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Microbial quality assessment methods for fresh-cut fruits and vegetables

Article  in  Stewart Postharvest Review · June 2008

DOI: 10.2212/spr.2008.3.10

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 Stewart Postharvest Review
An international journal for reviews in postharvest biology and technology

Microbial quality assessment methods for fresh-cut fruits and vegetables

Lihua Fan* and Jun Song

Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, Nova Scotia, Canada

Abstract
Purpose of review: The microbial population of fresh-cut fruits and vegetables is determined to a large extent by the origin of fruits
and vegetables, agricultural practices, conditions of harvesting, processing and storage. During distribution and storage, high humidity
in packaged fresh-cut products and cut surfaces provide favourable conditions for the growth of spoilage microorganisms. Microbial
quality assessment should be taken into consideration in evaluating the impact of handling, processing and treatment procedures on
maintaining the quality of fresh-cut fruits and vegetables and determining their shelf-life.
Findings: In performing microbial quality assessment, it is important to follow standardised methods that define conditions such as
incubation temperature and duration, media composition and growth atmosphere in order to be able to compare data from within the
same laboratory and between different laboratories. In this review, we describe microbial assessment methods commonly used for
fresh-cut fruit and vegetable microbial quality evaluation. These include the enumeration and identification of aerobic mesophilic bac-
teria (aerobic plate count), psychrotrophic microorganisms, lactic acid bacteria, yeast and mould, and coliforms. Some rapid methods
for enumerating microorganisms from food samples are also discussed.
Directions for future research: Well integrated quality assessments that include microbial quality, physiological and sensory quality
are important to determine the shelf-life of fresh-cut fruits and vegetables. Microbial quality data need to be correlated with shelf-life
outcomes in order to understand the linkage between microbial population dynamics and quality changes in response to pre and post-
processing treatments. The role of microbial contaminant-induced deterioration versus produce physiologic deterioration in fresh-cut
products needs to be determined. Such will enable the development of better approaches for maintaining good quality and ensuring the
safety of products.

Keywords: fresh-cut fruits and vegetables; microbial quality; spoilage microorganisms; microbial assessment; microbial enumeration
and identification

Abbreviations *Correspondence to: Dr L Fan, Agriculture and Agri-Food

ACC Aerobic Colony Count Canada, Atlantic Food and Horticulture Research Centre, 32
Main Street, Kentville, NS, B4N 1J5, Canada. Tel: +1 902
APC Aerobic Plate Count 6795550; Fax: +1 902 6792311; email: fanl@agr.gc.ca
CFU Colony Forming Units
Gram- Gram Negative Stewart Postharvest Review 2008, 3:10
Gram+ Gram Positive Published online 01 June 2008
LAB Lactic Acid Bacteria doi: 10.2212/spr.2008.3.10
MS Mass Spectrometry
MPN Most Probable Number
MRS DeMan, Rogosa, Sharpe
PCA Plate Count Agar
PCR Polymerase Chain Reaction
PDA Potato Dextrose Agar

© 2008 Stewart Postharvest Solutions (UK) Ltd.

Online ISSN:1945-9656
www.stewartpostharvest.com
Fan and Song / Stewart Postharvest Review 2008, 3:10
Introduction
Fruits and vegetables provide consumers with vitamins, min-
erals and fibre. They are an essential part of the human diet.
The United States Department of Agriculture recommends
five to nine servings of fruits and vegetables daily [1]. In
recent years, fresh-cut produce has become more popular due
to their convenience, freshness, taste and assumed
“healthiness”. Sales of fresh-cut produce have increased dra-
matically from near zero in 1985 to $12.5 billion in 2004, and
sales for fresh-cut fruits are expected to reach $2 billion by
2008 in USA [2].
Consumers expect that no further washing of these products
is required, which creates a great challenge to processors to
ensure the quality and safety of fresh-cut products since fruits
and vegetables are produced in a natural environment and
they are easily contaminated by spoilage microorganisms and
human pathogens [3]. The main characteristics of fresh-cut
fruits and vegetables that serve to increase the probability of
microbial spoilage and contamination are the presence of cut
surfaces or damaged plant tissues, the fact that sterility or
microbial stability of these products cannot be ensured, the
active metabolism of plant tissue, and the confinement of the
product in different packs [4**].
The major microbiological concerns related to fresh-cut pro-
duce are mesophilic and psychrotrophic microorganisms. In
most cases, the microorganisms present on fresh-cut fruits
and vegetables are similar to those found in the field. Thus,
microbial decay is mostly associated with the growth of mi-
croorganisms that originate in the preharvest environment
[5*]. Nguyen-the and Carlin [4**] indicated that mesophilic
bacteria counts on different fresh-cut vegetables range from
103 to 109 CFU/g, whereas densities on products evaluated
soon after processing range from 103 to 106 CFU/g. Approxi-
mately 80–90% of the mesophilic microflora of produce is
composed of Erwinia spp, Enterobacter spp and Pseudomo-
nas spp. Most pseudomonads identified were P. fluorescens,
which represented 50–90% of the pseudomonads. Lactic acid
bacteria (LAB) such as Leuconostoc mesenteroides and Lac-
tobacillus spp., and several species of yeast and moulds are
also commonly found on fresh-cut fruits and vegetables [4**,
6, 7*].
Human pathogens such as Salmonella spp, Escherichia coli
O157:H7 and Listeria monocytogenes have been linked to
cases of human illness that occurred following consumption
of contaminated produce [8]. Identification and detection
methods used for human pathogens are not included in this
review. Information regarding detection methods such as
immunological testing, genetic testing as well as biosensors
can be found in a chapter written by Fung [9**].
Knowing the initial microbial population and its species com-
position is of use in determining what sanitising practices
should be implemented for fresh-cut fruits and vegetables.
Maintaining a low microbial population is of value in mini-
mising spoilage microorganisms and their development, ex-
tending shelf-life and protecting human health. Therefore, the
objective of this review is to discuss the inter-connections
between microbial quality, product quality and shelf-life, and
microbial quality assessment methods.
Relationship between microbial population, qual-
ity changes and shelf-life of fresh-cut products
To determine produce quality, consumers frequently assess
appearance and aroma. However, microbial populations may
exceed safe levels on produce that are of acceptable visual
quality. According to French regulations, an aerobic plate
count (APC) of 5x107 colony forming units (CFU)/g is the
maximum acceptable value at the end of the microbiological
shelf-life of numerous fresh-cut vegetables [10*]. The shelf-
life of fresh-cut vegetable salads utilised by manufacturers is
usually 7–14 days depending on the types of vegetables
[11*]. Barriga et al. [12] found that even after psychrotrophic
microorganism counts reached 106–107 CFU/g, high visual
quality ratings were still given to fresh-cut lettuce. Again,
although bacteria counts on fresh-cut lettuce stored in modi-
fied atmosphere packaging exceeded 108 CFU/g after 7 days,
visual quality evaluation still suggested that the product was
acceptable for sale [13]. O’Connor-Shaw et al. [14] found
that senescence and unsatisfactory quality level of fresh-cut
kiwifruit, papaya and pineapple stored at 4°C were not corre-
lated with increased microbial densities. Zagory [7*] indi-
cated that bacterial populations may be not directly associ-
ated with product quality or shelf-life.
Generally speaking, leafy vegetables contain only small
amounts of sugars that promote the growth and proliferation
of Gram negative (Gram-) microorganisms. Fruits, on the
other hand, form readily utilisable substrates that permit the
development of spoilage caused by very rapid and predomi-
nant growth of LAB and yeast. This growth is often related to
the increased production of secondary metabolites such as
lactic, propionic and acetic acids, which are readily detected
as off-flavour. Some root vegetables, such as carrots, which
have higher sugar contents likely undergo microbial fermen-
tation [5*]. LAB increased in shredded or sliced carrots
achieving counts of 108 CFU/g. L. mesenteroides seems to be
the primary organism responsible for carrot spoilage [4**].
Other root vegetables such as chicory and potato, which have
more complex carbohydrate content, develop soft-rot symp-
toms [5*]. Ahvenainen [15] reported that the predominant
microflora of fresh leafy vegetables are Pseudomonas and
Erwinia spp. that display initial counts of approximately
105 CFU/g. Low numbers of mould and yeasts were also pre-
sent. During cold storage, pectinolytic strains of Pseudomo-
nas are responsible for bacterial soft rot. An increase in the
storage temperature and the carbon dioxide concentration in
the package will shift the composition of the microflora so
that LAB tend to predominate.
Manvell and Ackland [16] reported that rapid microbial
growth was detected in vegetable salads at chill and abuse
2
Fan and Song / Stewart Postharvest Review 2008, 3:10
temperatures. The isolates were Gram- rods or Gram positive
(Gram+) cocci, and were mainly psychrotrophs with some
mesophiles. In salad packs held at 7°C, LAB formed a low
proportion of the total population; whereas, at 30°C LAB
formed the dominant population. These temperature-related
differences in population composition led to the development
of tests useful in indicating shelf-life or temperature abuse. A
rapid test for detecting lactic acid and a technique based on the
Gram stain were described that enabled an indication of tem-
perature abuse prior to the occurrence of visual spoilage. The
limulus lysate test, an assay for Gram- bacterial endotoxin, was
also shown to be a simple and rapid method for assessing the
microbial quality of salads stored at chill temperature and thus
could provide a useful indicator of shelf-life [16].
Antes and Allende [17] reviewed the most critical steps in the
production chain and storage conditions of minimally-
processed leafy vegetables that might influence final microbial
and sensory quality. They discussed the advantages and disad-
vantages of recent strategies and improvements in washing and
sanitising agents and included an evaluation of emerging pres-
ervation techniques such as super atmospheric O2 atmospheres,
hot water treatments and UV-C radiation as feasible alterna-
tives for improving the final microbial quality and sensory
quality of products. Gould [18] also summarised new and
emerging preservation techniques for improving food quality
and safety, and extending shelf-life. Sapers [19] described re-
cent improvements in washing and disinfection technology for
fresh fruit and vegetable products. They concluded that effec-
tive washing and sanitising treatments play important roles in
reducing the microbial population on fresh fruits and vegeta-
bles used either for the fresh market or fresh-cut products.
In recent years, much research has focused on developing
novel approaches for determining the shelf-life of fresh-cut
fruits and vegetables [10*, 20*, 21]. Lavelli et al. [21] used
physicochemical, microbial and sensory parameters as indi-
ces to evaluate the quality of fresh-cut carrot. Microbial indi-
ces included total bacterial count, coliforms, LAB and yeasts.
Results showed that microbial population growth fitted the
Gompertz model (mathematical model). Here APC and total
coliforms reached the threshold concentration at day 7 at 4°C
and day 3 at 10°C. Allende et al. [13] evaluated psychrotro-
phic and mesophilic bacteria, coliforms and LAB, as well as
the sensory quality of fresh processed red lettuce throughout
the production chain and on the shelf. Based on microbial
quality and sensory attributes assessment, a shelf-life of less
than 7 days was considered the norm for fresh processed let-
tuce. Corbo et al. [10*] investigated the effect of temperature
on the shelf-life and microbial population of lightly proc-
essed cactus pear fruit. In this study, the shelf-life of the pro-
duce was kinetically modelled to determine the effects of
storage temperature as well as to assess the most relevant
microbial indices for product quality. The results suggested
that mathematical modelling might allow industry to use
more objective measurements to determine the shelf-life of
the products.
Microbial methods used for quality evaluation of
fresh-cut products: Enumeration of microorgan-
isms on fresh-cut products
Standard plate counts for aerobic mesophilic bacteria
Mesophilic microorganisms can persist at refrigeration tem-
peratures and may begin to grow during temperature abuse.
APC has been one of the most common tests applied to deter-
mine the level of mesophilic bacteria in raw or fresh-cut fruits
and vegetables [10*, 22–26]. The term aerobic colony count
(ACC) has been suggested to replace the APC for the reason
that ACC can describe the test undertaken more accurately
[27]. The APC quantifies bacterial densities using growth me-
dia that contains no selective or differential additives. The me-
dia most commonly used for this purpose is plate count agar
(PCA). APC can be performed using pour plate, spread plate
or spiral plating techniques. When using pour plate and spread
plates, fixed volumes of a series of dilutions of the test solution
are always plated in order to find the dilutions at which count-
able colonies are obtained in the range from 25 to 250 CFU/
plate. Spiral plating utilises a special syringe to apply liquid
extracts of fruit or vegetable material in a spiral track to the
surface of a rotating agar plate. The dispensing stylus deposits
decreasing amounts of test sample as it moves from near the
centre of the plate to the edge. This technique does not require
the numerous dilutions usually associated with total plate
counts, thereby speeding up the inoculation procedure and
eliminating manual errors. A colony counter is used with a
spiral plater for determining CFU/g. Regardless of the inocula-
tion method chosen, each dilution should be plated in duplicate
or triplicate, and incubated at 30 or 35°C for 48 h. Peptone
water (0.1% w/v peptone) is often used as the dilutant in order
to maintain the osmotic stability of diluted cells. For microbial
quality analyses, a 25-g sample of fresh-cut fruit or vegetable
is usually weighed into a sterile blender cup or a stomacher
bag. Based on the sample, either sterile bags or sterile filter
bags are used. Then, peptone water (225 mL) is added and the
sample is either blended or pummelled in laboratory stom-
acher. Alternatively, 10 g of vegetable or fruit sample is placed
in 90 mL of 0.1% peptone solution [10*, 28–30*]. Mesophilic
bacteria counts are usually expressed as log10 CFU/g of the
fresh-cut fruit or vegetable.
It is noted that the guidelines of APC is used to determine the
bacteriological quality of ready-to-eat foods at the point of
sale and indicate the level of contamination that is considered
to represent a significant potential risk to health. Therefore, it
is not applicable to fresh-cut fruits and vegetables [27, 31].
However, APC numbers are often used to evaluate product
quality, or to predict the shelf-life of fresh-cut fruits and
vegetables.
Standard plate counts for psychrotrophic bacteria
Psychrotrophic microorganisms can grow at refrigeration
temperatures. The numbers of psychrotrophic bacteria on
fresh-cut fruits and vegetables may increase when the chill-
ing process starts, especially during cold storage at 0-4°C. To
3
Fan and Song / Stewart Postharvest Review 2008, 3:10
enumerate these types of bacteria, the same procedures de-
scribed for aerobic mesophilic bacteria are used, but PCA
plates need to be incubated at 5–7°C for 7–10 days [10*, 24,
30*].
Counts for lactic acid bacteria
LAB such as L. mesenteroides are the primary organisms
responsible for spoilage of some fresh-cut produce. In pack-
aged mixed vegetables, the development of LAB is related to
temperature increases, and LAB counts seem to be high in
shredded carrots [4**, 6, 11*]. Their densities can be esti-
mated on DeMan, Rogosa, Sharpe (MRS) agar adjusted to
pH 5.6 and incubated under anaerobic conditions at 30°C for
48 h. Although an anaerobic system provides an oxygen-free
work chamber, a simple and easy way to achieve anaerobic
conditions is to use GasPakTM gas generating systems. These
are multi-use systems that produce atmospheres suitable for
isolating and cultivating anaerobic and microaerophilic bac-
teria by using gas generating sachets inside the system. An-
aerobic conditions for incubation may also be obtained by
using sterile butyl rubber stoppers on test tubes so that an
anaerobic gas headspace is retained. Garcia-Gimeno and Zur-
era-Cosano [11*] recovered LAB by placing food samples on
Bacto lactobacilli MRS agar (Difco Laboratories, Detroit,
MI), then covering them with an agar layer to obtain anaero-
bic conditions and incubating at 30°C for 48 h.
Enumeration of yeasts and moulds
A diverse population of yeasts and moulds can be found on
raw or fresh- cut fruits and vegetables prior to harvest, during
its storage and on the shelf. They form the predominant mi-
croorganisms associated with the spoilage of fresh-cut fruit
products. However, in fresh-cut fruit with a neutral pH such
as cantaloupe bacteria were the main source of spoilage [32].
Some yeasts and moulds can cause spoilage and some are a
public health concern due to their production of mycotoxins.
Among the yeast isolated from fresh-cut products are Can-
dida spp, Cryptococcus spp, Rhodotorula spp, Trichosporon
spp, Pichia spp and Torulaspora spp; fungi belong to the
genera Sclerotinia, Mucor, Aspergillus, Cladosporium,
Phoma and Rhizopus [4**].
Selective media and lower incubation temperatures are often
used to slow or inhibit bacterial growth and thereby select for
the outgrowth of yeasts and moulds. Selective bacterial inhi-
bition can be achieved using antibiotics such as chloram-
phenicol at 100 µg/mL, or through acidification of media
such as potato dextrose agar (PDA) with tartaric acid to
pH 3.5. Incubation is conducted at 22–25°C for up to
5 days. Jackson et al. [33] reported using oxytetracycline
gentamycin yeast extract (OGY) agar, and Corbo et al. [10*]
chose Sabouraud Dextrose Agar incubated at 28°C for
2 days for yeasts and Malt Extract Agar (MEA) incubated at
25°C for 4 days for moulds. Dichloran rose bengal chloramp-
enicol agar (DRBC) incubated at 25°C for 5 days was
used to recover and enumerate yeasts/moulds by Simmons et
al. [34].
Quantification of coliform bacteria
In conducting the microbiologic analysis of food in order to
assess the health risk associated with its consumption, indica-
tor organisms are often utilised. These usually are present in
higher numbers than true pathogens and are easier to detect.
The growth and survival characteristics of these organisms
are similar to those of pathogens. The most commonly used
indicator organisms are the coliforms. Coliform bacteria are a
group of Gram-, non-spore forming, aerobic or facultative
anaerobic rods that ferment lactose forming acid and gas
within 48 h at 35°C. E. coli is a member of the coliform
group. Most coliforms are members of the Enterobacteri-
aceae family. The presence of high numbers of coliforms in
foods is often assumed to be indicative of the co-presence of
intestinal pathogens, which are usually more difficult to de-
tect and quantify. In addition, their presence has been used to
indicate the absence of proper sanitation. However, as our
understanding of the ecology of coliform bacteria and their
behaviour in the environment increases, it has become appar-
ent that the enumeration of this group is a less than reliable
indicator of food quality and sanitation. This is especially so
for produce grown in natural soil, irrigated with untreated
surface water and packaged with minimal processing.
When enumerating coliforms, E. coli may be present among
the mixed population measured. Selective or differential me-
dia can be used for estimating E. coli levels under these cir-
cumstances. Among these is the most probable number
(MPN) method [35]. It is a statistical method that is useful
when working with materials carrying low numbers of organ-
isms (<100/g). The MPN procedure is a multiple tube fer-
mentation technique. In this method, samples are serially
diluted and at each dilution, multiple volumes are transferred
into three, five, or 10 tubes of liquid test medium. The tubes
are incubated and positive or negative results are recorded
based on bacteria growth (turbidity) in combination with gas
formation or acid production within the tubes. The number of
tubes indicating the presence or absence of the test groups of
organisms is compared to a statistical MPN table that gives
MPNs plus confidence intervals for various combinations of
positive tubes. The MPN approach can be used to obtain esti-
mates of the levels of coliforms, faecal coliforms and E. coli.
A field study conducted by Johnston et al. [36] evaluated the
microbiological quality of fresh produce. A total of 398 pro-
duce samples of leafy greens, herbs and cantaloupe were col-
lected through production and the packing shed and assayed
for total aerobic bacteria, coliforms, Enterococcus and E.
coli. This study demonstrated that each step from production
to consumption may affect the microbial population of pro-
duce. The microbial quality of five types of fresh produce
obtained at the retail stage was determined by Thunberg et al.
[37]. They used three different methods to determine total
coliforms including MacConkey agar plate counts, Colicom-
plete MPN and Petrifilm E. coli plated counts. Giménez et al.
[24] used brilliant green bile lactose broth (BGBL) (Difco)
incubated at 44°C for 48 h to determine the MPNs of faecal
4
Fan and Song / Stewart Postharvest Review 2008, 3:10
coliforms in fresh-cut artichoke. By subculturing positive
tubes onto a Levine agar (Merck) then incubating at 37°C for
48 h, suspected E. coli colonies could be recovered. Quantifi-
cation of the total and faecal coliform counts associated with
fresh-cut sliced carrots were performed on Violet Red Bile
Agar with incubation at 37 and 44.5°C for 24 h, respectively
[28]. If the numbers of coliform bacteria are expected to be
low, success in detecting and enumerating E. coli associated
with leafy vegetables such as lettuce and spinach has been
achieved by using a modification of the membrane filtration
method employed in water quality analysis. In this manner,
the entire contents of 1–50 mL of undiluted plant extract can
be screened by incubating the membrane on selective agar
[38].
Aerobic and anaerobic spore-forming microorganisms
Aerobic spore-forming microorganisms can be enumerated
on PCA via the pour plate method subsequent to heat treat-
ment (80°C for 10 min) to destroy the vegetative cells. In this
case, plates are incubated at 30°C for 3 days prior to count-
ing. For anaerobic spores plates prepared in the same way are
incubated under anaerobic conditions. Giménez et al. [24]
used this approach to show that the population of aerobic and
anaerobic spore-forming microorganisms on fresh-cut arti-
chokes remained constantly lower at 2.5 and 2 log10 CFU/g,
respectively, during 15 days storage at 4°C. The low storage
temperature seemed to inhibit spore germination of these
kinds of microorganisms.
Petrifilm enumeration of different microorganisms from
fresh-cut products
Petrifilm detection and enumeration involves adding plant
extracts (or dilutions) to rehydratable nutrients embedded in a
series of films formed within a self-sealing incubation unit. It
is available in a number of different formulations (3M Co.,
St. Paul, MN). A maximum of 1 mL of the liquid food sam-
ple is used to re-hydrate the media. The medium will support
the growth of microorganisms under suitable incubation con-
ditions. Resultant colonies are counted directly in the unit.
Recently a Petrifilm counter has been introduced which auto-
matically counts and records colony numbers. The advan-
tages of using Pertrifilm are: (1) that there is no need for me-
dia preparation and sterilisation; (2) its compact size requires
less incubation space; and (3) gas bubbles, which are espe-
cially useful in identifying coliforms, can be detected after
incubation. However, a small colony size makes counting
more difficult and small particles of fresh-cut product may be
mistakenly counted as microorganism colonies when low
dilutions are plated.
Petrifilms were used to count aerobic mesophilic and psy-
chrotrophic bacteria, yeast and moulds, and coliforms present
on strawberries [39]. Total ACCs on fresh-cut apples were
determined using PetrifilmTM after incubation at 37°C for
48 h, and counts ranging from 20–200 CFU/plate were con-
sidered to be within the countable range [40]. Strawberries
were tested for their E. coli carriage using PetrifilmTM coli-
form/E. coli plates and incubated at 35°C for 24 h [41]. In
addition, the Redigel system (3M Co., St. Paul, MN) consists
of tubes of sterile nutrient with a pectin gel in the tube and no
conventional agar, which are also used for microbial counts.
The above described methods are used to achieve the enu-
meration of different types of microorganisms. The resultant
data can be reported either as CFU/g or CFU/cm2 of the fruits
and vegetables tested.
Identification of microorganisms recovered from
fresh-cut products
The identification of spoilage organisms carried by fresh-cut
fruits and vegetables is an important step in developing ap-
proaches to inhibiting and controlling these organisms. Nu-
merous monitors and test kits are available commercially for
the microbiological analysis and testing of food, raw materi-
als and finished product. Many ready-to-use culture media
have been developed specifically for the simultaneous recov-
ery and identification of microorganisms. For example, the
selective media are developed to meet analytical test needs.
Bennik et al. [23] reported that Pseudomonas agar base con-
taining CFC selective agar supplement enabled the detection
of pseudomonads on minimally-processed, modified-
atmosphere packaged chicory endive, while Violet Red Bile
Glucose Agar gave suitable recoveries of the enterobacteria.
Selective media are also available in convenient, single test,
plastic ampoules; especially, for the growth and detection of
microorganisms collected on membrane filters. Thus, m-
Endo Total Coliform Broth is used to recover and identify
total coliforms while Pseudomonas Selective Broth is used
for Pseudomonas species bacteria. Currently, many diagnos-
tic systems including API, Minitek, Biolog, RapID, MicroID,
Crystal ID and VITEK systems are available for identifying
different microorganisms. These, however, have been devel-
oped primarily for application to clinical isolates. Their use
with environmental bacteria has only just been initiated. One
consequence of this is that data banks used to complete the
identification do not always recognise the biochemical prop-
erties detected and an incomplete or erroneous identification
is made. The use of the API (bioMérieux) and Biolog (Biolog
Inc) systems to identify microorganisms isolated from fresh-
cut fruits and vegetables is discussed in this review.
Phenotypic identification: API and Biolog test sys-
tems
Isolates recovered from fresh-cut fruits and vegetables are
usually initially grouped according to their morphology and
Gram-stain reaction. Their genera may subsequently be es-
tablished using a series of general tests including the oxidase
and catalase reactions, the indole test, oxidative fermentative
metabolism of glucose, motility and spore formation. Kongo
et al. [42] used both biochemical and ribotyping methods to
identify isolates from cheese. Following microscopic obser-
vation, Gram-staining, catalase testing, growth in MRS broth
at 15°C for 7 days and at 45°C for 2 days, and determining
the ability to produce CO2 from glucose in MRS broth con-
taining Durham tubes, further identification to the species
level was achieved with the API test systems.
5
Fan and Song / Stewart Postharvest Review 2008, 3:10
API system
The API system is one of several systems commercially
available for identification of microorganisms. Strips are
available for the identification ranging from Gram-, Gram+
bacteria, anaerobes and yeasts. For example, the API 50 CH
strip consists of 50 microtubes used to study fermentation of
substrates belonging to the carbohydrate family and its de-
rivatives. API 50 CH system associates 50 biochemical tests
for the carbohydrate metabolism of microorganisms, which is
used in conjunction with API CHL medium for the identifica-
tion of Lactobacillus and related genera, and with API 50
CHB/E medium for the identification of Bacillus and related
genera. The API system is supported by an extensive data-
base. The reactions are well standardised and in most cases,
easy to read after suitable usage.
Kongo et al. [42] used API CHL 50 and API 20 strip kits
(bioMérieux) to identify isolates from foods and incubated at
30°C for 24 and 48 h, respectively. They further character-
ised the identified strains using ribotyping, with EcoRI as the
cutting enzyme via the Riboprinter Microbial Characteriza-
tion System (DuPont- Qualicon). Garcia-Gimeno and Zurera-
Cosano [11*] reported using the API 50 CHL gallery to iden-
tify LAB isolated from ready-to-eat vegetable salads, while
aerobic psychrotrophic strains were identified using both API
20 E and API 20 EN strips. Yeast and mould isolates were
identified with API 20 C AUX. To complete the identifica-
tion of bacteria and yeast recovered from lightly-processed
cactus pear fruit, Corbo et al. [10*] selected colonies repre-
sentative of each different bacterial morphological type from
cultures and kept them on PCA at 4°C until they were identi-
fied. All bacteria strains were first grouped on the basis of
their staining reactions (as being Gram+ and Gram-), catalase
test, capacity for oxidative fermentative metabolism of glu-
cose, motility, cell shape and for spore formation (by heating
cultures at 80°C for 10 min and successive plating on PCA).
The isolates were identified to the species level using the
appropriate API identification system. Different yeast mor-
phologic types were also selected from the cultures and kept
on Sabouraud dextrose agar at 4°C until they were identified
using the API ATB ID32C system. Jacxsens et al. [43*]
noted that the isolates from industrially-prepared minimally-
processed mixed lettuce were identified by using API NE for
Gram- microorganisms, and API ID32 C for yeasts and API
50 CH for LAB. The identification was confirmed by LMG
Culture Collection applying protein gel electrophoresis
(SDS-PAGE) for LAB. In comparing API 50 CH strips with
whole-chromosomal DNA probes for their use in the identifi-
cation of Lactobacillus species, the use of genomic methods
such as 16S rRNA sequencing was suggested [44].
Biolog system
Biolog’s innovative technology is based on each microbe’s
ability to use particular carbon sources to produce a unique
pattern or fingerprint for that microbe. As a microorganism
begins to use the carbon sources contained in certain wells of
a commercially-prepared microplate, it respires. As respira-
tion continues a tetrazolium redox dye is reduced and the
well solution changes colour to purple. The end result is a
pattern of coloured wells within the microplate that is charac-
teristic of that particular microorganism. The microplate is
read in a plate-scanning device, then the fingerprint data is
fed to a data bank, which searches its extensive content for a
corresponding and pattern and identification is made. All
isolates to be identified using the Biolog systems are sub-
jected to Gram staining prior to identification. Jacxsens et al.
[43*] used Biolog GN plates to confirm the identity of bacte-
ria isolated from minimally processed mixed lettuce. These
plates identify Gram- microorganisms based on their ability to
oxidise 95 different carbon sources. Poubol and Izumi [30*]
were able to identify yeast strains present on mango cubes
using the Biolog system. Test isolates were first sub-cultured
to Biolog Universal Yeast agar (BUY; Biolog Inc) and incu-
bated at 26°C for 24–48 h. The resultant growth was then
suspended in sterile distilled water and adjusted to 47% T
using a Biolog YT turbidity standard. A 100 µL portion of
these was used to inoculate each well of the YT MicroPlate
prior to its incubation at 26°C. Each plate was then read by
the MicroStation Reader at 24, 48 and 72 h at a wavelength
of 590 nm. In our lab, many bacteria isolated from fresh-cut
onions during storage and on the shelf were identified to the
species level using Biolog GP 2 and GN 2 plates (unpub-
lished data).
Above growth-dependent methods are described. However,
there is increasing evidence for viable but non-culturable
microbes, particularly in the stressing environment [45]. In
this state, both Gram+ and Gram- bacteria are still viable and
show metabolic activity and respiration, but cannot be cul-
tured as CFUs by the conventional plate counts. Therefore,
standard methods should be supplemented by selected
growth-independent assay procedures for monitoring a viable
but non-culturable state. Currently, various methods have
been adapted to enable detection of viable but non-culturable
bacteria such as epiflourescent microscopy combined with
flow cytometry and molecular biology techniques [45].
Molecular technologies used in the identification
of targeted microorganisms
Nucleic acid-based systems designed for the detection of
genomic DNA specific to particular microorganisms are ca-
pable of achieving rapidly and highly sensitive identification
even when the target microbe is present in low numbers. The
polymerase chain reaction (PCR) is particularly useful in this
regard [30*, 46, 47*].
In fresh-cut fruits and vegetable research, Poubol and Izumi
[30*] used MicroSeq sequence analysis of 16 S rDNA to
identify bacteria isolated from mango cubes. The first 527
base pairs of the 16S rRNA gene were amplified by PCR,
then the product was sequenced with a set of proprietary
primers coupled with an ABI PRISMTM 310 Genetic Ana-
lyzer (PE Applied Biosystems, Foster City, Calif, USA) us-
ing the BigDyeTM terminator set. The results were analysed
6
Fan and Song / Stewart Postharvest Review 2008, 3:10
using the MicroSeqTM Analysis Software and MicroSeqTM 16
S rDNA Sequence Databases for bacterial species identifica-
tion. Watanabe et al. [48] used a similar molecular biological
approach to identify LAB. Analyses of DNA sequence reac-
tions were performed using an Applied Biosystems 310 se-
quencer. The homologies of the obtained 16 S rRNA se-
quences were assessed using the Basic Local Alignment
Search Tool (BLAST).
Description of the microbial composition and communities
on plant tissues are useful for assessing product quality and
predicting or following changes in shelf-life quality of fresh-
cut products. Biolog Eco plates (growth-dependent method)
can be used to analyse whole microbial communities. Hovda
et al. [47*] used denaturing gradient gel electrophoresis
(DGGE) of a PCR-amplified 16 S rDNA sequence to charac-
terise changes in the microbial flora of farmed cod treated by
ozone. Rudi et al. [49] developed a 16 S ribosomal DNA
array-based method (growth-independent method) for the
direct description of complex microbial communities in
modified atmosphere packaged vegetable salads.
Although the 16 S rRNA gene is widely used to identify bac-
terial species, the copy numbers of the chromosomal DNA
are different among species. Therefore, this is not a suitable
target for a real-time PCR assay. A newly developed real-
time PCR assay (primers targeting the rpoB gene) can enu-
merate the dominant bacterial species in ready-to-eat fruits
and vegetables [50].
Penicillium expansum causes postharvest fruit and vegetable
decay and is also of potential public health significance, since
it produces patulin, a mycotoxin. Rapid and specific detec-
tion of P. expansum is important for ensuring microbiological
quality and safety of fruits and vegetables. Marek et al. [46]
reported that the PCR amplified a 404-bp DNA product from
all the P. expansum isolates tested and determined the sensi-
tivity of the PCR. They concluded that PCR could potentially
be used as a rapid tool for screening fruits for the presence of
this fungus.
Fluorescent dyes for microbial visualisation and
assessment
Fluorescent stains are valuable tools that can be used to im-
prove the contrast between microbes and their surrounding
environment, indicate metabolic activity, assist with the de-
termination of structural composition and integrity, and per-
form rapid microbe-specific identification. There are various
analytical platforms including flow cytometry, fluorescence
microplate analysis, and fluorometry and fluorescence-based
microscopy. For example, various live/dead cell kits provide
a viability read-out based on membrane integrity. Intact live
bacteria exhibit green fluorescence upon their exposure to
certain dyes while dead bacteria exhibit red fluorescence
after their incubation in the presence of propidium iodide,
even when the population contains a mixture of bacterial
types.
Proteomic technology for microbial study
In the past decade, mass spectrometry methods have been
developed for the purpose of obtaining qualitative and quan-
titative analysis of proteins in biological samples. When
proteins are digested with trypsin, the resultant peptides can
be identified by mass spectrometry, either via matrix as-
sisted laser desorption/ionisation–time of flight (MALDI-
TOF) or nano-spray liquid chromatography–mass spec-
trometry (MS)/MS. The experimental spectrum is compared
with theoretical ones computed from protein sequence data-
bases to create “similarity scores” for each candidate pro-
tein. Alternatively, peptides are further isolated and frag-
mented within the mass spectrometer by MS/MS analysis,
resulting in a peptide fragment fingerprint. The peptide frag-
ment fingerprint approach is similar to the peptide mass fin-
gerprint approach, but is based on the MS/MS spectra and
correlates experimental peptide spectra with theoretical pep-
tide spectra from a MS/MS database [51]. With the estab-
lishment of more than 1,000 prokaryotic and eukaryotic ge-
nome sequences, the proteomic technique has been strongly
enhanced with rich genomic information. A review summa-
rising the application of proteomic technology to microbial
investigations was completed by Phillips and Bogyo [52].
Proteomic approaches have been employed to differentiate
bacteria in a community of 25 microorganisms [53]. When
capillary electrophoresis separation was applied in combina-
tion with a selective MS/MS, 34 specimens of bacteria were
identified with a 97% rate of success [54]. In a recent publi-
cation, Padliya et al. [55] pointed out the challenges of
pathogen identification using liquid chromatography/MS/
MS and a protein database for un-sequenced pathogens.
These authors suggested that un-interpreted mass spectra
should be used as a biomarker for pathogen identification.
Nevertheless, with the ever-increasing availability of ge-
nome sequence information and continued improvements of
MS technology, the MS-based proteomic technique holds
promise to overcome the limitations of traditional technolo-
gies, including intensive labour requirements, slow output
and unspecific (subjective) identification.
Conclusions
Review of published microbial quality assessments of fresh-
cut fruits and vegetables indicates that their microbial loads
contribute significantly to product quality and shelf-life.
Since the predominant microorganisms associated with spoil-
age differ between produce types, microbial quality assess-
ment methods should be considered and modified accord-
ingly. Evaluation of the microbiological quality of fresh-cut
fruits and vegetables should based on APC and other micro-
biological tests and should consider identifying the predomi-
nated microorganisms. When conducting microbial quality
assessment, it is important to follow standardised methods.
Integrated quality assessments, including physiologic, sen-
sory and microbial components, are needed to optimise shelf-
life. There are needs for future research to expand our under-
standing of the linkage between microbial population compo-
7
Fan and Song / Stewart Postharvest Review 2008, 3:10
sition and dynamics and quality changes relating to different
pre- and post-processing treatments in fresh-cut products.
Importantly, good pre- and post-processing practices and
temperature control during storage and the retail display of
fresh-cut fruits and vegetables are essential for maintaining
the quality of fresh-cut products. New technologies and ap-
proaches could focus on treatment steps and handling condi-
tions that inhibit the natural tendency of native microflora to
persist and multiply on plant tissues.
Acknowledgements
The authors would like to thank our research center reviewers
Drs. Greg Bezanson and John DeLong for their helpful sug-
gestions in the preparation of this review article. Contribution
no. 2353 of the Atlantic Food and Horticulture Research
Centre, Agriculture and Agri-Food Canada.
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