A review of modified atmosphere packaging of fish and fishery products - significance of microbial...

Review

A review of modified atmosphere packaging of fish

and fishery products – significance of microbial

growth, activities and safety

Morten Sivertsvik,1* Willy K. Jeksrud2 & J. Thomas Rosnes1

1 NORCONSERV, Institute of Fish Processing and Preservation Technology, Stavanger, Norway

2 Agricultural University of Norway, Department of Agricultural Engineering, As, Norway

(Received 8 January 2000; Accepted in revised form 9 September 2001)

Summary Modified atmosphere packaging (MAP) extends shelf-life of most fishery products by

inhibiting bacterial growth and oxidative reactions. The achievable extension of shelf-life

depends on species, fat content, initial microbial population, gas mixture, the ratio of gas

volume to product volume, and most importantly, storage temperature. The shelf-life of

fishery products is usually limited by microbial activity, although for some fatty fishes or at

superchilled storage, it can be limited by nonmicrobial activity. Packaging of fishery

products under modified atmospheres (MA) increases shelf-life compared with those

packaged under air, but confers little or no additional shelf-life increase compared with

vacuum packaging. The specific spoilage organism (SSO) of MA packaged cod at 0 °C has

been found to be Photobacterium phosphoreum. Whether or not this bacterium is the

general SSO for all marine temperate fishes at different storage temperatures and under

various CO2/N2/O2 mixtures needs to be resolved. Without proper control of storage

temperature, the benefits of MAP may be lost. Higher temperatures inevitably lead to less

dissolved CO2 in the product and consequently loss of inhibitory effect, which may result in

higher microbial and enzymatic activity, and uncertainties concerning the microbial safety,

as food-borne pathogens might be present in the product.

Keywords Bacteria, carbon dioxide, gas packaging, pathogenic bacteria, specific spoilage.

Introduction

Modified atmosphere (MA) packaged foods have

become increasingly more available, as food

manufacturers have attempted to meet consumer

demands for fresh, refrigerated foods with exten-

ded shelf-life. The use of an MA with an

enhanced carbon dioxide level has been shown

to extend the shelf-life of foods by retarding

microbial growth (Stiles, 1991). Fish and shellfish

are highly perishable and their deterioration is

primarily because of bacterial action (Colby

et al., 1993). Typical shelf-life under current icing

and refrigerated storage conditions ranges from 2

to 14 days (Stammen et al., 1990). Modified

atmosphere packaging (MAP) of fishery products

has been shown to inhibit the normal spoilage

flora and increase shelf-life significantly. How-

ever, the possibility that Clostridium botulinum

type E and non-proteolytic type B strains will

grow and produce toxin in low-oxygen atmos-

pheres at refrigerated temperatures has caused

great concern in studies on MAP of seafood

(Church, 1994). This review examines the effect

of the MAP technology used for fresh fishery

products on the spoilage microbiological flora

and on the food-borne pathogens that may be

present in these products.*Correspondent: Fax: +47 51 84 46 50;

e-mail: [email protected]

International Journal of Food Science and Technology 2002, 37, 107–127 107

Ó 2002 Blackwell Science Ltd

Microbial spoilage of fresh fish in air

Food spoilage can be considered as any change that

renders the product unacceptable for human con-

sumption (Huis in’t Veld, 1996). Spoilage of fish

and shellfish results from changes caused by

oxidation of lipids, reactions caused by activities

of the fish’s own enzymes, and the metabolic

activities of micro-organisms (Ashie et al., 1996).

Fish and shellfish are highly perishable, because of

their high aW, neutral pH, and presence of autolytic

enzymes. The rate of deterioration is highly tem-

perature dependent and can be inhibited by the use

of low storage temperature (e.g. fish stored on ice).

The spoilage of fresh fish is usually microbial.

However, in some cases chemical changes, such as

autooxidation or enzymatic hydrolysis of the lipid

fraction may result in off-odours and -flavours and,

in other cases, tissue enzyme activity can lead to

unacceptable softening of the fish (Huss et al.,

1997). The degree of processing and preservation,

together with storage temperature, will decide

whether the fish undergoes microbial spoilage,

biochemical spoilage or a combination of both.

Several investigators have concluded that the

micro-organisms associated with most fishery

products reflect the microbial population in their

aquatic environment (Liston, 1980; Colby et al.,

1993; Ashie et al., 1996; Gram & Huss, 1996). The

microflora of fish from temperate waters is dom-

inated by psychrotrophic, aerobic or facultative

anaerobic Gram-negative, rod-shaped bacteria,

and in particular by Pseudomonas, Moraxella,

Acinetobacter, Shewanella putrefaciens, Flavobac-

terium, Cytophaga, Vibrio, Photobacterium and

Aeromonas (Stammen et al., 1990; Gram & Huss,

1996; Huis in’t Veld, 1996). Vibrio, Photobacterium

and S. putrefaciens require sodium for growth and

are typical of marine waters, whereas Aeromonas

spp. are typical of freshwater fish. However,

S. putrefaciens has been isolated from freshwater

environments (Huss, 1995). Varying proportions

of Gram-positive organisms (Bacillus, Micrococ-

cus, Clostridium, Lactobacillus, Cornynebacterium

and Brochothrix thermosphacta) have been isola-

ted from seafood (Stammen et al., 1990; Gram &

Huss, 1996; Huis in’t Veld, 1996). The flora on

tropical fish often carries a slightly higher load of

Gram-positive bacteria compared with fish from

colder waters (Liston, 1980).

Micro-organisms are found on all the outer

surfaces (skin and gills) and in the intestines of live

and newly caught fish. The total numbers of

organisms vary enormously and Liston (1980)

states a normal range of 102–107 cfu (colony

forming units) cm)2 on the skin surface. The gills

and the intestines both contain between 103 and

109 cfu g)1 (Huss, 1995). The fish muscle is sterile

at the time of slaughtering/catch, but becomes

quickly contaminated by surface and intestinal

bacteria, and from equipment and humans during

handling and processing. During chilled storage,

there is a shift in bacterial types. Psychrotrophic

Pseudomonas and Shewanella dominate the micro-

flora after 1–2 weeks of storage. At higher tem-

peratures (25 °C), the microflora at the point of

spoilage is dominated by mesophilic Vibrionaceae

and, particularly if the fish are caught in polluted

waters, Enterobacteriaceae (Huss, 1995). The part

of the microflora which will ultimately grow on the

products is determined by the intrinsic [for exam-

ple, postmortem pH in the flesh, the poikilother-

mic nature of fish, and presence of trimethylamine

oxide (TMAO) and other non-protein–nitrogen

(NPN) components] and extrinsic parameters (for

example, temperature, processing and packaging

atmosphere) (Huss et al., 1997).

The sequence of spoilage of fresh fish can be

considered in seven steps (Liston, 1980):

1. Spoilage bacteria naturally present on fish;

2. Amino acids and other NPN substrates present;

3. Selective growth of organisms (mostly Pseudo-

monas) that actively oxidatively deaminate

amino acids;

4. Repression of proteinase production

de-repressed by selective use of amino acids

by Pseudomonas bacteria;

5. Amino acid recruitment to substrate pool by

bacterial hydrolysis of protein;

6. Ammonia and volatile fatty acid production

sharply increases because of the above men-

tioned point (step 5);

7. Specific spoilage organisms (SSO) producing

sulphur-containing and other odorous com-

pounds.

The composition of fresh fish flesh makes it

favourable tomicrobial growth (Colby et al., 1993).

TMAO in fish muscle can be degraded to trimeth-

ylamine (TMA) by endogenous enzymes, but at

chill storage temperatures TMA is produced by the

Modified atmosphere packaging of fish M. Sivertsvik et al.108

International Journal of Food Science and Technology 2002, 37, 107–127 Ó 2002 Blackwell Science Ltd

bacterial enzyme TMAoxidase (Ashie et al., 1996).

TMA is recognized as the characteristic ‘fishy’

odour of spoiled fish. When the oxygen level is

depleted, many of the spoilage bacteria utilize

TMAO as a terminal hydrogen acceptor, thus

allowing them to grow under anoxic conditions.

Towards the end of shelf-life various odorous low

molecular weight sulphur compounds such as H2S

and CH3SH, together with volatile fatty acids and

ammonia are producedbecause of bacterial growth.

Principle of MAP

The principle of MAP is the replacement of air in

the package with a different fixed gas mixture.

Once the gas mixture is introduced, no further

control of the gas composition is performed, and

the composition will inevitably change. CO2 is the

most important gas used in MAP of fish, because

of its bacteriostatic and fungistatic properties. It

inhibits growth of many spoilage bacteria and the

inhibition is increased with increased CO2-con-

centration in the atmosphere. The use of CO2 to

inhibit bacterial growth is not a new technology.

In 1877 Pasteur and Joubert observed that Bacillus

anthracis could be killed by CO2 (Valley, 1928)

and 5 years later the first article on the preserva-

tive effect of carbon dioxide on food was published

(Kolbe, 1882), showing extended storage life for

ox meat placed inside a cylinder filled with a

carbon dioxide atmosphere.

CO2 is highly soluble in water and fat, and the

solubility increases greatly with decreased tempera-

ture. The solubility in water at 0 °C and 1 atmo-

sphere is 3.38 g CO2/kgH2O, however, at 20 °C the

solubility is reduced to 1.73 g CO2/kg H2O

(Knoche, 1980). Therefore, the effectiveness of the

gas is always conditioned by the storage tempera-

ture with increased inhibition of bacterial growth as

temperature is decreased (Haines, 1933; Gill & Tan,

1980; Ogrydziak & Brown, 1982). The solubility of

CO2 leads to dissolved CO2 in the food product

(Knoche, 1980), according to equation (1):

CO2ðgÞþH2Oð1Þ$HCOÿ3 þHþ$CO2ÿ3 þ2Hþ

ð1Þ

(or for pH values <8, typical of seafood, the

concentration of carbonate ions may be neglected;

Dixon & Kell, 1989) (2):

CO2 þH2O$ H2CO3 $ HCOÿ3 þHþ ð2Þ

The concentration of CO2 in the food is dependent

on the product’s water and fat content, and of the

partial pressure of CO2 in the atmosphere,

according to Henry’s law (Ho et al., 1987). Dev-

lieghere et al. (1998a, 1998b) have demonstrated

that the growth inhibition of micro-organisms in

MA is determined by the concentration of dis-

solved CO2 in the product. After the packaging

has been opened, the CO2 is slowly released by the

product and continues to exert a useful preserva-

tive effect for a certain period of time, referred to

as CO2’s residual effect (Stammen et al., 1990).

The action of CO2 on the preservation of foods

was originally thought to be caused by displace-

ment of some or all of the O2 available for

bacterial metabolism, thus slowing growth (Dan-

iels et al., 1985). However, experiments with

storage of bacon and pork showed a considerable

increase in shelf-life under 100% CO2 compared

with storage in normal air atmospheres (Callow,

1932), but the preservative effect was not because

of the exclusion of O2, as storage in 100% nitrogen

(N2) offered no advantage over normal air storage.

The same results were also seen on pure cultures of

micro-organisms isolated from spoiled pork.

A drop in surface pH is observed in MA

products because of the acidic effect of dissolved

CO2, but this could not entirely explain all of

CO2’s bacteriostatic effect (Coyne, 1933). It was

shown that CO2 was more effective at lower

temperatures and that the change in pH caused

by the CO2 did not account for the retardation of

growth. In a study on several pure cultures of

bacteria isolated from fish products, CO2 atmos-

pheres were found to inhibit the growth of

bacteria markedly, whereas normal growth pat-

terns were observed under air or N2 atmospheres

(Coyne, 1932). It was also observed that bacterial

growth was inhibited even after the cultures were

removed from the CO2 atmosphere and trans-

ferred to an air environment, interpreted as a

residual effect of CO2 treatment. Bacterial growth

was distinctly inhibited under 25% CO2 and

almost no growth was observed under higher

CO2 concentrations for 4 days at 15 °C. The

results obtained could neither be explained by

the lack of O2 nor the pH effect. Coyne suggested

the possibility that intracellular accumulation of

Modified atmosphere packaging of fish M. Sivertsvik et al. 109

Ó 2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 107–127

CO2 would upset the normal physiological equi-

librium in other ways, i.e. by slowing down

enzymatic processes that normally result in pro-

duction of CO2. Thus, the effect of CO2 on

bacterial growth is complex and four activity

mechanisms of CO2 on micro-organisms has been

identified (Parkin & Brown, 1982; Daniels et al.,

1985; Dixon & Kell, 1989; Farber, 1991):

1. Alteration of cell membrane function including

effects on nutrient uptake and absorption;

2. Direct inhibition of enzymes or decreases in the

rate of enzyme reactions;

3. Penetration of bacterial membranes, leading to

intracellular pH changes;

4. Direct changes in the physico-chemical prop-

erties of proteins.

Probably a combination of all these activities

account for the bacteriostatic effect.

A certain amount (depending on the foodstuff)

of CO2 has to dissolve into the product to inhibit

bacterial growth (Gill & Penney, 1988). The ratio

between the volume of gas and volume of food

product (G/P ratio) should usually be 2 : 1 or 3 : 1

(volume of gas two or three times the volume of

food). This high G/P ratio is also necessary to

prevent package collapse because of the CO2

solubility in wet foods. Dissolved CO2 takes up

much less volume compared with CO2 gas, and

after packaging a product in CO2 atmosphere,

under-pressure is developed within the package

and package collapse may occur. The CO2 solu-

bility could also alter the food–water holding

capacity and thus increase drip (Davis, 1998).

Exudate from MAP fish can be reduced signifi-

cantly by dipping fillets in NaCl solution prior to

packaging (Bjerkeng et al., 1995; Pastoriza et al.,

1998). In fishery products eaten without prior

heating, such as crab and cooked fish, an acidic,

sherbet-like flavour can be observed when high

partial pressures of CO2 is used.

Nitrogen (N2) is an inert and tasteless gas, and

is mostly used as a filler gas in MAP, because of its

low solubility in water and fat (Church & Parsons,

1995). N2 is used to replace O2 in packages to

delay oxidative rancidity and inhibit growth of

aerobic micro-organisms, as an alternative to

vacuum packaging. The use of oxygen in MAP is

normally set as low as possible to inhibit the

growth of aerobic spoilage bacteria. Its presence is

reported to increase oxidative rancidity (Chen

et al., 1984), although others claim that rancidity

caused by presence of O2 in the atmosphere is no

problem (Haard, 1992). However, for some prod-

ucts oxygen could or should be used. High levels

of oxygen are used in red meat and red fish meat

(tunas, yellowtails, etc.) to maintain the red colour

of the meat, to reduce and retard browning caused

by formation of metmyoglobin (Oka, 1989). O2 in

MA-packages of fresh fish will also inhibit reduc-

tion of TMAO to TMA (Boskou & Debevere,

1997).

Modified atmosphere packaging of fishery

products

The effect of MAP on the shelf-life of foods in

general (Wolfe, 1980; Finne, 1982; Daniels et al.,

1985; Dixon & Kell, 1989; Smith et al., 1990;

Farber, 1991; Church, 1994; Church & Parsons,

1995; Davies, 1995; Phillips, 1996) and fish in

particular (Ogrydziak & Brown, 1982; Parkin &

Brown, 1982; Brody, 1989; Pedrosa-Menabrito &

Regenstein, 1990; Stammen et al., 1990; Skura,

1991; Reddy et al., 1992) has been reviewed by

several authors in the last decade.

The first extensive research on seafood stored in

CO2 was first reported in the early 1930s in the UK,

USA and Russia (Stansby & Griffiths, 1935). In a

100% CO2 atmosphere, fish kept fresh 2–3 times

longer than control fish in air at the same tempera-

ture (Killeffer, 1930). Even at 27 °C,MA-packaged

cod was found to be in good condition after several

days. The absorption of CO2 altered the pH of fish

from 6.6 to 6.2, but it was reversed on subsequent

exposure to air. Fresh haddock, cod, sole, whiting

and plaice were very effectively preserved under 20–

100% CO2 atmospheres, with the optimal condi-

tions under 40–50% CO2 (Coyne, 1933). The flat

fish had better keeping quality at 0 °C and 80%

CO2 compared with the other fish species. Haddock

stored under 25% CO2 had a shelf-life of approxi-

mately twice that of products handled by conven-

tional methods (Stansby & Griffiths, 1935). CO2

storage was most beneficial during prolonged

storage and when the best sanitary conditions

during filleting were used. Since these early inves-

tigations, numerous articles has been written in this

topic, some reporting a tremendous increase in

shelf-life, others reporting little of no shelf-life

extension, but more often an extension in the range



Table 1 Shelf-life of fresh fishery products packaged under MA, vacuum or air

Type of fishery

product*

Storage

temperature (°C)

Atmosphere†

CO2 : N2 : O2

G/P

ratio‡

Shelf-life

(days)§Reference

(comments)

Catfish, channel (Icatalurus

punctatus) fillet strips

2 80 : 20 air ns 28 Silva & White (1994)

Catfish (ns) fillets 16 Air ns 3 Reddy et al. (1997a)

16 75 : 25 : 0 ns 4

16 Vacuum – 3

8 Air ns 6

8 75 : 25 : 0 ns 13

8 Vacuum – 6

4 Air ns 13

4 75 : 25 : 0 ns 38–40

4 Vacuum – 20–24

Cod (Gadus morhua) fillets 1 CA 60 : 40 : 0 – 12 Woyewoda et al. (1984)

1 60 : 40 : 0 ns 12

1 Air ns 9

Cod (G. morhua) fillets 2 40 : 60 : 0 2 11 Guldager et al. (1998)

2

2

40 : 60 : 0

40 : 40 : 20

2

2

20¶

13

¶Thawed shelf-life after

2 months –20° storage

Cod (ns) fillets 16 Air ns 3–4 Reddy et al. (1999)

16 75 : 25 : 0 ns 6

16 Vacuum – 3–4

8 Air ns 13–17

8 75 : 25 : 0 ns 24–27

8 Vacuum – 13

4 Air ns 20–24

4 75 : 25 : 0 ns 55–60

4 Vacuum – 24–27

Cod (ns) fillets 0 40 : 30 : 30 3 12.5 Cann et al. (1983)

5 40 : 30 : 30 3 <7

10 40 : 30 : 30 3 3

0 Vacuum – 9

5 Vacuum – <4

10 Vacuum – 2

Cod (G. morhua) whole 2 100 : 0 : 0 ns 10 Jensen et al. (1980)

2 60 : 40 : 0 ns 10

2 40 : 60 : 0 ns 9–10

2 Vacuum – 8–9

2 Air ns ~7

Cod (G. morhua) whole/fillets 0 Air ns 12–13 Villemure et al. (1986)

0 25 : 75 : 0 ns 20

Cod (G. morhua) fillets 0 2 : 98 : 0 2 14 Dalgaard et al. (1993)

0 3 : 97 : 0 2 13

0 29 : 71 : 0 2 16

0 48 : 52 : 0 2 20

0 97 : 3 : 0 2 15–16

Cod (G. morhua) fillets 26 100 : 0 : 0 ns 2–3 Post et al. (1985a)

26 Other atmos¶ ns 2 ¶All packaging types

at 26 °C (air, vacuum,

12 Air ns 6 100% N2, 90 : 8 : 2,

and 65 : 31 : 4)



Table 1 Continued

Type of fishery

product*

Storage

temperature (°C)

Atmosphere†

CO2 : N2 : O2

G/P

ratio‡

Shelf-life

(days)§

Reference

(comments)

12 Vacuum – 10 except 100% CO2 had

a shelf-life of 2 days

12 0 : 100 : 0 ns 13

12 100 : 0 : 0 ns 11

8 Air ns 6

8 Vacuum – 16

8 0 : 100 : 0 ns 17

8 100 : 0 : 0 ns 23

8 90 : 8 : 2 ns 17

8 65 : 31 : 4 ns 16

4 100 : 0 : 0 ns 40–53

Cod, blue (Arapercis colias)

fillets commercially smoked

3

3

100 : 0 : 0

Vacuum

2

2

49

14

Penney et al. (1994)

3 Air 2 14

)1.5 100 : 0 : 0 2 113

)1.5 Vacuum 2 35

)1.5 Air 2 28

Crayfish (Pacifastacus

leniusxulus) whole cooked

4

4

80 : 20 air

air

ns

ns

21

14

Wang & Brown (1983)

Flounder (Limanda ferrugina) 26 Air ns 2 Post et al. (1985)

fillets 26 Vacuum – 2

26 0 : 100 : 0 ns 4

26 100 : 0 : 0 ns 1

12 Air ns 5

12 Vacuum – 8

12 0 : 100 : 0 ns 7

12 100 : 0 : 0 ns 8

8 Air ns 5

8 Vacuum – 7

8 0 : 100 : 0 ns 4

8 100 : 0 : 0 ns 10

Haddock (Melanogrammus

aeglefinus) whole

0

0

40 : 30 : 30

Air

ns

ns

10

8

Dhananjaya & Stroud

(1994)

5 40 : 30 : 30 ns 7

5 Air ns 7

10 40 : 30 : 30 ns 4

10 Air ns 4

Haddock (M. aeglefinus) fillets 0

0

60 : 20 : 20

Air

ns

ns

14

10

Dhananjaya & Stroud

(1994)

Hake (Merluccius 2 50 : 45 : 5 2 14 Pastoriza et al. (1998)

merluccius) slices 2 50 : 45 : 5 2 16¶ ¶5 min 5% NaCl-dip

2 Air 2 7–8

Herring, Baltic (ns) fillets 2 20 : 80 : 0 0.4 3 Randell et al. (1995)

2 20 : 80 : 0 1 3

2 40 : 60 : 0 0.4 6

2 40 : 60 : 0 1 8

2 Vacuum – 3

Herring (Clupea harengus) fillets 0

0

60 : 40 : 0

Air

ns

ns

14

12

Dhananjaya & Stroud

(1994)



Table 1 Continued

Type of fishery

product*

Storage

temperature (°C)

Atmosphere†

CO2 : N2 : O2

G/P

ratio‡

Shelf-life

(days)§Reference

(comments)

Herring (C. harengus) whole 0

0

60 : 40 : 0

Air

ns

ns

14

12

Dhananjaya & Stroud

(1994)

Hybrid striped bass (Morone

saxatilis · M. chrysops) strips

2

2

60 : 34 : 6

Air

ns

ns

13

7

Handumrongkul & Silva

(1994)

Mackerel (Scombrus

scombrus L.) fillets

)2 100 : 0 : 0 3 > 21 Hong et al. (1996)

Rockfish (Sebastes spp.) fillets 1.7 CA 80 : 20 air – 13 Parkin et al. (1981)

1.7 Air – 6

Salmon, atlantic (Salmo

salar) slices

2 100 : 0 : 0 1.5 18 Pastoriza et al. (1996)

2 Air 1.5 8

Salmon, king (Oncorhynchus 4.4 60 : 15 : 25 ns 12 Stier et al. (1981)

tshawytscha) fillets 4.4 Air ns 6

22.2 60 : 15 : 25 ns 2

22.2 Air ns 1

Salmon (ns) fillets 16 Air ns 4 Reddy et al. (1997b)

16 75 : 25 : 0 ns 5–6

16 Vacuum – 3

8 Air ns 13–17

8 75 : 25 : 0 ns 20–24

8 Vacuum – > 6, < 10

4 Air ns 24–27

4 75 : 25 : 0 ns 55–62

4 Vacuum – 34–38

Salmon (ns) steaks 0 60 : 40 : 0 3 12.9 Cann et al. (1984)

5 60 : 40 : 0 3 7.1

10 60 : 40 : 0 3 3.4

0 Vacuum – 11.8

5 Vacuum – 8

10 Vacuum – 3

Salmon (S. salar) fillets 2 60 : 40 : 0 1 17 Randell et al. (1999)

2 40 : 60 : 0 1 17

2 Vacuum – 17

2 Air – 11

Sardines (Sardinops 5 80 : 20 : 0 ns 4 Fujii et al. (1989)

melanostictus) 5 20 : 80 : 0 ns 4

5 Air ns 2

Shrimp, spotted (Pandalus

platyceros) Whole Head on/off

0

0

CA 100 : 0 : 0

Air

–

–

> 14

7

Matches & Layrisse

(1985)

0 Air – 7

Snapper (Chrysophrys 3 100 : 0 : 0 ns 6–8 Scott et al. (1984)

auratus) fillets 3 Vacuum – 3¶ ¶No/medium O2-barrier

mat.

3 Vacuum – 6¶ ¶High O2-barrier mat.

3 Air ns 3

Snapper (C. auratus) fillets )1 40 : 60 : 0 5 9 Scott et al. (1986)

)1 Air ns 9

)1 100 : 0 : 0 ns 18



of 30–60% for fresh fishery products using atmos-

pheres with elevated levels of CO2 is observed.

Table 1 summarizes some of the more recent

published articles about MAP and fish.

From Table 1, and as reported by others

(Schvester, 1990; Stammen et al., 1990), many of

the publications about MAP do not state the G/P

ratio. Changes in the CO2 and O2 levels inside the

Table 1 Continued

Type of fishery

product*

Storage

temperature (°C)

Atmosphere†

CO2 : N2 : O2

G/P

ratio‡

Shelf-life

(days)§

Reference

(comments)

Swordfish (Xiphias 2 Air – 6¶ Oberlender et al. (1983)

gladius) steaks 2

2

CA 100 : 0 : 0

CA 70 : 0 : 30

–

–

>22¶

>22¶

¶Shelf-life based on

microbial data only

2 CA 40 : 0 : 60 – 14¶

2 CA 70 : 30 : 0 – >22¶

2 CA 40 : 60 : 0 – 20¶

Tilapia (Tilapia spp.) fillets 4 75 : 25 : 0 ns >25 Reddy et al. (1995)

8 75 : 25 : 0 ns 13–16

16 75 : 25 : 0 ns 9–13

4 Air ns 9–13

8 Air ns 6–9

16 Air ns 3–6

Trout (ns) whole 0 60 : 40 : 0 3 8 Cann et al. (1984)

5 60 : 40 : 0 3 8

10 60 : 40 : 0 3 3.8

0 Vacuum – 9

5 Vacuum – 6.5

10 Vacuum – 3.7

Trout (Salmo gairdneri) fillets 1.7 80 : 20 : 0 ns 20 Barnett et al. (1987)

1.7

1.7

80 : 20 : 0

Air

ns

ns

20¶

10

¶Treated with 2%

potassium-sorbate dip

Trout, rainbow 2 20 : 80 : 0 0.4 6 Randell et al. (1995)

(Oncorhynchus mykiss) fillets 2 20 : 80 : 0 1 9

2 40 : 60 : 0 0.4 6

2 40 : 60 : 0 1 9

2 Vacuum – 6

Whiting (Merluccius 26 All atmosph.¶ ns 2¶ Post et al. (1985)

bilinearis) fillets 12

12

Air

Vacuum

ns

–

5

9

¶All packaging types at

26 °C (air, vacuum,

12

12

0 : 100 : 0

100 : 0 : 0

ns

ns

9

12

100% N2, 100% CO2,

90 : 8 : 2, and

3

8

Air

Vacuum

ns

–

4

10

65 : 31 : 4) had a shelf-life

of 2 days

8 0 : 100 : 0 ns 10

8 100 : 0 : 0 ns 15

8 90 : 8 : 2 ns 13

8 65 : 31 : 4 ns 7

4 100 : 0 : 0 ns 15

ns¼Not stated in article.

*Type of fish (species).†Initial atmosphere in percent CO2 : N2 : O2. If mixtures of CO2 and air is used this is reported as %CO2 : % air (e.g. 80 : 20 air), if

initial atmosphere is maintained during storage either in controlled atmosphere tank or by reflushing, this is indicated with CA.àG/P ratio¼Volume of gas to volume of product (assuming density ~1 kg L)1 for fish); (–) not relevant (vacuum/CA).§Shelf-life in days after packaging as determined by sensory analysis is not otherwise noted in comments.¶See comments in reference field to the right of asterisk for information on additional treatments, etc.



package head-space, or amount of dissolved CO2,

during storage are also seldom measured. This

makes comparison between different studies diffi-

cult. Various qualities of the raw material, species,

different storage conditions, type of atmosphere

(CA vs. MA), packaging material, analytical

methods used for shelf-life assessments and cri-

teria for endpoint of shelf-life, further increases

the difficulties of comparing results from different

experiments (Skura, 1991).

In a study with herring and trout different G/P

ratios were investigated (Randell et al., 1995). The

results (Table 1) showed increased shelf-life for a

G/P ratio of 1 compared with 0.4, the latter giving

no additional shelf-life extension as compared

with vacuum packaging. In the same study 40%

CO2 gave longer shelf-life compared with 20% in

the initial gas atmosphere. A G/P ratio of 2 and

50% CO2 doubled the shelf-life of hake (Pastoriza

et al., 1998) compared with air. The gas mixture,

combined with NaCl dipping prior to packaging,

gave an additional 2 days extension of shelf-life.

The study of Scott et al. (1984) showed the

importance of using a barrier packaging material

for MA- and vacuum-packaged products. Vacuum

packaged snapper fillets doubled their shelf-life

(from 3 to 6 days) when vacuum packed in high

O2-barrier bags compared with no- or medium-

barrier bags, all other variables were kept con-

stant. A barrier packaging material is not only

important to keep O2 from permeating into the

MA-package during storage, but also to prevent

CO2 permeating out of the package.

Vacuum packaging and MAP, with high CO2

levels (25–100%), extends the shelf-life of meat

products by several weeks or months (Gill &

Penney, 1988). Most results of MAP fish indicates

a shelf-life extension from a few days and up to a

week or more compared with air storage, depend-

ing on species and temperature. Differences in

spoilage microflora and in pH are mainly respon-

sible for the observed differences in the shelf-life of

fish and meat product (Huss, 1995). Strict aerobic

Gram-negative organisms, primarily Pseudomonas

spp., cause spoilage of meat under aerobic condi-

tions. These organisms are strongly inhibited by

anaerobic conditions and by CO2. Consequently,

they do not play any role in the spoilage of packed

meat. Instead the microflora of MAP meat prod-

ucts tends to be dominated by Gram-positive

organisms, which are much more resistant to CO2

(Huss, 1995). The best effect of MAP storage on

shelf-life has been obtained with fish from warm

waters and the spoilage flora on some packed fish

products was found to be dominated by Gram-

positive micro-organisms and in this way the

microflora was similar to the flora on packed

meats (Banks et al., 1980; Huss, 1995).

Stefansson & Lauzon (1999) recently summar-

ized 18 years of MAP fish research at the Icelandic

Fisheries Laboratories. Shelf-life increases of 28–

52% for retail packaged and 32–73% for bulk

packaged haddock fillets was observed. Retail

packaged cod fillets had a shelf-life extension of 32

to over 52%, while 42–74% increase was observed

for ocean perch fillets. The use of O2 with CO2 was

preferable to N2 as a filling gas, providing a

slightly longer shelf-life, for these lean fishes.

Cod (Gadus morhua) is probably the most

frequently studied fish under MA conditions.

Shelf-life under MA conditions has been reported

to be up to 40–60 days at 4 °C (Post et al., 1985;

Reddy et al., 1999), but these investigations were

C. botulinum challenge tests and shelf-life was

based solely on appearance, development of off-

odours and texture changes. The reported shelf-

lives of cod under MA conditions of 14–20 days at

0–3 °C (Table 1) are probably a more realistic

estimate of shelf-life.

In a work often sited in the MAP of fish

literature (Stenstrom, 1985), homofermentative

Lactobacillus spp. was found to dominate the

spoilage microflora of cod fillets stored in high

CO2 concentrations, accounting for 80, 62 and

85% of the microflora in 100% CO2, 90%

CO2 : 10% N2, and 90% CO2 : 10% O2, respect-

ively. In addition, S. putrefaciens (12, 30, and

12%) was found, together with Vibrionaceae,

Micrococcus spp., and B. thermosphacta. In the

atmosphere recommended by Stenstrom, 50%

CO2 and 50% O2, the microflora consisted of

44% Lactobacillus spp., 38% S. putrefaciens,

10% B. thermosphacta. This result is in conflict

with the results found by Dalgaard and

co-workers (see below). However, this may be

because organisms were isolated after 34 or

26 days of storage at 2 °C when spoilage of the

fish must have been severe. Also, in this study the

packages of cod were reflushed with the different

gas mixtures after 24 h of storage to compensate



for CO2 dissolved into the fish muscle, giving

some sort of quasi-controlled atmosphere.

Specific spoilage organisms of MA

packaged and air-stored fish

When a product is microbially spoiled, the spoil-

age microflora will consist of a mixture of species

(Huss et al., 1997), many of which can be com-

pletely harmless, both in terms of health hazards

and in terms of ability to produce off-odours and

off-flavours. The bacterial group causing the

important chemical changes during fish spoilage

often consists of a single species. The knowledge of

SSOs of different fishes from various aquatic

environments and under different packaging

conditions is still limited. However, for cod stored

under aerobic conditions in ice Shewanella putre-

faciens [formerly known as both Pseudomonas

putrefaciens and Alteromonas putrefaciens (Debe-

vere & Boskou, 1996)] has been identified as the

main spoilage bacterium (Gram et al., 1987).

Shewanella putrefaciens produce very intense and

unpleasant off-odours, reduces TMAO to TMA

and produces H2S, and is generally recognized as

the fish spoilage bacterium for iced cod fish (0 °C).Strains of Vibrionaceae were found to dominate

the spoilage of cod at 20 °C (Gram et al., 1987).

Shewanella putrefaciens is also reported as being

an important spoilage bacteria of cod and sole

together with Lactobacillus spp. in MAP (Stam-

men et al., 1990). For packed cod stored at 0 °C,however, the Gram-negative organism P. phos-

phoreum has been identified as the organism

responsible for spoilage in vacuum packs and in

MA packs with mixtures of CO2 and N2 (Dalg-

aard et al., 1993b; Dalgaard, 1995a). The growth

rate of this organism is increased under anaerobic

conditions and this may explain the importance of

the organism in vacuum packaging and MAP of

cod. In CO2-packed fish, the growth of S. putre-

faciens and of many other micro-organisms found

on live fish is strongly inhibited. In contrast,

P. phosphoreum was shown to be highly resistant

to CO2 (Dalgaard, 1995b). It was also shown that

the growth of this bacterium corresponds very well

with the shelf-life of packed fresh cod. Photobac-

terium phosphoreum reduces TMAO to TMA at

10–100 times the amount per cell than

S. putrefaciens, probably because of the large size

of the former (diameter 5 lm) while very little H2S

is produced during growth in fish substrates

(Dalgaard, 1995a; Dalgaard et al., 1996). Spoiled

MAP cod is characterized by high levels of TMA,

but little or no development of the putrid or H2S

odours typical for some aerobically stored spoiled

fish (Dalgaard, 1995a).

Photobacterium phosphoreum is sensitive to

freezing and was totally inactivated in thawed-

chilled MAP cod fillets after frozen storage at )20and )30 °C for 6–8 weeks (Bøknæs et al., 2000).

This approach has been used to extend the shelf-

life of MAP cod from 11 to 13 days up to around

20 days at 2 °C (Guldager et al., 1998). However,

an increased drip as a result of the frozen storage

was observed from the thawed MAP fish.

Photobacterium phosphoreum is widespread in the

marine environment and it seems likely that this

organism or other highly CO2 resistant micro-

organisms are responsible for spoilage of packed

fish products (Dalgaard et al., 1993b). In contrast,

a Shewanella-like bacteria was found to constitute

a major part of the microflora of gas packed cod

fillets stored at 7 °C (Boskou & Debevere, 1997),

and this bacterium was found to be less inhibited

by CO2 compared with Shewanella putrefaciens.

S. putrefaciens was unable to develop if high

concentrations of CO2 were applied (> 50%) and

high concentrations of O2 (70%) seemed to have

some inhibitory effect on the growth of S. putre-

faciens as well (Boskou & Debevere, 1998). No

production of TMA was observed when sufficient

O2 (>10%) for aerobic respiration of S. putrefac-

iens was included in the packaging atmosphere

(Boskou & Debevere, 1998). Lowered TMA pro-

duction in MA atmosphere with O2 (40 : 40 : 20

CO2 : N2 : O2) compared with atmosphere with-

out O2 (40 : 60 CO2 : N2) for cod fillets stored at

2 °C was also observed by Guldager et al. (1998).

The combined effect of CO2 and

temperature on the microbial spoilage

As mentioned earlier, temperature is of primary

importance in all fresh fish storage, including

MAP and vacuum packaging, as both enzymatic

and microbiological activity are greatly influenced

by temperature. Many bacteria are unable to

grow at temperatures below 10 °C and even

psychrotrophic organisms grow very slowly, and



sometimes with extended lag phases, as tempera-

tures approach 0 °C. Increased solubility of CO2

at lower temperatures will relatively increase the

effect of MAP. Studies of haddock in MAP and

air (Dhananjaya & Stroud, 1994) showed no

benefit of 40 or 60% CO2 atmosphere compared

with air at 5 and 10 °C, whereas the MA packaged

haddock had 2–4 days longer shelf-life at 0 °C(Table 1). Several other studies on different stor-

age temperatures confirm these results (Table 1).

MAP can be combined with superchilling to

further extend the shelf-life and safety of fresh fish.

In this technique, also known as partial freezing,

the temperature of the fish is reduced to 1–2 °Cbelow the initial freezing point and some ice is

formed inside the product (Haard, 1992; Sikorski

& Sun, 1994), and can be used to extend prime

quality of fresh fish. The freezing points of foods

are dependent on the water content in the prod-

ucts (Chang & Tao, 1981). Freezing points for

fishery products vary from about )1 to )2.5 °C,e.g. salmon, shrimp, and mackerel ~ )2.2 °C, carp~ )1.0 °C (Rahman & Driscoll, 1994). A typical

shelf-life extension of about 7 days is obtained on

superchilling fish as compared with traditional ice

stored fish of the same type. Bulk packaged

superchilled salmon combined with high CO2-

atmosphere maintained a high microbiological

and sensory quality, similar to whole salmon

(Salmo salar) for more than 3 weeks (Sivertsvik

et al., 1999). Bulk storage of salmon fillets

(Oncorhynchus kisutch and O. keta) in air-tight

bulk containers has been reported to have an

acceptable sensory shelf-life of 21 days in 90%

CO2 atmosphere at 0 °C (Barnett et al., 1982). The

same shelf-life was observed for mackerel at )2 °Cin 100% CO2 (Hong et al., 1996) and for smoked

blue cod a doubling of shelf-life was observed

when lowering the storage temperature from 3 to

)1.5 °C (Penney et al., 1994).

Shellfish and cooked fish in MAP

Crustacean shellfish keep up to 30% longer at

0 °C in a MA than in other types of packaging,

and the onset of blackspot in shell-on products is

delayed (Cann, 1988). An MA of 80% CO2 and

20% air was found to be the preferred atmosphere

for storage of freshwater crawfish (Procambaris

clarkii) tail meat compared with 100% CO2 or air

(Gerdes et al., 1989). CO2-enriched atmosphere

was found to increase the shelf-life of whole

cooked shrimps by 200%, compared with shrimps

stored on ice and exposed to air (Sivertsvik et al.,

1997). MA-packaged shrimps were evaluated as

fresh shrimps after 16 days of storage. CO2

atmospheres has also been found beneficial for

storage of fish-cakes (Yokoseki et al., 1956), and

also for chilled storage of raw squid and white

octopus (Morales et al., 1997).

Effect of CO2 on common spoilage

indicators

Total volatile nitrogen (TVN) analyses, often used

as spoilage indicator for fresh seafood held on ice,

was shown to have low values for fish stored in

CO2 atmosphere, even after sensory evaluation

indicated spoilage. This indicates a different

spoilage pattern for fish in CO2 atmosphere

compared with fish held on ice, and that TVN is

not suitable as a spoilage indicator (Banks et al.,

1980). K-value is an index for predicting shelf-life

and is calculated from concentrations of the nuc-

leotide breakdown products, hypoxanthine (Hx),

inosine (HxR), and inosine monophosphate (IMP)

as Ki¼ 100 · (Hx + HxR)/(IMP + HxR + Hx)

(Colby et al., 1993). Effects of 100% CO2 atmo-

sphere compared with air storage on the degrada-

tion of adenine nucleotides in chill-stored whitefish

(Coregonus clupeaformis) and rainbow trout

(S. gairdneri) were studied by Boyle et al. (1991)

and K-values were determined during storage up

to 26 days. Results indicated that CO2 atmos-

pheres did not alter K-values compared with those

observed for aerobically held fish. K-values of

tilapia fillets increased during storage of air and

MA (75 : 25 CO2 : N2), however, the MA-pack-

aged fillets were still sensory acceptable even at

high K-values (Reddy et al., 1995b). K-values were

independent of sensory spoilage and correlated

only with the length of storage of MA-packaged

(75 : 25 CO2 : N2) tilapia fillets at 4 °C. Little

relation between K-values and sensory quality had

also been reported on shelf-life studies of fresh

sardines in CO2-atmospheres (Fujii et al., 1989).

K-values were reported as not being suitable

predictors of shelf-life (Handumrongkul & Silva,

1994), and the authors proposed the use of a

modified K-value calculated without including



inosine content for MA products. Davis (1998)

suggests that this is caused by the influence of CO2

on tissue pH and the greater persistence of IMP in

fish of intrinsically low pH.

Nonmicrobial effect of MAP on fish

Low bacterial levels (105–106 cfu g)1) have been

found at the time of sensory rejection of some

MAP fish products, for example, salmon stored

under superchilling conditions (Sivertsvik et al.,

2000). In these cases, nonmicrobial reactions may

have been responsible for spoilage. O2 is prefer-

entially excluded from the packaging atmosphere

when packing fatty fish in order to inhibit oxida-

tive rancidity. Vacuum packaging could also be an

alternative to MAP of fatty fishes, such as salmon

and trout, giving similar sensory shelf-life (see

Table 1), the primary sensory spoilage parameter

being oxidative rancidity (Rosnes et al., 1997;

Randell et al., 1999). Usually, at low storage

temperature the shelf-life of vacuum packaged or

MAP salmon and trout is doubled compared with

air storage. In contrast, the salmon study of

Reddy et al. (1997b) observed extended shelf-life

of MAP as compared with both vacuum and air. It

should be noted that in this study the reported

shelf-life was very long for all storage types

compared with other studies. Although oxygen

containing MAs have been used, the development

of rancid off-odours in fatty fish species has not

been registered as a problem in other studies

(Haard, 1992). The colour of the belly flaps,

cornea and the skin may be altered for whole fish

stored in high CO2 concentrations (Haard, 1992),

and Coyne (1933) observed browning of gills of

whole fishes and some tissue softening in fillets

stored under pure CO2. Whether these texture

changes, observed for packaged fish in 100% CO2,

is caused by high amounts of dissolved CO2, pH

drop, excessive exudate, or a combination of these

is not certain. Hundred per cent CO2 has been

used for bulk transportation of salmon, without

negative effects on fish texture or skin (Sivertsvik

et al., 1999), however, softening of trout texture in

CO2 atmosphere has been observed (Chen et al.,

1984). Discoloration of the gills, because of an O2

free environment could be avoided by using small

amount of CO in the atmospheres (Rosnes et al.,

1998).

Microbial safety of MA packaged product

It is useful to distinguish between two categories of

MA packaged products. Those eaten without any

heat treatment immediately prior to consumption,

such as ready to eat products, sashimi/sushi,

smoked salmon, cooked shellfish and those prod-

ucts that will be subjected to heat treatment

sufficient to kill all vegetative pathogens before

serving (e.g. most fresh fish). Safety concerns

regarding pathogenic micro-organisms are of pri-

mary importance and deserve first priority during

manufacture.

Fish and shellfish are vehicles for transmission

of foodborne diseases (Huss et al., 1997). Path-

ogens found on fishes are either naturally present

in the fish from the aquatic environment (C.

botulinum type E and non-proteolytic types B

and F; pathogenic Vibrio spp.; A. hydrophila;

Plesiomonas shigelloides), or frequently present

(Listeria monocytogenes, C. botulinum proteolytic

types A, B; C. perfringens; Bacillus spp.), or

originating from the animal/human reservoir

(Salmonella, Shigella, Escherichia coli, Staphylo-

coccus aureus).

Storage of products under MA may not increase

the risks from Salmonella, Staphylococcus,

C. perfringens, Yersinia, Campylobacter, Vibrio

parahaemolyticus, and Enterococcus above those

expected for air-stored products (Silliker & Wolfe,

1980; Reddy et al., 1992). The same is observed

for A. hydrophila whereas growth of Plesiomonas

shigelloides is completely inhibited by MAP

(Kirov, 1997). No growth and difference in survi-

val of E. coli, S. aureus, V. parahaemolyticus, and

C. perfringens was observed in Jack mackerel

(Trachurus japonicus) during storage under air,

100% N2 or 40% CO2–60% N2 atmospheres at

5 °C (Kimura & Murakami, 1993). Slade &

Davies (1997) studied the fate of antibiotic resist-

ant strains of Y. enterocolitica, Aeromonas spp.,

and Salmonella typhimurium inoculated in fish

stored at 0 or 5 °C under air or MAP conditions

(60 : 40 : 0 and 40 : 30 : 30 CO2 : N2 : O2 atmo-

sphere for cod and 60 : 40 and 80 : 20 CO2 : N2

atmospheres for trout). A CO2 dependent bacte-

riostasis of Salmonella at chilled temperatures was

found. Growth of both Aeromonas and Yersinia

were observed, but the MA conditions inhibited

growth compared with air storage.



Hence, the pathogen of major concerns when

packaging fish under anaerobic conditions has been

and still is C. botulinum type E and non-proteolytic

type B. If MA packaged fish products are stored at

temperature below 10 °C, the other pathogen of

concern, above that for aerobically stored fish, is

Listeria monocytogenes (Gibson & Davis, 1995).

Listeria monocytogenes

Growth of L. monocytogenes is apparently not

significantly affected by MAP (Rocourt & Cossart,

1997), and it can grow under anaerobic, chilled

conditions. However inhibition of L. monocytog-

enes growth under 100% CO2 atmospheres (Avery

et al., 1994; Szabo & Cahill, 1998) or in atmos-

pheres with 50% CO2 levels in combination with

additional hurdles like bacteriocins (Nilsson et al.,

1997; Szabo & Cahill, 1998) and/or salt and

lowered pH (Farber et al., 1996) has been repor-

ted. Pothuri et al. (1996) observed an 8 days

extension of the lag phase of L. monocytogenes

in samples of crayfish tail meat treated with 1%

lactic acid and packaged under MA conditions

(75% CO2 : 10% O2, 15% N2) as compared with

that in air or vacuum packaging. 100% CO2

delayed, but did not inhibit growth of L. mono-

cytogenes as compared with air in inoculated

(103 cfu g)1) raw and cooked seafood nuggets

(Lyver et al., 1998a), and was able to reach

106 cfu g)1 before spoilage occurred in all pack-

aging methods. Packaging under enhanced CO2

atmospheres will delay the growth of L. monocyto-

genes, but the MA atmospheres and chilled

storage alone are not sufficient to control the

growth of the pathogen in some products. To

ensure the safety, especially for value-added sea-

food products with extended shelf-life, additional

hurdles must be applied.

Non-proteolytic C. botulinum

Non-proteolytic C. botulinum in MA packaged

fresh fishery products represents a potential risk

for several reasons (Reddy et al., 1999):

(a) non-proteolytic type strains of C. botulinum

are widely distributed in freshwater, estuarine,

and marine environments and therefore may

be found on fishery products harvested in

these areas;

(b) they can grow and produce toxins at tempera-

ture as low as 3.3 °C [and recently reported

even down to 3.0 °C (Graham et al., 1997)];

(c) the inhibition of the normal aerobic spoilage

bacteria by MAP reduces bacterial competi-

tion that may permit non-proteolytic

C. botulinum growth and toxin production

during prolonged refrigerated or temperature

abuse storage; and

(d) products contaminated with C. botulinum and

packaged under MA may become toxic and

yet remain acceptable with respect to odour

and appearance to the consumer.

The risks from botulism in MAP fish have been

widely reviewed (Genigeorgis, 1985; Baker &

Genigeorgis, 1990; Stammen et al., 1990; Reddy

et al., 1992). Irrespective of the actual minimum

growth temperature, production of neurotoxin

generally requires weeks at the low temperature

limit (Dodds & Austin, 1997). Graham et al.

(1997) found growth and toxin production from

spores of C. botulinum type B, E and F after

5 weeks at 3 °C, 3–4 weeks at 4 °C and 2–3 weeks

at 5 °C. Growth occurred more frequently from

spores of type F strains than for types B and E.

Results from recent publications on growth and

toxin production by C. botulinum in packaged

fishery products are shown in Table 2 (see

Stammen et al., 1990; Reddy et al., 1992 for older

publications). Toxin has been detected in MAP

fish prior to the products being considered spoiled

(Post et al., 1985; Garcia & Genigeorgis, 1987;

Taylor et al., 1990). However, other challenge

tests of C. botulinum and fish has shown MA or

vacuum packaged fish to spoil prior to or in

coincidence with toxin production (Cann et al.,

1984; Reddy et al., 1996, 1997a,b, 1999; Cai et al.,

1997). Even, if the results are not conclusive, there

is a potential threat for a packaged fish product to

become toxic prior to spoilage at storage tempera-

ture of 8 °C or above.

Originally, oxygen was introduced into the

package of selected products to reduce the risk of

anaerobic pathogenic bacterial growth, but this has

been discredited (Davies, 1995). It is now recog-

nized that the growth of C. botulinum in foods does

not depend upon the total exclusion of oxygen, nor

does the inclusion of oxygen as a packaging gas

ensure that growth of C. botulinum is prevented. In

the studies of Stier et al. (1981), Post et al. (1985)



andGarcia et al. (1987), O2was included in theMA

gas mixture, without providing further safety than

the MA gas mixtures without O2. Enhanced toxin

production of C. botulinum was not observed in

smoked herring packed in an atmosphere of high

concentrations of CO2 compared with other forms

of packaging where gaseous oxygen was removed

(Huss et al., 1980). The safest packaging method

Table 2 Recent publications on growth and toxin production by C. botulinum in packaged fishery products (see Reddy et al.,

1992; Stammen et al., 1990 for earlier publications)

Type of fishery

product

Storage

temperature

(°C)

Atmosphere

CO2 : N2 : O2

Inoculum

level

(spores g)1)

Toxin

detection

(days)

Shelf-life

(days) Reference

Salmon fillet 16 Air 100 4 4 Reddy et al. (1997b)

16 75 : 25 : 0 100 4 5–6

16 Vacuum 100 3 3

8 Air 100 17 13–17

8 75 : 25 : 0 100 24 20–24

8 Vacuum 100 10 > 6, < 10

4 Air 100 > 66 24–27

4 75 : 25 : 0 100 > 80 55–62

4 Vacuum 100 > 66 34–38

Tilapia fillets 16 Air 100 4 3 Reddy et al. (1996)

16 75 : 25 : 0 100 4 4

16 Vacuum 100 3 3

8 Air 100 20 6

8 75 : 25 : 0 100 40 17

8 Vacuum 100 17 10

4 Air 100 > 47 10

4 75 : 25 : 0 100 > 90 80

4 Vacuum 100 > 90 47

Catfish fillets 16 Air 100 3 3 Reddy et al. (1997a)

16 75 : 25 : 0 100 4 4

16 Vacuum 100 3 3

8 Air 100 9 6

8 75 : 25 : 0 100 18 13

8 Vacuum 100 6 6

4 Air 100 > 54 13

4 75 : 25 : 0 100 > 75 38–40

4 Vacuum 100 46 20–24

Cod fillets 16 Air 100 > 7 3–4 Reddy et al. (1999)

16 75 : 25 : 0 100 7 6

16 Vacuum 100 7 3–4

8 Air 100 > 41 13–17

8 75 : 25 : 0 100 > 60 24–27

8 Vacuum 100 17 13

4 Air 100 > 60 20–24

4 75 : 25 : 0 100 > 90 55–60

4 Vacuum 100 > 5 24–27

Rainbow trout fillets 10 Vacuum-skin packaging 103–104 6 3 Garren et al. (1995)

4 Vacuum-skin packaging 103–104 > 21 12

Channel catfish 10 Air 100–2000 4 4–6 Cai et al. (1997)

10 80 : 20 : 0 Masterbag 100–2000 6 6

10 80 : 20 : 0 100–2000 4 2–6

4 Air 100–2000 9 9

4 80 : 20 : 0 Masterbag 100–2000 > 30 9–12

4 80 : 20 : 0 100–2000 18 9–12



with regard to toxin production was found to be a

mixture of equal parts ofO2 andCO2. Even smoked

herring, packed with air in open bags or even in an

atmosphere of pure oxygen, and contaminated on

the surface with only approximately 102 g)1 type E

spores may be toxic in 6–9 days at 15 °C. Under

these circumstances, reduced oxygen tension or

even anaerobic conditions may exist as minute foci

or a thin film on the surface of the fish and below,

dependent on the degree of contamination with

oxygen-consuming micro-organisms. C. botulinum

grows optimally at a redox potential (Eh) of

)350 mV, but growth initiation may occur in the

Eh range of +30 to +250 mV. The presence of

other inhibitory factors lowers this upper limit.

Once growth is initiated, the Eh declines rapidly

(Dodds & Austin, 1997). The importance of redox

potential on the growth of C. botulinum is not fully

understood, for example, the complex interactions

that occur between redox levels, anaerobiosis and

inoculum levels, at temperatures approaching the

minimum for growth of nonproteolytic C. botuli-

num (Gibson et al., 2000).

Gibson et al. (2000) demonstrated that 100%

CO2 couldhave an inhibitory effect on the growthof

C. botulinum at chill temperatures, and an increased

inhibitory effect was observed when combining

100%CO2 with increasedNaCl level and decreased

pH level. Lyver & Smith (1998b) and Lyver et al.

(1998c) showed that growth and toxin production

by C. botulinum in raw and cooked surimi nuggets

could be controlled with competitive inhibition by

Bacillus species. The fat content may influence the

margin of safety of MA packaged aquacultured

fresh fish fillets during storage. The safety margin

(days from sensory spoilage to toxin detection)

being less for fattier fishes (salmon, catfish) when

compared with leaner fishes (tilapia, cod) (Reddy

et al., 1999). Trout and salmon inoculated with

spores of C. botulinum type B and E (2-log

spores g)1) and stored in MA and vacuum at 10,

15 and 20 °C spoiled clearly before they became

toxic (Cann et al., 1984). Salmonwere less toxigenic

compared with trout, which could be attributed to

lower pH in salmonduring storage.No difference in

toxin production was observed between MAP and

vacuum packaging. Comparison of surface and

deep inoculated fish showed a definite trend for the

latter to become toxic earlier in storage. Similar

results were also obtained for hot-smokedmackerel

(Cann et al., 1983). Treatment of the fish with the

bacteriocin nisin prior to packaging inMA delayed

the onset ofC. botulinum toxin production, without

affecting the spoilage microflora (Taylor et al.,

1990).

A mathematical model has been developed for

prediction of lag time prior to C. botulinum

toxigenesis (Baker & Genigeorgis, 1990). The

model revealed that about 75% of experimental

variation was explained by storage temperature

while the size of the C. botulinum spore inoculum

explained 7.5%. Collectively factors such as MA

compositions, type of fish and C. botulinum spore

type attributed to only 2.3% of the variation. The

data confirms the importance of temperature

control, and under temperature abuse conditions,

most fish independent of packaging method can

become toxic. Extensive research has been done to

predict growth of food pathogens, and

C. botulinum in particular. This has resulted in

commercial computer programs, like Food

MicroModelTM, to help assess the risk of food

pathogen growth under various packaging and

storage conditions (Graham et al., 1996). How-

ever, these software program have limited facility

to model alternative atmospheres, for example,

not including the G/P ratio in the package.

Clearly, the risk for C. botulinum to grow and

produce toxin is low at chilled temperature under

anaerobic conditions. In a study of 1074 test

samples of commercial, vacuum-packaged fresh

fish, none of the marginally organoleptic accept-

able samples was positive for C. botulinum toxin

after 12 days at 12 °C (Lilly & Kautter, 1990). The

authors concluded that either the fish did not

contain C. botulinum spores, or the spores were

unable to grow out and produce toxin before

spoilage made the product marginally unaccepta-

ble. However, an increased use of MA and other

minimal processing technologies combined with

improper cold chains may represent an increased

risk of botulism. The Advisory Committee on the

Microbiological Safety of Food (ACMSF) in the

UK published in 1992 a Report on Vacuum

Packaging and Associated Processes. Their con-

clusion was that, if the storage temperature and

the shelf-life are less than, respectively, 10 °Cand 10 days, the risk is low. For products with a

shelf-life of longer than 10 days, which are not

subjected to a heat treatment that will sufficiently



inactivate the spores of psychrotrophic C. botul-

inum, combinations of preservative factors such as

pH (<5), water activity (<0.97), and salt (>5%)

should be established to prevent the growth of

psychrotrophic C. botulinum (Gibson & Davis,

1995). As temperature abuse is common through-

out the distribution and retail chain and even more

frequent among consumer handling post-retail

(Reddy et al., 1992), ACMSF introduced even

stricter recommendations in 1994, even if there

was no evidence of illness caused by vacuum

packaging or MAP chilled foods (Davis, 1998).

When chilled storage is the only controlling factor,

storage at temperatures between 5 and 10 °Cshould be limited to 5 days, and shelf lives up to

10 days could be assigned for storage tempera-

tures at 5 °C or below.

The future of MAP

Several novel technologies offer the potential of

further increase in safety and shelf-life of MAP

products, including the use of active packaging

and hurdle technology. Active packaging is an

emerging technology in which food, package and

environment interact, resulting in an extended

shelf-life, including different kinds of gas emitters

and absorbers (Rooney, 1995). For many food

products the most relevant are O2 absorbers and

carbon dioxide producers used either alone to

develop a MA or in combination with a gas

mixture. O2 absorbers could be used to maintain a

low level of O2 inside the package even when using

packaging materials of inadequate barrier proper-

ties (Ashie et al., 1996).

The CO2 is usually introduced into the MA

package by evacuating the air and flushing the

appropriate gas mixture into the package prior to

sealing. Two other approaches to create a MA for

a product is either to generate the CO2 inside the

package after packaging or to dissolve the CO2

into the product prior to packaging. Both methods

can give appropriate packages with smaller G/P

ratio, and thus decrease the package size. Exam-

ples of the former is the use of either CO2

generators, using the O2 in the package head-

space to produce CO2 (Ashie et al., 1996) or

allowing dry ice (solid CO2) to sublime inside a

vacuum package generating close to 100% CO2

atmosphere (Sivertsvik et al., 1999). CO2 could

also be produced inside the packages after pack-

aging by allowing the exudate from the products

react with a mixture of sodium carbonate and

citric acid inside the drip pad (Bjerkeng et al.,

1995). Dissolving CO2 in the product prior to

packaging under elevated pressure and low tem-

perature is a novel approach to generate an MA

and has shown promising results on cottage cheese

(Chen & Hotchkiss, 1991) and salmon (Sivertsvik,

2000).

Hurdle technology (combined processes) means

the intentional combination of preservation tech-

niques in order to establish a series of preservative

factors (hurdles) that any micro-organisms present

should not be able to overcome. These hurdles

may be storage temperature, water activity, pH,

redox potential, preservatives and novel tech-

niques including MAP, bioconservation, bacte-

riocins, ultrahigh pressure treatment and edible

coatings (Leistner & Gorris, 1995). Potassium

sorbate is for example a preservative used in

combination with MAP of fish (Reddy et al.,

1992), and may give additional shelf-life increase

of MAP fish (Fey & Regenstein, 1982), while other

reports show no additional effect (Barnett et al.,

1987). The use of combination processes with

MAP is still in its development phase, but in the

future this technology could be the way to further

increase shelf-life and control pathogen growth,

ensuring safe products.

Smart packaging, such as Time Temperature

Indicators (TTI) is a technology that appears to

have a potential, especially with chill-stored MAP

products. To ensure microbial safety strict tem-

perature control is needed, and temperature abuse

should be avoided. TTIs could be applied to

monitor the temperature and to detect tempera-

ture abused packages (Labuza, 1993, 1996).

Conclusions

Only the highest quality fish and seafood products

should be used to benefit from the extended shelf-

life advantages of MAP. The extended shelf-life

will depend on the species, fat content, initial

microbial load, gas mixture, the ratio of G/P, and

most importantly temperature of storage. The

SSO of MAP cod at 0 °C has been found to be

P. phosphoreum. Whether this bacteria is the

general SSO for all marine fishes at different



storage temperatures and under various CO2/N2/

O2 mixtures needs to be resolved.

Without proper control of storage tempera-

tures, the benefits of MAP may be lost. Higher

storage temperature will inevitably lead to loss of

dissolved CO2 in the product and consequently

loss of inhibitory effect; higher microbial and

enzymatic activity; and uncertainties around the

microbial safety of the product.

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