CN101487035A - Method for preparing dextran with Gluconobacter oxydans as strain - Google Patents
Method for preparing dextran with Gluconobacter oxydans as strain Download PDFInfo
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- CN101487035A CN101487035A CNA2009100466301A CN200910046630A CN101487035A CN 101487035 A CN101487035 A CN 101487035A CN A2009100466301 A CNA2009100466301 A CN A2009100466301A CN 200910046630 A CN200910046630 A CN 200910046630A CN 101487035 A CN101487035 A CN 101487035A
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Abstract
The invention discloses a dextran preparation method that uses Gluconabacterium axydans as a strain and comprises the following steps: (1) the Gluconabacterium axydans strain is cultivated on a slant culture medium; (2) culture fluid that is obtained by the step (1) is inoculated into a fermented culture medium and fermentation cultivated, thus obtaining fermented fluid that contains dextran dextrinase (DDase); and (3) the dextran dextrinase is extracted from the fermented fluid that is obtained in the step (2), and dextrine is transformed into the dextran through an enzyme transformation experiment. The dextran that is obtained by the dextran preparation method has lower viscosity and consequently has lower calorific value, and the dextran with low calorific value is called as an indigestible dextrine and is a water-soluble dietary fiber. With the characteristics of water holding capacity and viscosity, the indigestible dextrine can be used as a food additive with low calorific value and can be widely applied to the production in food industry.
Description
Technical field
The present invention relates to the preparation method of dextran, relate in particular to the method that a kind of enzyme process prepares dextran.
Background technology
Dextran (dextran), have another name called dextran, it is a kind of high molecular polymer that forms by the glucose unit dehydration, as a kind of extracellular products that generates by the enzyme catalysis of secretion property, it be find the earliest in the world and early as the microbial polysaccharide of main plasma substitute, its chemical structure mainly is by the α of glucose-1, the linear long molecular chain that 6-key head and the tail dehydrating condensation forms, the α-1 that in molecular structure, contains different ratios, 2, α-1,3 and α-1, the side chain of 4-glycosidic link, molecular formula is (C
6H
10O
5) n.
Dextran has been widely used in a plurality of fields such as medicine, food, stratographic analysis because of multiple advantages such as it is safe, nontoxic, good biocompatibilities.The dextran colloidal solution has expanding blood volume, keeps the effect of blood pressure, and first aid usefulness can be used as plasma substitute when confessing blood and traumatic shock.China's pharmacopeia 1990 editions is listed dextran and compound preparation thereof in.That uses clinically often has three kinds of specifications: macrodex, Dextran 40, Dextran 20.In foodstuffs industry, dextran as low calorie foodstuff additive, has been used for the production of multiple drink and food because of its retentiveness, toughness.Also but instead of part maltose is made the weighting material of soft heart chocolate or is added in the brewer's malt and go, and improves the foaminess of goods.In petroleum industry, dextran can be used as oil well sludge additive.It also can be applicable to fine chemistry industry, makes sephadex, has been widely used in the molecular sieve chromatography technology at present.In addition, in food, animal-feed and cosmetics production, received suitable concern as potential benefit source of students material.
At present, the production of dextran has two kinds of methods: microorganism direct fermentation and enzymic synthesis method, industrial production is mainly based on direct fermentation.
Abroad, the commercial production bacterial strain of dextran is leuconostoc mesentroides (Leuconostocmesenteroides) NRRL B-512F.
And through the leuconostoc mesentroides (Leuconostoc mesenteroides) the 0326th of Chinese Academy of Medical Sciences blood transfusion and Blood Research Institute seed selection, present domestic production dextran is used more bacterial classification.
During direct fermentation production dextran, the product molecular size is difficult to control, preparation process is pinched through ethanol and is washed, hydrochloric acid hydrolysis, ethanol division, separation, drying and other steps, the ethanol consumption is big, production environment is abominable, and because of its fermentation back thalline is difficult to separate with product, impurity such as the nitrogen of introducing in the production, chlorine are difficult to control, cause the dextran quality low, clinical side reaction is many.
Utilize the Production by Enzymes dextran, particularly immobilized enzyme method production dextran can overcome these deficiencies.Advantages such as utilizing enzyme engineering technology to produce dextran is that more Technology is paid close attention in comparatively advanced in the world at present, research, and this method has been compared with traditional zymotechnique and can be continuously produced, molecular weight product is controlled, even molecular weight distribution, foreign matter content are low.
Utilization induces isolated dextransucrase to prepare dextran, has improved quality product, has improved production environment.Therefore, obtain highly active dextranase for realize above-mentioned many application, to improve product quality significant.
But, the method for present enzyme method for preparing dextral glycoanhydride, because the problem in enzyme source, can't be from improving the quality of products in essence.Therefore, studying a kind of new enzyme source, be used to prepare dextran, is one of relevant scientific and technical personnel's research emphasis.
Summary of the invention
The technical issues that need to address of the present invention are that disclosing a kind of is the method for strain preparation dextran with the bacillus of oxidizing glucose, to overcome the above-mentioned defective that prior art exists.
Method of the present invention comprises the steps:
(1) bacillus of oxidizing glucose (Gluconobacter oxydans) bacterial strain is cultivated on slant medium, be inoculated into then to cultivate in the seed culture medium and increase, temperature is 22-40 ℃, and incubation time is 18-168h;
(2) nutrient solution that step (1) is obtained, press the inoculum size of 2-20% (v/v), be inoculated into fermentation culture in the fermention medium, temperature is 22-40 ℃, mixing speed is 100-1000rpm, incubation time is 18-168h, obtains to contain dextran dextrinase (dextran dextrinase, tunning DDase);
(3) then from the fermented liquid of step (2), extract dextran dextrinase,, dextrin is converted into dextran through enzymatic conversion reaction.
The method of step (3) is a kind of method of routine, and the method as Bioscience BiotechnologyBiochemistry 56 (1992) 169-173 bibliographical informations is summarized as follows:
Fermented liquid with step (2), carry out frozen centrifugation successively, collect thalline, the washing thalline, resuspended to acetate buffer (5-30mM, pH3-6), add water-saturated n-butanol, 4 ℃ of vibration 1h, high speed frozen centrifugation, enzymes are distributed in aqueous phase (crude enzyme liquid); Crude enzyme liquid is through dialysis, hydrophobic chromatography, gel-filtration purifying; The configuration 50mL conversion fluid [acetate buffer (10mM that contains 2% dextrin, pH4-6)] place 250mL to shake bottle, pure enzyme liquid is added in the reaction system (final concentration is 0.001-0.1U/mL), place in the biochemical incubator 35-45 ℃, 100-400r/min shaking culture when viscosity reaches peak value (generally at 18-48h), add the equal-volume ethanol sedimentation and obtain dextran.
Such dextran only contains α-1,6 and α-1, and 4-glycosidic link, main chain are α-1, and the 6-key connects and have the branched structure of height, and branched structure makes the bacillus of oxidizing glucose dextran have lower viscosity under the close situation of molecular weight more.
Wherein:
The component of slant medium and content are: D-sorbyl alcohol 10-100g/L, yeast extract paste 5-100g/L, KH
2PO
40.5-10g/L, MgSO
47H
2O 0.1-5g/L, agar 5-30g/L;
The component of seed culture medium and content are (g/L): D-sorbyl alcohol 10-100g/L, yeast extract paste 5-100g/L, KH
2PO
40.5-10g/L, MgSO
47H
2O 0.1-5g/L;
The component of fermention medium and content are: carbon source 5-80g/L, yeast extract paste 5-100g/L, KH
2PO
40.5-10g/L, MgSO
47H
2O 0.1-5g/L.PH is controlled between the 3.5-8.0,
Described carbon source is selected from dextrin, D-sorbyl alcohol carbon source or glycerine;
Described bacillus of oxidizing glucose (Gluconobacter oxydans), performance to this bacterial classification in Critical Reviews inBiotechnology 27 (2007) 147-171 documents has detailed report, can select for use bacillus of oxidizing glucose to belong to the microorganism of (Gluconobacter oxydans sp.), described microorganism all can have been bought at Chinese microbial preservation center.
The invention has the beneficial effects as follows:
The applicant finds, dextran dextrinase (the dextran dextrinase that bacillus of oxidizing glucose (Gluconobacter oxydans) is produced, DDase), can utilize maltodextrin and starch partial hydrolysate to synthesize dextran, utilize the dextran of bacillus of oxidizing glucose preparation to compare with the dextran that utilizes the Leuconostoc mesenteroides preparation, viscosity is lower, thereby causes its calorific value lower, the dextran of low-heat claims difficult digestion dextrin again, is a kind of of water-soluble foodstuff fibre.Utilize the characteristics of its retentiveness, toughness, can be used as low calorie foodstuff additive, can be widely used in the production of food service industry.
Embodiment
The preservation of embodiment 1 bacterial classification
Slant preservation, the slant culture based component is: D-sorbyl alcohol 80g/L, yeast extract paste 20g/L, KH
2PO
42g/L, MgSO
47H
2O 0.5g/L, agar 20g/L.With the inoculation of fresh inclined-plane, put under 28 ℃ of conditions and cultivated thalline 1-2 days, be placed on 4 ℃ of refrigerators and preserve, standby.
The cultivation of embodiment 2 seeds
The liquid nutrient medium of configuration is poured 500mL into and is shaken bottle, every bottle of 100mL, and 115 ℃, 30min moist heat sterilization, the cooling back is inoculated from solid medium, places 30 ℃ of shaking table 200r/min isothermal vibrations to cultivate, and obtains the activatory seed culture fluid.
The component of seed culture medium and content are: D-sorbyl alcohol 80g/L, yeast extract paste 20g/L, KH
2PO
42g/L, MgSO
47H
2O 0.5g/L.
Embodiment 3 fermentation culture and product enzymatic process (the D-sorbyl alcohol is as carbon source)
Fermention medium: D-sorbyl alcohol 40g/L, yeast extract paste 20g/L, KH
2PO
42g/L, MgSO
47H
2O 0.5g/L adopts the NaOH of 2M to transfer pH to 5.5;
With the nutrient solution of embodiment 2, the volume inoculum size by 10% inserts liquid fermentation medium, 30 ℃, 200r/min constant temperature culture are to 96h, put bottle, adopt Process Biochemistry 39 (2004) 1299-1304 document disclosed methods, detecting enzyme work is 7.67 * 10
-4The U/ml fermented liquid.
Then frozen centrifugation, collect thalline, washing thalline, resuspended to acetate buffer (20mM, pH4), add water-saturated n-butanol, 4 ℃ of vibration 1h, high speed frozen centrifugation, enzymes are distributed in aqueous phase (crude enzyme liquid); Crude enzyme liquid obtains the higher enzyme liquid of purity through dialysis, hydrophobic chromatography, gel-filtration purifying; The configuration 50mL conversion fluid [acetate buffer (10mM that contains 2% dextrin, pH5)] place 250mL to shake bottle, pure enzyme liquid is added in the reaction system (final concentration is 0.05U/mL), place in the biochemical incubator 37 ℃, 250r/min shaking culture when viscosity reaches peak value (generally at 24h), add the equal-volume ethanol sedimentation and obtain dextran.
Adopt Bioscience Biotechnology Biochemistry 56 (1992) 169-173 document disclosed methods to detect, the dextran productive rate is 11%.
Embodiment 4, fermentation culture and product enzymatic process (glycerine is as carbon source)
Fermention medium: glycerine 20g/L, yeast extract paste 20g/L, KH
2PO
42g/L, MgSO
47H
2O0.5g/L, transfer pH5.5g/L, product with embodiment 2, volume ratio inoculum size by 10%, insert liquid fermentation medium, 30 ℃, 200r/min constant temperature culture are put bottle to 96h, adopt ProcessBiochemistry 39 (2004) 1299-1304 document disclosed methods, detecting enzyme work is 7.24 * 10
-4The U/ml fermented liquid.
Adopt the identical method of embodiment 3 then, separation and Extraction from above-mentioned fermented liquid, obtain highly purified dextran dextrinase, and conversion dextrins generates dextran in reaction system, adopt Bioscience Biotechnology Biochemistry 56 (1992) 169-173 document disclosed methods to detect, the dextran productive rate is 9.6%.
Embodiment 5, fermentation culture and product enzymatic process (dextrin is as carbon source)
Fermention medium: dextrin 20g/L, yeast extract paste 20g/L, KH
2PO
42g/L, MgSO
47H
2O0.5g/L transfers pH5.5, the volume inoculum size by 10%, insert liquid fermentation medium, 30 ℃, 200r/min constant temperature culture are put bottle to 96h, adopt Process Biochemistry 39 (2004) 1299-1304 document disclosed methods, detecting enzyme work is 35.3 * 10
-4The U/ml fermented liquid.
Adopt the identical method of embodiment 3 then, separation and Extraction from above-mentioned fermented liquid, obtain highly purified dextran dextrinase, and conversion dextrins generates dextran in reaction system, adopt Bioscience Biotechnology Biochemistry 56 (1992) 169-173 document disclosed methods to detect, the dextran productive rate is 23.1%.
Claims (8)
1. the application of bacillus of oxidizing glucose (Gluconobacter oxydans) in the preparation dextran.
2. be the method for strain preparation dextran with the bacillus of oxidizing glucose, it is characterized in that, comprise the steps:
(1) bacillus of oxidizing glucose (Gluconobacter oxydans) bacterial strain is cultivated on slant medium;
(2) nutrient solution that step (1) is obtained is inoculated into fermentation culture in the fermention medium, obtains to contain dextran dextrinase (dextran dextrinase, fermented liquid DDase);
(3) then from the fermented liquid that step (2) obtains, extract dextran dextrinase, through the enzymatic conversion experiment, dextrin is converted into dextran.
3. method according to claim 1 is characterized in that, when cultivating in the seed culture medium, temperature is 22-40 ℃, and incubation time is 18-168h.
4. method according to claim 1 is characterized in that, with the nutrient solution of step (1) acquisition, press the volume inoculum size of 2-20%, be inoculated into fermentation culture in the fermention medium, temperature is 22-40 ℃, mixing speed is 100-1000rpm, and incubation time is 18-168h.
5. method according to claim 1 is characterized in that, the component of slant medium and content are: D-sorbyl alcohol 10-100g/L, yeast extract paste 5-100g/L, KH
2PO
40.5-10g/L, MgSO
47H
2O 0.1-5g/L, agar 5-30g/L.
6. method according to claim 1 is characterized in that, the component of seed culture medium and content are: the component of seed culture medium and content are: D-sorbyl alcohol 10-100g/L, yeast extract paste 5-100g/L, KH
2PO
40.5-10g/L, MgSO
47H
2O 0.1-5g/L.
7. method according to claim 1 is characterized in that, the component of fermention medium and content are: carbon source 5-80g/L, yeast extract paste 5-100g/L, KH
2PO
40.5-10g/L, MgSO
47H
2O0.1-5g/L, pH is controlled between the 3.5-8.0.
8. method according to claim 7 is characterized in that described carbon source is selected from dextrin, D-sorbyl alcohol carbon source or glycerine.
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| CNA2009100466301A CN101487035A (en) | 2009-02-25 | 2009-02-25 | Method for preparing dextran with Gluconobacter oxydans as strain |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321597A (en) * | 2011-10-09 | 2012-01-18 | 中国海洋大学 | Dextran dextrinase for preparing dextran |
| CN102559566A (en) * | 2010-12-29 | 2012-07-11 | 华北制药集团新药研究开发有限责任公司 | Genetic engineering oxidized glucose bacillus and preparation method and application thereof |
-
2009
- 2009-02-25 CN CNA2009100466301A patent/CN101487035A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559566A (en) * | 2010-12-29 | 2012-07-11 | 华北制药集团新药研究开发有限责任公司 | Genetic engineering oxidized glucose bacillus and preparation method and application thereof |
| CN102559566B (en) * | 2010-12-29 | 2014-01-08 | 华北制药集团新药研究开发有限责任公司 | Genetic engineering oxidized glucose bacillus and preparation method and application thereof |
| CN102321597A (en) * | 2011-10-09 | 2012-01-18 | 中国海洋大学 | Dextran dextrinase for preparing dextran |
| CN102321597B (en) * | 2011-10-09 | 2013-10-02 | 中国海洋大学 | Dextran dextrinase for preparing dextran |
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Open date: 20090722 |