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����������������APPLICATION OF ANTIBROWNING AGENTS IN MINIMALLY PROCESSED CABBAGE

Eleni Manolopoulou1, Theodoros Varzakas 2* 
1Department of Plant Production, School of Agricultural Technology, Technological Educational Institute of Peloponnese, Greece; 
2Department of Food Technology, School of Agricultural Technology, Technological Educational Institute of Peloponnese, Greece. Email: *theovarzakas@yahoo.gr


Abstract

The aim of this study was to investigate the effect of storage at 0�C and 5�C on the color and organoleptic quality, of fresh-cut cabbage during the addition of ascorbic acid, citric acid and calcium chloride and during modified atmosphere packaging (MAP) with low �2 concentrations (1.5%) and high concentrations of C�2 (17%).
Of the two studied temperatures (0 and 5�C) the temperature of 0�C maintained better quality parameters (color, organoleptic quality) and increased the shelf life of minimally processed cabbage. Among the treatments (MAP, citric acid, ascorbic acid, calcium chloride) MAP and citric acid showed the best results in terms of color and total visual quality. The combination of low temperature storage (0�C) with MAP or citric acid treatment prolonged the shelf life of minimally processed cabbage for 22 days, time sufficient for acceptable marketing of the product.

Introduction
Fresh-cut produce is defined as any fresh fruit or vegetable or any combination thereof that has been physically altered from its original form, but remains in a fresh state. Regardless of commodity, it has been trimmed, peeled, washed and cut into 100% usable product that is subsequently bagged or packaged to offer consumers high nutrition, convenience and value while still maintaining freshness (IFPA). In recent years, consumers have become more health conscious in their food choices, but have had less time to prepare healthful meals. As a result the market demand for fresh-cut fruits and legumes has rapidly increased. 
�t is well known that minimally processed fruits and vegetables are generally more perishable than the original raw materials (Gorny et al. 1998; Pittia et al.1999), because the application of partial processing increases perishability due to increased metabolic activities and decompartmentalization of enzymes and substrates. This may cause browning, softening, and off-flavour development (Watada et al, 1990). 
Successful marketing of minimally processed fresh produce is linked to several factors as the maintenance of high level of sensory quality for an adequate duration subsequent to harvest (Gertmenian, 1992), the harvest timing to achieve peak quality, quality control for postharvest treatments and effective packaging.
Colour is an important quality attribute in the food and bioprocess industries, both for its aesthetic value and for quality judgement. Colour influences consumer�s choice and preferences. An important consideration during fresh-cut fruit processing is the preservation of normal tissue colour and control of discoloration or surface browning. 
Browning colouration is an important phenomenon in food handling and processing it affects appearance quality. Enzymatic browning involves the interaction of oxygen, phenolic compounds and polyphenol oxidases (PPOs). To prevent browning at least one component must be removed from the system. Browning can be prevented by exclusion or removal of one or both of the substrates (O2 and phenols), lowering the pH to 2 or more units below the optimum, or adding compounds that inhibit PPO or prevent melanin formation (Whitaker and Lee, 1995).
In leafy vegetables browning is considered as one of the most important defects because it is easily noticeable. It does not occur in intact plant cells since phenolic compounds in cell vacuoles are separated from the PPO that is present in the cytoplasm. Once tissues are damaged by slicing or cutting, the mixture of PPO and phenolic compounds consequently results in a rapid browning reaction. Enzymatic browning remains a costly concern for the fruit and vegetable industry, as it is one of the primary causes of quality degradation.
Enzymatic browning may be controlled through the use of physical and chemical methods and in most cases both are employed. Physical methods may include reduction of temperature and /or oxygen, use of MAP or edible coatings or treatment with gamma irradiation or high pressure. Chemical methods utilize compounds that act to inhibit the enzyme, remove its substrates (O2, phenolics) or function as preferred substrate (Soliva-Fortuny and Martin-Belloso, 2003).
Among the compounds that have been shown to inhibit browning are sulphites, and andioxidants which are grouped in accordance to their mode of action (i.e., as acidulants- such as citric, malic, and phosphoric acids-, reducing agents- such as ascorbic acid or the erythrobate isomer of ascorbic acid-, chelating agents- such as sorbic acid, polycarboxylic acids-,inorganic salts �such as calcium, zinc and sodium-, complexing, enzymes, and inhibitors) (Whitaker, 1994; Rojas-Grau et al., 2007; He and Luo, 2007; Lamikanra, 2002; Sapers et al., 1989; Sapers and Miller1992, 1993; Monsalve-Gonzalez et al., 1993; Dorantes-Alvarez and Chiralt, 2000; Alzamora et al., 2000; Drake and Spayd, 1983; Bolin and Huxsoll, 1989).
	Temperature is the most important factor in the preservation of fresh-cut fruit quality. The use of low temperatures during the storage, handling and processing of minimal processing fruits and vegetables can control the metabolic rates and the enzymatic activity that affect quality. Refrigeration slows down both PPO and POD activities, since low temperatures (0-4�C) are far from optimal for these enzymes (Tom�s-Barberan and Espin, 2001)
CA, MAP reduce oxygen and increase the carbon dioxide content of the atmosphere surrounding the commodity and can produce direct effects on phenolic composition. High levels of oxygen are particularly effective in inhibiting enzymatic discoloration. It has been hypothesized that high oxygen levels may cause substrate inhibition of PPO, or alternatively, that high levels of colourless quinones subsequently formed may cause feedback inhibition of PPO (James and Ngarmsak, 2010).
Cabbage is an important member of cruciferous vegetables being also an important dietary vegetable consumed as fresh cut salad or cooked. Recently it was widely marketed as a minimally processed product. Vegetables in the Brassica family are reported to possess cancer-preventive properties (Beecher 1994) attributed to their glucosinolates (Stoewsand 1995) and phenolic compounds (Le Marchand 2002; Galati and O�Brien 2004). Glucosinolates in Brassica vegetables confer defence functions to the plants and provide a source of bioactive compounds that are important to human nutrition and health. Brassica vegetables are therefore of great interest for their potential use in disease risk reduction and are considered as functional foods (Cartea and Velasco 2008). Cabbage and broccoli, excellent sources of indoles, dithiolthiones, isothiocyanates, and chlorophyllins, are also effective in minimizing the risk of heart attack (Howard and Kritchevsky 1999).
The objective of this study was to investigate the potential of ascorbic acid, citric acid, calcium chloride and MAP, individually, to extend the shelf life and maintain the quality of fresh-cut cabbage.

Materials and Methods
White Cabbage (Brassica oleracea L. var. capitata) var. Bunner harvested on Mars and April from a farm located in Messinia (Peloponnese, Greece) was minimally processed using the best preparation techniques. The leaves were cut into quarters and then sliced into 3 cm wide strips with a sharp stainless steel knife sterilized in ethanol. The shredded cabbage was washed for 4 min in chlorinated (100 ppm NaOCl) water at 5�C (at a solid/ liquid ratio of 1:3) to remove released nutrients. Then the cabbage was rinsed with tap water for 4 min and centrifuged for 1 min in a 23 cm diameter basked rotating at 500 rpm.
The treatments were conducted in parallel and prepared for the same batch of product. To minimize product heterogeneity, processed cabbage was pooled, mixed and subsequently divided in five equal parts. The first part was dipped in distilled water and was used as control sample. The second portion was dipped in ascorbic acid solution; the third was dipped in citric acid solution while the fourth portion was dipped in calcium chloride solution. The fifth and final portion was packaged in MAP, which was made by injection of a mixture with 5% �2 -10% C�2 balanced with N2.
Dipping treatments with solutions of ascorbic acid (Serva,Germany), citric acid (Merck, Germany) and calcium chloride (Carlo Erba, Italy) were applied separately for 3 min, after draining. All dipping solutions were made with distilled water at 5�C and the concentrations were 1% (w/v) in all cases.
One hundred � 2 g of sliced cabbages treated with ascorbic acid, citric acid, calcium chloride or distilled water (control) were placed in clear trays of polystyrene (15.5 x 22.5 x 3 cm) covered with PVC film (13 �m) and stored at 0�C and 5�C and 90% H.R. in darkness. The same quantity of sliced cabbage was placed in 30 �m thick MDPE packages (30 x 24 cm), (O2 permeability 6.170�730 mL m-2 d-1 bar-1 and CO2 permeability 41.520�1830 mL m-2 d-1 bar-1 , S=PCO2/PO2= 6.8 at 20 �C according to data provided by the film manufacturer) since gas flushing was applied. Gas flushing was carried out using a gas mixer (MAP Mix 9000, Denmark) and a vacuum compensation chamber (Multivac A, 300/16, Germany). All tested packages treatments mentioned above were stored at 0�C and 5�C (except the MAP maintained only at 0�C) and 90% relative humidity in the dark for 22 days. Ten packages were prepared per sampling date and treatment (ascorbic acid, citric acid, calcium chloride, control, MAP). Measurements were taken during the 0, 7th, 14th, and 22rd day. Every experiment was conducted in twice.
Parameters studied were: gas analysis (MAP) and color .Organoleptic control was carried out for discoloration (browning) of cut surfaces and total visual quality. The in�package atmosphere (�2, C�2) was measured with a headspace gas analyzer (CheckMate 9000, PBI Densensor Co., Denmark) drawing up to 2 mL of air samples Sampling took place with a hypodermic needle through a septum pasted on the packaging Colour was measured with a Minolta CR-300 chromameter (Minolta Corp. Japan) on CIE L*, a*, b* chromatic space. The instrument was initially calibrated using a white ceramic tile (Y=92.6 X=0.313 y=0.319). The L* chromatic variable, ranges from 0 (black) to 100 (white) and is an indicator of the lightening or darkening due to the physicochemical changes taking place during storage. The a* measures the degree of redness (+a*) and greenness (-a*) while the b* the degree of yellowness (+b*) and blueness (-b*). Colour changes were determined by parameters (L*), and hue angle [h�= arctan-1(b*/a*)]. 
Quality evaluation (cut surface browning, and overall visual acceptance) was assessed in 6 packs per treatment, by a panel of six semi trained judges adopting a hedonic scale from 1 to 5, rating with 5= very fresh with no browning on the cut surfaces, 3= slight browning on the cut surfaces with marginal marketability, 1= not to be consumed (Kim and Klieber, 1997). A score of 3 was considered as the marketable limit. 

Statistical analysis
The experiment was performed according to a full factorial design (treatments x storage time) and subjected to analysis of variance (ANOVA) using the Statgraphics Plus 5.1 (Statpoint Technologies, Inc, VA, USA) statistical software. Mean values were subjected to Fisher�s Least Significant Difference test (LSD) at p=0.05. This statistical test is liberal with respect to the comparison wise error rate, but is powerful for detecting true difference (Gacula & Sing, 1984). The presented data in the figures are the mean value from 2 experimental series since no significant difference between the series was found.

Results and discussion
Gas composition.
The composition of the atmosphere (O2 and CO2) inside the package of shredded cabbage is presented in Fig.1.The O2 concentration was reduced during the first days, whereas C�2 concentration increased.

Figure 1. Changes in O2 and CO2 of packaged shredded cabbage stored at 0�C. (N=20) (I=LSD)

After 6 days of storage the composition of the atmosphere in the packages was stabilized and remained at these levels until the end of storage (22rd day). �2 concentration stabilized at 1.5% (from an initial concentration of 5%) and C�2 concentration  remained at 17% (from an initial concentration of 10%). This change in gases could be attributed to the increase in respiratory activity of tissues due to the cutting (Surjadinata and Cinseros-Zevallos, 2003). According to Hiroaki et al (1993) and Gorny (2001), the ideal gas concentration for shredded cabbage storage is 5-7.5% �2 and 15% C�2, whereas Hu et al (2007) suggested a 2% �2 and 13% C�2 concentration. The sensitivity of minimally processed vegetables to modified atmospheres might be quite different compared to the whole product, withstanding more extreme concentrations of �2 and C�2 compared to whole vegetables and this is due to the fact that they find fewer obstacles in the diffusion of gases (Kader et al. 1989).

Colour
The L* colour value and hue angle (h�) were selected as the most suitable parameters for discolouration of cut surfaces of minimally processed fruits and vegetables (Sapers and Douglas, 1987).When the colour became darker, a decrease in L* and hue angle (h�) was observed. Hue angle (h�), considered the qualitative attribute of colour. A higher hue angle represents a lesser yellow character in the assays. It has been extensively used in the evaluation of colour parameters in green vegetables (Barreiro et al, 1997; Lopez et al, 1997). The ANOVA showed that: temperature, treatments, storage time and their interaction had a significant effect on L* and h� (p=0.005).
The lightness of minimally processed cabbage decreased linearly (Fig.2) from 70.94 �6 to 63.8�8.5 -61.3�8 for the chemical treatments, to 60.28�7 for the MAP and to 59.4�6 for the control, during 22 days at 0�C.

Figure 2.Effect of chemical (ascorbic acid, citric acid, calcium chloride) and physical (MAP) treatments on changes in lightness (L* ) of minimally processed cabbage stored at 0�C and 5�C for 22 days (Mean values N=2 repetitions X 10 samples=20) (I=LSD).

At 5�C the lightness decreased from 70.94�6 to 63.6�7.9-62.07�8.2 at all cases during 22 days of storage (Manolopoulou and Varzakas, 2011). At the end of storage at 0�C lightness change in treatments (ascorbic acid, citric acid, calcium chloride, MAP) and control varied between 10-16.3%, whereas at 5�C this change ranged between 10-13%.
Among the treatments at 0�C, citric acid proved to be the most effective in maintaining the lightness in minimally processed cabbage presenting the smallest change of L*value. At 5�C no significant difference in L* values between the control and treated samples was observed after 22 days of storage. Temperature is the most influential factor on pigment degradation. For many vegetables, shelf-life can be better extended by lowering the storage temperature to slightly above freezing point of the tissue. Citric acid lowers the product pH, helps control enzymatic browning (He and Luo, 2007), and maintains the lightness of cabbage.
Hue angle values (Fig.3) during storage were significantly affected by treatments (chemicals and physical) mainly at 0�C. The same observation was made by Manolopoulou and Varzakas (2011) for Hue angle values and chemical treatments.

Figure 3. Changes in hue angle (h�) of shredded cabbage stored at 0�C and 5�C which has undergone chemical and physical treatments (�ean values �=2 repetitions � 10 samples =20) (I=LSD)

Hue angle control value decreased rapidly at 0�C. Among the treatments applied, MAP retained better the values of hue angle. Modified atmosphere packaging and low temperature storage have been used for many years to reduce respiration rates and the deterioration of fresh-cut produce. MAP can decrease the rate of browning reactions due to reduced O2 level and elevated CO2 level in the surrounding atmosphere (Herner, 1987). The decrease in hue according to Bolin & Huxsoll (1991) could be due to chlorophyll degradation from the action of chlorophyllase. High concentrations in C�2 keep chlorophyll in cabbage (Isenberg and Sayles, 1969) and reduce polyphenoloxidase and phenolic compounds concentration (Buescher and Henderson 1977). Among the chemical treatments citric acid had the highest antioxidant capacity. Citric acid has been reported extensively for its inhibitory activity on polyphenol oxidase, PPO (Langdon, 1987) and its antibrowning activity in minimally processed fruits and vegetables (Ahvenainen, 1996). Our results come in agreement with Kim and Klieber (1997) who worked on minimally processed Chinese cabbage and Kaur and Kapoor (2001) working on minimally processed Indian cabbage. Ascorbic acid and calcium chloride are intermediate inhibitors as seen by their hue angle values (Fig.3). 

Overall organoleptic quality
Freshness is the most important quality criterion that consumers use to evaluate organoleptic quality of fruits and vegetables. Browning of cut surfaces is one of the most limiting factors on the shelf life of fresh-cut products and a significant factor of quality deterioration of shredded cabbage (Pirovani et al. 1997). Organoleptic quality evaluation with quantifiable physical measurements, determine the shelf-life of fresh cut vegetables. Changes in quality characteristics (overall acceptance and browning of cut surfaces), are shown in Fig.4 and 5. 

 
Figure 4. Changes in overall acceptance of minimally processed cabbage with different physical and chemical treatments during storage at 0�C and 5�C (Mean values N=2 repetitions X 6 samples per repetition X 6 panellists=72) (I=LSD)


Figure 5. Changes of cut surface browning of minimally processed cabbage packaged in MAP and treated with different chemical treatments during storage at 0�C (Mean values N=2 repetitions X 6 samples per repetition X6 panellists=72) (I=LSD).

Overall acceptance together with cut surface browning determined the shelf life of control samples which was 7 days at both storage temperatures (Figs 4,5) using score 3 as the limit. Among the different treatments (physical and chemical) shredded cabbage packaged in MAP and stored at 0�C, presented the highest score of overall acceptance and the smallest browning of cut surfaces. Hence, shredded cabbage in package was judged to be marketable until the 22rd day. High concentrations in C�2 protect shredded vegetables from browning of cut surfaces because they limit the action of polyphenoloxidase (PP�) and the concentration in phenolic substances (Buescher and Henderson, 1977). Our results come in agreement with those reported by Kaji et al. (1993), who reported that browning was suppressed as CO2 concentration increased (0% to 15%), with no influence of O2 concentration (2.5% to 10%). Treatment with citric acid extended the shelf life of shredded cabbage stored at 0�C marginally to 22 days. During marketing the predominant temperature is 5�C and at this temperature treatment with citric acid extended the shelf life from 7 days (control) to 14 days. Ascorbic acid maintained the overall quality for 14 days at 0�C and 7 days at 5�C. Similar results were reported by Rosen and Kader (1989) in pear slices. Calcium chloride maintained the overall quality and cut surface browning for 14 days at both storage temperatures. Similar results in apples were reported by Drake and Spayed (1983), however, Bolin and Huxoll (1989) reported that calcium chloride is not effective in preventing browning in peaches. 
Hence, based on overall acceptance and cut surfaces browning and what was described above it arises that package MAP retained browning of cut surfaces at acceptable levels as well as visual quality extending the shelf life of the product to 22 days at 0�C. Citric acid 1% can be used for minimally processed cabbage. Citric acid is the most commonly used acidifier in combination with other types of antibrowning agents to control browning in fruits and vegetables (He and Luo, 2007).


Conclusions
MAP with low �2 concentrations (1.5%) and high concentrations of C�2 (17%) retained the quality of shredded cabbage by reducing browning of the cut surfaces and, retaining total visual quality. MAP increased shelf life of shredded cabbage to 22 days at 0�C making it easily marketable. From chemical treatments citric acid better retains the overall acceptance of shredded cabbage. It can reduce cut surface browning. Citric acid treatment combined with low temperature storage (0�C) prolonged the shelf life of minimally processed cabbage for 22 days. 

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Isenberg F.M. & Sayles R.M.,1969. Modified atmosphere storage of DanisABCUVkmnop���������3	L	Z	e	o	�	���̸̸̩�������tiai�R�iGh�+Bh�5�mH	sH	h�5�h�+BB*mH	phsH	h�5�mH	sH	h�5�h�5�mH	sH	h�5�h�5�H*mH	sH	h�5�B*mH	phsH	h�+BB*mH	phsH	 h�5�h�5�B*H*mH	phsH	h�5�h�5�B*mH	phsH	&h�5�h�5�5�B*H*\�mH	phsH	#h�5�h�5�5�B*\�mH	phsH	h�V�h�5�mH	sH	h]:'5�mH	sH	*h�V�h�V�5�mH	sH	BCo��	�	�	�	�	�$49w������������������$��7$8$H$`��a$gd�4V$��7$8$H$`��a$gd�K�$��7$8$H$`��a$gd�i[$a$gdN}�$a$gd�gd�$a$gd]:'$a$gd�5�$a$gd�5�gd�5�$a$gd]:'����������	�	�	�	�	�	�	�	�	�	




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