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Factors affecting microbial spoilage and shelf-life of chilled vacuum-packedlamb transported to distant markets: a review

John Mills, Andrea Donnison, Gale Brightwell

PII: S0309-1740(14)00114-4DOI: doi: 10.1016/j.meatsci.2014.05.002Reference: MESC 6408

To appear in: Meat Science

Received date: 24 July 2013Revised date: 30 April 2014Accepted date: 2 May 2014

Please cite this article as: Mills, J., Donnison, A. & Brightwell, G., Factors affectingmicrobial spoilage and shelf-life of chilled vacuum-packed lamb transported to distantmarkets: a review, Meat Science (2014), doi: 10.1016/j.meatsci.2014.05.002

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Factors affecting microbial spoilage and shelf-life of chilled vacuum-packed lamb

transported to distant markets: a review

John Mills1, Andrea Donnison

1 and Gale Brightwell

2*

1Food Assurance and Meat Quality, AgResearch Ltd, Ruakura Research Centre, Private Bag

3123, Hamilton, New Zealand.

2Food Assurance and Meat Quality, AgResearch Ltd, Hopkirk Research Institute, Cnr

University Ave and Library Road, Massey University, Palmerston North 4442, New Zealand.

*Corresponding author. Tel.: +64 351 8678; fax: +64 353 7853.

E-mail address: gale.brightwell@agresearch.co.nz (G. Brightwell).

Key words

Vacuum-packed chilled lamb

Spoilage

Shelf-life

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Abstract

Vacuum-packaging and stringent control of storage temperatures enables the export of

meat to distant markets, supplying a chilled product that can favourably compete with local

fresh meats. Recently, in order to save fuel and reduce emissions, the speed of ships traveling

to international markets has decreased resulting in requirement for the shelf-life of chilled

vacuum-packed lamb to be extended beyond the recognised time of 60-70 days as described

in the scientific literature. Growth of microorganisms and their ability to cause spoilage of

vacuum-packed lamb is dependent on many factors, including the type and initial

concentration of spoilage bacteria, meat pH, water activity, availability of substrates, oxygen

availability and, most importantly, storage time and temperature of the packaged product.

This paper reviews existing knowledge of the spoilage bacteria affecting vacuum-packed

lamb, discusses the impact of these bacteria on product quality, shelf-life and spoilage, and

concludes that under specified conditions the shelf-life of chilled lamb can be extended to

beyond 70 days.

1. Introduction

One of the key challenges for today’s meat export industries is to get “fresh” product

of superior quality to distant markets. The most commonly used method of preserving meat

that provides the necessary product-life (without recourse to freezing or the addition of

preservatives), is to vacuum-pack larger “primal” cuts, thereby excluding oxygen and

preventing the growth of oxygen requiring spoilage bacteria (Gill, 1989). In order to further

minimise decrease in product quality, storage life, due either to either spoilage by bacteria

capable of anaerobic growth or to biochemical processes affecting colour stability, a storage

temperature of -1.5°C has been recommended and is, for example, applied routinely to all

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chilled product shipped from New Zealand to overseas markets (Gill et al., 1988a; Jeremiah,

& Gibson, 2001). For these storage conditions, temperatures above 0°C would be considered

abusive. These conditions are different to those employed for chilled storage of fresh meats

close to retail outlets, where cuts may also be stored in air or modified atmospheres at

temperatures of around 2°C, and in this situation temperatures above 5°C are considered

abusive (Tewari et al., 1999; James, & James, 2004a).

To avoid economic losses to the retailer, each meat product is given a specific storage

lifetime, which is the period for which that product is expected to remain safe and there is no

appreciable loss of quality; that is, the point at which colour and texture changes, and

bacterial metabolic activities make the meat offensive to the senses of the consumer (Gill,

1983). Currently, the storage lifetime for vacuum packed lamb held at -1.5°C has been

estimated to be between 60 and 70 days (Bell, 2001; James, & James, 2002). However,

pressure on shipping companies to reduce their environmental impact and fuel costs has

resulted in slowing their vessels by up to 20% (“slow steaming”), thus leading to increased

shipping times (Psaraftis, & Kontovas, 2013). This means that the storage life of vacuum-

packed meat must be sufficient to allow for this change, so that quality standards are met

when the product is sold to the consumer. At present, this can be achieved based on a generic

storage life of 70 days at -1.5°C. However, further decreases in shipping speeds may make

the trade unsustainable without further extension of storage life (MIA, 2012). Further, as a

consequence of these prolonged transport times, the meat microbiota of vacuum-packed lamb

is likely to be substantially different to that of locally-sourced product, which may result in

inappropriate and unnecessary rejection or downgrades. To ensure that chilled lamb subjected

to prolonged transport is not discarded unnecessarily, the characteristics of spoilage bacteria

over these longer storage period need to be investigated. Existing literature concerning the

microbiology of chilled meat has primarily focused on beef (Grau, 1980; Grau,1981; Jones,

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2004), with very little recent literature on the expected product life of vacuum-packed chilled

lamb. Considerable progress has been made over the last decade to extend the shelf-life of

chilled lamb, including a better understanding of the impacts of meat pH and water activity

(aw) on microbial growth. Furthermore, process hygiene has been improved in order to ensure

that the initial number of microorganisms on meat is as low as possible. Technical advances

have reduced the oxygen permeability of barrier films and allowed greater control of

temperature throughout processing and transport. As a result, with careful control a product

shelf-life of up to 12 weeks is now attainable for some cuts, particularly those of low pH (5.5-

5.8) (Kiermeier et al., 2013). This review discusses current understanding of the

microbiology of vacuum-packed chilled lamb and focuses on how the microbiota impact on

expected shelf-life and microbiological criteria set by specific customers.

2. Vacuum-packaging

Vacuum-packaging refers to meat that has been placed into a bag of low oxygen

permeability and a vacuum applied prior to sealing (Kropf, 2004a). As the vacuum is applied

the packaging collapses ensuring close contact between the film and meat that can be further

enhanced by shrink wrapping. Alternatively, vacuum skin packaging may be used on retail-

sized cuts, the “skin” being thermoformed around the meat by drawing a high vacuum on both

sides of the heated packaging film, then venting the upper side to air, forcing the film tightly

over the product, removing the void around (though not within) the product. When meat is

sealed with little headspace in oxygen-impermeable materials, the residual oxygen at the

meat surface/package interface will be rapidly converted to carbon dioxide by the respiratory

activity of the meat (Bell, 2001). In oxygen-depleted atmospheres, growth of aerobic spoilage

bacteria is prevented and the microflora changes to one that is dominated by slow growing,

CO2 tolerant bacteria (Borch et al., 1996). Although the shelf-life of lamb is greatly extended by

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vacuum-packaging, it will eventually be spoiled. Spoilage indicators include off-odours and

discolouration (Bell, 2001).

Most of the published research on the microbiology of chilled lamb was carried out on

meat vacuum-packed in a plastic bag with a low but measureable rate of oxygen transmission

(Gill, 1996). The transmission rates for films routinely used for vacuum-packaging have

improved, from the 30-40 cc/m²/24h at 25°C reported in 1985 (Gill, & Penney, 1985) to 18.6

cc/m²/24h at 23°C available today (Kiermeier et al., 2013). These transmission rates decrease

further with temperature, particularly below 0°C (Lambden et al., 1985), making it difficult to

determine the precise oxygen transmission into a chilled vacuum-packed sample or to

determine what impact this has on the development of the bacterial microbiota at the

meat/package interface long-term. Nevertheless, Gill, & Penney (1985) showed that storage

life was improved when lamb loins were stored at 0-0.5°C in foil laminates of immeasurably

low permeability compared to loins stored in the 30-40 cc/m²/24h at 25°C plastic films.

Premature spoilage of vacuum-packed meat is usually due to “leaky” packaging - e.g.

from the sharp ends of bones or poor seals (CSIRO, 2003). Most meat producers now check

pack seals prior to shipment, and specially-designed bags with thicker walls are now

available to pack bone-in product. Kiermeier et al. (2013) compared the storage life of bone-

in versus bone-out lamb shoulders at -0.3°C and found no significant differences between

microbial communities or sensory test scores.

3. Microbiology of vacuum-packed chilled lamb

It is generally accepted that it is not feasible to produce meat without some degree of

bacterial contamination (Mills, 2012a). A variety of bacterial species can be isolated from

lamb carcasses post slaughter, although the majority are poorly adapted to growth on the

meat matrix under chilled anaerobic storage conditions (Marshall, & Bal’a, 2001).

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Consequently, whilst aerobic spoilage bacteria such as Pseudomonas spp., and mesophilic

bacteria such as Escherichia coli, can sometimes be detected, they are unable to rapidly

proliferate. Rather, following a period of chilled storage, the resulting microbiota of vacuum-

packed lamb is dominated by some strains of lactic acid bacteria (LAB), notably Leuconostoc

spp. and Carnobacterium spp. (Jones et al., 2008). Other bacteria that grow on chilled

vacuum-packed lamb include some environmental species of psychrotropic

Enterobacteriaceae, notably Serratia spp., Hafnia alvei, Rahnella aquatilis and avirulent

members of the Y. enterocolitica group, and specific spoilage organisms Brochothrix

thermosphacta, Shewanella putrefaciens and psychrophilic “blown-pack” Clostridium spp.

(e.g. Clostridium estertheticum, Clostridium gasigenes) (Pennachia et al., 2011; Brighwell et

al., 2007; Gill, 2004; Seelye, & Yearbury, 1979). Not all species are implicated in spoilage.

For example, the avirulent Y. enterocolitica-like bacteria are not associated with spoilage

events (Gill, & Newton, 1979). Packaging and storage strategies aim to produce a microflora

dominated by LAB to maximise shelf-life. If lamb is produced under good manufacturing

practice, the initial count of microbes on the product surfaces is likely to be 103/cm

2 or less

(Gill, 2004; Phillips et al., 2013), then psychrotrophic organisms able to grow below 7°C

will be fewer still (Bell, 2001). If this condition is met and packaging material has low gas

permeability (<30 cc O2/m²/24h at 25°C) and there is very good temperature control (± 0.5

°C) lamb cuts should have a storage life of 10-12 weeks (Kiermeier et al., 2013).

3.1 Pathogenic bacteria

The majority of meat-borne pathogens are mesophiles (e.g. Salmonella,

Campylobacter jejuni, E. coli O157:H7) and require temperatures above 7° C for growth.

Therefore, the health hazard from these bacteria is not increased during vacuum-packed

storage at -1.5° (Bell, 2001) but neither is it necessarily decreased. For example, no loss of

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viability was observed for Campylobacter jejuni, E. coli O157:H7 or Salmonella on vacuum-

packed beef that was stored at -1.5° over 41 days (Dykes, & Moorhead, 2001; Dykes et al.,

2001). Further, when compared to a significant reduction in numbers on sterile beef and pork

under chilled, vacuum-packed retail display conditions, survival of C. jejuni was significantly

enhanced in the presence of a natural meat microbiota (Balamurugan et al., 2011).

There are four psychrotrophic pathogens that could be of concern on chilled vacuum-

packed lamb; spore-forming non-proteolytic Clostridium botulinum, Yersinia enterocolitica,

Listeria monocytogenes and Aeromonas hydrophila (Bell, 2001). Non-proteolytic C.

botulinum is associated with an extremely serious foodborne disease with as little as 30 ng of

neurotoxin sufficient to cause illness or even death (Lund, & Peck, 2000). It is an obligate

anaerobic bacterium (minimum growth temperature 2.5 - 3° C) that is unable to grow or

produce neurotoxin after 12 weeks at 2.1 – 2.5° C (Peck, 2009), temperatures that are

considerably higher than the -1.5° C at which vacuum-packed chilled lamb is transported. It

is normal practise for temperature data loggers to accompany all shipments (Gill et al.,

1988a), and in the author’s experience of the trade between New Zealand and the UK, the

data from these are examined by importers to ensure that the temperature did not exceed 2°C

during transit, thereby ensuring prevention of toxin production. At the retail end of the supply

chain the consumer is protected from C. botulinum by the requirement for chilled storage at

≤8° C and a shelf-life of ≤10 days (i.e. the “10 day rule”) (Peck et al., 2008).

The growth of the other three psychrotrophic pathogens mentioned above can be

controlled by adequate refrigeration. In general, the minimum temperature for growth of

these bacteria increases with lower meat pH and increasing levels of CO2 (Bell, 2001; Garcia

de Fernando et al., 1995). Further, competing bacteria may play an important role in

restricting the growth of pathogens. Kiermeier et al. (2013) observed a large increase in total

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viable count and lactic acid bacteria (particularly Carnobacterium spp.) on vacuum-packed

lamb shoulders stored at -0.3° C for 12 weeks that was considered to reflect selection for

protective microbial populations. Y. enterocolitica infection in humans is primarily associated

with consumption of pork, and whilst the prevalence of virulent strains varies among regions,

it is usually low or absent in cattle and sheep (Bailey et al., 2003; Schmid et al. 2013, Sierra

et al., 1995). Whilst Y. enterocolitica is capable of growth at -2 °C (EFSA, 2014), the

literature is inconsistent regarding the growth of virulent strains on meat under chilled

conditions. Some reports indicate that the ability of virulent Y. enterocolitica to compete with

other psychrotrophic bacteria is poor (Fukushima, & Gomyoda, 1986; Kleinlein, &

Untermann, 1990), whilst others suggest these strains can multiply under these conditions and

compete successfully (Bredholt et al., 1999; Gill, & Reichel, 1989). Listeriosis is usually

associated with ready-to-eat rather than fresh meats (EFSA 2014), and no L. monocytogenes

was recovered from Australian cattle, sheep or sheep meat (Bailey et al., 2003; Phillips et al.,

2013). L. monocytogenes has been shown to grow at -1°C, but not -2°C (EFSA, 2014; Gill, &

Reichel, 1989), and several studies have reported a reduction on beef and pork carcasses

during chilled storage (Moorhead, & Dykes, 2004; Prendergast et al., 2007). The role of

Aeromonas spp. as human pathogens remains contestable as definitive proof of the

organism’s pathogenicity is still absent, although several putative mechanisms of infection

have been proposed (Janda, & Abbott, 2010). Furthermore, the taxonomic boundary among

species typically related to human infection (A. hydrophila, A. caviae and A. veronii biovar

sobria) and others is difficult to define (Martino et al., 2014). Aeromonas spp. are members

of the normal faecal flora of food animals. These organisms have been isolated from sheep

and beef carcasses at prevalence of 71% and 35%, respectively, and have been reported at

numbers varying from <102 to >10

4 cfu g

-1 in various raw chicken, beef and pork samples

(Hudson 2004). Growth has been reported at -1.5°C in vacuum packed roast beef, and at -2°C

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in vacuum-packed high pH beef, with similar lag times to competing microbiota (Gill, &

Reichel, 1989; Hudson et al., 1994). Doherty et al. (1996) showed that numbers of

Aeromonas hydrophila decreased on low pH (5.5 – 5.8) lamb, but increased on high pH

(>6.0) lamb, at 0 and 5 °C under vacuum-pack.

As the focus of this review is on microorganisms that prevent or cause spoilage,

pathogens are not discussed further.

3.2 Spoilage bacteria

The initial microbiological objective of vacuum packaging was inhibition of the strictly

aerobic rapidly-growing pseudomonads, which are the principal cause of spoilage in fresh

and shrink wrapped meats (Gill, 2004). Consequently, the microbiota of vacuum-packed

chill-stored lamb is determined by conditions in the vacuum pack including temperature,

relative humidity and the partial pressure of O2 and CO2. It comprises anaerobic and

facultatively anaerobic bacteria, usually dominated by psychrotrophic LAB (Gill, & Newton,

1978). A summary of growth and spoilage characteristics of the six main groups of chilled-

meat spoilage bacteria is given in Table 1.

LAB are oxygen-tolerant anaerobes, which grow readily in the absence of O2 and are

not inhibited by CO2. There are a large number of species, many of which have undergone

reclassification and name changes (Stiles, & Holzapfel, 1997); the most common isolates

from chilled meats are Lactobacillus, Leuconostoc and Carnobacterium spp. (Jones, 2004;

Jones et al., 2008). Based on studies with vacuum-packed beef, LAB can only ferment

glucose and a limited number of the other carbohydrates that are present in lesser amounts on

vacuum-packed meat muscle tissues. Growth ceases when the bacterial numbers at the meat

surface are such that organisms consume glucose more rapidly than it can diffuse from within

the tissue, this substrate limitation typically occurring when numbers reach about 108 cfu/cm²

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(Gill, 2004). LAB have been classified according to the end-products of glucose

fermentation; obligate homofermenters produce only lactic acid (resulting in the slightly

acidic taste of aged meat) but heterofermenters can produce a range of other end products.

Some heterofermentative species, e.g. Carnobacterium and Leuconostoc spp., produce

compounds associated with spoilage including ethanol, butyric acid and sulphides (Jones,

2004). For many species within the LAB group, fermentation products are relatively

innocuous and so spoilage does not occur until sometime after maximum numbers (i.e. 108

cfu/cm²) have been reached (Jones, 2004). After packaging, the population of LAB is

generally low, below the limit of detection (ca. 10 LAB/cm2), but it increases during storage

until growth stops upon substrate depletion. At -1.5°C, ascendant LAB populations have been

shown to be displaced by succeeding populations without a decline in observable LAB

numbers. Towards the end of shelf-life, these populations usually consist of Leuconostoc or

Carnobacterium spp. (Jones, 2004).

Lamb meat has a higher pH than that of beef, and most vacuum-packed lamb products

include both fat and muscle tissues. This means that the pH of many cuts will be ≥ 5.8,

conditions that may enable bacteria with a higher spoilage potential to grow. However, LAB

will still have a growth rate advantage at chilled storage temperatures, and may continue to

dominate the population if the initial numbers of other spoilage bacteria are low. The lower

the chill temperature, the slower the growth rate of spoilage bacteria, with the optimum

temperature for the storage of vacuum-packed meat for long periods being -1.5°C (Gill et al.,

1988a & b). The levels of muscle metabolites such as glucose, and the rate at which these

metabolites become available to microbes, are less well documented for lamb than beef,

although the overall glucose concentration at normal pH is known to be higher for lamb (Bell,

2001; Gill, & Newton, 1978). Consequently, the threshold that bacteria can reach before

substrates become limiting may be higher for lamb than beef.

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Some species of phsychrotrophic Enterobacteriaceae cause deterioration of vacuum-

packed meat, characterised by unpleasant odours and greening. These facultative anaerobes

also attack glucose preferentially, but when this substrate becomes limiting then utilise amino

acids, producing amines, dimethyl sulphide and mercaptans which indicate putrefaction, and

ammonia, which raises the pH and can cause a pink-red colouration (Bell, 2001). Greening is

caused by the formation of hydrogen sulphide, with subsequent formation of green

sulphmyoglobin anaerobically; this oxidises to the red pigment metsulphmyoglobin on

exposure to air (Fox, 1966; Mills, 2012b). Although these bacteria are more likely to cause

spoilage under temperature abuse conditions (>5°C), there are a few species that can grow at

chill temperatures in vacuum-packed meat, including Hafnia alvei, Serratia spp., some

Enterobacter spp., and Rahnella aquatilis (Borch et al., 1996; Pennacchia et al., 2011).

Spoilage may be due to more than one organism. For example, spoilage of lamb primals

stored in vacuum-packs at 0°C was attributed to growth of both B. thermosphacta and

Enterobacteriaceae (Sheridan et al., 1997).

Growth of Brochothrix thermosphacta (producing cheesy or dairy odours), Shewanella

putrefaciens (producing hydrogen sulphide and greening) and psychrotrophic Enterobacteria

(producing sulphurous odours) can occur under various conditions. For vacuum-packed meat

these conditions include one or more of the following; a relatively high pH (greater than 6.0),

storage temperature of 5°C to 10°C, or packaging conditions resulting in the presence of

residual oxygen (e.g. presence of large amounts of surface adipose tissue with only limited

oxygen-scavenging potential (Gill, 2004)). Recently we have demonstrated that B.

thermosphacta, B. campestris, Serratia proteamaculans and Rahnella aquatilis were able to

grow on vacuum-packed lamb at pH values between 5.5 and 6.4 when stored chilled (-1.5, 0,

+2 and +7°C) and examined every 2-3 weeks for up to 84 days (Gribble, & Brightwell, 2013;

Gribble et al., 2014). B. thermosphacta and S. proteamaculans caused spoilage under these

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conditions whilst R. aquatilis only spoiled high pH (>6.0) meat at 7°C. These results differ

from previous work with beef, which indicates that B. thermosphacta and Enterobacteriacae

species cannot grow on or cause spoilage of low pH meat in the absence of oxygen (Bell,

2001). In the case of Brochothrix, it has been shown that the amount of undissociated lactic

acid is the effective inhibitor, rather than the pH itself, with around 50% and 90% reductions

in growth rate reported in the presence of 0.5mM and 2.0mM undissociated acid, respectively

(Campbell et al., 1979; Grau, 1980; Grau, 1981), although how this might influence the

different results seen on beef and lamb has yet to be determined.

Shewanella putrefaciens has been reported to cause extensive spoilage of chilled beef

(Gill, & Newton, 1979), but to our knowledge has not been reported as a cause of spoilage of

chilled lamb in any of the culture or culture-independent studies reported in the last 10 years

(Kiermeier et al., 2013; Pennacchia et al., 2011). The reason for this remains unclear,

although we have demonstrated that Shewanella putrifaciens strain CDC B5944 (NZRM825)

failed to grow on lamb shank meat (pH >6-2) at -1.5°C after 12 weeks (unpublished data).

Blown pack spoilage of vacuum-packed chilled lamb can occur even if the temperature

has been strictly maintained at the target temperature of -1.5°C. The use of molecular

methods has enabled an association between Enterobacteriaceae and blown pack spoilage to

be identified (Brightwell et al., 2007) but the bacteria generally responsible are gas-producing

cold-tolerant Clostridium spp., usually C. estertheticum or C. gasigenes (Broda et al., 1996 &

2009). Yang et al. (2009) demonstrated that growth of C. estertheticum on vacuum-packed

beef was limited by the availability of glucose (as for LAB and Enterobacteriaceae), but

fermentation of lactate continued, and it is this fermentation that liberates CO2, resulting in

blowing of the pack. Other cold-tolerant clostridia have be associated with other (off-odours,

but not blown) forms of lamb spoilage (Cavill et al., 2011) including C. putrefaciens and

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other species not previously associated with spoiled meat, although this has yet to be

confirmed by re-inoculation onto meat. It was also shown that more than one species may be

present in a single pack. Although in this study C. estertheticum remained the most common

species found on lamb. Using molecular methods, Broda et al. (2009) ascertained that spores

of cold-tolerant clostridia could be detected on animal fleeces and the slaughter room floor,

and that these were also detectable on chilled dressed carcasses.

The time of onset of blown pack was investigated in laboratory-based studies of vacuum-

packed lamb legs and beef silverside inoculated with different levels of C. estertheticum and

C. gasigenes strains at -1.5, 1 and 4 °C. In this study, no significant difference was reported

in the time to onset of gas production between lamb and beef for the strains tested. Only C.

estertheticum strains were found to be capable of blowing packs at -1.5°C, whilst C.

gasigenes caused slower onset of spoilage at 1 and 4 °C. Whilst spoilage was demonstrated

even with low levels of inoculated spores of C. estertheticum (≤10 cfu/cm²), meat inoculated

with 102-10

3 cfu/cm² and stored at -1.5°C blew within 40 days (Moschonas et al., 2010).

Consequently, to store lamb for longer periods at -1.5°C, considerable attention must be paid

to minimise contamination, for example by applying sporicidal agents to contact surfaces

during cleaning of equipment and contact surfaces.

Some species of pyschrotolerant clostridia, for example C. algidicarnis can cause

surface spoilage of chilled vacuum-packed lamb, as well as beef and venison (Adam et al.,

2010). Whilst little or no gas accumulated, on opening the pack sickly spoilage odours were

detected and clostridia were isolated from both the drip and surface swabs.

Vacuum-packed meat can briefly be exposed to high temperatures if a heat-shrink

process is applied. Bell et al. (2001) found that the heat shrink treatment accelerated the onset

of blown pack spoilage due to C. estertheticum, presumably due to stimulation of spore-

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germination. More recently, Adam et al. (2011) determined that spore germination in C.

frigidicarnis using artificial media was triggered by anaerobiosis in the presence of L-valine

(or L -norvaline) and L -lactate, conditions that are all present in vacuum-packed red meat.

Spores exposed to L -valine and L -lactate germinated in the absence of heat, although spores

that were heat treated and exposed to L -valine and L -lactate germinated in greater numbers

than spores exposed to L -valine and L-lactate alone.

Methods to reduce spoilage of lamb by psychrotrophic clostridia were investigated by

Adam et al. (2013). The exposure of vegetative cells to oxygen had little effect on C.

estertheticum, ruling this out as a practical method for reducing spoilage. Hot and cold water

washing of lamb inoculated with spores of C. estertheticum, was however shown to extend

the shelf-life of vacuum packs by 12 to 13 days at -1.5°C.

4. Microbiology of stored vacuum-packed lamb that is repacked in Modified Atmosphere

Packaging.

Following chilled storage and transportation, chilled vacuum-packed lamb packs are often

opened in a central facility and butchered into retail cuts for distribution in retail-ready packs

in modified atmosphere packaging (MAP). This type of packaging is superior to cling film

overwrap, as the assured product life of stored meat is 2-6 days, compared to only 1-3 days in

overwrap (Bell, 2001). MAP involves putting meat in a barrier package, evacuating the air

and filling the pack with a specific gas mixture, after which the pack is sealed with barrier

film. The packaging system consists of a material that is an effective barrier to transmission

of gases and water vapour (Kropf, 2004b). High-oxygen MAP (Hi-Ox MAP) is a widely used

system with gas mixes containing up to 80% oxygen, carbon dioxide in the range of 15-30%

and optionally up to 20% nitrogen (Kropf, 2004b). The CO2 concentration of Hi-Ox MAP is

high enough to inhibit the growth of pseudomonads, but does not affect the growth of LAB.

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Under Hi-Ox MAP B. thermosphacta, which is only moderately affected by CO2, can cause

early spoilage and although initially suppressed by CO2, Enterobacteriaceae will begin to

cause spoilage as their numbers increase and the rate of glucose diffusion from the underlying

tissues becomes inadequate to meet the bacterial demand and amino acids are utilised (Gill,

1986). In MAP for retail display (20-30% CO2 and 70-80% O2), chosen to enhance red colour

in muscle myoglobin and inhibit pseudomonads, LAB predominate and in particular, the

heterofermentative species Leuconostoc gasicomitatum have been shown to grow well in this

environment, producing acidic and buttery odours, and greening of the meat (Johansson et al.,

2011).

Temperature is a very important factor in determining species dominance in meat stored

under MAP. For example, on beef, an increase in diversity among the Enterobacteriaceae

was reported at abuse temperatures where S. liquefaciens can dominate but at low

temperatures (≤5°C) psychrotrophic Hafnia alvei is often the dominant species (Doulgeraki et

al., 2011). High pH has also been shown to be related to an increased risk of spoilage for

lamb that is stored at 0 or 5°C in a MAP atmosphere.

Spoilage after re-packaging in aerobic environments can also be due to microorganisms

that can cause “greening”. This is usually attributed to high numbers of LAB that produce

hydrogen peroxide (Borch et al., 1996), resulting in the oxidation of myoglobin to

cholemyoglobin. When present, greening is typically observed within a short time in chilled

lamb that has endured long storage in vacuum-packs and is then repacked in MAP.

5. Evaluating the microbiological status of vacuum-packed lamb

The hygienic status of meat in general is controlled during slaughter, dressing and

butchery by good manufacturing practice (GMP), achieved through adherence to appropriate

Hazard Analysis at Critical Control Point (HACCP) plans, following analysis of the risks

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posed by particular microbes under known manufacturing situations (Hathaway, & Cook,

1997). This may be confirmed during the fabrication process by evaluation of Total Viable

Count (TVC) to determine hygienic status, and an enteric indicator (e.g. Enterobacteriaceae)

to assess sanitary condition.

These indicators cease to be effective measures of sanitary condition after meat is

subjected to chilled, vacuum pack storage, due to the growth of non-pathogenic

psychrotrophic bacteria. Under these conditions, Escherichia coli has been proposed to be the

most useful indicator of unsatisfactory sanitary conditions due to its ability to identify faecal

contamination (McEvoy et al., 2004; Struijk, & Mossel, 2005). If GMP validation prior to

packaging has shown that hygiene standards have been met then the microbiological criteria

applied to vacuum-packed stored meats should be the same as for fresh meats based on E. coli

as the indicator. Generic indicators (e.g. TVC) also have limited value as they cannot predict

the presence of all the causative organisms, particularly spoilage bacteria, such as Brochothrix

and blown pack Clostridium, which can be difficult to detect (either by being outgrown by

LAB, or their anaerobic nature) and which must be considered separately.

The use of rapid molecular methods for specific spoilage organisms has become a cost-

effective alternative to culture based methods. Direct Real-time TaqMan PCR assays (i.e.

assays to detect bacteria without enrichment by concentrating and identifying targeted DNA

sequences) for the detection of B. thermosphacta and C. estertheticum have been developed

and validated (Brightwell, & Clemens 2012; Gribble et al., 2013). The minimum level of

detection of the Brochothrix assay was determined to be 7 cfu per PCR reaction. For C.

estertheticum, the minimum level of detection on meat, hide, blood/drip and environmental

swabs was approximately 3 spores/ml by direct PCR (without pre-enrichment of the

samples). These rapid molecular tests are proving to be useful tools for evaluating process

hygiene and effectiveness of automated clean-in-place (CIP) systems.

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Approaches based on the analysis of specific compounds produced during bacterial

growth are also being developed to aid in the detection and identification of spoilage,

although reports to date refer to testing carried out on beef. Argyri et al. (2011) reported that

analysis of organic acid profiles in vacuum- or modified atmosphere-packed beef (using high

performance liquid chromatography; HPLC) has potential as a rapid and sensitive method to

indicate spoilage. Ercolini et al. (2009) reported that some bacterial species produce specific

metabolic products that can be identified by gas chromatography/mass spectrophotometery

(GC/MS) and used this technique to identify the relationship between specific bacteria and

GC/MS profiles in vacuum-packed beef. Hernandez-Macedo et al. (2011, & 2012) used

GC/MS to analyse head space samples from vacuum-packed beef that had suffered blown

pack spoilage. These workers reported good correlation between volatile organic compounds

(VOCs) and gaseous compounds with the spoilage microorganisms that had been identified

by 16S rRNA clone sequencing.

Confinement odour is defined as a slight sour-acid, cheesy or milky odour that dissipates

within a few minutes of the pack being opened. It has been attributed to the growth of LAB in

the pack, and depends upon the species of LAB that predominate in the particular pack. This

has sometimes been misinterpreted as spoilage, but is actually considered an indicator that all

is well (Johnson, 1991). Jones (2004) noted that acetic acid was associated with increasing

LAB populations on beef, whilst butyric acid was produced by Leuconostoc, and sulphide

production with some strains of Lactobacillus sakei (Borch et al., 1996; Egan et al., 1989).

However, the threshold associated with any particular LAB species has yet to be established.

No data are currently available on the linkage between bacterial enumeration and sensory

outcome (Kiermeier et al., 2013), and for the more common causes of spoilage (LAB and

Enterobacteriaceae), these bacteria can have attained the maximum possible numbers on the

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meat surface several weeks before onset of spoilage (Gill, 2004). In addition, not all species

that have been isolated from vacuum-packed chilled meat have the same spoilage potential

(Bell, 2001; Gill, & Newton, 1979). Consequently, it is generally accepted that routine

microbiological testing of chilled fresh meats is unnecessary (ICMSF, 2011).

6. Controlling microbial contamination of vacuum-packed lamb

The shelf-life of chilled vacuum-packed lamb is dependent on many factors. These

include the amount and type of contaminating microbiota that is present on the meat surface

at packing, the pH of the individual meat cuts, the oxygen transmission rates of the packaging

films, and the temperatures applied during the chilling of the carcasses before de-boning, and

after vacuum-packing and stacking in cartons. Thereafter, the cold-chain of transportation to

market and storage prior to retailing also has a significant impact on the product-life. The use

of additives is normally not permitted when meat is to be labelled and sold as a fresh product.

Therefore, the only technologies currently available to preserve meat during chilled storage

involve the use of preservative packaging and refrigeration to as low a temperature as

possible without the meat actually freezing. It has been shown that optimal results with these

technologies are achieved when packing whole primal rather than consumer cuts (Gill, 1989).

The following factors have been shown to have an impact on the bacteriological condition

of chilled lamb:

6.1 Slaughter and dressing practises.

Washing of lambs, using swim or spray wash systems, is a controversial practice, its

stated purpose being to reduce the levels of visible contamination requiring trimming on

inspected carcasses. It is now being phased out in New Zealand due to issues with both

hygiene and animal welfare. Le Roux (2000) observed that swim washing achieved little or

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no immediate reductions in bacterial numbers on the fleece and was followed by significant

post-wash microbial growth on the fleece (up to 3 Log10 cfu/cm2 in 20 hours), predominated

by Enterobacteriaceae, that had a high potential for transfer onto the carcass during dressing.

It has been shown that microbial levels on the carcasses from clean, shorn animals are

significantly lower than from animals with a full fleece, providing that shearing was

performed on-farm and not immediately pre-slaughter (Biss, & Hathaway, 1995).

The use of inverted dressing (that is, dressing with all four legs shackled and ventrum

uppermost, with an opening Y-cut performed across the forelegs and neck) coupled with

stringent application of hygienic practices, has been shown to offer significant improvements

over traditional dressing when processing lamb carcasses (Bell, & Hathaway, 1996). In this

study, 12/15 sample sites on lamb carcasses dressed inverted showed reduced counts of E.

coli compared to traditional dressing methods. When inverted dressing is used, with the

exception of the peri-anal region the aerobic plate counts and E. coli numbers at different

positions along the dressing line indicated that little carcass contamination occurred after pelt

removal was complete.

6.2 Cross-contamination

During meat production, there are processes where contact between the meat and

environmental surfaces, workers’ hands and equipment is unavoidable (Gill, & McGinnis,

2000 & 2003). Prevention of cross-contamination by workers is largely achieved through

education and good hygienic discipline. Contamination from environmental surfaces is of

particular concern during the cutting of carcasses into primals for packing, as this operation

occurs at chilled temperatures which encourage the growth of psychrotrophic bacteria,

including those with high spoilage potential. It has been shown that routine cleaning may not

completely remove accumulated detritus from these areas, and that visibly clean surfaces may

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still contain films of protein and fat (Brightwell et al., 2006). Further, application of water in

further processing is undesirable as it can enhance bacterial growth on contact surfaces and

contribute to cross-contamination, if the surfaces are not adequately cleaned at end-of-day.

6.3 Influence of water activity (aw)

During pre-bone chilling, the aw on the carcass surface (where contaminating bacteria are

located) has been shown to be equivalent to the relative humidity in the surrounding

environment; i.e. a carcass held in a chiller at 90% RH would undergo surface desiccation

and in time resulting in a surface aw of 0.90 would be achieved (Lovatt, & Hill, 1998). The

situation is dynamic, and the timeframes concerned vary considerably depending on the size

and loading of the chiller. Humidity readings of 80-90 %RH typically recorded in these

chillers if spray-chilling is not used (Mills, unpublished data). Moisture loss from lamb

carcasses under these conditions has been reported to be around 2.2%, which was reduced to

0.86% using spray chilling (Brown et al., 1993).

Species of Enterobacteriaceae are inhibited when aw falls below ca. 0.94, whilst L.

monocytogenes is inhibited below 0.92 and toxin production is inhibited in Staphylococcus

aureus below 0.87 (ICMSF, 1996). A study was undertaken to determine the effect of

chilling carcasses artificially contaminated with E. coli O157:H7 (ATCC 12900) at 90%

relative humidity (RH). The reduction in count was 2.17 log10 MPN/cm², a significant

reduction (P ≤0.01) (Mills, & Withers, 2010). When meat is vacuum-packed, further drying

at the surface is prevented, and moisture from within the meat allows the surface aw to

equilibrate to above 0.98. Consequently, there is no inhibitory effect on bacteria once the

meat has been packed and stored (Bell, 2001).

6.4 Meat pH

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In beef, the pH of the meat has been shown to affect the growth of bacteria in two ways

(Gill, 2004). Firstly, the growth of some bacteria is reduced, or inhibited completely, when

the pH falls below a certain level. In addition, the glycolytic processes that determines the

ultimate post-rigor pH also determines the concentration of residual glucose in the meat and,

therefore, the point at which this preferred growth substrate becomes exhausted and amino-

acids start to be metabolised by LAB and Enterobacteriaceae, thereby resulting in spoilage.

It is generally believed that Enterobactericeae and B. thermosphacta are inhibited by pH

values of < 5.8, and Shewanella putrefaciens by values of < 6.0, whilst LAB are not affected

by meat pH (Gill, 1986; Bell, 2001; Gill, 2004). Beef has a normal pH post-rigor (i.e.

ultimate pH, pHu, when all available glycogen has been utilised) of around 5.5, whilst high

pH (referred to as dark, firm and dry; DFD) beef is considered to have a pHu of > 6.2

(Lawrie, 1998).

In lamb, the large muscle meats (e.g. rump, backstrap) have a mean pHu of 5.6-5.7, whilst

the smaller muscles of the shoulder and shank have a mean pHu of >6.0. The spoilage

potential of B. thermosphacta, Serratia proteamaculans and Rahnella aquatilis has recently

been investigated in vacuum-packed lamb shank (pH 5.9-6.4) and loin (pH 5.4-5.8) (Gribble

et al., 2014). 100 ±10 cfu of each bacterial species (strains previously isolated from vacuum-

packed meat) were inoculated individually onto separate 50 ±5 cm2 meat coupons and packed

in barrier bags of oxygen permeability 20cc/m2/24h at 23 °C. Five replicates from each group

were examined at 3-weekly intervals. All three bacterial species grew on vacuum-packed

lamb at both low and high pH values when stored chilled (-1.5, 0, 2 and 7°C) for up to 84

days. Growth rates were reduced, however, on pH 5-4-5.8 lamb when stored below 0°C.

Consequently, shoulder and shank meats may have a shorter shelf-life than leg and loin meats

if contaminated with these bacteria and stored under chilled conditions.

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The pHu of chilled meats may be influenced at meat premises through attention to stock

selection, transportation to the meat premises, in-plant animal handling and carcass selection.

The pH of individual meat cuts will influence the growth of spoilage bacteria, which should

be taken into account when estimating shelf-life (Lawrie, 1998).

6.5. Temperature

The optimum storage temperature for packaged meat is considered to be -1.5°C (Gill, et

al., 1988a). Although not completely inhibited, growth of both S. proteamaculans and B.

thermosphacta is significantly suppressed by storage at sub-zero temperatures (Gribble et al.,

2014). Clemens et al. (2010) reported a similar finding with Clostridium estertheticum on

beef and lamb. Sumner, & Jenson (2011) discussed how small increases in temperature from

-1.5°C can have significant effects on storage life; thus at temperatures of 0°, 2° and 5 °C, the

storage life could be reduced by approximately 30, 50 and 70% respectively. Figure 1

illustrates the effect of storage temperature on time to spoilage as a consequence of the

anaerobic growth on high pH lamb of a strain of psychrotrophic Serratia liquefaciens. The

more conservative model of Bell (2001) where shelf-life decreases by 10% for every 1 °C

increase above -1.5 in the -1.5 to 5 °C range, predicts a loss of shelf-life of 15, 35 and 65% at

these temperatures. It should be noted that temperature does not just affect the microbial

condition of the meat. Colour stability is also affected, with retail appearance significantly

affected by storage at 2 and 5 °C, compared to storage at below-zero temperatures (Jeremiah,

& Gibson, 2001).

These factors place considerable emphasis on ensuring that the temperature inside each

carton of meat in a refrigerated shipping container (reefer) remains relatively constant (-1.5 ±

0.5 °C) during transportation to an overseas market. The refrigeration systems inside these

containers are not designed to extract heat from the load, but rather to maintain it at a

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constant temperature. This means that it is essential that each carton of meat is equilibrated

and loaded into the container at the correct temperature before it is closed for shipment

(James, & James 2004b). Containers should be loaded to capacity using pallets and dunnage

such that there is unimpeded air-flow under and around the load, and sufficient gap between

the top of the load and the container ceiling (the ‘headspace’) to allow unimpeded flow of air

back to the refrigeration unit (Meat Industry Services, 2006). If these conditions are strictly

adhered to, the temperature difference between the delivery and return air to and from the

refrigeration unit has been recorded to be less than 0.8°C (James, & James, 2004b).

7. Summary of processing and supply chain issues

In the absence of animal disease, the majority of contaminating microorganisms on meat

derive from extrinsic sources that include the slaughtered animals themselves, meat plant

workers and the processing environment (Lawrie, 1998; Bell, 2001). In healthy animals

microbial presence in internal tissues such as muscle has been considered insignificant (Gill,

1979). There is, however, evidence for intrinsic sources of Clostridium algidicarnis, the

cause of stifle joint taint in lamb (Boerema et al., 2002). For lambs, the fleece is the most

significant source of contaminating microorganisms. Most are of faecal origin, with some

originating from the farm environment (Mills, 2012a). A significant relationship was found

between the level of dirt on fleeces and the level of total plate count bacteria,

Enterobacteriaceae and coliforms on separate sites (brisket, shoulder, flank and rump) after

pelt removal, irrespective of whether the fleece was wet or dry (Byrne et al., 2007). Although

some transfer of microorganisms is inevitable when the fleece is cut and removed,

contamination can be reduced if the number of opening cuts is minimised and carcasses

inverted before dressing (Bell, & Hathaway, 1996). During carcass dressing there are further

possibilities for contamination (Mills, 2012a), including accidental releases of faecal material

or intestinal contents that can occur if GMP is not strictly followed (Gill, 2005a & b). Cross-

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contamination from workers and the fabrication environment can also occur unless good

hygienic practice is maintained.

Vacuum-packaging and chill storage at -1.5°C allows transport of lamb to distant

markets. Meat processors adopt a range of protocols and adhere to regulations to ensure that

meat is safe for consumers, and the chance of spoilage is minimised. Thus, for lamb produced

under GMP, the initial count of microbes on the product surfaces is likely to be 103/cm

2 or

less, and organisms able to grow below 7°C fewer still. If this condition is met and packaging

material with low gas permeability and it is transported with very good temperature control (-

1.5 ± 0.5 °C) then lamb cuts should have a storage life of 10-12 weeks, as described by

James, & James (2002) and Kiermeier et al. (2013).

Acknowledgements

The authors would like to acknowledge Silver Fern Farms for its contribution to

industry context of this review paper; in particular, Grant Pearson, Neil Smith and Mark Bull.

We would also like to thank Dr Andrew Hudson (ESR Christchurch Science Centre),

Professor Mike Peck (Institute of Food Research, Norwich Research Park) and Dr Rex

Munday (AgResearch) for their editorial assistance during the preparation of this review

paper.

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Table 1. Conditions for growth and the spoilage characteristics of the six main groups of chilled-meat

spoilage bacteria on vacuum-packed lamb.

Microorganism1

Preferre

d

substrate

s for

anaerobi

c growth2

Oxygen

requirement

pH

requiremen

t

(anaerobic)

Reductio

n in

growth

during

storage

in

saturated

CO2

Spoilage

potential1

Common

Spoilage

Characteristic

s

Threshold

s of

spoilage

General

Remarks

Pseudomonas spp.1

glucose, aceti

c acid

pyruvate and

amin

o acids

Note: only

some

spp. can

grow

Aerobe High High Sulphurous off odours

107 /cm² at packing

Dominant in all

aerobic spoilage

flora

Shewanella putrefaciens

formate

amino acids

glucose

Aerobe

Utilises S2 as electron

receptor

anaerobically

No growth below pH

6.01

Moderate Very high

Sulphurous off odours and

greening under

vacuum-pack1

Major spoilage

organism of high-pH

beef

Brochothrix

thermosphacta

glucose Facultative

anaerobe

Growth

reduced below pH

5.8 at -2 to 0

°C2

Moderate High Some green

drip, meat discolouration

and pungent

dairy odours, poor bloom,

slight loss of

vacuum, and

bubbles2

~106/g2,5

Occasional

major spoilage

organism

on vacuum-

packaged

meat

Psychrotrophic

Enterobacteriaceae

glucose

glucose-6-P

amino acids

Facultative anaerobe

Growth reduced

below pH 5.8 at -2 to 0

°C2

Moderate High Some green drip, meat

discolouration and strong

sulphurous

odours, poor bloom, slight

loss of

vacuum,

bubbles2

Delayed, after

achieving ~108cfu/g

Specific species

only5

Major spoilage

organisms of

vacuum-

packaged high-pH

meat

LAB glucose

glucose-6-P

amino acids

Aerotolerant anaerobe

Low Low Off-flavours include,

cheesy, malty, acidic, or liver-

like and

production of slime.

Greening of

meat on exposure to air

Delayed, after

achieving

~108/

g

Specific

species

only5

Usually the

dominant organisms

of

vacuum-packaged

meat

“Blown Pack” Clostridium spp.

Glucose

Lactate4

Anaerobe but will survive

in cooler

aerobic

environment.4

Low High Softening of meat,

production of

large amounts

of exudates

and offensive

odours (dairy

or sulphurous)4

Initial loading of

1-10/g

spoilage

potential

dependent

on temperatur

e control 3

Sporadic major

spoilage

organism

in the

absence of

temperature abuse or

packaging

failure

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Data sourced from Bell, 2001 unless otherwise specified: 1 – Nychas et al., 2008; 2 – Gribble et al., 2013; 3 –

Clemens et al., 2010; 4 – Adam et al., 2010, 2013; 5 – Gill, 2004

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Figure 1 Effect of storage temperature on the time to spoilage as a consequence of the anaerobic

growth on high pH lamb of a strain of psychrotrophic Serratia liquifaciens.

1

2

3

4

5

6

7

8

9

0 10 20 30 40 50 60 70

bac

teri

a lo

g 10

/ cm

²

days

spoilage

-1.5°C2°C10°C20°C 0°C

Storage Temperature

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Highlights

1. Reviews existing knowledge of the spoilage bacteria affecting vacuum-packed lamb

2. Discusses impact of these bacteria on product quality, shelf-life and market access

3. Microbiology of chilled lamb and spoilage potential is different to that of chilled beef

4. Under specified conditions the shelf-life of chilled lamb can be extended to 70 days

and beyond