CN105420160B - A kind of Curdlan producing bacterial strain and its application - Google Patents
A kind of Curdlan producing bacterial strain and its application Download PDFInfo
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Abstract
The present invention relates to a kind of Curdlan producing bacterial strain and its application, bacterial strain is rhizobium (Rhizobium sp.) DH-6, and deposit number is CGMCC No.11547, and nucleotide sequence is as shown in SEQ ID NO.1.By rhizobium (Rhizobium sp.) DH-6 through seed culture and liquid deep layer fermenting, Curdlan is made.Curdlan producing bacterial strain provided by the invention has the characteristics such as high, the big, high yield of molecular weight of produced curdlan gel strength.
Description
Technical field
The invention belongs to polysaccharide producing bacterial strain and its application field, in particular to a kind of Curdlan producing bacterial strain and its
Using.
Background technique
Curdlan is a kind of exocellular polysaccharide generated by microorganism, and by glucose residue by β -1,3 glycosidic bonds connect
Glucan made of connecing has the characteristic by thermosetting gel.The glue that Curdlan is formed not only has very strong thermostabilization
Property, and there is freeze-thaw stability, be resistant to take turns freeze thawing treatment more and still keep stable structure, can be used as food solid agent, increasing
Thick dose, edible film etc., also can be used as functional materials improves the qualities such as viscoplasticity and the palatability of food.As a kind of safety
Polysaccharide, Curdlan by United States Food and Drag Administration (FDA) approval can be used for food additives, in food industry
It has broad application prospects.Curdlan also has important pharmacological properties, and sulfonated Curdlan has AntiHIV1 RT activity
The activity of virus has huge market potential in pharmaceuticals industry.
Curdlan industrialized production has succeeded in Japan at present, and China's associated production and research are still in starting
Stage.Though China has made some progress in terms of Curdlan generates strain and zymotechnique in recent years, heat setting is improved
The yield of glue polysaccharide, but Curdlan gel strength produced is still lower, and properties of product are poor, lack competition on the market
Power.
Curdlan, which generates strain, mainly the thin of the categories such as Agrobacterium, rhizobium and cellulomonas cartae Pseudomonas
Bacterium, producing strains type are less.Non-correlation between the amount of strain heat production curdlan and the gel strength of polysaccharide.Some strain heat settings
Glue polysaccharide yield is high, but gelation is poor, and gel strength is low；Some strain heat production curdlan gel strengths are high, but low output.Mesh
Preceding research is focused on improving yield by strain improvement and process optimization, and seldom attached gel characteristic carries out breeding species and excellent
Chemical industry skill, so that the performance of product is not able to satisfy application demand.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of Curdlan producing bacterial strain and its application, which is produced
Raw Curdlan molecular weight is big, gel strength is high, yield is high, has industrial applications prospect.
A kind of Curdlan producing bacterial strain of the invention, the bacterial strain are rhizobium (Rhizobium sp.) DH-6, are protected
Hiding number is CGMCC No.11547, and nucleotide sequence is as shown in SEQ ID NO.1.
A kind of application of Curdlan producing bacterial strain of the invention, comprising:
Rhizobium (Rhizobium sp.) DH-6 is sent out for starting strain through seed culture and liquid deep layer fermenting
Zymotic fluid, then separating-purifying, dry, and Curdlan is made；
Wherein seed culture are as follows: cultivated in seed culture medium, temperature is 28-30 DEG C, shaking speed 180-200rpm, training
Support time 16-20h；Liquid deep layer fermenting are as follows: ferment in the fermentation medium, temperature is 28 DEG C -30 DEG C, inoculum concentration 3%-10%
(v/v), shaking speed 200-240rpm, incubation time 3d-5d.
The seed culture medium are as follows: sucrose 20g/L, (NH)2HPO4 3g/L、KH2PO4 1g/L、MgSO4.7H2O 0.5g/
L, yeast extract 1g/L, CaCO33g/L, pH=6.8-7.2.
The fermentation medium are as follows: carbon source 60g/L, (NH)2HPO4 2.3g/L、KH2PO4 1g/L、MgSO4.7H2O
0.5g/L, organic nitrogen source 1g/L, CaCO33g/L, pH=6.8-7.2.
The carbon source is glucose or/and sucrose.
The organic nitrogen source is yeast extract and/or corn pulp.
The separating-purifying specifically: take fermentation liquid to be centrifuged 3-5min in 4500-5000rpm, remove supernatant, add water
It is supplemented to original volume, then 0.3-0.6M NaOH solution is added in 1:1 by volume, stirs evenly, static placement 0.5-1h, so
It is centrifuged 5-10min in 9000-12000rpm afterwards, takes supernatant, 0.3M HCl is added and is adjusted to final ph as neutrality, then with 80 mesh
Filter cloth is filtered, and the water of 1-2 times of volume is added in resulting solid content, is refiltered, the solid content being finally obtained by filtration
Middle 95% ethyl alcohol that 2-3 times of volume is added, is centrifuged 5-10min in 7000-9000rpm after stirring evenly.
The drying temperature is 50-60 DEG C.
The molecular weight of the Curdlan is 2.25 × 106-2.50×106Da, purity are 88-92% (w/w).
The gel strength that 2% 95 DEG C of (w/v) Curdlan heats 10min is 760-900g/cm2。
Curdlan producing bacterial strain rhizobium DH-6 (Rhizobium sp.DH-6) of the invention was October 28 in 2015
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: BeiChen West Road, Chaoyang District, BeiJing City 1
Number No. 3 Institute of Microorganism, Academia Sinica, institute), it is that separation screening obtains from soil that deposit number, which is CGMCC No.11547,
It arrives.
The screening technique of Curdlan producing bacterial strain is from soil through flat-plate bacterial colony primary dcreening operation, shake flask fermentation secondary screening, warp
It measures Curdlan yield and gel strength and obtains.
Screening flat board culture medium (g/L): 10g glucose, 5g yeast extract, 0.05g aniline blue, 20g agar,
PH7.2.30 DEG C of 2~3d of culture.
The bacterium colony for selecting screening flat board blue, is inoculated in fermentation medium, after 30 DEG C of culture 4d, takes 95 DEG C of fermentation liquid
Heating 10 minutes, can observation form gel.To its curdlan of the broth extraction of gel can be formed.
The measuring method of Curdlan yield: taking a certain amount of fermentation liquid to be centrifuged 5min in 5000rpm, removes supernatant,
It adds water and is supplemented to original volume.Then 0.6M NaOH solution is added in 1:1 by volume, stirs evenly, static to place 1 hour.So
It is centrifuged 10min in 9000rpm afterwards, takes supernatant, 0.3M HCl is added and is neutralized to pH=7.Then it is filtered with 80 mesh filter clothes,
The water that 2 times of volumes are added in gained solid content, refilters, and 3 times of volumes will finally be added in the solid content being obtained by filtration
95% ethyl alcohol is centrifuged 10min in 9000rpm after stirring evenly, precipitating is taken to be put into 60 DEG C of dryings in baking oven.
Gel strength measuring method: taking 0.3g sample in 15mL water, disperses sample in water with high-speed mixer
It is even, suspension is made, suspension is transferred in the test tube of 18mm × 180mm, is evacuated 3min under vacuum conditions, it then will examination
Pipe is put into 95 DEG C of water and heats 10min, cools down 30min after taking-up in cold water.Gel is taken out from test tube, is taken from bottom 20mm
With the gel of one section of 10mm at 30mm, (probe: P5, diameter 0.5cm stainless steel piston type cylinder is measured with Texture instrument
Body.Probe movement speed: 250mm/min).
Rhizobium (Rhizobium sp.) DH-6 of the invention, is preserved in China Microbiological on October 28th, 2015
Culture presevation administration committee common micro-organisms center, culture presevation number are CGMCC No.11547.
Beneficial effect
The present invention provides one plant of Curdlan producing strains, the Curdlan molecular weight which generates is big, gel
Intensity is high, yield is high, has industrial applications prospect.The method for preparing Curdlan using the present invention, Curdlan produce
Amount reaches 23.9g/L, and molecular weight is 2.25 × 106-2.50×106Da, 2% 95 DEG C of Curdlan heat the gel strength of 10min
For 760~900g/cm2。
Detailed description of the invention
Fig. 1 Curdlan1H NMR spectra.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
The screening of bacterial strain
(1) soil sample 10g is weighed, 90mL sterile water is added, a small amount of bead is put into conical flask, and shaking table vibrates 10min, system
At soil suspension.
(2) after standing 10min, 0.1mL is taken to be added in 0.9mL sterile saline, through serial dilutions to 10-4、
10-5、10-6Three gradients take 0.1mL to apply screening flat board respectively.
(3) it is observed after plate being placed in 30 DEG C of culture carton upside down culture 2-3d.
(4) choosing colony becomes bacterium colony blue and that color is deeper, in flat lining out, picking single colonie preservation.It will be on plate
Screen obtained bacterial strain and be further inoculated into progress shake flask fermentation secondary screening in fermentation medium, condition of culture is 30 DEG C, 240rpm,
4d。
(5) take 5ml fermentation liquid in 95 DEG C of heating water bath 10min, can observation form gel.Fermentation to gel can be formed
Liquid extracts its curdlan, measures thermal gels content and gel strength.Finally obtain that one plant of yield height, gel strength be big, performance
Stable bacterial strain DH-6.
Screening flat board culture medium (g/L): 10g glucose, 5g yeast extract, 0.05g aniline blue, 20g agar, pH7.2.
Fermentation medium (g/L): sucrose 60, (NH)2HPO4 2.3、KH2PO4 1、MgSO4.7H2O 0.5, yeast extract 1, CaCO3
3, pH=7.0.
Embodiment 2
The identification of isolated strains DH-6
(1) Morphological Identification
After LB plate culture 1d, bacterium colony is round, and surface is smooth wet, and neat in edge, bacterium colony is slightly raised, canescence.
Microscope hypothallus thin rod shape, Gram-negative.
(2) Physiology and biochemistry is identified
The physiological and biochemical property of bacterial strain DH-6 is shown in Table 1.
Table 1: bacterial strain DH-6 physiological and biochemical property
Wherein, "+" expression is positive, and "-" indicates negative.
(3) 16SrDNA Sequence Identification
The total genomic dna of bacterial strain DH-6 is extracted, and as template, expands the 16S rDNA sequence of the bacterial strain, draws
Object sequence is SEQ ID No.1, F:5'-CAGAGTTTGATCCTGGCT-3', SEQ ID No.2, R:5'-
AGGAGGTGATCCAGCCGCA-3', SEQ ID No.3.The 16SrDNA sequence of strain comparison result in ncbi database
It has been shown that, the bacterial strain and Rhizobium sp. are closest, which is rhizobium (Rhizobium sp.) bacterium.
It is final to determine that bacterial strain DH-6 should belong to root in conjunction with the form of the bacterial strain, physiological and biochemical property and 16S rDNA sequence
One kind (Rhizobium sp.) of tumor Pseudomonas, is named as Rhizobium sp.DH-6.This bacterial strain was protected on October 28th, 2015
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation number is CGMCC No.11547.
Embodiment 3
A ring strain (rhizobium (Rhizobium sp.) DH-6) is scraped from inclined-plane, is inoculated in and is trained equipped with 30ml seed
In the 250ml triangular flask for supporting base, 30 DEG C, 180r/min, shaking table culture 20h obtain seed liquor.Take seed liquor with 8% inoculation
Amount access is equipped in the 500ml triangular flask of 100ml fermentation medium, in 30 DEG C, 240r/min shaking table culture 4d.
Seed culture medium (g/L): sucrose 20, (NH)2HPO4 3、KH2PO4 1、MgSO4.7H2O 0.5, yeast extract 1,
CaCO33, pH=7.0.
Fermentation medium (g/L): sucrose 60, (NH)2HPO4 2.3、KH2PO4 1、MgSO4.7H2O 0.5, corn pulp 1,
CaCO33, pH=7.0.
It takes fermentation liquid to be centrifuged 5min in 5000rpm, removes supernatant, add water and be supplemented to original volume.Then by volume 1
: 1 is added 0.6M NaOH solution, stirs evenly, static to place 1 hour.Then it is centrifuged 10min in 10000rpm, takes supernatant,
0.3M HCl is added and is adjusted to final ph as neutrality.Then it is filtered with 80 mesh filter clothes, 2 times is added in resulting solid content
The water of volume, refilters, and 95% ethyl alcohol of 3 times of volumes will finally be added in the solid content being obtained by filtration, in 9000rpm after stirring evenly
It is centrifuged 10min, solid content is taken to be put into 60 DEG C of dryings in baking oven.Curdlan yield is 23.9g/L.
Embodiment 4
A ring strain (rhizobium (Rhizobium sp.) DH-6) is scraped from inclined-plane, is inoculated in and is trained equipped with 30ml seed
In the 250ml triangular flask for supporting base, 30 DEG C, 200r/min, shaking table culture 16h obtain seed liquor.Take seed liquor with 5% inoculation
Amount access is equipped in the 500ml triangular flask of 100ml fermentation medium, in 28 DEG C, 220r/min shaking table culture 5d.
Seed culture medium (g/L): sucrose 20, (NH)2HPO4 3、KH2PO4 1、MgSO4.7H2O 0.5, yeast extract 1,
CaCO33, pH=7.0.
Fermentation medium (g/L): glucose 60, (NH)2HPO4 2.3、KH2PO4 1、MgSO4.7H2O 0.5, yeast extract
Object 1, CaCO33, pH=7.0.
It takes fermentation liquid to be centrifuged 5min in 5000rpm, removes supernatant, add water and be supplemented to original volume.Then by volume 1
: 1 is added 0.6M NaOH solution, stirs evenly, static to place 1 hour.Then it is centrifuged 5min in 12000rpm, takes supernatant, adds
Enter 0.3M HCl and is adjusted to final ph as neutrality.Then it is filtered with 80 mesh filter clothes, 2 times of bodies is added in resulting solid content
Long-pending water, refilters, finally will in the solid content that be obtained by filtration be added 3 times of volumes 95% ethyl alcohol, after stirring evenly in 9000rpm from
Heart 5min takes solid content to be put into 60 DEG C of dryings in baking oven.Curdlan yield is 22.2g/L.
Embodiment 5
A ring strain (rhizobium (Rhizobium sp.) DH-6) is scraped from inclined-plane, is inoculated in and is trained equipped with 30ml seed
In the 250ml triangular flask for supporting base, 30 DEG C, 190r/min, shaking table culture 18h obtain seed liquor.Take seed liquor with 3% inoculation
Amount access is equipped in the 500ml triangular flask of 100ml fermentation medium, in 30 DEG C, 240r/min shaking table culture 3d.
Seed culture medium (g/L): sucrose 20, (NH)2HPO4 3、KH2PO4 1、MgSO4.7H2O 0.5, yeast extract 1,
CaCO33, pH=7.0.
Fermentation medium (g/L): glucose 60, (NH)2HPO4 2.3、KH2PO4 1、MgSO4.7H2O 0.5, yeast extract
Object 0.5, corn pulp 0.5, CaCO33, pH=7.0.
It takes fermentation liquid to be centrifuged 5min in 5000rpm, removes supernatant, add water and be supplemented to original volume.Then by volume 1
: 1 is added 0.6M NaOH solution, stirs evenly, static to place 1 hour.Then it is centrifuged 10min in 9000rpm, takes supernatant, adds
Enter 0.3M HCl and is adjusted to final ph as neutrality.Then it is filtered with 80 mesh filter clothes, 2 times of bodies is added in resulting solid content
Long-pending water, refilters, finally will in the solid content that be obtained by filtration be added 3 times of volumes 95% ethyl alcohol, after stirring evenly in 7000rpm from
Heart 10min takes solid content to be put into 60 DEG C of dryings in baking oven.Curdlan yield is 13.2g/L.
Embodiment 6
A ring strain (rhizobium (Rhizobium sp.) DH-6) is scraped from inclined-plane, is inoculated in and is trained equipped with 30ml seed
In the 250ml triangular flask for supporting base, 30 DEG C, 200r/min, shaking table culture 20h obtain seed liquor.Take seed liquor with 10% inoculation
Amount access is equipped in the 500ml triangular flask of 100ml fermentation medium, in 30 DEG C, 220r/min shaking table culture 4d.
Seed culture medium (g/L): sucrose 20, (NH)2HPO4 3、KH2PO4 1、MgSO4.7H2O 0.5, yeast extract 1,
CaCO33, pH=7.0.
Fermentation medium (g/L): glucose 30, sucrose 30, (NH)2HPO4 2.3、KH2PO4 1、MgSO4.7H2O 0.5、
Yeast extract 1, CaCO33, pH=7.0.
It takes fermentation liquid to be centrifuged 5min in 5000rpm, removes supernatant, add water and be supplemented to original volume.Then by volume 1
: 1 is added 0.6M NaOH solution, stirs evenly, static to place 1 hour.Then it is centrifuged 10min in 9000rpm, takes supernatant, adds
Enter 0.3M HCl and is adjusted to final ph as neutrality.Then it is filtered with 80 mesh filter clothes, 2 times of bodies is added in resulting solid content
Long-pending water, refilters, finally will in the solid content that be obtained by filtration be added 3 times of volumes 95% ethyl alcohol, after stirring evenly in 8000rpm from
Heart 10min takes solid content to be put into 60 DEG C of dryings in baking oven.Curdlan yield is 21.6g/L.
Embodiment 7
The property of Curdlan
It is 88~92% with the purity that phend-sulphuric acid measures gained Curdlan, it is more measures this with dark type viscometer
The molecular weight of sugar is 2.25 × 106~2.50 × 106Da.Curdlan1H NMR spectra is shown in Fig. 1, show the polysaccharide be by
Glucose is polymerized by β -1,3 glucosides key connection.The gel strength of 2% (w/v) Curdlan is 760~900g/
cm2.The gel TPA analysis that 6% (w/v) Curdlan is formed is shown in Table 2, compared with commercially available import, what which generated
Slightly below commercial samples, elasticity and cohesion are slightly above commercial samples, the nozzle of the two on the gel hardness that Curdlan is formed
Chewing property is close.
The TPA of 2 6% gel of table is analyzed
Claims (10)
1. a kind of Curdlan producing bacterial strain, it is characterised in that: the bacterial strain is rhizobium (Rhizobium sp.) DH-6,
Deposit number is CGMCC No.11547, and nucleotide sequence is as shown in SEQ ID NO.1；Wherein the Curdlan bacterial strain produces
The purity of Curdlan is 88-92% (w/w), and molecular weight is 2.25 × 106-2.50×106Da, 2% (w/v) thermal gels are more
The gel strength of 95 DEG C of heating 10min of sugar is 760-900g/cm2。
2. a kind of application of Curdlan producing bacterial strain as described in claim 1, comprising:
By rhizobium (Rhizobium sp.) DH-6 through seed culture and liquid deep layer fermenting, fermentation liquid is obtained, then separation mentions
It is pure, it is dry, Curdlan is made；
Wherein seed culture are as follows: cultivated in seed culture medium, temperature is 28-30 DEG C, shaking speed 180-200rpm, when culture
Between 16-20h；Liquid deep layer fermenting are as follows: ferment in the fermentation medium, temperature is 28 DEG C -30 DEG C, inoculum concentration 3%-10% (v/
V), shaking speed 200-240rpm, incubation time 3d-5d.
3. a kind of application of Curdlan producing bacterial strain according to claim 2, it is characterised in that: the seed culture
Base are as follows: sucrose 20g/L, (NH)2HPO4 3g/L、KH2PO4 1g/L、MgSO4.7H2O 0.5g/L, yeast extract 1g/L,
CaCO33g/L, pH=6.8-7.2.
4. a kind of application of Curdlan producing bacterial strain according to claim 2, it is characterised in that: the fermented and cultured
Base are as follows: carbon source 60g/L, (NH)2HPO4 2.3g/L、KH2PO4 1g/L、MgSO4.7H2O 0.5g/L, organic nitrogen source 1g/L,
CaCO33g/L, pH=6.8-7.2.
5. a kind of application of Curdlan producing bacterial strain according to claim 4, it is characterised in that: the carbon source is Portugal
Grape sugar or/and sucrose.
6. a kind of application of Curdlan producing bacterial strain according to claim 4, it is characterised in that: the organic nitrogen source
For yeast extract and/or corn pulp.
7. a kind of application of Curdlan producing bacterial strain according to claim 2, it is characterised in that: the separating-purifying
Specifically: it takes fermentation liquid to be centrifuged 3-5min in 4500-5000rpm, removes supernatant, add water and be supplemented to original volume, then press
0.6M NaOH solution is added in volume ratio 1:1, stirs evenly, static placement 0.5-1h, is then centrifuged 5- in 9000-12000rpm
10min takes supernatant, and 0.3M HCl is added and is adjusted to final ph as neutrality, then is filtered with 80 mesh filter clothes, resulting solid
The water that 1-2 times of volume is added in object, refilters, and 95% second of 2-3 times of volume will finally be added in the solid content being obtained by filtration
Alcohol is centrifuged 5-10min in 7000-9000rpm after stirring evenly.
8. a kind of application of Curdlan producing bacterial strain according to claim 2, it is characterised in that: the drying temperature
It is 50-60 DEG C.
9. a kind of application of Curdlan producing bacterial strain according to claim 2, it is characterised in that: the thermal gels are more
The molecular weight of sugar is 2.25 × 106-2.50×106Da, purity are 88-92% (w/w).
10. a kind of application of Curdlan producing bacterial strain according to claim 2, it is characterised in that: 2% (w/v) heat
The gel strength of 95 DEG C of curdlan heating 10min is 760-900g/cm2。
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| CN108752499A (en) * | 2018-05-08 | 2018-11-06 | 南京农业大学 | A method of separating-purifying can obtain right polysaccharide from microbial fermentation solution |
| CN113943761B (en) * | 2021-12-22 | 2022-03-22 | 山东国力生物科技有限公司 | Preparation method of micromolecular beta-1, 3-glucan |
| CN113957024B (en) * | 2021-12-22 | 2022-04-19 | 山东国力生物科技有限公司 | Rhizobium GL-1803 and application thereof in preparation of insoluble beta-glucan |
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| Extracellular polysaccharides of Rhizobium from the bangkok mircen collection;Footrakul P et al;《Journal of fermentation technology》;19810225;第59卷(第1期);摘要 * |
| 放射性土壤杆菌产可得然胶最佳条件的研究;赵双枝 等;《山东食品发酵》;20101020(第3期);28-32 * |
| 放射性土壤杆菌产可得然胶液体发酵工艺研究;张海龙 等;《中国食物与营养》;20110228;第17卷(第2期);42-45 * |
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