Combined approaches to differentiate the common Mr ... Combined approaches to differentiate the common

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  • Combined approaches to differentiate

    the common Mr Bacillus and the Super Spoiler

    AG Mathot, E Cozien, A Lochardet, L Coroller, N Desriac,

    V Huchet, D Sohier, F Postollec

    SPORE-RISK Joint Technological Unit

  • SPORE-RISK

    A JOINT TECHNOLOGICAL UNIT ON RISKS LINKED TO SPORE-FORMING BACTERIA

    The University Lab on Sporeformers & Moulds

    The Food Technology Institute on Food Safety & Quality

  • Bacillus species

     sporeforming bacteria are ubiquitous and exhibit a wide range of

    diversity in phenotypic behavior

     Persistance of sporeformers in industrial plants

    & huge economical losses

     high prevalence in raw materials and ingredients as spore,

    dormant but highly resistant contaminant

     in food, when conditions are favourable for germination and

    growth, cell multiplication of specific strains may lead to food

    poisoning or food spoilage

  • Prevalence & biodiversity

    0%

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    90%

    100%

    1 2 3

    Série6

    Série5

    Série4

    Série3

    Série2

    Série1

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    0%

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    raw material dehydrated ingredient

    processed food

    c o

    n ta

    m in

    a ti o

    n

    Moorella

    Anoxyb. flavithermus

    Geob. stearothermophilus

    Paenibacillus

    Bacillus

    Clostridium

    0%

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    raw material dehydrated ingredient

    processed food

    c o n ta

    m in

    a ti o n

    Brevib. laterosporus

    B. pumilus

    B. cereus

    B. licheniformis

    B. sporothermodurans

    B. subtilis

    B. weihenstephanensis

     diversity of sporeforming bacteria encountered in food and food

    industries remains largely unknown

     Multiple contamination & increased diversity in processed food

  • Industrial issues

    To adjust treatment and control Bacillus related contamination,

    the selection of targeted hazards to be used for challenge tests is crucial

     Super Spoiler, showing high ability

    to spoil, adhere & survive hurdles

     Common Mr Bacillus with

    classical phenotypic features 

  • 01 Growth ability of Bacillus strains

    involved in ropy bread

    Looking for the common Mr Bacillus and Super Spoiler

    Bacillus diversity on a phenotypic & genotypic aspect02

    Impact of cell history on resistance & recovery03

  •  bread spoilage occuring when climate is warm (25-30°C) and humid

     bread crumb becomes sticky and

    stringy due to proteolytic/amylolytic

    enzymatic activities

     mainly caused by B. subtilis, B.

    amyloliquefaciens and related species

    B. amyloliquefaciens & ropy bread 01

  • Methods 01

     Determination of growth cardinal values and spoilage potential

     selection of isolates based on molecular Rep-PCR fingerprints,

    heat resistance and amylase production screening (Valerio et al.

    2012)

     determination of growth rate in BHI broth for different temperature (4-55°C), pH (4.5-9.5), aw(0.964-0.994)

     artificial spore inoculation in bread with different formulations

    (WWB, WBB, WBB+LbBio) to determine growth rate in food

  • Determination of growth ability 01

    different T

    different pH

    different aw

    pHmin pHopt pHmax

    awmin awopt awmax

    µ m

    ax (

    h- 1 )

    Temperature (°C)

    Secondary models

    Lo g

    C F

    U /m

    l

    Time (h)

    Tmin Topt Tmax

    µmax : growth rate

    Lag : lag phase

    Log No : initial population

    Log Nmax : max population

    Primary models

    µmax

  •  ATCC8473 Type strain, isolated from ropy bread

    Determination of growth ability 01

     ISPA S109.3 isolated from wheat grain

     Evaluation of strain growth ability

    for different bread formulations, storage temperature

    aw: 0.962

  • Challenge test in bread 01

    Growth kinetic in a food

    matrice under defined

    conditions

    Challenge test at 30°C 1 growth kinetic in bread

     µmax

     Based on growth cardinal values, 1 challenge test is done in bread

    to account for the impact of food matrix on growth for ISPA S109.3

    Mathematical

    models

    In silico growth

    predictions for

    industrial relevant

    scenarii

  • Challenge test in bread 01

     Based on growth rate obtained in bread for given

    condition, in silico predictions may be done

  •  ISPA S109.3 in WBB, 18°C, 7days

    In silico testing of various scenari 01

     ISPA S109.3 in WBB, 30°C,

     In a few clic, growth may be predicted for different

    storage and formulation scenarii

  • Conclusions & perspectives

     identification of common Mr Bacillus (ATCC8473) and Super Spoiler (ISPA S109.3)

     cardinal values enables to account for growth abilities

     strain selection to perform challenge test in food is crucial

    Valerio et al. 2014

    01 Growth ability of Bacillus strains

    involved in ropy bread 01

  • B. licheniformis biodiversity 02

     Bacillus licheniformis shows high

    prevalence along food process, i.e. from raw

    materials to final products

     B. licheniformis show high spoilage

    potential with a large range of enzymatic

    abilities

    &

    Which diversity is encountered in milk&dairy?

    Is there similar features in closely related strains that

    could explain process selection/adaptation?

  •  collection of isolates from dairy food (48%), industrial environment

    (20%), or diverse environment (32%)

     determination of genetic relatedness using Madslien MLST

    scheme targeting 6 housekeeping genes

     determination of phenotypic features i) heat resistance, ii) growth,

    iii) ability to adhere on surfaces, iv) enzymatic abilities

    Methods 02

     Determination of genetic relatedness using the MLST scheme and phenotypic characterisation to

    identify potential clusters

  • Biodiversity

     Genetic relatedness is based on the comparison of gene

    sequences by MLST

    MLST CLUSTERS

    Major group

    Minor group

    02

    # locus difference

    1 1 1

    1

    1 1 1

    1 1 1 1

    1 1 1 1 1

    1

    1 1 1

    6

    6

    5 2 2 2

    18

    12

    3

    2 1

    2

    2

    2

    1 1

    1 1

    1

    1 1 1

    1

    1

    1

    1

    1

    1 1 1

    1 1

    1

    # strains per ST

    02

  • Milk & dairy products

    Deshydrated ingredient

    Canned food

    Industrial environmental samples

    Other food products

    Ground & environment

    Biodiversity 02

     Most milk&dairy isolates belongs to the major group of closely related strains

  • Biodiversity 02

     Projection of phenotypic features among MLST clusters

  • High adhesion

     Projection of highly adhering strains within MLST clusters

    Biodiversity 02

  • >1.5 mm amylase

     Projection of high amylase producing strains

    Biodiversity 02

  • >1.5 mm lipoprotease

     Projection of high lipoprotease producing strains

    Biodiversity 02

  • >1.25 mm protease

     Projection of high protease producing strains

    Biodiversity 02

  • >9 mm gelatinase

     Projection of high gelatinase producing strains

    Biodiversity 02

  • Biodiversity 02

    MLST CLUSTERS

    Major group

    Minor group  Minor group seems to gather isolates

    with lower heat resistance, lower Tmin,

    lower Tmax, lower gelatinase production

     Minor group would gather strains showing lower lichenysine production,

    and slower germination ability (Madslien et al. 2014)

     Most milk&dairy isolates belongs to

    the major group

     Adhesion and wide enzymatic

    production seems to be common

  • Conclusions & perspectives

     contaminants encountered in milk&dairy industry are

    closely related strains

     identification of Mr Bacillus and Super S