LYSOZYME LAB REPORT

Lysozyme Lab Report



Lysozyme Lab Report

Introduction

Lysozyme is an enzyme present in mucus secretion, blood and other areas of virtually all eukaryotic organisms. It is a hydrolytic enzyme able to cleave the cell wall of gram + and some gram - bacteria. Cleavage of the peptidoglycan well at the ß1-4 linkage between N-acetyl-glucosamine and N acetyl galactosamine (muramate) results in lysis of the bacteria. The role of lysozyme in innate immunity is clear. Comparison of lysozyme activity in various species may reveal interesting evolutionary aspects of the immune system. The activity of lysozyme can be measured in a variety of ways. This experiment works toward providing comparison of the various published techniques to each other so that a more standard assessment of lysozyme activity can be obtained by examination of diverse literature. We will examine the analysis of lysozyme activity by comparing two different techniques, a lysoplate and a turbidimetric. The turbidimetric assay results can be reported by two methods; relative lysozyme activity measured in Units/min, and by comparison to a standard curve generated using purified Hen egg white lysozyme (HEWL). We will examine activity of lysozyme in various species (including oyster, fish, dog, cat, iguana (perhaps), and human) using freeze dried Micrococcus lysodeiltikus (M.l.) as the substrate. We will also examine the reproducibility of the assay in the classroom, and what the effect of freezing is on evaluating lysozyme activity.

Lysozyme is effective against certain Gram-positive bacteria, including LAB. It has been used in the cheese industry as a bio-protectant for more than 20 years to prevent the butyric spoilage which causes the late blowing of semi-hard cheeses by Clostridium tyrobutyricum. Several studies have been done on the use of lysozyme to regulate or to inhibit MLF, and on the impact of some winemaking factors on the efficacy of lysozyme. Although lysozyme has been shown to inhibit wine LAB, little, if any, research has been focused on the use of lysozyme to inhibit the growth of spoilage LAB and to prevent spoilage problems caused by these microorganisms during winemaking.

Materials and Methods

Lysozyme

Lysozyme (hydrochloride form) was extracted from chicken egg white (Canadian Inovatech Inc., Abbotsford, BC, Canada). It had a purity of minimum 98% protein, with an activity of 24,000 units/mg as defined by the Shugar method. Spoilage lactic acid bacteria Four spoilage LAB cultures were used in this study. L. kunkeei (ATCC 700308, strain YH-15) and P. parvulus (WS12) were generously provided by Dr Charles Edwards from Washington State University, Pullman, WA, USA. P. damnosus (C5) was supplied by Ms Lisa van de Water from The Wine Lab, Napa, CA, USA. The strain of L. brevis was isolated from a contaminated red wine from California, USA and was identified by partial sequencing of the 16S rRNA. Prior to inoculation, all the bacterial cultures were acclimatised in sterile Chardonnay juice at pH 3.8. The inoculation levels were 106 to 108 CFU/mL. Chemical analysis and microbial enumeration Total sugar (glucose and fructose), L-malic acid, L- and Dlactic acid, acetic acid, and ethanol ...