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
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
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