Microbiota characterization of a Belgian protected designation of origin cheese, Herve cheese, using metagenomic analysis

Abstract

Herve cheese is a Belgian soft cheese with a washed rind, and is made from raw or pasteurized milk. The specific microbiota of this cheese has never previously been fully explored and the use of raw or pasteurized milk in addition to starters is assumed to affect the microbiota of the rind and the heart. The aim of the study was to analyze the bacterial microbiota of Herve cheese using classical microbiology and a metagenomic approach based on 16S ribosomal DNA pyrosequencing. Using classical microbiology, the total counts of bacteria were comparable for the 11 samples of tested raw and pasteurized milk cheeses, reaching almost 8 log cfu/g. Using the metagenomic approach, 207 different phylotypes were identified. The rind of both the raw and pasteurized milk cheeses was found to be highly diversified. However, 96.3 and 97.9% of the total microbiota of the raw milk and pasteurized cheese rind, respectively, were composed of species present in both types of cheese, such as Corynebacterium casei, Psychrobacter spp., Lactococcus lactis ssp. cremoris, Staphylococcus equorum, Vagococcus salmoninarum, and other species present at levels below 5%. Brevibacterium linens were present at low levels (0.5 and 1.6%, respectively) on the rind of both the raw and the pasteurized milk cheeses, even though this bacterium had been inoculated during the manufacturing process. Interestingly, Psychroflexus casei, also described as giving a red smear to Raclette-type cheese, was identified in small proportions in the composition of the rind of both the raw and pasteurized milk cheeses (0.17 and 0.5%, respectively). In the heart of the cheeses, the common species of bacteria reached more than 99%. The main species identified were Lactococcus lactis ssp. cremoris, Psychrobacter spp., and Staphylococcus equorum ssp. equorum. Interestingly, 93 phylotypes were present only in the raw milk cheeses and 29 only in the pasteurized milk cheeses, showing the high diversity of the microbiota. Corynebacterium casei and Enterococcus faecalis were more prevalent in the raw milk cheeses, whereas Psychrobacter celer was present in the pasteurized milk cheeses. However, this specific microbiota represented a low proportion of the cheese microbiota. This study demonstrated that Herve cheese microbiota is rich and that pasteurized milk cheeses are microbiologically very close to raw milk cheeses, probably due to the similar manufacturing process. The characterization of the microbiota of this particular protected designation of origin cheese was useful in enabling us to gain a better knowledge of the bacteria responsible for the character of this cheese.

Temporal and Spatial Differences in Microbial Composition during the Manufacture of a Continental-Type Cheese

“…While the significance of the presence of these populations is not yet clear, particularly at the levels present in the cheese, they may play a role in ripening (38,49,54). Psychrotrophic bacteria, including Psychrobacter and Pseudomonas, have previously been isolated from a variety of cheeses as well as raw milk and are particularly adapted to lowtemperature milk storage conditions (49,55,56).…”

Isolation and identification of lactic acid bacteria from Brazilian Minas artisanal cheese

“…It could be assumed that these LAB originated from raw milk and the endogenous culture, as they presented high counts of LAB at the time of cheese manufacture, 6.4 log units for raw milk (both seasons) and 7.0 and 8.0 for endogenous culture (rainy and dry seasons, respectively). Delcenserie et al (2014) reported that the average count of LAB in a Belgian protected designation of origin cheese was 7.5 log units when made with raw milk. The ripening time only influenced the mean counts of LAB in the M17 medium during the dry season (Table 1).…”

Metagenomic analysis of the bacterial microbiota linked to the traditional Algerian date product “Btana”

“…PCR reactions were carried out according to Delcenserie et al (2014) in a final volume of 100 μl containing 5 U of FastStart high-fidelity polymerase (Roche Diagnostics Belgium NV), 1× enzyme reaction buffer, 200 μM of dNTP mix (Eurogentec S.A., Seraing, Belgium), 0.2 μM of each primer, and 100 ng of template DNA. The PCR reactions were performed in a Mastercycler ep gradient apparatus (Eppendorf), with the following program: an initial denaturation for 15 min at 94°C, followed by 25 cycles of denaturation at 95°C for 40 s, annealing at 56°C for 40 s, and extension at 72°C for 1 min, and a final extension step at 72°C for 7 min (Delcenserie et al 2014). PCR products were then run on a 1 % agarose electrophoresis gel, and the DNA fragments were extracted and purified with the Wizard SV PCR purification kit (Promega Corporation, Belgium).…”