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CN103468762A - Oxygen carrying agent for increasing fermentation yield of welan gum and application thereof - Google Patents

Oxygen carrying agent for increasing fermentation yield of welan gum and application thereof Download PDF

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Publication number
CN103468762A
CN103468762A CN2013104409817A CN201310440981A CN103468762A CN 103468762 A CN103468762 A CN 103468762A CN 2013104409817 A CN2013104409817 A CN 2013104409817A CN 201310440981 A CN201310440981 A CN 201310440981A CN 103468762 A CN103468762 A CN 103468762A
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China
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fermentation
tween
welan gum
wei lan
lan glue
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CN2013104409817A
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Chinese (zh)
Inventor
马挺
李国强
罗影
吴萌萌
周洁芳
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Nankai University
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Nankai University
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Abstract

The invention relates to an oxygen carrying agent for increasing the fermentation yield of welan gum and application thereof. The welan gum is macromolecular microbial polysaccharides which are synthesized and secreted into the extracellular space by sphingomonas. Due to excellent fake plastic rheological property, suspension and emulsification properties, compatibility and stability, a growing concern on the welan gum exists. At the initial stage of the fermentation of sphingomonas TP-5 (collection number: CGMCC (China General Microbiological Culture Collection Center) No. 3097), 0.05% (m/v) of TritonX-100, 0.2% (m/v) of Tween-20, 0.2% (m/v) of Tween-40 or 0.3% (m/v) of Tween-80 are respectively added into a fermentation culture medium, and then, the yield of the welan gum can be respectively increased by 21.80%, 22.73%, 36.62% and 28.12%. Surfactant type oxygen carrying agents can be expected to be applied to the fermentation industry of high-viscosity polysaccharides, such as the welan gum and the like.

Description

Take oxygen agent and application thereof for increasing Wei Lan glue fermentation yield
Technical field
The invention belongs to the microbial fermentation technology field, specifically, it relates in several fermenting processs the function of carrying oxygen material that may affect Wei Lan glue output, and the application in high viscosity biological polyoses fermentative production.
Background technology
Wei Lan glue (welan gum) is also referred to as welan gum, welan gum, Welan gum or Warren glue, be Sphingol single-cell ( sphingomonassp. TP-5) take carbohydrate as main raw material a kind of sphingosine glue base polymer synthetic through aerobic fermentation.The structural framework of Wei Lan glue is comprised of the unit of D-Glucose, D-glucuronic acid, D-Glucose and L-rhamnosyl, and side chain consists of the L-seminose of strand or the L-rhamnosyl of strand.Due to its false creeping sex change, suspension and emulsifying property, ph stability and temperature tolerance with good tackifying, uniqueness, make Wei Lan glue be with a wide range of applications in a plurality of fields such as oil production, food, medicine, makeup and building trade.
Wei Lan glue be U.S. Kelco company after xanthan gum, gelling gum, develop the eighties one of microbial polysaccharide of market outlook arranged most, the Kelco company of the U.S. is the globally unique production of Wei Lan glue, supplier up to now, and a year supply only has 10,000 tons, far do not reach the actual demand amount of Wei Lan glue, supply-less-than-demand, make the market value of Wei Lan glue high.Simultaneously huge commercial profit is monopolized the production technology of Wei Lan glue, about paper and the patent of its industrial production technology rarely has report.And the external report for Wei Lan glue concentrates on structure, character and application facet mostly at present, and the report aspect fermentation and production seldom.China starts the fermentative production of Wei Lan glue and characteristic are studied in recent years, yet its process of industrialization is but very slow, and this is mainly due to the factors such as fermentation costs due to Wei Lan glue is high, the unit productive rate is low.
Summary of the invention
The objective of the invention is to solve the problem that the fermentative production cost is high, output is on the low side of the high viscosity polysaccharide such as Wei Lan glue, provide several be adapted at adding in substratum for increasing Wei Lan glue fermentation yield take the oxygen agent, and in the application in the fermentative production field of high viscosity polysaccharide.
A strain Sphingol single-cell TP-5(preserving number CGMCC No.3097 provided by the invention, this bacterial strain is open in the ZL200910069960.2 patent) add in fermentative production Wei Lan glue process take the oxygen agent, it is described that to take the oxygen agent be surfactant-based oxygen agent TritonX-100, Tween-20, Tween-40 or the Tween-80 of taking.
The fermentation initial stage, at fermention medium (m/v) (sucrose 44.68g/L, Na 2hPO 49.28g/L, CaCO 34.65g/L, Zein powder 1g/L, NaNO 34g/L, MgSO 40.8g/L, and by FeCl 20.0036mmol/L, ZnSO 40.01mmol/L, MnCl 20.01mmol/L and NiSO 40.02mmol/L add respectively 0.05%(m/v the trace element solution 1mL formed)) TritonX-100, Tween-20 0.2%(m/v), Tween-40 0.2%(m/v) or 0.3%(m/v) Tween-80, can make the output of Wei Lan glue improve respectively 21.80%, 22.73%, 36.62% and 28.12%.
 
advantage of the present invention and beneficial effect:
The present invention is mainly the angle from fermentation engineering, and the function of carrying oxygen material that may affect Wei Lan glue output in a class fermenting process is provided, and is intended to improve to greatest extent output, for the large-scale industrialization production that finally realizes Wei Lan glue provides reference.Along with the continuous progress of people to high viscosity biological polyoses fermentation technique, application of the present invention can make the output of Wei Lan glue improve constantly, and contributes to further to reduce production costs, and therefore the broadened application scope has good development prospect.
 
The accompanying drawing explanation
Fig. 1 is Sphingol single-cell sphingomonassp. the shake flask fermentation curve of TP-5;
Fig. 2 is the impacts of different oxygen carrier on the fermentation of Wei Lan glue;
Fig. 3 adds the impact of n-dodecane on the fermentation of Wei Lan glue;
Fig. 4 is the impact of different surfaces promoting agent on the fermentation of Wei Lan glue;
Fig. 5 is the impact of different surfaces promoting agent add-on on the fermentation of Wei Lan glue;
Fig. 6 is H 2o 2the interpolation time on the impact of Wei Lan glue fermentation;
Fig. 7 is H 2o 2add-on on the impact of Wei Lan glue fermentation.
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Embodiment
the fermentative production of embodiment 1 Wei Lan glue
Get the Sphingol single-cell of-80 ℃ of glycerine pipe preservations sphingomonassp. the TP-5(preserving number is CGMCC No.3097, and this bacterial strain is open in the ZL200910069960.2 patent) plate streaking, be placed in 30 ℃ of constant incubators, standing cultivation 3d; The single bacterium colony grown fine with aseptic inoculation ring picking be equipped with LB slant medium (m/v) (yeast extract paste 0.5%, peptone 1%, sodium-chlor 1%, agar 1.5%, pH 7.2; 121 ℃ of sterilizings after 20 minutes, be down to after 50 20 degree that test tube is tilted to be cooled after room temperature inclined-plane) test tube in draw zigzag, 30 ℃ of constant temperature culture 3d; From the activation the LB slant medium choose two ring thalline to be equipped with 5mL LB liquid nutrient medium (m/v) (yeast extract paste 0.5%, peptone 1%, sodium-chlor 1%, pH 7.2; 121 ℃ of sterilizings 20 minutes) shaking culture in test tube, become muddy to bacterium liquid in test tube, usings this as primary seed solution, receives in-built 200mL seed culture medium (m/v) (sucrose 15g/L, peptone 2.5 g/L, yeast powder 1.5 g/L, (NH 4) 2hPO 41.5 g/L, K 2hPO 42.5 g/L, MgSO 40.01 g/L, 7,121 ℃ of sterilizing 30min of pH), in 500mL triangular flask, shaking table is set to 30 ℃ of temperature, rotating speed 200rpm, shaking culture 24 hours; Cultured seed liquor is equipped with to 100mL fermention medium (m/v) (sucrose 44.68g/L, Na with 10% inoculum size access 2hPO 49.28g/L, CaCO 34.65g/L, Zein powder 1g/L, NaNO 34g/L, MgSO 40.8g/L, and by FeCl 20.0036mmol/L, ZnSO 40.01mmol/L, MnCl 20.01mmol/L and NiSO 40.02mmol/L in the 500mL triangular flask trace element solution 1mL formed), be placed in reciprocating constant-temperature table, 30 ℃, 115rpm, shaking culture is about 3 days.
From Fig. 1 shake flask fermentation curve, in whole fermenting process, pH slightly reduces; 0 ~ 14h is mainly the growth and breeding of TP-5 thalline and is the preparatory stage of synthetic product, synthetic Wei Lan glue, and to start fermented liquid be culture medium solution, and viscosity is lower; In the stage of the main Wei Lan glue of 14 ~ 40h output rapid growth, the 14h left and right starts synthetic Wei Lan glue, and fermentation broth viscosity starts to increase thereupon, with the output of Wei Lan glue, is proportionate; After 24h, bacterial strain TP-5 product glue rate increases sharply, and fermentation broth viscosity also increases rapidly thereupon; Enter fermentation later stage fermentation fluid viscosity after 40h higher, the dissolved oxygen deficiency, the output of Wei Lan glue starts slow growth; Fermentation proceeds to 66h product amount and reaches maximum value, and viscosity number also approaches maximum value, and glucose almost exhausts, and after 72h, viscosity no longer enlarges markedly, and maximum value reaches 3588mPas; Continue again fermentation and be unfavorable for the synthetic of Wei Lan glue, and can increase production cost.Therefore, now output is 18.05g/L in about 66h the controlled fermentation cycle.
 
embodiment 2alkanes the impact of oxygen carrier on the fermentation of Wei Lan glue
Ferment when initial, at fermention medium (m/v) (sucrose 44.68g/L, Na 2hPO 49.28g/L, CaCO 34.65g/L, Zein powder 1g/L, NaNO 34g/L, MgSO 40.8g/L, and by FeCl 20.0036mmol/L, ZnSO 40.01mmol/L, MnCl 20.01mmol/L and NiSO 40.02mmol/L add different oxygen carrier the trace element solution 1mL formed), probe into their impacts on Wei Lan glue fermentation yield under different concns, it is oxygen carrier that liquid alkane (comprising normal heptane, normal hexane, n-dodecane, n-hexadecane) and soya-bean oil are take in this experiment, add-on is controlled between 0 ~ 20%, fermentation 70h, with the extraction using alcohol of 3 times of volumes.N-hexadecane and soya-bean oil are insoluble to ethanol, and n-hexadecane and the soya-bean oil of understanding some after extraction using alcohol are adsorbed in product, then are removed with appropriate n-hexane extraction.
Result as shown in Figure 2, is added the output that normal hexane can not improve Wei Lan glue in fermentation system, and add in normal heptane secondary fermentation liquid, produce hardly glue, illustrates that normal hexane and normal heptane all are not suitable for the oxygen carrier as the fermentation of Wei Lan glue.N-dodecane, n-hexadecane and soya-bean oil can improve the output of Wei Lan glue to a great extent, and add the best results of n-dodecane.Add respectively 10% n-dodecane, 10% n-hexadecane, 5% soya-bean oil all can play effect of increasing production preferably, and output has improved respectively 75.63%, 51.15% and 69.93%.
Ferment when initial, add the n-dodecanes of different amounts, add-on is controlled at 5 ~ 10%, after investigating and adding n-dodecane, on the impact of Wei Lan glue output and fermented liquid apparent viscosity.As shown in Figure 3, after adding n-dodecane on the whole, the output of Wei Lan glue and fermented liquid apparent viscosity all increase result to some extent, and while wherein adding 9% n-dodecane, fermented liquid apparent viscosity maximum, reach 5629mPas; Yield values maximum while adding 10%, reach 28.5 g/L.
 
the impact of embodiment 3 tensio-active agents on the fermentation of Wei Lan glue
The fermentation initial stage, at fermention medium (m/v) (sucrose 44.68g/L, Na 2hPO 49.28g/L, CaCO 34.65g/L, Zein powder 1g/L, NaNO 34g/L, MgSO 40.8g/L, and by FeCl 20.0036mmol/L, ZnSO 40.01mmol/L, MnCl 20.01mmol/L and NiSO 40.02mmol/L add 0.1% Span-20, Tween-20, Tween-40, Tween-80 and TritonX-100 the trace element solution 1mL formed), probe into the impact of different types of tensio-active agent on Wei Lan glue fermentation yield.Result as shown in Figure 4, fails to improve Wei Lan glue output after adding Span-20, the output of the raising Wei Lan glue that other several tensio-active agent Tween-20, Tween-40, Tween-80 and TritonX-100 all can be in various degree.
Initial in fermentation, add several different tensio-active agents, add-on is controlled between 0.01% ~ 0.3%, probe into the impact on Wei Lan glue output when different concns of different types of tensio-active agent.As shown in Figure 5, these several tensio-active agents all can effectively improve the output of Wei Lan glue when suitable add-on, and the effect of Tween-40 is more obvious.Add respectively 0.05% TritonX-100,0.2% Tween-20,0.2% Tween-40,0.3% Tween-80 all can play good effect of increasing production to the fermentation of Wei Lan glue, and yield increase rate is respectively 21.80%, 22.73%, 36.62% and 28.12%.
 
embodiment 4 adds H 2 o 2 impact on the fermentation of Wei Lan glue
According to the different characteristics of different steps Wei Lan glue fermentation, while being chosen in 0h, 11h, 23h, 35h, 48h and 58h respectively at fermention medium (m/v) (sucrose 44.68g/L, Na 2hPO 49.28g/L, CaCO 34.65g/L, Zein powder 1g/L, NaNO 34g/L, MgSO 40.8g/L, and by FeCl 20.0036mmol/L, ZnSO 40.01mmol/L, MnCl 20.01mmol/L and NiSO 40.02mmol/L add 0.1% H the trace element solution 1mL formed) 2o 2, probe into different time and add H 2o 2impact on output and the fermented liquid apparent viscosity of Wei Lan glue.Result as shown in Figure 6, and is not added H 2o 2control group compare, no matter which stage to add H in 2o 2, the output of Wei Lan glue has increase by a small margin, and little on the impact of fermented liquid apparent viscosity.Wherein in the middle and later periods of 35h and 48h fermentation, add, better to the impact effect of output.
By H 2o 2add-on be controlled in 0.025% ~ 1% scope, add the H of different amounts when fermentation 35h 2o 2, probe into the H of different amounts 2o 2impact on output and the viscosity of Wei Lan glue.Result as shown in Figure 7, after add-on surpasses 0.5%, after fermentation 70h, does not have the generation of glue material and fermented liquid still to clarify in fermented liquid.This shows that excessive not only can the inhibition of the amount added also can affect the survival and reproduction of thalline synthesizing of Wei Lan glue.Control add-on in 0.075%, along with the increase of add-on, output has increase by a small margin, and the viscosity number impact is very unobvious, the H added 2o 2while being 0.075%, it is the most obvious that Wei Lan glue output improves, and increase rate is 16.42%.

Claims (3)

  1. For increasing Wei Lan glue fermentation yield take the oxygen agent, it is described that to take the oxygen agent be surfactant-based oxygen agent TritonX-100, Tween-20, Tween-40 or the Tween-80 of taking.
  2. 2. the surfactant-based application of oxygen agent in the fermentation of Wei Lan glue of taking as claimed in claim 1, it is characterized in that, the fermentation initial stage, add respectively 0.05%(m/v in fermention medium) TritonX-100,0.2%(m/v) Tween-20,0.2%(m/v) Tween-40 or 0.3%(m/v) Tween-80, can make the output of Wei Lan glue improve respectively 21.80%, 22.73%, 36.62% and 28.12%.
  3. 3. according to the surfactant-based application of taking the oxygen agent described in claim 2, it is characterized in that consist of (m/v) of described fermention medium: sucrose 44.68g/L, Na 2hPO 49.28g/L, CaCO 34.65g/L, Zein powder 1g/L, NaNO 34g/L, MgSO 40.8g/L, and by FeCl 20.0036mmol/L, ZnSO 40.01mmol/L, MnCl 20.01mmol/L and NiSO 40.02mmol/L the trace element solution 1mL formed.
CN2013104409817A 2013-09-25 2013-09-25 Oxygen carrying agent for increasing fermentation yield of welan gum and application thereof Pending CN103468762A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104845896A (en) * 2014-09-23 2015-08-19 中国石油大学(华东) Strain and method for producing Welan gum
CN105385174A (en) * 2015-10-27 2016-03-09 武汉理工大学 Asphalt thickener and asphalt cement

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1635132A (en) * 2004-11-19 2005-07-06 张禹 Process for preparing microbiological polysaccharide Gellan gum
CN101619300A (en) * 2009-07-31 2010-01-06 南开大学 Sphingomonas sp. TP-5 and method and application of same for producing welan gum

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1635132A (en) * 2004-11-19 2005-07-06 张禹 Process for preparing microbiological polysaccharide Gellan gum
CN101619300A (en) * 2009-07-31 2010-01-06 南开大学 Sphingomonas sp. TP-5 and method and application of same for producing welan gum

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
徐浩: "威兰胶发酵工艺研究", 《万方学位论文》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104845896A (en) * 2014-09-23 2015-08-19 中国石油大学(华东) Strain and method for producing Welan gum
CN105385174A (en) * 2015-10-27 2016-03-09 武汉理工大学 Asphalt thickener and asphalt cement
CN105385174B (en) * 2015-10-27 2017-10-24 武汉理工大学 A kind of pitch thickener and bituminous cements

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Application publication date: 20131225