Method for producing elastase preparation by utilizing liquid-state fermentation of microbes

Method for producing elastase preparation by utilizing liquid-state fermentation of microbes

  • CN 103,627,689 A
  • Filed: 09/05/2013
  • Published: 03/12/2014
  • Est. Priority Date: 09/05/2013
  • Status: Active Application
First Claim
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1. utilize a method for microorganism liquid state fermentation production elastoser zymin, it is characterized in that selecting Bacillus licheniformis ( baclicus lincheniformis) bacterial classification produced as elastoser of ELAT4 (CGMCC 5700), through Bacillus licheniformis strain activation, seed liquor preparation, fermented liquid cultivation, deep-layer liquid ventilating fermentation and elastoser zymin finished product preparation process, and make elastoser zymin, concrete steps are as follows:

  • Step 1, actication of culture;

    the Bacillus licheniformis of preservation is rule twice on strain activation and culture base, all in 37 ℃

    of incubators, cultivate 18-24h, the single bacterium colony obtaining of ruling for the second time to rule is for the first time bacterial classification, then from the flat board of line for the second time, select single bacterium colony access liquid spawn activation medium or solid spawn activation medium, 37 ℃

    of culture temperature, time 180-200r/min, shake-flask culture OD 600to lD 600;

    Step 2, seed liquor preparation;

    inoculate bacterium liquid to seed culture medium from the liquid tube activating, culture temperature 35-37 ℃

    , shaking table concussion frequency 160-180r/min, shaking table concussion cultivation 18-24h, become seed liquor;

    Plate streak, with transfering loop, from the strain inclined plane of preserving, be inoculated in actication of culture solid medium, place in 37 ℃

    of incubators and cultivate 18-24h, and then from activation solid medium picking list colony inoculation to another actication of culture solid medium, carry out re-activation, in 37 ℃

    of incubators, cultivate 18-24h;

    From flat board, select in the test tube of single bacterium colony access containing 5mL liquid spawn activation medium or solid spawn activation medium, test tube is placed in to shaking table, 37 ℃

    of culture temperature, time 180-200r/min, shake-flask culture OD 600to lD 600;

    Step 3, shake flask fermentation liquid is cultivated;

    the inoculum size by cultured seed culture fluid with 2%-8% (v/v), be seeded in 250ml triangular flask, the concussion of 160-180r/min shaking table is cultivated;

    Before Medium of shaking flask fermentation fermentation, to control temperature be 35-37 ℃

    to 6-10h, switches to 26-30 ℃ and

    remain to fermentation ends after 10h;

    Controlled fermentation liquid pH7.0-7.5 in fermentation 0-10h process remains on 7.5-8.5 by pH after 10h to fermentation ends process;

    Wherein, pH remains the method that drips acid-base solution that adopts;

    Step 4, deep-layer liquid ventilating fermentation;

    before fermentor tank sterilizing, add defoamer according to the ratio of l;

    1000, the tank pressure in fermenting process maintains 0.04Mpa;

    Inoculum size 3-6% (v/v), the initial fermention medium of fermentor tank ferment certainly initially to the 14h that ferments, ventilation 0.55vvm;

    Fermentation 14-24h, ventilation 0.75vvm;

    25h is to fermentation ends in fermentation, ventilation 0.6vvm;

    Fermentation 0-30h, temperature 34-36 ℃

    ;

    Temperature 25-30 ℃

    after fermentation 30h;

    Fermenting process pH7.5-8.0;

    Fermentation reaches 25h and adds the glucose solution 80ml that concentration is 100g/L;

    Fermentation reaches 30h, again adds the glucose 100ml of concentration 100g/L;

    Fermentation reaches 35h, and adding concentration is the soybean cake powder emulsion 150ml of 100g/L;

    Step 5, the preparation of elastoser zymin finished product;

    (1), fermented liquid degerming;

    4 ℃

    of fermented liquids, the centrifugal 10min of 8000r/min remove thalline;

    (2), ultrafiltration and concentration;

    adopting for the first time trapped molecular weight is the ultrafiltration membrance filter of 60kDa, retains filtrate;

    Use for the second time the ultrafiltration membrance filter of 20kDa, the concentrated 3-5 of filtrate doubly, preserves trapped fluid;

    (3), ammonium sulfate precipitation;

    the filtrate after ultrafiltration is regulated to pH to 6.5, add ammonium sulfate to 25%, 4 ℃

    of standing 8-10h of saturation ratio in filtrate, then the centrifugal 10min of 8000r/min whizzer at 4 ℃

    , removes bottom settlings, collects supernatant liquor;

    Regulate supernatant liquor pH8.5, add ammonium sulfate to freezing and centrifugal collecting precipitation albumen after 70%, 4 ℃

    of standing 8-10h of saturation ratio;

    The Tris-HCl buffered soln dissolution precipitation of 0.02mol/L, pH7.5, and fully dialysis;

    (4), vacuum lyophilization;

    by the ultra-filtration membrane ultrafiltration of the enzyme liquid 20kDa after dialysis, concentrated 6-10 doubly, adds protective material 1-2% sodium alginate, 2-3% sucrose, 3-5% maltodextrin, 0.1-0.25% potassium sorbate and 0.1-0.4% Sodium Benzoate;

    After fully disperseing ,-20 ℃

    of freezing 8-10h, vacuum tightness 30-50Pa, temperature-35 °

    C is following, freezing 15-18h, more makes elastoser zymin finished product through pulverizing.

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