Effect of atmospheres combining high oxygen and carbon dioxide levels on microbial spoilage and sensory quality of fresh-cut pineapple

Postharvest Biology and Technology 86 (2013) 73–84

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Effect of atmospheres combining high oxygen and carbon dioxide levels on microbial spoilage and sensory quality of fresh-cut pineapple Bao-Yu Zhang a , Simbarashe Samapundo a , Vasileios Pothakos a , Ilse de Baenst a , Göknur Sürengil b , Bert Noseda a , Frank Devlieghere a,∗ a Ghent University, Food2Know, Faculty of Bioscience Engineering, Department of Food Safety and Food Quality, Laboratory of Food Microbiology and Food Preservation, Coupure Links 653, 9000 Gent, Belgium b Ege University, Fisheries Faculty, Fish Processing Technology Department, Bornova-Izmir 35100, Turkey

a r t i c l e

i n f o

Article history: Received 6 March 2013 Accepted 8 June 2013 Keywords: Fresh-cut pineapple Oxygen Carbon dioxide Modified atmosphere packaging Yeasts Volatile organic compounds

a b s t r a c t Fresh-cut fruit such as pineapple have a very limited shelf-life. The study aims at prolonging the shelf-life of fresh-cut pineapple by means of modified atmospheres (MAs). The effect of MAs combining high O2 (21–70%) and CO2 (21–50%) levels on microbial spoilage and sensory quality of fresh-cut pineapple was therefore evaluated. In the first part of the study, the behaviour of two spoilage yeasts (Candida sake and Candida argentea) and one lactic acid bacterium (Leuconostoc citreum), which had previously been isolated from spoiled commercial fresh-cut pineapple cubes, were monitored on pineapple agar separately. In the second part of the study, the shelf-life of commercial fresh-cut pineapple cubes packaged in selected MAs was evaluated at 7 ◦ C. The results showed that MAs combining high O2 and high CO2 levels had a large inhibitory effect on the growth and volatile metabolite production of C. sake and C. argentea on pineapple agar. A MA with 50% O2 and 50% CO2 was in both cases the most inhibitive. Although MAs induced the production of ethyl acetate by the yeasts, the quantity of ethyl acetate was much lower in high O2 and high CO2 than that in air due to lower yeast population density in MAs. With regards to growth, L. citreum was not sensitive to high O2 and CO2 levels. The fresh-cut pineapple packaged in air had deteriorated and were not acceptable any more by day 7, while those packaged in 50% O2 combined with 50% CO2 , which also retarded the growth of aerobes and yeasts on pineapple cubes during storage, were still acceptable. It can be concluded that a MA with 50% O2 and 50% CO2 shows the best potential for extension of the shelf-life of fresh-cut pineapple. © 2013 Elsevier B.V. All rights reserved.

1. Introduction With unprecedented consumer demand for fresh-cut produce, this has become the fastest-growing segment in the food industry consistently achieving double digit growth rates (Gorny, 2005). Fresh-cut pineapple has become more and more popular as it is considered to be convenient to use compared to whole pineapple (Bierhals et al., 2011). However, the minimal processing employed may increase microbial spoilage of fruit through transfer of skin (surface) microflora to the fruit flesh where they can grow rapidly upon exposure to nutrient laden juices (O’Connor-Shaw et al., 1994). Current commercial fresh-cut pineapple products have a shelf-life of 5–7 days at 1–7 ◦ C, limited largely by the development of off-flavours and off-odours from physiological processes

∗ Corresponding author. Tel.: +32 9 264 6164; fax: +32 9 225 5510. E-mail addresses: [email protected], [email protected] (B.-Y. Zhang), [email protected] (F. Devlieghere). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.06.019

and microbiological spoilage (Liu et al., 2007; Montero-Calderon et al., 2010a). Modified atmosphere packaging (MAP), typically consisting of a combination of a lowered level of O2 (2–6%) and an elevated level of CO2 (7–15%), is often used for extending the shelf-life of freshcut pineapple by inhibiting fast-growing aerobes and slowing the respiration of living tissues (Martinez-Ferrer et al., 2002; Sandhya, 2010). However, the low O2 levels may stimulate the proliferation of anaerobic psychrotrophic microorganisms (Rojas-Grau et al., 2009). High O2 MAP has recently been suggested as an alternative for those using low O2 levels to extend the shelf-life by inhibiting the growth of naturally occurring spoilage microorganisms, preventing undesired anoxic respirative processes and decay of fresh and fresh-cut produce (Wszelaki and Mitcham, 2000; Day, 2001; Jacxsens et al., 2001; Oms-Oliu et al., 2008). However, the sensitivity of different organisms to O2 may vary greatly. Additionally, high MAP with about 99% O2 cannot prevent the growth of Pseudomonas fragi, Aeromonas hydrophila, Yersinia enterocolitica, and Listeria monocytogenes (Kader and Ben-Yehoshua, 2000). To date, only a handful of studies have been performed in which the positive

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effect of atmospheres combining high O2 and CO2 on the shelf-life of fresh-cut vegetables has been reported (Amanatidou et al., 2000; Conesa et al., 2007). No studies have yet been published regarding the effect of atmospheres combining elevated O2 and CO2 levels on the shelf-life of fresh-cut fruit. Volatile compounds are primarily responsible for the unique flavour characteristics that distinguish different fruit and vegetables and determine their desirability to the consumer (Forney et al., 2009). Spoilage microorganisms may produce volatile metabolites which can have an effect on the sensory quality of fresh-cut produce during storage (Ragaert et al., 2006; Amaro et al., 2012). However, as far as we know, little has been reported on the effect of atmospheres combining elevated O2 and CO2 levels on the behaviour of spoilage microorganisms and their volatile organic metabolite production and the sensory quality of fresh-cut pineapple during storage. Earlier research (Spanier et al., 1998) reported that freshcut pineapple chunks in the lower portion of containers (where the availability of O2 may be limited) developed off-flavours associated with microbial fermentation after seven to 10 days of storage. The objective of this study was to investigate the effect of modified atmospheres (MAs) consisting of various levels of O2 (21–70%) and CO2 (21–50%) on the growth and volatile organic compound (VOC) production of specific spoilage organisms (SSOs) on pineapple agar. Additionally, the effects of the headspace O2 and CO2 levels on the shelf-life of fresh-cut pineapple cubes were evaluated. 2. Materials and methods This study was performed in two parts. The first part of the study evaluated the effect of initial headspace (IH) air and MAs comprising of different combinations of O2 (21–70%) and CO2 (21–50%) levels on the growth and volatile metabolite production of SSOs on pineapple agar at 7 ◦ C. The second part of the study evaluated the effect of air and selected MAs on the microbial and sensory quality of commercial fresh-cut pineapple cubes stored at 7 ◦ C. The atmospheres applied in the second part were selected on the basis of the results of the experiments on pineapple agar. The methods used in each part of this study are described in detail below. 2.1. Part 1 – effect of MAs on growth and volatile metabolite production on pineapple agar of the specific spoilage organisms of fresh-cut pineapple 2.1.1. Isolates Two yeasts (Candida argentea and Candida sake) and one lactic acid bacterium (Leuconostoc citreum), previously isolated from spoiled commercial fresh-cut pineapple, were used in this study. All three isolates are maintained in the culture collection of the Laboratory of Food Microbiology and Food Preservation (Ghent University, Gent, Belgium). The identification of the yeasts was performed at BCCM/MUCL (BCCM/MUCL Agro (Industrial) Fungi and Yeasts Collection, Louvain-la-Neuve, Belgium) based on morphological, physiological and molecular analysis (sequencing of the large-subunit rDNA D1/D2 domain and the internal transcribed spacer, or ITS rDNA). The identification of lactic acid bacteria (LAB) was done at BCCM/LMG (Laboratorium voor Microbiologie, Faculty of Sciences, Ghent University, Gent, Belgium) based on amplified fragment length polymorphism (AFLP) analysis. 2.1.2. Preparation and inoculation of pineapple agar Pineapple agar was used as a simulation medium for fresh-cut pineapple. Pure pineapple juice (Materne, Belgium) supplemented with 1.5% Bacteriological Agar [Oxoid (Hampshire, UK)] was boiled over a Bunsen burner flame for 2 min in Schott bottles after which the bottles were placed in a water bath at 48 ± 1 ◦ C. Thereafter, when the agar had cooled to 48 ◦ C, 71 ± 0.2 g of pineapple

agar were poured into trays (volume = 269 mL, O2 transmission rate (OTR) = 0.5–13 cm3 /m2 day bar at 23 ◦ C, 0% relative humidity (RH), polypropylene (PP)/ethyl vinyl alcohol (EVOH), DECAPAC NV, Herentals, Belgium). The initial water activity (aw ) and pH of the pineapple agar were then measured in three duplicates by means of aw -kryometer (NAGY, Gaeufelden, Germany) and a SevenEasy pH metre (Mettler Toledo GmbH, Schwerzenbach, Switzerland), respectively. The aw (0.9903 ± 0.0003) and pH (3.7 ± 0.1) of the simulation agar did not differ from that of the pineapple juice. To prepare the inoculum, the yeasts were individually subcultured in 10 mL of sterile Sabouraud Broth [SB; Oxoid (Hampshire, UK)] whilst the LAB was sub-cultured in 10 mL of de Man Rogosa Sharpe broth [MRS broth, Oxoid (Hampshire, UK)] at 22 ± 1 ◦ C for 2 days. Second sub-cultures were prepared in the same media as the first sub-cultures and incubated for the same duration at the same temperature. Thereafter, the second sub-cultures were transferred to a refrigerator at 7 ± 1 ◦ C for 7 h to adapt the SSOs to the final incubation temperature used in the experiments. The temperature adapted SSOs were inoculated on the pineapple agar trays. Inoculated trays were individually sealed by a tray sealer (MECA 900, DECAPAC NV, Herentals, Belgium) using a high O2 barrier film (OTR = 5 cm3 /m2 day bar at 23 ◦ C, 50% RH. OPAEVOH (polyamide ethyl vinyl alcohol)/PE (polyethylene)/PP, BEMIS EUROPE Flexible Packaging, Monceau-sur-Sambre, Belgium) in the following initial conditions: 21% O2 + 21% CO2 , 50% O2 + 30% CO2 , 50% O2 + 50% CO2 , 70% O2 + 30% CO2 and 21% O2 (air), all balanced with N2 and stored at 7 ± 1 ◦ C. Two sealed trays per condition per SSOs were prepared for microbial growth, headspace gas composition and volatile organic compounds (VOCs), pH and sugar determination. The headspace O2 and CO2 levels in the tray were first measured by a headspace analyzer (CheckMate 9900 O2 , O2 /CO2 Headspace Analyzer, PBI – Dansensor, Denmark) before the packages were opened. Subsequently, the packages were opened aseptically and a quantity of 20 ± 0.1 g of pineapple agar was immediately transferred to a sterile plastic container (60 mL) and closed quickly. This sample was used for quantification of VOCs by means of SIFT-MS (Selected Ion Flow Tube Mass Spectrometer, Voice 200, Syft Technologies). The rest of the agar in the tray was used for the measurement of the pH, sugars and microbial analysis. The analysis of these parameters was performed over a 12- or 14-day incubation period. 2.1.3. Microbial analysis Microbial analysis was performed on days 0, 2, 4, 6, 8, 10 and 12 for yeast growth, while it was on days 0, 2, 4, 6, 9, 12 and 14 for LAB of incubation. On each day of analysis a piece of inoculated agar (ca. 10 g) was aseptically transferred to a sterile stomacher bag. Primary decimal dilutions of each sample were prepared by adding an appropriate volume of peptone saline solution [PSS, 8.5 g NaCl; 1 g peptone per litter, Oxoid (Hampshire, UK)]. The samples were homogenized for 30 s in a stomacher (Stomacher Lab-Blender 400, Led Techno, Eksel, Belgium). Subsequent decimal dilutions were then prepared from the primary decimal dilution in test-tubes containing 9 mL of sterile PSS. The decimal dilutions were then spread plated on Yeast Glucose Chloramphenicol agar [YGC, BioRad (Marnes-la-Coquette, France)] for the spoilage yeasts whilst the LAB was determined by pour plating on de Man Rogosa Sharpe agar [MRS agar, Oxoid (Hampshire, UK)]. The plates were then incubated at 22 ± 1 ◦ C until the colonies were sufficiently large for enumeration (2–3 days). 2.1.4. Quantification of volatile organic compounds The VOCs produced on pineapple agar by C. argentea and C. sake were previously identified by GC–MS (see Table 1). The identified VOCs were then used to develop a quantification method in the SIFT-MS. The underlying principle of SIFT-MS is well described by

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Table 1 Volatile organic compounds identified by GC–MS in the headspace of packaged pineapple agar inoculated with C. argentea and C. sake and spoiled commercial pineapple cubes and quantified by SIFT-MS, with the characteristic product ions, the coinciding precursor ions, mass-to-charge ratio (m/z), branching ratio (b) and reaction rate coefficient (K). The corresponding human olfactory threshold (OT) was obtained from Devos (1990). VOC’s Alcohols Ethanola , b 3-Methyl-1-butanola , b Butan-1-ola 2-Methyl-1-butanola Pentan-1-ola Propan-1-ola

Precursor

m/z

b (%)

K

Characteristic product ion

OT (mg/m3 ) 54.95 0.16 1.51 0.85 1.70 6.03

O2 + O2 + O2 + O2 + NO+ H3 O+ NO+

45 59 56 28 87 43 59

75 85 80 5 85 90 100

2.30E−09 2.10E−09 2.50E−09 2.30E−09 2.50E−09 2.70E−09 2.30E−09

C2 H5 O+ C3 H7 O+ C4 H8 + C2 H4 + C5 H11 O+ C3 H7 + C3 H7 O+

NO+ H3 O+ O2 + NO+ H3 O+ NO+

43 173 116 160 145 174

10 90 10 100 100 95

2.10E−09 3.00E−09 2.50E−09 2.50E−09 3.00E−09 2.50E−09

CH3 CO+ C10 H21 O2 + C6 H12 O2 + C7 H14 O2 ·NO+ C8 H16 O2 ·H+ C8 H16 O2 ·NO+

9.77 0.00 0.11 – 0.01

Acids Phenylacetic acida Hexanoic acidb Propanoic acidb 2-Methylbutanoic acidb 3-Methylbutanoic acidb Pentanoic acid b

NO+ H3 O+ NO+ NO+ NO+ O2 +

91 99 104 132 132 60

50 25 70 65 70 80

2.50E−09 3.00E−09 1.50E−09 2.50E−09 2.50E−09 2.40E−09

C7 H7 + C6 H11 O+ NO+ ·C2 H5 COOH·H2 O C5 H10 O2 ·NO+ C5 H10 O2 ·NO+ CH3 COOH+

0.59 0.06 0.11 0.01 0.01 0.02

Aldehydes Nonanalb 2-Methylpropanala 3-Methylbutanala 2-Methylbutanala

O2 + O2 + O2 + H3 O+

69 72 44 87

10 70 35 94

3.20E−09 3.00E−09 2.40E−09 3.70E−09

C5 H9 + C4 H8 O+ C2 H4 O+ C5 H10 O·H+

0.01 0.12 0.01 –

Esters Ethyl acetatea,b Ethyl octanoateb Ethyl butanoateb 3-Methylbut-1-yl ethanoatea Ethyl hexanoateb

a b

Detected in the headspace of inoculated agar. Detected in the headspace of spoiled pineapple cubes.

Olivares et al. (2010), Davis et al. (2005) and Noseda et al. (2010). Samples intended for the quantification of the VOCs (duplicates per sample analysis day) were stored in a freezer at −18 ± 1 ◦ C until the analysis date. On the day of analysis, each sample (20 ± 0.1 g) was initially repackaged in 0.9 L of N2 by the MULTIVAC packaging machine (Sepp Haggenmüller KG, Wolfertschwenden, Germany) and stored at 4 ◦ C for at least 2 h, to allow the liquid and gas phases to equilibrate (Noseda et al., 2010). Thereafter the target VOCs were measured by the SIFT-MS using the multiple ion monitoring mode (MIM). Quantification of the target VOCs occurred by using the reaction rate coefficients (K) and the branching ratios (b) of the reaction between the precursor ions (H3 O+ , NO+ and O2 + ) and the VOCs. The ionized masses used for quantification are presented in Table 1.

2.1.5. Sugar analysis HPLC was used to determine the concentration of sucrose, glucose and fructose in the pineapple agar, based on the method developed by Ragaert et al. (2006). For the extraction, 10 g of pineapple agar was homogenized for 30 s with 10 g of distilled water. After filtration (Ø125 mm, Schleicher & Schwell, Dassel, Germany), the filtrate was held at 80 ◦ C for 15 min to denature proteins including enzymes. After centrifugation for 15 min at 8050 × g (Centrifuge 5415C, Eppendorf, Hamburg, Germany) and filtration through a 0.2 ␮m filter (HPLC Syringe filter; GRACE, Deerfield, IL, USA), 200 ␮L of the filtrate was injected in the HPLC coupled to a RI detector (Refractive Index Detector 133, Gilson). The HPLC consisted of a pump (Pump 307, Gilson, Wisconsin, USA), an injector (Rheodyne 9096, Bensheim, Germany), a pre-column (MetaCarb 87H Guard cartridge, Varian, USA), a column (MetaCarb 87H, length 300 mm, ID 7.8 mm, Varian, USA) with column oven (35 ◦ C) (Model 7990, Jones Chromatography, California, USA) and a RI detector. 0.005 M H2 SO4 (VWR, Leuven, Belgium) was used as mobile phase which was at a flow rate of 0.6 mL/min.

The concentrations of the sugars were calculated using standard curves for sucrose (UCB, Leuven, Belgium), glucose (Sigma–Aldrich, St. Louis, MO, USA) and fructose (Sigma–Aldrich, St. Louis, MO, USA) which were made by adding each of these compounds in different known concentrations to distilled water. 2.2. Part 2 – effect of MAs on the shelf-life of fresh-cut pineapple 2.2.1. Packaging of fresh-cut pineapple To validate the experiments performed on pineapple agar, the growth and VOC production of the natural spoilage flora of commercial fresh-cut pineapple was evaluated in air (the atmosphere currently used by the commercial processor who supplied the fresh-cut pineapple in this study), IH 50% O2 + 30% CO2 and IH 50% O2 + 50% CO2 . Additionally, triangle tests and physical parameters (colour, juice leakage, firmness and juiciness) were followed to determine the effect of the IH O2 and CO2 levels on the sensory quality of fresh-cut pineapple. Fresh-cut pineapple (cubes, 1 ± 0.2 cm thick, 7–9 g, each) used in the experiment were delivered to our laboratory within 2 h of processing by the commercial processor. 120 ± 2 g of pineapple cubes were aseptically transferred into trays (volume = 443 mL, O2 transmission rate = 0.5–13 cm3 /m2 day bar at 23 ◦ C, 0% RH, PP/EVOH, DECAPAC NV, Herentals, Belgium) before being sealed in the desired atmospheres. A high O2 barrier film (OTR = 5 cm3 /m2 day bar at 23 ◦ C, 50% RH. OPAEVOH/PE/PP, BEMIS EUROPE Flexible Packaging, Monceau-sur-Sambre, Belgium) was used to seal the trays. The trays were then stored at 7 ◦ C for up to 7 days. Two trays per modified atmosphere (MA) evaluated were randomly selected at each sampling date for headspace gas composition and VOC analysis, pH, sugars and microbial quality. Twenty trays packaged in air and 10 trays packaged in each of the two MAs evaluated each were randomly chosen for sensory evaluation on each analysis date.

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2.2.2. Effect of MAs on the microbial quality of fresh-cut pineapple The microbiological quality (total aerobic, LAB and yeast counts) of the cut pineapple samples packaged in the different IH O2 and CO2 levels was determined in duplicate on days 0, 1, 3, 5 and 7 of storage at 7 ◦ C. As in the experiments on pineapple agar, the headspace O2 and CO2 levels in two packages per condition were first measured before the packages were opened. Subsequently, the packages were opened and samples were aseptically collected for measurement of the pH (ca. 15 g) and the microbiological quality (ca. 30 g). The samples for assessment of the microbiological quality were transferred to a sterile stomacher bag and primary and subsequent decimal dilutions were prepared in PSS as described above for the experiments on pineapple agar. To enumerate the yeasts, the decimal dilutions were spread plated on YGC, whilst the total aerobes were determined by pour plating the dilutions on Plate Count Agar [PCA, Oxoid (Hampshire, UK)]. LAB were enumerated by pour plating the decimal dilutions MRS agar adjusted to pH 5.7 by adding 1.4 g/L sorbic acid (Sigma–Aldrich, St. Louis, MO, USA) before boiling and autoclaving. The plates were then incubated at 22 ± 1 ◦ C until the colonies were sufficiently large for enumeration (2–3 days). 2.2.3. Volatile organic compound and sugar content of fresh-cut pineapple On each day of sample analysis, 20 g of pineapple cubes were aseptically collected from each tray and put in a 60 mL plastic container and then stored in a freezer at −18 ± 1 ◦ C until analysis. VOCs quantification by SYFT-MS was performed as is described in Section 2.1.4. The samples used for sugars (approx. 20 g each) were thoroughly homogenized in a Stomacher bag with an equal amount of distilled water for 2 min. Further preparation of the samples as well as the analysis with HPLC was performed as is described in Section 2.1.5. 2.2.4. Assessment of colour and juice leakage Juice leakage from pineapple cubes was measured according to the method of Montero-Calderon et al. (2008). In brief, the packages were tilted at a 20◦ angle for 5 min after which the accumulated liquid was recovered with a 5 mL syringe. Results were reported as liquid volume (mL) recovered per 100 g of fresh-cut pineapple in the package. The colour of the pineapple cubes was determined on 5 randomly chosen points on the surface of each cube with a Spectrophotometer Minolta Model CM-2500d (Konica Minolta Sensing Inc., Osaka, Japan). Zero calibration and white calibration were performed before the measurements were taken. Each reading consisted of L* (lightness), a* (green chromaticity) and b* (yellow chromaticity) coordinates. 6 pieces of pineapple cubes were measured from each pair of trays for each modified atmosphere condition. 2.2.5. Sensory quality of fresh-cut pineapple 2.2.5.1. Triangle tests. Triangle tests were used to determine if the modified atmospheres with high O2 and high CO2 levels could have an impact on the sensory quality of fresh-cut pineapple cubes. The tests evaluated potential differences between samples packaged in air (reference condition) to those packaged in IH 50% O2 + 30% CO2 and IH 50% O2 + 50% CO2 . The procedures followed were according to those described in ISO 4120:2004 (Sensory analysis – Methodology – Triangle test) (ISO, 2004). Pineapple cubes (ca. 25 g) were transferred into plastic containers labelled with three-digit number codes. The plastic containers were closed and kept at 7 ◦ C for one hour before the triangle tests were performed. The sensory evaluation was performed in a purpose built sensory analysis room with isolated booths (UGent Sensolab, Ghent University) by 16 untrained assessors (age range: 24–44 years, average age: 29 years, 8 female

assessors and 8 male assessors). For each test three coded samples, of which one was different from the other two, were presented to the assessors. Additionally, water was provided for cleansing the palate and the assessors recorded their responses on paper scorecards. Each assessor was asked to identify the different sample based on colour, odour and taste. The assessor were instructed to only assess the taste of the samples if they thought the colour or the odour were still acceptable. When the assessors finished the triangle test, they were asked to select the sample(s) they preferred and to indicate the reasons of why they considered a sample to be unacceptable or preferable. The test was always performed between 10.30 am and 11.30 am in a single repetition. 2.2.5.2. Firmness and Juiciness assessment. Sample preparation was the same as the triangle taste. Firmness and juiciness were evaluated on day 0 (initial condition), 3, 5 and 7 of storage in the sensory analysis room by 15 assessors (age: 24–44 years, average age: 29 years, 8 female assessors and 7 male assessors). Samples were coded with three-digit number and presented to assessors in randomized order. The samples were presented under red-light to mask the colour the samples. Water was used to cleanse the palate between samples. Firmness and juiciness were scored separately on a 5 point scale where 1 = very soft (mushy)/very dry, 2 = soft/dry, 3 = neither firm nor soft/neither juicy nor dry, 4 = firm/juicy, 5 = very firm/very juicy. Firmness and juiciness were always evaluated between 10.30 am and 11.30 am in single repetition. The assessors recorded their responses on paper scorecards. 2.2.6. Statistical analysis An individual tray was used as one replicate on each sampling day, per treatment. The results of triangle tests were analyzed according to ISO 4120:2004 (ISO, 2004). For 16 assessors, the critical number of correct responses required to obtain a statistically significant (˛ = 0.05) difference are 9. The maximum growth rates (MAX , log10 CFU/cm2 /day) of the SSOs were estimated by fitting the Baranyi growth model to the data (Baranyi et al., 1993) using the non-linear regression function of SPSS version 21 (IBM, SPSS, Chicago, IL, USA). To determine the effect of storage time and MAs on colour and juice leakage, proper ANOVA contrasts were used, with significant differences being established at P < 0.05. Significant differences in juiciness and firmness were analyzed by Kruskal–Wallis test due to the ordinal nature of these data. These statistical analyses were also performed in SPSS. Where applicable, the air packages functioned as the reference condition by employing suitable contrasts in the statistical analyses. 3. Results and discussion 3.1. Effect of MAs on the growth of the SSOs of fresh-cut pineapple on pineapple agar The effect of high O2 combined with high CO2 modified atmospheres on the growth of spoilage yeasts (C. argentea and C. sake) was large, but it was minor for the growth of lactic acid bacterium (L. citreum) at 7 ◦ C (see Table 2 and Fig. 1). For instance, the initial population density of SSOs on pineapple agar was 2.4–3.2 log10 CFU/cm2 and the population of yeasts increased to over 5 log10 CFU/cm2 , the level at which yeasts are generally known to start causing spoilage (Fleet, 1992), after about two days in air, whilst it took 3–10 days in MAs with high O2 and CO2 levels. The longest time to reach 5 log10 CFU/cm2 occurred in an initial atmosphere with 50% O2 + 50% CO2 . However, the population density of L. citreum only reached 5.9–6.2 log10 CFU/cm2 over a 14-day period on pineapple agar, irrespective of air and all MAs evaluated. The final population

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Table 2 Growth character of isolates on pineapple agar, stored under different high O2 combined with high CO2 at 7 ◦ C for 12–14 days, lag phase  (day), maximum specific growth rate max (log10 (CFU/cm2 )/day), initial count N0 (log10 CFU/cm2 ) and maximum population density Nmax (log10 CFU/cm2 ) with standard deviation (95% confidence interval) estimated via the Baranyi model (1993). Isolates

Package condition



max

C. argentea

21% O2 21% O2 + 21% CO2 50% O2 + 30% CO2 50% O2 + 50% CO2 70% O2 + 30% CO2

0.3 ± 0.2 0.5 ± 0.9 1.1 ± 1.6 2.4 ± 0.18 0

1.2 0.6 0.5 0.3 0.3

± ± ± ± ±

0.1 0.1 0.1 0.2 0.0

2.7 2.4 2.5 2.4 2.4

± ± ± ± ±

0.1 0.2 0.3 0.2 0.1

8.1 8.0 8.7 9.0 9.0

± ± ± ± ±

0.1 0.3 1.7 1.8 1.6

0.999 0.994 0.987 0.987 0.999

C. sake

21% O2 21% O2 + 21% CO2 50% O2 + 30% CO2 50% O2 + 50% CO2 70% O2 + 30% CO2

1.2 ± 0.1 1.2 ± 0.4 1.4 ± 0.5 2.7 ± 0.3 1.2 ± 0.6

1.4 0.8 0.6 0.5 0.5

± ± ± ± ±

0.1 0.1 0.1 0.0 0.1

3.1 3.1 3.2 3.1 3.1

± ± ± ± ±

0.1 0.1 0.1 0.0 0.1

7.9 7.7 7.7 5.7 5.8

± ± ± ± ±

0.0 0.1 0.1 0.1 0.1

0.999 0.997 0.997 0.999 0.997

L. citreum

21% O2 21% O2 + 21% CO2 50% O2 + 30% CO2 50% O2 + 50% CO2 70% O2 + 30% CO2

0 0 0 0 0

0.1 0.1 0.1 0.1 0.1

± ± ± ± ±

0.2 0.2 0.2 1.2 1.6

3.0 3.0 3.0 2.9 2.9

± ± ± ± ±

0.2 0.2 0.2 0.2 0.2

7.0 7.1 7.0 7.2 7.3

± ± ± ± ±

2.6 2.3 2.8 2.3 2.7

0.981 0.980 0.983 0.978 0.998

density did not exceed the limit level (7–8 log10 CFU/g) for fresh-cut fruit (Uyttendaele et al., 2010). As can be seen in Table 2 and Fig. 1, the fastest growth of C. argentea and C. sake occurred in air at which the stationary phase (approx. 8 log10 CFU/cm2 ) was reached after 6 days incubation at 7 ◦ C. Greater retardation of the growth of the C. sake and C. argentea occurred when the IH O2 and CO2 levels were increased. The inhibitory effect was greatest in IH 50% O2 + 50% CO2 for the growth of C. sake and C. argentea with the MAX , which were approx. 1/3 than those in air, of 0.3 ± 0.2 and 0.5 ± 0.1 log10 CFU/cm2 /day respectively. However, the growth of spoilage yeasts was inhibited when the IH O2 increased, combining with high IH CO2 , as seen

R2

Nmax

when growth in IH 70% O2 + 30% CO2 and IH 50% O2 + 30% CO2 are compared. The inhibitory effect of CO2 on the yeasts evaluated in the study increased when the IH CO2 level was increased. This was observed when the growth in IH 50% O2 + 50% CO2 was compared to growth in IH 50% O2 + 30% CO2 (see Table 2 and Fig. 1). Although the inhibitory effects of high O2 atmospheres to yeasts have also been reported in some studies (Conesa et al., 2007; Zheng et al., 2008), the mechanism(s) are yet unclear. According to MoradasFerreira et al. (1996), reactive oxygen species (ROS), notably superoxide (O2 − ) and hydroxyl (OH− ) radicals, hydrogen peroxide (H2 O2 ) and singlet oxygen (1 O2 ), can originate in high O2 partial pressures resulting in damage to cellular components 9

C. argentea

8

8

7

7

log10 CFU/cm2

log10 CFU/cm2

9

N0

6 5

4 3

C. sake

6 5 4 3

2

2 0

2

4

6

8

10

12

0

2

4

6

8

10

12

Time (days)

Time (days)

L. citreum

9

log10 CFU/cm2

8 7 6 5 4 3 2

0

2

4

6

8

10

12

14

Time (days) Fig. 1. Growth curves of C. argentea, C. sake and L. citreum during incubation on pineapple agar in different initial headspace O2 and CO2 levels ((♦) air, () IH 21% O2 + 21% CO2 , () IH 50% O2 + 30% CO2 , (䊉) IH 50% O2 + 50% CO2 and () IH 70% O2 + 30% CO2 ) at 7 ◦ C. Data points are the means, with bars representing the two measured values.

78

B.-Y. Zhang et al. / Postharvest Biology and Technology 86 (2013) 73–84

(via oxidation of lipids, proteins and nucleic acids) when ROS levels exceed the antioxidant capacity of yeast cells (oxidative stress). On the other hand, higher CO2 levels in the headspace result in higher concentrations of dissolved CO2 (dCO2 ) in the media, thereby increasing the inhibition (=decreasing the growth rate) of spoilage microorganisms (Jones and Greenfield, 1982; Devlieghere and Debevere, 2000). Proposed mechanisms for the inhibitory effect of CO2 include alterations in membrane fluidity, direct inhibition of certain enzyme activities and internal acidification by the hydration of CO2 into carbonic acid (H2 CO3 ), but all of these are still essentially hypothetical (Dixon and Kell, 1989; Aguilera et al., 2005; Garcia-Gonzalez et al., 2007). Aguilera et al. (2005) reported that the growth of Saccharomyces cerevisiae was more sensitive to CO2 in presence of O2 than O2 absence in glucose-limited chemostat cultures. Therefore, high O2 combined with high CO2 can achieve stronger inhibition to yeasts than high O2 or high CO2 used alone. The CO2 production and O2 consumption by spoilage yeasts on pineapple agar in the package during the incubation were also delayed markedly when IH O2 level and/or IH CO2 level in the package were raised (see Fig. 2). Headspace O2 levels and CO2 levels in the packages containing pineapple agar inoculated with C. argentea started to decrease and increase, respectively, on days 4, 6 and 8 when growth took place in air, IH 21% O2 + 21% CO2 and IH 50% O2 + 30% CO2 , respectively. No large changes occurred in the packages with IH 50% O2 + 50% CO2 and IH 70% O2 + 30% CO2 during 12 days of incubation. The headspace gas composition in the packages containing agar inoculated with L. citreum did not change during the incubation, except for a slight increase in the headspace O2 levels and a slight decrease of the headspace CO2 levels at the beginning of incubation due to the dissolution of CO2 in the water-phase of the pineapple agar (data not shown). This can also be seen in Fig. 2 during the first two days of incubation. A sharp increase in CO2 production has been reported to be a signal of the end of the shelf-life of fresh-cut pineapple (Marrero and Kader, 2006). Additionally, the

No volatile metabolites in the headspace changed appreciably throughout incubation of L. citreum in all atmospheres (data not shown) which could be due most part to the low population densities on pineapple agar and the low pervasive character of the metabolites of the LAB (Jacxsens et al., 2003). Nevertheless, various alcohols, esters, acids and aldehydes were detected during incubation of the spoilage yeasts evaluated on pineapple agar in the MAs investigated (see Table 1). Whilst VOC production by the yeasts in air took place after about only 4 days, it was retarded for 12 days in the other MAs evaluated during storage at 7 ◦ C as shown for some alcohols and esters in Figs. 3 and 4, respectively. The lowest quantities of VOCs were produced in IH 50% O2 + 50% CO2 , followed by IH 70% O2 + 30% CO2 and IH 50% O2 + 30% CO2 . The highest quantities of VOCs were produced in air and IH 21% O2 + 21% CO2 . As an example, whilst the quantity of ethanol produced by C. sake and C. argentea increased to 138–200 mg/m3 after 8 days incubation in air, it was only 4.8–7.6 mg/m3 in MA with IH 50% O2 + 50% CO2 after 12 days incubation. The quantity of volatile metabolites (including 3-methyl-1butanol, butan-1-ol, ethyl acetate, ethyl octanoate, ethyl butanoate, hexanoic acid, propanoic acid, nonanal, 2-methylpropanal and 3methylbutanal) produced by C. sake on pineapple agar were higher in IH 21% O2 + 21% CO2 than in air for the same period of incubation at 7 ◦ C, despite lower population densities occurring in IH 21% O2 + 21% CO2 compared to air. Large amounts of volatile metabolites (except ethyl acetate) were produced by the yeasts in air and all MAs when the population densities of C. argentea and C. sake on pineapple agar were grown to more than 6–7 log10 CFU/cm2 .

80

80

60

60

CO2 (%)

O2 (%)

3.2. Effect of MAs on volatile metabolite production and sugar consumption of SSOs

100

A

100

pH of pineapple agar (3.7 ± 0.1) did not change appreciably during incubation of the yeasts and lactic acid bacterium.

40

B

40

20

20

0

0 2

0

4

6

8

10

0

12

2

4

100

C

80

80

60

60

CO2 (%)

O2 (%)

100

6

8

10

12

10

12

Time (days)

Time (days)

40 20

D

40

20

0

0 0

2

4

6

8

Time (days)

10

12

0

2

4

6

8

Time (days)

Fig. 2. Changes in headspace O2 and CO2 concentrations in the packages with pineapple agar inoculated with C. argentea (A and B) and C. sake (C and D) during incubation in different initial headspace O2 and CO2 levels ((♦) air, () IH 21% O2 + 21% CO2 , () IH 50% O2 + 30% CO2 , (䊉) IH 50% O2 + 50% CO2 and () IH 70% O2 + 30% CO2 ) at 7 ◦ C. Data points are the means, with bars representing two measured values.

B.-Y. Zhang et al. / Postharvest Biology and Technology 86 (2013) 73–84

160

240

A

80 40

0 0

2

4 6 8 Time (days)

10

12

0

120

C

30 15

4 6 8 Time (days)

10

12

2

4 6 8 Time (days)

10

12

2

4 6 8 Time (days)

10

12

2

4 6 8 Time (days)

10

12

D

60 30

0

0 0

2

4 6 8 Time (days)

10

12

0 120

E

40

F

90

mg/m3

mg/m3

2

90

mg/m3

mg/m3

45

50

120 60

0

60

B

180

mg/m3

mg/m3

120

79

30

60

20 30

10 0

0 0

15

2

4 6 8 Time (days)

10

12

0 24

G

18

mg/m3

mg/m3

12

H

9

12

6 6

3 0

0 0

2

4 6 8 Time (days)

10

12

0

Fig. 3. Ethanol (A and B), 3-methyl-1-butanol (C and D), butan-1-ol (E and F) and 2-methyl-1-butanol (G and H) production in the headspace during incubation of C. argentea (A, C, E and G) and C. sake (B, D, F and H) in different initial headspace O2 and CO2 levels ((♦) air, () IH 21% O2 + 21% CO2 , () IH 50% O2 + 30% CO2 , (䊉) IH 50% O2 + 50% CO2 and () IH 70% O2 + 30% CO2 ) at 7 ◦ C. Data points are the means, with bars representing two measurement values.

For example, it can be clearly seen in Fig. 5 that large amounts of ethanol accumulated when the population densities of C. sake and C. argentea were over approx. 6.6 log10 CFU/cm2 . Hence, one of the possible explanations of volatile metabolite production is that when the yeast population densities are over 6–7 log10 CFU/cm2 , they are at numbers where they can alter the headspace gas composition (rapid O2 consumption coupled to CO2 production, see Fig. 2), which results in a conversion to respiration-fermentative and fermentative metabolism of spoilage yeasts. Additionally, the high glucose concentration in pineapple agar (3.2–3.7 g/100 g) may also lead to respiration-fermentative glucose metabolism of spoilage yeasts resulting in volatile metabolite production, even though in

the presence of O2 in the headspace (see Table 3). In previous studies on S. cerevisiae, the glucose sensitive yeast exhibits complete respiratory metabolism in low-glucose conditions (Gonzalez-Siso et al., 2000), while in the presence of surplus glucose (>0.15 g per L) it converts to respiration-fermentative glucose metabolism, where respiration and fermentation coexist (Kappeli, 1986; Fiechter and Seghezzi, 1992; Jouhten et al., 2012). The effect of MAs on VOC production of yeasts seems to follow the same trend as that observed on the growth. It has been observed by Renger et al. (1992) that the reduction in the formation of esters and higher alcohols when the carbon dioxide partial pressure was increased was partially caused by the inhibition of S. cerevisiae

80

B.-Y. Zhang et al. / Postharvest Biology and Technology 86 (2013) 73–84

250

800

A

600

mg/m3

mg/m3

200

B

150

400

100 200

50 0

0 0

40

2

4 6 8 Time (days)

10

12

0 40

C

20 10

12

2

4 6 8 Time (days)

10

12

2

4 6 8 Time (days)

10

12

D

20

0 0

2

4 6 8 Time (days)

10

12

0 120

E

40

F

90

mg/m3

mg/m3

10

10

0

50

4 6 8 Time (days)

30

mg/m3

mg/m3

30

2

30

60

20 30

10 0

0 0

2

4 6 8 Time (days)

10

12

0

Fig. 4. Ethyl acetate (A and B), ethyl octanoate (C and D) and ethyl butanoate (E and F) production in the headspace during incubation of C. argentea (A, C and E) and C. sake (B, D, and F) in different initial headspace O2 and CO2 levels ((♦) air, () IH 21% O2 + 21% CO2 , () IH 50% O2 + 30% CO2 , (䊉) IH 50% O2 + 50% CO2 and () IH 70% O2 + 30% CO2 ) at 7 ◦ C. Data points are the means, with bars representing two measurement values.

growth. However, the mechanisms by which MAs inhibit growth and volatile metabolite of yeasts could be different (Jones and Greenfield, 1982). Among VOCs produced by C. argentea and C. sake, ethanol, ethyl acetate and ethyl butanoate are the pyruvate metabolites produced via pyruvate decarboxylase (with conversion of NADH

250

Ethanol

mg/m3

200

150

100

50

0 2

3

4

5

6

7

8

log10 CFU/cm2 Fig. 5. The production of ethanol during the growth of C. argentea and C. sake in different initial headspace O2 and CO2 levels ((♦) air, () IH 21% O2 + 21% CO2 , () IH 50% O2 + 30% CO2 , (䊉) IH 50% O2 + 50% CO2 and () IH 70% O2 + 30% CO2 ) at 7 ◦ C.

to NAD+ ) (Pronk et al., 1996; Ragaert et al., 2006). 3-Methyl1-butanol, 2-methyl-butan-1-ol, propan-1-ol, 2-methylbutanoic acid, 2-methylbutanoic acid, 2-methylpropanal, 3-methylbutanal and 2-methylbutanal are intermediates of the Ehrlich pathway, formed by the successive deamination, decarboxylation and hydrogenation (with conversion of NADH to NAD+ ) of the amino acids valine, isoleucine and leucine, respectively (Ragaert et al., 2006; Hazelwood et al., 2008). Those compounds are also known as fusel alcohols, fusel acids and fusel aldehydes. In addition, phenylacetic acid was also related to amino acid catabolism (Hammer et al., 1996). As can be seen in Figs. 3 and 4, volatile metabolites related to Ehrlich pathway were formed at the same time as the formation of fermentative metabolites. The explanation of the correlation could be that pyruvate can be converted to isoleucine, valine and leucine by pyruvate acetohydroxy-acid synthase (Boumba et al., 2008). Hence, volatile metabolites were produced by oxidizing NADH to NAD+ , which is needed for glycolysis in anaerobic conditions for yeasts, during the ethanol formation and Ehrlich pathway. Ethyl acetate produced by C. argentea and C. sake were largely accumulated after the population densities were over 4–5 log10 CFU/cm2 . Notably, the quantity of ethyl acetate produced was more in MA conditions than in air conditions when the population densities of yeasts were at the same level. However, the quantity of ethyl acetate was much higher in air than that in high O2 and high CO2 due to lower yeast population density in MAs (see Fig. 4A and B). Ethyl acetate can be synthesized via two pathways. Both pathways use ethanol as one of the substrates, either

B.-Y. Zhang et al. / Postharvest Biology and Technology 86 (2013) 73–84

81

Table 3 Yeast counts and concentrations of sucrose, glucose, fructose and lactic acid during storage of pineapple agar inoculated with Candida argentea and Candida sake in air at 7 ◦ C. Yeast count (log10 CFU/cm2 )

Day

Sucrose (g/100 g)

Glucose (g/100 g)

Fructose (g/100 g)

Lactic acid (g/100 g)

Candida argentea 0 4 8 12

2.37–2.97a 6.33–6.85 7.94–7.96 8.01–8.13

5.71–6.57 5.79–6.12 3.54–5.52 3.74–4.01

2.77–3.15 2.78–2.95 1.46–2.26 1.10–1.23

2.77–3.19 2.86–2.91 1.74–2.69 1.82–2.10

– – – –

Candida sake 0 4 8 12

3.01–3.17 6.59–6.75 7.83–7.91 7.80–7.85

5.74–6.56 5.91–5.97 4.92–7.15 5.85–6.52

2.70–3.05 2.63–2.75 0.00–0.00 0.00–0.00

2.74–3.11 2.72–2.80 0.87–0.96 0.35–0.42

0.00–0.00 0.00–0.00 0.15–0.21 0.22–0.25

a

Values are mean of two measurements.

together with acetyl co-enzyme A, catalyzed by the enzyme alcohol acetyl transferase or with acetate, by the reverse reaction of enzyme-catalyzed hydrolysis (esterase activity) (Fredlund, 2004). However, it should be noted that both spoilage yeasts produced large quantities of ethyl acetate in IH 50% O2 + 50% CO2 at the end of incubation, whilst the quantity of ethanol produced was very low. Tabachnick and Joslyn (1953) reported that ethyl acetate formation by Hanisenila anomtiala is most probably an energy coupled reaction and is not the result of a reversal of a simple hydrolysis mediated by an esterase. Besides, Manimegalai et al. (1998) determined that the concentration (on dry weight basis) of iron (Fe) was 0.99–4.82 mg/100 g in pineapple juices. Loser et al. (2012) found that at a high Fe dosage (400–12,000 ␮g/L in the fed medium), higher formation rates were fund for ethyl acetate, pyruvate, and glycerol of Kluyveromyces marxianus while ethanol and acetate were formed in small amount. VOCs of ethanol, ethyl acetate, phenylacetic acid and nonanal were produced in the largest quantities by spoilage yeasts evaluated on pineapple agar at the end of incubation. Ethanol and ethyl acetate have a higher human olfactory thresholds (OT) while phenylacetic acid and nonanal have a lower OT (Devos, 1990). Besides, metabolites could be produced differently by different yeasts. It can be seen from Figs. 3 and 4 that C. sake produced lower amounts of VOCs than C. argentea in air at the end of incubation. The content of glucose, fructose and sucrose in pineapple agar inoculated with C. argentea decreased gradually during incubation in air at 7 ◦ C (see Table 3). However, these sugars did not change appreciably in the other atmospheres evaluated (data not shown). For C. sake, the concentrations of glucose and fructose decreased throughout incubation in air (see Table 3) and unlike C. argentea, also in the other atmospheres evaluated (data not shown). Moreover, the production of lactic acid coincided with the onset of depletion of glucose during incubation of C. sake in air on pineapple

agar. In difference to C. argentea, sucrose levels in the pineapple agar inoculated with C. sake did not change during incubation in all the atmospheres evaluated at 7 ◦ C. C. argentea and C. sake consumed glucose and fructose simultaneous with an apparently higher rate of glucose uptake than fructose. Damore et al. (1989) reported that glucose and fructose utilization by yeast share the same membrane transport components. However, Emmerich and Radler (1983) observed that two different mechanisms or carrier systems exist for the uptake of glucose and fructose in S. bailii, one resistant and the other very sensitive to uranyl ions. 3.3. Microbial growth of fresh-cut pineapple In agreement with the trends observed on pineapple agar, the growth of yeasts and aerobes contaminating the pineapple cubes was retarded markedly by IH high O2 and CO2 levels (see Table 4). The initial total aerobic counts on pineapple cubes was 4.0–4.2 log10 CFU/g. Growth of aerobes on the pineapple cubes took place after a lag phase of one day, irrespective of the packaging condition. At the end of storage (day 7), pineapple cubes packaged in IH 50% O2 + 50% CO2 had the lowest total aerobic counts (4.0–4.4 log10 CFU/g), followed by those packaged in IH 50% O2 + 30% CO2 (5.6–5.8 log10 CFU/g). Pineapple cubes packaged in air had the highest total aerobic counts (6.9–7.3 log10 CFU/g) on packaged pineapple cubes (see Table 4). The same trend was observed for the yeasts, with counts of ca. 7.5, 6.0 and 5.3 log10 CFU/g on pineapple cubes packaged in air, IH 50% O2 + 30% CO2 and IH 50% O2 + 50% CO2 , respectively, on day 7 of storage (see Table 4). Although high numbers of bacteria as well as yeasts and moulds have been previously found on fresh-cut pineapple during refrigerated storage (Liu et al., 2007; Montero-Calderon et al., 2008; Di Egidio et al., 2009; Rocculi et al., 2009), the population density of LAB in our study remained < 3 log10 CFU/g of pineapple in all the

Table 4 Total aerobic and yeast counts, and concentrations of sucrose, glucose, fructose and volatile metabolites during storage of fresh-cut pineapple cubes in IH air (A), IH 50% O2 + 30% CO2 (B) and IH 50% O2 + 50% CO2 (C) at 7 ◦ C. IH Day TAC (log10 CFU/g)

Yeasts (log10 CFU/g)

Sucrose (g/100 g)

Glucose (g/100 g)

Fructose (g/100 g)

Ethanol (mg/m3 )

Ethyl acetate (mg/m3 )

Phenylacetic acid (mg/m3 )

3-Methylbut1-yl ethanoate (mg/m3 )

0

4.0–4.2a

3.9–4.1

5.51–6.17

2.07–2.66

2.33–2.93

0.22–0.28

1.49–2.74

0.00–0.02

3.32–2.40

A

3 5 7

4.9–5.2 6.4–6.8 6.9–7.3

5.3–5.4 6.9–6.9 7.3–7.7

7.45–5.56 6.08–6.48 3.51–5.30

2.89–3.10 3.03–3.07 1.49–2.56

3.29–3.83 3.28–3.45 1.79–2.95

0.49–0.99 5.14–5.66 7.41–14.20

2.55–4.95 31.15–31.67 39.98–65.45

0.06–0.34 8.71–13.15 14.54–60.74

1.82–2.05 2.78–2.93 2.83–3.32

B

3 5 7

4.6–5.0 4.7–5.1 5.6–5.8

4.7–5.1 4.8–5.0 5.9–6.1

3.19–6.60 5.53–5.89 3.36–4.51

2.02–3.02 2.78–3.16 1.56–2.32

2.49–3.52 3.12–3.45 1.81–2.56

1.45–1.99 2.21–3.00 3.95–4.68

9.39–13.22 8.55–12.91 16.50–24.67

0.27–0.94 1.04–2.17 3.95–6.74

1.57–2.34 1.59–1.61 1.65–2.56

C

3 5 7

4.6–4.8 4.1–4.1 4.0–4.4

4.6–4.6 4.2–4.6 4.5–6.1

4.23–5.23 5.12–5.49 4.38–4.95

1.95–3.48 2.92–3.40 2.11–3.43

2.44–4.17 3.39–3.73 2.36–3.95

3.14–3.18 3.87–4.19 6.48–7.10

32.71–42.84 30.33–36.30 56.35–67.23

3.26–4.27 4.93–6.90 9.55–13.54

1.95–2.22 2.09–2.73 2.12–2.68

a

Values are mean of two measurements.

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B.-Y. Zhang et al. / Postharvest Biology and Technology 86 (2013) 73–84

Table 5 Changes in the colour, juice leakage (mL/100 g), firmness and juiciness of pineapple cubes packaged under in IH air (A), IH 50% O2 + 30% CO2 (B) and IH 50% O2 + 50% CO2 (C) at 7 ◦ C. Packaging conditions

Storage time (days) 0

1

3

5

7

L* A B C

69.8 ± 5.4A,a 69.8 ± 5.4A,a 69.8 ± 5.4A,a

69.8 ± 5.4A,ab 62.9 ± 7.7A,b 61.9 ± 6.7A,b

69.4 ± 5.0A,b 68.8 ± 3.8A,a 64.4 ± 4.8A,b

61.0 ± 5.4A,c 61.5 ± 8.2A,b 63.7 ± 4.8A,b

60.6 ± 5.9A,c 60.7 ± 8.7A,b 62.9 ± 6.6A,b

a*␤ A B C

1.6 ± 1.4A,a 1.6 ± 1.4A,a 1.6 ± 1.4A,a

1.1 ± 1.5A,a 0.3 ± 1.3A,b 0.8 ± 0.8A,bc

1.3 ± 1.1A,a 0.9 ± 0.8A,ab 1.2 ± 1.0A,ab

1.0 ± 0.9A,a 0.3 ± 0.8A,b 0.7 ± 0.9A,bc

−0.3 ± 0.9A,b 0.03 ± 0.89A,b 0.3 ± 0.9A,c

b*␤ A B C

43.9 ± 3.6A,a 43.9 ± 3.6A,a 43.9 ± 3.6A,a

38.6 ± 4.9A,b 32.0 ± 6.2A,bc 33.2 ± 6.2A,bc

37.2 ± 4.7A,b 35.6 ± 3.4A,b 35.9 ± 4.0A,b

31.3 ± 4.8A,c 29.5 ± 6.8A,c 31.4 ± 4.7A,c

32.9 ± 3.5A,c 30.1 ± 7.1A,c 30.4 ± 4.4A,c

4.0 ± 1.1A,a 3.2 ± 1.0A,a 3.9 ± 0.9A,a

5.1 ± 1.4A,a 5.5 ± 1.7A,a 5.2 ± 1.8A,a

6.3 ± 0.4A,a 5.6 ± 1.9A,a 4.8 ± 1.8A,a

6.1 ± 1.2A,a 6.1 ± 2.1A,a 5.7 ± 2.7A,a

␤

Juice leakage␥ A B C Firmness␦ A B C

3 (2–4)A,a 3 (2–4)A,a 3 (2–4)A,a

3 (2–3)A,a 3 (2–4)A,a 4 (3–4)A,a

2.5 (2–3)A,a 2 (2–3.5)A,a 3 (2–4)A,a

2.5 (2–3)A,a 3 (2–4)A,a 3 (2.5–4)A,a

Juiciness␦ A B C

5 (4–5)A,a 5 (4–5)A,a 5 (4–5)A,a

4 (4–5)A,a 4 (4–5)A,b 4 (4–5)A,b

4 (4–5)A,b 4 (3.5–4)A,bc 4 (3.5–5)A,b

3.5 (3–4)A,b 4 (3.5–4)A,c 4 (3–4)A,b

Values followed by the different letters within columns (capital letters) and within lines (small letters) are significantly different (P < 0.05) according to ANOVA contrasts and Kruskal–Wallis test. ␤ Mean ± standard deviation, n = 30. ␥ Mean ± standard deviation, n = 3. ␦ The median value (50th percentile) with values of 25th percentile and 75th percentile. Respectively 50, 75 and 25 percent (rounded down) of the assessors gave at least this value or greater. Fractional values were indicated by assessors as “in between two classes”.

atmospheres evaluated during storage which is consistent with the quite slow growth rate of L. citreum on pineapple agar. Generally, LAB may affect the organoleptic qualities of fresh-cut produce when the count is reached 7 or even up to 8 log10 CFU/g due to the low pervasive character of the metabolites as discussed above (Jacxsens et al., 2003). Overall, the results in present study are in agreement with Iversen et al. (1989) and Fleet (1992) who reported that yeasts are the predominant factor causing the spoilage of fresh-cut pineapple. The production of CO2 and consumption of O2 during storage occurred in all atmospheres evaluated (data not shown), due to the respiration of living tissues of pineapple (Rocculi et al., 2009) and microbial growth (Marrero and Kader, 2006). The pH of the pineapple cubes (data not shown) did not change significantly throughout storage from an initial value of 3.3 ± 0.1, irrespective of the packaging condition. Montero-Calderon et al. (2008) found similar results for fresh-cut pineapple (pH 3.58 ± 0.04) stored in high (40%) or low (11.4%) oxygen atmospheres. 3.4. Volatile metabolite production and sugars of fresh-cut pineapple Most of the VOCs detected in the headspace of the trays containing the pineapple cubes, including butan-1-ol, 3-methyl1-butanol, ethyl octanoate, 2-methylpropanal, hexanoic acid, increased slightly during storage of pineapple cubes in MAs and air (data not shown). The concentrations of ethanol, ethyl acetate, phenylacetic acid and 3-methylbut-1-yl ethanoate (=isopentyl acetate) during storage are shown in Table 4. It should be noted that some of the VOCs are produced not only by spoilage yeasts (see Table 1) but also by living tissues of the pineapple cubes.

These include ethyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, phenylacetic acid, nonanal, pentan-1-ol and butan-1-ol, 3-methylbut-1-yl ethanoate (Elss et al., 2005; Montero-Calderon et al., 2010b; Wei et al., 2011; Kaewtathip and Charoenrein, 2012). Some of the components such as ethyl acetate, ethyl octanoate, ethyl butanoate and ethyl hexanoate, are important contributors of pineapple aroma (Takeoka et al., 1989). The lower quantities of VOCs in the headspace of trays of pineapple cubes compared to those detected in pineapple agar could be attributed to the relatively low population densities of native yeasts (approx. 7.5 log10 CFU/g) at the end of storage of the packaged pineapple cubes in air compared to the final population density of specific spoilage yeasts on pineapple agar. The sugar levels in pineapple cubes packaged in these atmospheres did not change appreciably during storage. Hence, the quantities of ethyl acetate detected in the headspace could be largely produced by the living tissues of pineapple cubes and to a lesser extent by the native yeasts of the pineapple cubes. Furthermore, as observed on pineapple agar, the amount of ethyl acetate detected was higher in IH 50% O2 + 50% CO2 than in air. However, the amount of ethanol and phenylacetic acid were higher in air than in IH 50% O2 + 30% CO2 or IH 50% O2 + 50% CO2 , resulting from the growth of native spoilage yeasts. 3.5. Physical parameters of fresh-cut pineapple Although all physical parameters evaluated did not differ significantly between the atmospheres investigated, the colour parameters of L* (lightness), b* (yellow chromaticity) and a* (green chromaticity) values and juiciness significantly decreased (P < 0.05) during storage in all atmospheres (see Table 5). Additionally, the difference seemed to be occur earlier on pineapple cubes packaged

B.-Y. Zhang et al. / Postharvest Biology and Technology 86 (2013) 73–84 Table 6 The results of sensory evaluation of fresh-cut pineapple cubes packaged in different initial headspace high O2 combined with high CO2 levels during the storage at 7 ◦ C. Initial headspace O2 and CO2 levels

Day of storage 3

a

21% O2 – 50% O2 + 30% CO2 21% O2 – 50% O2 + 50% CO2

b

c

6 /16 6/16

5

7

5/16 6/16

7/16 10/16

a

21% O2 is the control in both tests. Number of people who correctly identified the odd sample. The critical number of correct responses required to obtain a statistically significant (˛ = 0.05) difference are at least 9. c Total number of panel. b

in IH 50% O2 + 30% CO2 and IH 50% O2 + 50% CO2 than packaged in air. Juice leakage slightly increased during storage, whilst firmness did not change significantly (P > 0.05) during storage. The decrease of colour (L* and b*) and increase of juice leakage of fresh-cut pineapple during storage in this study has also been reported by previous studies (Marrero and Kader, 2006; MonteroCalderon et al., 2008). According to Montero-Calderon et al. (2008) the decreases of L* and b* were due to development of translucency, rather than tissue browning. 3.6. Sensory quality of fresh-cut pineapple As can be seen in Table 6, the triangle test results showed that no significant sensory difference occurred between the samples packaged in air and those packaged in IH 50% O2 + 30% CO2 . However, a significant difference (P < 0.05) was found between samples packaged in air and those packaged in IH 50% O2 + 50% CO2 on day 7 of storage at 7 ◦ C (see Table 6). Sensory preference in this case was for the samples packaged in an IH 50% O2 + 50% CO2 . O’Connor-Shaw et al. (1994) reported that spoilage of pineapple pieces and other fresh-cut fruit stored at 4 ◦ C was not a consequence of microbial growth. However, during the sampling for microbiological and sensory evaluation it was apparent that differences in gas production by natural flora were evident as most of the packages in air were blown on day 7, whilst those packaged in IH 50% O2 + 50% CO2 and IH 50% O2 + 30% CO2 were not yet in this condition. Our results are in agreement with those of Torri et al. (2010) who observed the same trend with regards to the increase in the levels of volatile compounds during incubation as a result of the metabolism of the micro-flora of fresh-cut pineapple stored in air at 7–8 ◦ C. Additionally, the panellists indicated that differences between pineapple cubes packaged in different MAs were mainly based on the odour, indicating the importance of volatile metabolite production (and storage atmosphere) on the shelf-life of fresh-cut pineapple products. Although lower quantities of VOCs were detected in the headspace at the end of storage in pineapple cubes packaged in air, they were described as being less fresh and having a fermented odour, especially banana-odour. This was probably due to the slight increase in 3-methylbut-1-yl ethanoate concentrations in the headspace (see Table 4) and the low human olfactory thresholds (OT) for other volatile metabolites (Devos, 1990; Renger et al., 1992). Methylbut-1-yl ethanoate has a typically strong banana odour (Fahlbusch et al., 2003). 4. Conclusions The microbial spoilage of fresh-cut pineapple was mainly due to the growth and volatile metabolite production of yeasts under respiration-fermentative and fermentative metabolism during storage. MA combining high O2 and CO2 levels was shown to strongly retard the growth of yeasts and their production of volatile organic compounds. However, the effect of the MAs evaluated was

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minor on colour retention, firmness, juiciness and juice leakage of fresh-cut pineapple cubes during storage at 7 ◦ C. Overall, the application of MAP with high O2 and high CO2 (e.g. 50% O2 + 50% CO2 ) could be used to maintain the quality of fresh-cut pineapple. Acknowledgements The authors would like to acknowledge Flanders’ FOOD (Kunstlaan 43, 1040 Brussels) and China Scholarship Council (Chegongzhuang Avenue 9, 100044 Beijing) for financially supporting this study. The Ghent University ‘Geconcerteerde Onderzoeks Actie’ (GOA project) ‘Fast and Convenient Mass Spectrometry-Bases Real-Time Monitoring of Volatile Organic Compounds of Biological Origin’ of the Flemish government is gratefully acknowledged for the support in this research through instrumentation credits and by financial means. References Aguilera, J., Petit, T., de Winde, J.H., Pronk, J.T., 2005. Physiological and genome-wide transcriptional responses of saccharomyees cerevisiae to high carbon dioxide concentrations. FEMS Yeast Res. 5, 579–593. Amanatidou, A., Slump, R.A., Gorris, L.G.M., Smid, E.J., 2000. High oxygen and high carbon dioxide modified atmospheres for shelf-life extension of minimally processed carrots. J. Food Sci. 65, 61–66. Amaro, A.L., Beaulieu, J.C., Grimm, C.C., Stein, R.E., Almeida, D.P.F., 2012. Effect of oxygen on aroma volatiles and quality of fresh-cut cantaloupe and honeydew melons. Food Chem. 130, 49–57. Baranyi, J., Roberts, T.A., McClure, P., 1993. A nonautonomous differential-equation to model bacterial-growth. Food Microbiol. 10, 43–59. Bierhals, V.S., Chiumarelli, M., Hubinger, M.D., 2011. Effect of cassava starch coating on quality and shelf life of fresh-cut pineapple (ananas comosus l. Merril cv Perola). J. Food Sci. 76, E62–E72. Boumba, V.A., Ziavrou, K.S., Vougiouklakis, T., 2008. Biochemical pathways generating post-mortem volatile compounds co-detected during forensic ethanol analyses. Forensic Sci. Int. 174, 133–151. Forney, C.F., Mattheis, J.P., Baldwin, E.A., 2009. Effects on flavor. In: Yahia, E.M. (Ed.), Modified and Controlled Atmospheres for the Storage, Transportation, and Packaging of Horticultural Commodities. CRC Press/Taylor & Francis, Boca Raton, pp. 119–158. Conesa, A., Artes-Hernandez, F., Geysen, S., Nicolai, B., Artes, F., 2007. High oxygen combined with high carbon dioxide improves microbial and sensory quality of fresh-cut peppers. Postharvest Biol. Technol. 43, 230–237. Damore, T., Russell, I., Stewart, G.G., 1989. Sugar utilization by yeast during fermentation. J. Ind. Microbiol. 4, 315–324. Davis, B.M., Senthilmohan, S.T., Wilson, P.F., McEwan, M.J., 2005. Major volatile compounds in head-space above olive oil analysed by selected ion flow tube mass spectrometry. Rapid Commun. Mass Spectrom. 19, 2272–2278. Day, B.P.F., 2001. Modified atmosphere packaging of fresh fruit and vegetables – an overview. In: BenArie, R., PhilosophHadas, S. (Eds.), Proceedings of the 4th International Conference on Postharvest Science, vols. 1 and 2. , pp. 585–590. Devlieghere, F., Debevere, J., 2000. Influence of dissolved carbon dioxide on the growth of spoilage bacteria. Food Sci. Technol./Lebensm. Wiss. Technol. 33, 531–537. Devos, M., 1990. Standardized Human Olfactory Thresholds. IRL Press at Oxford University Press, Oxford. Di Egidio, V., Sinelli, N., Limbo, S., Torri, L., Franzetti, L., Casiraghi, E., 2009. Evaluation of shelf-life of fresh-cut pineapple using FT-NIR and FT-IR spectroscopy. Postharvest Biol. Technol. 54, 87–92. Dixon, N.M., Kell, D.B., 1989. The inhibition by CO2 of the growth and metabolism of micro-organisms. J. Appl. Bacteriol. 67, 109–136. Elss, S., Preston, C., Hertzig, C., Heckel, F., Richling, E., Schreier, P., 2005. Aroma profiles of pineapple fruit (ananas comosus l. Merr.) and pineapple products. Food Sci. Technol.-Leb. 38, 263–274. Emmerich, W., Radler, F., 1983. The anaerobic metabolism of glucose and fructose by saccharomyces-bailii. J. Gen. Microbiol. 129, 3311–3318. Fahlbusch, K.G., Hammerschmidt, F.J., Panten, J., Pickenhagen, W., Schatkowski, D., Bauer, K., Garbe, D., Surburg, H., 2003. Flavors and Fragrances, Ullmann’s Encyclopedia of Industrial Chemistry. Wiley-VCH, Weinheim. Fiechter, A., Seghezzi, W., 1992. Regulation of glucose-metabolism in growing yeastcells. J. Biotechnol. 27, 27–45. Fleet, G., 1992. Spoilage yeasts. Crit. Rev. Biotechnol. 12, 1–44. Fredlund, E., 2004. Central Carbon Metabolism of the Biocontrol Yeast Pichia anomala: Influence of Oxygen Limitation. Dept. of Microbiology, Swedish Univ. of Agricultural Sciences, Uppsala. Garcia-Gonzalez, L., Geeraerd, A.H., Spilimbergo, S., Elst, K., Van Ginneken, L., Debevere, J., Van Impe, J.F., Devlieghere, F., 2007. High pressure carbon dioxide inactivation of microorganisms in foods: the past, the present and the future. Int. J. Food Microbiol. 117, 1–28.

84

B.-Y. Zhang et al. / Postharvest Biology and Technology 86 (2013) 73–84

Gonzalez-Siso, M.I., Freire-Picos, M.A., Ramil, E., Gonzalez-Dominguez, M., Torres, A.R., Cerdan, M.E., 2000. Respirofermentative metabolism in kluyveromyces lactis: insights and perspectives. Enzyme Microb. Technol. 26, 699–705. Gorny, J.R., 2005. Leveraging innovative fresh-cut technologies for competitive advantage. In: Proceedings of the International Conference Postharvest Unlimited Downunder 2004, pp. 141–147. Hammer, E., Kneifel, H., Hofmann, K., Schauer, F., 1996. Enhanced excretion of intermediates of aromatic amino acid catabolism during chlorophenol degradation due to nutrient limitation in the yeast candida maltosa. J. Basic Microbiol. 36, 239–243. Hazelwood, L.A., Daran, J.-M., van Maris, A.J.A., Pronk, J.T., Dickinson, J.R., 2008. The ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl. Environ. Microbiol. 74, 2259–2266. ISO, 2004. Sensory analysis – methodology – triangle test, BS ISO 4120. Iversen, E., Wilhelmsen, E., Criddle, R.S., 1989. Calorimetric examination of cut fresh pineapple metabolism. J. Food Sci. 54, 1246–1249. Jacxsens, L., Devlieghere, F., Ragaert, P., Vanneste, E., Debevere, J., 2003. Relation between microbiological quality, metabolite production and sensory quality of equilibrium modified atmosphere packaged fresh-cut produce. Int. J. Food Microbiol. 83, 263–280. Jacxsens, L., Devlieghere, F., Van der Steen, C., Debevere, J., 2001. Effect of high oxygen modified atmosphere packaging on microbial growth and sensorial qualities of fresh-cut produce. Int. J. Food Microbiol. 71, 197–210. Jones, R.P., Greenfield, P.F., 1982. Effect of carbon-dioxide on yeast growth and fermentation. Enzyme Microb. Technol. 4, 210–222. Jouhten, P., Wiebe, M., Penttila, M., 2012. Dynamic flux balance analysis of the metabolism of Saccharomyces cerevisiae during the shift from fully respirative or respirofermentative metabolic states to anaerobiosis. FEBS J. 279, 3338–3354. Kader, A.A., Ben-Yehoshua, S., 2000. Effects of superatmospheric oxygen levels on postharvest physiology and quality of fresh fruits and vegetables. Postharvest Biol. Technol. 20, 1–13. Kaewtathip, T., Charoenrein, S., 2012. Changes in volatile aroma compounds of pineapple (Ananas comosus) during freezing and thawing. Int. J. Food Sci. Technol. 47, 985–990. Kappeli, O., 1986. Regulation of carbon metabolism in saccharomyces-cerevisiae and related yeasts. Adv. Microb. Physiol. 28, 181–209. Liu, C.L., Hsu, C.K., Hsu, M.M., 2007. Improving the quality of fresh-cut pineapples with ascorbic acid/sucrose pretreatment and modified atmosphere packaging. Packag. Technol. Sci. 20, 337–343. Loser, C., Urit, T., Forster, S., Stukert, A., Bley, T., 2012. Formation of ethyl acetate by kluyveromyces marxianus on whey during aerobic batch and chemostat cultivation at iron limitation. Appl. Microbiol. Biotechnol. 96, 685–696. Manimegalai, G., Neelakantan, S., Vennila, P., 1998. Changes in the trace elements content during pulping of fruits in different mixies. J. Food Sci. Technol. (Mysore) 35, 262–264. Marrero, A., Kader, A.A., 2006. Optimal temperature and modified atmosphere for keeping quality of fresh-cut pineapples. Postharvest Biol. Technol. 39, 163–168. Martinez-Ferrer, M., Harper, C., Perez-Muroz, F., Chaparro, M., 2002. Modified atmosphere packaging of minimally processed mango and pineapple fruits. J. Food Sci. 67, 3365–3371. Montero-Calderon, M., Rojas-Grau, M.A., Aguilo-Aguayo, I., Soliva-Fortuny, R., Martin-Belloso, O., 2010a. Influence of modified atmosphere packaging on volatile compounds and physicochemical and antioxidant attributes of fresh-cut pineapple (Ananas comosus). J. Agric. Food Chem. 58, 5042–5049. Montero-Calderon, M., Rojas-Grau, M.A., Martin-Belloso, O., 2008. Effect of packaging conditions on quality and shelf-life of fresh-cut pineapple (Ananas comosus). Postharvest Biol. Technol. 50, 182–189.

Montero-Calderon, M., Rojas-Grau, M.A., Martin-Belloso, O., 2010b. Aroma profile and volatiles odor activity along gold cultivar pineapple flesh. J. Food Sci. 75, S506–S512. MoradasFerreira, P., Costa, V., Piper, P., Mager, W., 1996. The molecular defences against reactive oxygen species in yeast. Mol. Microbiol. 19, 651–658. Noseda, B., Ragaert, P., Pauwels, D., Anthierens, T., Van Langenhove, H., Dewulf, J., Devlieghere, F., 2010. Validation of selective ion flow tube mass spectrometry for fast quantification of volatile bases produced on atlantic cod (Gadus morhua). J. Agric. Food Chem. 58, 5213–5219. O’Connor-Shaw, R., Roberts, R., Ford, A., Nottingham, S., 1994. Shelf-life of minimally processed honeydew, kiwifruit, papaya, pineapple and cantaloupe. J. Food Sci. 59, 1202–1207. Olivares, A., Dryahina, K., Navarro, J.L., Flores, M., Smith, D., Spanel, P., 2010. Selected ion flow tube-mass spectrometry for absolute quantification of aroma compounds in the headspace of dry fermented sausages. Anal. Chem. 82, 5819–5829. Oms-Oliu, G., Soliva-Fortuny, R., Martin-Belloso, O., 2008. Modeling changes of headspace gas concentrations to describe the respiration of fresh-cut melon under low or superatmospheric oxygen atmospheres. J. Food Eng. 85, 401–409. Pronk, J.T., Steensma, H.Y., vanDijken, J.P., 1996. Pyruvate metabolism in Saccharomyces cerevisiae. Yeast 12, 1607–1633. Ragaert, P., Devlieghere, F., Loos, S., Dewulf, J., Van Langenhove, H., Debevere, J., 2006. Metabolite production of yeasts on a strawberry-agar during storage at 7 ◦ C in air and low oxygen atmosphere. Food Microbiol. 23, 154–161. Renger, R.S., Vanhateren, S.H., Luyben, K., 1992. The formation of esters and higher alcohols during brewery fermentation – the effect of carbon-dioxide pressure. J. Inst. Brewing 98, 509–513. Rocculi, P., Emiliano, C., Romani, S., Sacchetti, G., Rosa, M.D., 2009. Effect of 1mcp treatment and N2 O map on physiological and quality changes of fresh-cut pineapple. Postharvest Biol. Technol. 51, 371–377. Rojas-Grau, M.A., Oms-Oliu, G., Soliva-Fortuny, R., Martin-Belloso, O., 2009. The use of packaging techniques to maintain freshness in fresh-cut fruits and vegetables: a review. Int. J. Food Sci. Technol. 44, 875–889. Sandhya, 2010. Modified atmosphere packaging of fresh produce: current status and future needs. Food Sci. Technol. Leb. 43, 381–392. Spanier, A.M., Flores, M., James, C., Lasater, J., Lloyd, S., Miller, J.A., 1998. Freshcut pineapple (Ananas sp.) flavor. In: Effect of storage. Food Flavors: Formation, Analysis, and Packaging Influences, 40., pp. 331–343. Tabachnick, J., Joslyn, M.A., 1953. Formation of esters by yeast. Ii. Investigations with cellular suspensions of Hansenula anomala. Plant Physiol. 28, 681–692. Takeoka, G., Buttery, R.G., Flath, R.A., Teranishi, R., Wheeler, E., Wieczorek, R., Guentert, M.,1989. Volatile constituents of pineapple (Ananas comosus [l.] merr.). In: ACS Symposium Series American Chemical Society (USA). ACS Publications, pp. 223–237. Torri, L., Sinelli, N., Limbo, S., 2010. Shelf life evaluation of fresh-cut pineapple by using an electronic nose. Postharvest Biol. Technol. 56, 239–245. Uyttendaele, M., Jacxsens, L., De Loy-Hendrickx, A., Devlieghere, F., Debevere, J., 2010. Microbiologische richtwaarden & wettelijke microbiologische criteria, Laboratorium voor Levensmiddelenmicrobiologie—en conservering. Laboratory of Food Microbiology and Food Preservation, Ghent University, Ghent, Belgium. Wei, C.B., Liu, S.H., Liu, Y.G., Lv, L.L., Yang, W.X., Sun, G.M., 2011. Characteristic aroma compounds from different pineapple parts. Molecules 16, 5104–5112. Wszelaki, A.L., Mitcham, E.J., 2000. Effects of superatmospheric oxygen on strawberry fruit quality and decay. Postharvest Biol. Technol. 20, 125–133. Zheng, Y., Yang, Z., Chen, X., 2008. Effect of high oxygen atmospheres on fruit decay and quality in Chinese bayberries, strawberries and blueberries. Food Control 19, 470–474.