International Journal of Food Science and Technology 1999, 34, 122 1

Review article
The microbiological safety of minimally processed
vegetables

Gillian A. Francis*, Christopher Thomas & David OBeirne

Food Science Research Centre, Department of Life Sciences, University of Limerick, Limerick, Ireland

Summary Demand for fresh, convenient, minimally processed vegetables has led to an increase in
the quantity and variety of products available to the consumer. Modified atmosphere
packaging, in combination with refrigeration, is increasingly being employed as a mild
preservation technique to ensure quality and storage-life. The fresh nature of these prod-
ucts, together with the mild processing techniques and subsequent storage conditions,
have presented indigenous and pathogenic microorganisms with new ecosystems and
potential infection vehicles; a number of outbreaks of foodborne disease being attributed
to their consumption. Psychrotrophic pathogens and pathogens which are capable of
maintaining an infectious potential under mild preservation regimes are of particular con-
cern; Listeria monocytogenes, Aeromonas hydrophila and Clostridium botulinum being
amongst the most notable. This review describes the processing, packaging and storage
procedures involved in the production of minimally processed vegetables, and details
their impact upon the survival and growth of associated pathogens. Gaps in our current
understanding of the consequences of this novel technology for microbiological safety are
highlighted.
Keywords Disinfection, food safety, foodborne pathogens, modified atmosphere packaging, processing.

mal processing. Pathogens may form part of this

Introduction
microflora, posing a potential safety problem.
Demand for fresh, minimally processed vegeta- Several studies have been done to determine the
bles has led to an increase in the quantity and
variety of products available to the consumer.
Minimally processed vegetables may be simply
trimmed vegetables (e.g. whole lettuce stripped of
its outer leaves) or may consist of trimmed,
peeled, sliced/shredded and washed and/or disin-
fected vegetables (e.g. dry coleslaw mix). The veg-
etables may be combined with cooked vegetables,
meats and pasta and/or salad dressings. The
products are packaged and stored at refrigeration
temperatures (Fig. 1).
Such ready-to-use (RTU) vegetables retain
much of their indigenous microflora after mini-

*Correspondent: Fax 13534 6133 1490; Figure 1 A flow diagram for the production of minimally
e-mail Jillian.Francis @UL.IE processed vegetables.

1999 Blackwell Science Ltd

2 Microbiological safety of vegetables Gillian A. Francis et al.
survival/growth characteristics of pathogens on a
range of RTU vegetables. This review examines
effects of minimal processing, packaging, and
storage conditions on the potential for survival
and growth of pathogens which might form part
of the indigenous microflora or be introduced as
contaminants during processing and packaging.
The need for continued research in a number of
areas, especially in combinations of RTU prod-
ucts, is highlighted.
Production of RTU vegetables
The first step in reducing contamination of the
raw vegetable is removal of outer layers or sur-
face dirt. After removal of contaminated external
layers, the vegetables are sliced/shredded.
Washing and antimicrobial dipping
RTU vegetables are thoroughly washed and are
often dipped in antimicrobial solutions. These
procedures reduce initial microbiological load,
thus reducing the rate of subsequent microbial
spoilage and minimising populations of potential
pathogens. The washing agent can be water
alone, but efficacy of washing is improved by
including antimicrobials, typically chlorine (100
ppm) or citric and ascorbic acid (1%) in the wash
water.
The final step in minimal processing is removal
of excess water added during washing, usually by
centrifuging (Bolin et al., 1977; Reyes, 1996).
Modified atmosphere (MA) packaging
RTU vegetables are usually sealed within semi-
permeable packages. When a sliced vegetable is
packaged, it respires, modifying the gas atmos-
phere inside the package. Ideally, O2 levels will
fall from 21% to 25%, and CO2 levels will
increase from 0.03% to 310% (OBeirne, 1996).
These gas levels, combined with refrigeration,
slow down product respiration, slow microbial
growth, delay physiological ageing and thus
extend shelf-life.
Storage temperature
RTU vegetables are stored at refrigeration tem-
International Journal of Food Science and Technology 1999, 34, 122
peratures (25 8C). Strict control of refrigeration
and a conservatively short shelf-life limit the
opportunity for growth by pathogenic and
spoilage microorganisms.
The microbiology of RTU vegetables
Indigenous microflora
RTU vegetables harbour large and diverse popu-
lations of microorganisms, and counts of 105107
CFU g21 are frequently present. Eighty to 90% of
bacteria are Gram-negative rods, predominantly
Pseudomonas, Enterobacter or Erwinia species
(Manvell & Ackland, 1986; Brocklehurst et al.,
1987; Nguyen-the & Prunier, 1989; Garg et al.,
1990; Magnuson et al., 1990; Marchetti et al.,
1992).
Lactic acid bacteria have been detected in
mixed salads and grated carrots and may pre-
dominate in salads when held at abuse (30 8C)
temperatures (Manvell & Ackland, 1986).
Yeasts which were commonly isolated included
Cryptococcus, Rhodotorula, and Candida
(Brackett, 1994). Webb & Mundt (1978) surveyed
14 different vegetables for moulds. The most
commonly isolated genera were Aureobasidium,
Fusarium, Mucor, Phoma, Rhizopus, and
Penicillium.
Pathogens
RTU vegetables have a good track record in
terms of food safety. However, foodborne
pathogens may be present, and a number of out-
breaks of foodborne disease have been traced to
RTU vegetables (Table 1). The pathogens most
frequently associated with RTU vegetables are
discussed below.
Listeria monocytogenes
L. monocytogenes is a Gram-positive rod-shaped
bacterium. The pathogen causes several diseases
in man including meningitis, septicaemia, still-
births and abortions (ICMSF, 1996).
Of particular concern with L. monocytogenes is
its ability to grow at refrigeration temperatures;
the minimum temperature for growth is reported
to be 20.4 8C (Walker & Stringer, 1987). It is also
facultatively anaerobic, capable of survival/
1999 Blackwell Science Ltd

Table 1 Examples of foodborne infections linked to the consumption of raw vegetables
Microorganism Product suspected
L. monocytogenes Shredded cabbage in coleslaw
Raw vegetables in salads
Salted mushrooms
Cl. botulinum Shredded cabbage in coleslaw
Salmonella Beansprouts
Enterohaemorrhagicic E. coli Salads of raw vegetables
Vibrio cholerae Cabbage
Virus hepatitis A Lettuce
growth under the low O2 concentrations within
MA-packages of RTU vegetables.
Sources and rates of contamination
L. monocytogenes is considered ubiquitous in the
environment, being isolated from soil, faeces,
sewage, silage, manure, water, mud, hay, animal
feeds, dust, birds, animals and man (Gray &
Killinger, 1966; Welshimer, 1968; Al-Ghazali &
Al-Azawi, 1990; Nguyen-the & Carlin, 1994;
Gunasena et al., 1995). It is also associated with
plant material including shrubs, wild grasses,
corn, cereals, and decaying vegetation (Welshimer
& Donker-Voet, 1971).
Since L. monocytogenes occurs widely in soil
and in the agricultural environment generally, it
is present naturally on many vegetables. Con-
tamination of vegetables by L. monocytogenes
may occur through agricultural practices, such as
irrigation with polluted water or fertilisation with
contaminated manure (Geldreich & Bordner,
1971; Nguyen-the & Carlin, 1994).
L. monocytogenes has been isolated from a
wide range of intact vegetables (Steinbruegge
et al., 1988; Heisick et al., 1989a; McLauchlin &
Gilbert, 1990; De Simon et al., 1992; Vahidy,
1992; Arumugaswamy et al., 1994; Tang et al.,
1994; Gunasena et al., 1995). Reports of recent
occurrences of L. monocytogenes on produce are
summarised in Table 2, although none of these
products were associated with outbreaks of food
poisoning.
L. monocytogenes has also been isolated from
chilled, RTU vegetables at rates ranging from 0%
(Petran et al., 1988; Farber et al., 1989; Gohil
et al., 1995; Fenlon et al., 1996) to 44% (Sizmur
& Walker, 1988; Beckers et al., 1989; McLauchlin
& Gilbert, 1990; Velani & Roberts, 1991; Harvey
1999 Blackwell Science Ltd International Journal of Food Science and Technology 1999, 34, 122
Microbiological safety of vegetables Gillian A. Francis et al. 3
Country Reference
Canada Schlech et al., 1983
US Ho et al., 1986
Sweden Nguyen-the & Carlin, 1994
US Solomon et al., 1990
UK OMahony et al., 1990
Mexico Nguyen-the & Carlin, 1994
Peru Nguyen-the & Carlin, 1994
US Nguyen-the & Carlin, 1994
& Gilmour, 1993; Arumugaswamy et al., 1994;
MacGowan et al., 1994; Doris & Seah, 1995).
In France, microbiological criteria for prepared
vegetables state that it is desirable to test for the
presence and levels of L. monocytogenes. The
French authorities have recently proposed the use
of a limit of 102 CFU g21 for RTU vegetables at
their use-by-date (Nguyen-the & Carlin, 1994).
In Germany, official recommendations for
microbiological criteria have been published by
the Federal Health Office (Table 3; Lund, 1993).
These include a quantitative test for L. monocy-
togenes. If the organism is present but numbers
are ,102 g21 no further action is taken. If num-
bers are .102 g21 further investigations are made.
The current target in the UK and USA is absence
of L. monocytogenes in 25 g of ready-to-eat foods.
Clostridium botulinum
Cl. botulinum is a member of the genus
Clostridium, characterised as Gram-positive, rod-
shaped, endospore forming, obligate anaerobes
(Varnum & Evans, 1991). The severity and fatal-
ity rate of botulism have been a significant worry
to food processors and consumers since the late
1800s and, as a result, discussion will be restrict-
ed to this species. The foodborne Clostridia are
comprehensively reviewed by McClane (1997)
and Dodds & Austin (1997).
Cl. botulinum is divided into numerous sub-
divisions, based on the serological specificity of
the neurotoxin produced and physiological differ-
ences between strains. Human botulism is nor-
mally attributed to sub-species antigenic types A,
B, E and occasionally type F. Following its
release by the cell, the neurotoxin requires activa-
tion via proteolysis. Proteases may be produced
4 Microbiological safety of vegetables Gillian A. Francis et al.

Table 2 Occurrence of L. monocytogenes on fresh, intact and RTU vegetables

Vegetable Number (and %) of Country and Reference

positive samples observations

Intact vegetables
Beansprouts 6/7 (85%) Malaysia Arumugaswamy et al., 1994
Cabbage 6/18 (33%) Sri Lanka Gunasena et al., 1995
1/92 (1.1%) US Heisick et al., 1989a
Cucumber 2/92 (2.2%) US Heisick et al., 1989a
1/15 (6.7%) Pakistan Vahidy, 1992
Lettuce 10/20 (50%) Sri Lanka Gunasena et al., 1995
1/28 (3.6%) Kuala Lumpur Tang et al., 1994
Potatoes 28/132 (21.2%) US Heisick et al., 1989a
19/70 (27.1%) US Heisick et al., 1989b
Radish 19/132 (14.4%) US Heisick et al., 1989a
25/68 (36.8%) US Heisick et al., 1989b
Salad vegetables 2/108 (1.8%) UK Velani & Roberts, 1991
Tomato 2/15 (13.3%) Pakistan Vahidy, 1992
Various vegetables 8/103 (7.8%) Spain De Simon et al., 1992

RTU vegetables
Cucumber slices 4/5 (80%) Malaysia Arumugaswamy et al., 1994
Coleslaw 2/92 (2.2%) Canada Schlech et al., 1983
2/50 (4%) Singapore Doris & Seah, 1995
3/39 (7.7%) UK MacGowan et al., 1994
Pre-packed mixed salads 3/21 (14.3%) Northern Ireland Harvey & Gilmour, 1993
4/60 (6.7%) UK Sizmur & Walker, 1988
Prepared mixed vegetables 8/42 (19%) UK Velani & Roberts, 1991
(contamination during
processing suspected;
,200 g21 present)
Fresh cut salad vegetables 11/25 (44%) The Netherlands Beckers et al., 1989
(,102 g21 present)
Chicory salads (8.8%) France Nguyen-the & Carlin, 1994
(,1 g21 present)
Prepared vegetables 1/26 (3.8%) UK MacGowan et al., 1994
Salad vegetables 4/16 (25%) Northern Ireland Harvey & Gilmour, 1993
Processed vegetables and salads (13%) UK McLauchlin & Gilbert, 1990

by the bacterial cell itself (proteolytic strains-anti-
genic types A, some B and some F), while non-
proteolytic strains (antigenic types E, some B and
some F) rely on external proteases for neurotox-
in activation (Dodds & Austin, 1997).
Sources and rates of contamination
Endospores of Cl. botulinum which provide pro-
tection from adverse conditions are ubiquitous,
being distributed in soils, aquatic sediments and
the digestive tract of animals and birds.
Vegetables are potentially contaminated during
growth, harvesting and processing (Rhodehamel,
1992). Despite their ubiquity, a recent study iden-
tified only 0.36% of pre-cut MA-packaged veg-
etables to be contaminated with Cl. botulinum
spores (Lilly et al., 1996). In the case of mush-
rooms, a much lower incidence of Cl. botulinum
was reported (Notermans et al., 1989) than had
been reported previously (Hauschild et al., 1978),
a change attributed to hygienic improvements in
growing techniques.
Aeromonas hydrophila
Aeromonas hydrophila is a motile, Gram-negative,
rod-shaped bacterium in the family Vibrionaceae.
A. hydrophila causes a broad spectrum of infec-
tions (septicaemia, meningitis, endocarditis) in
humans, often in immunocompromised hosts,
and Aeromonas spp. have been associated epi-
demiologically with travellers diarrhoea.

International Journal of Food Science and Technology 1999, 34, 122 1999 Blackwell Science Ltd

Table 3 Officially recommended microbiological criteria
for prepared, mixed salad vegetables in Germany (Lund,
1993)
Microorganism Limit g21
Total counts at production ,5 3 106
Total counts at retail ,5 3 107
E. coli ,102
Salmonella Absent from 25g
L. monocytogenes ,102 g21, no action
.102 g21, further
investigations made
Recommended shelf life 7 days (including day of
production)
Recommended temperature during 27 8C
transport and sale
Aeromonas as a human pathogen has been
reviewed by Altwegg & Geiss (1989).
A. hydrophila possesses a number of character-
istics of concern in RTU vegetables. It is a
psychrotroph. It grows slowly at 0 8C, but tem-
peratures of 45 8C will support growth in foods.
It is a facultative anaerobe, capable of growing in
atmospheres containing low concentrations of
oxygen.
Sources and rates of contamination
A. hydrophila is considered to be ubiquitous and
has been isolated from many sources. The best
known sources are treated and untreated water,
and animals associated with water, such as fish
and shellfish (ICMSF, 1996). Hazen et al. (1978)
isolated A. hydrophila from the vast majority of
aquatic environments.
A. hydrophila is also associated with soil
(Brandi et al., 1996) and with a range of foods
including fresh vegetables. Foods in which A.
hydrophila was isolated were most likely contam-
inated by water, soil or animal faeces.
The literature contains numerous reports of the
presence of A. hydrophila on vegetables. Callister
& Agger (1987) reported that grocery store pro-
duce may represent an important vehicle in trans-
mission of A. hydrophila gastroenteritis. These
authors detected A. hydrophila on virtually 100%
of 12 different produce items surveyed. Berrang
et al. (1989a) found A. hydrophila to be present
on fresh asparagus, broccoli and cauliflower.
Motile Aeromonas spp. were detected in 47.8% of
vegetable samples (Saad et al., 1995).
1999 Blackwell Science Ltd International Journal of Food Science and Technology 1999, 34, 122
Microbiological safety of vegetables Gillian A. Francis et al. 5
Marchetti et al. (1992) isolated high counts
(103106 g21) of A. hydrophila in commercial RTU
vegetable salads. Aeromonas spp. were also recov-
ered form green salad, coleslaw (Hudson & De
Lacy, 1991), pre-made salad samples (Fricker &
Tompsett, 1989), mayonnaise salad samples
(Knchel & Jeppesen, 1990) and commercial
mixed vegetable salads (Garca-Gimeno et al.,
1996). By contrast, none of the vegetable samples
from shops in Sweden were positive for
Aeromonas spp. (Krovacek et al., 1992).
Escherichia coli 0157:H7
E. coli, type species of the type Entero-
bacteriaceae genus, Escherichia, is a common
inhabitant of the gastrointestinal tract of mam-
mals. Despite the commensal status of the major-
ity of strains, pathogenic strains, particularly
enterohaemorrhagic E. coli 0157:H7, have
emerged as highly significant foodborne
pathogens. Gastroenteritis and haemorrhagic col-
itis are classical symptoms, while complications
including thrombocytopenic purpura and
haemolytic uraemic syndrome have been docu-
mented (Martin et al., 1986), the latter potential-
ly leading to renal failure and death in 35% of
juvenile cases (Karmali et al., 1983; Griffin &
Tauxe, 1991).
Sources and rates of contamination
The principal reservoir of E. coli 0157:H7 is
believed to be the bovine gastrointestinal tract
(Wells et al., 1991; Doyle et al., 1997). Hence,
contamination of associated food products with
faeces is a significant risk factor. Contamination
of, and survival of the organism in, natural water
sources make these potential sources in the distri-
bution of infection, particularly if untreated water
is consumed directly or is used to wash uncooked
foods. Information regarding contamination rates
from RTU vegetables is limited.
Salmonella
Salmonella, a genus of the family Entero-
bacteriaceae, are characterized as Gram-negative,
rod-shaped bacteria. Some pathogenic species of
Salmonella include S. typhimurium, S. enteritidis,
S. heidelberg, S. saint-paul, and S. montevideo.
6 Microbiological safety of vegetables Gillian A. Francis et al.
Normal symptoms of gastroenteritis include diar-
rhoea, nausea, abdominal pain, vomiting, mild
fever and chills (ICMSF, 1996).
Salmonellae are mesophiles, with optimum
temperatures for growth of 3543 8C. The growth
rate is substantially reduced at ,15 8C, while the
growth of most salmonellae is prevented at
,7 8C. Salmonella are facultatively anaerobic,
capable of survival in low O2 atmospheres.
Sources and rates of contamination
Salmonella are abundant in faecal material,
sewage and sewage-polluted water; consequently
they may contaminate soil and crops with which
they come into contact. Sewage sludge may con-
tain high numbers of salmonellae and, if used for
agricultural practices, will disseminate the bacteri-
um. Once introduced into the environment, sal-
monellae remain viable for months (ICMSF,
1996).
Salmonellae are associated naturally with the
bodies of all animals. Salmonellae have been iso-
lated from many mammals, poultry, cattle, birds,
reptiles, fish, amphibians and insects. Food is the
primary vector of Salmonella towards humans,
especially foods of animal origin and those subject
to sewage pollution.
Salmonellae have been isolated from several
fresh intact vegetables (Doyle, 1990; Tauxe, 1991).
In a study of whole vegetables sampled in the field
or in retail outlets in Spain, 7.5% of total samples
were contaminated with Salmonella spp. (Garcia-
Villanova Ruiz et al., 1987). In a survey of veg-
etables from shops in Italy, 68% of lettuce and
72% of fennel samples carried Salmonella spp.
(Ercolani, 1976). Salmonella were isolated from
leafy vegetables (4%) and beansprouts (20%) in
Malaysia (Arumugaswamy et al., 1994). In Egypt,
vegetables and salads were found contaminated
with Salmonella spp. (Saddik et al., 1985).
Yersinia enterocolitica
Yersinia enterocolitica is currently considered to
be the most significant genus member with respect
to foodborne disease (Varnum & Evans, 1991).
Traditional gastrointestinal symptoms, potential-
ly mediated through the activity of a heat-stable
enterotoxin, may develop into suppurative and
autoimmune complications (Robins-Browne,
1997). The psychrotrophic status of Y. enterocol-
International Journal of Food Science and Technology 1999, 34, 122
itica is of great significance with regard to refrig-
erated RTU vegetables.
Sources and rates of contamination
Y. enterocolitica occupies a broad range of
ecosystems including the intestinal tract, birds,
flies, fish and a variety of terrestrial and aquatic
ecosystems. However, most environmental iso-
lates lack virulence markers and are of doubtful
significance for human or animal health (Delmas
& Vidon, 1985). Isolation of Yersinia spp. from
raw vegetables has been reported at rates ranging
from 3.3% (Tassinari et al., 1994) to 46.1%
(Delmas & Vidon, 1985), although specific isola-
tion rates of pathogenic Y. enterocolitica strains
are likely to be significantly lower.
Campylobacter jejuni
Since their principal identification as human gas-
trointestinal pathogens in the 1970s (Butzler et al.,
1973; Skirrow, 1977) members of the thermophilic
campylobacters, e.g. Camp. jejuni, have emerged as
major human gastrointestinal pathogens (Ketley,
1997). The disease symptoms associated with these
small, Gram-negative, spiral microaerophilic bac-
teria vary, from self limiting gastrointestinal symp-
toms to Guillain Barre syndrome, the latter being
characterised by symmetrical ascending paralysis
(Ho et al., 1995). Despite fastidious growth
requirements, members of the genus survive at
refrigeration temperatures for extended periods
within nutrient limited environments. This proper-
ty, combined with the low infective dose
(Robinson, 1981) and their microaerophilic nature
indicates the potential significance of the genus
with respect to refrigerated RTU vegetables.
Sources and rates of contamination
Campylobacter are zoonotic pathogens, being pri-
marily associated with the intestinal tracts of wild
and domestic animals (Thomas et al., 1995) and
are distributed throughout the environment via
vehicles including birds, surface water and flies.
Inappropriate food preparation and handling
procedures may lead to the cross-contamination
of fresh produce with Campylobacter from
uncooked meats, and such errors could have
resulted in the identification of RTU vegetable
products as sources of infection (Bean & Griffin,
1999 Blackwell Science Ltd

1990; Altrkruse et al., 1994). However, despite
this, no comprehensive evaluation of the con-
tamination rates of RTU vegetable with
Campylobacter spp. has been published.
Viral pathogens
Foodborne viral infection is generally propagated
through the faecal-oral route, via direct contact
or via consumption of contaminated food and
water. The significance of the group with respect
to foodborne disease is clear with the inclusion of
Norwalk virus, Hepatitis A virus and other virus-
es within the top ten causes of foodborne disease
outbreaks in the USA (19831987; Cliver, 1997).
However, despite their significance, data regard-
ing the effects of food production environments,
food preparation and storage conditions on the
survival and infectivity of viruses is extremely lim-
ited, partly through the complexity of viral detec-
tion assays. Nonetheless, outbreaks including the
infection of 110 individuals with Hepatitis A, fol-
lowing the consumption of contaminated lettuce
and the potential of virus particles to survive for
extensive periods on vegetables stored at 4 and
20 8C (Sattar et al., 1994) have been identified.
Protozoan pathogens
One group of foodborne pathogens which have
received little attention in developed countries is
the parasites (Brackett, 1987a). Common para-
sites that occur on fresh vegetables include
Giardia lamblia, Entamoeba histolytica and
Ascaris spp. These organisms normally gain
access to vegetables before harvest, usually as a
result of contaminated irrigation water and poor
hygiene practices. In one outbreak of giardiasis,
epidemiologic evidence has implicated an asymp-
tomatic food handler as the probable source of G.
lamblia and raw sliced vegetables as the vehicle of
transmission (Beuchat, 1996).
Outbreaks of foodborne disease associated
with vegetables
The first conclusively documented foodborne out-
break of listeriosis occurred in the Maritime
provinces of Canada in 1981 and involved 34
perinatal cases and seven adult cases. The vehicle
of transmission was coleslaw and the source of L.
1999 Blackwell Science Ltd International Journal of Food Science and Technology 1999, 34, 122
Microbiological safety of vegetables Gillian A. Francis et al. 7
monocytogenes was stored cabbage (Schlech et al.,
1983).
An outbreak of L. monocytogenes infection
during the summer of 1979, involving 23 patients
from eight Boston hospitals, was reported by Ho
et al. (1986). This outbreak was attributed to the
consumption of raw salad vegetables (celery,
tomatoes, lettuce), but no attempt was made to
isolate L. monocytogenes from the vegetables at
the time of the outbreak. It was suspected that an
elderly patient in a London hospital acquired lis-
teric septicaemia after consuming contaminated
lettuce (Fain, 1996).
Outbreaks of botulism have been attributed to
vegetables packaged under MAs. Fresh agaric
mushrooms packaged in plastic films were impli-
cated in an outbreak (Sugiyama & Yang, 1975).
Shredded cabbage, asparagus, broccoli, tomatoes,
lettuce and cabbage have also been considered as
potential vehicles of botulinal toxin (Solomon
et al., 1990; Hotchkiss & Banco, 1992; Petran
et al., 1995).
The majority (32%) of food associated E. coli
0157:H7 outbreaks in the USA, between 1982 and
1994, were attributed to the consumption of
ground beef, however, approximately 6% of out-
breaks were associated with the consumption of
vegetables or salads (Doyle et al., 1997).
Recently, a large outbreak was associated with
alfalfa sprouts (Como-Sabetti et al., 1997). The
origin of contamination in such cases was likely
to result from the application of contaminated
manure/water to the crops, coupled with inade-
quate washing procedures.
Salmonellae are one of the most frequently
reported causes of foodborne outbreaks of gas-
troenteritis. A number of outbreaks of salmonel-
losis have been attributed to vegetables. An
outbreak of salmonellosis in the UK in 1988 was
associated with consumption of beansprouts
(attributed to S. saint-paul and S. virchow PT34;
OMahony et al., 1990). Epidemiological studies
have linked two multi-state outbreaks of salmo-
nellosis to raw tomatoes (Wood et al., 1991;
Hedberg et al., 1994). A number of general out-
breaks of infectious intestinal disease, in England
and Wales between 1992 and 1996, have been
associated with coleslaw and other salads conta-
minated with S. enteritidis and S. enteritidis PT4
(Evans, personal communication).
8 Microbiological safety of vegetables Gillian A. Francis et al.
In England and Wales, no reported outbreaks
of yersiniosis were attributed to raw vegetables
between the period of 19921996. Nonetheless, an
outbreak involving 16 individuals which occurred
in Pennsylvania in 1982 was strongly associated
with beansprouts and well water (Cover & Aber,
1989). Salads were implicated with a large
outbreak of campylobacteriosis in Canada
(Anonymous, 1985).
Factors affecting the growth of
pathogens on RTU vegetables
Raw vegetables may be contaminated with
pathogens when they enter the processing chain
(Nguyen-the & Carlin, 1994). The product itself
and its source will often determine the numbers
and types of microorganisms initially present.
Analyses at different stages of processing show
that the end product is frequently less contami-
nated than the raw vegetable (King et al., 1991).
However, contamination with pathogens may
occur during processing and distribution; scrupu-
lous hygiene must be applied at all levels of pro-
cessing.
The development of food poisoning microor-
ganisms on RTU vegetables depends on the prop-
erties of the microorganisms, on the intrinsic
properties of vegetables and on the effects of pro-
cessing, packaging and storage. Each produce
item may go through a series of processing steps,
including handling, equipment contact (slicing),
washing, packaging and storage. Each of these
treatments may affect microbial colonisation and
survival/growth. The more unit operations a
product undergoes, the more its flora will reflect
that of the environment in which it is produced.
Processing
Handling, particularly if done improperly, can
damage fresh produce, rendering it more suitable
for growth/survival of spoilage and pathogenic
microorganisms (Brackett, 1994). Damage of veg-
etables can occur during harvesting, packaging,
transportation and storage.
Contamination during processing may occur
from unhygienic personnel or through product-
to-machine impacts (e.g. inadequately cleaned
equipment). Slicing and shredding destroy surface
International Journal of Food Science and Technology 1999, 34, 122
cells, bruise underlying layers and allow juices to
leak from inner tissues on to equipment and on
to fresh-cut products. Cutters and slicers can be
potent sources of contamination because they
usually provide inaccessible sites which harbour
bacteria (Garg et al., 1990). The presence of cut
surfaces allows microbial infiltration and provides
an increased surface area for contamination and
growth (Brackett, 1994). Moisture and exudate
on cut surfaces and on surfaces of utensils and
equipment provide excellent media for rapid
growth of microorganisms. Therefore, during the
processes of slicing or shredding, there may be an
increase in the number of bacteria present.
Exposing vegetables to various types of cutting
resulted in a six- to seven-fold increase in micro-
bial populations (Brackett, 1994). Garg et al.
(1990) reported that shredders used to prepare
shredded lettuce and cabbage were major sources
of contamination in a factory producing RTU
vegetables.
L. monocytogenes may be present and can sur-
vive in the environment of many types of food
processing plants, particularly in damp, moist
conditions (Walker & Stringer, 1990; Farber &
Peterkin, 1991). It has been recovered from the
environment of processing operations used to pre-
pare RTU vegetables (Zhang & Farber, 1996).
Slicing operations may increase contamination of
RTU vegetables by L. monocytogenes. For exam-
ple, the pathogen was detected in 19% of pre-
pared mixed vegetables examined, whereas only
1.8% of individual ingredients were contaminated
(Velani & Roberts, 1991). The authors concluded
that substantial contamination by L. monocyto-
genes occurred during chopping, mixing and
packaging. A case was reported where L. mono-
cytogenes was regularly found in shredded cab-
bage and on shredding machines in the processing
line (Nguyen-the & Carlin, 1994). Nguyen-the
et al. (1996) reported the microbiological conse-
quences of mixing cooked vegetables with raw
vegetable salads. These authors observed that L.
monocytogenes grew better at 6 and 9 8C in a
mixed salad (raw endive and sweetcorn) than on
raw endive alone.
Salmonellae can become established and multi-
ply in the environment and equipment of a vari-
ety of food-processing facilities, potentially
cross-contaminating raw foods during processing
1999 Blackwell Science Ltd

Table 4 Microbial specifications
Microorganisms
for prepared salad vegetables in
France (Nguyen-the & Carlin,
1994).c: maximum allowable
Total counts at production
number of samples between m
Salads & shredded vegetables
and M for acceptance. m000:
Parsley, watercress &
value separating good quality
aromatic herbs
from marginally acceptable
Total counts at use-by-date
quality
Salads
Coliforms at 44.5 8C
Salmonella in 25g
(ICMSF, 1996). However, foods of animal origin
are the primary vector of Salmonella towards
humans, and therefore, a factory dedicated to
producing RTU vegetables would not present the
same potential contamination opportunities as
one producing products in which meats might be
included. Unfortunately, very little information is
available on effects of processing on the sur-
vival/growth of pathogens on RTU vegetables.
Recommendations implemented to ensure good
hygiene and quality of RTU vegetables relate to
manufacturing conditions, microbial specifica-
tions for the processed product, and storage
(Nguyen-the & Carlin, 1994). Conditions for pro-
duction of RTU vegetables can be regulated by
good manufacturing practices (GMP) as recom-
mended by Koek et al. (1983) to minimise conta-
mination.
Microbiological criteria for prepared, mixed
salad vegetables have been published in some
European countries, for example France and
Germany. In France (Table 4), microbial specifi-
cations for mesophilic flora and coliforms are
defined for RTU vegetables at the date of pro-
cessing and at the use-by-date (after storage at
the recommended temperature i.e. between 0 and
4 8C). In a revision of these criteria it is proposed
to replace the limit for coliforms growing at
44.5 8C by the same limit for E. coli (Nguyen-the
& Carlin 1994).
Washing and antimicrobial dip treatments
An important step in the processing of RTU veg-
etables is thorough washing, often accompanied
by dipping in an antimicrobial solution, to reduce
microbiological load. Washing vegetables with
water containing a free chlorine level of 100
mg l21 has been reported to reduce the number of
1999 Blackwell Science Ltd International Journal of Food Science and Technology 1999, 34, 122
Microbiological safety of vegetables Gillian A. Francis et al. 9
Sample size c Limit per gram
m M
5 2 5 3 105 5 3 106
5 2 5 3 106 5 3 107
5 a a 5 3 107
5 2 10 103
5 None None None
bacteria on lettuce leaves by up to 100-fold
(Adams et al., 1989). Dipping produce in ascor-
bic/citric acid solutions prior to packaging great-
ly reduced aerobic and anaerobic plate counts
and these differences persisted during storage
(McLachlan & Stark, 1985; OBeirne &
Ballantyne, 1987). Strict hygiene is critical during
washing and disinfection in order to avoid conta-
mination of produce. Chlorinated water from the
washing bath on a line processing chicory salads
was found to contain 103 bacteria ml21 (Nguyen-
the & Prunier, 1989). Washing produce in such a
washing bath may result in the transfer of
microorganisms, due to the re-use of wash water.
Ultraviolet radiation may be useful in inactivat-
ing vegetative bacterial cells in wash water, avoid-
ing cross-contamination.
There are theoretical concerns regarding the
safety of the use of antimicrobial dips. For exam-
ple, pathogens if present on raw vegetables, may
not be fully eliminated by commercial disinfection
procedures, while at the same time the effects of
disinfection on the indigenous microflora may be
to reduce or remove natural competitive organ-
isms. As a result, disinfection may produce con-
ditions which favour survival/growth of the
pathogen.
Effects of sodium hypochlorite on L. monocy-
togenes have been widely studied in vitro and were
shown to be very effective (Brackett, 1987b; El-
Kest & Marth, 1988a,b; Tuncan, 1993). However,
the elimination of L. monocytogenes from the sur-
face of vegetables by disinfection is limited and
unpredictable (Nguyen-the & Carlin, 1994). This
ineffectiveness may be due to a number of fac-
tors; an aqueous hypochlorite solution may not
wet the hydrophobic surface of the waxy cuticle
or penetrate into the crevices, creases, and natur-
al openings of the vegetable (Adams et al., 1989).
10 Microbiological safety of vegetables Gillian A. Francis et al.
The most useful effect of chlorine may be in inac-
tivating vegetative cells in washing water and on
equipment during processing as part of a hazard
analysis critical control points (HACCP) system,
thus avoiding build-up of bacteria and cross-con-
tamination (Wilcox et al., 1994).
Brackett (1987b) examined effects of chlorine
on Brussel sprouts contaminated with L. mono-
cytogenes. Brussel sprouts were inoculated with
L. monocytogenes, dried and immersed in a
200 mg l21 chlorinated solution. The counts of L.
monocytogenes were reduced approximately 100-
fold, 10-fold more than those treated with water.
In addition, L. monocytogenes was undetectable
in the chlorinated solution or on Brussel sprouts
subsequently immersed in the same chlorinated
solution after inoculated samples. Zhang &
Farber (1996) examined the effects of various dis-
infectants against L. monocytogenes on fresh-cut
vegetables. For chlorine (200 ppm, 10 min), the
maximum observed log10 reductions of L. mono-
cytogenes at 4 8C and 22 8C, respectively, were 1.3
and 1.7 for lettuce and 0.9 and 1.2 for cabbage.
Thus, the bactericidal effect on L. monocytogenes
was higher at 22 8C than at 4 8C, and was higher
on lettuce than on cabbage.
In relation to post-disinfection contamination,
some authors have reported that disinfection of
salad leaves or tomato slices with chlorine prior
to inoculation with L. monocytogenes did not
affect its subsequent growth during storage
(Beuchat & Brackett, 1990; 1991).
By contrast, Carlin et al. (1996b) found that L.
monocytogenes inoculated on to disinfected (10%
hydrogen peroxide) endive leaves, grew better
than on water-rinsed produce, and gave higher
counts after 7 days at 10 8C than on leaves rinsed
with water. Similarly, Francis & OBeirne (1997)
found that dipping shredded lettuce in a chlorine
(100 ppm) or citric acid (1%) solution followed by
storage at 8 8C, resulted in significant increases in
Listeria populations compared with undipped
samples.
Effects of organic acids as disinfectants on the
growth of L. monocytogenes on produce, has
received little attention. Lactic and acetic acid
(1%) reduced numbers of L. monocytogenes on
lettuce by only 0.5 and 0.2 log cycles respectively,
and were therefore not very effective (Zhang &
Farber, 1996).
International Journal of Food Science and Technology 1999, 34, 122
Salmonellae are readily killed by disinfectants
commonly used in the food industry (ICMSF,
1996). Zhuang et al. (1995) examined the efficacy
of chlorine treatment on inactivation of S. mon-
tevideo on and in tomatoes. Populations of S.
montevideo on the surface and in the core tissues
were substantially reduced by dipping tomatoes in
solutions containing 60 or 110 ppm chlorine,
respectively; however, treatment in solutions con-
taining 320 ppm chlorine did not result in com-
plete inactivation.
The efficacy of buffered sodium hypochlorite
solution (Bionox), in controlling S. enteritidis
contamination on vegetables (carrot, zucchini)
was studied by Park et al. (1991). These authors
found that the sanitizing solution was very effec-
tive in reducing Salmonella levels on vegetables
(counts following 20 min exposure to the sanitiz-
ing solution were ,2 g21).
Beuchat & Ryu (1997) examined the efficacy of
chlorine in removing Salmonella from alfalfa
sprouts. Sprouts were dipped in solutions con-
taining 200, 500 or 2,000 ppm chlorine for 2 min.
The pathogen was reduced by about 2 log10 CFU
g21 after treatment with 500 ppm chlorine, com-
pared with the control, and to undetectable levels
after treatment with 2000 ppm chlorine.
The response of Aeromonas spp. to sodium
hypochlorite, iodoform and 2-chlorophenol
revealed the sensitivity of these organisms to dis-
infectants. Treatment with 5 ppm hypochlorite
inactivated Aeromonas cells in 1 min at 25 8C
(ICMSF, 1996). However, the impact of disinfec-
tion on the survival of A. hydrophila, E. coli
0157:H7 or other pathogens on RTU vegetables
is poorly understood.
The tolerance of E. coli 0157:H7 to acidic pH
is well documented (Zhao et al., 1993; Conner &
Kotrola, 1995), particularly with respect to
organic acid sprays applied to beef surfaces
(Brackett et al., 1994). Survival of the organism
for 35 days when held at 5 8C in mayonnaise
based sauces (pH ,4.0) has been reported
(Weagant et al., 1994). Hence, following cross-
contamination, such foods may act as significant
vehicles of infection, particularly in the context of
salad bars.
The sensitivity of Y. enterocolitica to low pH
conditions, with a reported growth range of pH
4.49.0 (Stern et al., 1980), has led to the pro-
1999 Blackwell Science Ltd

posal that an environment ,pH 4.0 as a growth
controlling factor in foods (Neilsen & Zeuthen,
1985). Indeed, the organism demonstrates poor
survival characteristics in artificially inoculated
acid salad dressings (Aldova et al., 1975;
Brackett, 1986).
Camp. jejuni demonstrates growth down to a
pH of 4.9 in laboratory media and the rate of
vegetative cell inactivation below this point is
temperature dependent, being slowest at refriger-
ation temperatures (Gill & Harris, 1982).
However, the impact of pH and disinfection
treatments on the subsequent survival of this and
other pathogens in foods at refrigeration temper-
atures requires further evaluation.
MA-packaging
Potential effects of MA technology on
survival/growth of pathogens are poorly under-
stood. MA-packaging of refrigerated RTU veg-
etables might be cause for public health concern
in at least three ways. The gas atmospheres and
refrigeration temperatures employed may inhibit
the development of some spoilage aerobic
microorganisms (Daniels et al., 1985; Farber,
1991). Some of these microorganisms may be nat-
ural competitors of pathogens and their suppres-
sion may facilitate pathogen survival/growth,
without the product showing obvious signs of
spoilage. Secondly, MA-packaging increases the
shelf-life of products, thus increasing the time
available for pathogens to grow. Over-extending
the shelf-life may allow development of significant
populations. Thirdly, although the low levels of
oxygen (25%) within MA-packages of vegetables
(e.g. at 4 8C) should inhibit growth of obligate
anaerobes such as Cl. botulinum, if MA-packages
are subjected to temperature abuse, they may
become anaerobic as a result of increased product
respiration. This could enable growth and toxin
production by Cl. botulinum to occur. Of particu-
lar concern is the growth of psychrotrophic, fac-
ultative anaerobes, which can tolerate low O2
modified atmospheres (Bennik et al., 1995). A
number of studies have indicated that MA-pack-
aging may select for such psychrotrophic
pathogens (Hintlian & Hotchkiss, 1986; Beuchat
& Brackett, 1990; Kallender et al., 1991; Brackett,
1994).
1999 Blackwell Science Ltd International Journal of Food Science and Technology 1999, 34, 122
Microbiological safety of vegetables Gillian A. Francis et al. 11
Beuchat & Brackett (1990) showed that popu-
lations of L. monocytogenes on lettuce significant-
ly increased during storage but MA-packaging
(3% O2, 97% N2) did not influence the rate of
growth. Nonetheless, because MA-packaging
reduced the rate of organoleptic deterioration, the
risk of listeriosis may be increased due to the
increased time available for growth. Similarly,
survival/growth of L. monocytogenes on vegeta-
bles was not influenced by MA-packaging
(97% N2, 3% O2) compared with those packaged
in air (Berrang et al., 1989b; Beuchat & Brackett,
1991).
By contrast, Ringl et al. (1991) found that L.
monocytogenes did not grow at either 8 or 4 8C on
shredded chicory salads packaged in air using a
semi-permeable film, but did grow when the prod-
uct was packaged after flushing with nitrogen.
Similarly, Francis & OBeirne (1997) reported
that nitrogen flushing combined with storage at
8 8C, enhanced the growth of L. monocytogenes
on shredded lettuce. In work by Carlin et al.
(1996a), chicory leaves inoculated with L. mono-
cytogenes were stored at 10 8C in air, or under
10%, 30% or 50% CO2, with 10% O2. L. monocy-
togenes grew better as the concentration of CO2
increased. Some of the inconsistencies in the liter-
ature cited above highlight the need for more
research in order to better understand the poten-
tial effects of MA packaging on the survival of
L. monocytogenes.
Several studies have demonstrated that A.
hydrophila can grow on vegetables stored at
45 8C under MAs. For example, Berrang et al.
(1989a) examined the effects of gas atmospheres
on growth of A. hydrophila on fresh asparagus,
broccoli and cauliflower. A. hydrophila reached
high populations on all three vegetables during
storage at 4 and 15 8C, but growth was not sig-
nificantly affected by gas atmosphere. However,
the authors noted that controlled atmosphere
storage extended the length of time the vegetables
were considered acceptable for consumption.
Bennik et al. (1995) studied effects of gas atmos-
pheres on growth of A. hydrophila, using a solid
surface model system. The maximum specific
growth rate of A. hydrophila decreased with
increasing CO2 concentrations, but maximum
population densities were not affected by CO2
concentrations up to 50%. Growth patterns of A.
12 Microbiological safety of vegetables Gillian A. Francis et al.
hydrophila were virtually the same under 1.5%
and 21% O2.
Although it is generally accepted that Cl. botu-
linum cannot grow in food exposed to atmos-
pheric oxygen levels, the redox potential of most
foods is usually sufficiently low to facilitate
growth and hence toxigenesis (Kim & Foegeding,
1993). Optimal growth is evident at Eh of
2350mV (Smoot & Pierson, 1979) but is possible
in the range of 130 to 1250mV. The growth of
the aerobic spoilage microflora rapidly decreases
food Eh, improving conditions for the growth of
Cl. botulinum. Hyperbaric CO2 levels inhibit Cl.
botulinum, while growth stimulation under atmos-
pheric CO2 levels has been described (Sperber,
1982). Atmospheres modified passively through
the respiration of fresh mushrooms packed in
polyvinylchloride potentially create conditions
suitable for growth (Sugiyama & Yang, 1975).
These findings prompted the USFDA to recom-
mend the puncturing of packaging films applied
to mushrooms. More recently Larson et al. (1997)
identified a probability of ,1 in 105 for toxin pro-
duction to occur in MA-packaged vegetables.
Little work has been done on effects of atmos-
phere composition on growth of other foodborne
pathogens on RTU vegetables. CO2 had no
inhibitory effect on growth of E. coli 0157:H7 on
shredded lettuce stored at 13 or 22 8C, and
growth potential was increased in an atmosphere
of O2/CO2/N2: 5/30/65, compared with growth in
air (Abdul-Raouf et al., 1993; Diaz & Hotchkiss,
1996). This illustrates the failure of 10% CO2 to
inhibit E. coli 0157:H7 at refrigeration tempera-
tures (Hao & Brackett, 1993).
Piagentini et al. (1997) showed that S. hadar
grew well on minimally processed cabbage, pack-
aged in mono-oriented polypropylene bags.
Tamminga et al. (1978) showed that S. typhimuri-
um developed well on peeled, steamed, vacuum-
packed potatoes, at 5, 14 and 22 8C. The
literature contains many reports on the ability of
Salmonella to grow under MAs on meat products
(Silliker & Wolfe, 1980; Gray et al., 1984; Baker
et al., 1986) In general, increased CO2 concentra-
tions (50100%), reduced growth but these con-
centrations would cause damage to most RTU
vegetables.
Information describing the survival characteris-
tics of Y. enterocolitica on RTU vegetables stored
International Journal of Food Science and Technology 1999, 34, 122
under differing atmospheric conditions remains
extremely limited. On meat products, Y. entero-
colitica has been shown to rapidly proliferate at
4 8C in air, and under vacuum and MAs and
atmospheres containing 4050% CO2 were
described to have minimal inhibitory effects
(Manu-Tawiah et al., 1993; Shenoy & Murano,
1996;). These findings are in agreement with those
of Gill & Reichel (1989), who demonstrated the
ability of Y. enterocolitica to grow at 5 8C under
CO2, although conflicting evidence has been
reported (Bodnaruk & Draughon, 1998). Storage
of products below 0 8C under vacuum or below
2 8C under CO2 should be sufficient to inhibit
Yersinia (Gill & Reichel, 1989). The severity of
these conditions may exclude their application to
many RTU vegetables but the lower availability
of nutrients, compared to meat, is likely to
enhance the beneficial influence.
Conflicting results are evident in the literature
on the growth and survival of Campylobacter in
food products packaged under different atmos-
pheres at different temperatures (Farber, 1991).
At refrigeration temperatures, survival on inocu-
lated turkey was extended in atmospheres of
4060% CO2 or 100% N2 in comparison to air
(Phebus et al., 1991). However, studies to date
have been limited to poultry and other meat
products.
MA-packaging of RTU vegetables is a novel
technology, and more research is needed to
determine its effects on growth of pathogens and
spoilage microorganisms and their interactions in
these products.
Storage temperature
Storage temperature is probably the single most
important factor affecting the growth of micro-
organisms in RTU vegetables. Results obtained
on shredded chicory salads (Nguyen-the & Carlin,
1994), carrot slices (Buick & Damoglou, 1987),
and shredded lettuce (Bolin et al., 1977; Beuchat
& Brackett, 1990), showed that growth of the
mesophilic microflora was significantly reduced as
storage temperature decreased.
French regulations imposed 8 8C as a maxi-
mum storage temperature for RTU vegetables in
1987. This limit was lowered to 4 8C in 1988, but
RTU vegetables are often stored or distributed at
1999 Blackwell Science Ltd

higher temperatures. In the UK, according to the
Food Hygiene Regulations, retail packs of pre-
pared salad vegetables should be maintained at
temperatures below 8 8C. Guidelines for handling
chilled foods, published by the UK Institute of
Food Science and Technology (IFST, 1990), rec-
ommend a storage temperature range of 05 8C
for prepared salad vegetables, noting that some
vegetables may suffer damage if kept at the lower
end of this temperature range.
Storage of RTU vegetables at adequate refrig-
eration temperatures, will limit pathogen growth
to those which are psychrotrophic. Previous work
has shown that L. monocytogenes survives or
grows on a range of RTU vegetables, such as ice-
berg lettuce (Steinbruegge et al., 1988; Beuchat &
Brackett, 1990), shredded cabbage (Kallender
et al., 1991) and on a range of intact fresh veg-
etables (Berrang et al., 1989b) at refrigeration
temperatures. Although capable of growth at low
temperatures, reducing the storage temperature
will extend the lag phase and reduce the rate of
growth (Beuchat & Brackett, 1990; Carlin et al.,
1995). Conversely, temperature abuse during stor-
age will markedly reduce the lag and generation
times and permit more rapid growth of L. mono-
cytogenes.
Temperature profiles of Cl. botulinum vary with
physiological group. Proteolytic strains have opti-
mal growth temperatures of 3540 8C and demon-
strate a growth and toxigenic potential at
temperatures between 10 and 50 8C (Pierson &
Reddy, 1988; Hatheway, 1993;). Non-proteolytic
strains have an optimal temperature of 2530 8C,
with a range of 3.345 8C, defining them as psy-
chrotrophs and hence capable of growth under
refrigeration conditions. Furthermore, toxigenesis
by non-proteolytic strains was detected within
1034 days at 8 8C and 1720 days at 5 8C fol-
lowing the inoculation of cooked vegetables
(Carlin & Peck, 1996).
Aeromonads grow at temperatures between 2
and 45 8C (ICMSF, 1996). Eddy (1960) observed
the growth of several environmental and food
strains at 1 8C. Growth of A. hydrophila on fresh
cauliflower, broccoli and asparagus stored under
a controlled atmosphere at 4 and 15 8C has been
reported by Berrang et al. (1989a). Growth was
slower at 4 8C than at 15 8C. Garca-Gimeno
et al. (1996) followed survival of A. hydrophila on
1999 Blackwell Science Ltd International Journal of Food Science and Technology 1999, 34, 122
Microbiological safety of vegetables Gillian A. Francis et al. 13
vegetable salads stored under MAs at 4 and
15 8C. A. hydrophila survived but did not grow on
samples stored at 4 8C. By contrast, it grew on
salads stored at 15 8C, from ,103108 CFU g21 in
the first 24 h, with a subsequent decline in popu-
lation. Callister & Agger (1987) observed that
populations of Aeromonas spp. on various veg-
etables increased 10- to 1000-fold during two
weeks storage at 5 8C. Counts of A. hydrophila
on RTU vegetables increased from 103 to 104
CFU g21 and 106 CFU g21 in 7 days at 53 8C
(Marchetti et al., 1992).
Y. enterocolitica is a psychrotroph of potential-
ly great significance with respect to food safety. A
minimum growth temperature of 4 8C is general-
ly accepted but growth at 1 8C, with a doubling
time of ,40 h, was observed (Schiemann, 1989).
This illustrates the essential requirement for strict
temperature control, particularly in the case of
extended shelf-life products devoid of additional
growth limiting hurdles.
Growth of Campylobacter spp. within foods at
,30 8C has not been reported. However, the
organism can survive at refrigeration tempera-
tures in both nutrient rich (ground beef) and
nutrient limited (de-ionised water) conditions for
extended periods (Christopher et al., 1982;
Thomas & Mabey, 1996).
Mesophilic pathogens are unable to grow
where temperature control is adequate (i.e. 4 8C
or less). The minimum growth temperature of sal-
monellae is reported to be 5.2 8C (ICMSF, 1996).
Survival and slow growth in produce stored for
extended periods in chilled conditions (within the
growth range for Salmonella) may be of concern.
On a range of vegetables, Salmonella consistently
survived for more than 28 days at 24 8C
(ICMSF, 1996). Populations of S. hadar on min-
imally processed cabbage were not different from
initial levels after 10 days storage at 4 8C
(Piagentini et al., 1997). On samples stored at 12
and 20 8C, Salmonella populations increased by 4
and 6 logs respectively after 10 days storage
(Piagentini et al., 1997). Populations of S. monte-
video on the surfaces of inoculated tomatoes
stored at 10 8C did not change significantly over
18 days. However, significant increases in popu-
lation occurred within 7 days when tomatoes
were stored at 20 8C (Zhuang et al., 1995).
The mesophilic status of E. coli 0157:H7 makes
14 Microbiological safety of vegetables Gillian A. Francis et al.
growth in RTU vegetables unlikely under good
refrigeration. Growth has been reported at 8 8C
(Weeratna & Doyle, 1991) on minced beef. The
substantial amounts of nutrients provided
through the processing (shredding) of lettuce may
facilitate growth of E. coli 0157:H7 on such prod-
ucts (Abdul-Raouf et al., 1993).
Besides its direct effect on microbial growth,
storage temperature also determines respiration
rate of the product and, therefore, changes in
gaseous atmospheres within the package, and this
in turn may influence the behaviour of pathogens.
In general, inhibitory effects of MAs increase as
the storage temperature is lowered (Dixon & Kell,
1989).
Because of difficulties in maintaining low tem-
peratures in the chill-chain, additional barriers to
control the growth of pathogens (particularly psy-
chrotrophs) are required. Combining beneficial
intrinsic, processing and extrinsic inhibitory
factors results in considerable improvement in
the microbial safety of the product. Suitable
combinations of growth-limiting factors at sub-
inhibitory levels can inhibit certain microorgan-
isms through the Hurdles Concept (Scott, 1989).
Theoretically, each hurdle contributes to the
eventual inhibition of a microorganism and all
the hurdles must be overcome in order to grow.
A diagrammatical representation of this concept
is presented in Fig. 2.
Interactions between the indigenous
microflora and pathogen
Interactions between pathogens and background
microflora have been studied in meat and dairy
products (Nguyen-the & Carlin, 1994), but there
is little data on vegetable products.
The antagonistic properties of lactic acid bac-
teria have been reviewed recently (Piard &
Desmazeaud, 1992). Lactic acid bacteria exert
antimicrobial effects due to one or more of the
following mechanisms: lowering pH (Raccach &
Baker, 1979); generating hydrogen peroxide
(Price & Lee, 1970); competing for nutrients
(Iandolo et al., 1965); and possibly by producing
antimicrobial compounds, such as bacteriocins or
antibiotics (Klaenhammer, 1988; Harris et al.,
1989; Arihara et al., 1993). Lactic acid bacteria
are naturally present on vegetables, though usu-
International Journal of Food Science and Technology 1999, 34, 122
Figure 2 The hurdle concept (Scott, 1989).
aw: water activity; MAP: modified atmosphere packaging;
mf: presence of competitive microflora; t: storage
temperature.
Here each hurdle is of different intensity, and the main
hurdles are MA-packaging and storage temperature.
ally at low numbers. They can reach high counts
on some RTU products, in particular products
within MA-packages containing high CO2 con-
centrations (Carlin et al., 1990), where they may
grow faster than the aerobic spoilage bacteria.
Thus, the use of a particular gas atmosphere may
select for proliferation of desired subpopulations,
resulting in suppression of undesirable pathogens.
Competitive effects of lactic acid bacteria may
limit pathogen growth on RTU vegetables, but
there is insufficient data available to prove this
conclusively.
Pseudomonads are major components of the
indigenous microflora of RTU vegetables
(Nguyen-the & Carlin, 1994) and are capable of
excreting cell wall degrading enzymes and thus
releasing nutrients (Liao et al., 1993). Since
pseudomonads are relatively sensitive to CO2
(Enfors & Molin, 1980), their suppression can
influence nutrient availability for other microor-
ganisms (e.g. pathogens). Marshall & Schmidt
(1991), working with milk, found that addition of
pseudomonads increased the multiplication of L.
monocytogenes. These authors proposed that pro-
teolytic pseudomonads supplied free amino acids.
There are no similar results for vegetables. Using
an endive leaf medium, Bennik et al. (1996) found
that the addition of strains of fluorescent
pseudomonads very slightly reduced the final
population density of L. monocytogenes. The
authors also reported that competition of the
background microflora with L. monocytogenes
resided mostly with enterobacteria and not with
pseudomonads. Francis & OBeirne (1998)
reported that competition of the background
microflora of lettuce with L. monocytogenes
resided primarily with Enterobacter species.
The growth of the background microflora also
significantly affected the growth and toxigenesis
of Cl. botulinum in refrigerated foods (Hauschild,
1999 Blackwell Science Ltd

1989; Hutton et al., 1991). Competition for nutri-
ents and the production of inhibitory substances
(fermentation end products, bacteriocins and
fatty acids) play a role in limiting toxigenesis.
However, the growth of acid tolerant yeast and
mould populations in low acid products increases
product pH, making the environment more suit-
able for toxigenesis (Huhtanen et al., 1976).
Growth rates of Y. enterocolitica are compara-
ble to those of the indigenous psychrotrophic
flora of meat under a variety of atmospheric con-
ditions (Manu-Tawiah et al., 1993). These obser-
vations indicate the potential of Y. enterocolitica
to grow successfully in competition with other
psychrotrophs, including lactic acid bacteria,
pseudomonads and members of the Entero-
bacteriaceae.
Little information has been published with
regard to interactions between E. coli 0157:H7
and other microorganisms. E. coli 0157:H7 is
more competitive against spoilage microorganisms
than Salmonella (Varnum & Evans, 1991), and,
through its acid tolerance properties, is hypothet-
ically more resistant to the acidic fermentation
end-products of lactic acid populations.
Complex interactions between elements of the
microflora may have significant effects on sur-
vival/growth of pathogens. These need to be fully
understood in order that novel mild preservation
technology can continue to be applied safely.
Future developments in mild preservation
technology
Novel mild preservation techniques applied to
RTU vegetables have been reviewed in detail by
Ohlsson (1994) and Ahvenainen (1996).
Significant efforts are continuing in a number of
fields.
For example, work on moderate vacuum pack-
aging (MVP) involves the packaging of respiring
produce in airtight containers under approxi-
mately 40kPa of atmospheric pressure and stor-
age under refrigeration conditions. This treatment
reduces the partial pressure of atmospheric gases,
prolonging product quality through a reduced
product respiration and growth of spoilage
microflora and has the potential to reduce patho-
genic populations. Indeed L. monocytogenes, S.
typhimurium, Y. enterocolitica and Bacillus cereus
1999 Blackwell Science Ltd International Journal of Food Science and Technology 1999, 34, 122
Microbiological safety of vegetables Gillian A. Francis et al. 15
populations demonstrate a rapid and complete
loss of viability following storage at 7 8C in MV-
packaged beansprouts (Gorris et al., 1994).
Active packaging employs the gas absorbent
and emitting properties of various chemical com-
pounds including potassium permanganate. This
technology is still in its infancy and its effects
upon the survival of foodborne bacterial
pathogens remains to be established.
The application of biodegradable edible coat-
ings, including sucrose polyesters of fatty acids
and cellulose derivatives, to the surface of foods
is attracting increasing attention from researchers
and commercial interest. Such processes extend
shelf-life and, with the inclusion of antimicro-
bials, reduce the potential growth of pathogens.
This area has been reviewed by Guilbert et al.
(1995) and Baldwin et al. (1995).
The application of high pressure treatments as
a food preservation technique potentially increas-
es the shelf-life and safety of vegetables through
the inactivation of enzymes and the destruction/
inactivation of spoilage and pathogenic microor-
ganisms. Vegetative cells are readily destroyed by
a single high pressure pulse, while spore-forming
bacteria, like Cl. botulinum, are more resistant
and require multiple pulses of approximately 600
MPa. Some side affects have been noted follow-
ing application, including the softening of cab-
bage and increased enzymatic browning rates on
mushrooms and potatoes.
Despite the efficiency, safety and suitability to
products with surface contamination (OBeirne,
1989) the application of irradiation to foods is
still restricted through consumer scepticism and
the limits of legislation.
This review identifies areas where more
research is needed to ensure the safety of RTU
vegetables. The potential hazards introduced by
combinations of RTU products and the microbial
implications of product-package incompatibility
needs to be addressed. The effects of the com-
petitive microflora on the survival/growth of
pathogens under MAs will also be important.
Conclusions
Demand for RTU vegetables has led to a contin-
ual increase in the quantity and diversity of prod-
ucts available to the consumer. Mild preservation
16 Microbiological safety of vegetables Gillian A. Francis et al.
technology, particularly refrigeration and MA-
packaging, is increasingly being relied upon to
ensure the safety and quality of RTU vegetables.
Such technology has resulted in the increased sig-
nificance of psychrotrophic and facultative anaer-
obic pathogens including L. monocytogenes, A.
hydrophila and Cl. botulinum. The emergence of
such organisms, combined with continual product
evolution, presents numerous questions with
regard to the microbial safety of these products.
Only through continued investigation and coop-
eration and education of all parties involved in
the food chain can the microbial safety of RTU
vegetables be achieved.
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