International Dairy Journal 27 (2012) 13e21

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International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Quantitative and qualitative microbial analysis of raw milk reveals substantial

diversity inuenced by herd management practices
Adrien Mallet a,1, Micheline Guguen a, Franois Kauffmann b, Christophe Chesneau b, Andr Sesbou b,
Nathalie Desmasures a, *,1
a
b

Unit des Microorganismes dIntrt Laitier et Alimentaire (MILA), EA 3213, IFR 146 ICORE, Universit de Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen Cedex, France
Laboratoire de Mathmatiques Nicolas Oresme, UMR CNRS 6139, UFR des Sciences, Campus 2, Universit de Caen Basse-Normandie, 14032 Caen Cedex, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 24 February 2012
Received in revised form
1 June 2012
Accepted 17 July 2012

The quantitative (n 260) and qualitative (n 24) microbial compositions of raw milk samples were
assessed using culture analyses and a molecular inventory based on the rDNA sequences of 1697 isolates.
A statistical analysis was also performed to correlate farm practices with the microbial populations
present in the milk samples. Ripening bacteria and Pseudomonas were the dominant groups. Milk
samples were distinguishable by the different proportions of their microbial groups, which varied among
producers. Individual practices inuenced the levels of technologically important microorganisms, for
example, pre-dipping of teats reduced levels of lactococci. Standard plate counts correlated with the
levels of Gram-negative bacteria, Pseudomonas, lactococci and ripening bacteria, depending on the
milking season. Lactic acid bacteria and yeasts, which were poorly represented, nevertheless seemed to
constitute a resilient basal ora. Qualitative species diversity was considerable, with more than 100
bacterial species identied, dominated by Gram-positive bacteria, notably staphylococci and
Corynebacterium.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
The microbiological quality of raw milk is central to a number of
public health, socio-economical and technological issues. In the
interest of public health, and to prevent food-borne diseases, many
efforts have been made in industrialised countries to improve the
sanitary quality of raw milk. Such advancements have included
maintaining freshly produced milk in cold storage, the development of udder-cleaning and teat-disinfecting procedures, and
monitoring the health status of dairy herds. Consequently, the
microbial content of raw milk has decreased over the past several
decades (Beuvier & Buchin, 2004). As expected, enhanced safety
measures have reduced the levels of undesirable microorganisms
(spoilage ora and potential pathogens). However, the entire
natural microbiota spectrum has also been affected (Michel,

* Corresponding author. Tel.: 33 231 56 55 22.

E-mail address: nathalie.desmasures@unicaen.fr (N. Desmasures).
1
Present address: E.A. 4651 Aliments Bioprocds Toxicologie Environnements
(ABTE), Universit de Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen
Cedex, France.
0958-6946/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.idairyj.2012.07.009

Hauwuy, & Chamba, 2001), such that some microorganisms or

their phenotypic traits are no longer found in raw milk in industrialised countries (Corroler, Mangin, Desmasures, & Guguen,
1998; Panoff, Desmasures, Thammavongs, & Guguen, 2002;
Salama, Musaja-Jeknic, Sandine, & Giovannoni, 1995).
Of all the ripened cheese produced in France in 2010, 14.5% was
made with raw milk (180,000 tons). Thus, raw milk cheeses make
a substantial contribution to the French cheese market
(Anonymous, 2010). At the European level, it is estimated that farm
house-made cheeses (made mainly from raw milk) generate
signicant incomes for thousands of farming households (Pujol,
1997). For these products, the sanitary quality of raw milk is
essential; however, the natural microora are also of primary
technological importance for the making of cheese.
Raw milk microora contribute greatly to the sensory characteristics of raw milk cheeses in terms of the particular avours and
aromas they generate (Awad, 2006; Bouton, Buchin, Duboz, Pochet,
& Beuvier, 2009; Burbank & Qian, 2008; Callon, Berdagu, Dufour, &
Montel, 2005; Chambers, Esteve, & Retiveau, 2010). The natural
ora of raw milk and raw milk cheeses may also contribute to food
biopreservation (Callon, Saubusse, Didienne, Buchin, & Montel,
2011; Eppert, Valdes-Stauber, Gotz, Busse, & Scherer, 1997; Gay &
Amgar, 2005; Imran, Desmasures, & Vernoux, 2010; Retureau,

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A. Mallet et al. / International Dairy Journal 27 (2012) 13e21

Callon, Didienne, & Montel, 2010). Thus, the microbial diversity of

raw milk directly impacts the quality of dairy products, including
raw milk cheeses (natural microora), but it also attracts interest as
a source of indigenous cheese starters.
Given these considerations, the preservation of raw milk
microbial diversity, and perhaps its reconstruction, may be necessary in some production areas to sustain the consistency of traditional dairy products and to prevent pathogen proliferation.
Although several studies have focused on the quantitative assessment of various microbial groups in raw milk and on the evolution
of microbial communities during lactation periods (Callon et al.,
2007; Dalmasso, Prestoz, Rigobello, & Demarigny, 2008;
Desmasures, Bazin, & Guguen, 1997; Michel et al., 2001; Tormo,
Agabriel, Lopez, Lekhal, & Roques, 2011), few studies have
addressed the qualitative diversity of raw milk microora.
Farm practices are known to impact the presence and levels of
spoilage and pathogenic microorganisms (Latorre et al., 2009;
Magnusson, Christiansson, & Svensson, 2007; Rasmussen, Galton, &
Petersson, 1991), but how global farm practices impact the microbial diversity of raw milk has not been well studied. Michel et al.
(2001) showed that milking hygiene and disinfection practices
inuence the microoral composition of raw milk, notably the
proportions of different groups of microorganisms. Verdier-Metz,
Michel, Delbs, and Montel (2009) used Single Strand Conformation Polymorphism analysis to show that efcient but less intensive
hygiene practices preserve higher bacterial diversities in raw milk
and contribute to its hygienic status.
The aim of the present study was to investigate the quantitative
and qualitative microbial diversity of the raw milk produced in the
Basse-Normandie region in France and to explore the relationships
between the quantitative microbial proles of raw milk and farm
practices.

2. Materials and methods

2.1. Collection of milk samples
Samples of bulk-tank raw milk from 130 dairy farms located in
various parts of the Basse-Normandie region of France were
collected in a winter period (JanuaryeFebruary 2008), when
dairy cows were housed indoors, and in a spring period
(MayeJune 2008), when the cows were generally kept outdoors
grazing on pastures. Raw milk was kept at 2e4  C on the farm, and
all samples were collected from the bulk tanks after agitation and
just before raw milk was transported to the factory. Depending on
the factory for which the milk was destined, bulk-tanks contained
the product of two (n 52) or four (n 208) successive milkings.
Approximately 150 mL of raw milk was collected aseptically in
sterile plastic containers. Samples were transported to the laboratory in refrigerated packs and kept at 4  C until microbial analysis,
which was performed on the same day.

2.2. Survey of farm practices

A survey, designed to study the relationships between counts of
microbial groups in raw milk and production practices, was performed at 130 farms. The survey was conducted on-farm via
personal interviews with the dairy farmers and collected information on the farm and its herd structure, the feeding practices,
and the milking and hygiene procedures for the two sampling
periods. Thirty-six qualitative variables and one quantitative variable were described for the farm practices and were retained for
statistical analysis.

2.3. Microbiological analyses

2.3.1. Quantitative analysis
Standard plate counts (SPC) were determined with plate count
agar (containing 1 g L1 skim milk). Plates were incubated at 30  C
for 72 h. Presumptive lactococci counts were obtained with plate
count agar containing bromocresol purple (BCP) as previously
described (Desmasures et al., 1997). Presumptive lactobacilli counts
were assessed using Rogosa agar incubated at 30  C for 72 h in
anaerobic conditions. Presumptive leuconostocs were counted
using Nichels and Leesment modied medium (with X-gal) incubated at 25  C for 96 h (Vogensen et al., 1987). Cheese ripening
bacteria medium (CRBM) (Denis, Guguen, Henry, & Levert, 2001)
was used to count presumptive ripening bacteria (staphylococci
and coryneform bacteria) after incubation at 25  C for 120 h. For
presumptive Gram-negative bacteria, plate count agar with crystal
violet (PCAI) was incubated at 30  C for 48 h as previously described
(Callon et al., 2007). Presumptive Pseudomonas counts were
determined with cetriminefucidinecephaloridin agar (CFC) incubated at 25  C for 48 h. Yeast and mould counts were obtained using
oxytetracycline glucose agar (OGA) after incubation at 25  C for
72 h.
2.3.2. Qualitative analysis
A spiral plater (Eddy Jet, IUL Instruments, Barcelona, Spain) was
used to inoculate milk samples (50 mL) onto three Petri dishes
containing tryptone soya agar with 6 g L1 yeast extract, a nal
concentration of 20 g L1 glucose tryptone soya agar yeast extract,
""(TSAYE) and a pH of 7.0  0.2. The three plates were incubated
at either 20  C for 72 h, 30  C for 48 h, or 40  C for 24 h. Two Petri
dishes containing TSAYE at pH 5.5 and pH 9.0, were each inoculated with 50 mL milk samples and incubated at 30  C for 48 h.
Twelve farms (24 milk samples) were selected based on the
various quantitative microbiological proles of their raw milk (i.e.,
high counts of technological microora, notably LAB, and/or Gramnegative bacteria) and to represent the various production practices (i.e., use/no use of pre and post-dipping of teats, type of
milking machine). For each of these milk samples, 10e15 bacterial
colonies representing the different morphotypes were selected on
each of the ve TSAYE plates, and all the yeasts colonies (typically
1e5) were selected on OGA plates. Subcultures of isolates were
done on the same media and pure cultures were kept at 80  C as
glycerol stocks. A total of 1562 bacterial isolates and 135 yeast
isolates were recovered.
2.4. Bacterial DNA extraction and PCR amplication of 16S rRNA
genes
DNA from bacterial isolates was extracted from 1 mL overnight
cultures using a Wizard Genomic DNA purication Kit (Promega,
Madison, WI, USA), following the manufacturers instructions.
Ribosomal 16S rRNA genes (w1500 bp) were amplied using the
universal primers W02 (50 -GNTACCTTGTTACGACTT-30 ) and W18
(50 -GAGTTTGATCMTGGCTCAG-30 ) as previously described (Callon
et al., 2007). Amplication was performed with a Veriti 96-Well
Thermal Cycler System (Applied Biosystems, Foster city, CA, USA).
Samples were incubated for 5 min at 94  C followed by 25 cycles at
the following conditions: 1 min at 94  C; 1 min at 50  C; 1 min at
72  C and a nal extension for 7 min at 72  C.
2.5. Partial 16S rRNA gene sequencing and bacteria identication
Bacterial 16S rDNA amplimers were sent to Genoscreen (Lille,
France) for sequencing using the W02 and/or W18 primers. The
partial sequences were compared to sequences available in the

A. Mallet et al. / International Dairy Journal 27 (2012) 13e21

15

mean for the 260 milk samples was 4.9  103 cfu mL1. Samples
having SPC <104 cfu mL1 accounted for 76.8% of total samples
collected in winter and 76.2% of those collected in spring. In addition, 5.4% of the winter and 10.0% of the spring raw milk samples
had SPC <103 cfu mL1 (paucimicrobial) (Fig. 1).
All groups of microorganisms exhibited wide ranges of counts
among the samples. Presumptive ripening bacteria and presumptive Pseudomonas (among Gram-negative bacteria) were predominant in the majority of samples, with geometric means of
6.0  102 cfu mL1 and 5.3  102 cfu mL1, respectively.
The proportion of samples with counts that contained greater
than 10% ripening bacteria decreased with increasing SPC (Fig. 2).
For paucimicrobial milk, ripening bacteria represented 58.8% and
41.9% of the microorganism found in the winter and spring samples,
respectively, whereas for all milk samples, these bacteria were
present at levels of 37.3% and 30.2% in the winter and spring
samples, respectively (data not shown).

National Center for Biotechnology Information (NCBI) Genbank

database using the BLASTN program or in the EBI (European Bioinformatics Institute) databases if denitive identication (>95%
sequence homology) could not be made using the GenBank
database.
2.6. Yeast identication
Yeasts isolates were sent to Genoscreen for direct sequencing
targeting the D1/D2 domain of the 26S rRNA gene.
2.7. Statistical analysis
A linear model was used to study the target variables (logarithm
to base 10 of each microbial group count) as functions of the 37
explanatory variables retained from the survey of farm practices.
The total number of degrees of freedom for the main effect model
was 56. A total of 108 farms were studied (among the 130 farms, 22
had missing microbial data and were not included). To select
a model, a minimal subset of explanatory variables was chosen
and an optimal model containing the Bayesian information
criteria (BIC) (Hastie, Tibshirani, & Friedman, 2009) that depended
on these selected variables was searched (Hofmann, Gatu, &
Kontoghiorghes, 2007).
First, a subset of admissible explanatory variables, that could
explain the target variable, was sought. An explanatory variable
was retained when the target variable depended on this explanatory variable, with a p-value lower than 20%. Variables that correlated to other variables (more than 75%) were discarded. Several
methods were used to model target variables with the selected set
of explanatory variables (i.e., step-by-step algorithms for the linear
model, random forest models, and recursive partitioning). Ten
variables were nally selected because they appeared most
frequently as signicant variables in the different models.
Second, an optimal linear model for the BIC was constructed for
the main effects that depended on these minimal subsets of
variables.
Finally, the selected model was validated. Particular attention was
paid to explanatory variables that were not included in the selected
subset of variables but were near one of the explanatory variables.
This analysis was carried out with the statistical software R
(Ihaka & Gentleman, 1996) with caret, leaps and bestglm packages.

3.1.2. Correlations between microbial groups

Fig. 3 is a graphic representation of the principal component
analysis (PCA) based on a covariance matrix for each season and
shows the position of milk samples on the factorial axes 1 and 2.
The two principal axes may be interpreted in the same way for the
two periods. The rst axis, which represents 42% of the total variability in the winter samples and 47% of the total variability in the
summer samples, classies milk samples according to decreasing
microbial counts, and so represents an index of microbial load. The
second axis, which represents 19% of the total variability in the
winter samples and 18% of the total variability in the summer
samples, classies milk samples according to the decreasing levels
of the microbial groups of technological interest (presumptive
lactococci, presumptive lactobacilli, presumptive leuconostocs and
yeasts) and the increasing levels of presumptive Gram-negative
bacteria and Pseudomonas.
Table 2 shows the correlation coefcients between counts of
microbial groups and between the sampling periods. The overall
microbial levels of raw milk (SPC) correlated with the levels of
Gram-negative bacteria and presumptive lactococci in winter. In
spring, the overall microbial levels were mainly correlated with the
levels of ripening bacteria, and secondly with the levels of
presumptive Pseudomonas, Gram-negative bacteria and lactococci.
Presumptive Gram-negative bacteria and Pseudomonas had the
highest correlation coefcients (0.71 in winter samples and 0.68 in
spring samples). There were also positive correlations between
presumptive yeasts and lactococci (0.53 and 0.43), presumptive
yeasts and lactobacilli (0.48 and 0.60) and between presumptive
lactobacilli and leuconostocs (0.45 and 0.60) in the winter and
spring samples, respectively. High winter/spring autocorrelations
were found for presumptive lactobacilli counts (0.62), presumptive
leuconostocs counts (0.58) and yeast counts (0.54). The lowest

3. Results
3.1. Quantitative results
3.1.1. Microbiological proles of milk samples
The microbial counts in the raw milk samples collected during
the two sampling periods are shown in Table 1. The SPC geometric

Table 1
Mean counts for nine microbial groups in 260 raw milk samples (130 in winter and 130 in spring).a
Microbial groups

Winter

Spring
1

Geometric mean (cfu mL

Standard plate count
Presumptive lactococci
Presumptive lactobacilli
Presumptive leuconostocs
Presumptive ripening bacteria
Presumptive Gram-negative bacteria
Presumptive Pseudomonas
Presumptive yeasts
Presumptive moulds
a

5.6
8.2
1.0
9.8
6.8
7.9
4.7
7.4
2.8











10
101
102
101
102
102
102
101
100

Min

Geometric mean (cfu mL1)

Max
3

6.8  10
1
1
1
2.0  101
5
<10 (n 8)
2
<1 (n 27)

2.0
1.4
3.2
2.5
1.1
1.3
1.6
3.0
0.5











10
104
104
103
105
105
105
104
101

4.1
7.4
2.0
1.2
5.3
7.2
5.9
9.5
2.4











103
101
102
102
102
102
102
101
100

Min

Max

3.2  103
<1 (n 3)
<1 (n 1)
1
1.0  101
<10 (n 2)
1.5  101
1
<1 (n 45)

3.1
2.5
2.7
5.0
1.2
3.8
3.9
1.4
4.5

Data <1 and <10 were changed to numerical data as 1 and 10, respectively, to calculate the geometric mean; n, number of samples below threshold.











105
104
104
103
105
105
105
104
102

16

A. Mallet et al. / International Dairy Journal 27 (2012) 13e21

Percentage of raw milk samples

60

50.0
50

43.4

40
30

24.0

20

16.2
10.0

10

12.4 12.3

7.8
6.2

5.4

7.0
5.4

0
1000

1001-5000

5001-10000

10001-20000

20001-50000

>50000

Standard plate counts (cfu mL-1)

Fig. 1. Distribution of standard plate counts of raw milk samples in winter (black bars) and spring (grey bars).

winter/spring autocorrelation coefcients were for presumptive

ripening bacteria (0.17) and Gram-negative bacteria (0.25).
3.2. Farm practices
Herds of the 130 farms studied were composed entirely of either
Prim Holstein cows (23.8%), Normandy cows (35.4%) or were a mix
of these two breeds (40.8%). For most farms (80%), milk was
collected by the factory every two days (four milkings in the bulktank). The most frequently used type of milking machine was the
looped milkline (80.7%). Milking hygiene practices consisted of predipping with iodine, polyhexamethylene biguanide, chlorine,
hydrogen peroxide or essential oil for 50% of all producers, whereas
post-dipping (chlorine, iodine or lactic acid) was used by almost all
producers in winter and spring (93.1% and 87.7%, respectively). For
further information, refer to the Supplementary data, Table S1.
3.3. Relation between microbial ora and farms practices

Percentage of raw milk samples

having 10% presumptive ripening
bacteria

Neither PCA (Fig. 3) nor multiple correspondence analysis (data

not shown) allowed the farms to be classied into specic groups
based on their microbiological prole, but rather they appeared to
form a continuum. An optimal log-linear model was used to
determine the inuence of farm practices on the levels of the
various microbial groups and detected several apparent tendencies

(Table 3). Samples obtained from four successive milkings exhibited higher SPC than samples from two successive milkings, as expected, but also had higher counts of presumptive lactococci,
lactobacilli and yeasts. The capacity of the tank and the herd size
inuenced microbial groups. Gram-negative bacteria counts and
mould counts were lower in milk obtained from large tanks
(>4000 L) than in milk taken from smaller tanks. Large herds were
associated with lower presumptive Pseudomonas and mould counts
than were smaller herds. Teat preparation practices negatively
inuenced the level of technological ora. Pre-dipping was
associated with lower levels of SPC, lactococci, yeasts and moulds
whereas post-dipping was associated with low levels of presumed
ripening bacteria and leuconostocs. The counts of Gram-negative
bacteria, presumed Pseudomonas and ripening bacteria associated
with Normandy cows were higher than those associated with Prim
Holstein herds (mixed herds composed of both breeds had intermediate levels for these three ora). Finally, counts of lactobacilli
and leuconostocs associated with dead-end milklines were higher
than those associated with looped milklines.
3.4. Qualitative microbial diversity in raw milk samples from 12
selected dairy farms
From the 24 samples collected during the two seasonal periods
from the 12 selected farms, 1562 bacteria isolates and 135 yeasts

100
90

85
74

80
70
60

50

50

35

33

10001-20000

>20000

40
30
20
10
0
<1000

1000-5000

5001-10000

Standard plate counts (cfu

mL-1)

Fig. 2. Distribution according to the standard plate count of raw milk samples containing 10% presumed ripening bacteria.

A. Mallet et al. / International Dairy Journal 27 (2012) 13e21

17

(45.8%). Several species rarely described in raw milk were also

identied: Clavibacter michiganensis, Comamonas testosteroni,
Enhydrobacter aerosaccus, Frigoribacterium sp., Ochrobactrum
anthropi and Renibacterium salmoninarum.
Similar numbers of bacterial species were found in each season:
85 in winter and 74 in spring. Gram-positive bacteria accounted for
70.6% of the bacteria species in the winter season, compared with
60.5% in spring.
The number of bacterial species recovered depended on the
temperature (20  C, 30  C, 40  C at pH 7.0) and on the pH (pH 5.5
and 9.0 at 30  C) of the culture conditions. The highest number of
different species (65) was obtained at 30  C/pH 7.0, 62 were obtained at 20  C/pH 7.0 and 56 at 30  C/pH 9.0. Forty-seven species
were recovered at 30  C/pH 5.5 and 36 species at 40  C/pH 7.0.
Forty-six species were identied in only one condition. Eleven of
these were isolated at 20  C/pH 7.0, 13 at 30  C/pH 7.0, seven each at
40  C/pH 7.0 and 30  C/pH 5.5, and eight species at 30  C/pH 9.0. For
example, Dietzia sp., Leucobacter sp. and Renibacterium sp. were
only recovered from 30  C/pH 9.0; Enhydrobacter sp., Gordonia
bronchialis, Plantibacter sp., Sanguibacter sp., and Tessaracoccus sp.
from 20  C/pH 7.0; and Lysinibacillus and Micrococcus luteus
from 30  C/pH 5.5. The Brevibacillus genus was only observed at
40  C/pH 7.0.
The two most prevalent yeast genera were Kluyveromyces,
which was isolated from 66.7% to 50.0% of the raw milk samples
collected in winter and spring, respectively, and Candida, which
was isolated from 41.7% to 33.3% of winter and spring samples,
respectively.

4. Discussion
4.1. Variability of quantitative microbiological prole of raw milk

Fig. 3. Principal component analysis: Representation of the variables and milk in

winter (A) and in spring (B).

isolates were cultured. The bacterial isolates represented 53

different genera and 112 species whereas the yeast isolates represented eight genera and 17 species. These are detailed in
Supplementary data, Tables S2 and S3 respectively.
Among the 112 bacterial species, about two-thirds corresponded to Gram-positive bacteria (66.1%). Of the ten species
detected with the highest frequencies among the milk samples,
eight were Gram-positive: Staphylococcus haemolyticus (87.5%),
Staphylococcus aureus (79.2%), Lactococcus lactis (70.8%), Enterococcus faecalis (66.7%), Staphylococcus saprophyticus (62.5%),
Staphylococcus hominis (58.3%), Staphylococcus epidermidis (50.0%)
and Kocuria rhizophila (50.0%); and two were Gram-negative:
Stenotrophomonas maltophilia (50.0%) and Acinetobacter johnsonii

The quantication of groups of microorganisms revealed the

hygienic qualities of the raw milk samples. SPCs are similar to those
found in other areas in France such as Savoie (5.8  103 cfu mL1;
Michel et al., 2001) and Franche-Comt (7.2  103 cfu mL1; Bouton,
Tessier, Guyot, & Beuvier, 2005), and in other European countries
such as Denmark (7.4  103 cfu mL1; Holm, Jepsen, Larsen, &
Jespersen, 2004).
Fifteen years ago, we used a similar approach to assess the
microbiological proles of raw milk from the same area
(Desmasures et al., 1997). The lactobacilli, yeast and Pseudomonas
counts in both studies were nearly identical; however, the mean
level of acidifying lactococci was found to be one order of magnitude lower in the present study. This result suggests that lactococci
are more sensitive to, or affected by, the evolution of farm practices
than the other three ora.
Although the mean SPC were similar for samples collected in the
two sampling periods, high variability was observed among the
counts for individual farms. Despite their low SPC, milk samples
exhibited substantial diversity in their microbial compositions,
notably in terms of the variable proportions of their positive ora
[ripening bacteria, lactic acid bacteria (LAB) and yeasts] and Gramnegative bacteria, as has been reported previously (Michel et al.,
2001). In some cases, intra-farm variability was also observed
between the two sampling periods, but in most cases the microbial
counts were similar for the two seasons.
Counts of presumptive lactobacilli appeared to be two-fold
higher in the spring sampling period than in the winter samples.
These differences may be due to seasonal variation. Similar levels of
seasonal differences were also observed for Lactobacillus in raw
milk used for Comt cheese (Bouton et al., 2005) and in raw ewes
milk (Salmeron, de Vega, Prez-Elortondo, Albisu, & Barrn, 2002),

18

A. Mallet et al. / International Dairy Journal 27 (2012) 13e21

Table 2
Pearson correlation coefcients established between raw milk microbial ora in winter and in spring.a
Winter
SPC
Winter
SPC
Lc
Lb
Leu
CRB
G neg
Ps
Y
Spring
SPC
Lc
Lb
Leu
CRB
G neg
Ps
Y

Spring
Lc

Lb

Leu

CRB

G neg

Ps

1.00
0.49
0.31
0.06
0.35
0.58
0.41
0.41

1.00
0.47
0.20
0.09
0.30
0.30
0.53

1.00
0.45
0.00
0.11
0.07
0.48

1.00
0.11
0.16
0.20
0.39

1.00
0.20
0.18
0.23

1.00
0.71
0.36

1.00
0.44

1.00

0.54
0.20
0.22
0.05
0.32
0.14
0.28
0.31

0.29
0.34
0.30
0.11
0.17
0.01
0.10
0.24

0.25
0.33
0.62
0.31
0.24
0.05
0.09
0.42

0.11
0.26
0.57
0.58
0.15
0.19
0.13
0.29

0.07
0.18
0.09
0.15
0.17
0.02
0.01
0.04

0.27
0.11
0.19
0.08
0.17
0.25
0.38
0.09

0.17
0.05
0.15
0.00
0.12
0.15
0.39
0.16

0.22
0.18
0.42
0.33
0.21
0.00
0.20
0.54

SPC

Lc

Lb

Leu

CRB

G neg

Ps

1.00
0.58
0.35
0.18
0.69
0.60
0.63
0.46

1.00
0.51
0.24
0.46
0.39
0.40
0.43

1.00
0.60
0.37
0.22
0.24
0.60

1.00
0.12
0.21
0.20
0.27

1.00
0.48
0.37
0.43

1.00
0.68
0.16

1.00
0.29

1.00

Abbreviations: SPC, standard plate counts; Lc, presumptive lactococci; Lb, presumptive lactobacilli; Leu, presumptive leuconostocs; CRB, presumptive cheese ripening
bacteria; G neg, presumptive Gram-negative bacteria; Ps, presumptive Pseudomonas; Y, presumptive yeasts.

suggesting that some seasonal factors may inuence counts of

lactobacilli.
4.2. Relationships between microbial groups
Merging results from the PCA and the correlation analysis, the
main microbial ora can be separated into three ecological groups:
Gram-negative bacteria (including Pseudomonas); ripening
bacteria; and LAB and yeasts. Indeed, it appears that levels of these
groups vary differently from each other. This suggests that they do
not share the same reservoirs and/or that they are inuenced by
different factors (probably linked to farm practices). Another
possibility is that the predominance of one group inhibits or
decreases the growth of the others during cold storage of milk.
The winter counts of presumptive lactococci, lactobacilli, leuconostocs and yeast were correlated with the spring ones. It is possible
that they constitute a resilient basal ora (as previously demonstrated for some farms by Desmasures & Guguen, 1997) that exists

in a small number of stable reservoirs. Although levels of lactococci

were quite low, they contributed to the SPC of both sampling periods
(Fig. 4). All Gram-negative bacteria also contributed to SPC, especially in winter, whereas SPC principally consisted of Pseudomonas in
the spring samples. Gram-negative bacteria and Pseudomonas
correlated well with each other, but did not autocorrelate between
the two sampling periods. These results suggest the existence of
a high number of potential reservoirs, with only some of them being
preferentially active at any given time, depending on the farms
practices, the season or on occasional irregular events or factors
(Piton & Richard, 1985). In the same way, levels of ripening bacteria
did not autocorrelate between seasons, but accounted for much of
the SPC of the spring samples and were among microbial groups
with the highest levels in milk from both periods. These results
suggest that these bacteria also have multiple reservoirs (Vacheyrou
et al., 2011; Verdier-Metz et al., 2012), which are probably different
from those of the Gram-negative bacteria and Pseudomonas or that
these bacteria are not sensitive to the same factors.

Table 3
Farm practices having signicant effects on microbial counts.a
Practices

Type

Compared with

Inuence on

Estimated effects (log)b

Season

Number of milkings

Tank capacity

>4000 L

<2000 L

Herd size

>60

<30

Teat preparation

Pre-dipping

No

Post-dipping

No

Cow breed

Normandy

Holstein

Milkline conguration

Dead-ended

Looped

SPC
Lactococci
Lactobacilli
Yeasts
Gram-negative
Moulds
Pseudomonas
Moulds
SPC
Lactococci
Yeasts
Moulds
CRB
Leuconostocs
Gram-negative
Pseudomonas
CRB
SPC
Lactobacilli
Leuconostocs

0.25*
0.69***
0.75***
0.41**
0.78***
0.44***/0.51**
1.16***
0.19*
0.27**
0.49**
0.29*
0.19*/0.35**
0.57**
0.31*
0.62**
0.54**
0.36**
0.28*
0.65**/0.46*
0.53**/0.38*

W
S
S
S
W
W/S
W
W
S
S
W
W/S
W
S
S
S
S
W
W/S
W/S

Abbreviations: W, Winter; S, Spring; degree of statistical difference: *p < 0.05; **p < 0.01; ***p < 0.001.
Interpretation: for example, for the number of milkings, the estimated effect of 0.25 is equivalent to an SPC increase of 0.25 log10 in the samples from four milkings (type of
practices) compared with the samples from two milkings in the winter season.
b

A. Mallet et al. / International Dairy Journal 27 (2012) 13e21

Fig. 4. Graphic representation of the hypothetical main relationships between raw

milk microbial groups in winter (A) and in spring (B) Simple black arrows represent the
correlation standard plate counts and the other presumptive microbial groups; double
arrows represent relationships between microbial groups; each small full square
represents one hypothetical reservoir. SPC, standard plate count; CRB, cheese ripening
bacteria; Ps, pseudomonas; G-, Gram-negative bacteria; Lc, lactococci; Leu, leuconostoc; Lb, lactobacilli; Y, yeasts.

The correlation between LAB levels and yeast levels probably

arises from the likelihood that these ora are inuenced by similar
conditions and practices in the farm environment, or because these
ora have a proximate origin source. The fact that some lactobacilli,
lactococci and yeasts grow on all three of the specic media used to
culture each group could also explain the correlation.
Globally, the different oras seem to correlate better with each
other in spring than in winter. This was especially true for those
that accounted for a high proportion of the SPC. The lower correlation in winter suggests that, may be due to indoor housing and to
the close proximity of faeces, there are more accidental contaminations than in spring.
4.3. Inuence of farm practices on microbial counts
The statistical analyses undertaken identied possible negative
and positive inuences of several farm practices. As the use of
a specic practice by producers is often due to the global
management system of the farm, it is often difcult to separate the
inuence of a single practice from the inuences of other practices
or groups of practices. For example, the inuences of tank capacity
and herd size (which were both associated with low levels of Gramnegative bacteria and moulds in raw milk if tank size was >4000 L
and herd size was >60 cows) are probably due to the overall large
size of these farms. Small farms and large farms are generally
operated differently, so it is possible that these results can be
explained by the globally enhanced hygiene practices of large

19

farms, which are generally more thorough than those of smaller

farms. Thus, even if some individual practices have substantial
impact on certain ora, in general, the larger farms tended to be
associated with lower overall microbial counts. An equivalent
argument can be made regarding the inuence of cow breed, as
small herds had higher proportions of Normandy cows.
Concerning teat care, pre-dipping and post-dipping practices
reduced the levels of several microbial groups in milk, but these
practices impact more the technologically important microbial
groups than the other groups. Certainly, mould levels were lower in
milk from farms that practiced teat-dipping, but these practices did
not appear to decreased the levels of Pseudomonas and Gramnegative bacteria in milk, even if they decrease the levels of these
microorganisms on teats (data not shown). Thus, the Pseudomonas
found in milk seemed not to mainly come from teats. The true
benets of these practices are unclear and the systematic application of pre- and post-dipping seems contraindicated for milk
destined for the manufacture of raw milk products. Higher levels of
cleanliness in the cows litter are likely to be more suitable than
teat-dipping for the production of raw milk products, and pre- and
post-dipping could be reserved for herds or cows with recurring
problems of contamination or in isolated cases (e.g., cows with
mastitis). Such examples of practices that inuence the levels of
technologically important bacteria in milk could be linked to the
observed decrease of Lactococcus (lactis) in raw milk over time
(Panoff et al., 2002; Salama et al., 1995) and to the likely high
sensitivity of lactococci to hygiene practices, as suggested by the
present study. It has been shown that milking machine types
inuence the levels of technologically important microorganisms
in milk, suggesting that these machines are microbiological
reservoirs.
4.4. Qualitative microbial diversity
The large number of genera and species detected in this study
demonstrates that raw milk harbours tremendous microbial
diversity. Thus, raw milk offers great potential as a reservoir of
microbial diversity, with possibly hundreds or thousands of
different strains. These results are comparable with those reported
by Vacheyrou et al. (2011), who identied 50 environmental
bacterial species in milk from 16 farms in Franche-Comt (France)
and by Callon et al. (2007) who identied forty different microbial
genera in goat milk samples from one farm in Rhne-Alpes
(France). The high frequency of Gram-positive bacteria found in raw
milk agrees with results reported by Fricker, Sknseng, Rudi, Stessl,
and Ehling-Schulz (2011), who used FTIR spectroscopy to analyse
milk from farms in Germany, Austria and Norway, and found 66% of
milk tank bacteria to be Gram-positive.
The highest number of species (65) was identied with the
30  C/pH 7.0 culture conditions; however, this represented only
58% of the total identied species (112). Some species were only
isolated in one culture condition. The use of multiple culture
conditions allowed identication of twice as many species as were
found at 30  C/pH 7.0, giving a more realistic picture of raw milk
microbial diversity, including subdominant microbial groups. The
complementarity of culture-dependent and culture-independent
methods in maximizing the number of species identied has
been shown previously (Verdier-Metz et al., 2012). In this study, the
culture-dependent approach was shown to be improved by using
multiple culture conditions and unselective media, which can
reveal higher microbial diversity.
Regarding bacterial species with the potential to be food-borne
pathogens, in these experimental conditions, the Listeria or the
Campylobacter genera were not detected. Pseudomonas aeruginosa
was recovered from ve samples and Salmonella enterica was found

20

A. Mallet et al. / International Dairy Journal 27 (2012) 13e21

in two samples. S. aureus was also frequently detected. In a previous

evaluation of the prevalence of four pathogens in raw milk
(DAmico & Donnelly, 2010), 67% of the farms tested were positive
for S. aureus in at least one milk sample, but Listeria monocytogenes,
Escherichia coli O157:H7, and Salmonella sp. were not detected. In
another study, S. aureus was found in 61.5% of raw milk samples
(Giannino, Marzotto, Dellaglio, & Feligini, 2009). Results obtained
by EFSA (2009, 2010) and Ramsey and Funk (2009) suggest that
cheeses made from pasteurised milk are not safer than cheese
made from raw milk. The hygiene practices and industrial controls
that have developed over many years have very much minimised
the risk of contamination. The high frequency of Staphylococcus
species found in the milk samples and, to a lesser extent, that of
Corynebacterium species (nine different species were identied for
each genus) was similar to the results reported by Fricker et al.
(2011). It is noteworthy that S. haemolyticus was found in nearly
all raw milk samples (87.5%), although it is rarely described in dairy
products (for example, see Serhan et al., 2009). As noted by
Bautista, Bermejo, and Nuez (1986), this species of staphylococci,
along with S. aureus, is also predominant in raw ewes milk.
S. haemolyticus, S. aureus, several other staphylococci, and Corynebacterium species are commonly found on teat skin, which is
a microbial reservoir for milk (Verdier-Metz et al., 2012).
Presumptive ripening bacteria appeared to be a dominant group in
raw milk obtained from two milkings, but the level and proportion
of these bacteria decreased in samples obtained from four milkings.
This could probably be caused by growth competition or by inhibition due to the growth of other ora, in particular Gram-negative
bacteria.
Psychrobacter sp. were detected mainly in winter samples. This
is a psychrotrophic genus also described in cheese (Coton et al.,
2012; Larpin-Laborde et al., 2011; Maoz, Mayr, & Scherer, 2003).
Gram-negative bacteria exhibited greater diversity in spring milk
than in winter milk. Although many species were identied that
have been frequently described in raw milk, a number of
uncommon species were also found, such as Clavibacter michiganensis and Ochrobactrum sp., which were recently identied on
teat skin (Verdier-Metz et al., 2012) and in raw milk (Coton et al.,
2012), respectively.
5. Conclusion
Considerable quantitative and qualitative microbial diversity in
the raw milk from Basse-Normandie was found and this diversity
(microbial load and probably the composition of raw milk) was
inuenced by combinations of milk harvesting and farm management practices. Raw milk is a complex microbial ecosystem. In fact,
the term raw milks seems to be more appropriate than raw
milk, because milk from each farm is different. Although the
sources of some of the species identied and their technological
aspects are uncertain, very few possible pathogens were detected.
Preserving the safety, quality and microbial diversity of raw milk
are probably the most important challenges for the future of raw
milk products. The reservoirs of the microbial ora of raw milk are
also complex because they are multifactorial. In future studies, we
plan to investigate the sources of raw milk microbial diversity in the
milking environment, and to dene or identify milking practices
that preserve this diversity.
Acknowledgements
This work was supported by a CIFRE grant from the Association
Nationale de la Recherche Technique, by the Regional Council of
Basse-Normandie, by European Funds for Regional Development
(FEDER), and by private funds. This project gets the VALORIAL label.

The authors would like to thank R. Cassier, L. Gargadennec,

K. Cruchon and M. Fleury for their assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.idairyj.2012.07.009.
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