651

Surface microbial consortia from Livarot, a French

smear-ripened cheese
Sandra Larpin-Laborde, Muhammad Imran, Catherine Bonaïti, Nagamani Bora,
Roberto Gelsomino, Stefanie Goerges, Françoise Irlinger, Michael Goodfellow,
Alan C. Ward, Marc Vancanneyt, Jean Swings, Siegfried Scherer,
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14

Micheline Guéguen, and Nathalie Desmasures

Abstract: The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening.
Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast
among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR
(rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG)5-PCR, and when appropriate by 16S
rDNA sequencing and SDS–PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly as-
signed to the genera Arthrobacter, Brevibacterium, Corynebacterium, and Staphylococcus. New taxa related to the genera
Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents
were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed
a high level of diversity and mainly included members of the genera Alcaligenes, Hafnia, Proteus, Pseudomonas, and Psy-
chrobacter. Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three
dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gram-
negative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of
their positive versus negative role should be objectively examined.
For personal use only.

Key words: smear cheese, bacteria, yeast, diversity, dynamics.

Résumé : La microflore de surface (902 isolats) de Livarots provenant de trois fromageries a été examinée au cours de l’af-
finage. Les levures ont été identifiées principalement par spectroscopie infrarouge à transformée de Fourier. Geotrichum
candidum était la levure dominante parmi 10 espèces. Les bactéries ont été identifiées par Biotype 100, dérépliquées par
rep-PCR et 156 souches représentatives ont été identifiées par BOX-PCR ou (GTG)5-PCR, et éventuellement par séquençage
de l’ADNr 16S et SDS–PAGE. Les bactéries à Gram positif représentaient 65 % des isolats et appartenaient principalement
aux genres Arthrobacter, Brevibacterium, Corynebacterium et Staphylococcus. De nouveaux taxons associés aux genres
Agrococcus et Leucobacter ont été mis en évidence. Les souches de levures et de bactéries à Gram positif ajoutées en tant
que levains de maturation n’étaient pas toujours détectables au cours de l’affinage. Trente deux pour cent des isolats bacté-
riens étaient à Gram négatif. Ils révélaient un haut niveau de diversité et incluaient principalement des membres des genres
Alcaligenes, Hafnia, Proteus, Pseudomonas et Psychrobacter. Quel que soit le lait utilisé (pasteurisé ou non), des niveaux
similaires de diversité ont été observés dans les trois ateliers, qui avaient tous des procédures de nettoyage efficaces ainsi
que de bonnes pratiques de fabrication. Certaines bactéries à Gram négatif pourraient avoir un rôle positif dans certaines
technologies fromagères. L’examen de leurs bénéfices et risques devrait être réalisé objectivement.
Mots‐clés : fromage à croûte lavée, bactéries, levures, diversité, dynamiques.

Received 27 July 2010. Revision received 15 April 2011. Accepted 19 April 2011. Published at www.nrcresearchpress.com/cjm on
4 August 2011.
S. Larpin-Laborde,* M. Imran, M. Guéguen, and N. Desmasures. Université de Caen Basse-Normandie, Unité des microorganismes
d’intérêt laitier et alimentaire, E.A. 3213, IFR 146 ICORE, 14032 Caen CEDEX, France.
C. Bonaïti and F. Irlinger. Laboratoire de génie et de microbiologie des procédés alimentaires, INRA, 78850 Thiverval-Grignon, France.
N. Bora†, M. Goodfellow, and A.C. Ward. School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK.
R. Gelsomino, M. Vancanneyt, and J. Swings. BCCM/LMG Bacteria Collection, Universiteit Gent, 9000 Gent, Belgium.
S. Goerges‡ and S. Scherer. Abteilung Mikrobiologie, Zentralinstitut für Ernhrungs und Lebensmittelforshung, 85350 Freising, Germany.
Corresponding author: Nathalie Desmasures (e-mail: nathalie.desmasures@unicaen.fr).
*Present address: Bioprocess division, Millipore Corporation, 67120 Molsheim, France.
†Present address: Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.
‡Present address: Naturkost Ernst Weber GmbH, 81371 München, Germany.

Can. J. Microbiol. 57: 651–660 (2011) doi:10.1139/W11-050 Published by NRC Research Press
 652
Introduction
Smear cheeses are so called because their surfaces are
washed during ripening, giving rise to a glistening rind.
Among them, red smear cheeses are characterized by the de-
velopment of an orangish red colour on the surface. Livarot
cheese is a traditional French red smear cheese with a Pro-
tected Designation of Origin. Milk production and cheese
making and ripening (minimum 3 weeks) are exclusively
done in a restricted area in Normandy. After standardization
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14
(to a 25–30 g/L concentration of fat) and addition of starter,
cow’s milk is ripened at 8–10 °C over 12–15 h and then
heated to 36 °C to be rennet-coagulated. After it is cut into
small pieces and slowly mixed, the curd is transferred in cy-
lindrical moulds (commonly 12 cm in diameter). Twenty-four
hours later, the curd is removed from the moulds. The salting
operation is done after a 24–48 h period. During ripening, at
least three rind-washing operations are done. Just before
packaging (17–18 ripening days), Livarot cheese is sur-
rounded by three to five characteristic bands and thereafter
kept at 4 °C for a minimal period of 35 days. Dry matter is
at least 40 g per 100 g of cheese after complete desiccation.
Some of the production is farmhouse-made but most is done
at the (semi)industrial scale. Although the same basic tech-
nology is used by all the cheese producers, variations occur
from one dairy to another. For example, (i) the milk used for
Livarot manufacturing can be either pasteurized or unpasteur-
For personal use only.
ized, (ii) cheese is either salted in brine or with dry salt, and
(iii) the surfaces of the Livarot cheeses are either washed
with brine or smeared three to five times during the first
3 weeks of ripening, using one or more combinations of cul-
tures of microorganisms, which may include Brevibacterium
linens, Debaryomyces hansenii, Geotrichum candidum, or
Kluyveromyces lactis. These variations, and probably many
others, result in diversity (in colour, taste, and aroma) among
the Livarot cheeses produced (http://www.fromage-normandie.
com/livarot/).
Investigating the surface microbiota of smear cheeses is
still scientifically relevant, as microbial populations are not
always well known; and consequently, several new microbial
species also need to be described from such substrates (Bora
et al. 2007, 2008; Schmidt et al. 2009). The dynamics of mi-
crobial communities during ripening are also important to de-
scribe in order to determine their specific role regarding
organoleptic properties of cheeses. It is well known that dur-
ing ripening, yeasts dominate initially and D. hansenii is the
major species found on the surface. Geotrichum candidum or
Yarrowia lipolytica have also been described on the surface
of Limburger, Münster, Tête de Moine (Wyder and Puhan
1999), and Livarot (Larpin et al. 2006) cheeses. The physico-
chemical conditions created by the yeasts allow the growth of
salt-tolerant, acid-sensitive bacteria. Diverse populations of
coryneform bacteria, micrococci, and staphylococci
(Eliskases-Lechner and Ginzinger 1995a; Abliz et al. 2004;
Rademaker et al. 2005; Rea et al. 2007) as well as “marine”
lactic acid bacteria (Roth et al. 2010) have been found on the
surface of smear cheeses. Recently, the presence of Gram-
negative bacteria has also been highlighted (Rea et al. 2007;
Fontana et al. 2010). While some information is available re-
garding the microbial diversity found on the surface of one
commercial Livarot cheese sold on the retail market (Mou-
nier et al. 2009) and on the dynamics of the main yeast spe-
Can. J. Microbiol. Vol. 57, 2011
cies, to our knowledge no data focus on the “whole” surface
microflora of Livarot cheese and its evolution during ripen-
ing.
In this study, which was part of the European project
QLK1-CT-2001-02228, we made an inventory of the bacte-
rial and yeast floras on the surface of Livarot cheese during
ripening. The objective was to determine the microbial
groups dominating in Livarot at various stages of ripening,
and hence, having a putative role in cheese technology.
Materials and methods
Cheese samples
Batches of Livarot cheese manufactured from either pas-
teurized or raw milk in three different dairies were studied.
The cheese samples were taken 1 day before the salting oper-
ation (∼2 days of ripening), 1 day before packaging
(∼21 days of ripening), and at the end of ripening
(∼60 days). Dairy A used thermized milk and Brevibacte-
rium aurantiacum (sold as Brevibacterium linens), Staphylo-
coccus sp., D. hansenii, G. candidum, and K. lactis as
commercial starter cultures; the ripening room temperature
was between 12 and 14 °C and humidity between 94% and
96%. Dairy B used raw milk and Brevibacterium aurantia-
cum (sold as B. linens), G. candidum, and K. lactis as com-
mercial starter cultures; the ripening room temperature was
between 13 and 15 °C and humidity between 93% and 95%.
Dairy C used pasteurized milk and Brevibacterium aurantia-
cum (sold as B. linens), Staphylococcus sp., D. hansenii,
G. candidum, and K. lactis as commercial starter cultures;
the ripening room temperature was between 12 and 15 °C
and humidity 97%.
Determination of viable counts
The surface microflora of each cheese was enumerated, as
described by Brennan et al. (2002), on yeast extract glucose
chloramphenicol agar (YGCA) for the yeasts and plate count
agar + 3% NaCl (PCA+) for bacteria. For each sample, start-
ing from a single dilution, about 50 bacteria and 50 yeast
colonies were isolated from PCA+ and YGCA, respectively,
with care taken to cultivate members of each observed mor-
photype while keeping its relative abundance. The resulting
450 bacteria and 452 yeasts were purified, subcultured three
times, and maintained at –80 °C.
Characterization of bacteria
Gram staining, motility, and catalase and oxidase activity
were performed on all of the bacterial isolates.
Genomic DNA was extracted from isolates as described in
Versalovic et al. (1994), and amplification of template DNA
was performed by repetitive extragenic palindromic (rep-
PCR), using primers RepIRI and Rep2I. The fingerprint pro-
files were analysed using BioNumerics software (Applied
Maths, Sint-Martins-Latem, Belgium). Cluster analysis was
performed using the Jacquard similarity coefficient and
Ward’s clustering method. In all clusters, isolates exhibiting
distinct profiles were chosen in conjunction with morphology
to represent the observed diversity. They are cited below as
dereplicated isolates.
Apart from Gram-positive and catalase-negative isolates,
which were considered to be lactic acid bacteria (LAB) and
Published by NRC Research Press
 Larpin-Laborde et al.
hence are not considered further, most isolates were also
characterized using Biotype 100 strips (Biomérieux, Marcy
l’Étoile, France) (Grimont et al. 1996), which were incubated
at 30 °C for up to 6 days. Recognizer® software (Taxolab,
Pasteur Institute, Paris, France) and reference databases for
Gram-negative and coryneform bacteria were used to identify
the isolates. In addition, 16S rDNA sequencing of 26 isolates
from the middle stage of ripening in dairy C was done to
confirm their identity.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14
Identification of dereplicated isolates
The dereplicated isolates were grown on tryptic soy broth
(TSB; BBL, Sparks, Maryland, USA) containing agar (15 g/L;
Oxoid, Basingstoke, England). Representative strains were
maintained at –80 °C and deposited in the public BCCM/
LMG Bacteria Collection (http://bccm.belspo.be/about/lmg.
php). All of the strains were grown for 1–2 days at 30 °C
on TSB (Difco, Detroit, Michigan, USA) containing agar at
15 g/L (Oxoid). DNA was extracted from rod-shaped iso-
lates according to Gevers et al. (2001) and from coccus-
shaped isolates using the same protocol but with lysostaphin
(5000 U/mL, Sigma, St. Louis, Missouri, USA) replacing
mutanolysin. A set of reference strains, known to be present
on red smear cheeses, was included for identification by
rep-PCR. PCR was performed according to Versalovic et
al. (1994) using the primers BOXA1R for rods and (GTG)5
For personal use only.
for cocci. If a strain gave an unsatisfactory band pattern
with one primer set, the other primer set was tested. Band
patterns were analysed using the Pearson coefficient and
UPGMA cluster analysis with BioNumerics software (Ap-
plied Maths). Dereplicated isolates were subjected to 16S
rRNA gene sequence analysis for identification, using pri-
mers described by Coenye et al. (1999). Representative
strains were subjected to SDS–PAGE analysis as described
by Pot et al. (1994), and numerical analysis was performed
using the Gel Compare version 4.2 (Applied Maths, Kor-
trijk, Belgium). Isolates giving an ambiguous Gram stain
were the subject of fatty acid analysis as described by
Vancanneyt et al. (1996).
Identification of yeasts
The yeasts were dereplicated and identified by Fourier
transform infrared (FTIR) spectroscopy. Sample preparation
and spectral evaluation were performed according to
Kümmerle et al. (1998) using an IFS-28B FTIR spectrometer
and opus 3.17 software from Windows (Bruker, Karlsruhe,
Germany). Identification was done as described by Goerges
et al. (2008). The threshold value for cluster delineation was
set at a spectral distance of 0.5–1.5, depending on the spe-
cies. One to six isolates were randomly selected as represen-
tatives of each cluster, depending on cluster size. The 46
resulting dereplicated yeast isolates were characterized using
physiological tests according to Yarrow (1998), including fer-
mentation of D-glucose, D-galactose, and lactose; assimilation
of 23 carbon sources and nitrite, ethylamine, lysine, cadaver-
ine, and glucosamine; tolerance to 1% (v/v) acetic acid; and
protease and lipase production. The resulting phenotypic pro-
files were compared with those described by Barnett et al.
(2000). Identity of the 46 dereplicated yeast isolates was con-
firmed using Random Amplified Microsatellites-PCR (RAM-
PCR) and PCR – restriction fragment length polymorphism
653
(PCR–RFLP) analysis. Isolates giving ambiguous results
were subjected to DNA sequencing. For these purposes,
DNA was prepared as described by Vasdinyei and Deak
(2003), except that the nucleic acid pellet was dissolved in
100 µL of water containing 2 µL of RNase (20 µg/µL). For
RAM-PCR, the oligonucleotide primer (CAC)5 was used to
amplify the intersingle sequence repeats according to Gente
et al. (2002), and for PCR–RFLP, ITS1 and ITS4 primers
were used to amplify the internal transcribed spacer region
(Guillamón et al. 1998). For sequencing purposes, the D1–D2
domain of the LSU rDNA was amplified with primers NL1
and NL4m (O’Donnell 1993), according to Abliz et al. (2004).
The amplified LSU rDNA products were sent to Genome Ex-
press (Meylan, France) for purification and direct sequencing.
The sequence data were blasted against the NCBI database.
For PCR–RFLP, CfoI, HaeIII, and HinfI restriction en-
zymes were used independently to digest the amplification
products of the internal transcribed spacer PCR. PCR prod-
ucts (25 µL) were digested in a 40 µL final volume consist-
ing of 0.5 µL of restriction enzyme (Invitrogen), 3.6 µL of
the buffer specific for that enzyme, and 0.4 µL of enzyme of
bovine serum albumin (Invitrogen), and the mixture was then
incubated at 37 °C for 2 h. The restriction fragments were
separated by electrophoresis on 3% (m/v) agarose (Invitrogen)
gels in 1× TAE (Tris – acetate – EDTA) buffer.
Results
Microbial counts
Surface bacterial counts prior to salting ranged from
106 cfu/cm2 cheese in dairy C to >108 cfu/cm2 cheese in
dairy A (Fig. 1). At the middle stage of ripening, bacterial
levels up to 109–1010 cfu/cm2 cheese were obtained. They
were then either quite stable or increased (dairy C) until the
end of ripening. The yeast population reached 107 cfu/cm2
cheese in the early stage of ripening, except for dairy C
where the level was only 105 cfu/cm2 cheese. Similar levels
(about 5 × 107 cfu/cm2) were reached for the three batches
at the middle stage of ripening, thereafter, the yeast popula-
tion decreased very slowly.
Biodiversity of surface bacteria
Of the 450 bacteria isolated, 292 (65%) were Gram-
positive, 145 (32%) were Gram-negative, and 13 (3%) were
not determined.
Three hundred and eighty-six of the 450 isolates were suc-
cessfully screened by rep-PCR and assigned to 17 clusters
(data not shown). Depending on the size of the cluster, one
to 20 bacteria were chosen per cluster, so that 156 (40%) iso-
lates were finally selected. Molecular analysis, using rep-PCR
(BOX or (GTG)5 primers) and (or) 16S rRNA gene sequence
analysis and (or) SDS–PAGE profiling, led to the assignment
of 10 out of the 156 isolates to putatively novel taxa. The
Gram-positive bacteria were identified mainly as Arthro-
bacter arilaitensis, Brevibacterium aurantiacum, Corynebac-
terium casei (Table 1) and as members of the Staphylococcus
saprophyticus group. Among the non-Enterobacteriaceae
Gram-negative bacteria, the genera Pseudomonas, Alcali-
genes, Acinetobacter, and Psychrobacter were identified.
Among the 450 isolates, 411 were subjected to phenotypic
analysis. Seventy-five Gram-positive and catalase-negative
Published by NRC Research Press
 654 Can. J. Microbiol. Vol. 57, 2011

Fig. 1. Changes in bacterial (solid lines) and yeast (broken lines) populations during ripening of Livarot cheese manufactured in three dairies:
•, dairy A; ▪, dairy B; ▴, dairy C.
11

10

9
Log of cells per cm2 cheese

8
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14

4
0 10 20 30 40 50 60

Days of ripening

Table 1. Identity of dereplicated bacterial strains, using molecu- contained strains identified as Candida catenulata, Debaryo-
lar analysis. myces hansenii, Geotrichum candidum, Kluyveromyces lactis
or Kluyveromyces marxianus, Yarrowia lipolytica, and Can-
Identity LMG No.
dida glabrata or Pichia jadinii. The five remaining isolates
For personal use only.

Agrococcus casei 22447 formed single-membered clusters, one of which was identi-
Arthrobacter arilaitensis 22417 fied as Cryptococcus humicolus and another as Torulaspora
Brachybacterium spp. 22434 delbrueckii. Using physiological tests and molecular tools,
Brevibacterium aurantiacum 22422 the identity of 46 dereplicated isolates was confirmed in
Corynebacterium casei 22430 most cases. One out of the eight isolates identified as Y. lip-
Lactic acid bacteria 22794 olytica using the phenotypic data was finally identified as
Macrococcus sp. 22445 Candida deformans, based on the percentage identity of its
Microbacterium gubbeenense 22428 LSU rDNA sequence. It was not possible to differentiate be-
New taxon close to the genus Leucobacter 22448, 22412 tween the type strains K. lactis and K. marxianus using phe-
Staphylococcus saprophyticus group 22436 notypic data. However, these strains were distinguished using
Acinetobacter spp. — RAM-PCR, and all of the Livarot isolates belonged to the
Alcaligenes spp. — species K. lactis (Fig. 2). Among four isolates identified by
Pseudomonas spp. — FTIR spectroscopy as Candida catenulata, one was finally
Psychrobacter spp. — identified as a strain of Candida natalensis, as it showed a
Enterobacteriaceae 22792 LSU rDNA sequence that was 99% similar to Candida nata-
Other Gram-negative bacteria 22790 lensis. One isolate, which was identified as Crytococcus hu-
Note: Numbers for each representative bacterium are from the Labor- micolus by both FTIR spectroscopy and by the physiological
atorium voor Microbiologie (LMG), Universiteit Gent. data, was found to show 99% identity with Cryptococcus
musci using the LSU rDNA data.
isolates were considered to be LAB and were not studied fur-
ther. The 336 remaining isolates were examined using the Population dynamics
Biotype 100 strips (Table 2), and 247 of them were success- By merging data obtained using (i) FTIR spectroscopy and
fully identified. Forty isolates were shown to be coagulase- molecular analysis for yeasts, (ii) Biotype 100 analysis of 247
negative staphylococci, which were mainly identified as bacterial isolates, (iii) additional 16S rDNA sequencing of 26
Staphylococcus xylosus or Staphylococcus spp. The 97 iso- of the bacterial isolates, and (iv) molecular analysis of 386
lates found to be coryneform bacteria were notably assigned bacterial isolates, including the 156 dereplicated ones, an
to two taxa, namely Arthrobacter arilaitensis and Brevibacte- overview of surface microbial diversity of Livarot cheese
rium spp. Gram-negative isolates were assigned to 11 species was obtained (Table 4).
or genera. The predominant taxa were identified as Alcali- For bacteria, members of 20 different taxa were identified
genes faecalis, Alcaligenes piechaudii, Proteus vulgaris, and at all stages of ripening for dairy A. The number of bacterial
Hafnia alvei. groups remained constant during ripening but dominant
groups changed. At the early stage of ripening the S. sapro-
Biodiversity of yeasts phyticus group dominated, P. vulgaris dominated at the mid-
Four hundred and fifty-two yeast isolates were assigned to dle stage, and S. xylosus and members of the new taxon close
28 clusters by FTIR spectroscopy (Table 3). The clusters to Leucobacter sp. were the most numerous groups at the late

Published by NRC Research Press

 Larpin-Laborde et al. 655

Table 2. Identification of bacterial strains isolated from Livarot Table 3. Yeast diversity assessed by Fourier transform infrared
cheese using Biotype 100 strips. spectroscopy.

No. of No. of No. of

Identification isolates Identified species clusters isolates (%)
Staphylococci Candida catenulata 4 23 (5)
Staphylococcus equorum 2 Debaryomyces hansenii 2 56 (12)
Staphylococcus spp. 17 Geotrichum candidum 10 278 (62)
Staphylococcus xylosus 21 Kluyveromyces lactis or K. marxianus 3 38 (8)
Total 40 Pichia jadinii or Candida glabrata 1 2 (<1)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14

Yarrowia lipolytica 8 50 (11)

Coryneform bacteria Cryptococcus humicolus 0 1 (<1)
Arthrobacter arilaitensis 32 Geotrichum candidum 0 3 (<1)
Brachybacterium alimentarium 10 Torulaspora delbrueckii 0 1 (<1)
Brachybacterium faecium 2
Brevibacterium spp./Arthrobacter spp. 1
Brevibacterium spp. /Corynebacterium spp. 1 Fig. 2. Random amplified microsatellites PCR patterns of isolates
Brevibacterium spp. 34 assigned to the genus Kluyveromyces, using phenotypic data, and of
Corynebacterium casei 15 the type strains Kluyveromyces lactis and Kluyveromyces marxianus,
Microbacterium gubbeenense 2 based on (CAC)5 primer. Lanes: 1, K. lactis CBS 683T; 2–5, yeasts
Total 97 strains isolated from Livarot cheese; and 6, K. marxianus CBS 712T.
1
Gram-negative bacteria
Alcaligenes eutrophus 1 2
Alcaligenes faecalis 30 3
Alcaligenes piechaudii 21
Alcaligenes spp. 2 4
For personal use only.

Citrobacter freundii sensu stricto 1 5

Escherichia coli 4
Hafnia alvei 16
Proteus vulgaris 21
Providencia heimbacheae 1 cheese, the diversity level was quite constant. Candida cate-
Pseudomonas putida 9 nulata and G. candidum were found throughout the ripening
Serratia liquefaciens 4 period. In contrast, K. lactis and P. jadinii were isolated only
Total 110
Not determined 19
No growth in biotype 100 medium 70
Total isolates 336
stage of ripening. In cheese from dairy B, 10 Gram-positive
and six Gram-negative groups were delineated from all stages
of ripening. The bacterial diversity increased at the middle
stage of ripening mainly with respect to the Gram-positive
flora. Hafnia alvei strains dominated the early stage of ripen-
ing, Corynebacterium casei the middle stage of ripening, and
Alcaligenes faecalis the late stage. In cheese from dairy C,
Gram-negative taxa were the most numerous. The greatest di-
versity was found at the middle stage of ripening. Arthro-
bacter arilaitensis and Pseudomonas putida isolates were
dominant before packaging. At the end of ripening, the bac-
terial flora was dominated by Arthrobacter arilaitensis, Bre-
vibacterium aurantiacum, and Psychrobacter sp.
The diversity of the yeast flora was lower than that of the
bacteria (Table 4). Members of five species were found at the
early stage of ripening in dairy A cheese, with only two and
three species detected at the middle and late stages, respec-
tively. Debaryomyces hansenii and G. candidum were present
at all stages of ripening. Cryptococcus musci and K. lactis
were present only at the beginning of ripening, and Y. lipoly-
tica was detected at the late ripening stage. In dairy B
6
at the beginning of ripening and Y. lipolytica at the middle
stage. Members of six different species were identified in
dairy C cheese. As for dairy A cheese, both D. hansenii and
G. candidum were present at all stages of ripening; K. lactis
was detected only at the first stage of ripening. The highest
diversity was recorded before packaging. Yarrowia lipolytica
was first detected at the middle stage of ripening and showed
a higher incidence at the end of ripening. Members of nine
taxa were common to the three cheeses: Arthrobacter arilai-
tensis, Brachybacterium alimentarium, Brevibacterium aur-
antiacum, Brevibacterium sp., the taxon closely related to
the genus Leucobacter, and Pseudomonas sp. for the bacte-
ria; and G. candidum, K. lactis, and Y. lipolytica for the
yeasts.
Discussion
Maximum viable bacterial counts obtained during the rip-
ening of Livarot cheese were 1–2 logs higher than those pre-
viously reported for Gubbeen cheese (108 cfu/cm2) (Brennan
et al. 2002) or one Livarot cheese from the retail market
(Mounier et al. 2009) but similar to those of Tilsit, which
had 109 cfu/cm2 (Eliskases-Lechner and Ginzinger 1995a).
A variety of morphologically different bacterial colonies
grew on PCA+. The Gram-positive bacteria isolated from
the surface of Livarot cheese belonged either to the LAB or
to commonly described genera, such as Arthrobacter, Brevi-
bacterium, Corynebacterium, Microbacterium, and Staphylo-

Published by NRC Research Press

 656 Can. J. Microbiol. Vol. 57, 2011

Table 4. Identification of isolates at different stages of ripening based on samples taken from the three dairies.

Dairy A Dairy B Dairy C

Early Middle Late Early Middle Late Early Middle Late Total
Bacteria
Gram-positive bacteria
Agrococcus casei 1 1
Arthrobacter arilaitensis 3 6 1 5 9 10 34
Arthrobacter spp. 1 1
Brachybacterium alimentarium 1 1 1 5 1 1 10
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14

Brachybacterium spp. 2 2
Brevibacterium aurantiacum 5 1 2 5 3 1 8 25
Brevibacterium spp. 4 3 3 1 2 1 3 17
Corynebacterium casei 3 1 16 20
Macrococcus sp. 1 1
Microbacterium gubbeenense 1 3 4
New taxon close to Leucobacter spp. 6 11 1 2 1 21
Staphylococcus epidermidis group 1 1
Staphylococcus equorum 1 1
Staphylococcus saprophyticus group 14 3 1 1 19
Staphylococcus pasteuri 2 2
Staphylococcus xylosus 2 2 17 21
Staphylococcus spp. 1 1 2
Unidentified Gram-positive bacteria 1 1 12 5 2 9 5 35
Gram-negative bacteria
Acinetobacter johnsonii 1 1
For personal use only.

Acinetobacter spp. 1 1
Alcaligenes faecalis 2 26 28
Alcaligenes piechaudii 1 1
Alcaligenes spp. 1 1 3 5
Enterobacteriaceae 1 1 2 1 5
Escherichia coli 2 1 3
Ewingella americana 2 2
Hafnia alvei 14 2 1 17
Marinomonas spp. 3 3
Proteus vulgaris 14 2 1 17
Proteus spp. 2 2
Pseudomonas putida 8 8
Pseudomonas stutzeri 1 1
Pseudomonas spp. 2 1 1 1 1 6
Psychrobacter celer 2 2
Psychrobacter spp. 1 1 15 17
Raoultella planticola 1 1
Serratia grimesii 1 1
Serratia liquefaciens 2 1 3
Unidentified Gram-negative bacteria 7 2 2 1 1 4 2 2 21
Other unidentified bacteria 3 4 4 2 13

Yeasts
Candida catenulata 11 7 3 21
Candida natalensis 2 2
Cryptococcus musci 1 1
Debaryomyces hansenii 12 14 3 21 3 3 56
Geotrichum candidum 23 36 40 30 42 34 6 42 28 281
Kluyveromyces lactis 10 5 23 38
Pichia jadinii 2 2
Torulaspora delbrueckii 1 1
Yarrowia lipolytica 4 7 1 18 1 19 50

Total microbial isolates 92 94 98 71 99 100 78 99 96 827

Published by NRC Research Press

 Larpin-Laborde et al.
coccus (Beresford et al. 2001). Less common bacteria were
also found on Livarot cheese, e.g., Brachybacterium alimen-
tarium, as described for hard cheeses (Schubert et al. 1996),
but also bacteria belonging to the genera Agrococcus and
Macrococcus. The latter have been encountered in cow’s and
goat’s milks and were detected in the non-smear-ripened Si-
cilian cheese Ragusano (Randazzo et al. 2002). Agrococcus
casei sp. nov. has been identified not only from Livarot
cheese but also from two other smear-ripened cheeses,
namely, Gubbeen and Tilsit (Bora et al. 2007; Rea et al.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14
2007). The technological impact of these bacteria remains to
be understood.
Unlike what has been done in most previous studies focus-
ing on surface bacteria from cheese (Eliskases-Lechner and
Ginzinger 1995a; Valdés-Stauber et al. 1997; Rademaker et
al. 2005), the bacterial isolates in this investigation were
chosen to represent the macroscopic diversity irrespective of
whether strains were Gram-positive or Gram-negative. Little
is known about the occurrence of Gram-negative bacteria,
which are mainly regarded as indicators of either poor hy-
giene (e.g., coliform bacteria) (Jay et al. 2005) or as spoil-
age-contaminating bacteria (e.g., Pseudomonas fluorescens)
(Neugebauer and Gilliland 2005). Milk used for making the
Livarot cheeses was either pasteurized — and can be consid-
ered as free from or as containing very low levels of non-
thermoresistant bacteria such as coliform bacteria or pseudo-
For personal use only.
monads — or unpasteurised. Raw milk used in France nowa-
days commonly has total bacterial counts of less than
5000 cfu/mL (Beuvier and Buchin 2004).
Hygiene practices in the three dairy plants were very satis-
factory, all having efficient cleaning procedures, good manu-
facturing practices, and personnel hygiene policy. The
sanitary and hygienic qualities of Livarot cheese can be con-
sidered satisfactory, as potential pathogenic bacteria, such as
Listeria monocytogenes, Staphylococcus aureus or other co-
agulase positive staphylococci, and Salmonella sp., were not
identified amongst the isolates. Some Escherichia coli strains
were isolated from the early stage but not from the later stage
of ripening in dairy A and C cheeses. It is interesting, there-
fore, that a high population of Gram-negative bacteria be-
longing to 20 identified taxa was detected on the surface of
all Livarot cheeses. Enterobacteriaceae have been isolated
from various cheeses (Tornadijo et al. 1993; Duthoit et al.
2005), while H. alvei has been shown to be the major Gram-
negative species during ripening on the surface of Camem-
bert, a soft cow’s milk cheese (Richard and Zadi 1983). A
similar result was reported for a soft goat’s milk cheese
(Sablé et al. 1997).
Some strains of H. alvei have been implicated as agents of
gastroenteritis (Rodríguez et al. 1999), but others are sold as
ripening agents for cheesemaking (Skovmose 2003). In Li-
varot, H. alvei was only found on dairy B cheese, represent-
ing 33% of its Gram-negative flora. It has been suggested
that H. alvei is involved in the synthesis of volatile aromatic
compounds in Camembert (Richard and Gratadoux 1984)
and shown that Enterobacteriaceae strains of dairy origin
are able to produce volatile compounds in experimental His-
pánico (Morales et al. 2004) and red smear (Deetae et al.
2009b) cheeses. Several studies have demonstrated that coli-
forms and fecal coliforms exist in many raw food materials
657
where their presence has no relationship to food safety (Jay
et al. 2005).
Pseudomonas and Psychrobacter strains were found at the
surface of Livarot cheese. Members of these genera have
been described as spoilage bacteria responsible for the ap-
pearance of sticky or fatty rind on blue-veined cheeses and
of a slimy film on cottage-cheese (Bergère and Lenoir 1997)
or for the development of a fluorescent stain on the cheese
surface. No defects were recorded on Livarot cheeses used in
this study. Some of these Gram-negative bacteria are well
known for exhibiting strong proteolytic and lipolytic activ-
ities. They also produce in food (e.g., Pseudomonas putida)
(Morales et al. 2004; Jay et al. 2005), including cheese (Dee-
tae et al. 2009a), volatile sulphur compounds such as di-
methyl disulfide, which significantly contribute to the aroma
of cheese (Kagkli et al. 2006). Such activities may have a
positive effect during cheese ripening. Given such results,
we think that it would be interesting to explore the contribu-
tion of Gram-negative bacteria to cheese ripening, provided it
is balanced by safety assessments.
Maximum viable yeast counts reached levels (∼107 cfu/cm2)
comparable to those reported for other red smear cheeses,
such as Limburger and Münster (Wyder and Puhan 1999).
The yeast flora consisted mainly of D. hansenii, G. candi-
dum, K. lactis, and Y. lipolytica. These species have been
described to as agents of lipolytic, proteolytic, or esterase
activities during ripening of smear cheeses (Choisy et al.
1997). Geotrichum candidum, D. hansenii, and K. lactis
can be used as commercial starter cultures in the manufac-
turing of Livarot cheese but not, to our knowledge, Y. lip-
olytica. Nevertheless this organism was present in greater
numbers than were K. lactis strains. The predominant yeasts
isolated from Livarot cheese have been found in this and
other smear cheeses, such as brick cheeses (Valdés-Stauber
et al. 1997), Limburger and Münster cheeses (Wyder and
Puhan 1999), Tilsit cheese (Eliskases-Lechner and
Ginzinger 1995b), and Münster and Maroilles cheeses
(Mounier et al. 2010). Nevertheless, if D. hansenii is con-
sidered to be by far the dominant yeast occurring in virtu-
ally all cheeses (Beresford et al. 2001), it was not as
dominant as G. candidum at the end of ripening of Livarot
cheese. It would seem that Livarot cheese is a biotope to
which G. candidum is well adapted.
High diversity, with as much as 46 different microbial
groups, has been demonstrated at the surface of Livarot
cheese using culture-dependent polyphasic analysis. While it
can be inferred from various studies done on German cheeses
(Maoz et al. 2003) and Camembert cheese (Henri-Dubernet
et al. 2008) that uncultivable microorganisms are not so com-
mon in cheese and milk, an approach coupling both culture-
dependent and culture-independent rRNA analysis would
have probably yielded more diversity.
Several bacterial and yeast commercial cultures are used in
Livarot cheese making. A strain of Staphylococcus sp., which
was added as a commercial culture in dairy C, was not recov-
ered from the surface microbial flora at any point during rip-
ening of Livarot cheese. A similar phenomenon has been
reported for Gubbeen cheese (Brennan et al. 2002) for a
commercial strain of Brevibacterium linens and for Limbur-
ger cheese (Goerges et al. 2008). In the same way, Feurer et
al. (2004) concluded that the bacterial composition of the
Published by NRC Research Press
 658
surface flora of a French red smear cheese at the end of rip-
ening did not reflect the composition of the smearing inocu-
lum. In contrast, isolates belonging to the same taxon as
another commercial Staphylococcus sp. strain were recovered
at two sampling points from cheese manufactured in dairy A,
using this commercial strain. While commercial strains of
K. lactis were added to the three cheeses, this yeast was only
detected in the early stage of ripening and thus seemed un-
able to establish in Livarot cheese, as confirmed using real-
time PCR (Larpin et al. 2006). Thus it seems clear that
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14
microorganisms, deliberately added as smearing agents, do
not necessarily become dominant, depending on the strain
and maybe on the species (K. lactis). This last point needs to
be further investigated by cheese makers to select, if neces-
sary, appropriate commercial strains.
Whatever the milk used (pasteurized or unpasteurized),
similar levels of biodiversity were observed in the three dair-
ies, suggesting a nonnegligible role of the resident microflora
of the cheese dairies. Members of some species were found
in the cheeses from all three dairies, as exemplified by Ar-
throbacter arilaitensis, Brevibacterium aurantiacum, the pre-
sumptive novel taxon closely related to the genus
Leucobacter, G. candidum, K. lactis, and Y. lipolytica. In
contrast, members of other species were specific to a particu-
lar dairy. Each cheese had its own microbial flora at species
level, a phenomenon that has been reported for LAB and cor-
For personal use only.
yneform bacteria in Salers cheese (Duthoit et al. 2003) and,
at the strain level, for lactobacilli in Comté cheese (Depouilly
et al. 2004) and Camembert cheese (Henri-Dubernet et al.
2008). The microbiological differences between the three
cheeses may be influenced by milk composition, practices or
technology, and interactions between starters and nonstarter
microorganisms (Buchin et al. 1998; Tessier et al. 2003). For
all three cheeses (although the effect is not as strong in dairy
A cheese), the middle sample was characterized by a high
bacterial diversity. This can be explained by the fact that the
smear was not developed at an early stage of ripening, so
many bacterial isolates particularly in dairies B and C were
LAB. Few species (probably commercial strains) dominated
at the early stage (mainly in dairies A and B) when the pH
was low. In the middle stage, when the pH was more favor-
able, it can be assumed that the cheese surface was colonized
by microorganisms originating from milk and from the resi-
dent dairy microbiota. Selective forces (competition for nu-
trient, lowering of temperature) probably occur at the end of
ripening. Finally, regarding dairy C, as 26 additional isolates
were subjected to rRNA phylogenetic study, a bias was prob-
ably introduced in the level of diversity compared with other
sampling points and other dairies.
Compared with other smear cheeses, Livarot cheese seems
to be characterized by the presence of G. candidum as the
dominant yeast, by members of a new taxon that is most
closely related to the genus Leucobacter, and by high num-
bers of Gram-negative bacteria. Describing microbial diver-
sity and dynamics can lead to improvements in cheese
technology. Indeed, members of all these taxa may contribute
to the aromatic properties of the cheese, the hurdle effect re-
garding pathogenic bacteria, and cheese typicality. Knowing
these data, technological parameters can be used to influence
the balance of microbial communities. It appears that some
Gram-negative bacteria should now be regarded as potentially
Can. J. Microbiol. Vol. 57, 2011
useful bacteria in some cheese technologies. The assessment
of their positive versus negative role should be objectively
done.
Acknowledgements
This study was supported in part by the European Union
(project QLK1-CT-2001-02228: Biodiversity and anti-listerial
activity of surface microbial consortia from Limburger, Re-
blochon, Livarot, Tilsit and Gubbeen cheese). Part of this
work was made possible by a Ph.D. scholarship from Higher
Education Commission (HEC) of Pakistan and received a fi-
nancial support from the Conseil Régional de Basse-Normandie.
References
Abliz, P., Fukushima, K., Takizawa, K., and Nishimura, K. 2004.
Identification of pathogenic dematiaceous fungi and related taxa
based on large subunit ribosomal DNA D1/D2 domain sequence
analysis. FEMS Immunol. Med. Microbiol. 40(1): 41–49. doi:10.
1016/S0928-8244(03)00275-X. PMID:14734185.
Barnett, J.A., Payne, R.W., and Yarrow, D. 2000. Yeasts: character-
isation and identification. Cambridge University Press, Cam-
bridge, UK.
Beresford, T.P., Fitzsimons, N.A., Brennan, N.L., and Cogan, T.M.
2001. Recent advances in cheese microbiology. Int. Dairy J. 11(4–
7): 259–274. doi:10.1016/S0958-6946(01)00056-5.
Bergère, J.-L., and Lenoir, J. 1997. Les accidents de fromagerie et les
défauts des fromages. In Le fromage. Edited by A. Eck and J.-C.
Gillis. Lavoisier Tec and Doc, Paris, France. pp. 509–543.
Beuvier, E., and Buchin, S. 2004. Raw milk cheeses. In Cheese —
chemistry, physics and microbiology. Vol 1. 3rd ed. Edited by P.F.
Fox, P.L.H. McSweeney, T.M. Cogan, and T.P. Guinee. Elsevier
Academic Press, London, UK. pp. 319–345.
Bora, N., Vancanneyt, M., Gelsomino, R., Swings, J., Brennan, N.,
Cogan, T.M., et al. 2007. Agrococcus casei sp. nov., isolated from
the surfaces of smear-ripened cheeses. Int. J. Syst. Evol. Microbiol.
57(1): 92–97. doi:10.1099/ijs.0.64270-0. PMID:17220448.
Bora, N., Vancanneyt, M., Gelsomino, R., Snauwaert, C., Swings, J.,
Jones, A., et al. 2008. Mycetocola reblochoni sp. nov., isolated
from the surface microbial flora of Reblochon cheese. Int. J. Syst.
Evol. Microbiol. 58(12): 2687–2693. doi:10.1099/ijs.0.64201-0.
PMID:19060041.
Brennan, N.M., Ward, A.C., Beresford, T.P., Fox, P.F., Goodfellow,
M., and Cogan, T.M. 2002. Biodiversity of the bacterial flora on the
surface of a smear cheese. Appl. Environ. Microbiol. 68(2): 820–
830. doi:10.1128/AEM.68.2.820-830.2002. PMID:11823224.
Buchin, S., Delague, V., Duboz, G., Berdagué, J.-L., Beuvier, E.,
Pochet, S., and Grappin, R. 1998. Influence of pasteurization and
fat composition of milk on the volatile compounds and flavor
characteristics of a semi-hard cheese. J. Dairy Sci. 81(12): 3097–
3108. doi:10.3168/jds.S0022-0302(98)75874-6.
Choisy, C., Desmazeaud, M., Gripon, J.C., Lamberet, G., and Lenoir,
J. 1997. La biochimie de l’affinage. In Le fromage. Edited by A.
Eck and J.-C. Gillis. Lavoisier Tec and Doc, Paris, France. pp. 86–
105.
Coenye, T., Falsen, E., Vancanneyt, M., Hoste, B., Govan, J.R.W.,
Kersters, K., and Vandamme, P. 1999. Classification of Alcali-
genes faecalis-like isolates from the environment and human
clinical samples as Ralstonia gilardii sp. nov. Int. J. Syst.
Bacteriol. 49(2): 405–413. doi:10.1099/00207713-49-2-405.
PMID:10319461.
Deetae, P., Spinnler, H.E., Bonnarme, P., and Helinck, S. 2009a.
Growth and aroma contribution of Microbacterium foliorum,
Proteus vulgaris and Psychrobacter sp. during ripening in a cheese
Published by NRC Research Press
 Larpin-Laborde et al.
model medium. Appl. Microbiol. Biotechnol. 82(1): 169–177.
doi:10.1007/s00253-008-1805-7. PMID:19083231.
Deetae, P., Mounier, J., Bonnarme, P., Spinnler, H.E., Irlinger, F., and
Helinck, S. 2009b. Effects of Proteus vulgaris growth on the
establishment of a cheese microbial community and on the
production of volatile aroma compounds in a model cheese. J.
Appl. Microbiol. 107(4): 1404–1413. doi:10.1111/j.1365-2672.
2009.04315.x. PMID:19426267.
Depouilly, A., Dufrene, É., Beuvier, É., and Berthier, F. 2004.
Genotypic characterisation of the dynamics of the lactic acid
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14
bacterial population of Comté cheese. Lait, 84(1–2): 155–167.
doi:10.1051/lait:2003036.
Duthoit, F., Godon, J.J., and Montel, M.C. 2003. Bacterial
community dynamics during production of registered designation
of origin Salers cheese as evaluated by 16S rRNA gene single-
strand conformation polymorphism analysis. Appl. Environ.
Microbiol. 69(7): 3840–3848. doi:10.1128/AEM.69.7.3840-3848.
2003. PMID:12839752.
Duthoit, F., Tessier, L., and Montel, M.C. 2005. Diversity, dynamics
and activity of bacterial populations in ‘Registered Designation of
Origin’ Salers cheese by single-strand conformation polymorph-
ism analysis of 16S rRNA genes. J. Appl. Microbiol. 98(5): 1198–
1208. doi:10.1111/j.1365-2672.2005.02575.x. PMID:15836490.
Eliskases-Lechner, F., and Ginzinger, W. 1995a. The bacterial flora
of surface ripened cheese with special regard to coryneforms. Lait,
75(6): 571–584. doi:10.1051/lait:1995644.
Eliskases-Lechner, F., and Ginzinger, W. 1995b. The yeast flora of
For personal use only.
surface ripened cheese. Milchwissenschaft, 50: 458–462.
Feurer, C., Vallaeys, T., Corrieu, G., and Irlinger, F. 2004. Does
smearing inoculum reflect the bacterial composition of the smear
at the end of the ripening of a French soft, red-smear cheese? J.
Dairy Sci. 87(10): 3189–3197. doi:10.3168/jds.S0022-0302(04)
73454-2. PMID:15377597.
Fontana, C., Cappa, F., Rebecchi, A., and Cocconcelli, P.S. 2010.
Surface microbiota analysis of Taleggio, Gorgonzola, Casera,
Scimudin and Formaggio di Fossa Italian cheeses. Int. J. Food
Microbiol. 138(3): 205–211. doi:10.1016/j.ijfoodmicro.2010.01.
017. PMID:20167385.
Gente, S., Desmasures, N., Panoff, J.M., and Guéguen, M. 2002.
Genetic diversity among Geotrichum candidum strains from
various substrates studied using RAM and RAPD-PCR. J. Appl.
Microbiol. 92(3): 491–501. doi:10.1046/j.1365-2672.2002.01553.x.
PMID:11872125.
Gevers, D., Huys, G., and Swings, J. 2001. Applicability of rep-PCR
fingerprinting for identification of Lactobacillus species. FEMS
Microbiol. Lett. 205(1): 31–36. doi:10.1111/j.1574-6968.2001.
tb10921.x. PMID:11728712.
Goerges, S., Mounier, J., Rea, M.C., Gelsomino, R., Heise, V.,
Beduhn, R., et al. 2008. Commercial ripening starter microorgan-
isms inoculated into cheese milk do not successfully establish
themselves in the resident microbial ripening consortia of a South
German red smear cheese. Appl. Environ. Microbiol. 74(7): 2210–
2217. doi:10.1128/AEM.01663-07. PMID:18281427.
Grimont, P.A.D., Vancanneyt, M., Lefevre, M., Vandemeulebroecke,
K., Vauterin, L., Brosch, R., et al. 1996. Ability of Biolog and
Biotype-100 systems to reveal the taxonomic diversity of the
pseudomonads. Syst. Appl. Microbiol. 19: 510–527.
Guillamón, J.M., Sabaté, J., Barrio, E., Cano, J., and Querol, A. 1998.
Rapid identification of wine yeast species based on RFLP analysis
of the ribosomal internal transcribed spacer (ITS) region. Arch.
Microbiol. 169(5): 387–392. doi:10.1007/s002030050587. PMID:
9560418.
Henri-Dubernet, S., Desmasures, N., and Guéguen, M. 2008.
Diversity and dynamics of lactobacilli populations during ripening
659
of RDO Camembert cheese. Can. J. Microbiol. 54(3): 218–228.
doi:10.1139/W07-137. PMID:18388993.
Jay, J.M., Loessner, M.J., and Golden, D.A. 2005. Modern food
microbiology. 7th ed. Springer Science, New York, USA.
Kagkli, D.M., Bonnarme, P., Neuveglise, C., Cogan, T.M., and
Casaregola, S. 2006. L-Methionine degradation pathway in
Kluyveromyces lactis: identification and functional analysis of
the genes encoding L-methionine aminotransferase. Appl. Environ.
Microbiol. 72(5): 3330–3335. doi:10.1128/AEM.72.5.3330-3335.
2006. PMID:16672474.
Kümmerle, M., Scherer, S., and Seiler, H. 1998. Rapid and reliable
identification of food-borne yeasts by Fourier-transform infrared
spectroscopy. Appl. Environ. Microbiol. 64(6): 2207–2214.
PMID:9603836.
Larpin, S., Mondoloni, C., Goerges, S., Vernoux, J.-P., Guéguen, M.,
and Desmasures, N. 2006. Geotrichum candidum dominates in
yeast population dynamics in Livarot, a French red-smear cheese.
FEMS Yeast Res. 6(8): 1243–1253. doi:10.1111/j.1567-1364.
2006.00127.x. PMID:17156021.
Maoz, A., Mayr, R., and Scherer, S. 2003. Temporal stability and
biodiversity of two complex antilisterial cheese-ripening microbial
consortia. Appl. Environ. Microbiol. 69(7): 4012–4018. doi:10.
1128/AEM.69.7.4012-4018.2003. PMID:12839776.
Morales, P., Feliu, I., Fernández-García, E., and Nuñez, M. 2004.
Volatile compounds produced in cheese by Enterobacteriaceae
strains of dairy origin. J. Food Prot. 67(3): 567–573. PMID:
15035375.
Mounier, J., Monnet, C., Jacques, N., Antoinette, A., and Irlinger, F.
2009. Assessment of the microbial diversity at the surface of
Livarot cheese using culture-dependent and independent ap-
proaches. Int. J. Food Microbiol. 133(1-2): 31–37. doi:10.1016/j.
ijfoodmicro.2009.04.020. PMID:19481828.
Mounier, J., Le Blay, G., Vasseur, V., Le Floch, G., Jany, J., and
Barbier, G. 2010. Application of denaturing high-performance
liquid chromatography (DHPLC) for yeasts identification in red
smear cheese surfaces. Lett. Appl. Microbiol. 51(1): 18–23.
PMID:20477955.
Neugebauer, K.A., and Gilliland, S.E. 2005. Antagonistic action of
Lactobacillus delbrueckii ssp. lactis RM2-5 toward spoilage
organisms in Cottage cheese. J. Dairy Sci. 88(4): 1335–1341.
doi:10.3168/jds.S0022-0302(05)72799-5. PMID:15778300.
O’Donnell, K. 1993. Fusarium and its near relatives. In The fungal
holomorph: mitotic, meiotic and pleomorphic speciation in fungal
systematics. Edited by D.R. Reynolds and J.W. Taylor. CAB
International, Wallingford, UK. pp. 225–233.
Pot, B., Vandamme, P., and Kersters, K. 1994. Analysis of
electrophoretic whole-organism protein fingerprints. In Chemical
methods in procaryotic systematics. Edited by M. Goodfellow and
A.G. O’Donnell. John Wiley and Sons, Ltd., Chichester, UK. pp.
493–521.
Rademaker, J.L.W., Peinhopf, M., Rijnen, L., Bockelmann, W., and
Noordman, W.H. 2005. The surface microflora dynamics of
bacterial smear-ripened Tilsit cheese determined by T-RFLP DNA
population fingerprint analysis. Int. Dairy J. 15(6–9): 785–794.
doi:10.1016/j.idairyj.2004.08.027.
Randazzo, C.L., Torriani, S., Akkermans, A.D.L., de Vos, W.M., and
Vaughan, E.E. 2002. Diversity, dynamics, and activity of bacterial
communities during production of an artisanal Sicilian cheese as
evaluated by 16S rRNA analysis. Appl. Environ. Microbiol. 68(4):
1882–1892. doi:10.1128/AEM.68.4.1882-1892.2002. PMID:
11916708.
Rea, M.C., Görges, S., Gelsomino, R., Brennan, N.M., Mounier, J.,
Vancanneyt, M., et al. 2007. Stability of the biodiversity of the
Published by NRC Research Press
 660
surface consortia of Gubbeen, a red-smear cheese. J. Dairy Sci. 90(5):
2200–2210. doi:10.3168/jds.2006-377. PMID:17430918.
Richard, J., and Gratadoux, J.J. 1984. Evolution de la flore
microbienne à la surface des Camemberts fabriqués avec du lait
cru. Lait, 64(645–646): 496–520. doi:10.1051/lait:1984645-64638.
Richard, J., and Zadi, H. 1983. Inventaire de la flore bactérienne
dominante des Camemberts fabriqués avec du lait cru. Lait, 63(623–
624): 25–42. doi:10.1051/lait:1983623-6243.
Rodríguez, L.A., Vivas, J., Gallardo, C.S., Acosta, F., Barbeyto, L.,
and Real, F. 1999. Identification of Hafnia alvei with the
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 12/26/14
Microscan WalkAway System. J. Clin. Microbiol. 37(12): 4186–
4188. PMID:10565961.
Roth, E., Miescher Schwenninger, S., Hasler, M., Eugster-Meier, E.,
and Lacroix, C. 2010. Population dynamics of two antilisterial
cheese surface consortia revealed by temporal temperature
gradient gel electrophoresis. BMC Microbiol. 10(1): 74. doi:10.
1186/1471-2180-10-74. PMID:20222967.
Sablé, S., Portrait, V., Gautier, V., Letellier, F., and Cottenceau, G.
1997. Microbiological changes in a soft raw goat’s milk cheese
during ripening. Enzyme Microb. Technol. 21(3): 212–220.
doi:10.1016/S0141-0229(97)00271-8.
Schmidt, V.S.J., Mayr, R., Wenning, M., Glöckner, J., Busse, H.J.,
and Scherer, S. 2009. Bavariicoccus seileri gen. nov., sp. nov.,
isolated from the surface and smear water of German red smear
soft cheese. Int. J. Syst. Evol. Microbiol. 59(10): 2437–2443.
doi:10.1099/ijs.0.006601-0. PMID:19622668.
Schubert, K., Ludwig, W., Springer, N., Kroppenstedt, R.M.,
For personal use only.
Accolas, J.P., and Fiedler, F. 1996. Two coryneform bacteria
isolated from the surface of French Gruyère and Beaufort cheese
are new species of the genus Brachybacterium: Brachybacterium
alimentarium sp. nov., and Brachybacterium tyrofermentans sp.
nov. Int. J. Syst. Bacteriol. 46(1): 81–87. doi:10.1099/00207713-
46-1-81. PMID:8573524.
Can. J. Microbiol. Vol. 57, 2011
Skovmose, E. 2003. Cheese flavour options. Available from http://
www.danlac.com/news/cheese-flavour-options [accessed 20 July
2010].
Tessier, L., Bugaud, C., and Buchin, S. 2003. Tentative hierarchization
of the influence of milk properties and technological practices on
rheological properties of Abondance cheese. J. Dairy Res. 70(4):
403–411. doi:10.1017/S0022029903006228. PMID:14649411.
Tornadijo, E., Fresno, J.M., Carballo, J., and Martin-Sarmiento, R.
1993. Study of Enterobacteriaceae throughout the manufacturing
and ripening of hard goat’s cheese. J. Appl. Bacteriol. 75(3): 240–
246. PMID:8244902.
Valdés-Stauber, N., Scherer, S., and Seiler, H. 1997. Identification of
yeasts and coryneform bacteria from the surface microflora of
brick cheeses. Int. J. Food Microbiol. 34(2): 115–129. doi:10.
1016/S0168-1605(96)01171-3. PMID:9039559.
Vancanneyt, M., Witt, S., Abraham, W.R., Kersters, K., and
Fredrickson, H.L. 1996. Fatty acid content in whole-cell hydro-
lysates and phospholipid fractions of Pseudomonas: a taxonomic
evaluation. Syst. Appl. Microbiol. 19: 528–540.
Vasdinyei, R., and Deak, T. 2003. Characterization of yeast isolates
originating from Hungarian dairy products using traditional and
molecular identification techniques. Int. J. Food Microbiol. 86(1–2):
123–130. doi:10.1016/S0168-1605(03)00251-4. PMID:12892927.
Versalovic, J., Schneider, M., de Bruijn, F.J., and Lupski, J.R. 1994.
Genomic fingerprinting of bacteria using repetitive sequence-
based polymerase chain reaction. Methods Cell Biol. 5: 25–40.
Wyder, M.T., and Puhan, Z. 1999. Investigation in the yeast flora in
smear ripened cheeses. Milchwissenschaft, 54: 330–333.
Yarrow, D. 1998. Methods for the isolation, maintenance and
identification of yeasts. In The yeasts: a taxonomic study. 4th
ed. Edited by C.P. Kurtzman and J.W. Fell. Elsevier Science B.V.,
Amsterdam, the Netherlands. pp. 77–100.
Published by NRC Research Press