Antimicrobial Properties And Mechanisms Of Action Of Egg Albumen Collected From The Early Incubation Stages

Egg albumen has unique physical and chemical properties and contains a network of defenses against microbial infection. It was considered to play an important role in antibacterial defence of the egg yolk and the avian embryo. The antimicrobial system can be activated by incubation due to the changes of physical and chemical properties of egg albumen during egg incubation. The research object of this diploma paper was egg albumen collected from the early incubation stages. And the purpose of this research was to reveal the underline antimicrobial mechanisms and the interaction relationship of egg albumen proteins during early incubation. Including the studies:(1) The changes of antibacterial system and antibacterial activity of egg albumen during egg incubation.(2) The antimicrobial mechanisms of action of egg albumen during egg incubation.(3) In vitro study of the antimicrobial proteins network of egg albumen. Specific content and the results are as follows:The changes in basic properties and in vitro antimicrobial activity of egg albumen during early incubation were determined. The result has shown that the pH of egg albumen was increased and reached its highest level after48hours incubation while the albumen height decreased to the lowest. Afterwards, the pH was decreased slowly and the viscosity of thick and thin egg albumen increased rapidly. The antimicrobial activity of egg albumen was also changed during the early incubation. The egg albumen collected from the second day of incubation has shown a significant increase of antimicrobial activity, especially to Salmonella enteritidis. However, the antimicrobial activity of egg albumen was decreased as incubation progressed. The antimicrobial effects of egg albumen on gram-negative bacteria would almost disappear after5days of incubation.The most abundant and well characterized antimicrobials in egg albumen are ovotransferrin and lysozyme. The changes of ovotransferrin and lysozyme content in egg albumen were quantified via high-performance liquid chromatography (HPLC). The results showed that the concentration of ovotransferrin in egg albumen was increased closely with the time of incubation but that of lysozyme remained substantially unchanged and was very similar to that of the non-incubated eggs. We supposed that egg incubation conditions may result in the degradation of lysozyme content. In contrast to lysozyme, ovotransferrin content has shown higher stability during the early incubation periods. The iron ion binding capacity and specific lysozyme activity of the egg albumen during early incubation stages was also investigated. The concentration of ovotransferrin determined on the iron-binding capacity was increased gradually and reached nearly23.78±0.65mg/mL after5days of incubation, which implied an increased iron binding capacity. The specific lysozyme activity in egg albumen decreased from30397±2280to15036±2574U/mg during the first two days of incubation. However, in the later stages of incubation, an increase of the enzymatic activity has been found. We inferred that the possible reason for the changes of lysozyme activity in egg albumen during incubation may be due to the dynamic changes of pH in egg albumen.To further understand the stability of egg albumen proteins during early incubation, an off-line peptidomics approach combining gel chromatography and MALDI-TOF MS was used to map the low molecular weight (LMW) protein profile of egg albumen during early incubation. The observed changes in the LMW protein profile during development were highly dynamic.114mass signal peaks of LMW proteins ranging from1,035.88to7,112.91m/z were detected during the early incubation stages (0-5d). Among them,28,26,35,35,20, and17peaks of proteins were present specifically in egg albumen within0,1,2,3,4,5days of incubation. The research result has confirmed that some large molecular weight proteins would degraded into small fragments during the early incubation stages.Carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) were used in this study to determine the changes of physiological state of Salmeonella enteritidis cells treated by egg albumen. Following egg albumen treatment, Salmonella enteritidis was discriminated into three subpopulations by Fluorescence microscopy and Flow cytometric assessment:viable, dead and membrane-compromised cells. The incubation temperature and pH were both significant factors in anti-Salmonella enteritidis properties of egg albumen. Besides, the main target for antimicrobial factors in egg albumen on Salmonella enteritidis cells would be membrane integrity and the action mechanism of egg white was bacteriostatic rather than bactericidal effect during early incubation.The morphological changes of Salmeonella enteritidis cells treated with egg albumen were investigated by using SEM. The result showed that bacteria cells grown in incubated egg albumen were abnormal in shape and size indicating a disruption of the membrane integrity. As to Salmeonella enteritidis cells treated with incubated egg albumen and double-stained with SYTO9and PI, both green and red fluorescence can be detected. These studies confirmed previously mentioned idea that the main target for antimicrobial factors in incubated egg albumen on Salmonella enteritidis cells would be membrane integrity. Flow cytometric technique was used to quantify different bacteria subpopulations treated with egg albumen incubated for different periods. The result showed that the ability of egg albumen to disrupt bacteria membrane integrity would changed as incubation progressed and the egg albumen incubated for two days has shown to possess the most strong ability. At the same time, egg albumen from the early incubation stages could inhibit the expression of hilA gene of Salmonella enteritidis and minimize its adhesion and invasion activity to Caco-2cell.The synergistic antimicrobial activity of ovotransferrin and lysozyme to Salmonella enteritidis was determined by using the SYTO9/PI double staining technique in combination with flow cytometry. The result showed that ovotransferrin could disrupt the membrane integrity of Salmonella enteritidis cells and the inhibitory effect of lysozyme to Salmonella enteritidis was weak. Alkaline pH would enhance the antimicrobial activities of ovotransferrin and lysozyme to Salmonella enteritidis and promote the synergistic antimicrobial activity of them. Ovalbumin was the most abundant protein in egg albumen which constitutes a special protein environment for the antimicrobial proteins. It showed little antimicrobial activity on Salmonella enteritidis. However, we found that ovalbumin increased the synergistic activities of ovotransferrin and lysozyme under alkaline pH, which indicated that a complicated interaction relationship was existed between egg albumen proteins and albumen physicochemical environment during egg incubation.