candelilla wax coating containing potassium sorbate on microbiological and physicochemical attributes of pears

candelilla wax coating containing potassium sorbate on microbiological and physicochemical attributes of pears

Scientia Horticulturae 218 (2017) 326–333 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate...

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Scientia Horticulturae 218 (2017) 326–333

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Effect of carboxymethylcellulose/candelilla wax coating containing potassium sorbate on microbiological and physicochemical attributes of pears ˛ c , Barbara Baraniak a Dariusz Kowalczyk a,∗ , Monika Kordowska-Wiater b , Emil Zieba a

Department of Biochemistry and Food Chemistry, University of Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland c Laboratory of Confocal and Electron Microscopy, Centre for Interdisciplinary Research, John Paul II Catholic University of Lublin, Al. Kra´snicka 102, 20-718 Lublin, Poland b

a r t i c l e

i n f o

Article history: Received 2 January 2017 Received in revised form 17 February 2017 Accepted 25 February 2017 Keywords: Pears Coating CMC Potassium sorbate Fungi

a b s t r a c t The main objective of this study was to assess the efficacy of a coating composed of carboxymethylcellulose (CMC), candelilla wax and potassium sorbate (KS) as a post-cold-storage treatment to prevent fungal infections in pears stored under simulated retail display conditions. Moreover, the effect of coating on the physiology and biochemistry of pears was investigated. The coating was very effective against Botrytis cinerea and Monilinia fructigena, while Rhizopus nigricans was the most resistant to KS. The KS-free coating also delayed the fungal growth rate, probably due to modification of the gaseous atmosphere within the fruit tissues. Coated pears showed slower ripening than the uncoated samples, as indicated by unaffected green skin color and inhibited loss of firmness. Unfortunately, coating induced anaerobic respiration and the symptoms of superficial scald in pears. Overall, the results showed that KS can be added into a coating formulation to control fungal growth; however, CMC-based emulsion is not a suitable carrier for KS, when coating is intended to be applied to pears exposed to postharvest cold storage. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Pears can be harvested almost ripe and sold for immediate consumption for fresh markets. New storage techniques, however, have made pears accessible for consumers even 6–8 months after harvest. Rapid cooling and cold storage (−1 to 0 ◦ C) at high humidity, at least 90–95%, is the most important tool for preserving the quality of pears. Controlled atmosphere (CA) storage can extend shelf life, using O2 and CO2 concentrations ranging in 1–2% and 0–2%, respectively, depending on pear cultivar (Kader, 2007). After the refrigeration it is best to let the fruit ripen at ∼25 ◦ C. Since pears are very perishable once they are ripe, in recent years, some authors have begun to take an interest in the protection of quality of pears using the edible coatings as a conservation technique (Amarante et al. 2001; Hasan and Nicolai, 2014; Hussain et al., 2010; Moraes et al., 2012). The results showed that coating has potential to extend the shelf life and maintain quality of pears, since give the same

∗ Corresponding author. E-mail address: [email protected] (D. Kowalczyk). http://dx.doi.org/10.1016/j.scienta.2017.02.040 0304-4238/© 2017 Elsevier B.V. All rights reserved.

effect as modified atmosphere storage with respect to modifying the internal gas composition (Dhall, 2013). Pears should be handled very carefully during and after harvest since wounds and bruises act as infection sites for fungi. The great advantage of edible coatings is that their protective function can be easily improved by the incorporation of additives. Enrichment of coating with antimicrobial agents results in the creation of active packaging systems, where the gradual migration of the active substance from the carrier matrix to the surface layers of product provides high efficiency. In this respect, an antimicrobial coating could reduce fungus populations, improve the handling characteristics and, consequently, limit the amount of postharvest decay in fruits. Sorbic acid (2,4-hexadienoic acid) and its salts are wellknown preservatives suitable for many applications. They exhibit high processing and storage stability, are considered generally recognized as safe, show effectiveness against numerous spoilage causing yeasts, molds and bacteria, and their typical use levels normally do not alert the taste or odor of a food product (Theron and Rykers, 2011). It has been demonstrated that dipping of pears in 2% potassium sorbate (KS) solution was effective method to control postharvest diseases at cold storage or during marketing at room temperature. Furthermore, KS treatment significantly reduced the

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loss in fruit weight and firmness, kept the content of chlorophyll, decreased juice acidity, and increased total sugar content (El-Eryan and El-Metwally, 2014). Mehyar et al. (2014) showed that incorporation of KS into edible coatings reduced its quick depletion from the surface of the product and consequently prolonged the antifungal protection. Previous study has demonstrated that the CMC/candelilla wax (CnW) film activated with KS can be recommended for use as easily washable coating for the treatment of food surfaces, the typical point of entry for pathogens and a likely location of maximum microbial contamination (Kowalczyk et al., 2015). To˘grul and Arslan (2004) showed that coating of pears with CMC-based emulsion extended the shelf-lives of fruits for up to 16 days. Also, Hussain et al. (2010) found that CMC coating was effective in giving 6 and 2 day extension in shelf life of pear following 45 and 60 days of refrigeration, respectively. Considering these results, the aim of this study was to assess the efficacy of a coating composed of CMC, CnW, and KS as a post-cold-storage treatment to prevent fungal infections in pears stored under simulated retail display conditions. Moreover, effect of coating on the physiology and biochemistry of pears was investigated. 2. Materials and methods 2.1. Materials Pears (Pyrus communis L., cv Lukasówka) were purchased from a local market in Lublin, Poland, 24 h before treatments. Until the moment of purchase, fruits were cold-stored for 2 months in a commercial chamber (−1 ◦ C, 95% RH) at normal atmosphere. Fruits selection criteria were homogeneous size, absence of skin damage, physiological maturity, and intense green color. The coatings were made from sodium carboxymethylcellulose 30 GA (Dow-Wolff Cellulosics, Germany), candelilla wax SP-75 (Strahl & Pitsch Inc, USA), sorbitol, potassium sorbate (KS), and Tween 40 (Sigma-Aldrich). 2.2. Coating formulations Coating-forming solutions (CFSs) were produced according to the procedure outlined by Kowalczyk et al. (2015). Aqueous CFS comprised of CMC (5% w/w), sorbitol (3% w/w), CnW (0.5% w/w), Tween 40 (0.35% w/w), and KS (3%w/w). CFS without KS (denoted by CMC-CnW) was also prepared. Uncoated fruits were used as the control. 2.3. Light microscopy Examination of the thickness of coatings on surface of pears was performed using an light inverted microscope (Olympus IX70, Japan). To best visualize the coatings, CFSs were colored with Ponceau 4R (Libella, Poland). Microscopic slides (thickness ∼ = 20 ␮m) were obtained using a cryostat (Hyrax C25, Zeiss, Germany) at −30 ◦ C. The cross-sections were made from three fruits. The thickness of coating was determined at four different locations and measured from microscopic images by the software AxioVision 4.8.1 (Carl Zeiss). 2.4. Effect on fungal infection in fruits The phytopathogenic fungi: Botrytis cinerea Pers. ex Nocca & Balb , Monilinia fructigena (Aderhold et Ruhland) Honey , Alternaria alternata (Fr.) Keissler and Rhizopus nigricans were maintained on potato dextrose agar (BTL, Poland) slants at 4 ◦ C. Spores suspensions were prepared by adding sterile saline to the cultures. The suspensions were diluted to a final concentration of 105 spores/ml. Pears were washed and disinfected with ethanol solution (70% v/v) for 30 s. Then, fruits were coated by dipping in CFS for 30 s and

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allowed to dry under aseptic conditions in a laminar flow cabinet for 2 h at room temperature. Fruits were first wounded (3 mm deep and 4 mm wide) with a sterile needle in opposite sides. 20 ␮L of the spore suspension was dispensed into the each hole and 20 min later the wounds were covered with the CFS (except for uncoated samples). The control and coated fruits were placed in duplicates in sterile plastic vessels, loosely closed with lids so as to allow air access, and stored at 22 ◦ C and ∼50% RH. Rotting zone diameters surrounding the wounds were measured and the means were used to calculate the inhibition of rot surface lesions (%). 2.5. Effect on physicochemical parameters of fruits Pears were washed with tap water, air-dried and disinfected with a solution of sodium hypochlorite (0.1 g L−1 ) for 2 min. Subsequently, fruits were re-washed, re-dried, dipped in the CFSs for 30 s, and air-dried for 2 h. Physicochemical analyses of samples were performed at least in triplicate on the 0, 3rd, 6th and 9th day of storage under simulated retail display conditions (22 ± 1 ◦ C, 50 ± 5% RH). Lots of 10 fruits per treatment were used to measure weight loss (WL). The results were expressed as the percentage loss of initial weight. Respiration rate of fruits was measured in a static system. Three fruits (∼400 g) were sealed in a container (3.6 L) and the concentrations of CO2 and O2 in the atmosphere (22 ◦ C, ∼50% RH) were measured with an infrared gas analyzer (AirTECH duo, Gazex, Poland) over 30 min. Respiration rates in terms of CO2 accumulation (RCO2 ) and O2 consumption (RO2 ), both expressed as mL kg−1 h−1 , were determined according to Eq. (1) and Eq. (2), respectively (Fonseca et al., 2002):



RCO2 = and RO2 =



pCO2 Vv

100 × W × t



(1)



pO2 Vv

100 × W × t

(2)

Where pCO2 is an increase in partial pressure of carbon dioxide (%), pO2 is a decrease in partial pressure of oxygen (%), Vv is void volume (ml), W is weight of the sample (kg), t is sampling period (h). Moisture content (MC) determination was done by drying the samples (∼20 g) at 105 ◦ C until the weight became constant. Determination of active acidity was performed using a pH meter (Elmetron CPC 401, Poland) according to ISO 1842 (1991). Determination of total acidity (TA) was performed according to ISO 750:1998 (potentiometric titration) and the results were given in terms of malic acid. The content of reducing sugars (SC) was determined using Lane–Eynon method as described by Ranganna (2000). Firmness was measured on the opposite sides of ten fruits with a TA-XT2i texture analyzer (Stable Micro Systems, UK) with a load cell of 50 kg and a 2 mm diameter cylindrical probe. Test speed and penetration were 1 mm/s and 5 mm, respectively. Skin color values (CIE L*a*b) were measured using a colorimeter (NH310, 3nh, China) with a D65/8◦ /SCE lighting system. Three replicates of 5 fruits were used per treatment. 2.6. Sensory analysis Fruits were evaluated by a trained and experienced panel qualified as experts according to PN-EN ISO 8586:2014–03. Sensory parameters were assessed on a quantitative descriptive ranking method with 5 point scale (1 = worst, 5 = best). The assessed attributes included overall appearance, gloss, aroma, taste, juiciness, and firmness.

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2.7. Statistical analysis All data were expressed as mean ± standard deviation (n ≥ 3). Differences among data mean values were tested for statistical significance at the p < 0.05 level using analysis of variance (STATISTICA 6.0, StatSoft Inc., Tulsa, USA) and the Fisher’s test.

3. Results and discussion 3.1. Fungal growth on the infected fruits Table 1 shows mean values for rot lesions caused by the phytopathogenic fungi on the control and coated pears stored at room temperature. Fruits that were not coated exhibited typical signs of infection such as browning, softening, or necrosis on the 2nd (R. nigricans) or the 3rd/4rd day (A. alternata, B. cinerea, M. fructigena) of storage. The slowest development of infection was observed in pears inoculated with A. alternata. The application of coatings reduced rot lesions caused by the fungi; however, in the case of A. alternate and B. cinerea the longer incubation periods were needed to detect a statistically significant difference. In general, the coating containing KS showed more potent antifungal activities compared to antimicrobial-free coating formulation (Table 1, Fig. 1). The inhibition of A. alternata and B. cinerea infections by the coatings was enhanced over time. The opposite tendency was observed for the fast-growing fungi (R. nigricans and M. fructigena). These results confirm that the efficiency of antimicrobial packaging material depends on the fungal growth rate (Kowalczyk et al., 2015). R. nigricans was the most resistant, whereas M. fructigena and B. cinerea were found the most susceptible to KS supported in the coating (the inhibitions of rot lesions were 22.83-48.84, 67.16-100.00%, and 43.75-70.25%, respectively). These observations are in agreement with previous in vitro results obtained by the disc diffusion method (Kowalczyk et al., 2015). In contrast to in vitro study (Kowalczyk et al., 2015), that has demonstrated that KS-free films do not show any appreciable inhibitory effect on the tested fungi, in this study the coating without antimicrobial compound delayed the growth rate of fungi. This result is likely to be related to modification of the gaseous atmosphere within the fruit tissues. As can be seen from Table 2, the coated fruits exhibited increased CO2 production and reduced O2 consumption. Since most fungi are obligate aerobes, requiring oxygen to survive, it could be speculated that the gas barrier properties of the coating prevented oxygen access to the infection sites, giving inhibitory effect on the germination and mycelium growth of the fungi. Additionally, at high levels of CO2 , most pathogens are suppressed by reduction in the rate of various metabolic functions (Barkai-Golan, 1990). Antifungal performance of KS supported in edible coatings has been also verified by other researchers. Park et al. (2005) showed that hydroxypropylmethylcellulose (HPMC) coating containing 0.3% of KS reduced the visible mold incidence in strawberries inoculated with Rhizopus sp. or Cladosporium sp. Moreover, the coating reduced total aerobic count and coliforms, however, its antimicrobial effectiveness was lower than that of chitosan/KS-based. In the analysis of plums inoculated with Monilinia fructicola, Karaca et al. (2014) found that KS, used at 1.0% in HPMC-based coating, was the most effective antimicrobial agent in reduction postharvest brown rot. Similarly, Mari et al. (2004) found that KS was the only antimicrobial substance, that used in form of 1.5% aqueous solution significantly reduced infection rates on the stone fruits wounded and inoculated with Monilinia laxa and Rhizopus stolonifer. The average level of natural yeasts and molds on apples, tomatoes, and cucumbers was reduced by KS incorporated into coating formulations based on guar gum, pea starch, and potato starch (Mehyar

et al., 2014). The greatest inhibition of fungi was obtained on apples and tomatoes, whereas the surface roughness of cucumbers reduced effectiveness of KS. High negative correlation existed between KS surface concentration and the yeast and mold counts. The coatings enhanced retention of KS on the fruit surface, which led to the higher antifungal effectiveness compared with spraying and dipping of the food products with aqueous solution of KS (Mehyar et al., 2014). Sayanjali et al. (2011) found that pistachios coated with CMC-based films containing various concentrations of KS (0.25–1 g/100 mL film solution) showed no growth of mycotoxigenic Aspergillus species. Interestingly, the coating without KS also inhibited mold growth. As already mentioned, a similar effect was observed in our study (Table 1, Fig. 1).

3.2. Physicochemical attributes of pears 3.2.1. Weight loss and respiration rate The WL of pears, both control and coated, increased during simulated retail display (Table 2). Contrary to expectations, WL of the coated fruits was significantly higher than those of the uncoated. These results differ from previously published studies (Hussain et al., 2010; To˘grul and Arslan, 2004). The highest WL was observed for pears covered with KS-added coating; i.e. on the 9th day the WL of fruits was nearly double that of control. Since the coating did not affect the MC of pears (Table 2), it can be concluded that the increased WL was mainly due to an increased respiration rate of fruits rather than transpiration. A strong correlation (r = 0.71) existed between WL and CO2 production, but no linear relationship was found between WL and MC. Aerobic respiration is the chemical process by which fruits convert sugars and O2 into CO2 , water, and heat. In the absence of O2 (in anaerobic respiration), CO2 is produced without the concomitant consumption of equal amounts of oxygen. As can be seen from Table 2, the coated pears produced more CO2 and simultaneously consumed less O2 in comparison with the uncoated fruits. Moreover, the production of CO2 by coated fruits increased by increasing storage time, whereas the respiration rate of control pears was time-independent. This discrepancy could be attributed to anaerobic respiration of the coated pears. Previous studies have also reported that coating could induce undesirable fermentation reactions and, consequently, physiological disorder of fruits (Park et al., 1994; Baldwin et al., 1999). It is associated with the modification of internal atmosphere of plant tissues (too low O2 and high CO2 concentrations) as a result of too high gas barrier properties of coating material. Climacterictype fruits, to which European pears belong, usually do not tolerate high-performance gas coatings, especially if these are applied at a preclimacteric state (Bai and Plotto, 2012). Both CMC and CnW represent the materials witch relatively good oxygen barrier properties (Donhowe and Fennema, 1993; Shin et al., 2014) and their combined use likely resulted in an inordinately increase in the pear skin resistance to gas diffusion. Apart from the type of filmogenic agent, the barrier properties of the films are also strongly affected by their thickness. Thick coatings can extremely modify fruit internal atmosphere (Amarante et al., 2001; Maqbool et al., 2011). Nevertheless, the emulsion coatings formed on the fruits were relatively thin; their average thickness was 19.00 and 21.42 ␮m for CMC-CnW and CMC-CnW-KS formulations, respectively (Fig. 2), which is only double that of the cuticlar layer on pears (Konarska, 2013). Therefore, it can be ruled out that the induction of fermentative metabolism was caused by the excessive coating thickness. In previous studies there were no sign of anaerobic respiration in fruits covered by the polymer-based layers of about 156–692 ␮m in thickness (RojasGraü et al., 2007; Valenzuela et al., 2015). In general, the pears coated with CMC-CnW and CMC-CnW-KS exhibited similar rate of anaerobic respiration. Only on the 6th day the fruits coated with

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Table 1 Efficacy of the coatings in reducing rot lesions caused by fungi on infected pears. Fungi

A. alternata

B. cinerea

M. fructigena

R. nigricans

Storage time (day)

4 5 6 7 8 4 5 6 7 8 4 5 6 7 8 4 5 6 7 8

Diameter of rot (mm)

Inhibition of lesion (%)

Control

CMC-CnW

CMC-CnW-KS

CMC-CnW

CMC-CnW-KS

8.00 ± 2.45a 11.00 ± 4.65a 22.50 ± 8.42bcd 28.00 ± 14.70de 41.75 ± 5.78f 12.00 ± 16.08a 35.5 ± 9.50bcd 49.25 ± 12.07cde 55.00 ± 7.79de 60.50 ± 7.05e 17.25 ± 3.60cde 38.25 ± 5.90g 55.00 ± 4.40h 73.50 ± 7.50i 83.75± 2.99i 53.75 ± 12.50cde 73.75 ± 14.90g 79.00 ± 4.08gh 89.50 ± 6.14h 92.00 ± 4.97h

7.75 ± 2.22a 9.00 ± 0.82a 13.25 ± 3.40ab 24.75 ± 15.04cd 37.50 ± 15.50ef 8.00 ± 9.60a 13.75 ± 17.01ab 20.00 ± 23.45ab 22.50 ± 21.01ab 28.25 ± 16.60abc 5.25 ± 6.40ab 14.75 ± 11.60bc 25.75 ± 10.11def 30.75 ± 9.25fg 39.25 ± 3.60g 27.50 ± 11.90a 41.50 ± 14.57bc 58.25 ± 10.43ef 72.50 ± 5.07g 83.50 ± 2.65gh

8.00 ± 1.41a 9.00 ± 0.82a 10.5 ± 0.57a 12.25 ± 0.96ab 15.50 ± 1.29abc 6.75 ± 7.80a 13.25 ± 15.35ab 17.50 ± 20.21ab 17.75 ± 20.50ab 18.00 ± 20.80ab 0.00 ± 0.00a 8.50 ± 5.80abc 16.50 ± 1.73cd 25.25 ± 10.11def 27.50 ± 14.10ef 27.50 ±6.45a 39.75 ± 7.80ab 43.75 ± 9.18bcd 55.50 ± 8.43de 71.00 ± 10.42fg

3.13 18.18 30.00 18.75 34.13 33.33 61.27 59.39 59.09 53.31 69.57 61.44 53.18 58.16 53.13 48.84 43.73 26.27 18.99 9.24

0.00 18.18 53.33 56.25 62.87 43.75 62.68 64.47 67.73 70.25 100.00 77.78 70.00 65.65 67.16 48.84 46.10 44.62 37.99 22.83

Values with the same superscript letters within a fungus are not significantly different (p < 0.05).

Fig. 1. Rot lesions caused by A. alternata (a), B. cinerea (b), M. fructigena (c), and R. nigricans (d) on infected control and coated pears observed on the 8th day of storage under simulated retail display conditions.

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Table 2 Weight loss (WL), respiration rate (CO2 production and O2 consumption), moisture content (MC), titratable acidity (TA), pH, sugar content (SC), firmness and color parameters (L*a*b*) of control and coated pears during simulated commercial storage.

WL (%)

CO2 production (ml kg−1 h−1 )

O2 consumption (ml kg−1 h−1 )

MC (%)

TA (%)

pH

SC (%)

Firmness (N)

L*

a*

b*

a–h

Storage time (days)

Control

CMC-CnW

CMC-CnW-KS

0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9

– 1.14 ± 0.09a 2.22 ± 0.16bc 3.22 ± 0.21de 17.25 ± 0.21ab 18.84 ± 0.66bc 17.32 ± 0.83ab 17.74 ± 0.24ab 50.33 ± 6.61bc 46.37 ± 12.21bc 54.85 ± 13.56c 45.43 ± 6.47bc 84.77 ± 0.06a 85.01 ± 0.06ab 85.17 ± 0.04abc 85.54 ± 0.97abc 0.25 ± 0.01e 0.25 ± 0.00e 0.21 ± 0.01d 0.20 ± 0.00cd 4.55 ± 0.01ab 4.50 ± 0.01ab 4.48 ± 0.01a 4.51 ± 0.01ab 4.25 ± 0.11ab 4.25 ± 0.07ab 4.26 ± 0.07abc 4.78 ± 0.39d 7.58 ± 1.74bc 3.60 ± 0.78a 2.97 ± 0.65a 2.51 ± 0.55a 58.81± 2.44c 62.46 ± 2.96d 65.28 ± 1.48d 65.00 ± 1.98d −5.02 ± 0.98a 0.77 ± 2.07b 4.61 ± 1.78c 6.14 ± 1.62c 45.43 ± 1.56ab 46.79 ± 1.39abc 49.03 ± 1.67c 48.26 ± 1.75c

– 1.64 ± 0.42ab 3.32 ± 0.65e 4.86 ± 0.89f 21.12 ± 0.14cd 22.90 ± 0.47de 25.77 ± 1.32f 34.65 ± 0.16h 46.91 ± 9.77bc 19.20 ± 2.55a 22.23 ± 15.25a 23.83 ± 5.39a 85.07 ± 0.18abc 85.73 ± 0.12bc 85.74 ± 0.13bc 84.84 ± 0.06ab 0.17 ± 0.00b 0.14 ± 0.01a 0.15 ± 0.00a 0.14 ± 0.01a 4.57 ± 0.01b 4.88± 0.04d 4.89 ± 0.01d 4.89 ± 0.02d 4.28 ± 0.41abc 4.25 ± 0.04ab 3.95 ± 0.03ab 3.83 ± 0.31a 8.40 ± 1.15bcd 8.71 ± 1.54cd 7.14 ± 0.83b 8.02 ± 0.92bc 56.82± 3.09abc 57.50 ± 2.88bc 57.31 ± 2.79abc 54.56 ± 2.34a −5.91 ± 2.33a −5.81 ± 1.37a −5.73 ± 1.53a −3.99 ± 3.88a 47.36 ± 1.80abc 48.04 ± 1.69bc 48.68 ± 1.50c 47.66 ± 2.505bc

– 2.59 ± 0.88cd 4.34 ± 0.44f 6.37 ± 0.60g 22.23 ± 0.78de 24.06 ± 0.71ef 32.09 ± 2.52g 35.13 ± 0.77h 47.00 ± 5.66bc 22.50 ± 3.54a 19.06 ± 0.18a 22.36 ± 13.85a 85.20 ± 0.09abc 84.75 ± 0.08a 85.30 ± 0.09abc 85.26 ± 0.96abc 0.18 ± 0.00bc 0.15 ± 0.01a 0.15 ± 0.01a 0.15 ± 0.00a 4.70 ± 0.04c 4.86 ± 0.01d 5.11 ± 0.01e 5.12 ± 0.01e 4.42 ± 0.31bc 4.08 ± 0.09ab 3.82 ± 0.06a 3.88 ± 0.15a 7.89 ± 0.72bc 7.94 ± 1.26bc 8.55 ± 1.64cd 8.10 ± 1.60bc 55.78 ± 2.33ab 57.56 ± 1.70bc 57.41 ± 1.61abc 54.75 ± 3.21ab −5.65 ± 0.82a −5.57 ± 1.07a −5.63 ± 0.92a −4.01 ± 7.34a 47.52 ± 0.81abc 47.55 ± 1.23bc 48.05 ± 1.08bc 45.01 ± 5.03a

Values within a parameter with different superscript letters are significantly different (p < 0.05).

addition of preservative produced significantly more CO2 , which could explain their higher WL (Table 2). 3.2.2. Sugar content, titratable acidity and pH Acids and sugars are two main components that greatly influence flavor properties of fruits. Generally, during the ripening process organic acids as a respiratory substrate are degraded, while the sugar content increases. As shown in Table 2, this dependence was observed only for uncoated pears. In the case of the coated fruits, both SC and TA decreased over time. The reduction in SC may be explained by the fact that coating promoted anaerobic fermentation, i.e. sugars were converted into ethanol and CO2 . Interestingly, the coated pears, regardless of storage time, were characterized by lower TA compared to the control fruits. The reason for this is not clear but it may have something to do with presence of coating components. To˘grul and Arslan (2004) have observed that the loss in TA of coated pears increased with increasing content of CMC in the coating formulation. The authors suggested that CMC can interfere with the titration. According to El-Eryan and El-Metwally (2014), the application of KS also produces a lower acidity in pears. During storage, there was a significant increase in the pH of coated fruits and as a consequence the coated samples differed significantly (p < 0.05) from the control (Table 2). This result is likely

to be related to more intensive loss of TA in the coated fruits as compared to uncoated samples. In general, pears coated with CMCCnW-KS exhibited the highest pH. A possible explanation for this might be that KS had an alkalizing effect on the fruit homogenates. The previous study have shown that the increase in KS concentration led to gradual increase in pH of film-forming solutions based on the different biopolymers, including CMC (Kowalczyk et al., 2015). It is because KS is the reaction product of a weak acid and a strong base. 3.2.3. Firmness and color The firmness of uncoated pears decreased from 7.58 to 2.51 N after 9 days of storage (Table 2). In contrast, the coated samples, regardless of the coating composition, kept their initial firmness values through the whole storage period. It suggests that the coating treatment effectively prevented the ripening of pears apparently due to indirect inhibition of the catalytic activity of carbohydrate-degrading enzymes. In pears a key role in conversion of cell wall pectic polysaccharides to water-soluble forms is played by polygalacturonase (PG). Activity of PG increases in parallel with respiration and ethylene production, ultimately reaching 10–12 times the initial level (Ahmed and Labavitch, 1980), which could explain the observed dramatic drop in the firmness of control

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Fig. 2. The thickness of the coatings on the surface of pears determined from the micrographs (a). Light micrographs (x 400 magnification) showing cross-sections of uncoated pear (b), pear coated with CMC-CnW formulation (c), and pear coated with CMC-CnW-KS formulation (d).

pears (Table 2). Climacteric fruits are characterized by an extraordinary increment in ethylene production which accompanies the respiratory peak during ripening (Chaves and Mello-Farias, 2006). Absence of O2 inhibits the biosynthesis of ethylene (Thompson, 2014) and, consequently, alterations in the fruit texture. Consequently, it has been demonstrated that CA and modified active packaging (MAP) storage (i.e., high CO2 and low O2 ) considerably delayed the softening rate of pears (Ke et al.,1990; Geeson et al., 1991; Wang and Sugar, 2013). Edible coatings influence fruit quality by the same mechanisms as modified atmosphere storage, i.e. by restriction of O2 entering, thus modification of internal gas composition and reduction of oxidative metabolism (Dhall, 2013). Delayed softening of coated pears has also been observed by Moraes et al. (2012) for alginate covered fruits. Uncoated pears showed faster ripening than the coated samples, which was also indicated by the measurement of changes in skin color. As can be seen in Table 2, the coated fruits, unlike that of coated ones, showed an increase in L* (lightness) and lost in a* (greenness) parameters during storage. The change in the color of skin in ripening pears results from degradation of chlorophyll and accumulation of carotenes. The coating treatment preserved green color of pears (Table 2, Fig. 3) most likely by reducing climacteric rise in respiration. It has been shown that modified atmosphere could decrease chlorophyllase activity (Guevara et al., 2001). Inhibition of chlorophyll breakdown as a result of coating application has also been observed in previous studies for various horticultural products, including pears (Bai and Plotto, 2012; Hussain et al., 2010). After chlorophyll degradation, yellow carotenoid pigments become visible (Fig. 3). In this study uncoated pears stored at room temperature reached full yellow color within 6 days. Neverthe-

Fig. 3. Overall appearance of pears observed on the 9th day of storage under simulated retail display conditions. From left to right: control, CMC-CnW, CMC-CnW-KS.

less, the color measurement did not reveal significant differences between +b* values (yellowness) of the control and coated samples (Table 2). At the end of storage, the brown patches occurred on the skin of coated fruits (Fig. 3), which resulted in large spread (a high standard deviation) of a* and b* values. The cross-sections of fruits revealed that the discoloration was only skin deep. These symptoms indicate a physiological storage disorder called superficial scald. Its development is an expression of necrosis of the hypodermal cortical tissue. Based on the fact that superficial scald can be induced by different etiological factors, including CO2 injury (Lurie and Watkins, 2012), the observed symptoms were most likely the result of accumulation of CO2 (up to a critical level) in the internal atmosphere of coated fruits (Table 2). High CO2 level may contribute to the production of reactive oxygen species (Larrigaudière et al., 2004), which are highly active and may indiscriminately cause lipid peroxidation, resulting in the further release of free radicals. Oxidative stress is the generally accepted cause of super-

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Fig. 4. Scores for sensory analysis of control and coated pears on the 0 (a), 3rd (b), 6th (c), and 9th (d) day of storage under simulated retail display conditions.

ficial scald, although other factors are clearly involved (Whitaker et al., 2009). It should be noted that the pears used in this study, prior to coating treatment, were exposed to the postharvest cold storage. The superficial scald usually develops after fruit have been removed from cold storage (Lurie and Watkins, 2012). The obtained results seem to be consistent with other research which found that ® Semperfresh coating increased scald incidence in early harvested apples upon ripening in 20 ◦ C following several months storage at 0 ◦ C (Kerbel et al., 1989). It is interesting to note that the opposite effect can also occur, i.e. emulsion coatings alleviated scald symptoms in pears and apples (Lau and Meheriuk 1994; Ju et al., 2001).

parameters of coated fruits were judged as worse than uncoated ones. The scores of sensory attributes of samples coated without and with the addition of KS were relatively the same. Data from sensory evaluation indicated serve symptoms of anaerobic respiration and CO2 injury in the coated fruit. The main sensory attribute damaged by the coating treatment was taste (scores around 1) (Fig. 4), however, the judges also perceived the development of “off-flavors” due to accumulation of fermentative metabolites, such as acetaldehyde, ethanol, and ethyl acetate. The unpleasant taste, together with the brown discoloration (Fig. 3), caused the coated samples to be hardly edible. The highest scores for firmness were given to coated pears, which is in agreement with data obtained by the instrumental method (Table 2).

3.3. Sensorial quality 4. Conclusion During maturation biochemical and physiological changes occur in fruits which give them desirable external aspect and eating quality. Fig. 4. shows the overall appearance, gloss, aroma, taste, juiciness, and firmness scores for the control and coated pears. It can be observed that initially (up to 3 days) the coated samples had better overall appearance than the untreated samples, which may be explained by the fact that coating provided shine to fruits. The panellist detected the rapid loss of marketable quality attributes in the coated pears. From the 6th day of storage, most of the evaluated

The coating was effective method to control fungal growth in pears stored under simulated retail display conditions, but failed as a conservation technique, since induced anaerobic respiration, which led to rapid loss of the marketable quality attributes. In conclusion, CMC-based emulsion is not suitable carrier for KS, when coating is intended to be applied to pears as post-cold-storage treatment. A selection of other coating material with appropriate gas permeability should be considered in future research in order to

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