Antibiotic Resistance Breeding of Bacillus Subtilis NJA133and Its Meat Application
|School||Nanjing Agricultural College|
|Course||Of Food Science|
|Keywords||Bacillus subtilis nja133 Ribosome engineering Optimization offermentation Chilled meat Preservation|
Ribosome-egineering was a newly developed simple microbial resource exploration and industrial microbial breeding method, as it uncomplicated and practicable to proceed, convenient to assemble, this method showed prosperous application future on industrial microbial breeding. During the earlier stage of our work in this laboratory, a Bacillus subtilis strain (named nja133) was separated which can produce anti-microbial lipopeptide, especially showed a strong anti-microbial activity on the main germ Pseudomonas sp. those induce rottenness of cooling meat. This study focused on the refinement of Bacillus subtilis njal33at the aim of a strain whose anti-microbial substance yield was improved, fermentation conditions was optimized, the bio-potency of anti-microbial substance was determined, further efforts was the study of the application of anti-microbial substance on preservation of cooling meat, key achievements was as follows:1. According to ribosome-egineering technology, five kinds of antibiotic:streptomycin, kanamycin, chloromycetin, rifampicine and gentamycin have been chosen for the treatment of wild strain and to select those strains with high antimicrobial substances productivity, productivity and inhibition effect was evaluated by inhibition zone. Bacillus subtilis nja117is breeded by Streptomycin,whose inhibition zone improve17.59%than the wild strain; Based on this, the strain K1-24treated by kanamycin whose inhibition zone improve19.70%than Bacillus subtilis nja717;Finally, Bacillus subtilis K1-24was breeded with different multi-antibiotic combination treatment. Through there rounds of antibiotic tolerance screening, higher productivity strain Bacillus subtilis A1B2-2was selected and showed an improved antimicrobial activity by35.27%compared to starting strain.2. The study of Bacillus subtilis AlB2-2’s flask fermentation conditions show that adding carbon source, include corn flour, molasses, and rhamnose and nitrogen include soybean flour and beef extract in the initial medium have a certain role in promoting strains antimicrobial substances production. Metal ions as Mg2+, Mn2+, Ca2+and K+also promote the production of antimicrobial substances. Fructose, urea and metal ions include Fe3+, Cu2+, Zn2+and Co2+takes on inhibitory effect on antimicrobial substance fermentation of Bacillus subtilis A1B2-2. The optimum fermentation conditions for Bacillus subtilis A1B2-2is that the initial medium pH7.0,2%of the inoculum’s size, liquid volume100mL, temperature33℃,incubation time26h.Under this condition, strains have a higher productivity of antimicrobial substances.In this study, Calibration curve was constructed by adopting microbial detection method, inhibitory zone diameter reflected the potency of antimicrobial activity. Pseudomonas was used as indicating bacteria, the potency of gentamycin was measured by drilling method, and then biological potency of antimicrobial substance produced by Bacillus subtilis can be calculated through calibration curve. Calibration curve equation was y=0.0828x-0.7218, R2=0.9955, range from2.46U/mL to11.72U/mL.3. Antimicrobial substance produced by Bacillus subtilis A1B2-2can inhibit growth of Pseudomonas effectively, in this study fermentation products was added to cooling meat as preservative and evaluated it’s freshness retaining effect. Compared to nisin, different concentration of fermentation product addition was proceed, result showed that fermentation product addition under5.6U/mL was less freshness retaining effective than nisin; while when concentration reached up to280U/mL the freshness retaining effect became prior to nisin, corresponding shelf life can be prolonged from original3days to11-12days.