Françoise Berthier

Traditional cheeses: Rich and diverse microbiota with associated benefits

The risks and benefits of traditional cheeses, mainly raw milk cheeses, are rarely set out objectively, whence the recurrent confused debate over their pros and cons. This review starts by emphasizing the particularities of the microbiota in traditional cheeses. It then describes the sensory, hygiene, and possible health benefits associated with traditional cheeses. The microbial diversity underlying the benefits of raw milk cheese depends on both the milk microbiota and on traditional practices, including inoculation practices. Traditional know-how from farming to cheese processing helps to maintain both the richness of the microbiota in individual cheeses and the diversity between cheeses throughout processing. All in all more than 400 species of lactic acid bacteria, Gram and catalase-positive bacteria, Gram-negative bacteria, yeasts and moulds have been detected in raw milk. This biodiversity decreases in cheese cores, where a small number of lactic acid bacteria species are numerically dominant, but persists on the cheese surfaces, which harbour numerous species of bacteria, yeasts and moulds. Diversity between cheeses is due particularly to wide variations in the dynamics of the same species in different cheeses. Flavour is more intense and rich in raw milk cheeses than in processed ones. This is mainly because an abundant native microbiota can express in raw milk cheeses, which is not the case in cheeses made from pasteurized or microfiltered milk. Compared to commercial strains, indigenous lactic acid bacteria isolated from milk/cheese, and surface bacteria and yeasts isolated from traditional brines, were associated with more complex volatile profiles and higher scores for some sensorial attributes. The ability of traditional cheeses to combat pathogens is related more to native antipathogenic strains or microbial consortia than to natural non-microbial inhibitor(s) from milk. Quite different native microbiota can protect against Listeria monocytogenes in cheeses (in both core and surface) and on the wooden surfaces of traditional equipment. The inhibition seems to be associated with their qualitative and quantitative composition rather than with their degree of diversity. The inhibitory mechanisms are not well elucidated. Both cross-sectional and cohort studies have evidenced a strong association of raw-milk consumption with protection against allergic/atopic diseases; further studies are needed to determine whether such association extends to traditional raw-milk cheese consumption. In the future, the use of meta-omics methods should help to decipher how traditional cheese ecosystems form and function, opening the way to new methods of risk-benefit management from farm to ripened cheese.

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

The objectives of this work were to describe the diversity of mesophilic lactobacilli in Comte 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 Comte 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.

Biodiversity and growth dynamics of lactic acid bacteria in artisanal PDO Ossau-Iraty cheeses made from raw ewe's milk with different starters

The biodiversity and growth dynamics of Lactic Acid Bacteria (LAB) in farm-house Ossau-Iraty cheeses were investigated from vat milk to 180 days of ripening in six independent batches made from six raw ewes milks using five typical cheese-making methods. Commercial starter S1 was used for three batches, starter S1 combined with S2 for one batch and no starter for two batches. Up to ten LAB species from five genera and up to two strains per species were identified per milk; up to eleven species from five genera and up to three strains per species were identified per cheese. Lactococcus lactis, Lactobacillus paracasei, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, and Leuconostoc mesenteroides were detected in all cheeses. Lactococci reached the highest counts irrespective of the milk and starter used. Lactococci and enterococci increased during manufacture, and mesophilic lactobacilli increased during ripening. Strain and species numbers, the percentage of isolates originating from the raw milk, maximum counts of each genus/species and time for reaching them, all varied according to whether or not a starter was used and the composition of the starter. The genotypes of strains within species varied according to the raw milk used. This generated distinct LAB microbiotas throughout manufacture and ripening that will certainly impact on the characteristics of the ripened cheeses.

Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples

The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R(2) > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei.

On the screening of hydrogen peroxide‐generating lactic acid bacteria

The published plate methods for the detection of hydrogen peroxide‐producing lactic acid bacteria, which employ horseradish peroxidase and a chromogen, clearly fail to detect all the organisms that produce H2O2. Whilst keeping the same principle, the use of a novel growth medium and of the chromogen tetramethyl‐benzidine allows the detection of H2O2 production by strains previously classified as non‐producers.

Multiple interactions between Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii strongly affect their growth kinetics during the making of hard cooked cheeses.

In hard cooked cheeses, any interactions between the thermophilic starters as they grow during the cheese-making are critical, since they modify bacterial growth kinetics and acidification kinetics, so affecting the ripening process and the final characteristics of the cheese. Twenty-four experimental hard cooked cheeses were made under controlled conditions, the milk being inoculated with various combinations of thermophilic strains of Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii. Over the first day of manufacturing we recorded a wide range of different growth kinetics for each starter species used, and a wide range of pH kinetics, depending on the starter combination. Most of the bacterial variability could be statistically explained by the nature, quantity, and/or presence or absence of the different strains inoculated. Four main interactions between the three species were evidenced during cheese-making. There was antagonism between L. helveticus and L. delbrueckii. The lactobacilli had a positive effect on S. thermophilus, which was reciprocal for L. helveticus. L. helveticus had a negative effect on S. thermophilus cultivability. And the combination of S. thermophilus inoculated in large quantities and L. helveticus strain H2 had a negative effect on the growth of the L. delbrueckii strain D2. While the positive effect of L. delbrueckii on S. thermophilus probably corresponds to interactions in milk that have already been described and published, the other interactions were hitherto unknown. These interactions are of major importance for the growth kinetics of streptococci and thermophilic lactobacilli during cheese-making.

Expression of six mitochondrial genes during Drosophila oogenesis: analysis by in situ hybridization

We have done a comparative analysis of RNA from six mitochondrial genes (rDNA, ND2, COI, COIII, ND4-ND5, Cyt b) during Drosophila oogenesis, using in situ hybridization. This study showed the same variation for each of these transcripts, which is similar to that obtained with the total mitochondrial RNA (Tourmente et al. (1990) Biol. Cell 60, 119-127). A constant RNA density until stage 9, followed by a rapid decline, was observed in follicle and nurse cells. These results confirm those previously obtained (Tourmente et al., (1990) Biol. Cell 60, 119-127), in favor of the existence of a correlation between the mtRNA level and the cell volume and/or the nuclear DNA content, and suggest a global extra-mitochondrial, transcriptional control mechanism. We also show that the relative proportions of the different RNA are similar, whatever the stage and cell type examined, even though the total mtRNA quantity is different. They are comparable to those previously obtained by Northern analysis of Drosophila embryos (Berthier et al. (1986) Nucleic Acids Res. 14, 1400-1412, suggesting a posttranscriptional control independent of the cell type. Surprisingly, we have detected an extra-mitochondrial hybridization for COIII, both in light and electron microscopy. Northern analysis of poly(A)+RNA from ovaries or cultured cells revealed an 1.7 kb extra-mitochondrial RNA, which is probably of nuclear origin.

Suitability of a new mixed-strain starter for manufacturing uncooked raw ewe's milk cheeses

Most raw milk Ossau-Iraty cheeses are currently manufactured on-farm using the same commercial streptococcal-lactococcal starter (S1). One way to enhance the microbial diversity that gives raw milk its advantages for cheese-making is to formulate new starters combining diverse, characterized strains. A new starter (OI) combining 6 raw milk strains of lactococci, recently isolated and characterized, was tested in parallel with the current starter by making 12 Ossau-Iraty raw milk cheeses at 3 farmhouses under the conditions prevailing at each farm. Compliance of the sensory characteristics with those expected by the Ossau-Iraty professionals, physicochemical parameters and coliforms were quantified at key manufacturing steps. The new starter OI gave cheeses having proper compliance but having lower compliance than the S1 cheeses under most manufacturing conditions, while managing coliform levels equally well as starter S1. This lower compliance relied more on the absence of Streptococcus thermophilus in starter OI, than on the nature of the lactoccocal strains present in starter OI. The study also shows that variations in 5 technological parameters during the first day of manufacture, within the range of values applied in the 3 farmhouses, are powerful tools for diversifying the scores for the sensory characteristics investigated.