Free Access
Issue
Dairy Sci. Technol.
Volume 89, Number 6, November-December 2009
Page(s) 569 - 581
DOI https://doi.org/10.1051/dst/2009032
Published online 23 September 2009

© INRA, EDP Sciences, 2009

1. INTRODUCTION

Large-scale industrial processes relying on the use of selected starter cultures led to a low variability in the dairy microflora. Moreover, sanitation processes, such as milk pasteurization, which has a fundamental role in the control of pathogenic bacteria, also resulted in a significant reduction of the natural bacterial populations involved in naturally fermented and ripened cheese production. However, some traditional dairy products are still fermented and ripened using unselected starters and, therefore, correspond to a wide range of products with different flavors, texture and microbiological qualities [6, 29]. Moreover, the importance of raw milk as a source of strains harboring genetic diversity has been outlined in traditional cheese produced without pasteurization [9]. Finally, the existence of area typical wild strains would account for the recognized area particularity, allowing cheese labeling according to PDO (protected designation of origin). Thus, these products have been proposed as sources for new strains of interest for use in food fermentation and ripening.

Several species, Debaryomyces hansenii, Kluyveromyces lactis, Kluyveromyces marxianus and Yarrowia lipolytica, mainly constitute the yeast flora in dairy products and cheeses [12] where they contribute to the development of texture and flavor during the ripening process [27]. The need for new strains in the dairy industry and for a deeper knowledge of the natural microflora present in typical dairy products led to the study of the biodiversity of the most common yeast species involved in traditional cheese ripening.

To assess this biodiversity, several molecular approaches were used. For years, Saccharomyces cerevisiae strains were routinely characterized with RFLP (restriction fragment length polymorphism) analysis of chromosomal or mitochondrial DNA or electrophoretic karyotyping [1, 3, 32]. Techniques based on the PCR amplification of known sequences rather than repeated sequences have proved to be faster and just as efficient as RFLP analysis [14]. Yet, molecular methods for typing most non-conventional yeast species lack, mainly because of the paucity of available sequences. Repeated sequences within microsatellites [13, 28] or tRNA [23] were used as primers to generate strain-specific patterns.

Sequencing data on some yeast species that contribute to cheesemaking such as D. hansenii var. hansenii [17] and K. marxianus var. marxianus [18], referred to further on as D. hansenii and K. marxianus, respectively, are now available. These sequence data were used to detect and describe retrotransposons [22]. Retrotransposons are mobile elements responsible for genomic polymorphism. These elements transpose via mRNA intermediates [4]. In yeasts, the large majority of retrotransposons consist of long terminal repeat (LTR) retrotransposons, the so-called Ty in S. cerevisiae. The most common LTR retrotransposon of D. hansenii is Tdh5, a member of the Ty5 family. In K. marxianus, only one LTR retrotransposon has been identified, Tkm1, a member of the Ty1/copia family [22]. Excision of the retrotransposon through a homologous recombination at the bordering LTRs leaves an isolated, or so called, solo LTR. Solo LTRs outnumber the full-length elements in the genome. These repeated sequences were successfully used for the typing of S. cerevisiae strains [16, 21] and of other organisms [15], through the PCR amplification of implicated sequences. Estimation of the number of LTR retrotransposons in D. hansenii and K. marxianus [11, 22] indicated that an inter-LTR PCR fingerprinting method could be developed for these species. In this work, sequences of retrotransposons present in K. marxianus and D. hansenii [22] were used to develop a method based on the PCR amplification of sequences separating LTRs in the genome, using oligonucleotide primers designed within these LTRs. The developed inter-LTR PCR method was used to carry out genomic fingerprinting of strains isolated from traditional cheeses.

2. MATERIALS AND METHODS

2.1. Yeast strains and growth conditions

Strains were obtained from the Centre International de Ressources Microbiennes (CIRM-Levures, http://www.inra.fr/cirmlevures) and are listed in Table I. Most of the strains were isolated during the ripening of different types of traditional French cheeses from different regions: Camembert from Normandy, Chevrotin des Aravis from the Alps (Haute-Savoie) and Saint-Nectaire from Massif-Central [2, 10, 20]. Few strains were from Spanish Roncal cheese [28]. Strains were cultured at 28 °C overnight with agitation in liquid YPD medium (glucose 1% – Sigma Aldrich, St. Quentin, France; Bacto yeast extract 1% and Bacto peptone 1% – BD, Le Pont de Claix, France).

2.2. Oligonucleotidic primers

LTR sequences were aligned using the ClustalX program (http://bips.u-strasbg.fr/fr/Documentation/ClustalX/) and primers were designed in the conserved regions, i.e. oligonucleotides DH8 and DH9 from the D. hansenii LTR retrotransposon Tdh5 (Accession No. AJ439552) and oligonucleotides KM1 and KM2 from the K. marxianus LTR retrotransposon Tkm1 (Accession No. AJ439546). Primers used in this study are described in Table II.

Table IA.

List of the 56 D. hansenii strains used in this study.

Table IB.

List of the 61 K. marxianus strains used in this study.

Table II.

Oligonucleotidic primers used in this study and the associated Simpson’s diversity index.

2.3. Fingerprinting conditions

Genomic DNA was extracted using the Dneasy Plant Kit (Qiagen, Les Ulis, France) and quantified by fluorimetry with PicoGreen (Invitrogen, Cergy-Pontoise, France) following the manufacturer’s instructions. Primers were synthesized and purified by HPLC (Proligo, Évry, France). Amplification reactions were performed in a 50-μL volume containing the total genomic DNA quantity required: 1 μmol·L−1 of each primer, 500 μmol·L−1 of dNTP, 1.25 U of Taq DNA polymerase and 5 μL of 10 X PCR buffer (Q-Biogen, Illkirch, France). Total genomic DNA quantities corresponded to 20 ± 5 ng for D. hansenii strains and to 45 ± 5 ng for K. marxianus strains. PCR conditions using the primer pair DH8/DH9 were as follows: 94 °C for 4 min, 4 cycles of 94 °C for 1 min, 51 °C for 1 min and 72 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 44 °C for 30 s and 72 °C for 2 min with a final extension completed at 72 °C for 4 min. PCR conditions with the primer pair KM1/KM2 were as follows: 94 °C for 4 min, four cycles of 94 °C for 30 s, 38 °C for 30 s and 72 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 41 °C for 30 s and 72 °C for 2 min with a final extension at 72 °C for 4 min. PCR amplification was performed with an I-Cycler thermocycler (BIORAD, Les Ulis, France). A total of 35 μL of each reaction mixture was loaded on a 2% agarose gel (wt/vol) (Q-Biogen, France) with 1 X TBE electrophoresis buffer (Q-Biogen, Illkirch, France) containing 0.2 mg·mL−1 ethidium bromide and run at 120 V in a SUB-CELL GT electrophoresis system (BIORAD, Les Ulis, France) for 3 h.

2.4. Data analysis

All PCR amplification profiles were analyzed with the Bionumerics program (Applied Maths, Ghent, Belgium) [31]. The performed analysis included (i) normalization of electrophoresis patterns to compensate for minor differences in migration, (ii) subtraction of a non-linear background from the patterns and comparison based on the rolling disk principle, (iii) calculation of Pearson’s coefficient for similarity between patterns and (iv) clustering of the patterns using the unweighted pair group method with arithmetic averages [30]. The inter-LTR PCR discriminatory level was evaluated using Simpson’s diversity index D (D = 1 − 1/N (N − 1) Σxj (xj − 1)), where N is the number of strains and xj is the number of strains per group [31].

3. RESULTS

3.1. Inter-LTR PCR amplification discriminating performances

Oligonucleotidic primers were designed to match conserved regions of the LTRs aligned with ClustalX (data not shown) and used to PCR amplify genomic DNA from various strains of D. hansenii and K. marxianus species. Different primer pairs were tested; those leading to the most discriminating results were selected and used throughout this study (Tab. II). To ensure repeatability of the PCR inter-LTR fingerprinting method, different conditions of amplification were tested with the genomic DNA of three strains in independent experiments as described by Gente et al. [13] (data not shown).

Fingerprinting profiles were then generated for 56 D. hansenii strains and 61 K. marxianus strains mainly isolated from various traditional French cheeses [2, 20]. The genetic diversity was then assessed by examining the clustering of the typing profiles obtained (Figs. 1 and 2). The selected primer pairs ensure a Simpson’s diversity index higher than 95% (Tab. II). The amplified bands ranged from 400 to 1300 bp for the genomic fingerprints of D. hansenii strains and from 300 to 1500 bp for the genomic fingerprints of all K. marxianus strains. The patterns of the various strains differed in fragment number, size and intensity.

thumbnail Figure 1.

D. hansenii strains inter-LTR fingerprinting. Origin and biotope of isolation of the 56 studied D. hansenii strains is indicated. Stars indicate strains displaying a unique profile. Strains with similar profiles are boxed. The vertical bar indicates the 75% similarity cut-off. Note that, although the strains CLIB 239 and CLIB 677 display similarity below the 75% threshold, they were not grouped in a cluster as their respective profiles are clearly different. N/A: not available.

thumbnail Figure 2.

K. marxianus strains inter-LTR fingerprinting. Origin and biotope of isolation of the 61 studied K. marxianus strains is indicated. Stars indicate strains displaying a unique profile. Strains with similar profiles are boxed. The vertical bar indicates the 85% similarity cut-off.

3.2. Genetic diversity within D. hansenii strains in French cheese

For D. hansenii, grouping of the profiles led to seven clusters and 25 unique profiles with a similarity coefficient of 75%. Two observations can be made from the obtained inter-LTR profile dendrogram (Fig. 1). A number of strains share very similar profiles such as CLIB 665, CLIB 690, CLIB 664, CLIB 692, CLIB 698 and CLIB 617. These strains were isolated from the surface of Chevrotin des Aravis in the same batch between 17 and 25 days after the start of the ripening process; their classification into the same cluster is therefore not surprising (Figs. 1 and 3). This is also true for the cluster including CLIB 607, CLIB 608, CLIB 684, CLIB 685 and CLIB 702; these strains were isolated from a Camembert at different times during the first steps of the cheese-making process or even in the dairy factory atmosphere (Fig. 1). These sets of strains are thus associated to a dairy factory and a batch.

thumbnail Figure 3.

Clustering of the D. hansenii strain patterns from the same geographical origin. The inter-LTR fingerprinting profiles of a total of 33 strains originating from the Haute-Savoie area and isolated from cheese during ripening are compared. Stars indicate strains displaying a unique profile. Strains with similar profiles are boxed. The vertical bar indicates the 75% similarity cut-off.

The second observation is that, for most of the D. hansenii strains studied, a wide genetic diversity was observed no matter what geographical region the strains were from, the type of cheese analyzed or the process step. Overall, the 56 inter-LTR profiles obtained corresponded to seven clusters and 25 individual patterns (Fig. 1). This is highlighted in Figure 3 by the clustering of the patterns obtained with 33 strains isolated from the surface of Chevrotin des Aravis between 17 and 25 days after the start of ripening. These strains, isolated from Haute-Savoie, exhibited four clusters and 14 unique patterns. Although some of the strains were clearly related if not identical (CLIB 691 and CLIB 695, or the already mentioned clusters CLIB 676, CLIB 696, CLIB 663, CLIB 659, CLIB 594 and CLIB 665, CLIB 690, CLIB 664, CLIB 692, CLIB 698 and CLIB 617), these results showed an important genetic diversity among the D. hansenii strains isolated from the same batch. Furthermore, we found that eight strains isolated from the Alençon area generated a cluster of five strains constituted of CLIB 607, CLIB 608, CLIB 684, CLIB 685 and CLIB 702 and three individual patterns (CLIB 609, CLIB 656 and CLIB 686) (Fig. 1), indicating that strains isolated from the processing environment were genetically closely related to the strains isolated from the cheese of this region.

3.3. Genetic diversity within K. marxianus strains in French cheese

Inter-LTR PCR was performed for 61 strains of K. marxianus isolated from dairy environment and cheese and camembert from several dairy factories in Normandy [2, 20]. Data analysis revealed an important genetic diversity among the studied strains seven clusters and 43 unique patterns with a similarity coefficient of 85% (Fig. 2). Some strains grouped in clusters of two, exhibiting similar profiles such as the groups TL 298/CLIB 788 and TL 202/CLIB 775. Two larger clusters of eight strains (TL 225, TL 269, TL 297, TL 291, TL 294, CLIB 784, TL 285 and CLIB 785), consisted of strains all isolated from the same batch few days after the start of ripening. These K. marxianus strains are very likely identical or closely related. However, the overall diversity was really high. This is illustrated in Figure 4 in which clustering of the inter-LTR genetic profiles of 38 strains of the Alençon area showed the existence of three clusters and 32 unique patterns.

thumbnail Figure 4.

Clustering of the K. marxianus strain patterns from the same geographical origin. The inter-LTR fingerprinting profiles of a total of the 38 K. marxianus strains originating from the Alençon area are compared. Stars indicate strains displaying a unique profile. Strains with similar profiles are boxed. The vertical bar indicates the 85% similarity cut-off.

As for D. hansenii, no correlation was observed between inter-LTR PCR profiles and batch variety, process time, sampling time or place (surface or inside). It has to be noted that we had no example of strains from different origins like geographical localization, batch type or dairy plant sharing a similar profile. Thus, this high specificity displayed by the patterns we have generated indicates that it may be used in the future to associate the strains to the varieties of cheese to ease PDO labeling or to correlate strain technological properties to the type of cheese.

4. DISCUSSION

Although the availability of sequences of retrotransposons and LTRs is a prerequisite, these elements proved to be of real interest for the design of primers dedicated to yeast strain typing in species other than S. cerevisiae. In this study, a PCR-based fingerprint method was developed to assess genetic diversity among D. hansenii and K. marxianus strains, based on the variability of the insertion of the LTR retrotransposons, Tdh5 and Tkm1, respectively.

Previous works on D. hansenii have shown that a large genetic variability existed at the chromosomal structure level as monitored by Pulse Field Gel Electrophoresis (PFGE) [7, 24] and at the DNA sequence level [8, 28]. By comparing, for some strains, the typing results of PFGE and inter-LTR PCR methods, we were able to confirm that strains, which were shown to share similar electrophoretic karyotypes in a previous study [7], were closely related using the proposed approach, i.e. CLIB 626, CLIB 627 and CLIB 628 (data not shown). Although transposition monitoring cannot reflect physiological properties of the strains, it can nevertheless indicate that strains are closely related. It is less true for changes in chromosomal structures which are considered to be due to frequent recombination events between repeated sequences leading to reciprocal translocations [5, 26].

As observed for D. hansenii, this work showed the extreme diversity among K. marxianus strains. This is certainly linked to the high estimated number of transposons in this species [22], but this diversity at the level of transposon distribution must clearly reflect intra-specific genetic diversity. This result indicating a probable high transposition activity is interesting, as a very closely related species K. lactis, another major yeast in cheese, does not seem to carry any active transposon [11, 22]. This work has to be further carried out to evaluate whether the genetic diversity based on the transposition history of the strains tested and observed in this study is correlated in any way with physiological or technological properties.

The fact that most of the strains of our study were isolated from cheese during the ripening process emphasizes the observed diversity; this is especially true for the K. marxianus strains originating from the Normandy Alençon area. A widespread genetic diversity was observed among cheese yeasts isolated from the studied traditional cheese, as previously described for Y. lipolytica and Geotrichum candidum [19]. The persistence of a high genetic diversity among cheese yeast flora could suggest that traditional cheeses may require the presence of a complex flora for their elaboration.

Although the large majority of the strains displayed a specific profile, we could find a number of groups of two to six strains, sharing a very similar profile. It has to be noted that the strains belonging to these groups were isolated from the same batch or from the same facilities. We found that for D. hansenii, strains isolated from the processing environment were genetically closely related to the strains isolated from the cheese of this region. A similar observation was made with strains involved in the processing of a Danish cheese [25]. This type of strains may be prevalent in the dairy factory, as it was found in the atmosphere of the dairy house, in the milk and in the cheese after draining. In agreement, with these observations, a dominant strain was also found during the production of Danish Danbo type of cheese [25]. One can object to this observation that the typing method used in this work, mtDNA RFLP, is not very discriminant (see [28]). A study assessing technological properties of over 20 K. marxianus strains from water buffalo mozzarella did not differentiate these strains on the basis of the production of end metabolites such as sulfur dioxide, higher alcohols, ethyl acetate and acetaldehyde [29]. The type of cheese, i.e. of fermentation, may of course be essential with regard to technological properties.

The case of the very close strains of K. marxianus TL 202 and CLIB 775 (Fig. 2) is particularly interesting. Both strains were isolated from brine at different moments, suggesting an adaptation of a certain genotype to these environmental conditions. This indicates that the method described here should therefore allow for following a strain during the cheese-making process environment, materials and ingredients.

In conclusion, inter-LTR PCR fingerprinting is easy and rapid to perform and therefore provides a real alternative to more time- and labor-consuming methods (i.e. PFGE) or less discriminating methods (mitochondrial DNA RFLP). In this study, the inter-LTR PCR characterization of D. hansenii and K. marxianus strains from fermentation and ripening of French cheeses indicates that strains may be specific to traditional cheese type or to an area. These facts are in full agreement with the notions of “terroir” and typicity promoted by the PDO, although further studies are required to evaluate the role of these strains in the cheese typicity and how they can be used by the cheese manufacturers.

Acknowledgments

This work was funded by the Ministère de la Recherche (Program ACTIA 01.4), the GDR/CNRS 2354 “Genolevures II”, ARILAIT Recherches, the Bureau des Ressources Génétiques, INRA, ADRIA Développement and ADRIA Normandie.

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All Tables

Table IA.

List of the 56 D. hansenii strains used in this study.

Table IB.

List of the 61 K. marxianus strains used in this study.

Table II.

Oligonucleotidic primers used in this study and the associated Simpson’s diversity index.

All Figures

thumbnail Figure 1.

D. hansenii strains inter-LTR fingerprinting. Origin and biotope of isolation of the 56 studied D. hansenii strains is indicated. Stars indicate strains displaying a unique profile. Strains with similar profiles are boxed. The vertical bar indicates the 75% similarity cut-off. Note that, although the strains CLIB 239 and CLIB 677 display similarity below the 75% threshold, they were not grouped in a cluster as their respective profiles are clearly different. N/A: not available.

In the text
thumbnail Figure 2.

K. marxianus strains inter-LTR fingerprinting. Origin and biotope of isolation of the 61 studied K. marxianus strains is indicated. Stars indicate strains displaying a unique profile. Strains with similar profiles are boxed. The vertical bar indicates the 85% similarity cut-off.

In the text
thumbnail Figure 3.

Clustering of the D. hansenii strain patterns from the same geographical origin. The inter-LTR fingerprinting profiles of a total of 33 strains originating from the Haute-Savoie area and isolated from cheese during ripening are compared. Stars indicate strains displaying a unique profile. Strains with similar profiles are boxed. The vertical bar indicates the 75% similarity cut-off.

In the text
thumbnail Figure 4.

Clustering of the K. marxianus strain patterns from the same geographical origin. The inter-LTR fingerprinting profiles of a total of the 38 K. marxianus strains originating from the Alençon area are compared. Stars indicate strains displaying a unique profile. Strains with similar profiles are boxed. The vertical bar indicates the 85% similarity cut-off.

In the text

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