Taxonomic structure of the yeasts and lactic acid bacteria microbiota of pineapple (Ananas comosus L. Merr.) and use of autochthonous starters for minimally processing

Food Microbiology 27 (2010) 381e389

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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 identified yeast in pineapple fruits. Lactobacillus plantarum and Lactobacillus rossiae were the main identified 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 acidification. 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 Profiles of processed pineapples indirectly confirmed the capacity of autochthonous starters to dominate during fermentation. HSP and SP also showed the highest antioxidant activity and firmness, the better preservation of the natural colours and were preferred for odour and overall acceptability. Ó 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 flavour (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.: þ39 080 5442949; fax: þ39 080 5442911. E-mail address: [email protected] (M. Gobbetti). 0740-0020/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2009.11.012

fibres (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 antiinflammatory agents in rheumatoid arthritis, ulcerative colitis, colon inflammation, 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 benefits associated with their consumption. Public health institutions recommend the consumption of at least five 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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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-flavours 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 fluctuations 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 identification 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 amplified 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-Gavilán 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 identification 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 amplified 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 amplification 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 amplification (Di Cagno et al., 2008b). The molecular weight of the amplified 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 amplified fragments were scored, with faint bands being ignored. The three series of RAPD-PCR profiles were evaluated and combined to obtain a unique dendrogram, calculating an index of genetic similarity by the Simple Matching coefficient (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 acidification

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 purified 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 acidification were determined and modelled according to the Gompertz equation as modified by Zwietering et al. (1990). Growth data were modelled according to the equation: y ¼ k þ A exp { exp[(mmax e/A)(l  t) þ 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

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length of the lag phase expressed in hours; and t is the time. Acidification data were modelled according to the equation: y ¼ k þ A exp { exp[(Vmax e/A)(l  t) þ 1]}; where y is the acidification 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 acidification 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 fingerprinting

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 filtration on 0.22 mm membrane filters (Millipore Corporation, Bedford, MA 01730). Slices of pineapple used to fill 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, five 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 72°C for 15 sec)

Heating (at 72°C for 15 sec)

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

Heated Pineapple Fermented and Heated Pineapple (HP) (FHP)

Spontaneous fermentation (at 25°C 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 25°C for 24 h)

Fermentation (at 25°C for 24 h)

Heated and Started Pineapple (HSP)

Fermented Pineapple (FP)

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

Started Pineapple (SP)

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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 ¼  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 defined as the equitability of activities across all utilized substrates: E ¼ 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. Identification 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 ÄKTA Purifier 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 flow 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 identification 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. Identification 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 final 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 identified by partial sequencing of the 16S rRNA. Apart from the layer, only three species were identified. 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 coefficient 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, Forlì, 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 modifications. Appeararance, flavour, 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; flavour: 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 acidification (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 acidification (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 ¼ 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 acidification. 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)

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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 amplification 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 coefficient 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 significantly (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 confirmed 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 acidification 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 ¼ 3). Growth and acidification data were modelled according to the Gompertz equation, as modified 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

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Table 2 Cell numbers (log cfu g1) of lactic acid bacteria and yeasts, and Biolog fingerprinting 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

S

E

1

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 significantly different (P < 0.05). Each value was expressed as the mean  standard deviations (n ¼ 3) analyzed in duplicate.

3.4. Biolog fingerprinting The catabolic profiles 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 significantly (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 significance (equitability) of the values of H0 and S indices, confirmed the significant (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 acidification. 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 significantly (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_ significantly (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, firmness 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 firmness significantly (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 significantly different (P < 0.05). Each value was expressed as the mean  standard deviations (n ¼ 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

0

5

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 ¼ 3).

fermentation. It was 1.45  0.06 and 1.64  0.04 kg cm2e1 for FHP and FP, respectively. The firmness of started pineapples HSP and SP (1.79  0.05 and 1.83  0.03 kg cm2e1, respectively) did not significantly (P > 0.05) vary with respect to HP. During storage, the firmness of all processed pineapples further decreased. Nevertheless, the firmness 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 significantly (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 significantly (P < 0.05) scored the highest for pineapples started by autochthonous lactic acid bacteria (Table 4). Due to lowest value of pH, flavour 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 identified. 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 significantly different (P < 0.05). Each value was expressed as the mean  standard deviations (n ¼ 3) analyzed in duplicate.

The microbial population of fruits and vegetables is estimated to fluctuate 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 (Buckenhüskes, 1997). Although recently discovered (Corsetti et al., 2005), Lb. rossiae was identified 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 identified, the RAPD typing grouped isolates of lactic acid bacteria in several clusters which may reflect 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 finding 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 Profiles 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 modified the profile of organic acids leading to the decrease of the concentration of malic acid, and synthesizing lactic and acetic acids. This modification might had direct (pH) or indirect repercussions (redox potential) on the activity of endogenous browning enzymes, oxidation and sensory properties (colour, flavour and aroma) of pineapples (Hernández 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 (Buckenhüskes, 1997). By determining the sum of the

scores for sensory properties, pineapples started with autochthonous lactic acid bacteria were mainly classified 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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