Purification of Lysozyme from Shell Liquor of Eastern Oysters (Crassostrea virginica) and Its Use in Antimicrobial Films to Preserve Smoked Fish
The objective of our study was to purify lysozyme from the fluid filling cavity (shell liquor) of the eastern oysters (Crassostrea virginica) and determine the antimicrobial activity of purified lysozyme against foodborne pathogens in smoked salmon samples.
Oyster shell liquor was collected over four seasons namely summer 2003, fall 2003, winter 2004, and spring 2004. Spring season showed the highest lysozyme concentration. Lysozyme was purified from 300 liters of oyster shell liquor by a two-step ion exchange chromatography using SP- and CM- Sepharose Fast Flow columns, respectively. Two hundred five mg of pure lysozyme was obtained which represented a recovery of 11.1%. The purity and molecular size of the isolated lysozyme showed a single band of about 18 KDa by SDS-PAGE.
MIC assays of the oyster lysozyme were carried out by serially diluting oyster and hen egg white lysozyme to get a concentration range of 160-2.5?g/ml. Twenty four hours bacterial suspension, Brain heart Infusion or APT broth (as required) was added to each well of a 96-well plate followed by incubating the microtiter plates and measuring absorbance at 640 nm with a microplate reader. MIC results showed that oyster lysozyme had antimicrobial activity against both gram-positive and gram-negative bacteria causing food spoilage and poisoning.
Smoked salmon samples were cut into 1 g pieces, inoculated with 24 h broth cultures of L. monocytogenes and S. anatum, dipped into zein propylene glycol, 0.75% agar gel, and calcium alginate coating. Calcium alginate coating was chosen as the best coating out of the three coatings. Various treatments of calcium alginate edible coatings were incorporated with oyster lysozyme or hen egg white lysozyme on the surface of the smoked salmon, allowed to air dry for 20 min and refrigerated at 4°C. Bacterial counts were determined at 0, 7, 14, 21, 28, and 35 days at 37°C for 24 h and CFU/g were determined. Combination of 160 ?g/ml of oyster lysozyme and 1000 IU/g of nisin treatment was found to be the most effective treatment and was shown to retain its antimicrobial activity inside the calcium alginate coating for 35 day period.
Advisor:Marlene Janes; Jerome La Peyre; Jack Losso; Witoon Prinyawiwatkul
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Date of Publication:07/18/2005