the accurate mass of sample automatically.Set determination condition as：Nitrogen carrier gas flow is 20mL/min, is risen
Warm 10 DEG C/min of rate, start-stop temperature are 25~800 DEG C.
3.2.5.5 Zeta potential measures
The end of the whole generation molecule of COS/PAMAM dendrimer derivatives is all active-NH2Beginning, and per pickup branch
There will be the variation of amino, amino is positively charged active group, using its potential change of Zeta potential Observable, is come with this
Making up grafting repeatedly, repeatedly rear infrared signature absorption peak can not judge the deficiency of its structure feature without significant change.By sample
The aqueous solution of 0.05mg/mL is configured to, then measures its Zeta electricity three times using nano-particle size analysis instrument continuous scanning at 25 DEG C
Place value.
3.3.1 the single factor test conditional FP tree of G0.5 synthesis
3.3.1.1 influence of the reaction molar ratio to G0.5 grafting rates
Under normal circumstances, only just meeting is reacted when reactivity site and grafting functional base content reach proper proportion
It carries out to the maximum extent.As can be seen from Figure 2, in a certain range, grafting rate increases with the content of reaction monomers and is increased, this master
If because with the increase of graft monomer contents, the diffusion velocity of reactivity function base is accelerated, and is allowed to and is grafted object
The effective collision number of active site increases, and reacts and is carried out to positive direction.When reaching reaction saturation ratio, i.e., shell is few in figure
The ratio of sugar and methyl acrylate reaches 1：When 8, since content of monomer excessively causes system viscosity to increase, function base diffusion speed
Degree slows down, and vies each other active site between reaction monomers and forms inhibiting effect, and since methyl acrylate easily occurs
Self-polymerization causes the increase of side reaction, eventually leads to grafting rate reduction.
3.3.1.2 influence of the reaction temperature to G0.5 grafting rates
Most of synthetic reactions belong to the endothermic reaction, and appropriate temperature can give energy necessary to reaction, push
Reaction generates gibbs free energy change and then reacts.As can be seen from Figure 3：Reaction the starting stage, grafting rate with
The raising of reaction temperature and increase, improve temperature not only can provocative reaction site to the maximum extent activity, but also this experiment
Initiator ammonium ceric nitrate used could only meet the bond dissociation energy needed for reaction under the conditions of higher than 40 DEG C, and it is few to cause shell
Active group generates free radicals in glycan molecule.When temperature reaches 45 DEG C, the primary group of free radicals concentration of system reaches maximum, connects
Branch rate highest.When continuing to improve temperature, the side reaction in reaction system increases, and reactive functional group quantity is reduced, it is suppressed that main anti-
The progress answered, causes grafting rate to decline.
3.3.1.3 influence of the initiator additive amount to G0.5 grafting rates
In this experiment, the additive amount of initiator ammonium ceric nitrate is one of principal element for influencing reactive grafting rate.In nitric acid
Under the Ce+ effects of cerium ammonium, a large amount of free radicals can be generated in chitosan oligosaccharide unit molecule, enhance the electricity to C=C in methyl acrylate
Sub- sucking action, under the attack of free radical, double bond is opened, and with the intermolecular formation chemical bond of chitosan oligosaccharide, graft reaction occurs.
The principle of reaction is generated free radicals according to ammonium ceric nitrate initiation chitosan oligosaccharide, a certain amount of chitosan oligosaccharide molecule needs corresponding dosage
Cerium ion causes, and by Fig. 4 it is found that as ammonium ceric nitrate additive amount increases, reactive grafting rate gradually increases, when adding in 1.5g
During ammonium ceric nitrate, reactive grafting rate reaches maximum.But after reaction proceeds to half, due to increasing for side reaction, reaction
Difficulty increases, then increases the additive amount of initiator and will not play positive effect.
3.3.1.4 influence of the reaction time to G0.5 grafting rates
Radical reaction includes three chain initiation, chain growth and chain termination processes, and the sufficient reaction time is the reaction institute
It is necessary.Fig. 5 is influence of the reaction time to G0.5 grafting rates.It can be seen from the figure that in initial reaction stage, due in system
Existing reactant concentration is larger, and grafting rate is made to be increased rapidly in 6h, and over time, graft reaction orderly carries out,
Grafting amount gradually increases.But after reaction carries out 8h, in reaction system side reaction increase, certain influence is caused on product, is led
Grafting rate is caused seriously to reduce.
3.3.2 the response surface optimization analysis of G0.5 synthesis
3.3.2.1 response surface designing scheme and result of the test
On the basis of single factor experiment result, three reaction molar ratio, initiator additive amount and reaction temperature masters are chosen
Factor is wanted to carry out response surface analysis and optimization.Following 17 groups of experiments are designed using the principle of the Center of Box-Benhnken,
It is worth in response with grafting rate, synthesis condition is advanced optimized by the response phase method (RSM) of Three factors-levels, is investigated
The responsiveness of each factor and the result of the effect of cross influence two-by-two.Experimental design scheme is with result as shown in following table 3-4：
Table 3-4 response surfaces designing scheme and experimental result
3.3.2.2 multiple regression Fitting Analysis is established with mathematical model
Multiple regression Fitting Analysis is carried out to the experimental data in table 3-4 by DesignExpert8.0.4 softwares, is obtained
Grafting rate and influence factor initiator additive amount react and meet polynary quadratic linear equation between molar ratio, reaction temperature,
Mathematical model is shown in formula 3-3：
Y=+52.62+9.70*A+1.96*B+3.08*C-1.03*A*B-0.030*A*C-1.30*B* C
-13.74*A2-2.05*B2-2.89*C2 (3-3)
Wherein, Y-- grafting rates；A-initiator additive amount；B-reaction molar ratio；C- reaction temperatures.
Table 3-5 response surface quadratic form variance analyses
Whether variance analysis is conversion ratio to be had a significant impact to illustrate that above-mentioned mathematical model is by determination process parameter
It is no significant.Response surface quadratic form variance analysis the results are shown in Table 3-5, show that the response develops by F value=106.58 of model
Mathematical model be extremely significant, and it is since noise generates that F values, which only have 0.01% chance,.Lose intend item F values be
2.55, and only 19.42% chance is as caused by noise, is inapparent relative to pure error, these all meet polynary
The requirement of linear fit.The coefficient R of equation actual value2=the 0.9928 and R of predicted value2=0.9200 is close, and
Both close to 1, show that there are good degrees of fitting between the actual value and predicted value of the grafting rate of G0.5.Correction determines
Coefficient AdjR2The variation distribution that=0.9834, which illustrates response, 98.34% can be explained by the equation.In addition, the model
The coefficient of variation (C.V=3.09%) and signal-to-noise ratio (Adeq Precision=28.356 ＞ 4) are all in the range of model permission.
To sum up, the mathematical model that multilinear fitting obtains has effective meaning for the analysis of experimental result with prediction.
In A, B, C, AB, AC, BC, A2、B2、C2In nine model terms, A, B, C, A2、B2、C2Pr ＞ F ＜ 0.0500, say
These bright model terms are significant, and the Pr ＞ F ＞ 0.1000 of excess-three item, are inapparent.For unessential model terms
(model terms for not including support model hierarchical structure), which can be simplified, carrys out improved model.Meanwhile the F values for comparing A, B, C can obtain
Go out, the influence size of initiator additive amount, reaction molar ratio and reaction temperature to the grafting rate of G0.5 is followed successively by：Initiator adds
Dosage ＞ reaction temperatures ＞ reacts molar ratio, and the F values performance extremely significantly (Pr of A>F values<0.0001), factor A2It is also pole
It is significant, it is the primary motivating factor that methyl acrylate grafts on chitosan oligosaccharide to show initiator.This point with it is existing research into
Fruit is consistent, and some researches show that in the presence of no initiator, methyl acrylate is difficult to occur instead with chitosan
Should, then the methyl acrylate that generally to methylate can be just grafted on chitosan or in physical factor by alkylating
Being reacted under such as plasma, gamma-rays induction can just carry out.
3.3.2.3 the cross action of influence factor
Initiator additive amount, reaction molar ratio, the one of condition of reaction temperature are kept in optimum level, drafting 3D songs
Face figure and contour map can obtain other two factor and the cross influence of grafting rate acted on.The central point of contour is three-dimensional
Peak in surface chart, i.e., the maximum value of the grafting rate reached under the interaction of two independent variables.Contour
Shape can reflect the power of two variable cross actions, more tend to be oval, represent that cross action is stronger, more round, represent to hand over
Fork effect is weaker.The response surface 3D figures and contour map of the cross action of AB, AC, BC are as shown in fig. 6, shape from contour
It can be seen that cross action power is followed successively by：BC ＞ AB ＞ AC, the result of this and variance analysis are consistent.Due to factor A's
Independence is especially strong, so the cross action of it and other two variables dies down, the interactional work between factor BC instead
It is firmly most strong.Illustrate ensureing that initiator is suitable, with the increase of reaction molar ratio and the liter of reaction temperature
Height, grafting rate show as the trend reduced after increase, this is because appropriate reaction molar ratio and temperature can increase reaction list
Effective collision between body and chitosan oligosaccharide molecule improves reactive grafting rate, when reaction molar ratio is excessive or reaction temperature is excessively high
When, the easy autohemagglutination of methyl acrylate causes the side reaction of reaction system to increase, and grafting rate reduces.
3.3.2.4 optimum synthesis condition determining and verifying
The optimum synthesis condition that G0.5 can be obtained by response surface optimization analytic approach is：Initiator additive amount is 1.67g, is reacted
Molar ratio COS:MA=I:8.48, reaction temperature is 47.39 DEG C, and the grafting rate predicted value of the G0.5 synthesized under this condition is
55.2522%.Experiment condition is extremely difficult to accurate as above-mentioned optimum condition in practical operation, so doing following adjustment：Draw
Agent additive amount 1.65g is sent out, reacts molar ratio 1：8.5,47.5 DEG C of reaction temperature.It is repeated according to the experimental program after adjustment
Experiment, it is 52.38% to measure average grafting rate, is consistent substantially with predicted value, illustrates the quadratic regression equation established in this experiment
It can more efficiently predict grafting rates of the G0.5 under certain synthesis condition.
Conclusion：It is obtained by the single factor experiment to G0.5 synthesis conditions：Reaction molar ratio is COS:MA (g/mL)=1:
8, reaction temperature is 45 DEG C, and initiator additive amount is 1.5g, reaction time 8h.In fixed reaction on the basis of single factor test
Between for 8h, choose reaction molar ratio, three principal elements of initiator additive amount and reaction temperature carry out response surface optimizations, obtain
Optimum synthesis condition is：Initiator additive amount 1.65g, reaction molar ratio COS:MA (g/mL)=1:8.5, reaction temperature 47.5
℃.And it is obtained by variance analysis：Three factors influence grafting rate strong and weak sequence and are followed successively by：Initiator additive amount>Reaction
Molar ratio>Reaction temperature, wherein, as a result show that the influence power of initiator additive amount is the most notable.
3.3.3.1 the amino content of G1.0
The whole generation product amino content of the bis- jumping potential measurements of table 3-7
Table 3-7 is shown：With the increase of grafting algebraically, amino content constantly reduces, and this is mainly due to reactions to be mainly
Increasing overall molecule amount, the increased amplitude of increased Amplitude Ratio wherein amino of the overall molecule amount of product is big, so performance
Go out the trend of amino content reduction.But, according to reaction line map, with the progress of reaction, the quantity of terminal amino group is continuous
Increase.
3.3.3.2G0.5-G3.0 carbon-nitrogen ratio
The methyl acrylate of this experiment grafting relates generally to the variation of carbon and nitrogen element content with ethylenediamine, so can
With with carbon nitrogen percentage composition than estimating reaction situation about carrying out.Fig. 7 is carbon nitrogen percentage composition ratio in different algebraically product molecules
Block diagram, as can be seen from Fig., the continuous variation of adjacent algebraically carbon-nitrogen ratio illustrate to be chemically reacted.Whole generation molecule G1.0
Carbon-nitrogen ratio with grafting algebraically increase and increase, this is mainly due to reaction when carbon incrementss be more than nitrogen
Incrementss.Half for molecule grafting is methyl acrylate, only can increase carbon element content.
3.3.3.2 thermal stability analysis
It can show that modified each thermal stability for COS/PAMAM derivatives has not compared with chitosan oligosaccharide by Fig. 8
With the variation of degree.They all mainly undergo two zero-g periods：The weight that initial temperature is lost to 100 DEG C or so is by sample
In product caused by the evaporation of moisture and crystal structure initial breakdown；150 DEG C to the 400 DEG C drastically declines of weight nearby are mainly
Since the main framing of chitosan oligosaccharide and its sugar unit molecule of derivative thermally decomposes, the chemical bond in pyranose ring is opened,
Crystal structure is totally disrupted；Slow reduce of weight may be by the decomposition of ashes residue remaining after degrading after 400 DEG C
Caused by.The thermal stability of wherein G0.5 and G1.0 increases compared with chitosan oligosaccharide, and the quality of final remaining ashes residue compares shell
Oligosaccharides is more, illustrates that first two steps are very successful to the branched modification of chitosan oligosaccharide.But with the increase of grafting algebraically, grafting rate gradually drops
Low, side reaction increases, and causes the variation of its thermal stability little, nearly close to chitosan oligosaccharide.
4 infrared structures parse
4.1 G0.5 are parsed for infared spectrum
Fig. 9 show the infrared spectrum comparison diagram of chitosan oligosaccharide, methyl acrylate and G0.5.In the G0.5 spectrograms of synthesis,
3404.7cm in protoconch oligosaccharides bands of a spectrum-1O-H the and N-H stretching vibrations at place absorb overlap peak and significantly become smaller, and have occurred certain
Offset, by original 3404cm- 1The broad peak at place is to lower wave number 3385cm- 1It is mobile.Illustrate under the initiation of ammonium ceric nitrate
C in chitosan oligosaccharide2- NH on position2Or C3Radical reaction has occurred in-OH on position.1590cm in chitosan oligosaccharide-1Locate the deformation of N-H
Vibration absorption peak also has the tendency that being obviously reduced in the corresponding positions of G0.5, further illustrates that reaction is probably happened at shell
Oligosaccharides C2On the active amino in position.Compared with the spectrogram of methyl acrylate, wherein 1654cm-1Locate strong stretching vibration in C=C
Absorption peak completely disappears in the spectrogram of G0.5, and in 1384cm- 1It may be propylene new stronger characteristic absorption peak occur
- CH in sour methyl esters3Characteristic absorption peak, it is also possible to graft reaction is had occurred by the C=C in methyl acrylate and chitosan oligosaccharide
Cause caused by its C-C skeleton change.In addition, 1075cm- 1The enhancing for locating C-O-C vibration absorption peaks is likely to be propylene
Sour methyl esters graft on it is that ester group after chitosan oligosaccharide acts on as a result, these all to illustrate that with methyl acrylate grafting has occurred in chitosan oligosaccharide anti-
It should.
4.2 G1.0 are parsed for infared spectrum
The infrared spectrum comparison diagram of G0.5, ethylenediamine and G1.0 are as shown in Figure 10, as can be seen from the figure：With G0.5 phases
Than it is the most significantly 1629cm in former absorption band to change in G1.0 bands of a spectrum- 1The C=O stretching vibrations for locating amide Ⅰ absorb
Peak increases, and-CH3In 1384cm-1The characteristic absorption peak at place significantly becomes smaller, and illustrates that the ester group on G0.5 occurs with ethylenediamine
Michael addition reaction.2923cm- 1And 2878cm- 1Respectively-CH2Asymmetry and symmetrical stretching vibration enhancing,
3385cm- 1The stretching vibration absworption peak peak shape of place N-H deepens and offset relative to having in G0.5, also demonstrates G1.0 and is closed
Into.
In this experiment, Michael addition reaction relates generally to the amino in the ester group and ethylenediamine in former methyl acrylate
Amidated process, in the process in order to prevent in molecule bridge architecture formation, the ethylenediamine of addition is enough, but
With reactant competitive reaction easily occurs for excessive ethylenediamine, and height is made to reduce product for low generation molecule is contained in dendrimer
Monodispersity, so need to remove unreacted ethylenediamine with a large amount of absolute ethyl alcohol and distilled water after the completion of each reaction, with
This improves the purity of product.
4.3 G0.5-G1.0 infrared spectrum comparison diagrams
Figure 11 is the infrared spectrum comparison diagram of G0.5, G1.0, as can be seen from the figure：With grafting algebraically increase,
3400cm-1Locate the stretching vibration peak size alternating of N-H in amino, 1390cm- 1Place-CH3The variation of characteristic absorption peak is all propylene
The result of sour methyl esters and ethylenediamine alternate grafting.2880cm-1Locate the gradual reinforcement of methylene absorption peak, 1640cm-1Locate C=O
The increase of stretching vibration peak is due to the function and effect that group is accumulated in building-up process.And characteristic absorption peak at 1070cm-1
Variation is then by caused after C-O-C grafting in methyl acrylate.Because only exist methyl acrylate in entire reaction process
With the checker of group in ethylenediamine, the introducing without other characteristic groups, so the infrared signature of G0.5, G1.0 absorb
The shape and tendency of the spectral line at peak are basically identical, they only exist the difference of peak shape size power.With reference to elemental analysis
As a result it can show that graft reaction has occurred with branching molecule in chitosan oligosaccharide.
5 thermal stability analysis
5.1 Zeta potential measurement results
Free amino group can be with the H in water when being dissolved in water+Protonation can occur, generate extra OH-Ion causes molten
The pH of liquid is reduced, and potential value also reduces therewith.As shown in figure 12 for the COS/PAMAM derivatives of different algebraically in aqueous solution
Zeta potential variation diagram, consume amino since methyl acrylate is reacted with amino and subtract its content when synthesis half is for molecule
It is few, protonation occurs and reduces, H in solution+Concentration increases, and Zeta potential value is caused to increase；It connects when synthesizing whole generation molecule
Branch is ethylenediamine, can generate a large amount of amino, enhances protonation in solution, OH-Concentration increases, the entirety of solution
Potential value, which reduces, is even up to negative value.
The bactericidal property test material and equipment of 6.1 G1.0 bacteriostatic agents
6.1.1 experiment material and reagent
Table 4-1 strain subjects
Table 4-2 main agents
6.1.2 laboratory apparatus and equipment
Table 4-3 key instrument equipment
6.2.1 the bactericidal property test of G1.0 bacteriostatic agents
6.2.1.1 the preparation of actication of culture and bacteria suspension
It pipettes the strain that 50 μ L are deposited in glycerol tube and is seeded to (the hereinafter referred to as LB cultures of 50ml nutrient broth mediums
Base), shaken cultivation 16-18h in 37 DEG C of constant temperature oscillators is placed in, bacterial strain is activated to exponential phase.After taking appropriate activation
Bacterium solution is outwelled supernatant, is then used under above-mentioned centrifugal condition in 10mL centrifuge tubes, 4000rpm/min centrifugation 10min
0.85% brine white bacterial plaque 1-2 times, remaining glycerine when removing preservation.Pipette 1ml treated bacterium solution in
In test tube, it is diluted to the bacteria suspension of various concentration gradient step by step according to decimal dilution method with 0.85% physiological saline, takes 0.2mL
It is diluted to 10-4CFU·mL-1And the bacterium solution of following concentration carries out plate count.The concentration gradient that clump count is chosen at 4-5 is true
It is set to the most suitable growth concentration of the bacterium, at this time a concentration of 10-8cfu·mL-1.The bacterium that the concentration is configured according to the method described above is hanged
Liquid, it is spare.
6.2.1.2 MIC is measured
With reference to the Zhou Deqing second editions《Microbiology Experiment study course》The assay method of middle fungicide MIC, utilizes liquid diluting
Method carries out bactericidal property test to the prepared COS/PAMAM fungicide of this experiment.Concrete operations：First sample is dissolved in
0.1mol/L CH3COONa-0.2mol/L CH3COOH buffer solutions are configured to the solution of 1000 μ g/mL, according to shown in table 4-4
Method prepare the different solution containing concentration in test tube, then add in what is prepared in 0.2mL 6.2.1.1 in each pipe
10-8cfu·mL-1Bacterium solution is tested, using containing only bacterium and only culture solution with sample as blank control.Above-mentioned test tube is placed in 37
DEG C, cultivate 12h in the constant temperature oscillation case of 200rpm/min, observe by the naked eye the turbidity of each test tube, if sample sets from certain
Test tube starts to become the clear as control group, shows a concentration of minimal inhibitory concentration (MIC, the minimum
inhibitory concentration)。
The preparation of the test tube of table 4-4 difference sample sizes
6.2.1.3 MBC is measured
The measure of minimum bactericidal concentration (MBC) is carried out on the basis of the MIC measurement results of 4.2.2.2.MIC is tested
In it is all become clear test tube solutions and be inoculated into solid medium, the growing state of bacterium colony is observed, most at last in tablet without apparent
The minimal sample concentration of bacterium colony growth is defined as minimum bactericidal concentration (MBC, minimum of the sample to test bacterium
bactericidal concentration)。
6.2.1.4 sterilizing rate curve
The tested bacterium of 2mL are inoculated in the fluid nutrient medium of 50mL, small test tube is dispensed into, is divided into experimental group and control
Group, adds in the sample solution of a concentration of MIC in experimental group, and control group adds in the physiological saline of same volume, shakes up, make antibacterial
Agent and tested bacterium come into full contact with.According to the method that GB/T4789.3-2010 is provided, above-mentioned each group solution is diluted step by step respectively
To suitable concentration, then pipette 1mL with liquid-transfering gun and be placed on agar medium, with coated with glass rod coating it is uniform after, be inverted flat
Plate is put into 37 DEG C of constant incubator and cultivates different time, and viable plate count is carried out in setting time point.By following
Formula calculates[95]The sterilizing rate of microballoon：
Wherein：B% represents sterilizing rate；N0For control group viable count；N1For experimental group viable count.
6.2.2 the preparation of COS/PAMAM dendrimers absorption Ag+ and Cu2+ compound preservatives
It is accurate to weigh COS/PAMAM samples prepared by 15.0mg in the iodine flask of 100mL, it is different to add in 25.0mL
The HAc-NaAc buffer solutions of pH make it fully dissolve.A concentration of 3.0mgmL of 5.0mL are added in into iodine flask again-1Ag+、
Cu2+Solution to be added without sample sets as blank control test, is placed in 25 DEG C of 100rpmmin-1Constant temperature oscillation case oscillation
24h.After balance to be adsorbed, residual ion content in solution is measured with supernatant is taken.Adsorbance Q is calculated according to the following formula[96]。
Wherein：CoConcentration (the mgmL of metal ion before absorption-1)；CeThe concentration of metal ion after adsorption equilibrium
(mg·mL-1)；Static saturated adsorption capacity (the mgg of Q-COS/PAMAM molecules-1)；V- liquor capacities (mL)；M-COS/
The dry weight (g) of PAMAM samples.
6.2.3 the bactericidal property test of COS/PAMAM/Cu2+ bacteriostatic agents
The same 6.2.1 of experimental method.
6.2.4 COS/PAMAM and COS/PAMAM/Cu2+ sterilization mechanisms are probed into
6.2.4.1 extracellular dna, RNA are measured
The mechanism of action of fungicide mainly has at following 2 points：One can strike off subunit's tie point on mycoderm for fungicide
Hydrophobic bond and metal bridge, film surface is caused crack or gap occur, loses physiological function；Secondly for fungicide institute band just
Charge can generate electrostatic attraction with the negative electrical charge of bacterium surface institute band and form electrovalent bond, cause bacteriolysis, destroy the thin of bacterium
Cell wall and membrane structure leak so as to cause cellular content such as DNA, RNA etc., eventually lead to cell death.Therefore pass through
The content of DNA, RNA are measured to judge the level of breakage of cell, indirectly, ultraviolet specrophotometer can be utilized at 260nm
Colorimetric estimation absorbance value come judge test bacterium cell level of breakage.
6.2.4.2 TTC ldh assay
TTC (red tetrazolium) can receive the H (H generated during cellular respiration by dehydrogenase+/ e), generate red three
Phenyl first Beam (TF), reaction equation is as follows：
TTC can enter cell interior across cell wall and cell membrane in the case where viable bacteria acts on from environment.Therefore, TTC can be used
Measure the vigor of tested bacterium cell, i.e. viable count is more, and metabolism is stronger, and the TTC absorbed from environment is more, generation
TF amounts it is also more., whereas if fungicide leads to viable bacteria effect, quantity is reduced or death, the production quantity of TF will become
It is few.So the antibacterial effect of fungicide can be investigated by measuring TF yields, its Bactericidal Mechanism is further studied.
6.2.4.3 scanning electron microscope morphological observation
Relatively the mechanism of action of the cationic germicide of accreditation is mainly to pass through positive charge and the bacterium of itself institute's band at present
The negative electrical charge of surface institute band generates electrostatic attraction, upsets the distribution of charges of cell membrane surface, destroys its physiological activity, and then ooze
Bacteriolysis is generated to intracellular thoroughly, the cellular contents such as DNA, RNA is caused to flow out, eventually lead to cell death.Cell death
Its cell fragment can be adsorbed on fungicide surface afterwards, it is possible to surface topography observation is carried out to the sample after sterilization and is come directly
See the function and effect of ground reaction fungicide.
6.3.1 the bactericidal effect of COS/PAMAM derivatives
6.3.1.1 MIC measurement results
The MIC value of table 4-5 G1.0 tested bacterium effects to four kinds
By testing G1.0 to Escherichia coli, two kinds of Gram-negative bacterias of pseudomonas aeruginosa and staphylococcus aureus
It can be obtained with the MIC value of two kinds of gram-positive bacterias of bacillus subtilis：G1.0 is respectively provided with the 4 kinds of tested bacterium surveyed aobvious
The fungistatic effect of work.It is analyzed from tested bacterium angle, three kinds of fungicide are better than other strains to the bacteriostasis property of Escherichia coli,
Bacillus subtilis takes second place, and staphylococcus aureus is in third, and they are worst to the biocidal property of pseudomonas aeruginosa, this
Mainly since tested bacterium has very strong drug resistance in itself.
Claims (8)
1. a kind of chitosan oligosaccharide graft copolymer G1.0, it is characterised in that its molecular formula is as follows：
Wherein n is any integer value.
A kind of 2. preparation method of chitosan oligosaccharide graft copolymer G1.0 described in claim 1, it is characterised in that the preparation side
Method is as follows：
1) it weighs a certain amount of chitosan oligosaccharide, adds in the acetic acid of 1% (v/v) as reaction dissolvent, acetic acid addition and chitosan oligosaccharide
Aggregate relation is as follows：Above-mentioned acetic acid solution 25ml is added in per 1g chitosan oligosaccharides；Fully after dissolving, nitrogen is passed through into bottle and removes oxygen
Gas；A certain amount of ammonium ceric nitrate is added under nitrogen protection as initiator, the addition of initiator and the mass ratio of chitosan oligosaccharide
It is 1.6:1-2.5:1；Then appropriate methyl acrylate is slowly added dropwise；The reaction of above-mentioned chitosan oligosaccharide and methyl acrylate rubs
You are than being 1：4—1：10；Setting mixing speed is 200rpm/min, 2-10h of successive reaction at a temperature of 40 DEG C-55 DEG C；It treats anti-
After the completion of answering, reaction gains G0.5 is filtered out, the reaction gains G0.5 filtered out is first then used into reaction dissolvent 1% (v/v)
Acetic acid filters 3 times, then acetone, ether, absolute ethyl alcohol rinse 3 times repeatedly successively, remove unreacted methyl acrylate, finally put
Enter 50 DEG C of vacuum drying chambers to be dried for standby；
2) G0.5 that above-mentioned steps are made spare is weighed, the methanol solvate for adding in chromatographic grade impregnates 12h, controlling reaction temperature 20
DEG C, the pure ethylenediamine of analysis, the lower reaction 8h of N2 protections is added dropwise；The addition of methanol and the aggregate relation of G0.5 are as follows：Often
1mgG0.5 add in 0.5ml methanol；The addition of ethylenediamine and the aggregate relation of G0.5 are as follows：0.08ml second is added in per 1mgG0.5
Diamines；It waits after the completion of reacting, reaction gains G1.0 is filtered out, is rinsed 3 times with reaction dissolvent methanol, then again successively with anhydrous
Ethyl alcohol, distilled water wash 3 times repeatedly, are placed in 50 DEG C and are dried in vacuo to obtain the final product.
3. the preparation method of chitosan oligosaccharide graft copolymer G1.0 as claimed in claim 2, it is characterised in that：In the step 1)
Containing preventing the polymerization inhibitor of self-polymerization to methoxyl group phenol MEHQ in the methyl acrylate of addition.
4. the preparation method of chitosan oligosaccharide graft copolymer G1.0 as claimed in claim 2, it is characterised in that：Shell is few in step 1)
The reaction molar ratio of sugar and methyl acrylate is 1:8—1:8.5.
5. the preparation method of chitosan oligosaccharide graft copolymer G1.0 as claimed in claim 2, it is characterised in that：It is set in step 1)
Good mixing speed is 200rpm/min, and continuous reaction time is 8h at a temperature of 40 DEG C -55 DEG C.
6. the preparation method of chitosan oligosaccharide graft copolymer G1.0 as claimed in claim 2, it is characterised in that：The reaction raw materials
The preparation process of chitosan oligosaccharide is as follows：
1), weigh a certain amount of chitosan makes it be completely dissolved in a certain amount of 1% (v/v) acetic acid solution by high-speed stirred
In, then solution is transferred in quartzy three-necked bottle, adds a certain amount of 30% (v/v) H2O2, correctly it is installed on microwave synthesizer
In cavity, ultrasonic probe is made to be less than below the interface of solution；Microwave power 300w is set, 30min is reacted, opens ultraviolet light
Row assistant degradation is shone into, to the end of experiment, then for 24 hours, i.e., water-bath concentrated by rotary evaporation under solution is spent at 40 DEG C is freeze-dried
It can obtain chitooligosaccharide- samples of the viscosity average molecular weigh M η 25000 or so；The aggregate relation of above-mentioned acetic acid addition and chitosan
It is as follows：The above-mentioned acetic acid solutions of 100mL, H are added in per 3g chitosans2O2The aggregate relation of addition and chitosan is as follows：Gather per 1g shells
Sugar adds in 0.1mL30% (v/v) H2O2；
2) it is, 1 according to mass ratio：1：1 ratio weighs a certain amount of papain, pectase and cellulase and is dissolved in pH=
In the buffer solution of 5.5 HAC-NaAC, the enzyme solution of 1mg/mL is configured to, for use；The above-mentioned shell after microwave degradation is oligomeric
The pH of sugar juice is adjusted to 5.5, is 1 according to the ratio between concentration of enzyme-to-substrate：10, above-mentioned for use enzyme solution is added in, is stirred evenly, and
It is placed in 45 DEG C of constant temperature water bath and digests 4h；Boiling heating 10min after the completion makes enzyme-deactivating, is evaporated under reduced pressure and is concentrated to give at 40 DEG C
To the reaction raw materials chitosan oligosaccharide sample solution needed for preparation.
7. the preparation method of chitosan oligosaccharide graft copolymer G1.0 as claimed in claim 6, it is characterised in that：The reaction raw materials
The preparation process of chitosan oligosaccharide is further included carries out molecular-weight gradation to the chitosan oligosaccharide sample solution for preparing gained, and choosing molecular weight is
The chitosan oligosaccharide of 3500-7000 is as raw material；Specific stage division is as follows：A diameter of 7000 He of 25mm molecular cut offs is chosen respectively
The bag filter that diameter 34mm molecular cut offs are 3500 carries out pre-treatment, removes metal ion；By the smaller bag filter set of diameter
Enter in the bag filter being relatively large in diameter, wherein one end bag filter is clamping fixed, and distilled water flushing is clean, then with the liquid-transfering gun of 5mL
The chitosan oligosaccharide sample solution for pipetting claim 6 preparation is packed into inner layer bag filter, between the double-deck bag filter of distilled water filling
Gap, bag filter clamp opening are then placed in the large beaker equipped with distilled water the 5d that dialyses；By double-deck intermembrane space after the completion of dialysis
Liquid takes out, freeze-drying, you can obtain the reaction raw materials chitosan oligosaccharide of 3500-7000 molecular weight.
8. applications of the chitosan oligosaccharide graft copolymer G1.0 described in claim 1 as food antiseptic bacteriostatic agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810033904.2A CN108192000B (en) | 2018-01-14 | 2018-01-14 | Chitosan oligosaccharide graft copolymer G1.0 and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810033904.2A CN108192000B (en) | 2018-01-14 | 2018-01-14 | Chitosan oligosaccharide graft copolymer G1.0 and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108192000A true CN108192000A (en) | 2018-06-22 |
| CN108192000B CN108192000B (en) | 2021-06-01 |
Family
ID=62589140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810033904.2A Active CN108192000B (en) | 2018-01-14 | 2018-01-14 | Chitosan oligosaccharide graft copolymer G1.0 and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108192000B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115521960A (en) * | 2022-09-20 | 2022-12-27 | 山东海锋生物工程有限公司 | Production process for reducing non-enzymatic browning of chitosan oligosaccharide |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1803849A (en) * | 2005-12-16 | 2006-07-19 | 武汉大学 | Method for preparing complete water soluble low molecular weight chitosan/chitooligosaccharace |
| CN102604115A (en) * | 2012-02-22 | 2012-07-25 | 天津大学 | Carboxymethyl chitosan quaternary ammonium salt/PAMAM(Polyamidoamine) core-shell nanoparticles and preparation method |
| CN104744608A (en) * | 2013-12-25 | 2015-07-01 | 殷国铭 | Method for preparing chitosan into water-soluble chitosan oligosaccharide |
| CN105037736A (en) * | 2015-06-05 | 2015-11-11 | 青岛科技大学 | Chitosan grafted hyperbranched polymer polyamide-amine (PAMAM), and preparation method and application thereof |
-
2018
- 2018-01-14 CN CN201810033904.2A patent/CN108192000B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1803849A (en) * | 2005-12-16 | 2006-07-19 | 武汉大学 | Method for preparing complete water soluble low molecular weight chitosan/chitooligosaccharace |
| CN102604115A (en) * | 2012-02-22 | 2012-07-25 | 天津大学 | Carboxymethyl chitosan quaternary ammonium salt/PAMAM(Polyamidoamine) core-shell nanoparticles and preparation method |
| CN104744608A (en) * | 2013-12-25 | 2015-07-01 | 殷国铭 | Method for preparing chitosan into water-soluble chitosan oligosaccharide |
| CN105037736A (en) * | 2015-06-05 | 2015-11-11 | 青岛科技大学 | Chitosan grafted hyperbranched polymer polyamide-amine (PAMAM), and preparation method and application thereof |
Non-Patent Citations (9)
| Title |
|---|
| NORIO TSUBOKAWA ET AL.: "Surface modification of chitosan powder by grafting of ‘dendrimer-like’ hyperbranched polymer onto the surface", 《 REACTIVE & FUNCTIONAL POLYMERS》 * |
| NORIO TSUBOKAWA ET AL.: "Surface modification of chitosan powder by grafting of ‘dendrimer-like’ hyperbranched polymer onto the surface", 《REACTIVE & FUNCTIONAL POLYMERS》 * |
| 于广利等: "《糖药物学》", 31 October 2012, 中国海洋大学出版社 * |
| 何佳等: "《微生物工程概论》", 30 April 2008, 兵器工业出版社 * |
| 岳武: "壳寡糖基聚合物的合成与应用研究", 《中国优秀博硕士学位论文全文数据库 (硕士) 工程科技Ⅰ辑》 * |
| 张军林等: "《简明生物化学》", 30 June 2014, 华中师范大学出版社 * |
| 朱婉萍等: "《甲壳素及其衍生物的研究与应用》", 30 November 2014, 浙江大学出版社 * |
| 李恒等: "微波-过氧化氢协同降黏辅助酶法制备壳寡糖", 《现代化工》 * |
| 黎厚斌主编: "《包装应用化学》", 31 January 2014, 印刷工业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115521960A (en) * | 2022-09-20 | 2022-12-27 | 山东海锋生物工程有限公司 | Production process for reducing non-enzymatic browning of chitosan oligosaccharide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108192000B (en) | 2021-06-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108440763A (en) | A kind of chitosan oligosaccharide graft copolymer G2.0 and its preparation method and application | |
| Kasaai | Various methods for determination of the degree of N-acetylation of chitin and chitosan: a review | |
| Skorik et al. | Evaluation of various chitin-glucan derivatives from Aspergillus niger as transition metal adsorbents | |
| Xu et al. | Chitosan film acylation and effects on biodegradability | |
| Badawy | Structure and antimicrobial activity relationship of quaternary N‐alkyl chitosan derivatives against some plant pathogens | |
| Kildeeva et al. | About mechanism of chitosan cross-linking with glutaraldehyde | |
| Macquarrie et al. | Applications of functionalized chitosan in catalysis | |
| Mikhailov et al. | Crosslinking of chitosan with dialdehyde derivatives of nucleosides and nucleotides. Mechanism and comparison with glutaraldehyde | |
| Hussain et al. | Preparation of chitosan oligomers and characterization: Their antifungal activities and decay resistance | |
| CN110256607A (en) | A kind of anionic 2- hydroxypropyltrimethylammonium chloride chitosan and preparation method and application | |
| CN108192000A (en) | A kind of chitosan oligosaccharide graft copolymer G1.0 and its preparation method and application | |
| CN109576256A (en) | Method for encapsulating double enzymes by magnetic DNA hydrogel | |
| Bhuniya et al. | Novel route to synthesis of allyl starch and biodegradable hydrogel by copolymerizing allyl‐modified starch with methacrylic acid and acrylamide | |
| Song et al. | Efficient digestion of chitosan using chitosanase immobilized on silica-gel for the production of multisize chitooligosaccharides | |
| CN108276535A (en) | A kind of chitosan oligosaccharide graft copolymer G3.0 and its preparation method and application | |
| Linhorst et al. | Chitin deacetylase as a biocatalyst for the selective N-acylation of chitosan oligo-and polymers | |
| Wang et al. | Improve plant photosynthesis by a new slow-release carbon dioxide gas fertilizer | |
| Bohlmann et al. | Exploring bacterial heparinase II activities with defined substrates | |
| Zhang et al. | The Antioxidant and Antibacterial Activities of the Pyridine‐4‐Aldehyde Schiff Bases Grafted Chloracetyl Chitosan Oligosaccharide Derivatives | |
| Rohaeti et al. | Mechanical properties and antibacterial activity of cellulose composite based coconut water with addition glycerol, chitosan, and silver nanoparticle | |
| Tanaka et al. | Synthesis and Biological Evaluation of the Forssman Antigen Pentasaccharide and Derivatives by a One‐Pot Glycosylation Procedure | |
| Ali et al. | Synthesis of schiff bases based on chitosan and heterocyclic moiety: Evaluation of antimicrobial activity | |
| Qin et al. | Moisture retention and antibacterial activity of modified chitosan by hydrogen peroxide | |
| CN105154070A (en) | Light-click-reaction-based DNA marker with fluorescent responses, preparing method thereof and application thereof | |
| Patil et al. | Synthesis, Characterization, and Antibacterial Studies of Mixed Ligand Dioxouranium Complexes with 8‐Hydroxyquinoline and Some Amino Acids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |