Challenge testing with Brochothrix thermosphacta on minced pork meat shows interest to couple metagenetics to metabolomics to study food spoilage

(1)

Challenge testing with Brochothrix thermosphacta

on minced pork meat shows interest to couple

metagenetics to metabolomics to study food spoilage

Baré, G.

1*

_{, Cauchie, E.}

1

_{, Leenders, J.}

2

_{, Tahiri, A.}

1

_{, Delhalle, L.}

3

_,

Korsak Koulagenko, N.

1

_{, de Tullio, P.}

2

_{& Daube, G.}

1

1 _{Fundamental and Applied Research for Animal and Health (FARAH), Food Sciences Department, Faculty of Veterinary Medicine, University of Liège, Quartier Vallée 2, Avenue de Cureghem 10,}

B-4000 Liege, Belgium.

2 _{Laboratory of Medicinal Chemistry, Department of Pharmacy, Center for Interdisciplinary Research on Medicines (CIRM), University of Liege, 4000 Liège, Belgium.}

3 _{KeyFood plateform, Rue Auguste Piccard, 20, Maison de l’Industrie, 6040 Gosselies, Belgium}

Challenge testing with Brochothrix thermosphacta

on minced pork meat shows interest to couple

metagenetics to metabolomics to study food spoilage

Baré, G.

1*

_{, Cauchie, E.}

1

_{, Leenders, J.}

2

_{, Tahiri, A.}

1

_{, Delhalle, L.}

3

_,

Korsak Koulagenko, N.

1

_{, de Tullio, P.}

2

_{& Daube, G.}

1

1 _{Fundamental and Applied Research for Animal and Health (FARAH), Food Sciences Department, Faculty of Veterinary Medicine, University of Liège, Quartier Vallée 2, Avenue de Cureghem 10,}

B-4000 Liege, Belgium.

2 _{Laboratory of Medicinal Chemistry, Department of Pharmacy, Center for Interdisciplinary Research on Medicines (CIRM), University of Liege, 4000 Liège, Belgium.}

3 _{KeyFood plateform, Rue Auguste Piccard, 20, Maison de l’Industrie, 6040 Gosselies, Belgium}

INTRODUCTION

RESULTS

ACKNOWLEDGMENTS

OBJECTIVES

CONCLUSIONS

* g.bare@ulg.ac.be

The spoilage of perishable foods is

mainly caused by bacterial activity.

The risk of unwanted bacterial growth

is particularly high in the minced pork

meat.

1. Performing aging tests of minced pork meat at different temperatures

2. Studying the growth of the microorganism which was dominant during aging tests : this growth is followed

at different temperature, in minced pork meat previously sterilized ("Challenge testing")

3. Identifying the metabolites used and produced by this microorganism during testing challenge

("metabolomic study")



This work showed that -omics

technologies

(metagenetics

and

metabolomics)

can

be

used

conclusively

to

study

microbial

spoilage of minced pork meat

The authors acknowledge the General Operational Direction of

Economy, Employment and Research (Walloon Public Service) of the

Walloon Region (Belgium).

MATERIALS AND METHODS

1. Aging test

_{2. Challenge test}

_{3. Metabolomics}

minced pork meat Packaging

30 % O₂ : 70 % CO₂ Storage 1) +2 ˚C 2) +8 ˚C 3) +12 ˚C Samples taken

after 6 days 16S rDNA-targeted metagenomics (metagenetics)

irradiated (sterile) minced pork meat

Inoculation B. thermosphacta 102 _{bacteria/g of} meat Storage 1) +2 ˚C 2) +8 ˚C 3) +12 ˚C Counting (PCA, 22 ˚C , 48h) Packaging 30 % O₂ : 70 % CO₂ Samples taken over time

1. Aging tests :

Minced pork meat samples are placed in

modified atmosphere packs (70% of CO

₂

and

30%

d’O

₂

)

and

incubated

at

different

temperatures (2°C, 8°C and 12°C). Samples

are taken after 6 days.

2. Challenge tests:

Minced pork meat is sterilized by gamma

irradiation and inoculated in depth with aliquots

of a culture of B. thermosphacta MM008.

Minced pork samples are placed in modified

atmosphere packs (70% of CO2 and 30% O2)

and incubated at different temperatures (2°C,

8°C and 12°C). Samples are taken over time:

cell concentration of B. thermosphacta is

determined by counting the colonies on PCA

medium

3. Metabolomic study :

Meat samples are extracted twice by D

₂

O and

analyzed by 1H-NMR.

Centrifugation 15 min 13000 rpm Supernatant analysis by 1H-NMR Vortex 1 min 15 min at rest Addition of 300 µl D₂O 100 mg meat + 500 µl D₂O Vortex 1 min Centrifugation 2 x 30 seconds 5000 rpm

Brochothrix thermosphacta is the

dominant microorganism in all aging

conditions (in modified atmosphere packs)

As expected, the higher the

incubation temperature, the faster the growth of this strain is

All samples can be splitted into 3 groups according to their spectral profile:

1) samples taken at time 0 (in green);

2) samples inoculated with B. thermosphacta and taken at final time (in pink);

3) samples uninoculated, taken at final time (in red).

RESULTS

An increased production of creatine, acetone and acetate was found in the minced pork meat samples inoculated with B.

thermosphacta. These molecules come likely from the

catabolism of protein from meat and the degradation of metabolites such as lactate, glucose, carnitine, betaine, ...

Metabolites without

inoculation (at final time)

Metabolites after inoculation

with B. thermosphacta (at final time)

Glycerol, Glucose, Taurine,

Lactate, Carnitine, Betaine, Glycine

Creatine, Acetate, Acetone

3. Metabolomics (continuation)

Metagenome of minced pork meat at time zero and at final

time (6 days) after aging tests at different temperatures

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% pas d'incu bation 2 8 12 Temperature (°C) R e la ti v e a b u n d a n c e (% ) Others unclassified Psychrobacter sp. Photobacterium sp. Photobacterium kishitanii Leuconostoc inhae Leuconostoc gasicomitatum Lactococcus piscium Lactobacillus algidus Brochothrix thermosphacta Brochothrix sp. Acinetobacter sp.

Metagenome of minced pork meat at time zero and at final time (6 days) after aging tests at different temperatures