Download pdf - Combined approaches to differentiate the common Mr ... · Combined approaches to differentiate the common Mr Bacillus and the Super Spoiler AG Mathot, E Cozien, A Lochardet, L Coroller,

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%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 3

Série6

Série5

Série4

Série3

Série2

Série1

1

2

3

4

5

6

1

2

3

4

5

6

1

2

3

4

5

6

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

raw material dehydrated ingredient

processed food

co

nta

min

atio

n

Moorella

Anoxyb. flavithermus

Geob. stearothermophilus

Paenibacillus

Bacillus

Clostridium

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

raw material dehydrated ingredient

processed food

conta

min

ation

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

01Growth 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 historyon 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

µm

ax (

h-1)

Temperature (°C)

Secondary models

Log

CF

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


involved in ropy bread01

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


contaminants encountered in milk&dairy industry are

closely related strains

identification of Mr Bacillus and Super Spoiler that show

either common or peculiar behavior

No clear correlation between phenotypic and genetic

features that could explain observed clusters

Bacillus diversity on a phenotypic & genotypic aspect02

Impact of cell history

Food safety & quality

issues during shelf-life

Spore contamination in raw material,

ingredients, environnement, surfaces

> Process & sanitation

Avoid germination, outgrowth and cell

multiplication in food during shelf-life

> Food recipe, food storage

03

Impact of cell history

Food safety & quality

issues during shelf-life

03

per treatment

pre treatment

post treatment

B. weihenstephanensis KBAB4 spore production (5-50°C, pH5-8)

Pre-treatment conditions: sporulation kinetics based on agar

spore counts to quantify the impact of sporulation conditions on

spore resistance

Post-treatment conditions: germination kinetics based on agar

counts to quantify the impact of temperature and pH on recovery

Quantification and modelling of the impact of

pre-, per- and post-treatment

on spore resistance and recovery

Methods 03

Heat treatment in capillary tube at 85-90°C

Time (hours)

Log C

FU

/ml

Impact of cell history 03

strong impact of temperature encountered during sporulation on

spore heat resistance and spore load

Sporulation temperature (°C)

Log

(d 9

0°C

)



log(

d) Sporulation

0 10 20 30 40 50

-1

-0.5

0

0.5

1

1.5

2

Recovery Temperature (°C)

log

10(d

)

Recovery temperature (°C)

log(

d)

Recovery

strong impact of temperature encountered during sporulation on

spore heat resistance and spore load


0 10 20 30 40 50

-1

-0.5

0

0.5

1

1.5

2

Recovery Temperature (°C)

log

10(d

)

Recovery temperature (°C)

Growth temperature (°C)


µm

ax(h

-1)

log(

d)

log(

d)

Growth

Sporulation

Recovery

Modeling

Tmin , Topt , Tmax pHmin , pHopt , pHmax

Physiological parameters

Baril et al. + trunet et al.


pre-, per-, post-treatment conditions strongly impact spore

heat resistance and recovery

generic concept observed for B. licheniformis

and G. stearothermophilus

Spore used in challenge test maximise the risk

Baril et al. 2012, Trunet et al. 2015


Take home message

Cardinal values may be used to account for

strain diversity



involved in ropy breadBetter knowledge and integration of biodiversity is an added value to control contamination

Bacillus diversity on a phenotypic & genotypic aspect02 Selection of common Mr Bacillus or Super Spoiler

used for food testing respond to FBO’s requests

Acknowledgements

Regional & dairy food industrial fundings Collaborations

Ron Ronimus, university of Waikato (Nz),

Line Thorsen, university of Copenhaguen (Dk),

Francesca Valerio, CNR ISPA (It),

Elisabeth Madslien, NVH (No),

Robert Nout, Wageningen university (Nl)

Acknowledgements

The University Lab on Sporeformers & Moulds

The Food Technology Institute on Food Safety & Quality

Thank you for your attention!

Many thanks to the

organizing committee