Food and Nutrition Sciences, 2013, 4, 49-58
http://dx.doi.org/10.4236/fns.2013.49A2008 Published Online September 2013 (http://www.scirp.org/journal/fns)
Selective Lactococcus Enumeration in Raw Milk*
Laetitia Gemelas, Véronique Rigobello, Maï Huong Ly-Chatain, Yann Demarigny
Isara-Lyon, Unité BioDyMIA, Lyon, France.
Received February 13th, 2013; revised March 13th, 2013; accepted March 20th, 2013
Copyright © 2013 Laetitia Gemelas et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The Lactococcus diversity in cow and goat raw milk was investigated. To do so, a protocol had to be established for the
specific enumeration of lactococci. Eight agar media and one control medium were analysed to compare their profi-
ciency in evaluating the Lactococcus population in raw milk: M17 Nal, Elliker, modified Elliker, PCA + milk, modified
KCA, modified Chalmers, Turner, FSDA. The M17 medium was used as reference. Eighteen pure strains were tested
on these media for their selectivity towards lactococci: six Lactococcus species or subspecies, three Leuconostoc, three
Enterococcus, two Lactobacillus, one Streptococcus thermophilus, one Pseudomonas fluorescens, one Escherichia coli
and one Staphylococcus aureus. All these bacteria were chosen for their regular presence in raw milk. The KCA me-
dium proved to be the most selective towards lactococci, on condition that 1) we discriminated the colonies using the
catalase test and 2) we subtracted the Enterococcus population counted on BEA. However, it was not possible to sepa-
rate the Streptococcus from the Lactococcus colonies on KCA. The “Lactococcus-like” population including these two
genera was estimated at a mean level of 3.18 log(cfu)/mL and 4.14 log(cfu)/mL in cow and goat raw milk respectively.
This is consistent with the data already published.
Keywords: Lactococcus; Culture Media; Raw Milk; LAB; Modified KCA
1. Introduction
Raw milk include different microflora traditionally
grouped into three categories: positive, negative and neu-
tral. Technological microflora is considered as positive.
Among them, lactic acid bacteria (LAB), i.e. Lactococ-
cus, Leuconostoc, homo and heterofermentative Lacto-
bacilli, Pediococcus, and cheese surface bacteria—Mi-
crococcus, non-pathogenic Staphylococcus, yeasts and
moulds—are generally referred to. Spoiling bacteria—
negative microflora—include for instance the Pseudo-
monas and Escherichia genera. Neutral bacteria, among
them many Archaebacteria, are considered to have no
effect on milk quality [1,2]. Raw milk LAB are known to
contribute positively to cheese making. Despite being at
a much lower level compared with the starter bacteria (3
log(cfu)/mL vs 6 to 7 log(cfu)/mL), wild LAB are able to
participate in the acidification step and in the ripening
[3,4] reaching levels as high as 7 - 8 log(cfu)/g [5]. Their
enzymes help to modify the physico-chemical and bio-
chemical environment of the cheese which allows the
aromatic balance to develop [6]. In light of these obser-
vations, cheese makers are divided over the necessity to
control pathogens, on the one hand, by lowering the total
bacterial count and their will, on the other, to favour
positive microflora [7,8]. This position which has proven
to be antinomic until now can be justified by two expla-
nations. The microbial flux within the farm which leads
to the microbial enrichment of the milk is still unknown.
A plate medium designed for the specific enumeration of
positive microflora is lacking. This is particularly true for
the Lactococcus population.
Lactococci, Qualified Presumption of Safety (QPS)
microorganisms, are a technological microflora of impor-
tance. Wild lactococcal strains rapidly grow during the
first steps of cheese making due to their proteolytic en-
zymes. Through the production of lactic acid, they par-
ticipate in curd formation and contribute positively to the
taste and texture characteristics of the cheeses [9]. In
spite of these interesting technological abilities, the level
of lactococci in raw milk is still unknown. No specific
medium in which to count them is currently available
because of the complex nutritional requirements of these
bacteria. Consequently, rich non-selective media are used
to study them. But these agar plates allow many other
bacteria to form colonies exhibiting the same morpho-
*Diversity of the Lactococcus type population in cow and goat’s raw
milks.

Selective Lactococcus Enumeration in Raw Milk 50
type as that of lactococci.
Among the media mentioned in the literature to culti-
vate lactococci, M17 is frequently put forward as plate
count agar and broth [10]. But the reducing power of
lactococci linked with organic acid production is also
tested on Turner agar and modified KCA [11,12]. Prote-
olytic and non-proteolytic strains are separated on FSDA
agar (Fast Slow Differential Agar) and PCA (Plate Count
Agar) supplemented with milk (1%) [13]. Inhibitors of
gram negative bacteria (i.e. sodium azide) and acidity
indicator (i.e. bromocresol purple) are sometimes added
to PCA or M17 agar in order to improve their efficiency
in detecting LAB and especially lactococci [14]. Modi-
fied Elliker agar and modified Chalmers agar are selec-
tive media which bring to light the acidifying bacteria
[15,16].
2. Materials and Methods
2.1. Reference Strains, Culture Maintenance and
Reference Media
Eighteen known strains were used. Lactococcus garvieae
CIP 102507, Lc plantarum CIP 102506, Enterococcus
faecalis CIP103631, Ec faecium CIP106742, Leucono-
stoc mesenteroides subsp. cremoris CIP103009 (referred
to hereafter as Ln cremoris), Ln mesenteroides subsp.
dextranicum CIP102423 (Ln dextranicum), Ln mesenter-
oides subsp. mesenteroides CIP54178 (Ln mesenter-
oides), Staphylococcus aureus CIP103429, Escherichia
coli CIP7624 were purchased from the “Collection de
l’Institut Pasteur”. Ec durans, Lc lactis subsp. lactis (Lc
lactis), Lc lactis subsp. cremoris (Lc cremoris), Lc lactis
subsp. hordniae (Lc hordniae), Lc lactis subsp. lactis
biovar. diacetylactis (Lc diacetylactis), three Lc planta-
rum strains, Streptococcus thermophilus, Lactobacillus
paracasei, Lb plantarum and Pseudomonas fluorescens
originated from our laboratory collection. These strains
had been previously isolated from raw milk samples and
carefully characterized and identified [17]. All the strains
were kept frozen at −80˚C in a mixture of the culture
medium and glycerol 30% (Sigma-Aldrich).
After thawing, the strains were cultured in their spe-
cific maintenance broth, that is:
 Lactococcus, Enteroco ccus and St thermophilus: M17
broth (Biokar) for 24 h at 30˚C, 30˚C and 44˚C re-
spectively;
 Leuconostoc and Lactobacillus: MRS broth (Biokar)
for 24 h at 30˚C and 37˚C respectively;
 Ps fluorescens, S aureus and E. coli: BHI broth (Bio-
kar) for 24 h at 30˚C, 37˚C and 37˚C respectively.
Purity tests were carried out in aerobic conditions at
the same temperatures and on the same media, but in
Petri dishes. Incubation lasted 24 h, except for Leucono-
stoc strains which were incubated for 72 h and for Lac-
tobacillus strains, which were incubated for 48 h in an-
aerobic conditions.
2.2. Agar Media and Culture Conditions
Nine agar culture media were tested. Two elective media
were dedicated to the enumeration of the mesophilic
aerobic lactic acid bacteria, PCA (Biokar) with 1 g/L
skim milk powder (Oxoid) and Elliker (Biokar). Three
selective media were used: basic M17 agar (Biokar) was
amended with 0.04 g/L nalidixic acid (Sigma-Aldrich);
modified Elliker agar contained 1 g/L thallium acetate
(Merk Group) and 25 mg/L bromocresol purple (Chimie-
Plus) to underline acidifying strains; Chalmers media in-
cluded polymixin-β-sulfate (100 iu·mL−1, Sigma-Aldrich)
and a redox indicator (TTC, triphenyl tetrazolium chlo-
ride, Sigma-Aldrich). Three media were chosen to high-
light specific biochemical features: the proteolytic active-
ity was checked on FSDA and the reducing power of
strains on Turner and KCA agar. These three media and
the Chalmers medium were all prepared in the lab. Basic
M17 agar was employed as control. The characteristics
of each medium are displayed on Table 1. The nine me-
dia were incubated for 48 h at 30˚C aerobically. These
conditions are supposed to be optimal for the growth of
the Lactococcus population.
2.3. Analytical Design
The nine media quoted above were checked for their
ability to favour the growth of the different Lactococcus
species and for their selectivity when in the presence of
other bacterial populations in pure and mixed cultures.
On the basis of these two experiments, the most suitable
medium was then kept for further use on raw milk. Some
presumed lactococcal isolates were collected for phenol-
typic and genomic identification.
2.3.1. Microbi ological An al y s i s
1) Tests on Reference Strains
The eighteen strains were cultured in optimal condi-
tions. As soon as optical density corresponded to the
growth phase, a sample was removed for enumeration on
the nine media listed above. The results obtained were
compared with one another and with the control medium
(M17). Each experiment was duplicated.
2) Test on Re-Seeded Model Milk (RMM)
Re-seeded model milks (RMM) were performed as de-
scribed by Dalmasso et al. [17]: pasteurized milk was
seeded with the eighteen strains quoted above so as to
mimic the habitual levels found in raw milk (Table 2).
The RMM allowed us to test the proficiency of the media
in evaluating the Lactococcus population in a mix of
bacteria.
3) Milk Sampling
Milk samples were taken from the milk tank just after

Selective Lactococcus Enumeration in Raw Milk 51
Table 1. Characteristics and composition of nine media dedicated to the enumeration of the Lactococcus population.
Constituents
(g·L−1, except steril whey)
M17a
[10] M17 Nala PCAb + milk 1% [14]
Ellikerb
[18]
Modified
Ellikerb [15]
FSDAb
[13]
Modified
Chalmersb [16]
Modified
KCAb [12]
Turnerb
[11]
Tryptone 2.5 2.5 5 20 20 - - 17 5
Polypeptone 2.5 2.5 - - - - 3 - -
Soytone 5 5 - - - - - -
Yeast extract 2.5 2.5 2.5 5 5 - 3 4.25 5
Meat extract 5 5 - - - - 3 - -
Gelatine - - - 2.5 2.5 - - 2.1 -
Milkc - - 10 - - 100 - - -
Glucose - - 1 5 5 - 20 4.25 0.5
Lactose 5 5 - 5 5 - 20 4.25 -
Saccharose - - - 5 5 - - - -
Ascorbic acid 0.5 0.5 - 0.5 0.5 - - - -
NaCl - - - 4 4 - - 3.4 -
MgSO4 0.25 0.25 - - - - - - -
β-glycerophosphate disodium 19 19 - - - 19 - - -
K2HPO4 - - - - - - - - 2
Calcium lactate - - - - - - - 6.8 -
Sodium citrate - - - - - - - 1.7 -
Calcium citrated - - - - - - - 5 -
Whey (v/v)e - - - - - - - 0.1 -
L-arginin - - - - - - - - 2
Bromocresol purple - - - - 0.025 - - - -
TTCf - - - - - - - 0.1 0.05
Litmus - - - - - 1 - - -
Neutral red - - - - - - 0.005 - -
CaCO3 - - - - - - 20 - -
Thallium acetate - - - 1 - - - -
Nalidixic acid - 0.04 - - - - - - -
Sodium acetate - - - 1.5 1.5 - - - -
Polymixin-β-sulfatef - - - - - - 100 i.u./mL - -
Agar 15 15 12 15 15 10 15 12.8 15
Buffered pH 7.1 - 7.2 7.1 - 7.2 - - - - 6 - -
M17, PCA, Elliker are ready to use (Biokar), the other media are laboratory-made. aMedia sterilised at 115˚C, 20 min; bMedia sterilised at 121˚C, 15 min; cRe-
formed skim milk powder separately sterilised at 110˚C, 10 min; dCalcium citrate separately sterilised at 121˚C, 15 min; eWhey separately sterilised at 110˚C,
10 min; fTTC (triphényl tetrazolium chloride) and Polymixin-β-sulfate sterilised by filtration (0.45 µm).
milking. Eight milk samples were collected on eight
farms from the Rhône-Alpes Region (France): Cf1, Cf2,
Cf3, Cf4, Cf5, Cf6, Gf1 and Gf2 (“C”, “G” and “f” refer
respectively to cow, goat and fresh milk); three raw cow
milk samples came from three farms in the Massif Cen-
tral Region: Cf7, Cf8 and Cf9. Six raw cow milk and two

Selective Lactococcus Enumeration in Raw Milk 52
Table 2. Theoretical bacterial concentrations in the re-seeded milk samples. Data expressed in log(cfu)/mL.
Lc lactis
Lc cremoris Lc hordniae
Lc plantarum
Lc garvieae
Lc diacetylactis
Ln mesenteroides
Ln dextranicum
Ln cremoris
Ec faecalis Ec faecalis
Ec durans
Lb plantarum
Lb paracasei
St thermophilus
E coli
Ps fluorescens
S aureus
Total population
Level 2.3 2.3 1.4 1.4 1.4 1.4 2.2 2.2 2.2 1.5 1.5 1.5 1.7 1.7 2.7 2.7 2.0 2.3 3.4
raw goat milk samples were collected from eight farms in
the Franche-Comté Region: Ct1, Ct2, Ct3, Ct4, Ct5, Ct6,
Gf3 and Gf4 (“t” means that the milk was thawed before
use).
Thawed samples were kept at −80˚C until analysis
(within one week). Fresh samples were stored at 0/+4˚C
and analysed within 12 hours after milk sampling.
The colonies from the six samples Cf1, Cf2, Cf3, Gf1,
Ct1 and Ct2 were first counted. Some catalase negative
colonies thought to belong to the Lactococcus genus
were then purified on M17 agar in view of phenotypic
and genotypic characterisation. Each isolate was stored at
−80˚C in a mix of medium and 30% glycerol. The other
samples were just checked for the enumeration of the
Lactococcus population.
2.3.2. Phenot y pi c Ch aracteriz ati on
Four tests were chosen for their aptness in identifying the
Lactococcus genus. The absence of the catalase was at-
tested by pouring a drop of H2O2 directly on each colony
(no gas production). Microscopic observation after Gram
stain indicated the shape (cocci), the arrangement (small
chains) and the position of the bacteria (Gram+). The
type of lactic acid isomer produced was determined using
the D-/L-lactate enzymatic test (ENZYTEC, R-Biopharm).
This test was performed on the supernatant of a 24 h
culture of each bacterium. Lactococcus, Streptococcus
and Ente roco ccus strains produce L-lactate whereas Leu-
conostoc strains produce D-lactate, and heterofermenta-
tive Lactobacillus bacteria produce D-, L- or a mix of D-
and L-lactate. BEA (Biokar) was used to separate pre-
sumed lactococci from enterococci. Lactococci are gen-
erally unable to develop on BEA due to bile salt inhi-
bition. BEA is dedicated to the specific count of entero-
cocci (this was checked on the eighteen strains, although
the results are not reproduced here).
2.3.3. Genotypic Identification
The isolates presumed to belong to the Lactococcus ge-
nus—catalase negative, producing L-lactate, unable to
grow on BEA—were cultured at 30˚C for 24 h in 5 mL
of M17 broth. The total DNA was extracted using the
Nucleospinb tissue kit (Machery-Nagel). Pure DNA was
stored at −20˚C until use. A multiplex PCR assay was
performed as described by Pu et al. [19], using the fol-
lowing primers: 1RL, LacreR, LgR and PilpraR. PCR
products were electrophoresed through 1% (w/v) agarose
gel (Sigma-Aldrich) in TBE buffer (Tris—Borate—
EDTA pH 8, Sigma-Aldrich) at 100V for 3 h. The DNA
fragments were stained with ethidium bromide (Sigma-
Aldrich), viewed under UV light (302 nm, Biorad) and
photographed on a digital camera (Camedia C-5060).
The band patterns were normalized and processed using
the GelCompar 3.1 software (Applied Maths). The size
of PCR products was determined with a DNA size
marker (Sigma-Aldrich).
Some isolates were subjected to gene sequencing, hav-
ing been chosen as representative strains of each cluster
obtained from the multiplex PCR dendrogram. The par-
tial 16 S rRNA gene sequence analysis was performed by
the company BACT UP (France) and the complete 16 S
rRNA gene sequence analysis was performed by Idymik
Company (France).
2.4. Statistical Analysis
A repeatability test was performed with three Lactococ-
cus strains. They were incubated at 30˚C in M17 broth.
During their exponential growth phase, each of them was
enumerated on ten M17 agar dishes. Standard errors were
calculated with the Student number for a 5% threshold
value for a (n − 1) degree of freedom. The standard-error
was evaluated to  0.10 log(cfu)/mL for populations be-
tween 7 and 9 log(cfu)/mL. Thus, two results were con-
sidered as significantly different if the variation was su-
perior to 0.10 log(cfu)/mL. Statistics—variance analysis,
means, etc.—were performed by means of the XLSTAT
software (2011, version 5.01).
3. Results
3.1. Growth Test of Reference Strains
3.1.1. Mediu m Specificity towards Each Strain
None of the four Lc plantarum strains grew on modified
Elliker (Table 3). Lactococcus colonies were difficult to
count on FSDA, irrespective of the species. The enu-
meration of lactococci on PCA + milk, Elliker, M17 Nal
(+ nalidixic acid), Turner, KCA, and Chalmers did not
produce significantly different results from those ob-
tained on the control medium (M17 agar), differences

Selective Lactococcus Enumeration in Raw Milk 53
Table 3. Results of growth tests of eighteen strains—Lc lactis, Lc cremoris, Lc hordniae, Lc plantarum, Lc garvieae, Lc diacety-
lactis, Ln mesenteroides, Ln dextranicum, Ln cremoris, Ec durans, Lb plantarum, Lb paracasei, St thermophilus, E. coli, Ps
fluorescens, S. aureus—on eight media—FSDA, modified Elliker, M17 Nal, PCA + milk, Elliker, modified Chalmers, modi-
fied KCA, Turner—and on the control medium—M17.
Lc lactis
Lc cremoris
Lc hordniae
Lc plantarum
Lc garvieae
Lc diacetylactis
Ln mesenteroides
Ln dextranicum
Ln cremoris
Ec faecalis Ec faecium
Ec durans
Lb plantarum
Lb paracasei
St thermophilus
E. coli
Ps fluorescens
S aureus
FSDA A A A A A A
Modified elliker A A A A A
M17
M17 Nal A A
PCA + milk
Elliker
Modified chalmers A A A A
Modified KCA A A A A A A A
Turner A A A A A A A
A: absence of growth. Gray-coloured: growth.
being less than or equal to 0.10 log(cfu)/mL (p < 0.05).
Therefore, modified Elliker and FSDA media were set
aside at this step of the study.
The eighteen strains tested were all able to develop on
M17, PCA + milk and Elliker media. E. coli and Ps fluo-
rescens were inhibited on M17 Nal. On these four media,
the colony’s morphotype was identical—small, circular,
smooth, shiny and creamy, whatever the genus or the
species. Therefore, these media did not appear suitable
for the specific enumeration of lactococci. On modified
Chalmers, if we consider the “non Lactococcus” strains,
eight strains out of twelve were able to form colonies,
their morphotype being identical to the one of lactococci.
Consequently, this media was also set aside. On Turner
and KCA, only four strains out of the twelve “non Lac-
tococcus” strains were able to form pink or red Lacto-
coccus type colonies: Ec faecium, Ec faecalis, E. coli and
Ps fluorescens. Ec durans was only observed on Turner
and S aureus on KCA.
To sum up, growth on KCA and Turner gave the most
convincing results for the selective enumeration of pre-
sumed Lactococcus and Enterococcus genera. These two
media were kept for further analysis whereas the others
were set aside. Since enterococci were able to develop on
these two media, we proposed to specifically count en-
terococci on BEA medium and to subtract the entero-
coccal count from the Turner or KCA count.
3.1.2. Species Recovery from RMM
A sample of RMM was plated on BEA agar, Turner and
KCA. Catalase negative colonies with a pink or a red
colour were enumerated specifically on these last two
media. The result obtained corresponded to the level of
presumed lactococci + enterococci. The count result ob-
tained on BEA was then subtracted from the KCA and
the Turner results. This gave an estimation of the pre-
sumed lactococcal microflora.
The microbial results gathered from three RMM are
displayed on Tabl e 4. The “KCA protocol” led to a fairly
accurate estimation of the Lactococcus population, the
discrepancy between objective and estimated data being
inferior to 0.21  0.09 log(cfu)/mL. A variance analysis
was performed on these data. No statistical difference
was observed whatever the milk sample or strain consid-
ered. On the contrary, the Turner medium systematically
gave unusable results, enterococci and lactococci both
being underestimated. Consequently this medium was
not retained for the following analyses.
3.2. Protocol Design for Estimating the
Lactococcus Population
The microbial results obtained on six raw cow milks—
Cf1, Cf2, Cf3, Gf1, Ct1, Ct2—are shown on Table 5.
Between 50% and 100% of the catalase negative isolates
were picked up from KCA Petri dishes. The phenotypic
and genotypic characterisation of these isolates allowed
presumed Lactococcus colonies to be separated from
other contaminants—namely enterococci. All the Lacto-
coccus isolates were Gram + cocci producing L-lactate
and were inhibited by bile salts. On the basis of these

Selective Lactococcus Enumeration in Raw Milk 54
Table 4. Estimation of the Lactococcus population in re-seeded model milk (RMM) using the KCA and turner media. The
Enterococcus population (enumerated on BEA) has been subtracted from the results obtained on these two media. Results are
expressed in log(cfu)/mL.
Lc species Control (M17) KCA TurnerKCA (difference with the control) Turner (difference with the control)
RMM 1 Lc cremoris 3.31 3.44 A 0.13 A
Lc garvieae 3.41 ND ND ND ND
Lc hordniae 3.60 3.81 2.88 0.21 0.7
Lc lactis 3.82 3.93 2.24 0.11 1.6
Lc plantarum 3.82 3.91 1.82 0.09 2.0
RMM 2 Lc cremoris 4.69 4.79 A 0.10 A
Lc garvieae 4.39 4.38 A 0.01 A
Lc hordniae 4.38 4.42 A 0.04 A
Lc lactis 4.69 4.80 A 0.11 A
Lc plantarum ND ND ND ND A
RMM 3 Lc cremoris 4.69 4.60 A 0.09 A
Lc garvieae 4.39 4.20 A 0.19 A
Lc hordniae 4.38 4.50 A 0.12 A
Lc lactis 4.69 4.80 A 0.11 A
Lc plantarum ND ND ND ND ND
ND: not determined; A: absence of growth.
Table 5. Analysis of raw cow milk samples from the Rhône-Alpes region (Cf1, Cf2, Cf3, Cf4, Cf5, Cf6), from the Massif Cen-
tral region (Cf7, Cf8, Cf9), and from the Franche-Comté region (Ct1, Ct2, Ct3, Ct4, Ct5, Ct6) and raw goat milk samples
from the Rhône-Alpes region (Gf1, Gf2), and from the Franche-Comté region (Gf3, Gf4). “C”, “G”, “f” and “t” refer respec-
tively to cow, goat, fresh and thawed milks. Results are expressed in log(cfu)/mL of raw milk.
Medium and microflora enumeratedCf1 Cf2 Cf3 Gf1 Ct1 Ct2 Ct3 Ct4 Ct5 Ct6Cf4Cf5Cf6 Gf2 Cf7 Cf8 Cf9Gf3Gf4
Level on KCA 3.15 4.33 4.37 3.732.462.791.952.303.323.333.19- - - - - - - -
Level of the catalase negative
population on KCA 2.61 3.87 2.26 3.19 2.29 2.641.90 1.78 2.49 2.30 2.87 3.64 3.40 4.15 3.12 2.54 2.41 3.28 4.60
Level of the Enterococcus
population on BEA 2.11 2.11 2.68 2.89 2.04 2.71 1.00 2.28 2.46 2.36 2.00 2.181.93.03 2.75 1.7 1.00 1.00 2.51
Level of the presumed Lc-like
phenotype population 2.45 3.87 * 2.89 1.92* 1.85 * 1.30 * 2.81 3.63 3.38 4.11 2.88 2.48 2.40 3.28 4.60
% of catalase-isolates
picked up on KCA 50% 100% - 100%100%67% - - - - - - - - - - -
Level of Lc-like phenotype estimed
with genotypic identification 2.30 3.86 - 2.411.891.74 - - - - - - - - - - -
*Inferior to detection threshold.
tests, the level of the Lactococcus population was esti-
mated at between 1.74 to 3.86 log(cfu)/mL.
These results were compared with those obtained by
subtracting the enumeration on BEA (Enterococcus po-
pulation) to the catalase negative microflora count ob-
tained on KCA. The difference between the two estima-
tions of the Lactococcus population was inferior or equal
to 0.48 log(cfu)/mL. No Lactococci were detected in
milk Cf3. This result was consistent with the methodol-
ogy used for estimating the lactococci because this latter
population was inferior to the detection threshold. The
enterococcal count on BEA agar was superior to the cata-
lase negative colony count obtained on KCA.
3.3. Lactococcus Diversity
The diversity of the lactococcal isolates was investigated
by multiplex PCR (Figure 1). The 46 isolates picked
from the five samples—Cf1 Cf2, Gf1, Ct1, Ct2—were ,

Selective Lactococcus Enumeration in Raw Milk 55
100908070
200.00 400.00 600.00 800.00 1000 1200 1400 1600 1800 2000 4.00E+3
Cluster 1 - Lc Lactis [1]
Cluster 2 - Lc Lactis [1]
Cluster 3 - Streptococcus spp. [31]
Custer 4 - unidentified [2]
*
Cluster 5 - Lc Lactis [9]
Cluster 6 - Enterococcus spp. [1]
Cluster 7 - Lc raffinolactis [1]
*
Figure 1. Dendrogram drawn by UPGMA of correlation value of normalized multiplex-PCR patterns from lactococci. Each
pattern is identified by a cluster number and by a number between brackets referring to the number of strains which dis-
played this profile. The seven clusters from 1 to 7 are defined at a coefficient of similarity of 80% materialized by a bold ver-
tical line. Strains coming from goat’s raw milk are identified by a *.
arranged in 7 clusters (80% similarity threshold). Only
one profile is observed in each cluster. Cluster 6 is an
example of an enterococcal profile. The isolate picked
from a raw goat milk sample Cf2 was identified as En-
terococcus spp by DNA sequencing. Nine isolates picked
from samples Cf1, Ct1 and Ct2 form cluster 5. A 228 pb
band is observed. This size is specific to the Lactococcus
lactis species, a result confirmed by DNA partial se-
quencing of the 16 S rRNA genes of one isolate coming
from the Ct1 sample. The remaining 36 isolates could not
be identified by multiplex PCR. One representative of
each cluster was thus identified by sequencing. With the
exception of cluster 4, formed by 2 isolates collected
from raw goat milk, all these strains were able to be
identified. Clusters 1 and 2 were associated with Lc lactis.
These two isolates were taken from the Cf1 sample.
Cluster 7 included one strain identified as Lc raffinolactis.
This isolate came from raw goat milk (sample Gf1). It is
noteworthy that these three isolates did not present the
typical DNA bands observed for Lc lactis (238 pb) and
Lc raffinolactis (860 pb), although sequencing identifica-
tion was doubtless (>97%). Cluster 3 included 31 isolates
coming from the milk Cf2. Among them, one isolate was
assigned to Streptococcus ssp. by partial 16S rRNA gene
sequencing. This result was partially confirmed by a se-
quencing test made by another lab which established the
genus assignment. This isolate was inhibited by bile salts,
a characteristic of lactococci. The presence of strepto-
cocci on the surface of KCA, although not surprising, led
us to limit the protocol here developed to the evaluation
of the Lactococcus and the Streptococcus population.
Thereafter in this article, this microflora will be design-
nated as “Lactococcus-like” microflora, implying that the
distinction between lactococci and streptococci by the
KCA culture-dependant methodology is not possible.
3.4. Lactococcus-Like Microflora Levels in
Raw Milk
The Lactococcus-like population ranged from 1.30 to
3.87 log(cfu)/mL for raw cow milk and from 2.89 to 4.60
log(cfu)/mL for raw goat milk. In four raw cow milk
samples, this population was inferior to the detection
threshold. In this case, the enterococcal microflora pro-
bably overwhelmed lactococci. No differences were ob-
served considering the geographical origin of the raw
milk.
4. Discussion
The bacteria from the Lactococcus genus are used as
starters to ferment many different dairy products. They
are also present at low levels in raw milk, coming from
plants and biofilms present in the milking machine [17].
Lactococci have been studied extensively for many years
and the yearly number of publications varies between
less than 300 to more than 400 articles. Surprisingly,
whereas the knowledge—genetic, metabolic, transcryp-
tomic, etc.—of this microorganism has been greatly im-
proved, no attempts have been made to develop media
for its specific enumeration in a complex food matrix.
Moreover, knowledge has specifically focused on Lc
lactis, and little is known about the other species or sub-
species. In this work, we tried to take stock of the level
and the diversity of lactococci in raw milk. The prelimi-
nary step in reaching this objective involved developing
a methodology that would allow the Lactococcus popula-
tion to be estimated in raw milk, whatever the species or
the subspecies present.
Many media have been proposed in the past for the
enumeration of lactococci. In spite of substantial efforts,
none of them proved to be selective. Among them, M17,
PCA + milk and Elliker enable the growth of mesophilic
aero tolerant LAB [10,20]. The addition of 0.04 g·L−1
nalidixic acid to these three media allows the Gram
negative bacteria to be depressed. But other Gram posi-
tive bacteria are still able to grow, an observation also
made by Corroler et al. [21]. The use of thallium acetate
in the modified Elliker medium [19], another Gram

Selective Lactococcus Enumeration in Raw Milk 56
negative inhibitor, proved to hinder the growth of the
four Lactococcus plantarum strains we tested. Even if
few references exist on this species, Demarigny et al. [22]
regularly found this bacterium in natural whey starters.
The observations we made on FSDA were identical to
those of Grattepanche et al. [23]. If the protease positive
colony morphotype—round, regular, smooth—was easily
observed, protease negative colonies were never detected.
Modified Chalmers did not enable us to clearly distin-
guish the different lactic acid bacteria genera on the basis
of their morphotype. In fact, two media were found to be
of interest, Turner agar and KCA. Among other compo-
nents, these plate count agar include TTC, a redox poten-
tial indicator. Acid fermentation by lactococci generally
leads to the concomitant decrease in the redox potential.
Colonies then appear red/pink. In pure culture, KCA and
Turner agar gave similar enumerations to those obtained
with the reference M17 medium, whatever the Lacto-
coccus species or subspecies. But in mixed-culture, the
Turner agar underestimated the Lactococcus population.
Among several hypotheses, we can propose as a possible
explanation that microorganisms competed for nutrients.
Indeed, Lactococcus is a nutritionally demanding bacte-
rium. For instance for Lc lactis, the amount of amino
acid auxotrophies varies between 6 to 8 [24]. The Turner
composition is less rich than KCA (less nitrogen and
glucidic sources). The resulting nutritional stress that can
occur on Turner may explain the discrepancy between
control (M17) and test (Turner) data. KCA proved, then,
to be the most suitable medium. On KCA, a catalase test
must be added to distinguish catalase negative colonies
supposed to be assigned to Lactococcus or Enterococ cus.
At the same time, Enterococcus bacteria have to be
counted on BEA. This result is then subtracted from the
result obtain on KCA. This gives a rather good estima-
tion of the Lactococcus population in pure or mixed cul-
tures.
Six raw cow milk samples were used to evaluate the
diversity of the Lactococcus species. The isolates col-
lected from these raw milk samples were subjected to
multiplex PCR [19] and partial 16 S rRNA gene se-
quence analysis. The multiplex PCR allowed 9 isolates
out of 44 to be accurately identified. The relevance of
this method when applied on wild strains appears here
debatable. Indeed, the work of Pu et al. [19] only focused
on selected starter strains. While the multiplex PCR gave
pertinent results on these strains, our own observations
would appear to cast doubt on the pertinence of this
methodology when applied to wild bacteria. Although
many reasons for this could be put forward, at this time,
we have no satisfactory explanation to offer. The partial
gene sequencing did, however, bring other information.
Lc lactis was detected without any doubt in three raw
cow milk samples and Lc raffinolactis in one raw goat
milk sample. Two isolates picked from one raw goat
milk could not be identified by DNA sequencing. More-
over, sequencing allowed us to discover Streptococcus
strains which had first been assigned to Lactococcus on
the basis of their phenotype.
Streptococcus has already been observed in raw cow
milk. Thus, the presence of this genus is not surprising.
Franciosi et al. [25] identified St. dysgalactiae, St. parau-
beris, St. suis, St. macedonicus in raw cow milk. Desma-
sures et Beuvier [26] also reported Streptococcus ssp. in
raw cow and goat milk. Jans et al. [27] designated the Strep-
tococcus bovis/Streptococcus equinus complex (SBSEC)
“as the predominant LAB in spontaneously fermented
African milk products, […] Mexican, Greek, and Italian
cheese”.
Some of the streptococcal strains show a lot of simili-
tudes with lactococci, among them, the bile salt inhibi-
tion. In our methodology, it would seem to be impossible
to distinguish Lactococcus from Streptococcus strains.
This could lead us to question the actual identification of
the enumerated microflora already published in the lit-
erature. Indeed, our results suggest that some Strepto-
coccus strains may have been confused with the Lacto-
coccus genus. The mean level of Lactococcus-like mi-
croflora in eleven raw cow milk samples was estimated
at 3.18 log(cfu)/mL with a minimum of 1.30 log(cfu)/mL
and a maximum of 3.38 log(cfu)/mL. In four raw goat
milk samples, the mean was superior: 4.14 log(cfu)/mL
with a minimum of 2.89 log(cfu)/mL and a maximum of
4.60 log(cfu)/mL. These results are consistent with those
published on raw cow and goat milk in the literature [26].
No differences were observed between raw milk coming
from the three different regions. Published data taking
into account the geographical origin of milk samples is
still lacking. Moreover, the number of samples analysed
in this study was probably not sufficient to assess the
possible influence of this factor.
The study of lactococcal diversity brings out, unsure-
prisingly, the presence of Lc lactis in the majority of raw
cow milk [25,26,28]. On the other hand, Lc raffinolactis,
found in raw goat milk, is reported to a lesser extent in
the literature on raw goat milk [22,29].
5. Conclusion
Our methodology is getting close to being able to enume-
rate lactococci. The results obtained were congruent with
those in the literature. However, it was not possible to
separate streptococci, which are phenotypically similar to
lactococci, unless the whole catalase negative isolates on
KCA were to be gene sequenced. This is unsuitable in a
dairy lab for routine analyses. Our results confirm the
impossibility of developing a methodology dedicated to
selectively enumerating the lactococcal microflora. How-
ever, it would be interesting to extend this work to en-

Selective Lactococcus Enumeration in Raw Milk 57
compass more raw milk samples and to examine in par-
ticular the streptococcal microflora.
6. Acknowledgements
The authors would like to thank Carl Holland for English
corrections. This project was supported by the RMT
(“Réseau Mixte Technologique”) “Fromages de Terroir”
and the CASDAR project “FloracQ” (Ministère de
l’Agriculture et de la Pêche, Chambre d’agriculture du
cantal).
REFERENCES
[1] L. Quigley, O. O’Sullivan, T. P. Beresford, R. P. Ross, G.
F. Fitzgerald and P. D. Cotter, “Molecular Approaches to
Analysing the Microbial Composition of Raw Milk and
Raw Milk Cheese,” International Journal of Food Mi-
crobiology, Vol. 150, No. 2-3, 2011, pp. 81-94.
doi:10.1016/j.ijfoodmicro.2011.08.001
[2] M. Vacheyrou, A.-C. Normand, P. Guyot, C. Cassagne, R.
Piarroux and Y. Bouton, “Cultivable Microbial Commu-
nities in Raw Cow Milk and Potential Transfers from Sta-
bles of Sixteen French Farms,” International Journal of
Food Microbiology, Vol. 146, No. 3, 2011, pp. 253-262.
doi:10.1016/j.ijfoodmicro.2011.02.033
[3] S. Buchin, V. Delague, G. Duboz, J. L. Berdague, E.
Beuvier, S. Pochet and R. Grappin, “Influence of Pas-
teurization and Fat Composition of Milk on the Volatile
Compounds and Flavor Characteristics of a Semi-Hard
Cheese,” Journal of Dairy Science, Vol. 81, No. 12, 1997,
pp. 3097-3108. doi:10.3168/jds.S0022-0302(98)75874-6
[4] E. Beuvier, K. Berthaud, S. Cegarra, A. Dasen, S. Pochet,
S. Buchin and G. Duboza, “Ripening and Quality of
Swiss-Type Cheese Made from Raw, Pasteurized or Mi-
crofiltered Milk,” International Dairy Journal, Vol. 7, No.
5, 1997, pp. 311-323.
doi:10.1016/S0958-6946(97)00015-0
[5] Y. Bouton, P. Guyot and R. Grappin, “Preliminary Char-
acterization of Microflora of Comté Cheese,” Journal of
Applied Microbiology, Vol. 85, No. 1, 1998, pp. 123-131.
doi:10.1046/j.1365-2672.1998.00476.x
[6] Y. Demarigny, E. Beuvier, S. Buchin, S. Pochet and R
Grappin, “Influence of Raw Milk Microflora on the Char-
acteristics of Swiss-Type Cheeses: II. Biochemical and
Sensory Characteristics,” Le Lait, Vol. 77, No. 1, 1997,
pp. 151-167. doi:10.1051/lait:1997110
[7] N. Desmasures, “Etude de laits de Haute Qualité: Carac-
téristiques Microbiologiques et Aptitude à la Transforma-
tion en Camembert au lait cru,” Ph.D. Dissertation, Uni-
versity of Caen, Caen, 1995.
[8] E. Beuvier and S. Buchin, “Raw Milk Cheeses,” In: P. F.
Fox, P. L. H. McSweeney, T. M. Cogan and T. P. Guinee,
Eds., Cheese: Chemistry, Physics and Microbiology, 3rd
Edition, Tec & Doc Editions, Vol. 1, Paris, 2004, pp. 319-
345. doi:10.1016/S1874-558X(04)80072-1
[9] E. Casalta, R. Zennaro, M.-X. Maroselli and R. Legouar,
“Effect of Specific Starters on Microbiological, Bio-
chemical and Sensory Characteristics of Venaco, a Cor-
sican Soft Cheese,” Sciences de Aliments, Vol. 17, No. 1,
1997, pp. 79-94.
[10] B. E. Terzaghi and W. E. Sandine, “Improved Medium
for Lactic Streptococci and Their bacteriophages,” Ap-
plied and Environmental Microbiology, Vol. 29, No. 6,
1975, pp. 807-813.
[11] N. Turner, W. E. Sandine, P. R. Elliker and E. A. Day,
“Use of Tetrazolium Dyes in an Agar Medium for Dif-
ferentiation of Streptococcus lactis and Streptococcus
cremoris,” Journal of Dairy Science, Vol. 46, No. 5, 1963,
pp. 380-385. doi:10.3168/jds.S0022-0302(63)89059-1
[12] G. Waes, “The Enumeration of Aromabacteria in BD
Starters,” Netherlands Milk and Dairy Journal, Vol. 22,
1968, pp. 29-39.
[13] A. M. Huggins and W. E. Sandine, “Differenciation of
Fast and Slow Milk Coagulating Isolates in Strains of
Streptococci,” Journal of Dairy Science, Vol. 67, No. 8,
1984, pp. 1674-1679.
doi:10.3168/jds.S0022-0302(84)81491-5
[14] N. Desmasures and M. Gueguen, “Monitoring the Micro-
biology of High Quality Milk by Monthly Sampling over
2 Years,” Journal of Dairy Research, Vol. 64, No. 2,
1997, pp. 271-280. doi:10.1017/S0022029996002130
[15] J. F. Chamba, G. Bonnaz and P. Bourg, “Comparaisons
de Diverses Méthodes de Dénombrement de la Flore
Acidifiante du lait cru,” Le Lait, Vol. 61, No. 609-610,
1981, pp. 555-567. doi:10.1051/lait:1981609-61035
[16] V. Vanos and L. Cox, “Rapid Routine Method for the
Detection of Lactic Acid Bacteria among Competitive
Flora,” Food Microbiology, Vol. 3, No. 3, 1986, pp. 223-
234. doi:10.1016/0740-0020(86)90003-1
[17] M. Dalmasso, C. Hennequin, D. Duc and Y. Demarigny,
“Influence of Backslopping on the Acidifications Curves
of ‘Tomme’ Type Cheeses Made during 10 Successive
Days,” Journal of Food Engineering, Vol. 92, No. 1,
2009, pp. 50-55. doi:10.1016/j.jfoodeng.2008.10.019
[18] P. R. Elliker, A. W. Anderson and G. Hannesson, “An
Agar Culture Medium for Lactic Acid Streptococci and
Lactobacilli,” Journal of Dairy Science, Vol. 39, No. 11,
1956, pp. 1611-1612.
doi:10.3168/jds.S0022-0302(56)94896-2
[19] Z. Y. Pu, M. Dobos, G. K. Y. Limsowtin and I. B. Powell,
“Integrated Polymerase Chain Reaction-Based Procedures
for the Detection and Identification of Species and Sub-
species of the Gram-Positive Bacterial Genus Lactococ-
cus,” Journal of Applied Microbiology, Vol. 93, No. 2,
2002, pp. 353-361.
doi:10.1046/j.1365-2672.2002.01688.x
[20] V. Michel, A. Hauwuy and J. F. Chamba, “La Flore
Microbienne de laits Crus Vache: Diversité et Influence
des Conditions de Production,” Le Lait, Vol. 81, No. 5,
2001, pp. 575-592. doi:10.1051/lait:2001151
[21] D. Corroler, I. Mangin, N. Desmasures and M. Gueguen,
“An Ecological Study of Lactococci Isolated from Raw
Milk in the Camembert Cheese Registered Designation of
Origin Area,” Applied and Environmental Microbiology,
Vol. 64, No. 2, 1998, pp. 4729-4735.

Selective Lactococcus Enumeration in Raw Milk 58
[22] Y. Demarigny, C. Sabatier, N. Laurent, S. Prestoz, V.
Rigobello and M. J. Blachier, “Microbiological Diversity
in Natural Whey Starters Used to Make Traditional Ro-
camadour Goat Cheese and Possible Relationships with
Its Bitterness,” Italian Journal of Food Science, Vol. 18.
No. 3, 2006, pp. 251-266.
[23] F. Grattepanche, P. Audet and C. Lacroix, “Milk Fer-
mentation by Functional Mixed Culture Producing Nisin
Z and Exopolysaccharides in a Fresh Cheese Model,” In-
ternational Dairy Journal, Vol. 17, No. 2, 2007, pp. 123-
132.
[24] V. Monnet, D. Atlan, C. Beal, M. C. Champomier-Verges,
M. P. Chapot-Chartier, H. Chouayekh, M. Cogain-Bous-
quet, M. Deghorain, P. Gaudu, C. Gilbert, E. Guedon, I.
Guillouard, P. Goffin, J. Guzzu, P. Hols, V. Juillard, V.
Ladero, N. Lindley, S. Lortal, P. Loubiere, E. Maguin, C.
Monnet, F. Rul, R. Tourdot-Marechal and M. Yvon, “Mé-
tabolisme et Ingénierie Métabolique,” In: G. Corrieu and
F. M. Luquet, Eds., Bactéries Lactiques—De la Géné-
tique aux Ferments, Tec & Doc Editions, Paris, 2008, pp.
271-509.
[25] E. Franciosi, L. Settanni, A. Cavazza and E. Poznanski,
“Biodiversity and Technological Potential of Wild Lactic
Acid Bacteria from Raw Cows’ Milk,” International
Dairy Journal, Vol. 19, No. 1, 2009, pp. 3-11.
doi:10.1016/j.idairyj.2008.07.008
[26] N. Desmasures and E. Beuvier, “Ce qu’il Faut Savoir
Avant d’Intervenir sur les Microflores des laits,” In:
Microflore du lait cru, CNAOL and GIS Alpes Jura, 2011,
pp. 13-74.
[27] C. Jans, C. Lacroix and L. Meile, “A Novel Multiplex
PCR/RFLP Assay for the Identification of Streptococcus
bovis/Streptococcus Equinus Complex Members from
Dairy Microbial Communities Based on the 16S rRNA
Gene,” FEMS Microbiology Letters, Vol. 326, No. 2,
2012, pp. 144-150.
doi:10.1111/j.1574-6968.2011.02443.x
[28] A. Mallet, M. Guéguen, F. Kauffmann, C. Chesneau, A.
Sesboué and N. Desmasures “Quantitative and Qualita-
tive Microbial Analysis of Raw Milk Reveals Substantial
Diversity Influenced by Herd Management Practices,”
International Dairy Journal, Vol. 27, No. 1-2, 2012, pp.
13-21. doi:10.1016/j.idairyj.2012.07.009
[29] A. Badis, D. Guetarni, B. Moussa Boudjemac, D. E. Hen-
nic and M. Kihalc, “Identification and Technological Pro-
perties of Lactic Acid Bacteria Isolated from Raw Goat
Milk of Four Algerian Races,” Food Microbiology, Vol.
21, No. 5, 2004, pp. 579-588.
doi:10.1016/j.fm.2003.11.006