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New developments in the knowledge of
cheese microbial ecosystems.
Prof. HE Spinnler,
AgroParisTech / INRA
78850 Thiverval Grignon
France
www.agroparistech.fr
Goals and outlines
• The cheese curd and the ripening environment determines
the growth of a microbial ecosystem inside the cheese and at
the cheese surface.
• This growth :
• Determines the expression of metabolisms
• And consequently the sensory caracteristics of the ripened cheeses
• Even if relatively complex, these microbial communities are
composed of a limited number of cultivable moulds, yeasts
and bacteria.
• In soft cheeses where the ratio S/V is high the rôle of the
surface microbial ecosystem is particularly important.
2
Methods used to understand the development
and expression of the ecosystem
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Milk
ecosystems
Factories
ecosystems
Direct ecosystem approach
-
Effect of microbial combination
- Effect of species omission on functions
- Understanding interactions and modelling
- Sequencing the genome of complete
ecosystems ( High throuput sequencing)
-Transcriptomics and expression of complete
ecosystems ( High Throuput sequencing)
- Metabolite analysis (flavour produced)
-Sensory analysis
Cheese
ecosystems
Individual species studies
- Complete Genomes
- Transcriptomics and regulation of
expression
- Proteomics
- Metabolite analysis (flavour produced,
metabolomics)
- Physiology (régulations)
PLAN
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1. Questions about Cheese Microbial
Ecosystems
2. Diversity observed and main functions
3. Important parameters for the selection
of the important species
4. Relations with organoleptic properties
From Where these micro-organisms
are coming from?
The diversity of flora occuring in/on cheese are collected all along
the process from paddock to plate
Farm
plants
cheese technology
cheese
Starters
Milk
Coagulation
and Moulding
Salting
and Surface drying
Ripening
5
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Wolfe et al, 2014
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Geographic distribution
Wolfe et al, 2014
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Geographic distribution
Wolfe et al, 2014
Inoculated Diversity
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• Some species necessary to make a pasteurised milk
camembert cheese
Lactococcus (Lc) lactis ssp lactis
Lc lactis ssp lactis biovar
diacetylactis,
Lc lactis ssp cremoris
Leuconostoc mesenteroïdes
Kluyveromyces lactis
Debaryomyces hansenii
Geotrichum candidum
(Yarrowia lipolytica)
Penicillium camemberti
Brevibacterium linens
(Arthrobacter spp.)
Hafnia alvei
Factory : Microbial diversity in Munster cheeses
(Single Strand Conformation Polymorphism)
January
*
* **
*
*
April
Brevibacterium linens
Arthrobacter arilaitensis
Carnobacterium maltaromaticum
Lactobacillus sakei
Streptococcus thermophilus
L.lactis subsp.lactis
??
: pic b
: pic a
: pic e
: pic i
: pic g
: pic h
: pic c
??
: pic d
h
Milk +
starters
Octob
er
Feurer C. et al. JDS, 2004
b
Massage
solution
g
Inoculum
i
Quite stable and résilient flora:
50 % resident flora (*)
50 % inoculated flora ( )
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What are they?
On 137 cheese microbial communities
Most of them are consistantly convergents
Wolfe et al, 2014, Cell, 422-433
Why do they grow?
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• What favours selection of bacteria and
yeast in cheese?
• How they adapt to dairy curds and
their environment e.g. how to:
- promote ripening starters with growth
rates more competitive than pathogens
- use ecosystems more resistant to
unexpected germs and be more
resilient if a technological factor (Dry
matter, salt level, distribution of fat) is
different to the one expected
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When are they growing in the cheese?
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1. Lactic acid bacteria are normally the first to grow and they
produce the lactic acid that will protect the curd for a few
days
2. As the curd is acid the yeasts and moulds are the only one
able to grow for a while (4-8
days) at the surface.
3. Inside the cheese only anaerobic bacteria such as
Propionibacterium are able to grow when the temperature
is over .
4. When the pH is not too low (over 5.3) Ripening bacteria
start to grow but it is only over pH= 5.8 that they will grow
significantly.
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Succession within a Natural Rind Community Is Highly Reproducible (A and B) Reproducible
succession in an in situ rind community was observed as three batches of a natural rind
cheese aged (1–63 days). (A) Relative abundance of community members...
Wolfe et al, Cell, Volume 158,
Issue 2, 2014, 422 - 433
PLAN
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1. Questions about Cheese Microbial
Ecosystems
2. Diversity observed and main functions
3. Important parameters for the selection
of the important species
4. Relations with organoleptic properties
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What are their functions?
1. Protection against the other micro-organisms
2. Aspect and colour :
White : Penicillium camemberti, Penicillium nalgiovensis
Toad skin: G. candidum
Orange : B. linens
3. Odour : Debaryomyces hansenii, Kluyveromyces lactis,
Brevibacterium linens, B. aurantiacum, Pseudoalteromonas
haloplanctis
4. Taste : Penicillium camemberti, Lc lactis, Pseudomonas sp.
(bitterness)
5. Flavour : B. linens, G.candidum,P.c. , P.r.
What is doing what?
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But the relative expression of their genomes in cheese is still
poorly known and this knowledge could help :
To prioritize the research and consider the organisms that are
more active
To establish the relationship between the expression and the
sensory properties
To get better control of cheese organoleptic properties
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PLAN
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1. Questions about Cheese Microbial
Ecosystems
2. Diversity observed and main functions
3. Important parameters for the selection
of the important species
4. Relations with organoleptic properties
3.1. Moisture
•Consistancy of the moiture level is probably the more
important parameter for the cheese ripening and properties.
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•To much moisture accelarate the ripening processes
•The stucture of the curd and the regularity of the porosity
around the fat globule is also important. Pockets of whey can
be the source of spoilage (in pressed cheese mainly)
•The important parameter is the relative Humidity of the
cheese but more informative is the Moisture in the Defated
Cheese (MDC). The consistancy of this value integrate the
variability of milk composition and of the process, up to the
end of moulding.
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Humidity from surface to inner cheeese
Water concentration profiles at different times of salting.
J. Santapaola , S. Maldonado , J.L. Medina
NaCl diffusion kinetics in dry salting of goat cheese
Journal of Food Engineering, Volume 118, Issue 2, 2013, 172 - 177
Correlations between genius accurence and
moisture level
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Taxonomic groups show
different responses to
gradients in moisture
across cheese rinds.
 A plot of Pearson’s r
depicts significant (p <
0.05, with false discovery
rate correction) negative
and positive correlations
between abundance of
particular genera and
percent moisture.
Wolfe et al, 2014
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Correlations between Bacteria and Fungi?
Wolfe et al, 2014
3.2. Salt
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-Salt will be mainly selective when the dry salt or brine is
applied :many species are killed some are just
« chocked ». (e.g. : Geotrichum candidum) and need a few
days before to grow again.
- The salt diffuse inside the cheese and its concentration in
the cheese is finally low (1.5 to 3%)
-However if you consider salt/moisture, this quantity is
sensitive especially for the bacterial development –
bacteria are very sensitive to salt
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Salt in cheeses
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Correlation between salt level and moisture
Bloomy
Natural
Wash rind
Ratio Salt/moisture is a parameter that
can be useful to control the ripening
Wolfe et al, 2014
a) In cheddar cheese
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Starter Bacteria
Non Starter Bacteria
Starter bacteria count (a) and non-starter bacteria count (b) in cheeses with different
NaCl addition levels throughout ripening: ♦, 3.0%; ■, 2.5%; ▴, 2.25%; □, 1.8%; ▵,
1.25%; ○, 0.5%. Values presented are the means of triplicate trials.
A. Rulikowska et al.
International Dairy Journal,
Volume 28, Issue 2, 2013, 45 55
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b) Adaptation of reconstituted microbial ecosystem of
washed rind cheeses - Effect of salt
1.7 % salt
2.8 % salt
Straub et al, submitted
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3.3. pH
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 Context : Important changes in curd, composition, structure,
aspect, texture, colour and taste due to biological activity and
transport phenomena.
CO2 O2
Yeasts, Penicillium, Ripening bacteria
Surface
Water
core
Lactose
+
-
NH4+
Lactate
+
+
+
Flavour
compoun
ds
-
+
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3.3.
pH
Cheese pH
Soft cheeses
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Solubilised
cheeses
PPNC : Non
cooked pressed
cheeses
PPC : Pressed
cooked cheeses
Cheese pH
Limit of pH for the
growth of different
groups
Evolution of the pH in différent cheeese during
ripening (B. Mietton)
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pH
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3.3.
Wolfe et al, 2014
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On these 3 parameters moisture is the more important selection factor for the
cheese surface diversity- Salt cc is the least significant
Wolfe et al, 2014
3.4. Adaptation to the chemical environment
Genetic adaptation => increase in fitness
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a) Data from complete genomes
- Arthrobacter arilaitensis (Monnet et al, 2010, Plos1)
• Adaptation to low iron concentration (siderophores)
• High capabilities to catabolize fatty acids
• Adaptation to osmotic shocks (glycyl betaïne transporters)
- Corynebacterium casei (Monnet et al, 2012, J.
Bacteriol., 194(3):738)
• Adaptation to low iron concentration (siderophores, low
number of enzymes needing iron as cofactors)
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Capture of Iron through siderophores
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Monnet et al, 2012, AEM, 78:3185
Arthrobacter arilaitensis (7/7), Corynebacterium casei ( 2/2), C. variabile (1/2)
Brevibacterium aurantiacum (4/5), B. antiquum (1/1) B. linens (1/1) are stimulated by
FeCl3, 6H20 (1mg/kg of cheese), Growth of all the strains of these species were
stimulated with 50mmol/kg of cheese of the siderophore desferrioxamine B.
The expression of Iron ABC Siderophore transporters AARI_02870, AARI_3970,
AARI_3980 are also amplified by the desferroxiamine B.
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% of DNA mapped unique reads
(70% of total reads)
(Sequenced by SOLiD™, INRA MetaQuant
Platform, 35bp)
Straub et al, submitted
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Expression (RNA) of the different genomes
Straub et al, submitted
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PLAN
www.agroparistech.fr
1. Questions about Cheese Microbial
Ecosystems
2. Diversity observed and main functions
3. Important Parameters for the selection
of the important species
4. Relations with organoleptic properties
www.agroparistech.fr
Main functions
with impact
•
•
•
•
•
Lactic acid production or lactic acid uptake
Proteolysis amino acid breakdown
Lipolysis and free fatty acid accumulation
Flavour production
Production of colouring molecule
Example of the expression of flavour properties in Camembert
(Molimard et al, 1997)
Cabage,
Cow shed
5
4
DIM 2 (26.7%)
0
P4G2
P3G3
-2
P4G1
P2G1
P4
P3G1
P2
P1G2
P2G3
P4G3
-1
Intensity, Blue
Cheese
P3G2
3
Butter, Milk
2 Cream,
1 Mushroom
P2G2
P1G3
Cardboard,
Plastic
P1G1
P3
P1
-3
-4
-6
-4
-2
0
2
DIM 1 (45.8%)
4
6
8
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L-methionine degradation pathways in cheeseripening micro-organisms
L-methionine
(CH3-S-CH2-CH2-CH(NH2)-COOH)
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L-methionine
g-Lyase (MGL)
a-HB
Methylthioacetaldehyde
a-KB
(CH 3-CH 2-CO-CO 2H)
+
Acyl-CoA +
METHANETHIOL
(CH 3SH)
Glu
KMBA
(CH 3S-CO-R)
reduction
(CH 3S-CH 2-CH 2-CO-CO 2H)
KMBAdemethiolase
a-KB +
Thioesters
Aminotransferase
(+ acceptor)
GDH
NH3
reduction
a-KG
HMBA
KMBAdecarboxylase
Methional
(CH 3S-CH 2-CH 2-CHO)
METHANETHIOL
(CH3SH)
Auto-oxidation
AA, FFA, Sugars
DMDS, DMTS
Other VSC
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Wolfe et al, 2014
Conclusions :
There is a large diversity of microbes in cheese and at the cheese
surface but the selection factors of their development is not clear
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The data from genomics help to understand adaptation of bacteria,
yeasts and moulds to the chemical environment of dairy products
This knowledge may help control contaminant outbreaks and permit a
« microbial driven ecology »
The new information obtained could help to increase a range of starters
available, more adapted to cheese technologies.
Which means :
- a better growth and propagation in the cheese environment
For example with starter with a better
moisture/salt
Or more adapted to low iron environment
-a
adaptation to a range of
better functional expression : for flavour, colour, texture, …
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Conclusions :
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The transcriptomic approaches are helping to understand the base of
the development of specific qualities in pure culture and in
ecosystems of limited complexity.
Understanding ecosystems means also understanding the basis of
microbial interactions. Genomics or transcriptomics are, at the
moment, insufficient to elucidate the types of interactions between
micro-organisms
Other constraints related to differential kinetics of mass transfer are
still not quantitative. These depend on the curd composition, structure
and on environmental parameters.
.
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