Methods to Determine Proteolytic Activity of Lactic Acid Bacteria.

SAVOY DE GIORI, G.; HEBERT, E.M.

Food Microbiology Protocols. Series Methods in Biotechnology.

Humana Press Inc

Lugar: Totowa, New Jersey; Año: 2001; p. 197 - 202

The lactic acid bacteria (LAB) are considered weakly proleolytic when compared with many other groups of bacteria (e.g. Bacillus, Proteus, Pseudomonas). However, most strains of LAB rely on a complex proteolytic system that allow them to liberate essential and growth-stimulatory amino acids and small peptides from the protein-rich substrates such as milk, meat and vegetables in which they are primarily found. The proteolytic system of dairy LAB is composed of an extracellular, cell-envelope proteinase, various intracellular peptidases, and amino acid and peptide transport systems for uptake of the products of proteolysis. The action of the cell-envelope proteinase is not only crucial to the growth of the organism in milk but also to secondary proteolysis and flavour development in cheese (1). Many procedures have been used to detect proteolysis of LAB. Traditionally, the method most widely used is that described by Hull (2). The Hull method relies on the release of tyrosine-and tryptophan-containing peptides that react with Folin-Ciocalteau reagent; consecuentely, this method lacks sensitivity. In this chapter we describe different procedures, more sensitive and rapid, to determine the proteolytic activity of LAB, that are routinely used in our laboratory. These techniques include the use of o-pthaldialdehyde (OPA), chromogenic peptide (S-Ala) or fluorescein isothiocyanate (FITC)-casein as substrate. The lactic acid bacteria (LAB) are considered weakly proleolytic when compared with many other groups of bacteria (e.g. Bacillus, Proteus, Pseudomonas). However, most strains of LAB rely on a complex proteolytic system that allow them to liberate essential and growth-stimulatory amino acids and small peptides from the protein-rich substrates such as milk, meat and vegetables in which they are primarily found. The proteolytic system of dairy LAB is composed of an extracellular, cell-envelope proteinase, various intracellular peptidases, and amino acid and peptide transport systems for uptake of the products of proteolysis. The action of the cell-envelope proteinase is not only crucial to the growth of the organism in milk but also to secondary proteolysis and flavour development in cheese (1). Many procedures have been used to detect proteolysis of LAB. Traditionally, the method most widely used is that described by Hull (2). The Hull method relies on the release of tyrosine-and tryptophan-containing peptides that react with Folin-Ciocalteau reagent; consecuentely, this method lacks sensitivity. In this chapter we describe different procedures, more sensitive and rapid, to determine the proteolytic activity of LAB, that are routinely used in our laboratory. These techniques include the use of o-pthaldialdehyde (OPA), chromogenic peptide (S-Ala) or fluorescein isothiocyanate (FITC)-casein as substrate.