| Bacitracin is a broad-spectrum polypeptide antibiotic produced by Bacillus licheniformis or Bacillus subtilis,and is a 12-cyclic peptide formed by non-ribosomal synthetase(NRPS)catalysis.Bacitracin has an extremely wide application range,and can be applied to the treatment of livestock and poultry diseases,skin diseases,and other areas.It can be used as a feed additive in Southeast Asia,Africa and other regions,so its demand at home and abroad is relatively large.In the research both in China and abroad,scholars have selected or modified the strain by means of strain mutagenesis,metabolic regulation,genetic engineering,etc.,to achieve the purpose of bacitracin production.In this paper,the room temperature and atmospheric pressure plasma mutagenesis technology was used to breed bacillus licheniformis producing bacitracin,and the obtained target strain was adaptively evolved to obtain a product-tolerant strain.Optimization of fermentation conditions and fermentation medium is carried out immediately;On this basis,the pilot scale-up study was conducted and the main research results were as follows:1.Strain breed: That bacillus licheniformis producing bacitracin is used as an original strain to carry out room temperature and normal pressure plasma mutagenesis,primary screening is carried out through an agar transfer block method,and then the breed is carried out through shaking flask fermentation secondary screening.After 30 rounds of screening,a mutagenic strain with high yield,named F115,was screened out from nearly two thousand mutagenic strains.And the product tolerance of F115 was studied.It was found that the higher the concentration of bacitracin in the fermentation medium was,the lower the final cell concentration and the lower the output of bacitracin.Then the F115 strain was subjected to product adaptive evolution by using a micro-droplet culture system,and finally a strain resistant to bacitracin,namely strain F115.8,was screened.After 10 times of continuous passage,the genetic stability of strain F115.8 was verified to be good.The final yield of bacitracin was 845.23±8.24 U/m L,24.81%higher than that of the original strain.2.Optimization of fermentation parameters: The optimum fermentation conditions were determined by single factor experiment as follows: pH 7.2,rotation speed 300 r/min,temperature 35℃,and liquid loading 30/250 m L.The optimal fermentation medium composition was confirmed as follows: 6.14%soybean cake powder,3.2% corn flour,0.072% ammonium sulfate,0.06% calcium carbonate,0.0008% magnesium sulfate and 0.0008% manganese sulfate.The final bacitracin titer was 958.45±10.48 U/m L,13.37% higher than that before optimization and 41.5% higher than that of the original strain.3.Pilot-scale research: Based on the above research,the pilot-scale research of F115.8 strain was carried out,and its growth trend was determined by preliminary exploration in a 50 L fermentation tank;On this basis,the fermentation conditions were optimized in a 1 m~3 fermentor,and the changes of bacitracin yield and the aerobic capacity of the strain were investigated by using different air flow rates.Finally,the fermentation process was determined as follows: the air flow rate in 0～8 h was 43 m/h,the air flow rate in 8～22 h was 48 m/h,and the final bacitracin titer unit was 991.4 U/mL. |
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