Accepted Manuscript

Case studies of packaging and processing solutions to improve

meat quality and safety

Mari Ann Tørngren, Mianne Darré, Annemarie Gunvig,

Alexander Bardenshtein

PII: S0309-1740(18)30242-0
DOI: doi:10.1016/j.meatsci.2018.06.018
Reference: MESC 7591
To appear in: Meat Science
Received date: 28 February 2018
Revised date: 16 June 2018
Accepted date: 18 June 2018

Please cite this article as: Mari Ann Tørngren, Mianne Darré, Annemarie Gunvig,
Alexander Bardenshtein , Case studies of packaging and processing solutions to improve
meat quality and safety. Mesc (2018), doi:10.1016/j.meatsci.2018.06.018

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Case studies of packaging and processing solutions to improve meat quality and safety

Mari Ann Tørngrena*, Mianne Darréa, Annemarie Gunviga, Alexander Bardenshteinb

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Danish Meat Research Institute, Danish Technological Institute, Gregersensvej 9, DK-2630
Taastrup, Denmark
b
Materials, Plastic and Packaging Technology, Danish Technological Institute, Gregersensvej

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6, DK-2630 Taastrup, Denmark

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Mari Ann Tørngren: matn@teknologisk.dk, orcid.org/0000-0001-8396-8532, Mianne Darré:

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mtde@teknologisk.dk, Annemarie Gunvig: agg@teknologisk.dk, Alexander Bardenshtein:
alb@teknologisk.dk, orcid.org/0000-0003-1359-9449.
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*Corresponding author: Mari Ann Tørngren, phone +45 72202682
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Abstract

A significant amount of the meat is wasted due to spoilage or safety risks. Active packaging
systems have a great potential to reduce waste through chemical and microbial control of the
product and/or the storage environment. Although commercial products are already available,
active packaging is far from being fully developed. In contrast, passive packaging, such as
modified atmosphere packaging (MAP) and vacuum packaging, have been fully
implemented. Research conducted at the Danish Meat Research Institute (DMRI),

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demonstrates that it is possible to create new opportunities for the meat industry by
modifying MAP or combining microwave treatment with vacuum packaging. Predictive shelf

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life models can be used to estimate the shelf life in MAP or vacuum under dynamic
temperature conditions. Using the tri-gas guidelines, the industry can benefit from the

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increased eating quality, and the in-package decontamination process using vacuum
packaging in combination with 5.8 GHz microwaves eliminates C. botulinum spores,
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resulting in increased food safety and an extended shelf life.

Keywords: Fresh meat, Shelf life, Packaging, Tri-gas MAP, Vacuum, Microwave
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1. Introduction

Meat sustainability can be improved by increasing productivity or by reducing waste and

spoilage (Saucier, 2016). Shelf life can be defined by time to discolouration (appearance)
which is related to myoglobin oxidation, and odour, which is related to bacterial metabolism.
Shelf life of meat can also be defined by the storage time until spoilage, which mainly
depends on the number and type of microorganisms. Bacterial growth is determined by the

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storage conditions, i.e. temperature and packaging type (Borch, Kant-Muermans & Blixt,
1996). However, enzymatic degradation (Meinert et al., 2009) and oxidation of lipids and

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proteins will also have an impact on meat quality during storage by impairing colour stability,

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flavour and tenderness (Skibsted, Mikkelsen & Bertelsen, 1998). For decades, researchers
have worked extensively to gain a better understanding of shelf life, focusing on the essential
parameters that can result in maximum freshness of meat.
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To achieve industrial value, scientific knowledge must be translated into best practice
guidelines and then be implemented. One way to transfer knowledge on best practice of shelf
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life estimation between researchers and the meat industry is the ongoing development and
implementation of predictive models (Tenenhaus-Aziza & Ellouze, 2015) that can estimate,
for example, spoilage (Meinert, 2009), microbial growth (Braun & Sutherland, 2004) and
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autoxidation of myoglobin (Tofteskov, Hansen, & Bailey, 2017).

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Historically, packaging has been one of the most important breakthroughs for shelf life
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extension of fresh meat, although the function of food packaging is evolving from simple
passive preservation methods to active and intelligent packaging methods that go beyond the
traditional functions. In active and intelligent packaging, product, package and its
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environment interact to extend shelf life, improve safety and/or quality. In addition,
intelligent packaging monitor and communicate information about the state of the product
(Morris, Padmanabhan, Cruz-Romero, Cummins, & Kerry, 2017; Realini & Marcos, 2014).
Products such as oxygen scavengers, antimicrobial sachets, films and trays, carbon dioxide
emitters, moisture regulators and miscellaneous systems such as microwave susceptors and
steam valves are already commercially available for application (Ahmed et al., 2017; Fang,
Zhao, Warner, & Johnson, 2017). Nevertheless, new technologies are still being developed
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for active and intelligent packaging, and they need to be refined before implementation is
feasible and cost effective (McMillin, 2017).

The aim of this article is to present results on fresh meat shelf life modelling, tri-gas modified
atmosphere packaging, and in-package microwave decontamination. All three solutions are
examples of industrially driven research conducted at DMRI based on modification of
existing passive packaging systems and their interactions with well-established processing
technologies to create new quality and safety improving solutions.

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2. Boosting meat quality technology

The purpose of packaging is to maintain the desired properties of the meat during storage and

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display (McMillin, 2008). Deterioration of meat quality during storage includes
discolouration, off-flavour and off-odour development, loss of nutrients, texture changes,
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pathogenicity and spoilage (Skibsted, Mikkelsen & Bertelsen, 1998). The following sections
describe the results from experiments concerning the optimisation or extension of shelf life
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and the benefits of using predictive models to determine the shelf life of meat.
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2.1. Predicting shelf life

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The purpose of shelf life modelling is to have a fast and dynamic tool to predict the
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significance of events (e.g. unexpected temperature deviation) or to be able to predict shelf

life with as narrow tolerances as possible to avoid either too optimistic or too pessimistic use-
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by dates (McMeekin, 2004).

Shelf life can be defined as the period of time a product can be stored without becoming
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sensorially unacceptable or becoming a health risk. The predominant reason for meat
spoilage is microbial activity, oxidation and enzymatic autolysis (Dave & Ghaly). In some
cases, spoilage is caused by one specific organism (Gram & Dalgaard, 2002; Kalchayanad,
Ray, & Field, 1993), but spoilage mainly depends on the composition of a heterogeneous
microflora. However, other reasons for spoilage exist, since even sterile vacuum-packed meat
has a limited shelf life and becomes bitter over time, probably due to the proteolytic
deterioration of meat proteins by intrinsic enzymes (Meinert et al., 2009). Odorous
impression (scale anchored to the extremes ‘fresh’ and ‘spoiled’) is a suitable attribute for
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describing spoilage of pork, beef (Meinert et al., 2009) and chicken (Franke, Höll,
Langowski, Petermeier & Vogel, 2017), because the development of deviating raw meat
odour and psychrotrophic growth follows the same pattern (Meinert et al., 2009).

Several research groups have developed mathematical models that describe the growth of
specific spoilage bacteria, such as Brochothrix thermosphacta (Baranyi et al., 1996; Braun &
Sutherland, 2004) and Pseudomonas spp. (Braun & Sutherland, 2003), although they provide

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no link to the sensorial quality of the meat (available online at www.combase.cc). Others
have worked on modelling the shelf life of fresh meat, predicting the sensory shelf life from a
combination of specific microorganisms (Koutsounanis, Stamatiou, Skandamis, & Nychas,

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2006), while other models have been developed for risk assessment or inactivation of

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pathogens (Tenenhaus-Aziza & Ellouze, 2015). In 2008, the first shelf life model was
developed at DMRI (www.dmripredict.dk) to meet the Nordic meat industry’s demand for a
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tool to estimate use-by dates. The first model covered a broad segment of pork cuts on the
market, packed aerobically, in modified atmosphere (MA) or in vacuum. Shelf life models for
vacuum-packed beef, MA-packed chicken, bacon and minced meat (pork and beef) have
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subsequently been developed (Table 1). For all models, predictions can be made in the
temperature range of -1 to +7 °C and for an initial psychrotrophic plate count of between 0
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and 4 log CFU/cm2.

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2.1.1 Method
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At www.dmripredict.dk the industry has access to six different shelf life models that are
developed on data from a large number of meat samples randomly collected over time at
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commercial plants in three countries. The real-life variation in the microbial flora is therefore
included, which is essential for the robustness and applicability of the models. All the storage
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experiments were conducted under controlled and standardised conditions until complete
spoilage. Table 1 summarises cuts, number of samples, storage conditions, packaging
methods and additives included in the experiments.

Table 1. Overview of cuts, storage temperatures, packaging methods, additives and number
of samples included in storage trials for the six shelf life models
Model Cut Temperature Packaging Additives No. of
[°C] samples
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Pork cuts Tenderloin -1.5; 1; 2; 4; 7 Aerobic None 1500

Belly Vacuum
Loin with and MAP2
without rind
Riblet
Pork neck filet
Sparerib
Minced pork -0.5; 2; 5; 7 MAP2 None 680

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Beef cuts Silverside -1.5; -0.5; 2; 3.5; Vacuum None 850
Cuvette 5; 7

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Minced beef -0.5; 2; 5; 7 MAP2 None 680
Chicken cuts Breast 0.8; 4; 5; 7 MAP2 ≤ 0.8% salt 1080

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Fresh/brine- Leg
injected
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Cured pork1 Streaky bacon -1; 2; 5; 7 Vacuum 2.5% -5.5% 1080
Back bacon salt in aqueous
phase
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1
The products contained 60-120 ppm nitrite and were not smoked.
2
MAP atmosphere: 70% O2 and 30% CO2
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Throughout the storage trials, five meat samples were taken 10-14 times during storage. The
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sampling frequency depended on storage temperature and meat type. At each sampling, the
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raw meat odour was evaluated as: 1. fresh, 2. slightly deviating but acceptable, 3. deviating
and unacceptable or 4. putrid/rotten. Furthermore, the psychrotrophic counts, were analysed,
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as described in Meinert et al., 2009. The fitting of both the microbiological growth curves and
the sensory assessments of the raw meat odour was performed using nonlinear regression
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(Baranyi & Roberts, 1994). The fitted data for microbiological and sensorial results followed
the same S-formed shape, typical for a bacterial growth curve, whereas the parameters
characterising the growth and development of spoilage differed. Parallel to modelling the
psychrotrophic growth, this finding made it possible to model shelf life based on changes in
fresh meat odour, which was the first sensorial parameter to change during storage. The
number of psychrotrophic counts at the time for packaging was found to influence the
development of raw meat odour.
The equations developed for the growth of psychrotrophic bacteria and the changes in the
odour of the raw meat in relation to storage temperature and packaging method have been
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implemented in a user-friendly interface. The user enters the number of psychrotrophic

bacteria (log CFU/g) and the storage temperature, and the predicted shelf life is equivalent to
the time (days) until the odour score has reached five.

2.1.2 DMRI fresh meat models

The shelf life models for fresh meat include specific simulation procedures for cuts of beef,
pork and chicken, minced beef and pork as well as bacon. In the models, the packaging

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method is mainly fixed, whereas the procedure for pork cuts has the option to choose the
packaging method. It is even possible to choose a temperature profile, i.e. a given
temperature for a given amount of days, such as four days at 0 °C and then continuing at 4

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°C. Figure 1 shows some of the outputs from the shelf life models, where the shelf life of

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retail-packed pork (wrap/aerobic, MAP and vacuum) and beef (only vacuum-packed) is
predicted based on storage temperatures of 0 °C, 3 °C and 5 °C. As expected, the shelf life
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was extended by decreasing the temperature; for example, the shelf life of over-wrapped
aerobic) pork can be doubled by lowering the storage temperature from 5 °C to 0 °C, and
vacuum packaging and storage at 0 °C can extend shelf life five times compared with
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wrapped pork stored at 5 °C. Furthermore, the shelf life of vacuum-packed beef is twice as
long as that of vacuum-packed pork. This illustrates that the predictive models are tools that
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can be used to estimate the importance of changes in temperature and packaging method for
the shelf life of different meat types.
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DMRI’s shelf life models are available, at no cost, at www.dmripredict.dk and are an
essential tool that helps the industry to predict the shelf life of meat. For example, the
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industry uses the models to recalculate the shelf life if products have been exposed to
temperature abuse for a period of time, which means that the shelf life is more realistic in
relation to the actual storage conditions.

During the last decade, the dmripredict modelling tools has reached more than 1700 users
from more than 30 countries and has thereby proven its applicability for the meat industry in
general. Training courses and a user manual with examples are important methods to increase
the use of predictive models, and the experience from DMRI training courses is that attendees
appreciate both the theoretical content and the practical exercises. And the result has enabled
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the industry to focus its efforts on optimising temperature, packaging and hygiene, since
these are the most effective parameters when it comes to extending the shelf life of fresh
meat.

2.2 Modified Atmosphere Packaging for fresh meat (MAP)

MAP is widely used for retail packaging of fresh meat (McMillin, 2008; Singh, Wani, &
Saengerlaub, 2011). However, the recommended gas compositions should be reconsidered,

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since, the traditional high-O2 MAP with 70-80% O2 and 20-30% CO2 retains an attractive
bloom colour, but also initiates oxidative deterioration, resulting in decreased eating quality

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shortly after packaging.

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It has been widely reported that high-O2 MAP results in less tender and less juicy meat with
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increased rancid flavour and extended premature browning (PMB) for several meat types, e.g.
pork (Lund, Lametsch, Hviid, Jensen & Skibsted, 2007), beef (Clausen, Jakobsen, Ertbjerg &
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Madsen, 2009; Lagerstedt, Lundström & Lindahl, 2011; Tørngren, 2003), chicken (Jongberg,
Wen, Tørngren & Lund, 2014) and lamb (Frank et al., 2017).

This reduced eating quality is most likely caused by oxidative modifications of lipids giving
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rise to rancid flavour, and oxidation of structural proteins causing reduced tenderness and
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juiciness (Bao & Ertbjerg, 2015; Estévez, 2011; Geesink, Robertson & Ball, 2015; Jongberg,
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Skov, Tørngren, Skibsted & Lund, 2011). To avoid undesirable changes in the eating quality
traits, several studies recommend oxygen-free packaging instead of high-O2 MAP (Tørngren,
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2003; Cayuela, Gil, Banon, & Garrido, 2004; Clausen et al., 2009). The drawback of this
approach is the less attractive non-bloomed colour of the fresh meat surface (Hunt, Sörheim, &
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Slinde, 1999).

2.2.1. Eating quality – the role of CO2

Traditionally, fresh meat in MAP is packed with 20-30% CO2 to extend microbiological shelf
life (Franke et al., 2017; Spanos, Baussá, Baron & Tørngren, 2014), whereas O2 is responsible
for the blooming of the meat colour and for the oxidative deterioration of lipids and proteins
during retail storage. However, some studies indicate that CO2 might also have a significant
impact on the quality traits. For instance, beef steaks packed in 50% O2 and 50% CO2 have
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more pinholes (small air bubbles in the meat structure) and premature browning after cooking,
and are less juicy than steaks packed in high-O2 MAP (Clausen, 2004). An increasing number
of pinholes is also found in brine-enhanced pork chops packed in tri-gas MAP with 40% CO2
(Tørngren & Darré, 2014), and sliced pork belly was crispier and had a higher cooking loss
when using 40% CO2 instead of 20% CO2 (Tørngren, Spanos, Baron, Christensen & Hofer,
2016). Furthermore, Esmer et al. (2011) reported decreasing oxidative stability and loss of
redness in minced beef when the CO2 concentration was raised (Esmer, Irkin, Degirmencioglu,

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& Degirmencioglu, 2011). Increasing the concentration of CO2 while reducing the O2
concentration will have consequences, and therefore, in order to maintain the quality, it is
advisable to introduce a filler gas, such as nitrogen (N2), to balance the volume of the

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headspace. In the following sections, this is referred to as tri-gas MAP.

2.2.2 Optimising eating quality using tri-gas MAP

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In 2012, the Danish meat industry initiated research activities to develop new guidelines for
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MAP aimed at improving eating quality and achieving uncompromised shelf life. The purpose
of this work was to document the benefits and drawbacks of using tri-gas MAP and to develop
new guidelines for Danish pork and beef in MAP (Table 4). High priority retail cuts were
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selected, including cuts of fresh pork and beef, minced pork and beef and injection-enhanced
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pork cuts. In tri-gas MAP, the O2 concentration is reduced to 30-40% and balanced with an
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intermediate CO2 concentration (20-40%), and the third gas (N2) acts as a filler gas.

In the following section, the DMRI tri-gas MAP guidelines are demonstrated for Australian
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beef exported to European markets.

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2.2.3 Method

The following protocol explains in detail the testing of tri-gas MAP on Australian beef
exported to the EU, although the same protocol was also used for the trials performed on
Danish pork and beef. The results from the Danish trials are shown in section 2.2.5.

The experiment was designed as two sub-trials: one trial for documentation of the shelf life
and the other trial for documentation of the eating quality (Figure 2). For both sub-trials,
Longissimus thoracis et lumborum (LTL) was selected on the same day of slaughter (grass-
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fed British cross-breed cattle, 250-350 kg carcass weight, fat score 1-3, meat colour score 1C-
4) (AUS-MEAT 2005) at an Australian beef processing plant, vacuum-packed and exported
to Denmark by boat (75 days’ shipping time, average temperature of 0.2 °C+/-1°C). On
reaching the DMRI pilotplant in Denmark, the LTL was cut into 1.5 cm steaks and repacked
for retail display using one of the following four packaging methods: (1) tri-gas MAP (30%
O2 + 30% CO2 + 40% N2), (2) high-O2 MAP (70% O2 + 30% CO2), (3) vacuum and (4) over-
wrap (MAPET trays wrapped with oxygen-permeable PVC film). During retail display (5 °C,

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1200 lux, up to 35 days), samples were analysed to quantitate effects on sensorial quality and
microbial growth of the raw meat (day 0 plus eight sampling days for up to 120% of the
expected shelf life for each type of packaging). Sensory attributes were analysed twice during

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storage (day 0 and close to the expected use-by date, i.e. day 3 for over-wrap, day 7 for MAP

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and day 18 for vacuum-packed meat). The expected use-by date was determined by using
shelf life predictive models and performing a pre-study on re-packed Australian beef (LTL),
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in MAP (70% O2 + 30% CO2) and vacuum. The meat for the pre-study was purchased from
the Danish supermarket chain that sells meat from Australia similar to that used in the main
trial. This ensured comparable selection criteria, slaughter conditions and distribution.
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(Figure 2)
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Shelf life was determined by a combination of sensorial evaluation of the raw meat and
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microbial analysis (psychrotrophic plate count, PCA, 6.5 °C for 10 days). Furthermore, the
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gas composition was measured in the headspace of MAP samples. The sensorial evaluation of
the raw meat odour and appearance was performed by a panel of three to five experts, all
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trained in assessing the shelf life of meat products, on a 4-point scale (1 = fresh odour/colour,
2 = slightly diverging odour/colour, 3 = diverging odour/colour, 4 = rotten). Shelf life is
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defined as ‘time until odour or appearance is unacceptable’ and is reached when the average
score is 2.5.

Samples for sensory evaluation were cooked on a frying pan at 170 °C to a core temperature
of 63-64 °C. The samples (3.5 x 2.5 x 1.5 cm) were served on pre-heated plates and were
evaluated by an accredited sensory panel of trained assessors using a 15-point unstructured
line scale anchored at the extremes (0 = low intensity and 15 = high intensity). The training
was based on ISO 8586-1 “General guidance for selection, training and monitoring of
assessors” and the descriptive sensory analysis was performed according to “DMRI analysis
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instruction SM 305-11”, based on ASTM-MNL 13, ISO 4121 and ISO 13299. The
descriptive attributes for appearance (internal cooked colour, pinholes), flavour (roasted
meat, metallic, rancid and warmed-over flavour (WOF)), taste (acid, sour, bitter) and texture
(hardness, juiciness, tenderness) were determined and tested during two training sessions.

2.2.4 Results

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Figure 3 shows changes in the psychrotrophic plate count (log CFU/cm2) during display storage
of exported beef steaks after a total shipping time of 75 days and re-packing. As expected from

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the beef cut model (www.dmripredict.dk, Figure 1), the microbial counts (log (CFU/g)
depended on the type of packaging in the following order: over-wrap > MAP > vacuum. No

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differences in counts were observed between tri-gas MAP and high-O2 MAP.
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(Figure 3)
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The measured shelf life, as defined by odour and appearance, of the retail-packed beef steaks
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is shown in Table 2 and depended on the packaging method used for retail display. Wrap-
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packed steaks had a shelf life of approx. six to seven days, MA-packed steaks, regardless of
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gas mixture, had a shelf life of approx. eight to nine days, and vacuum-packed steaks had a
shelf life of approx. 17 days. Both microbial counts and the sensorial evaluation indicated
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that tri-gas MAP did not compromise shelf life compared to high-O2 MAP, and this is
supported by other studies (Esmer et al., 2011; Gammariello, Incoronato, Conte, & Nobile,
2015; Muhlisin et al., 2014; Xiaoyin et al., 2016).
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Table 2. Retail shelf life (odour and appearance) of retail-packed Australian beef steaks,
previously shipped from Australia to Denmark at 0.2 °C for 75 days, then put in retail display
at 5 °C (lighting of 1200 lux ).
Over-wrap Tri-gas MAP High-O2 MAP Vacuum
Odour 6.5 days 9.5 days 9 days 17 days
Appearance 7.5 days 8 days 9.5 days 27 days
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Furthermore, sensory data showed that beef steaks packed in tri-gas MAP (30% O2 + 30%
CO2 + 40% N2) had higher trained panel scores for roasted meat flavour and lower scores for
internal cooked colour, warmed-over flavour and rancid flavour relative to high-O2 MAP
(day 7), whereas scores for juiciness and tenderness were equal to high-O2 MAP (day 7) and
vacuum-packed steaks (day 18). In fact, all packaging methods experienced a decline in
texture when evaluated relative to the expected use by date. As shown in the DMRI
guidelines (table 4), higher texture scores were expected for tri-gas MAP relative to high-O2

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MAP. This unexpected result might be explained by differences in the antioxidative capacity
of the meat, due to differences in the diets (Duckett et al., 2009; de Zawadzki et al. 2017),
resulting in less radical formation and protein oxidation of the meat from Australian grass fed

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cattle.

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Table 3. LSmeans and levels of significance for sensory attributes of retail-packed Australian
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beef steaks, previously shipped from Australia to Denmark at 0.2 °C for 75 days, then put in
retail display at 5 °C (lighting of 1200 lux). Gas composition in tri-gas MAP was 30% O2 +
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30% CO2 + 40% N2, and in high-O2 MAP (70% O2 + 30% CO2).

Retail-packaging None Tri-gas High- Vacuu Over- p-values

MAP O2 m wrap
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MAP
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Storage time 0 7 days 7 18 days 3 days Packagin Time

days days g
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Cooked colour 4.9a 7.6c 10.4d 6.7b 4.7a <0.0001 0.0186

Pinholes 0.9a 2.4b 2.0b 2.1b 0.9a 0.0002 0.0007
Roasted meat flavour 7.7c 5.6b 4.1a 4.6a 6.3b 0.0015 0.0021
4.3c 1.9a 1.6a 2.8b 3.6c
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Metallic flavour <0.0001 0.0168

Warmed-over 0.1a 3.2c 5.9d 5.8d 2.0b 0.0003 <0.000
flavour 1
Rancid flavour 0.0a 1.4a 3.4b 3.8b 0.7a 0.0092 0.0036
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Acid taste 5.1d 2.7b 1.9a 2.3ab 3.6c 0.0029 0.0011

Sour taste 0.0a 2.3b 4.6c 5.1c 1.2ab 0.0004 0.0013
Bitter taste 4.0a 4.1bc 4.5c 5.5d 3.8ab 0.0004 0.2252
Hardness 3.9a 4.9cd 4.7bc 5.4d 4.3ab 0.0506 0.0175
Juiciness 9.0b 7.6a 7.7a 7.2a 8.6b 0.0985 0.0025
Tenderness 8.7c 7.1a 7.3ab 7.5ab 7.8b 0.4011 0.0010

To obtain maximum eating quality of retail-packed beef after long time distribution, it is
recommended to retail-pack steaks in over-wrap, since over-wrap preserves the fresh meat
flavour characteristics and minimises the development of off-flavours throughout retail
display. If a longer shelf life is required, modified atmosphere packaging in low O2 tri-gas
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MAP (30% O2, 30% CO2 + 40% N2) is recommended, since rancid flavour, warmed-over
flavour and premature browning are reduced compared with the use of traditional high-O2
MAP.

This study also shows the importance of the trial design. Sampling for sensory evaluation is
often carried out on one or two specific sampling days. In this case, it was decided to conduct
the second evaluation close to the use-by date (day 3 for over-wrap, day 7 for MAP and day

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18 for vacuum-packed steaks) to obtain knowledge on how the packaging systems performed
at the beginning and at the end of the expected shelf life. Instead of seeing the difference

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between aerobic storage (evaluated close to the use-by date) and anoxic packaging (evaluated

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half-way to the use-by date), it was clear that, regardless of the packaging, eating quality will
decrease close to the use-by date (Tørngren & Darré, 2016). The experimental design could
be improved by including both set time points and use-by dates as a basis for the comparison
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of samples.
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2.2.5 Guidelines for Tri-gas MAP

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The DMRI guidelines showed that tri-gas MAP is also recommended for pork. To achieve
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enhanced tenderness, less rancid flavour and PMB, pork chops can be packed in a tri-gas MAP
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containing a gas mixture of 40% O2 + 20-30% CO2 + 30-40% N2. The improved eating quality
might be due to less lipid oxidation and less cross-linking of the structural proteins. This is
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supported by Bao & Ertbjerg (2015), who found that myosin heavy chain (MHC) cross-linking
is influenced by the O2 concentration as follows: 0% < 20-40% < 60% < 80% O2.
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The only cut that benefits from a higher CO2 concentration is sliced, fried pork belly (Danish
national dish), which becomes crispier when retail-packed in 50% O2 + 40% CO2 + 10% N2
compared with high-O2 MAP and other tri-gas MAP gas mixtures with only 20% CO2.
Crispiness is not affected when increasing only O2 (from 0-80% O2 balanced with 20% CO2
and N2), but the combination of intermediate levels of both O2 and CO2 had a significant effect
on the texture. This gas mixture also resulted in the highest cooking loss, and it is therefore
suggested that the increased crispiness is due to decreased water-holding capacity (WHC)
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(Tørngren, Spanos, Baron, Christensen, & Hofer, 2016), probably caused by protein cross-
linking and aggregation of myofibrils (Bao, Boeren, & Ertbjerg, 2018).

The overall results from the tri-gas MAP trials on Danish beef and pork are summarised as
guidelines in Table 4 and indicate the need for product-specific MAP to meet the intersection
point for shelf life and eating quality.

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Table 4. DMRI guidelines for tri-gas MAP with improved eating quality and uncompromised
shelf life compared with high-O2 MAP.

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% O2 % % N2 Tri-gas MAP compared to References
CO2 High O2-MAP

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Pork, chops 40 20-30 30-40 Unchanged: juiciness Tørngren et al., 2013
(loin and More: tender, colour stable
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neck) Less: rancid, PMB
Pork, belly 40-50 40-50 0-20 Unchanged: colour stability Tørngren et al., 2013
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More: crispy Tørngren et al., 2016

Less: stale and piggy flavour
Beef, LTL 30 30 40 More: tender, juicy, meat Tørngren & Darré,
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steaks flavour 2015

Less: PMB, WOF, hardness
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Tri-gas MAP is not recommended for all kinds of retail cuts. Minced meat is more sensitive to
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oxidation and must be stored in an anoxic atmosphere to obtain improved eating quality. This is
supported by studies showing less free thiol groups in minced beef stored in 20-80% O2 for six
days compared to anoxic storage (Bao, Puolanne, & Ertbjerg, 2016; Spanos, Baussá, Baron &
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Tørngren, 2014) and by higher TBARS and less free thiols in minced pork (Spanos, Tørngren,
Christensen, & Baron, 2016). Furthermore, it is difficult to meet the intersection point for shelf
life and eating quality for minced meat, since the microbial shelf life of minced meat packed in
MAP decreases significantly if the O2 level in the headspace is lowered to ≤ 40% compared to
50-80% (Esmer et al., 2011; Spanos, Baussá, Baron, Tørngren, 2014), whereas ≤ 40% O2 is
needed to prevent lipid oxidation in the meat.

2.2.6. Discussion on MAP

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The DMRI tri-gas MAP guidelines and the Australian tri-gas MAP study suggest the need for
product-specific MAP and are supported by the fact that animal, breed and muscle type have a
significant impact on oxidative stability (Min, Nam, Cordray, & Ahn, 2008) and colour
stability in oxygen-containing packaging systems (Seyfert, Mancini, Hunt, Tang, & Faustman,
2007; Spanos, Christensen, Tørngren, & Baron, 2016; Warner, Kearney, Hopkins, & Jacob,
2017). Recommendations should not be based solely on chemical analyses, since these show
the chemical stage and not the perceived appearance or eating quality. This argument is

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supported by Jongberg et al. (2014), who showed that there was a stronger tendency for lipid
and protein oxidation to occur in chicken thighs compared to chicken breast but concluded that
thighs were more suitable for high-O2 MAP, because rancidity was less pronounced in thighs

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after cooking (Jongberg et al., 2014).

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There is no doubt that trained assessors can differentiate between meat packed in high-O2
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MAP and non-O2 packaging, but the interesting questions for the industry are: “What do
consumers perceive?” and, since MAP has been available to consumers in many countries for
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the past four decades, “Are MAP flavour and texture the new normal?”

Several consumer studies have been conducted the past decade. Scandinavian consumers
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(Danish, n = 382, Swedish, n = 374 and Norwegian, n = 316) compared non-O2 packed beef
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steaks to high-O2 MAP prepared and evaluated at home. Independent of country, the
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Scandinavian consumers clearly preferred steaks packed without O2, both in terms of
tenderness, juiciness, flavour and overall liking (Aaslyng, Tørngren, & Madsen, 2010).
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Likewise, Australian consumers (n = 40) rated skin-packed LTL beef steaks with higher
scores for juiciness, flavour and overall liking compared to high-O2 MAP (Geesink et al.,
2015), confirmed by a larger study (Australian consumers, n = 1440) evaluating retail-packed
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beef steaks from three different muscles (LTL, gluteus medius and psoas major) aged for 5,
12 and 40 days. The steaks were packed in over-wrap (oxygen permeable film), VSP and
high-O2 MAP and, independent of muscle and aging, steaks packed in high-O2 MAP were
rated much lower compared to VSP and over-wrap for tenderness, juciness, liking of flavour
and overall acceptabilitity (Polkinghorne et al., 2018).

In contrast, Irish consumers (n = 134) were given beef packed in tri-gas MAP with 40, 50, 60,
70 and 80% O2 (balanced with 20% CO2 and N2) and expressed sensory preference for beef
packed in tri-gas MAP with 40% O2 followed by 80% O2 (Zakrys, O'Sullivan, Allen, &
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Kerry, 2009). Even though Irish consumers showed a preference for 80% O2 relative to 50-
70% O2, it is evident that consumers, in the studies referred to, prefer meat with low or no O2
in the headspace, indicating that MAP flavour is not the new normal.

3. Boosting meat safety technology

Preservation of meat and meat products is essential for the meat industry. Several thermal and

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non-thermal methods are known and have been tested for decontaminating effects on bacteria
in packed products to obtain increased shelf life and safety (Aymerich, Picouet, & Monfort,

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2008). An example of a thermal method is steam pasteurisation, and examples of non-
thermal methods are hydrostatic pressure (HPP), bio-preservation and active packaging

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(Aymerich et al., 2008), cold plasma (Bauer et al., 2017; Leipold, Kusano, Hansen, &
Jacobsen, 2010), UV light (Loretz, Stephan, & Zweifel, 2011), pulsed light (Singh, Kumar,
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Kumar, Bhat, 2012) and chemical methods using organic acids (Buncic, McKinstry, Reid, &
Anil, 2002; Gill & Landers, 2004; Lawson, Jensen, Christiansen, & Lund, 2009; Loretz et al.,
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2011; Mani-López, García, & López-Malo, 2012; Youssef, Yang, Badoni, & Gill, 2012).

The Food Standard Agency (FSA) recommends that the shelf life of fresh vacuum-packed
food is less than ten days for storage temperatures of +3-8°C (FSA, 2008), even though
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sensorial indicators of spoilage may not occur before 25 days for pork and 47 days for beef at
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+3 °C (Figure 1). These cautious recommendations are due to the growth risk of C.
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botulinum. At temperatures above 3.3 °C and under anaerobic conditions, C. botulinum can
produce a very harmful toxin and cause a fatal form of food poisoning (Peck, 1997).
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3.1 Decontamination of vacuum-packed meat

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To reduce the risk of growth of C. botulinum, FSA recommends a heat treatment at 90 °C for
10 minutes, or a treatment time/temperature combination sufficient to achieve a 6 log
reduction of C. botulinum spores. In practice, this means that meat cuts must be at least semi-
processed to extend shelf life beyond ten days. To overcome this potential market barrier,
DMRI tested different methods for decontamination of meat surfaces, including high-
frequency microwaves, hot water and steam treatment.

3.1.1. Development of high frequency microwave test rig

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Microwave frequencies allocated for industrial, scientific and medical (ISM) use are 433,
915, 2450 and 5800 MHz (Aymerich et al., 2008). However, state-of-the-art commercial
microwave food decontamination systems have so far used only two of these frequencies,
namely 915 MHz and 2.45 GHz (Tang, 2015). In Europe, researchers in Alfa-Laval (Sweden)
designed a 2.45 GHz system in which packaged foods were microwaved while being
immersed in water (Stenstrom 1972, 1974). This design reduced heating non-homogeneity
and made it possible to implement high temperature and short-time thermal processes.

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Waterless systems are also commercially available, but the food suffers from severe heating
non-homogeneity that may compromise decontamination. In spite of this challenge, three
European designs of 2.45 GHz microwave systems were commercialised by Officine

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Meccaniche Attrezzature per Ceramiche (OMAC) (Harlfinger 1992), Berstorff (Schlegel

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1992), and Gustosi (http://www.gusto-si.it/engnew/tecnologia.html). Recently, a consortium
led by Washington State University developed and commercialised a decontamination system
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that operates at 915 MHz for processing of ready-to-eat meals, enabling penetration of the
whole meal, and uses water immersion (Tang 2015).
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However, none of these microwave systems can be used for surface decontamination of raw
meat, since they penetrate too deeply and therefore denaturate a too thick layer of the
product. This is where 5800-MHz microwave processing promises a better performance.
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Although 5800 MHz (5.8 GHz) microwaves have not yet been used for food processing
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applications (Tang, 2015), this frequency enables smaller penetration depth and higher
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energy absorptivity of microwaves compared with those of 2.45 GHz, 915 and 433 MHz
microwaves (Cole & Cole, 1941). The power penetration depths of 2.45 GHz and 5.8 GHz
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microwaves in raw lean beef are respectively ~ 8 mm and ~ 2.5 mm, and the energy
absorptivity at 5.8 GHz is 1.42 times higher than that at 2.45 GHz.
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In order to implement and explore this higher-frequency microwave processing, a laboratory

5.8 GHz microwave processing test rig, based on a commercial magnetron M5801J, 750 W
(MUEGGE GmbH, Germany), attached to a standard 30-litre cavity of a household
microwave oven was built at the Danish Meat Research Institute. It was equipped with
specialised control and power supply units that enable precise continuous settings of the
treatment time and generated microwave power.

3.1.2 Method for decontamination of meat surfaces

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Meat samples

M. semimembranosus of random origin was cut into cubes (4.5×4.5×4.5 cm) weighing
approx. 100 g ± 10 g and dried in a LAF bench (Laminar Flow Cabinet) for 15 minutes.
Then, 1 ml of a C. botulinum spore cocktail (107 spores/ml) was added to the samples,
corresponding to approx. 105 spores per cm2 of the meat surface. The cocktail contained four
non-toxigenic and gas-producing strains of psychrotrophic C. botulinum (further described in
Gunvig (2016). Finally, the meat pieces were vacuum-packed in plastic bags made of a film

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laminate PETP 12/PEP LDPE 75. When meat samples are vacuum-packed, a thin liquid film
will be formed at the interface of the meat and the packaging film, due to mechanical

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compression of the tissue and vacuum extraction of the water.

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Microwave treatment

For microwave processing, the vacuum-packed meat samples were suspended from hooks
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placed at the top of a circular plastic device, which ensures exposure of the entire surface
leaving no cold spots on the surface (Figure 4 A). This circular plastic device was placed in
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the microwave cavity where it replaced the standard glass turntable. This arrangement
enabled circular movement of the samples through the pattern of 5.8 GHz standing waves
formed in the cavity, and the elevated vertical position resulted in a reasonably homogenous
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microwave heating (Figure 4 C). Two microwave-treatment experiments were carried out.
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In the first experiment, 4 x 3 samples were treated at full power for 1, 2, 3 or 4 minutes from
an initial surface temperature of 21 °C.
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In the second experiment, a fractionated treatment was implemented to check the

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controllability of the process and prove the feasibility of a multi-stage treatment. In this
experiment, the initial surface temperature was increased to 45 °C by immersing the samples
in a water bath at 55 °C for five minutes. Immediately after pre-heating the surface in the
water bath, the samples were treated in the microwave test rig one by one at full power for
one minute. The treatment then continued at only 25% of full power for 1, 2 or 2.5 minutes.
Five replicates were performed for each treatment time.

Steam flow treatment: To be able to separate impacts of “pure” microwave and steam
processing, steam treatments of unpacked samples were carried out using an approx. 2-litre
plastic container. A steam nozzle equipped with a heat-insulated handle was inserted into the
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container through the lid and connected to a desktop steam generator (Trevil, Type 1624,
Italy). The meat samples were suspended from hooks attached to the lid using sterile threads
connected to a corner of each sample. The samples were treated one by one in a closed
container with full immersion in a steam flow at 2 bar (g) of the overpressure applied to the
nozzle. The treatment times were 20, 40, 60, 80 and 100 seconds. Three replicates were
performed for each treatment time.

Water bath treatment

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Vacuum-packed meat samples were immersed in a water bath at 90 °C for 0, 2, 4 or 7
minutes; at 95 °C for 0, 0.5, 1 and 2 minutes; at 97 °C for 0, 0.5, 1 and 1.5 minutes and at 99

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°C for 0, 0.5, 1, 2 and 4 minutes. Three samples were analysed for each treatment time,

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except for the treatment at 99 °C, where five samples were used. The treatment for 0 min
corresponds to untreated samples for all decontamination experiments. In all experiments, the
samples were immersed in ice water immediately after heat treatment.
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Microbiological analysis
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The samples were ten-fold diluted in 0.86% saline with peptone. The spore samples were heat
treated at 75 °C for 20 minutes before plating on Shahidi Fergusson Perfringens (SFP) agar
and incubated for three days at 30 °C. The decimal reduction time (D-value) and the
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treatment time necessary to obtain a 6 log reduction of C. botulinum spores were determined
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for each treatment. The D-values are equal to the slope of the regression line for the
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logarithm of spore counts versus treatment time.

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3.1.3 Results for decontamination of meat surfaces

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Microbiological analyses of 5.8 GHz microwave-treated samples are shown in Figure 5, and
it is seen that the spore counts of C. botulinum decrease over time. After 3.5 minutes, the
number of C. botulinum spores on the surface is reduced by approx. 5 log. The D-value for
the microwave treatment was calculated to be 0.79 minutes.

D-values for microwave processing and water bath treatments that were capable of a 6 log
reduction are listed in Table 4. A 6 log reduction in the number of heat-resistant C. botulinum
spores can be achieved after approx. four minutes of treatment with microwaves (5.8 GHz) at
100% effect. At a reduced effect (100% for 1 min + 25% for 1, 2 or 2.5 minutes) and with
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preheating of the meat to 45 °C, 5.6 minutes is required to achieve a reduction of 6 log. The
advantage of using microwaves is that the thin water film between the packaging material and
the meat surface is immediately heated to 100 °C without heating of the centre of the meat
(Figure 4B). This results in an increase in the death rate of the spores. In a water bath at 90
°C, 95 °C, 97 °C and 99 °C, 23.9, 21.8, 13.3 and 8.3 minutes are required to gain a reduction
of 6 log.

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In Table 5, it is seen that the D-values for the water bath treatment decrease with increasing
temperatures, as expected. The D90°C value of 3.99 minutes is in good agreement with the

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D90°C value of 4.4 minutes, which was determined for the strains in a laboratory meat system
with momentary heating (Jakobsen, 2005).

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Table 5: D-values for C. botulinum spores and time necessary to reduce the counts by 6 log
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on the surface of fresh meat, when heat treating with a water bath (90 °C, 95 °C, 97 °C and
99 °C) and high frequency microwaves.
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Treatment D-value, min Time for 6 log, min

Water bath, 90 °C 3.99 23.9
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Water bath, 95 °C 3.64 21.8

Water bath, 97 °C 2.22 13.3
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Water bath, 99 °C 1.39 8.3

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Microwave, continuous 0.68 4.1

Microwave, fractionated 0.79 4.7
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It is important to note that the water bath treatment at 90 °C results in a 6 log reduction of C.
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botulinum spores after 23.9 min, which is much longer than the ten minutes recommended by
FSA. Higher heat resistance of the applied spores could be the reason for this extension of the
treatment time.

D-values for steam flow treatment are not shown in Table 5, as this method resulted in only a
slight reduction of C. botulinum spores. A reduction of 1 log CFU/cm2 was achieved during
the first 20 seconds of the treatment. However, longer treatments resulted in minimal
reductions, if any at all. This phenomenon is called ‘tailing’, and it is most likely due to steam
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condensation creating a microscopically thin condensed water film at the surface that screens
microorganisms from the heat provided by the steam condensation.

The results show that the time taken to obtain a 6 log reduction of C. botulinum spores can be
reduced six times when using 5.8 GHz microwaves compared to hot water at 99 °C. The
shorter treatment time with 5.8 GHz microwaves is due to a faster heating of the thin water
film between the packaging material and the meat surface. Steam flow treatment was not

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capable of reducing C. botulinum by a 6 log reduction in this set-up.

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3.1.4 Discussion

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High efficacy of 5.8-GHz microwave processing of vacuum-packed fresh meat has been
demonstrated in laboratory experiments using small meat cuts. As for the commercialisation
of the developed technology, the main barrier to overcome is upscaling of the microwave
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power available for decontamination to the level required for typical industrial meat
processing throughputs. For instance, our estimations show that the power required for 5.8-
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GHz microwave processing at a commercially relevant throughput of raw meat of 300

kg/hour is approx. 3.2 kW. This requirement is neither easy nor cheap to fulfil using, for
instance, commercially available but expensive (at least EUR 12,300 per unit) 5.8 GHz
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magnetrons of maximum generated power of 700-750 W.

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4. Conclusions
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Three industry-initiated developments involving passive packaging systems are exemplified

by DMRIs shelf life models, tri-gas MAP packaging and high frequency microwave surface
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decontamination of vacuum-packed meat.

Predictive shelf life models for fresh meat are tools that provide the industry with the
possibility to quickly and easily obtain, an estimate of the shelf life of different meat cuts
packed in MAP and vacuum, stored under dynamic temperature conditions. These shelf life
models have been used worldwide, and more than 1700 users have signed up, showing that
the shelf life models are applicable.
High-O2 MAP has been used by the industry for four decades, but, in order to obtain minimal
changes in the eating quality during display and an uncompromised shelf life, it is
recommended to retail-pack fresh meat anoxically or in a low oxygen tri-gas MAP (O2, CO2
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and N2). It is not known whether the industry has implemented tri-gas MAP, but the
increasing interest shown by packaging companies and the meat industry indicates that the
solution to obtain increased eating quality is significant to the industry, and that current
barriers regarding adjustment of the equipment might be manageable.

A higher-frequency 5.8-GHz microwave treatment applies a thermal shock to the meat

surface, with a penetration of only a few millimetres. This processing leaves the centre of the
meat red and is therefore a significant step beyond state-of-the-art microwave

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decontamination of food that is represented by commercial systems that make use of lower-
frequency microwaves at 915 MHz and 2.45 GHz, which are capable of penetrating the core

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of a product. The main advantage of the higher-frequency microwave processing compared
with the hot water bath treatment commonly used in the industry is that the treatment time for

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a 6 log reduction of C. botulinum is reduced by a factor of two (compared with hot water at
99 °C). Furthermore, after the microwave processing resulting in a 6 log reduction of C.
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botulinum, it will be possible to extend the shelf life at 5 °C to more than the ten days
recommended by the FSA. Before implementation of the technology in the industry the
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equipment must be up-scaled and further experiments performed on cuts relevant to the
industry regarding dimensions and volume.
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Acknowledgement
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This work was supported by the Danish Pig Levy Fund and a grant from the Danish
Technological Institute’s performance contract 2016-2018, entered into with the Danish
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Agency for Institutions and Educational Grants, under The Ministry of Higher Education and
Science. Australian collaborators and The Australian Meat Processor Corporation (AMPC)
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are gratefully acknowledged for a successful performance during the execution of the tasks
and for funding.
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Figure 1. Predicted shelf life of fresh pork retail-packed in wrap, MAP (70% O2 + 30% CO2)
and vacuum, and fresh beef in vacuum stored at 0 °C (black bars), 3 °C (grey bars) and 5 °C
(white bars), respectively. Predicted by www.DMRIpredict.dk. Version 5.1. For all
predictions, the psychrotrophic count (6.5 °C for 10 days) at packaging is fixed at 2.5
log/cm2, and the standard deviation (std.) is fixed at 0.9.

Figure 2. Experimental design of the two sub-trials for documentation of shelf life and eating

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quality of Australian longissimus et lumborum exported to the EU. Retail-packed in wrap,
vacuum, tri-gas MAP (30% O2 + 30% CO2 + 40% N2) and high-O2 MAP (70% O2 +30%

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CO2) and stored in retail display (5 °C, lux 1200) for up to 35 days.

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Figure 3. Psychrotrophic plate count (log cfu/cm2) of beef steaks from Australian
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longissimus et lumborum exported to the EU (vacuum for 75 days at 0.2 °C) and retail-
packed in wrap (×), , tri-gas MAP (◊), high-O2 MAP (□) and vacuum (∆) stored in retail
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display (5 °C, lux 1200). Assessments were performed at day: 0, 3, 4, 7, 8, 9, 10, 11 and 13
for over-wrap; 0, 4, 7, 9, 10, 14, 15, 18, 21 for MAP and 0, 10, 14, 18, 25, 28, 32 and 35 for
vacuum n=5 (M. A. Tørngren & Darré, 2016).
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Figure 4. Microwave processing of meat samples. A. vacuum packed unprocessed meat

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sample suspended from hooks on a circular plastic device. B. Microwave processing showing
boiling meat juice in blown packaging, C. Cross-section view of a meat samples after 5
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minutes of 5.8 GHz microwave processing.

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Figure 5 C. botulinum spore counts (log spore/cm2) as a function of time of fractionated

microwave treatment. Full power for 1 minute plus 25% of full power for 1, 2 or 2.5 minutes
( ● = Number C. botulinum (log spores/cm2) in one sample, n = 5, ----- = regression line).
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