CN103952450B - A kind of method utilizing microbial fermentation technology to produce guar gum - Google Patents
A kind of method utilizing microbial fermentation technology to produce guar gum Download PDFInfo
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Abstract
The invention discloses a kind of method utilizing microbial fermentation technology to produce guar gum.The method utilizes Extracellular polymers sphingosine unit cell strain fermentation to prepare guar gum, and its step includes actication of culture, preparation seed liquor, fermentation, Deproteinization and post processing.The deposit number of described Extracellular polymers Sphingol single-cell is CGMCC NO.1.6417.Present invention optimizes the separating and purifying technology of guar gum and technological parameter, establish a set of down-stream processing methods, it is simple that the method has technique, it is easy to the advantages such as operation；Have with short production cycle simultaneously, be not affected by waiting the restriction with geographical environmental condition, it is easy to the characteristics such as extraction and wide application, be suitable for industrialized production.The present invention utilizes glucose for raw material, prepares guar gum by microbial fermentation technology, and fermentation liquid component is simple, and product is present in fermentation liquid, and impurity is few, it is easy to purification, and the guar gum prepared is consistent with guar gum standard substance.
Description
Technical field
The present invention relates to the production of guar gum, in particular a kind of method utilizing microbial fermentation technology to produce guar gum.
Background technology
Guar gum, English " guargum " by name, is from extensively planting a kind of High Purity natural polysaccharide extracted in the endosperm of a kind of leguminous plant-Guar beans (CyamopsistetragonolobaL.Taub) in Indian-Pakistani subcontinent.Molecular structure feature and natural sex due to its uniqueness so that it is rapidly become the novel environment friendly paper making additive of superior performance；It is also extensively used for food, oil, medicine and other fields simultaneously.Guar gum is a kind of nonionic polysaccharide with regard to molecular structure, and it is with poly-mannose for molecular backbone, connects with β (1-4) glycosidic bond between D-mannopyranose units.D-galactopyranose is then connected on poly-mannose backbone with α (1-6) key.In guar gum, mannose is 2: 1 with the mol ratio of galactose units, is namely connected to a galactose branches every a mannosyl units, and the molecular weight of guar gum is about 220000.Guar gum is macromole Natural hydrophilic colloid, and outward appearance is from white to yellowish free flowing powder, can be dissolved in cold water or hot water, meets after water and forms colloid substance, reaching effect of rapid thickening.It is widely used in the industries such as the thickening purpose such as oil fracturing, drilling well and food additive, printing and dyeing and building coating.The natural polymer that most effective and water solublity that guar gum is known is best, at low concentrations, can form highly viscous solution, show non newtonian rheological behavior, forms acid reversible gel with Borax.Due to its special performance, the industries such as food, pharmacy, cosmetics, individual health care, oil, viscous mosquito agent, papermaking and textile printing and dyeing it are applied at present.
Prior art is all rely on chemical method extract from Guar beans and prepare guar gum, add Guar beans cultivation and environmental condition is required also very strict, although now in the ground such as Yunnan Province of China successful introduction, but ubiquity yields poorly, extracted amount is few, region requires the defects such as height, seasonal supply, also occupy substantial amounts of arable land simultaneously, cause the current guar gum of China also to rely primarily on the present situation of import.This not only greatly limit its application, and increases the production cost of guar gum.How just can efficiently solve the defect of above-mentioned prior art, developmental research go out a kind of have the greens of feature such as applied widely, production technology is simple, economic benefit is good, non-secondary pollution, environmentally friendly brand-new guar gum produce and technology of preparing be a problem demanding prompt solution.And, Microbial exopolysaccharides is except character specific to the high molecular polymer with most plants polysaccharide and synthesis, also have under low concentration in high viscosity, pseudoplastic behavior, heat stability, can with multiple salt compatibility and the film being formed, have with short production cycle simultaneously, it is not affected by waiting the restriction with geographical environmental condition, it is easy to the characteristics such as extraction and wide application, so there is vast potential for future development in many fields of commercial production Yu life.And current existing patent and document are all about utilizing chemical means to change its physicochemical property, meet the report of commercial production demand.Up to now, there is not yet the report that microbial fermentation technology is applied to produces guar gum method.
Therefore, prior art existing defects, it is necessary to improve.
Summary of the invention
The technical problem to be solved is for the deficiencies in the prior art, it is provided that a kind of method utilizing microbial fermentation technology to produce guar gum.
Technical scheme is as follows:
A kind of method utilizing microbial fermentation technology to produce guar gum, its step is as follows:
(1) actication of culture
The Extracellular polymers Sphingomonas preserved at 4 DEG C is transferred in solid medium by inclined-plane, at 30 DEG C, cultivates 12～20h at constant incubator；
(2) seed liquor is prepared
Take activated in (1) after strain, be inoculated in seed culture medium, liquid amount 100mL/500mL triangular flask, shaking speed 180r/min, 30 DEG C concussion cultivation 12～20h；
(3) fermentation
The inoculum concentration that seed liquor by volume mark is 2%～4% of preparation in (2) is linked into the fermentation cylinder for fermentation equipped with culture medium, coefficient 0.75, fermentation temperature is 28～31 DEG C, pH is 7.0～7.2, V: V of ventilation is 0.7-0.9: 1, and mixing speed is 180-190r/min, keeps dissolved oxygen saturation more than 12%, cultivate 5d, collect fermentation liquid；
(4) Deproteinization
The n-butyl alcohol mixing vibration half an hour of the chloroform and 0.04 times of fermentating liquid volume that add 0.2 times of fermentating liquid volume in the fermentation liquid collected in (3), is easily separated, repeat process 2～3 times, till the interface of both organic facies and aqueous phase is without precipitation, effectively remove the protein in guar gum；
(5) post processing
The guar gum component of Deproteinization in (4) through decolouring, filtration, low pressure concentration, crystallization and is obtained product guar gum after drying.
Described Extracellular polymers Sphingol single-cell (Sphingomonassanxanigehens) is purchased from General Microbiological Culture preservation administrative center of China Committee for Culture Collection of Microorganisms, and deposit number is CGMCCNO.1.6417.
Described method, in step (1), according to mass volume ratio, consisting of of solid medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, 1.5% agar, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
Described method, in step (2), according to mass volume ratio, consisting of of seed culture medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
Described method, in step (3), according to mass volume ratio, consisting of of culture medium: 4%～6% glucose, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
In the present invention, decolouring, filter, low pressure concentration, crystallization and dry belong to prior art, those skilled in the art can need rationally to select according to product；After fermentation liquid is preferably collected by the present invention, 5min is adsorbed with activated carbon 5g/100mL, filter through filter membrane (aperture is 0.45 μm), taking its filtrate adopts rotating thin film evaporimeter 48 DEG C to be evaporated to and steam without moisture, and the mass percentage concentration adding triploid long-pending in the guar gum solution of concentration is 95% ethanol, standing 12h at 4 DEG C, take its precipitation, and in vacuum drying oven, 45 DEG C of vacuum drying 6h, obtain guar products.
The assay method of glucose and guar gum in the present invention:
1) determination of maximum absorption wavelength
Prepare 80 μ g/mL Glucose standards solution, 80 μ g/mL polysaccharide crude solution, anthrone-colorimetry colour developing, within the scope of wavelength 550-700nm, measure light absorption value, obtain light absorption value curve.The obtained the maximum absorption corresponding wavelength of light absorption value curve is as the absorbing wavelength of standard curve.
2) foundation of standard curve
Glucose is dried to constant weight, and precision weighs this glucose 1.0g, and constant volume, in 100mL volumetric flask, takes 1mL constant volume in 100mL volumetric flask after shaking up, i.e. 100 μ g/mL Glucose standards solution.Take 20mL test tube with ground stopper, numbering, from 0.2mg/mL, by the glucose standard of the value preparation series concentration increasing 0.2mg/mL concentration every time, until maximum solution concentration 1.8mg/mL.Then in every test tube, add the 10mL anthrone sulfuric acid solution (ice-water bath) prepared in advance respectively, mixing, cover stopper, boiling water bath boils 10min (timing after water-bath reboiling), takes out, be water-cooled to room temperature immediately, with the first pipe for blank, zeroing, under above-mentioned measured maximum absorption wavelength, measures the light absorption value of each pipe respectively.Thinking glucose content abscissa, corresponding light absorption value is vertical coordinate, sets up glucose standard curve.
3) guar gum assay
Precision weighs 100.0mg sample, and constant volume, in 1000mL volumetric flask, measures this sample liquid 2.0mL after mixing, repeat 3 groups, is then respectively adding the 10mL By Anthrone Sulphuric acid solution prepared in advance, shakes up, boiling water bath heating 10min.Respectively with the first pipe of glucose standard curve for blank, zeroing, measure light absorption value in maximum absorption wave strong point.The content of guar gum in fermentation liquid is checked in by standard curve.
The assay method of bacterial strain fermentation liquor viscosity: by the fermentation liquid in step (3) with the centrifugal 20min of 3000rpm, supernatant viscosity NDJ-79 type Rotary Viscosimeter is measured.
The present invention has started the large-scale production new way utilizing microbial fermentation processes to prepare guar gum；Present invention optimizes the separating and purifying technology of guar gum and technological parameter, establish a set of down-stream processing methods, it is simple that the method has technique, it is easy to the advantages such as operation；Have with short production cycle simultaneously, be not affected by waiting the restriction with geographical environmental condition, it is easy to the characteristics such as extraction and wide application, be suitable for industrialized production.The present invention utilizes glucose for raw material, prepares guar gum by microbial fermentation technology, and fermentation liquid component is simple, and product is present in fermentation liquid, and impurity is few, it is easy to purification, and the guar gum prepared is consistent with guar gum standard substance.
Accompanying drawing explanation
Fig. 1 is glucose standard curve.
Fig. 2 is fermentation time and guar gum Relationship with Yield figure.
Fig. 3 is the graph of a relation of guar gum yield and fermentation liquid viscosity.
Fig. 4 is the Fourier transform infrared spectroscopy figure of guar gum and guar gum standard substance；Note: upper figure is guar gum, figure below is guar gum standard substance.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
Extracellular polymers Sphingol single-cell (Sphingomonassanxanigenens) is purchased from General Microbiological Culture preservation administrative center of China Committee for Culture Collection of Microorganisms, and deposit number is CGMCCNO.1.6417.
Embodiment 1 is with glucose for carbon source, by the fermentation tank batch fermentation pilot production of 8L
1, actication of culture
The Extracellular polymers Sphingomonas preserved at 4 DEG C is transferred in solid medium by inclined-plane, at 30 DEG C, 16h is cultivated at constant incubator, wherein, according to mass volume ratio, consisting of of culture medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, 1.5% agar, all the other are water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
2, preparation seed liquor
Take activated in 1 after strain, be inoculated in seed culture medium (liquid amount 100mL/500mL triangular flask), shaking speed 180r/min, 30 DEG C concussion cultivate 16h.Wherein, according to mass volume ratio, consisting of of seed culture medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
3, the fermentation tank batch fermentation of 8L
The seed liquor of preparation in 2 is linked into the fermentation cylinder for fermentation equipped with culture medium, coefficient 0.75 by the inoculum concentration of 2%～4% (volume fraction), controls temperature 28～31 DEG C, pH7.0～7.2, keep dissolved oxygen saturation more than 12%, cultivate 5d, collect fermentation liquid.Wherein, according to mass volume ratio, consisting of of culture medium: 5% glucose, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, all the other are water；PH7.0～7.2,115 DEG C of sterilizing 25min.Fermentation temperature 28～31 DEG C, ventilation 0.8: 1 (V: V), mixing speed 180-190r/min, pH7.0～7.2.
4, the extraction of guar gum
5min is adsorbed with activated carbon 5g/100mL, filter through filter membrane (aperture is 0.45 μm), taking its filtrate adopts rotating thin film evaporimeter 48 DEG C to be evaporated to and steam without moisture, the mass percentage concentration adding triploid long-pending in the guar gum solution of concentration is 95% ethanol, after standing 12h in 4 DEG C, take its precipitation, and in vacuum drying oven, 45 DEG C of vacuum drying 6h, obtain guar products.After testing, fermentation is to 58h viscosity up to 9860MPa s, and the output of sugar of this bacterial strain is up to 21.246g/L, and its purity is 95%.
Embodiment 2
1, actication of culture
The Extracellular polymers Sphingomonas preserved at 4 DEG C is transferred in solid medium by inclined-plane, at 30 DEG C, 16h is cultivated at constant incubator, wherein, according to mass volume ratio, consisting of of culture medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, 1.5% agar, all the other are water.PH7.0～7.2,115 DEG C of sterilizing 25min.
2, preparation seed liquor,
Take activated in 1 after strain, be inoculated in seed culture medium (liquid amount 100mL/500mL triangular flask), shaking speed 180r/min, 30 DEG C concussion cultivate 16h.Wherein, according to mass volume ratio, consisting of of seed culture medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
3, the fermentation tank batch fermentation of 8L
The seed liquor of preparation in 2 is linked into the fermentation cylinder for fermentation equipped with culture medium, coefficient 0.75 by the inoculum concentration of 2%～4% (volume fraction), controls temperature 28～31 DEG C, pH7.0～7.2, keep dissolved oxygen saturation more than 12%, cultivate 5d, collect fermentation liquid.Wherein, according to mass volume ratio, consisting of of culture medium: 5% glucose, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, all the other are water.PH7.0～7.2,115 DEG C of sterilizing 25min.Fermentation temperature 28～31 DEG C, ventilation 0.8: 1 (V: V), mixing speed 180-190r/min, pH7.0～7.2.
4, the extraction of guar gum
5min is adsorbed with activated carbon 5g/100mL, filter through filter membrane (aperture is 0.45 μm), taking its filtrate adopts rotating thin film evaporimeter 48 DEG C to be evaporated to and steam without moisture, the mass percentage concentration adding triploid long-pending in the guar gum solution of concentration is 95% ethanol, after standing 12h in 4 DEG C, take its precipitation, and in vacuum drying oven, 45 DEG C of vacuum drying 6h, obtain guar products.
Accompanying drawing 2 is the graph of relation of guar gum average content, Biomass and fermentation time in above-mentioned fermentation tank.By this bacterial strain known of curve in figure in the incipient stage fermented, then thalline through the period of delay of 4h, must quickly enter exponential phase, starts to synthesize guar gum from 6h.After 26h, thalline enters stable phase, enters the stable phase later stage at 44h, and As time goes on its yield constantly accelerated, and when fermentation is to 58h, it is 21.246g/L that guar gum reaches maximum output.
Embodiment 3
1, actication of culture
The Extracellular polymers Sphingomonas preserved at 4 DEG C is transferred in solid medium by inclined-plane, at 30 DEG C, 16h is cultivated at constant incubator, wherein, according to mass volume ratio, consisting of of culture medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, 1.5% agar, all the other are water.PH7.0～7.2,115 DEG C of sterilizing 25min.
2, preparation seed liquor,
Take activated in 1 after strain, be inoculated in seed culture medium (liquid amount 100mL/500mL triangular flask), shaking speed 180r/min, 30 DEG C concussion cultivate 16h.Wherein, according to mass volume ratio, consisting of of seed culture medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
3, the fermentation tank batch fermentation of 8L
The seed liquor of preparation in 2 is linked into the fermentation cylinder for fermentation equipped with culture medium, coefficient 0.75 by the inoculum concentration of 2%～4% (volume fraction), controls temperature 28～31 DEG C, pH7.0～7.2, keep dissolved oxygen saturation more than 12%, cultivate 5d, collect fermentation liquid.Wherein, according to mass volume ratio, consisting of of culture medium: 5% glucose, 0.06% ammonium nitrate, 0.5%KH2PO4, 0.01%MgSO4, all the other are water.PH7.0～7.2,115 DEG C of sterilizing 25min.Fermentation temperature 28～31 DEG C, ventilation 0.8: 1 (V: V), mixing speed 180-190r/min, pH7.0～7.2.
4, the extraction of guar gum
5min is adsorbed with activated carbon 5g/100mL, filter through filter membrane (aperture is 0.45 μm), taking its filtrate adopts rotating thin film evaporimeter 48 DEG C to be evaporated to and steam without moisture, the guar gum solution of concentration adds mass percentage concentration 95% ethanol that triploid is long-pending, after standing 12h in 4 DEG C, take its precipitation, and in vacuum drying oven, 45 DEG C of vacuum drying 6h, obtain guar products.
Accompanying drawing 3 is the graph of a relation of guar gum yield and fermentation liquid viscosity.Being shown by figure, the model equation between guar gum content and fermentation liquid viscosity is: y=0.0011x2-0.0038x+4.3262, R2=0.9789.In formula, x is guar gum content, and y is fermentation liquid viscosity, and R is regression coefficient.The viscosity of fermented supernatant fluid increases along with the increase of guar gum content, thus illustrating that guar gum yield and fermented supernatant fluid viscosity exist extremely significant linear positive correlation to a certain extent.
The detection of guar gum prepared by embodiment 4 present invention
(1) guar gum of preparation in Example 3, carries out examination of infrared spectrum.Accompanying drawing 4 is the infrared spectrogram of guar gum and guar gum standard substance, basically identical by the position of each main peaks of both analyses and form, it is possible to prove that product prepared by the present invention is high-purity guar gum.
(2) other physicochemical property of guar gum
The guar gum that the present invention extracts is light brown crystalline, odorlessness, the organic solvent such as ethanol insoluble in 95%, acetone, ether, is dissolved in hot water；IKI reaction is feminine gender, illustrates that this polysaccharide is non-starch class and cellulose substances；Polysaccharide and fehling reagent react for positive, have brick red precipitation to generate, illustrate wherein to contain reducing sugar；React for negative and ninhydrin reaction feminine gender, biuret reaction feminine gender with Coomassie brilliant G-250, it was demonstrated that wherein do not contain protein component.
It should be appreciated that for those of ordinary skills, it is possible to improved according to the above description or converted, and all these are improved and convert the protection domain that all should belong to claims of the present invention.
Claims (4)
1. the method utilizing microbial fermentation technology to produce guar gum, is characterized in that, its step is as follows:
(1) actication of culture
The Extracellular polymers Sphingomonas preserved at 4 DEG C is transferred in solid medium by inclined-plane, at 30 DEG C, cultivates 12～20h at constant incubator；The deposit number of described Extracellular polymers Sphingomonas is CGMCCNO.1.6417；
(2) seed liquor is prepared
Take activated in (1) after strain, be inoculated in seed culture medium, liquid amount 100mL/500mL triangular flask, shaking speed 180r/min, 30 DEG C concussion cultivation 12～20h；
(3) fermentation
The inoculum concentration that seed liquor by volume mark is 2%～4% of preparation in (2) is linked into the fermentation cylinder for fermentation equipped with culture medium, coefficient 0.75, fermentation temperature is 28～31 DEG C, pH is 7.0～7.2, V: V of ventilation is 0.7-0.9: 1, and mixing speed is 180-190r/min, keeps dissolved oxygen saturation more than 12%, cultivate 5d, collect fermentation liquid；
(4) Deproteinization
The n-butyl alcohol mixing vibration half an hour of the chloroform and 0.04 times of fermentating liquid volume that add 0.2 times of fermentating liquid volume in the fermentation liquid collected in (3), is easily separated, repeat process 2～3 times, in fermentation liquid, the interface of both organic facies and aqueous phase is without, till precipitation, effectively removing the protein in guar gum；
(5) post processing
The guar gum component of Deproteinization in (4) through decolouring, filtration, low pressure concentration, crystallization and is obtained product guar gum after drying.
2. method according to claim 1, it is characterized in that, in step (1), according to mass volume ratio, consisting of of solid medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4,0.01%MgSO4,1.5% agar, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
3. method according to claim 1, it is characterized in that, in step (2), according to mass volume ratio, consisting of of seed culture medium: 1% glucose, 0.25% yeast powder, 0.5% peptone, 0.06% ammonium nitrate, 0.5%KH2PO4,0.01%MgSO4, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
4. method according to claim 1, is characterized in that, in step (3), according to mass volume ratio, consisting of of culture medium: 4%～6% glucose, 0.06% ammonium nitrate, 0.5%KH2PO4,0.01%MgSO4, all the other be water；PH is 7.0～7.2,115 DEG C of sterilizing 25min.
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| CN1995326A (en) * | 2006-09-26 | 2007-07-11 | 张国沛 | Sphingol single-cell bacteria and method for producing microorganism polysaccharide- 'sanzan' gum using the strain |
| US7868167B2 (en) * | 2005-11-01 | 2011-01-11 | Cp Kelco U.S., Inc. | High viscosity diutan gums |
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| US7868167B2 (en) * | 2005-11-01 | 2011-01-11 | Cp Kelco U.S., Inc. | High viscosity diutan gums |
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| 合成生物聚合物的重要微生物资源-鞘氨醇单胞菌;黄海东等;《微生物学报》;20090504;第49卷(第5期);560-566 * |
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