Microbial shelf-life of vacuum packed wild boar meat
cuts at two different storage temperatures.
Koomen J.I.
Supervisor: Paulsen P. Institute of Meat Hygiene, University of
Veterinary Medicine Vienna, Austria
Date: 13-3-2014
Student number: 3582094
Time span: January 2014 – March 2014
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Table of contents
2
1. Abstract
p. 3
2. Introduction
p. 4
2.1 Game as food source
p. 4
2.2 Microbiology, hygiene & shelf life
p. 4
2.3 Effect of storage conditions on microbial shelf life
p. 5
2.4 Responsibility for ‘safe’ food, with a focus on Austria
p. 6
2.5 Wild boar in central Europe
p. 6
3. Material and methods
p. 8
3.1 Material
p. 8
3.1.1 Samples from wild boar carcasses processed by untrained staff
p. 8
3.1.2 Samples from wild boar carcasses processed by trained staff in a small-scale processing unit
p. 9
3.1.3 Samples from wild boar carcasses processed by trained staff in an approved game-handling-establishment
p. 9
3.2 Methods
p. 9
3.2.1 pH
p. 10
3.2.2 Lactic acid bacteria
p. 10
3.2.3 Total viable count (TVC)
p. 10
3.2.4 (presumptive) Pseudomonas
p. 10
3.2.5 E.coli and enterobacteriaceae
p. 10
3.2.6 Surfaces samples
p. 10
3.2.7 Trichinella spp. and Alaria alata
p. 10
3.3 Statistical analysis
p. 11
4. Results and discussion
p. 12
4.1 Trichinella spp and Alaria alata
p. 12
4.2 Microbiological condition of carcass surface samples
p. 12
4.3 Temperature
p. 12
4.4 pH-values before during and after storage
p. 12
4.5 E.coli counts before, during and after storage
p. 14
4.6 Enterobacteriaceae counts before, during and after storage
p. 16
4.7 TVC before, during and after storage
p. 19
4.8 Lactic acid bacterial counts before, during and after storage
p. 21
4.9 Pseudomonas counts before, during and after storage
p. 24
5. Conclusion
p. 27
References
p. 29
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
1. Abstract
In order to collect data about the use-by date of meat cuts from wild boar carcasses, 7 carcasses
were cut, in 3 different settings (untrained staff; trained staff at small-scale unit; trained staff at
approved game-handling-establishment). Meat cuts were vacuum-packed, stored at two different
temperatures (2°C and 3.8°C) and examined in defined time intervals (up to 14 days, 3-4 replicates
per cut). At days 0, 3, 7, 10 and 14, samples were tested for pH, Total Viable Count (TVC),
presumptive pseudomonas, E.coli, enterobacteriaceae and lactic acid bacteria.
E.coli values decreased independent from storage temperature over time. Temperature exerted a
significant effect on TVC. At 2°C storage temperature an average increase of 1.5 log cfu/g and at
3.8°C an increase of 2 log cfu/g was observed, depending on type of meat cut and experimental
series, the differences could exceed 1 log unit by far. This suggested that a difference of merely 1.8°C
storage temperature caused significant differences in TVC. Result of pseudomonas and
enterobacteriaceae counts were in the same trend, increase was 1 log cfu/g and 1.5 log cfu/g for 2°C
and 3.8°C storage temperature, respectively. Lactic acid bacteria counts increased with 2 log cfu/g at
2°C storage temperature and increased with 3 log cfu/g at 3.8°C storage temperature.
A difference in microbial numbers between the samples from the three settings was expected, due
to presumed differences in processing hygiene during cutting, but no evidence for such factor was
found.
3
Meat from approved game handling-establishment had a short use-by date (based on the level of
bacterial contamination). In the two other settings the use-by date was slightly higher. Use-by date at
3.8°C was consistently shorter than at 2°C. Findings demonstrate that both initial bacterial numbers
as well as storage temperature have a significant impact on shelf-life.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
2. Introduction
2.1 Game as food source
Game meat, also wild boar meat, is highly valued by consumers.(Paulsen et al. 2011) Consumers tend
to buy and eat healthier and safer food. They tend to follow a number of trends in food
consumption, eat more chicken and pork, less beef. They want products with less cholesterol and
kilojoule and they are more and more concerned about the environment and production methods.
These are reasons consumers buy and eat more meat from wild game than they did before.(Hoffman
& Wiklund 2006) Bertolini also describes an increasing trend in game consumption from 1970 to
1990 to the current level. (Bertolini et al. 2005) It is estimated that in Austria the total carcass weight
of wild game is ca. 9 million kg/year. This corresponds to an average game meat consumption of 0.5
kg/year/person. (Winkelmayer et al. 2011)
2.2 Microbiology, hygiene & shelf life
4
A lot of waste is produced due to microbial spoilage of food. Meat is especially susceptible for
spoilage due to its chemical composition; this can cause visible, textural, odour and colour changes.
Spoilage is characterised by any change in food that makes it unacceptable for consumers to eat.
(Gram et al. 2002) Although muscle tissue of healthy slaughter animals is considered as sterile(Gill et
al. 1978), bacteria from skin, intestines, the environment and workers hands etc. are transferred on
meat surfaces during slaughter and further processing. Consequently, bacterial genera occurring on
fresh meat cuts including Acinetobacter, Pseudomonas, Enterobacteriaceae, lactic acid bacteria,
Staphylococcus, Micrococcus and Flavobacterium, are described. (Doulgeraki et al. 2012) Despite the
same chemical composition of meat cuts, different storage or preservation conditions provided
different bacteria the conditions to grow, become dominant and cause spoilage. Not all bacteria
present on meat cuts need to cause spoilage. Only the bacteria able to produce metabolites
associated with spoilage are so called spoilage bacteria. (Doulgeraki et al. 2012)(Gram et al. 2002)
Deutz reported the importance of good hygiene practice (GHP) in order to keep microbial counts on
game meat surfaces as low as possible. The hygienic quality and use-by date are highly depended on
bacterial counts on the carcass, place were the animal was shot, time between shooting, evisceration
and cutting and the practical hygiene during cutting. Bacterial counts on exposed muscle surfaces of
eviscerated carcasses of large game processed under GHP were 3.6 * 105 cfu/cm2 and 2.4 * 105
cfu/cm2 whereas bacterial counts under average conditions were 2.0 * 106 cfu/cm2 and 6.6 * 105
cfu/cm2 (Deutz et al. 2000) Another study pointed out the importance of cooling during the entire
meat chain. Paulsen and Winkelmayer found in 2004 that pre-cooling of the carcass at 10°C was
associated with Total viable Count (TVC) of 4.1 log cfu/cm2 and pre-cooling at 18 °C with 5.7 log
cfu/cm2 on exposed muscle surfaces. Subsequent cooling at 0.4°C preserved these numbers for 96 h.
(Paulsen 2011)(Paulsen & Winkelmayer 2004)
European legislation No. 2073/2005 (EC 2005) provides some microbiological criteria for food stuff.
However legislation on process hygiene criteria refers to carcasses from farm animals, not to meat
from free living game. In the past years some studies have been conducted with a view to elaborate
microbial limits for game carcasses or to provide an estimate which magnitude of microbial load
could be expected. (Atanassova et al. 2008)(Deutz et al. 2000)(Avagnina et al. 2012)(Obwegeser et al.
2012). Some of these data have been collated in Table 1.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
TVC
Enterobacteriaceae
E. coli
Reference
6.0
2.0
(Deutz et al. 2000)
6.0
(Paulsen et al. 2003)
5.0
3.0
2.0
(Lagrange & Schmidt 2005)
Table 1 Suggested microbial limits on wild game carcasses ( log cfu/g) collected by Paulsen (Paulsen 2011)
Paulsen (Paulsen 2011) also summarized data (Table 2) about the microbial conditions of game meat
cuts. He concluded that average microbial count of 3-4 log cfu/g TVC, 2-3 log cfu/g
enterobacteriaceae and 1-2 log cfu/g E. coli can be achieved under GHP conditions.
Samples
Conditions
n
Obtained at1
Hare
Frozen
79
GHE
Hare
Frozen, larded
85
GHE
Roe deer
Frozen
44
GHE
Red deer
Frozen
491
GHE
Wild boar
Frozen
224
GHE
Various
Frozen
63
GHE
Various
Chilled, vac.packed
Chilled
30
LSH-sc
43
LSH-training
Fresh after
cutting
49
Various
5
Roe deer
TVC (log
cfu/g)
4.0
(2.2-6.2)
4.0
(3.4-8.3)
5.7
(3.5-7.3)
4.3
(1.1-6.6)
6.0
(2.6-8.6)
6.4*
(4.3-7.3)
4.4
(2.0-7.7)
5.1
(3.0-7.7)
5.7
EB (log
cfu/g)
2.0
(1.0-3.8)
2.7
(1.0-6.0)
3.6
(1.0-6.3)
2.2
(1.0-4.2)
3.5
(1.0-5.8)
6.0*
(4.3-7.0)
2.3
(1.0-5.7)
2.7
(2.0-5.7)
3.9
E. coli log
cfu/g)
-
Reference
-
(Türck 2008)
2.3
(1.0-3.8)
1.7
(1.0-3.7)
1.0
(1.0-4.9)
3.1*
(2.0-4.4)
1.0
(1.0-3.5)
1.0
(1.0-3.0)
2.0
(Türck 2008)
(Türck 2008)
(Türck 2008)
(Türck 2008)
(Wacheck 2008)
(Fettinger 2011)
(Fettinger 2011)
Experimental from
Irschik, unpublished
visually
data
contaminated
carcasses
Table 2 Microbial counts in game meat cuts; studies conducted in Germany and Austria (data are mean* or median plus range)
1GHE: sampled at game handling establishment; LSH-sc: microbial self-control samples from hunters supplying directly to consumers;
LSH-training: samples taken at training courses for supplying directly to consumers.
2.3 Effect of storage conditions on microbial shelf life
In 2010 Membré et al. studied the microbiological conditions of packed game meat on the French
market. They gathered information about meat cut, shooting season, storage conditions and country
of origin. (Membré et al. 2011) They report:’ although not statistically significant, storage
temperature seems to have an impact on meat hygiene quality.’ Other studies confirm this thought.
(Paulsen 2011)(Doulgeraki et al. 2012)
It is now generally recognized that storage temperature is one of the key factors effecting shelf life of
vacuum packed meat. Storage temperature of ca. 0°C keeps microbial numbers nearly constant for
during a week. (Paulsen 2011) Common bacteria found in meat at chilled temperatures are
Pseudomonas spp., Enterobacteriaceae and lactic acid bacteria. Pseudomonas spp. are particularly
involved in spoilage at chill temperature. Whereas vacuum packing of meat cuts favours the
dominance of facultative anaerobe bacteria like lactic acid bacteria. More species of lactobacilli were
found under storage temperatures of 4°C than at 1°C, this indicates the selective effect of storage
temperature on bacterial growth. (Doulgeraki et al. 2012) Fettinger stored hare meat cuts for 7 days
at 0°C and 4°C. She found circa 2 log units increase in enterobacteriaceae and TVC at 4°C storage
temperature. In contrast, less than 1 log unit increase by 0°C storage temperature. (Fettinger et al.
2010)
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
European legislation specifies the maximum storage temperature of meat from free-living large game
(including wild boar) at 7 °C. (EC 2004) Unfortunately survey studies have shown that temperature
abuse is very common. Temperatures higher than 10°C are not uncommon during transportation,
retail storage or consumer handling. This can lead to unexpected loss of quality and a significantly
reduced meat shelf-life. (Koutsoumanis et al. 2006)
2.4 Responsibility for ‘safe’ food, with a focus on Austria
Hunters are food producers and thus have the responsibility to provide ‘safe’ food, like in other food
producing sectors. (Paulsen 2011) In order to provide safe game food for the consumer, Austria
implemented in 1994 an inspection system in accordance to EU directive 92/45/EEC. (Winkelmayer
et al. 2011)(EC 1992) The system underwent a few changes and the implementation of ‘Good
Hygiene Practice’ (GHP), in 2006. In essence the system includes inspection ante mortem (before
shooting) done by the hunter, inspection post mortem (after shooting and eviscerating ) which has to
be done by a trained person (hunter with specific training) or a veterinarian. When one of both
would find some abnormalities, an official veterinarian needs be consulted, if the meat is intended
for placing on the market. When game is marketed via game-handling-establishments an official
veterinarian always has to check the carcasses, taking into account attached written information
provided by the hunter and a trained person. When game is shot for self-supply no checking of the
carcass by a person with specific training is necessary. In order to enable the producer (hunter or
professional) to fulfil the obligation to act according GHP and produce ‘safe’ food, training courses
for hygiene and direct marketing of fresh game meat are offered. (Fettinger et al. 2011) (Paulsen et
al. 2011)
6
2.5 Wild boar in central Europe
Throughout whole Europe, wild boar populations are increasing, they cause agricultural and
economical damage. Moreover they increase the risk of epidemics. Hunting strategies used now a
day appeared not to be appropriate to reduce wild boar population increase. Higher hunting rates of
piglets and adult females are recommended. (Keuling et al. 2013)
Hunting statistics from Austria shows that the number of boar shot in 2012/2013 increased with
88.5% compared to the previous hunting season. A number of 49 734 was shot in season 2012/2013
compared to 26 380 wild boars shot in 2011/2012.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Figure 1 Wild boar shot in Austria from 1913 - 2010. (Maurer n.d.)
Most of the animals, 31 593 were shot in the province Niederösterreich near the border with the
Czech Republic, Slovakia and Hungary. (Neumann 2012) Interpreting the number of wild boars shot
throughout the years, see figure 1 and 2, it is clear that number of boar shot during a season is
increasing. From 1960 to 2010 the number of wild boars increased significantly.
7
Figure 2 Number of wild boar shot throughout the years in Austria (Anonymous n.d.)
In Austria around 120 000 hunters perform their hobby or profession at nearly 840 000m2 hunting
area. (Anon n.d.) That hobby does not only involve the hunting part, also an increasing amount of
hunters want to process their game and place meat cuts on the market instead of whole carcasses.
Some reasons are the higher price they get for processed meat and the increasing demand by
consumers for wild boar meat.
This, and the increase of wild boar population will results in larger quantities of such meat being
processed and retailed in the next years. The aim of this study is to obtain data on the
(microbiological) shelf life of wild boar meat in order to give some guidance to hunters and small
businesses that place such meat on the market and have to define the best before date/use-by date.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
3 Material and methods
3.1 Material
All boars used in this study were shot in the Lower Austria, in January and February 2014. Animals
were eviscerated in the field and placed in a cool storage room within 6 h post mortem. Animals
were inspected by the hunter and a trained person, and written evidence that no serious abnormities
had been detected, was provided with the carcasses.
Three carcasses were obtained as a whole, and processed in the cutting room of the Institute of
Meat Hygiene, University of Veterinary Medicine Vienna. From another three carcasses, meat cuts
were produced and packed in an agricultural school; finally, one batch of freshly cut and packed wild
boar meat was collected from an approved middle-scale game meat cutting plant (approved gamehandling-establishment) (Table 3).
8
Boar Number
Processing method
1
processed by untrained staff
2
processed by untrained staff
3
processed by trained staff in a small-scale processing unit
4
processed by trained staff in a small-scale processing unit
5
processed by trained staff in a small-scale processing unit
6
processed by untrained staff
7
processed by trained staff in an approved game-handlingestablishment
Table 3 Boar number and processing method/setting
3.1.1 Samples from wild boar carcasses processed by untrained staff
Carcasses one, two and six were skinned, deboned and cut by the investigators, simulating a hunter
who shoots and butchers wild game occasionally. The boars were brought to the institution in
eviscerated condition, and were stored in the refrigerator at around 0-2°C. The body cavity was kept
open using body cavity spanners. 72 hours after shooting the carcasses from boar 1 and 2 were
suspended from the hocks on a metal frame. After 96 hours the same was done for wild boar 6. Meat
surface samples (i.e. a 1 cm thick slice) were taken from the exposed surfaces of the abductor
muscles of the hind legs and were tested for TVC, lactic acid bacteria, E.coli and pseudomonas.
Skinning and cutting was performed as described in the book “Wildbretdirektvermarktung”
(Winkelmayer et al. 2007), a training book for hunters producing meat cuts from game. After
skinning and cutting the carcasses in the major cuts: shoulders, hind legs, back, belly, neck (done at
ambient temperature), these were stored again in the refrigerator for over an hour to reach
temperatures below 7 °C. Further deboning and trimming was also done following the descriptions in
the book. (Winkelmayer et al. 2007) All trimming work was done at average temperature, 18-20°C.
Loin and hind leg were cut in 2-3 cm thick slices. Shoulder and goulash (i.e. meat chunks cut from the
antebrachial and crural muscles) were cut to chunks of ca. 100 gram. All meat cuts were vacuum
sealed, randomly allocated to one of two refrigerator facilities and stored for up to fourteen days
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
(Table 4). Temperature was registered in 15 min. intervals with a data logger according EN 12830
(Testo H175 series, Testo, Lenzkirch, Germany).
3.1.2 Samples from wild boar carcasses processed by trained staff in a small-scale processing unit
Boars number three, four and five were skinned, deboned and cut at an agricultural school in the
course of a training session for hunters supplying game meat cuts to the consumer. The samples
used for this experiment however were cut by the teacher, a butcher specialized in processing of wild
game. The numbers of samples (ca. 100 g -portions of goulash) were 15, 17 and 11 for boars number
3,4,5. All samples were vacuum sealed at the school. Samples were stored at the institution at ca. 2°C
for up to fourteen days (Table 4). Temperature was registered with a datalogger (Testo H-175 series).
3.1.3 Samples from wild boar carcasses processed by trained staff in an approved game handling
establishment
Batch 7 obtained from carcasses that had been skinned, deboned and cut in an approved gamehandling-establishment (AGHE) in the lower part of Austria. In this establishment more than one
animal was processed at once by three trained staff members. Skinning, deboning and further
processing were all performed in separated rooms. Another staff member packed 45 portions of
around 250 gram with goulash meat. At the institution the 45 packages were randomly assigned to
one of two refrigerator facilities and stored for 14 days (Table 4). Temperature was registered with a
datalogger (Testo H-175 series).
3.2 Methods
9
Per type of meat cut, sampling day and storage temperature, 3 to 4 packages were examined (Table
3). The samples were removed from the package under aseptic conditions, and transferred into a
sterile Stomacher 400 bag. A 1:1 suspension (weight based) was prepared with isotonic NaCl 0,85%
solution (NaCl Merck number 1.06404). The stomacher bag was pummelled for 120 seconds using a
bagmixer 400 model (Interscience, F). From this suspension, a serial decimal dilution was made in
Maximum Recovery Diluent (0.85% NaCl Merck 1.06404 and 0.1% peptone Oxoid L34).
Day 0
Day 3
0°C
5°C
0°C
5°C
Boar 1
Hind leg
3
3
3
3
Goulash
3
3
3
3
Shoulder
3
3
3
3
Loin
3
3
3
3
Boar 2
Hind leg
3
3
3
3
Goulash
3
3
Shoulder
3
3
3
3
Loin
3
3
3
3
Boar 6
Hind leg
3
3
3
3
Goulash
3
3
3
3
Shoulder
3
3
3
3
Loin
3
3
3
3
Boar 3
Goulash
3
3
Boar 4
Goulash
3
3
Boar 5
Goulash
3
3
Boar 7
Goulash
4
4
4
4
Table 4 Number of samples taken on each day for each temperature
Day 7
0°C
3
3
3
3
3
4
3
3
3
3
3
3
3
3
2
4
5°C
3
3
3
3
3
4
3
3
3
3
3
3
4
Day 10
0°C
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
5°C
3
3
3
3
3
3
3
3
3
3
3
4
Day 14
0°C
3
5°C
3
3
3
3
3
3
3
3
5
3
3
3
3
3
4
4
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
3.2.1 pH
The pH values were determined in the 1:1 suspensions of the samples by means of a penetrating
electrode (Schott blue line pH27; Schott, Mainz, Germany).
3.2.2 Lactic acid bacteria
In order to count the lactic acid bacterial colonies an aliquot of 0.1 mL dilution was streaked out on a
MRS agar plate (Merck number 1.10660). After at least 48 hours incubation at 30°C colonies were
counted and multiplied by the dilution number.
3.2.3 Total viable count (TVC)
0.1 mL of dilution was streaked out on plate count agar (Merck number 1.05463). After at least 48
hours incubation at 30°C, colonies were counted and multiplied by the dilution number.
3.2.4 (presumptive) Pseudomonas
0.1 mL of dilution was streaked out on GSP (glutamate-starch-penecilline; Merck 1.10230). After at
least 48 hours incubation at 25°C typical colonies (i.e. >1 mm diameter, and violet to blueish colour)
were counted and multiplied by the dilution number.
3.2.5 E. coli and enterobacteriaceae
10
E.coli and enterobacteriaceae were enumerated using a semi-automated MPN technique (Tempo ®,
Biomérieux, F).
3.2.6 Surfaces samples
From boar 1, 2, 6 and 7 surfaces sample were tested for TVC, E.coli, enterobacteriaceae and
pseudomonas. Surface samples from boar 1, 2 and 6 were taken from the exposed abductor muscle
surfaces. From two boars in the handling establishment each one abdominal surface sample was
taken. E.coli, enterobacteriaceae, pseudomonas and lactic acid bacteria colonies were counted using
methods described above.
3.2.7 Trichinella sp. and Alaria alata
Boar 1, 2 and 6 were examined for Trichinella sp. with the magnetic stirrer method according to
Regulation No. 2075/2005 (EC 2005) and checked for Alaria alata mesocercariae using a special
migration method. (Riehn et al. 2010)
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
3.3 Statistical analysis
Numbers for microbial analyses were converted to log units. Numbers below the limit of detection
were set to the limit of detection. Descriptive statistics was done by MS Excel. Inferent statistics
included univariate analysis of variance (log microbial number or pH as dependent variable), with
least-significant-difference (LSD) test to discriminate among means. Two-factor interactions were
considered. Non-normal distributed sample sets were tested with the Kruskal-Wallis test
(Statgraphics 3.0 software, Statistical Graphics Inc., USA). All incomplete cases were removed in
order to perform statistical analysis.
11
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
4 Results and discussion
4.1 Trichinella sp. and Alaria alata
None of the samples tested positive for the abovementioned parasites.
Boar 1
sample a
Boar 1
sample b
Boar 2
sample b
Boar 6
sample a
Boar 6
sample b
Boar 7
sample a
Boar 7
sample b
TVC (log cfu/g)
6.93
6.96
7.08
6.89
8.88
8.80
8.49
9.82
Enterobactericeae (log
cfu/g)
Pseudomanas spp. (log
cfu/g)
E. coli (log cfu/g)
5.65
5.79
6.73
6.69
3.88
3.76
2.00
5.04
6.92
5.93
7.01
6.81
8.85
8.78
8.40
9.64
5.18
5.23
6.72
6.68
3.20
2.85
2.00
1.00
Boar 2
sample a
Surface
samples
4.2 Microbiological condition of carcass surface samples
Table 5 Microbial conditions from surfaces samples
12
In Table 5 it can be seen that bacterial numbers on surfaces differed between wild boars 1 & 2 on the
one, and 6 & 7 on the other hand. The latter had higher TVC and pseudomonas counts, but lower
numbers in E. coli. These carcasses had been stored already for ca. 1 week in the chilling room before
cutting, so the growth of pseudomonads was not unexpected. Conversely, wild boars 1 & 2
demonstrated lower TVCs, but higher E. coli numbers. Carcasses 1 & 2 originated from one hunting
event, were eviscerated and cooled down immediately after the hunt, and tested 72 h post mortem,
so the numbers of E. coli and Enterobacteriaceae are most likely explained by contamination of meat
surfaces in the course of evisceration and/or transport.
4.3 Temperature
Meat cuts were stored in two different refrigerator facilities. The first possibility was a cold room
refrigerator with a mean temperature of 2.0°C and standard deviation of 0.4°C. The second
possibility was a household refrigerator with a mean temperature of 3.8°C and standard deviation of
0.8°C.
4.4 pH values before, during and after storage
All pH values determined in the meat cuts before storage were in the range from 5.43 to 6.08 (Tables
6 and 7). In all wild boar series with different meat cuts (1, 2 and 6), significant differences of pH
values were observed between the meat cuts. In wild boar 1 and 6, shoulder and goulash meat cuts
formed a homogeneous group which differed significantly from the loin and hind leg meat cuts. The
results of wild boar 2 were in the same trend, loin and hind leg were not significantly different; the
same applied for shoulder and goulash meat cuts, but these combinations did differ significantly.
During the whole experiment pH values were in a range of 4.87 to 6.08. Values below pH 5 were only
observed in wild boar 7 at day 14. This could be due to high lactic acid bacteria counts, because these
bacteria metabolise sugar in lactic acid and lower the pH. (Gill & Gill 2005) pH changes during
storage, in general 14 days, did not exceed 0.8 pH units.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Boar 1
Boar 2
Boar 6
Hindleg
5.50±0.01
5.52±0.03
5.67±0.08
Goulash
5.64±0.08
5.67±0.05
5.88±0.08
Shoulder
5.68±0.04
5.68±0.07
5.91±0.10
Loin
5.45±0.02
5.48±0.02
5.59±0.07
Table 6 Mean pH values ± standard deviation in meat cuts from wild boar 1, 2 and 6 before storage (day 0)
Goulash
Boar 3
Boar 4
Boar 5
Boar 7
5.65±0.05
5.74±0.20
5.94±0.12
5.67±0.04
Table 7 Mean pH values ± standard deviation in meat cuts from wild boar 3, 4, 5 and 7 before storage (day 0)
After statistical analysis of pH values during the storage days, various significant differences were
found, which are summarized in Table 8.
Wild boar
Temperature
Storage time
Meat cuts
Interaction
1
+(p=0.0003)
+(p=0.0000)
+(p=0.0000)
time X
temperature
2
-(p=0.4657)
-(p=0.2855)
+(p=0.0000)
6
-(p=0.6816)
+(p=0.0432)
+(p=0.0000)
13
3
- (p=0.8419)
4
-(p=0.6177)
5
-(p=0.6883)
7
+(p=0,0031)
+(p=0,0000)
time X
temperature
time X
temperature
Table 8 Statistically significant (p<0.05) effects on pH values
Significant differences in meat cuts form wild boar 1,2 and 6 are mentioned above. In wild boar series
1 and 7 temperature effectuated a significant difference in pH during storage time, whereas in series
2 and 6, temperature did not effectuate any difference. This inconsistency could be due to the minor
difference in the two storage temperatures (1.8°C). In three of the four wild boar series with two
storage temperatures (boar 1, 6 and 7), the interaction between temperature and time was
associated with a significant difference in pH values. In wild boar 1, 6 and 7, storage time caused a
significant change in pH value. Interestingly, in all boars from the small-scale production
establishment, time effectuated no significant difference in pH values until day 10 or 14 (last day of
experiment). Meat cuts from wild boars 6 and 7 showed the same tendency, as days 0, 3 and 7
formed a homogeneous group which differed significantly from the homogeneous group of days 10
and 14. Wild boar 1 showed the interesting fact that day 3 differed significantly from the
homogeneous group constituted by days 0, 7 and 10.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
The amount of glycogen in the muscle before slaughter/death determines the pH of the meat
immediately after slaughter (Lawrie & Ledward 2006). Bacteria like lactic acid bacteria and
pseudomonas first metabolise glucose, when glucose is depleted they start to metabolise amino
acids into alkaline compounds and therefore lower the pH. The more glucose is present in post
mortem tissue the longer it takes for pH to drop due to bacterial action. When glucose is metabolised
no harmful metabolites are produced, however when amino acid are metabolised harming
metabolites are produced and spoilage starts (Gill & Gill 2005). Stress, physical exercise or fasting
before slaughter decreases glycogen amount and cause therefore a higher ultimate pH. Wild boars
are likely to be exposed more to stress factors prior to death and therefore the carcass could have a
higher pH after death. (Gill 2007) (Lawrie & Ledward 2006). A similar observation has been reported
for pH in muscles of hunted and slaughtered pheasant. A higher pH was observed in the hunted,
probably due to increased physical activity prior to death. (Paulsen et al. 2008) Ultimate pH reached
due to post-mortem glycolysis after normal (without glycogen loss) death is 5.4-5.5. (Lawrie &
Ledward 2006) All final pH values in this study were slightly higher which could be due to stress prior
to death. In general a higher pH means increasing rate of spoilage. (Lawrie & Ledward 2006)
Mean ultimate pH values of 5.77 were observed by Avagnina (Avagnina et al. 2012) in 72 wild boar
carcasses. In minced wild boar meat (an experiment with wild boar salami) a mean pH of 5.8 was
determined. (Paulsen et al. 2011) All these values were in the same range as found in this study
which can point out that slightly higher pH value for wild boar compared to domestic pig can be
expected.
14
In a storage study with pheasant meat cuts time did not affect pH values whereas storage
temperature did. Lower temperature was associated with lower pH. (Paulsen et al. 2008) Considering
the results found in this study no clear trend for all carcasses could be observed in pH values in
relation to temperature, storage time and meat cuts.
4.5 E.coli counts before, during and after storage
E.coli numbers on carcass surfaces were reflected in numbers of E. coli on the freshly prepared meat
cuts. Wild boar 1 and 2 with higher E.coli numbers on carcass surfaces provided meat cuts with
higher E. coli values. Whereas wild boar 6 and 7 with lower E. coli values on carcass surfaces provided
meat cuts with lower E. coli values (Tables 5 and 9). For series 3, 4, 5, no corresponding data for
carcass surfaces were available. E.coli values in meat cuts from wild boar 4 and 5 were under the
limit of detection and therefore not displayed (Table 9).
Hind leg
Goulash
Boar 1
Boar 2
Boar 6
Boar 3
Boar 7
2.35±0.32 (day 0)
3.80±0.36 (day 0)
1.16±0.51 (day 0)
2.28±0.34 (day 14)
4.30±0.32 (day 10)
1.01±0.14 (day 14)
2.26±0.32 (day 0)
4.13±0.47 (day 0)
1.48±0.57 (day 0)
1.10±0.17 (day 0)
0.96±0.002 (day 0)
1.85±0.32 (day 10)
3.65±0.34 (day 7)
1.45±0.94 (day 14)
0.96±0.03 (day 14)
1.26±0.34 (day 14)
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Shoulder
Loin
1.49±0.49 (day 0)
3.77±1.15 (day 0)
1.05±0.23 (day 0)
1.45±0.47 (day 10)
3.21±0.10 (day 10)
1.12±0.35 (day 14)
1.21±0.33 (day 0)
3.89±0.45 (day 0)
0.95±0 (day 0)
0.97±0.02 (day 10)
4.00±0.29 (day 14)
0.96±0.02 (day 14)
Table 9 Mean ± standard deviation (log cfu/g) E.coli before and after storage
It could be shown that storage temperature had no significant influence on E.coli numbers during
storage, whereas duration of storage had a significant influence on E.coli numbers in some series
(Table 9 and Figure 3). In Figure 3 can be seen that values from E.coli fluctuate during storage, but
generally show a minor decrease over time.
15
Figure 3 E.coli values over storage (log cfu/g) from wild boar 1,2 and 6
This effect has already been reported by Anasay (Anasay et al. 1999). Over fourteen days of storage
they reported 0.93; 1.08; 0.97; 0.29; 0.66; 0.61; 1.67 and 1.93 log units decrease in E.coli. The
decrease in E.coli colonies could be due to the mesophilic character of E.coli (Prescott & Klein 2002)
or the minimal growth temperature of 4.9-11.2 °C (Gonthier & Tilly 2001). Storage conditions used in
this experiment and the cited studies were below this minimum growth temperature. Contrary, in
studied pheasants (shot in either muscle tissue or in abdominal cavity) E.coli values increased over
storage time. On day 7, less than 1 log cfu/g E.coli was observed and on day 14 values of 2.0 and 3.3
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
log cfu/g and 3.2 and 4.1 cfu/g were seen for 0°C and 4°C storage temperature, respectively. (Paulsen
et al. 2008)
Microbial limits for E.coli laid down in European law (EC 2005) are 2.7 log cfu/g (“m”) and 3.7 log
cfu/g units (“M”) for minced meat. Although these values are applicable for meat from domestic pig,
they could be used as limit for wild boar meat preparations. Only in wild boar 2 the “M” value was
exceeded, what could be due to higher E.coli number on surface samples, as suggested before.
It is generally known that E.coli is a good parameter for slaughter hygiene and faecal contamination
of meat cuts. E.coli numbers might therefore be used to compare the three processing methods in
order to assess processing hygiene. It was assumed to find any difference between the processing
methods, but results did not provide evidence for such a factor.
16
Wild boar number
Storage time
Temperature
meat cuts
1
+ (p=0.0006)
- (p=0.2110)
+ (p=0.0000)
2
+ (p= 0.0048)
- (p=0.2027)
- (p=0.1914)
6
- (p=0.3473)
- (p=0.3358)
- (p=0.1163)
3
- (p=0.5247)
4
(-)
5
(-)
7
+ (p=0.0000)
- (p=0.5807)
Table 10 Statistically significant (p<0.05) effects on E.coli values
Because most of the values from wild boar 4 and 5 meat cuts were under the limit of detection no
statistical analysis was done for these meat cut series. As mentioned above temperature was never
significantly affecting E. coli values. This could be due to the minor difference in storage temperature
(1.8°C) and the mesophilic nature of this bacterium. In three of seven series storage time exerted a
significant difference. In wild boar 1 days 0-10 and 0-3 formed two homogeneous groups . All other
possible combinations were significantly different. In wild boar 2 only day 10 differed significantly
from the homogeneous days 0, 3 and 7. In wild boar 7 samples, day 7 and 14 differed significantly
from days 0, 3 and 10. This is in contrast to the expected rather linear or exponential growth of the
colonies. Only in wild boar 1, meat cuts exerted a significant difference in E.coli values. Hind leg and
goulash meat cuts differed from loin and shoulder cuts.
4.6 Enterobacteriaceae counts before, during and after storage
Comparing enterobacteriaceae numbers in surfaces samples from wild boar 1, 2, 6 and 7 with
enterobacteriaceae numbers in freshly prepared meat cuts, the data suggest that carcasses with
higher enterobacteriaceae numbers provided meat cuts with higher enterobacteriaceae numbers
(Table 5 and 11). For series 3, 4 and 5 no corresponding data for carcass surfaces were available. In
general, over time enterobacteriaceae count increases, increase is clearer at storage temperature of
3.8°C (Table 11). Increase can be up to 3 log units at both 2°C and 3.8°C storage temperature
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
(shoulder, wild boar 6). More general an increase of 1 log unit and 1,5 log unit for 2°C and 3.8°C
storage temperature were seen in wild boar 1, 2 and 6. In one of the series enterobacteriaceae count
decreased from 2.9 log cfu/g to 2.0 log cfu/g, but due to the high standard deviation the significance
of this finding is doubtful. In meat cuts from wild boar 7, no significant difference between the two
different storage temperatures was found as regards enterobacteriaceae count; therefore no
distinction has been made between temperatures after storage in Table 12. In meat cuts from boar 5
and 7 a high increase in enterobacteriaceae count was found (circa 2 log units). In meat cuts from
boar 3 and 4 an increases of circa 0.2 log units was found.
Boar 1
Boar 2
Boar 6
Hind leg before storage
2.33±0.32 (day 0)
3.46±0.09 (day 0)
2.91±0.88 (day 0)
Hind leg after storage 2°C
2.68±0.85 (day 14)
2.53±0.48 (day 10)
2.00±0 (day 14)
Hind leg after storage 3.8°C
3.78±0.71 (day 14)
2.61±0.08 (day 10)
4.42±0.21 (day 14)
Goulash
2.48±0.31 (day 0)
3.90±0.42 (day 0)
2.82±0.57 (day 0)
Goulash after storage 2°C
3.18±0.59 (day 10)
3.82±0.36 (day 7)
4.82±1.32 (day 10)
Goulash after storage 3.8°C
3.75±0.35 (day 10)
4.28±1.68 (day 7)
4.47±0.98 (day 14)
Shoulder
1.48±0.48 (day 0)
4.12±0.47 (day 0)
0.98±0.02 (day 0)
Shoulder after storage 2°C
2.44±0.76 (day 10)
3.19±0.48 (day 10)
3.99±1.03 (day 14)
Shoulder after storage 3.8°C
3.55±0.27 (day 10)
4.37±0.05 (day 10)
3.77±1.54 (day 14)
Loin
1.33±0.19 (day 0)
3.73±0.54 (day 0)
1.27±0.34 (day 0)
Loin after storage 2°C
2.00±0(day 10)
3.73±0.16 (day 14)
2.00±0 (day 14)
Loin after storage 3.8°C
2.85±1.21 (day 10)
4.15±0.36 (day 14)
2.85±1.48 (day 14)
17
Table 11 Mean ± standard deviation (log cfu/g) enterobacteriaceae before and after storage in meat cuts from wild boar 1, 2 and 6
Boar 3
Boar 4
Boar 5
Boar 7
Goulash before storage
2.00±0 (day 0)
2.28±0.77 (day 0)
0.95±0 (day 0)
2.39±0.36 (day 0)
Goulash after storage
2.26±0.24 (day 14)
2.44±1.29 (day 14)
3.16±0.83 (day 10)
4.08±0.43 (day 14)
Table 32 Mean ± standard deviation (log cfu/g) enterobacteriaceae before and after storage in meat cuts from wild boar 3, 4, 5 and 7
In European legislation (EC 2005) limits for enterobacteriaceae for carcasses of domestic pigs (postslaughter, pre-chilling) are laid down. These limits are 2.0 log cfu/cm2 (“m”) to 3.0 log cfu/cm2(“M”).
No specific microbial limits for wild boar exist, but, as mentioned in the introduction, a limit for game
carcasses has been proposed at 3.0 log cfu/g. (Paulsen 2011)
Atanassova and coworkers (Atanassova et al. 2008) reported an enterobacteriaceae count in freshly
shot wild boar in a range of 1.7 to 3.5 log cfu/g. In a storage experiment with meat cuts from hares,
enterobacteriaceae numbers on day 0 of 2.2 log cfu/g (hares shot without any damage to the meat)
and 2.7 log cfu/g (hares shot with damage to the meat) were found. After 7 days of storage at 0°
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
numbers increased to 3.1 and 4.2 log cfu/g. After 7 days of storage at 7°C, numbers increased to 3.1
and 5.3 log cfu/g, respectively. (Fettinger et al. 2010) Experiments with wild boar meat cuts and with
hare meat cuts showed the same trend, i.e. meat cuts stored at higher temperature reach suggested
microbiological limits earlier.
Comparing the suggested limit and the data from the wild boar meat cut series in this study, it is
clear that wild boar 2 already exceeds these limits before storage (Table 11). This could be due to the
higher numbers in carcass surface samples from wild boar 2 (Table 5). In wild boar 1 and 6 no clear
trend between the observed values and the suggested limit can be seen, some meat cuts have higher
values and others have lower (Figure 4). As will be pointed out later, certain meat cuts seem to have
a higher level of bacterial contamination in terms of enterobacteriaceae. This is probably caused by
more hand contact during preparation of the meat cuts or caused by the higher initial bacterial
numbers on carcass surfaces. In meat cuts from wild boar 3, 4 and 5, numbers were below or near to
the suggested limit. In wild boar 7, values after storage are higher than the suggested limit.
18
Figure 4 Enterobacteriaceae counts (log cfu/g) in the different meat cuts from wild boar 1 and 6 over storage time compared to the
suggested limit (red line, 3 log cfu/g)
It is generally known that enterobacteriaceae count, as E.coli count, is a good parameter for
slaughter hygiene and faecal contamination of meat cuts. Enterobacteriaceae numbers might
therefore be used to compare the three processing methods in order to assess processing hygiene. It
was assumed to find any difference between the processing methods, but results did not provide
evidence for such a factor.
Wild boar
Storage time
Temperature
Meat cuts
interaction
1
+(p=0.0000)
+(p=0.0000)
+(p=0.0000)
storage time X temperature
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
2
+(p=0.0004)
+(p=0.0327)
+(p=0.0005)
storage time X temperature
meat cut X storage time
6
+(p=0.0000)
3*
+(p=0.0262)
4*
-(p=0.05105)
5*
+(p=0.0434)
7
+(p=0.0000)
+(p=0.0000)
-(p=0.6237)
+(p=0.0000)
storage time X temperature
Table 13 Statistically significant (p<0.05) effects (+) on Enterobacteriaceae values; * Kruskal Wallis test used because inhomogeneity of
variances.
In Table 13 the statistical analysis of enterobacteriaceae values is summarized. In 6 of the 7 series,
storage time exerted a significant influence in enterobacteriaceae count. In boar 1, counts on all days
significantly differed from each other. In wild boar 2, days 0, 3 and 7 were not significantly different
but they significantly differed from day 10. In boar 6, day 0 and 3 were not significantly different but
they significantly differed from a homogeneous group formed by days 7, 10 and 14. Interestingly, in
wild boar 3, days 0, 3 and 10 were not significantly different but they significantly differed from days
14 and 7. In wild boar 5, day 0 significantly differed from the homogeneous group days 3, 7 and 10.
And in wild boar 7, day 0 significantly differed from the not significantly different days 3, 7 and 10
and from day 14.
19
In three of the four series with different storage temperatures, temperature effectuated a significant
difference on enterobacteriaceae count which indicates the importance of temperature on
enterobacteriaceae growth.
As regards differences between meat cuts, significant differences between meat cuts were found,
but the pattern was not really consistent: In wild boar 1, loin and shoulder cuts were not significantly
different, whereas goulash and hind leg meat cuts were significantly different. In wild boar 2 hind leg
and loin meat cuts were not significantly different, but they significantly differed from shoulder cuts.
Goulash meat cuts were (due to the incomplete data set ) not included in the statistical analysis. In
wild boar 6, all meat cuts significantly differed from each other.
4.7 TVC before, during and after storage
TVC on carcass surfaces samples from wild boar 6 & 7 were higher than those on surfaces from wild
boar 1 & 2. This corresponds to the numbers found in freshly prepared meat cuts, which suggests
that carcasses with higher TVC provide meat cuts with higher TVC. In general TVC increase was circa
1.5 log units when samples were stored at 2°C and circa 2 log units when samples were stored at
3.8°C. Because storage temperature did not effectuate a significant difference in wild boar 7, no
distinction in temperature is made in Table 15 as regards TVC from meat cuts from wild boar 7. In
samples from boars 3, 4, 5 and 7, an increase of circa 2 log units was observed after 10 and 14 days
of storage at 2°C (and 3.8°) temperature.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Boar 1
Boar 2
Boar 6
Hind leg before storage
4.17±0.46 (day 0)
4.21±0.18 (day 0)
6.49±0.40 (day 0)
Hind leg after storage 2°C
6.72±0.35 (day 14)
4.79±1.04 (day 10)
7.49±0.41 (day 14)
Hind leg after storage 3.8°C
7.52±0.60 (day 14)
7.01±0.41 (day 10)
8.16±0.62 (day 14)
Goulash
4.42±0.62 (day 0)
4.56±0.64 (day 0)
6.56±0.20 (day 0)
Goulash after storage 2°C
6.93±0.56 (day 10)
5.43±0.45 (day 7)
8.17±0.40 (day 10)
Goulash after storage 3.8°C
8.36±0.10 (day 10)
5.73±0.41 (day 7)
8.77±0.09 (day 14)
Shoulder
3.79±0.27 (day 0)
4.54±0.47 (day 0)
6.22±0.08 (day 0)
Shoulder after storage 2°C
5.66±0.90 (day 10)
5.08±0.36 (day 10)
8.38±0.22 (day 14)
Shoulder after storage 3.8°C
7.52±0.18 (day 10)
7.11±0.04 (day 10)
8.36±0.12 (day 14)
Loin
4.61±0.30 (day 0)
3.82±0.54 (day 0)
6.52±0.34 (day 0)
Loin after storage 2°C
4.80±0.20 (day 10)
5.79±0.37 (day 14)
6.68±0.84 (day 14)
Loin after storage 3.8°C
7.10±0.53 (day 10)
7.58±0.34 (day 14)
7.78±0.63 (day 14)
Table 44 Mean ± standard deviation (log cfu/g) TVC before and after storage in meat cuts from wild boar 1, 2 and 6
20
Boar 3
Boar 4
Boar 5
Boar 7
Goulash before storage
4.37±0.48 (day 0)
4.20±0.28 (day 0)
4.58±0.34 (day 0)
4.86±0.30 (day 0)
Goulash after storage
6.66±0.90 (day 14)
6.84±0.59 (day 14)
8.61±0.12 (day 10)
7.85±0.19 (day 14)
Table 55 Mean ± standard deviation (log cfu/g) TVC before and after storage in meat cuts from wild boar 3, 4, 5 and 7
Limits (4.0 log cfu/cm2 (“m”) to 5.0 log cfu/cm2 (“M”)) for TVC are laid done in European legislation
(EC 2005) , although these values are not specific for wild boar or other wild animal, and refer to
carcasses pre-chill, they could be taken as a tentative guide line for microbial limits in game meat.
Suggested limits especially for game, as mentioned before, are 5.0 log or 6.0 log cfu/g (Paulsen
2011). TVC in wild boar 1 reached this limit (6.0 log cfu/g) in at least one sample after 10 days of
storage at 2°C, and 7 days of storage at 3.8°C. Corresponding data for wild boar 2 were day 14 at 2°C
and day 7 at 3.8°C storage temperature. Initial values in wild boar 6 already exceed 6 log cfu/g. It can
be said that a difference of merely 1.8°C in storage temperature caused a significant difference in
TVC.
Meat cuts from wild boar 3, 4 and 5 reached the limit of 6 log cfu/g at days 14, 10 and 10 (2°C). In
meat cuts from series 7, higher bacterial counts on carcass surfaces and therefore on the freshly
prepared cuts might have contributed to the finding that TVC levels of 6 log cfu/g were already
exceeded at day 3 of storage (both temperatures). The results suggest that initial TVC were lowest in
wild boar processed in low-scale units with trained staff and thus, resulting cuts had a longer shelf
life than cuts obtained from untrained staff or from medium- to large-scale units.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Fettinger and coworkers (Fettinger et al. 2010) showed in a study with hare meat cuts the same
tendency. Meat cuts stored at 3-4°C reached levels of 6 log cfu/g at day 7, whereas meat cuts stored
at 0°C only reached 4 log cfu/g at day 7. Starting values were 4 log cfu/g. (Fettinger et al. 2010)
Paulsen (Paulsen et al. 2008) observed an increase of 1.25 and 1.45 log units from day 3 to day 14 in
pheasant meat cuts.
TVC values tell a lot about the general state of the meat cuts, in terms of hygiene during cutting and
the use-by date. After analysing the data it can be said that only a minor temperature increase
reduces the possible storage time and use-by date considerably.
21
Wild boar
Storage time
Temperature
Meat cuts
interaction
1
+ (p=0.0000)
+ (p=0.0000)
+ (p=0.0000)
meat cut X temperature,
storage time X
temperature
2
+ (p=0.0000)
+ (p=0.0000)
+ (p=0.0000)
storage time X
temperature
6
+ (p=0.0000)
+ (p=0.0000)
+ (p=0.0000)
meat cut X storage time
3
- (p=0.0695)
4
+ (p=0.0003)
5
+ (p=0.0001)
7
+ (p=0.0000)
- (p=0.3307)
Table 66 Statistically significant (p<0.05) effects on TVC.
In almost all (boar 1, 2, 6, 4, 5 and 7) series storage time exerted a significant influence on TVC. In
wild boar 3 series storage time effectuated no significant difference.
In three of the four series with different storage temperatures, temperature effectuated significant
differences in TVC. Only in boar 7 no significant importance of temperature in TVC changes was
observed.
In all series with different meat cuts, significant differences were found between the TVC of different
cuts. In wild boar 2 and 6 hind leg and shoulder cuts formed a homogenous group differing
significantly from the group loin and goulash meat cuts. In wild boar 1, loin and shoulder cuts did not
significantly differ, but they did differ significantly from goulash and hind leg meat cuts.
4.8 Lactic acid bacterial counts before, during and after storage
When values of lactic acid bacteria before and after storage are compared it is evident that the lactic
acid bacterial count increase over time. (Table 17) It can be seen that lactic acid bacterial growth is
higher at 3.8°C storage temperature than at 2°C. At 3.8°C, a circa 3 log units increase between day 0
and days 10/14 was observed. At storage temperature of 2°C this increase was lower; circa 2 log
units (Figure 5). The same trend was seen in the other series 3, 4 and 5(2°C). Circa 2 log units increase
over storage time could be observed (Table 18 and Figure 6). These data suggest an influence of
temperature on lactic acid bacterial growth.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Figure 5 Lactic acid bacteria growth over storage time in meat cuts from wild boar 1, 2, 6 and 7 at storage temperature of 2°C and 3.8°C
22
Figure 6 Lactic acid bacteria growth over storage time in meat cuts from wild boar 3, 4 and 5 at storage temperature of 2°C
Boar 1
Boar 2
Boar 6
Boar 7
Hind leg before storage
2.30±0.27 (day 0)
3.32±0.40 (day 0)
2.84±0.23 (day 0)
Hind leg after storage 2°C
6.39±0.40 (day 14)
4.72±0.66 (day 10)
5.18±0.35 (day 14)
Hind leg after storage 3.8°C
7.37±0.06 (day 14)
6.87±0.32 (day 10)
7.28±0.14 (day 14)
Goulash
2.46±0.33 (day 0)
3.19±0.44 (day 0)
2.81±0.46 (day 0)
3.54±0.19 (day 0)
Goulash after storage 2°C
5.38±0.16 (day 10)
4.70±0.26 (day 7)
5.43±0.35 (day 14)
6.19±0.24 (day 14)
Goulash after storage 3.8°C
6.61±0.21 (day 10)
5.72±0.59 (day 7)
6.77±0.12 (day 14)
6.06±0.93 9day 14)
Shoulder
2.50±0.37 (day 0)
2.90±0.34 (day 0)
2.47±0.30 (day 0)
Shoulder after storage 2°C
4.08±0.74 (day 10)
4.28±0.28 (day 10)
4.69±0.21 (day 14)
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
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Shoulder after storage
3.8°C
5.79±0.43 (day 10)
6.89±0.16 (day 10)
6.86±0.52 (day 14)
Loin
2.58±0.16 (day 0)
2.58±0.42 (day 0)
2.41±0.33 (day 0)
Loin after storage 2°C
4.13±0.39 (day 10)
5.59±0.26 (day 14)
4.30±0.00 (day 14)
Loin after storage 3.8°C
6.47±0.07 (day 10)
7.25±0.37 (day 14)
6.63±0.55 (day 14)
Table 77 Mean ± standard deviation (log cfu/g) lactic acid bacterial counts before and after storage in meat cuts from wild boar 1, 2, 6
and 7
Boar 3
Boar 4
Boar 5
Goulash before storage
2.9±0.05 (day 0)
2.5±0.17 (day 0)
2.7±0.15 (day 0)
Goulash after storage
4.78±0.09 (day 14)
4.08±0.19 (day 10)
6.57±0.40 (day 14)
Table 88 Mean ± standard deviation (log cfu/g) lactic acid bacterial counts before and after storage in meat cuts from wild boar 3, 4 and
5
In a study on beef meat cuts, no significant influence of storage temperature was observed. In this
study lactic acid bacteria increased over storage time regardless of storage conditions (vacuum, open
air, temperature). (Pennacchia et al. 2011) Because lactic acid bacteria can grow well under anaerobe
conditions it usually dominates microbial flora in vacuum packed meat. (Gill & Gill, 2005)
23
Wild boar
Storage time
Temperature
Meat cuts
interaction
1
+ (p=0.0000)
+ (p=0.0000)
+ (p=0.0000)
Meat cut X storage time,
storage time X
temperature
2
+ (p=0.0000)
+ (p=0.0000)
+ (p=0.0000)
Temperature X storage
time
6
+ (p=0.0000)
+ (p=0.0000)
+ (p=0.0000)
Temperature X storage
time
3*
+ (p=0.0219
4*
+ (p=0.0146)
5
+ (p=0.0000)
7
+ (p=0.0426)
+ (p=0.0000)
Table 99 Statistically significant (p<0.05) effects on lactic acid bacteria values; * Kruskal Wallis test used because of inhomogeneity of
variances.
In Table 19 data from the statistical analysis of lactic acid bacteria are summarized. In all series all
possible factors exerted a significant influence on the lactic acid bacteria numbers. In wild boar 1, 2,
6, 3, 5 and 7 a linear change in lactic acid bacterial count during storage time was seen. All LAB
counts of the storage days in wild boar 1, 2, 5 and 6 were significantly different. In wild boar 7
however day 7, 10 and 14 were not significantly different, but they differed significantly from days 0
and 3. In wild boar 3 the days 0, 3, 7 were significantly different from 10 and 14. In wild boar 4 days
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
0, 3 and 10 were not significantly different, but this homogeneous group differed significantly from
day 7. Day 7 was not significantly different from day 14. And day 14 was not significantly different
from day 10. In all cases with different storage temperatures, temperature (2°C and 3.8°C) exerted a
significant influence on lactic acid bacteria values. Numbers in loin and goulash meat cuts in wild
boar 1 did not differ significantly, whereas they did significantly differ from numbers in shoulder and
hind leg cuts. In wild boar 2, loin, hind leg and shoulder meat cuts differed significantly from each
other. Goulash meat cuts were not included in the statistical analysis because this data set was
incomplete. In wild boar 6, goulash and shoulder meat cuts did not differ significantly, but these
meat cuts differed significantly from loin and hind leg cuts.
4.9 Pseudomonas counts before, during and after storage
Pseudomonas values on carcasses from wild boar 1 & 2 were lower than the values on the carcasses
of wild boar 6 & 7. The same relation was observed in freshly prepared meat cuts, as meat cuts from
wild boar 1 & 2 had lower pseudomonas counts than those from wild boar 6 & 7 (Table 20 & 21). As
can be seen in Table 20, pseudomonas values increased over storage time. Pseudomonas values
increased with circa 1 log unit at a storage temperature of 2°C. At 3.8°C, pseudomonas values
increased with around 1.5 log units from day 0 to day 7, 10 or 14. This suggests that only 1.8°C
temperature difference can be of importance in pseudomonas bacterial growth. In wild boar 3, 4 and
5, around 2.5 log units increase of pseudomonas values could be observed (Table 21). In meat
samples from wild boar 7, no increase was observed, but initial levels were already over 6 log cfu/g.
Boar 1
Boar 2
Boar 6
Hind leg before storage
4.49±0.26 (day 0)
3.78±0.19 (day 0)
6.43±0.21 (day 0)
Hind leg after storage 2°C
5.59±0.61 (day 14)
3.88±0.19 (day 7)
6.43±0.26 (day 14)
Hind leg after storage 3.8°C
6.11±0.06 (day 14)
4.53±0.92 (day 7)
6.97±0.45 (day 14)
Goulash
4.40±0.36 (day 0)
3.59±0.40 (day 0)
6.47±0.18 (day 0)
Goulash after storage 2°C
5.92±0.26 (day 10)
4.53±0.59 (day 7)
7.37±0.13 (day 10)
Goulash after storage 3.8°C
6.12±0.39 (day 10)
5.01±0.80 (day 7)
8.10±0.07 (day 14)
Shoulder
3.63±0.27 (day 0)
3.69±0.21 (day 0)
6.10±0.34 (day 0)
Shoulder after storage 2°C
4.57±0.40 (day 10)
4.28±0.36 (day 10)
6.27±0.37 (day 14)
Shoulder after storage 3.8°C
5.30±0.30 (day 10)
5.17±0.38 (day 10)
7.01±0.29 (day 14)
Loin
3.86±0.14 (day 0)
3.63±0.22 (day 0)
6.38±0.27 (day 0)
Loin after storage 2°C
3.82±0.47 (day 10)
4.92±0.22 (day 14)
5.82±0.29 (day 14)
Loin after storage 3.8°C
5.17±0.47 (day 10)
5.26±0.24 (day 14)
6.84±0.34 (day 14)
24
Table 20 Mean ± standard deviation (log cfu/g) pseudomonas counts before and after storage in meat cuts from wild boar 1, 2 and 6
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Boar 3
Boar 4
Boar 5
Boar 7
Goulash before storage
4.06±0.57 (day 0)
3.70±0.49 (day 0)
3.10±0.16 (day 0)
6.51±0.22 (day 0)
Goulash after storage
6.30±0.43 (day 14)
6.33±0.42 (day 14)
6.44±0.41 (day 10)
6.73±0.11 (day 14)
Table 21 Mean ± standard deviation (log cfu/g) pseudomonas counts before and after storage in meat cuts from wild boar 3, 4, 5 and 7
Pseudomonas spp. are one of the sub sections of TVC, but is in vacuum packed meat of particular
importance due to the growth conditions. Pseudomonas can grow between 0-7°C and has its
optimum between 20-30°C.(Prescott & Klein 2002) Pseudomonads are aerobe bacteria, under high
oxygen conditions pseudomonads grow fast. (Gill & Gill 2005) An increase in beef of 2 log units over
20 days of storage under aerobic conditions at 4°C storage temperature is reported. (Pennacchia et
al. 2011) Under conditions of 1% oxygen or less pseudomonads grow slower but they do not stop
growing. (Gill & Gill 2005)(Crowley et al. 2010) Sometimes in vacuum packs not all oxygen is
removed, therefore pseudomonads spp. can still grow, but they will be dominated by lactic acid
bacteria. (Gill & Gill 2005)(Fettinger et al. 2010)
Wild boar
Storage time
Temperature
Meat cuts
Interaction
1
+ (p=0.0000)
+ (p=0.0000)
+ (p=0.0000)
meat cut X storage time,
temperature X storage
time
2
- (p=0.3634
- (p=0.1277)
+ (p=0.0009)
meat cut X storage time
6
+ (p=0.0000)
+ (p=0.0011)
+ (p=0.0000)
meat cut X storage time,
temperature X storage
time, meat cut X
temperature
3
+ (p=0.0100)
4
+ (p=0.0029)
5*
+ (p=0.0303)
7
+ (p=0.0000)
25
- (p=0.3582)
Table 102 Statistically significant (p<0.05) effects on pseudomonas values; *Kruskal-Wallis test performed because of inhomogeneity in
variances.
Statistical analysis from pseudomonas data are summarized in Table 22. Only in wild boar 2, no
significant effect of storage time on the number of pseudomonas colonies was found. In other meat
cut series, the results were not in the same trend. Differences in initial contamination or in packaging
(pockets containing residual oxygen) might have accounted for this. In wild boar 1, results at days 0
and 3 formed one, and results at days 7 and 10 the other homogenous group. In wild boar 6, day 0
significantly differed from days 3,7,10 and 14. Days 3 and 7 did not significantly differ from each
other. In wild boar 3, days 0, 3 and 7 did not significantly differ, also days 0, 7 and 10 did not. These
combinations differed significantly from each other and from day 14. In wild boar 4, days 0, 3 and 7
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
did not significantly differ, like days 3, 10 and 14 did not differ significantly. These combinations
differed significantly from each other. In wild boar 5, days 0, 3 and 7 did not significantly differ but
they differ from the not significantly different days 7 and 10. In wild boar 7, interestingly days 0 and
14 did not significantly differ, like days 14, 7 and 10. But these combinations differed significantly
from each other and from day 3.
Wild boar 1
Wild boar 6
Wild boar 3
Day 1
X
X
X
Day 3
X
Day 7
X
Day
10
X
Day
14
X
X
X
X
X
X
Wild boar 5
Wild boar 7
X
X
X
X
X
X
X
X
X
Wild boar 4
X
X
X
X
X
X
X
X
X
X
X
X
Table 23 Visualisation of significant (p<0.05) differences between influence pseudomonas counts on the different days of storage, every
colour represents a homogeneous group (i.e. results are not significantly different from each other) of days within one wild boar meat
sample series
26
Temperature exerted only in 2 of 4 meat cut series a significant influence. In all series with different
meat cuts, meat cuts had a significant influence on pseudomonas counts. Wild boar 1 and 6 showed
the same outcome; loin and shoulder meat cuts did not differ significantly but this combination
differed significantly from hind leg and goulash cuts. In wild boar 2 loin and hind leg meat cuts did
not significantly differ but they differed significantly from the shoulder cut. Goulash meat cuts were
not included, because the data set was not complete.
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
5. Conclusion
It has been suggested that game meat produced and stored under GHP conditions should not exceed
6 log cfu/g in terms of TVC (Paulsen 2011) Although it could be argued that for vacuum-packed meat
cuts, limits could be set higher, as lactic acid bacteria should be the dominant taxa in the total flora, a
number of references and also this study demonstrated the abundance of spoilage-associated
Pseudomonas sp. Thus, it was reasonable to apply the 6 log cfu/g limit also in this study.
In Table 24 the number of samples that reached 6 log cfu/g at storage temperature of 2°C are listed.
Interesting is the fact that all sample from wild boar 6 at day 1 had values higher than 6 log cfu/g,
whereas at day 3 only one sample had a value higher than 6 log cfu/g. Such inhomogeneity
complicates the proper assessment of results and prompts for examination of more than one
packaging unit per batch.
Generally it can be said that meat cuts processed by untrained staff or processed in a low scale
processing unit by trained staff reached the use-by date between 7 and 10 days of storage. Samples
from an AGHE reached the use-by date already between days 0 and 3. Notably, the business
operators mentioned that they would not store such meat cuts under refrigeration, but deep-freeze
the packages, store and deliver the packs in deep-frozen condition. At storage temperature of 3.8°C
meat samples reached the use by date earlier (Table 25). Also in this case samples from the AGHE
reached use by date already between 0 and 3 days.
27
In order to prolong the use-by date it is important that processing is done in a hygienic way, that
microbial numbers on the exposed surfaces of the carcasses are as low as possible and that meat
cuts stored as cool as possible.
In order to allow the establishment of a use-by date, the abovementioned process factors need to be
standardized and kept under control, e.g. in the framework of GHP.
Wild boar day 0
day 3
day 7
day 10
day 14
1 0/12
0/12
3/12
7/12
3/3
2 0/12
0/9
1/12
0/9
2/6
6 12/12
1/12
12/12
12/12
11/11
3 0/3
0/3
0/3
2/3
2/3
4 0/3
0/3
1/3
2/3
5/5
5 0/3
0/3
1/2
3/3
7 0/4
4/4
4/4
4/4
4/4
Table 24 Number of samples that reached 6 log units TVC at 2°C storage temperature, compared to the total number of samples
examined.( > 50% sample had values over 6 log cfu/g, 50% of the samples had values of more than 6 log cfu/g, < 50% of the samples had
values of more than 6 log cfu/g.)
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
Wild boar
day 0
day 3
day 7
day 10
day 14
1 0/12
1/12
10/12
12/12
3/3
2 0/12
0/9
5/12
9/9
3/3
6 12/12
2/12
12/12
12/12
12/12
7 0/4
4/4
4/4
4/4
4/4
Table 25 Number of samples that reached 6 log units TVC at 3.8°C storage temperature, compared to the total number of samples. ( >
50% sample had values over 6 log cfu/g, < 50% of the samples had values of more than 6 log cfu/g.)
28
Microbial shelf-life of vacuum packed wild boar meat cuts at two different storage temperatures
Koomen J.I.
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