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Factors Affecting Quality and Safety of Fresh-Cut Produce

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Critical Reviews in Food Science and Nutrition, 52:595–610 (2012)
Copyright
C Taylor and Francis Group, LLC
ISSN: 1040-8398 / 1549-7852 online
DOI: 10.1080/10408398.2010.503685

Factors Affecting Quality and Safety

of Fresh-Cut Produce

G. A. FRANCIS,1 A. GALLONE,2 G. J. NYCHAS,3 J. N. SOFOS,4 G. COLELLI,5

M. L. AMODIO,5 and G. SPANO6
1
Food Science Research Centre, Department of Life Sciences, University of Limerick, Limerick, Ireland
2
Department of Basic Medical Sciences, University of Bari, “Aldo Moro”, Piazza Giulio Cesare, Bari
3
Agricultural University of Athens, Iera Odos, Athens, Greece
4
Colorado State University, Fort Collins, Colorado, USA
5
PRIME, University of Foggia, via Napoli, Foggia, Italy
6
DiSA, University of Foggia, via Napoli, Foggia, Italy

The quality of fresh-cut fruit and vegetable products includes a combination of attributes, such as appearance, texture, and
flavor, as well as nutritional and safety aspects that determine their value to the consumer. Nutritionally, fruit and vegetables
represent a good source of vitamins, minerals, and dietary fiber, and fresh-cut produce satisfies consumer demand for freshly
prepared, convenient, healthy food. However, fresh-cut produce deteriorates faster than corresponding intact produce, as a
result of damage caused by minimal processing, which accelerates many physiological changes that lead to a reduction in
produce quality and shelf-life. The symptoms of produce deterioration include discoloration, increased oxidative browning
at cut surfaces, flaccidity as a result of loss of water, and decreased nutritional value. Damaged plant tissues also represent
a better substrate for growth of microorganisms, including spoilage microorganisms and foodborne pathogens. The risk of
pathogen contamination and growth is one of the main safety concerns associated with fresh-cut produce, as highlighted
by the increasing number of produce-linked foodborne outbreaks in recent years. The pathogens of major concern in fresh-
cut produce are Listeria monocytogenes, pathogenic Escherichia coli mainly O157:H7, and Salmonella spp. This article
describes the quality of fresh-cut produce, factors affecting quality, and various techniques for evaluating quality. In addition,
the microbiological safety of fresh-cut produce and factors affecting pathogen survival and growth on fresh-cut produce are
discussed in detail.

Keywords Ready to eat, Escherichia coli O157:H7, Listeria monocytogenes, stress, food quality

INTRODUCTION tissues (Brecht, 1995), expose cytoplasm, and provide a poten-

The worldwide fresh-cut produce industry has grown rapidly
in recent years to a multi-billion dollar sector, largely driven
by increasing consumer demand for healthy, freshly prepared,
convenient fruits and vegetables. Ohlsson (2002) suggested that
minimal processing techniques have emerged to replace tradi-
tional harsher methods of food preservation as they retain nu-
tritional and sensory quality better. Fresh-cut produce may con-
sist of peeled, sliced, shredded, trimmed, and/or washed fruits
or vegetables. These products are usually sealed within semi-
permeable packages and stored at refrigeration temperatures.
The unit operations employed during processing (i.e., peeling,
slicing, shredding) cause the destruction of surface cells, stress
Address correspondence to G. Spano, DiSA, University of Foggia, via
Napoli 25, 71100, Foggia, Italy. E-mail: g.spano@unifg.it
tially richer source of nutrients for microorganisms than intact
produce (Brackett, 1994; Barry-Ryan et al., 2000). These factors
combined with high Aw and either close to neutral (vegetables)
or low acidic (many fruits) tissue pH, and facilitate rapid micro-
bial growth (Beuchat, 1996; Parish et al., 2003). A number of
important human pathogens can contaminate fresh-cut produce
and there has been an increase in the number of produce-linked
foodborne outbreaks in recent years, including the spinach-
associated Esherichia coli O157:H7 outbreak in 2006, which
resulted in 205 illnesses and 3 deaths (FDA, 2007). Produce con-
tamination can occur during agricultural production (via animals
or insects, soil, water, dirty equipment, and human handling),
harvesting, processing (cutting, shredding, washing, contami-
nated work surfaces/equipment, hygiene practices of workers),
packaging (contaminated packaging materials/equipment), and
transportation and distribution. In addition to microbial hazards,

595
596 G. A. FRANCIS ET AL.
other aspects related to the safety of fresh-cut produce include
the potential presence of external chemical contaminants, natu-
rally occurring toxic compounds, and foreign bodies.
This review will discuss the sensory quality of fresh-cut pro-
duce, factors affecting quality and methods used to evaluate
quality. In addition, the microbiological safety of fresh-cut pro-
duce and factors affecting pathogen survival and growth will be
reviewed in detail. Moreover, current and emerging molecular
technologies for the detection of common bacterial foodborne
pathogens will be discussed.
QUALITY OF FRESH-CUT PRODUCE
The quality of fresh-cut produce is related to several at-
tributes, including appearance, texture, flavor, and nutritional
and safety aspects.
Appearance is the main factor affecting consumer choice in
the first phase of purchase (Lund and Snowdon, 2000). However,
consumer satisfaction in terms of other organoleptic characteris-
tics (e.g., aroma, taste, and texture) strongly affects subsequent
purchases. Fresh-cut pieces should appear freshly cut, with a
bright color surface, and be free of defects and decay. Appear-
ance characteristics can be subjectively or objectively measured,
using the human eye or specific instruments, respectively. In the
case of visual evaluation, color charts or guides may be used as
references for evaluation; however, results may be affected by
human error and evaluation conditions (e.g., type of lighting).
Many authors use subjective rating scales to evaluate general
appearance of fresh-cut produce such as peaches and nectarines
(Gorny et al., 1999; Aguayo et al., 2006; Amodio and Colelli,
2008), vegetable mix (Amodio et al., 2006), and mint leaves
(Kenigsbuch et al., 2007). Kader and Cantwell (2004) used rat-
ing scales and color charts for the evaluation of the appearance
of both whole and fresh-cut produce.
Produce color may also be measured instrumentally, on the
basis of light reflected off or transmitted through the commodity.
The CIE L∗ a∗ b∗ color scale is the most frequently used scale for
color references, based on L∗ , a∗ , and b∗ parameters and their
derivative measurements (hue and chroma). Usually, an increase
in L∗ value is correlated with the development of whiteness in
samples, and a decrease in this parameter indicates browning
development (Rico et al., 2007). Colorimetric or spectrophoto-
metric measures of sample homogenates may also be used (Viňa
and Chaves, 2006). Finally, analytical determinations of enzyme
activity may be used to describe browning development, as it
has been shown that phenylalanine ammonia lyase (PAL) ac-
tivity, a key enzyme in the biosynthesis of phenol compounds,
was related to browning of fresh-cut lettuce (Saltveit, 1997).
Similarly, activity of polyphenol oxidase (PPO) was related to
browning of phenol compounds in many fruit and vegetables
(Martinez and Whitaker, 1995; Amiot et al., 1997).
Texture is very tightly linked to tissue deterioration and can be
used as an index of freshness and quality decline (Cantwell and
Suslow, 2002). Consumers expect fresh-cut products to main-
tain firm and crunchy texture (Fillion and Kilcast, 2002), and
very often soft or flaccid produce is revealed by the appear-
ance. Softening of fruit tissues is one of the most important fac-
tors limiting shelf-life (Agar et al., 1999; Karakurt and Huber,
2003), and firmness losses in fruits are primarily caused by enzy-
matic degradation of pectins catalyzed by pectin methylesterase
(PME) and polygalacturonase (PG) (Karakurt and Huber, 2003).
In vegetable tissues, ageing processes and senescence, water
losses, and wounding effects, are the main causes of texture
changes. In products such as spinach, wilting can be one of the
major causes of deterioration in visual appearance and texture
(Piagentini et al., 2002), while in the case of fresh-cut asparagus,
celery, and carrot, shelf-life may be limited by lignification of
vascular tissues (Everson et al., 1992; Barry-Ryan and O’Beirne,
1998a; Viňa and Chaves, 2006).
Texture evaluation is not always easily assessed. In several
studies, the crispness of fresh-cut lettuce was quantified as the
maximum load required to break the sample using a Kramer cell
(Han et al., 2004; Baur et al., 2005; Martı́n-Diana et al., 2006).
Firmness of fruit or vegetable pieces can be measured using
a penetrometer with probes of different diameters (according
to dimension), that penetrate tissues a given distance (Gorny
et al., 2002; Lana et al., 2005; Rico et al., 2007). Amodio et al.
(2006) measured the firmness for pumpkin, zucchini, celery,
carrot, “Borlotti” beans, and peas with an Instron Universal
Testing machine. Quantification of lignin content or the activity
of enzymes involved in its biosynthesis (An et al., 2007), pheny-
lalanine ammonia lyase (PAL), peroxidise, and isoperoxidases,
(Zhang et al., 2001) may be correlated with the textural changes
of vascular tissues. Furthermore, PME or PG activities may
be correlated with flesh-tissue firmness of carambola (Teixeira
et al., 2007) or with lettuce crispness (Rico et al., 2007).
The perception of basic tastes (salty, sweet, sour, and bitter),
mouthfeel sensation, and aroma contribute to flavor definition
(Meilgaard et al., 1991). Sweetness is one of the most impor-
tant components of fruit and vegetable flavor, and its perception
is modified by sourness or acid levels and aroma compounds
(Baldwin, 2004). Aroma compounds contribute to flavor either
directly, or by retronasal stimulation in the nose during chewing.
Fruit aroma is characterized by a high content of volatile oils
and aliphatic esters, which have a higher threshold of perception,
compared to volatiles responsible for vegetable aroma, which
are mainly nitrogen and sulphur compounds. The complexity of
human perception of flavor and interactions between many com-
pounds makes it difficult to quantify flavor using instrumental
analysis, and to relate measurement of individual components to
human acceptance (O’Mahony, 1995). Generally, total soluble
solid content and titratable acidity are used to describe flavor
of fruits and vegetables (Artés et al., 1999; Rinaldi et al., 2004)
and as indicators of maturity stage (Crisosto et al., 2007). Sugar
and organic acid composition (Marsh et al., 2004; Amodio et al.,
2007), and volatile compound identification and quantification
(Burdon et al., 2005; Saftner et al., 2007) are more time consum-
ing evaluations. In addition, other parameters may be estimated
 QUALITY AND SAFETY ISSUES IN FRESH-CUT PRODUCE
including the pungency of onion (Allium cepa L.) measured
with enzymatic determination of pyruvic acid (Hamilton et al.,
1996), and bitterness in chicory was related to sesquiterpene
lactones concentration (Peters and van Amerongen, 1998).
Generally, flavors in fresh-cut produce decline more rapidly
than does appearance, as processes such as cutting and shred-
ding cause mixing of enzymes and substrates, contributing to
flavor deterioration due to volatile losses and synthesis of stress
related off-flavor volatiles (Hodges and Toivonen, 2007). Low
oxygen conditions, that may be generated using modified at-
mosphere packaging, may lead to accumulation of off-flavor
compounds such as ethanol and acetaldehyde (Burdon et al.,
2006). The maturity stage of fruits used for fresh-cut process-
ing has a strong impact on fresh-cut product quality and flavor.
The fresh-cut industry often prefers to process firmer and less
mature fruit to improve shelf-life duration; however, riper fruit
have higher organoleptic quality as reported for peach and nec-
tarine slices (Gorny et al., 1999). Similarly, mature cantaloupe
fruit contained greater quantities of flavor-related compounds
(Beaulieu, 2006).
Flavor quality attributes may be assessed by taste panels us-
ing the traditional 9-point hedonic scale, or a simple 3-point
scale, including the descriptive terms excellent, acceptable, and
unacceptable (Artés et al., 1999; Allende et al., 2007), or us-
ing structured (Saftner et al., 2005) or unstructured line scales
(O’Mahony, 1995).
Fresh-cut products satisfy the consumer demand for easy-
to-use, convenient, healthy food. Fruit and vegetables represent
a good source of vitamins, minerals, and dietary fiber. More-
over, they are rich in constituents known as phytochemicals or
phytonutrients (e.g., carotenoids and phenols) that have been
shown to have a positive effect on human health (Block et al.,
1992; Cox et al., 1996; Craig and Beck, 1999). However, pro-
cess wounding induces a number of physiological disorders
which may accelerate nutrient losses and these losses need to
be minimized (Soliva-Fortuny and Martı́n-Belloso, 2003). Af-
ter cutting, antioxidants were more susceptible to degradation
as they were exposed to oxygen or light. Vitamin C losses in
fresh-cut products, after 6 days of storage at 5◦ C, ranged from
5% in mango to 25% in cantaloupe pieces, compared to whole
fruits, while light promoted vitamin C degradation in kiwi-fruit
slices (Gil et al., 2006). Oxidation of vitamins also occurred
on exposure to acidic pH and sanitization treatments (Wright
and Kader, 1997). Post-cutting interaction of product substrates
with enzymes, such as ascorbate oxidase, polyphenol oxidase,
and peroxidase, may enhance degradation of phytonutrients. In
addition, browning due to oxidation of phenols may reduce nu-
trient content.
By contrast, an increase in nutritional value of wounded tis-
sues, as a result of induced synthesis of phenolic compounds,
has been reported for cut lettuce, celery, carrot, parsnips, and
sweetpotato, while in the same study a decrease of phenols was
observed in cut zucchini, radish, potato, and red cabbage. Other
factors affecting the amount and profile of wound-induced sol-
uble phenolics, other than type of tissue, were initial levels of
597
reduced ascorbic acid and soluble phenolic compounds (Reyes
et al., 2007).
Among all the factors to be considered when addressing
safety issues for fresh-cut produce, the potential for pathogen
contamination and foodborne outbreaks is the one receiving
most attention. The psychrotrophic pathogen Listeria monocyto-
genes and the mesophilic pathogens Salmonella and Escherichia
coli O157:H7 are among the most important pathogens involved
in human illness associated with consumption of produce (FDA,
2008; Sivapalasingam, 2004; Sagoo et al., 2003; Rangel et al.,
2005). Indeed, outbreaks of salmonellosis and listeriosis due
to consumption of vegetables have been reported (Nguyen and
Carlin, 1994), while enterohaemorrhagic E. coli O157:H7 and
Salmonella made major news headlines in the United States in
2006 and 2007 due to outbreaks associated with the consumption
of pre-prepared leafy green salad vegetables and other produce
(Anon. 2006).
In addition to microbial risk, other aspects related to the
safety of fresh-cut produce include the potential presence
of external chemical contaminants, naturally occurring toxic
compounds, and foreign bodies. Chemical contaminants from
external origin may include residues of pesticides and other
pre-harvest and/or postharvest chemical application. In addi-
tion, degradation of sanitizing agents (i.e., chlorine) may lead
to the accumulation of chlorinated trihalomethanes, haloacetic
acids, and chloramines (Wei et al., 1999). Natural toxic com-
pounds may include quercitin (Dunnick and Halley, 1992) and
alkaloids, such as alpha-chaconine and alpha-solanin in potatoes
(Friedman et al., 1992), and psoralen in celery (Aharoni et al.,
1996). Finally, the potential presence of foreign bodies is a very
important issue in the fresh-cut produce industry (Edwards and
Stringer, 2007). Among foreign bodies, insects are one of the
most important issues, especially for fresh salads; they are gen-
erally represented by field pests, although storage pests might
infest the harvested or fully manufactured product during stor-
age. Other foreign bodies include glass, plastic, and bodies of
animal origin, wood, and other contaminants from processing
plants and/or operators (Edwards and Stringer, 2007).
FACTORS AFFECTING QUALITY AND PATHOGEN
SURVIVAL AND GROWTH
The quality of fresh-cut produce and pathogen survival and
growth on fresh-cut produce is influenced by a number of
interdependent factors including product type, minimal pro-
cessing operations (e.g., slicing, washing, antimicrobial, and
anti-browning treatments), packaging, and storage temperature
conditions.
Product Type
Each product type has a unique combination of compositional
and physical characteristics and will have specific growing,
598 G. A. FRANCIS ET AL.
harvesting and processing practices, and storage conditions. Dif-
ferent cultivars vary in several attributes including size, color,
flavor, texture, nutritive value, pest resistance, processing suit-
ability, eating quality, and yield. The cultivar selected for fresh-
cut processing has a very critical effect on product shelf-life
and overall quality. Susceptibility to browning may also widely
differ from one cultivar to another. Differences in browning po-
tential may be related to differences in phenol content and in
the rate of enzymatic activities of PPO (Martinez and Whitaker,
1995; Amiot et al., 1997), PAL (Saltveit, 1997), and phenol
peroxidase (POD) (Degl’Innocenti et al., 2005), and to differ-
ences in the content of antioxidant compounds (i.e., ascorbic
acid) which prevent oxidation of phenol compounds (Cocci
et al., 2006; Degl’Innocenti et al., 2007). Variety selection may
also affect the nutritional value of fresh-cut produce, and plant
breeders have been successful in selecting carrot and tomato
cultivars with higher carotenoid and vitamin A contents, and
sweet corn cultivars that maintain their sweetness for longer
after harvest. Furthermore, there are cantaloupe cultivars which
have higher sugar content than others, and pineapple cultivars
with higher contents of ascorbic acid, carotenoids, and sugars
(Kader, 2004).
In addition to the effects of product type on quality and
composition, pathogen survival and growth also varies signifi-
cantly with the type of product (Austin et al., 1998; Carlin and
Nguyen, 1994). Dry coleslaw mix was largely unsuitable for L.
monocytogenes and E. coli O157:H7 growth while significant
growth of the pathogens occurred on shredded lettuce (Francis
and O’Beirne, 2001a; 2001b). Product factors that may affect
pathogen survival and/or growth include: pH, presence of nat-
ural antimicrobials and/or competitive spoilage microflora, and
respiration rate/packaging interactions.
Product pH strongly influences the survival and growth of
pathogens. Most vegetables have a pH of ≥5.0, and conse-
quently support the growth of most foodborne bacteria. Many
fruits have acidic pH; however, a number of soft fruits/melons
have pH values ≥5.0 and will support growth of many pathogens
(Beuchat, 1996; Escartin et al., 1989; Nguyen and Carlin, 1994).
L. monocytogenes survived and grew on apple slices, cantaloupe
melon (Conway et al., 2000; Ukuku and Fett, 2002), and toma-
toes (Beuchat and Brackett, 1991). Acid tolerance is common in
E. coli O157:H7 and Salmonella serotypes and these organisms
can survive/grow in more acidic produce (Dingman, 2000; Liao
and Sapers, 2000; Ukuku and Sapers, 2001).
Some fruit and vegetable tissues have naturally occurring
antimicrobials that provide varying levels of protection against
pathogens (Sofos et al., 1998). The inhibitory effects of raw
carrots and carrot juice on the growth of L. monocytogenes were
reported (Beuchat et al., 1994; Beuchat and Brackett, 1990b;
Nguyen and Carlin, 1994; Jacxsens et al., 1999). Natural com-
pounds from edible plants, such as oilseeds, herbs, and spices,
may have potential as alternative or complementary antimicro-
bial agents for fresh-cut produce (Burt, 2004). Nychas (1995)
reported antimicrobial activity from oregano, thyme, sage, rose-
mary, clove, coriander, garlic, and onions against both bacteria
and molds. In addition, oils of oregano, thyme, and clove were
very effective against E. coli (Hammer et al., 1999; Smith-
Palmer et al., 1998).
Fresh-cut produce harbor large populations of microorgan-
isms including Pseudomonas, lactic acid bacteria (LAB), and
Enterobacteriaceae (Francis et al., 1999). The background mi-
croflora provide indicators of temperature abuse largely by caus-
ing detectable spoilage, and levels can vary significantly for each
product type and during storage. Various researchers have re-
ported antagonism by the native microflora of vegetables against
L. monocytogenes, S. typhimurium and E. coli O157:H7 (Fran-
cis and O’Beirne, 1996; Francis and Carlin et al., 1996; Francis
et al., 1998; Duffy et al., 1999; Liao and Cooke, 2001). A mixed
bacterial population isolated from endive or lettuce reduced Lis-
teria growth in vegetable media (Carlin et al., 1996; Francis and
O’Beirne, 1998a; 1998b), and P. fluorescens and P. viridiflava
inhibited growth of L. monocytogenes on potato slices (Liao
and Sapers, 1999). Enterobacter isolates significantly reduced
the L. monocytogenes growth during storage on a model lettuce
medium; however, the inhibitory activities of Enterobacter spp.
decreased as the concentration of CO2 increased (Francis and
O’Beirne, 1998a). The native microflora of cantaloupe melon,
especially the yeast and mold populations, might have out-
competed L. monocytogenes for colonizable space and available
nutrients, thus resulting in the decline of populations of L. mono-
cytogenes (Ukuku and Fett, 2002). In addition, strains of LAB
inhibited A. hydrophila, L. monocytogenes, and S. typhimurium
on vegetable salads (Vescovo et al., 1996). Salmonellae grew
better on vegetables when co-cultured with Erwinia carotovora
or P. viridiflava, two major causes of bacterial soft-rot (Wells
and Butterfield, 1997). The importance of the natural microflora
in controlling populations of pathogens, either by direct compe-
tition or through the production of antimicrobials, may provide
biocontrol approaches for controlling pathogen contamination,
survival, and growth on fresh-cut produce.
Processing Operations
The unit operations employed during processing of mini-
mally processed produce (i.e., peeling, slicing, shredding) cause
the destruction of surface cells, stress tissues (Brecht, 1995),
and in the case of fruits, remove natural barriers such as
cuticles and skins, which make tissues more susceptible to water
loss and decay (Altekruse et al., 1997; Agar et al., 1999).
As a consequence of damage caused by processing, there is
an increase in respiration rate and ethylene production which
may accelerate deterioration of non-climateric tissues and pro-
mote ripening in climacteric fruits (Saltveit, 1997; Saltveit et al.,
2005). Phenols may also increase as a consequence of wound-
ing due to the activation of PAL enzyme (Saltveit, 1997).
In addition, membrane lipid degradation may cause loss of
lipid components and of cell compartmentalization; this may
cause enzymes and substrates to interact resulting in detrimen-
tal effects on final quality (Marangoni et al., 1996). Browning
 QUALITY AND SAFETY ISSUES IN FRESH-CUT PRODUCE
discoloration due to the interaction of phenols with PPO en-
zyme, and chlorophyll degradation in green tissues are some
examples of these interactions (Martinez and Whitaker, 1995;
Heaton and Marangoni, 1996). Lipoxygenase activity may pro-
mote synthesis of desirable or undesirable aroma volatiles
(Myung et al., 2006). The extent of wounding is affected by the
number of cuts and the severity of the cutting treatments or the
sharpness of cutting blades. Portela and Cantwell (2001) showed
that melon pieces cut with a blunt blade exhibited increased
ethanol concentrations, off-odors, and electrolyte leakage com-
pared to pieces processed with a sharp blade. Similarly, the use
of sharp cutting blades reduced the wound response, lignin accu-
mulation, white blush, softening, and microbial growth in fresh-
cut carrots (Bolin and Huxsoll, 1991; Barry-Ryan and O’Beirne,
1998). Generally, normally metabolic activity increases with the
number of cuts, as shown for whole, half, sliced potato and
potato sticks, and for shredded and sliced radicchio (Saavedra
del Aguila et al., 2006).
There are numerous preservation strategies that can be ap-
plied to maintain fresh-cut produce quality, focusing on reduc-
ing browning (Garcia and Barret, 2002), and tissue softening
after cutting (Gorny et al., 2002). Methods to control enzymatic
browning of tissues include lowering or increasing the temper-
ature, modifying the atmosphere, the use of enzyme inhibitors,
or the removal/substitution of substrates (Garcia and Barret,
2002). Several types of chemicals are used to control brown-
ing; some act directly as inhibitors of oxidative enzymes, such
as chelating agents, while others act by rendering the medium
inadequate for the development of the browning reaction, such
as acidulants. Other chemicals such as reducing and complex-
ing agents react with the products of the PPO reaction before
these products form dark pigments (Garcia and Barret, 2002).
Ascorbic acid, the most widely used antioxidant compound, is
a reducing substance that reduces colorless intermediate prod-
ucts, such as o-quinones to o-diphenols and also acts as a weak
acidulant. Cisteyne behaves similary to ascorbic acid (Gunes
and Lee, 1997). Other substances which inhibit oxidation in-
clude 4-hexylresorcinol, sodium chloride, and calcium com-
pounds (Drake and Spayd, 1983). Mixtures of anti-oxidants
have been investigated on several products; Gorny et al. (2002)
observed a significant increase in shelf-life of “Bartlett” pear
slices after post-cutting immersion in a solution of 2% ascorbate,
1% calcium lactate, and 0.5% cisteyne at pH 7. Soliva-Fortuny
et al. (2002) reported a consistent browning reduction in apple
cubes after 3 month of storage, when treated with 1% ascorbic
acid and 0.5% calcium chloride and stored in opportune gas
atmospheres.
Dips with calcium compounds have been used as a firming
treatment for several fresh-cut fruits and vegetables, such as zuc-
chini slices (Izumi and Watada, 1995), shredded carrots, apples,
sliced pears, and strawberries. The role of Ca2+ on firmness
has been attributed to the stabilization of membrane systems
and formation of Ca-pectates, which increase the rigidity of the
middle lamella and cell wall. Dipping in 1% calcium chloride
or 2% calcium lactate, in combination with a controlled atmo-
599
sphere of 2–4% O2 and 5-10% CO2 , can extend the shelf-life of
fresh-cut kiwi-fruits from 9 to 12 days (Agar et al., 1999).
The use of edible coatings to improve the quality of fresh-cut
produce and to minimize changes due to processing is another
important alternative strategy. Edible coatings provide a par-
tial barrier to gas exchange (including water vapor), and help to
maintain or improve color, texture, mechanical integrity, volatile
flavors, and to reduce microbial growth (Baldwin et al., 1995).
The choice of coating used on fresh-cut products is very crit-
ical due to the hydrophilic nature of cut surfaces where some
coatings may not adhere. In addition, some coatings may not
resist water vapor pressure (Baldwin et al., 1995). Lipids confer
important water barrier characteristics but may give a gummy
mouthfeel to the product, while hydrophilic polymers have less
barrier properties, especially with high RH values (Baldwin
et al., 1996). In addition, antioxidants, fungicides, and preserva-
tives may be added to the emulsion mix (Baldwin et al., 1995;
Pranoto et al., 2005) to increase the coating performance, while
adding minerals or vitamins may enhance the nutritional value
of fresh-cut products (Dong et al., 2000; Han et al., 2004).
Moreover, cutting/slicing processes release nutrients and
possibly antimicrobial substances from plant cells, which will in
turn affect the behavior of pathogens (Barry-Ryan and O’Beirne,
1998; Brackett, 1994). In general, pathogens grow slowly on un-
injured surfaces of fresh intact produce; however, bruising, cut-
ting, or slicing facilitates microbial contamination and growth
(Seo and Frank, 1999; Han et al., 2000; 2001; Takeuchi et al.,
2000). Starting at harvest, bruising and cutting should be min-
imized prior to processing (Liao and Cooke, 2001). Peeling,
slicing, and shredding, should be carried out with equipment
designed to cause the minimum of tissue disruption, as severe
processing facilitates more effective penetration and subsequent
growth of pathogens (Gleeson and O’Beirne, 2005). Shredding
and slicing processes are important sources of contamination of
fresh-cut produce. Pathogens can become attached to process-
ing equipment (e.g., slicers, shredders) and once attached are
very difficult to remove by chemical sanitizers (Bremer et al.,
2001; Nguyen and Carlin, 1994). Indeed, L. monocytogenes
has been recovered from the environment of processing op-
erations used to prepare fresh-cut vegetables (Zhang and Far-
ber, 1996), highlighting the importance of strict hygiene during
processing. GMPs should include effective work surface and
machine sanitizing to eliminate the risk of pathogen contami-
nation from the processing environment or from machines used
in processing (Nguyen and Carlin, 1994; Zhang and Farber,
1996).
Washing in water removes soil and other debris, some of
the surface microflora, and cell contents and nutrients released
during slicing that help support the growth of microorganisms.
However, while washing in tap water removed bacteria from
exposed surfaces, substantial numbers remained in hollows
at the junction of epidermal cells and in folds in the epider-
mis (Beuchat, 1992; Brackett, 1987; Izumi, 1999; Nguyen and
Carlin, 1994). In addition, due to the re-use of wash water in in-
dustry, washing may result in cross-contamination of products
600 G. A. FRANCIS ET AL.
rather than decontamination (Beuchat, 1996; Brackett, 1992;
Beuchat and Ryu, 1997).
A variety of antimicrobial wash solutions have been used to
reduce populations of microorganisms on fresh produce. The
effectiveness of washing/antimicrobial dipping depends on a
number of factors including: (1) type of treatment, (2) type,
numbers, physiological growth phase, and stress resistance of
the target microorganism(s), (3) product type, (4) antimicrobial
concentration, (5) pH of the solution, (6) contact time, (7) tem-
perature of washing water, and (8) general sanitation of plant
and equipment (Best et al., 1990).
Chlorine is the most frequently used disinfectant for fresh
fruits and vegetables; added to water as a solid, liquid, or gas
(Beuchat and Ryu, 1997). Generally, no more than 2-log10 re-
ductions of bacteria on produce after chlorine treatment have
been reported (Beuchat, 1999). The maximum log10 reductions
of L. monocytogenes, after treatment with chlorine (200-ppm),
were 1.7 for lettuce and 1.2 for cabbage (Zhang and Farber,
1996). Dipping coleslaw and lettuce in a chlorine solution (100-
ppm) reduced initial L. innocua and E. coli populations, but
resulted in enhanced survival during extended storage at 8◦ C
(Francis and O’Beirne, 2002). Chlorine (100- to 200-ppm) was
only marginally effective at reducing E. coli levels on lettuce
tissue surfaces (Beuchat, 1999), and broccoli florets (Behrsing
et al., 2000), while Salmonella populations on alfalfa sprouts
were reduced by about 2 log10 CFU/g after treatment with 500-
ppm chlorine (Beuchat and Ryu, 1997).
Chlorine, used at concentrations currently permitted by the
industry to wash fresh produce, cannot be relied upon to elim-
inate pathogens. The ineffectiveness of chlorine treatment may
be due to a number of factors. For instance, the efficacy of
disinfection treatments also depends on the nature of bacterial
attachment (Han et al., 2000; 2000; Liao and Cooke, 2001),
and microbial cells that become internalized or embedded in
crevices, creases, or injured tissues may be inaccessible to dis-
infection treatments (Koseki et al., 2001; Seo and Frank, 1999).
Biofilms occur naturally on field crops and bacterial cells within
biofilms were shown to be more resistant to removal by wash-
ing and inactivation by sanitizers and disinfectants (Jones and
Heaton, 2006). It is important to sanitize injured surfaces be-
fore cutting as once cut or injured surfaces are contaminated by
pathogens, it is very difficult to remove attached microorgan-
isms.
Another concern regarding the use of antimicrobial dips
is that pathogens may not be fully eliminated by commer-
cial treatments, while at the same time natural competitive or-
ganisms may be removed. L. monocytogenes inoculated onto
disinfected (10% hydrogen peroxide) endive leaves grew bet-
ter than on water-rinsed produce (Carlin et al., 1996), and
dipping lettuce in a chlorine (100-ppm) solution followed by
storage at 8◦ C, significantly enhanced Listeria growth com-
pared with undipped samples (Francis and O’Beirne, 1997).
Disinfection before contamination with the pathogen occurs
which may increase growth of the pathogen because pop-
ulations of competing microflora have been removed (Ben-
nik et al., 1996). Therefore, temperature management (i.e.,
≤4◦ C) after reduction of microbial populations is crucial for
safety.
Viruses and protozoan cysts on fruits and vegetables gener-
ally exhibit higher resistance to disinfectants than do bacteria
or fungi (Beuchat, 1998). Feline caliciviruses were very resis-
tant to commercial disinfectants; however, peroxyacetic acid
and H2 O2 were effective at decontaminating strawberries and
lettuce when used at four-fold higher concentrations than those
recommended by the manufacturers (Gulati et al., 2001). Treat-
ment of Cryptosporidium parvum oocysts with 1-ppm ozone for
5 minutes resulted in <1 log10 inactivation (Korich et al., 1990).
Due to the ineffectiveness of chlorine in removing pathogens
from produce and the increasing concern over the production
of chlorinated organic compounds and their impact on human
and environmental safety, a variety of other disinfectants, in-
cluding acidic electrolyzed water (Park et al., 2001), peroxy-
acetic acid (Park and Beuchat, 1999), chlorine dioxide (Zhang
and Farber, 1996), hydrogen peroxide (Sapers and Simmons,
1998), and trisodium phosphate (Zhang and Farber, 1996) have
been evaluated. However, none of the disinfectant treatments
are likely to ensure elimination of all pathogens, and behavior
of surviving pathogens during subsequent storage remains un-
predictable (Behrsing et al., 2000; Beuchat and Ryu, 1997; Park
and Beuchat, 1999; Zhang and Farber, 1996).
Current interest in alternatives to chlorine dipping is likely
to result in novel, more natural antimicrobial treatments, and
the application of additional interventions beyond the washing
step. Natural antimicrobials, including those from edible plants
such as oilseeds, herbs, spices, fruit, and vegetables, have been
studied for their potential as possible replacements for chem-
ical additives because of their safety for human consumption
and wide acceptance from consumers (Skandamis and Nychas,
2001). Phenolic compounds present in plant essential oils (EOs)
have been shown to possess antimicrobial activity and some are
classified as generally recognized as safe (GRAS); they may be
useful to prevent post-harvest growth of spoilage and pathogenic
bacteria (Singh et al., 2002). The susceptibility of bacteria to the
antimicrobial effect of EOs appears to increase with a decrease
in the pH of the product, storage temperature, and the amount
of oxygen within the package (Burt, 2004). Other natural an-
timicrobial steps may be introduced, including inoculation of
produce with organisms inhibitory to one or more pathogens
(Vescovo et al., 1996).
Application of physical treatments, including mild heat, may
improve the overall effectiveness of the washing step. Novel de-
contamination techniques, including UV, pulsed electric fields,
microwave, high intensity pulsed light, and thermal destruction
using condensing steam, warrant further investigation (James,
2007). Ionizing radiation may also be used either alone or in
combination with other treatments. Doses in the range of <1
to 3 kGy have been shown to reduce or eliminate populations
of pathogens and post-harvest spoilage organisms on produce
(Farkas, 1997) and salmonellae were not recovered from alfalfa
sprouts irradiated with 0.5 kGy (Rajkowski and Thayer, 2000).
 QUALITY AND SAFETY ISSUES IN FRESH-CUT PRODUCE
However, despite the efficiency, the safety and suitability to
products with surface contamination, the use of irradiation will
depend on its acceptance by consumers.
Packaging
Modified atmosphere packaging (MAP) uses low oxygen and
enriched CO2 levels to preserve quality of fresh-cut produce.
The beneficial effects of MAP include a reduction in respira-
tion rate, ethylene production, enzymatic reactions, and of some
physiological disorders, thereby enhancing product quality and
shelf-life (Ahvenainen, 1996; Solomos, 1997). MAP aims to
create an ideal gas composition within the packaging, which
can be generated directly by the commodity within the packag-
ing or actively, by flushing a gas mixture through the packaging
before sealing. Once the package is closed, the gas composition
will inevitably change due to produce respiration and gas perme-
ability properties of the packaging film (Sivertsvik et al., 2002).
Appropriate gas compositions for each product type should be
used, since tolerance to low oxygen and high CO2 levels vary
depending on the product. Outside of these limits, anaerobic
respiration with the production of undesirable metabolites and
other physiological disorders may occur (Soliva-Fortuny et al.,
2002; Solomos, 1997). A gas atmosphere with 0.5% O2 and
10% CO2 reduced respiration rate and ethylene production, de-
cay, and weight loss in carrot slices, shreds, and sticks and dis-
coloration on shreds (Izumi et al., 1996). An atmosphere with
3% O2 and 10% CO2 increased the overall quality of fresh-cut
“Iceberg” lettuce (Lopéz-Gálvez et al., 1996b), while 1–3.8%
O2 and 3-6% CO2 preserved the quality of fresh-cut “Savoy” let-
tuce for 10 days (Kim et al., 2004). Amodio et al. (2006) found
that an atmosphere of 3% O2 and 20% CO2 was beneficial for a
retaining quality of a vegetable mix of thirteen species includ-
ing re-hydrated peas and beans, and for controlling yellowing
of fresh-cut spinach, parsley, and beet. Carbon dioxide-enriched
atmospheres (15%) significantly delayed decay occurrence and
reduced color changes and the development of off-odors in
“Honeydew” melon pieces, while flushing packages with 4%
O2 and 10% CO2 maintained the quality of fresh-cut “Can-
taloupe” melon better than passive modified atmospheres (Bai
et al., 2001).
In addition to the use of traditional atmospheres, Day (1996)
discussed the rationale behind and potential applications for
novel gas mixtures (i.e., high oxygen, argon, and nitrous oxide)
for modified atmosphere packaging (MAP) of fresh prepared
produce. High O2 levels were effective at inhibiting enzymatic
discoloration, preventing anaerobic fermentation reactions, and
influencing aerobic and anaerobic microbial growth. High O2
atmospheres improved sensory shelf-life of raspberries and
strawberries, by inhibiting the development of molds and by
maintaining fresher sensory properties, as long as atmospheric
conditions did not induce fermentation (Van der Steen et al.,
2002). Escalona et al. (2006) recommended the use of 80 kPa
O2 in MAP of fresh-cut Butter lettuce to avoid fermentation,
601
in combination with 10–20 kPa CO2 for reducing its respi-
ration. The use of argon as a major component in MAP has
been reported to reduce microbial growth and improve product
quality retention (Day, 1996: 1998). Replacing nitrogen in air
with helium or argon, enhances gas diffusion and reduces the
concentration gradient of O2 between the inside and outside
of a commodity, and should allow the storage of commodi-
ties at lower O2 concentrations than they would tolerate in N2
atmospheres. Continuous nitrous oxide (N2 O) gas treatment in-
hibited ripening and delayed color changes in pre-climateric
fruits of tomatoes and avocados and inhibited ethylene action
and synthesis in higher plants (Leshem and Wills, 1998). In
addition, argon and nitrous oxide are known to improve san-
itization by making micro-organisms more sensitive to other
anti-microbial agents (Qadir and Hashinaga, 2001). More re-
search on this topic and on the application of novel gas atmo-
spheres for fresh-cut produce is needed; some authors found that
non-conventional atmospheres of argon, nitrous oxide, with a
low percentage of CO2 and O2 maintained quality of minimally
processed apples for 12 days, in terms of PPO inhibition and
lowered metabolic activities (Rocculi et al., 2004). By con-
trast, Jamie and Saltvait (2002) did not find any improvement
in produce quality due to the replacement of N2 with helium or
argon.
In addition to the effects of MAP on produce quality,
MAP may also influence the survival and growth of pathogens
(O’Beirne and Francis, 2003). Of particular concern with re-
frigerated MAP produce is the growth of psychrotrophic, fac-
ultative anaerobic, and microaerophilic microorganisms, which
can tolerate refrigeration temperatures and low O2 atmospheres
(Bennik et al., 1995), and a number of studies have indicated
that MAP may select for such pathogens (Beuchat and Brackett,
1990a; Brackett, 1994; Hintlian and Hotchkiss, 1986; Kallender
et al., 1991). Numerous researchers have shown that survival and
growth of L. monocytogenes on produce is not reduced or af-
fected by MAP (Amanatidou et al., 1999; Berrang et al., 1989b;
Beuchat and Brackett, 1990a; 1991; Jacxsens et al., 1999). How-
ever, in other work, nitrogen flushing combined with storage at
8◦ C enhanced the growth of L. monocytogenes on shredded let-
tuce (Francis and O’Beirne, 1997) and chicory salads. Carlin
et al. (1996) examined the survival of L. monocytogenes on
chicory leaves stored at 10◦ C in air, or under 10%, 30%, or 50%
CO2 , with 10% O2 and found that L. monocytogenes grew better
as the concentration of CO2 increased.
Salmonella and E. coli O157:H7 can grow under MAP con-
ditions; however, there is insufficient information available on
whether gas atmospheres inhibit or enhance their growth. CO2
had little or no inhibitory effect on growth of E. coli O157:H7 on
shredded lettuce stored at 13 or 22◦ C, and growth potential was
increased in an atmosphere of O2 /CO2 /N2 : 5/30/65, compared
with growth in air (Diaz and Hotchkiss, 1996). MAP did not
influence the survival rate of hepatitis A virus on lettuce stored
at 4◦ C, but an improvement in virus survival was observed on
lettuce stored under high CO2 levels (70% CO2 /30% N2 ) at
room temperature (Bidawid et al., 2001).
602 G. A. FRANCIS ET AL.
The effects of novel gas atmospheres including the use of
high O2 levels (i.e., >70% O2 ) and noble gases (Day, 1996)
on pathogen growth have also been examined. In an agar-based
study to investigate the effects of high O2 (90%) and moder-
ate CO2 (10–20%) concentrations on foodborne pathogens at
8◦ C, Amanatidou et al. (1999) noted inhibitory action against
L. monocytogenes, S. typhimurium, S. enteritidis, and E. coli.
Studies to determine the effects of gas atmospheres on protozoan
parasites are limited. Therefore, more research to determine the
survival of these as well as other pathogens on MAP produce is
warranted.
Storage Temperatures
Maintaining low temperature during transport and storage of
fresh-cut product is a very critical aspect of produce quality due
to the impact of low temperature on metabolic reactions. Tem-
perature strongly affects respiration rate, permeability of gases
through packaging films and therefore, changes in atmospheres
within the EMA-packages (Exama et al., 1993; Hertog et al.,
1998; Jacxsens et al., 2000). Enzymatic browning and shrivel-
ling, being the most limiting of sensory disorders in fresh-cut
produce, are also inhibited by low storage temperature (Garcia-
Gimeno et al., 1998).
Moreover, storage temperature is one of the most impor-
tant of factors affecting survival and growth of pathogens on
fresh-cut produce. Storage of produce at adequate refrigera-
tion temperatures will limit pathogen growth to those that are
psychrotrophic, for example, L. monocytogenes. While psy-
chrotrophic organisms, such as L. monocytogenes, are capable
of growth at low temperatures, reducing the storage temperature
(≤4◦ C) will significantly reduce the rate of growth (Beuchat and
Brackett, 1990a; Carlin et al., 1995). L. monocytogenes popu-
lations remained constant or decreased on packaged vegetables
stored at 4◦ C, while at 8◦ C the growth of L. monocytogenes was
supported on all vegetables, with the exception of coleslaw mix
(Francis and O’Beirne, 2001a).
Mesophilic pathogens, such as Salmonella and E. coli
O157:H7, are unable to grow where temperature control is
adequate (i.e., ≤4◦ C). However, if temperature abuse occurs,
they may then grow. Survival of Salmonella in produce stored
for extended periods in chilled conditions may be of concern;
Salmonella survived on a range of vegetables for more than
28 days at 2–4◦ C (ICMSF, 1996). E. coli O157:H7 popula-
tions survived on produce stored at 4◦ C and proliferated rapidly
when stored at 15◦ C (Richert et al., 2000). Reducing the stor-
age temperature from 8 to 4◦ C significantly reduced growth of
E. coli O157:H7 on MAP vegetables; however, viable popula-
tions remained at the end of the storage period at 4◦ C (Francis
and O’Beirne, 2001a). Ensuring that temperatures are kept at
or below 4◦ C throughout the cold-chain is essential for micro-
bial safety and requires considerable attention to detail. Time-
temperature indicators embedded in the packaging may have a
significant role in ensuring that safe storage temperatures are
used.
Bacterial Cross Protection
Stress can be defined as a change in the genome, the pro-
teome, or the environment, producing a decrease in the growth
or survival rate. Stress responses are of particular importance
to microorganisms, because their habitats are subjected to con-
tinual changes (such as temperature, osmotic pressure, and sub-
strate availability). Stressors or stress factors may be of chemi-
cal, physical, or biological nature. Although some stresses (e.g.,
high/low temperature, high/low pH value) are induced by en-
vironmental or processing conditions, natural stresses such as
acidity and, sometimes, starvation are generated by cell growth
itself. In addition, many bacteria form reactive oxygen species
(ROSs) that may cause oxidation of macromolecules, protein
denaturing, and induce breaks in nucleic acid chains.
During production of fresh-cut produce, cumulative mild pro-
cessing steps are employed, to control microbiological growth.
Pathogens on plant surfaces are already stressed and stress may
be increased during the multiple mild processing steps, poten-
tially leading to very hardy bacteria geared towards enhanced
survival (Jones and Heaton, 2006). Cross-protection can occur
because the overlapping stress responses enable bacteria ex-
posed to one stress to become resistant to another stress. A
number of stresses have been shown to induce cross protection,
including heat, cold, acid, and osmotic stress. Among other fac-
tors, adaptation to heat stress appears to provide bacterial cells
with more pronounced cross protection against several other
stresses. Heat adaptation of Salmonella was reported to pro-
vide protection against subsequent heat treatments and low pH
conditions (Foster, 1995). Similarly, S. typhimurium showed in-
creased resistance to heat and salt following adaptation to acidic
conditions (Leyer and Johnson, 1993). Acid adapted L. mono-
cytogenes, Salmonella, and E. coli O157:H7 survived signifi-
cantly better in acidic foods such as fruit juices, when compared
to non-adapted cells (Gahan et al., 1996). Acid adaptation en-
hanced survival of L. monocytogenes during storage in packages
of vegetables which had relatively high in-pack CO2 levels (25-
30% in MAP coleslaw and bean sprouts) (Francis and O’Beirne,
2001b).
Understanding how pathogens sense and respond to mild
stresses are essential in order to design safe and effective min-
imal processing regimes. The ability of pathogens to survive
stress requires specific, co-ordinated responses, which induce
resistance to the stressful conditions. The molecular mecha-
nisms involved are complex and there are a number of genes
involved in bacterial stress response (Abee and Wouters, 1999;
Rowbury, 2002). For instance, the ability of L. monocytogenes
and several Gram-positive bacteria (such as B. subtilis and
S. aureus) to resist many adverse environmental conditions has
been attributed in part to activation of the alternative sigma fac-
tor σ B, encoded by the sigB gene (Giotis et al., 2008). More
than 150 general stress proteins/genes belong to the σ B regulon,
which is believed to provide cells with non-specific, multiple,
and preventive stress resistance. Survival under stress involves
adaptive responses mediated by a set of conserved proteins
 QUALITY AND SAFETY ISSUES IN FRESH-CUT PRODUCE
(usualy called heat-shock proteins), that are upregulated upon
exposure to heat shock, low pH, oxidative agents, toxic chemical
compounds, starvation, and in general, any situation in which
bacterial growth is arrested indicating a protective role in the
general stress response. For example, expression of approxi-
mately half the acid shock proteins induced following exposure
to acidic conditions were also stimulated by subjecting cells to
a heat shock treatment (Foster, 1995).
Apart from the enhanced survival in foods and increased re-
sistance to subsequent food processing treatments, adapted or
hardened pathogens may also have enhanced virulence (Abee
and Wouters, 1999; Archer, 1996; Gahan and Hill, 1999). Stress
response and cross-protection must be considered when cur-
rent processing technologies are being modified or when new
preservation technologies are being developed for fresh-cut pro-
duce. These responses are particularly significant in minimal
processing technologies used in preparation of fresh-cut pro-
duce, where the imposition of one sub-lethal stress may lead
to the induction of multiple stress responses that may reduce
the efficacy of subsequent treatments (Hill et al., 1995). More
research on how to use cumulative sub-lethal hurdles and prac-
tical interventions safely without inducing stress response is
needed.
MOLECULAR TOOLS FOR MICROBIAL DIAGNOSIS
PCR – Based Technologies for Foodborne Pathogen
Detection
Detection and identification of a microbial species in a com-
plex sample like clinical, environmental, and food matrices, is
conventionally based upon isolation of the microrganism in pure
culture and examination of its morphological and physiological
properties. By this approach, microbiologists have been able to
identify, characterize, type, and quantify a wide range of micro-
bial species. Notwithstanding the remarkable advancement in
microbial diagnostics, particularly for pathogenic bacteria, the
limits of conventional culture-dependent and phenotypic char-
acterization are nowadays considered a severe constraint for
precise, fast, and reliable diagnostics. The ability of many bac-
teria to develop a viable but non-culturable state, the significant
shift in some biochemical properties of microrganisms when
exposed to environmental stresses and the high virulence of
some pathogenic bacteria (often below the sensitivity threshold
of conventional culture methods) are just some examples of the
limitations of traditional microbial techniques. Moreover, con-
ventional methods to detect foodborne pathogens are usually
labor intensive, expensive, and may take up to several days in
order to predict or identify bacterial pathogens. Considering the
limited shelf life of produce, rapid and accurate methods for
pathogen detection are required.
The basis of any Nucleic Acid Diagnostic (NAD) assay is
detection of a specific nucleic acid target sequence, unique to
603
the bacterial pathogen of interest. Even within a given bacterial
genus, there are unique sequences specific to each species and
these can be exploited to determine the presence of those bacte-
rial species in a sample. DNA is a very stable molecule, which
makes it particularly suitable as a molecular target for NAD
assays as it can be isolated relatively simply from a variety of
complex biological samples (Beneduce et al., 2007; 2007). Most
of the gene-probe systems focus on detection of specific DNA
or rRNA sequences and utilize PCR or Reverse transcription-
PCR (RT-PCR) for signal amplification. Although it has been
suggested that the detection of only viable cells via their ribo-
somal RNA (rRNA) (Sheridan et al., 1998), neither DNA nor
rRNA are appropriate indicators of viability (Uyttendaele et al.,
1997). In contrast to DNA and rRNA, mRNA molecules are
degraded rapidly in living bacterial cells. Most mRNA species
have a half-life of only a few minutes, because of their rapid
degradation by enzymes (RNase), which are very stable even in
harsh environments. Thus, specific mRNA sequences represent
an excellent target for the detection of viable pathogens (Glynn,
2008).
Several PCR-based methods have been developed for the de-
tection of Campylobacter spp., Salmonella spp., and L. mono-
cytogenes in foods (Lübeck et al., 2003; Malorny et al., 2004;
D’Agostino et al., 2004; Naravaneni and Jamil, 2005). More-
over, commercially available conventional NAD assays include
the BAX
R
(DuPont Qualicon, DE, USA) and Dr FoodTM. (Dr
Chip Biotech Inc., Miao-Li, Taiwan) kits. BAX
R
kits are avail-
able for the identification of Salmonella, E. coli O157:H7, Lis-
teria, L. monocytogenes, C. jejuni, C. coli, S. aureus, and En-
terobacter sakazakii. BAX
R
tests involve culture-based enrich-
ment of the food sample, cell lysis to release the DNA, followed
by PCR amplification with gel-based detection of the PCR prod-
ucts. In addition, Pulse field gel electrophoresis (PFGE) has
been reported to be a useful molecular tools in order to trace
food borne pathogens or to subtyping pathogenic strains (Fran-
cis and O’Bernie, 2006).
RT-PCR has been used to measure mRNA for confirming
an organism’s viability. However, despite potential advantages,
RT-PCR is difficult to exploit, because of the complexity of the
method to measure low levels of intact mRNA from a few vi-
able cells and small numbers of DNA molecules can produce
false positive signals. Nucleic acid sequence-based amplifica-
tion (NASBA) can be used to achieve mRNA-based technology
as an alternative method. NASBA has several advantages over
RT-PCR. It exclusively amplifies RNA rapidly and isothermally
and DNA cannot be used as template in NASBA (Simpkins et al.,
2000; Baeumner et al., 2001; Cook 2003; Rodriguez-Lazaro
et al., 2004). Recently, the development of quantitative real-
time PCR (qPCR and qRT-PCR) technology has enhanced the
sensitivity and specificity of PCR-based assays in the detection
of pathogenic microrganisms in food and the environment,
allowing quantitative analysis and avoiding the use of time-
consuming electrophoresis (Fukushima et al., 2003; Malorny
et al., 2004; Spano et al., 2005; Mercanoglu and Griffiths, 2005;
Beneduce et al., 2007).
604 G. A. FRANCIS ET AL.
Lab on Chip
DNA microarray techniques have been rapidly developed
for gene expression analysis. DNA microarrays consist of large
numbers of probes (either oligonucleotides or cDNAs) immo-
bilized on a solid surface such as specially treated glass. The
first practical demonstrations of microarrays were treated glass
slides with probes deposited in spots onto the surface. Hy-
bridizations are performed by application of labelled nucleic
acid target in a liquid state to the microarray surface. Follow-
ing appropriate hybridization and washing steps, target nucleic
acid bound to probes on the array surface are visualized us-
ing a microarray scanner. Some progress has been made with
the identification of foodborne pathogens from genomic DNA
using microarrays under laboratory conditions (Borucki et al.,
2005; Ahn and Walt, 2005). Microarrays have been used for
the molecular identification of E. coli O157:H7 (Wu et al.,
2003) and Campylobacter spp. (Volokhov et al., 2003) fol-
lowing PCR amplification of target genes. Direct detection of
bacterial RNA using oligonucleotide microarrays has been
demonstrated (Small et al., 2001; Anthony et al., 2005). A
microarray-based assay incorporating signal amplification and
suspension microarray technologies has been reported for the
identification and subtyping of L. monocytogenes from genomic
DNA (Borucki et al., 2005). Microbead type arrays have been
developed for identification of Salmonella spp. targeting the
invA2 and spvB genes (Ahn and Walt, 2005). A multiplex PCR
for E. coli O157:H7 (eaeA, hlyA, stx1, and stx2) and Salmonella
(invA) combined with a suspension microarray detection sys-
tem had a similar sensitivity for each species (Straub et al.,
2005).
Biosensors
A recent frontier in pathogen detection is represented by
biosensors. Nucleic acid based sensors, play an increasingly im-
portant role in the detection of pathogenic organisms in health
care, environment monitoring, and food safety (Ivnitski et al.,
2000; Moldenhauer, 2008). Diagnostic biosensors are a group of
devices and technologies that use a biologically derived material
immobilized on a detection platform to measure the presence
of one or more analyte (Mascini et al., 2005). For applications
in food microbiological analysis, an ideal biosensor would be
a self-contained, automated system capable of pathogen detec-
tion directly from a food matrix without pre-enrichment and
also capable of differentiating live from dead cells (Ivnitski
et al., 2000). To date, biosensor technologies developed for the
specific detection of pathogenic bacteria in food samples have
included metabolism-based, antibody-based, and DNA-based
systems. The current generation of optical biosensors use sur-
face plasmon resonance (SPR) to monitor biomolecular inter-
actions on a surface in real time (Rand et al., 2002) and targets
have also included whole-cell detection of L. monocytogenes
(Leonard et al., 2004).
CONCLUSION
The recent rapid growth in the volume of produce consump-
tion and in the globalization of sourcing can be expected to con-
tinue. Future research may be oriented to increase organoleptic
quality and nutritional value of fresh-cut produce. To enhance
the nutritional and organoleptic quality of fresh-cut produce
more attention should be directed towards obtaining raw mate-
rials with superior quality in terms of flavor, firmness, and nutri-
tional value. As for processing, research is needed on the effects
of abiotic stress applications, such as UV and gamma radia-
tions, heat treatments, and alternative atmospheres on enhanc-
ing organoleptic and nutritional quality of fresh-cut products.
Both producers and consumers will benefit from the availability
of alternative nondestructive techniques for the prediction of
organoleptic and nutritional attributes.
Several critical gaps in knowledge on the microbiological
safety of fresh-cut produce are identified, notably uncertainty
about the location of contaminating cells on or in plant tis-
sues and the influence of environmental stresses on growth and
infectivity (Delaquis et al., 2007). Stress response and cross-
protection must be considered when current food processing
technologies are being modified or new control measures de-
veloped. These responses are particularly significant in minimal
processing technology where the imposition of one sub-lethal
stress may lead to the induction of multiple stress responses that
may reduce the efficacy of subsequent treatments (Hill et al.,
1995). More research on how to use cumulative sub-lethal hur-
dles and practical interventions safely without inducing stress
response is needed. Furthermore, research on the mechanisms
of resistance of pathogens to multiple stresses is required. Other
research trends driven by the needs of this sector include greater
understanding of pertinent and emerging pathogens, particu-
larly E. coli O157:H7, viruses, and protozoan parasites; greater
understanding of processes of produce contamination gener-
ally, and of how to prevent them; application of alternative
approaches to assuring the safety of produce and the devel-
opment of more effective decontamination technologies; and
enhanced foodborne disease surveillance and epidemiological
systems and techniques (Todd et al., 2007). Recent outbreaks
have highlighted the need for better traceability and an early
warning system in the produce industry. These technologies can
help establish the history of food items, sources of contami-
nation, and links between geographically isolated outbreaks of
food poisoning with a common source.
The development of molecular tools for microbial diag-
nosis of pathogenic bacteria has dramatically improved both
diagnostic efficiency and knowledge about spread of virulent
microrganisms in the environment, routes of transmission,
etc. The main advantage of microbial identification by genetic
markers is the relative stability of the genotype rather than the
phenotype. Nucleic acids and proteins can act as a “fingerprint”
of a microbial species, allowing more precise identification as
well as traceability. Although the cost of molecular diagnosis is
still too high to be supported by small and medium companies
 QUALITY AND SAFETY ISSUES IN FRESH-CUT PRODUCE
involved in the fresh-cut produce market, surveillance and
investigation of fresh produce may be easily performed by
National or Regional “check points” (e.g., universities) in order
to improve the safety of produce on the national and/or interna-
tional market. Foodborne prevention can be built into industry
practices by identifying where in the chain contamination can
occur or be eliminated. A combined working strategy, essen-
tially based on a scientific analysis of risk in the supply chain
is the adoption of Hazard Analysis and Critical Control Points
(HACCP) and a close collaboration with both the food industry
and the farms and/or the farmers involved in fresh cut production
is crucial to further ensure food safety for consumers.
ACKNOWLEDGEMENTS
This work was partially funded by MIUR in the frame-
work of—Progetti di Ricerca industriale/PON R&C 2007–2013,
PRODOTTI ORTOFRUTTICOLI AD ALTO CONTENUTO
IN SERVIZIO: TECNOLOGIE PER LA QUALITÀ E NUOVI
PRODOTTI (OFR.AL.SER.)
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