CN1635132A - Process for preparing microbiological polysaccharide Gellan gum - Google Patents

Process for preparing microbiological polysaccharide Gellan gum Download PDF

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Publication number
CN1635132A
CN1635132A CN 200410064546 CN200410064546A CN1635132A CN 1635132 A CN1635132 A CN 1635132A CN 200410064546 CN200410064546 CN 200410064546 CN 200410064546 A CN200410064546 A CN 200410064546A CN 1635132 A CN1635132 A CN 1635132A
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enlarged culturing
preparation
gellan gum
substratum
shake
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CN1269964C (en
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张禹
张国佩
张茶
张少华
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Hebei Henbo Bio Technology Co ltd
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张禹
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Abstract

The invention relates to a preparation method of microbe polysaccharide Gellan gum belonging to the microbe fermentation production field. The method comprises steps of preparing the slope strains, preparing the seed liquid, inoculating the seed liquid into the fermentation culture medium to ferment. The invention solves the problems of high production cost, low product yield (the density of the Gellan gum coarse polysaccharide is 1.3g/100ml) exiting in known technology. The invention employs starch as the carbon source for the second level expanding culture medium and fermentation culture medium, and has advantages of low cost, high yield. The density of the Gellan gum coarse polysaccharide is 1.5-1.6g/100ml when the fermentation is finished.

Description

The preparation method of microbiological polysaccharide Gellan gum
Technical field
The present invention relates to the microbial fermentation production field, be meant a kind of preparation method of microbiological polysaccharide Gellan gum especially.
Background technology
Gelling gum is to utilize modern biotechnology to carry out aerobic fermentation by the bacterial classification that monospore bacillus waterweed belongs in (pseudomonas elodea), and a kind of transparent gelifying agent through special refinement technique is produced has good thermostability.Consumption is few, and it is good, acidproof, alkaline-resisting to hold flavor property, anti-biological enzyme, and gel-strength can adjust as required, is widely used in industries such as food, medicine, fine chemistry industry.Have only U.S. CP keclo company to realize industrial production at present in the world, production and sales are in monopoly position, and production technology difficulty own is big, so cost an arm and a leg.More domestic universities and colleges also once attempted to produce gelling gum with comparatively easy production technology, but effect is not ideal always.The patent No. is the production technique that discloses a kind of new microbiological polysaccharide Gellan gum in 00125858.3 the patent, but above-mentioned prior art exists the production cost height, the shortcoming of product yield low (concentration of gelling gum Crude polysaccharides is 1.3g/100ml).
Summary of the invention
The preparation method who the object of the present invention is to provide the microbiological polysaccharide Gellan gum that a kind of production cost is low, the product yield is high is to overcome the deficiency that prior art exists.
Overall technology design of the present invention is:
The preparation method of microbiological polysaccharide Gellan gum, comprise in the aseptic culture medium of sphingomonas paucimobilis Sphingomonaspaucimobilis bacterial classification inoculation carbonaceous sources, nitrogenous source, inorganic salt and the water of selecting for use monospore bacillus waterweed to belong to (pseudomonas elodea) and carry out aerated culture, comprise following processing step:
A, with original strain bevel bacterial classification;
B, the slant strains for preparing in the A step is made seed liquor through enlarged culturing;
C, with the seed liquor for preparing in the B step by seed liquor: fermention medium is that the inoculum size of 7%-10% inserts fermention medium and inserts and ferment, and wherein fermention medium comprises following component (weight part):
Starch 2.5-3.0 dregs of beans 0.2 NH 4NO 30.1 MgSO 40.01
K 2HPO 40.15 CaCO 30.05 water 96.49-96.99
The temperature of fermention medium is that 26-36 ℃, PH are 6.5-6.8, ventilation 0.1-0.3vvm, and fermentation period is 50-60 hour.Selected bacterial classification is that monospore bacillus waterweed belongs to (pseudomonas elodea) sphingomonas paucimobilis Sphingomonaspaucimobilis.
Processing condition among the present invention in each processing step are:
Enlarged culturing in the B step comprises shake-flask culture, one-level enlarged culturing and secondary enlarged culturing, slant strains is inserted the shake-flask culture base make shake-flask seed through fermentation, shake-flask seed inserts the substratum of one-level enlarged culturing and makes first order seed through fermentation, and the substratum of the substratum of slant strains, shake-flask culture base, one-level enlarged culturing comprises following component (weight part) in A, the B step:
Disaccharides 0.8-1.0 yeast extract paste 0.18-0.2 extractum carnis 0.25-0.3
Peptone 0.1-0.15 agar 2-2.5 water 95.85-96.67
The culture condition of slant strains is temperature 26-36 ℃, 72 hours time; The condition of one-level enlarged culturing is inoculum size 1-3%, culture temperature 26-36 ℃, and pH value 6.5-6.8, ventilation 0.1-0.15vvm, cycle 20-24h.
The substratum of secondary enlarged culturing comprises following component (weight part):
Disaccharides 0.3-0.5 starch 0.5 ammonium nitrate 0.25-0.3
Bean powder 0.2 K 2HPO 40.15 KH 2PO 40.08 MgSO 40.01
Water 98.26-98.51 pH6.5-7.0
The condition of secondary enlarged culturing is inoculum size 7-10%, culture temperature 26-36 ℃, and pH value 6.5-6.8, ventilation 0.1-0.15vvm, cycle 18-20h.
After the viscosity of the fermented liquid in the C step surpasses 2000CP, add the tensio-active agent of 80-100ppm in fermented liquid, tensio-active agent is selected from polysorbate60, Triton X-100.
Reaction end in the one-level enlarged culturing is for having a net increase of the OD value more than 0.5, and the mensuration of OD value is selected 622 spectrophotometers, 650nm wavelength for use.
Reaction end in the secondary enlarged culturing is for having a net increase of the OD value more than 1.0, and the mensuration of OD value is selected 622 spectrophotometers, 650nm wavelength for use.
Residual sugar is below 0.2% in the fermenting process.
Shaking speed in one-level enlarged culturing and the secondary enlarged culturing is 120-220 rev/min, and the fermentor tank tank diameter is than 2-2.5: 1, and rotating speed is 180-200 rev/min, also can adopt no stirring-type to add air-distributor; Fermentor tank in the fermenting process, stirring rake are 4-6 group, and whipped form adopts at interval installation shaft to pusher and disk curved blade type paddle radially, and rotating speed is 120-180 rev/min.
Starch in secondary enlarged culturing substratum and the fermention medium can replace with hydrolysis sugar, and the disaccharides in the substratum of slant medium, shake-flask culture base, one-level enlarged culturing and the substratum of secondary enlarged culturing is selected sucrose, fructose or the mixture of the two for use.
Substantive distinguishing features that the present invention possessed and the technical progress that obtains are:
Integrated artistic of the present invention is reasonable in design, is easy to realize, with the carbon source of starch as secondary enlarged culturing substratum and fermention medium, production cost reduces greatly, product yield height, after testing, fermenting at the end, gelling gum Crude polysaccharides concentration is 1.5-1.6g/l00ml.
Embodiment
Below in conjunction with embodiment the present invention is described further:
Produce bacterial classification in the present embodiment and select for use monospore bacillus waterweed to belong to the sphingomonas paucimobilis Sphingomonaspaucimobilis bacterial classification of (pseudomonaselodea), production technique comprises the steps:
A, with original strain bevel bacterial classification;
B, the slant strains for preparing in the A step is made seed liquor through enlarged culturing;
C, with the seed liquor for preparing in the B step by seed liquor: fermention medium is that the inoculum size of 7%-10% inserts fermention medium and inserts and ferment, and wherein fermention medium comprises following component (weight part):
Starch 2.8 dregs of beans 0.2 NH 4NO 30.1 MgSO 40.01
K 2HPO 40.15 CaCO 30.05 water 96.6
The temperature of fermention medium is that 28 ℃, PH are 6.7, ventilation 0.2vvm, and fermentation period is 50 hours.
Processing condition in each processing step are:
Enlarged culturing in the B step comprises shake-flask culture, one-level enlarged culturing and secondary enlarged culturing, slant strains is inserted the shake-flask culture base make shake-flask seed through fermentation, shake-flask seed inserts the substratum of one-level enlarged culturing and makes first order seed through fermentation, the substratum of slant strains in A, the B step, shake-flask culture base,
The substratum of one-level enlarged culturing comprises following component (weight part):
White sugar 0.8 yeast extract paste 0.19 extractum carnis 0.28
Peptone 0.13 agar 2 water 96
The culture condition of slant strains is 28 ℃ of temperature, 72 hours time; The condition of one-level enlarged culturing is an inoculum size 2%, 30 ℃ of culture temperature, pH value 6.7, ventilation 0.14vvm, cycle 23h.
The substratum of secondary enlarged culturing comprises following component (weight part):
White sugar 0.4 starch 0.5 ammonium nitrate 0.28
Bean powder 0.2 K 2HPO 40.15 KH 2PO 40.08 MgSO 40.01
Water 98.3 pH6.5-7.0
The condition of secondary enlarged culturing is an inoculum size 8%, 31 ℃ of culture temperature, pH value 6.7, ventilation 0.12vvm, cycle 19h.
After the viscosity of the fermented liquid in the C step surpasses 2000CP, add the tensio-active agent of 90ppm in fermented liquid, tensio-active agent is selected polysorbate60 for use.
Reaction end in the one-level enlarged culturing is for having a net increase of the OD value more than 0.5, and the mensuration of OD value is selected 622 type spectrophotometers, 650nm wavelength for use.
Reaction end in the secondary enlarged culturing is for having a net increase of the OD value more than 1.0, and the mensuration of OD value is selected 622 type spectrophotometers, 650nm wavelength for use.
Residual sugar is below 0.2% in the fermenting process.
Shaking speed in one-level enlarged culturing and the secondary enlarged culturing is 130 rev/mins, and the fermentor tank tank diameter was than 2.2: 1, and rotating speed is 190 rev/mins, also can adopt no stirring-type to add air-distributor; Fermentor tank in the fermenting process, stirring rake are 4-6 group, and whipped form adopts at interval installation shaft to pusher and disk curved blade type paddle radially, and rotating speed is 15 rev/mins.

Claims (10)

1, the preparation method of microbiological polysaccharide Gellan gum comprises and carries out aerated culture in the aseptic culture medium of selecting bacterial classification inoculation carbonaceous sources, nitrogenous source, inorganic salt and water for use, it is characterized in that it comprises following processing step:
A, with original strain bevel bacterial classification;
B, the slant strains for preparing in the A step is made seed liquor through enlarged culturing;
C, with the seed liquor for preparing in the B step by seed liquor: fermention medium is that the inoculum size of 7%-10% inserts fermention medium and inserts and ferment, and wherein fermention medium comprises following component (weight part):
Starch 2.5-3.0 dregs of beans 0.2 NH 4NO 30.1 MgSO 40.01
K 2HPO 40.15 CaCO 30.05 water 96.49-96.99
Selected bacterial classification is that monospore bacillus waterweed belongs to (pseudomonas elodea) sphingomonaspaucimobilis Sphingomonaspaucimobilis.
The temperature of fermention medium is that 26-36 ℃, PH are 6.5-6.8, ventilation 0.1-0.3vvm, and fermentation period is 50-60 hour.
2, the preparation method of microbiological polysaccharide Gellan gum according to claim 1, it is characterized in that the enlarged culturing in the described B step comprises shake-flask culture, one-level enlarged culturing and secondary enlarged culturing, slant strains is inserted the shake-flask culture base make shake-flask seed through fermentation, shake-flask seed inserts the substratum of one-level enlarged culturing and makes first order seed through fermentation, and the substratum of the substratum of slant strains, shake-flask culture base, one-level enlarged culturing comprises following component (weight part) in A, the B step:
Disaccharides 0.8-1.0 yeast extract paste 0.18-0.2 extractum carnis 0.25-0.3
Peptone 0.1-0.15 agar 2-2.5 water 95.85-96.67
The culture condition of slant strains is temperature 26-36 ℃, 72 hours time; The condition of one-level enlarged culturing is inoculum size 1-3%, culture temperature 26-36 ℃, and pH value 6.5-6.8, ventilation 0.1-0.15vvm, cycle 20-24h.
3, the preparation method of microbiological polysaccharide Gellan gum according to claim 2 is characterized in that the substratum of described secondary enlarged culturing comprises following component (weight part):
Disaccharides 0.3-0.5 starch 0.5 ammonium nitrate 0.25-0.3
Bean powder 0.2 K 2HPO 40.15 KH 2PO 40.08 MgSO 40.01
Water 98.26-98.51 pH6.5-7.0
The condition of secondary enlarged culturing is inoculum size 7-10%, culture temperature 26-36 ℃, and pH value 6.5-6.8, ventilation 0.1-0.15vvm, cycle 18-20h.
4, the preparation method of microbiological polysaccharide Gellan gum according to claim 1, the viscosity that it is characterized in that the fermented liquid in the described C step adds the tensio-active agent of 80-100ppm above behind the 2000CP in fermented liquid.
5, the preparation method of microbiological polysaccharide Gellan gum according to claim 4 is characterized in that described tensio-active agent is selected from polysorbate60, Triton X-100.
6, the preparation method of microbiological polysaccharide Gellan gum according to claim 2 is characterized in that reaction end in the described one-level enlarged culturing for having a net increase of the OD value more than 0.5, and the mensuration of OD value is selected 622 type spectrophotometers, 650nm wavelength for use.
7, according to the preparation method of claim 1 or 3 described microbiological polysaccharide Gellan gums, it is characterized in that reaction end in the described secondary enlarged culturing for having a net increase of the OD value more than 1.0, the mensuration of OD value is selected 622 type spectrophotometers, 650nm wavelength for use.
8,, it is characterized in that residual sugar is below 0.2% in the described fermenting process according to the preparation method of claim 1 or 4 described microbiological polysaccharide Gellan gums.
9, according to the preparation method of claim 1,2 or 3 described microbiological polysaccharide Gellan gums, it is characterized in that the shaking speed in described one-level enlarged culturing and the secondary enlarged culturing is 120-220 rev/min, the fermentor tank tank diameter is than 2-2.5: 1, rotating speed is 180-200 rev/min, also can adopt no stirring-type to add air-distributor; Fermentor tank in the fermenting process, stirring rake are 4-6 group, and whipped form adopts at interval installation shaft to pusher and disk curved blade type paddle radially, and rotating speed is 120-180 rev/min.
10, according to the preparation method of claim 1,2 or 3 described microbiological polysaccharide Gellan gums, it is characterized in that the starch in described secondary enlarged culturing substratum and the fermention medium can replace with hydrolysis sugar, the disaccharides in the substratum of slant medium, shake-flask culture base, one-level enlarged culturing and the substratum of secondary enlarged culturing is selected sucrose, fructose or the mixture of the two for use.
CN 200410064546 2004-11-19 2004-11-19 Process for preparing microbiological polysaccharide Gellan gum Expired - Fee Related CN1269964C (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011035530A1 (en) * 2009-09-25 2011-03-31 浙江大学 Yellow pigments generation deficient sphingomonas strain and application thereof in gellan gum production
CN101240307B (en) * 2008-03-25 2011-11-09 张禹 Method for preparing gellan gum by using waste glucose mother liquor
CN102605017A (en) * 2012-03-30 2012-07-25 杭州健恒生物技术有限公司 Method for preparing gellan gum by fermenting
CN102747016A (en) * 2012-06-28 2012-10-24 福建省农业科学院农业生物资源研究所 Gellan gum efficient production strain and its application
CN103468762A (en) * 2013-09-25 2013-12-25 南开大学 Oxygen carrying agent for increasing fermentation yield of welan gum and application thereof
CN107805649A (en) * 2017-11-10 2018-03-16 浙江帝斯曼中肯生物科技有限公司 A kind of novel fermentation technique for producing gellan gum
CN109251950A (en) * 2018-10-18 2019-01-22 河北鑫合生物化工有限公司 With high-gel strength, high viscosity, high acyl group gellan gum preparation method

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100451106C (en) * 2006-09-12 2009-01-14 山东大学 Sphingomonaspaucimobilis of high-yield gellan gum and use therefor

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101240307B (en) * 2008-03-25 2011-11-09 张禹 Method for preparing gellan gum by using waste glucose mother liquor
WO2011035530A1 (en) * 2009-09-25 2011-03-31 浙江大学 Yellow pigments generation deficient sphingomonas strain and application thereof in gellan gum production
US8685698B2 (en) 2009-09-25 2014-04-01 Zhejiang University Yellow pigments generation deficient Sphingomonas strain and application thereof in gellan gum production
CN102605017A (en) * 2012-03-30 2012-07-25 杭州健恒生物技术有限公司 Method for preparing gellan gum by fermenting
CN102747016A (en) * 2012-06-28 2012-10-24 福建省农业科学院农业生物资源研究所 Gellan gum efficient production strain and its application
CN103468762A (en) * 2013-09-25 2013-12-25 南开大学 Oxygen carrying agent for increasing fermentation yield of welan gum and application thereof
CN107805649A (en) * 2017-11-10 2018-03-16 浙江帝斯曼中肯生物科技有限公司 A kind of novel fermentation technique for producing gellan gum
US11149293B2 (en) 2017-11-10 2021-10-19 Zhejiang Dsm Zhongken Biotechnology Co. Ltd Fermentation method for producing gellan gum
CN109251950A (en) * 2018-10-18 2019-01-22 河北鑫合生物化工有限公司 With high-gel strength, high viscosity, high acyl group gellan gum preparation method
CN109251950B (en) * 2018-10-18 2022-07-01 河北鑫合生物化工有限公司 Preparation method of gellan gum with high gel strength, high viscosity and high acyl

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