Desirable Somatic Cell Role on Swiss-type
Cheese Making Properties and Final Quality
Na Li1,2,3, R. Richoux3, N. Leconte1,2, C. Bevilacqua4,5,6, M.B. Maillard1,2, S. Parayre1,2,
L. Aubert-Frogerais3, J. Warlouzel3, E. Moya-Leclair4,5, C. Denis4,5,6, P. Martin4,5, V. Gagnaire1,2
INRA, UMR 1253, Science et Technologie du Lait et de l’Œuf, 65 rue de Saint Brieuc, 35042 Rennes, France
Agrocampus Ouest, UMR 1253, Science et Technologie du Lait et de l’Œuf, 65 rue de Saint Brieuc, 35042 Rennes, France
3 Actalia, BP 50915, 35009 Rennes Cedex, France
4 INRA, UMR 1313, Génétique Animale et Biologie Intégrative, 78350 Jouy-en-Josas, France
5 AgroParisTech, UMR 1313, Génétique Animale et Biologie Intégrative, 78350 Jouy-en-Josas, France
6 Plateforme @BRIDGe, 78350 Jouy-en-Josas, France
1
2
[email protected]
water
Milk Somatic Cells: an element naturally present in milk

Milk Somatic Cells: macrophages, PMNs*, lymphocytes, epithelial cells

Participation in udder defense
proteins
lipids
lactose
minerals
ACCEPTED
Bovine milk grading in France
Healthy
Normal
Infected
Abnormal
Somatic cell count x103 /mL
<100
100 – 250
250-400
>400
For dairy industries
Good
Acceptable
Acceptable
(+ penalty)
Refused

Concomitant  udder inflammation/infection
 bacterial contamination
 modification of milk components

Somatic cell count the indicator of udder health and milk quality
one criterion of milk payment system
What are the effects of somatic cells
in dairy processing and products ?
*PMNs: Polymorphonuclear neutrophils
1
Numerous concomitant factors
with the presence of somatic cells in milk
Mac

PMN
Lym
Mac
Literature[1-3]:“Effects
of somatic cells”

- on the quality of milk, cream and powder
Methods used:
- mixed milk from healthy and mastitic cows
Conclusion: worsened dairy processing and products
Lym
Source of enzymes
lipases

- individual milk with different somatic cell counts
PMN
e.g. proteases
- on the cheese-making properties and quality …

Somatic cell role?
Participation in cheese
making and quality [4-5]
Positive
Negative
Are Somatic Cells Foe or Friend
Real role of somatic cells in cheese making and quality
without the biases of other co-factors ?
References: 1 (Chen et al., 2010); 2 (Fernandes et al., 2008); 3 (Ma et al., 2000); 4 (Sanchez-Macias et al. 2013); 5 (Souza et al. 2012)
2
Strategy
Somatic cell role?
Adding controlled somatic cell counts in cell-free cheese milk
Swiss-type Cheeses
without addition of
propionibacteria
Permeate: partially skimmed milk
without
Microfiltration
5µm
CTL
LSCC
×3
HSCC
×3
×3
Cheese milk
somatic cells
0
Skimmed milk
Skimming
400 000
1 000 000 cells/mL
Standardisation in fat content
Cream
Raw milk
Retentate: Milk Fraction
Standardisation in somatic cells
enriched in somatic cells
Total somatic cell count:
Reference method + RT qPCR (DNA)

Characterization of somatic cells
Flow cytometry:
Identification/distribution/viability

Cheese making properties
Drainage
Acidification

Cheese ripening (70 days)
Proteolysis
lipolysis
3
Results
new
Characterization of somatic cells during cheese manufacture
Raw milk
Cheese milk
Retentate
Cheeses
qPCR-cell DNA
×1 000 cell/mL
×12.3
4.5×105
5.5×106
Retentate
Raw milk
Units: cells/mL
Target Data
CTL
0
2.3
LSCC 400 405.7
HSCC 1000 935.3
LSCC:83%
HSCC:73%
Trapped in cheese
at day 1 of ripening
Cheese milk
Macrophages
33±3%
68±3%
49±3%
52±2%
Raw milk
10±1%
14±1%
22±4%
37±2%
Retentate
Lymphocytes
Cheese milk
Cell viability
Flow cytometry
Cell viability (%)
60
PMNs
Distribution
Flow cytometry
15±1%
Total Count
Reference method: cell counter
Dramatic decrease in cell viability
- microfiltration
- heat treatment
20
Viability
40
0
Raw Milk
Retentate
Cheese milk
4
Results
Parameters of cheese making: drainage and acidification
CTL
LSCC
HSCC
Acidification (pH)
5.1
5.1
5.2
Dry Matter (%)
61.7
61.4
61.7
Fat in DM (%)
46.5
46.0
45.7
MNFS* (%)
1.16
1.16
1.14
Cultivability of lactic acid bacteria
S.Thermophilus + L.helveticus
Cheese Ripening: 0, 1, 8, 21, 35, 49, 70 days
Literature:
Acidification: ↗2-21%
[1]
Rennet time : ↗4-24%
[2-3]
Dry matter: ↘3.4-6.7%
[4]
Somatic cells
DO NOT worsen
the cheese making
*MNFS: Moisture in No Fat Substance
1 (Politis et al., 1988); 2(Rogers et al., 1994); 3 (Mazal et al., 2007); 4 (Barbano et al., 1991)
5
Results
Lipolysis indices in cheese ripening
Individual free fatty acids
Total free fatty acids
CTL
g/kg cheese
aa
0.15
0.6
0.4
28.3±4.4 %
LSCC HSCC
a
a
b
∆24%
∆33%
0.2
b
b
*
0
400
900
Somatic cell count in cheese milk (x1000 cells/mL)
a
b
0.1
0.05
0
a
NS
b aa b aa
b
aa
NS
cb a
NS
aa
NS
0
C9
C10
C12
C13
C14
C15
C16 C16:1 C18 C18:1 C18:2 C18:3 C19
#
#
#
# #
C20
#
- Increase in lipolysis for cell-added cheeses
- High level of long-chain free fatty acids for HSCC cheese assay
This increase in lipolysis was due to
the lipoprotein lipase of somatic cells.
* Similar data: 0.39 (Dherbécourt et al., 2010)
# Confirmed by Deckelbaum et al., 1990
6
Results
Proteolysis indices in cheese ripening
(1/2)
Non-casein and non-protein nitrogen content (%)
30
CTL
LSCC 4×105
HSCC 9×105
25
20
a
b
c
∆6.8%
Non-casein nitrogen content (%)
15
a
b
b
∆7.9%
10
Non-protein nitrogen content (%)
5
0
0
20
40
Cheese ripening (days)
60
Slight increase in proteolysis for cell-added cheeses
80
7
Results
Proteolysis indices in cheese ripening
α-casein degradation nitrogen content
(%)
casein production
αs1-casein
40
No≠activity plasmin
30
CTL
LSCC 4×105
HSCC 9×105
β-casein
β-casein degradation nitrogen content
(%)
↗αs1-I
50
(2/2)
20
30
a
b
b
20
10
αs1-I casein
10
αs2-casein
0
γ1-, γ2- and γ3-caseins
0
0
20
40
60
Cheese ripening (days)
Hypothesis: Cathepsins D
-Endogenous enzymes
-Active on s-caseins [1-2]
-Optimum pH value: acid
-Thermal resistant [3]
80
0
20
40
60
80
Cheese ripening (days)
Somatic cell count
correlation 0.7 [4]
Plasmin activity
1 (Hurley et al. 2000); 2 (McSweeney et al. 1995); 3(Hayes et al. 2001); 4(Le Roux et al., 1995).
8
Somatic Cell Role
Foe or Friend ?
Take home message

Microfiltration approach to collect somatic cells from raw milk

Itinerary of somatic cells during cheese manufacture
New
New
- flow cytometry approach
- qPCR to measure the DNA amount of somatic cells

Somatic cell role on Swiss-type cheese manufacture

NOT FOE: No impact on cheese making

New
slight increase in lipolysis and proteolysis

Maybe FRIEND

Somatic cells themselves are only the indicator of milk quality
Somatic cells
Mac
PMN
Lym
9
Acknowledgements
V. Gagnaire, R. Richoux,
N. Leconte, M.B. Maillard, S. Parayre,
L. Aubert-Frogerais, J. Warlouzel,
C. Bevilacqua, E. Moya-Leclair,
C. Denis, P. Martin