High-density fermentation technique of powdered pichia pastoris

A powdery Pichia pastoris, high-density fermentation technology, applied in the field of microorganisms, can solve the problems of long fermentation period, low concentration of fermentation liquid bacteria, high cost of medium, etc., achieve low production cost, high biomass, and ease the growth of bacteria slow effect

Active Publication Date: 2015-04-29
武汉光华时代生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Pichia powder has a series of problems such as low concentration of fermentation broth, long fermentation period, and high cost of medium. To develop a low-cost and

Method used

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  • High-density fermentation technique of powdered pichia pastoris
  • High-density fermentation technique of powdered pichia pastoris
  • High-density fermentation technique of powdered pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: powdery Pichia pastoris ACCC21153 shake flask fermentation culture

[0050] 1. Preparation of powdery Pichia ACCC21153 high-density fermentation medium

[0051] (1) Prepare materials according to the following formula

[0052] Mix 15g of peptone, 1.5g of potassium dihydrogen phosphate, 0.45g of magnesium sulfate, 15g of corn steep liquor (industrial grade, protein content ≥ 45%, acidity ≤ 14%, sulfite ≤ 0.3%, ash ≤ 10%), and distilled water Dissolve all the solids and make up to 1000mL, adjust the pH to 6.0 with 5-8mol / L sodium hydroxide, dispense into 250mL shaker flasks, the filling volume is 30mL, and sterilize at 103.4kPa steam pressure and 115℃ for 30min;

[0053] (2) Weigh 48g of glucose, dissolve it with distilled water and replenish to 100mL, and sterilize at 103.4kPa steam pressure and 115°C for 30min to obtain glucose solution;

[0054] (3) Take 2.0 mL of the glucose solution in step (2) and add it to the sterilized medium in step (1) to obtain...

Embodiment 2

[0062] Embodiment 2: powdery Pichia pastoris ACCC21153 fermentation tank batch fermentation culture

[0063] 1. Preparation of powdery Pichia ACCC21153 high-density fermentation medium

[0064] (1) Prepare materials according to the following formula

[0065] Mix 8g of peptone, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, and 15g of corn steep liquor, dissolve all the solids with distilled water and make up to 900mL, adjust the pH to 6.4 with 5-8mol / L sodium hydroxide, and adjust the pH to 6.4 at 103.4kPa steam pressure , Sterilize at 121°C for 20 minutes;

[0066] (2) Glucose solution configuration and sterilization are the same as in Example 1;

[0067] (3) Add all the glucose solution in step (2) to the sterilized medium in step (1) to obtain a powdery Pichia ACCC21153 high-density fermentation medium.

[0068] 2. Fermentation tank fermentation culture

[0069] A. Inclined seed activation

[0070] With embodiment 1;

[0071] B. Shake flask seed pre...

Embodiment 3

[0075] Embodiment 3: Powdered Pichia pastoris ACCC21153 fermentation tank fed-batch fermentation culture

[0076] 1. Preparation of powdery Pichia ACCC21153 high-density fermentation medium

[0077] (1) Prepare materials according to the following formula

[0078] Mix 12 g of peptone, 3.5 g of potassium dihydrogen phosphate, 0.45 g of magnesium sulfate, and 15 g of corn steep liquor. The preparation method is the same as in Example 2, and the pH is adjusted to 6.4. Sterilize at 103.4 kPa steam pressure and 121 ° C for 20 min;

[0079] (2) Weigh 40g of glucose, dissolve it with distilled water and replenish to 100mL, and sterilize at 103.4kPa steam pressure and 115°C for 30min to obtain glucose solution;

[0080] (3) Add all the glucose solution in step (2) to the sterilized medium in step (1) to obtain a powdery Pichia ACCC21153 high-density fermentation medium.

[0081] 2. Fermentation tank fermentation culture

[0082] A. Inclined seed activation

[0083] With embodiment...

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Abstract

The invention belongs to the technical field of agricultural microbiology, and particularly relates to a high-density fermentation technique of powdered pichia pastoris. The technique comprises the steps of seed preparation and fermentor culture. The conditions of the fermentor culture are as follows: the loaded liquid volume is 40-60% (V/V), the inoculation amount is 1-5% (V/V), the control temperature is 28-40 DEG C, the ventilatory capacity is 1-3.5 VVM, the startup rotating speed is 300 r/min, the rotating speed is increased by 100 r/min in every 4h, after being increased to 700r/min, the rotating speed is kept unchanged, and the fermentation cycle is 20-30h. In the process of fermentation, by dropwise adding a NaOH solution, the pH value of fermentation liquor is adjusted to about 6.0. After 13h of fermentation, a certain amount of glucose is added at certain interval, the fermentation is completed after 20-30h, and the effective viable count is 30.0-3.5 hundred million/mL. By adopting the fermentation technique disclosed by the invention, the biomass is significantly increased, the process is simple, the efficiency is superior to that in the prior art, and the production cost is lower.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a high-density fermentation process of powdery Pichia pastoris. The fermentation medium has simple components and low cost, adopts the method of feeding glucose to promote bacteria accumulation, and is suitable for the production of powdery Pichia pastoris High density fermentation production. Background technique [0002] Powdered Pichia pastoris has very special advantages as a fermentation industry strain. It can not only provide high-quality active protein sources for the feed industry, provide important strains for the bio-fertilizer industry, but also efficiently secrete glycerin and other industrial raw materials and products. Therefore, vigorously developing the powdered Pichia pastoris industry has a very broad market prospect. The powdery Pichia inoculum currently on the market is mainly powder, and a large amount of powdery live Pichia is required for inoculation. Th...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12R1/84
CPCC12N1/16C12N1/165C12R2001/84
Inventor 陈守文徐迪红杨欢冀志霞马昕
Owner 武汉光华时代生物科技有限公司
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