Meat Science 131 (2017) 34–39

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Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Effect of abattoir and cut on variations in microbial communities of vacuum- MARK

packaged beef
Mandeep Kaur⁎, John P. Bowman, Bianca Porteus, Alison L. Dann, Mark Tamplin
Tasmanian Institute of Agriculture, School of Land and Food, University of Tasmania, Sandy Bay Campus, Hobart, Tasmania 7005, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: This report builds on the earlier studies of the shelf-life of chilled Australian vacuum packaged (VP) beef primals
Shelf-life (striploin and cube roll), products distinguished in the global marketplace for unusually long shelf-life. Notable
Beef primal findings in those studies were a shelf-life of at least 26 weeks at −0.5 °C, low microbial counts, and relatively
Bacterial communities high sensory scores. However, growth rates for total viable counts (TVC) and lactic acid bacteria (LAB) varied
Vacuum-packaged
among the different abattoirs. The present study adds to these findings, by providing greater definition about
temporal changes in bacterial communities using terminal restriction fragment length polymorphism (TRFLP)
and clone library analyses of 16S ribosomal RNA (16S rRNA) gene, and measuring statistical associations among
abattoir, beef cut, storage time and sensory attributes. Bacterial communities changed over time, with
Carnobacterium spp. typically predominating (29–97%) at the end of storage. Variation in TRFLP profiles
showed that different Carnobacterium strains predominated in different abattoirs, and that additional variation
was due to the presence of other taxa typical of VP meat microbiomes. TRFLP-based community structure
correlated significantly (P ≤ 0.01) with sensorial characteristics, such as vacuum integrity, confinement odour,
and intact pack appearance of beef. This study shows that Carnobacterium spp. predominate on extended shelf-
life VP beef primals, while other taxa may produce subtle effects on shelf-life duration.

1. Introduction
Fresh meat is a niche to a relatively definable microbial community,
which originates from varied sources within meat processing facilities
(Doulgeraki, Ercolini, Villani, & Nychas, 2012; García-López,
Prieto, & Otero, 1998). The animals that present at the abattoir are
the primary sources of bacterial contamination of otherwise sterile beef
tissues. However, once in the processing facilities, microbes can
establish biofilms and form aerosols facilitating transmission to multi-
ple batches of meat (Brooks & Flint, 2008; De Filippis, La Storia,
Villani, & Ercolini, 2013; Pothakos, Devlieghere, Villani,
Björkroth, & Ercolini, 2015). The specific processing and sanitation
protocols adopted in different abattoirs may further influence the initial
diverse colonizing community (Pothakos et al., 2015; Stellato et al.,
2016). Thereafter, packaging conditions, storage temperature and
interactions among different microbes can exert selection pressure
affecting the subsequent development of meat bacterial community
structure, as well as determining bacterial growth kinetics and rates of
meat spoilage. In addition, the inherent biochemistry and anatomy of
individual muscles influences the dominant microbial consortium, the
specific spoilage organisms (SSO), and overall meat spoilage process
(Corry, 2007; Mills, Donnison, & Brightwell, 2014; Nychas, Skandamis,
Tassou, & Koutsoumanis, 2008; Wheatley, Giotis, & McKevitt, 2014).
Once established, SSO produce metabolites that reduce meat quality
and shelf-life (Casaburi, Piombino, Nychas, Villani, & Ercolini, 2015;
Ercolini et al., 2011). The quantity of these undesirable compounds
increases as microbes grow to higher levels. Certain bacteria, particu-
larly those that grow in aerobic atmospheres, have a greater negative
effect on meat quality. In contrast, some species, such as lactic acid
bacteria (LAB), that preferentially grow in low oxygen atmospheres
(e.g. vacuum and modified atmosphere packaging) produce substances
that have comparatively less impact on meat quality. In fact, some LAB
by-products have been shown to enhance consumer acceptance of
certain meat products (Leisner, Laursen, Prévost, Drider, & Dalgaard,
2007; Pothakos et al., 2015).
It has been known for many years that meat shelf-life can be
extended by reducing levels of aerobic bacteria through vacuum-
packaging (VP) and effective cold storage (Gill, 1986). However, more
recent surveys of commercial raw beef show some VP beef products
have an unusually long shelf-life. Australian VP beef primals with low
initial total viable counts (TVC) (Phillips, Sumner, Alexander, & Dutton,
2001; Vanderlinde, Shay, & Murray, 1998), rarely reached 7 log TVC/

⁎
Corresponding author.
E-mail address: Mandeep.Kaur@utas.edu.au (M. Kaur).

http://dx.doi.org/10.1016/j.meatsci.2017.04.021
Received 19 November 2016; Received in revised form 30 March 2017; Accepted 20 April 2017
Available online 22 April 2017
0309-1740/ © 2017 Elsevier Ltd. All rights reserved.
M. Kaur et al.
cm2 and largely maintained acceptable sensory evaluation scores for at
least 26 weeks of storage at −0.5 °C (Small, Jenson,
Kiermeier, & Sumner, 2012; Small, O'Callaghan, & Beilken, 2011).
Authors of these studies also observed that growth rates and net
changes in levels of TVC and LAB varied among similar products
produced at different abattoirs. However, the reason(s) for these
differences remains unknown. One hypothesis to explain this variability
in counts is that combined hurdles, such as low temperature, pH and
low oxygen, subject bacteria to stress conditions at the growth-no-
growth boundary. Other possible explanations are that low initial
numbers, and the types and proportions of bacteria that normally
contaminate meat during slaughter and processing, differ among
abattoirs and fluctuate during storage.
The aim of the present study was to measure temporal changes in
bacterial communities on VP beef primals using cultivation indepen-
dent techniques. Terminal restriction fragment length polymorphism
(TRFLP), and cloning and sequencing, were used to assess bacterial
community structure on beef striploins and cube rolls from six
Australian export abattoirs, for samples stored for 30 weeks at
− 0.5 °C. The samples examined here are the same studied by Small
et al. (2012) obtained from six different abattoirs and stored as rinsates
at − 80 °C. Small et al. (2012) found substantial variations in aerobic
TVC and LAB growth rates for the samples, suggesting possible
variability in bacterial community structure. For a sensorial perspec-
tive, Small et al. (2012) reported the samples scored highly for vacuum
integrity and post bloom appearance up until week-28 of storage and
for intact pack appearance up to week 26. Persistent off odour was
noted by week-28 of storage.
The overall goal of the present report was to establish the primary
community components of long shelf-life of VP beef primals, to
determine if these communities varied among abattoirs, and to
investigate relationships with sensorial attributes.
2. Material and methods
2.1. Sample collection
Frozen (− 80 °C) 100 ml rinsate samples from a previous study on
VP beef primals were analyzed here. Detailed description of sample
collection and processing is provided in Small et al. (2012). Briefly, VP
beef striploins and cube rolls collected from six Australian export
abattoirs (A through F) and stored at − 0.5 °C for 30 weeks were used.
These abattoirs represent a wide geographic range from Tasmania to
Queensland, with regard to their location and cattle source. Further,
they vary in their capacity (process 600 and 3500 cattle per day) and
processing protocols (abattoirs C and D utilized hot-water pasteuriza-
tion on final carcasses prior to chilling). Microbiological analyses were
done at the time of packaging (time 0) and at 8, 12, 16, 20, 24, 26 and
30 weeks. Triplicate samples of striploin and cube roll were used at
each sampling point, where individual primal was placed in a sterile
plastic bag and massaged for 2 min with 500 ml of 0.85% saline. From a
200 ml aliquot, a portion (100 ml) was frozen and used for microbial
community analysis in the present study, and other portion used for
TVC and LAB counts as described in Small et al. (2012). Triplicate
rinsate samples from storage times of 0, 16, and 30 weeks were
included in the present study. These time intervals were chosen as
they represented points in TVC and LAB growth curves for initial levels,
and exponential and stationary growth phases.
2.2. Sensory analysis
Sensory qualities of meat samples were assessed by a six-member
informal sensory panel based at the Commonwealth Scientific and
Industrial Research Organization (CSIRO) at weeks 8, 12, 16, 20, 24, 26
and 30 (Small et al., 2012). In brief the analysis involved a nine-point
hedonic scale for assessing vacuum integrity (0 = no vacuum, to
35
Meat Science 131 (2017) 34–39
8 = complete/tight vacuum), intact pack appearance and post bloom
appearance of the meat (0 = severe discolouration, to 8 = fresh/no
discolouration) and confinement odour upon pack opening (0 = ex-
treme off odour, to 8 = fresh no off/confinement odour) (Small et al.,
2012).
2.3. Terminal restriction fragment length polymorphism (TRFLP) analysis
Triplicate rinsate samples from weeks 0, 16 and 30 from all
abattoirs, were thawed and aliquoted into 4 × 1 ml subsamples in
1.5 μ centrifuge tubes. Samples were centrifuged at 5000 × g for
10 min, 0.75 ml of supernatant removed, the pellet resuspended in
the remaining supernatant, and the four subsamples combined. These
samples were centrifuged at 5000 × g for 10 min, the supernatant
removed, and DNA extracted using the DNeasy™ Blood and Tissue kit
(Qiagen, Valencia, CA) as per manufacturer's instructions. Genomic
DNA was quantified using a NanoDrop 8000 spectrophotometer
(Thermo Scientific, Wilmington, Detroit, Michigan).
The V1–V8 region of the 16S rRNA gene sequence was amplified
using the universal primers 27F (5′-GAGTTTGATCCTGGCTCAG) (Lane,
1991) and 1492R (5′-TACGGYTACCTTGTTACGACTT) (Turner, Pryer,
Miao, & Palmer, 1999). Primer 27F was labelled with the Beckman
Coulter WellRED fluorescent dye D3, and 1492R with D4 WellRED dye
(Sigma Aldrich, St Louis, Missouri, USA). The PCR amplification
mixture contained 10 μl of Immomix Mastermix (Bioline, Alexandria,
NSW), 0.5 μM of each primer, and approximately 10 ng of template
DNA and PCR-grade water, to make a final volume of 20 μl. The PCR
amplification program was as follows: 1 cycle of 95 °C for 10 min;
35 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, with a
final elongation of 72 °C for 7 min. PCR products were visually
inspected with agarose gel electrophoresis.
Duplicate PCRs were performed and pooled to reduce PCR bias, and
purified by ethanol precipitation. Aliquots of purified PCR products
were digested individually with HinfI, MspI and RsaI restriction
endonucleases (New England Biolabs Inc., Ipswich, MA), according to
the manufacturer's instructions. The digests were visually checked with
agarose gel electrophoresis. Three enzymes were chosen to mitigate the
effects of different bacterial groups having similar profiles with a
particular enzyme. Digested PCR samples were then desalted and
purified using a 96-well ethanol precipitation method (Beckman
Coulter CEQ8000 Handbook) and wrapped in aluminium foil to
minimize light exposure.
Purified digest products (1–3 μl) were mixed with 30 μl sample
loading solution (Beckman Coulter, Brea, California) and 0.3 μl of
600 bp DNA size standard (Beckman Coulter), and analyzed on the
Beckman Coulter CEQ8800 Genetic Analyser System using the Frag-4
method (injection 2.0 kV/20 s, capillary 50 °C/4.8 kV for 60 min).
Fragment profiles were sorted by size and any peak
height < 1000 DFU (Dye Fluorescence Units), < 60 bp, and > 600 bp
were discarded and filtered for spurious peaks using CEQ8000
Fragment Analysis software (Beckman Coulter). Peaks were standar-
dized to percent relative peak height; any peak with < 1% of the total
peak area was discarded and the peaks that contributed < 2% of the
total peak area were removed to minimize the effect of baseline noise
from variable amounts of DNA loaded (Osborne, Rees,
Bernstein, & Janssen, 2006).
2.4. 16S rRNA gene clone libraries
Rinsate samples from cube roll and striploin from abattoirs A, C, D
and F, for weeks 0, 16 and 30, were used for clone library analysis. The
V1–V4 region of the 16S rRNA gene was amplified using the forward
primer 27F (5′-GAGTTTGATCCTGGCTCAG) and reverse primer 907R
(5′-CCGTCAATTCCTTTAAGTTT); the PCR conditions were used as
above for TRFLP. The PCR product was then purified using an
UltraClean® PCR Clean-Up Kit (Mobio, Carlsbad, CA). Purified PCR
M. Kaur et al. Meat Science 131 (2017) 34–39

Transform: Square root

products were cloned using an Invitrogen pCR®4 TOPO TA vector kit Resemblance: S17 Bray Curtis similarity
(Life Technologies, Grand Island, NY) as per the manufacturer's 0.3 Abattoir
protocol. Clones were screened using the vector primers M13F (5′- A
GTAAAACGACGGCCAG) and M13R (5′-CAGGAAACAGCTAGGAC). B
Clones (60 for each sample) with inserts were sent to Macrogen C
0.2
D
(Seoul, Korea) for sequencing. Sequences were quality checked and
E
vector sequence removed using ChromasPro v1.32 (Technelysium Pty F
Ltd). The results were checked for chimeric sequences using Pintail v1.1 0.1
(Ashelford, Chuzhanov, Fry, Jones, & Weightman, 2005).

CAP2
2.5. Statistical analysis 0

The TRFLP peak height data from all the enzyme digests for all
samples were collated into an Excel™ spreadsheet and analyzed in -0.1
PRIMER v6 software (Primer-E Ltd., Plymouth Marine laboratory, UK).
A similarity matrix using the Bray-Curtis coefficient was calculated
(Bray & Curtis, 1957). PERMANOVA (permutational multivariate ANO- -0.2
VA) (add-in to PRIMER) (Anderson & Gribble, 1998) was used to test for
differences in bacterial communities under the fixed factors (abattoir,
cut, storage time). Differences were considered significant at P < 0.01 -0.3
(using 9999 permutations). Canonical analysis of principal coordinates -0.3 -0.2 -0.1 0 0.1 0.2 0.3
CAP1
(CAP) (Anderson & Willis, 2003) was then used to visualize multi-
dimensional community differences plotted along simple axes expres- Fig. 1. Canonical analysis of principal coordinates (CAP) of bacterial TRFLP peak data
sing similarity and dissimilarity between samples and viewed in terms from vacuum packaged beef primals (cube roll and striploin both) based on Bray Curtis
for the factors abattoir, cut and week of storage. similarity matrix and grouped by abattoir.

The relationship of bacterial terminal restriction fragments (TRFs)

and meat sensory characteristics (data obtained from Small et al., 2012)
was investigated using a distance-based linear model (DISTLM) and
distance-based redundancy analysis (dbRDA) (Anderson & Gribble,
1998). For DISTLM, the forward selection option and 9999 permuta-
tions were used to identify variables that were significantly correlated
with the observed bacterial community structure. The vectors on the
dbRDA ordination diagram show strength and direction of the relation-
ship between individual vector (sensory characteristics) and construc-
tion of the constrained ordination plot. Further, the bacterial TRF data
were employed to explore differences in community diversity (Shannon
index, richness and evenness) in individual abattoirs, storage time and
primal cuts, by analysis of variance using Calypso (http://bioinfo.qimr.
edu.au/calypso/).
The 16S rRNA gene clone library taxonomic affiliations were
assigned with the naïve Bayesian classifier of the Ribosomal Database
Project (RDP) with an 80% bootstrap confidence cut-off (Wang, Garrity,
Tiedje, & Cole, 2007). Counts were converted to percent (%) relative
abundance to genus level. Taxonomy results were removed for any taxa
that contributed < 1% to overall abundance. Differences among bac-
terial communities for different factors (abattoir, cut, storage time)
based on phylogenetic distance were calculated using UniFrac
(Lozupone & Knight, 2005).
3. Results
3.1. TRFLP analysis of VP beef primal communities from different abattoirs
A–F, B–C, B–D, C–F, D–F after 16 weeks, and between A–F, C–F, D–F,
E–F at the end of shelf-life at 30 weeks.
3.2. TRFLP analysis of VP beef communities over storage and between cuts
When the same data were analyzed by storage period (weeks),
significant differences (P = 0.0011) were observed with three distinct
clusters corresponding to three time points obtained in CAP plots
(Fig. 2). Significant differences were also observed in striploin and cube
roll primal cuts during storage under VP conditions for 30 weeks
(P = 0.0001) (Fig. 3). Furthermore, within each abattoir and at each
time point, significant differences (P ≤ 0.1) in striploins and cube rolls
were observed, except for abattoirs B (week 16), E (week 0 and 30) and
F (week 30). A significance level of ≤ 10% was considered significantly
different as the maximum possible permutations with three replicates
were 10.
3.3. Changes in VP beef primal bacterial diversity during storage
Significant differences were observed among abattoirs (P = 0.006)
Transform: Square root
Resemblance: S17 Bray Curtis similarity
0.2 Week
0
16
30
0.1

In order to test the impact of abattoir on community structure

variation, the bacterial TRFLP profiles generated for each sample were
CAP2

0
compared using PERMANOVA. Significant differences in bacterial
community profiles among all abattoirs (P = 0.0001) were found,
except for abattoirs A and C where the difference was marginal -0.1
(P = 0.0128), and for abattoirs B and F, which had near complete
overlap (P = 0.2709). These results are visualized in the CAP plot
(Fig. 1). The plot showed distinct grouping of VP meat samples by -0.2
abattoir, with the first canonical axis separating abattoirs B, E and F -0.3 -0.2 -0.1 0 0.1 0.2 0.3
from abattoirs A and C, while the second axis separated abattoir D from CAP1

abattoirs A, B, C and F. Differences among abattoirs changed over a Fig. 2. Canonical analysis of principal coordinates (CAP) of bacterial TRFLP peak data
period of 30 weeks. Differences were significant (P ≤ 0.01) between from vacuum packaged beef primals (cube roll and striploin both) based on Bray Curtis
abattoirs A–B, B–E, C–F, D–F, E–F at the start, between A–B, A–D, A–E, similarity matrix and grouped by the storage period at −0.5 °C.

36
M. Kaur et al.
Transform: Square root
Resemblance: S17 Bray Curtis similarity
0.3
0.2
0.1
CAP2
0 0
-0.1
-0.2
-0.3
-0.3 -0.2 -0.1 0 0.1 0.2
CAP1
Fig. 3. Canonical analysis of principal coordinates (CAP) of bacterial TRFLP peak data
from vacuum packaged beef primals (cube roll and striploin both) based on Bray Curtis
similarity matrix and grouped by the primal cut × storage time at − 0.5 °C.
Fig. 4. Bacterial diversity analysis of TRFs associated with vacuum packaged beef primals
processed at six different abattoirs (A, B, C, D, E and F) and stored at − 0.5 °C for
30 weeks.
for bacterial community diversity (Fig. 4). Abattoir D showed the
highest diversity followed by abattoir A. The higher diversity observed
at abattoir D resulted from a greater number of bacterial species
(measured as number of TRFs) present, and which were evenly
distributed. The lowest diversity was observed at abattoir F. Striploins
showed greater bacterial diversity, compared to cube rolls, and
diversity decreased with storage time (data not shown).
3.4. Relationship between meat sensory quality parameters and TRFs
The DISTLM analysis showed that relationships among sensory
attributes and bacterial TRFs were significant (Fig. 5), with positive
correlations (P ≤ 0.01) observed between TRFs and vacuum integrity,
confinement odour, and intact pack appearance. No correlation with
post bloom appearance was observed.
3.5. Community structure of VP cube rolls over 30 weeks
The 16S rRNA gene clone library analysis showed that initial
bacterial communities on cube rolls from different abattoirs were
generally dominated by members of classes Betaproteobacteria and
Gammaproteobacteria, except for abattoir F where members of
Firmicutes predominated (86%) from the outset (Supplementary Table
S1). All abattoirs had Pseudomonas spp. in the initial population (6–22%
of clones). Janthinobacterium spp. were abundant initially on samples
Meat Science 131 (2017) 34–39
Resemblance: S17 Bray Curtis similarity
60 Week
CutWeek 0
C0 16
Post bloom appearance
C16 30
C30 40
S0
S16
dbRDA2 (4.4% of fitted, 1.4% of total variation)
S30
20
Confinement odour
Intact pack appearance
-20 Vacuum integrity
-40
0.3 -60
-60 -40 -20 0 20 40 60
dbRDA1 (95.2% of f itted, 29.7% of total variation)
Fig. 5. dbRDA analysis of variation of TRFLP bacterial community structure constrained
at storage week as explained by meat sensory characteristics. Vectors represent correla-
tions of variables with community structure along the two dbRDA axes by Pearson's
correlations.
from abattoirs A (76%), C (22%) and D (44%). By week 16, C. divergens
was present on samples across all abattoirs (44–74% of clones).
Abattoirs A, C and F also contained other Carnobacterium spp. as well
at this stage. Brochothrix thermoshacta was found on samples at abattoir
D (48%). At 30 weeks, C. divergens was dominant on samples from
abattoir A (85%) and C (68%), and to lesser extent from D (32%) and F
(48%). Abattoirs A and D still contained detectable levels of
Janthinobacterium spp., though relative abundance was substantially
lower. Abattoirs D and F samples were the only abattoirs at 30 weeks to
contain C. maltaromaticum (21 and 42%). Abattoir C also contained
Serratia proteamaculans (27%). Overall, after 30 weeks of storage at
− 0.5 °C, Carnobacterium became clearly the most abundant taxon on
VP primals across all abattoirs (53–93% of total clones), with the most
consistent and predominant species being Carnobacterium divergens.
UniFrac analysis showed differences between abattoir cube roll
sample pairs over 30 weeks of storage. A Bonferroni-corrected P value
of < 0.05 was considered significantly different; however results
showed that P values for other abattoir comparisons were 0.06, and
thus could be designated “suggestively different”. Over 30 weeks,
bacterial communities in abattoirs measured by clone library were
different by most comparisons (data not shown). A summary statistical
analysis showed that all abattoirs for each week were significantly
different (P ≤ 0.01). Overall, this was in agreement with TRFLP
analysis.
3.6. Community structure of VP striploins over 30 weeks
Similar to cube rolls, bacterial communities on striploins across all
abattoirs over 30 weeks were generally dominated by members of the
phyla Proteobacteria and Firmicutes as assessed by clone libraries.
Initially, striploins from abattoirs A, C and D contained
Janthinobacterium, and represented 84 and 94% of clones from abattoirs
A and D, respectively. In abattoir F striploin samples, Carnobacterium
was again the dominant species, as found for cube rolls (87% of clones).
At week 16, all dominant species were Carnobacterium spp., either C.
maltaromaticum or C. divergens. Besides Carnobacterium, other genera
were detected at lower levels, including Clostridium (14% and 8%,
abattoir A and D, respectively), and Lactobacillus (7–16%, abattoirs A, C
37
M. Kaur et al.
and D), Lactococcus (16%, abattoir C), Leuconostoc (9.5%, abattoir F).
By week-30, Carnobacterium predominated on samples from abattoirs A,
C and D (77–97%), whereas for abattoir F, Leuconostoc gelidum (63%)
and Carnobacterium (29%) were the predominant bacteria.
UniFrac analysis showed more similarities for striploins among
abattoirs compared to cube rolls. Abattoirs A and D were not
significantly different at 0 and 16 weeks, most likely due to similar
predominance of Janthinobacterium spp. All abattoirs at 16 weeks were
less different to each other, likely due to having high abundances of
Carnobacterium spp. There was a significant difference between abat-
toirs D and F at 30 weeks due to clustering of species at each abattoir.
Abattoir D samples had a dominant population of C. divergens (89%)
whereas Leuconostoc gelidum (63% of clones) and C. maltaromaticum
(26%) predominated in abattoir F samples. As with cube rolls, striploin
samples were significantly different among abattoirs (P ≤ 0.01).
Analysis between striploin and cube rolls at each abattoir showed no
significant differences over the 30 weeks of storage. For abattoir C at
0 week and abattoir F at 16 weeks, the strongest similarity was between
striploin and cube roll samples (P = 1). At 0 weeks, abattoir C cube
rolls and striploins shared similar populations of Pseudomonas,
Acidovorax, C. divergens, Janthinobacterium and Sphingomonas. For
abattoir F, at 16 weeks similarity between cube rolls and striploin
could be explained by having Pseudomonas, C. maltaromaticum and C.
divergens populations.
4. Discussion
This study builds on the information gained in previous studies on
VP beef primal shelf-life (Small et al., 2011; Small et al., 2012). Notable
findings in those studies were the extremely long shelf-life of Australian
VP beef primals (26 weeks) at − 0.5 °C, low microbial counts, relatively
high sensory scores, and variable growth rates of TVC and LAB among
the six abattoirs surveyed. The variability in TVC and LAB counts were
sometimes observed for different primal cuts produced by the same
processor.
This earlier study defined growth kinetics of two common microbial
parameters of meat quality, i.e. TVC and LAB. While each of these tests
have long historical use and are primary tests for domestic and
international market standards, they measure changes broadly in
meat-associated bacteria; more data are needed to develop fundamental
understanding of growth dynamics, in particular to explain the
observed variability.
In the present study, we used PCR-based molecular techniques to
explore temporal and spatial changes in bacterial communities on VP
primals, and to determine associations with changes in TVC and LAB
levels. Exploring spatial and temporal microbial community dynamics
via TRFLP also enables meaningful insights into the effect of these
changes, in relation to food quality parameters (Kaur, Bowman,
Stewart, & Evans, 2015). The discriminative ability of the approach
(TRFLP and associated multivariate statistical analyses) used in this
study was demonstrated from significant correlations between impor-
tant meat sensory quality attributes (confinement odour, post bloom
appearance and overall vacuum pack appearance) and bacterial com-
munity dynamics during storage. As storage progressed, changes in
bacterial community structure occurred, resulting in sensorial dete-
rioration of the meat.
TRFLP analysis showed that bacterial communities on beef primals
produced at six abattoirs were different. These differences might arise
due to dissimilarities in in-house bacterial communities residing in each
abattoir environment. These localized populations presumably origi-
nate from incoming bacteria on animals transported from local areas.
The six abattoirs, studied here are dispersed across diverse geographical
locations in Australia, ranging from tropical far north Queensland to
temperate Tasmania, each region being a unique ecosystem in itself
with regard to cattle breed, vegetation and rainfall pattern. Although,
sampling was done at the same time in all abattoirs, the cattle
38
Meat Science 131 (2017) 34–39
slaughtered in abattoir F experienced tropical rains, and thereby might
carry different microbiota into the processing environment. The selec-
tion and establishment of the resident bacteria could be further
influenced by different processing protocols adopted in each abattoir,
such as hot-water pasteurization of final carcasses prior to chilling at
abattoir C and D. Once established, these abattoir microbiomes act as
source of inoculum for subsequent production batches (De Filippis
et al., 2013; Hultman, Rahkila, Ali, Rousu, & Björkroth, 2015; Stellato
et al., 2016).
The differences among abattoirs were both qualitative, i.e. the type
of bacterial species/strains, and quantitative, i.e. the relative percen-
tage of different species/strains. Although, non-LAB at the start, and
LAB during storage, dominated primals from different abattoirs, there
still were differences in microbial diversity at each abattoir. These
differences did not appear to be associated with highly unique genera or
species, indicating that the factors influencing different growth rates of
TVC and LAB at six abattoirs may lie at the strain level. To test this
hypothesis, strains from different abattoirs could be analyzed for
temperature and pH growth/no-growth boundaries, production of
growth-limiting factors (e.g. types and levels of organic acids, quorum
factors, bacteriocins) and for the inherent ability to utilize meat-based
nutrients. In this regard, Zhang, Baranyi, and Tamplin (2015) found
high variation in intra- and inter-species inhibitory activities among
bacteria isolated from the same samples used in this present study, thus
indicating this property might influence inter-abattoir variation in
bacterial community structure.
Bacterial communities on VP primals changed over storage. These
results agreed with published literature (Ercolini, Russo, Torrieri,
Masi, & Villani, 2006; Ercolini et al., 2011; Jones, 2004; Kiermeier
et al., 2013), reflecting a transition in bacterial species that predomi-
nate in aerobic atmospheres, to one comprised of LAB in VP meat. The
differences observed in cube rolls and striploins in this study were not
anticipated as it was assumed that both primal cuts would likely have
been exposed to similar environmental surfaces during slaughter and
processing, especially within the same abattoir. However, it is possible
that anatomical and physiological differences between these types of
cuts promote different types and levels of bacteria. Further, the manner
in which these cuts are prepared, and the time and extent they are
exposed to different surfaces, might also affect their microbial composi-
tion. In this regard, Holder, Corry, and Hinton (1997) found a
correlation between microbial counts on chicken carcass sampling sites
and exposure time. Sites with higher exposure had significantly higher
number of microbes than less exposed ones.
The results from clone libraries show that in all abattoirs bacterial
communities changed during storage. When beef primals were initially
packaged at the abattoirs, they were contaminated mostly with non-
LAB species (e.g. Pseudomonas, Janthinobacterium), with the exception
of abattoir F, which had a dominate community of Carnobacterium.
With time, LAB species viz. C. maltaromaticum, C. divergens, Lactobacillus
spp., Lactococcus spp., and Leuconostoc spp. became dominant, with the
occasional occurrence of Acidovorax spp., Serratia spp., B. thermoshacta,
and Clostridium spp. All of the bacterial genera observed in the current
study are commonly found in meat and meat products (Borch, Kant-
Muermans, & Blixt, 1996; Casaburi et al., 2015; Doulgeraki et al., 2012;
Jones, 2004; Jones, Hussein, Zagorec, Brightwell, & Tagg, 2008;
Labadie, 1999). However, species/strains and further, their relative
propositions, vary from study to study, and sometimes within a study,
such as differences among abattoirs, time and type of meat primal.
Overall, these results agree with the TRFLP studies. Although clone
library assigns 16S rRNA sequences to a genera/species, this method is
less discriminatory than TRFLP that detects intra-species (strain)
differences.
High phenotypic and metabolic intra-species variability in both C.
maltaromaticum and C. divergens has been reported in the literature
(Leisner et al., 2007; Zhang et al., 2015). Zhang et al. (2015) studied the
interactions among diverse group of bacteria isolated from the same VP
M. Kaur et al.
beef primals in the previous and current study, and found different
levels of interactions among different isolates. In total, C. maltaroma-
ticum and C. divergens isolates displayed a large inhibition spectrum,
both intra- and interspecies specific. As such, C. maltaromaticum, and C.
divergens may have strong influences on bacterial community structure
in VP beef, and the long shelf-life as observed by Small et al. (2012).
The species of Carnobacterium, especially C. maltaromaticum and C.
divergens, have been found in VP lamb and beef primals with extra-
ordinary long shelf-life (Kiermeier et al., 2013; Youssef, Gill,
Tran, & Yang, 2014).
The development of bacterial communities on VP beef is a dynamic
process, in which the origin of meat plays an important role. Culture-
independent DNA-based techniques hold potential for providing greater
resolution into community analysis. In this study, LAB, especially
Carnobacterium spp., appeared to dominate on meat. However, factors
contributing to this genus' dominance, its interaction with other
members of the meat microbiome and also among its own species
and strains, and their role in meat sensory attributes, requires further
study. Understanding the universality of these interactions, if present
under different storage conditions, muscle and meat types can lead to a
better apprehension of the spoilage process and shelf-life prediction,
thereby, overall supply chain management.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.meatsci.2017.04.021.
Acknowledgements
This research was funded by Meat and Livestock Australia
(A.MFS.0194). Useful advise and discussions with Ian Jenson,
Manager, Market Access Science and Technology, MLA and John
Sumner, consultant, MLA are gratefully acknowledged. We acknowl-
edge the receipt of meat rinsate samples and sensory data from Alison H
Small, Commonwealth Scientific and Industrial Research Organization.
The authors would like to thank Adam Smolenski (Central Science
Laboratory — Research, University of Tasmania, Australia) for his
technical assistance with TRFLP analysis.
References
Anderson, M. J., & Gribble, N. A. (1998). Partitioning the variation among spatial,
temporal and environmental components in a multivariate data set. Australian Journal
of Ecology, 23, 158–167.
Anderson, M. J., & Willis, T. J. (2003). Canonical analysis of principal coordinates: A
useful method of constrained ordination for ecology. Ecology, 84, 511–525.
Ashelford, K. E., Chuzhanov, A. N. A., Fry, J. C., Jones, A. J., & Weightman, A. J. (2005).
At least 1 in 20 16S rRNA sequence records currently held in public repositories is
estimated to contain substantial anomalies. Applied and Environmental Microbiology,
71, 7724–7736.
Borch, E., Kant-Muermans, M. L., & Blixt, Y. (1996). Bacterial spoilage of meat and cured
meat products. International Journal of Food Microbiology, 33, 103–120.
Bray, J. R., & Curtis, J. T. (1957). An ordination of the upland forest communities of
southern Wisconsin. Ecological Monographs, 27, 325–349.
Brooks, J. D., & Flint, S. H. (2008). Biofilms in the food industry: Problems and potential
solutions. International Journal of Food Science and Technology, 43, 2163–2176.
Casaburi, A., Piombino, P., Nychas, G., Villani, F., & Ercolini, D. (2015). Bacterial
populations and the volatilome associated to meat spoilage. Food Microbiology, 45,
83–102.
Corry, J. E. L. (2007). Spoilage organisms of red meat and poultry. In G. C. Mead (Ed.),
Microbiological analysis of red meat, poultry and eggs (pp. 101–122). Cambridge:
Woodhead.
De Filippis, F., La Storia, A., Villani, F., & Ercolini, D. (2013). Exploring the sources of
bacterial spoilers in beefsteaks by culture-independent high-throughput sequencing.
PLoS ONE, 8e70222. http://dx.doi.org/10.1371/journal.pone.0070222.
Doulgeraki, A. I., Ercolini, D., Villani, F., & Nychas, G. J. (2012). Spoilage microbiota
associated to the storage of raw meat in different conditions. International Journal of
Food Microbiology, 157, 130–141.
Ercolini, D., Ferrocino, I., Nasi, A., Ndagijimana, M., Vernocchi, P., La Storia, A., ...
Villani, F. (2011). Monitoring of microbial metabolites and bacterial diversity in beef
39
Meat Science 131 (2017) 34–39
stored under different packaging conditions. Applied and Environmental Microbiology,
77, 7372–7381.
Ercolini, D., Russo, F., Torrieri, E., Masi, P., & Villani, F. (2006). Changes in the spoilage-
related microbiota of beef during refrigerated storage under different packaging
conditions. Applied and Environmental Microbiology, 72, 4663–4671.
García-López, M. L., Prieto, M., & Otero, A. (1998). The physiological attributes of Gram-
negative bacteria associated with spoilage of meat and meat products. In A. Davies, &
R. Board (Eds.), The microbiology of meat and poultry (pp. 1–34). London: Blackie
Academic and Professional.
Gill, C. O. (1986). The control of microbial spoilage in fresh meats. In A. M. Pearson, & T.
R. Dutson (Eds.), Advances in meat research: Meat and poultry microbiology (pp. 49–88).
AVI: Westport, NY.
Holder, J. S., Corry, J. E. L., & Hinton, M. H. (1997). Microbial status of chicken portions
and portioning equipment. British Poultry Science, 38, 505–511.
Hultman, J., Rahkila, R., Ali, J., Rousu, J., & Björkroth, K. J. (2015). Meat processing
plant microbiome and contamination patterns of cold-tolerant bacteria causing food
safety and spoilage risks in the manufacture of vacuum-packaged cooked sausages.
Applied and Environmental Microbiology, 81, 7088–7097.
Jones, R. J. (2004). Observations on the succession dynamics of lactic acid bacteria
populations in chill-stored vacuum-packaged beef. International Journal of Food
Microbiology, 90, 273–282.
Jones, R. J., Hussein, H. M., Zagorec, M., Brightwell, G., & Tagg, J. R. (2008). Isolation of
lactic acid bacteria with inhibitory activity against pathogens and spoilage organisms
associated with fresh meat. Food Microbiology, 25, 228–234.
Kaur, K., Bowman, J. P., Stewart, D. C., & Evans, D. E. (2015). The fungal community
structure of barley malts from diverse geographical regions correlates with malt
quality parameters. International Journal of Food Microbiology, 215, 71–78.
Kiermeier, A., Tamplin, M., May, D., Holds, G., Williams, M., & Dann, A. (2013).
Microbial growth, communities and sensory characteristics of vacuum and modified
atmosphere packaged lamb shoulders. Food Microbiology, 36, 305–315.
Labadie, J. (1999). Consequences of packaging on bacterial growth. Meat is an ecological
niche. Meat Science, 52, 299–305.
Lane, J. (1991). 16S/23S rRNA sequencing. In E. Stackebrandt, & M. Goodfellow (Eds.),
Nucleic acid techniques in bacterial systematics (pp. 115–175). NY: John Wiley and
Sons.
Leisner, J. J., Laursen, B. G., Prévost, H., Drider, D., & Dalgaard, P. (2007).
Carnobacterium: Positive and negative effects in the environment and in foods. FEMS
Microbiology Reviews, 31, 592–613.
Lozupone, C., & Knight, R. (2005). UniFrac: A new phylogenetic method for comparing
microbial communities. Applied and Environmental Microbiology, 71, 8228–8235.
Mills, J., Donnison, A., & Brightwell, G. (2014). Factors affecting microbial spoilage and
shelf-life of chilled vacuum-packed lamb transported to distant markets: A review.
Meat Science, 98, 71–80.
Nychas, G. E., Skandamis, P. N., Tassou, C. C., & Koutsoumanis, K. P. (2008). Meat
spoilage during distribution. Meat Science, 78, 77–89.
Osborne, C. A., Rees, G. N., Bernstein, Y., & Janssen, P. H. (2006). New threshold and
confidence estimates for terminal restriction fragment length polymorphism analysis
of complex bacterial communities. Applied and Environmental Microbiology, 72,
1270–1278.
Phillips, D., Sumner, J., Alexander, J. F., & Dutton, K. M. (2001). Microbiological quality
of Australian beef. Journal of Food Protection, 64, 692–696.
Pothakos, V., Devlieghere, F., Villani, F., Björkroth, J., & Ercolini, D. (2015). Lactic acid
bacteria and their controversial role in fresh meat spoilage. Meat Science, 109, 66–74.
Small, A. H., Jenson, I., Kiermeier, A., & Sumner, J. (2012). Vacuum-packed beef primals
with extremely long shelf life have unusual microbiological counts. Journal of Food
Protection, 75, 1524–1527.
Small, A., O'Callaghan, D., & Beilken, S. (2011). Shelf-life of chilled vacuum packed beef.
Final report. Project code A.MFS.0166. Sydney, NSW: Meat and Livestock Australia.
Stellato, G., La Storia, A., De Filippis, F., Borriello, G., Villani, F., & Ercolini, D. (2016).
Overlap of spoilage-associated microbiota between meat and the meat processing
environment in small-scale and large-scale retail distributions. Applied and
Environmental Microbiology, 82, 4045–4054.
Turner, S., Pryer, K. M., Miao, V. P., & Palmer, J. D. (1999). Investigating deep
phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA
sequence analysis. The Journal of Eukaryotic Microbiology, 46, 327–338.
Vanderlinde, P. B., Shay, B., & Murray, J. (1998). Microbiological quality of Australian
beef carcass meat and frozen bulk packed beef. Journal of Food Protection, 61,
437–443.
Wang, Q., Garrity, G. M., Tiedje, J. M., & Cole, J. R. (2007). Naïve bayesian classifier for
rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and
Environmental Microbiology, 73, 5261–5267.
Wheatley, P., Giotis, E. S., & McKevitt, A. (2014). Effects of slaughtering operations on
carcass contamination in an Irish pork production plant. Irish Veterinary Journal.
http://dx.doi.org/10.1186/2046-0481-67-1.
Youssef, M. K., Gill, C. O., Tran, F., & Yang, X. (2014). Unusual composition of microflora
of vacuum-packaged beef primal cuts of very long storage life. Journal of Food
Protection, 77, 2161–2167.
Zhang, P., Baranyi, J., & Tamplin, M. (2015). Interstrain interactions between bacteria
isolated from vacuum-packaged refrigerated beef. Applied and Environmental
Microbiology, 81, 2753–2761.