CN103509733A - Klebsiella sugarcanna used for isomaltulose production - Google Patents
Klebsiella sugarcanna used for isomaltulose production Download PDFInfo
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- CN103509733A CN103509733A CN201310142534.3A CN201310142534A CN103509733A CN 103509733 A CN103509733 A CN 103509733A CN 201310142534 A CN201310142534 A CN 201310142534A CN 103509733 A CN103509733 A CN 103509733A
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- sucrose
- klebsiella
- sugarcane
- palatinose
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Abstract
The invention discloses Klebsiella sugarcanna which is obtained by isolation of root soil samples of sugarcane, and is used for isomaltulose production. Klebsiella sugarcanna is obtained by following steps: the soil samples are collected from sugarcane root; the soil samples are diluted, and sucrose solid spread plates are coated with the diluted soil samples so as to obtain single colonies by separation; sucrose fluid medium is used for culturing; isomaltulose is detected by thin layer chromatography; and component ratio of a plurality of sugars in the medium are analyzed by high performance liquid chromatography. Klebsiella sugarcanna is capable of transforming sucrose into isomaltulose via isomerization, and the content of glucose and fructose by-product in the product is less than 1%. An enzyme used for catalyzing transformation of sucrose into isomaltulose is alpha-glucosyltransferase, and is obtained by inducement of Klebsiella sugarcanna, wherein sucrose is taken as an inductive agent.
Description
Technical field
Patent of the present invention relates to a kind of microbial bacteria, can synthesize α-glucose based transferase, and the bio-transformation of catalysis sucrose is Palatinose, for Palatinose, produces.
Background technology
Palatinose (α-D-glucopyranosyl-1,6-D-fructose Isomaltulose), claim again palatinose, be a kind of glucose and fructose with α-1,6-glycosidic link is in conjunction with the reducing disaccharides forming, the natural honey that is present in, but content is extremely low.As the isomer of sucrose, its mouthfeel and physical properties and sucrose are closely similar, and sugariness only has sucrose half.Due to the special physiological function of Palatinose, as a kind of heath food additive, can be used as under given conditions sucrose substitute and be applied to foodstuffs industry.
Palatinose almost can not by plaque form bacterium especially variable chains coccus utilize, and can reduce the generation of insoluble glucan and acid thereof, thereby prevent that variable chains coccus is adsorbed onto dental surface and reduces adamantine local decalcification, has prevention of dental caries activity.Palatinose is in human body, can only be resolved into glucose and fructose and digested absorption by the enteric microorganism at small intestine place, but because its decomposition rate is very slow, take in after Palatinose, energy can be slowly provided for a long time, and can not cause the rising rapidly of glucose in blood and Regular Insulin, thereby become diabetic subject's heath food.Meanwhile, Healthy People or patients with diabetes mellitus Palatinose can not cause any gastrointestinal upset and Blood Lipid.
Palatinose is to take sucrose as raw material, after synthetic α-glucose based transferase (EC.5.4.99.11) the catalysis sucrose isomerization of microorganism, obtain, found that so far certain micro-organisms can be Palatinose by sucrose inversion, microbial species during bacteriums as several in Serratia, Erwinia, Protaminobacter and Klebsiella etc. belong to, representative bacterial classification mainly comprises Protaminobacter rubrum, Serratia plymuthica and Klebsiella planticola.In the microorganism having been found that, most catalysates be take Palatinose as main (75-80%), but be also attended by trehalulose and glucose and fructose simultaneously, form.Glucose wherein and the existence of fructose have a strong impact on the quality product of Palatinose and functional, must adopt the technical points such as resin isolation from removing, cause production cost significantly to improve, therefore, the diversity of α-glucose based transferase catalysis sucrose isomerization product becomes the industrial major obstacle of Palatinose.
Summary of the invention
The microbial bacteria that the object of this invention is to provide a kind of High-efficient Production Palatinose, can be converted into Palatinose by sucrose, and in product, only have glucose and the fructose of extremely low concentration.
The present invention is the sugarcane klebsiella of a kind of called after LI8, it is a strain pure culture bacterial strain, taxonomy is accredited as sugarcane klebsiella (Klebsiella sugarcanna), deposit number is: CCTCC M 2013153, this bacterial strain can synthesize α-glucose based transferase, at catalysis sucrose isomery, turns in the product of Palatinose, and Palatinose content is 88.9%, trehalulose is 10.3%, and glucose and fructose are 0.8%.Its technical solution is:
By steps such as various sugar component ratios from sugarcane root collection pedotheque, cultivation in sucrose solids flat board dilution painting plate isolation list bacterium colony, liquid sucrose substratum, thin-layer chromatography (TLC) detect synthesizing of Palatinose and high performance liquid chromatography (HPLC) method analyzing and testing sucrose isomerization product, obtain Palatinose production bacterium.
Sugarcane klebsiella described in the present invention (Klebsiella sugarcanna), on April 19th, 2013, be preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC M 2013153, and depositary institution address is: Luo Jia Shan, wuchang, wuhan Wuhan University, postcode: 430072.
In technique scheme, the described step of separation of sugarcane klebsiella from soil comprises:
The pedotheque of collecting from sugarcane root is suspended in stroke-physiological saline solution, on sucrose solids flat board, carrying out serial dilution is coated with dull and stereotyped, in 30 ° of incubators, cultivate 24 hours, picking list colony inoculation is in liquid sucrose substratum, and shaking culture (150 revs/min) is spent the night in 30 ° of C shaking tables, thin layer chromatography detects in fermented supernatant fluid whether contain Palatinose.With efficient liquid phase chromatographic analysis, detect the sugar component ratio in the culture supernatant that produces Palatinose, choose a strain and can produce single bacterium colony that maximum concentration Palatinose and glucose and fructose content are lower for the present invention, and called after LI8, the morphological specificity of this bacterium colony on sucrose solids flat board is circular, smooth, White-opalescent.Wherein,
Described sucrose solids plate culture medium consists of: 20～25g sucrose, 4.5～5.5g yeast powder, 0.45～0.55g sal epsom, 0.8-1.0g dipotassium hydrogen phosphate, 15-20g agar, 1000g water, pH7.0～7.2.
Described liquid sucrose substratum consists of: 38～42g sucrose, 4.5～5.5g peptone, 3.5～4.5g yeast powder, 1000g water, pH7.0～7.2.
Described thin layer chromatography is: get 0.5 μ l fermented supernatant fluid point sample on silica gel thin-layer chromatography plate, colour developing after launching in separation chamber, developping agent consists of the volume ratio 4:3:0.8 of ethyl acetate-acetic acid-water, developer is the volume ratio 5:5:1 of this amine (4%)-bis-amine (4%)-phosphoric acid (85%), colour temp is 80 ° of C, developing time is 5 minutes, and Palatinose forms typical yellow-green colour spot.
Described high performance liquid chromatography is: adopt Waters 2690 systems, chromatography column is glycan analysis dedicated columns (4.6 * 250 millimeters), detector is Waters 2410 refractive index detectors, 70% second cyanogen aqueous solution wash-out for sample, eluent flow rate is 1 ml/min, according to calculated by peak area sugar content and proportion of products.
Other correlation analysis methods in the present invention are:
α-glucose based transferase measuring method: get 0.1 milliliter of nutrient solution and mix with 0.4 milliliter of 2wt% sucrose solution that is dissolved in 20mM phosphate buffer solution (pH7.0), in 30 ° of C water-baths, react 10 minutes, add immediately 1.5 milliliter 3,5-edlefsen's reagent stops enzyme reaction, and in boiling water bath, heat after 5 minutes cooling, at wavelength 520nm place, measure absorbance, by Palatinose typical curve, determine reducing sugar content.An enzyme activity unit is defined as: under above-mentioned reaction conditions, the isomerization of per minute catalysis sucrose forms the needed enzyme amount of 1 μ mol Palatinose.
3,5-edlefsen's reagent compound method: 6.9g crystalline phenol is dissolved in 15.2mL10% sodium hydroxide, and is diluted to 69mL, add 6.9g sodium bisulfite, make first liquid; 255g sodium tartrate is dissolved in 300mL10% sodium hydroxide, then adds 3 of 880mL1%, 5-dinitrosalicylic acid solution, makes second liquid; After the first and second liquid are mixed, and store in brown reagent bottle, under room temperature, place 7～10 days, make 3,5-edlefsen's reagent.
Beneficial effect of the present invention
While adopting the sugarcane klebsiella production Palatinose in the present invention, in product, only contain glucose and fructose by product lower than 1%, thereby in follow-up sugar refining technology without separation and purification direct condensing crystal just, both can significantly reduce production costs, save again a large amount of water of productive use.
Embodiment:
Embodiment 1
From sugarcane root, collect pedotheque, getting 1g is suspended in 100mL stroke-physiological saline solution, plate dilution method is coated on sucrose solids flat board, in 30 ° of C incubators, cultivate 24 hours, random single bacterium colony of selecting on flat board, a part for picking colony is inoculated on fresh sucrose solids flat board, in 30 ° of C incubators, cultivates 16 hours, for bacterial classification, preserves.An other part for bacterium colony is inoculated in liquid sucrose substratum, in 30 ° of C concussions, cultivates (150 revs/min) 16 hours, and whether thin layer chromatography detects in nutrient solution has Palatinose to generate.The nutrient solution that selection contains Palatinose, high-efficient liquid phase chromatogram technique analysis detects the sugar composition minute in nutrient solution, select to produce that Palatinose content is the highest, glucose and the minimum bacterium of fructose content be for the present invention, in order to guarantee the purity of bacterium of the present invention, on sucrose solids flat board, adopt method of scoring to cultivate, picking one strain list bacterium colony is for the present invention, and called after LI8.This bacterium has the whole crucial physiological and biochemical property that klebsiella spp has: Gram-negative, amphimicrobian, oxidase negative, do not move, shaft-like, produce pod membrane, and the homology of the 16S rDNA gene order in the present invention and other known klebsiellas is all between 97～99.2%, illustrates that LI8 Pseudomonas in the present invention is in klebsiella.But the LI8 bacterium in the present invention is compared with each kind in known klebsiella spp, there is again obvious difference, comprise while producing indoles and 10 ° of C and growing, and with the DNA-DNA hybrid rate of other klebsiellas all in 3.4-28.2% scope, illustrate that the LI8 in the present invention is different from other known klebsiellas, so called after sugarcane klebsiella (Klebsiella sugarcanna), expression is separated acquisition from sugarcane root soil.
Above-mentioned physiological and biochemical property, DNA hybridization and 16S rDNA sequence more all adopt microbiology evaluation ordinary method, and coherent reference book can be looked into.
Embodiment 2
By sugarcane klebsiella by preserving inclined plane inoculating in sucrose solids medium slant, after in 30 ° of C incubators, activation culture is spent the night, be inoculated in 50mL seed culture medium, 30 ° of C shaking table concussions (150 revs/min) were cultivated after 6 hours, by 1% inoculum size, be inoculated in 100mL fermention medium, under 30 ° of C and 150 revs/min of conditions, cultivate 10 hours, high performance liquid chromatography detects the product component in nutrient solution.Result demonstration, the sugar component ratio in nutrient solution is: 88.9% Palatinose, 10.3% trehalulose, 0.8% glucose and fructose.
Described seed culture medium consists of: 1.5g sucrose, 0.3g yeast powder, 0.2g peptone, 0.05g sal epsom, 0.07g SODIUM PHOSPHATE, MONOBASIC, 0.06g SODIUMNITRATE, 100g water, pH7.0～7.2.
Described fermentative medium formula is: 4g sucrose, 0.2g yeast soaks powder, 0.15g peptone, 100g water, pH7.0～7.2.
Above-mentioned high performance liquid chromatography used is: adopt Waters 2690 systems, chromatography column is glycan analysis dedicated columns (4.6 * 250 millimeters), detector is Waters 2410 refractive index detectors, 70% second cyanogen aqueous solution wash-out for sample, eluent flow rate is 1 ml/min, according to calculated by peak area sugar content and proportion of products.
Embodiment 3
Press after embodiment 2 method activated seed nutrient solutions, by 1% inoculum size, be inoculated in the basic medium (50mL) that contains different carbon sources, under 30 ° of C and 150 revs/min of conditions, cultivate 10 hours, detect the alpha-glucose-based transferase active in nutrient solution.Found that in the nutrient solution that only contains sucrose, raffinose, fructose and Palatinose and have α-glucose based transferase to generate, wherein sucrose induction generates the α-glucose based transferase of maximum vigor.In various carbon source substratum, alpha-glucose-based transferase active is as shown in table 1.
| Carbon source | Alpha-glucose-based transferase active (U/mL) |
| Sucrose | 15.8 |
| Raffinose | 13.9 |
| Fructose | 11.2 |
| Palatinose | 4.5 |
| Glucose | 0 |
| Lactose | 0 |
| Wood sugar | 0 |
| Pectinose | 0 |
| Cellobiose | 0 |
| Semi-lactosi | 0 |
| Melibiose | 1.7 |
Table 1
Described basic medium consists of: 1.5g peptone, 0.5g sal epsom, 1g dipotassium hydrogen phosphate, 1g sodium-chlor, 1000g water, pH7.0～7.2.
Above-mentioned carbon source used comprises: sucrose, raffinose, fructose, Palatinose, glucose, lactose, wood sugar, pectinose, cellobiose, melibiose and semi-lactosi.
Claims (7)
1. the sugarcane klebsiella that a strain is produced for Palatinose, its deposit number is CCTCC M 2013153.
2. sugarcane klebsiella according to claim 1, is characterized in that being Palatinose by sucrose inversion.
3. sugarcane klebsiella according to claim 1, is characterized in that culture condition is that 30 ° of C and 150 revs/min of concussions are cultivated.
4. sugarcane klebsiella according to claim 1, is characterized in that inducing the synthetic substratum of a-glucanotransferase to consist of: 4g sucrose, 0.2g yeast soaks powder, 0.15g peptone, 100g water, pH7.0-7.2.
5. sugarcane klebsiella according to claim 1, the sugared proportion of composing that it is characterized in that sucrose inversion product is 88.9% Palatinose, 10.3% trehalulose, 0.8% glucose and fructose.
6. sugarcane klebsiella according to claim 1, is characterized in that synthesizing a-glucanotransferase.
7. the synthetic a-glucanotransferase of sugarcane klebsiella according to claim 6, is characterized in that a-glucanotransferase synthesis condition is to take sucrose as inductor.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849395A (en) * | 2015-06-11 | 2015-08-19 | 广州甘蔗糖业研究所 | Method for detecting isomaltulose in food |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575629A (en) * | 2009-06-11 | 2009-11-11 | 大连工业大学 | Method for producing isomaltulose without purification step |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101575629A (en) * | 2009-06-11 | 2009-11-11 | 大连工业大学 | Method for producing isomaltulose without purification step |
Non-Patent Citations (2)
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| 罗霆: "1株有固氮能力的甘蔗克雷伯氏菌的分离鉴定及固氮特性", 《热带作物学报》 * |
| 许波: "环状糊精葡萄糖基转移酶的研究进展", 《食品科学》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104849395A (en) * | 2015-06-11 | 2015-08-19 | 广州甘蔗糖业研究所 | Method for detecting isomaltulose in food |
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