The Studies Of Fresh-Keeping Effect And Preliminary Antimicrobial Mechanism Of Protamine On Grass Carp Fillets

Protamine as a natural bacteriostatic agent in China is mainly used in the field of medicine,and it has been used in food preservation in foreign countries.In this paper,the effects of protamine on the freshness of the grass carp fillets were studied,and the relationship between the antibacterial activity of the protamine and the secondary structure of protein was studied,which laid the foundation for the study of the mechanism of protamine inhibition and provide a new way for further application of protamine in production.The results indicated:1.The effects of protamine on the preservation effect of grass carp fillets were determined by pH value,total number of colonies,TVB-N value and TBARS value.With the extension of storage time,it was found that the pH value decreased first and then increased during the whole storage process;the changes of the total number of colonies were strictly increased and then maintain stablity,and the TVB-N values were increasing all the time;TBARS did not show regularity in the whole storage process.Through the determination of the above indicators,we found that protamine has a better effect on prolonging shelf life of grass carp fillets,and the preservation effect is similar to Nisin.2.The isolates were isolated and purified from the spoilage microorganisms in the grass carp fillets,and 21 strains were isolated and purified.21 strains were conducted colony morphological observation(Shape,margin,color,degree of uplift etc.),individual morphology observation(flagellum,capsules,spores etc.)and physiological and biochemical reactions(Gram staining,oxidase test,catalase test etc.),the results were recorded: we founded that group 2 was Staphylococcus;group 5 was Rhizoctonia;group 6 was Bacillus,group 7 was Vibrio,group 8 was Pseudomonas,group 10 was Enterobacteriaceae,group 11 was Achromobacter,group 12 was Acinetobacter,group 14 was Aeromonas,group 20 was Plesiomonas,group 21 was Pseudomonas.At the same time,the growth characteristics of spoilage microorganisms were studied.It was found that pH and salt had a great effect on spoilage microorganisms,and the effect of sugar concentration was small.3.Selection of bacteriostatic effect of protamine by Bacillus,Vibrio,Enterobacteriaceae,Aeromonas and Pseudomonas.Protamine had obvious inhibitory effects on Bacillus,Enterobacteriaceae and Aeromonas,for Vibrio and Pseudomonas and the mixed strains with poor antibacterial effects.When the bacterial suspension concentration were 106CFU/ml respectively,the minimum inhibitory concentration of Bacillus,Enterobacteriaceae and Pseudomonas were 40 ?g/m L,40 ?g/m L,80 ?g/mL respectively.4.The effects of pH value,salt concentration,temperature and metal ion on the secondary structure and antibacterial activity of protamine were measured,the results showed that the acidic environment had a great effect on the antibacterial activity of protamine,the effect of alkaline environment were small,and there were certain relationship between the antibacterial activity of protamine and the secondary structure,part of the ?-turn will be transformed into random in the acid environment;100 ? high temperature treatment will reduce the antibacterial activity of protamine,and treated with 30 min its antibacterial activity decreased significant more than treated with 15 min,and the secondary structure did not changed;with the increase of salt concentration,the diameter of the inhibition zone decreased first and then increased,the secondary structure of protamine tranformed ?-turn,?-sheet and random into ?-sheet and random,the elliptic spectrum peaks appear regularly.The effect of different metal ions on the antibacterial activity of protamine is different,the effect of divalent cations on the antibacterial activity was higher than that of monovalent cations,and when the Ca2+ concentration was 20 mmol/L,protamine lost the antibacterial activity,while the Na+?K+?Mg2+ was 40 mmol/L,the protamine remains its properities.5.Preliminary study on the effect of protamine on cell wall membrane: in the bacteria suspensions by adding protamine,the conductivity of the first rised and then declined,protein content did not appear obvious regular changes,nucleic acid content has been increased,which indicating that protamine protein on the role of microbial cells will not damage the integrity of the cell membrane;the membrane protein,FDA experiments and the observation of the surface charge of the bacteria,showed that protamine could alter the permeability of the cell membrane,but didn't change its structure,it similar to the foregoing rule of proteins,nucleic acids and derived conductivity.