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Marie-Christine Montel, Unité de Recherches fromagères
INRA Aurillac
Ecological approaches for managing microbial diversity to improve safety of traditional food.
Example of cheese from experiments performed in WP2A of Truefood project
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Improvement of microbial safetyWP2A Truefood project
Task 2A.3Improvement of environmental conditions governing cheese ripening taking account process efficiency and cheese quality (INRA, DRI)
Task 2A.2
Management of microbial diversity for inhibiting pathogenic bacteria (L. monocytogenes, S. aureus) in traditional cheeses
( INRA, UL, TUM, DRI)
Task 2A.1 Improvement microbial safety of milks through reduction of mastitis and the use of antibiotics by feeding regimes and other management practices ( ISS, INRA,)
Task 2A.4
Development of a bio-preservation (using lactic acid bacteria) for inhibiting food pathogens on pork muscle tissues (ADIV)
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Why an ecological approach?
Because Cheeses and milk are microbial ecosystems
– Cheese is a system in which microbial populations (living or biotic part ) live and have relationships between them and with their surrounding environments ( abiotic part) .
» Microbial populations interact each other in synergy or antagonism and their life dpends on nutriments, temperature, humididity, oxygen.
–The life of these microbes is determinant for the qualities of cheeses.
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Microbial safety
Pathogenic bacteria : • commercialised raw milk cheeses have to comply with EC regulation
•Listeria monocytogenes•Salmonella •Staphylococus aureus ( no enterotoxins production ) •Eschercihia coli
Opportunist pathogenic bacteria
Metabolite productions•Enterotoxins•Mycotoxins•Biogenic amines
Transfert of antibioresistance
-
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Above all,
Benefits refer to
pleasure and happiness of consumers who require authenticity , traceability, sensorial
properties, safety and health values.
great taste,
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Why to manage microbial diversity ?
A challenge for industrial producing Traditional Fermented products (Cheese, sausage…) for conciliating all these aspects and meet all these requirements
To eliminate pathogenic bacteria
EC regulation for 4 species
To preserve microbial community:
during manufacturing and ripening
Microbial Safety Gustative pleasure Health
Minimise
RisksIncrease
Benefits
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Our behaviour in managment of microbial diversity
for microbial safety must have always in mind the
benefits of microbial diversity for cheeses
-Healthy aspects?
-Contribution in hurdle technology
-Diversity of their sensorial properties
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Management of microbial diversity
Elimination of pathogenic micro-organisms and maintain of microbial diversity having an interest through all the process control of raw material : microbial quality of
milk, meat... control of microbial dynamics during ripening
Tools for tracking and monitoring microbial diversity
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How to identify and monitor microbial community
Picture depending on culture media, cultavibility of strainsIdentification by
phenotypic tests, genomic tests (speciesPCR, 16s or 23s DNAr sequencing)Physical spectra ( FTIR)
Monitor microbial dynamicsPicture biased by •Dominant population detected•DNA Extraction •PCR amplification•Coelution in the same peaks
Approaches get rich each other
ClassicalMolecular
PCR-SSCP,TGGE, TTGE, DGGE, lH-PCR…
Milk , cheeses
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What approaches for biopreservation of traditional products ?
1. Selection of strains or metabolites (example : nisine)
2. Understanding microbial ecosystems having antagonist activities • Example of milk and raw milk cheese ecosystems
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Validation at industrial scale
Test on agar well
In vitro screening of inhibitory activity
Challenge tests in experimental cheese
Identification of inhibiting substances bacteriocins,
H2O2,
Strains Collections
Milks , cheeses
Coculture in milk or other media
Selection of strains or inhibiting metabolites
Agar diffusion Bioluminescence
Detection of bacteriocin genes
1
GAP here!!!!
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Time consuming and fastidious
Frequent fails due to gap between the results obtained in vitro and those obtained in cheeses Consortia with high antilisteria activities by
in vitro test but not at the surface of cheesesLow number of strains selected for example
in Truefood project able to inhibit and without effect on sensorial properties
Selection of strain inhibiting
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Strategies by ecological approach
Rely on a general principle in ecology indicating that: the whole is more than the sum of each individu as
many interactions - synergy, antagonism, competition..- can occur
Hypothesis that preservation of the microbial community with the wholeness of its diversity is important for the different functions -inhibition of pathogens, production of aromatic compounds
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Understanding microbial ecosystem having antilisteria activity
Test of simplified microbial consortia in experimental cheese
Screening of milks, or cheese surface on experimental cheeses
Analysis of the most inhibitory microbial consortia
Identification of microbial and biochemical part of the ecosystem
Advice for milk production or cheese ripening
proposal of consortium
4
23
14
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Microbial consortia from Surfaces of cheesesSmear cheeses: Munster
Farm Saint-nectaire cheeses
Microbial consortium from Raw milk from Saint-Nectaire area
Examples of microbial cheese ecosystems with antilisteria activities
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First example consortia of microbes from the surface of
Saint-Nectaire
Study of Inhibition of Listeria monocytogenes at the surface
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Antilisteria activities of surfaces of Saint-Nectaire cheese microbial consortia
-2,5
-2
-1,5
-1
-0,5
0
0,5
SN
15
SN
11
SN
26
SN
19
SN
13
SN
9S
N2
2S
N3
0S
N2
4S
N2
5S
N2
3S
N1
2S
N2
9S
N6
SN
8S
N3
4S
N2
0S
N2
8S
N1
6S
N3
2S
N2
SN
17
SN
1S
N3
5S
N3
1S
N2
7S
N3
SN
14
SN
33
SN
18
SN
10
SN
5S
N4
SN
7
Great diversity in the antilisteria activities observed without relation with pH values
o 5.9<pH28d<8.4
Selection of one consortium SN15
Comparison of inhibitory effect of consortia (34) selected from farm Saint-Nectaire surface cheeses. (Trials on surface of non cooked pressed cheeses ripened at 7°C during 28 days)
Log
Inhi
bitio
n
High inhibition
No inhibition
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Stability over storage of the inhibiting effect of SN15 consortia against Listeria monocytogenes
Time (months)
ΔL
og
Lm
(u
fc/c
m²)
ΔLog Lm = Log (Lm SN15) - Log (Lm control)Microbial consortium SN15 still
inhibitory after 24 months storage at -20°C
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Identification by phenotypic test, RFLP and 16S DNAr sequencing
B= Micrococcaceae/ Corynebacteriaceae (7 species) Arthrobacter nicotianae Arthrobacter bergeri Staphylococcus pulvereri Staphylococcus xylosusBrevibacterium linens or casei/ Brevibacterium antiquum Brachybacterium
A=Lactic acid bacteria (6 species)Lb. casei Lb. curvatusLn. mesenteroides Carnobacterium mobile Marinilactibacillus psychrotolerans E. faecalis
D=Yeasts (4 species)
Candida sake Yarrowia lipolytica Debaryomyces hanseniiGeotrichum sp.
C=Gram negative bacteria (3 species)
Proteus vulgarisSerratia proteomaculans Pseudomonas fluorescens or syrinqae
D A
B
C
D
Identification of microbial consortium (SN15) inhibiting L. monocytogenes
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Reconstitution of complex consortium SN15 from the surface
Constitution of consortium with 19 strains (one strain by species) inoculated at 2 Log /cm2 at the surface of cheeses
Comparison of L. monocytogenes growth at the surface of cheeses with the natural complex or reconstituted consortia
Complex more inhibitory than the reconstituted
Study in progress for understanding why?
Log Complex
reconstituted
Inhi
bitio
n
D
3 yeasts
3 Gram negative
6 Lactic acid bacteria
7 Micrococcaceae/ Corynebacteriaceae
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Second example microbial consortium from raw milk
Study Inhibition of L. monocytogenes in the core of cheeses
Complex consortium selected in previous study : Millet al, 2006; Saubusse et al, 2007
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Composition of Microbial consortium from raw milk having antisteria activty
( Saubusse et al, 2007)
Great diversity : 29 microbial species
B= Micrococcaceae/ Corynebacteriaceae (10 species) Staph. saprophyticus/ Staph. equorum/ Staph haemolyticusCorynebacterium casei/Coryne. flavescensArthrobacter nicotianaeBrevibacterium linensExiguobacterium sp.Brachybacterium rhamnosusMacrococcus caseolyticus
A=Lactic acid bacteria (9 species)Lb. casei / Lb. farciminisLb. curvatus Lb. plantarumLn. pseudomesenteroides Ln. citreumE. hiraeE. FaecalisA. viridans
D=Yeasts (5 species)
Rhodosporium babjevae Debaryomyces hansenii
Candida pseudointermediaC. pararugosa C. deformans
C=Gram negative bacteria (5 species)
Pseudomonas putidaEnterobacter amnigenus Acinetobacter sp.Chryseobacterium spStenotrophomonas maltophilia
D A
B
C
D
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Simplification of reconstituted
consortia from raw milk A B
C D
A B
C
A B
D
D
C
B
D
A
C D
A B
A B A D B D
yeastGram negative
Lactic acid bacteria Micrococcaceae/ Corynebacteriaceae
Inoculation in pasteurised milk
with S. thermophilus (St)
Manufacturing non coocked
pressed cheeses
Comparison of L. monocytogenes
to a control with only Stthermophilus
Microbial and biochemical analysis
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Inhibition of L. monocytogenes with simplified consortia in core of cheeses
-2,5
-2
-1,5
-1
-0,5
0
0,5
1
1,5ABCD AB AD A ABC ABD BDC BD B
Synergy between lactic acid bacteria and non lactic acid bacteria in the inhibitionLoss of inhibition in simplified consortia without lactic acid bacteria
ΔLog Lm = Log (Lm assay) - Log (Lm control)
ΔLo
g Lm
D=yeast
C=Gram negative
A=Lactic acid bacteria
B=Micrococcaceae/ Corynebacteriaceae
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Active
ABCD
AB
AD
A B
control
-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6
Fact. 1 : 51,77%
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5F
act.
2 :
32,
90%
pH
High count of listeria
L lactateD lactateAcetic acid
3-methylbutyric acid
Butyric acid
Hexanoic acid
ALCOOLS2-heptanol2-butanol2-pentanol
ESTERSEthyl formiateEthyl butanoate
ALDEHYDES
2-methyl-propanalbutanal
KETONES2-hexanone4-methyl-2-pentanone2,3-pentanedione2,3-butanedione2-butanone
Link between [L. monocytogenes] and [organic acids]- [volatil compounds]- pH
Hypothesis on the role of acetic acid and D lactate in the inhibition of L. monocytogenes in cheeses Important variation in volatiles profils according to consortia inoculated
Low count of listeria
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Inhibition by several hurdles during ripening
H202Bacteriocins
pH Ac. lactic L
Ethyl esters
Ac. acetic
AlcoholsAc. lactic D
pH>5.3 <8mg/g <2mg/g <0.3mg/g
Besides these microbial factors, environnemental factors can also limit the development of pathogenes
Temperature, relative humidity during ripening
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What are the populations involved in the production ?
Microbial dynamics studied by culture methods associated with molecular toolsExample of microbial balance in cheese with
consortium AB the most inhibitory
Leuconostoc
Enteroccoccus
CorynebacteriaceaeMicrococcaceae
Nmax
Log
UFC
/g
Lactobacillus
5
6
7
8
9 Leuconostoc
Enteroccoccus
CorynebacteriaceaeMicrococcaceae
Nmax
Log
UFC
/g
Lactobacillus
5
6
7
8
9
19 species inoculated still present
BUT
Difficulties to quantifye species of this group
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In conclusion, what applications for traditional fermented food?
Arguments for maintaining microbial diversity as a thumb for safety of fermented products ( ex cheese)
High inhibitory potentialities of complex microbial ecosytem from the surface of Cheese but application still limited by difficuties to reconsitute it Further studies needed to understand why
Scientific data to think about suitable balance between microbial populations in milk Further studies to adapt milk production practices
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In conclusion, what applications for traditional fermented food?
Proposal a simplified microbial consortium still complex associating lactic acid bacteria ( 8 species) and non lactic acid bacteria ( 12 species?) for inhibiting L. monocytogenes in the core of cheese but its industrial use need some improvmentsOptimise the preparation of the consortium
and insure its stability overtime Validate it use at industrial scaleEvaluate its effect on sensorial propertiesDevelop rapid methods to quantifye non lactic acid
bacteria in cheese
WP6 of Truefood
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Thank you for your attention and coming instead of visiting the museum
Partners of WP2A of Truefood project and especially Cécile Callon for her helpful contribution in this presentation
Financial support : European Commission under the 6th Framework programme for RTD ( contrat N° Food CT-2006-016264)