Preparation, Purification, Flocculating And Antimicrobial Activity Of Protamine

Protamine is a kind of polyphosphate cationic peptide which is existed in the in mature testis of a variety of animals.It is a low-molecular-weight protein fraction that is rich in basic amino acids such as arginine and lysine. Current research on protamine mainly focused on the field of food and medicine. The research on flocculating activity of protamine was not previously reported. In this paper, the protamine extracted from salmon and carp testis were studied. The antibacterial activity of two kinds of protamine were compared. At the same time, the flocculating effect of protamine on microalgae was investigated. Exploring the correlation between the antibacterial activity and flocculation activity of protamine is necessary.1. First, the nutrients of salmon milt and carp milt were measured. Two kinds of milt contain large amounts of proteins and nucleic acids, as well as ash and a small amount of fat. Two kinds of protamine were extracted from milt through the method of NaCl washing, sulfuric acid solution extraction and ethanol precipitation. The antimicrobial activity of salmine was higher than carp protamine through the method of agar disc diffusion. Salmine had a broader antimicrobial spectrum than carp protamine. The amino acid composition analysis of two kinds of milt showed that salmon milt was rich in 7.62% arginine and 3.76% lysine, respectively and carp milt was rich in 6.08% arginine and 4.45% lysine, respectively.2. Salmine and carp protamine were used to flocculate microalgae and microbial cells. Salmine showed the best flocculating effect on Dunaliella salina. The flocculating rate on D.salina could reach 85.51% at 20 ug/mL. Carp protamine showed the best flocculating effect on Platymonas subcordiformis. The flocculating rate on P. subcordiformis could reach 85% at 20μg/mL. The precipitated cells of D.salina and supernatant after flocculation were separated and the residual cells in the supernatant were re-cultured and re-flocculated. Re-cultured residual cells could be flocculated by salmine at the similar flocculating rate (85.89%). The precipitated cells were observed by the microscope, which could maintain good vitality and swimming state. Meanwhile, the precipitated cells also could continue to be re-cultured. The re-cultured and re-flocculated condition of P. subcordiformis was similar to that of D.salina. Further, the flocculating rate of D. salina was not influenced even at the highest temperature treatment of salmine. But when the treatment temperature of carp protamine was over 100℃, the flocculating rate of P. subcordiformis was diminished. Protease treatment could made the flocculating activity of two kinds of protamine lost completely.3. Protamine which had better antibacterial activity was researched, it showed that the minimum antibacterial concentration of salmine on gram negative bacteria was lower than that of gram positive bacteria. When the pH is over 6.0, salmine has better antibacterial activity. Salmine was suitable for the basic foods but acidic foods. Temperature treatment had no effect on the antimicrobial activity of salmine. Therefore salmine could be used for pasteurization or high pressure sterilization in food, greatly making up for the shortcomings in the application of food preservatives. Protease (neutral protease, alkaline protease, trypsin, animal protease, and flavor protease) treatment made the antimicrobial activities of salmine completely lost.4. Salmine and carp protamine extracted from milt testis was successfully separated and purified by CM Sepharose FF ion exchange chromatography. And then they were verificated with the help of specific arginine reaction-Sakaguchi reaction and SDS precipitation reaction. Salmine and carp protamine were obtained two active peaks and an active peak, respectively after separation and purification. Amino acid analysis showed that PEAK Ⅲ and PEAK IV of salmine were rich in lysine and arginine, respectively. Based on the peak areas, PEAK IV of salmine was arginine-rich (73.46%), PEAK Ⅲ of salmine was lysine-rich (37.36%), and PEAK Ⅲ of carp protamine was lysine-rich (39.24%). In addition, PEAK Ⅲ and PEAK Ⅳ of salmine both showed antimicrobial activity, but the antimicrobial activity of PEAK Ⅲ was much lower than that of PEAK Ⅳ. Antimicrobial activity of PEAK Ⅲ of carp protamine after chromatography got increased. After salmine being purified by CM Sepharose Fast Flow ion exchange chromatography, PEAK IV showed a better flocculating activity, whereas PEAK Ⅲ showed no flocculating activity. The PEAK Ⅲ of carp protamine after chromatography also showed no flocculating activity.Protamine had good flocculating activity and antimicrobial activity which could provide a reference for applications of flocculant and preservative in the future. It is also provide some thoughts for application of polypeptide or protein as a polycationic peptide. Chemical composition and structure of salmine especially the amino acid composition on antimicrobial and flocculating activity requires more research.