JFS: Food Chemistry and Toxicology

Effect of Solid Content and Lipid Content of Whey Protein Isolate-Beeswax Edible Coatings on Color Change of Fresh-cut Apples
M.B. PEREZ-GAGO, M. SERRA, M. ALONSO, M. MATEOS, AND M.A. DEL RO

Food Chemistry and Toxicology

ABSTRACT: Edible coatings from whey protein isolate (WPI)-beeswax (BW) were made with various total solid (8%, 12%, 16%, 20%) and BW content (0%, 20%, 40%, 60%, dry bases). Apple pieces were coated with the emulsion coatings; weight loss and color (CIE L*, a*, b*, and browning index [BI]) were measured during storage. Results show that coated apples had higher L* and presented lower BI than uncoated apples, indicating that whey proteins exert an antibrowning effect. The BI decreased as the solid content of the coating emulsions increased. Increasing BW content decreased enzymatic browning. Coating application did not reduce weight loss in fresh-cut apples, probably due to the products high relative humidity. Keywords: fresh-cut apple, edible composite coatings, whey protein, color change

Introduction

HE MARKET DEMAND FOR FRESH- CUT FRUITS AND VEGETABLES

has experienced a rapid expansion due to the increased health consciousness of consumers, busy lifestyles, and purchase power (Baldwin and others 1995a). Shelf life of fresh-cut products is greatly reduced compared with whole fruits and vegetables because of the rapid metabolism of the wound tissue. Browning is a major problem in this industry. The main approach to inhibiting browning is the use of antibrowning agents that are based on citric acid or ascorbic acid. However, the use of high concentrations of acid or other chemical agents could significantly affect flavor and odor (Le Tien and others 2001). Another technique is the use of modified atmosphere packaging. However, it is necessary to avoid excessive reduction of oxygen, which could induce anaerobic metabolism. Edible films and coatings can offer a possibility to extend the shelf life of fresh-cut produce by providing a semipermeable barrier to gases and water vapor, and therefore, reducing respiration, enzymatic browning, and water loss (Baldwin and others 1995b); Guilbert and others 1996). In addition, such coatings can carry preservatives and antioxidants, reducing the amount required of these ingredients if used in combination. Some of the results showing the potential of edible coatings to extend the shelf life of fresh-cut fruit and vegetables have been summarized by Wong and others (1994), Baldwin and others (1995a, b), and Ahvenainen (1996). Most of the literature describes coating formulations that include some preservative and/or antioxidant. However, little is know on the effect of edible coating formulations without carrying any additive. Factors that have been proven to affect performance of edible coatings on postharvest life of whole fruits, such as solid content and lipid content, have not been studied in fresh-cut products. The study of these factors previous to the incorporation of additives, such as

antioxidants and/or antimicrobials, is very important to optimize coating performance on fresh-cut produce. Whey protein has excellent nutritional and functional properties and the ability to form films. Whey protein has been shown to produce transparent, bland, flexible, water-based edible films with excellent oxygen, aroma, and oil barrier properties at low relative humidity (McHugh and Krochta 1994; Miller and Krochta 1997; De Mulder-Johnson 1999). On the other hand, whey protein films provide a poor moisture barrier. However, incorporation of lipids reduces their water vapor permeability (McHugh and Krochta 1994; Shellhammer and Krochta 1997; Prez-Gago and Krochta 2000). Le Tien and others (2001) showed that whey protein coatings containing antibrowning agents are effective at preventing oxidative browning of apples and potatoes. The objective of this work was to study the effect of solid content and beeswax (BW) content of whey protein isolate (WPI)-BW edible composite coatings without antibrowning agents in the effectiveness in postponing enzymatic browning of fresh-cut apples.

Materials and Methods

Materials
Beeswax (Brillocera S.A., Valencia, Spain) was selected as the lipid phase of the WPI emulsion films. The BW was emulsified with 10% whey protein solution made from WPI (Davisco Foods Intl., Le Sueur, Minn., U.S.A.). Glycerol and stearic acid were from Panreac Quimica, S.A. (Barcelona, Spain).

Coating formulations
Two experiments were done to study the effect of solid content and BW content of WPIBW edible coatings on color change of fresh-cut apples. In the first experiment, 30% BW emulsions (dry solid basis) were prepared with 8%, 12%, 16%, and 20% total solid content. In the second experiment, WPIBW emulsion coatings at 16% total solid content were prepared containing 0%, 20%, 40%, and 60% BW (dry solid basis). Aqueous solutions of 10% (wt/wt) WPI were prepared and heated for 30 min in a 90 C water bath to denature the whey protein.
2003 Institute of Food Technologists
Further reproduction prohibited without permission

MS 20030173 Submitted 4/2/03, Revised 6/3/03, Accepted 7/1/03. Authors Perez-Gago, Alonso, and del Rio are with Dept. de Postcosecha, Inst. Valenciano de Investigaciones Agrarias, 46113 Moncada, Valencia, Spain. Authors Serra and Mateos are with Dept. de Biotecnologa, Inst. Tecnolgico Agroalimentario, Valencia, Spain. Direct inquiries to author Perez-Gago (E-mail: mbperez@ivia.es).

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The lipid was melted in the hot protein solution, and glycerol and stearic acid were added in the amount required to get the final film composition. The final solid content of the emulsion solutions was reached by adding water to reach the desired value. The proteinplasticizer ratio selected was 3 parts WPI to 1 part glycerol (dry solid basis), and this ratio was kept constant throughout the study. The BW-stearic acid ratio was also kept constant and consisted of 4 parts BW to 1 part fatty acid (dry solid basis). Samples were homogenized with a high-shear probe mixer (PolyTron, Model PT 2100, Kinematica AG Inc., Lucerne, Switzerland) for 4 min at 30000 rpm. After homogenization, the emulsions were placed in an ice bath to prevent further denaturation of the whey protein and to crystallize the lipid particles. The emulsions were degassed at room temperature with a vacuum pump and stored at 5 C until application. presented in a random order, and the judges had to rank each sample from highest to lowest degree of browning. The visual quality in each treatment based on general visual appearance (color plus effect of coating) was also determined on the basis of the following visual (hedonic) scale: 9 = excellent, just sliced; 7 = very good; 5 = good, limit of marketability; 3 = fair, limit of usability; and 1 = poor, inedible (Gorny and others 1999). A color photograph of samples rated with this scale was used by 3 judges to score the samples. Results for ranking based on color and visual quality were expressed as an average of the quality scores.

Statistical analyses

Fruit selection and preparation

Golden Delicious apples were purchased from a local wholesale produce distributor. The fruits were store briefly at 4 C until processing. The apples were cleaned, peeled, immersed in citric acid to avoid initial browning, cut into rectangular pieces (approximately 5.5 cm 3.5 cm 1.5 cm), and sanitized by immersion into a 100 ppm sodium hypochlorite solution for 2 min. After being drained for 3 min, the fresh-cut apple pieces were completely dipped into the coating solution for 5 min. After dipping, apple pieces were removed from the solution, drained of excess coating for 15 min, and air-dried with circulating air at 10 C. A maximum of 15 apples were processed at the same time to minimize excessive exposure to oxygen. A sharp stainless-steel knife was used throughout the process to reduce mechanical bruising and samples were processed in a temperature-controlled room at 10 1 C. Four apple pieces were placed in each polypropylene tray and 3 trays per treatment were prepared, which corresponded to 3 replicates. Half of the trays were heat-sealed with micro-perforated polypropylene films to ensure no modification of the surrounding atmosphere (35-m thickness) (35 Pa. 200, Amcor Flexibles, Barcelona, Spain), and the rest remained opened. Finally, samples were stored 1 d at 20 C or 7 d at 5 C.

Results and Discussion

Weight loss
Weight loss of coated and uncoated apple pieces was measured at the end of the storage periods for samples stored 1 d at 20 C or 7 d at 5 C. Samples remained either uncovered or covered with polypropylene films as a secondary package. Covering the samples with polypropylene films significantly reduced weight loss of apple pieces (P 0.05). However, coating application did not significantly improve moisture loss (P 0.05) (data not shown). Few differences were observed between coating treatments and results based on coating composition, and solid content varied with storage conditions. The lack of uniformity in the behavior of the treatments leads to no conclusions based on the effect of solid content or BW content on weight loss. These results contrast with the behavior of stand-alone films or when coatings are applied to whole fruits. Results found in the literature reported that water vapor permeability of WPI stand-alone films significantly decreased with increased lipid content (Shellhammer and Krochta 1997; Perez-Gago and Krochta 2000). Similarly, increasing the content of hydrophobic components in edible composite coatings also reduced weight loss of whole fresh fruits (Perez-Gago and others 2002). Few articles have reported the effect of coatings on weight loss of fresh-cut produce. McHugh and Senesi (2000) reported no effect of apple puree coatings, containing ascorbic or citric acid, on moisture loss from either unpacked or packed apple samples. When coatings contained vegetable oil and the samples were covered with polyvinyl films, moisture loss of apple pieces was not reduced after 44 h of storage at 5 C, and a slight reduction was observed after 68 h of storage. However, the effect of wrapping the apple pieces with apple puree stand-alone films containing 27% vegetable oil or 23% BW was significantly more effective in reducing moisture loss during storage for 12 d at 5 C. These results indicate the greater effect of stand-alone films in reducing weight loss on fresh-cut fruits compared with coating the fruit, which could be due to the inability of forming a uniform coating in the wet surface of the broken cellular material. The moisture of the fruit surface affects the degree of adhesion of the hydrophobic and hydrophilic coating materials (Baldwin and others 1995a, 1995b).

Weight loss determination

Weight loss was measured at the end of the storage period by weighting the trays containing 4 apple pieces. Measurements were in triplicates and the results were expressed as the percentage loss of initial weight.

Colorimetric measurements
Color measurements were made periodically with a Minolta Chromameter (Model CR-300, Ramsey, N.Y., U.S.A.) on 12 apple slices per treatment using the CIELAB color parameters, L*, a*, and b*. Each measurement was taken at 3 locations for each sample piece. A standard white calibration plate was used to calibrate the spectrophotometer. Results were also reported as brown index (BI), defined as brown color purity, which is usually used as an indicator of the browning extent in sugar-containing food products (Buera and others 1986). The following equation was used to determine BI:

where x is the cromaticity coordinate calculated from the XYZ tristimulus values according to the following formula x = X/(X + Y + Z).

Sensory evaluation

Color changes depending on solid content of WPI At the beginning and at the end of each experiment, apple fruit BW edible coatings
Increases in enzymatic browning that occurred in apple pieces
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slices were evaluated visually. Each treatment was coded and was
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Food Chemistry and Toxicology

Statistical analysis was performed using STATGRAPHICS Plus 2.1 (Manugistics, Inc., Rockville, Md., U.S.A.). Specific differences between means were determined by least significant difference (LSD). Specific differences for color obtained by sensory evaluation were determined by the Friedman test, which is recommended with ranking (UNE 87 023 1995). Significance of differences was defined at P 0.05.

Whey protein isolate-beeswax edible coatings . . .

during storage were accompanied by an increase in colorimetric a* and b* values and a decrease in lightness (white to black) and hue values. The BI was calculated and used as a good indicative of color change during storage. Figure 1 shows the BI of fresh-cut apples coated with WPIBW emulsion coatings having different solid content and stored either unpacked or packed 7 d at 5 C or 1 d at 20 C. Application of WPI BW emulsion coatings significantly reduced browning compared with control apples deep in sodium hypochlorite and citric acid solutions (P 0.05). At time zero, right after coating application, the control samples presented a higher BI than coated ones, which could indicate that the coating emulsions possibly had a bleaching effect. Solid content had a significant effect on browning under all storage conditions. As solid content of emulsion coatings increased, browning decreased. The results show 2 different groups inhibiting enzymatic browning of apple pieces with significant difference (P 0.05) between them: (1) WPIBW coatings containing 8% and 12% solid content, and (2) WPIBW coatings containing 16% and 20% solid content. Apples coated with 20% solid content coatings had significantly higher L* values and lower a* and b* values than samples coated with 8% and 12% solid content coatings. However, they did not show a fresh appearance. The surface showed a whitish appearance, which could be due to the higher level of BW in the coating formulations. This result indicates that, in the present conditions, 16% solid content would be more appropriate to reduce enzymatic browning. Le Tien and others (2001) studied the antioxidant capacity for film formulations based on calcium caseinate or whey protein concentrate with or without carboxymethyl cellulose sodium salt, and observed that whey proteins exhibit higher antioxidative power than calcium caseinate. These results were attributed to the coatings acting as an oxygen barrier and/or the antioxidative properties of whey protein films. The presence of several cysteine side residue amino acids in whey proteins might provide them with the ability to reduce enzymatic browning because cysteine inhibits polyphenol oxidasesmediated enzymatic reactions (Gorny and others 2002).

Food Chemistry and Toxicology

Color changes that depend on BW content of WPI BW edible coatings

To study the effect of BW content, emulsion coatings with 16% solid content were prepared because this solid content was not visually appreciated in the previous work. Similarly, to the previous results, coating application decreased enzymatic browning of apple pieces (P 0.05) (Figure 2). At time zero, the apple pieces that were coated with WPIBW emulsion coatings presented lower BI and higher L* values than samples coated with WPI solutions and uncoated samples, which indicates that BW gives a whitish appear-

Figure 1Effect of solid content of whey protein isolate-beeswax coating on the browning index of fresh-cut apple slices. Samples were stored at 5 C and 20 C, either uncovered or covered with polypropylene films. Vertical bar = pooled least significant difference at the 5% level. 2188

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Whey protein isolate-beeswax edible coatings . . .

ance that helps reduce visual browning in fresh-cut apples. Increasing BW content decreased BI under all storages conditions (P 0.05), the 60% BW coatings being the ones that decreased browning the most. However, the surface showed a whitish appearance that was not considered fresh looking by sensory evaluation. No significant differences in BI were found between apples coated with 20% BW and 40% BW content coatings (P 0.05). Low storage temperature and modified atmosphere packaging are extensively used to reduce rates of respiration and cut surface browning of many fresh-cut fruit and vegetable products (Gorny and others 1999). As expected, storage at 5 C and covered samples improved the quality of the fresh-cut apples by reducing enzymatic browning (Figure 1 and 2). At 5 C storage, covered samples had significantly lower BI than uncovered samples (P 0.05) because of the reduced exposure to atmospheric oxygen, whereas at 20 C, there were no differences between covered and uncovered samples, probably due to the faster rate of browning observed at this storage temperature. The level of BI after 7 d of storage at 5 C was

Figure 2Effect of beeswax content of whey protein isolate-beeswax coating on the browning index of fresh-cut apple slices. Samples were stored at 5 C and 20 C, either uncovered or covered with polypropylene films. Vertical bar = pooled least significant difference at the 5% level.
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not significantly different than samples stored 1 d at 20 C, which shows the effectiveness of reducing temperature in prolonging shelf life of fresh-cut fruits. the apple slices, based on color and general appearance. The control samples were rated as the highest browning value at all storage conditions, and they were considered inedible by the judges (Figure 3a). An increase of the solid content of the WPIBW coatings applied to the apple slices reduced the visual browning. The judges gave 16% and 20% solid content coated apple slides the lowest browning values, and no differences were found between these 2 treatments considering color ranking. However, the formulations with 20% solid content gave a whitish appearance to the fresh-cut apple slices, which made appearance unacceptable from the commercial point of view (Figure 3b). Samples coated with 8% and 12% solid content coatings were grouped together and ranked between the control and samples coated with higher solid content coatings (Figure 3a). These results are consistent with the ones found by the colorimeter, that showed significant differences

Sensory evaluation
Browning of fresh-cut fruits, such as apples, is the main inconvenience for their marketability. Color differences are generally assessed by the colorimeter; however, small differences observed instrumentally might not be observed visually. For this reason, the results found by the colorimeter were compared with browning appreciation by a sensory panel. The judges were asked to rank the apple slices from lowest (1) to highest (5) degree of browning and were allowed to group those samples that were considered to have similar color. In addition, the judges also evaluated the effect of coating application on the visual appearance of

Food Chemistry and Toxicology

Figure 3Effect of solid content of whey protein isolatebeeswax coating on visual browning (a) and sensory evaluation (b) of fresh-cut apple slices. Judges ranked the apple pieces from 5 (highest browning) to 1 (lowest browning) and were allowed to group those treatments that were considered similar. Visual quality (color plus effect of the coating) was based on a 9-point category scale. Means within each storage time with the same letter are not different (P < 0.05). 2190

Figure 4Effect of beeswax content of whey protein isolate-beeswax coating on visual browning (a) and sensory evaluation (b) of fresh-cut apple slices. Judges ranked the apple pieces from 5 (highest browning) to 1 (lowest browning) and were allowed to group those treatments that were considered similar. Visual quality (color plus effect of the coating) was based on a 9-point category scale. Means within each storage time with the same letter are not different (P < 0.05).
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between these 2 groups. However, the colorimeter was not able to detect the whitish appearance related to coating application with 20% solid content coatings. In general, when samples were evaluated for general visual appearance, values ranked between fair (limit of usability) and poor (inedible), which indicated the need of adding antibrowning agents to the coatings formulations to improve the quality (Figure 3b). Samples coated with WPIBW coatings at 16% solid content and having different BW content were also ranked with lower browning values than the uncoated samples (Figure 4a). In general, the apple slices coated with 60% BW content coatings were ranked with the lowest browning value, which confirms the results obtained by the colorimeter. However, these samples were also characterized by a whitish appearance, probably due to the high BW content of the sample, which made appearance unacceptable from the commercial point of view (Figure 4b). This whitish effect was reduced as BW content was reduced. Apple slices coated with 20% BW content were rated as good (natural apple appearance and limit of marketability) by the judges, and the different scores were only influenced by the storage conditions. In general, covered samples were scored higher than uncovered samples because of the higher exposure to oxygen of uncovered samples. In addition, samples stored at 20 C for 1 d reached similar evaluations to samples stored at 5 C for 7 d, which shows the benefit of using low-temperature storage to help reduce browning. appropriate to reduce browning. Nevertheless, these coatings should be improved with the addition of antibrowning agents, so further research is needed for this purpose.

Acknowledgments
This work was funded by the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria through project CAL01-021-C2-1.

References
Ahvenainen R. 1996. New approaches in improving the shelf life of minimally processed fruit and vegetables. Trends Food Sci Technol 7:17987. Baldwin EA, Nisperos-Carriedo MO, Baker RA. 1995a. Edible coatings for lightly processed fruits and vegetables. HortSci 30:358. Baldwin EA, Nisperos-Carriedo MO, Baker RA. 1995b. Use of edible coatings to preserve quality of lightly (and slightly) processed products. Critical Rev Food Sci Nutr 35:50924. Buera MP, Lozano RD, Petriella C. 1986. Definition of color in the non enzymatic browning process. Die Farbe 32:31822. De Mulder-Johnson C. 1999. Thermal analysis of, and oil migration through films from whey protein isolate [DPhil dissertation]. Davis, Calif.: Univ. of California. Available from: Shields Library Microcopy Collection LD781.D5j 1999 D468. p 3357. Gorny JR, Hess-Pierce B, Kader AA. 1999. Quality changes in fresh-cut and nectarine slices as affected by cultivar, storage atmosphere and chemical treatments. J Food Sci 64:42932. Gorny JR, Hess-Pierce B, Cifuentes RA, Kader AA. 2002. Quality changes in freshcut pear slices as affected by control atmosphere and chemical preservatives. Postharv Biol Technol 24:2718. Guilbert S. 1986. Technology and application of edible protective films. In: Matholouthi M, editor. Food packaging and preservation: theory and practice. London: Elsevier Applied Science Publishing Co. p 37193. Le Tien C, Vachon C, Mateescu MA, Lacroix M. 2001. Milk protein coatings prevent oxidative browning of apples and potatoes. J Food Sci 66:5126. McHugh TH, Krochta JM. 1994. Water vapor permeability properties of whey protein-lipid emulsion films. J Am Oil Chem Soc 71:30712. McHugh TH, Senesi E. 2000. Apple wraps: a novel method to improve the quality and extend the shelf life of fresh-cut apples. J Food Sci 65:4805. Miller S, Krochta JM. 1997. Oxygen and aroma barrier properties of edible films: a review. Trends Food Sci Technol 8:22837. Prez-Gago MB, Krochta JM. 2000. Drying temperature effect on water vapor permeability and mechanical properties of whey protein-lipid emulsion films. J Agric Food Chem 48:268792. Prez-Gago MB, Rojas C, del Ro MA. 2002. Effect of type and amount of edible hydroxypropyl methylcellulose-lipid composite coatings used to protect postharvest quality of mandarins cv. Fortune. J Food Sci 67:290310. Shellhammer TH, Krochta JM. 1997. Whey protein emulsion film performance: effect of lipid type and amount. J Food Sci 62:3904. UNE 87 023. 1995. Normas espaolas para el ensayo de clasificacin por ordenacin. In: Associatin Espaola de Normalizacin y Certificacin, AENOR. Anlisis Sensorial. Tomo 1: Alimentacin. Spain: AENOR. p 15166. Wong DW, Tillin SJ, Hudson JS, Pavlath AE. 1994. Gas exchange in cut apples with bilayer coatings. J Agric Food Chem 42:2278-85.

Conclusions

HE USE OF WPIBW COATINGS REDUCED ENZYMATIC BROWNING

of Golden Delicious apples significantly, probably due to the antioxidant effect of amino acids, such as cysteine. Changes in solid content and BW content of the emulsion coatings affected the degree of browning of the apple slices. An increase of coating solid content and BW content reduced browning significantly. However, emulsion coatings with 20% solid content gave a whitish appearance that made the apples unacceptable. Similarly, high levels of BW content in the WPIBW emulsion coatings reduced browning but increased the whitish appearance of the samples. Results observed by sensory evaluation indicated that WPIBW emulsion coatings at 16% solid content and with 20% BW content are the most

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