CN105420108A - Preparation and use methods of cellulose aerogel culture medium - Google Patents
Preparation and use methods of cellulose aerogel culture medium Download PDFInfo
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- CN105420108A CN105420108A CN201510967115.2A CN201510967115A CN105420108A CN 105420108 A CN105420108 A CN 105420108A CN 201510967115 A CN201510967115 A CN 201510967115A CN 105420108 A CN105420108 A CN 105420108A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention belongs to the field of microculture, and in particular discloses preparation and use methods of a cellulose aerogel culture medium. The preparation method comprises the following steps: cooling an NaOH/urea system at minus 8 DEG C to minus 12 DEG C for 15min; weighing cellulose with a certain mass, adding into the NaOH/urea system in a stirring process, and continuously stirring until the cellulose is dissolved, thus forming a uniform cellulose solution; pouring the cellulose solution with a certain volume into a container, adding a precipitator to obtain cellulose hydrogel, washing the cellulose hydrogel, freezing under minus 20 DEG C to form frozen gel, and freeze-drying to obtain the cellulose aerogel culture medium. A preparation process of the cellulose aerogel culture medium is simple, the operation is easy, the raw materials are economical and environment-friendly, the bacteria growth velocity is increased while a carbon source is provided, and a good application prospect is achieved.
Description
Technical field
The invention belongs to microorganism culturing field, be specifically related to a kind of method of preparation and use of cellulose aerogels substratum.
Background technology
Mierocrystalline cellulose is that occurring in nature content is the abundantest, distribution resource the most widely, can reach hundreds of hundred million tons every year by photosynthesis generation.As a kind of natural polymer of green, Mierocrystalline cellulose has the advantages such as security, stability, nontoxicity and biocompatibility, alternative petrochemical materials, thus alleviates world energy sources crisis to a certain extent.In cellulose aerogels preparation process, cellulose aerogels is as the third generation aerogel of new life, the premium propertiess such as the high porosity of silica aerogel and polymeric aerogels, high-specific surface area are had concurrently, incorporated the excellent properties of self simultaneously, as good biocompatibility, degradability, have great application prospect in pharmacy, the crude oil field such as to sew, but also do not developed preferably in this field of microorganism culturing.
Along with the development of bio-science, biological culture technigue has been widely used in the every field such as medical science, plant.Substratum is the nutriment of the artificial preparation growing for microorganism, plant tissue and animal tissues and maintain, containing the nutritive substance that microorganism growth needs.Traditional microorganism culturing, because microorganism is from the surface growth of substratum, also exists the problems such as antimicrobial absorbent nutritive substance is uneven, growth speed is comparatively slow.
Summary of the invention
For solving the shortcoming and defect part of prior art, primary and foremost purpose of the present invention is the preparation method providing a kind of economic environmental protection, efficiently cellulose aerogels substratum.
Another object of the present invention is to the using method of the cellulose aerogels substratum providing above-mentioned preparation method to obtain.
The object of the invention is achieved through the following technical solutions:
A preparation method for cellulose aerogels substratum, comprises the following steps:
(1) cellulosic dissolving: by NaOH/ urea solvent system in-8 DEG C ~-12 DEG C cooling 15min; Take the Mierocrystalline cellulose of certain mass, add in NaOH/ urea system under the condition stirred, continue stirring until cellulose dissolution, form uniform cellulose solution;
(2) preparation of cellulose aerogels: the cellulose solution of certain volume is poured in container, adds precipitation agent, obtain cellulose aquagel, cellulose aquagel is washed, at-20 DEG C, freezing 18 ~ 24h forms frozen glue, and lyophilize, namely obtains cellulose aerogels substratum.
Preferably, in described NaOH/ urea solvent system, the mass ratio of NaOH and urea is 7:12.
Preferably, described Mierocrystalline cellulose refers to the shorter cellulosic material of staple length <100 μm.
Preferably, described precipitation agent is selected as ethanol, acetic acid or mass concentration are the sulfuric acid of 3% ~ 12%, and the add-on of precipitation agent is 2 ~ 3 times of cellulose solution volume.
Preferably, described sublimation drying is 48h.
The using method of the cellulose aerogels substratum that above-mentioned preparation method obtains, step is as follows:
(1) nutrient solution is prepared: select nutrient solution, and add glucose 1g/L ~ 5g/L, extractum carnis 4g/L ~ 20g/L, regulates pH with phosphate buffer solution, stirs;
(2) inoculation culture: 2h will be cultivated in the nutrient solution after stirring in strain inoculation to step (1) under optimal temperature, speed conditions;
(3) by the cellulose aerogels substratum of certain volume at 120 DEG C of sterilizing 30min, in gnotobasis, cellulose aerogels substratum after sterilizing is immersed in the nutrient solution after step (2) inoculation culture, soak time is 40 ~ 60min, take out in the culture dish placed after sterilization, cultivate under optimal temperature.
The pH value of indication in above-mentioned using method, optimal temperature, speed conditions refer to pH value, optimal temperature, the speed conditions of the required microorganism cultivated; The cultivation of bacterial classification is carried out in an aseptic environment.
Described nutrient solution comprises the micro-components such as potassium primary phosphate, ferrous sulfate, ammonium sulfate, cobalt chloride, magnesium sulfate, zinc sulfate, calcium chloride, manganous sulfate, and can modify according to different fostering requirement.
Compared with prior art, the present invention has the following advantages and beneficial effect:
Cellulose aerogels medium preparing technique of the present invention is simple, and easy handling, raw material economics environmental protection, while providing carbon source, improves the growth velocity of bacterial classification, have a good application prospect.
The biocompatibility good due to cellulose aerogels and cavernous structure, can be applied to the preparation of substratum.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
(1) take 4g Mierocrystalline cellulose and join in the NaOH/ urea solvent system of-10 DEG C of precooling 15min that (NaOH quality is 7g, urea quality is 12g, quality is 77g), under the rotating speed of 600r/min, mechanical stirring 10min obtains cellulose solution, cellulose solution being poured into volume is in the small beaker of 50mL, is highly 5mm.
(2) ethanol is added dropwise in cellulose solution along beaker inwall, leaves standstill 5h, obtain cellulose aquagel.Use distilled water repetitive scrubbing again, the salt that removing is remaining, pure cellulose aquagel can be obtained.
(3) the pure cellulose aquagel freezing 20h at-20 DEG C step (2) obtained, obtains Mierocrystalline cellulose frozen glue, then obtains cellulose aerogels substratum through lyophilize.Wherein cryodesiccated temperature is-50 DEG C, and the time is 48h.
(4) green wooden enzyme nutrient solution is configured:
Nutrient solution is selected, and composition shown in table 1 is nutrient solution Trace Elements, adds glucose 1g/L; Extractum carnis 15g/L; Regulate pH to be 7.0 with phosphate buffer solution, magnetic agitation is to even.
Table 1 nutrient solution trace element component
| Material | Addition (g) | Material | Addition (g) |
| Potassium primary phosphate | 6 | Ferrous sulfate | 0.03 |
| Ammonium sulfate | 4.2 | Cobalt chloride | 0.01 |
| Magnesium sulfate | 1.0 | Zinc sulfate | 0.01 |
| Calcium chloride | 0.9 | Manganous sulfate | 0.008 |
Viride bacterial classification to be inoculated in the nutrient solution after stirring and to cultivate 2h under optimal temperature, speed conditions;
(5) cellulose aerogels substratum step (3) prepared is at 120 DEG C of sterilizing 30min, aseptically the cellulose aerogels substratum after sterilizing is immersed in the nutrient solution after step (4) inoculation culture, soaks 60min.Take out in the culture dish placed after sterilization, at 30 DEG C, carry out cultivation 48h.
After cultivation terminates, naked eyes can see the mycelial growth of dense viride; Collect the mycelia that viride produces, weigh, obtaining dry mycelial weight is 700 μ g, compares with traditional agar potato substratum, promotes more than 2 times.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1. a preparation method for cellulose aerogels substratum, is characterized in that, comprises the following steps:
(1) NaOH/ urea solvent system is cooled 15min in-8 DEG C ~-12 DEG C; Take the Mierocrystalline cellulose of certain mass, add in NaOH/ urea system under the condition stirred, continue stirring until cellulose dissolution, form uniform cellulose solution;
(2) pour in container by the cellulose solution of certain volume, add precipitation agent, obtain cellulose aquagel, washed by cellulose aquagel, freezing formation frozen glue at-20 DEG C, lyophilize, obtains cellulose aerogels substratum.
2. the preparation method of cellulose aerogels substratum according to claim 1, is characterized in that, in described NaOH/ urea solvent system, the mass ratio of NaOH and urea is 7:12.
3. the preparation method of cellulose aerogels substratum according to claim 1, is characterized in that, described Mierocrystalline cellulose refers to the cellulosic material of staple length <100 μm.
4. the preparation method of cellulose aerogels substratum according to claim 1, it is characterized in that, described precipitation agent is selected as ethanol, acetic acid or mass concentration are the sulfuric acid of 3% ~ 12%, and the add-on of precipitation agent is 2 ~ 3 times of cellulose solution volume.
5. the preparation method of cellulose aerogels substratum according to claim 1, is characterized in that, the described cryodesiccated time is 48h.
6. the using method of cellulose aerogels substratum that obtains of the preparation method of cellulose aerogels substratum according to claim 1, it is characterized in that, described using method comprises the following steps:
(1) select nutrient solution, and add glucose 1g/L ~ 5g/L, extractum carnis 4g/L ~ 20g/L, regulates pH with phosphate buffer solution, stirs;
(2) 2h will be cultivated in the nutrient solution after stirring in strain inoculation to step (1) under optimum pH, optimal temperature, speed conditions;
(3) by the cellulose aerogels substratum of certain volume at 120 DEG C of sterilizing 30min, in gnotobasis, cellulose aerogels substratum after sterilizing is immersed in the nutrient solution after step (2) inoculation culture, take out in the culture dish placed after sterilization, cultivate under optimal temperature.
7. using method according to claim 6, is characterized in that, the time of step (3) described immersion is 40 ~ 60min.
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| CN201510967115.2A CN105420108B (en) | 2015-12-18 | 2015-12-18 | The method of preparation and use of cellulose aerogels culture medium |
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| CN201510967115.2A CN105420108B (en) | 2015-12-18 | 2015-12-18 | The method of preparation and use of cellulose aerogels culture medium |
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| CN105420108A true CN105420108A (en) | 2016-03-23 |
| CN105420108B CN105420108B (en) | 2019-05-14 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019190379A1 (en) | 2018-03-28 | 2019-10-03 | Rise Innventia Ab | Cellulose containing hybrid aerogels and hybrid xerogels, and process for their production |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880410A (en) * | 2010-07-21 | 2010-11-10 | 武汉大学 | High-strength transparent cellulose material and preparation method thereof |
| CN103937010A (en) * | 2014-04-28 | 2014-07-23 | 华南理工大学 | High-performance graphene/cellulose composite hydrogel and aerogel and preparation methods thereof |
| WO2014178797A1 (en) * | 2013-05-03 | 2014-11-06 | National University Of Singapore | A Polysaccharide Aerogel |
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2015
- 2015-12-18 CN CN201510967115.2A patent/CN105420108B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880410A (en) * | 2010-07-21 | 2010-11-10 | 武汉大学 | High-strength transparent cellulose material and preparation method thereof |
| WO2014178797A1 (en) * | 2013-05-03 | 2014-11-06 | National University Of Singapore | A Polysaccharide Aerogel |
| CN103937010A (en) * | 2014-04-28 | 2014-07-23 | 华南理工大学 | High-performance graphene/cellulose composite hydrogel and aerogel and preparation methods thereof |
Non-Patent Citations (2)
| Title |
|---|
| HONGLI CAI等: "Aerogel Microspheres from Natural Cellulose Nanofibrils and Their Application as Cell Culture Scaffold", 《BIOMACROMOLECULES》 * |
| 陶丹丹等: "纤维素气凝胶材料的研究进展", 《纤维素科学与技术》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019190379A1 (en) | 2018-03-28 | 2019-10-03 | Rise Innventia Ab | Cellulose containing hybrid aerogels and hybrid xerogels, and process for their production |
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