CN108624524A - The bacterial strain and its separating screening method of one plant of production bacteria cellulose - Google Patents
The bacterial strain and its separating screening method of one plant of production bacteria cellulose Download PDFInfo
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Abstract
The invention discloses the separating screening methods of one plant of new bacteria cellulose producing bacterial strain and bacterial strain, the Strain Designation is Han Shi coltfoal shapes bacillus (170518 hww of Komagataeibacter hansenii Man), it is preserved in China General Microbiological culture presevation administrative center, deposit number is CGMCC No.15468.The strain isolation from mango, be Gram-negative, rod-short aerobic bacteria, there is stable, high-yielding ability, can when baffle flask ferments mass production bacteria cellulose.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to the bacterial strain and its separation screening of one plant of production bacteria cellulose
Method.
Background technology
Bacteria cellulose is the ultrapure ultra-microfibers element synthesized by bacterium, is by D-Glucose with β-Isosorbide-5-Nitrae glucosides key connection
Made of chain macromolecule.Compared with plant cellulose present in nature, in crystallinity, chemical purity, tensile strength, bullet
Property modulus, water imbibition and biocompatibility etc. are superior to plant cellulose, it is considered to be a kind of novel day haveing excellent performance
Right Bio-Nano-Materials.In recent years, the research of bacteria cellulose becomes one of the hot spot of current Microbe synthesis Material Field, and
It is latent that huge application is shown in fields such as biological medicine, tissue engineering bracket material, acoustics equipment, food, cosmetics, papermaking
Power.
However, up to the present, the large-scale production of bacteria cellulose still has some problems, mainly fermentation production rate
It is low, production cost is high etc..Further to expand the range of production cellulose strain, production cost is reduced, bacteria cellulose production is obtained
The bacterial strain of high, the hereditary metabolic stability of amount is an important and feasible approach.
Invention content
In order to overcome the deficiencies of existing technologies, the present invention provides one plant, and bacteria cellulose isolated from mango produces
Raw bacterium Han Shi coltfoal shapes bacillus (Komagataeibacter hansenii Man-170518-hww) is now preserved in Chinese common micro-
Biological inoculum preservation administrative center CGMCC No.15468, address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, when preservation
Between be on March 21st, 2018, the bacterial strain can band baffle flask fermentation in generate a large amount of bacteria cellulose.
The form of the Han Shi coltfoal shapes bacillus (Komagataeibacter hansenii Man-170518-hww) of the present invention
It is characterized as：Bacterium colony is in milk yellow, random circle partially, protrusion, rough surface.The bacterium is Gram-negative aerobic bacteria, does not have fortune
Dynamic property, is bacteria cellulose production bacterium, and physiological and biochemical property is shown in Table one.
Han Shi coltfoal shapes bacillus (Komagataeibacter hansenii Man-170518- are obtained by sequencing analysis
Hww 16S rDNA sequences), overall length 1403bp, using BLAST in the 16S rDNA genes and GenBank of the bacterial strain oneself
The bacterium of typing carries out sequence analysis, analysis shows the bacterium and Komagataeibacter hansenii strain NBRC
14820 sequence homologies 99%.In conjunction with the systematic growth result of study of 16S rDNA complete sequence analysis, it is by the Strain Designation
Komagataeibacter hansenii Man-170518-hww。
The present invention Han Shi coltfoal shapes bacillus (Komagataeibacter hansenii Man-170518-hww) it is main
Physiological and biochemical property it is as shown in Table 1：
One physiology biochemical results of table
Note：+ indicate positive ,-indicate negative；
Han Shi coltfoals shape bacillus (Komagataeibacter hansenii Man-170518-hww) of the present invention
16S rDNA gene orders (1,403bp) such as SEQ ID NO：Shown in 1：
GAACCGGAACGGTGAGTCGGCTGCGTCCTTGCGGTTCGCTCACCGGCTTAAGGTCAAACCAACTCCCATGGTGTG
ACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCACCTTCA
TG CACTCGAGTTGCAGAGTGCAATCCGAACTGAGACGGCTTTTGGAGATCCGCTCGGCATCGCTGCCTGGCTTCCC
ACTGT CACCGCCATTGTAGCACGTGTGTAGCCCAGGACATAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCC
TCCGGCTT GTCACCGGCAGTTCCTTTAGAGTGCCCACCCAGACGTGCTGGCAACTAAAGGCGAGGGTTGCGCTCGT
TGCGGGACTTA ACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGCTGGAGGTCTCTTGCGA
GAAATGTCCATCTC TGGACACAGCCTCCGCATGTCAAGTCCTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCA
CATGCTCCACCGCTTGT GCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTGT
GCTTATCGCGTTAACTACGA CACTGAATGGCCAAGCCATCCAACATCCAGCACACATCGTTTACAGCGTGGACTAC
CAGGGTATCTAATCCTGTTTGCT CCCCACGCTTTCGCGCCTCAGCGTCAGTCATGAGCCAGGTTGCCGCCTTCGCC
ACCGGTGTTCTTCCCAATATCTACGA ATTTCACCTCTACACTGGGAATTCCACAACCCTCTCTCACACTCTAGTCG
TCACGTATCAAATGCAGCCCCCAGGTTAA GCCCGGGAATTTCACATCTGACTGTAACAACCGCCTACGCGCCCTTT
ACGCCCAGTCATTCCGAGCAACGCTTGCCCCC TTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTCT
TCTGCGGGTACCGTCATCATCGTCCCCGCTGAAAG TGCTTTACAATCCGAAAACCTTCTTCACACACGCGGCATTG
CTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCAC TGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCC
CAGTGTGGCTGATCATCCTCTCAGACCAGCTATCGATCATC GCCTTGGTAGGCCTTTACCCCACCAACTAGCTAAT
CGAACGCAGGTTCCTCCACAGGCGACTCGCGCCTTTGACCCTCA GGTGTCATGCGGTATTAGCTTCAGTTTCCCAA
AGTTATCCCCCACCCATGGACAGATCCCTACGCGTTACTCACCCGTC CGCCACTAACCCCGAAAGGTTCGTGCGAC
TTGCATGTGTAAGCATGCCGTCCACCGGTTTTTCC
The purpose of the invention is achieved by the following technical solution：
The invention also provides bacterial cellulose strain Han Shi coltfoal shape bacillus (Komagataeibacter hansenii
Man-170518-hww separating screening method), is as follows：
The first step：Take fruit rot part in the test tube of an enriched medium, static culture waits for long on intraluminal fluid face
There is milky colloid membrane.
Second step：Primary screening：After taking the film described in the first step to be washed with sterile saline, shreds and be placed in dress
There are oscillation and gradient dilution in an enriched medium test tube described in the first step, is coated with HS fluorescent screening tablet cultures, so
Picking single bacterium falls in the small test tube equipped with secondary enriched medium and cultivates afterwards, finally selects bacterial strain.
Third walks：The bacterial strain of picking described in second step is transferred to and isolates and purifies tablet, is cultivated in the incubator.
4th step：Above-mentioned third is taken to walk cultured tablet, 5 single bacterium colonies of each bacterial strain picking are linked into liquid screening
Stationary culture in culture medium.
5th step：Postsearch screening：Picking colony further does fermentation verification, further obtains the Han Shi coltfoals shape bacillus.
In the first step, the fruit is mango.
In the first step, an enriched medium is：Glycerine 1.33%-3%, peptone 0.33%-0.75%, yeast
Powder 0.33%-0.75%, disodium hydrogen phosphate 0.18%-0.41%, citric acid 0.08%-0.17%, natamycin 0.2%-
0.45%, adjusting pH are 4.0-7.0；Preferably, it is glycerine 2%, peptone 0.5%, yeast powder 0.5%, disodium hydrogen phosphate
0.27%, citric acid 0.115%, natamycin 0.3%, it is 4.0 to adjust pH.
In the first step, the temperature of the static culture is 25 DEG C -30 DEG C；Preferably, it is 30 DEG C.
In the first step, the time of the static culture is 3d-7d；Preferably, it is 5d.
In second step, the film is the chip-proof film of the positive.
In second step, the HS fluorescent screenings tablet is：Glucose 1.33%-3%, peptone 0.33%-0.75%, ferment
Female powder 0.33%-0.75%, disodium hydrogen phosphate 0.18%-0.41%, citric acid 0.08%-0.17%, corn steep liquor 0.67%-
1.5%, 28 0.01%-0.03% of fluorescent whitening agent, adjusting pH are 4.0-6.0, agar 1.8%-2.0%.Preferably, it is Portugal
Grape sugar 2%, peptone 0.5%, yeast powder 0.5%, disodium hydrogen phosphate 0.27%, citric acid 0.115%, corn steep liquor 1%, fluorescence
Brightening agent 28 0.02%, it is 6.0 to adjust pH, agar 1.8%.
In second step, the time of the tablet culture is 3d-7d；Preferably, it is 5d.
In second step, the tablet is cultivated under 365nm ultra violet lamps.
In second step, the single bacterium colony is apparent, the larger single bacterium colony of fluorescence.
In second step, the bacterial strain be capable of long film and medium pH value be more than 6.5.
In second step, described with the number that sterile saline is washed is 2-4 times；Preferably, it is 3 times.
In second step, the secondary enriched medium is glycerine 1.33%-3%, peptone 0.33%-0.75%, yeast
Powder 0.33%-0.75%, disodium hydrogen phosphate 0.18%-0.41%, citric acid 0.08%-0.17%, natamycin 0.2%-
0.45%, adjusting pH are 4.0-7.0；Preferably, it is glycerine 2%, peptone 0.5%, yeast powder 0.5%, disodium hydrogen phosphate
0.27%, citric acid 0.115%, natamycin 0.3%, it is 7.0 to adjust pH.
In third step, the plating medium that isolates and purifies is：Glucose 1.33%-3%, peptone 0.33%-
0.75%, yeast powder 0.33%-0.75%, disodium hydrogen phosphate 0.18%-0.41%, citric acid 0.08%-0.17%, corn
0.67%-1.5% is starched, it is 5.5-6.5, agar 1.8%-2.0% to adjust pH with dilute hydrochloric acid；Preferably, it is glucose 2%, egg
White peptone 0.5%, yeast powder 0.5%, disodium hydrogen phosphate 0.27%, citric acid 0.115%, corn steep liquor 1%, it is 6.0 to adjust pH, fine jade
Fat 1.8%.
In third step, the temperature of the incubator is 25 DEG C -30 DEG C；Preferably, it is 30 DEG C.
In third step, the time of the culture is 3~5d；Preferably, it is 5d.
In 4th step, the liquid screening medium is：Glucose 2-6%, yeast powder 0.2-1.0%, peptone
0.2-1.0%, disodium hydrogen phosphate 0.1-0.5%, citric acid 0.05-0.2%, it is 7.0 to adjust pH.Preferably, it is glucose
4%, yeast powder 0.5%, peptone 0.5%, disodium hydrogen phosphate 0.27%, citric acid 0.115%, it is 7.0 to adjust pH.
In 4th step, the temperature of the culture is 25 DEG C -30 DEG C；Preferably, it is 30 DEG C.
In 4th step, the time of the culture is 3~9d；Preferably, it is 7d.
In 5th step, the fermentation medium is：Sucrose 3.3%-7.5%, yeast powder 0.6%-1.05%, peptone
0.33%-0.75%, dipotassium hydrogen phosphate 0.2%-0.45%, acetic acid 0.13%-3% adjust pH after 5.5-6.5, to add
Bitter salt 1.33%-3%；Preferably, it is sucrose 5%, yeast powder 0.9%, peptone 0.5%, dipotassium hydrogen phosphate
0.3%, acetic acid 0.2% adds bitter salt 2% after adjusting pH is 6.0.
In 5th step, the fermentation condition is：100-150r/min, 250mL baffle flask liquid amount 50mL, 28-32
DEG C shaken cultivation 3-4d；Preferably, it is 120r/min, 250mL baffle flask liquid amounts 50mL, 30 DEG C of shaken cultivation 4d.
In 5th step, the bacterium colony is larger bacterium colony.
According to above method, the present invention is selected to one plant of suitable dynamic cultivation, stable, high-yielding production bacteria cellulose bacterium
Strain.The bacterial strain is preserved in China General Microbiological Culture Collection Center, and preserving number is：CGMCC No.15468.
The beneficial effects of the present invention are：One plant of new bacteria cellulose producing bacterial strain, the bacterium at separation screening of the present invention
Strain is Gram-negative, the aerobic bacteria of rod-short eliminates the sour energy of production by the high bacterial strain of secondary enriched medium screening pH value
The high bacterial strain of power, the bacterial strain screened have metabolic advantage, and fingerprinting stress, the high bacterium of dispersibility are screened by baffle flask
Strain, and have stable, high-yielding ability, can when baffle flask ferments mass production bacteria cellulose.
Description of the drawings
Fig. 1 is the yield of each bacterial strain of primary screening first batch in embodiment 1.
Fig. 2 is the yield of each bacterial strain of primary screening second lot in embodiment 1.
Fig. 3 is primary screening producing strain in embodiment 2.
Fig. 4 is baffle flask postsearch screening producing strain.
Fig. 5 is common triangular flask postsearch screening producing strain.
Fig. 6 is the further secondary screening yield of bacterial strain of postsearch screening.
Fig. 7 is the dynamic fermentation product form observation chart of bacterial strain, wherein figure A is bacterial strain Man-170518-hww of the present invention
Dynamic fermentation product form observation chart, figure B be bacterial strain CGMCC 1.1812 dynamic fermentation product form observation chart.
Fig. 8 is baffle flask.
Specific implementation mode
Further accurate specific description is carried out to the present invention below in conjunction with example and attached drawing, these embodiments are only
For the present invention, but the present invention is not limited thereto.
Embodiment 1：The primary screening of high yield bacteria cellulose
Take the decayed portion about 1g of fruit, the sterilized 18mm × 180mm examinations equipped with enriched medium of 10mL of access
Guan Zhong, 30 DEG C of static culture 5d, on intraluminal fluid face with milky colloid membrane and film will not fragmentation person be the positive.Film is taken to use
After 0.9% sterile saline is washed 3 times, shreds to be placed in 18mm × 180mm test tubes equipped with 10mL enriched mediums and shake
Swing 1h, take oscillation liquid HS fluorescent screening tablet cultures 5d is coated with after gradient dilution after, tablet under 365nm ultra violet lamps,
Apparent, the larger single bacterium of picking fluorescence falls in the small test tube equipped with 2mLpH values for 7 secondary enriched medium and cultivates 5d, and
The pH for measuring culture solution selects the bacterial strain that be capable of long film and medium pH value is more than 6.5, and the bacterium colony of picking, which is transferred to, to be isolated and purified
Tablet cultivates 3~5d in 30 DEG C of incubators, selects larger bacterium colony and draws slant preservation.This step is known as primary screening.
Embodiment 2：The primary screening (screening pH reference examples) of high yield bacteria cellulose
Take the decayed portion about 1g of fruit, the sterilized 18mm × 180mm examinations equipped with enriched medium of 10mL of access
Guan Zhong, 30 DEG C of static culture 5d, on intraluminal fluid face with milky colloid membrane and film will not fragmentation person be the positive.Film is taken to use
After 0.9% sterile saline is washed 3 times, shreds to be placed in 18mm × 180mm test tubes equipped with 10mL enriched mediums and shake
Swing 1h, take oscillation liquid HS fluorescent screening tablet cultures 5d is coated with after gradient dilution after, tablet under 365nm ultra violet lamps,
Apparent, the larger single bacterium of picking fluorescence falls in the small test tube equipped with 2mLpH values for 5 secondary enriched medium and cultivates 5d, and
The pH for measuring culture solution selects the bacterial strain that be capable of long film and medium pH value is more than 4.0, and the bacterium colony of picking, which is transferred to, to be isolated and purified
Tablet cultivates 3~5d in 30 DEG C of incubators.
The fermentation of the primary screening bacterial strain of 3 embodiment 1 of embodiment
(1) activation of strain：Take out the control strain (Gluconacetobacter of the production bacteria cellulose of slant preservation
Xylinus CGMCC 1.1812) and 74 plants of embodiment 1 production celluloses bacterial strains.Strain is transferred to from cryopreservation tube respectively
In 250mL triangular flasks equipped with 30mL seed liquors, 30 DEG C, 200r/min shaken cultivations for 24 hours.
(2) separation screening obtains single bacterium colony：Seed liquor is taken out, dips bacterium solution with oese, is then carried out on tablet
Sectional streak.Tablet is placed in 30 DEG C of incubators and is cultivated 5 days.
(3) static culture：With oese, 5 single bacterium colonies of picking from tablet, access are trained containing 15mL liquid screenings respectively
Support base, in the centrifuge tube that capacity is 50mL, each 5, bacterial strain work is parallel, and ensures to be inoculated with the bacterium colony size phase in centrifuge tube
Closely.Centrifuge tube is placed in stationary culture 7 days in 30 DEG C of incubator.
(4) extraction of bacteria cellulose：Cellulose is washed twice, is then submerged in the NaOH solution of 0.2M, 100
Isometric glacial acetic acid is added in DEG C heating water bath 30min, stands 10min after stirring 5min, discards supernatant, by cellulose film water
It washes three times.
(5) drying is weighed：The gel bacteria cellulose of acquisition is transferred on tablet, and is positioned over 60 DEG C of baking oven
In, it dries for 24 hours.By minusing, the dry weight of bacteria cellulose is obtained.
Seed culture medium：Glucose 2%, yeast powder 0.5%, peptone 0.5%, disodium hydrogen phosphate 0.27%, citric acid
0.115%, corn steep liquor 1%；
Liquid screening medium：Glucose 4%, yeast powder 0.5%, peptone 0.5%, disodium hydrogen phosphate 0.27%, lemon
Lemon acid 0.115%, it is 7.0 to adjust pH.
All 74 plants production cellulose producing strains are compared, find the production of bacterial strain Man-170518-hww of the present invention
Amount is highest in all production cellulose bacterial strains, is control strain Gluconacetobacter xylinus CGMCC 1.1812
2.30 times (Fig. 1 and Fig. 2) of yield.Bacterial strain more than control is 24 plants, and ratio is 32.4% therefore bacterial strain Man- of the present invention
The yield that 170518-hww static fermentations produce bacteria cellulose is very high, is advantage superior strain.
Embodiment 4：The fermentation of the primary screening bacterial strain of embodiment 2
Take out control strain (the Gluconacetobacter xylinus CGMCC of the production bacteria cellulose of slant preservation
1.1812) it is activated with the bacterial strain of 42 plants of production celluloses of embodiment 2, other operations are the same as embodiment 3.To 42 plants of gained and right
It is compared according to 1.1812 yield of bacterial strain Gluconacetobacter xylinus CGMCC, is more than only 4 plants of control strain, ratio
It is 9.5%.(Fig. 3)
No. 1 two enriched medium pH of embodiment are 7, select the bacterial strain that be capable of long film and medium pH value is more than 6.5,
The ratio in selected bacterial strain being more than control is 32.4%；No. 2 two enriched medium pH of embodiment be 7, select be capable of long film and
Medium pH value is more than 4 bacterial strain, and the ratio in selected bacterial strain being more than control is 9.5%；Therefore the method for embodiment 1 wants excellent
In the method for embodiment 2.
Embodiment 5：Postsearch screening superior strain
(1) preparation of seed：Take out Man-170518-hww bacterial strains, the control strain of slant preservation
Gluconacetobacter xylinus CGMCC 1.1812 are with other by produce bacteria cellulose 24 of primary screening
Bacterial strain.Respectively by strain from being transferred in cryopreservation tube in the 250mL triangular flasks equipped with 30mL seed liquors, 30 DEG C, 200r/min shakes
Swing culture for 24 hours.
(2) OD values are measured, determine inoculum concentration：It takes the bacterium solution after 200 μ L activation in 96 orifice plates respectively, uses spectrophotometer
OD values are measured, according to different OD values, being inoculated with the bacterium solutions of different volumes, (standard is in zymotic fluid：OD values are inoculated with when being 0.8
In 2mL to the 250mL baffle flasks equipped with 50mL zymotic fluids (Fig. 8)).Since baffle flask is compared with common triangular flask,
Evenly, mass transfer is more abundant for mixing, has clear advantage.The bacterial strain for producing bacteria cellulose is all stringent aerobic bacteria, is being grown
Good mass transfer and a large amount of oxygen are needed with during production bacteria cellulose, therefore compared to common triangular flask, baffle triangle
Bottle is advantageously.Moreover, because baffle itself has strong mixing left and right, is screened with baffle flask, be easy to get production
The more dispersed bacterial strain of bacteria cellulose.
(3) dynamic fermentation：Triangular flask is positioned in shaking table, 30 DEG C, 200r/min shaken cultivations 4 days.
(4) extraction of bacteria cellulose：Cellulose is washed twice, is then submerged in the NaOH solution of 0.2M, 100
Isometric glacial acetic acid is added in DEG C heating water bath 30min, stands 10min after stirring 5min, discards supernatant, by cellulose film water
It washes three times.
(5) drying is weighed：The bacteria cellulose of acquisition is transferred on tablet, and is positioned in 60 DEG C of baking oven, is dried for 24 hours
It is dry.By minusing, the dry weight of bacteria cellulose is obtained.
Seed culture medium：Glucose 2%, yeast powder 0.5%, peptone 0.5%, disodium hydrogen phosphate 0.27%, citric acid
0.115%, corn steep liquor 1%；
Fermentation medium：Sucrose 4%, yeast powder 0.9%, peptone 0.5%, disodium hydrogen phosphate 0.27%, acetic acid
0.2%, after adjusting pH is 6.0, add bitter salt 2%.
All 24 plants production cellulose producing strains are compared, find the production of bacterial strain Man-170518-hww of the present invention
Amount is highest in all production cellulose bacterial strains, is control strain Gluconacetobacter xylinus CGMCC 1.1812
2 times (Fig. 4) of yield.Wherein be more than control strain bacterial strain be 18, ratio be 75% (Fig. 4) therefore, bacterial strain Man- of the present invention
170518-hww is advantage superior strain in the yield also highest of baffle flask dynamic fermentation.
Embodiment 6：Postsearch screening superior strain (baffle bottle comparative examples)
(1) preparation of seed：Take out slant preservation control strain Gluconacetobacter xylinus CGMCC
1.1812 pass through 40 bacterial strains for producing bacteria cellulose of primary screening with other.Strain is transferred to dress from cryopreservation tube respectively
Have in the 250mL triangular flasks of 30mL seed liquors, 30 DEG C, 200r/min shaken cultivations for 24 hours.
(2) OD values are measured, determine inoculum concentration：It takes the bacterium solution after 200 μ L activation in 96 orifice plates respectively, uses spectrophotometer
OD values are measured, according to different OD values, being inoculated with the bacterium solutions of different volumes, (standard is in zymotic fluid：OD values are inoculated with when being 0.8
In 2mL to the common triangular flasks of 250mL equipped with 50mL zymotic fluids).
Other methods are the same as embodiment 5.
All 40 plants production cellulose producing strains are compared, wherein the bacterial strain more than control strain is 5 plants, ratio is
12.5% (Fig. 5)
The secondary screening of 7 bacterium of embodiment
4 plants of superior strains that the 5 plants of superior strains and embodiment 6 that Example 5 screens respectively screen, according to embodiment 6
Method, fermented with baffle flask, yield is shown in Fig. 6.
From fig. 6 it can be seen that the producing strain screened from embodiment 5 is apparently higher than the bacterial strain screened from embodiment 6.
Embodiment 8：The observation of tunning form
(1) activation of strain：Take out the control bacterium of the Man-170518-hww bacterial strains and production bacteria cellulose of slant preservation
Strain Gluconacetobacter xylinus CGMCC 1.1812, are inoculated into the 250mL containing 30mL seed culture mediums respectively
In triangular flask, 30 DEG C, 200r/min shaken cultivations for 24 hours.
(2) OD values are measured, determine inoculum concentration：It takes the bacterium solution after 200 μ L activation in 96 orifice plates respectively, uses spectrophotometer
OD values are measured, according to different OD values, being inoculated with the bacterium solutions of different volumes, (standard is in zymotic fluid：OD values are inoculated with when being 0.8
In 2mL to the 250mL triangular flasks equipped with 50mL zymotic fluids).
(3) dynamic fermentation：Zymotic fluid after inoculation is positioned in shaking table, 30 DEG C, 200r/min shaken cultivations 4 days.
It can be found that the type strain CGMCC 1.1812 of bacterial strain Man-170518-hww of the present invention and production bacteria cellulose
Morphologically there is differ greatly from for the bacteria cellulose that dynamic fermentation generates.1.1812 dynamic fermentations of type strain CGMCC
The bacteria cellulose of generation flocks together, and is lumps.And Man-170518-hww bacterium dynamic fermentation generation of the present invention is thin
Fungin scatter, and is form of spherical particles (Fig. 7 A).Spherical particle bacteria cellulose has more nano size voids and bigger
Specific surface area imparts its better cellular affinity and adsorption capacity, anti-in bio-separation, slow releasing carrier of medication and immobilization
Answer etc. has more unique application prospect.In large-scale industry fermentation process, due to the needs of air agitation, spherical
Granular bacterial strain has prodigious advantage than pockets of bacterial strain.Therefore bacterial strain Man-170518-hww of the present invention is very suitable for moving
State fermented and cultured, has broad application prospects.
Embodiment 9：The measurement of zymotic fluid pH
(1) activation of strain：Take out the pattern bacterium of the Man-170518-hww bacterial strains and production bacteria cellulose of slant preservation
Strain ATCC 31749, ATCC 31750.Strain is transferred to 250mL capacity from cryopreservation tube respectively, containing 30ml seed liquors
In triangular flask, 30 DEG C, 200r/min shaken cultivations for 24 hours.
(2) OD values are measured, determine inoculum concentration：It takes the bacterium solution after 200 μ L activation in 96 orifice plates respectively, uses spectrophotometer
OD values are measured, according to different OD values, being inoculated with the bacterium solutions of different volumes, (standard is in zymotic fluid：OD values are inoculated with when being 0.8
In 2mL to the triangular flask with baffle equipped with 50mL zymotic fluids).
(3) dynamic fermentation：Triangular flask is positioned in shaking table, 30 DEG C, 200r/min shaken cultivations 4 days.
(4) pH value determination：The measurement of pH is carried out to zymotic fluid in fermentation termination.
Type strain ATCC31749 and ATCC31750 the fermentation termination pH value for producing bacteria cellulose is respectively 4.2 Hes
4.0, the pH value of bacterial strain Man-170518-hww fermentation termination zymotic fluids of the present invention is 6.5.
The large-scale production of bacteria cellulose there is a problem of one it is very severe：When using carbohydrate as carbon source, meeting
A large amount of by-product is generated, these by-products are in acidity so that the pH of zymotic fluid is reduced, and causes fermentation condition to change, no
The efficiency that bacteria cellulose is produced conducive to the later stage, reduces the yield of cellulose.
Bacterial strain Man-170518-hww bacterium of the present invention do not produce acid, are made good use of to carbon source, nitrogen source, and cellulose output is high, and
Production is stablized, and has fabulous character.
Embodiment 10：The comparison of dynamic fermentation yield
(1) activation of strain：Take out Man-170518-hww bacterial strains, the control strain of slant preservation
Type strain ATCC 31749, the ATCC of Gluconacetobacter xylinus CGMCC 1.1812 and production bacteria cellulose
31750.Be inoculated into respectively in the 250mL triangular flasks containing 30ml seed liquors, 30 DEG C, 200r/min shaken cultivations for 24 hours.
(2) OD values are measured, determine inoculum concentration：It takes the bacterium solution after 200 μ L activation in 96 orifice plates respectively, uses spectrophotometer
OD values are measured, according to different OD values, being inoculated with the bacterium solutions of different volumes, (standard is in zymotic fluid：OD values are inoculated with when being 0.8
In the band baffle flask of 2mL to the 250mL equipped with 50mL zymotic fluids).
(3) dynamic fermentation：Triangular flask is positioned in shaking table, 30 DEG C, 200r/min shaken cultivations 4 days.
(4) extraction of bacteria cellulose：Cellulose is washed twice, is then submerged in the NaOH solution of 0.2M, 100
Isometric glacial acetic acid is added in DEG C heating water bath 30min, stands 10min after stirring 5min, discards supernatant, by cellulose film water
It washes three times.
(5) drying is weighed：The bacteria cellulose of acquisition is transferred on tablet, and is positioned in 60 DEG C of baking oven, is dried for 24 hours
It is dry.By minusing, the dry weight of bacteria cellulose is obtained.
It can be found that with triangular flask dynamic fermentation bacterial strain Man-170518-hww and control strain with baffle
Gluconacetobacter xylinus CGMCC 1.1812 and type strain ATCC 31749, the ATCC for producing bacteria cellulose
31750 compare, and the bacteria cellulose output of bacterial strain Man-170518-hww is 10.2g/L, and CGMCC 1.1812, ATCC
The yield of 31749 and ATCC 31750 is respectively 4.8g/L, 2.5g/L and 3.2g/L.Therefore, it is shaken with the triangular flask with baffle
When bottle fermentation, bacterial strain Man-170518-hww of the present invention is with the obvious advantage, and yield is significantly higher than other bacterial strains, is suitble to dynamic cultivation.
Above-described embodiment simply to illustrate that the present invention technical concepts and features, its object is to allow the common of this field
Technical staff cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.Every
According to the equivalent change or modification that the essence of the content of present invention is made, should all cover within the scope of the present invention.
SEQUENCE LISTING
<110>East China Normal University
<120>The bacterial strain and its separating screening method of one plant of production bacteria cellulose
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1403
<212> DNA
<213>Artificial sequence
<400> 1
gaaccggaac ggtgagtcgg ctgcgtcctt gcggttcgct caccggctta aggtcaaacc 60
aactcccatg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg 120
ctgatccgcg attactagcg attccacctt catgcactcg agttgcagag tgcaatccga 180
actgagacgg cttttggaga tccgctcggc atcgctgcct ggcttcccac tgtcaccgcc 240
attgtagcac gtgtgtagcc caggacataa gggccatgag gacttgacgt catccccacc 300
ttcctccggc ttgtcaccgg cagttccttt agagtgccca cccagacgtg ctggcaacta 360
aaggcgaggg ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga 420
cagccatgca gcacctgtgc tggaggtctc ttgcgagaaa tgtccatctc tggacacagc 480
ctccgcatgt caagtcctgg taaggttctg cgcgttgctt cgaattaaac cacatgctcc 540
accgcttgtg cgggcccccg tcaattcctt tgagtttcaa ccttgcggcc gtactcccca 600
ggcggtgtgc ttatcgcgtt aactacgaca ctgaatggcc aagccatcca acatccagca 660
cacatcgttt acagcgtgga ctaccagggt atctaatcct gtttgctccc cacgctttcg 720
cgcctcagcg tcagtcatga gccaggttgc cgccttcgcc accggtgttc ttcccaatat 780
ctacgaattt cacctctaca ctgggaattc cacaaccctc tctcacactc tagtcgtcac 840
gtatcaaatg cagcccccag gttaagcccg ggaatttcac atctgactgt aacaaccgcc 900
tacgcgccct ttacgcccag tcattccgag caacgcttgc ccccttcgta ttaccgcggc 960
tgctggcacg aagttagccg gggcttcttc tgcgggtacc gtcatcatcg tccccgctga 1020
aagtgcttta caatccgaaa accttcttca cacacgcggc attgctggat caggcttgcg 1080
cccattgtcc aatattcccc actgctgcct cccgtaggag tctgggccgt gtctcagtcc 1140
cagtgtggct gatcatcctc tcagaccagc tatcgatcat cgccttggta ggcctttacc 1200
ccaccaacta gctaatcgaa cgcaggttcc tccacaggcg actcgcgcct ttgaccctca 1260
ggtgtcatgc ggtattagct tcagtttccc aaagttatcc cccacccatg gacagatccc 1320
tacgcgttac tcacccgtcc gccactaacc ccgaaaggtt cgtgcgactt gcatgtgtaa 1380
gcatgccgtc caccggtttt tcc 1403
Claims (14)
1. a kind of bacterial cellulose strain Han Shi coltfoal shapes bacillus (Komagataeibacter hansenii) Man-170518-
Hww is preserved in China General Microbiological culture presevation administrative center, and the deposit date is on March 21st, 2018, deposit number was
CGMCC No.15468, which is characterized in that it is Gram-negative aerobic bacteria, does not have motility, is bacteria cellulose production
Bacterium.
2. bacterial strain as described in claim 1, which is characterized in that the 16S rDNA gene orders such as SEQ ID NO of the bacterial strain：
Shown in 1.
3. bacterial strain as described in claim 1, which is characterized in that the bacterium colony of the bacterial strain in milk yellow, it is random partially circle, it is convex
It rises, rough surface.
4. a kind of separating screening method of bacterial cellulose strain, which is characterized in that specifically include following steps：
The first step：Take fruit rot part in the test tube of an enriched medium, static culture waits on intraluminal fluid face with breast
White colloid membrane；
Second step：Primary screening：After taking the film described in the first step to be washed with sterile saline, shreds and be placed in equipped with
Oscillation and gradient dilution in an enriched medium test tube described in one step, are coated with HS fluorescent screening tablet cultures, then choose
It takes single bacterium to fall in the small test tube equipped with secondary enriched medium to cultivate, finally selects bacterial strain；
Third walks：The bacterial strain of picking described in second step is transferred to and isolates and purifies tablet, is cultivated in the incubator；
4th step：Above-mentioned third is taken to walk cultured tablet, 5 single bacterium colonies of each bacterial strain picking are linked into liquid screening culture
Stationary culture in base；
5th step：Postsearch screening：Picking colony further does fermentation verification, further obtains the Han Shi coltfoals shape bacillus.
5. screening technique according to claim 4, which is characterized in that an enriched medium and secondary enrichment culture
Base is：Glycerine 1.33%-3%, peptone 0.33%-0.75%, yeast powder 0.33%-0.75%, disodium hydrogen phosphate
0.18%-0.41%, citric acid 0.08%-0.17%, natamycin 0.2%-0.45%, adjusting pH are 4.0-7.0.
6. screening technique according to claim 4, which is characterized in that in the first step, the temperature of the static culture is 25
℃-30℃；The time of the static culture is 3d-7d.
7. screening technique according to claim 4, which is characterized in that in second step, the HS fluorescent screenings tablet is：Portugal
Grape sugar 1.33%-3%, peptone 0.33%-0.75%, yeast powder 0.33%-0.75%, disodium hydrogen phosphate 0.18%-
0.41%, citric acid 0.08%-0.17%, corn steep liquor 0.67%-1.5%, 28 0.01%-0.03% of fluorescent whitening agent are adjusted
PH is 4.0-6.0, agar 1.8%-2.0%.
8. screening technique according to claim 4, which is characterized in that described to be washed with sterile saline in second step
Number be 2-4 times；The film is the chip-proof film of the positive；The time of the tablet culture is 3d-7d；The tablet
It is cultivated under 365nm ultra violet lamps；The bacterial strain be capable of long film and medium pH value be more than 6.5.
9. screening technique according to claim 4, which is characterized in that described to isolate and purify plating medium in third step
For：Glucose 1.33%-3%, peptone 0.33%-0.75%, yeast powder 0.33%-0.75%, disodium hydrogen phosphate 0.18%-
0.41%, citric acid 0.08%-0.17%, corn steep liquor 0.67%-1.5%, adjusting pH are 5.5-6.5, agar 1.8%-
2.0%.
10. screening technique according to claim 4, which is characterized in that in third step, the temperature of the incubator is 25
℃-30℃；The time of the culture is 3~5d.
11. screening technique according to claim 4, which is characterized in that in the 4th step, the liquid screening medium is：
Glucose 2-6%, yeast powder 0.2-1.0%, peptone 0.2-1.0%, disodium hydrogen phosphate 0.1-0.5%, citric acid 0.05-
0.2%, it is 7.0 to adjust pH.
12. screening technique according to claim 4, which is characterized in that in the 4th step, the temperature of the culture is 25 DEG C-
30℃；The time of the culture is 3~9d；Preferably, it is 7d.
13. screening technique according to claim 4, which is characterized in that in the 5th step, the fermentation medium is：Sucrose
3.3%-7.5%, yeast powder 0.6%-1.05%, peptone 0.33%-0.75%, dipotassium hydrogen phosphate 0.2%-0.45%, second
Sour 0.13%-3% adjusts pH after 5.5-6.5, to add bitter salt 1.33%-3%.
14. screening technique according to claim 4, which is characterized in that in the 5th step, the fermentation condition is：100r/
Min-150r/min, 250mL baffle flask liquid amount 50mL, 28 DEG C~32 DEG C 3~4d of shaken cultivation.
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| CN112824524A (en) * | 2019-11-21 | 2021-05-21 | 中国科学院微生物研究所 | Bacterial cellulose producing strain and separation identification and yield analysis thereof |
| CN112961793A (en) * | 2019-12-12 | 2021-06-15 | 华东师范大学 | Bacterial cellulose producing strain, screening method and preparation of bacterial cellulose |
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| CN112824524B (en) * | 2019-11-21 | 2022-11-15 | 中国科学院微生物研究所 | Bacterial cellulose producing strain and separation identification and yield analysis thereof |
| CN112961793A (en) * | 2019-12-12 | 2021-06-15 | 华东师范大学 | Bacterial cellulose producing strain, screening method and preparation of bacterial cellulose |
| CN113718001A (en) * | 2020-05-25 | 2021-11-30 | 华东师范大学 | Method for producing bacterial cellulose by using bacterial fermentation waste glycerol |
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