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Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating
LWT - Food Science and Technology xxx (2016) 1e9
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Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating € € kmen* Kübra S. Ozdemir, Vural Go Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering, Hacettepe University, 06800 Beytepe Campus, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2016 Received in revised form 26 October 2016 Accepted 27 October 2016 Available online xxx
The aim of this study was to evaluate the mixture of chitosan and ascorbic acid as an edible coating to extend the shelf-life of pomegranate arils. Pomegranate arils coated with varying concentrations of chitosan and ascorbic acid were stored at 5 ± 1 C for 28 days. Physical, chemical, microbiological and sensory quality attributes of the arils were determined during storage. There were no significant differences in the contents of anthocyanins, organic acids and sugars for coated and control (uncoated) samples during storage. Chitosan-ascorbic coating helped keeping the visual quality of arils during storage as confirmed by their surface color measurement. Chitosan-ascorbic coating inhibited bacterial and fungal growth on arils. Furthermore, the chitosan-ascorbic acid solution inhibited the mesophilic aerobic bacteria immediately after coating and coated arils presented no growth during storage. The bacterial and fungal growth were analyzed by using the Gompertz model to estimate the microbiological shelf-life of samples. The results revealed that chitosan-ascorbic coating can prolong the lag time of microorganisms and extend the shelf-life of arils up to 21 days during storage at 5 C. Sensory scores (color, taste, aroma) were also higher in chitosan-ascorbic acid coated arils that were quite acceptable even after 25 days of refrigerated storage. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Punica granatum Edible coating Chitosan film Anthocyanin Gompertz model Chemical compounds studied in this article: Ascorbic acid (PubChem CID: 54670067) Chitosan (PubChem CID:71853) Cyanidin-3,5-di-O-glucoside (PubChem CID: 164999) cyanidin-3-glucoside (PubChem CID:197081) pelargonidin-3,5-diglucoside (PubChem CID: 13988280) pelargonidin-3-glucoside (PubChem CID: 3080714)
1. Introduction Pomegranate is an important source of anthocyanins, phenolic du & Yılmaz, 2012; compounds, vitamins and minerals (Gündog s, 2000; O'Grady, Sigge, Caleb, & Opara, Melgarejo, Salazar, & Arte 2014). It has been reported to have many positive health benefits due to its anti-inflammatory and anti-atherosclerotic properties as well as other benefits such as chemoprevention (Aviram & Dornfeld, 2001; Faria & Calhau, 2011; Malik et al., 2005). The edible part of the fruit is arils and constitutes 52% of total fruit (w/ w), comprising 78% juice and 22% seeds (Elnemr, Ismail, & Ragab, 1990). However the difficulty in peeling the fruit and separation of arils limit its consumption. Therefore, ready-to-eat fresh arils
* Corresponding author. €kmen). E-mail address: [email protected] (V. Go
could be an alternative to increase consumption of pomegranates. Ready-to-eat fresh arils have an important commercial value due to their healthiness and convenience. Nevertheless, the arils are highly perishable and quickly deteriorate during storage. Most of the studies in literature about pomegranates focus on chemical composition, chemical and physical changes during ripening of whole fruit and postharvest treatments for improving s, Villaescusa, & Tudela, 2000; quality and shelf-life of fruit (Arte Fawole & Opara, 2013; Varasteh, Arzani, Barzegar, & Zamani, 2012). However, there are few studies on the preservation of pomegranate arils. Shelf-life of pomegranate arils could be extended by the application of edible coatings instead of using chemical preservatives or modified atmosphere packaging. Edible coatings maintain a semi-permeable membrane on coated fruits so this membrane decreases the exchange of O2 and CO2 between coated fruit and environment (Olivas & Barbosa-Canovas, 2005; Park, 1999). Moreover, some edible coatings improve the
http://dx.doi.org/10.1016/j.lwt.2016.10.057 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
€ €kmen / LWT - Food Science and Technology xxx (2016) 1e9 K.S. Ozdemir, V. Go
2
appearance of food and have potential to delay or even inhibit the growth of pathogenic and spoilage microorganisms (Dutta, Tripathi, Mehrotra, & Dutta, 2009; Quintavalla & Vicini, 2002). Chitosan is one of the edible coating materials which is a natural carbohydrate polymer obtained by the deacetylation of chitin [poly-b-(1e4)-N-acetyl-D-glucosamine] that has been generally recognized as safe. Chitosan possesses excellent film-forming properties and can be applied as an edible surface coating to fruits and vegetables. Chitosan also possess antimicrobial properties that depend on several factors like deacetylation degree, molecular weight, pH and temperature (Devlieghere, Vermeulen, & Debevere, 2004; Vasconez, Flores, Campos, Alvarado, & Gerschenson, 2009). It has been successfully used to prolong shelf-life of longan fruit, fresh cut broccoli, raspberry and many other fruits and vegetables (Jiang & Li, 2001; Moreira, Roura, & Ponce, 2011; Tezotto-Uliana, Fargoni, Geerdink, & Kluge, 2014). The application of chitosan is restricted to some extent because it is insoluble at neutral pH (Ge & Luo, 2005). However chitosan is soluble in acidic environment so acetic, formic and hydrochloric acids were used to prepare chitosan solutions in several publications (Chien, Sheu, & Yang, 2007; Jiang & Li, 2001; Tezotto-Uliana et al., 2014). Acetic acid has strong unpleasant and pungent smell with sour vinegar taste, and it adversely affects the sensory properties of coated fruits. Similarly, hydrochloric and formic acids have a highly pungent and penetrating odor. In this study, ascorbic acid was used as an alternative organic acid to aid dissolving chitosan for the preparation of chitosan film coatings. Ascorbic acid is essential for human health and it is found naturally in many fruits. Application of chitosan and ascorbic acid combinations in varying concentrations were investigated for the first time in order to improve the shelf-life of pomegranate arils during cold storage. For this purpose, microbial growth, weight loss, anthocyanins, sugar and organic acids, pH, titratable acidity, total soluble solids (TSS) content and CIE L* a* b* color parameters and sensory quality were determined during storage time. Moreover, microbial growth was also modelled as a way of predicting the shelf-life of pomegranate arils. 2. Materials and methods
was applied for 1 h and a translucent solution was obtained. Pomegranates were washed in tap water then arils were separated and the peel was carefully removed manually. Arils were randomly distributed into four groups and immersed in the coating solutions for 5 min. Arils (100 g) were immersed in 200 mL of solution for each time. After the immersion they were left to dry at air conditioned laboratory at 25 C for 2 h. For analyses, 35 packages (10 packages for microbiological analyses, 3 packages for weight loss, 2 packages for color analyses, 10 packages for sensory analysis and 10 packages for chemical analyses) were prepared for each group. Packaging material was sterile polypropylene jars (30 mL) covered with BOPP (biaxially oriented polypropylene) film (thickness of 30 mm and O2 permeability of 1600 cm3 m2 per day). Each package consists of 10 g of sample. Packaged samples were stored at 5 ± 1 C for 28 days and sampling was carried on 0, 7, 14, 21 and 28th days of storage. Microbial quality, visual quality, sensory analysis and weight loss were analyzed in control group, 1% AA treated arils, 1%CH-1%AA and 2%CH-2%CH coated arils. Analyses of pH, titratable acidity, total soluble solid content, sugar, organic acids and anthocyanins were only tested on control group and 1%CH-1%AA treated arils. 2.3. Weight loss Pomegranate arils were weighed at the end of each sampling day. Weight loss was calculated as the percentage difference between the initial weight and the final weight of the pomegranate arils. 2.4. Color measurement Color analyses were performed by using a computer vision € kmen & Süg üt, 2007). A color based image analysis technique (Go image obtained by a digital camera, under controlled and defined illumination conditions. Illumination was achieved with 2 Philips, Natural Daylight 18 W fluorescent lamps with color temperature of 6500 K. The images were analyzed using a software developed for this purpose by using Matlab R2011a (The MathWorks Inc., USA). Images were captured at a resolution of 20.2 megapixels and stored in a personal computer in JPEG format.
2.1. Chemicals and consumables Fresh pomegranates (Punica granatum L.) were purchased from a local wholesale distributor. Cyanidin-3,5-diglucoside (Cy-3,5-dG, 90%), cyanidin-3-glucoside (Cy-3-G 95%), pelargonidin-3,5diglucoside (Pg-3,5-dG, 90%), pelargonidin-3-glucoside (Pg-3-G, 97%) and acetonitrile (HPLC grade), ascorbic acid (AA, analytical grade), chitosan (CH) (crab shells, degree of deacetylation 75e85% and medium molecular weight) were purchased from SigmaAldrich (Steinheim, Germany). Formic acid (98%), plate count agar (PCA) and potato dextrose agar (PDA) were obtained from Merck (Darmstadt, Germany). A Phenomenex Polyphenol C12 (Synergi Max-RP 80 Å, 250 4.6 mm, i.d., 4 mm, Waldbronn, Germany) LC Column was used for the analysis of anthocyanins. Nylon syringe filters (pore size 0.45 mm) and OASIS HLB cartridges were supplied by Waters (Milford, MA, USA). 2.2. Preparation of chitosan-ascorbic acid film formulations and treatments Aqueous solutions (w/v) of 1% chitosan and 1% ascorbic acid (1% CH-1%AA), 2% chitosan and 2% ascorbic acid (2%CH-2%AA), 1% ascorbic acid were prepared and distilled water was used as control. To achieve complete dissolution of chitosan, ultrasonification
2.5. Determination of pH, titratable acidity and total soluble solid content The aril juice was obtained using a kitchen type press. The pH, titratable acidity (TA) and total soluble solid (TSS) of the juices were determined. The pH value of the juice was measured by using a pHmeter (Hanna instruments, USA). TA was measured on 5 g juice by adjusting the pH to 8.2 with 0.1 M NaOH and it was expressed as citric acid equivalent. TSS content of pomegranate arils was determined by using a digital refractometer (Pal-1, Atago, Tokyo, Japan). 2.6. Analyses of sugars and organic acids Five grams of pomegranate arils were extracted with 50 mL of the mixture of acetonitrile: water in 1% formic acid (20:80, v/v) by homogenization at 10 000 rpm for 2 min (Heidolph Silent Crusher M, Schwabach, Germany). After centrifugation at 7500g for 5 min, 1 mL of the supernatant was passed through a preconditioned Oasis HLB cartridge. The first eight drops of the eluent were discarded, and the rest was collected into an HPLC vial. Concentrations of sugars and organic acids were determined by using an Agilent Technologies (Waldbronn, Germany) 1100 HPLC system equipped with a refractive index (RI) detector, diode array detector (DAD), quaternary pump, auto-sampler and column oven.
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
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An isocratic elution with a mobile phase consisting 0.1% phosphoric acid in water (v/v) at a flow rate of 1 mL$min1 was used. A 0.01 mL of sample was injected into a Shodex Rspak KC-811 column (300 mm 7.8 mm i.d., 7 mm, Tokyo, Japan) at 20 C. Analysis of sugar was performed by using RI detector and organic acid was performed by DAD at 220 nm, on the same run. Identification of sugars and organic acids were accomplished by comparing the retention time of the peaks in pomegranate samples to those of standard compounds. The quantification of sugars and organic acids were based on external calibration curves of each compound. 2.7. Analysis of anthocyanins Five grams of pomegranate arils (pith and aril) were extracted with 50 mL of the mixture of acetonitrile: water in 1% formic acid (20:80, v/v) by homogenization at 10 000 rpm for 2 min (Heidolph Silent Crusher M, Schwabach, Germany). After centrifugation at 7500g for 5 min, supernatant was filtered through a 0.45 mm syringe filter. Anthocyanins were determined by using an Agilent 1200 series HPLC-DAD system coupled to an Agilent 6130 single quadrupole mass spectrometer. Chromatographic separation was carried out on a Phenomenex Polyphenol C12 (Synergi Max-RP 80 Å, 250 4.6 mm, i.d., 4 mm, Waldbronn, Germany) LC Column. A gradient mixture of solvent A (1% formic acid in water) and solvent B (1% formic acid in acetonitrile) was used at a flow rate of 0.7 mL$min1 at 30 C with the following gradient profile: 0e12 min, linear gradient elution from 10 to 30% B; 12e15 min, isocratic elution of 30% B; 15e18 min, linear gradient elution from 30 to 10% B; and 18e25 min, isocratic elution of 10% B. The injection volume was 0.01 mL. Data acquisition was performed in scan mode using the following interface parameters: drying gas (N2), flow of 130 mL$min1; nebulizer pressure, 275 kPa; drying gas temperature, 350 C; capillary voltage, 4 kV; fragmentor voltage, 1.93e-17 J. MS spectra were recorded in positive ion mode between m/z 100e500. Quantification of individual anthocyanins acquired from their DAD chromatograms by using external calibration curves of each standard compound built in a range between 0.1 and 10 mg/L. Confirmation of anthocyanins were based on comparison of the peaks to the retention times of standard compounds. Mass spectra of the peaks assigned as anthocyanins were also used for confirmation.
3
CFU g1) is the maximum growth at the stationary phase and t is the time (day). The experimental data were modelled using the curve fitting module in MATLAB. 2.9. Sensory evaluation Sensory evaluation of pomegranate arils was performed during storage by a sensory panel. The panel consisted of 8 members with sensory evaluation experience in fruit quality and they were trained in a pre-test for evaluating the aroma of pomegranate arils to be familiar with its quality attributes. Panelists were asked to evaluate color, freshness, taste, aroma, texture and overall acceptance using a 5-point scale described by Martínez-Romero et al., 2013. The rating for each characteristic was based on a five-point scale (5e1) with 5 corresponded to extremely liked (very characteristic of the fruit), 4 ¼ like moderately, 3 ¼ neither like nor dislike, 2 ¼ dislike moderately and 1 corresponded to extremely disliked (non-characteristic of the product). Scores of 3 and above were considered as acceptable for commercial purposes. The sensory evaluation was done in 1st, 7th, 14th and 25th days of storage. 2.10. Statistical analysis The results were reported as average ± standard deviations. Differences were estimated for all treatments by analysis of variance (ANOVA) followed by Tukey's Honest Significant Difference test (p < 0.05). Independent sample t-test were used to compare control group and 1%CH-1%AA coated samples in the results of chemical analyses. All statistical analyses were performed using the SPSS 18.0 version (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Evaluation of changes in weight loss, color-visual quality and chemical composition Chitosan-ascorbic acid coating did not affect weight loss as control, 1% ascorbic acid and coated fruit lost similar weight during the 28 days of storage (Fig. 1). The commercial weight loss limit is 4e6% for minimally processed fruits. In all treatments, the weight loss is under this commercial limit during storage time. Also, titratable acidity and pH did not change significantly during storage in both control and 1%CH-1%AA coated samples (Table 1). Vargas, Albors, Chiralt, and Gonzalez-Martinez (2006) coated
2.8. Total microbial counts and kinetic modelling Two samples were taken from each treatment on each sampling day. Aerobic mesophilic, yeast and mold counts on pomegranate arils subjected to different treatments were evaluated throughout storage (0, 7, 14, 21, 28th day). Total aerobic mesophiles were determined using PCA after incubation at 35 C for 48 h. Yeast and mold counts were performed on PDA by using the spread plate method after incubation at 25 C for 5e7 days. Colony forming units (CFU) per gram of arils were calculated and expressed in terms of log CFU g1. Additionally, the cell load data from each treatment were modelled according to the Gompertz equation modified by Zwietering, Jongenburger, Rombouts, and Van't Riet (1990):
log10
N N0
m e ¼ A exp exp m ðʎ tÞ þ 1 A
(1)
where N is the log CFU g1 at a given time t (day), N0 is the log CFU g1 at the beginning of storage, mm [D log (CFU g1) per day ] is the maximal growth rate, ʎ (days) is the lag time, A (log
Fig. 1. Weight loss (%) of pomegranate arils during storage at 5 C (C) control; (B) 1% AA, (D) 1%CH-1%AA, () 2%CH-2%AA. Vertical bars represent the standard deviation (n ¼ 3).
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
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Table 1 Titratable acidity (TA), pH, total soluble solids (TSS), organic acids and sugars in control group and 1%CHe 1% AA coated arils during storage at 5 C. Treatment
Day
pH
Control
0 7 14 21 28 0 7 14 21 28
3.86 3.89 3.86 3.91 3.86 3.93 3.96 3.99 4.01 4.05
1%CH-1%AA
T.A. (Citric acid eq. %) ± ± ± ± ± ± ± ± ± ±
0.01a 0.04a 0.00a 0.01a 0.04a 0.04a 0.01a 0.09a 0.05a 0.01a*
1.41 1.23 1.41 1.37 1.44 1.15 1.23 1.16 1.18 1.09
± ± ± ± ± ± ± ± ± ±
0.04a 0.01b 0.00a 0.00a 0.14a 0.99a 0.01a 0.09a 0.04a* 0.00a
T.S.S. (brix) 16.15 16.80 16.75 16.40 16.55 16.25 16.50 16.55 17.05 17.00
± ± ± ± ± ± ± ± ± ±
0.07a 0.14ab 0.07b 0.14ab 0.21ab 0.07a 0.00b 0.07b 0.07c* 0.00c
Total Organic Acids (g$kg1) 18.01 17.14 18.97 17.62 15.48 16.23 16.16 15.25 17.81 17.34
± ± ± ± ± ± ± ± ± ±
0.51a* 0.91a 2.47a 2.05a 1.44a 0.09a* 0.21a 0.37a 1.77a 0.62a
Total Sugars (g$kg1) 125.94 127.37 119.69 133.44 121.74 126.36 125.48 118.29 126.77 122.20
± ± ± ± ± ± ± ± ± ±
2.47a 0.98a 9.40a 7.57a 2.31a 3.35a 3.62a 3.13a 6.97a 5.34a
Values are given in average ± standard deviation (n ¼ 2). Different letters within the same column in each treatment indicates a significant difference (p < 0.05). (*) indicates a significant difference determined between chitosan-ascorbic coated and control samples in the same storage day (p < 0.05).
Fig. 2. Photographs of control arils, 1%AA treated arils, 1%CH-1%AA, and 2%CH-2%AA coated arils at the 0th, 7th and 14th day of storage.
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
€ €kmen / LWT - Food Science and Technology xxx (2016) 1e9 K.S. Ozdemir, V. Go Table 2 Changes in CIE L*a*b* color values of arils during storage at 5 C. Treatment
Day
L*
Control
1 7 14 21 28 1 7 14 21 28 1 7 14 21 28 1 7 14 21 28
35.0 34.3 34.8 30.5 38.4 36.0 34.0 37.5 39.4 38.7 33.2 32.2 36.7 35.4 40.4 35.5 37.9 32.9 35.7 41.1
1%AA
1%CH-1%AA
2%CH-2%AA
a* ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.7ab 3.9abAB 4.9ab 3.3aA 2.5b 3.2ab 2.6aAB 4.4a 4.0bB 3.6b 3.0a 3.4aA 4.7ab 4.8aAB 2.3b 4.4a 3.2bB 2.9a 4.9aAB 4.1b
b*
41.3 41.7 38.3 37.5 27.5 46.2 43.3 43.7 45.7 29.8 41.3 42.1 42.4 43.2 36.7 42.7 44.7 39.7 38.0 33.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.6aA* 4.2a 7.0a 6.1aA 5.4bA 2.0aB 3.7a 2.9a 5.2aB 4.6bAB 2.8abA 3.2a 4.6a 3.5aB 4.3bC 3.5abAB 2.6a 1.7ab 3.7bcA 3.3cBC
28.2 29.1 26.7 27.6 16.7 30.4 29.6 28.1 31.7 15.5 27.0 29.1 27.3 29.0 19.0 28.1 29.7 26.7 26.4 18.8
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.5a 3.7a 4.5a 4.7aAB 2.8b 1.8a 2.9a 2.8a 3.5aA 3.9b 2.7a 2.6a 4.4a 3.7aAB 2.2b 2.6a 2.04a 1.7a 4.6aB 2.9b
Values are mean ± standard deviation (n ¼ 10). Values in the same column within the same treatment having different superscript lowercase letters are significantly different (p < 0.05). Values in the same column on the same storage day having different superscript uppercase letters are significantly different (p < 0.05).
strawberries with 1% chitosan and 1%, 2%, 4% oleic acid and stored at 4±1 C for 10 days. They reported that soluble solid content, acidity and pH did not change significantly during storage and were not affected by coating application. Our pH and titratable acidity results are in agreement with the results of Vargas et al. (2006). However, throughout 28 days of storage the total soluble solid content increased to 17% in coated arils while it was 16.55% in control (Table 1). In control samples, there was a gradual increase in TSS content until the 7th day of storage and after that a decline occurred. The TSS increased in the early stage of storage was attributed to the activity of microorganisms that decomposes polymeric carbohydrates into their sub-units. The decline occurred in TSS after 7th day of storage due to the high metabolism of the arils and senescence processes. The color characteristics of pomegranate arils and their visual qualities are presented in Fig. 2 and Table 2. Pomegranate arils coated with chitosan and ascorbic acid showed no signs of deterioration and therefore was acceptable for consumption. Chitosanascorbic acid coated arils and control samples showed stability in L* parameter during storage at 5 C, however chitosan-ascorbic acid coated arils showed significantly higher lightness (p < 0.05) on day
5
21 compared to control. The findings also showed that a* value, which is related to redness and color stability, was significantly higher in chitosan coated arils compared to the control samples after 28 days of storage. This can be confirmed visually from the images of the samples presented in Fig. 2. Parameter b* showed stability in all samples during 21 days of storage, however it was significantly reduced on the 28th day (p < 0.05). The change in b* value points to decrease in yellowness and consequently lower saturation (Kapetanakou, Stragkas, & Skandamis, 2015). Glucose and fructose were the main sugars in pomegranate arils (Table 3). Fructose content did not change in coated samples and it was also quite stable in control samples with an average content of 70.4 g$kg1 during 28 days of storage. Similarly, glucose whose overall content was slightly lower than fructose (55.2 g$kg1) showed slight fluctuations, but no significant reduction occurred. Melgarejo et al. (2000) determined sugar contents of 40 Spanish pomegranate cultivars. Fructose and glucose content ranged between 59.6 and 70.4 g$kg1 and 56.6e64.5 g$kg1, respectively. The levels of fructose and glucose contents in the present work are comparable with literature. Citric, malic and lactic acids were the main organic acids determined in pomegranate arils. Citric acid was the dominant acid in control samples with 11.14 g$kg1 in comparison with 6.05 g$kg1 malic acid and 0.83 g$kg1 lactic acid. Similarly, 9.95 g$kg1 citric, 5.56 g$kg1 malic acid and 0.72 g$kg1 lactic acid were determined in chitosan-ascorbic acid coated arils in the beginning of storage. These amounts did not change during the lu, Go €kmen, and Artık (2002) determined organic storage. Poyrazog acid composition of thirteen pomegranate varieties and they found citric, malic, tartaric, oxalic, D-(-)quinic and succinic acids in pomegranate juices. Similarly to our results, citric acid was the predominant acid with a range of 0.33e8.96 g$L1. Citric acid and malic acid concentrations found relatively higher in our study than the reported levels in literature. Anthocyanins are phenolic compounds responsible for typical red color of pomegranates. The main anthocyanin pigments found in pomegranate arils were determined as Cy-3-G, Cy-3,5-dG, Pg-3G and Pg-3,5-dG. The most predominant anthocyanin was Cy-3-G, while Pg-3-G was present in the lowest amount in all samples (Fig. 3). Individual anthocyanin contents showed stability during 21 days of storage in both control and 1%CH-1%AA coated samples. However, after 28 days of shelf-life, Cy-3-G decreased in coated samples (Fig. 3). The concentration of Cy-3-G was determined as 821.85 mg$kg1 and 956.21 mg$kg1 in control samples and 1%CH1%AA coated samples respectively, in the beginning of the storage time. It was 982.46 mg$kg1 and 708.41 mg$kg1 for control and 1% CH-1%AA coated samples at the end of storage. Anthocyanin
Table 3 Sugar and organic acid composition of control group and 1%CH-1%AA coated arils during storage at 5 C. Treatment
Day
Sugars (g$kg1)
Control
0 7 14 21 28 0 7 14 21 28
55.40 56.13 52.87 58.32 53.29 56.53 56.12 52.19 53.56 53.09
Glucose
1%CH-1%AA
± ± ± ± ± ± ± ± ± ±
Organic acids (g$kg1) Fructose
0.89a 0.28a 3.50a 2.62a 1.18a 0.45a 0.25a 1.19a 2.96a 1.79a
70.54 71.23 66.82 75.12 68.46 69.84 69.36 66.10 73.21 69.11
± 1.58a ± 1.26a ± 5.89a ± 4.95a þ 1.12a ± 2.91a ± 3.87a ± 1.95a ± 4.01a ± 3.54a
Citric acid
Malic acid
11.14 ± 0.06a* 9.74 ± 0.23a 11.06 ± 0.45a* 9.87 ± 0.70a 9.53 ± 0.53a 9.95 ± 0.20ab* 9.88 ± 0.04ab 9.38 ± 0.06a* 11.08 ± 0.66b 9.70 ± 0.11a
6.05 6.58 7.08 6.96 5.15 5.56 5.46 4.98 5.81 6.68
± ± ± ± ± ± ± ± ± ±
0.45a 0.71a 1.90a 1.20a 0.95a 0.26a 0.21a 0.03a 1.13a 0.78a
Lactic acid 0.83 0.82 0.83 0.80 0.81 0.72 0.82 0.89 0.92 0.97
± ± ± ± ± ± ± ± ± ±
0.12a 0.03a 0.11a 0.16a 0.04a 0.03a 0.04a 0.28a 0.03a 0.05a
Values are given in mean ± standard deviation (n ¼ 2). Different superscript letters within the same column in each treatment indicates a significant difference (p < 0.05). (*) indicates a significant difference determined between chitosan-ascorbic coated and control samples in the same storage day (p < 0.05).
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
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Fig. 3. Changes of individual anthocyanin concentrations (mg$kg1) of 1%CH-1%AA coated arils ( ) and control ( )during storage at 5 C. a) cyanidin-3,5-diglucoside, b) cyanidin-3-glucoside, c) pelargonidin-3,5-diglucoside, d) pelargonidin-3-glucoside. Vertical bars represent the standard deviation (n ¼ 2).
synthesis could continue during postharvest storage of pomegranates. Increases in anthocyanin concentration after harvest during cold storage have been reported for fruits other than pomegranates such as raspberry (Han, Zhao, Leonard, & Traber, 2004). Miguel, Fontes, Antunes, Neves, and Martins (2004) stated that this was correlated with the activity of the anthocyanin biosynthesis enzymes. Chitosan films form a very effective gas barrier, probably due to the dense structure of the film (Wong, Gastineau, Gregorski, Tillin, & Pavlath, 1992). It is possible that anthocyanin synthesis was reduced by the reduction in gas metabolism and significantly inhibited with the combination of chitosan and ascorbic acid barrier. Similar results were found for raspberries coated with chitosan after harvest and stored at 0 C for 12 days (Tezotto-Uliana et al., 2014). According to these results, we expected that a similar trend will be observed in 2%CH-2%AA coated samples. So these chemical analyses were not carried out for 2%CH2%AA coated arils. 3.2. Microbial stability of pomegranate arils The influence of different coatings on the growth of aerobic mesophilic bacteria, yeast and mold populations during storage are
shown in Fig. 4. Coating with chitosan and ascorbic acid significantly reduced bacteria, yeast and mold populations throughout the storage time (Fig. 4a and b). In the first day, initial aerobic mesophilic bacteria were below 1 log CFU g1, yeast and mold counts on fresh arils were below 2 log CFU g1. Treatments with 1%CH-1%AA and 2%CH-2%AA were helpful in reducing the fungal load throughout storage time. In 2%CH-2%AA treated arils, yeast and mold counts were under 2 log CFU g1 until 21st day, while it was higher in control and 1%AA treated arils throughout storage. Aerobic mesophilic count was determined as 6.8 log CFU g1 and 5.9 log CFU g1 in control samples and 1%AA treated samples at the end of storage respectively. However, there was no growth of aerobic mesophilic bacteria in 1%CH-1%AA and 2%CH-2%AA coated pomegranate arils at the end of storage. Antimicrobial activity of chitosan has been confirmed by previous studies on other fresh fruits and vegetables (Alvarez, Ponce, & Moreira, 2013; Devlieghere et al., 2004; Jiang & Li, 2001; Moreira, Ponce, Ansorena, & Roura, 2011). Pushkala, Parvathy, and Srividya (2012) enhanced the microbiological quality of carrot shreds by using chitosan powder coating. Jiang and Li (2001) delayed the increase in decay of stored longan fruit by chitosan coating. Besides chitosan antimicrobial activity, the type of solvent could affect its
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
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resulted in easy penetration of the chitosan molecule into the cytoplasmic membrane of bacterial cell wall and changing the internal pH of the bacteria (Cadogan, Lee, Popuri, & Lin, 2014). The microbial counts were also modelled by using modified Gompertz equation. The model appropriately fitted the experimental data since the coefficient of determination ranged from 0.98 to 1.00. Maximal growth rate, maximum growth at the stationary phase, and lag phase defined by the models are given in Table 4. The lag phase time and the maximal growth rate were highly influenced by the composition of coating solutions. The growth of yeasts and molds was delayed by chitosan-ascorbic acid coating hence 1% CH-1%AA and 2%CH-2%AA coated arils exhibiting longer lag time (ʎ) (9.90 and 17.04 days) than in control group and 1% AA treated arils (7.32 and 4.14 days, respectively). More rapid microbial growth were observed for the populations in control group and 1%AA treated arils. Gompertz model described the evolution of aerobic mesophilic counts as well as yeast and mold counts. The lag phase time and the maximal growth rate were determined for control and 1%AA treated arils but these parameters cannot be calculated for chitosan-ascorbic acid coated arils due to lack of growth. In arils treated with 1%AA, lag time (ʎ) for aerobic mesophilic populations was lower and maximal growth rate (mmax) was higher compared to the control. 1%AA treatment increased the acidity on the aril surface which provided favorable environment for microorganisms. In contrast, no microbial growth was observed in 1%AA-1%CH and 2% AA-2%CH coated arils. Moreira et al. (2011) found similar patterns for aerobic mesophilic bacteria, yeast and mold growth in fresh cut broccoli coated with chitosan. The application of chitosan coating extended the lag phase in comparison to the control sample of fresh-cut broccoli. Longer lag time and lower maximum specific growth rates are preferred by the industry (Zimmermann, Miorelli, Massaguer, & Aragao, 2011). 3.3. Sensory quality Table 5 shows the effects of different coatings on the sensory attributes and acceptance of pomegranate arils during cold storage. Control and 1%AA showed similar scores as the coated arils at the beginning of the storage (on day 1 and 7), in all sensory attributes (p > 0.05). However, on 14th day of storage control and 1%AA treated arils were microbiologically rejected. Contrary to that, coated samples were still organoleptically acceptable with scores above 3, regardless of the chitosan and ascorbic acid concentration (Table 5). Moreover, 1%CH-1%AA coating maintained product attributes such as aril color, taste, aroma and texture even on the 25th day of storage. In addition, the panelists did not perceive any offflavor in pomegranate arils as a consequence of chitosan and ascorbic acid treatment.
Fig. 4. Growth of (a) yeasts and moulds, and (b) aerobic mesophilic bacteria on Control (*) samples, 1%AA (), 1%CH -1%AA (þ), 2%CH-2%AA (:) coated pomegranate arils during storage with Gompertz model fits.
antimicrobial action due to the pH of chitosan/solvent solution. According to a study, ascorbic acid solvated chitosan membrane showed a higher antibacterial activity than citric, maleic, oxalic and tartaric acid due to its crosslinking effect which shrinks the intersegmental space of the polymer chains and reduction its size. This
Table 4 Gompertz parameters for yeast and mould and mesophilic aerobic microbial growth in arils treated with different coating solutions. Population
Dipping Condition
Gompertz model parameters
mm
A Yeast and mold
Mesophilic aerobic bacteria
Control (water) 1%AA 1%CH-1%AA 2%CH-2%AA Control (water) 1%AA 1%CH-1%AA 2%CH-2%AA
7.31 7.69 5.29 5.45 6.89 4.43 e e
± ± ± ± ± ±
0.10 0.37 0.06 0.07 0.02 0.66
1.15 0.89 0.50 1.19 0.55 0.71 e e
± ± ± ± ± ±
0.25 0.27 0.03 0.14 0.24 0.40
ʎ
R2
7.32 ± 1.86 4.14 ± 1.26 9.90 ± 0.55 17.04 ± 0.31 10.42 ± 1.37 5.59 ± 0.71 e e
1.00 0.99 1.00 1.00 0.98 1.00 e e
Gompertz parameters: A, maximum growth attained at the stationary phase; mm, maximal growth rate; ʎ, lag phase period; R2, regression coefficient. Significance level at p < 0.05. Values are given in average ± standard deviation (n ¼ 4). () Indicates no growth.
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Table 5 Sensory evaluation during storage of coated and uncoated pomegranate arils. Application
Panelist scores Day 1
Color
Freshness
Taste
Aroma
Texture
Overall acceptance
Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA
4.1 3.9 4.4 4.3 4.1 3.9 4.1 3.9 3.4 3.8 3.8 3.5 4.1 4.0 4.1 3.8 4.5 4.5 4.6 4.6 4.0 3.8 4.4 4.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Day 7 a
1.1 1.4a 0.9a 1.2a 0.8a 0.8a 1.0a 1.1a 1.2a 1.2a 1.3a 1.2a 1.0a 1.1a 0.8a 1.6a 0.8a 0.5a 0.5a 0.5a 0.5a 1.2a 0.7a 1.0a
3.6 4.4 4.0 2.9 3.3 3.8 3.8 3.4 3.3 3.5 4.0 3.9 3.3 3.3 3.4 3.5 3.8 4.0 3.9 3.3 3.0 3.4 4.0 3.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Day 14 ab
0.7 0.7a 0.8a 0.8b 1.0a 0.9a 1.0a 0.9a 1.4a 0.9a 1.1a 0.4a 0.7a 0.7a 0.7a 0.5a 0.9a 0.5a 1.0a 0.7a 0.8a 0.5a 0.9a 0.9a
3.2 3.5 4.0 3.0 e e 3.4 3.3 e e 3.5 3.1 e e 3.2 3.1 e e 3.4 3.1 e e 3.4 3.0
± ± ± ±
Day 25 ab
1.0 0.8ab 0.9a 0.7b
± 0.7b ± 0.5ab
± 0.9a ± 0.7a
± 0.7a ± 0.7a
± 0.7a ± 0.7a
± 0.5a ± 0.9a
-* e 3.4 2.3 e e 2.1 2.3 e e 3.0 2.5 e e 3.4 3.0 e e 2.9 2.4 e e 2.6 2.3
± 0.7a ± 0.9b
± 1.0a ± 1.0a
± 1.4a ± 0.7a
± 0.7a ± 1.1a
± 0.8a ± 0.9a
± 1.2a ± 0.9a
Data are the mean ± standard deviation (n ¼ 8). For each parameter, values followed by different superscript letters denote a significant difference at p < 0.05.; comparison was done across treatments on the same storage day. Scores of 3 and above were considered as acceptable. (*) Data was not obtained due to the microbiological spoilage of sample.
4. Conclusion In conclusion, the results revealed that microbiological shelf-life of pomegranate arils can be significantly improved by means chitosan-ascorbic acid coating during cold storage, owing to antimicrobial activity of chitosan. In this way, color visual quality of pomegranate arils was also protected because ascorbic acid in the binary coating mixture is very effective anti-browning agent. As a practical guiding, coating of the pomegranate arils with an aqueous mixture of 1% chitosan and %1 ascorbic acid is recommended to ensure a shelf-life of 3 weeks under cold storage conditions (5 C). However, uncoated arils (control) could not reach this far (<10 days). By the end of storage, 1% chitosan and %1 ascorbic acid coated arils preserved their color, taste and aroma. Overall, the use of chitosan-ascorbic acid coating could be applied on perishable minimally processed fruits to extend shelf-life by improving microbiological safety, preserving nutritional and sensory quality. References Alvarez, M. V., Ponce, A. G., & Moreira, M. D. (2013). Antimicrobial efficiency of chitosan coating enriched with bioactive compounds to improve the safety of fresh cut broccoli. LWT-Food Science and Technology, 50(1), 78e87. s, F., Villaescusa, R., & Tudela, J. A. (2000). Modified atmosphere packaging of Arte pomegranate. Journal of Food Science, 65(7), 1112e1116. Aviram, M., & Dornfeld, L. (2001). Pomegranate juice consumption inhibits serum angiotensin converting enzyme activity and reduces systolic blood pressure. Atherosclerosis, 158(1), 195e198. Cadogan, E. I., Lee, C. H., Popuri, S. R., & Lin, H. Y. (2014). Effect of solvent on physicochemical properties and antibacterial activity of chitosan membranes. International Journal of Polymeric Materials and Polymeric Biomaterials, 63(14), 708e715. Chien, P. J., Sheu, F., & Yang, F. H. (2007). Effects of edible chitosan coating on quality and shelf life of sliced mango fruit. Journal of Food Engineering, 78(1), 225e229. Devlieghere, F., Vermeulen, A., & Debevere, J. (2004). Chitosan: Antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables. Food Microbiology, 21(6), 703e714. Dutta, P. K., Tripathi, S., Mehrotra, G. K., & Dutta, J. (2009). Perspectives for chitosan based antimicrobial films in food applications. Food Chemistry, 114(4), 1173e1182.
Elnemr, S. E., Ismail, I. A., & Ragab, M. (1990). Chemical-composition of juice and seeds of pomegranate fruit. Nahrung-Food, 34(7), 601e606. Faria, A., & Calhau, C. (2011). The bioactivity of Pomegranate: Impact on health and disease. Critical Reviews in Food Science and Nutrition, 51(7), 626e634. Fawole, O. A., & Opara, U. L. (2013). Harvest discrimination of pomegranate Fruit: Postharvest quality changes and relationships between instrumental and sensory attributes during shelf life. Journal of Food Science, 78(8), S1264eS1272. Ge, H. C., & Luo, D. K. (2005). Preparation of carboxymethyl chitosan in aqueous solution under microwave irradiation. Carbohydrate Research, 340(7), 1351e1356. €kmen, V., & Süg üt, I. (2007). A non-contact computer vision based analysis of Go color in foods. International Journal of Food Engineering, 3(5). du, M., & Yılmaz, H. (2012). Organic acid, phenolic profile and antioxidant Gündog capacities of pomegranate (Punica granatum L.) cultivars and selected genotypes. Scientia Horticulturae, 143, 38e42. Han, C., Zhao, Y., Leonard, S. W., & Traber, M. G. (2004). Edible coatings to improve storability and enhance nutritional value of fresh and frozen strawberries (Fragaria x ananassa) and raspberries (Rubus ideaus). Postharvest Biology and Technology, 33(1), 67e78. Jiang, Y. M., & Li, Y. B. (2001). Effects of chitosan coating on postharvest life and quality of longan fruit. Food Chemistry, 73(2), 139e143. Kapetanakou, A. E., Stragkas, I. G., & Skandamis, P. N. (2015). Developing an antimicrobial packaging of Ready-To-Eat pomegranate arils based on vapors of brandy or distillery ethanol. Food Research International, 69, 141e150. Malik, A., Afaq, F., Sarfaraz, S., Adhami, V. M., Syed, D. N., & Mukhtar, H. (2005). Pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer. Proceedings of the National Academy of Sciences of the United States of America, 102(41), 14813e14818. n, F., Díaz-Mula, H. M., Zapata, P. J., Valero, D., Martínez-Romero, D., Castillo, S., Guille et al. (2013). Aloe vera gel coating maintains quality and safety of ready-to-eat pomegranate arils. Postharvest Biology and Technology, 86, 107e112. s, F. (2000). Organic acids and sugars composiMelgarejo, P., Salazar, D. M., & Arte tion of harvested pomegranate fruits. European Food Research and Technology, 211(3), 185e190. Miguel, G. A., Fontes, C., Antunes, D., Neves, A., & Martins, D. (2004). Anthocyanin concentration of “Assaria” pomegranate fruits during different cold storage conditions. Journal of Biomedicine and Biotechnology, 5, 338e342. Moreira, M. D., Ponce, A., Ansorena, R., & Roura, S. I. (2011). Effectiveness of edible coatings combined with mild heat shocks on microbial spoilage and sensory quality of fresh cut broccoli (Brassica oleracea L.). Journal of Food Science, 76(6), M367eM374. Moreira, M. D., Roura, S. I., & Ponce, A. (2011). Effectiveness of chitosan edible coatings to improve microbiological and sensory quality of fresh cut broccoli. LWT-Food Science and Technology, 44(10), 2335e2341. O'Grady, L., Sigge, G., Caleb, O. J., & Opara, U. L. (2014). Effects of storage temperature and duration on chemical properties, proximate composition and selected
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€ €kmen / LWT - Food Science and Technology xxx (2016) 1e9 K.S. Ozdemir, V. Go bioactive components of pomegranate (Punica granatum L.) arils. LWT-Food Science and Technology, 57(2), 508e515. Olivas, G. I., & Barbosa-Canovas, G. V. (2005). Edible coatings for fresh-cut fruits. Critical Reviews in Food Science and Nutrition, 45(7e8), 657e670. Park, H. J. (1999). Development of advanced edible coatings for fruits. Trends in Food Science & Technology, 10(8), 254e260. lu, E., Go €kmen, V., & Artık, N. (2002). Organic acids and phenolic comPoyrazog pounds in pomegranates (Punica granatum L.) grown in Turkey. Journal of Food Composition and Analysis, 15(5), 567e575. Pushkala, R., Parvathy, K. R., & Srividya, N. (2012). Chitosan powder coating, a novel simple technique for enhancement of shelf life quality of carrot shreds stored in macro perforated LDPE packs. Innovative Food Science & Emerging Technologies, 16, 11e20. Quintavalla, S., & Vicini, L. (2002). Antimicrobial food packaging in meat industry. Meat Science, 62(3), 373e380. Tezotto-Uliana, J. V., Fargoni, G. P., Geerdink, G. M., & Kluge, R. A. (2014). Chitosan applications pre- or postharvest prolong raspberry shelf-life quality. Postharvest Biology and Technology, 91, 72e77.
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Varasteh, F., Arzani, K., Barzegar, M., & Zamani, Z. (2012). Changes in anthocyanins in arils of chitosan-coated pomegranate (Punica granatum L. cv. Rabbab-eNeyriz) fruit during cold storage. Food Chemistry, 130(2), 267e272. Vargas, M., Albors, A., Chiralt, A., & Gonzalez-Martinez, C. (2006). Quality of coldstored strawberries as affected by chitosan-oleic acid edible coatings. Postharvest Biology and Technology, 41(2), 164e171. Vasconez, M. B., Flores, S. K., Campos, C. A., Alvarado, J., & Gerschenson, L. N. (2009). Antimicrobial activity and physical properties of chitosan-tapioca starch based edible films and coatings. Food Research International, 42(7), 762e769. Wong, D., Gastineau, F., Gregorski, K. S., Tillin, S. J., & Pavlath, A. E. (1992). Chitosanelipid films microstructure and surface energy. Journal of Agricultural and Food Chemistry, 40(4), 540e544. Zimmermann, M., Miorelli, S., Massaguer, P. R., & Aragao, G. M. F. (2011). Growth of Byssochlamys nivea in pineapple juice under the effect of water activity and ascospore age. Brazilian Journal of Microbiology, 42(1), 203e210. Zwietering, M. H., Jongenburger, I., Rombouts, F. M., & Van’t Riet, K. (1990). Modelling of the bacterial growth curve. Applied and Environmental Microbiology, 56, 1875e1881.
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
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Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating € € kmen* Kübra S. Ozdemir, Vural Go Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering, Hacettepe University, 06800 Beytepe Campus, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2016 Received in revised form 26 October 2016 Accepted 27 October 2016 Available online xxx
The aim of this study was to evaluate the mixture of chitosan and ascorbic acid as an edible coating to extend the shelf-life of pomegranate arils. Pomegranate arils coated with varying concentrations of chitosan and ascorbic acid were stored at 5 ± 1 C for 28 days. Physical, chemical, microbiological and sensory quality attributes of the arils were determined during storage. There were no significant differences in the contents of anthocyanins, organic acids and sugars for coated and control (uncoated) samples during storage. Chitosan-ascorbic coating helped keeping the visual quality of arils during storage as confirmed by their surface color measurement. Chitosan-ascorbic coating inhibited bacterial and fungal growth on arils. Furthermore, the chitosan-ascorbic acid solution inhibited the mesophilic aerobic bacteria immediately after coating and coated arils presented no growth during storage. The bacterial and fungal growth were analyzed by using the Gompertz model to estimate the microbiological shelf-life of samples. The results revealed that chitosan-ascorbic coating can prolong the lag time of microorganisms and extend the shelf-life of arils up to 21 days during storage at 5 C. Sensory scores (color, taste, aroma) were also higher in chitosan-ascorbic acid coated arils that were quite acceptable even after 25 days of refrigerated storage. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Punica granatum Edible coating Chitosan film Anthocyanin Gompertz model Chemical compounds studied in this article: Ascorbic acid (PubChem CID: 54670067) Chitosan (PubChem CID:71853) Cyanidin-3,5-di-O-glucoside (PubChem CID: 164999) cyanidin-3-glucoside (PubChem CID:197081) pelargonidin-3,5-diglucoside (PubChem CID: 13988280) pelargonidin-3-glucoside (PubChem CID: 3080714)
1. Introduction Pomegranate is an important source of anthocyanins, phenolic du & Yılmaz, 2012; compounds, vitamins and minerals (Gündog s, 2000; O'Grady, Sigge, Caleb, & Opara, Melgarejo, Salazar, & Arte 2014). It has been reported to have many positive health benefits due to its anti-inflammatory and anti-atherosclerotic properties as well as other benefits such as chemoprevention (Aviram & Dornfeld, 2001; Faria & Calhau, 2011; Malik et al., 2005). The edible part of the fruit is arils and constitutes 52% of total fruit (w/ w), comprising 78% juice and 22% seeds (Elnemr, Ismail, & Ragab, 1990). However the difficulty in peeling the fruit and separation of arils limit its consumption. Therefore, ready-to-eat fresh arils
* Corresponding author. €kmen). E-mail address: [email protected] (V. Go
could be an alternative to increase consumption of pomegranates. Ready-to-eat fresh arils have an important commercial value due to their healthiness and convenience. Nevertheless, the arils are highly perishable and quickly deteriorate during storage. Most of the studies in literature about pomegranates focus on chemical composition, chemical and physical changes during ripening of whole fruit and postharvest treatments for improving s, Villaescusa, & Tudela, 2000; quality and shelf-life of fruit (Arte Fawole & Opara, 2013; Varasteh, Arzani, Barzegar, & Zamani, 2012). However, there are few studies on the preservation of pomegranate arils. Shelf-life of pomegranate arils could be extended by the application of edible coatings instead of using chemical preservatives or modified atmosphere packaging. Edible coatings maintain a semi-permeable membrane on coated fruits so this membrane decreases the exchange of O2 and CO2 between coated fruit and environment (Olivas & Barbosa-Canovas, 2005; Park, 1999). Moreover, some edible coatings improve the
http://dx.doi.org/10.1016/j.lwt.2016.10.057 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
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2
appearance of food and have potential to delay or even inhibit the growth of pathogenic and spoilage microorganisms (Dutta, Tripathi, Mehrotra, & Dutta, 2009; Quintavalla & Vicini, 2002). Chitosan is one of the edible coating materials which is a natural carbohydrate polymer obtained by the deacetylation of chitin [poly-b-(1e4)-N-acetyl-D-glucosamine] that has been generally recognized as safe. Chitosan possesses excellent film-forming properties and can be applied as an edible surface coating to fruits and vegetables. Chitosan also possess antimicrobial properties that depend on several factors like deacetylation degree, molecular weight, pH and temperature (Devlieghere, Vermeulen, & Debevere, 2004; Vasconez, Flores, Campos, Alvarado, & Gerschenson, 2009). It has been successfully used to prolong shelf-life of longan fruit, fresh cut broccoli, raspberry and many other fruits and vegetables (Jiang & Li, 2001; Moreira, Roura, & Ponce, 2011; Tezotto-Uliana, Fargoni, Geerdink, & Kluge, 2014). The application of chitosan is restricted to some extent because it is insoluble at neutral pH (Ge & Luo, 2005). However chitosan is soluble in acidic environment so acetic, formic and hydrochloric acids were used to prepare chitosan solutions in several publications (Chien, Sheu, & Yang, 2007; Jiang & Li, 2001; Tezotto-Uliana et al., 2014). Acetic acid has strong unpleasant and pungent smell with sour vinegar taste, and it adversely affects the sensory properties of coated fruits. Similarly, hydrochloric and formic acids have a highly pungent and penetrating odor. In this study, ascorbic acid was used as an alternative organic acid to aid dissolving chitosan for the preparation of chitosan film coatings. Ascorbic acid is essential for human health and it is found naturally in many fruits. Application of chitosan and ascorbic acid combinations in varying concentrations were investigated for the first time in order to improve the shelf-life of pomegranate arils during cold storage. For this purpose, microbial growth, weight loss, anthocyanins, sugar and organic acids, pH, titratable acidity, total soluble solids (TSS) content and CIE L* a* b* color parameters and sensory quality were determined during storage time. Moreover, microbial growth was also modelled as a way of predicting the shelf-life of pomegranate arils. 2. Materials and methods
was applied for 1 h and a translucent solution was obtained. Pomegranates were washed in tap water then arils were separated and the peel was carefully removed manually. Arils were randomly distributed into four groups and immersed in the coating solutions for 5 min. Arils (100 g) were immersed in 200 mL of solution for each time. After the immersion they were left to dry at air conditioned laboratory at 25 C for 2 h. For analyses, 35 packages (10 packages for microbiological analyses, 3 packages for weight loss, 2 packages for color analyses, 10 packages for sensory analysis and 10 packages for chemical analyses) were prepared for each group. Packaging material was sterile polypropylene jars (30 mL) covered with BOPP (biaxially oriented polypropylene) film (thickness of 30 mm and O2 permeability of 1600 cm3 m2 per day). Each package consists of 10 g of sample. Packaged samples were stored at 5 ± 1 C for 28 days and sampling was carried on 0, 7, 14, 21 and 28th days of storage. Microbial quality, visual quality, sensory analysis and weight loss were analyzed in control group, 1% AA treated arils, 1%CH-1%AA and 2%CH-2%CH coated arils. Analyses of pH, titratable acidity, total soluble solid content, sugar, organic acids and anthocyanins were only tested on control group and 1%CH-1%AA treated arils. 2.3. Weight loss Pomegranate arils were weighed at the end of each sampling day. Weight loss was calculated as the percentage difference between the initial weight and the final weight of the pomegranate arils. 2.4. Color measurement Color analyses were performed by using a computer vision € kmen & Süg üt, 2007). A color based image analysis technique (Go image obtained by a digital camera, under controlled and defined illumination conditions. Illumination was achieved with 2 Philips, Natural Daylight 18 W fluorescent lamps with color temperature of 6500 K. The images were analyzed using a software developed for this purpose by using Matlab R2011a (The MathWorks Inc., USA). Images were captured at a resolution of 20.2 megapixels and stored in a personal computer in JPEG format.
2.1. Chemicals and consumables Fresh pomegranates (Punica granatum L.) were purchased from a local wholesale distributor. Cyanidin-3,5-diglucoside (Cy-3,5-dG, 90%), cyanidin-3-glucoside (Cy-3-G 95%), pelargonidin-3,5diglucoside (Pg-3,5-dG, 90%), pelargonidin-3-glucoside (Pg-3-G, 97%) and acetonitrile (HPLC grade), ascorbic acid (AA, analytical grade), chitosan (CH) (crab shells, degree of deacetylation 75e85% and medium molecular weight) were purchased from SigmaAldrich (Steinheim, Germany). Formic acid (98%), plate count agar (PCA) and potato dextrose agar (PDA) were obtained from Merck (Darmstadt, Germany). A Phenomenex Polyphenol C12 (Synergi Max-RP 80 Å, 250 4.6 mm, i.d., 4 mm, Waldbronn, Germany) LC Column was used for the analysis of anthocyanins. Nylon syringe filters (pore size 0.45 mm) and OASIS HLB cartridges were supplied by Waters (Milford, MA, USA). 2.2. Preparation of chitosan-ascorbic acid film formulations and treatments Aqueous solutions (w/v) of 1% chitosan and 1% ascorbic acid (1% CH-1%AA), 2% chitosan and 2% ascorbic acid (2%CH-2%AA), 1% ascorbic acid were prepared and distilled water was used as control. To achieve complete dissolution of chitosan, ultrasonification
2.5. Determination of pH, titratable acidity and total soluble solid content The aril juice was obtained using a kitchen type press. The pH, titratable acidity (TA) and total soluble solid (TSS) of the juices were determined. The pH value of the juice was measured by using a pHmeter (Hanna instruments, USA). TA was measured on 5 g juice by adjusting the pH to 8.2 with 0.1 M NaOH and it was expressed as citric acid equivalent. TSS content of pomegranate arils was determined by using a digital refractometer (Pal-1, Atago, Tokyo, Japan). 2.6. Analyses of sugars and organic acids Five grams of pomegranate arils were extracted with 50 mL of the mixture of acetonitrile: water in 1% formic acid (20:80, v/v) by homogenization at 10 000 rpm for 2 min (Heidolph Silent Crusher M, Schwabach, Germany). After centrifugation at 7500g for 5 min, 1 mL of the supernatant was passed through a preconditioned Oasis HLB cartridge. The first eight drops of the eluent were discarded, and the rest was collected into an HPLC vial. Concentrations of sugars and organic acids were determined by using an Agilent Technologies (Waldbronn, Germany) 1100 HPLC system equipped with a refractive index (RI) detector, diode array detector (DAD), quaternary pump, auto-sampler and column oven.
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An isocratic elution with a mobile phase consisting 0.1% phosphoric acid in water (v/v) at a flow rate of 1 mL$min1 was used. A 0.01 mL of sample was injected into a Shodex Rspak KC-811 column (300 mm 7.8 mm i.d., 7 mm, Tokyo, Japan) at 20 C. Analysis of sugar was performed by using RI detector and organic acid was performed by DAD at 220 nm, on the same run. Identification of sugars and organic acids were accomplished by comparing the retention time of the peaks in pomegranate samples to those of standard compounds. The quantification of sugars and organic acids were based on external calibration curves of each compound. 2.7. Analysis of anthocyanins Five grams of pomegranate arils (pith and aril) were extracted with 50 mL of the mixture of acetonitrile: water in 1% formic acid (20:80, v/v) by homogenization at 10 000 rpm for 2 min (Heidolph Silent Crusher M, Schwabach, Germany). After centrifugation at 7500g for 5 min, supernatant was filtered through a 0.45 mm syringe filter. Anthocyanins were determined by using an Agilent 1200 series HPLC-DAD system coupled to an Agilent 6130 single quadrupole mass spectrometer. Chromatographic separation was carried out on a Phenomenex Polyphenol C12 (Synergi Max-RP 80 Å, 250 4.6 mm, i.d., 4 mm, Waldbronn, Germany) LC Column. A gradient mixture of solvent A (1% formic acid in water) and solvent B (1% formic acid in acetonitrile) was used at a flow rate of 0.7 mL$min1 at 30 C with the following gradient profile: 0e12 min, linear gradient elution from 10 to 30% B; 12e15 min, isocratic elution of 30% B; 15e18 min, linear gradient elution from 30 to 10% B; and 18e25 min, isocratic elution of 10% B. The injection volume was 0.01 mL. Data acquisition was performed in scan mode using the following interface parameters: drying gas (N2), flow of 130 mL$min1; nebulizer pressure, 275 kPa; drying gas temperature, 350 C; capillary voltage, 4 kV; fragmentor voltage, 1.93e-17 J. MS spectra were recorded in positive ion mode between m/z 100e500. Quantification of individual anthocyanins acquired from their DAD chromatograms by using external calibration curves of each standard compound built in a range between 0.1 and 10 mg/L. Confirmation of anthocyanins were based on comparison of the peaks to the retention times of standard compounds. Mass spectra of the peaks assigned as anthocyanins were also used for confirmation.
3
CFU g1) is the maximum growth at the stationary phase and t is the time (day). The experimental data were modelled using the curve fitting module in MATLAB. 2.9. Sensory evaluation Sensory evaluation of pomegranate arils was performed during storage by a sensory panel. The panel consisted of 8 members with sensory evaluation experience in fruit quality and they were trained in a pre-test for evaluating the aroma of pomegranate arils to be familiar with its quality attributes. Panelists were asked to evaluate color, freshness, taste, aroma, texture and overall acceptance using a 5-point scale described by Martínez-Romero et al., 2013. The rating for each characteristic was based on a five-point scale (5e1) with 5 corresponded to extremely liked (very characteristic of the fruit), 4 ¼ like moderately, 3 ¼ neither like nor dislike, 2 ¼ dislike moderately and 1 corresponded to extremely disliked (non-characteristic of the product). Scores of 3 and above were considered as acceptable for commercial purposes. The sensory evaluation was done in 1st, 7th, 14th and 25th days of storage. 2.10. Statistical analysis The results were reported as average ± standard deviations. Differences were estimated for all treatments by analysis of variance (ANOVA) followed by Tukey's Honest Significant Difference test (p < 0.05). Independent sample t-test were used to compare control group and 1%CH-1%AA coated samples in the results of chemical analyses. All statistical analyses were performed using the SPSS 18.0 version (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Evaluation of changes in weight loss, color-visual quality and chemical composition Chitosan-ascorbic acid coating did not affect weight loss as control, 1% ascorbic acid and coated fruit lost similar weight during the 28 days of storage (Fig. 1). The commercial weight loss limit is 4e6% for minimally processed fruits. In all treatments, the weight loss is under this commercial limit during storage time. Also, titratable acidity and pH did not change significantly during storage in both control and 1%CH-1%AA coated samples (Table 1). Vargas, Albors, Chiralt, and Gonzalez-Martinez (2006) coated
2.8. Total microbial counts and kinetic modelling Two samples were taken from each treatment on each sampling day. Aerobic mesophilic, yeast and mold counts on pomegranate arils subjected to different treatments were evaluated throughout storage (0, 7, 14, 21, 28th day). Total aerobic mesophiles were determined using PCA after incubation at 35 C for 48 h. Yeast and mold counts were performed on PDA by using the spread plate method after incubation at 25 C for 5e7 days. Colony forming units (CFU) per gram of arils were calculated and expressed in terms of log CFU g1. Additionally, the cell load data from each treatment were modelled according to the Gompertz equation modified by Zwietering, Jongenburger, Rombouts, and Van't Riet (1990):
log10
N N0
m e ¼ A exp exp m ðʎ tÞ þ 1 A
(1)
where N is the log CFU g1 at a given time t (day), N0 is the log CFU g1 at the beginning of storage, mm [D log (CFU g1) per day ] is the maximal growth rate, ʎ (days) is the lag time, A (log
Fig. 1. Weight loss (%) of pomegranate arils during storage at 5 C (C) control; (B) 1% AA, (D) 1%CH-1%AA, () 2%CH-2%AA. Vertical bars represent the standard deviation (n ¼ 3).
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Table 1 Titratable acidity (TA), pH, total soluble solids (TSS), organic acids and sugars in control group and 1%CHe 1% AA coated arils during storage at 5 C. Treatment
Day
pH
Control
0 7 14 21 28 0 7 14 21 28
3.86 3.89 3.86 3.91 3.86 3.93 3.96 3.99 4.01 4.05
1%CH-1%AA
T.A. (Citric acid eq. %) ± ± ± ± ± ± ± ± ± ±
0.01a 0.04a 0.00a 0.01a 0.04a 0.04a 0.01a 0.09a 0.05a 0.01a*
1.41 1.23 1.41 1.37 1.44 1.15 1.23 1.16 1.18 1.09
± ± ± ± ± ± ± ± ± ±
0.04a 0.01b 0.00a 0.00a 0.14a 0.99a 0.01a 0.09a 0.04a* 0.00a
T.S.S. (brix) 16.15 16.80 16.75 16.40 16.55 16.25 16.50 16.55 17.05 17.00
± ± ± ± ± ± ± ± ± ±
0.07a 0.14ab 0.07b 0.14ab 0.21ab 0.07a 0.00b 0.07b 0.07c* 0.00c
Total Organic Acids (g$kg1) 18.01 17.14 18.97 17.62 15.48 16.23 16.16 15.25 17.81 17.34
± ± ± ± ± ± ± ± ± ±
0.51a* 0.91a 2.47a 2.05a 1.44a 0.09a* 0.21a 0.37a 1.77a 0.62a
Total Sugars (g$kg1) 125.94 127.37 119.69 133.44 121.74 126.36 125.48 118.29 126.77 122.20
± ± ± ± ± ± ± ± ± ±
2.47a 0.98a 9.40a 7.57a 2.31a 3.35a 3.62a 3.13a 6.97a 5.34a
Values are given in average ± standard deviation (n ¼ 2). Different letters within the same column in each treatment indicates a significant difference (p < 0.05). (*) indicates a significant difference determined between chitosan-ascorbic coated and control samples in the same storage day (p < 0.05).
Fig. 2. Photographs of control arils, 1%AA treated arils, 1%CH-1%AA, and 2%CH-2%AA coated arils at the 0th, 7th and 14th day of storage.
€ €kmen, V., Extending the shelf-life of pomegranate arils with chitosan-ascorbic acid coating, Please cite this article in press as: Ozdemir, K. S., & Go LWT - Food Science and Technology (2016), http://dx.doi.org/10.1016/j.lwt.2016.10.057
€ €kmen / LWT - Food Science and Technology xxx (2016) 1e9 K.S. Ozdemir, V. Go Table 2 Changes in CIE L*a*b* color values of arils during storage at 5 C. Treatment
Day
L*
Control
1 7 14 21 28 1 7 14 21 28 1 7 14 21 28 1 7 14 21 28
35.0 34.3 34.8 30.5 38.4 36.0 34.0 37.5 39.4 38.7 33.2 32.2 36.7 35.4 40.4 35.5 37.9 32.9 35.7 41.1
1%AA
1%CH-1%AA
2%CH-2%AA
a* ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.7ab 3.9abAB 4.9ab 3.3aA 2.5b 3.2ab 2.6aAB 4.4a 4.0bB 3.6b 3.0a 3.4aA 4.7ab 4.8aAB 2.3b 4.4a 3.2bB 2.9a 4.9aAB 4.1b
b*
41.3 41.7 38.3 37.5 27.5 46.2 43.3 43.7 45.7 29.8 41.3 42.1 42.4 43.2 36.7 42.7 44.7 39.7 38.0 33.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.6aA* 4.2a 7.0a 6.1aA 5.4bA 2.0aB 3.7a 2.9a 5.2aB 4.6bAB 2.8abA 3.2a 4.6a 3.5aB 4.3bC 3.5abAB 2.6a 1.7ab 3.7bcA 3.3cBC
28.2 29.1 26.7 27.6 16.7 30.4 29.6 28.1 31.7 15.5 27.0 29.1 27.3 29.0 19.0 28.1 29.7 26.7 26.4 18.8
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.5a 3.7a 4.5a 4.7aAB 2.8b 1.8a 2.9a 2.8a 3.5aA 3.9b 2.7a 2.6a 4.4a 3.7aAB 2.2b 2.6a 2.04a 1.7a 4.6aB 2.9b
Values are mean ± standard deviation (n ¼ 10). Values in the same column within the same treatment having different superscript lowercase letters are significantly different (p < 0.05). Values in the same column on the same storage day having different superscript uppercase letters are significantly different (p < 0.05).
strawberries with 1% chitosan and 1%, 2%, 4% oleic acid and stored at 4±1 C for 10 days. They reported that soluble solid content, acidity and pH did not change significantly during storage and were not affected by coating application. Our pH and titratable acidity results are in agreement with the results of Vargas et al. (2006). However, throughout 28 days of storage the total soluble solid content increased to 17% in coated arils while it was 16.55% in control (Table 1). In control samples, there was a gradual increase in TSS content until the 7th day of storage and after that a decline occurred. The TSS increased in the early stage of storage was attributed to the activity of microorganisms that decomposes polymeric carbohydrates into their sub-units. The decline occurred in TSS after 7th day of storage due to the high metabolism of the arils and senescence processes. The color characteristics of pomegranate arils and their visual qualities are presented in Fig. 2 and Table 2. Pomegranate arils coated with chitosan and ascorbic acid showed no signs of deterioration and therefore was acceptable for consumption. Chitosanascorbic acid coated arils and control samples showed stability in L* parameter during storage at 5 C, however chitosan-ascorbic acid coated arils showed significantly higher lightness (p < 0.05) on day
5
21 compared to control. The findings also showed that a* value, which is related to redness and color stability, was significantly higher in chitosan coated arils compared to the control samples after 28 days of storage. This can be confirmed visually from the images of the samples presented in Fig. 2. Parameter b* showed stability in all samples during 21 days of storage, however it was significantly reduced on the 28th day (p < 0.05). The change in b* value points to decrease in yellowness and consequently lower saturation (Kapetanakou, Stragkas, & Skandamis, 2015). Glucose and fructose were the main sugars in pomegranate arils (Table 3). Fructose content did not change in coated samples and it was also quite stable in control samples with an average content of 70.4 g$kg1 during 28 days of storage. Similarly, glucose whose overall content was slightly lower than fructose (55.2 g$kg1) showed slight fluctuations, but no significant reduction occurred. Melgarejo et al. (2000) determined sugar contents of 40 Spanish pomegranate cultivars. Fructose and glucose content ranged between 59.6 and 70.4 g$kg1 and 56.6e64.5 g$kg1, respectively. The levels of fructose and glucose contents in the present work are comparable with literature. Citric, malic and lactic acids were the main organic acids determined in pomegranate arils. Citric acid was the dominant acid in control samples with 11.14 g$kg1 in comparison with 6.05 g$kg1 malic acid and 0.83 g$kg1 lactic acid. Similarly, 9.95 g$kg1 citric, 5.56 g$kg1 malic acid and 0.72 g$kg1 lactic acid were determined in chitosan-ascorbic acid coated arils in the beginning of storage. These amounts did not change during the lu, Go €kmen, and Artık (2002) determined organic storage. Poyrazog acid composition of thirteen pomegranate varieties and they found citric, malic, tartaric, oxalic, D-(-)quinic and succinic acids in pomegranate juices. Similarly to our results, citric acid was the predominant acid with a range of 0.33e8.96 g$L1. Citric acid and malic acid concentrations found relatively higher in our study than the reported levels in literature. Anthocyanins are phenolic compounds responsible for typical red color of pomegranates. The main anthocyanin pigments found in pomegranate arils were determined as Cy-3-G, Cy-3,5-dG, Pg-3G and Pg-3,5-dG. The most predominant anthocyanin was Cy-3-G, while Pg-3-G was present in the lowest amount in all samples (Fig. 3). Individual anthocyanin contents showed stability during 21 days of storage in both control and 1%CH-1%AA coated samples. However, after 28 days of shelf-life, Cy-3-G decreased in coated samples (Fig. 3). The concentration of Cy-3-G was determined as 821.85 mg$kg1 and 956.21 mg$kg1 in control samples and 1%CH1%AA coated samples respectively, in the beginning of the storage time. It was 982.46 mg$kg1 and 708.41 mg$kg1 for control and 1% CH-1%AA coated samples at the end of storage. Anthocyanin
Table 3 Sugar and organic acid composition of control group and 1%CH-1%AA coated arils during storage at 5 C. Treatment
Day
Sugars (g$kg1)
Control
0 7 14 21 28 0 7 14 21 28
55.40 56.13 52.87 58.32 53.29 56.53 56.12 52.19 53.56 53.09
Glucose
1%CH-1%AA
± ± ± ± ± ± ± ± ± ±
Organic acids (g$kg1) Fructose
0.89a 0.28a 3.50a 2.62a 1.18a 0.45a 0.25a 1.19a 2.96a 1.79a
70.54 71.23 66.82 75.12 68.46 69.84 69.36 66.10 73.21 69.11
± 1.58a ± 1.26a ± 5.89a ± 4.95a þ 1.12a ± 2.91a ± 3.87a ± 1.95a ± 4.01a ± 3.54a
Citric acid
Malic acid
11.14 ± 0.06a* 9.74 ± 0.23a 11.06 ± 0.45a* 9.87 ± 0.70a 9.53 ± 0.53a 9.95 ± 0.20ab* 9.88 ± 0.04ab 9.38 ± 0.06a* 11.08 ± 0.66b 9.70 ± 0.11a
6.05 6.58 7.08 6.96 5.15 5.56 5.46 4.98 5.81 6.68
± ± ± ± ± ± ± ± ± ±
0.45a 0.71a 1.90a 1.20a 0.95a 0.26a 0.21a 0.03a 1.13a 0.78a
Lactic acid 0.83 0.82 0.83 0.80 0.81 0.72 0.82 0.89 0.92 0.97
± ± ± ± ± ± ± ± ± ±
0.12a 0.03a 0.11a 0.16a 0.04a 0.03a 0.04a 0.28a 0.03a 0.05a
Values are given in mean ± standard deviation (n ¼ 2). Different superscript letters within the same column in each treatment indicates a significant difference (p < 0.05). (*) indicates a significant difference determined between chitosan-ascorbic coated and control samples in the same storage day (p < 0.05).
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Fig. 3. Changes of individual anthocyanin concentrations (mg$kg1) of 1%CH-1%AA coated arils ( ) and control ( )during storage at 5 C. a) cyanidin-3,5-diglucoside, b) cyanidin-3-glucoside, c) pelargonidin-3,5-diglucoside, d) pelargonidin-3-glucoside. Vertical bars represent the standard deviation (n ¼ 2).
synthesis could continue during postharvest storage of pomegranates. Increases in anthocyanin concentration after harvest during cold storage have been reported for fruits other than pomegranates such as raspberry (Han, Zhao, Leonard, & Traber, 2004). Miguel, Fontes, Antunes, Neves, and Martins (2004) stated that this was correlated with the activity of the anthocyanin biosynthesis enzymes. Chitosan films form a very effective gas barrier, probably due to the dense structure of the film (Wong, Gastineau, Gregorski, Tillin, & Pavlath, 1992). It is possible that anthocyanin synthesis was reduced by the reduction in gas metabolism and significantly inhibited with the combination of chitosan and ascorbic acid barrier. Similar results were found for raspberries coated with chitosan after harvest and stored at 0 C for 12 days (Tezotto-Uliana et al., 2014). According to these results, we expected that a similar trend will be observed in 2%CH-2%AA coated samples. So these chemical analyses were not carried out for 2%CH2%AA coated arils. 3.2. Microbial stability of pomegranate arils The influence of different coatings on the growth of aerobic mesophilic bacteria, yeast and mold populations during storage are
shown in Fig. 4. Coating with chitosan and ascorbic acid significantly reduced bacteria, yeast and mold populations throughout the storage time (Fig. 4a and b). In the first day, initial aerobic mesophilic bacteria were below 1 log CFU g1, yeast and mold counts on fresh arils were below 2 log CFU g1. Treatments with 1%CH-1%AA and 2%CH-2%AA were helpful in reducing the fungal load throughout storage time. In 2%CH-2%AA treated arils, yeast and mold counts were under 2 log CFU g1 until 21st day, while it was higher in control and 1%AA treated arils throughout storage. Aerobic mesophilic count was determined as 6.8 log CFU g1 and 5.9 log CFU g1 in control samples and 1%AA treated samples at the end of storage respectively. However, there was no growth of aerobic mesophilic bacteria in 1%CH-1%AA and 2%CH-2%AA coated pomegranate arils at the end of storage. Antimicrobial activity of chitosan has been confirmed by previous studies on other fresh fruits and vegetables (Alvarez, Ponce, & Moreira, 2013; Devlieghere et al., 2004; Jiang & Li, 2001; Moreira, Ponce, Ansorena, & Roura, 2011). Pushkala, Parvathy, and Srividya (2012) enhanced the microbiological quality of carrot shreds by using chitosan powder coating. Jiang and Li (2001) delayed the increase in decay of stored longan fruit by chitosan coating. Besides chitosan antimicrobial activity, the type of solvent could affect its
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resulted in easy penetration of the chitosan molecule into the cytoplasmic membrane of bacterial cell wall and changing the internal pH of the bacteria (Cadogan, Lee, Popuri, & Lin, 2014). The microbial counts were also modelled by using modified Gompertz equation. The model appropriately fitted the experimental data since the coefficient of determination ranged from 0.98 to 1.00. Maximal growth rate, maximum growth at the stationary phase, and lag phase defined by the models are given in Table 4. The lag phase time and the maximal growth rate were highly influenced by the composition of coating solutions. The growth of yeasts and molds was delayed by chitosan-ascorbic acid coating hence 1% CH-1%AA and 2%CH-2%AA coated arils exhibiting longer lag time (ʎ) (9.90 and 17.04 days) than in control group and 1% AA treated arils (7.32 and 4.14 days, respectively). More rapid microbial growth were observed for the populations in control group and 1%AA treated arils. Gompertz model described the evolution of aerobic mesophilic counts as well as yeast and mold counts. The lag phase time and the maximal growth rate were determined for control and 1%AA treated arils but these parameters cannot be calculated for chitosan-ascorbic acid coated arils due to lack of growth. In arils treated with 1%AA, lag time (ʎ) for aerobic mesophilic populations was lower and maximal growth rate (mmax) was higher compared to the control. 1%AA treatment increased the acidity on the aril surface which provided favorable environment for microorganisms. In contrast, no microbial growth was observed in 1%AA-1%CH and 2% AA-2%CH coated arils. Moreira et al. (2011) found similar patterns for aerobic mesophilic bacteria, yeast and mold growth in fresh cut broccoli coated with chitosan. The application of chitosan coating extended the lag phase in comparison to the control sample of fresh-cut broccoli. Longer lag time and lower maximum specific growth rates are preferred by the industry (Zimmermann, Miorelli, Massaguer, & Aragao, 2011). 3.3. Sensory quality Table 5 shows the effects of different coatings on the sensory attributes and acceptance of pomegranate arils during cold storage. Control and 1%AA showed similar scores as the coated arils at the beginning of the storage (on day 1 and 7), in all sensory attributes (p > 0.05). However, on 14th day of storage control and 1%AA treated arils were microbiologically rejected. Contrary to that, coated samples were still organoleptically acceptable with scores above 3, regardless of the chitosan and ascorbic acid concentration (Table 5). Moreover, 1%CH-1%AA coating maintained product attributes such as aril color, taste, aroma and texture even on the 25th day of storage. In addition, the panelists did not perceive any offflavor in pomegranate arils as a consequence of chitosan and ascorbic acid treatment.
Fig. 4. Growth of (a) yeasts and moulds, and (b) aerobic mesophilic bacteria on Control (*) samples, 1%AA (), 1%CH -1%AA (þ), 2%CH-2%AA (:) coated pomegranate arils during storage with Gompertz model fits.
antimicrobial action due to the pH of chitosan/solvent solution. According to a study, ascorbic acid solvated chitosan membrane showed a higher antibacterial activity than citric, maleic, oxalic and tartaric acid due to its crosslinking effect which shrinks the intersegmental space of the polymer chains and reduction its size. This
Table 4 Gompertz parameters for yeast and mould and mesophilic aerobic microbial growth in arils treated with different coating solutions. Population
Dipping Condition
Gompertz model parameters
mm
A Yeast and mold
Mesophilic aerobic bacteria
Control (water) 1%AA 1%CH-1%AA 2%CH-2%AA Control (water) 1%AA 1%CH-1%AA 2%CH-2%AA
7.31 7.69 5.29 5.45 6.89 4.43 e e
± ± ± ± ± ±
0.10 0.37 0.06 0.07 0.02 0.66
1.15 0.89 0.50 1.19 0.55 0.71 e e
± ± ± ± ± ±
0.25 0.27 0.03 0.14 0.24 0.40
ʎ
R2
7.32 ± 1.86 4.14 ± 1.26 9.90 ± 0.55 17.04 ± 0.31 10.42 ± 1.37 5.59 ± 0.71 e e
1.00 0.99 1.00 1.00 0.98 1.00 e e
Gompertz parameters: A, maximum growth attained at the stationary phase; mm, maximal growth rate; ʎ, lag phase period; R2, regression coefficient. Significance level at p < 0.05. Values are given in average ± standard deviation (n ¼ 4). () Indicates no growth.
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Table 5 Sensory evaluation during storage of coated and uncoated pomegranate arils. Application
Panelist scores Day 1
Color
Freshness
Taste
Aroma
Texture
Overall acceptance
Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA Control 1%AA 1%CH-1%AA 2%CH-2%AA
4.1 3.9 4.4 4.3 4.1 3.9 4.1 3.9 3.4 3.8 3.8 3.5 4.1 4.0 4.1 3.8 4.5 4.5 4.6 4.6 4.0 3.8 4.4 4.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Day 7 a
1.1 1.4a 0.9a 1.2a 0.8a 0.8a 1.0a 1.1a 1.2a 1.2a 1.3a 1.2a 1.0a 1.1a 0.8a 1.6a 0.8a 0.5a 0.5a 0.5a 0.5a 1.2a 0.7a 1.0a
3.6 4.4 4.0 2.9 3.3 3.8 3.8 3.4 3.3 3.5 4.0 3.9 3.3 3.3 3.4 3.5 3.8 4.0 3.9 3.3 3.0 3.4 4.0 3.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Day 14 ab
0.7 0.7a 0.8a 0.8b 1.0a 0.9a 1.0a 0.9a 1.4a 0.9a 1.1a 0.4a 0.7a 0.7a 0.7a 0.5a 0.9a 0.5a 1.0a 0.7a 0.8a 0.5a 0.9a 0.9a
3.2 3.5 4.0 3.0 e e 3.4 3.3 e e 3.5 3.1 e e 3.2 3.1 e e 3.4 3.1 e e 3.4 3.0
± ± ± ±
Day 25 ab
1.0 0.8ab 0.9a 0.7b
± 0.7b ± 0.5ab
± 0.9a ± 0.7a
± 0.7a ± 0.7a
± 0.7a ± 0.7a
± 0.5a ± 0.9a
-* e 3.4 2.3 e e 2.1 2.3 e e 3.0 2.5 e e 3.4 3.0 e e 2.9 2.4 e e 2.6 2.3
± 0.7a ± 0.9b
± 1.0a ± 1.0a
± 1.4a ± 0.7a
± 0.7a ± 1.1a
± 0.8a ± 0.9a
± 1.2a ± 0.9a
Data are the mean ± standard deviation (n ¼ 8). For each parameter, values followed by different superscript letters denote a significant difference at p < 0.05.; comparison was done across treatments on the same storage day. Scores of 3 and above were considered as acceptable. (*) Data was not obtained due to the microbiological spoilage of sample.
4. Conclusion In conclusion, the results revealed that microbiological shelf-life of pomegranate arils can be significantly improved by means chitosan-ascorbic acid coating during cold storage, owing to antimicrobial activity of chitosan. In this way, color visual quality of pomegranate arils was also protected because ascorbic acid in the binary coating mixture is very effective anti-browning agent. As a practical guiding, coating of the pomegranate arils with an aqueous mixture of 1% chitosan and %1 ascorbic acid is recommended to ensure a shelf-life of 3 weeks under cold storage conditions (5 C). However, uncoated arils (control) could not reach this far (<10 days). By the end of storage, 1% chitosan and %1 ascorbic acid coated arils preserved their color, taste and aroma. Overall, the use of chitosan-ascorbic acid coating could be applied on perishable minimally processed fruits to extend shelf-life by improving microbiological safety, preserving nutritional and sensory quality. References Alvarez, M. V., Ponce, A. G., & Moreira, M. D. (2013). Antimicrobial efficiency of chitosan coating enriched with bioactive compounds to improve the safety of fresh cut broccoli. LWT-Food Science and Technology, 50(1), 78e87. s, F., Villaescusa, R., & Tudela, J. A. (2000). Modified atmosphere packaging of Arte pomegranate. Journal of Food Science, 65(7), 1112e1116. Aviram, M., & Dornfeld, L. (2001). Pomegranate juice consumption inhibits serum angiotensin converting enzyme activity and reduces systolic blood pressure. Atherosclerosis, 158(1), 195e198. Cadogan, E. I., Lee, C. H., Popuri, S. R., & Lin, H. Y. (2014). Effect of solvent on physicochemical properties and antibacterial activity of chitosan membranes. International Journal of Polymeric Materials and Polymeric Biomaterials, 63(14), 708e715. Chien, P. J., Sheu, F., & Yang, F. H. (2007). Effects of edible chitosan coating on quality and shelf life of sliced mango fruit. Journal of Food Engineering, 78(1), 225e229. Devlieghere, F., Vermeulen, A., & Debevere, J. (2004). Chitosan: Antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables. Food Microbiology, 21(6), 703e714. Dutta, P. K., Tripathi, S., Mehrotra, G. K., & Dutta, J. (2009). Perspectives for chitosan based antimicrobial films in food applications. Food Chemistry, 114(4), 1173e1182.
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