CN101993847A - Bacterial cellulose strain - Google Patents
Bacterial cellulose strain Download PDFInfo
- Publication number
- CN101993847A CN101993847A CN2010105159463A CN201010515946A CN101993847A CN 101993847 A CN101993847 A CN 101993847A CN 2010105159463 A CN2010105159463 A CN 2010105159463A CN 201010515946 A CN201010515946 A CN 201010515946A CN 101993847 A CN101993847 A CN 101993847A
- Authority
- CN
- China
- Prior art keywords
- bacterial strain
- bacteria cellulose
- bacteria
- medium
- glucose
- 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
Images
Abstract
The invention discloses a new bacterial cellulose strain which is named Gluconacetobacter.sp.M438 and has the Collection number of CGMCC No. 3917 in China General Microbiological Culture Collection Center (CGMCC). The strain is obtained through the processes of separating, selecting and carrying out high-pressure mutagenesis of buckwheat vinegar mashes, is identified as a subspecies of Ga. hansenii, and is the new bacterial cellulose strain, wherein the cellulose yield of the strain can reach up to 40g/100mL culture medium. The bacterial cellulose strain has the advantage of high and stable yield and was collected in CGMCC on June 10, 2010.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of bacterial strain, be specifically related to a kind of bacteria cellulose bacterial strain (
Gluconacetobacter. Sp ) separation, mutagenesis, evaluation and cultivation.
Background technology
Can synthetic cellulose except plant and animal, some bacteriums can produce born of the same parents' outer fiber element than plant more efficiently in the heterotrophism mode.Usually microorganism synthetic Mierocrystalline cellulose is called " biology cellulose (Bio-cellulose) " or " bacteria cellulose (Bacterial cellulose is called for short BC) ".Bacteria cellulose has many excellent characteristic, and is ultra-fine as high purity, high-crystallinity, high water-holding power, high strength, advantages such as good biodegradability and building-up process Modulatory character.As a kind of new microbial synthetic materials, bacteria cellulose has been applied to a plurality of fields such as food, papermaking, medicine.
Yet, producing yielding poorly of bacteria cellulose bacterial strain at present, the production cost height far can not practical requirement, and seeking a kind of bacteria cellulose bacterial strain stable, high yield is that those skilled in the art are desired.
At present, induced mutations is the effective way that obtains superior strain.For many years, physics and chemomorphosis are the industrial micro breeding efficient ways always.Wherein physical mutagenesis, effect simple and practical because of it obviously is used widely.In recent years, along with the development of high pressure theory and technology, the biological research of high pressure has been deep into biological a plurality of field, and the high pressure induced-mutation technique has had new progress aspect Microbial Breeding.Gao Xiangs etc. have obtained withstand voltage mutant strain TG1P, DH5 α P and HB101P with 220MPa autoclaving e. coli tg1, DH5 α and HB101, and their survival rates under the 220MPa high pressure have improved 2～3 orders of magnitude.The virus (VSV) of inactivation still keeps high immunogenicity to first observed such as Silva to subunit dissociation and polymerization under high pressure take place.Wang Suilou etc. utilize ultraviolet ray and 150MPa pressure mutagenesis laccase to produce Ganoderma (G.lucidum Karst), find that ultra-high voltage has mutagenesis significantly to mycelia form and fermentation character.Wang Hua etc. discover that high pressure has tangible influence to survival rate, morphological specificity, proteolytic enzyme and the amylase activity of aspergillus oryzae; Obtain a strain fast growth, spore quantity is many, proteinase activity is high desirable dissociant HP300a after the autoclaving, the leading indicator of its Cheng Qu all is better than contrasting strain.
Summary of the invention
At lower, the unsettled defective of bacteria cellulose bacterial strain rate ratio of present report, the object of the invention is, by to the separation of bacteria cellulose bacterial strain, the new bacterial strain M438 of bacteria cellulose that the high pressure mutafacient system has obtained a kind of high yield.Its cellulose output of this bacterial strain (wet film) can reach the 40g/100mL substratum, far above the Production by Bacteria cellulose amount (30g/100mL substratum) of present report.
In order to realize above-mentioned task, method of the present invention is to be achieved by the following technical programs:
A kind of bacteria cellulose bacterial strain is characterized in that, this bacteria cellulose bacterial strain called after bacteria cellulose bacterial strain M438(
Gluconacetobacter.sp.M438), the preserving number in Chinese common micro-organisms DSMZ is CGMCC No.3917.
The 16S rRNA gene complete sequence of above-mentioned bacteria cellulose bacterial strain M438 (1,403bp, GenBank accession GU227424) is as follows:
AGGGAATGGGGGCATGCTTACACATGCAAGTCGCACGAACCTTTCGGGGTTAGTGGCGGACGGGTGAGTAACGCGTAGGGATCTGTCCATGGGTGGGGGATAACTTTGGGAAACTGAAGCTAATACCGCATGACACCTGAGGGTCAAAGGCGCGAGTCGCCTGTGGAGGAACCTGCGTTCGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGATGATCGATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGTTTTCGGATTGTAAAGCACTTTCAGCGGGGACGATGATGACGGTACCCGCAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCAAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGCGTAGGCGGTTGTTACAGTCAGATGTGAAATTCCCGGGCTTAACCTGGGGGCTGCATTTGATACGTGACGACTAGAGTGTGAGAGAGGGTTGTGGAATTCCCAGTGTAGAGGTGAAATTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCAACCTGGCTCATGACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTGTGCTGGATGTTGGATGGCTTGGCCATTCAGTGTCGTAGTTAACGCGATAAGCACACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGGACTTGACATGCGGAGGCTGTGTCCAGAGATGGGCATTTCTCGCAAGAGACCTCCAGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCTTTAGTTGCCAGCACGTCTGGGTGGGCACTCTAAAGGAACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGTCCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCCAGGCAGCGATGCCGAGCGGATCTCCAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGGTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTGACCTTAAGCCGGTGAGCGAACCGCAAGGACGCAGCCGACCACGGTCGTTCCCACGGGGTG。
The preparation method of above-mentioned bacteria cellulose bacterial strain is characterized in that, this method comprises the following steps:
The separation of bacterial Mierocrystalline cellulose produces bacterium J from the buckwheat vinegar wine with dregs of brew
2, its separation method is: allow buckwheat vinegar wine with dregs on enrichment medium, carry out the long film of enrichment earlier, treat that bacteria cellulose film grows after, get the 1g cellulose membrane with the method for sterile sampling, join in the 9mL sterile saline after aseptic mortar grinds and make 10
-1Bacteria suspension, stepwise dilution gets 10 then
-5, 10
-6, 10
-7Three dilution bacteria suspension 0.2mL are applied on the acetic bacteria special culture media, 30 ℃ of constant temperature culture 3～4d, picking grows fine, and bacterium colony is full evenly, and single bacterium colony of the suitable flat board of extent of dilution, be inoculated in the slant medium respectively, after waiting to grow abundant lawn, each picking one ring slant strains is inoculated in respectively in the test tube that 10mL basis fermention medium is housed, 30 ℃ of constant temperature culture 5d filter out the bacterial strain that can produce gel-film; Can produce the bacterial strain of gel-film, the separation of ruling on the isolation medium flat board rules twice continuously, and after microscopy was the bacterial strain pure growth, slant preservation was in 4 ℃ of refrigerators;
The enrichment medium main component is: glucose: 2%, and yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, nysfungin: 50mg/L;
Acetic bacteria special culture media main component is: glucose: 10%, and yeast extract paste 1.0%, CaCO
32.0%, agar: 1.5%, pH6.8;
Isolation medium and slant preservation substratum main component are: glucose: 2%, and yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, agar: 1.7%, the pH nature;
Basis fermention medium main component is: glucose: 2%, and yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O 1%, ethanol: 2%;
2) the high hydrostatic pressure mutagenesis of bacteria cellulose bacterial strain:
Get the strain cultured solution that cellulose membrane output is the highest and output is more stable of liquid culture 18h～24h, add sterilized liquid nutrient medium dilution, cell concentration is adjusted into 10
7About individual/ml, make bacteria suspension; Getting the bacteria suspension 15mL for preparing and pack under aseptic condition and seal in the aluminium foil Polypropylene Bag, is high pressure mutagenesis 15min under 250MPa, 25 ℃ of conditions of temperature at pressure; Obtain bacteria cellulose bacterial strain M438, bacteria cellulose bacterial strain M438 is transferred to 4 ℃ of preservations of slant medium;
The slant medium main component is: glucose: 2%, and yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, agar: 1.7%;
The liquid nutrient medium main component is: glucose: 2%, and yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%;
The cultural method of above-mentioned bacteria cellulose bacterial strain M438 is, the bacteria cellulose bacterial strain M438 high pressure mutagenic strain of preservation is gone in the seed culture medium by 2% ~ 4% inoculum size (v/v) switching, in 30 ℃, 150rpm constant temperature shaking table shaking culture 18 ~ 24h obtains fresh seeds liquid.Again M438 fresh seeds liquid is gone into to be equipped with in the 250mL wide-mouth fermentation flask of 100mL fermention medium according to 9% inoculum size (v/v) switching, leave standstill in 30 ℃ and cultivate 7d, cellulose output (wet film) can reach 40g/100mL.
The main component of seed culture medium is: glucose: 7%, and yeast extract paste: 1%, K
2HPO
4: 0.5%, MgSO
47H
2O:1.5%, ethanol: 2%;
The fermention medium main component is: and carbon source (glucose: sucrose=2:1): 3%, yeast extract paste: 0.5%, K
2HPO
4: 0.08%, MgSO
47H
2O:1.5%, FeSO
4: 0.4%, ZnSO
4: 0.03%, citric acid: 0.05%, ethanol: 0.7%.
The bacteria cellulose bacterial strain M438 that the present invention obtains compares with present report bacterial strain, has high yield, stable yields characteristic, combining form, Physiology and biochemistry and Phylogenetic Analysis, and M438 is
Ga.hanseniiSubspecies, be the new high yield bacteria cellulose bacterial strain of a strain.This bacterial classification is preserved in Chinese common micro-organisms DSMZ on June 10th, 2010, is called for short CGMCC, and registers on the books, and this biological inoculum was preserved 30 years on June 10th, 2010.
Description of drawings
Fig. 1 is the Phylogenetic Analysis figure that is M438 of MEGA 4.1.
The present invention is described in further detail below in conjunction with accompanying drawing.
Embodiment
In order to obtain high yield, stable bacteria cellulose bacterial strain, improve the output of bacteria cellulose, the present invention is on the basis of early-stage Study, utilize superhighpressure technology that the bacteria cellulose bacterial strain that filters out is carried out mutagenic treatment, obtained high yield bacteria cellulose bacterial strain M438, its cellulose output (wet film) can reach 40g/100mL, and the highest bacteria cellulose output of report is no more than 30g/100mL at present.
1) preparation of bacteria cellulose bacterial strain M438 bacterial classification:
Present embodiment relates to the bacteria cellulose bacterial strain M438 bacterial classification of screening, from buckwheat vinegar wine with dregs separation screening, after high hydrostatic pressure mutagenesis, obtain, specifically comprise the following steps:
(1) isolation and purification of bacteria cellulose bacterial strain:
The separation of bacterial Mierocrystalline cellulose produces bacterium from the buckwheat vinegar wine with dregs of laboratory brew, its separation method is: allow buckwheat vinegar wine with dregs carry out the long film of enrichment earlier on enrichment medium, after treating that bacteria cellulose film grows, get the 1g cellulose membrane with the method for sterile sampling, join in the 9mL sterile saline after aseptic mortar grinds and make 10
-1Bacteria suspension, stepwise dilution gets 10 then
-5, 10
-6, 10
-7Three dilution bacteria suspension 0.2mL are applied on the acetic bacteria special culture media, 30 ℃ of constant temperature culture 3-4d, picking grows fine, and bacterium colony is full evenly, and single bacterium colony of the suitable flat board of extent of dilution, be inoculated in the slant medium respectively, after waiting to grow abundant lawn, each picking one ring slant strains is inoculated in the test tube that 10mL basis fermention medium is housed respectively, 30 ℃ of constant temperature culture 5d filter out the bacterial strain that can produce gel-film.Can produce the bacterial strain of gel-film, the separation of ruling on the isolation medium flat board rules twice continuously, and after microscopy was the bacterial strain pure growth, slant preservation was in 4 ℃ of refrigerators.
Enrichment medium main component: glucose: 2%, yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, nysfungin: 50mg/L, pH nature;
Acetic bacteria special culture media main component: glucose: 10%, yeast extract paste: 1.0%, CaCO
3: 2.0%, agar: 1.5%, pH6.8;
Isolation medium, slant preservation substratum main component: glucose: 2%, yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, agar: 1.7%, the pH nature;
Basis fermention medium main component: glucose: 2%, yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, the pH nature.
(2) the high hydrostatic pressure mutagenesis of bacteria cellulose bacterial strain:
Get the bacterial strain pure culture liquid that cellulose membrane output is the highest and output is more stable of liquid culture 18-24h, add sterilized liquid nutrient medium dilution bacterium liquid, cell concentration is adjusted into 10
7About individual/ml, make bacteria suspension.Getting the bacteria suspension 15mL for preparing packs under aseptic condition and seals in the aluminium foil Polypropylene Bag, at pressure is to carry out high hydrostatic pressure mutagenesis under 250MPa, time 15min, 25 ℃ of conditions of temperature, obtain bacteria cellulose bacterial strain M438, bacteria cellulose bacterial strain M438 is transferred to 4 ℃ of preservations of slant medium.
Slant medium main component: glucose: 2%, yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, agar: 1.7%, the pH nature;
Liquid nutrient medium main component: glucose 2%, yeast extract paste 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, the pH nature.
2) cultivation of bacteria cellulose bacterial strain M438:
The bacteria cellulose bacterial strain M438 of preservation is gone in the seed culture medium by 2% ~ 4% inoculum size (v/v) switching, and in 30 ℃, 150rpm constant temperature shaking table shaking culture 18 ~ 24h obtains M438 fresh seeds liquid.Again M438 fresh seeds liquid is gone into to be equipped with in the 250mL wide-mouth fermentation flask of 100mL fermention medium according to 9% inoculum size (v/v) switching, leave standstill in 30 ℃ and cultivate 7d, cellulose output (wet film) can reach 40g/100mL.
The main component of seed culture medium is: glucose: 7%, and yeast extract paste: 1%, K
2HPO
4: 0.5%, MgSO
47H
2O: 1.5%, ethanol: 2%.
The fermention medium main component is: and carbon source (glucose: sucrose=2:1): 3%, yeast extract paste: 0.5%, K
2HPO
4: 0.08%, MgSO
47H
2O:1.5%, FeSO
4: 0.4%, ZnSO
4: 0.03%, citric acid: 0.05%, ethanol: 0.7%.
3) evaluation of bacteria cellulose superior strain M438
1. morphology is identified:
Individual morphology: somatic cells is shaft-like, and is single or paired, and the thalline size is between the μ m of (2.08～4.16) * (0.83～0.94), and the bacterial strain gramstaining is negative.
Colony's form: the flat-plate bacterial colony size between 0.8～1.5mm, circle, smooth surface, projection, opaque, be creamy white.The inclined-plane lawn is thread, and smooth surface is creamy white, and is opaque.Liquid culture 3d, fluid surface have epistasis to produce, and be haze-free, do not have precipitation, no bubble, and the nutrient solution color and luster does not become.
2. physiology, biochemical character are identified as shown in the table.
Table 1: bacterial strain
M 438 Physiological and biochemical property
Table 2: bacterial strain
M 438 Product acid test
Table 3: bacterial strain
M 438 Utilization of carbon source test
Annotate: "-" expression does not utilize, and "+" expression utilizes, and " ++ " expression utilizes by force
Table 4: bacterial strain
M 438 Nitrogenous source utilization test
Annotate: "-" expression does not utilize, and "+" expression utilizes, and " ++ " expression utilizes by force
3. Phylogenetic Analysis (16S rRNA sequential analysis):
M
43816S rRNA sequencing result (1403bp, GenBank accession GU227424):
AGGGAATGGGGGCATGCTTACACATGCAAGTCGCACGAACCTTTCGGGGTTAGTGGCGGACGGGTGAGTAACGCGTAGGGATCTGTCCATGGGTGGGGGATAACTTTGGGAAACTGAAGCTAATACCGCATGACACCTGAGGGTCAAAGGCGCGAGTCGCCTGTGGAGGAACCTGCGTTCGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGATGATCGATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGTTTTCGGATTGTAAAGCACTTTCAGCGGGGACGATGATGACGGTACCCGCAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCAAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGCGTAGGCGGTTGTTACAGTCAGATGTGAAATTCCCGGGCTTAACCTGGGGGCTGCATTTGATACGTGACGACTAGAGTGTGAGAGAGGGTTGTGGAATTCCCAGTGTAGAGGTGAAATTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCAACCTGGCTCATGACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTGTGCTGGATGTTGGATGGCTTGGCCATTCAGTGTCGTAGTTAACGCGATAAGCACACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGGACTTGACATGCGGAGGCTGTGTCCAGAGATGGGCATTTCTCGCAAGAGACCTCCAGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCTTTAGTTGCCAGCACGTCTGGGTGGGCACTCTAAAGGAACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGTCCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCCAGGCAGCGATGCCGAGCGGATCTCCAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGGTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTGACCTTAAGCCGGTGAGCGAACCGCAAGGACGCAGCCGACCACGGTCGTTCCCACGGGGTG
Do the Phylogenetic Analysis (Fig. 1) of M438 with MEGA 4.1, it and Chinese Salmonella (Ga.hansenii) homology are the highest.
Combining form, Physiology and biochemistry and Phylogenetic Analysis, M
438Think
Ga.hanseniiSubspecies.
Claims (5)
1. a bacteria cellulose bacterial strain is characterized in that, this bacteria cellulose bacterial strain called after bacteria cellulose bacterial strain M438(
Gluconacetobacter.sp.M438), the preserving number in Chinese common micro-organisms DSMZ is CGMCC No.3917.
2. bacteria cellulose bacterial strain as claimed in claim 1 is characterized in that, the gene order of described bacteria cellulose bacterial strain M438 is as follows:
AGGGAATGGGGGCATGCTTACACATGCAAGTCGCACGAACCTTTCGGGGTTAGTGGCGGACGGGTGAGTAACGCGTAGGGATCTGTCCATGGGTGGGGGATAACTTTGGGAAACTGAAGCTAATACCGCATGACACCTGAGGGTCAAAGGCGCGAGTCGCCTGTGGAGGAACCTGCGTTCGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGATGATCGATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGTTTTCGGATTGTAAAGCACTTTCAGCGGGGACGATGATGACGGTACCCGCAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCAAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGCGTAGGCGGTTGTTACAGTCAGATGTGAAATTCCCGGGCTTAACCTGGGGGCTGCATTTGATACGTGACGACTAGAGTGTGAGAGAGGGTTGTGGAATTCCCAGTGTAGAGGTGAAATTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCAACCTGGCTCATGACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTGTGCTGGATGTTGGATGGCTTGGCCATTCAGTGTCGTAGTTAACGCGATAAGCACACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGGACTTGACATGCGGAGGCTGTGTCCAGAGATGGGCATTTCTCGCAAGAGACCTCCAGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCTTTAGTTGCCAGCACGTCTGGGTGGGCACTCTAAAGGAACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGTCCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCCAGGCAGCGATGCCGAGCGGATCTCCAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGGTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTGACCTTAAGCCGGTGAGCGAACCGCAAGGACGCAGCCGACCACGGTCGTTCCCACGGGGTG。
3. the preparation method of the described bacteria cellulose bacterial strain of claim 1 is characterized in that, this method comprises the following steps:
1) isolation and purification of bacteria cellulose bacterial strain:
The separation of bacterial Mierocrystalline cellulose produces bacterium from the buckwheat vinegar wine with dregs of brew, its separation method is: allow buckwheat vinegar wine with dregs carry out the long film of enrichment earlier on enrichment medium, after treating that bacteria cellulose film grows, get the 1g cellulose membrane with the method for sterile sampling, join in the 9mL sterile saline after aseptic mortar grinds and make 10
-1Bacteria suspension, stepwise dilution gets 10 then
-5, 10
-6, 10
-7Three dilution bacteria suspension 0.2mL are applied on the acetic bacteria special culture media, 30 ℃ of constant temperature culture 3～4d, picking grows fine, and bacterium colony is full evenly, and single bacterium colony of the suitable flat board of extent of dilution, be inoculated in the slant medium respectively, after waiting to grow abundant lawn, each picking one ring slant strains is inoculated in respectively in the test tube that 10mL basis fermention medium is housed, 30 ℃ of constant temperature culture 5d filter out the bacterial strain that can produce gel-film; Can produce the bacterial strain of gel-film, the separation of ruling on the isolation medium flat board rules twice continuously, and after microscopy was the bacterial strain pure growth, slant preservation was in 4 ℃ of refrigerators;
The enrichment medium main component is: glucose: 2%, and yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, nysfungin: 50mg/L;
Acetic bacteria special culture media main component is: glucose: 10%, and yeast extract paste 1.0%, CaCO
32.0%, agar: 1.5%;
Isolation medium and slant preservation substratum main component are: glucose: 2%, and yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, agar: 1.7%, the pH nature;
Basis fermention medium main component is: glucose: 2%, and yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O 1%, ethanol: 2%;
2) the high hydrostatic pressure mutagenesis of bacteria cellulose bacterial strain:
Get the strain cultured solution that cellulose membrane output is the highest and output is more stable of liquid culture 18h～24h, add sterilized liquid nutrient medium dilution, cell concentration is adjusted into 10
7About individual/ml, making bacteria suspension, get the bacteria suspension 15mL for preparing and pack under aseptic condition and seal in the aluminium foil Polypropylene Bag, is high hydrostatic pressure mutagenesis 15min under 250MPa, 25 ℃ of conditions of temperature at pressure; Obtain bacteria cellulose bacterial strain M438, bacteria cellulose bacterial strain M438 is transferred to 4 ℃ of preservations of slant medium;
Slant medium main component: glucose: 2%, yeast extract paste: 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%, agar: 1.7%;
Liquid nutrient medium main component: glucose 2%, yeast extract paste 0.5%, K
2HPO
4: 0.1%, MgSO
47H
2O:1%, ethanol: 2%.
4. the cultural method of the described bacteria cellulose bacterial strain of claim 1, it is characterized in that, the bacteria cellulose bacterial strain M438 of preservation is transferred in the seed culture medium by the inoculum size of 2% ~ 4% v/v, in 30 ℃, 150rpm constant temperature shaking table shaking culture 18 ~ 24h obtains M438 fresh seeds liquid, M438 fresh seeds liquid is transferred in the 250mL wide-mouth fermentation flask that the 100mL fermention medium is housed according to the inoculum size of 9% v/v again, leave standstill in 30 ℃ and cultivate 7d, cellulose output reaches 40g/100mL;
The seed culture medium main component is: glucose: 7%, and yeast extract paste: 1%, K
2HPO
4: 0.5%, MgSO
47H
2O: 1.5%, ethanol: 2%.
The fermention medium main component is: carbon source: 3%, and yeast extract paste: 0.5%, K
2HPO
4: 0.08%, MgSO
47H
2O:1.5%, FeSO
4: 0.4%, ZnSO
4: 0.03%, citric acid: 0.05%, ethanol: 0.7%.
5. method as claimed in claim 4 is characterized in that the mass ratio of described carbon source consists of: glucose: sucrose=2:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010515946A CN101993847B (en) | 2010-10-22 | 2010-10-22 | Bacterial cellulose strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010515946A CN101993847B (en) | 2010-10-22 | 2010-10-22 | Bacterial cellulose strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101993847A true CN101993847A (en) | 2011-03-30 |
| CN101993847B CN101993847B (en) | 2012-09-26 |
Family
ID=43784602
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010515946A Expired - Fee Related CN101993847B (en) | 2010-10-22 | 2010-10-22 | Bacterial cellulose strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101993847B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250774A (en) * | 2011-07-22 | 2011-11-23 | 北京大学 | Algae engineering bacteria secreting crystalline cellulose and preparation method and use thereof |
| CN104164380A (en) * | 2014-04-23 | 2014-11-26 | 黑龙江大学 | Bacterial cellulose high-yielding strain |
| CN105802896A (en) * | 2016-05-23 | 2016-07-27 | 江南大学 | Komagataeibacter rhaeticus for producing saccharic acid-1,4-lactone |
| CN106497789A (en) * | 2016-10-21 | 2017-03-15 | 天津工业大学 | A kind of screening technique of the bacterium bacterial strain with bacteria cellulose high productive capacity |
| CN106497824A (en) * | 2016-09-27 | 2017-03-15 | 陈福生 | One plant height produces the preparation method of the Acetobacter gluconicum and its phenyllactic acid of phenyllactic acid |
| CN110144370A (en) * | 2019-05-24 | 2019-08-20 | 江南大学 | A kind of matrix circulation is continuously fermented the method for producing bacteria cellulose |
| WO2020206522A1 (en) * | 2019-04-12 | 2020-10-15 | Biocelltis Pesquisa, Desenvolvimento E Inovação Em Bioengenharia Ltda - Me | Biocellulose film for topical use and manufacturing method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381694A (en) * | 2008-10-21 | 2009-03-11 | 广东省微生物研究所 | Bacteria cellulose producing bacteria and method for preparing bacteria cellulose using above bacterial strain |
| CN101591626A (en) * | 2009-06-30 | 2009-12-02 | 南开大学 | One strain of gluconacetobacter and application thereof |
-
2010
- 2010-10-22 CN CN201010515946A patent/CN101993847B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381694A (en) * | 2008-10-21 | 2009-03-11 | 广东省微生物研究所 | Bacteria cellulose producing bacteria and method for preparing bacteria cellulose using above bacterial strain |
| CN101591626A (en) * | 2009-06-30 | 2009-12-02 | 南开大学 | One strain of gluconacetobacter and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李志西等: "高产细菌纤维素菌株的分离及初步鉴定", 《中国酿造》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250774A (en) * | 2011-07-22 | 2011-11-23 | 北京大学 | Algae engineering bacteria secreting crystalline cellulose and preparation method and use thereof |
| CN104164380A (en) * | 2014-04-23 | 2014-11-26 | 黑龙江大学 | Bacterial cellulose high-yielding strain |
| CN105802896A (en) * | 2016-05-23 | 2016-07-27 | 江南大学 | Komagataeibacter rhaeticus for producing saccharic acid-1,4-lactone |
| CN105802896B (en) * | 2016-05-23 | 2019-07-02 | 江南大学 | A kind of glucose vinegar bacillus of malaga saccharic acid -1,4- lactone |
| CN106497824A (en) * | 2016-09-27 | 2017-03-15 | 陈福生 | One plant height produces the preparation method of the Acetobacter gluconicum and its phenyllactic acid of phenyllactic acid |
| CN106497824B (en) * | 2016-09-27 | 2019-10-01 | 陈福生 | One plant height produces the gluconacetobacter of phenyllactic acid and its preparation method of phenyllactic acid |
| CN106497789A (en) * | 2016-10-21 | 2017-03-15 | 天津工业大学 | A kind of screening technique of the bacterium bacterial strain with bacteria cellulose high productive capacity |
| WO2020206522A1 (en) * | 2019-04-12 | 2020-10-15 | Biocelltis Pesquisa, Desenvolvimento E Inovação Em Bioengenharia Ltda - Me | Biocellulose film for topical use and manufacturing method |
| CN110144370A (en) * | 2019-05-24 | 2019-08-20 | 江南大学 | A kind of matrix circulation is continuously fermented the method for producing bacteria cellulose |
| CN110144370B (en) * | 2019-05-24 | 2021-03-30 | 江南大学 | Method for producing bacterial cellulose by substrate circulating continuous fermentation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101993847B (en) | 2012-09-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107245457B (en) | The extracting method and application of the exocellular polysaccharide and the exocellular polysaccharide of a kind of dendrobium candidum endogenetic fungal bacterial strain and its generation | |
| CN101993847B (en) | Bacterial cellulose strain | |
| CN101724587B (en) | Lactobacillus brevis L2 bacterial strain of high yield gamma-aminobutyrique and screening method and applications thereof | |
| CN105017086A (en) | Separation and purification method for L-citrulline | |
| CN105132331A (en) | Komagataeibacter nataicola and application thereof | |
| CN102119631B (en) | Grifola frondosa strain for producing polysaccharide with composite raw material of rice bran and wheat bran | |
| CN113308392B (en) | Application of Nori endophytic Siamese bacillus | |
| CN104694424A (en) | Bacillus amyloliquefaciens separated from fermented soya beans and used for producing protease | |
| CN103060208B (en) | Trichoderma engineering strain capable of efficiently expressing beta-1, 4-glucanase coding gene and application thereof | |
| CN110129216A (en) | A kind of bacillus subtilis mutagenic strain and its cultural method suitable for solid fermentation production gamma-polyglutamic acid | |
| CN107964516B (en) | Acinetobacter and application thereof in degrading quorum sensing signal molecule DSF | |
| CN103484500B (en) | Bacterial CD-126 fermentation solution and application thereof | |
| CN103849581B (en) | Raw brevibacterium and screen purification process and purposes in a kind of Pericarpium Zanthoxyli | |
| CN102994430A (en) | Bacterial cellulose production strain and application thereof | |
| CN105176859A (en) | Strain MQO-153 for production of arginine deiminase | |
| CN103992953A (en) | Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide | |
| CN104745654B (en) | A kind of method for preparing nimoctin and its production bacterial strain | |
| CN107937315A (en) | A kind of DSF colony induction signalings degradation bacteria and its application in control of plant disease | |
| CN102719502A (en) | Method for producing L-alanine by mutating lactate-production bacteria | |
| CN104404016A (en) | Naringinase production method | |
| CN104480021A (en) | Hirsutella sinensis capable of high-yielding cordyceps polysaccharide and cultivating method thereof | |
| CN105177075A (en) | Method for preparation of L-citrulline with arginine as raw material | |
| CN108611294B (en) | Bacterium and its application is quenched in a kind of colony induction signaling molecule DSF | |
| CN109486731A (en) | A kind of resistance to acetate ethanol bacterium and its application | |
| CN106701638B (en) | A kind of fermentation preparation of bdellovibrio bacteriovorus ecological preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120926 Termination date: 20131022 |