Origin and diversity of mesophilic lactobacilli in Comté cheese, as revealed by PCR with repetitive and species-specific primers

Abstract

The objectives of this work were to describe the diversity of mesophilic lactobacilli in Comté cheese at the strain and species levels, to determine the origin(s) of this non-starter microflora, and to get a collection of well characterised strains from Comté cheeses. Strains were isolated from milks, starter cultures and eight cheeses from two factories, with four cheeses made from the same vat in each factory. Strain and species assignations were performed with a combination of two PCR-based methods, amplification with the pairs of repetitive primers ERIC1/ERIC2 and REP–1R-Dt/REP2-D, and amplification with specific primers for Lactobacillus zeae, Lactobacillus paracasei and Lactobacillus rhamnosus. The reliability and reproducibility of these methods were assessed using 49 collection strains of mesophilic lactobacilli commonly detected in cheeses. A total of 488 isolates of mesophilic lactobacilli was collected and was assigned to 44 different strains and three different species. Lactobacillus paracasei and Lactobacillus rhamnosus were the predominant species in milks, starter cultures and cheeses, and constituted 98.7% of the isolates. Strain diversity was found at both individual cheese and factory levels. Thirteen and fifteen different strains were detected throughout cheesemaking and ripening in two individual cheeses made in different factories; only 11 different strains were detected in the two corresponding mature cheeses. The data strongly suggest that most mesophilic lactobacilli strains originate from raw milk.

Introduction

Comté cheese is a hard-cooked ripened cheese variety manufactured from raw cow's milk in a limited region in the East of France, and labelled “Appellation d’Origine Protégée” (AOP) (Beuvier, 1996). Thermophilic and mesophilic whey starter cultures, including selected strains of Lactobacillus helveticus, Streptococcus thermophilus and Lactococcus lactis, are added during the cheesemaking process. Mesophilic lactobacilli are detected as a dominant non-starter microflora in Comté cheese, where their viable numbers increased from 10³ to 10⁴ cfu g⁻¹ cheese at the beginning of ripening to 10⁸ cfu g⁻¹ after four weeks of ripening, and remain at this level throughout a ripening period of at least five months (Grappin, Beuvier, Bouton, & Pochet, 1999).

Mature Comté cheeses exhibit complex and varied sensory properties (Stèvenot, Bérodier, & Schlich, 1997), which could originate from various mechanisms, including the activities of the microbial ecosystem. This was demonstrated with experimental mini Comté-type cheeses, where changes in the level or origin of the milk microflora were shown to affect notably the sensory properties of the mature cheeses (Beuvier, Berthaud, Cegarra, Dasen, Pochet, & Duboz, 1997; Demarigny, Beuvier, Dasen, & Duboz, 1996). The mesophilic lactobacilli could participate to the elaboration of the sensory properties of mature Comté cheese because of their abundance and time of presence during ripening, as suggested and investigated in other cheese varieties (Fox, McSweeney, & Lynch, 1998; Sollberger, 1990).

To investigate this aspect, and especially to explain and understand the diversity of the sensory properties in mature Comté cheese, there is a need to know the origin and to characterise the microflora of Comté cheese at the strain level, as different strains of a same species often have different enzymatic potentialities in terms of flavour compound production (Williams, Felipe, & Banks, 1998). A collection of well-characterised strains from Comté cheese is also essential to conduct experiments in cheesemaking.

The objectives of this study were to evaluate the diversity of mesophilic lactobacilli in Comté cheese according to cheesemaking and ripening conditions, and to investigate the origin(s) of the strains found in cheese to elucidate at least partially the role of the raw milk microflora on the sensory properties of mature cheese. In these respects, a new and reliable approach which allowed a rapid and easy assignment of isolates at the strain and species levels was developed and applied to isolates of mesophilic lactobacilli from milks, starter cultures and Comté cheeses collected in two cheese factories.