CN109097418A - The method that antibiotic property bacteria cellulose film is prepared in situ - Google Patents
The method that antibiotic property bacteria cellulose film is prepared in situ Download PDFInfo
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Abstract
The invention discloses a kind of methods that antibiotic property bacteria cellulose film is prepared in situ.Mulberry leaf are prepared protein of folium mori with the method that alkali carries acid precipitates by the method, and remaining residue is hydrolyzed into monosaccharide with dilute sulfuric acid, become the mulberry leaf hydrolyzate containing antibacterial material after mixing, again using mulberry leaf hydrolyzate as carbon and nitrogen sources and antibacterial raw material, the bacteria cellulose film of inoculation acetobacter xylinum fermentation preparation antibiotic property.The method of the present invention is simple, at low cost, and antibacterial material is evenly distributed in bacteria cellulose through in-situ fermentation, and antibiotic property is stablized, and antibacterial material is not easily runed off, can be widely used on medical dressing.
Description
Technical field
The present invention relates to a kind of methods that antibiotic property bacteria cellulose film is prepared in situ, and belong to anti-biotic material technical field.
Background technique
It is well known that antibiotic property is one of most important function in hygrometric state dressing.General medical dressing is all in nothing
The basis material applied atop antibacterial material of the not no antibiotic property such as woven fabric, nylon, thus achieve the purpose that antibacterial, antibacterial material one
As use silver nano-grain.(Wei B, Yang G, the Hong F.Preparation and evaluation of of document 1
a kind of bacterial cellulose dry films with antibacterial properties[J]
.Carbohydrate Polymers, 2011,84 (1): 533-538.) it is after being freeze-dried bacteria cellulose film, to be immersed in
To achieve the purpose that antibacterial in Benza, but since antibacterial agent is that physical absorption is entered, so the medical dressing
The stability of antibacterial is simultaneously bad, is limited to storage time, concentration etc..Document 2 (XiujuZhang, YingFang,
WenbinChen.Preparation of Silver/Bacterial Cellulose Composite Membrane and
Study on Its Antimicrobial Activity[J].Synthesis and Reactivity in Inorganic
And Metal-Organic Chemistry, 2013,43 (7): 907-913.) it is that bacteria cellulose is immersed in high concentration
In silver nitrate solution, then reduction silver, is prepared for Ag/BC composite membrane, has good antibacterial effect, but involves great expense, silver
The shortcomings that particle is easy to fall off is also to be difficult to avoid that.
Summary of the invention
It is at high cost for existing medical dressing, complicated deficiency is prepared, the present invention provides one kind, and antibacterial is prepared in situ
The method of property bacteria cellulose film.Mulberry leaf are prepared protein of folium mori with the method that alkali carries acid precipitates by this method, and will be remaining
Residue is hydrolyzed into monosaccharide with dilute sulfuric acid, becomes mulberry leaf hydrolyzate after mixing.Antibiotic property is prepared by carbon and nitrogen sources of mulberry leaf hydrolyzate
Bacteria cellulose film.
Technical scheme is as follows:
The method that antibiotic property bacteria cellulose film is prepared in situ, by mulberry leaf hydrolysis become mulberry leaf hydrolyzate, using its as carbon,
Nitrogen source prepares bacteria cellulose, and bacteria cellulose is purified, the specific steps are as follows:
Step 1, mulberry leaf being placed in 4~6g/LNaOH solution, ultrasonic mixing is uniform, and water-bath at 40~60 DEG C, centrifugation,
Collect supernatant and solid residue；
Step 2, HCl solution adjusts the pH to 3 ± 0.2 of supernatant, is freeze-dried after the precipitating of generation is cleaned, obtains mulberry
Protein of folium mori is added in 6~7mol/L HCl solution and dissolves by leaf protein, filters, and neutralizes, obtains protein of folium mori hydrolyzate；
Step 3, the H that mass concentration is 2%~15% is added in solid residue2SO4In, 80~120 DEG C of 1~5h of acidolysis, from
After the heart, by Ca (OH)2It is added in supernatant, is adjusted to pH=10 ± 0.5, carry out detoxification, be centrifuged after detoxification, add in supernatant
Enter H2SO4Solution is adjusted to pH=6 ± 0.5, and 0.1~0.2g/L Na is added2SO3, boil 5~it is centrifuged after ten minutes, in supernatant
Activated carbon adsorption is added in liquid, mulberry leaf fiber hydrolysate is obtained after centrifugal concentrating；
Step 4, protein of folium mori hydrolyzate is mixed to get with mulberry leaf fiber hydrolysate and mixes mulberry leaf hydrolyzate, by mulberry leaf
The ratio of quality and the volume of fermentation medium is 20~25:100, g:mL, prepares fermentation training as raw material to mix mulberry leaf hydrolyzate
Base is supported, acetobacter xylinum is inoculated with, fermentation impregnates in water after fermentation and removes residual media ingredient, and it is fine to obtain antibiotic property bacterium
Tie up plain film.
Preferably, in step 1, the concentration of the NaOH solution is 4~5g/L.
Preferably, in step 1, the ultrasonic time is 20min or more.
Preferably, in step 1, the water bath time is 1~2h, and centrifugation time is 15min or more.
Preferably, in step 3, the H2SO4The concentration of solution is 7%~15%.
Preferably, in step 3, the acidolysis temperature is 100 DEG C, and the acidolysis time is 1.5h.
Preferably, in step 4, the fermentation medium is every 100mL mulberry leaf hydrolyzate, and ammonium sulfate 0.10g, phosphorus is added
Acid dihydride potassium 0.50g, magnesium sulfate 0.07g, calcium lactate 0.02g, glacial acetic acid 0.15g, citric acid 0.06g, sodium carboxymethylcellulose
0.04g adjusts pH=6.0.
Preferably, in step 4, fermented and cultured 14 days.
The present invention prepares protein of folium mori in the method precipitated with alkali carries acid, and remaining residue is hydrolyzed into dilute sulfuric acid
Monosaccharide, it is former at the mulberry leaf hydrolyzate containing antibacterial material after mixing, then using mulberry leaf hydrolyzate as carbon source and nitrogen source and antibacterial material
Position is prepared for the bacteria cellulose film with antibiotic property.The method of the present invention is simple, at low cost, and antibiotic property is stablized, and can answer extensively
With on medical dressing.
Figure of description
Fig. 1 be conventional method preparation bacteria cellulose film (LA-BC) and embodiment 1 in mulberry leaf hydrolyzate preparation it is thin
The infrared spectrogram of fungin film (MH-BC).
Fig. 2 is the SEM figure of MH-BC obtained in LA-BC and embodiment 1.
Fig. 3 is the XRD diagram of MH-BC obtained in LA-BC and embodiment 1.
Fig. 4 is MH-BCD antibacterial effect figure obtained in LA-BC and embodiment 1.
Fig. 5 is that the bacterium of MH-BC obtained in LA-BC and embodiment 1 holds concentration OD600Figure.
Specific embodiment
Below with reference to embodiment and attached drawing, the invention will be further described.
The formula (w/v, g/100ml) of the fermentation medium in existing laboratory: glucose 2.25, sucrose 2.75, peptone
1.00, yeast extract 0.75, ammonium sulfate 0.10, potassium dihydrogen phosphate 0.50, magnesium sulfate 0.07, calcium lactate 0.02, glacial acetic acid 0.15,
Citric acid 0.06, sodium carboxymethylcellulose 0.04 adjust pH=6.0, are inoculated with Acetobacter xylinum after high-temperature sterilization
NUST 4.2。
Embodiment 1
The preparation of mulberry leaf hydrolyzate:
Mulberry leaf are smashed it through into 60 meshes, are placed in 5g/L NaOH solution, ultrasonic 20min, and water-bath 1h at 45 DEG C, from
The heart 15 minutes, supernatant and solid residue were collected respectively；Supernatant is mixed with 1mol/L HCl, is adjusted to PH=3, it will be produced
Precipitating cleaning after be freeze-dried, obtain protein of folium mori.Protein of folium mori is added in 6mol/L HCl, is filtered, neutralizes, obtains
Protein of folium mori hydrolyzate.Solid residue is added to 7% H2SO4In, 100 DEG C of acidolysis 1.5h, by Ca (OH) after centrifugation2It is added to
In supernatant, it is adjusted to PH=10, detoxification is carried out, is centrifuged after detoxification, 5mol/LH is added in supernatant2SO4, it is adjusted to PH=6, and
0.1g/LNa is added2SO3, it boils and is centrifuged after five minutes, a small amount of active carbon is added in supernatant, adsorb 12h, after centrifugal concentrating
To mulberry leaf fiber hydrolysate.Two parts hydrolyzate is mixed up to mulberry leaf hydrolyzate.
The preparation and purification of antibiotic property bacteria cellulose film:
Mulberry leaf hydrolyzate 100mL is taken, ammonium sulfate 0.10g, potassium dihydrogen phosphate 0.50g, magnesium sulfate 0.07g, calcium lactate is added
0.02g, glacial acetic acid 0.15g, citric acid 0.06g, sodium carboxymethylcellulose 0.04g adjust pH=6.0, are inoculated with after high-temperature sterilization
Acetobacter xylinum NUST 4.2, fermented and cultured 14 days.The bacteria cellulose film of preparation is taken out, clear water is immersed in
Middle immersion 48h every two hours changes a water, and the residual media component permutation in gel film is come out.Up to antibacterial
The bacteria cellulose film of property.
By can see in the Fourier transform infrared (FT-IR) of Fig. 1, the bacteria cellulose film of mulberry leaf hydrolyzate preparation
(MH-BC) characteristic peak of the bacteria cellulose film (LA-BC) with the formula preparation of this laboratory.In 3340cm-1Place is stretching for-OH
Contracting vibration absorption peak, peak bandwidth and strong illustrate that surface both all contains a large amount of-OH；2910cm-1Place is bacteria cellulose
C-H stretching vibration peak；1650cm-1Caused by place is the hemiacetal group held due to glucide 4 '；1425cm-1The absorption at place
Peak is-CH2Symmetric curvature vibration peak；1040cm-1The absorption peak at place is the stretching vibration peak of the C-O-C and C-O-H of saccharide ring,
And 890cm-1The absorption peak at place is then the characteristic absorption peak of cellulose β -1,4 glycosidic bond.Unlike, MH-BC is in 1650-
1040cm-1Between there are many sharp new peaks, main absorption peak has: 1600cm-1And 1500cm-1Place is phenyl ring bone
The characteristic absorption peak of frame, 1170cm-1For the absorption peak of C-O-H on phenyl ring, in addition, MH-BC is in 1650cm-1Peak type ratio LA-BC
Peak type here is sharp, and intensity is big, and conjecture may be caused by the C=O that Flavonoid substances contain in hydrolyzate.
(a) and (c) is respectively the SEM figure of LA-BC and MH-BC in Fig. 2, is both had as we can see from the figure by crisscross
Complicated fiber is mutually wound and the tridimensional network that is formed, and the BC pattern observed under low power number is mainly by fiber filament band
It is desultorily mutually intertwined, the difference is that fiber filament band relatively figure (a) is loose in figure (c), and schemes some skies in (c)
Gap is capped；The mainly the two fiber ribbon layer shape overlapping observed under high magnification numbe, and all there are many apparent holes
Hole structure, while it can be concluded that MH-BC fibre diameter will obviously be coarser than LA-BC fibre diameter in figure (a) in figure (c), in figure (a)
LA-BC fibre diameter is about 60nm, and scheming MH-BC fibre diameter in (c) is about 200nm, furthermore schemes the mesh of MH-BC in (c)
Mesh diameter of the diameter obviously than LA-BC in figure (a) is big, this may be the tool of the Flavonoid substances as present in mulberry leaf hydrolyzate
Active force is weaker caused between the fiber filament band that some biocidal properties cause it to secrete.
Fig. 3 is the XRD diagram of MH-BC and LA-BC.14.65 ° of the vertex of the MH-BC and LA-BC angle of diffraction, 16.93 ° and
14.65 °, 10.75 ° of the angle of diffraction, and 12.41 ° and 27.39 ° of peak only has MH-BC to have.It can be seen that MH-BC is not only wrapped
Characteristic peak containing bacteria cellulose, also with the characteristic peak of flavone compound.
In fig. 4, it is seen that the Escherichia coli and staphylococcus aureus of MH-BC occur on solid agar medium
Apparent inhibition zone, and two class bacterial growths are good on the culture medium of blank control group and LA-BC, it means that LA-BC does not have
Antibiotic property.Simultaneously, it can be seen that MH-BC shows the antibiotic property different to two kinds of bacteriums, and MH-BC is coated with staphylococcus aureus
Culture medium have bigger inhibition zone compared with Escherichia coli, display MH-BC has better antibacterial to gram-positive bacterium
Ability.And the fermentation strain used is a kind of gramnegative bacterium, this inhibits the pollution in fermentation process to a certain extent
Rate and the successful chance of raising fermentation.
Shown in Fig. 5, the OD of the bacterial suspension of MH-BC600In 6h be it is minimum, it is very high anti-that it shows that MH-BC has
Bacterium efficiency.12h, the OD of MH-BC are extended to when the time600Percentage increases bacterial suspension and is lower than other two, and containing golden yellow
The staphylococcic OD of color600It is minimum.This shows that MH-BC has continuous anti-microbial property.In addition, MH-BC is after 12h is cultivated
The OD of bacterial suspension600Percentage value added staphylococcus aureus (0.43) is lower than Escherichia coli (0.52), shows MH-BC
There is better anti-microbial property to gram-positive bacterium.
The reaction condition and Reducing sugar of Examples 1 to 10 are shown in Table 1.
Table 1
Table 1 is the yield of reductive hydrolysis sugar under the conditions of differential responses.As can be seen from the table, the embodiment of the present invention 1 is made
Mulberry leaf hydrolyzate content of reducing sugar highest.
Claims (8)
1. the method that antibiotic property bacteria cellulose film is prepared in situ, which is characterized in that specific step is as follows:
Step 1, mulberry leaf are placed in 4~6g/LNaOH solution, ultrasonic mixing is uniform, and water-bath at 40~60 DEG C, is centrifuged, and collects
Supernatant and solid residue；
Step 2, HCl solution adjusts the pH to 3 ± 0.2 of supernatant, is freeze-dried after the precipitating of generation is cleaned, obtains mulberry leaf egg
It is white, protein of folium mori is added in 6~7mol/L HCl solution and is dissolved, is filtered, neutralizes, obtains protein of folium mori hydrolyzate；
Step 3, the H that mass concentration is 2%~15% is added in solid residue2SO4In, 80~120 DEG C of 1~5h of acidolysis, centrifugation
Afterwards, by Ca (OH)2It is added in supernatant, is adjusted to pH=10 ± 0.5, carry out detoxification, be centrifuged after detoxification, be added in supernatant
H2SO4Solution is adjusted to pH=6 ± 0.5, and 0.1~0.2g/L Na is added2SO3, boil 5~it is centrifuged after ten minutes, in supernatant
Middle addition activated carbon adsorption obtains mulberry leaf fiber hydrolysate after centrifugal concentrating；
Step 4, protein of folium mori hydrolyzate is mixed to get with mulberry leaf fiber hydrolysate and mixes mulberry leaf hydrolyzate, by the quality of mulberry leaf
Ratio with the volume of fermentation medium is 20~25:100, g:mL, prepares fermentation medium as raw material to mix mulberry leaf hydrolyzate,
It is inoculated with acetobacter xylinum, fermentation impregnates in water after fermentation and removes residual media ingredient, obtain antibiotic property bacteria cellulose
Film.
2. the method according to claim 1, wherein the concentration of the NaOH solution is 4~5g/ in step 1
L。
3. the method according to claim 1, wherein the ultrasonic time is 20min or more in step 1.
4. the method according to claim 1, wherein in step 1, the water bath time is 1~2h, when centrifugation
Between be 15min or more.
5. the method according to claim 1, wherein in step 3, the H2SO4The concentration of solution be 7%~
15%.
6. the method according to claim 1, wherein the acidolysis temperature is 100 DEG C, described in step 3
The acidolysis time is 1.5h.
7. the method according to claim 1, wherein the fermentation medium is every 100mL mulberry in step 4
Ammonium sulfate 0.10g, potassium dihydrogen phosphate 0.50g, magnesium sulfate 0.07g, calcium lactate 0.02g, glacial acetic acid 0.15g is added in leaf hydrolyzate,
Citric acid 0.06g, sodium carboxymethylcellulose 0.04g adjust pH=6.0.
8. the method according to claim 1, wherein in step 4, fermented and cultured 14 days.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113481256A (en) * | 2021-08-10 | 2021-10-08 | 武汉纺织大学 | Method for producing bacterial cellulose by jasmine flower culture |
| CN114561437A (en) * | 2022-03-24 | 2022-05-31 | 中国科学院广州能源研究所 | Method for preparing functional bacterial cellulose by in-situ fermentation of eucommia ulmoides hydrolysate |
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| CN105002231A (en) * | 2015-08-05 | 2015-10-28 | 南京理工大学 | Method for preparing bacterial cellulose by biotransforming mulberry leaves |
| CN106047776A (en) * | 2016-08-16 | 2016-10-26 | 南京理工大学 | Acetobacter xylinum improved fermentation culture medium |
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2018
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|---|---|---|---|---|
| CN105002231A (en) * | 2015-08-05 | 2015-10-28 | 南京理工大学 | Method for preparing bacterial cellulose by biotransforming mulberry leaves |
| CN106047776A (en) * | 2016-08-16 | 2016-10-26 | 南京理工大学 | Acetobacter xylinum improved fermentation culture medium |
Non-Patent Citations (2)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113481256A (en) * | 2021-08-10 | 2021-10-08 | 武汉纺织大学 | Method for producing bacterial cellulose by jasmine flower culture |
| CN114561437A (en) * | 2022-03-24 | 2022-05-31 | 中国科学院广州能源研究所 | Method for preparing functional bacterial cellulose by in-situ fermentation of eucommia ulmoides hydrolysate |
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