Accelerating cheese proteolysis by enriching Lactococcus lactis proteolytic system with lactobacilli peptidases

Abstract

The knowledge available on the genetics and proteolytic system of lactic acid bacteria makes it possible to genetically engineer starters with increased proteolytic properties. Our objective was to identify the best available strains capable of accelerating or modulating casein proteolysis during cheese ripening.

To attain this goal, we used Lactococcus lactis strains expressing 5 different Lactobacillus peptidases to ripen a cheese model. At the end of ripening, free amino acids were quantitatively and qualitatively analysed.

We identified the mixture of prolidase, PepQ, and X-prolyl dipeptidyl peptidase, PepX, as well as the peptidase PepW as the most efficient peptidases to increase, up to 3-fold, the overall level of amino acids at the end of ripening. The levels of threonine, asparagine, glycine, methionine, valine, glutamine, isoleucine and proline in particular increased (more than 3.5 fold). Grouping the amino acids produced according to the specific aroma compounds that each may give rise to following an enzymatic or chemical conversion, revealed that expression of PepW or PepX and PepQ increased the amounts of all groups of amino acids while expression of PepQ or PepN increased more especially those of aromatic amino acids/proline and glutamic acid, respectively.

The combination of increased proteolysis and conversion of amino acids into aroma compounds now needs to be tested. In addition, the role of proline and its derived compounds in the overall flavour of cheese should be investigated.

Introduction

Proteolysis occurring in cheese during ripening plays a major role in the development of organoleptic properties in hydrolysing bitter peptides and in releasing free amino acids, precursors of aroma compounds. Lactic acid bacteria, which constitute the main flora of dairy products participate in this proteolytic process especially in hydrolysing short and medium peptides into amino acids (Visser, 1977). The proteolytic systems of Lactococcus lactis and Lactobacillus helveticus and Lactobacillus delbrueckii are now well described. They are composed of a cell-wall proteinase and of more than ten intracellular peptidases exhibiting different specificities (for a review, see Christensen, Dudley, Pederson, & Steele, 1999). The overall proteolytic activity of lactobacilli has been found higher than that of L. lactis because lactobacilli possess additional peptidases and because their peptidases have higher expression levels (Khalid & Marth, 1990; Sasaki, Basman, & Tan, 1995).

It is now well accepted that free amino acids released in cheese are not solely responsible for the typical cheese flavour mainly because they are present in concentrations which are lower than their taste threshold values (Kubickova & Grosch, 1998). Moreover, the release of free amino acids is not a limiting step in the flavour development process as demonstrated by Wallace and Fox (1997) who added large amounts of free amino acids in cheese without strongly modifying its aroma. A few years ago, Yvon, Thiroin, Rijnen, Fromentier, and Gripon (1997) demonstrated that transamination, the first step of amino acid conversion into aroma compounds, restricts the development of aromatic compounds derived from proteins. Because increasing the rate of amino acid transamination is now possible by adding α-keto-glutarate as an amino group acceptor (Yvon, Berthelot, & Gripon, 1998) and because specific groups of amino acids (branched-chain, aromatic, sulphur) lead to specific groups of aromatic compounds with animal, floral or garlic notes (Yvon & Rijnen, 2002), it is especially interesting to increase proteolysis and favour the release of specific groups of amino acids. In this work, we evaluated the possibility of proteolysis intensification and modulation. The approach chosen was to enrich the proteolytic system of L. lactis with lactobacilli peptidases that either have or not counterparts in L. lactis (Luoma et al., 2001; Wegmann, Klein, Drumm, Kuipers, & Henrich, 1999). We decided to include the general aminopeptidase PepN and the proline specific peptidase PepX in the group of peptidases expressed in lactococci. In a previous work carried out with PepN and PepX negative mutants of lactococci, we pointed out the important role of these two peptidases during ripening (Guinec, Nardi, Matos, Gripon, & Monnet, 2000). The proteolytic potential of the modified L. lactis strains was tested in ripening conditions in a cheese model called pseudo-curd. The results demonstrated that several peptidases are either limiting or lacking for the degradation of caseins by L. lactis.