Food Microbiology 27 (2010) 381e389

Contents lists available at ScienceDirect

Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Taxonomic structure of the yeasts and lactic acid bacteria microbiota

of pineapple (Ananas comosus L. Merr.) and use of autochthonous
starters for minimally processing
Raffaella Di Cagno a, Gainluigi Cardinali b, Giovanna Minervini a, Livio Antonielli b,
Carlo Giuseppe Rizzello b, Patrizia Ricciuti c, Marco Gobbetti a, *
a
b
c

Department of Plant Protection and Applied Microbiology, University of Bari, via Amendola 165/a, 70126 Bari, Italy
Department of Applied Biology, University of Perugia, Italy
Department of Biologia e Chimica Agro-Forestale ed Ambientale, University of Bari, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 26 July 2009
Received in revised form
16 November 2009
Accepted 17 November 2009
Available online 26 November 2009

Pichia guilliermondii was the only identied yeast in pineapple fruits. Lactobacillus plantarum and
Lactobacillus rossiae were the main identied species of lactic acid bacteria. Typing of lactic acid bacteria
differentiated isolates depending on the layers. L. plantarum 1OR12 and L. rossiae 2MR10 were selected
within the lactic acid bacteria isolates based on the kinetics of growth and acidication. Five technological options, including minimal processing, were considered for pineapple: heating at 72
C for 15 s
(HP); spontaneous fermentation without (FP) or followed by heating (FHP), and fermentation by selected
autochthonous L. plantarum 1OR12 and L. rossiae 2MR10 without (SP) or preceded by heating (HSP). After
30 days of storage at 4
C, HSP and SP had a number of lactic acid bacteria 1000 to 1,000,000 times higher
than the other processed pineapples. The number of yeasts was the lowest in HSP and SP. The
Community Level Catabolic Proles of processed pineapples indirectly conrmed the capacity of
autochthonous starters to dominate during fermentation. HSP and SP also showed the highest antioxidant activity and rmness, the better preservation of the natural colours and were preferred for odour
and overall acceptability.
2009 Elsevier Ltd. All rights reserved.

Keywords:
Yeasts
Lactic acid bacteria
Fermented pineapple
Autochthonous starter

1. Introduction
Pineapple (Ananas comosus L Merr.) is the most representative
fruit of the Bromeliaceae family. It is mainly cultivated in the tropical and subtropical regions. In 2007, the production of pineapple
was estimated to be ca. 188,733,577 tonnes (www.fao.org). As
tropical fruit, its cultivation is only preceded by banana and citrus
(Bartholomew et al., 2002). Ripe fruits are consumed fresh, also as
topping in desserts and salads, cooked in pies, cakes and puddings
or processed. Canned pineapple and pineapple juice are generally
subjected sterilization and consumed throughout the world, mainly
because of their pleasant, and unique aroma and avour (Morton,
1987; Bartolome et al., 1995). The FAO organization estimated that
ca. 184,833 tonnes of pineapple per year are consumed in Italy
(www.fao.org). Besides sensory properties, the nutritional features
of pineapple also deserve an interest. Pineapple core is a source of

* Corresponding author. Tel.: 39 080 5442949; fax: 39 080 5442911.

E-mail address: gobbetti@agr.uniba.it (M. Gobbetti).
0740-0020/$ e see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2009.11.012

bres (Stuab et al., 1983) to be used as functional ingredients for

bakery and meat products (Prakongpan et al., 2006). Pineapple
fruits and their extracts (bromelain) are proposed as potential antiinammatory agents in rheumatoid arthritis, ulcerative colitis,
colon inammation, chronic pain and asthma (Secor et al., 2005;
Onken et al., 2008).
Overall, fresh fruits are essential components of the human diet
and there is considerable evidence of the health and nutritional
benets associated with their consumption. Public health institutions recommend the consumption of at least ve daily servings of
fruits. Especially, fresh and minimally processed pineapple (e.g.,
peeled, cut and packaged fruit salads), but also pineapple juice are
strongly recommended for the nutritional properties. Minimally
processed pineapples are present in the market under different
shapes: cubes, slices, chunks and cored whole fruit. Since the bulky
inedible crown and peel tissue are removed, they have the
commercial advantage of decreasing the weight for transport (Budu
and Joyce, 2005). Nevertheless, two major problems might affect
the quality of minimally processed pineapples. The shelf-life is very
limited (ca. 2e3 days) because of the pulp browning and

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R. Di Cagno et al. / Food Microbiology 27 (2010) 381e389

accumulation of liquid in the packaging (Antoniolli et al., 2007).

Because they are not heat-processed and without preservatives
added, minimally processed pineapple might also be contaminated
by yeasts and moulds which cause off-avours and -odours,
discoloration, and in the extreme cases human illness (Trindade
et al., 2002). In particular, yeasts are the common contaminants of
fruits and the inhibition of their growth represents the major task
for minimally processing (Devenport, 1996).
Recently (Di Cagno et al., 2008a,b, 2009a), it was shown that
the use of selected and autochthonous lactic acid bacteria starters
guaranteed the prolonged shelf-life of fermented carrots, French
beans, tomato juice, and red and yellow peppers which also
maintained agreeable nutritional, rheology and sensory properties. Autochthonous strains always had better performances than
allochthonous strains, originating from different fruits or vegetables. The epiphytic microbial population of plants is largely
subjected to uctuations of the physical and nutritional conditions (Lindow and Brandi, 2003). Nevertheless, analyses by
molecular methods showed that each species of vegetables and
fruits harbours a dominant and constant microbiota (Yang et al.,
2000). The autochthonous microbiota of fruits may have various
functions: (i) to exert intrinsic antagonistic activity towards
spoilage and pathogen microorganisms; (ii) to deliver health
relevant microorganisms to the gastrointestinal tract; and (iii) to
supply autochthonous lactic acid bacteria suitable to be re-used
as starters.
To the best of our knowledge, nothing is known about the yeasts
and lactic acid bacteria microbiota of pineapple fruits and the use of
selected autochthonous lactic acid bacteria starters has not yet
been considered for minimally processing. This work aimed at
describing the taxonomic structure of the yeasts and lactic acid
bacteria microbiota of pineapple fruits. Selected autochthonous
lactic acid bacteria starters were used for pineapple processing and
compared to other technological options to guarantee microbiological, antioxidant, texture, colour and sensory properties.
2. Materials and methods
2.1. Samples
Pineapple fruits (A. comosus L. Merr.) at commercial maturity
were purchased in triplicate from three supermarkets of Bari, Italy.
Prior to use, fruits were kept at 4
C. Outside spirals were removed
and fruits were cut into slices of ca. 1 cm thick and ca. 6e7 cm of
diameter. Each slice was subsequently divided into three layers: (i)
outer ring of ca. 2 cm of width (OR), (ii) middle ring of ca. 3 cm (MR)
and (iii) inner ring of ca. 1e2 cm (IR). Sterile knifes under sterile
conditions were used to prepare slices from the three layers.

(Yeast Extract 1% [w/v], Peptone 1% [w/v], Dextrose 2% [w/v] and

Agar 1.7% [w/v]). Gram-positive, catalase-negative and non-motile
rod isolates were cultivated in MRS broth at 30
C for 24 h, and restreaked onto MRS agar. Stock cultures were stored at 20
C in 10%
(v/v) glycerol.
2.3. Genotypic identication of yeasts and lactic acid bacteria
Genomic DNA of yeasts was extracted as previously described
(Bolano et al., 2001; Cardinali et al., 2001). The D1/D2 domain of the
26S rDNA was amplied and sequenced according to the procedure
previously described by Kurtzman and Robnett (1998). Analysis of
the sequences was carried out with Geneious (http://www.
geneious.com). Electropherograms were processed by BLAST
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) and sequences aligned
with clustalw algorithm (http://www.clustal.org/).
Genomic DNA of lactic acid bacteria was extracted according to
De Los Reyes-Gaviln et al. (1992). Two primer pairs (Invitrogen Life
Technologies, Milan, Italy), LacbF/LacbR and LpCoF/LpCoR, were
used to amplify 16S rRNA gene fragment of lactic acid bacteria (De
Angelis et al., 2006). Taxonomic identication was carried out by
comparing the sequence of each isolate with those reported in the
Basic BLAST database (http://www.ncbi.nlm.nih.gov). Lactobacillus
plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum
isolates were further characterized by partial sequencing of the
recA gene (Torriani et al., 2001). Weissella cibaria/confusa isolates
were further characterized by partial sequencing of the pheS gene
(Naser et al., 2005).
2.4. Randomly amplied polymorphic DNA-polymerase chain
reaction (RAPD-PCR) analysis of lactic acid bacteria
The PTC-100 Peltier Thermal Cycler (MJ Research Inc. Waltham, Massachusetts USA) was used for PCR amplication of
lactic acid bacteria. Three primers (Invitrogen), with arbitrarily
chosen sequences (M13, 50 -GAGGGTGGCGGTTCT-30 , P7 50
AGCAGCGTGG 30 and P4 50 CCGCAGCGTT 30 ), were used singly in
three series of amplication (Di Cagno et al., 2008b). The
molecular weight of the amplied DNA fragments was estimated
by comparison with 1 Kb Plus DNA Ladder (Invitrogen). For RAPD
analysis, the presence or absence of fragments was recorded as 1
or 0, respectively. Only reproducible well-marked amplied
fragments were scored, with faint bands being ignored. The three
series of RAPD-PCR proles were evaluated and combined to
obtain a unique dendrogram, calculating an index of genetic
similarity by the Simple Matching coefcient (Sokal and
Michener, 1958).

2.2. Isolation of yeasts and lactic acid bacteria

2.5. Selection of lactic acid bacteria based on the kinetics

of growth and acidication

Ninety millilitres of peptone-physiological solution (0.1% [w/v]

bacteriological peptone [Oxoid Basingstoke, Hampshire, United
Kingdom] and 0.85% [w/v] NaCl) were added to 10 g of each
pineapple layer and homogenized for 5 min. An aliquot of this
suspension was serially diluted and plated in triplicate on Malt
Extract Agar (MEA) (Oxoid) added of 150 ppm chloramphenicol
(Sigma) or on MRS agar (Oxoid), containing 0.1% (w/v) of cycloheximide (Sigma). MEA plates were incubated at 25
C for 48 h.
MRS agar plates were incubated at 30
C for 48e72 h under
anaerobiosis. After incubation, at least 15 colonies were isolated
from MEA and MRS plates of the highest dilution. Morphologically
distinct yeasts colonies were selected and puried by streaking on
MEA plates. The MEA plates were incubated at 25
C for 48 h.
Isolated yeasts were grown and maintained in YEPDA plates

All isolates of lactic acid bacteria were cultivated in MRS broth at

30
C for 24 h, harvested by centrifugation (10,000  g, 10 min, 4

C), washed twice in 50 mM sterile potassium phosphate buffer (pH
7.0) and used to inoculate (4% v/v, initial cell density ca. 7.0 log cfu
mL1) the sterile pineapple juice (see below for preparation).
Fermentations were carried out at 25
C for 24 h. Kinetics of growth
and acidication were determined and modelled according to the
Gompertz equation as modied by Zwietering et al. (1990). Growth
data were modelled according to the equation: y k A exp
{ exp[(mmax e/A)(l  t) 1]}; where y is the growth expressed as
log cfu mL1 h1 at the time t; k is the initial level of the dependent
variable to be modelled (log cfu mL1); A is the difference in cell
density between inoculation and the stationary phase; mmax is the
maximum growth rate expressed as D log cfu mL1 h1; l is the

R. Di Cagno et al. / Food Microbiology 27 (2010) 381e389

383

length of the lag phase expressed in hours; and t is the time.

Acidication data were modelled according to the equation: y k
A exp { exp[(Vmax e/A)(l  t) 1]}; where y is the acidication
extent expressed as dpH dt1 (units of pH h1) at the time t; k is the
initial pH (units); A is the difference in pH between inoculation and
the stationary phase; Vmax is the maximum acidication rate
expressed as dpH h1; l is the length of the lag phase expressed in
hours; and t is the time.

2.7. Microbiological analysis

2.6. Processing pineapples

2.8. BIOLOG community-level metabolic ngerprinting

The protocol for processing and storage of pineapples is

described in Fig. 1. Pineapple juice was prepared by homogenization (5 min at room temperature, PBI International) of the whole
slice of the fruit. After homogenization, the supernatant was
recovered by centrifugation (10,000  g, 10 min, 4
C) and sterilized
by ltration on 0.22 mm membrane lters (Millipore Corporation,
Bedford, MA 01730). Slices of pineapple used to ll the juice were
cut into four pieces. Each piece included all the three layers (OR, MR
and IR). The area of each piece was 10e12 cm2 L. plantarum 1LE12
and Lactobacillus rossiae 2LC10 were used as the mixed autochthonous starter to inoculate (4% v/v) the sterile pineapple juice.
According to the protocol of Fig. 1, ve samples were prepared and
stored for 30 days at 4
C: Heated Pineapple (HP), Fermented and
Heated Pineapple (FHP), Heated and Started Pineapple (HSP), Fermented Pineapple (FP), and Started Pineapple (SP).

Carbon source utilization patterns of the microbial communities

during fermentation and storage of pineapples were assessed by
using BIOLOG 96-well Eco-Microplates (Biolog, Inc., Hayward, CA,
USA) (Crecchio et al., 2004). Microplates contain 31 different
carbon-sources (carbohydrates, carboxylic acids, polymers, amino
acids, amines and miscellaneous substrates) and the control,
without carbon source, in triplicate. Ten grams of mixed pineapple
fruit and juice were homogenized with 90 mL of sterile sodium
chloride (0.9% w/v) solution and centrifuged at 10,000  g for 15
min at 4
C. The pellet was washed with sterile 50 mM TriseHCl (pH
8.8), further with sterile sodium chloride solution and then
centrifuged again. The cell suspension was diluted (1:100) in sterile
sodium chloride solution and dispensed (150 mL) into each of 96
wells of the BIOLOG Eco-Microplates. Incubation was at 30
C in the
dark and colour development was measured at 590 nm with

Microbiological analyses were carried out by mixing 10 g of

pineapple fruit and juice. Mesophilic lactic acid bacteria and yeasts
were determined on MRS agar (Oxoid), containing 0.1% of cycloheximide (Sigma), at 30
C for 48e72 h under anaerobiosis, and on
MEA (Oxoid), added of 150 ppm chloramphenicol, at 25
C for 48 h,
respectively.

Pineapple fruits

Removing the outside spirals and cutting in slices of ca. 1 cm thick.

Filling the sterile juice (ca. 100 g of pineapple fruits into 100 mL of sterile juice)
into 200 mL of fermentation glass jar

Heating
(at 72C for 15 sec)

Heating
(at 72C for 15 sec)

Spontaneous fermentation
(at 25C for 24 h)
Heating
(at 72C for 15 sec)

Heated Pineapple Fermented and Heated

Pineapple
(HP)
(FHP)

Spontaneous fermentation
(at 25C for 24 h)
Inocolulation of
autochthonous starter
(ca. 7.0 log cfu mL-1)
into sterile juice

Inocolulation of
autochthonous starter
(ca. 7.0 log cfu mL-1)
into sterile juice

Fermentation
(at 25C for 24 h)

Fermentation
(at 25C for 24 h)

Heated and Started

Pineapple
(HSP)

Fermented Pineapple
(FP)

Storage
(at 4C for 30 days)
Fig. 1. Protocol for processing pineapples.

Started Pineapple
(SP)

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R. Di Cagno et al. / Food Microbiology 27 (2010) 381e389

a microplate reader (BIOLOG Microstation) every 24 h up to 120 h.

Three indices were calculated (Shannon, 1948a,b). Shannon's
P
diversity (H0 ), indicating the substrate utilization pattern: H0 
pi ln (pi), where pi is the ratio of the activity of a particular substrate
to the sums of activities of all substrate activity at 120 h. Substrate
richness (S), measuring the number of different substrates used,
was calculated as the number of wells with a corrected absorbance
greater than 0.25. Substrate Evenness (E) was dened as the equitability of activities across all utilized substrates: E H0 /log S.

2.12. Statistical analysis

All analysis and fermentations were carried out in triplicate and
samples were twice analyzed (total of six analyses for each sample).
Data were subjected to one-way ANOVA (SAS, 1985); paircomparison of treatment means was achieved by Tukey's procedure at P < 0.05, using the statistical software, Statistica for
Windows (Statistica 6.0 per Windows 1998).
3. Results

2.9. Determination of pH, organic acids and ethanol

3.1. Identication of yeasts

The pH was measured by a Foodtrode electrode (Hamilton,

Bonaduz, Switzerland). Organic acids were determined on the
water-soluble extract by HPLC (High Performance Liquid Chromatography) using an KTA Purier system (GE Healthcare) equipped
with an Aminex HPX-87H column (ion exclusion, Biorad) and a UV
detector operating at 210 nm. Elution was at 60
C, with a ow rate
of 0.6 mL min1, using H2SO4 10 mM as mobile phase (Zeppa et al.,
2001). Organic acids used as standards were from Sigma Chemical
Co. The concentration of ethanol was determined by enzymatic
method (DIFF-CHAMB, Italia srl., Italy).

As estimated by plating on MEA agar, the cell density of yeasts

decreased from 5.01  0.05, 4.05  0.29 to 4.00  0.60 log cfu g1
from the outer ring (OR), middle ring (MR) to inner ring (IR),
respectively. Forty-eight isolates were recovered from the highest
dilutions of the MEA plates and subjected to identication by
sequencing the D1/D2 domain of the DNA encoding the 26S rRNA.
All isolates from the three layers belonged to the species Pichia
guilliermondii. Since considered to be contaminant and spoilage
microorganisms (Bolano et al., 2001; Cardinali et al., 2001), yeasts
were not included for further starter selection.
3.2. Identication and typing of lactic acid bacteria

2.10. Radical DPPH scavenging capacity

Extract was prepared by mixing 5 g of pineapple fruit and juice
with 50 mL of 80% (v/v) of methanol. The mixture was purged with
stream of nitrogen and mixed for 30 min, then centrifuged at 6000
 g for 20 min. The extract was transferred into test tubes, purged
with a stream of nitrogen and refrigerated before analysis. The free
radical scavenging capacity was determined using the stable 2,2diphenyl-1-picrylhydrazyl radical (DPPH_) as described by Yu et al.
(2002). The antioxidant reaction was started by transferring 1 mL of
the extract into a test tube, containing 4 mL of 80% (v/v) methanol
and 1 mL of freshly prepared DPPH_ solution. The nal concentration of DPPH_ in the reaction mixture was 100 mM. The reaction was
monitored by reading the absorbance at 517 nm for 30 min at 2 min
intervals. A blank reagent was used to study stability of DPPH_ over
the test time. The mixture was purged with stream of nitrogen,
mixed for 30 min and centrifuged at 6000 rpm for 20 min. The
absorbance measured at 10 min was used to calculate the mmoles of
DPPH_ scavenged by pineapple extracts. The kinetics of the antioxidant reaction in the presence of pineapple fruit extracts were also
determined over a 30 min period and compared with 75 ppm
butylated hydroxytoluene (BHT) as an antioxidant reference.

As estimated by plating on MRS agar, presumptive mesophilic

lactic acid bacteria were 5.75  0.91, 5.18  0.22 and 4.32  0.64 log
cfu g1 in OR, MR and IR, respectively. One-hundred-four Grampositive, catalase-negative and non-motile rods isolates, able to
grow at 15
C and to acidify MRS broth, were identied by partial
sequencing of the 16S rRNA. Apart from the layer, only three species
were identied. L. plantarum was the dominant species (79 of the
104 isolates). L. plantarum (30 isolates) and L. rossiae (4) were found
in OR; L. plantarum (26), L. rossiae (15) and W. cibaria (2) in MR; and
L. plantarum (23) and L. rossiae (4) in IR.
All 104 isolates were subjected to RAPD-PCR analysis by primers
M13, P4 and P7. At the similarity level of 80%, isolates were grouped
into ten clusters (listed as T1 e T10). The 50% of all isolates
belonging to each cluster was subjected to the simple matching
coefcient analysis (Fig. 2). Clusters T1, T2 and T7 included isolates
from all three layers. T1 and T2 only grouped L. plantarum, and T7
only harboured L. rossiae. T3 and T6 included isolates of L. plantarum from contiguous layers MR and IR. T4 grouped isolates of
L. rossiae from contiguous layers OR and MR. Clusters T5, T8, T9
and T10 included isolates of L. rossiae from OR and IR, and W. cibaria
and L. plantarum from MR only.
3.3. Processing of pineapples

2.11. Texture, colour and sensory analyses

Texture of pineapple fruits was measured by Penetrometer
tester equipped with a plunger number 2 mm (TR Turoni, Forl,
Italy). Colour indices b and L were determined by a Chromameter
CR-200 Tristimulus Colorimeter (Minolta, Osaka, Japan). The
sensory analysis of processed pineapples was determined by using
the descriptive model of Bartolome et al. (1995) with a few modications. Appeararance, avour, odour, off-odours and overall
acceptability were the sensory attributes considered by using a 1e5
structured scale. A nontrained panel consisting of 10 judges was
used. For appearance: 1, good; 2, fairly good; 3, acceptable; 4,
slightly bad; 5, bad; avour: 1, sweet; 2, fairly sweet; 3, sweet-sour;
4, fairly sour; 5, sour; odour: 1, characteristic; 2, slightly characteristic; 3, off-odours; overall acceptability: 1, likes very much; 2,
likes slightly; 3, accepts; 4, dislikes slightly; 5, dislikes.

Preliminarily, all lactic acid bacteria isolates were screened

based on the kinetics of growth and acidication (Table 1). All
isolates were used as single starter for fermentation of pineapple
juice. The minimum (m) and maximum (M) values of the
parameters of growth and acidication (Table 1) refer to whole
number of isolates. After 24 h of fermentation at 25
C, all isolates
grew at least 1.23  0.1 log cfu mL1 (minimum value of A, m).
L. plantarum 1OR12 and L. rossiae 2MR10 showed almost the highest
increase of the cell yield (maximum value of A, M 2.55  0.3 log cfu
mL1). These two strains were also characterized by low and high
values of l (2.70  0.04 and 3.34  0.02 h) and mmax (0.88  0.05 and
0.64  0.03 D log cfu mL1 h1), respectively. The same behaviour
was found for the kinetic of acidication. The m and M values of DpH
were 0.35  0.03 and 0.85  0.02, respectively. In particular,
L. plantarum 1OR12 showed the highest (0.85  0.02 pH units)

R. Di Cagno et al. / Food Microbiology 27 (2010) 381e389

100

T1 Lactobacillus plantarum

T2

L. plantarum

T3

L. plantarum

T4

Lactobacillus rossiae

T5
T6

L. rossiae
L. plantarum

T7

L. rossiae

T8

L. rossiae

T9
T10

Weissella cibaria
L. plantarum

Similarity (%)
80

90

385

70

60

50

1OR1
1OR2
1OR4
1OR5
1OR11
1OR12
1OR13
1OR14
1MR1
1MR2
1MR3
1MR4
1MR14
1CD15
1CD20
1CD2
1CD18
1CD19
1CD10
1MR19
1CD8
2OR4
2OR18
2OR19
2MR3
2MR1
2OR6
2CD13
2MR11
2CD15
2OR9
2OR14
2MR6
2MR8
2MR10
2MR15
2MR20
2MR17
3XOR1
2MR22
2CD22
3XOR2
2MR28
2MR29
2CD21
2MR21
3XCD1
3XOR3
2CD23
3XMR3
3XMR5
3XMR4

Fig. 2. Dendrogram obtained by combined random amplication of polymorphic DNA patterns for the lactic acid bacteria isolates from pineapples using primers M13, P4 and P7.
Cluster analysis was based on the simple matching coefcient and unweighted pair group with arithmetic average. OR, isolates from outer ring (OR); MR, isolates from middle ring
(MR); and IR, isolates from inner ring (IR) of pineapple fruits.

and lowest (1.45  0.03 h) values of DpH and l, respectively.

Based on the above results, L. plantarum 1OR12, isolated from the
layer OR, and L. rossiae 2MR10, isolated from MR, were selected
as the autochthonous starters. These two species were also
undoubtedly the most numerically representative of the pineapple
microbiota.
Five different technology options were considered for processing of pineapples (Fig. 1). Before heating or fermentation, the
number of presumptive lactic acid bacteria in pineapple fruit and
juice was ca. 5.0  0.4 log cfu g1. After 24 h of spontaneous
fermentation at 25
C (fermented pineapple, FP), the cell number of
presumptive lactic acid bacteria did not increase (4.50  0.4 log cfu
g1) (Table 2). This number did not signicantly (P > 0.05) differ
from that found in fermented and heated pineapple (FHP) or in
heated pineapple (HP) (3.9  0.3 and 3.6  0.3 log cfu g1,
respectively). On the contrary, heated and started pineapple (HSP),
and started pineapple (SP) showed increases of the cell density of
both the autochthonous starters from ca. 7.0 to 8.5  0.4 and 9.4  0.2

log cfu g1, respectively. After 30 days of storage at 4
C, the cell

number of presumptive lactic acid bacteria only slightly decreased in
FHP. Although pineapples processed with autochthonous starters
(HSP and SP) showed the decrease of ca. 1 log cycle, at the end of
storage they contained a number of lactic acid bacteria 1000 to
1,000,000 times higher than the other processed pineapples. RAPDPCR analysis conrmed the presence of L. plantarum 1OR12 and
L. rossiae 2MR10 in HSP and SP.
Before heating or fermentation (Fig. 1), the number of yeasts in
pineapples was ca. 4.0  1.1 log cfu g1. After spontaneous
fermentation (FP), the cell density increased to 5.0  0.5 log cfu g1
(Table 2). On the contrary, HSP and SP showed decreases of the cell
number (2.1  0.2 and 2.6  0.2 log cfu g1, respectively). The other
processed pineapples contained a number of yeasts similar to that
found in the fruits. During storage, yeasts slightly increased in FP
and decreased in all other processed pineapples. After 30 days at 4

C, the lowest number of yeasts was found in HSP and SP (1.7  0.2
and 1.9  0.4 log cfu g1, respectively).

Table 1
Parameters of the kinetics of growth and acidication of pineapple juice started (24 h at 25
C) with single isolates of lactic acid bacteria. The minimum (m) and maximum (M)
refer to whole number of isolates from pineapple fruits. Values for individual lactic acid bacteria further used as starters for pineapple processing are also included.

Pineapple juice started with single lactic acid bacteria

L. plantarum 1OR12
L. rossiae 2MR10

A (log cfu mL1)

l (h)

m 1.23 
M 2.55 
2.52 
2.45 

m 2.65 
M 6.15 
2.70 
3.34 

0.1
0.3
0.1
0.3

mmax (Dlog cfu mL1 h1) DpH (pH units) l (h)

0.04
0.1
0.04
0.02

m 0.35 
M 0.90 
0.88 
0.64 

0.01
0.05
0.05
0.03

m 0.35 
M 0.85 
0.85 
0.76 

The data are the means of three independent experiments  standard deviations (n 3).
Growth and acidication data were modelled according to the Gompertz equation, as modied by Zwietering et al. (1990).

0.03
0.02
0.02
0.02

m 1.45 
M 3.07 
1.45 
1.52 

Vmax (DpH h1)

0.03
0.05
0.03
0.02

m 0.28 
M 0.62 
0.58 
0.45 

0.01
0.01
0.01
0.01

386

R. Di Cagno et al. / Food Microbiology 27 (2010) 381e389

Table 2
Cell numbers (log cfu g1) of lactic acid bacteria and yeasts, and Biolog ngerprinting of processed pineapples before (1 day) and after 30 days of storage at 4
C.
Time (days)

Sample

Lactic acid bacteria (log cfu g1)

d

Yeasts (log cfu g1)

H0

cd

HP
FHP
HSP
FP
SP

3.6
3.9
8.5
4.5
9.4







0.3
0.3cd
0.4a
0.4c
0.2a

3.5
4.6
2.1
5.0
2.6







0.3
0.3b
0.2e
0.5ab
0.2d

2.77
2.91
0.52
2.94
1.45







0.1a
0.1a
0.4e
0.2a
0.1d

14.7
15.7
3.5
15.0
4.7







1.2b
1.5a
1.0e
1.0ab
0.6d

2.38
2.43
1.66
2.50
1.97







0.1b
0.2a
0.7d
0.1a
0.1c

30

HP
FHP
HSP
FP
SP

2.5
3.6
7.3
4.2
8.5







0.2e
0.4d
0.3b
0.4c
0.5b

3.0
3.8
1.7
5.6
1.9







0.2d
0.5c
0.2f
0.6a
0.4f

2.76
2.61
0.61
1.96
1.17







0.1a
0.4b
0.4e
0.1c
0.3d

15.3
15.5
4.7
7.3
4.0







2.1a
0.7a
2.1d
0c
1.0e

2.34
2.34
1.37
2.52
1.86







0.1b
0.2b
0.3e
0.1a
0.7c

HP, heated pineapple; FHP, fermented and heated pineapple; HSP, heated and started pineapple; FP, fermented pineapple; SP, started pineapple. For the details of processed
pineapples see Fig. 1 and Material and methods.
H0 , Shannon's diversity; S, substrate richness; E, substrate evenness.
aef
, Means within the column with different superscript letters are signicantly different (P < 0.05).
Each value was expressed as the mean  standard deviations (n 3) analyzed in duplicate.

3.4. Biolog ngerprinting

The catabolic proles of the microbiota of pineapples before and
after 30 days of storage was determined by calculating the indices
H0 , S and E (Table 2). According to the utilization pattern substrate
(H0 index), pineapple fruits processed with autochthonous starters
(HSP and SP) were signicantly (P < 0.05) different from HP, FPH
and FP. Before storage, HSP had the lowest H0 index (0.52  0.4)
followed by SP (1.45  0.1). These values remained almost constant
during 30 days of storage, meaning a constant and stable microbial
population. On the contrary, HP, FHP and FP had very high values of
H0 . These values remained almost constant during storage. The only
exception was FP which showed a decrease from 2.94  0.2 to 1.96
 0.1. The S index (substrate richness) had the same trend as the H0
index. The E index, giving a measure of the statistical signicance
(equitability) of the values of H0 and S indices, conrmed the
signicant (P < 0.05) differences between started and nonstarted
pineapples.

3.5. pH, organic acids and ethanol

As expected, pineapples fruit and juice had an inherent acidity
(pH ca. 3.5). Fermentation by autochthonous lactic acid bacteria
without (SP) or preceded by heating (HSP) caused a further
decrease of the pH to ca. 2.73  0.07. The other pineapples which
were subjected to spontaneous fermentation (FHP and FP) did not
show the same extent of acidication. The value of pH was ca. 3.25
 0.06. HP had almost the same value of pH of nonprocessed fruits.
The values of pH remained almot constant during storage.
The concentration of organic acids and ethanol found in HP was
almost similar to that found in fruits before processing. Compared
to HP (64.0  1.3 mM), the concentration of citric acid signicantly
(P < 0.05) decreased only in pineapples subjected to spontaneous
fermentation (Table 3). FHP and FP contained the highest cell
densities of yeasts (Table 2) and all isolates of P. guilliermondii were
positive for citrate fermentation/assimilation. Compared to HP
(50.7  1.1 mM), the concentration of malic acid markedly
decreased in HSP and SP. Both the autochthonous starters were
positive for malate fermentation. Lactic acid was only found in
processed pineapples with autochthonous starters: 37.8  1.6 and
37.3  1.4 mM for HSP and SP, respectively. The synthesis of lactic
acid was probably due to the conversion of malate and/or to the
fermentation of soluble carbohydrates. Also the acetic acid was only
found in the started pineapples. Especially, the obligately heterofermentative L. rossiae 2MR10 might be responsible for the
synthesis of acetic acid. The concentration of ethanol was the

highest in processed pineapples FHP and FP. The concentration of

organic acids and ethanol of all processed pineapples was almost
constant during storage (data not shown).
3.6. Antioxidant activity
The antioxidant properties were determined based on the
scavenging activity towards DPPH radical. During assay, the coloured stable DPPH radical is reduced to non-radical DPPH-H when
in the presence of an antioxidant or a hydrogen donor. DPPH radical
without antioxidants or pineapple extracts was stable over the time
(data not shown). The colour intensity of DPPH_ showed a logarithmic decline when in the presence of BHT (75 ppm) (Fig. 3). All
processed pineapples caused a sharp drop of the DPPH_ colour
intensity, indicating the rapid and high capacity to quench DPPH
radical. After 10 min of reaction, the remaining colour intensity of
DPPH_in the presence of BHT was 19.1  0.2%. Compared to HP (48.2
 0.1%), the colour intensity of DPPH_ signicantly (P < 0.05)
increased only in FP (66.6  0.1%). On the contrary, the colour
intensity of DPPH_ decreased in HSP and SP (40.6  0.1 and 38.9 
0.1%, respectively). The scavenging activity towards DPPH radical of
all processed pineapples remained almost constant during storage.
3.7. Texture, colour and sensory analyses
Before storage, rmness of pineapple HP (1.85  0.06 kg cm2e1)
was almost the same of that found in fruits before processing (1.92
 0.06 kg cm2e1). Compared to HP, the rmness signicantly (P <
0.05) decreased in pineapples subjected to spontaneous

Table 3
Concentration (mM) of citric, malic, lactic and acetic acids, and ethanol of processed
pineapples before storage at 4
C.
Sample

Citric acid

HP
FHP
HSP
FP
SP

64.0
52.7
66.8
41.8
63.8







1. 3a
2.1b
1.4a
2.2c
1.6a

Malic acid
50.7
46.6
22.0
37.2
22.4







1.1a
1.5b
2.5d
2.4c
1.8d

Lactic acid

Acetic acid

Ethanol

N.D*
N.D.
37.8  1.6a
N.D.
37.3  1.4a

N.D.
N.D.
7.8  0.6b
N.D.
11.2  1.9a

7.5
17.2
6.7
19.5
8.2







1.0bc
3.1a
1.1c
2.4a
1.5b

HP, heated pineapple; FHP, fermented and heated pineapple; HSP, heated and
started pineapple; FP, fermented pineapple; SP, started pineapple. For the details of
processed pineapples see Fig. 1 and Material and methods.
*N.D., not detected.
aed
, Means within the column with different superscript letters are signicantly
different (P < 0.05).
Each value was expressed as the mean  standard deviations (n 3) analyzed in
duplicate.

R. Di Cagno et al. / Food Microbiology 27 (2010) 381e389

387

1.8
1.6

D.O. (Abs 517nm)

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

10

15
Time (min)

20

25

30

Fig. 3. Kinetics of the scavenging activity of BHT (C), HP, Heated Pineapple (;); FHP, Fermented and Heated Pineapple (-); HSP, Heated and Started pineapple (,); FP, Fermented
Pineapple (A); and SP, Started Pineapple (7) towards DPPH radical. The concentration of DPPH_ in the reaction mixture and control (B) was 100 mmol. Data are the means of three
independent experiments  standard deviation (n 3).

fermentation. It was 1.45  0.06 and 1.64  0.04 kg cm2e1 for FHP
and FP, respectively. The rmness of started pineapples HSP and SP
(1.79  0.05 and 1.83  0.03 kg cm2e1, respectively) did not
signicantly (P > 0.05) vary with respect to HP. During storage, the
rmness of all processed pineapples further decreased. Nevertheless, the rmness of HSP (1.66  0.06 kg cm2e1) and SP (1.75  0.07
kg cm2e1) was higher than that found in HP (1.50  0.03 kg cm2e1),
and FHP and FP (1.31  0.02 and 1.54  0.04 kg cm2e1).
Before storage, yellow (b) and bright indices (L) of processed
pineapple HP were 26.5  1.2 and 85.2  3.4. These values were
lower than those found in fruits before processing (28.1  1.4 and
90.3  3.8). Both indices decreased during processing of all processed pineapples. The indices were 19.82  1.4 and 20.6  1.6, and
69.56  2.7 and 72.35  3.0 for FHP and FP, respectively. The same
indices for HSP and SP were signicantly (P < 0.05) higher (24.7 
1.9 and 25.4  2.1, and 81.5  3.9 and 83.4  3.5) and approached
those of HP. Although decreasing, the differences for yellow and
bright indices between started and nonstarted samples remained
constant during storage (data not shown).
At the end of storage, all the sensory attributes were signicantly (P < 0.05) scored the highest for pineapples started by
autochthonous lactic acid bacteria (Table 4). Due to lowest value of
pH, avour was the sensory attribute which mainly differentiated
started pineapples. Overall, fermentation of pineapples by
autochthonous lactic acid bacteria without (SP) or preceded by
heating (HSP) determined an agreeable odour (1.37  0.8 and 1.45
 0.9) and overall acceptability (1.24  0.4 and 1.38  0.9). These
attributes were not appreciated for FHP (2.75  1.1 and 4.04  1.7)
and FP (3.00  1.5 and 4.65  1.9).

Di Cagno et al., 2008a,b). Pineapple fruits harboured autochthonous

yeasts and lactic acid bacteria at cell densities of ca. 4.5 and 5.0 log
cfu g1, which corresponded to those usually found in tropical fruits
(Spurr, 1994; Postmaster et al., 1997). Cell densities were the
highest in the outer ring but yeasts and lactic acid bacteria were
also found in the middle and inner rings of pineapples. The outside
spirals of pineapple is a good barrier to prevent microbial
contamination. Nevertheless, the nectariferous glands of the spirals
have a deep central cavity which is open onto the base of the style
through three separate canals. These canals should provide an
access for microorganisms which tolerate this hostile environment.
Instead of the large microbial biodiversity of tropical fruits and
other vegetables (Postmaster et al., 1997; Nielsen et al., 2007; Di
Cagno et al., 2008b, 2009a), pineapples represented a very selective
environment. Only a single species of yeast, P. guilliermondii, and
two main species of lactic acid bacteria, L. plantarum and L. rossiae
were identied. P. guilliermondii was already isolated from the
surface of other fruits, including apples and pears (Bolano et al.,
2001; Cardinali et al., 2001; Cardinali, pers. Comm.). Likely to other
fruit or vegetable fermentations, also for pineapple processing,
yeasts and, in this case P. guilliermondii, was considered as

4. Discussion

HP, heated pineapple; FHP, fermented and heated pineapple; HSP, heated and
started pineapple; FP, fermented pineapple; SP, started pineapple. For the details of
processed pineapples see Fig. 1 and Material and methods.
aed
Means within a column with different superscript letters are signicantly
different (P < 0.05).
Each value was expressed as the mean  standard deviations (n 3) analyzed in
duplicate.

The microbial population of fruits and vegetables is estimated to

uctuate between 5 and 7 log cfu g1 (Spurr, 1994). The number of
yeasts and lactic acid bacteria may range between 2e6 and 3e5 log
cfu g1, respectively (Rosini et al., 1982; Nyanga et al., 2007;

Table 4
Sensory analysis of processed pineapples after 30 days of storage at 4
C.
Sample

Appearance

HP
FHP
HSP
FP
SP

1.54
2.95
1.65
3.85
2.00







1.0d
1.1b
0.9d
1.3a
1.2c

Flavour
2.75
3.04
3.87
3.15
3.74







1.1b
1.5b
1.0a
1.1b
1.2a

Odour
2.05
2.75
1.37
3.00
1.45







Acceptability
1.0b
1.1a
0.8c
1.5a
0.9c

2.46
4.04
1.24
4.65
1.38







1.3b
1.7a
0.4c
1.9a
0.9c

388

R. Di Cagno et al. / Food Microbiology 27 (2010) 381e389

a spoilage microorganism. L. plantarum is an ubiquitous and

metabolic versatile bacterium largely found in fruits and vegetables
(Buckenhskes, 1997). Although recently discovered (Corsetti et al.,
2005), Lb. rossiae was identied from different ecosystems such as
sourdough (Di Cagno et al., 2007), gastrointestinal tract (De Angelis
et al., 2006; Di Cagno et al., 2009b) and semolina (Valerio et al.,
2009). In spite of the very limited number of species identied, the
RAPD typing grouped isolates of lactic acid bacteria in several
clusters which may reect the way of entry. The number of isolates
of L. plantarum was almost equally distributed in the three layers.
As shown by RAPD typing (Fig. 2), clusters T1 and T2 were found in
the three layers. T3 and T6 was found in two contiguous layers (MR
and IR) and T10 derived from MR. Therefore, isolates of L. plantarum
might be considered as derivations of OR and/or IR layers. On the
contrary, most of the isolates of L. rossiae were mainly located into
MR, indicating this layer as the presumptive optimal niche.
Overall, fresh and minimally processed pineapples have a very
short shelf-life, and canned pineapples are subjected to very
intense heat treatments which in part decrease the sensory and
nutritional properties. This study aimed at nding a protocol for
minimally processing to increase the shelf-life and to maintain
agreeable sensory and nutritional features. Five technological
options were compared. They included only heating at 72
C for 15 s
(HP), spontaneous fermentation without (FP) or followed by heating (FHP), and fermentation by autochthonous L. plantarum 1OR12
and L. rossiae 2MR10 without (SP) or preceded by heating (HSP).
Both autochthonous lactic acid bacteria grew well in pineapple
without nutrient supplementation and pH adjustment. Heating
before inoculum of starters seemed to be not indispensible to
achieve high number of viable lactic acid bacteria, inhibition of
spoilage yeasts and agreeable sensory and nutritional properties.
After 30 days of storage at 4
C, SP harboured 7.3 log cfu g1 of
viable L. plantarum 1OR12 and L. rossiae 2MR10. This number
approached that of potential probiotic beverages (Yoon et al., 2004).
Usually, Gram-negative bacteria and yeasts dominate the microbiota of nonstarted fruits and vegetables. Their inhibition is achieved using elevated concentrations of NaCl, sugars, other chemical
compounds or because the rapid growth of autochthonous lactic
acid bacteria (Fleming et al., 1975). The cell density of spoilage
yeasts in pineapples started with autochthonous lactic acid bacteria
was markedly lower than that found in pineapple subjected to
spontaneous fermentation. In agreement, the Community Level
Catabolic Proles of the started pineapples were clearly different
from those of the other processed fruits. This, probably indicated
the capacity of autochthonous starters to limit the growth of other
bacteria and yeasts.
Fruit acidity and sweetness are two of the major factors determining the quality of pineapples (Paull and Chen, 2003). As
previously reported (Bartholomew et al., 2002; Paull and Chen,
2003), citrate and malate were the main organic acids found in
pineapple. Especially, autochthonous starters modied the prole
of organic acids leading to the decrease of the concentration of
malic acid, and synthesizing lactic and acetic acids. This modication might had direct (pH) or indirect repercussions (redox
potential) on the activity of endogenous browning enzymes,
oxidation and sensory properties (colour, avour and aroma) of
pineapples (Hernndez et al., 2009). Indeed, started pineapples had
the highest antioxidant activity throughout processing and storage.
Compared to spontaneous fermentation, SP also showed the better
preservation of the natural colours. Firmness and sensory properties of pineapples processed with autochthonous starters were also
preferable. The partial inhibition of endogenous pectinolytic
enzymes or lipoxygenases, responsible for negative changes in
texture and sensory properties might had also occurred in started
pineapples (Buckenhskes, 1997). By determining the sum of the

scores for sensory properties, pineapples started with autochthonous lactic acid bacteria were mainly classied as characteristic for
odour and likes very much for the overall acceptability.
This study describes the taxonomic structure of yeasts and lactic
acid bacteria microbiota of pineapple. The fermentation by
autochthonous lactic acid bacteria, also without any heat treatment, is an example of minimally processing which may guarantee
shelf-stable pineapples, containing an elevated number of viable
lactic acid bacteria and keeping agreeable antioxidant, texture,
colour and sensory properties.

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