Effect of Nitrogen Gas Packaging on the Quality and Microbial Growth of Fresh-Cut Vegetables under Low Temperatures

326 Journal of Food Protection, Vol. 65, No. 2, 2002, Pages 326 332 Copyright Q, International Association for Food Protection Effect of Nitrogen Gas Packaging on the Quality and Microbial Growth of Fresh-Cut Vegetables under Low Temperatures SHIGENOBU KOSEKI* AND KAZUHIKO ITOH Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan MS 01-213: Received 15 June 2001/Revised 10 September 2001 ABSTRACT Nitrogen (N 2 ) gas packaging for fresh-cut vegetables (lettuce and cabbage) has been examined as a means of modi ed atmosphere packaging (MAP) for extending the shelf life of cut vegetables. Gas composition in enclosed packages that contained cut vegetables and were lled with 100% N 2 had an oxygen (O 2 ) concentration of 1.2 to 5.0% and a carbon dioxide (CO 2 ) concentration of 0.5 to 3.5% after 5 days of storage. An atmosphere of low concentrationsof O 2 and high CO 2 conditions occurred naturally in the package lled with N 2 gas. Degradation of cut vegetables in terms of appearance was delayed by N 2 gas packaging. Because of this effect, the appearance of fresh-cut vegetables packaged with N 2 gas remained acceptable at temperatures below 58C after 5 days. Treatment with acidic electrolyzed water (AcEW) contributed to the acceptability of the vegetables appearance at 5 and 108C in the air-packaging system. N 2 gas packaging did not signi cantly affect the growth of microbial populations (total aerobic bacteria, coliform bacteria, Bacillus cereus, and psychrotrophic bacteria) in or on cut vegetables at 1, 5, and 108C for 5 days. Microbial growth in or on the cut vegetables was inhibited at 18C for 5 days regardless of atmospheric conditions. Inhibition of respiration plays an important role in extending the shelf life of fresh produce during storage and distribution. Controlled atmosphere storage or modi ed atmosphere packaging (MAP) is becoming an increasingly common approach to controlling respiration and extending the shelf life of fresh produce (5, 20). Lowering the oxygen (O 2 ) gas concentration and elevating the carbon dioxide (CO 2 ) gas concentration surrounding the produce will usually inhibit respiration effectively (7). The MAP system can create atmospheric conditions different from those in the air by the interaction of respiration and gas permeation through the packaging lm. Several mixed and homogeneous atmospheres have been proposed for various freshcut vegetables (12 14, 18, 19). Although the optimum gas composition differs according to vegetables, conventional research has shown that O 2 concentrations of around 0.5 to 5% and CO 2 concentrations of around 5 to 15% are effective (6). Nitrogen (N 2 ) gas packaging is a popular antioxidation technique in various food industries, including the beverage industry. This technology contributes to extending the shelf life of products. Since N 2 packaging is a simple method requiring only N 2 gas, it has many practical advantages. However, there have been very few reports on the application of N 2 packaging to fresh-cut vegetable storage (4). As fresh-cut vegetables stay alive in an N 2 atmosphere, i.e., in a 0% O 2 atmosphere, vegetables will start anaerobic respiration, and an odor will occur because of the production of ethanol. Thus, it has been considered dif cult to adopt N 2 packaging for fresh-cut vegetables. At present, the ad- * Author for correspondence. Tel: 181-11-706-2558; Fax: 181-11-706-3886; E-mail: koseki@bpe.agr.hokudai.ac.jp. justment of the initial gas composition for each vegetable is complicated and costly. Therefore, MAP systems are not yet in widespread practical use. In this study, we examined the effect of N 2 packaging on the quality of fresh-cut vegetables during storage. We used the gas permeability of plastic lm to facilitate the permeation of atmospheric gases after N 2 packaging and the respiration of fresh-cut vegetables in an attempt to produce an optimal gas composition. This technique could be used for extending the shelf life of, as well as controlling the microbial growth on, packaged fresh-cut vegetables. The objective of this study was to con rm the changes in gas composition in package-enclosed fresh-cut vegetables with 100% N 2 gas. Furthermore, we examined the appearance (i.e., browning) of the cut vegetables as well as the microbial growth in and on them and made a general evaluation as to the practicality of N 2 packaging for storing and distributing vegetables. MATERIALS AND METHODS Fresh-cut vegetables. Fresh lettuce and cabbage were purchased at a local supermarket in Sapporo, Hokkaido, Japan. These vegetables were harvested 2 days before retail sale. The storage temperature was kept below 108C during distribution. The outer leaves were removed and discarded. Lettuce leaves were then cut into ca. 5- by 5-cm squares, and cabbage leaves were shredded to a width of 2 to 3 mm. Samples of lettuce or cabbage (1,000 g) were washed by soaking in alkaline electrolyzed water (AlEW, 20 liters) for 1 min, and then disinfected by soaking in acidic electrolyzed water (AcEW, 20 liters) for 1 min. A batch-type electrolysis apparatus, Super Oxeed Labo (Model JED-020, AOI Engineering, Shizuoka, Japan), was used to prepare the electrolyzed water, which involved the electrolysis of a 0.1% sodium chloride solution at 9 to 12 direct current volts for 10 min at room tem-

J. Food Prot., Vol. 65, No. 2 NITROGEN GAS PACKAGING OF FRESH-CUT VEGETABLES 327 FIGURE 1. Changes in O 2 and CO 2 concentrations during storage of the cut lettuce treated with acidic electrolyzed water (AcEW) or tap water (TW) in packages lled with N 2 or air at 1, 5, and 108C for 5 days. Results are means 6 standard deviation, n 5 3. perature. As a control, samples of lettuce or cabbage (1,000 g) were washed by soaking in tap water (TW; 20 liters) for 2 min. The water adhering to the lettuce or cabbage was removed by centrifugation (40 3 g, 20 s). These treatments were conducted in duplicate, and a 2,000-g sample was prepared for each condition. Storage of treated fresh-cut vegetables. A 50-g sample of each fresh-cut vegetable was enclosed in a package (effective area, 700 cm 2 ) of low-density polyethylene lm (thickness, 40 mm). A square piece of silicon rubber (2.25 cm 2 ) was attached to the surface of the package as a septum to facilitate the withdrawal of gas samples. The air was removed from each package by vacuum, and then 300 ml of ashed N 2 or air was added, and the opening of each package was closed by heat sealing. Half of each vegetable was used for the N 2 -packaging system, and the rest was used for the air-packaging system (control). For the two packaging systems, each vegetable sample was divided at 1, 5, and 108C, respectively. Each of these samples was then divided into four additional samples to correspond to testing that would occur on days 0, 1, 3, and 5. These packages were prepared in duplicate. On each day of analysis, the gas composition in the package of each group was determined. After gas analysis, the package in each group was opened, and the contents were analyzed. Objective measurements of browning and microbial populations were made on day 0 (the day experiments were initiated) and after 1, 3, and 5 days of storage. Gas analysis. The O 2 and CO 2 concentrations in the polyethylene lm packages containing cut vegetables were determined by a packaging atmosphere analyzer (MAPTEST 3000, Hitech Instruments, Luton, UK). Twenty milliliters of gas was sampled with a gastight syringe through a silicone rubber septum glued on the package and was then analyzed with the packaging atmosphere analyzer. Microbiological analysis. To enumerate the microorganisms in and on the fresh-cut vegetables, a sample of each vegetable (25 g) was combined with 225 ml sterile 0.85% sodium chloride solution in a sterile polyethylene bag and pummeled with a stomacher for 2 min at high speed. The wash uid was then serially diluted. All microbiological media used in this study were purchased from Merck (Darmstadt, Germany). Total aerobic bacterial counts were determined by pouring 1 ml of diluted sample into plate count agar. Plates were incubated at 358C for 48 h, and the colonies were counted. Coliform counts were determined by pouring 1 ml of diluted sample into violet red bile agar. Plates were incubated at 358C for 24 h, and the colonies were counted. Bacillus cereus counts were quanti ed by direct plating 0.1 ml of diluted sample onto the surface of a mannitol-egg yolk-polymyxin agar plate. Plates were incubated at 358C for 24 to 48 h, and the colonies were counted. Psychrotrophic bacteria counts were quanti ed by direct plating 0.1 ml of diluted sample onto the surface of plate count agar. Plates were incubated at 78C for 10 days, and the colonies were counted. All pour and spread plate experiments

328 KOSEKI AND ITOH J. Food Prot., Vol. 65, No. 2 FIGURE 2. Changes in O 2 and CO 2 concentrations during storage of the cut cabbage treated with acidic electrolyzed water (AcEW) or tap water (TW) in packages lled with N 2 or air at 1, 5, and 108C for 5 days. Results are means 6 standard deviation, n 5 3. for quantitative analysis were carried out in duplicate at each relevant dilution. Browning analysis. Browning was determined according to the method of Kawano et al. (8). A 20-g sample of cut vegetable was homogenized with 80 ml of ethanol (99%) by a blender (SM- 26; Sanyo, Tokyo, Japan). The homogenate was ltrated by no. 6 lter paper (TOYO, Tokyo, Japan) with suction. Filtrated residues were vacuum dried (RLS-10NA; Kyowa, Tokyo, Japan) for 2 h. The residues were then milled with a crusher mill (KENIS, Tokyo, Japan) for 20 s. These milled residues were put into a quartz cell 1 cm in width and measured by colorimeter (DR-200b; Minolta, Tokyo, Japan). The color of the samples was represented by the L *, a *, and b * systems. The color tone before storage was assumed to be the original, and browning after storage was expressed as the chrominance (DE) from the initial color. DE was de ned as follows: DE 5 Ï (L* 2 L *) 1 (a* 2 a *) 1 (b* 2 b *) 2 2 2 0 0 0 where L * 0, a * 0, and b * 0 were the initial values, and L *, a *, and b * were the values after storage. Statistical analysis. Gas, microbial, and browning analyses were conducted in duplicate for each day of analysis during storage. Three independent replications of all experiments were conducted. Data were analyzed with statistical analysis software (MS- Excel 2000, Microsoft, Bothell, Wash.). All data were subjected to analysis of variance and a least signi cant difference test to determine signi cant differences (P # 0.05) between treatments. RESULTS Gas analysis. Headspace gas analysis of the packages during the storage period showed that there was moderate gas permeation. Figure 1 shows the changes with time in the O 2 and CO 2 concentrations during the storage of the cut lettuce in packages lled with N 2 and air. In the N 2 packages, O 2 and CO 2 concentrations were elevated by 4 to 5% and 0.5 to 1.7% after 5 days of storage, respectively. An atmosphere of low O 2 and high CO 2 occurred naturally in the packages lled with N 2. In the packages lled with air, O 2 concentrations fell moderately, and CO 2 concentrations rose greatly at 108C. Although the CO 2 concentration in packages lled with air was elevated to 1 to 3% at 5 and 108C, the O 2 concentration did not fall to a level appropriate (below 5%) for the control of respiration. The N 2 -packaging system more signi cantly (P # 0.05) inhibited the CO 2 evolution than did the air-packaging system at 5 and 108C. The respiration of lettuce samples treated with AcEW increased more than that of samples treated with TW under air-packaging conditions at 108C. There was a signi cant difference (P # 0.05) in CO 2 evolution between lettuce samples treated with AcEW and those treated with TW in the air-packaging system at 108C after 5 days of storage.

J. Food Prot., Vol. 65, No. 2 NITROGEN GAS PACKAGING OF FRESH-CUT VEGETABLES 329 FIGURE 3. Changes in populations of aerobic bacteria, coliform bacteria, Bacillus cereus, and psychrotrophic bacteria in/on the lettuce treated with acidic electrolyzed water (AcEW) or tap water (TW) during storage in packages lled with N 2 or air at 1, 5, and 108C for 5 days. Results are the mean value of three replications. Figure 2 shows the changes with time in the O 2 and CO 2 concentrations during the storage of cut cabbage in packages lled with N 2 and air. In the N 2 -packaging system, the O 2 concentration was elevated by 2.5 to 4.2% at every storage temperature on day 1, and then was reduced to 1.2 to 2.5% after 5 days at 5 and 108C. The O 2 concentration at 18C was approximately 4% from days 1 to 5 of storage. The CO 2 concentration was elevated to 1.5 to 3.6% at every storage temperature after 5 days. In the packages lled with air, the O 2 concentrations fell greatly, and the CO 2 concentrations rose greatly at both 5 and 108C after 5 days. The N 2 -packaging system inhibited CO 2 evolution signi cantly more (P # 0.05) than did the air-packaging system at all temperatures after 5 days. The elevation in CO 2 concentration was dependent on the storage temperature. As the storage temperature was increased, the CO 2 evolution increased. The cut cabbage samples produced more CO 2 than the cut lettuce samples. It is known that the cutting of fresh produce increases the respiration rate (16, 17). Therefore, cabbage shredded nely into 2- to 3-mm pieces increased the respiration rate more than lettuce. The cabbage treated with AcEW showed greater CO 2 evolution than did the cabbage treated with TW under air-packaging conditions at 108C. There was a signi cant difference (P # 0.05) in CO 2 evolution between cabbage samples treated with AcEW and those treated with TW in the air-packaging system at 108C after both 3 and 5 days of storage. Microbial analysis. The ph values for AcEW, AlEW, and TW were 2.4 6 0.1, 11.1 6 0.1, and 7.0 6 0.1, respectively. Although the oxidation-reduction potential of AcEW was high, such as 1,148 6 6 mv, AlEW showed a very low oxidation-reduction potential, such as 2872 6 8 mv. The oxidation-reduction potential of TW was 358 6 20 mv. The available chlorine concentration of AcEW and TW was 40.6 6 1.2 ppm and 0.3 6 0.1 ppm, respectively. Washing with AlEW for 1 min and then disinfecting with AcEW for 1 min reduced the total aerobic bacteria, coliform bacteria, B. cereus, and psychrotrophic bacteria in and on the lettuce and cabbage by 1 to 2 log 10 CFU/g, respectively (Figs. 3 and 4). These reductions are in agreement with those of our published results (10, 11). Changes in bacterial populations in or on the lettuce treated with AcEW and TW during storage in the N 2 - or air-packaging system at three temperatures are shown in Figure 3. No bacteria populations increased during storage at 18C for 5 days in either packaging system. Regardless of atmospheric composition, increases in the population of every bacterium were similar through the 5 days of storage at 5 or 108C. Increases in microbial populations were dependent on storage temperature, regardless of packaging conditions. Microbial populations in or on lettuce treated with TW followed a trend similar to that observed on lettuce treated with AcEW. All bacteria examined in this study grew more quickly in or on lettuce treated with AcEW than

330 KOSEKI AND ITOH J. Food Prot., Vol. 65, No. 2 FIGURE 4. Changes in populations of aerobic bacteria, coliform bacteria, Bacillus cereus, and psychrotrophic bacteria in/on the cabbage treated with acidic electrolyzed water (AcEW) or tap water (TW) during storage in packages lled with N 2 or air at 1, 5, and 108C for 5 days. Results are the mean value of three replications. on lettuce treated with TW at 5 and 108C. In other words, when the initial microbial populations were low, the growth rate quickened. This trend was particularly evident for aerobic bacteria and coliform bacteria. However, after 5 days of storage, the microbial populations in and on the lettuce treated with AcEW did not exceed those in and on the lettuce treated with TW at the same storage temperature. Overall growth of every microorganism examined in this study was not signi cantly in uenced by the type of atmosphere under which the lettuce was stored. Figure 4 shows the growth of aerobic bacteria, coliform bacteria, B. cereus, and psychrotrophic bacteria in and on the cabbage treated with AcEW and that treated with TW. Microbial growth in and on the cabbage was similar to that in and on the lettuce. Microbial growth in and on the cut cabbage was also dependent on the storage temperature. Overall growth of all microorganisms examined in this study was not signi cantly in uenced by the type of packaging system. Browning analysis. Increases in DE indicate the progression of browning. Figure 5 shows the browning of lettuce and cabbage treated with AcEW or TW and stored in packages with N 2 or air. Although the browning of both lettuce samples treated with AcEW and TW was kept to a DE value of,5 in the N 2 -packaging system after storage at 108C for 5 days, the browning of lettuce stored in the airpackaging system was increased by a DE value of 7 to 9. At 1 and 58C, the browning of the lettuce in the N 2 packaging was kept to a very low level below a DE value of 2. In the air-packaging system, the browning of the lettuce treated with TW at 58C progressed to a DE value of.3 after 5 days. The browning was minimal in lettuce treated with AcEW and stored in the air-packaging system at 58C. The browning of the lettuce was also minimal in the air-packaging system at 18C. There were signi cant differences (P # 0.05) in browning between lettuce samples treated with AcEW and those treated with TW in both packaging systems after 5 days of storage at 108C. There were also signi cant differences (P # 0.05) in browning between lettuce samples treated with AcEW and those treated with TW in the airpackaging system after 5 days of storage at 58C. The progression of the browning of the cut cabbage showed a trend similar to that of the cut lettuce. The degree of browning of cut cabbage was smaller than that of cut lettuce at 108C in the air packaging. The N 2 -packaging system restrained browning signi cantly more (P # 0.05) on cut cabbage at 5 and 108C after 5 days than did the airpackaging system. Treatment with AcEW also facilitated browning at the higher temperature in the air-packaging system. There were signi cant differences (P # 0.05) in browning between the cabbage samples treated with AcEW and those treated with TW in both packaging systems after 5 days of storage at 108C. In addition, there were signi cant differences (P # 0.05) in browning between cabbage sam-

J. Food Prot., Vol. 65, No. 2 NITROGEN GAS PACKAGING OF FRESH-CUT VEGETABLES 331 FIGURE 5. Changes in the browning (DE) of lettuce and cabbage treated with acidic electrolyzed water (AcEW) or tap water (TW) and then stored in packages lled with N 2 or air at 1, 5, and 108C for 5 days. Results are means 6 standard deviation, n 5 3. ples treated with AcEW and those treated with TW in the air-packaging system after 5 days of storage at 58C. DISCUSSION MAP storage extends the shelf life of fresh produce by controlling the atmospheric conditions in a polymeric lm package using the respiration activity of the fresh produce and the permeation of O 2 and CO 2 gases through the polymeric lm. Optimal O 2 and CO 2 concentrations in controlled atmosphere storage and MAP systems have been reported for various fresh produce items (12 14, 18, 19). For lettuce, an O 2 concentration of 5% and a CO 2 concentration of 0% have been reported (5), whereas an O 2 concentration,1% and a CO 2 concentration.10% have been estimated as an aerobic condition for cabbage (5). In this study, the N 2 -packaging system created conditions similar to those reported as optimum. Since the lm used in this study, which was recommended for cut cabbage packaging (8), had moderate gas permeability, respiration would be controlled by the low atmospheric O 2 concentration. From a practical point of view, a simpli ed packaging method for cut vegetables is desirable. Therefore, N 2 packaging will be a means of MAP for fresh produce. Degradation of the cut vegetables in terms of appearance (i.e., browning) was delayed by the N 2 -packaging system in this study. The browning of vegetables becomes noticeable at a DE of.2.9 (8). In the N 2 -packaging system, browning did not occur during storage at temperatures of 58C or below for 5 days. At 108C, the browning of the cut vegetables was more restrained in the N 2 -packaging system than in the air-packaging system. Since browning is principally caused by enzymes related to oxidation, O 2 concentrations greatly affect the progress of browning. Furthermore, as enzymatic reactions slow at low temperatures, browning should not progress at low temperatures, such as below 58C. N 2 packaging together with cryogenic temperature control should satisfy these conditions. Treatment with AcEW would also help limit browning. Regardless of the packaging system, the browning of the cut vegetables treated with AcEW was less than that of the vegetables treated with TW at both 5 and 108C storage. Enzymatic activity on the surface of cut vegetables may be reduced by treatment with AcEW. Since AcEW has a strong oxidation-reduction potential (9, 15), the enzymes may be oxidized and weakened. A signi cant observation in this study is that although N 2 packaging, which is a kind of MAP, extends the length of time that vegetables may be kept without browning, it does not affect the growth of microbial populations. All test strains of bacteria exhibited the same growth trend, whether on vegetables stored under N 2 or air packaging. Similar

332 KOSEKI AND ITOH J. Food Prot., Vol. 65, No. 2 observations have been reported regarding the effect of controlled atmosphere storage or MAP on the sensory qualities of micro ora naturally occurring on asparagus, broccoli, cauli ower, and bell peppers (1 3). Although the N 2 -packaging system extends the shelf life of cut vegetables in terms of appearance, it is necessary to recognize the risk of bacterial growth. Bacterial populations in and on vegetables stored above 58C are unaffected by atmospheric conditions. Even if vegetables are disinfected with AcEW or a similar disinfectant, when storage temperature is relatively high, such as above 58C, bacterial populations increase rapidly. Any vegetable may present a public health hazard if pathogenic microorganisms are present, regardless of the storage technique used. Moreover, the risk of foodborne illness when fresh vegetables are stored for longer periods would be high, because pathogens have an opportunity to reach greater populations. Therefore, the N 2 -packaging system should not be used alone to extend the shelf life of cut vegetables, because microbial growth is not affected. None of the bacteria examined in this study showed a population increase when stored at 18C. This temperature is a reasonable growth deterrent of most microorganisms; however, storage at this temperature is dif cult to manage with present distribution systems. Furthermore, low-temperature storage should not be relied on as the sole preservative technique because of the potential for abuse by distributors, retailers, and consumers. Therefore, we have proposed the combination of AcEW treatment, N 2 packaging, and cryogenic temperature control. First, AcEW is used for the decontamination of fresh-cut vegetables. 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