| Ovomucin, a high-molecular-weight sulfated glycoprotein, exist in egg white and only take up 2% to 4% of total egg white protein. Ovomucin shares genetic similarities with mammalian mucins, which had been reported to possess antimicrobial properties. There were few reports about antimicrobial activity of ovomucin in eggs. The present thesis focused on the antimicrobial activity and its mechanisms of ovomucin against the common bacteria in eggs. The main research contents and results are as follows:First, a new method of isolating ovomucin is established with polyethylene glycol(PEG) precipitation and then precipitation by Mg Cl2. The best extraction condition by PEG is explored. The results showed that the volume rate of egg white and water was 1:2,and the concentration of PEG 8000 was 2%(w/w), in which conditions, ovomucin could be separated from egg white. The extractive was then precipitated with 0.05mol/L Mg Cl2 to remove other proteins. The main co-precipitated proteins were ovalbumin,ovotransferrin, ovomucoid. Purity of ovomucin was 87%, and its yield almost reached80% via this improved two-step precipitation.The antimicrobial activity and stability of ovomucin against the common bacteria in eggs such as Staphylococcus aureus, Saprophyticus subsp, Bacillus subtilis, Escherichia coli, Salmonella enteritidis, and the decay-causing Streptococcus mutans were studied.Results indicated ovomucin had obvious inhibition zones when co-incubating with Staphylococcus aureus, Saprophyticus subsp and Streptococcus mutans. The minimum inhibitory concentration(MIC) against them were 62.5μg/m L, 125μg/m L and 1000μg/m L,respectively. On the other hand, the inhibitory effect against Bacillus subtilis, Escherichia coli, Salmonella enteritidis were poor without inhibition zones. The antimicrobial stability of ovomucin on Staphylococcus aureus under the influence of different factors such as p H, temperature and metal icons was discussed, the results showed that the relative lower temperature(20℃, 25℃) could inhibit the growth of Staphylococcus aureus when thermal treatment at 20℃, 25℃and 40℃. Ovomucin displayed the betterinhibition in higher concentration under the same temperature. The antimicrobial activity of ovomucin on Staphylococcus aureus varied with different p H. It increased under acid and alkaline condition, when compared with neutral p H. Addition of some sorts of metal ion such as Na+, K+, Ca2+, Mg2+(0.05mol/L~0.20mol/L) didn’t show any positive effect on antimicrobial activity of ovomucin. A positive effect was observed when added0.05mol/L of Cu2+, Zn2+, Mn2+, and while there were weak antimicrobial activity at a higher concentration. Fe3+strengthened antimicrobial effect of ovomucin only when the concentration was 0.05mol/L.The antimicrobial mechanism of ovomucin to Staphylococcus aureus, Saprophyticus subsp and Streptococcus mutans was investigated. The growth curve indicated that ovomucin slowed logarithmic phase or shortened stationary phase of above three bacteria,and the cell membrane permeability had changed. Observation under scanning electron microscope revealed that the structure of the bacteria cells were significantly destroyed by ovomucin, and the cells wall were melted, sticked and adhered together. The outline of cells got blur, and the cell membrane were damaged. These results showed that the antimicrobial activity of ovomucin against bacteria is relevant to the damage of cell wall,cytoplasmic membrane and the increased permeability of cytoplasmic membrane.Ovomucin was hydrolyzed with one of three proteases(pepsin, trypsin and alcalase),and the antimicrobial activity of ovomucin hydrolysate was evaluated. The antimicrobial activity on Staphylococcus aureus showed that 4mg/m L of ovomucin was hydrolyzed by pepsin, and 2mg/m L of ovomucin was hydrolyzed by trypsin, which had inhibition zone of(11.57±0.41)mm,(11.41±0.24)mm respectively. The hydrolysate from three proteases showed no inhibition zone on Saprophyticus subsp and Streptococcus mutans. The UV spectrum scanning showed the low molecular peptide increased after ovomucin was hydrolyzed by protease, and the absorbance peak was stronger at 220 nm. The conditions of hydrolysis with trypsin was further optimized, and the optimum antimicrobial activity with a hydrolysis condition of substrate concentration of 2mg/m L, enzyme dosage [E]/[S]5%(w/w), reaction time of 4h, reaction temperature of 40℃. SDS-PAGE and GFC showed that trypsin hydrolysate relative molecular mass mainly was from 10 k Da to15 k Da. The growth curve indicated that trypsin hydrolysate slowed logarithmic phase or shortened stationary phase on Staphylococcus aureus.In order to explore the relationship between structure and antimicrobial activity, the antimicrobial activities of subunit and carbohydrate chain of ovomucin were studied.Ovomucin was decomposed into subunits by SDS, β-ME and ultrasonication, and then purified by GFC to obtain O-glycan by β-elimination reaction. Results showed that subunit and sugar chain had no antimicrobial activity on Staphylococcus aureus,Saprophyticus subsp and Streptococcus mutans, and we confirm it through the growth curve. Thus the results showed that the antimicrobial activity of components of dissociated ovomucin dramatically decreased or disappeared. |
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