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International Dairy Journal 11 (2001) 293305
Origin and diversity of mesophilic lactobacilli in Comt!e cheese,
as revealed by PCR with repetitive and species-specific primers
Francoise Berthier*, Eric Beuvier, Andr!e Dasen, R!emy Grappin
INRA, Station de Recherches en Technologie et Analyses Laiti"eres, B.P. 89, 39801 Poligny Cedex, France
Abstract
The objectives of this work were to describe the diversity of mesophilic lactobacilli in Comt!e 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 Comt !e 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 REP1R-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. r 2001 Elsevier Science
Ltd. All rights reserved.
Keywords: Species-specific PCR; Strain typing; Lb. paracasei; Lb. rhamnosus; Lb. zeae; REP-PCR; ERIC-PCR; Comt!e cheese; Raw milk; Mesophilic
lactobacilli
1. Introduction
Comt!e cheese is a hard-cooked ripened cheese variety
manufactured from raw cows milk in a limited region in
the East of France, and labelled Appellation dOrigine
Prot!eg!ee (AOP) (Beuvier, 1996). Thermophilic and
mesophilic whey starter cultures, including selected
strains of Lactobacillus helveticus, Streptococcus thermo-
philus and Lactococcus lactis, are added during the
cheesemaking process. Mesophilic lactobacilli are de-
tected as a dominant non-starter microflora in Comt !e
cheese, where their viable numbers increased from 103 to
104 cfug1 cheese at the beginning of ripening to
108 cfug1 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 Comt!e cheeses exhibit complex and varied
sensory properties (St"evenot, B!erodier, & Schlich, 1997),
which could originate from various mechanisms, includ-
ing the activities of the microbial ecosystem. This was
demonstrated with experimental mini Comt!e-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 Comt!e 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 Comt!e cheese, there is a need to know the origin
and to characterise the microflora of Comt!e 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
*Corresponding author. Fax: +33-3-84-37-37-81.
E-mail address: [email protected] (F. Berthier).
0958-6946/01/$- see front matter r 2001 Elsevier Science Ltd. All rights reserved.
P I I : S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 0 5 9 - 0
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from Comt!e cheese is also essential to conduct experi-
ments in cheesemaking.
The objectives of this study were to evaluate the
diversity of mesophilic lactobacilli in Comt!e cheese
according to cheesemaking and ripening conditions, andto 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 Comt!e cheeses collected in two cheese
factories.
2. Materials and methods
2.1. Samples
Bacterial strains were isolated at the same time in two
different factories, 1 and 2, equipped with four cheese
vats. These factories were known to produce cheeses
with different sensory properties. Cheeses were ripened
between 5.6 and 9.3 months according to four different
schemes (ad) used in Comt!e technology. According to
Scheme a, cheeses were ripened at 131C for 2 weeks, at
171C for 5 weeks, and at 61C until their optimal ripening
time. According to Scheme b, cheeses were ripened at
131
C for 7 weeks, at 171
C for 6 weeks, and at 61
C untiltheir optimal ripening time. According to Scheme c,
cheeses were ripened at 131C for 2 weeks, at 101C for 5
weeks, at 171C for 4 weeks, and at 61C until their
optimal ripening time. According to Scheme d, cheeses
were ripened at 131C for 7 weeks, at 191C for 5 weeks,
and at 61C until their optimal ripening time. Cheeses
1ad and 2ad were graded by the same sensory analyst
to determine their ripening endpoint. Milk 1 and milk 2
were from factories 1 and 2, respectively.
Bacterial strains from the two cheeses 1b and 2b,
which were ripened under the same conditions, were
isolated at 1, 7, 21, 49, 63, 91, 122 days, and at their
optimal ripening time. Strains from the six other cheeses
were isolated only at their optimal ripening time. In
addition, strains were isolated from the two raw milks,
from the five starter cultures and from curds before
pressing. Cheese samples of 10 g without rind were taken
at the mid-radius of each Comt!e wheel. The isolate
numbers in each sample are given in Table 1.
2.2. Isolation of mesophilic lactobacilli
Milks, starter cultures, curds and cheeses were
aseptically sampled. Samples were emulsified in sterile
2% (wt/vol) trisodium citrate (pH 8.5), diluted, platedon MRS agar pH 6.5 (De Man, Rogosa, & Sharpe,
1960) and on FH agar ( Isolini, Grand, & Gl.attli, 1990)
and incubated anaerobically for 5 days at 201C and 3
days at 371C, respectively. About 20 different colonies
were randomly picked up from the MRS plates, and in a
few cases also from FH plates, and purified twice on
MRS agar plates. All isolates were checked for growth
at 151C and were examined microscopically prior to
storing. They were maintained at 201C i n a 1 : 1
glycerolMRS mixture and routinely streaked on MRS
plates before use.
2.3. Collection strains
A selection of 49 type strains or well-characterised
strains of mesophilic lactobacilli were obtained from
different culture collections, DSMZ (Deutsche Samm-
lung von Mikroorganismen und Zellkulturen, Braunsch-
weig, Germany), LMG (Laboratorium voor
Microbiologie, Universiteit Gent, Gent, Belgium),
ATCC (American Type Culture Collection, Rockville,
Md., USA), CNRZ (Centre National de Recherches
Zootechniques, INRA Jouy-en-Josas, France), NCFB
(National Collection of industrial Bacteria, Shinfield,
Reading, Berkshire, UK), NCDO (National Collectionof Dairy Organisms, Shinfield, Reading, Berkshire, UK)
Table 1
Isolation of mesophilic lactobacilli
Source of isolates No. of isolates
Factory 1 Factory 2
Milks 20 16a
Starter cultures:
Lb. culture 15 0
Lc. culture 16a F
St. culture 1a 1a
Cheese curd b 20 4a
Ripening cheese b
1 day 17 0
7 day 19 0
21 day 20 20
49 day 20 20
63 day 20 2091 day 20 20
122 day 20 20
Mature cheese
1a 20
1b 20
1c 20
1d 19
2a 19
2b 20
2c 20
2d 19
aIsolated on FH medium.
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or from different laboratories, Station de Recherches sur
la Viande, INRA Theix, France, Laboratoire de
Recherches sur la viande, INRA Jouy-enJosas, France
and IRTA Meat Technology Centre, Monells, Spain
(Berthier & Ehrlich, 1999). Details of the species andstrain numbers are given in Fig. 2.
2.4. DNA isolation from lactobacilli isolates
DNA was extracted from 1 mL samples of fresh MRS
cultures in the exponential growth phase. Total DNA
was either phenol extracted as described previously
(Berthier et al., 1999), or rapidly extracted with the
Instagene matrix as described by the manufacturer
(Biorad, Ivry sur Seine, France). Instagene isolated
DNA was ethanol precipitated and resuspended in 10 mL
10 mm Tris (pH 8.0), 1 mm EDTA. The quantity of
DNA obtained by the first method was estimated by
comparison with known standards in ethidium bromide-
stained 0.7% agarose gels.
2.5. Rep-PCR
Primer sets ERIC1R/ERIC2 and REP1R-Dt/REP2-
D (Versalovic, Koeuth, & Lupski, 1991) were used for
ERIC- and REP-PCR amplifications, respectively. They
were synthesised by Genosys Biotechnologies (Cam-
bridge, UK). PCR amplification was performed in a
final volume of 20 mL containing 1x PCR buffer
(Applig"ene), 420 ng phenol-extracted DNA, or 5mL
Instagene-extracted DNA, 1.0 mm MgCl2, 0.25mm each
primer, 200 mm each dNTP, and one unit Taq DNA
polymerase (Applig"ene Oncor, Illkirch, France). PCR
reactions were carried out in a thermal cycler Gene Amp
PCR system 9600 apparatus (PerkinElmer Applied
Biosystems) programmed for 30 cycles of amplification
of 1 min at 941C, 1 min at 401C, 6 min ramping to 721C,
and 1 m in at 721C, preceded by 5 min at 941C.
Electrophoresis and computer analysis were performed
as previously described (Berthier et al., 1999), except
that a GS 670 Molecular Imager System (Biorad, Ivry
sur Seine, France) and the version 4.0 instead of 3.1 of
the software package GelCompar were used.
2.6. Species-specific PCR
The oligonucleotide primers were obtained from
Genosys Biotechnologies (Cambridge, UK) and are
listed in Table 2, together with the references of their
description. The primers zeae16S and zeaeITS were
designed from the nucleotide sequences listed in Table 2.
The primer 16reverse was paired with primers para-
casei16S, rhamnosus16S or zeae16S. The primer 16 was
paired with primers paracaseiITS, rhamnosusITS or
zeaeITS. PCR reactions were performed in 10mL 1xPCR buffer (Applig"ene) supplemented with 1.0 mm T
able
2
Sequencesoftheoligonucleotideprimersusedfo
rspecies-specificPCRamplification
Primer
Location/Gene-Bankaccessionnumber
Oligonucleot
idesequence(50
-30)
Reference
Pr
imerspecificity
16
16SrRNAgene,50end,forw
ard
GCTGGATCACCTCCTTTC
(Berthier&Ehrlich,1999)
Universal
16rev
erse
16SrRNAgene,50end,reve
rsedprimer16S
GAAAGGA
GGTGATCCAGC
Universal
zeae1
6S
16SrRNAgeneofLb.zeae
typestrain,50end,
forward/d86516
GCATCGTG
ATTCAACTTAA
Lb
.zeae
rhamnosus16S
16SrRNAgeneofLb.rham
nosustypestrain,50end,
forward/d16552
TTGCATCT
TGATTTAATTTTG
(Ward&Timm
ins,1999),withan
additionalTat
50end
Lb
.rhamnosus
parac
asei16S
16SrRNAgeneofLb.caseiATCC334,50end,
forward/d86517
CACCGAGATTCAACATGG
(Wardetal.,1999)
Lb
.paracasei
zeaeITS
Short16S23Sintergenicsp
acerofLb.caseiATCC
393,50end,reverse/z75479
CGATGCGAATTTCTAAATT
Lb
.zeae
rhamnosusITS
Short16S23Sintergenicsp
acerofLb.rhamnosus
G1,50end,reverse/u32966
CGATGCGAATTTCTATTATT
(Tilsala-Timisja
rvi&Alatossava,1997)withoutG
withanadditio
nalTat50end
Lb
.rhamnosus
parac
aseiITS
Short16S23Sintergenicsp
acerofLb.paracasei
ATCC27092,50end,reverse/u32964
CGATGCGAATTTCTTTTTC
(Tilsala-Timisja
rvietal.,1997)
withoutthesecondCat50end
Lb
.paracasei
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MgCl2, 0.3 mm each primer, 200mm each dNTP, 0.5 unit
Taq DNA polymerase (Applig"ene Oncor, Illkirch,
France), and 210 ng phenol-extracted DNA, or 1.5mL
Instagene-extracted DNA. All the ingredients, except
specific primers, were first mixed together, aliquoted and
then specific primers were added. PCR reactions were
carried out in a thermal cycler Gene Amp PCR system
9600 apparatus (PerkinElmer Applied Biosystems)
programmed for 30 cycles of amplification of 1 min at
941C, 0 min at 551C (ITS pairs), or 0 min at 531C (16S
pairs), and 1 min at 721
C, preceded by 5 min at 941
C;The 10mL were electrophoresed in a 1% agarose gel and
were subsequently visualised by UV illumination after
ethidium bromide staining. Two PCR products of 350
and 185 bp were observed when DNA could be
amplified.
3. Results
3.1. Isolates
Altogether, 488 isolates of mesophilic lactobacilli
were collected, 287 from factory 1 and 201 from factory
2, with twenty isolates of mesophilic lactobacilli
collected from each sample, except from some of them
(Table 1). As shown in Table 1, mesophilic lactobacilli
were isolated from two out of the five starter cultures,
and from all the cheese and milk samples. MRS plating
at 201C was effective to select mesophilic lactobacilli
from most samples, but not all, selecting cocci in milk 2,
curd 2 and cheese 2b at 1day, or selecting no bacteria in
cheese 2b at 7 days. From the latter samples, FH plating
at 371C selected mesophilic lactobacilli in milk 2,
mesophilic lactobacilli together with thermophilic lacto-
bacilli in curd 2b and only thermophilic lactobacilli fromthe other samples, cheese 2b at 1 and 7 days.
MRS incubated at 201C selected almost exclusively
mesophilic lactobacilli in cheeses after seven days of
ripening, even if pediococci were occasionally isolated.
As shown in Fig. 1, MRS and FH enumerations were
systematically higher on MRS compared with FH forcheese 1b, mainly because one dominant strain in cheese
1, Lb. paracasei A12 (see below), was unable to grow at
371C, which is the incubation temperature recom-
mended for selecting Lb. paracasei on FH (Isolini
et al., 1990). Enumeration was higher on MRS
compared with FH for milk 2, curd 2b and cheese 2b
at 1 day because of the presence of cocci, presumably
enterococci according to their phenotypic characterisa-
tion. Lactococci were never isolated on MRS medium at
201C, even in the curds or the young cheeses, although
added as starter culture.
The isolates of mesophilic lactobacilli collected on FH
medium in factory 2 from milk, curd and young cheese
were included in the study because they will not impede
comments relating to strain diversity, despite the fact
that two different media at two different temperatures
were used. If present in older cheeses, they would indeed
have grown on MRS under the conditions used, and all
isolates from older cheeses of factory 2 were able to
grow on both MRS and FH under the conditions used.
3.2. Strain typing by Rep-PCR
MgCl2, primers and DNA concentrations, as well as
the temperature profile in the PCR cycle were optimisedto obtain reproducible fingerprints with a sufficient
number of bands. A ramping was thus introduced in the
original and usually used procedure (Versalovic et al.,
1991) to ensure the reliability of the method (Sobral &
Honeycutt, 1993). The annealing temperature recom-
mended for REP primers, 401C, was used with both
REP- and ERIC-primers. Finally, amplifications with
pairs of primers were found more informative than with
a single primer.
(i) Type strains and collection strains. The reproduci-
bility and the discriminatory power of Rep-PCR to
strain level was assessed. Rep-PCR analysis was applied
to 49 type strains or collection strains of mesophilic
lactobacilli (Table 3) which are commonly found in
cheese or closely related to them genetically. The strains
used are listed in Fig. 2. Most of these strains were
assigned to different species by DNA/DNA hybridisa-
tion (Bringel, Curk, & Hubert, 1996; Collins, Phillips, &
Zanoni, 1989; Dellaglio, Botazzi, & Vescovo, 1975;
Dellaglio, Dicks, du, & Torriani, 1991; Montel, Talon,
Fournaud, & Champomier, 1991) and/or species-specific
PCR amplification (Berthier & Ehrlich, 1998; Berthier
et al., 1999). As shown in Fig. 2, fingerprints which were
visually identical merged at the 88% similarity coeffi-
cient following cluster analysis of combined REP- andERIC-fingerprints, DNA being isolated by the phenol
Fig. 1. Comparison of enumeration after FH and MRS plating of
samples from identically ripened Comt!e cheeses 1b and 2b.
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method. Identical fingerprints with both REP- and
ERIC-primers were obtained for all strains provided
from different collections and known as identical, Lb.
plantarum ATCC 14917T/CNRZ 211T, Lb. paracasei
CNRZ 62T/NCDO 151T, Lb. rhamnosus CNRZ 212T/
LMG 6400T/DSM 20247T. Identical fingerprints were
also obtained for Lb. plantarum CNRZ 211T/CNRZ
1228 and Lb. pentosus CNRZ 1555/CNRZ 1537 as
found with RAPD fingerprints (Tailliez, Qu!en!ee, &
Chopin, 1996), and for Lb. rhamnosus CNRZ 442/
CNRZ 205, Lb. pentosus CNRZ 1570/CNRZ 1537, Lb.
rhamnosus DSM 20247/DSM 20711, and Lb. paracasei
subsp. tolerans DSM 20012/LMG 9191T. Different
fingerprints were obtained from the other 33 strains
with both REP- and ERIC-primers, except from three
strains, Lb. curvatus CTC 448, Lb. curvatus CTC 243
and Lb. pentosus CNRZ 1547, which exhibited different
fingerprints only with REP primers.
(ii) Isolates. According to the above results, we
decided to fingerprint all cheese isolates with REP
primers, and then to confirm REP-based fingerprintdiscrimination with ERIC-based fingerprint discrimina-
tion of isolates subsets representing each putative
strains. As Instagene-isolated DNA gave the same
fingerprints as phenol-extracted DNA from the collec-
tion strains (data not shown) when similar DNA
concentration were added in the PCR mixture, it was
used in Rep-PCR analysis of isolates because of rapidity
in isolating DNA. The similarity between different Rep
fingerprints of the same strain was lower with Instagene-
isolated DNA because of the more variable DNA
concentration in the PCR mixture, which sometimes
led to differences in band intensity between fingerprints,
less or more DNA increasing or decreasing the intensity
of bands with low molecular weight compared to the
intensity of bands with high molecular weight, respec-
tively. In that respect, and to avoid the erroneous
assessment of two different fingerprints instead of one,
fingerprint identity was deduced from both clustering
analysis and visual inspection, and new fingerprints were
generated changing the DNA concentration when
doubtful fingerprint were detected. REP-based finger-
print discrimination was further confirmed with ERIC-based fingerprint discrimination.
Table 3
Lb. paracasei, Lb. rhamnosus and Lb. zeae affiliation of collection strains
Strain Current name Species affiliation with Species affiliation
CNRZ 313 Lb. casei DNA/DNA hybridisationa,b Lb. zeae
Lb. zeae species-specific PCRcDSM 20178T Lb. zeae Lb. zeae species-specific PCRc Lb. zeae
LMG 9191T Lb. paracasei DNA/DNA hybridisationb Lb. paracasei
subsp. paratolerans Lb. paracaseispecies-specific PCRc
ATCC 334 Lb. casei Lb. paracasei species-specific PCRc Lb. paracasei
CNRZ 320 Lb. paracasei Lb. paracasei species-specific PCRc Lb. paracasei
CNRZ 383 Lb. paracasei Lb. paracasei species-specific PCRc Lb. paracasei
CNRZ 763 Lb. paracasei Lb. paracasei species-specific PCRc Lb. paracasei
DSM 4905 Lb. paracasei DNA/DNA hybridisationa Lb. paracasei
Lb. paracasei species-specific PCRc
DSM 20006 Lb. paracasei DNA/DNA hybridisationd Lb. paracasei
Lb. paracasei species-specific PCRc
DSM 20008 Lb. paracasei DNA/DNA hybridisationa,b Lb. paracasei
Lb. paracasei species-specific PCRc
DSM 20012 Lb. paracasei DNA/DNA hybridisationa,b Lb. paracasei
Lb. paracasei species-specific PCRc
DSM 20020 Lb. paracasei DNA/DNA hybridisationb Lb. paracasei
Lb. paracasei species-specific PCRc
DSM 20207 Lb. paracasei DNA/DNA hybridisationa,b Lb. paracasei
Lb. paracasei species-specific PCRc
NCDO 151T=CNRZ 62T Lb. paracasei DNA/DNA hybridisationd,b Lb. paracasei
Lb. paracasei species-specific PCRc
LMG 6400T=CNRZ 212T, DSM 20247T Lb. rhamnosus Lb. rhamnosus species-specific PCRc Lb. rhamnosus
CNRZ 205 Lb. rhamnosus Lb.rhamnosus species-specific PCRc Lb. rhamnosus
CNRZ 442 Lb. rhamnosus Lb. rhamnosus species-specific PCRc Lb. rhamnosus
DSM 20023 Lb. rhamnosus Lb. rhamnosus species-specific PCRc Lb. rhamnosus
DSM 20711 Lb. rhamnosus DNA/DNA hybridisationa Lb. rhamnosus
Lb. rhamnosus species-specific PCRc
aDellaglio et al. (1975).b
Collins et al. (1989).cThis work.dDellaglio et al. (1991).
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Forty-four different fingerprints among the 448
isolates collected were identified in this manner
(Fig. 3). All were different with REP primer amplifica-
tion alone, except five of them, A1, A31, A32, A33 A34
and A35, which were discriminated only with ERICprimer amplification.
3.3. Presumptive species assignation by Rep-PCR
fingerprint
Rep-PCR fingerprints could be used to give pre-
sumptive assignation of strains to species. Indeed, as canbe seen in Fig. 3, combined REP and ERIC fingerprints
Fig. 2. REP- and ERIC-PCR fingerprints of collection strains of mesophilic lactobacilli, and generated dendrogram from combined fingerprints. Lb.
paracasei CNRZ 62 and Lb. paracasei NCDO 151 are synonyms, as well as Lb. plantarum ATCC 14917 and Lb. plantarum CNRZ 211, and Lb.
rhamnosus LMG 6400, Lb. rhamnosus CNRZ 212 and Lb. rhamnosus DSM 20247. Species assignation after species-specific PCR amplification was
indicated in italics when there were discrepancies with the current name of strains.
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from strains assigned to the same species merged in most
cases at 40% or more similarity, while those of strains
assigned to different species merged at less than 40%
similarity. Nevertheless, in some cases, fingerprints from
species represented by a few strains, such as Lb. zeae,
merged with those from other species, leading to the
misclassification of species if the 40% similarity is taken
as the criteria for species discrimination. In contrast, all
fingerprints from strains assigned to the same species
sometimes merged at less than 40% similarity, as those
of different species. In this case, the presumptiveassignation depended on the identification library used.
The 49 fingerprints from collection strains were
compared to the 44 fingerprints from this work (results
not shown). Strains B1 and B3 merged at 72.4%
similarity into the cluster including Lb. rhamnosus
LMG 6400T, and strains B2 merged at 58.7% similarity
with Lb. rhamnosus DSM 20023; strains A1 to A34,
except three, merged at 38% similarity into the cluster
including Lb. casei ATCC 334. Strains C1, C2 and C3
merged at 49% similarity with Lb. parabuchneri LMG
17769. All fingerprints from these strains exhibited
several common bands with the fingerprints from thecollection strains they merged into, strengthening their
Fig. 3. REP- and ERIC-PCR fingerprints diversity among 488 isolates of mesophilic lactobacilli from raw milks, starter cultures and Comt!e cheeses
of factories 1 and 2, and generated dendrogram from combined fingerprints. The strains isolated from milk, starter cultures and cheeses of factory 2
are preceded by a black dot. A1A35, Lb. paracasei strains; B1B3, Lb. rhamnosus strains; C1C3, presumed Lb. parabuchneri strains; D1F1,
unassigned strains. The number of isolates per strain is given in bracket.
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presumptive assignation. Fingerprints from strains A11,
A27, A28, A35, D1, E1 and F1 exhibited similarity
coefficients of less than 40% with the fingerprints from
the 49 collection strains, as well as the type strains of
other mesophilic lactobacilli, Lb. farciminis, Lb. alimen-tarius, Lb. maltoromicus, Lb. coryneformis, Lb. amylo-
philus, Lb. bifermentans and Lb. sharpeae (results not
shown). Strains D1, E1 and F1 were obligatory
heterofermentative, whereas strains A11, A27, A28,
and A35 were facultatively heterofermentative.
3.4. Species assignation by species-specific PCR
We used PCR to specifically affiliate strains or isolates
to the three closely related species Lb. paracasei (type
strains: strains NCDO 151T; and ATCC 25599T;
synonym: LMG 9191T), Lb. rhamnosus (type strain
ATCC 7469T; synonyms: LMG 6400T and CNRZ
212T), and Lb. zeae (type strain ATCC 15820T;
synonym: DSM 20178T). Newly- designed primers were
used to amplify Lb. zeae DNA. The primers derived
from the 16S23S intergenic spacer were paired with
primer 16, which was formerly used in other species-
specific PCR amplification of Lactobacillus (Berthier
et al., 1998).
(i) Type and collection strains. The specificity of the
different PCR reactions, described in the method
section, was assessed as follows. The six sets of PCR
primers were used with each DNA of the Lb. casei, Lb.
rhamnosus, Lb. paracasei and Lb. zeae strains listed inTable 2. The 16reverse/rhamnosus16S and 16/rhamno-
susITS pairs amplified only Lb. rhamnosus DNA. The
16reverse/paracasei16S and 16/paracaseiITS pairs am-
plified Lb casei 334 and all Lb. paracasei DNA. The
16reverse/zeae16S and 16/zeaeITS pairs amplified Lb.
casei CNRZ 313 and Lb. zeae DSM 20178T DNA.
(ii) Isolates. Representative isolates of each strain
discriminated by Rep-PCR were subjected to Lb.
paracasei, Lb. rhamnosus and Lb. zeae specific PCR
amplification. 35 strains, A1A35, and three strains,
B1B3, were thus assigned to Lb. paracasei and Lb.
rhamnosus, respectively.
3.5. Strain diversity in factories 1 and 2
The strains isolated in factory 1 were genetically
different from those isolated in factory 2. As seen in
Fig. 3, no identical Rep fingerprints were indeed found
between the 23 and 21 fingerprints from factories 1 and
2, respectively, even if some were very similar, for
example A1 and A18.
As seen in Fig. 4, six and ten different strains were
isolated in milks 1 and 2. Strains B1 and A17 were the
dominant strains in milks 1 and 2, representing 55% and
25% of the milk isolates. Six and seven different strainswere isolated from the starter cultures of Lactococcus
and thermophilic lactobacilli used in factory 1, respec-
tively, with strain B1 being dominant in both. Thirteen
and fifteen different strains were isolated in cheeses 1b
and 2b throughout cheesemaking and ripening. Three
strains, B1, A12 and A31, and six strains, A14, A27,A22, A13 A15 and B3, dominated, 88% in cheese 1b
and 2b, respectively.
At the end of ripening, similar numbers of mesophilic
lactobacilli were found between differently ripened
cheeses from the same factory (for cheeses 1b and 2b,
Fig. 1), but the number of strains between cheeses varied
from two to nine, with an average of five strains (Fig. 5).
One to two strains represented between 80% and 85%
of the isolates in all cheeses, except one. Most of the
dominant strains in cheeses 1b and 2b throughout
ripening were dominant in the eight mature cheeses, but
some strains were only detected at the end of ripening.
In factory 1, only one strain, A12, was common to all
mature cheeses and was the most dominant, represent-
ing from 50% to 85% of the isolates. Strain B1 was the
second dominant strain in cheeses 1a and 1c. These two
strains represented from 80% to 85% of the isolates
from cheeses 1a to 1d. A more variable and complex
pattern was observed at the end of ripening of cheeses
manufactured in factory 2. In contrast to factory 1, three
strains, A13, A14 and A27, were common to all cheeses.
In cheeses 2a, 2b and 2d, strain A14 was dominant
representing between 30% and 65% of cheese isolates,
while in cheeses 2a, 2c and 2d A14 together with a
second strain represented 8085% of the cheese isolates.In cheese 2b, six strains made up 85% of the cheese
isolates.
3.6. Species diversity
The same two predominant species of mesophilic
lactobacilli, Lb. paracasei and Lb. rhamnosus, were
identified in milks, in two starter cultures, and in cheeses
from factories 1 and 2, but in very different proportions
between factories. For instance, in cheeses, Lb. rhamno-
sus and Lb. paracasei represented respectively 48% and
52% in cheese 1, and 90.9% and 8.3% in cheese 2b
throughout cheesemaking and ripening. Furthermore,
Lb. rhamnosus was only present in two of the eight
mature cheeses at the end of ripening. A minor species,
Lb. parabuchneri was detected in milk 2 and in two
cheeses from factory 1 at the end of ripening.
3.7. Strain origin(s)
Data from this study indicated that a large number of
the mesophilic lactobacilli strains in Comt!e cheese could
originate from raw milk. However, the data were not
conclusively proving because we did not detect all cheese
strains in the raw milks. Indeed, with 20 isolatescollected per sample, strains could only be detected
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from the different samples if they represented 5% or
more of the bacterial population enumerated after
plating.
As seen in Fig. 4, four different situations were
observed among the strains of cheese 1b: (i) one minor
strain, A2, was found only in the milk; (ii) two major
strains, A12 and B1, and two minor strains A1 and A11,
were found in both the milk and in at least one of the
starter culture; (iii) the third major strain, A31, was only
found in starter cultures; and (iv) the seven other minor
strains were not found in either the milk or the starter
cultures. These results suggest four different ways of
contamination, depending on the strains considered:
milk for A2, both milk and starter culture(s) for A12,
B1, A1 and A11, starter culture(s) for A31, and the
equipment and/or the factory environment for the
others. The initial levels of strains in milk, the relative
contribution of starter microflora and raw milk micro-
flora to these levels, and the levels they reached in the
curd, should be considered to choose between raw milk
and starter cultures, or both, as source of mesophilic
lactobacilli. Strain A2 was present at 7102 cfug1 in
curd 1b, a level corresponding to that postulated from
its level in milk, provided that no growth took place
during cheesemaking. Strain A2 could then originatefrom milk. The strains A1, A11, A12 and B1 most
probably originated also from milk rather than from
starter culture(s), as they were detected in curd 1b at
levels similar with those postulated from their respective
levels in milk without any growth, and as the contribu-
tion of starter cultures to the final cell number of
mesophilic lactobacilli in milk was 200 times less than
that of raw milk microflora. The relative contribution of
starter cultures to the initial level of mesophilic
lactobacilli strains favoured also a milk origin for the
major strain A31. To reach the 2.7103 cfug1
enumerated in curd 1 and if it originated from the
starter cultures, strain A31 should indeed grow with a
generation time of 20 min postulating that growth was
exponential from the beginning of cheesemaking (if not,
generation time would be still lower), which is impos-
sible. But, if inoculated from milk at a level below the
detection limit of the method, strain A31 should grow
with a minimum generation time of 7.4 h, which is
possible. If so, all the minor strains which were not
detected in milk could also originate from milk. The
mesophilic lactobacilli detected in starter cultures
probably originated from the whey which was used as
a growth medium for the starter cultures. This whey was
collected a day before the cheeses used in this study were
manufactured. All starter strains, except A4, B2 and F1,were also detected in curd 1 (not shown). Moreover
Fig. 4. Strain diversity among mesophilic lactobacilli isolates from milks, starter cultures, and Comt!e cheeses 1b and 2b throughout cheesemaking
and ripening. Milk 1, starter cultures 1 and cheese 1b were from factory 1. Milk 2 and cheese 2b were from factory 2. , strains which were detected
only in milk or curd. &, minor strains in cheese. All strains, except those from milk 2 and Lc. starter culture 1, were isolated on MRS medium.
Strains from milk 2 and Lc. starter culture 1 were isolated on FH medium. Strain A12 did not grow on FH medium in the incubation conditions used.
A1A35, Lb. paracasei strains; B1B3, Lb. rhamnosus strains; C1C3, presumed Lb. parabuchneristrains; D1F1, unassigned strains.
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strains A4, B2 and F1 were never detected in cheese 1
throughout cheesemaking and ripening. This indicates
that the same strains of mesophilic lactobacilli were
present in curd of cheeses manufactured on two
consecutive days, from daily collected milk.
Among strains of cheese 2b, only the situations (i) and
(iv) described above for cheese 1b were found for both
minor and major strains. The strains A13, A15, A16,
A17, A20 and B3 could thus originate from milk, while
the other strains could originate either from the milk,
where they were then below the detection limit of the
method, or from the equipment and/or the factory
environment. The small number of isolates we obtained
from curd 2b did not allow to choose definitively
between these two hypotheses. The four isolates we
obtained in curd were one of strain A15, two of strain
A16 and one of strain A17, reflecting the profile of milk
strains presented in Fig. 4, and favouring thus the milkorigin of all the strains of cheese 2b. The level of
mesophilic lactobacilli present in milk was very low,
1020 cfu mL1 milk.
4. Discussion
To describe in detail the diversity of mesophilic
lactobacilli in Comt!e cheeses, a new, rapid, easy and
reliable approach was developed to assign a large
number of uncharacterised isolates at both the strain
and the species levels. This approach was based on
strain typing and presumptive species assignation of the
isolates with Rep-PCR, followed by reliable species
assignation with species-specific PCR of representative
isolates for each strain. The present work showed
that Rep-PCR analysis was very well adapted to
strain discrimination of mesophilic lactobacilli. In
terms of rapidity and ease of performance, the otheravailable strain typing method is RAPD. Rep-PCR
Fig. 5. Strain diversity among mesophilic lactobacilli isolates from eight mature Comt!e cheeses manufactured in factories 1 and 2 according to four
different ripening Schemes, ad, used in Comt!e cheese technology.&, strains which were detected only in mature cheeses. All strains were isolated on
MRS medium. A5A35, Lb. paracasei strains; B1, Lb. rhamnosus strains; C1C2, presumed Lb. parabuchneristrains.
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reproducibility was the same as that for RAPD under
similar conditions of fingerprint analysis (Berthier et al.,
1999). However, Rep-PCR analysis presents three
advantages over RAPD analysis. First, contrary to
RAPD, the same primers can reliably discriminatestrains of many different Gram-positive and Gram-
negative species (Rademaker, Louws, & de Bruijn,
1998), which was not possible with RAPD primers,
even among mesophilic lactobacilli (Berthier, unpub-
lished); thus, Rep-PCR can be applied to completely
uncharacterised isolates. The second advantage of Rep-
PCR is that each REP- and ERIC-PCR fingerprints of
mesophilic lactobacilli strains contained more bands
than the single RAPD fingerprints previously obtained
for the same strains (Berthier et al., 1999; Tailliez et al.,
1996), as was previously observed for Listeria strains
(Jersek, Tcherneva, Rijpens, & Herman, 1996); thus, the
number of different PCR reactions to perform for
obtaining the same discrimination level is reduced. The
third advantage is that Rep-PCR amplification is less
sensitive than RAPD amplification to minor changes in
reaction conditions, because REP and ERIC primers are
longer (Gillings & Holley, 1997). The robustness and
reproducibility of Rep fingerprints was improved in this
work by increasing the ramp time from the annealing to
the extension steps (Sobral et al., 1993). In other
respects, two sets of primers designed from two different
sequences, the 16S rRNA coding sequence and the 16S
23S small intergenic spacer, are now available together
with reliable PCR conditions to specifically amplifyDNA from each of the three related species Lb.
paracasei , Lb. rhamnosus and Lb. zeae. Newly-designed
primer pairs, which combined previously-and newly-
designed primers, were used to specifically amplify with
PCR these three related species. The reliability of
species-specific PCR amplification was assessed using a
large selection of well-studied and different strains of
each species. Our optimised PCR conditions ensured a
species assignation in accordance with all previous
results (Collins et al., 1989; Dellaglio et al., 1975;
Dellaglio et al., 1991). In particular, the two strains Lb.
zeae DSM 20178T and Lb. caseiATCC 393, classified in
Lb. zeae after DNA/DNA hybridisation, but harbour-
ing different 16S rRNA coding sequences (Mori et al.,
1997) were identically assigned to the same species with
the two sets of primers. In this work, 0.06% of isolates
were not species assigned; and 0.06% were assigned to a
presumptive lactobacilli species. The successful applica-
tion of Rep-PCR combined with species-specific PCR to
assign directly the isolates of mesophilic lactobacilli at
the species level, without ambiguities or discrepancies
between the results of the two methods, avoided the use
of the ambiguous phenotypic species assignation (Col-
lins et al., 1989; Curk, Hubert, & Bringel, 1996).
It is striking to note that 35 different strains of Lb.paracasei, but only three different strains of Lb.
rhamnosus were detected in this work. Lb. paracasei
seems to exhibit a different intraspecies genomic
variability from that of Lb. rhamnosus. This difference
can be observed both among the collection and Comt !e
cheese strains. Lb. paracasei and Lb. rhamnosus strainscan exhibit very different Rep fingerprints. But Lb.
rhamnosus fingerprints varied discontinuously and were
grouped into three distinct subclusters, while Lb.
paracasei fingerprints varied more continuously and
were grouped in a main cluster, when the collection and
cheese strains were analysed together.
This work showed that mesophilic lactobacilli from
milk, starter cultures and Comt!e cheese at different ages
could be selectively isolated and enumerated on FH
medium incubated at 201C and not 371C as recom-
mended (Isolini et al., 1990). These lactobacilli can be
facultative or obligatory-heterofermentative lactobacilli.
The MRS medium was insufficient for selecting lacto-
bacilli from some milks, starter cultures and cheeses in
the early stages. The temperature of 201C, not 371C,
allowed the growth of all mesophilic lactobacilli strains.
The dynamics of mesophilic lactobacilli population
enumerated on FH incubated at 371C presented in this
work is typical of that found in Comt!e cheese (Grappin
et al., 1999).
A striking result of this work is that diversity among
mesophilic lactobacilli from Comt!e cheeses of two
different origins was found at the strain, but not at the
species level. Each Comt!e cheese origin could be
identified by its profile of mesophilic lactobacilli strains,with no strain overlapping between profiles. Qualita-
tively, the strain profile in individual mature cheese of
the same manufacturing batch varied little according to
four different ripening conditions used in Comt!e
technology, some minor strains appearing or disappear-
ing; the most important changes were in the different
predominant strains found in the mature cheeses of one
cheese batch. The stability of the strain profiles of
mesophilic lactobacilli over different time periods for the
same Comt!e factory remains to be explored. The origin-
specific strain profile, but not the species profile, of
mesophilic lactobacilli was recently observed in mature
Irish Cheddar cheese (Fitzsimons, Cogan, Condon, &
Beresford, 1999).
The same mean number of different mesophilic
lactobacilli strains were identified in Comt !e (this work)
and Irish Cheddar mature cheeses. However, twice as
many were identified throughout Comt!e cheese ripening.
Thus, there is a need to examine precisely the strain
dynamics throughout cheesemaking and ripening. That
will be developed in a further report.
As mentioned above, diversity among mesophilic
lactobacilli from different Comt!e cheeses was not found
in their species profile. Indeed, the same two species, Lb.
paracasei and Lb. rhamnosus, were predominant, albeitat different proportions, in the two Comt!e cheeses
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monitored throughout ripening. It should be noted
however that Lb. rhamnosus was absent in the dominant
population at the end of ripening. Whereas Lb.
paracasei was previously detected as a predominant
species in many different cheese varieties, Lb. rhamnosuswas only detected in some mature hard cheeses, Swiss-
type, Idia$zabal, Swedish, and Parmigiano Reggiano
cheeses (Coppola et al., 1997; Elortondo, Echobarria,
Albisu, & Barcina, 1998; Jimeno, Lazaro, & Sollberger,
1995; Lindberg, Christiansson, Rukke, Eklund, &
Molin, 1996). A third minor species, Lb. parabuchneri
was detected in two cheeses from one factory. This last
species was also mentioned in three English Cheddar
mature cheeses (Williams & Banks, 1997). The species
diversity of mesophilic lactobacilli in individual mature
Comt!e cheese was of the same magnitude order as that
reported for mature Irish Cheddar cheese (Fitzsimons
et al., 1999), but lower than that reported by in mature
English Cheddar cheeses (Williams et al., 1997). The
mesophilic lactobacilli population in Comt!e cheese was
almost exclusively composed of facultatively heterofer-
mentative lactobacilli, as in Irish Cheddar and Parmi-
giano Reggiano cheeses (Coppola et al., 1997;
Fitzsimons et al., 1999).
This work strongly suggests that a large number of
the mesophilic lactobacilli strains in Comt!e cheese
originated from the raw milk, and that this source was
probably more important than factory-environment,
processing equipment or starter culture. This result
supports the milk origin of mesophilic lactobacilli thatcould be previously hypothesised (Demarigny et al.,
1996). The origin, associated to the specific strain profile
of raw milk mesophilic lactobacilli according to milk
origin, could partly explain the differences in sensory
properties of experimental Swiss-type cheeses which
differed only by the origin of the raw milk microflora
that were present (Demarigny, Beuvier, Buchin, Pochet,
& Grappin, 1997). The origin of the other non-starter
populations present in Comt!e cheese throughout chee-
semaking and ripening remains to be elucidated. On the
other hand, it would be also interesting to understand
why all of the mesophilic lactobacilli strains detected in
the dominant population in milk were not detected in
the dominant mesophilic lactobacilli population in
cheese throughout cheesemaking and ripening.
Acknowledgements
The technical support of Franck Dufrene is greatly
appreciated. This work was financially supported by the
INRA grant A.I.P. Structure et dynamique des
"ecosyst"emes bact!eriens, and by the region council of
Franche-Comt!e and the European Community
contract no. 96/9 R00202003. The authors would liketo thank S. Pochet for critical reading of the manuscript,
and Helen Lamprell and Jean M. Banks for revising the
English language.
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