Optimization Of Fermentation Pocess For Two Antimicrobial Peptides From Aquatic Animals And Preliminary Study On Pharmacology And Pharmacodynamics

The research foundation of the paper included Escherichia coli expressionsystem for anti-lipopolysaccharide factors (ALF) cloned from Eriocheir sinensisand pichia pastoris expression system for penaeidin 3-2 cloned fromFenneropenaeus chinensis. The paper has set up a suitable system for theantimicrobial activities of antimicrobial peptides; culture medium and cultureconditions for the production of ALF were optimized and several cultureconditions were also optimized for recombinant penaeidin 3-2. On this basis,the two antimicrobial peptides were expressed in 20L fermentor. Thefermentation product penaeidin 3-2 was used in the diet of GIFT tilapia.Experimental diets contained different levels of antimicrobial peptide andflorfenicol. The paper compared growth performance, physiological andbiochemical indexes, intestinal flora and organizational structure in differentgroups, which enable the further research in mechanisms of antimicrobialpeptides and their pharmacology and pharmacodynamics. It tried to explorethe probability of antimicrobial peptides as a new kind of feed additives andsupplied the test data and theoretical basis for the large-scale application ofantimicrobialpeptides.Theresultswereasfollows:1. The highest level of ALF fusion protein was achieved in mediumcontaining 10g/L yeast extract, 15.0g/L K₂HPO₄.12H₂O, 1.89g/L KH₂PO₄,10g/L NaCl, 3.176g/L (NH₄)SO₄, 3.75g/L sucrose, 3mmol/L MgSO₄ and 0.1 mmol/L CaCl2 with a cell optical density of 0.8 at 600 nm before induction, anIPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7°C,and apost-induction timeof 4h.Thewholeoptimization strategy improved thetarget protein content to 13.2%, which was 2.16-fold of the yield beforeoptimization. The optimized medium and conditions were applied in high celldensitycultivation,andthesolubletargetprotein constituted 10.5% ofthetotalprotein.2. The culture conditions were optimized in shake flasks. The optimalinitial pH were 5.0 with the additive of 10g/L caseinacid hydrolysate in themedium. The pichia pastoris achived the highest productivity with apost-induction temperature of 28℃and a post-induction time of 96h. Thefermentation was carried out with methanol-glycerol fed and methanol fedduringtheperiodofinduction.Themethanol-glycerolfedwasmoresuitableforthe production of target protein. The recombinant protein showed activitiesagainstGram-positiveandnegativebacterias.3. The results showed that weight gain ratio, red blood cells and catalaseactivities were significantly higherthan those in control when 5mg/kg¹0mg/kgantimicrobial peptide were added. Superoxide dismutase activities in liver andlysozymeactivitiesinserumweresignificantlylowerthanthoseincontrolwhen50mg/kg antimicrobial peptide was added in diet. Besieds, most livercells weretransparentwithcapillaryvesselscongestion.With100mg/kgflorfenicolindiet,red blood cells was significantly improved, but there was no significantdifference in other indexes. When fishes were infected with Aeromonashydrophila, the death rate was significantly lower in groups with5mg/kg²0mg/kg antimicrobial peptide in diet. The other groups showed nosignificantdifferencecomparedwiththecontrolgroup.