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

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)

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.

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

-

Above all,

Benefits refer to

pleasure and happiness of consumers who require authenticity , traceability, sensorial

properties, safety and health values.

great taste,

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

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

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

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

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

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!!!!

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

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

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

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

First example consortia of microbes from the surface of

Saint-Nectaire

Study of Inhibition of Listeria monocytogenes at the surface

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

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

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

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

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

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

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

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

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

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

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

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

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

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)