CN101275154B

CN101275154B - Production method for microbial polysaccharide welan gum

Info

Publication number: CN101275154B
Application number: CN200810018721XA
Authority: CN
Inventors: 詹晓北; 郑志永; 贾薇; 朱莉; 吴剑荣
Original assignee: Jiangnan University
Current assignee: Anhui Lonking Biotechnology Co., Ltd.
Priority date: 2008-01-22
Filing date: 2008-01-22
Publication date: 2011-04-13
Anticipated expiration: 2028-01-22
Also published as: CN101275154A

Abstract

The present invention provides a preparing method of microbial polysaccharides Weilan gum, belonging to biotechnology field. The invention use an alcaligenes faecalis as a fermentation strain, using sucrose or glucose commonly used in food industry as carbon source for microbe cultivation, using conventional chemical fertilizer in the agricultural production as nitrogen source and inorganic salt, the medium of microbial growth is prepared by auxiliary yeast powder, aerobic fermentation is processed in the conditions of 30 DEG C, 1.0vvm, 500r/min ventilatiing and stirring, adding specially supplementary food fluid in the fermentation course achieves the following aims: (1) controlling the penetrating pressure of the broth in the hypotonicity condition; (2) controlling the pH of the broth between 4.0 and 7.0; (3) providing abundant nutritious matter such as carbon source and nitrogen source for microbial growth, obtaining Weilan gum broth after 72 hours, obtainin Weilan gum product by drying in 95-115 DEG C and atmospheric pressure. The production process of the Weilan gum product does not use organic solvent, the broth obtains Weilan gum product to correspond with the petroleum exploitation industry standard only processing one step drying.

Description

A kind of production method of microbial polysaccharide welan gum

Technical field

A kind of production method of microbial polysaccharide welan gum belongs to technical field of bioengineering.

Welan gum (welan gum) is obtained through fermentative production by the bar-shaped Alcaligenes of a kind of Gram-negative usually, can be used as good stablizer, thickening material, suspension agent, gelifying agent etc., is mainly used in industries such as geological and mineral and building.Its molecular structure is as follows; main chain is made up of glucose, glucuronic acid and rhamnosyl unit; ratio is 2: 1: 1; approximately a rhamnosyl or seminose are arranged with α-(1 every 4 unit; 3) glycosidic link branch is connected on the gluconic acid unit; there is 85% repeating unit on 2 carbon of β-(1,3) glucose, to be connected with ethanoyl.

The molecular structure of Welan gum

Welan gum has good thickening, pseudo-plasticity, thermostability and has acid and alkali-resistance, anti-brinish ability as a kind of good amendment.Welan gum can obtain more satisfactory permeability reduction coefficient as amendment in the oil oil reservoir, notable attribute is that at high temperature (120～150 ℃) still can keep high viscosity and ideal shear thinning effect, this feature makes Welan gum can be positioned the high permeability zones territory of oil reservoir effectively, selectivity forms high-viscosity gel and the low oil zone of shutoff high permeability, play intelligence and regulate the effect of oil reservoir permeability profile, thereby help the hyposmosis district that displacement of reservoir oil fluid enters high oil length, effectively improve the recovery ratio of tertiary oil recovery.

The production method of Welan gum is generally fermentative Production, and after the Welan gum that conventional fermentation process obtains need pass through organic solvent deposit, centrifugal or Plate Filtration and squeezing, drying technology obtained the Welan gum product again.Used organic solvent need be recycled through rectifying, but energy consumption is very big, and production plant with an organic solvent has requirement of explosion proof in design and production process, increased its cost of investment and production cost.The present invention adopts the thinking of hypotonic fermentation, adopt the fermention medium and the supplemented medium of formulated in process of production, accurately controlling microbial is grown and is produced Welan gum nutritional needs ratio and demand, control the osmotic pressure of fermentation and best pH condition well, not residual unnecessary nutritive ingredient when microbial fermentation is finished, used to make the best, so the gained fermented liquid promptly gets the Welan gum product that meets the oil production commercial size through a step convection drying.

Summary of the invention

The production method that the purpose of this invention is to provide a kind of microbial polysaccharide welan gum does not adopt organic solvent in the production process, make production process simple, saves cost of investment and production cost, increases market competition advantage.

Technical scheme of the present invention: a kind of production method of microbial polysaccharide welan gum, produce the bacterial strain Alcaligenes ATCC 31555 of Welan gum by aerobic cultivation one strain, stream adds the supplemented medium of formulated in the ventilating fermentation process, makes the osmotic pressure of (1) control fermented liquid be in hypotonic condition; (2) pH of control fermented liquid is between 4.0～7.0, and keeps constant; (3) for microorganism growth provides abundant carbon source and nitrogenous source nutritive substance, obtained the Welan gum fermented liquid through 72 hours:

([Food science] 2007.Vol.28.No.09 " the rheological characteristics Study on influencing factors of novel microorganism polysaccharide-Wei Lan glue " is open, and the applicant guarantees to provide to the public in 20 years for bacterial strain: Alcaligenes ATCC 31555.)

Used seed culture medium component comprises glucose, ammonium nitrate, yeast powder, peptone, KH ₂PO ₄, MgSO ₄, the pH of seed culture medium is 4～7, uses preceding through 121 ℃ of sterilization 20min;

Used fermention medium component comprises glucose, sucrose, yeast powder, urea, dipotassium hydrogen phosphate, sal epsom, and the initial pH of fermention medium is 4～7, uses preceding through 121 ℃ of sterilization 20min;

Used supplemented medium component comprises glucose, yeast powder, urea and/or ammonium nitrate, ammoniacal liquor;

The seed preparation: from the microbial strains of solid inclined-plane picking one ring fresh culture, insert in the seed culture medium, 30 ℃ of overnight incubation get seed liquor;

Welan gum fermentation: the inoculum size of cultured seed liquid by 5% (v/v) inserted in the fermention medium, at 30 ℃, keep tank pressure 0.01MPa, with aerobic fermentation under 1.0vvm, the 500r/min ventilation agitation condition, flow feeding substratum in the fermenting process, utilize the pH value of supplemented medium control fermented liquid, pH is controlled in 4～7 scopes in the fermenting process; Got the Welan gum fermented liquid through 72 hours;

The Welan gum fermented liquid of gained is constant pressure and dry under 105 ℃ of conditions, obtains pulverous Welan gum product through pulverizing and screening again;

The feed supplement control method in thalli growth stage: in the BIOSYNTHESIS OF MONOSE AND process, microorganism growth is certain proportionlity to the demand of carbon source and nitrogenous source, when utilizing physiological tart carbon source, the pH of fermented liquid will present long trend, utilize the nitrogenous source of the peristaltic pump auto-feeding physiological alkalescence of computer control: urea and/or nitrate, or itself just being the nitrogenous source ammoniacal liquor of alkalescence, the pH of control fermented liquid helps microbial growth in 4～7 scope;

Producing the feed supplement control method in Welan gum stage: Alcaligenes ATCC 31555 pH in the process of synthetic Welan gum remains unchanged substantially or is ascendant trend slightly, ability according to cell concentration and thalline consumption carbon source, slowly mend carbon source by given pace, the residual sugar in the fermented liquid is maintained in 1～5g/L scope.

The production method of described microbial polysaccharide welan gum, preferred substratum consists of:

Used seed culture medium is (g/L): glucose 10, ammonium nitrate 0.6, yeast powder 2.5, peptone 5, KH ₂PO ₄0.5, MgSO ₄0.1, pH4～7,121 ℃ sterilization 20min;

Used fermention medium (g/L): glucose 10, sucrose 1, yeast powder 1.0, urea 1, dipotassium hydrogen phosphate 0.5, MgSO ₄0.1, pH4～7,121 ℃ sterilization 20min;

Used supplemented medium (g/L): glucose 400, yeast powder 10, urea 40, and ammoniacal liquor 12.5% (v/v).

Welan gum analytical procedure: 80 ℃ of water-baths of fermented liquid 20 minutes, 95% ethanol that adds 2 times of volumes after cooling, magnetic stirrer is to there being precipitation to occur, after 4 ℃ of static spending the night, centrifugal 30 minutes of 8000r/min discards supernatant liquor, obtain the Welan gum crude product, wash throw out twice with dehydrated alcohol, dry to constant weight scales/electronic balance weighing for 95 ℃.

Beneficial effect of the present invention: Welan gum is produced bacterial strain and is grown in hypotonic substratum, stream adds the supplemented medium that satisfies strain growth just and produce needs in the fermenting process, best osmotic pressure and the pH condition of whole fermentation process control, mending the nutrition of going into is utilized fully by microorganism substantially, only residual minute quantity nutrition, fermented liquid main component after fermenting process finishes is a Welan gum, only contain a small amount of somatic cells and residual nutrition, but foreign matter content is within the scope of oil production industrial microbe polysaccharide product specification.Fermented liquid promptly gets the Welan gum product behind 105 ℃ of constant pressure and dries, after measured, the purity of Welan gum product is greater than 80% (w/w), and the oven dry loss is less than 10% (w/w).Do not adopt conventional organic solvent precipitation method in the production process, cost of investment and production cost have been reduced on the one hand, also promoted the green production of microbial polysaccharide on the other hand, reduced the pollution of organic solvent, will help to improve the microbial polysaccharide competitiveness of product in market environment.

Embodiment

The production of embodiment 1. microbial polysaccharide welan gums

Bacterial strain Alcaligenes ATCC 31555.

Used seed culture medium is (g/L): glucose 10, ammonium nitrate 0.6, yeast powder 2.5, peptone 5, KH ₂PO ₄0.5, MgSO ₄0.1, pH6.8,121 ℃ of sterilization 20min.

Used fermention medium (g/L): glucose 10, sucrose 1, yeast powder 1.0, urea 1, dipotassium hydrogen phosphate 0.5, MgSO ₄0.1, pH6.0,121 ℃ of sterilization 20min.

On 15L Biostat C10-3 mechanical agitator tank, ferment, inoculum size 5% (v/v), liquid amount is 9L, fermentor tank mixing speed 500r/min, strength ofdraft is 1.0vvm, keeps tank pressure 0.0 1MPa, 30 ℃ of leavening temperatures, utilize the pH value of supplemented medium control fermented liquid, pH is controlled in 4～7 scopes in the fermenting process.Fermenting got the Welan gum fermented liquid in 72 hours, measured Welan gum content 15g/L.

The Welan gum fermented liquid of gained is constant pressure and dry under 105 ℃ of conditions, obtains pulverous Welan gum product through pulverizing and screening again.

Claims

1. the production method of a microbial polysaccharide welan gum is characterized in that,

Bacterial strain: Alcaligenes ATCC 31555;

Used seed culture medium is (g/L): glucose 10, ammonium nitrate 0.6, yeast powder 2.5, peptone 5, KH ₂PO ₄0.5, MgSO ₄0.1 pH 4～7,121 ℃ of sterilization 20min;

Used fermention medium (g/L): glucose 10, sucrose 1, yeast powder 1.0, urea 1, dipotassium hydrogen phosphate 0.5, MgSO ₄0.1 pH 4～7,121 ℃ of sterilization 20min;