CN102618468B - Temperature resistant alcaligenes and application method of alcaligenes for producing Welan gum - Google Patents
Temperature resistant alcaligenes and application method of alcaligenes for producing Welan gum Download PDFInfo
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- CN102618468B CN102618468B CN201210086289.4A CN201210086289A CN102618468B CN 102618468 B CN102618468 B CN 102618468B CN 201210086289 A CN201210086289 A CN 201210086289A CN 102618468 B CN102618468 B CN 102618468B
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- 229920002310 Welan gum Polymers 0.000 title claims abstract description 39
- 241000588986 Alcaligenes Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title abstract description 13
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 239000007788 liquid Substances 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 8
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 8
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 7
- 230000012010 growth Effects 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 3
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- 238000003756 stirring Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 10
- 229910052799 carbon Inorganic materials 0.000 abstract description 10
- 241000588810 Alcaligenes sp. Species 0.000 abstract description 6
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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
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses provides a temperature resistant alcaligenes, is named as (Alcaligenes sp.) HT-1, and is preserved in CCTCC, wherein the preservation number is CCTCC NO: M 2012062. The invention further discloses an application method of the alcaligenes for producing Welan gum. The alcaligenes provided by the invention can ferment at high temperature to produce the Welan gum, and has the advantages that the fermentation time is remarkably shortened; the production intensity is improved; the purposes of energy conservation and environmental protection are achieved; various carbon sources and nitrogen sources can be utilized; the cultivation conditions are slack; and the gum yield and the quality are high.
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 rheological properties and the safety in utilization of its molecular structure diversity, uniqueness.Whole world microbial polysaccharide demand is about ten thousand ton/years of 15-20 at present, and annual value of production can reach hundred million dollars of 150-200, and output and annual increment are all more than 10%, and some novel polysaccharide annual increments are more than 30%.The eighties in last century, U.S. Kelco company found that successively one group of novel microbial polysaccharide is gelling gum class polysaccharide, has caused the great interest of scientific circles and industry member after xanthan gum.Welan gum is a member of gelling gum family, and its monomer composition is 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 the industries such as printing ink, food, textile printing and dyeing, agricultural chemicals, makeup, medicine.
At present, the production of Welan gum is mainly to adopt microbe fermentation method, and bacterial classification is the basis of fermentation, improves by strain 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 production level of microorganism also depends on best envrionment conditions, temperature is on the impact of fermentation and regulate to control be affect one of most important factor of organism growth breeding, is mainly manifested in that cell growth, product are synthesized, the aspects such as the physical properties of fermented liquid and biosynthesizing direction.In certain scope, along with the rising of temperature, in microbe, the biochemical reaction enzyme activity increases, and the growth and breeding speed of cell is accelerated; 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, the heat of fermentation that discharges due to thalline usually surpasses the suitableeest culture temperature of microorganism, and therefore generally need not heat needs cooling 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 a strain production Welan gum, be Alcaligenes NXG-3 (CGMCC No.1737), the temperature of the fermentative production Welan gum of this bacterial strain is 25～35 ℃, 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 be to provide the application of above-mentioned Alcaligenes in the preparation Welan gum.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
One strain heat-resistance type Alcaligenes, its Classification And Nomenclature are Alcaligenes (Alcaligenes sp.) HT-1, have been preserved in Chinese Typical Representative 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 as follows:
1.. the preparation of Alcaligenes CGMCC No.1737 bacteria suspension:
Alcaligenes CGMCC No.1737 is inoculated in aseptic seed culture medium, under the condition of 25～32 ℃, shaking culture 8-12 hour, seed liquor is repeatedly centrifugal and make bacteria suspension with after damping fluid washing, wherein in seed culture medium, each amounts of components is counted by each component and substratum percent weight in volume: 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 plasma body shone 1-10 minute, irradiation dose is 1-10SLM, and temperature is 20-25 ℃, the bacteria suspension of processing repeatedly after centrifuge washing dilution spread on flat board, divide to be arranged in 36-40 ℃ of incubator, establish a gradient for every 2 ℃, cultivated 2-3 days.
3.. the shaking flask screening:
The picking growth is very fast, and healthy and strong full moistening macrocolony carries out shake flask fermentation and sieves again.In fermention medium, each amounts of components is counted by each component and substratum percent weight in volume: the carbon source consumption is 2～6% of substratum, and the nitrogenous source consumption is 0.1～1% of substratum, and the inorganic salt consumption is 0.01～1% of substratum, and all the other are water.
Select viscosity higher after repeatedly going down to posterity, temperature tolerance is good, and lawn is plentiful, and is moistening, glossy, without the bacterium colony that irregular lawn generates, isolates above-mentioned Alcaligenes.
Heat-resistance type Alcaligenes CCTCC NO:M 2012062 bacterial strains that the present invention obtains have following character:
(1) colonial morphology feature:
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 biochemistry characteristic:
A. culture temperature: 36～40 ℃, optimum temperuture is 37 ℃;
B. grow in pH5～10 scopes;
C. pigment formation: produce yellow pigment;
D. anti-NaCl concentration: can grow in 5% concentration.
(3) antibiotic susceptibility test:
The HT-1 bacterial strain is as follows to following antibiotic minimal inhibitory concentration:
(4) nutritional character:
Do not need to add somatomedin in the substratum of Alcaligenes HT-1, can utilize multiple compounds as carbon source, these materials both can use separately, also can the composite in the proper ratio carbon source of serving as.Organonitrogen or inorganic nitrogen can use as nitrogenous source.In substratum, each amounts of components proportioning is counted by each component and substratum percent weight in volume: the carbon source consumption is that 2～7% of substratum (is to contain carbon source 2-7 gram in every 100ml substratum, lower same), the nitrogenous source consumption is 0.1～1% of substratum, the inorganic salt consumption is 0.01～1% of substratum, and all the other are water.Carbon source generally adopts one or more in glucose, sucrose, fructose, Zulkovsky starch, molasses, starch hydrolyzate; Nitrogenous source is peptone, yeast extract paste, corn steep liquor, bean cake powder, cottonseed meal, urea, (NH
4)
2SO
4, NH
4One or more in Cl; Inorganic salt are the 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: with preserving number be CCTCC NO:M 2012062 inoculation in carbonaceous sources, nitrogenous source, inorganic salt and water without bacteria fermentation culture medium in, ventilate under the condition of proper growth, 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 starch hydrolyzate, and concentration is 20～70g/L; Described nitrogenous source is peptone, yeast extract paste, corn steep liquor, bean cake powder, cottonseed meal, urea, (NH
4)
2SO
4And NH
4The mixture of any one or a few in Cl, concentration are 1～10g/L; Described inorganic salt are any one or a few in sylvite, magnesium salts, phosphoric acid salt and 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 extracting Welan gum from the Welan gum fermented liquid is techniques well known, but the extracting method of describing in concrete referenced patent ZL200610088356.0.
Specifically:
The method of utilizing temperature tolerance Alcaligenes HT-1 to prepare Welan gum provided by the invention, with CCTCC NO:M2012062 bacterial strain slant activation after one day (slant medium employing beef-protein medium), cultivated on the substratum that contains carbon source, nitrogenous source and inorganic salt 30～35 hours, can generate the Welan gum fermented liquid of 40～45g/L, usually fermentation condition also comprises: 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%, the content of each component is percent weight in volume, namely g/100ml, as follows.36～40 ℃ of leavening temperatures, better is 37 ℃; The initial pH scope of substratum is 6.5～8.0, and better 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 the organic solvents such as ethanol, Virahol.
(2) product has good temperature tolerance, processes half an hour under 150 ℃, and viscosity is constant; In the scope of pH2～13, stable in properties.
(3) product is after 4mol/L sulphuric acid hydrolysis, aldoononitrile acetate are processed, 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 as follows:
(4) adopt periodate oxidation method, identified that the glycosidic link type of this product 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 Starch Hydrolysis liquid glucose (place of glucose or sucrose) and bean cake powder (substituting yeast extract paste or peptone) as the Carbon and nitrogen sources of Welan gum fermention medium, the fermentation raw material cost to be declined to a great extent.
(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, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment only is used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
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 seed culture medium, under the condition of 25～32 ℃, shaking culture 10 hours, seed liquor is repeatedly centrifugal and make bacteria suspension with after 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 plasma body irradiation 5 minutes, and irradiation dose is 10SLM, and temperature is 25 ℃, and repeatedly dilution spread is on flat board after centrifuge washing for the bacteria suspension of processing, and under 30-40 ℃ of condition, every 2 ℃ arrange a thermograde, cultivate 2-3 days.
3.. the shaking flask screening:
Select after mutagenesis on culture dish healthy and strong full, moistening macrocolony and carry out shake flask fermentation and sieve again, fermentation shake flask substratum: glucose 40g/L, soybean cake powder 4g/L, peptone 1g/L, K
2HPO
42g/L, MgSO
40.1g/L, pH7.2.
Select viscosity higher after repeatedly going down to posterity, temperature tolerance is good, and lawn is plentiful, and is moistening, glossy, and the bacterium colony without irregular lawn generates obtains energy-conservation heat-resistance type Alcaligenes of the present invention.
Embodiment 2:
Slant medium: peptone 10g/L, extractum carnis 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 triangular flask of 250ml volume sterilized 15 minutes for 121 ℃.
Be Alcaligenes sp.HT-1 37 ℃ of cultivation 48-72h on slant medium of CCTCC NO:M 2012062 with preserving number, then connect this bacterium of ring in fermention medium, cultivate 35h for 37 ℃, shaking flask rotating speed 220r/min, in the fermented liquid that obtains at last, the content of Welan gum is 34.5g/L.
Embodiment 3:
Slant medium: peptone 10g/L, extractum carnis 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 triangular flask of 250ml volume sterilized 15 minutes for 121 ℃.Be Alcaligenes sp.HT-1 37 ℃ of cultivation 48-72h on slant medium of CCTCC NO:M2012062 with preserving number, then connect this bacterium of ring in fermention medium, cultivate 30h for 37 ℃, shaking flask rotating speed 220r/min, in the fermented liquid that obtains at last, the content of Welan gum is 40.2g/L.
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, seed liquor is pressed in the cooled fermented liquid of inoculum size access of fermentating liquid volume 5% (v/v), cultivate (ventilation 1vvm for 37 ℃, mixing speed is 600r/min) 32h, contain Welan gum 44.9g/L in fermented liquid, fermentation broth viscosity can reach 14000cp.
Embodiment 4:
1750 gram W-Gums are mixed with appropriate water, add appropriate high-temperatureα-amylase liquid, water is settled to 35L, and in the 50L mechanical agitating fermentation tank of packing into, then 90 ℃ of insulation liquefaction 10 minutes add bean cake powder 280g, K
2HPO
470g, MgSO
43.5g, pH7.2, then be warming up to 121 ℃ of steam sterilizings 10 minutes.Seed liquor is pressed in the cooled fermented liquid of inoculum size access of fermentating liquid volume 5% (v/v), cultivated 35 hours for 37 ℃, contain Welan gum 40g/L in fermented liquid, ripe fermented liquid is warming up to 80～90 ℃ of deactivations 15 minutes, the concentration that adds the fermentating liquid volume twice after cooling is 95% ethanol, 4 ℃ of incubated overnight, centrifugal removal supernatant liquor, throw out dries by the fire 1h to constant weight in 100 ℃ of baking ovens, namely get the Welan gum finished product.
Embodiment 5:
The rear extraction process of Welan gum:
A. coarse filtration: get the maturing fermentation liquid that 50mL obtains by embodiment 2, regulating pH with HCl is 6.5～7.5, carries out coarse filtration, and the coarse filtration mode is selected 110 order industrial filter cloths, adds 0.5g flocculating aids (diatomite) in fermented liquid, suction filtration.
B. ultrafiltration and concentration: will concentrate in ultra-fine filter through the Welan gum fermented liquid of coarse filtration, the ultra-filtration membrane molecular weight that dams is 10
6Dalton, working pressure is 0.2MPa, controls temperature 50 C, face speed 4m/s.
C. alcohol is analysed: the fermented liquid after ultrafiltration being added 95% industrial alcohol, ethanol consumption under constantly stirring is long-pending 1.2 times of Welan gum ultrafiltration and concentration liquid, standing formation precipitation.
D. dry and pulverize: will precipitate piece and blend, in the filter cloth of packing into, suction filtration to absence of liquid flows out, and 55 ℃ of vacuum of throw out are done oven dry 3 hours, pulverizes and is product.
Claims (3)
1. a strain heat-resistance type Alcaligenes, its Classification And Nomenclature be Alcaligenes (
AlcaligenesSp.) HT-1 has been preserved in Chinese Typical Representative culture collection center, preserving number CCTCC NO:M2012062.
2. the application of heat-resistance type Alcaligenes claimed in claim 1 in Welan gum is produced.
3. application according to claim 2, it is characterized in that, concrete grammar is: with preserving number be CCTCC NO:M2012062 inoculation in carbonaceous sources, nitrogenous source, inorganic salt and water without bacteria fermentation culture medium in, ventilate under the condition of proper growth, stir culture, the Welan gum fermented liquid of generation obtains Welan gum through extraction; 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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| CN1888054A (en) * | 2006-07-13 | 2007-01-03 | 南京工业大学 | Bacillus alcaligenes and its application in preparing Weilan gum |
| 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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|---|---|---|---|---|
| CN1888054A (en) * | 2006-07-13 | 2007-01-03 | 南京工业大学 | Bacillus alcaligenes and its application in preparing Weilan gum |
| 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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