CN107937307A - One plant of bacteria cellulose Producing Strain and its optimal fermentation condition - Google Patents
One plant of bacteria cellulose Producing Strain and its optimal fermentation condition Download PDFInfo
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- CN107937307A CN107937307A CN201711220732.1A CN201711220732A CN107937307A CN 107937307 A CN107937307 A CN 107937307A CN 201711220732 A CN201711220732 A CN 201711220732A CN 107937307 A CN107937307 A CN 107937307A
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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
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract
The invention discloses a kind of bacteria cellulose superior strain, is named as Komagataeibacter rhaeticus TJPU03.The bacterial strain is obtained by the corrupt tangerine peel of spontaneous fermentation, for one plant of new bacterial cellulose strain, its bacteria cellulose output (dry weight) may be up to 8.28g/L culture mediums, have the characteristics that stable high yield, have broad application prospects in bacteria cellulose production industry.
Description
Technical field
The present invention relates to microbial technology field, and in particular to one plant of bacteria cellulose in corrupt fruit remnants
(BC) separation, identification and its optimal conditions for producing bacteria cellulose (BC) of superior strain.
Background technology
Global fossil energy is increasingly in short supply, and the utilization of renewable resource increasingly attract attention.Cellulose is the earth
On it is most abundant, and there is biodegradable a kind of large biological molecule, its development and application is as regenerative resource
Research hotspot.Native cellulose can be synthesized by photosynthesis of plant at present and microorganism biological synthesizes, and plant cellulose is often
The impurity such as lignin, hemicellulose and pectin are associated with, need to be pre-processed before commercial Application, are so not only resulted in pair
The destruction of cellulosic molecule, and a large amount of contaminated wastewater environment can be produced.It is and fine for one bacterium of cellulose of Microbe synthesis
Dimension is plain (BC), exists with high-purity nano level Cellulosed molded article, using being before not required to pre-process, has the plant cellulose incomparable
Superiority, this make its numerous areas such as food industry, biomedicine, acoustics equipment, papermaking, environment exist widely should
With many high value added products have very tempting commercial application prospect.
Bacteria cellulose (BC) excellent performance, it is widely used, therefore numerous areas has it demand of magnanimity, but at present
There are superior strain, amplification production the defects of strain is few, low output, high production cost, are screened in a manner of inexpensive for the production of BC
The Pseudomonas of BC, becomes active demand, and this measure can fundamentally improve the BC situations that supply falls short of demand.To realize the industrial metaplasia of BC
Production, it is necessary first to screen excellent strain.By to current, production BC bacterium only have a few in research report nature, mainly
The microorganisms such as aceticanhydride Pseudomonas, Agrobacterium, false born of the same parents' Bacillus are concentrated on, the acetobacter xylinum wherein in acetobacter is found earliest,
Research is more thorough, is the strain that synthetic cellulose ability is most strong so far, is commonly used for that cellulose is basic and application
The type strain of research.The screening of excellent species, is mainly separated based on the raw material in nature.Such as horse rosy clouds are with length
The vinegar fermented grain of film isolates and purifies out one plant of acetobacter aceti for material, and the results show bacterium yield is high and performance is stablized.Hong Feng etc. with
Saccharine material, low value starch materials and waste cellulose class raw material are used for the raw material for screening production BC bacterial strains, Yang Guang etc. with agricultural
The raw material of discarded object and accessory substance as screening production BC bacterial strains, obtains good achievement in research.In view of China's fruit produce
It is abundant, and the production of fruit has obvious seasonal variety, easily generation fruit is a large amount of corrupt and the waste of discard processing shows
As choosing the raw material sources that corrupt fruit makees screening production BC bacterial strains, becoming the development trend hveing great potential.This kind of research can fill
Divide and utilize the components such as abundant carbon source contained in organic waste matter, and alleviate to a certain extent since a large amount of abandon buries institute's shape
Into SOLID ORGANIC pollutant rubbish and production waste exhaust emission, fully demonstrate environmentally protective sustainable principle.At present
Screening on BC superior strains also carries out unremitting effort by guidance, researchers of this environmentally protective principle substantially,
But the example for successfully filtering out high yield BC bacterial strains is still very few, and production cost is still high, screening high yield BC bacterial strains
Method need to be furtherd investigate.
On the other hand, the fermentation condition such as the composition of culture medium, pH value, temperature, dissolved oxygen and fermentation method, the fermentation to BC
Production has a significant impact, and the problem of worth further investigated.The species difference of BC bacterial strains is produced, it is poor that required nutriment exists
It is different, and the composition of culture medium has a significant impact the yield of BC.Both at home and abroad to having carried out substantial amounts of research in this respect,
Hestrin passes through optimum organization medium component to increase BC yield, it is determined that the basal medium being widely used：Glucose
2.0g/100mL, peptone 0.5g/100mL, yeast leachate 0.5g/100mL, disodium hydrogen phosphate 0.27g/100mL, citric acid
0.11g/100mL、pH 6.0.Later stage people are directed to different strains, and the component of culture medium is improved.Research more at present
Report thinks that fructose is the optimum carbon source for producing BC, secondly dextrose and saccharose, sorbierite and mannitol do the production of carbon source BC
Amount is also higher.But fructose is more expensive, glucuronic acid can be generated when dextrose and saccharose is as carbon source, reduces the pH of culture medium
Value, causes the BC underproduction.To reduce production cost, there is researcher to use trade waste, waste cotton fabric treating liquid, distiller's grains lixivium
Replace the expensive components in culture medium Deng inexpensive substance, although these raw materials reduce cost, but add pretreatment early period and
The drawbacks of later purification is difficult.Also research co-cultures fermentation with lactic acid bacteria using production BC bacterium, lactic acid or second is added into culture medium
The synergistic factors such as alcohol, to improve BC yield.In addition, suitable temperature, pH value, dissolved oxygen level, exist not for different bacterial strains
Same optimized scope, and different fermentation methods affects the structure, property and yield of BC.Therefore, BC superior strains are expanded,
Low-cost high-efficiency production BC is prerequisite and the task of top priority for realizing BC industrialized productions.
The content of the invention
For current production BC bacterial strains and BC production status and technical need, it is an object of the present invention to provide one plant of BC high
Bacterium is produced, which is named as Komagataeibacter rhaeticus TJPU03 in the corrupt tangerine peel of spontaneous fermentation
(K.rhaeticus TJPU03), for character as shown in Figure of description 1, thalline is rod-short, single or paired presence, is had bright
The pod membrane that aobvious exocellular polysaccharide is formed, thalline size is Gram's staining between (1.82~2.5) × (0.78~0.95) μm
Negative strain.Found by 16SrDNA sequencings and phylogenetic tree construction, the bacterial strain and Komagataeibacter
Rhaeticus is the most similar, and similarity reaches 99.7%, therefore we are strain was named K.rhaeticus TJPU03.
Shown in K.rhaeticus TJPU03 phylogenetic trees Figure of description 2, Query is strain to be tested in figure.
The second object of the present invention is to provide a kind of optimum process of screened K.rhaeticus TJPU03 productions BC
Condition, it is characterised in that best medium, which forms, is：Glucose 35g/L, peptone 5.5g/L, yeast extract 7g/L, phosphoric acid hydrogen two
Sodium 4g/L, citric acid 1.4g/L；Optimal fermentation condition is：10% inoculum concentration, 3.5,32 DEG C of pH.Optimum carbon source is glucose
And sucrose, optimum nitrogen source are peptone and yeast extract compound nitrogen source.In optimal conditions, BC yield (weight in wet base) may be up to 63g/
100mL culture mediums, and the BC yield (weight in wet base) of model bacterium acetobacter xylinum is 30g/mL culture mediums.
Brief description of the drawings
Fig. 1 is the optical microphotograph sem observation figure of K.rhaeticus TJPU03 and gluconate pyracetobacillus (G.xylinum).
Fig. 2 is the phylogenetic tree of K.rhaeticus TJPU03.
Fig. 3 is the Flied emission scanning electricity of K.rhaeticus TJPU03 and gluconate pyracetobacillus (G.xylinum) production BC
Mirror figure.
Fig. 4 is the infrared spectrum of K.rhaeticus TJPU03 and gluconate pyracetobacillus (G.xylinum) production BC.
Specific embodiment
The present invention is illustrated with reference to embodiment.
The isolation and purification of embodiment 1K.rhaeticus TJPU03
After the corrupt tangerine peel laboratory sample of spontaneous fermentation is carried out fully broken etc. pretreatment, 10g sterile waters are weighed
Rinse to small beaker, being sufficiently stirred ensures that strain therein is all dissolved in water, and sterilization treatment is seeded to after dilute filtration
Enriched medium in, 30 DEG C, bacteria suspension is made in 150r/min shaken cultivations 24h.Will bacteria suspension moderately dilution after be coated on through
Cross sterilization treatment, have acid-producing bacteria screening layer calcium carbonate isolation medium on, 150 μ L/ plates, in degerming super-clean bench
30 DEG C of 3~5d of culture, the bacterium colony that there is transparent region on picking periphery is rule repeatedly purifies culture, obtains producing sour single bacterium colony.Each strain
Acid-producing bacteria is seeded to inclined-plane Storaged media, is placed in -4 DEG C of refrigerators and preserves.The slant medium preserved during use from refrigerator
Inoculating strain treats colony growth to luxuriant to the slant medium of new sterilization treatment, and 2~3 ring lawns of inoculation are to having sterilized
The seed activation fluid nutrient medium of processing, 30 DEG C, 130r/min shaken cultivations 24h.Seed liquor is accessed into sterilized HS fermentations
Culture medium, obtains the bacterial strain that can produce gelatinous BC films.
The identification of embodiment 2K.rhaeticus TJPU03
(1) Morphological Identification
Thalline is rod-short, single or paired presence, has the pod membrane that obvious exocellular polysaccharide is formed, and thalline size exists
Between (1.82~2.5) × (0.78~0.95) μm, Gram's staining is feminine gender, as shown in Figure of description 1.
(2) Molecular Identification
Determine that the bacterial strain is negative bacterium by Gram-staining process first, pass through the Bacteria of Kang Wei ShiJi Co., Ltd
Genomic DNA kit(Cat：CW05525) kit extracts its genomic DNA, then with the universal primer pair of identification bacterium
The 16srDNA genes of 1492R (GGTTACCTTGTTACGACTT) and 27F8F (AGAGTTTGATCCTGGCTCA) genome carry out
Amplification.Amplification be Takara companies PrimeSTAR HS (Premix) hi-fi systems (Cat：R040).Amplification system
For：PrimeSTAR HS (Premix, 2X) 25ul, 1492R 0.5ul, 27F8F 0.5ul, template DNA 1ul, deionized water
23ul.Amplification condition is：98 DEG C of 10s, 60 DEG C of 5s, 72 DEG C of 2min, 30 circulations.PCR product is through gel electrophoresis, and purifying is simultaneously
Hua Da Gene science limited company is sent to be sequenced after recycling.Shown in phylogenetic tree Figure of description 2.
Embodiment 3 utilizes K.rhaeticus TJPU03 productions BC
By the K.rhaeticus TJPU03 after activation, with 10% inoculum concentration, it is inoculated into sterilized equipped with 100mL hairs
In the 250mL wide-mouth bottles of ferment culture medium, fermentation medium composition is：Glucose 35g/L, peptone 5.5g/L, yeast extract 7g/L,
Disodium hydrogen phosphate 4g/L, citric acid 1.4g/L, distilled water.PH to 3.5 is adjusted, after 8 layers of gauze sealing, in biochemical cultivation case 30
DEG C quiescent culture 15d.Treat after fermentation, the BC films (K-BC) of generation float on fermentation medium liquid level.BC films are taken out, with steaming
Distilled water oscillation cleaning for several times after, boil 30min in 80 DEG C of water-baths with 15% NaOH solution, remove the unnecessary culture medium in product
And thalline, use distilled water instead again afterwards and continue 80 DEG C of water-baths and boil and repeatedly, treat that BC films are cleaned to homogeneous milk white gel shape completely,
Taking-up is drained away the water with filter paper, then BC films are centrifuged 20min with 8000r/min rotating speeds, is then weighed and is obtained weight in wet base；BC films are put
Drying to constant weight in 65 DEG C of baking ovens, weighs to obtain its dry weight.BC films obtained by K.rhaeticus TJPU03 strain fermentations are average wet
Weight is 632.15g/L culture mediums, and wet film average thickness is 1.5cm, average dry weight 8.28g/L.
Embodiment 4 utilizes gluconate pyracetobacillus (Gluconacetobacter xylinum) production BC
Using the identical method of embodiment 3, BC is produced with G.xylinum, gained BC films (G-BC) weight in wet base that is averaged is
242.62g/L culture mediums, wet film average thickness are 1.0cm, average dry weight 3.64g/L.Pass through field emission scanning electron microscope
(FESEM) K-BC and G-BC film microscopic appearances are observed, as shown in Figure of description 3, it is known that K-BC films are than G-BC film
It is more loose.K-BC and G-BC film chemical compositions are analyzed by infrared spectrum, as shown in Figure of description 4, it is known that two
The chemical composition of person is identical, and the tunning for illustrating K.rhaeticus TJPU03 is BC really.
Claims (2)
1. one plant of bacteria cellulose Producing Strain, it is characterised in that strain was named Komagataeibacter rhaeticus
TJPU003。
2. bacterial strain according to claim 1, produces the Optimal technique process of bacteria cellulose, it is characterised in that best medium
Form and be：Glucose 35g/L, peptone 5.5g/L, yeast extract 7g/L, disodium hydrogen phosphate 4g/L, citric acid 1.4g/L；Optimal hair
Ferment condition is：10% inoculum concentration, 3.5,32 DEG C of pH；Optimum carbon source is dextrose and saccharose, and optimum nitrogen source is peptone and ferment
Female cream compound nitrogen source.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484471A (en) * | 2019-08-28 | 2019-11-22 | 北京爱发科技有限公司 | The method that one plant height produces the acidproof bacterial strain of bacteria cellulose and its produces bacteria cellulose |
| CN111684057A (en) * | 2018-09-17 | 2020-09-18 | 爱思开百朗德株式会社 | Bacterial cellulose high-yield strain and preparation method of cellulose tablet by using same |
| WO2021130299A1 (en) * | 2019-12-23 | 2021-07-01 | Kim Köster & Cord Henning Labuhn Gbr | Method for producing a nonwoven from bacterial nanocellulose |
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| CN105713860A (en) * | 2016-03-11 | 2016-06-29 | 江南大学 | Acid-resistant bacterial cellulose high-yielding strain and method for preparing edible bacterial cellulose with same |
| CN106265474A (en) * | 2016-08-31 | 2017-01-04 | 百朗德生物化学(海门)有限公司 | A kind of method utilizing microbial strains fermenting and producing facial film |
| US20170166908A1 (en) * | 2015-12-09 | 2017-06-15 | Samsung Electronics Co., Ltd. | Vector replicable in e. coli and cell of genus komagataeibacter, cell including the same, and method of using the same |
| CN107299073A (en) * | 2017-08-25 | 2017-10-27 | 南京工业大学 | Acetobacter xylinum gene recombinant strain and construction method and application thereof |
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2017
- 2017-11-27 CN CN201711220732.1A patent/CN107937307A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170166908A1 (en) * | 2015-12-09 | 2017-06-15 | Samsung Electronics Co., Ltd. | Vector replicable in e. coli and cell of genus komagataeibacter, cell including the same, and method of using the same |
| CN105713860A (en) * | 2016-03-11 | 2016-06-29 | 江南大学 | Acid-resistant bacterial cellulose high-yielding strain and method for preparing edible bacterial cellulose with same |
| CN106265474A (en) * | 2016-08-31 | 2017-01-04 | 百朗德生物化学(海门)有限公司 | A kind of method utilizing microbial strains fermenting and producing facial film |
| CN107299073A (en) * | 2017-08-25 | 2017-10-27 | 南京工业大学 | Acetobacter xylinum gene recombinant strain and construction method and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111684057A (en) * | 2018-09-17 | 2020-09-18 | 爱思开百朗德株式会社 | Bacterial cellulose high-yield strain and preparation method of cellulose tablet by using same |
| CN111684057B (en) * | 2018-09-17 | 2023-06-30 | 株式会社现代百朗德 | Bacterial cellulose high-yield bacterial strain and preparation method of cellulose tablet by using bacterial cellulose high-yield bacterial strain |
| CN110484471A (en) * | 2019-08-28 | 2019-11-22 | 北京爱发科技有限公司 | The method that one plant height produces the acidproof bacterial strain of bacteria cellulose and its produces bacteria cellulose |
| WO2021130299A1 (en) * | 2019-12-23 | 2021-07-01 | Kim Köster & Cord Henning Labuhn Gbr | Method for producing a nonwoven from bacterial nanocellulose |
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