Bacillus subtilis growth acceleration and endospore generation culture method

A Bacillus subtilis, culture method technology, applied in the field of promoting the growth of Bacillus subtilis and producing spores, can solve the problems of low spore formation rate, low number of viable bacteria, long fermentation period, etc., and achieve extended storage period and high density Cultivation improves and increases the effect of stabilizing activity

Inactive Publication Date: 2015-09-09

INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI

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AI-Extracted Technical Summary

Problems solved by technology

[0004] The invention solves the problems that the process conditions related to spore formation are not easy to control, the spore formation rate is low, the fermentation period is long, and the number of viable bacteria is low in the liquid fer...

Abstract

The invention discloses a bacillus subtilis growth acceleration and endospore generation culture method and aims to solve the problems of low viable count, long fermentation period, low endospore generation rate, difficulty in control of endospore generation related process conditions in a liquid fermentation process of bacillus subtilis. The method includes: activating strains; performing liquid fermentation in shake flasks for seed culture, carrying out fermentation cultivation until residual sugar content of the strains is 0.1%, adding supplementary liquid to control the residual sugar content to range from 0.1 to 0.2, fermenting, supplementing for 5-7h, stopping supplementation and continuing to ferment for 4h, adjusting the pH value to range from 8.5 to 9 and controlling dissolved oxygen to be more than 90% until fermentation is finished. A fermentation medium is composed of glucose, yeast extract, sodium chloride, K2HPO4, MgSO4, MnSO4 and water, and the initial pH value is 7.2-7.5; the supplementary liquid is composed of glucose and yeast extract, and the initial pH value is 7.2-7.5. The bacillus subtilis growth acceleration and endospore generation culture method is used for bacillus subtilis culture.

Application Domain

BacteriaMicroorganism based processes

Technology Topic

GROWTH ACCELERATIONSChloride sodium +10

Examples

Experimental program(1)

Example Embodiment

[0014] In this embodiment, the Bacillus subtilis with the deposit number CGMCC No. 10603 is used for cultivation, and the specific method is as follows:

[0015] Step 1. Activation of the slant seeds: pick a single colony strain on the slant and insert it into a test tube seed medium (5 mL), and culture with shaking (70 rpm) at 30°C for 16 hours. Test tube seed culture medium composition: glucose 8g/L, yeast extract 5g/L, sodium chloride 3g/L, K 2 HPO 4 2g/L, CaCO 3 2g/L, the initial pH is 7.2-7.5.

[0016] Step 2. Seed cultivation: Shake flask seed culture medium is filled with 50/500mL, and 2% inoculum is connected to test tube seed liquid, and cultured on a 30℃ shaker at 180 rpm for 16 hours to obtain seed liquid; shake flask seed medium is the same as test tube seed Medium.

[0017] Step three, batch fermentation: fermentation medium (glucose 8g/L, yeast extract 5g/L, sodium chloride 3g/L, K 2 HPO 4 2g/L, MgSO 4 0.2g/L, MnSO 4 0.1g/L, the initial pH is 7.2-7.5) The filling volume is 10/20L, and the 1% inoculum volume is connected to the shake flask seed solution, 30℃, pH value 7, ventilation volume 10-15L/min, tank pressure 0.01- 0.03MPa, stirring at 50-600rpm for fermentation, the dissolved oxygen is controlled above 70%;

[0018] Step 4. Fermentation to the mid-late logarithmic growth of the bacteria (residual sugar content is about 0.1%) 6h, start the feed fermentation, feed liquid (glucose 10%, yeast extract 4%, initial pH 7.2-7.5) flow acceleration 3mL/min, control the residual sugar content between 0.1-0.2, feed for 7h, stop feeding and continue fermentation for 4h, then adjust the pH to 8.5-9, increase the dissolved oxygen to more than 90%, until the end of the fermentation (3-5h ).

[0019] In this embodiment, the number of viable Bacillus subtilis can reach 9 billion/mL; the formation rate can reach more than 95%. The shelf life is more than 5 years, and the number of viable bacteria can reach 8 billion/mL in 5 years.

PUM

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