Evolution of the Raw Cow Milk Microflora, Especially Lactococci,Enterococci, Leuconostocs and Lactobacilli over a Successive 12 Day Milking Regime
This research was developed to track the microbial composition
of raw milk obtained from twelve successive milking operations from the
same farm with a special attention to lactococci, enterococci, lactobacilli
and leuconostocs. Enterococci, lactobacilli and leuconostocs were detected
at low levels. Lactococci represented the most abundant microflora. The
dominance of Lc lactis subsp. cremoris from days 1 to 5
was followed by the dominance of Lc lactis subsp. lactis.
Technological characterizations, Rep-PCR and whole-cell protein patterns
analyses revealed the existence of two groups of lactococci. The first
group designed as fundamental microflora included strongly acidifying
strains. They were present day after day. The second group-transitory
microflora-was observed occasionally and was less acidifying. A major
influence of the biofilms of the milking equipment was postulated to explain
the stability and the composition of the fundamental microflora. The implication
of these observations on natural whey starters` composition and cheese
particularity is discussed.
to cite this article:
Dalmasso Marion, Prestoz Sylvie, Rigobello Veronique and Demarigny Yann, 2008. Evolution of the Raw Cow Milk Microflora, Especially Lactococci,Enterococci, Leuconostocs and Lactobacilli over a Successive 12 Day Milking Regime. International Journal of Dairy Science, 3: 117-130.
Cheese making implies two main successive processes. The milk is firstly
clotted and the resulting curd acidified thereafter. Acidification results
from the metabolism of Lactic Acid Bacteria (LAB), which transform lactose
into lactic acid. This action is essential to allow the curd to reach
the proper pH value and to remove the whey. LAB can originate from four
different sources depending on the process: the raw milk, the cheese plant
environment, commercial starters and the whey from the previous cheese
making. This last practice called back slopping is still largely used
in many countries and for a great variety of processes. The whey is recovered
just after molding and incubated until the next cheese making process
(Gatti et al., 2003). During this culture, LAB level increases
dramatically as a consequence of the multiplicity of genus and species.
The resulting whey starter-designed thereafter as Natural Whey Starter
(NWS)-consists of complex microbial associations (Reinheimer et al.,
1996). The LAB population originates from three main sources, (1) the
previous cheese making, (2) the microbial environment of the cheese plant
and (3) the raw milk microflora (Bertoni et al., 2001).
The role of LAB is therefore mainly limited to the first steps of cheesemaking.
During the ripening process, starter LAB are less important than other
flora such as, for instance, Non Starter Lactic Acid Bacteria (NSLAB).
Nevertheless, they still contribute to the development of the typical
cheese aroma and texture. LAB possess many enzymes-namely endo and exopeptidases-which
can directly contribute to the appearance of sapid molecules, or ultimately
generate aromatic compounds precursors (Mauriello et al., 2001;
Herreros et al., 2003).
Natural Whey Starters (NWS) are particularly interesting for these two
enzymatic properties. The diversity of strains which composed these bacterial
communities is assumed to contribute to the enhancement of the typical
flavor of many traditional cheeses and among them many products of Controlled
Denomination of Origin (CDO).
If the microbial diversity of NWS is commonly described as positive,
a major problem may arise in case of any technological problem. In particular,
acidification defects are more and more reported by cheese makers using
NWS. Such problems may originate from diverse causes and generate a great
array of consequences among which, sudden stops or lengthening of the
milk acidification curves frequently described. However, it is at the
present time almost impossible to identify the origin of the problem and
therefore to propose an appropriate solution because of the microbial
complexity of NWS.
Among the four LAB sources which participate in the NWS diversity, raw
milk is often described as a major, although fluctuating, source of LAB
(Bachmann et al., 1996; Centeno et al., 1996; Manolopoulou
et al., 2003; Duthoit et al., 2005). The influence of the
raw milk microflora on the cheese characteristics has been demonstrated
in the past (Herreros et al., 2007), even if most of the research
was not specifically focussed on NWS. At the present time, no experiment
has been published on the specific influence of raw milk LAB on the complexity
of NWS ecosystems. Such work faces to two main difficulties. The raw milk
LAB microflora is quantitatively rather low and its contribution difficult
to assess. Moreover, the study of NWS implies a dynamic approach on several
consecutive days and little is known about the evolution of LAB from day
to day in raw milk of the same farm origin.
Consequently, the composition of raw cow milk samples coming from the
same farm was followed over 12 consecutive days. The aim of this study
was to evaluate LAB biodiversity, with a specific focus on lactococci,
leuconostocs, lactobacilli and enterococci, from day to day. In a future
work, we will study the influence of this LAB dynamic on the composition
of natural whey starters.
MATERIALS AND METHODS
Milk Sampling and Colony Enumeration
Raw cow milk samples were collected from the same farm over twelve
consecutive days in February 2006. Mesophilic aerobic microflora, leuconostocs,
enterococci, yeasts and moulds, total and faecal coliforms, Pseudomonas
were enumerated respectively on PCA, MSE, KF, GGC, VRBL and CFC agar as
described by Desmasures et al. (1997). Lactococci were checked
on Turner agar (Curk et al., 1994), facultative heterofermentative
lactobacilli on FH-agar (Isolini et al., 1990); Micrococcaceæ
on Mannitol Salt Agar (Demarigny, 1997) and corynebacteria on Cheese Ripening
Bacterial Medium (Denis et al., 2001).
After enumeration, a maximum of four colonies were selected from Turner,
MSE, KF and FH agar. After microscopic examination, cocci were purified
on M17 agar (Biokar diagnostics, 60000 Beauvais, France) (30 °C, 24
h) and bacilli strains were purified on MRS agar (Biokar diagnostics)
(anaerobiosis, 37 °C, 24 h). Each isolate was finally stored at -80
°C. One hundred and thirty six strains were collected and characterized
from raw milks -40, 40, 40 and 16 isolates respectively from MSE, Turner,
KF and FH agar.
After thawing, lactococci, leuconostocs and enterococci were cultivated
on M17 broth (Biokar diagnostics) for 24 h at 30 °C and lactobacilli
on MRS broth (Biokar diagnostics) for 24 h at 30 °C. Cells were then
harvested and washed twice in 0.09 g L-1 NaCl (Sigma) distilled
water. Optical density at 600 nm was adjusted between 0.2 and 0.3. This
final suspension was used to inoculate at 2% the characterization media.
All the incubations were made at 30 °C.
Biochemical Characterization of Lactococci and Leuconostocs
Salt resistance, growth at 15 and 37 °C, sugars fermentation,
arginine dihydrolase capability and citrate utilization were studied as
described by Demarigny (1997).
Biochemical Characterization of Enterococci
Sugar fermentation was tested on API 20A basic medium (BioMérieux,
69280 Marcy l`Etoile, France). 0.5 mL of a saccharose, glycerol or L-arabinose
solution (50 g L-1, Sigma) was added to 4.5 mL of medium. Growth
at 45 °C and pH = 9.6 was performed on M17 broth. Citrate utilization
was checked on KCA agar (Nickels and Leesment, 1964).
Biochemical Characterization of Lactobacilli
Catalase test, arginine dihydrolase capability, citrate utilization,
growth at 15 and 45 °C, production of CO2 and sugar fermentation
for the characterization of lactobacilli were performed as described by
Isolates were all analysed for the following technological aptitudes:
acidification abilities, proteolysis, autolysis and behaviour on lithmus
milk as described by Demarigny et al. (2006).
Whole-Cell Protein Patterns
The procedure followed was described by Demarigny et al. (2006)
to study the diversity of lactococci from natural whey starters.
Extraction of Total DNA
From a single culture broth incubated at 30 °C for 24 h, total
DNA was extracted by using the Nucleospin tissue kit (Macherey-Nagel,
67722 Hoerdt, France).
PCR Amplification and Gel Electrophoresis
The strains were confirmed to belong to Enterococcus, Leuconostoc
mesenteroides and Lactococcus lactis subsp. lactis
and cremoris by means of PCR-based methods. Enterococcal DNA were
amplified using primers E1 and E2 (Sigma), biding to positions 632-646
and 1353-1369, respectively and corresponding to positions in the
E. coli 16S rRNA sequence, according to Deasy et al. (2000).
16S rDNA fragments of Ln. mesenteroides were amplified using primers
Lnm1, corresponding to conserved E. coli 16S rRNA position 185
forward and Lnm2 (position 470 reverse) (Sigma) according to Cibik
et al. (2000). Lactococcus lactis subsp. lactis or subsp.
cremoris DNA amplification was performed using primers His1 and
His2 (Sigma), biding to positions 671-688 and 1587-1604, respectively
and corresponding to positions in Lc. lactis subsp. lactis
NCDO 2118 numbering, according to Corroler et al. (1998). Inter-Repetitive
Extragenic Palindromic sequences were amplified by means of two 18-mer
primers in combination (Rep1R-Dt, Rep2-D) (Sigma) as described by Bouton
et al. (2002) for REP-PCR. In all cases, amplification reactions were
performed in a final volume of 25 μL containing 1x reaction PCR buffer
(Sigma), 0.4 μM of each opposing primers, 1 mM MgCl2 (Sigma),
0.2 μM of each deoxynucleoside triphosphate (Sigma), 0.5U Tap DNA
Polymerase (Sigma) and 5 μL of DNA. The primer sequences and the
PCR amplification conditions applied are recapitulated on Table
1. Amplification cycles were performed with a Thermal Cycler (Biorad,
92430 Marne-la-Coquette, France). PCR products of 25 μL were electrophoresed
in a 10 g L-1 Seakem GTG agarose gel (Sigma) in TBE (Tris -
Borate - EDTA pH 8) at 100 V for 3 h. The 123-pb DNA ladder (Invitrogen,
95613 Cergy Pontoise, France) was used as a size standard. The DNA fragments
were stained with ethidium bromide (Sigma), viewed under UV light (302
nm, Biorad) and photographed on a digital camera (Camedia C-5060, Olympus).
|| Primer sequences, amplification and application of
Computer Analyses of REP-PCR Profiles
The band patterns were normalized and processed according to the same
procedure as whole-cell protein patterns. The similarity coefficient (80%)
of the Rep-PCR technique was evaluated by studying two strains six fold.
Correlations, Multiple Correspondence Factorial Analyses (MCFA) between
isolates and technological abilities and Hierarchical Classifications
(HC) were performed using the STATITCF software (5th version, 1995, Institut
Techniques des Céréales et des Fourrages, Paris, France).
The mesophilic aerobic microflora level ranged between 3.2 ±
0.08 and 5.2 ± 0.08 log cfu mL-1. Corynebacteria and
Micrococcaceæ were present systematically at constant levels, respectively
3.4 ± 0.12 and 2.2 ± 0.16 log cfu mL-1 (Fig.
1). Yeast counts were inferior to 1.0 ± 0.2 log cfu mL-1,
except on days 2, 8, 11 and 12 (2-3 ± 0.2 log cfu mL-1).
Moulds were detected irregularly in milk and at levels that barely exceeded
2.0 ± 0.66 log cfu mL-1, except on days 11 and 12 (over
3.0 ± 0.66 log cfu mL-1). Coliforms and Pseudomonas
were temporarily found at levels ranging from 1.2 to 4.4 ± 0.4
log cfu mL-1.
The evolution of the four main LAB microflora was based on the use of
selective-FH agar, KF-and elective-Turner, MSE-media. The selectivity
of these media being erratic, it is admitted to refer to a specific population
by adding the term presumed. In some cases however, the gap between presumed
and real levels is too large to be relevant. As a consequence, even the
additional use of presumed does not refer to any correct tendency. In
this study, this difficulty was overcome by characterizing isolates from
the four LAB media. 136 isolates were picked following the number of colonies
enumerated each day: 16 from FH agar, 40 from each other media. These
isolates were identified by means of phenotypic and PCR-based methods.
The isolates from Turner and FH media were all classified as respectively
Lactococcus and Lactobacillus. Thirteen isolates out 40
from MSE were identified as Leuconostoc, while the 27 remaining
isolates were further characterized as lactococci.
||Evolution of the non-LAB microflora- ()
mesophilic aerobic microflora, ()
total coliforms, ()
faecal coliforms, ()
Micrococaceae-in raw cow milk during twelve successive days of milking
||Evolution of the LAB population- ()
lactobacilli-in raw cow milk during twelve successive days of milking
Thirty-one isolates out of 40 from KF were referred to Enterococcus
genus. It was not possible to clearly link the nine other isolates with
an identified population. These results allowed to calculate the correct
levels corresponding to each population. For instance on day 1, if two
isolates from KF out of four were clearly identified as enterococci, the
number deduced from plate counting was weighted by a coefficient equal
to 2/4 (Fig. 2).
Lactococci level was relatively stable from day 1 to day 4 (between 3.0
and 5.0 ± 0.1 log cfu mL-1) and decreased thereafter
to reach 2.0 ± 0.1 log cfu mL-1 on days 9 and 10. Then,
the number of lactococci increased again. Enterococci were detected every
day but never overstepped 2.0 ± 0.4 log cfu mL-1.
||Main enterococcal groups- ()
III-obtained by hierarchical classification for twelve days of milking
Lactobacilli were only detected on days 2, 11 and 12. Leuconostocs were
detected everyday except on days 1 and 8. The levels of these two populations
ranged between 2.4 and 4.0 ± 0.3 log cfu mL-1. A strong
correlation was observed between leuconostocs and lactobacilli (r = 0.95).
It is noteworthy that lactococci, mesophilic aerobic microflora and total
coliforms were strongly correlated (r>0.82), inferring a potential
common origin for these three microflora.
Leuconostocs and Lactobacilli
The thirteen strains belonging to the Leuconostoc genus appeared
to have the phenotypic characteristics of Ln mesenteroides subsp.
mesenteroides. Leuconostocs mean acidifying and proteolytic activities
were rather weak, respectively 0.20 ± 0.02 pH units and 9.2 ±
4 μmoles of glycine mL-1.
Lactobacilli were characterized by phenotypic means. They all belonged
to the facultative heterofermentative lactobacilli group. Further identifications
indicated that the isolates were classified as Lb plantarum or
Lb paracasei subsp. paracasei.
Fourteen isolates were identified as Enterococcus fæcalis,
ten as Ec durans and two as Ec fæcium. Five enterococci
could not be accurately identified. Distribution of these three populations
during the 12 days did not reveal any specific trend. Ec fæcalis
strains were more commonly identified at the beginning of the period and
Ec durans more common at the end. Despite this dual distribution,
no correlation was observed between the three species.
Enterococci were divided into three groups-I, II, III-thanks to HC analysis
(Fig. 3). The mean acidifying and proteolytic activities
were equal to 0.66 ± 0.06 pH units and 3.6 ± 0.4 μmoles
of glycine mL-1 for group I strains, 0.56 ± 0.08 pH
units and 2.4 ± 0.7 μmoles of glycine mL-1 for
group II strains and 0.26 ± 0.13 pH units and 1.7 ± 0.9
μmoles of glycine mL-1 for group III strains. A negative
correlation was observed between III and II or III and I populations r
= -0.68. Group III strains were generally present when the strains of
the other groups were absent or at low rates.
||Main lactococcal groups- ()
C and ()
erratic strains-obtained by hierarchical classification for twelve
days of milking
Forty lactococci were picked from Tuner agar and 27 from MSE agar.
All these isolates displayed the phenotypic features of the subspecies
Lactococcus lactis subsp. lactis. For example, they
all showed positive arginine dihydrolase capability. They were, also,
able to grow in a 40 g L-1 NaCl culture broth and to use maltose
and ribose. Growth in litmus milk indicated that over 95% of the isolates
were able to reduce, acidify and clot the milk in less than 24 h. None
of the strains were able to use citrate to produce diacetyl.
The confirmation of the species identification was based on the identification
of the histidine biosynthesis operon region, because of its Lc lactis
species specificity. Based on its size, the resulting fragment was
subspecies specific. Lc lactis subsp. cremoris strains have
a DNA sequence of about 200 bp between the orf3 and hisC genes,
which is not present in Lc lactis subsp. lactis strains.
This work showed that all the strains belonged to Lc lactis species.
However, 50% of the strains were classified in the cremoris subspecies.
From day 1 to day 4, only Lc lactis subsp. cremoris was
present in the milk. On day 5, a change was observed, the two subspecies
being equally represented. Thereafter, Lc lactis subsp. cremoris
disappeared to be replaced by Lc lactis subsp. lactis.
MCFA and HC analysis allowed to separate lactococci into four main groups,
A, B, C and erratic strains (Fig. 4). If autolysis was
roughly identical irrespective of the strains, some slight differences
were observed on the basis of proteolysis and acidification aptitudes.
The mean acidifying and proteolytic activities were equal to 1.70 ±
0.01 pH units and 6.7 ± 0.6 μmoles of glycine mL-1
for group A strains, 1.58 ± 0.23 pH units and 5.1 ± 1.6
μmoles of glycine mL-1 for group B strains and 1.74 ±
0.14 and 6.4 ± 1.1 μmoles of glycine mL-1 for group
C strains. Strains which could not fall into one of these three groups
were designed as erratic strains.
From days 1 to 6, the proportion of A strains decreased gradually in
time. A strains represented 75% of the lactococci isolates on day 2 and
only 15% on day 6. At the same time, group C became more and more important
from day 2-25% of the isolates-to day 6-85% of the isolates. C strains
suddenly disappeared thereafter, to be replaced by B strains. Erratic
strains represented in some cases the dominant flora among lactococci
isolates. Their presence was rather congruent with that of B strains.
||Dendrogramme of 58 lactococcal whole-cell protein patterns
by SDS-PAGE. Each pattern is identified by a milk number indicating
the origin of the strain, e.g., all the lactococcal strains from milk
1 are labelled Milk 1 on the dendrogramme. Clusters are materialized
by bold horizontal lines and numbered from 1 to 11. The similarity
coefficient of 90% is materialized by a bold vertical line
The whole-cell protein patterns of the 67 lactococci were analyzed by
SDS-PAGE. Nine strains providing unusable profiles were not kept. The
58 remaining patterns were divided into 11 strain clusters, on the basis
of a similarity coefficient of 90% (Fig. 5). Clusters
6 and 8 grouped 21 and 19 profiles i.e., 69% of the lactococci strains.
Among these two clusters, strains originated from milks 1 to 11, meaning
that along this period, a fundamental microflora had settled durably.
The 18 other patterns were shared among the nine remaining clusters. They
corresponded with strains isolated from milks 3, 4, 6 and 8 to 12. Compared
with the leading microflora represented by clusters 6 and 8, this secondary
microflora could be designed as transitory.
It is interesting to note that these results partially confirmed the
observations deduced from acidifying abilities and proteolysis analysis.
C strains profiles were distributed among several clusters. A and B strains
profiles were essentially concentrated in clusters 6 and 8. These strains
could be linked with the fundamental microflora and C and erratic strains
to the transitory microflora. The distribution of A, B and C strains among
the clusters allowed to calculate the diversity coefficient H = Σ
Pi log Pi (with Pi = number of strains in cluster i/total number of strains)
(Shannon and Weaver, 1983). A, B and C populations diversity coefficients
were respectively equal to 30, 54 and 71%. A population was the less diversified,
strains being shared among clusters 6 and 8. C population included six
||Example of a dendrogramme of lactococcal profiles by
REP-PCR. Each pattern is identified by a milk number indicating the
origin of the strain, e.g., all the lactococcal strains from milk
1 are labelled Milk 1 on the dendrogramme. Clusters are materialized
by bold horizontal lines and numbered from 1 to 8. The similarity
coefficient of 80% is materialized by a bold vertical line
A second analysis was made by Rep-PCR. An example of the results obtained
with this technique is displayed on Fig. 6. Eight clusters
were obtained. 83% of the lactococci classified in the SDS-PAGE cluster
6 were again in the same cluster (3). This observation confirms the existence
of some form of stable leading microflora in the milk from day to day.
The other strains, in particular those from SDS-PAGE cluster 8, were not
grouped into a specific cluster and were scattered among many clusters.
The origin of the raw milk microflora is still currently under study.
The potential sources of contamination include the skin of the cow, feed
dusts, milking equipment and finally the environment of the cheese plant.
Among the different microflora which can be identified in raw milk, lactic
acid bacteria seem particularly attractive for the cheese maker. These
bacteria, namely lactococci and lactobacilli, are acknowledged to contribute
to curd acidification. They are also suspected to bring interesting features-directly
during the cheese making process or thereafter during the ripening stage-
to the cheese. Such elements enable to give the cheese its original flavour
typical of its geographical origin. These bacteria are mainly characterized
by their strong rusticity. This quality leads them to colonize the curd
at the end of the cheese making process, in spite of hard selective conditions:
their level is at least 10000 times lower than the starter, some nutrients
are rapidly falling in numbers-non protein nitrogen for instance-, acidification
is very intense and in some cases, the technological temperature is far
from their optimum.
This work was undertaken to monitor the evolution of the main LAB microflora
during twelve consecutive days of milking, with a specific focus on the
Lactococcus group. It allowed the study of the LAB microflora dynamic
from day to day, which had never been done before at such an extensive
scale. Three different methodologies were combined to study enterococci,
leuconostoc, lactobacilli and lactococci: technological aptitudes, PCR
methods and whole-cell protein patterns. Corsetti et al. (2003)
followed a similar approach-wall-cell protein patterns, RAPD-to study
sourdough lactic acid bacteria. They concluded to the effectiveness of
the combination of two different methods to classify invasive microbial
populations. In a recent study, we also underlined the necessity to cross
two different microbial techniques to observe NWS, each method giving
complementary results (Demarigny et al., 2006).
On a twelve-day period, the mean mesophilic aerobic flora was centered
around 4.0 ± 0.08 log cfu mL-1, with some variations.
These values are similar to the observations already made by many authors
on raw milk samples. The evidence of a correlation between lactococci,
coliforms and mesophilic aerobic flora emphasizes a potential common origin.
Lactococci levels, in particular, are frequently close to mesophilic aerobic
flora levels. Lactococci probably originate from biofilms present in the
milking equipment. This assertion is based on the results obtained by
Laithier et al. (2004), which showed that biofilms were mainly
composed of LAB, the other flora being generally-yet not systematically-far
less important. In a recent study, Kagkli et al. (2007) established
that milk enterococci and lactobacilli also originated from milking equipment.
In our case, however, no correlation was observed between the levels of
these two populations.
Although frequently counted, enterocci did not reach high levels in milk.
Isolates were identified as Ec faecium, Ec faecalis and
Ec durans, three species commonly found in raw milks (Aquilanti
et al., 2006). Their technological interest appeared limited because
of low milk acidifying and proteolytic abilities, which is in accordance
with Morandi et al. (2006). Some trends were observed following
the acidifying proteolytic characteristics of the strains isolated. Lactobacilli
were inconstant as observed by Bouton et al. (2005). These authors
indicated that variations in the levels of fermentative lactobacilli could
be partially explained by the type of feeding used by the dairy farmer,
farm practices and general hygiene of the premises.
The specific enumeration of leuconostocs was particularly difficult to
achieve. The MSE medium only allowed counting dextrane-producing strains
of leuconostocs. This lack of selectivity was made worse by the pressure
exerted by the other microorganisms able to grow on MSE (Corry et al.,
2007). In spite of this problem, we only found small numbers of Leuconostoc
mesenteroides subsp. mesenteroides. This observation reinforces
the assertion that bacteria from the Leuconostoc genus are a minor
microflora in milk (Zamfir et al., 2006).
Lactococci appeared to be the principal LAB population, their proportion
ranging between 93 and 100% of the total LAB microflora. They, also, constituted
the major part of the milk microflora. Their levels varied considerably,
from 2.0 to 5.0 ± 0.1 log cfu mL-1. These results are
congruent with former data. For instance, Corroler et al. (1998)
indicated that lactococci varied between 2 and 3 log cfu mL-1,
following the origin of the sample and the season.
All lactococci were confirmed to be part of the Lactococcus lactis
species. Most of strains exhibited phenotypic characteristics of the subspecies
lactis whereas they have been found to own the genetic profile
of the subspecies cremoris. This observation is in complete agreement
with the study completed by Ward et al. (1998) in which more than
70% of Lc lactis were found to have the phenotype of the subspecies
lactis and the genotype of the subspecies cremoris. This
ambiguous taxonomic assignment of Lc lactis strains can be directly
compared to the works of Salama et al. (1995). Actually, these
authors reported that phenotypic changes from lactis to cremoris
subspecies had been observed before. It is particularly true for the phenotypic
change from arginine negative to arginine positive capability, which is
attributed to transducing phage. Consequently, several methods have to
be used and new phenotypic characteristics have to be investigated in
order to discriminate properly the two subspecies lactis and cremoris.
For example, Nomura et al. (1999) proposed to test the presence
of γ-aminobutyric acid, a by-product only produced by the subspecies
lactis. We can wonder, however, if the phenotypic characterisation
is still a relevant method to identify lactococci.
During the 12 day period, we observed two different behaviors. From day
1 to day 6, Lc lactis subsp. cremoris was the only subspecies
identified, replaced later by Lc lactis subsp. lactis. If
we suppose a major influence of biofilms on the LAB population, this shift
appears surprising. Indeed, dairy farm practices were identical from day
to day during the twelve consecutive days. Otherwise, we can argue with
Salama et al. (1995) that Lc lactis subsp. cremoris
acquired its lactis characteristics inside the biofilm by transducing
phages. This hypothesis would be then fundamental. It would then imply
that the release of Lactococcus strains from biofilms would be
accompanied by the concurrent release of phages. If raw milk lactococci
are intended to settle and even to dominate in NWS, the origin of acidification
defects during cheese making would be possibly explained by these phages
Moreover, in a preceding study (Demarigny et al., 2004) NWS high
levels of Lc lactis subsp. cremoris were observed in cheese.
This population displayed the phenotypic features of the cremoris
subspecies. Considering that Lc lactis subsp. cremoris are
frequently added in commercial starter (Coppola et al., 2006),
it would indicate that NWS dominant populations partly originate from
the added starter.
The analysis of Lactococcus strains patterns indicated the occurrence
of a fundamental microflora, exhibiting stable and interesting technological
behaviors and a transitory microflora. This observation is always congruent
with the biofilm hypothesis and the observations made by Laithier et
al. (2005). The authors reported that lactococci and more generally
LAB from biofilms could be classified among ten different classes following
Our results were confirmed by using three methodological means at the
same time. Unlike Casalta et al. (2005), the Rep-PCR characterization
partially failed to disclose different patterns. This technique may therefore
be inappropriate to study the lactococci diversity from the same ecosystem.
Apart from the fundamental population, the presence of SDS-PAGE patterns
corresponding to particular technological features-less important acidification
ability-could indicate another source of lactococci, derived from the
milk bulk and not from biofilm. The diversity coefficients calculation
seemed to confirm that the transitory microflora was more diverse than
the fundamental microflora. This could confirm a different origin of the
The dynamics and the characteristics of the main LAB populations in raw
cow milk were studied on a twelve-day period of milking. It is allowed
to suppose that lactococci which dominated over the other LAB exerted
an inhibition effect. It will be interesting to test in another study
the ability of wild raw milk lactococci to produce antimicrobial compounds,
active against the other LAB microflora.
According to preceding research, we considered biofilms, which settle
in the milking equipment, as the main origin of lactococci. We postulated
the influence of transducing phages, as indicated by Salama et al.
(1995) to explain the stability of the lactococci characteristics. It
would imply that the study of biofilms phages would be of a great interest
to sort out cheese making defects. We can still wonder if the diversity
of raw milk microflora contributing to the cheese particularity is not
initially due to the exchanges of transducing phages.
Finally, it could be interesting to monitor the LAB microflora on many
farms for many weeks. The LAB dynamics and specific farm practices have
to be taken into account too.
The authors would like to thank Marc Chareyron for his help in the writing
of this paper and Fanny Moignard for her technical support.
1: Aquilanti, L., L. DellAquila, E. Zannini, A. Zocchetti and F. Clementi, 2006. Resident lactic acid bacteria in raw milk Canestrato Pugliese cheese. Lett. Applied Microbiol., 43: 161-167.
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2: Bachmann, H.P., D.A. McNulty, P.L.H. McSweeney and M. Rüegg, 1996. Experimental designs for studying the influence of the raw milk flora on cheese characteristics: A review. J. Soc. Dairy Tech., 49: 53-56.
Direct Link |
3: Bertoni, G., L. Calamari and M.G. Maianti, 2001. Producing specific milks for speciality cheeses. Proc. Nutr. Soc., 60: 231-246.
4: Bouton, Y., P. Guyot, E. Beuvier, P. Tailliez and R. Grappin, 2002. Use of PCR-based methods and PFGE for typing and monitoring homofermentative lactobacilli during Comte cheese ripening. Int. J. Food Microbiol., 76: 27-28.
Direct Link |
5: Casalta, E., J.M. Cachenaut, C. Aubert, F. Dufrene, Y. Noel and E. Beuvier, 2005. Application of specific starters for the manufacture of Venaco cheese. Lait, 85: 205-222.
Direct Link |
6: Centeno, J.A., S. Menendez and J.L. Rodriguez-Otero, 1996. Main microflora present as natural starters in Cebreiro raw cow's-milk cheese (Northwest Spain). Int. J. Food Microbiol., 33: 307-313.
Direct Link |
7: Cibik, R., E. Lepage and P. Tailliez, 2002. Molecular diversity of Leuconostoc mesenteroides and Leuconostoc citreum isolated from traditional french cheeses as revealed by RAPD fingerprinting, 16S rDNA sequencing and 16S rDNA fragment amplification. Syst. Applied Microbiol., 23: 267-278.
CrossRef | PubMed | Direct Link |
8: Coppola, S., V. Fusco, R. Andolfi, M. Aponte, G. Blaiotta, D. Ercolini and G. Moschetti, 2006. Evaluation of microbial diversity during the manufacture of Fior di Latte di Agerola, a traditional raw milk pasta-filata cheese of the Naples area. J. Dairy Res., 73: 264-272.
Direct Link |
9: Corroler, D., I. Mangin, N. Desmasures and M. Gueguen, 1998. An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area. Applied Environ. Microbiol., 64: 4729-4735.
Direct Link |
10: Corry, J.E.L., B. Jarvis, S. Passmore and A. Hedges, 2007. A critical review of measurement uncertainty in the enumeration of food micro-organisms. Food Microbiol., 24: 230-253.
Direct Link |
11: Corsetti, A., M. De Angelis, F. Dellaglio, A. Paparella, P.F. Fox, L. Settanni and M. Gobbetti, 2003. Characterization of sourdough lactic acid bacteria based on genotypic and cell-wall protein analyses. J. Applied Microbiol., 94: 641-654.
Direct Link |
12: Curk, M.C., J.M. Boeufgras, B. Decaris, F. Gavini, K. Kersters, J.P. Larpent, P. Le Bourgeois, P. Renault, H. de Roissart and C. Rouvier, 1994. Methods Used for the Identification of Lactic Acid Bacteria. Dans: Bactéries lactiques. In: Lactic Acid Bacteria, De Roissart, H., F.M. Luguet, H. Roissart and F.M. Luguet (Eds). Vol. 1. Uriage, Lorica, pp: 141-168
13: Deasy, B.M., M.C. Rea, G.F. Fitzgerald, T.M. Cogan and T.P. Beresford, 2000. A rapid PCR based method to distinguish between Lactococcus and Enterococcus. Syst. Applied Microbiol., 23: 510-522.
Direct Link |
14: Demarigny, Y., 1997. Influence of raw milk microflora and technological parameters (salt contents and ripening temperature) on the evolution of the biochemical, microbiological and sensorial characteristics of semi-hard cheese. Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation. Poligny, France.
15: Demarigny, Y., C. Sabatier and M.J. Blachier, 2004. Control of the use of natural whey starters in the making of Rocamadour cheese, a PDO raw goat milk cheese. In: Proceedings of the Produits Alimentaires Fermiers Conference, Clermont-Ferrand, France.
16: Demarigny, Y., C. Sabatier, N. Laurent, S. Prestoz, V. Rigobello and M.J. Blachier, 2006. Microbiological diversity in natural whey starters used to make traditional Rocamadour goat cheese and possible relationships with its bitterness. Italian J. Food Sci., 18: 261-276.
Direct Link |
17: Denis, C., M. Gueguen, E. Henry and D. Levert, 2001. New media for the enumeration of cheese surface bacteria. Lait, 81: 365-379.
Direct Link |
18: Desmasures, N., F. Bazin and M. Gueguin, 1997. Microbiological composition of raw milk from selected farms in the Camembert region of Normandy. J. Applied Microbiol., 83: 53-58.
Direct Link |
19: Duthoit, F., L. Tessier and M.C. Montel, 2005. Diversity, dynamics and activity of bacterial populations in registered designation of origin salers cheese by single-strand conformation polymorphism analysis of 16S rRNA genes. J. Applied Microbiol., 98: 1198-1208.
Direct Link |
20: Gatti, M., C. Lazzi, L. Rossetti, G. Mucchetti and E. Neviani, 2003. Biodiversity in Lactobacillus helveticus strains present in natural whey starter c J. Applied Microbiol., 95: 463-470.
Direct Link |
21: Herreros, M.A., J.M. Fresno, M.J. Gonzalez-Prieto and M.E. Tornadijo, 2003. Technological characterization of lactic acid bacteria isolated from Armada cheese (a Spanish goat's milk cheese). Int. Dairy J., 13: 469-479.
Direct Link |
22: Herreros, M.A., R. Arenas, M.H. Sandoval, J.M. Castro, J.M. Fresno and M.E. Tornadijo, 2007. Effect of addition of native cultures on characteristics of Armada cheese manufactured with pasteurised milk: A preliminary study. Int. Dairy J., 17: 328-335.
Direct Link |
23: Isolini, D., M. Grand and H. Glattli, 1990. Selective medium for the detection of obligatory and facultative hetefermentative Lactobacilli. Schweiz Milchw Forschung, 19: 57-59.
24: Kagkli, D.M., M. Vancanneyt, C. Hill, P. Vandamme and T.M. Cogan, 2007. Enterococcus and Lactobacillus contamination of raw milk in a farm dairy environment. Int. J. Food Microbiol., 114: 243-251.
Direct Link |
25: Laithier, C., Y.M. Chatelin, H. Tormo and Y. Lefrileux, 2004. Biofilms in farms producing goat cheese: Localisation, nature and role on products quality. Proceedings of the 11th Rencontres Recherche Ruminant Conference, December 8-9, 2004, Paris, France, pp: 112-112
26: Laithier, C., Y.M. Chatelin, R. Talon, J. Barral, H. Tormo and Y. Lefrileux, 2005. Effectiveness in laboratory then in farms of cleaning/desinfection processes on positive selection of biofilms. Proceedings of the 12th Rencontres Recherche Ruminants Conference, December 7-8, 2005, Paris, France, pp: 367-370
27: Manolopoulou, E., P. Sarantinopoulos, E. Zoidou, A. Aktypis, E. Moschopoulou, I.G. Kandarakis and E.M. Anifantakis, 2003. Evolution of microbial population during traditional feta cheese manufacture and ripening. Int. J. Food Microbiol., 82: 153-161.
Direct Link |
28: Mauriello, G., L. Moio, G. Moschetti, P. Piombino, F. Addeo and S. Coppola, 2001. Characterization of lactic acid bacteria strains on the basis of neutral volatile compounds produced in whey. J. Applied Microbiol., 90: 928-942.
Direct Link |
29: Morandi, S., M. Brasca, C. Andrighetto, A. Lombardi and R. Lodi, 2006. Technological and molecular characterization of enterococci isolated from North-West Italian dairy products. Int. Dairy J., 16: 867-875.
Direct Link |
30: Nickels, C. and H. Leesment, 1964. Methods for differentiation and quantitative determination of starter bacteria. Milchwissenschaft, 19: 374-378.
31: Nomura, M., H. Kimoto, Y. Someya and I. Suzuki, 1999. Novel characteristic for distinguishing Lactococcus lactis subsp lactis from subsp cremoris. Int. J. Syst. Bacteriol., 49: 163-166.
Direct Link |
32: Reinheimer, J.A., A. Quiberoni, P. Tailliez, A.G. Binetti and V.B. Suarez, 1996. The lactic acid microflora of natural whey starters used in Argentina for hard cheese production. Int. Dairy J., 6: 869-879.
CrossRef | Direct Link |
33: Salama, M.S., T. Musafija-Jeknic, W.E. Sandine and S.J. Giovannoni, 1995. An ecological study of lactic acid bacteria: Isolation of new strains of Lactococcus including Lactococcus lactis subspecies cremoris. J. Dairy Sci., 78: 1004-1017.
Direct Link |
34: Shannon, G.E. and W.W. Weaver, 1963. The Mathematical Theory of Communities. 1st Edn., University of Illinois Press, Urbana, ISBN: 978-0252725463, pp: 111-125
Direct Link |
35: Ward, L.J.H., J.C.S. Brown and G.P. Davey, 1998. Two methods for the genetic differentiation of Lactococcus lactis ssp. lactis and cremoris based on differences in the 16S rRNA gene sequence. FEMS. Microbiol. Lett., 166: 15-20.
Direct Link |
36: Zamfir, M., M. Vancanneyt, L. Makras, F. Vaningelgem and K. Lefebvre et al., 2006. Biodiversity of lactic acid bacteria in Romanian dairy products. Syst. Applied Microbiol., 29: 487-495.
CrossRef | PubMed |
37: Bouton, Y., L. Tessier, P. Guyot and E. Beuvier, 2005. Relationships between dairy farmer practices and the levels of microbiological populations in milk used for Comté cheese-making. Proceedings of the 12th Rencontres Recherche, (RR'05), Paris, France, pp: 403-403