CN105420160A - Curdlan producing strain and application thereof - Google Patents
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- CN105420160A CN105420160A CN201510960634.6A CN201510960634A CN105420160A CN 105420160 A CN105420160 A CN 105420160A CN 201510960634 A CN201510960634 A CN 201510960634A CN 105420160 A CN105420160 A CN 105420160A
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Abstract
The invention relates to a curdlan producing strain and application thereof. The strain is Phizobium sp. DH-6, the preservation number is CGMCC No.11547 and the nucleotide sequence is as shown by SEQ ID No.1. Curdlan is prepared by using the Phizobium sp. DH-6 through seed culture and liquid deep fermentation. The curdlan producing strain provided by the invention has the characteristics the gel strength of the produced curdlan is high, the molecular weight is great, the yield is high and the like.
Description
Technical field
The invention belongs to polysaccharide producing bacterial strain and Application Areas thereof, particularly a kind of Curdlan producing bacterial strain and application thereof.
Background technology
Curdlan is a kind of exocellular polysaccharide produced by microorganism, and the dextran be formed by connecting by β-1,3 glycosidic link by glucosyl residue, has the characteristic by thermosetting gel.The glue that Curdlan is formed not only has very strong thermostability, and there is freeze-thaw stability, tolerance many wheels freeze thawing treatment still can keep stable structure, can be used as food shaping agent, thickening material, edible film etc., also can be used as the quality such as visco-elasticity and palatability that functional substance improves food.As a kind of safe polysaccharide, Curdlan be can be used for foodstuff additive by U.S. food and drug administration (FDA) approval, has broad application prospects in foodstuffs industry.Curdlan also has important pharmacological properties, and Sulfonated Curdlan has the activity of anti HIV-1 virus, has huge market potential in pharmaceutical industry.
Current Curdlan suitability for industrialized production succeeds in Japan, and China's related production and research are still in the starting stage.Though China produces in bacterial classification and zymotechnique at Curdlan and has made some progress in recent years, improve the output of Curdlan, the Curdlan gel-strength of producing is still lower, and product performance are poor, commercially lack competitiveness.
Curdlan produces the bacterium that bacterial classification mainly contains the genus such as Agrobacterium, rhizobium and cellulomonas cartae Pseudomonas, and producing strains kind is less.Non-correlation between the amount of bacterial classification heat production curdlan and the gel-strength of polysaccharide.Some bacterial classification Curdlan output is high, but gelation is poor, and gel-strength is low; Some bacterial classification heat production curdlan gel-strengths are high, but yield poorly.Current research is focused on improving output by strain improvement and process optimization, and seldom attached gel characteristic carries out breeding species and Optimization Technology, makes the performance of product not meet application demand.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Curdlan producing bacterial strain and application thereof, and the Curdlan molecular weight that this bacterial strain produces is large, gel-strength is high, output is high, has industrial applications prospect.
A kind of Curdlan producing bacterial strain of the present invention, described bacterial strain is root nodule bacterium (Rhizobiumsp.) DH-6, and deposit number is CGMCCNo.11547, and nucleotide sequence is as shown in SEQIDNO.1.
The application of a kind of Curdlan producing bacterial strain of the present invention, comprising:
Be starting strain by root nodule bacterium (Rhizobiumsp.) DH-6, through seed culture and liquid submerged fermentation, obtain fermented liquid, then separating-purifying, dry, obtained Curdlan;
Wherein seed culture is: cultivate in seed culture medium, and temperature is 28-30 DEG C, shaking speed 180-200rpm, incubation time 16-20h; Liquid submerged fermentation is: ferment in the fermentation medium, and temperature is 28 DEG C-30 DEG C, inoculum size 3%-10% (v/v), shaking speed 200-240rpm, incubation time 3d-5d.
Described seed culture medium is: sucrose 20g/L, (NH)
2hPO
43g/L, KH
2pO
41g/L, MgSO
4.7H
2o0.5g/L, yeast extract 1g/L, CaCO
33g/L, pH=6.8-7.2.
Described fermention medium is: carbon source 60g/L, (NH)
2hPO
42.3g/L, KH
2pO
41g/L, MgSO
4.7H
2o0.5g/L, organic nitrogen source 1g/L, CaCO
33g/L, pH=6.8-7.2.
Described carbon source is that glucose is or/and sucrose.
Described organic nitrogen source is yeast extract and/or corn steep liquor.
Described separating-purifying is specially: get fermented liquid in the centrifugal 3-5min of 4500-5000rpm, removing supernatant liquor, add water again and be supplemented to original volume, then 1:1 adds 0.3-0.6MNaOH solution by volume, stir, static placement 0.5-1h, then in the centrifugal 5-10min of 9000-12000rpm, get supernatant liquor, add 0.3MHCl and be adjusted to final ph for neutral, filter with 80 order filter clothes again, the water of 1-2 times of volume is added again in the solid substance of gained, refilter, 95% ethanol of 2-3 times of volume is added in the solid substance finally filtration obtained, in the centrifugal 5-10min of 7000-9000rpm after stirring evenly.
Described drying temperature is 50-60 DEG C.
The molecular weight of described Curdlan is 2.25 × 10
6-2.50 × 10
6da, purity is 88-92% (w/w).
The gel-strength of 2% (w/v) Curdlan 95 DEG C heating 10min is 760-900g/cm
2.
Curdlan producing bacterial strain root nodule bacterium DH-6 (Rhizobiumsp.DH-6) of the present invention are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica) on October 28th, 2015, deposit number is CGMCCNo.11547, is that separation screening obtains from soil.
The screening method of Curdlan producing bacterial strain is that shake flask fermentation sieves again through flat-plate bacterial colony primary dcreening operation from soil, after measured Curdlan output and gel-strength and obtain.
Screening plate culture medium (g/L): 10g glucose, 5g yeast extract, 0.05g aniline blue, 20g agar, cultivates 2 ~ 3d for pH7.2.30 DEG C.
Select the bacterium colony screening dull and stereotyped blue, be inoculated in fermention medium, after 30 DEG C of cultivation 4d, get fermented liquid 95 DEG C heating 10 minutes, can observation form gel.To its curdlan of broth extraction that can form gel.
The measuring method of Curdlan output: get a certain amount of fermented liquid in the centrifugal 5min of 5000rpm, removing supernatant liquor, then add water and be supplemented to original volume.Then 1:1 adds 0.6MNaOH solution by volume, stirs, static placement 1 hour.Then in the centrifugal 10min of 9000rpm, get supernatant liquor, add 0.3MHCl and be neutralized to pH=7.Then filter with 80 order filter clothes, add the water of 2 times of volumes in gained solid substance again, refilter, add 95% ethanol of 3 times of volumes in the solid substance finally filtration obtained, in the centrifugal 10min of 9000rpm after stirring evenly, get precipitation and put into baking oven 60 DEG C of dryings.
Gel-strength measuring method: get 0.3g sample in 15mL water, with homogenizer, sample is uniformly dispersed in water, make suspension, suspension is transferred in the test tube of 18mm × 180mm, bleed 3min under vacuum conditions, then test tube is put into 95 DEG C of water and heat 10min, after taking-up, in cold water, cool 30min.From test tube, take out gel, get the gel of one section of 10mm from bottom 20mm and 30mm place, carry out measuring (probe: P5, diameter 0.5cm stainless steel piston-type right cylinder with Texture instrument.Probe translational speed: 250mm/min).
Root nodule bacterium of the present invention (Rhizobiumsp.) DH-6, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 28th, 2015, culture presevation number is CGMCCNo.11547.
beneficial effect
The invention provides a strain Curdlan producing strains, the Curdlan molecular weight that this bacterial strain produces is large, gel-strength is high, output is high, has industrial applications prospect.Adopt the present invention to prepare the method for Curdlan, Curdlan output reaches 23.9g/L, and molecular weight is 2.25 × 10
6-2.50 × 10
6da, the gel-strength of 2% Curdlan 95 DEG C heating 10min is 760 ~ 900g/cm
2.
Accompanying drawing explanation
Fig. 1 Curdlan
1hNMR collection of illustrative plates.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
The screening of bacterial strain
(1) take soil sample 10g, add 90mL sterilized water, a small amount of granulated glass sphere puts into Erlenmeyer flask, and shaking table vibration 10min, makes soil suspension liquid.
(2), after leaving standstill 10min, get 0.1mL and join in 0.9mL stroke-physiological saline solution, through serial dilutions to 10
-4, 10
-5, 10
-6three gradients, get 0.1mL respectively and are coated with screening flat board.
(3) flat board is placed in after cultivation 2-3d is inverted by 30 DEG C of incubators and observes.
(4) choosing colony becomes blue and that color is darker bacterium colony, at flat lining out, and the preservation of picking list bacterium colony.Be inoculated into further in fermention medium and carry out shake flask fermentation by flat board screening the bacterial strain obtained and sieve again, culture condition is 30 DEG C, 240rpm, 4d.
(5) get 5ml fermented liquid in 95 DEG C of heating in water bath 10min, can observation form gel.To its curdlan of broth extraction that can form gel, measure hot gel content and gel-strength.Finally obtain that a strain output is high, gel-strength be large, the bacterial strain DH-6 of stable performance.
Screening plate culture medium (g/L): 10g glucose, 5g yeast extract, 0.05g aniline blue, 20g agar, pH7.2. fermention medium (g/L): sucrose 60, (NH)
2hPO
42.3, KH
2pO
41, MgSO
4.7H
2o0.5, yeast extract 1, CaCO
33, pH=7.0.
Embodiment 2
The qualification of isolated strains DH-6
(1) identification of morphology
After LB slat chain conveyor 1d, bacterium colony is circular, and smooth surface is moistening, neat in edge, and bacterium colony is slightly protruding, canescence.Microscope hypothallus thin rod shape, Gram-negative.
(2) Physiology and biochemistry qualification
The physiological and biochemical property of bacterial strain DH-6 is in table 1.
Table 1: bacterial strain DH-6 physiological and biochemical property
Wherein, "+" expression is positive, and "-" represents the reaction that is negative.
(3) 16SrDNA Sequence Identification
Extract the total genomic dna of bacterial strain DH-6, and as template, the 16SrDNA sequence of this bacterial strain that increases, its primer sequence is SEQIDNo.1, F:5'-CAGAGTTTGATCCTGGCT-3', SEQIDNo.2, R:5'-AGGAGGTGATCCAGCCGCA-3', SEQIDNo.3.16SrDNA sequence comparison result display in ncbi database of this bacterial classification, this bacterial strain and Rhizobiumsp. are the most close, and this bacterial strain is rhizobium (Rhizobiumsp.) bacterium.
In conjunction with the form of this bacterial strain, physiological and biochemical property and 16SrDNA sequence, finally judge the one (Rhizobiumsp.) that bacterial strain DH-6 should belong to rhizobium, called after Rhizobiumsp.DH-6.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 28th, 2015, and culture presevation number is CGMCCNo.11547.
Embodiment 3
From inclined-plane, scraping one ring bacterial classification (root nodule bacterium (Rhizobiumsp.) DH-6), is inoculated in and is equipped with in the 250ml triangular flask of 30ml seed culture medium, 30 DEG C, 180r/min, and 20h cultivated by shaking table, obtains seed liquor.Get seed liquor with 8% inoculum size access be equipped with in the 500ml triangular flask of 100ml fermention medium, in 30 DEG C, 240r/min shaking table cultivate 4d.
Seed culture medium (g/L): sucrose 20, (NH)
2hPO
43, KH
2pO
41, MgSO
4.7H
2o0.5, yeast extract 1, CaCO
33, pH=7.0.
Fermention medium (g/L): sucrose 60, (NH)
2hPO
42.3, KH
2pO
41, MgSO
4.7H
2o0.5, corn steep liquor 1, CaCO
33, pH=7.0.
Get fermented liquid in the centrifugal 5min of 5000rpm, removing supernatant liquor, then add water and be supplemented to original volume.Then add 0.6MNaOH solution at 1: 1 by volume, stir, static placement 1 hour.Then in the centrifugal 10min of 10000rpm, get supernatant liquor, add 0.3MHCl and be adjusted to final ph for neutral.Then filter with 80 order filter clothes, add the water of 2 times of volumes in the solid substance of gained again, refilter, in the solid substance finally filtration obtained, add 95% ethanol of 3 times of volumes, in the centrifugal 10min of 9000rpm after stirring evenly, get solid substance and put into baking oven 60 DEG C of dryings.Curdlan output is 23.9g/L.
Embodiment 4
From inclined-plane, scraping one ring bacterial classification (root nodule bacterium (Rhizobiumsp.) DH-6), is inoculated in and is equipped with in the 250ml triangular flask of 30ml seed culture medium, 30 DEG C, 200r/min, and 16h cultivated by shaking table, obtains seed liquor.Get seed liquor with 5% inoculum size access be equipped with in the 500ml triangular flask of 100ml fermention medium, in 28 DEG C, 220r/min shaking table cultivate 5d.
Seed culture medium (g/L): sucrose 20, (NH)
2hPO
43, KH
2pO
41, MgSO
4.7H
2o0.5, yeast extract 1, CaCO
33, pH=7.0.
Fermention medium (g/L): glucose 60, (NH)
2hPO
42.3, KH
2pO
41, MgSO
4.7H
2o0.5, yeast extract 1, CaCO
33, pH=7.0.
Get fermented liquid in the centrifugal 5min of 5000rpm, removing supernatant liquor, then add water and be supplemented to original volume.Then add 0.6MNaOH solution at 1: 1 by volume, stir, static placement 1 hour.Then in the centrifugal 5min of 12000rpm, get supernatant liquor, add 0.3MHCl and be adjusted to final ph for neutral.Then filter with 80 order filter clothes, add the water of 2 times of volumes in the solid substance of gained again, refilter, in the solid substance finally filtration obtained, add 95% ethanol of 3 times of volumes, in the centrifugal 5min of 9000rpm after stirring evenly, get solid substance and put into baking oven 60 DEG C of dryings.Curdlan output is 22.2g/L.
Embodiment 5
From inclined-plane, scraping one ring bacterial classification (root nodule bacterium (Rhizobiumsp.) DH-6), is inoculated in and is equipped with in the 250ml triangular flask of 30ml seed culture medium, 30 DEG C, 190r/min, and 18h cultivated by shaking table, obtains seed liquor.Get seed liquor with 3% inoculum size access be equipped with in the 500ml triangular flask of 100ml fermention medium, in 30 DEG C, 240r/min shaking table cultivate 3d.
Seed culture medium (g/L): sucrose 20, (NH)
2hPO
43, KH
2pO
41, MgSO
4.7H
2o0.5, yeast extract 1, CaCO
33, pH=7.0.
Fermention medium (g/L): glucose 60, (NH)
2hPO
42.3, KH
2pO
41, MgSO
4.7H
2o0.5, yeast extract 0.5, corn steep liquor 0.5, CaCO
33, pH=7.0.
Get fermented liquid in the centrifugal 5min of 5000rpm, removing supernatant liquor, then add water and be supplemented to original volume.Then add 0.6MNaOH solution at 1: 1 by volume, stir, static placement 1 hour.Then in the centrifugal 10min of 9000rpm, get supernatant liquor, add 0.3MHCl and be adjusted to final ph for neutral.Then filter with 80 order filter clothes, add the water of 2 times of volumes in the solid substance of gained again, refilter, in the solid substance finally filtration obtained, add 95% ethanol of 3 times of volumes, in the centrifugal 10min of 7000rpm after stirring evenly, get solid substance and put into baking oven 60 DEG C of dryings.Curdlan output is 13.2g/L.
Embodiment 6
From inclined-plane, scraping one ring bacterial classification (root nodule bacterium (Rhizobiumsp.) DH-6), is inoculated in and is equipped with in the 250ml triangular flask of 30ml seed culture medium, 30 DEG C, 200r/min, and 20h cultivated by shaking table, obtains seed liquor.Get seed liquor with 10% inoculum size access be equipped with in the 500ml triangular flask of 100ml fermention medium, in 30 DEG C, 220r/min shaking table cultivate 4d.
Seed culture medium (g/L): sucrose 20, (NH)
2hPO
43, KH
2pO
41, MgSO
4.7H
2o0.5, yeast extract 1, CaCO
33, pH=7.0.
Fermention medium (g/L): glucose 30, sucrose 30, (NH)
2hPO
42.3, KH
2pO
41, MgSO
4.7H
2o0.5, yeast extract 1, CaCO
33, pH=7.0.
Get fermented liquid in the centrifugal 5min of 5000rpm, removing supernatant liquor, then add water and be supplemented to original volume.Then add 0.6MNaOH solution at 1: 1 by volume, stir, static placement 1 hour.Then in the centrifugal 10min of 9000rpm, get supernatant liquor, add 0.3MHCl and be adjusted to final ph for neutral.Then filter with 80 order filter clothes, add the water of 2 times of volumes in the solid substance of gained again, refilter, in the solid substance finally filtration obtained, add 95% ethanol of 3 times of volumes, in the centrifugal 10min of 8000rpm after stirring evenly, get solid substance and put into baking oven 60 DEG C of dryings.Curdlan output is 21.6g/L.
Embodiment 7
The character of Curdlan
The purity measuring gained Curdlan with phend-sulphuric acid is 88 ~ 92%, and the molecular weight measuring this polysaccharide with dark type viscometer is 2.25 × 10
6~ 2.50 × 10
6da.Curdlan
1fig. 1 is shown in by HNMR collection of illustrative plates, shows that this polysaccharide is polymerized by the connection of β-1,3 glycosidic link by glucose.The gel-strength of 2% (w/v) Curdlan is 760 ~ 900g/cm
2.The gel TPA analysis in table 2 that 6% (w/v) Curdlan is formed, compared with commercially available inward, a little less than commercial samples on the gel hardness that the Curdlan that this bacterial strain produces is formed, elasticity and cohesion are a little more than commercial samples, and both chewiness are close.
The TPA of table 26% gel analyzes
Claims (10)
1. a Curdlan producing bacterial strain, is characterized in that: described bacterial strain is root nodule bacterium (Rhizobiumsp.) DH-6, and deposit number is CGMCCNo.11547, and nucleotide sequence is as shown in SEQIDNO.1.
2. an application for Curdlan producing bacterial strain as claimed in claim 1, comprising:
Root nodule bacterium (Rhizobiumsp.) DH-6, through seed culture and liquid submerged fermentation, is obtained fermented liquid, then separating-purifying, dry, obtained Curdlan;
Wherein seed culture is: cultivate in seed culture medium, and temperature is 28-30 DEG C, shaking speed 180-200rpm, incubation time 16-20h; Liquid submerged fermentation is: ferment in the fermentation medium, and temperature is 28 DEG C-30 DEG C, inoculum size 3%-10% (v/v), shaking speed 200-240rpm, incubation time 3d-5d.
3. the application of a kind of Curdlan producing bacterial strain according to claim 2, is characterized in that: described seed culture medium is: sucrose 20g/L, (NH)
2hPO
43g/L, KH
2pO
41g/L, MgSO
47H
2o0.5g/L, yeast extract 1g/L, CaCO
33g/L, pH=6.8-7.2.
4. the application of a kind of Curdlan producing bacterial strain according to claim 2, is characterized in that: described fermention medium is: carbon source 60g/L, (NH)
2hPO
42.3g/L, KH
2pO
41g/L, MgSO
47H
2o0.5g/L, organic nitrogen source 1g/L, CaCO
33g/L, pH=6.8-7.2.
5. the application of a kind of Curdlan producing bacterial strain according to claim 4, is characterized in that: described carbon source is that glucose is or/and sucrose.
6. the application of a kind of Curdlan producing bacterial strain according to claim 4, is characterized in that: described organic nitrogen source is yeast extract and/or corn steep liquor.
7. the application of a kind of Curdlan producing bacterial strain according to claim 2, it is characterized in that: described separating-purifying is specially: get fermented liquid in the centrifugal 3-5min of 4500-5000rpm, removing supernatant liquor, add water again and be supplemented to original volume, then 1:1 adds 0.6MNaOH solution by volume, stir, static placement 0.5-1h, then in the centrifugal 5-10min of 9000-12000rpm, get supernatant liquor, add 0.3MHCl and be adjusted to final ph for neutral, filter with 80 order filter clothes again, the water of 1-2 times of volume is added again in the solid substance of gained, refilter, 95% ethanol of 2-3 times of volume is added in the solid substance finally filtration obtained, in the centrifugal 5-10min of 7000-9000rpm after stirring evenly.
8. the application of a kind of Curdlan producing bacterial strain according to claim 2, is characterized in that: described drying temperature is 50-60 DEG C.
9. the application of a kind of Curdlan producing bacterial strain according to claim 2, is characterized in that: the molecular weight of described Curdlan is 2.25 × 10
6-2.50 × 10
6da, purity is 88-92% (w/w).
10. the application of a kind of Curdlan producing bacterial strain according to claim 2, is characterized in that: the gel-strength of 2% (w/v) Curdlan 95 DEG C heating 10min is 760-900g/cm
2.
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| CN106434495A (en) * | 2016-12-02 | 2017-02-22 | 张星昊 | Rhizobium pusense and method for preparing beta-1,3 glucan fermenting liquid by rhizobium pusense |
| 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 |
| CN113943761A (en) * | 2021-12-22 | 2022-01-18 | 山东国力生物科技有限公司 | Preparation method of micromolecular beta-1, 3-glucan |
| CN113957024A (en) * | 2021-12-22 | 2022-01-21 | 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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| CN106434495B (en) * | 2016-12-02 | 2019-05-17 | 张星昊 | A kind of Bodhisattva rhizobium and its β -1 is produced, the method for 3 dextran fermentation liquid |
| 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 |
| CN113943761A (en) * | 2021-12-22 | 2022-01-18 | 山东国力生物科技有限公司 | Preparation method of micromolecular beta-1, 3-glucan |
| CN113957024A (en) * | 2021-12-22 | 2022-01-21 | 山东国力生物科技有限公司 | Rhizobium GL-1803 and application thereof in preparation of insoluble beta-glucan |
| 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 |
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