High-density culture method for bacilli and culture medium
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A technology for high-density culture and bacillus, which is applied to the high-density culture method and medium field of bacillus, and can solve the problem of low spore density and so on.
Active Publication Date: 2014-03-26
GUANGDONG HINAPHARM PHARMA CO LTD
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Problems solved by technology
[0008] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a high-density culture method and culture medium for bacillus, aiming to solve the problem that the obtained spore density is not high when the existing high-density fermentation culture technology cultivates bacillus
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Embodiment 1
[0060] Embodiment 1: the high-density cultivation of Bacillus subtilis
[0061] With Bacillus Subtilis as the strain, the following steps are carried out:
[0062] 1) Domestication of strains: In order to increase the sporulation rate of bacteria in the fermenter, the strains need to be domesticated. Dilute the strains preserved on the slant with normal saline, heat-treat it in an 80°C water bath for 10 minutes, spread it on the LB plate medium, culture it at 35°C for 48 hours, pick a single colony in sterile normal saline, and heat it at 80°C. C water bath heat treatment for 10 minutes, repeat the above steps once. Pick a single colony for subculture and the slant of the LB test tube as a spare strain.
[0063] 2) First-level shake flask seed culture: connect a ring of domesticated slant strains to the first-level shake flask medium, 100 ml of liquid in a 500 ml triangular flask, and shake the flask at 35°C and 200 rpm 30-36 hours. The formula of the primary shake flask m...
Embodiment 2
[0069] Embodiment 2: the cultivation of bacillus licheniformis
[0070] With Bacillus licheniformis as the strain, the following steps are carried out:
[0071] 1) Acclimatization of strains: Dilute the strains preserved on the slant with normal saline, heat-treat them in a water bath at 80°C for 10 minutes, spread them on LB plates, culture them at 35°C for 48 hours, and pick a single colony in a sterile Heat treatment in a water bath at 80°C for 10 minutes in normal saline, and repeat the above steps once.
[0072] 2) First-level shake flask seed culture: connect a loop of domesticated slant strains to the first-level shake flask culture medium, 100ml of liquid in a 500ml triangular flask, and shake the flask at 35°C and 200 rpm Cultivate for 36 hours, wherein, the formula of the primary shake flask medium is: peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, pH7.0; Heat treatment in 80°C water bath for 5 minutes and then set aside.
[0073] 3) Second-le...
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Abstract
The invention discloses a high-density culture method for bacilli and a culture medium. The high-density culture method for bacilli comprises the following steps including Step 1, strain domestication, Step 2, shake flask seed culture, Step 3, seed tank seed culture, and Step 4, fermentation tank high-density fermentation culture. Ingredients of a fermentation culture basic culture medium comprise peptone, yeast powder soaking, glucose, disodium hydrogen phosphate, monopotassium phosphate, magnesium sulfate, manganese sulfate, calcium chloride and bean pulp. A fermentation culture medium supplemental culture medium is glucose solution, the glucose content in the glucose solution is 3 to 4 percent of the total quantity of the fermentation liquid, and the high-density culture method for bacilli is applicable to the high-density culture of bacillus subtilis and bacillus licheniformis. The high-density culture method for bacilli provided by the invention has the advantages that the spore forming rate of the bacilli in the fermentation liquid can be higher than 90 percent, and the spore density is higher than 120 hundred million / milliliter to the lowest degree.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a high-density culture method and culture medium for bacillus. Background technique [0002] Practice has proved that with the use of antibiotic feed additives, while inhibiting pathogenic bacteria, it also inhibits the normal flora in the digestive tract of animals, destroying the ecological balance of flora, causing a large number of pathogenic bacteria to proliferate, causing secondary infections or endogenous sexual infection. Moreover, the frequent and large-scale use of antibiotics will lead to the emergence of drug-resistant strains, increase the susceptibility of animals, cause the decline of immune function and cause intestinal diseases and the resulting environmental pollution. In addition, the residues of antibiotics are also a great threat to human health, and their effects in disease prevention and treatment are becoming increasingly unsatisfactory. [0003] Therefore,...
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