CN102618468A - Temperature-resistant alcaligenes and application thereof in welan gum production - Google Patents
Temperature-resistant alcaligenes and application thereof in welan gum production Download PDFInfo
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- CN102618468A CN102618468A CN2012100862894A CN201210086289A CN102618468A CN 102618468 A CN102618468 A CN 102618468A CN 2012100862894 A CN2012100862894 A CN 2012100862894A CN 201210086289 A CN201210086289 A CN 201210086289A CN 102618468 A CN102618468 A CN 102618468A
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- 229920002310 Welan gum Polymers 0.000 title claims abstract description 39
- 241000588986 Alcaligenes Species 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- 239000001888 Peptone Substances 0.000 claims description 13
- 108010080698 Peptones Proteins 0.000 claims description 13
- 235000019319 peptone Nutrition 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 229920002472 Starch Polymers 0.000 claims description 7
- 241000588810 Alcaligenes sp. Species 0.000 claims description 6
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 6
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 6
- 239000008107 starch Substances 0.000 claims description 6
- 235000019698 starch Nutrition 0.000 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 229930091371 Fructose Natural products 0.000 claims description 3
- 239000005715 Fructose Substances 0.000 claims description 3
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 235000012343 cottonseed oil Nutrition 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 159000000003 magnesium salts Chemical class 0.000 claims description 3
- 235000012054 meals Nutrition 0.000 claims description 3
- 235000013379 molasses Nutrition 0.000 claims description 3
- 150000003016 phosphoric acids Chemical class 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 4
- 238000004321 preservation Methods 0.000 abstract description 3
- 238000004134 energy conservation Methods 0.000 abstract description 2
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 10
- 239000000725 suspension Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 6
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- 150000004676 glycans Chemical class 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 238000001914 filtration Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 2
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 235000019890 Amylum Nutrition 0.000 description 1
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- 102000004190 Enzymes Human genes 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000005842 biochemical reaction Methods 0.000 description 1
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- 238000009395 breeding Methods 0.000 description 1
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- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a strain of temperature-resistant Alcaligenes, which is classified and named as Alcaligenes HT-1, is preserved in China center for type culture Collection, and has a preservation number of CCTCC NO: m2012062. The invention also discloses application of the strain in welan gum production. The strain can be fermented at high temperature to produce welan gum, so that the fermentation time is obviously shortened, the production strength is improved, and the energy conservation and environmental protection are realized. The strain can utilize various carbon sources and nitrogen sources, the culture conditions are extensive, the gel yield is high, and the quality is good.
Description
Technical field
The invention belongs to the fermentation engineering field, relate to a kind of heat-resistance type Alcaligenes (Alcaligenes sp.) HT-1 and the application in welan gum (welan gum) is produced thereof.
Background technology
Microbial polysaccharide is with a wide range of applications in many fields of industrial production and life with physics and the rheological properties and the safety in utilization of its molecular structure variety, uniqueness.At present whole world microbial polysaccharide demand is about ten thousand tons/year of 15-20, and annual value of production can reach hundred million dollars of 150-200, and output and year increment be all more than 10%, and some in novel polysaccharide year increment more than 30%.The eighties in last century, U.S. Kelco company found that successively one group of new microbial polysaccharide is a gelling gum class polysaccharide, has caused the great interest of scientific circles and industry member after XG 550.Welan gum is a member of gelling gum family, and its set of monomers becomes glucose, glucuronic acid, seminose and rhamnosyl.Welan gum has many good and unique physicochemical properties, can be used as thickening material, suspension agent, stablizer etc. and is widely used in oil, concrete, coating industry, also can be used for industries such as printing ink, food, textile printing and dyeing, agricultural chemicals, makeup, medicine.
At present, the production of welan gum mainly is to adopt microbe fermentation method, and bacterial classification is the basis of fermentation, through the strain improvement improvement, filters out the production bacterium of high and stable yields, can make fermenting process reach the purpose of economical and efficient.In addition; The fermentative prodn level of mikrobe also depends on best envrionment conditions; Temperature is to the influence of fermentation and to regulate control be influence organism growth to breed one of most important factor, shows mainly that cell growth, product synthesize, aspects such as the physical properties of fermented liquid and biosynthesizing direction.In certain scope, along with the rising of temperature, the biochemical reaction enzyme activity increases in the microbe body, and the growth and breeding of cell speeds up; Along with the rising of temperature, the solubleness of gas in solution reduces, and the transfer rate of oxygen also can change.In industrialized fermenting process, because the heat of fermentation that thalline discharges usually surpasses the righttest culture temperature of mikrobe, therefore generally need not heat needs the refrigerative situation more on the contrary.Therefore the seed selection heat-resistance type is produced bacterial strain and not only is beneficial to the regulation and control fermenting process, and energy-conserving and environment-protective.
Chinese patent 200610088356.0 discloses the bacterial strain of strain production welan gum; Be Alcaligenes NXG-3 (CGMCC No.1737), the temperature of the fermentative prodn welan gum of this bacterial strain is 25～35 ℃, and optimum temperuture is 30 ℃; Incubation time is 36～72 hours, and the highest fermentation yield is 27g/L.
Summary of the invention
Technical problem to be solved by this invention is on the basis of Chinese patent 200610088356.0, and a strain heat-resistance type Alcaligenes is provided.
The present invention also has the technical problem that solves to provide the application of above-mentioned Alcaligenes in the preparation welan gum.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
One strain heat-resistance type Alcaligenes, its classification called after Alcaligenes (Alcaligenes sp.) HT-1 has been preserved in Chinese typical culture collection center; Preservation address: China. Wuhan. Wuhan University; Postcode 430072; Preserving number CCTCC NO:M 2012062.Preservation date is: on March 7th, 2012.With this bacterium as producing bacterial strain.
Heat-resistance type Alcaligenes of the present invention is to be obtained by Alcaligenes CGMCC No.1737 (ZL200610088356.0) mutagenesis screening.Concrete mutagenesis screening step is following:
1.. the preparation of Alcaligenes CGMCC No.1737 bacteria suspension:
Alcaligenes CGMCC No.1737 is inoculated in the aseptic seed culture medium; Under 25～32 ℃ condition, shaking culture 8-12 hour, that seed liquor is repeatedly centrifugal and process bacteria suspension with after the damping fluid washing; Wherein each amounts of components is counted by each component and substratum percent weight in volume in the seed culture medium: glucose 1～2%; Peptone 0.1～0.5%, yeast extract paste 0.2～0.8%, all the other are water.
2.. plasma body mutagenesis and thermograde screening:
Bacteria suspension is placed under the plasma body irradiation 1-10 minute, and irradiation dose is 1-10SLM, and temperature is 20-25 ℃; The bacteria suspension of handling repeatedly dilutes behind the centrifuge washing to be coated on the flat board; Divide to be arranged in the 36-40 ℃ of incubator, establish a gradient for per 2 ℃, cultivated 2-3 days.
3.. shake the bottle screening:
The picking growth is very fast, and healthy and strong full moistening macrocolony carries out shake flask fermentation and sieves again.Each amounts of components is counted by each component and substratum percent weight in volume in the fermention medium: the carbon source consumption is 2～6% of a substratum, and the nitrogenous source consumption is 0.1～1% of a substratum, and the inorganic salt consumption is 0.01～1% of a substratum, and all the other are water.
It is higher after repeatedly going down to posterity, to select viscosity, and temperature tolerance is good, and lawn is plentiful, and is moistening, glossy, does not have the bacterium colony that irregular lawn generates, and isolates above-mentioned Alcaligenes.
Heat-resistance type Alcaligenes CCTCC NO:M 2012062 bacterial strains that the present invention obtains have property:
(1) colonial morphology characteristic:
On nutrient agar medium, to cultivate 20-22h for 36 ℃ and yellow small colonies occurs, bacterium colony is rounded, smooth surface, sticking shape, center projections is opaque.Proper extension is cultivated, and colony colour is deepened, and the size and dimension of cell changes very little.
(2) physiology and biochemical characteristic:
A. culture temperature: 36～40 ℃, optimum temperuture is 37 ℃;
B. in pH5～10 scopes, grow;
C. pigment produces: produce yellow pigment;
D. anti-NaCl concentration: can grow in 5% concentration.
(3) antibiotic susceptibility test:
The HT-1 bacterial strain is following to following antibiotic minimal inhibitory concentration:
(4) nutritional character:
Need not add growth factor in the substratum of Alcaligenes HT-1, can utilize multiple compound as carbon source, these materials both can use separately, also can compositely in the proper ratio serve as carbon source.Organonitrogen or inorganic nitrogen can use as nitrogenous source.Each amounts of components proportioning is counted by each component and substratum percent weight in volume in the substratum: the carbon source consumption is that 2～7% of substratum (is to contain carbon source 2-7 gram in every 100ml substratum; Down together); The nitrogenous source consumption is 0.1～1% of a substratum; The inorganic salt consumption is 0.01～1% of a substratum, and all the other are water.Carbon source generally adopts one or more in glucose, sucrose, fructose, Zulkovsky starch, molasses, the starch hydrolyzate; Nitrogenous source is peptone, yeast extract paste, steeping water, bean cake powder, cottonseed meal, urea, (NH
4)
2SO
4, NH
4Among the Cl one or more; Inorganic salt are inorganic salts commonly used such as sylvite, magnesium salts, phosphoric acid salt, vitriol.
The application of above-mentioned heat-resistance type Alcaligenes CCTCC NO:M 2012062 in welan gum is produced.
Concrete grammar is: the inoculation that with preserving number is CCTCC NO:M 2012062 is in the no bacteria fermentation culture medium of carbonaceous sources, nitrogenous source, inorganic salt and water; Under the condition of proper growth, ventilate, stir culture, the welan gum fermented liquid of generation obtains welan gum through extraction.
Wherein, described carbon source is any one or a few the mixture in glucose, sucrose, fructose, Zulkovsky starch, molasses and the starch hydrolyzate, and concentration is 20～70g/L; Described nitrogenous source is peptone, yeast extract paste, steeping water, bean cake powder, cottonseed meal, urea, (NH
4)
2SO
4And NH
4The mixture of any one or a few among the Cl, concentration are 1～10g/L; Described inorganic salt are any one or a few in sylvite, magnesium salts, phosphoric acid salt and the vitriol, and concentration is 0.1～10g/L.
Wherein, the condition of described proper growth is: initial pH is 6.5～8.0, culture temperature is that 36～40 ℃, incubation time are 30～35 hours.
Wherein, the method for from the welan gum fermented liquid, extracting welan gum is a techniques well known, but the process for extracting of describing among the concrete referenced patent ZL200610088356.0.
Specifically:
The method of utilizing temperature tolerance Alcaligenes HT-1 to prepare welan gum provided by the invention; Be with CCTCC NO:M2012062 bacterial strain slant activation after one day (slant medium employing beef-protein medium); On the substratum that contains carbon source, nitrogenous source and inorganic salt, cultivated 30～35 hours, and can generate the welan gum fermented liquid of 40～45g/L, fermentation condition also comprises usually: substratum is: glucose or W-Gum hydrolyzed solution 1～6%; Peptone or bean cake powder 0.1～1%, MgSO
40.01～0.5%, K
2HPO
40.01～0.5%, each components contents is percent weight in volume, promptly g/100ml is as follows.36～40 ℃ of leavening temperatures, preferable is 37 ℃; The initial pH scope of substratum is 6.5～8.0, and preferable is 6.8～7.2; Rotating speed 100-200r/min.Such fermentation condition both had been applicable to shake flask fermentation, also can be amplified in the fermentor tank of 100L to use.
The welan gum of the present invention's preparation has following physico-chemical property:
(1) product is soluble in water, 1% welan gum solution viscosity can reach 2500-3500cp (condition determination: NDJ-1 type viscometer, No. 4 rotors, 60rpm), welan gum is insoluble to organic solvents such as ethanol, Virahol.
(2) product has good temperature tolerance, handles half a hour down at 150 ℃, and viscosity is constant; In the scope of pH2～13, stable in properties.
(3) product is after 4mol/L sulphuric acid hydrolysis, sugared nitrile acetic ester derivatization treatment, and the monosaccharide component that gas chromatographic detection gets welan gum is: glucose, seminose, rhamnosyl; Chemical method records and also contains glucuronic acid in this product.Structural formula is following:
(4) adopt periodate oxidation method, identified that the glycosidic link type of these article is mainly 1 → 4 key and 1 → 3 key.
Advantage of the present invention:
(1) the present invention screens a strain heat-resistance type welan gum and produces bacterium, and this bacterium can produce welan gum under 36-40 ℃ of condition, and fermentation time significantly shortens, energy-conserving and environment-protective.
(2) can use thick amylum hydrolysate of the sugar liquid (place of glucose or sucrose) and bean cake powder (substituting yeast extract paste or peptone), the fermentation raw material cost to be declined to a great extent as the carbon source and the nitrogenous source of welan gum fermention medium.
(3) fermentation yield of bacterial strain significantly improves, and reaches 40～45g/L welan gum output, is adapted to suitability for industrialized production.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described content of embodiment only is used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1: the heat-resistance type welan gum is produced the seed selection of bacterial strain
1.. the preparation of Alcaligenes bacteria suspension:
Alcaligenes CGMCC No.1737 is inoculated in the seed culture medium, under 25～32 ℃ condition, shaking culture 10 hours; Seed liquor is repeatedly centrifugal and process bacteria suspension with after the damping fluid washing, seed culture medium wherein: glucose 20g/L, peptone 3g/L; Yeast extract paste 1g/L, K
2HPO
42g/L, MgSO
40.1g/L, pH7.2.
2.. plasma body mutagenesis and thermograde seed selection:
Bacteria suspension is placed under the plasma body irradiation 5 minutes, and irradiation dose is 10SLM, and temperature is 25 ℃, and the bacteria suspension of handling repeatedly dilutes behind the centrifuge washing to be coated on the flat board, and under the 30-40 ℃ of condition, per 2 ℃ are provided with a thermograde, cultivate 2-3 days.
3.. shake the bottle screening:
Selecting after the mutagenesis on the petridish healthy and strong full, moistening macrocolony carries out shake flask fermentation and sieves the fermentation shake flask substratum again: glucose 40g/L, soybean cake powder 4g/L, peptone 1g/L, K
2HPO
42g/L, MgSO
40.1g/L, pH7.2.
It is higher after repeatedly going down to posterity, to select viscosity, and temperature tolerance is good, and lawn is plentiful, and is moistening, glossy, does not have the bacterium colony that irregular lawn generates, and obtains energy-conservation heat-resistance type Alcaligenes of the present invention.
Embodiment 2:
Slant medium: peptone 10g/L, Carnis Bovis seu Bubali cream 3g/L, NaCl 5g/L, agar 20g/L, pH7.2.
Fermention medium: glucose 50g/L, yeast extract paste 4g/L, peptone 1g/L, K
2HPO
42g/L, MgSO
40.1g/L, pH7.2.Liquid amount 50ml in the 250ml volumetrical triangular flask sterilized 15 minutes for 121 ℃.
With preserving number is Alcaligenes sp.HT-1 37 ℃ of cultivation 48-72h on slant medium of CCTCC NO:M 2012062; Connect this bacterium of ring then in fermention medium; Cultivate 35h for 37 ℃, shake a bottle rotating speed 220r/min, the content of welan gum is 34.5g/L in the fermented liquid that obtains at last.
Embodiment 3:
Slant medium: peptone 10g/L, Carnis Bovis seu Bubali cream 3g/L, NaCl 5g/L, agar 20g/L, pH7.2.
Fermention medium: glucose 60g/L, peptone 8g/L, K
2HPO
42g/L, MgSO
40.1g/L, pH7.2.Liquid amount 50ml in the 250ml volumetrical triangular flask sterilized 15 minutes for 121 ℃.With preserving number is Alcaligenes sp.HT-1 37 ℃ of cultivation 48-72h on slant medium of CCTCC NO:M2012062; Connect this bacterium of ring then in fermention medium; Cultivate 30h for 37 ℃, shake a bottle rotating speed 220r/min, the content of welan gum is 40.2g/L in the fermented liquid that obtains at last.
Embodiment 4:
With glucose 300g, soybean cake powder 40g/L, K
2HPO
410g, MgSO
40.5g, pH7.2, the water constant volume is made into fermented liquid to 5.0L, in the 7.5 liters of glass stirred fermentors of packing into, 121 ℃ of steam sterilizings 15 minutes.Preparation seed culture medium: glucose 20g/L, peptone 4g/L, yeast extract paste 1g/L, K
2HPO
42g/L, MgSO
40.1g/L, pH7.2.Meeting Alcaligenes sp.HT-1 two encircles in seed culture medium; Cultivate 14h for 37 ℃ and get seed liquor; The inoculum size of seed liquor by fermentating liquid volume 5% (v/v) inserted in the cooled fermented liquid 37 ℃ of cultivations (ventilation 1vvm, mixing speed are 600r/min) 32h; Contain welan gum 44.9g/L in the fermented liquid, fermentation broth viscosity can reach 14000cp.
Embodiment 4:
1750 gram W-Gums are mixed with an amount of water, add an amount of high-temperature liquid, water is settled to 35L, and in the 50L mechanical agitating fermentation tank of packing into, 90 ℃ of insulations were liquefied 10 minutes, added bean cake powder 280g then, K
2HPO
470g, MgSO
43.5g pH7.2 was warming up to 121 ℃ of steam sterilizings 10 minutes again.The inoculum size of seed liquor by fermentating liquid volume 5% (v/v) inserted in the cooled fermented liquid, cultivated 35 hours, and contained welan gum 40g/L in the fermented liquid for 37 ℃; Sophisticated fermented liquid is warming up to 80～90 ℃ of deactivations 15 minutes; The concentration of cooling back adding fermentating liquid volume twice is 95% ethanol, 4 ℃ of incubated overnight, centrifugal removal supernatant; Throw out dries by the fire 1h to constant weight in 100 ℃ of baking ovens, promptly get the welan gum finished product.
Embodiment 5:
The back extraction process of welan gum:
A. coarse filtration: get the maturing fermentation liquid that 50mL obtains by embodiment 2, using HCl to regulate pH is 6.5～7.5, carries out coarse filtration, and the coarse filtration mode is selected 110 order industrial filter cloths for use, adds 0.5g flocculating aids (zeyssatite) in the fermented liquid, suction filtration.
B. ultrafiltration and concentration: the welan gum fermented liquid that will pass through coarse filtration concentrates in ultra-fine filter, and the ultra-filtration membrane molecular weight that dams is 10
6Dalton, working pressure is 0.2MPa, 50 ℃ of controlled temperature, face speed 4m/s.
C. alcohol is analysed: with the industrial alcohol of the adding 95% under constantly stirring of the fermented liquid after the ultrafiltration, amount of ethanol is 1.2 times that welan gum ultrafiltration and concentration liquid amasss, leaves standstill and forms deposition.
D. dry and pulverize: pellet is blended, and suction filtration to absence of liq flows out in the filter cloth of packing into, and 55 ℃ of vacuum of throw out are done oven dry 3 hours, pulverizes and is product.
Claims (5)
1. a strain heat-resistance type Alcaligenes, its classification called after Alcaligenes (Alcaligenes sp.) HT-1 has been preserved in Chinese typical culture collection center, preserving number CCTCC NO:M 2012062.
2. the application of the described heat-resistance type Alcaligenes of claim 1 in welan gum is produced.
3. application according to claim 2; It is characterized in that; Concrete grammar is: the inoculation that with preserving number is CCTCC NO:M 2012062 is in the no bacteria fermentation culture medium of carbonaceous sources, nitrogenous source, inorganic salt and water; Under the condition of proper growth, ventilate, stir culture, the welan gum fermented liquid of generation obtains welan gum through extraction.
4. application according to claim 3 is characterized in that, described carbon source is any one or a few the mixture in glucose, sucrose, fructose, Zulkovsky starch, molasses and the starch hydrolyzate, and concentration is 20～70g/L; Described nitrogenous source is peptone, yeast extract paste, steeping water, bean cake powder, cottonseed meal, urea, (NH
4)
2SO
4And NH
4The mixture of any one or a few among the Cl, concentration are 1～10g/L; Described inorganic salt are any one or a few in sylvite, magnesium salts, phosphoric acid salt and the vitriol, and concentration is 0.1～10g/L.
5. application according to claim 3 is characterized in that, the condition of described proper growth is: initial pH is 6.5～8.0, culture temperature is that 36～40 ℃, incubation time are 30～35 hours.
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| CN104404101A (en) * | 2014-11-10 | 2015-03-11 | 河北恒标生物科技有限公司 | Preparation method of paste welan gum |
| CN110144318A (en) * | 2019-04-22 | 2019-08-20 | 南京工业大学 | Pigment-free low-molecular-weight welan gum production strain and construction method and application thereof |
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| CN101275154A (en) * | 2008-01-22 | 2008-10-01 | 江南大学 | Production method for microbial polysaccharide welan gum |
| CN101942491A (en) * | 2010-08-10 | 2011-01-12 | 江南大学 | Method for efficiently separating and extracting welan gum from welan gum fermentation liquor |
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| CN110144318A (en) * | 2019-04-22 | 2019-08-20 | 南京工业大学 | Pigment-free low-molecular-weight welan gum production strain and construction method and application thereof |
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