METAGENOMIC AND METATRANSCRIPTOMIC ANALYSIS OF NATURALLY CONTAMINATED BEEF MEAT PRODUCTS UNDER MODIFIED ATMOSPHERE PACKAGING DURING STORAGE

S. MACÉ*, G. KERGOURLAY, B. TAMINIAU & G. DAUBE

Fundamental and Applied Research for Animal & Health (FARAH), Department of Food Science, Faculty of Veterinary Medicine, University of Liège

METAGENOMIC AND METATRANSCRIPTOMIC ANALYSIS OF NATURALLY CONTAMINATED BEEF MEAT PRODUCTS UNDER MODIFIED ATMOSPHERE PACKAGING DURING STORAGE

S. MACÉ*, G. KERGOURLAY, B. TAMINIAU & G. DAUBE

Fundamental and Applied Research for Animal & Health (FARAH), Department of Food Science, Faculty of Veterinary Medicine, University of Liège

25th International ICFMH Conference - FoodMicro 2016Dublin, Ireland, July 19th – 22nd, 2016

INTRODUCTION

* sabrina.mace@ulg.ac.be

Meat is one of the most perishable food products on the market thus resulting ineconomic losses. Different parameters have an impact on the meat spoilage such asthe initial microbial load, post-harvest processing, packaging method and thestorage temperature. An approach combining classic microbiology methods andmetagenomic approach was used to monitor changes in bacterial populationsduring the refrigerated storage of 2 different batches of naturally contaminated rawbeef meat under modified atmosphere packaging.

Sabrina Macé is Marie Curie COFUND postdoctoral fellow at Ulg.

This research is funded through by European Union and Ulg

The purpose of this work is to characterize the dynamic of

MAP beef spoilage microbiota and highlight among the

identified species the most active populations during storage.

MATERIALS AND METHODS

Total bacterial genomic

DNA isolation and 16S

rRNA gene (V1-V3)

libraries construction

were conducted by

Genalyse Patner

(Liège, Belgium). High

throughput sequencing

was performed with the

Miseq Sequencing

System (Illumina).

RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

200 isolates were isolated and more than 60 were identified.

Leuconostoc gelidum, Carnobacteriumdivergens, C. maltaromaticum, Lactococcuspiscium, Brochothrix thermosphacta and

Pseudomonas spp

In batch 1, only 7 OTUs are identified at a treshold > 1% of the global population detected. Lactobacillus algidusdominated the microbiota at the beginning of the storage.

From day 5, the samples are dominated by Lc gelidum and Lb. algidus

CONCLUSIONS

Using conventional methods and 16S rRNA gene metagenetic analysis , LAB are the dominant bacteria

at the time of spoilage.

Among them, Lc. gelidum, Lb. algidus and Lc. piscium and C. divergens and C. maltaromaticum were

identified. B. thermosphacta and Pseudomonas spp were also isolated from the samples.

Focusing on the dominating species, metatranscriptomics analysis will be conducted on those 20

samples to highlight the most active bacterial populations and the bacterial genes involved in beef

meat spoilage.

2 different batches of MAP steak beef,

Belgian origin

Commercial shelf life limit = 7 days at 4˚C

STORAGE (1/3 à 4°C – 2/3 à 8°C)

Day 7 Day 1 Day 9Day 5Day 3

For each batch at each day, 2 samples were analyzed.

For this study, 10 samples for each of the 2 batches

Bacterial pellet was

stored at -80°C after a

treatment with bacteria

RNA protect solution

(Qiagen).

Then, total RNA sample

was extracted using

RNA easy mini Kit

(Qiagen).

Microbial plate counting using

different media :

Total bacterial count (PCA-

22°C), psychrophilic LAB

(Elliker -20°C), LAB (MRS -

25°C), Enterobacteriaceae

(VRBG -37°C), Brochothrix

(STAA-22°C), Pseudomonas

(CFC-25°C).

From the plates, bacterial

strains were isolated. Then

identification were conducted

using 16S rRNA gene Sanger

sequencing.

Sample preparation for

library and RNAseq

sequencing will be

perfomed using a Hiseq

Sequencing System (

Illumina) by a technical

sequencing platform.

ANALYSIS

For each of the 20 samples

SAMPLES

Relative abundance of bacterial species

detected by 16S rRNA gene metagenetic

analysis in 10 samples of batch 1 during

storage

Bioinformatic analysis

were performed using

the mothur software

package (Schloss et

al., 2009).

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Others

Brochothrix_thermosphacta

Lactococcus_piscium

Lactococcus_EU826673

Carnobacterium__divergens

Photobacterium_phosphoreum

Leuconostoc_gelidum

Lactobacillus_algidus

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Others

Chryseobacterium_sabrina2_16S_OTU40

Bacilli_sabrina2_16S_OTU111

Variovorax_AM696992

Lactobacillales_sabrina2_16S_OTU288

Erysipelothrix__rhusiopathiae

env.OPS_17_sabrina2_16S_OTU35

Flavobacterium_JX196644

Arthrobacter__oxydans

Leuconostoc_sabrina2_16S_OTU100

Pseudomonadaceae__caeni

Exiguobacterium__profundum

Acinetobacter_FJ774972

Arthrobacter_GU570654

Staphylococcus__equorum

Arthrobacter_sabrina2_16S_OTU27

Psychrobacter_FJ463826

Fusobacterium__necrophorum

Thermus_KC120646

Thermus__thermophilus

Pseudomonas_JN873210

Arthrobacter_FJ381999

Chryseobacterium_sabrina2_16S_OTU18

Acinetobacter_EF632916

Soonwooa_sabrina2_16S_OTU15

Lactobacillus_EU777805

Psychrobacter_psychrophilus

Psychrobacter_cibarius

Streptococcus_equinus/bovis

Carnobacterium__divergens

Janthinobacterium_EU536259

Myroides_EF509349

Escherichia_coli

Photobacterium_phosphoreum

Lactococcus_EU826673

Pseudomonas_fluorescens/poe/trivialis

Lactococcus_piscium

Brochothrix_thermosphacta

Leuconostoc_gelidum

Lactobacillus_algidus

Relative abundance of

bacterial species detected by

16S rRNA gene metagenetic

analysis in 10 samples of

batch 2 during storage

In batch 2, 39 OTUs are identified at a treshold > 1% of the global population detected. At the shelf life limit (7 days) days of storage B. thermosphacta and LAB: Lc. gelidium, Lactobacillus

algidus and Lc. piscium at the dominant species.

Changes in bacterial counts in batch 1

during storage. Arrows symbols indicate that count

values were below the detection threshold.

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

Day1-1 Day1-2 Day3-1 Day3-2 Day5-1 Day5-2 Day7-1 Day7-2 Day9-1 Day9-2

Total bacteria count

Pseudomonas

Brochothrix

LAB

Psychrophilic LAB

Enterobacteriaceae

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

Day1-1 Day1-2 Day3-1 Day3-2 Day5-1 Day5-2 Day7-1 Day7-2 Day9-1 Day9-2

Total bacteria count

Pseudomonas

Brochothrix

LAB

Psychrophilic LAB

Enterobacteriaceae

In both batches, psychrophilic LAB are dominant after the 5 days of storage and

until the end with more than 7 log (CFU/g) and LAB more between 6 and 7 log (CFU/g)

In bath 2, Pseudomonas and Brochothrixreach almost 6 log (CFU/g) at the time of

spoilage (7 days).

Changes in bacterial counts in batch 2

during storage. Arrows symbols indicate that count values were below the detection threshold.

Batch 2 samples, clearly present an important diversity at the beginning of storage compare to batch .In batch, the samples present a low diversity during

all the storage.

Biodiversity diversity index of the

20 samples

After 7 days of storage (shelf-life limit), both batches present sensory properties linked to spoilage : rancid odour, brown coloration and slime

According the relative abundance and alpha-diversity results , it seems that’s

the meat in batch 2 was more fresh than in batch 1

0

2

4

6

8

10

12

14

16

18

20

inverted simpson