Microbiological Quality and Shelf Life of Enzyme-peeled Fresh-cut Persimmon Slices Stored in High CO2 Atmospheres

The microbiological quality and shelf life of enzyme-peeled fresh-cut persimmon slices were evaluated during storage in a high CO2 controlled atmosphere (CA) and active modified atmosphere packaging (MAP) at 10 8C. Microbial counts of the enzymepeeled slices were lower in high CO2 atmospheres (10%, 15%, and 20%) than in air during CA storage for 6 days at 10 8C with the 20% CO2 atmosphere being most effective. High CO2 atmospheres did not affect the number of bacterial and fungal species detected in the persimmon slices. The surface color, expressed as C* values, of the peeled side of enzyme-peeled slices was lower in high CO2 than in air after 6 days of CA storage. In contrast, C* values at the cut side were higher for slices stored in 20% CO2 than in air on Day 6. High CO2 atmospheres did not affect other quality of enzyme-peeled slices such as texture, pH, sugar content, and total ascorbic acid content. Based on the optimum 20% CO2 concentration in a CA, enzyme-peeled slices were stored in a MAP flushed with either air or 20% CO2 for 4 days at 10 8C. The CO2 concentration approached an equilibrium of either 5% or 10% after 3 days of storage in packages flushed with either air or 20% CO2, respectively, and the O2 decreased to ’10% in both packages. Adding 20% CO2 to the MAP was effective in reducing the growth of mesophiles and coliforms but not fungi in enzyme-peeled persimmon slices throughout 4 days of storage. The diversity of bacterial and fungal flora was partially similar between packages flushed with air and 20% CO2. Texture, pH, surface color, sugar content, and total ascorbic acid content of enzyme-peeled persimmon slices were unaffected by air or 20% CO2 as the flushing gas, except that C* values of the enzymatically peeled side on Day 4 were lower for slices flushed with 20% CO2 than air. A 20% CO2 atmosphere is recommended for reducing the microbial population of enzyme-peeled persimmon slices stored at 10 8C and the shelf life of persimmon slices in an active MAP with 20% CO2 is 4 days at 10 8C. Peeling is an important process to maintain the overall quality of fresh-cut fruits during their production and distribution. However, the common methods of mechanical and chemical peeling often cause the loss and damage of flesh, and sometimes there is deterioration of the visual, physiological, and microbial quality of fresh-cut fruits. As an alternative to these methods, the enzymepeeling technology began with the first report of a method to remove the peel of grapefruit by dipping scored samples in an enzymatic solution under vacuum (Bruemmer et al., 1978). This method was then applied to various fruits such as grapefruit (Pao et al., 1996), oranges (Pinnavaia et al., 2006), mangoes (Sakho et al., 1998), apricots, nectarines, peaches (Toker and Bayindirli, 2003), and Japanese persimmon fruits (Ozaki et al., 2004). We reported that enzyme-peeled persimmon slices resulted in a substantial reduction of microbes in fresh-cut slices, and there was no difference in the physicochemical quality between enzyme-peeled and knifepeeled slices (Murakami et al., 2012). It is important to maintain the microbiological and physiological quality of fresh-cut fruits during storage. Because cells and tissue of fruits are wounded by peeling and cutting, a deterioration in quality resulting from biochemical changes usually occurs with fresh-cut produce (Watada and Qi, 1999). CA storage and MAP including reduced O2 and/or elevated CO2 have been shown to extend the shelf life of fresh-cut fruits as reviewed by Gorny (2003) and Izumi (2005). An atmosphere of 3% to 5% O2 + 5% to 8% CO2 is recommended for storing intact persimmon fruit, which maintains its quality at 0 to 5 C (Kader, 2003). The external appearance of fresh-cut persimmon slices remained unchanged while they were held for 8 d at 5 C in the presence of a 12% CO2 atmosphere (Wright and Kader, 1997). With other fresh-cut fruits, Aguayo et al. (2007) reported that a CA of 15% CO2 alone or 4% O2 + 15% CO2 reduced the microbial counts of freshcut melon stored at 5 C. Budu et al. (2007) showed that atmospheres of either 80% O2 + 15% CO2 or 5% O2 + 15% CO2 reduced the microbial growth on fresh-cut pineapple stored at 5 ± 1 C. When fresh-cut ‘Carabao’ and ‘Nam Dokmai’ mango cubes were stored under air or high CO2 atmospheres (3%, 5%, and 10%) at 5 or 13 C, a 10% CO2 atmosphere enhanced the bacteriostatic activity (Poubol and Izumi, 2005). These findings indicate that high CO2 atmospheres appear to be more beneficial for controlling microbial quality than low O2. The changes in quality of fresh-cut persimmons under high CO2 atmospheres need to be better understood to ensure quality enzyme-peeled persimmons during storage and distribution. In this study, we initially determined the optimum ranges of high CO2 in a CA based on the microbial, physicochemical, and nutrient quality of enzyme-peeled ‘Tone-wase’ persimmon slices during storage at 10 C. The microbiological quality and shelf life of enzyme-peeled persimmon slices during storage in an active MAP of 20% CO2 at 10 C were then evaluated. ‘Tone-wase’ persimmons were used, because we previously reported that an early-maturing cultivar, Tone-wase, was more suitable than a late-maturing cultivar, Fuyu, for the fresh-cut industry in Japan for an optimal microbial and physicochemical quality of enzymatically peeled slices (Murakami et al., 2012). Materials and Methods Fruit materials. Ninety-nine fruits of ‘Tonewase’ persimmon (Diospyros kaki Thunb.) were obtained from a commercial grower in Wakayama Prefecture, Japan, from Sept. to Oct. 2011. Fruit were treated to remove astringency by holding them in a closed chamber with a 95% CO2 atmosphere for 18 h at 25 C followed by storage for 0 to 2 d at room temperature. Treated fruit were then transported to the laboratory at Kinki University and stored at 20 C for 2 to 3 d before processing. Processing. Enzyme-peeled fruit samples were produced according to the method of Ozaki et al. (2004). Calyces of fruits were removed with a sterilized knife. The enzymatic peeling was then performed as follows. The peel was made porous with a needlepoint holder, and then the fruit were submerged in a hot water bath at 100 C for 45 s and then cooled in tap water for 1 min. Fruit were infused with enzyme by dipping them in 3% protopectinase (Pectinase-IGA; IGA Bio Research Ltd., Osaka, Japan) produced by Trichosporon penicillatum SNO-3 (Sakai, 1988) at 37 C for 3 h and then rinsed under running tap water for 1 min. The enzyme-peeled fruit was sliced radially into four sections using a sterilized knife. The enzymatic peeling treatment was replicated three times using three fruits for Received for publication 5 June 2012. Accepted for publication 10 Oct. 2012. This research was part of the Program for Fostering Regional Innovation (City Area Type) sponsored by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Dr. Kathy K. Kamo for reading the manuscript. To whom reprint requests should be addressed; e-mail [email protected]. 1758 HORTSCIENCE VOL. 47(12) DECEMBER 2012 each treatment of high CO2 CA storage, and active MAP. High CO2 controlled atmosphere storage. Twelve enzyme-peeled slices weighing 400 to 500 g were placed into a 2-L plastic container containing 5 mL of distilled water in a plastic beaker to maintain a high relative humidity. Three replicates were stored under a continuous flow of air or high CO2 atmosphere (10%, 15%, and 20%) with the balance being air at a flow rate of 15 mL·min for 6 d at 10 C. Active modified atmosphere packaging of high CO2. For active MAP, the optimum gas concentration was based on the results of CA storage. Four enzyme-peeled slices weighing 150 to 180 g were packaged in OPP film (30 mm thick, 21 3 23 cm, O2 permeability of 1170 mL·m·d·1013 hPa; Sumitomo Bakelite Co., Ltd., Tokyo, Japan) flushed with either air or 20% CO2. In this study, all enzyme-peeled slices flushed with air or 20% CO2 were found unmarketable after 6 d of storage, so three replicates of each treatment were monitored for 4 d at 10 C. Microbial counts. Slices of persimmon fruits during a CA and active MAP storage were taken periodically, and microbial analysis of each sample was performed as previously described (Izumi, 1999). A 10-g sample cut with a sterile scalpel was macerated in 90 mL sterile saline solution (0.85% NaCl in water) in a sterile stomacher bag with an Elmex stomacher (Promedia SH-001; ELMEX, Tokyo, Japan) for 4 min at room temperature. Serial dilutions from each sample were made using sterile saline solution and then poured on the following media: two standard method agar (SMA; Nissui Pharmaceutical, Tokyo, Japan) plates incubated at 37 C for 48 h for enumeration of mesophiles, two desoxycholate agar (Nissui Pharmaceutical) plates incubated at 37 C for 24 h for enumeration of coliform groups, and three potato dextrose agar (PDA; Nissui Pharmaceutical) plates with 100 ppm of chloramphenicol incubated at 26 C for 72 h for enumeration of fungi. The microbiological plate count data were converted to log cfu per gram of fruit. Microbial isolation and identification. The diluent of each sample was plated onto the surface of solidified SMA and PDA. Bacteria and fungi (molds and yeasts) were aseptically isolated from the SMA plates incubated at 37 C for 48 h and PDA plates incubated at 26 C for 72 h, respectively. One hundred thirty-five bacterial and 40 fungal (three mold and 37 yeast) isolates were selected from different-appearing types of colonies on petri plates. A MicroSeq microbial identification system (Applied Biosystems, Foster City, CA) was used to identify the bacteria and fungi. The sequencing data were analyzed using Analysis Software (MicroSeq analysis software Version 2.0, MicroSeq. 16S rDNA sequence databases Version 2.2 for bacteria, and MicroSeq. D2 LSU rDNA sequence databases Version 2.0 for fungi) as previously described (Poubol and Izumi, 2005). A cutoff of the lowest distance score from the sequence in the database was chosen for species identification. O2 and CO2 analyses. The O2 and CO2 concentrations in packages were measured daily during the MAP storage period using a gas chromatography equipped with a thermal conductivity detector (GSC-8AIT-TCD; Shimadzu, Kyoto, Japan). The columns used for O2 and CO2 analyses were Molecular Sieve SA at 60 C and Porapak Q at 90 C, respectively. Quality evaluation. Subsamples of slices were taken at scheduled time periods during CA storage or active MAP storage for evaluation of quality as previously described (Murakami et al., 2012; Poubol and Izumi, 2005). Surface color (C* value) of the peeled side and cut side of three slices per replication was measured with a Handy Colorimeter (Model NR-3000; Nippon Denshoku, Tokyo, Japan). Surface pH of three slices was determined with a compact pH meter (Model B113; Horiba, Tokyo, Japan). Texture (N) of the three slices was measured by the force required to shear the slice with a cutter blade using an EZ test texture analyzer (Model AR228; Shimadzu, Kyoto, Japan). Sugar content of the three slices was determined using a spectral photometer (Model ultraviolet-1600; Shimadzu) based on the method of SomogyiNelson (Chachin, 1981). Total ascorbic acid content of the three slices was determined using high-performance liquid chromatography (Model LC-10AD; Shimadzu) equipped with a PLRP-S 100A column (4.6 mm 3 25 cm, 5 mm; Polymer Laboratories, Varian, Inc., Amherst, MA) and an electro-chemical detector (Model ECD 300; Eicom, Kyoto, Japan). Statistical analysis. Statistically significant differences (P # 0.05) were determined for the microbial population, gas concentration, and quality evaluation data based on an analysis of variance using the SAS system, Release 6.12 (SAS Institute, Cary, NC). The mean values were compared using the Tukey’s honestly significant difference method when main effects were significant among the treatments in CA or active MAP storage. Results and Discussion Microflora of enzyme-peeled fresh-cut persimmon slices during controlled atmosphere storage. Initially, slices after the peeling and cutting treatment showed a count of 3.0 log cfu·g for mesophiles, and it was below the limit of detection for both coliforms (2.4 log cfu·g) and fungi (3.0 log cfu·g) (Fig. 1). Because persimmon fruit were submerged in hot water at 100 C to inactivate the substances in the peel that inhibit the pectolytic enzyme during the enzymatic peeling process, the heating treatment served as hot water pasteurization as shown in our previous report (Murakami et al., 2012). Fan et al. (2008) also reported that hot water treatment at 76 C for 3 min reduced the microbial population of cantaloupe. During storage in air, counts of mesophiles of persimmon slices increased to 6.4 log cfu·g by Day 4. The high CO2 (10%, 15%, and 20%) CA reduced the count increase with the reduction being greatest in 20% CO2 (4.8 log cfu·g ) on Day 4. Counts of coliforms and fungi of slices stored in air increased to 5.5 log cfu·g by Day 6. In comparison, 20% CO2 storage showed the lowest count of 3.4 log cfu·g for coliforms and 4.3 log cfu·g for fungi. Elevated CO2 has been shown to effectively reduce microbial growth on fresh-cut mango cubes (Poubol and Izumi, 2005), fresh-cut pineapple (Antoniolli et al., 2007; Budu et al., 2007), and fresh-cut apple slices (Gunes and Hotchkiss, 2002). Inhibition of microbial growth by CO2 may result from the presence of dissolved CO2 in the aqueous phase of fresh-cut products, which causes a decrease of the intercellular pH and enzymatically inhibits catalyzed reactions and enzyme synthesis (Molin, 2000). Poubol and Izumi (2005) reported that the microflora diversity was less for the fresh-cut mango cubes stored in 10% CO2 rather than in air or CO2 atmospheres of either 3% or 5%. However, in our study, high CO2 did not show the desired effects on the microbial diversity of enzyme-peeled slices during storage. No major differences were found in the number of bacterial and fungal species isolated from enzyme-peeled slices stored under either air or high CO2 atmospheres even after 6 d of storage (Table 1). Isolates from the enzyme-peeled samples included phytopathogenic bacteria such as Pseudomonas and Stenotrophomonas, soilborne bacteria such as Bacillus and Curtobacterium, and yeasts living in a plant–soil environment such as Cryptococcus, Pichia, and Rhodotorula. These bacteria and fungi are frequently found in other fruits, and they were mostly epiphytic microorganisms (Izumi et al., 2008a, 2008b). Only enzyme-peeled slices stored under 20% CO2 contained Weissella, lactic acid bacterium, and Aureobasidium, a mold. Quality of enzyme-peeled fresh-cut slices during controlled atmosphere storage. The surface color (C* value) of the peeled side significantly decreased by Day 6. There were no significant differences in the surface color of the peeled side when slices were stored in air or high CO2 atmospheres, except for Day 6 when C* values of the peeled surface were significantly higher for slices stored in air than in high CO2 although the cause is unknown (Fig. 2). In contrast, C* values of the cut side were higher for slices stored in high CO2, particularly 20% CO2, as compared with samples stored in air on Day 6. The peeled and cut surfaces of all samples became brown or dark-colored during storage as indicated by the decreasing C* values. Brown discoloration of the peeled surface of slices may have resulted from non-enzymatic browning that occurred during the heating treatment (100 C for 45 s) of the enzyme-peeling process, and browning of the cut surface may have been the result of enzymatic browning when phenolic compounds were exposed to polyphenol oxidase. Thus, high CO2 atmospheres would be helpful in reducing the development of brown discoloration only on cut surfaces. The texture and pH of all samples HORTSCIENCE VOL. 47(12) DECEMBER 2012 1759 | POSTHARVEST BIOLOGY AND TECHNOLOGY