Different Analytical Approaches in Assessing Antibacterial Activity and the Purity of Commercial Lysozyme Preparations for Dairy Application

Milena Brasca,Stefano Morandi,Tiziana Silvetti,Luisa Pellegrino,Veronica Rosi,Stefano Cattaneo

doi:10.3390/molecules18056008

Abstract

Hen egg-white lysozyme (LSZ) is currently used in the food industry to limit the proliferation of lactic acid bacteria spoilage in the production of wine and beer, and to inhibit butyric acid fermentation in hard and extra hard cheeses (late blowing) caused by the outgrowth of clostridial spores. The aim of this work was to evaluate how the enzyme activity in commercial preparations correlates to the enzyme concentration and can be affected by the presence of process-related impurities. Different analytical approaches, including turbidimetric assay, SDS-PAGE and HPLC were used to analyse 17 commercial preparations of LSZ marketed in different countries. The HPLC method adopted by ISO allowed the true LSZ concentration to be determined with accuracy. The turbidimetric assay was the most suitable method to evaluate LSZ activity, whereas SDS-PAGE allowed the presence of other egg proteins, which are potential allergens, to be detected. The analytical results showed that the purity of commercially available enzyme preparations can vary significantly, and evidenced the effectiveness of combining different analytical approaches in this type of control.

Highlights

Lysozyme (LSZ, muramidase, EC 3.2.1.17) is one of the most extensively studied antimicrobial enzymes. It occurs in several mammalian secretions and in hen egg white (HEW), which represents the raw material of choice for the production of LSZ on an industrial scale [1]
This work has demonstrated that the HPLC method adopted by ISO allows the LSZ concentration to be determined with accuracy and data are significantly correlated to those obtained by the turbidimetric assay advised by FAO/WHO
The HPLC method gives evidence of molecular modifications that can occur during the production process and conservation

Summary

Introduction

Lysozyme (LSZ, muramidase, EC 3.2.1.17) is one of the most extensively studied antimicrobial enzymes It occurs in several mammalian secretions (milk, saliva, tears) and in hen egg white (HEW), which represents the raw material of choice for the production of LSZ on an industrial scale [1]. This low molecular weight enzyme (14,307 Dalton) consists of 129 amino acids cross-linked by four disulphide bridges, and shows lytic activity on the β(1→4) glycosidic bond between N-acetyl-Dglucosamine and N-acetylmuramic acid in the cell wall of bacterial species, Gram-positive microorganisms such as lactic acid bacteria (LAB) and Clostridia. In 1981, was the first country to allow the industrial application of LSZ in cheese production [6], and nowadays LSZ is permitted as a preservative (E1105) in ripened cheeses, in accordance with current EU legislation [9]

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