International Dairy Journal
International Dairy Journal
International Dairy Journal
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,
14
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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).
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
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
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
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
(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
mL-1)
Fig. 2. Distribution according to the standard plate count of raw milk samples containing 10% presumed ripening bacteria.
17
4. Discussion
4.1. Variability of quantitative microbiological prole of raw milk
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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.
Table 3
Farm practices having signicant effects on microbial counts.a
Practices
Type
Compared with
Inuence on
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
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