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Screening of High Yield Nisin-Producing Strains and Optimizating of Its Fermentation Conditions

Author: LiHaiNa
Tutor: GuoXuePing；ZuoHengChang
School: Shandong University
Course: Pharmaceutical Engineering
Keywords: Nisin Mutation Breeding Fermentation conditions Fed-batch fermentation Downstream extract
CLC: TS202.3
Type: Master's thesis
Year: 2011
Downloads: 100
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Abstract


Nisin is a natural antimicrobial peptides A microbial metabolism, is composed of 34 amino acids, can effectively inhibit food spoilage caused by many Gram-positive bacteria and their spores growth, is approved for use by the World Health Organization and the UN Food and Agriculture food preservative, has been widely used in more than 50 countries and regions. Nisin is particularly sensitive to proteases in the gastrointestinal tract, be rapidly degraded by α-chymotrypsin, a non-toxic to human safety. Same time as the application of food preservatives and 40 years, Nisin did not induce resistance to a wide range of, which makes its biosynthesis widespread concern. In 1953, Nisin Aplin Barrett in the UK to achieve the earliest industrial production. After 50 years of development, it is reported that the British Nisin production has reached the level of 8000 IU / mL. Our country is relatively backward in this area the reported domestic Nisin production strains titer generally about 4000IU/mL, reported a big gap with foreign. Currently, most of the research on Nisin focus on two aspects of the medium optimization and optimization of fermentation conditions of Nisin production strains titer lower limit of the main reason for its wide application in the country. The study Nisin ATCC11454 as the original strain by ultraviolet, microwave, sodium nitrite and nitrosoguanidine composite mutagenic treatment and Nisin resistance screening agent, combined with the double agar diffusion method, eventually bred strain Nisin yield strains, named the the lactic streptococci E-15. The strain Nisin titer to reach 6200IU/mL, 5 times compared to the original strain. The passaged test prove the genetic characteristics of the strains. Flask fermentation conditions of strain, and determine the the Nisin best fermentation medium composition and culture conditions. Get the Nisin optimal medium formula for: peptone 20gL, the casein peptone 15gL, lactalbumin hydrolyzate 15gL, sucrose 30 g / L, MgSO4 · 7H2O 0.2 g / L, KH2PO410 g / L NaCl 2 g / L, Twain - 801mL / L, pH value of 6.8,121 ℃ sterilization 20min. The effects of inoculum size, fermentation time fermented nisin. Optimal culture conditions: inoculation amount of 5%, the fermentation time was 28 h. Streptococcus lactis E-15 amplification test using 10 L fermentation tank of the automatic control, the feedback control pH anaerobic batch fermentation. Use of the principle of feedback control pH complement to a certain speed, a mixed solution of sucrose with sodium hydroxide was added to the fermenter to maintain the pH at about 6.5, and a stable control of the sucrose content of the fermenter was maintained at 10 to 15gL, fermentation 28 h Nisin titer of 9600 IU / mL. Conducted a study to determine the recovery of the frame filter of the fermentation broth was filtered and sterilized after the fermentation broth using a pore size of an ultrafiltration membrane of 3000 Da ultrafiltration to obtain a concentrated liquid, spray-dried to obtain the finished product, the extraction process of Nisin up to 96.1%.

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