Food Packaging and Shelf Life 22 (2019) 100409

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Food Packaging and Shelf Life

journal homepage: www.elsevier.com/locate/fpsl

Starch-based edible coating formulation: Optimization and its application to T

improve the postharvest quality of “Cripps pink” apple under different
temperature regimes
Rahul Thakura, , Penta Pristijonoa, Christopher J. Scarletta, Michael Bowyera, S.P. Singhb,
⁎

Quan V. Vuonga,
⁎

a
School of Environmental and Life Sciences, University of Newcastle, Ourimbah, NSW 2258, Australia
b
NSW Department of Primary Industries, Ourimbah, NSW 2258, Australia

ARTICLE INFO ABSTRACT

Keywords: This study demonstrated Response Surface Methodology (RSM) optimisation of starch edible coating formulation and
Starch its application on apple fruit quality and storage life at two different temperatures (20 °C and 5 °C + 1 day at 20 °C).
Edible film Films were optimised for physical, mechanical and permeability properties and the optimal formulation was sprayed
Edible coating over the fruit surface. Fruit weight loss, respiration rate, total soluble solids (TSS), titratable acidity (TA), firmness,
Postharvest storage
greasiness and change in the fruit skin colour were measured. The effect of the coating treatment on changes to the
Shelf life
bioactive profile (phenolics and free radical scavenging activity) of the fruit was also analysed. The results obtained
from this study showed that the optimised formulation (rice starch 2.5% ι-car 1.5% sucrose fatty acid ester 2%
glycerol 1.5%) in combination with low temperature was effective in reducing weight loss and maintained tissue
firmness without affecting TSS, TA and the bioactive profile of the apple fruit during postharvest storage. A delay in
skin colour change and a significant reduction in fruit greasiness was observed in coated fruit (p < 0.05). These
results show the potential of starch-based coating formulation in the formulation in improving the visual appearance,
without affecting the fruit internal quality and nutritional properties of apple fruit during storage.

1. Introduction
Coating fruit prior to marketing is an important postharvest treat-
ment commonly practiced to enhance visual gloss and extend shelf life.
Shellac and carnauba wax are the most commonly used as commercial
coatings for the apple fruit to improve its fruit gloss, control water loss
and maintain the fruit quality through delayed ripening and senescence
(Bai, Alleyne, Hagenmaier, Mattheis, & Baldwin, 2003). These materials
have inherent problems including poor gas permeability, skin whitening
or blushing during cold storage conditions (Conforti & Totty, 2007), but
they successfully delay postharvest ripening and improve the postharvest
qualities of the fruit. A range of different coating formulations have been
tested in an effort to improve these limitations, including carboxymethyl
cellulose (Conforti & Totty, 2007), soy protein (Liu et al., 2017), chitosan
(Khalifa, Barakat, El-Mansy, & Soliman, 2017) and alginates (Olivas,
Mattinson, & Barbosa-Cánovas, 2007). To date, however, no single
coating thus far has proved comparable to current commercial coatings
in terms of shelf life extension and fruit quality improvement.
Surface edible coatings can control the fruit peel permeance, modify
the internal atmosphere, reduce the water loss and slow down the fruit
respiration rate (Embuscado & Huber, 2009; Pavlath & Orts, 2009).
Within the apple industry, when visual cues are not available, con-
sumers judge apple ripening by assessing skin greasiness, change in skin
background colour and firmness, perceiving through the touch of the
fruit skin (Dobrzanski & Rybezynski, 2002). Failure of the apple in-
dustry to meet these standards can result in product rejection and a
reduced financial return. Therefore, maintaining the postharvest
quality of apple fruit during storage and distribution is one of the major
challenges facing the apple industry.
According to Thakur et al. (2017), the efficiency of fruit coating
materials primarily depends on the nature of the coating ingredients
and their relative, optimal concentrations. Optimisation of coating
materials to develop edible films has been conducted in recent research
studies (Dave, Rao, & Nandane, 2016; Saberi et al., 2016). To determine
the optimum conditions, a model is usually used to create a validity
map (Li, Hu, & Xiao, 2017). A good approach to optimising coating

⁎
Corresponding author at: School of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Brush Road,
Ourimbah, NSW 2258, Australia.
E-mail addresses: Rahul.thakur@uon.edu.au (R. Thakur), Vanquan.Vuong@newcastle.edu.au (Q.V. Vuong).

https://doi.org/10.1016/j.fpsl.2019.100409
Received 7 November 2018; Received in revised form 11 May 2019; Accepted 26 September 2019
2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
R. Thakur, et al.
materials is the Response Surface Methodology (RSM) approach. RSM is
one of the most relevant multivariate techniques with the advantages of
reducing experimental runs and showing the interaction between
variables as well as optimising the conditions (Dave et al., 2016).
Edible coatings are more successful in a composite form as a single in-
gredient is not effective enough for improving shelf life and quality of fruit.
In previous studies, starch biopolymer has been successfully blended with
polysaccharides gums (Eom, Chang, Lee, Choi, & Han, 2018; Saberi et al.,
2018), gelatine (Fakhouri, Martelli, Caon, Velasco, & Mei, 2015), mixed
polysaccharides for e.g., carrageenan (Techawipharat, Suphantharika, &
BeMiller, 2008; Thakur et al., 2016) chitosan (Pinzon, Garcia, & Villa, 2018)
and proteins (Chinma, Ariahu, & Abu, 2012). The compatibility and strong
complex forming ability of starch with carrageenan have been also in-
vestigated in previous studies (Huc et al., 2014; Lascombes et al., 2017). The
composite has provided an economical and potential option for film de-
velopment. In our previously studies, we found that starch-ι-carrageenan
composites created films with improved attributes (Thakur, Pristijono et al.,
2017; Thakur et al., 2016). Rice starch-carrageenan composite matrices are
potential for fruit coating applications, none of the previous studies have
reported on the application of this composite for coating apple. Therefore,
the current study aimed to (i) obtain the optimised film formulation from
rice starch-carrageenan composite matrices using RSM and (ii) to apply this
optimised formulation to apple stored under two different storage tem-
peratures (20 °C and 5 °C + 1day at 20 °C).
2. Materials and methods
2.1. Materials
2.1.1. Procurement of fruit and film/coating material
Rice grains (Oryza sativa var. Doongara) were obtained from Sunrice
(Sun Rice, Leeton Australia). The ι-car (Chondrus crispus) was purchased
from Melbourne Food Ingredient Depot, Australia. Sucrose fatty acid
ester was obtained from the Tokyo Chemical Industry Co. Ltd Japan.
Glycerol was obtained from Ajax Finechem Pty. Ltd, Australia. Starch
isolation and characterisation of its chemical composition are described
elsewhere (Thakur et al., 2016). The uncoated apple fruit (variety
Cripps Pink) free from physical damage and contamination were col-
lected from a local shop and coated on the day of purchase.
2.2. Methods
2.2.1. Formation and conditioning of films
The film-forming solution was prepared according to the method
described by Thakur et al. (2016). Briefly, starch (3%) and ι-carra-
geenan (2%) were prepared separately with constant agitation on a hot
plate using a magnetic stirrer (200 rpm) for 15 min. Sucrose ester (2%)
was added to the warm (starch-ι-car) solution with the subsequent
addition of glycerol (1.5%) with continuous stirring. 20 mL of final film
solution was poured into Petri plates and dried in the oven for 24 h
under controlled conditions (35 °C, RH 50%). For evaluation, films were
peeled off and conditioned at 27 °C, RH 60% for 72 h.
2.3. Edible film properties
2.3.1. Film thickness, solubility and water vapour permeability
A digital micrometer (Mitutoyo, Co., Code No. 543-551-1, Model ID-
F125, 139 Japan; sensitivity = 0.001 mm) was used to measure the film
thickness (n = 3; 10 measurements per film). Average thickness value
was used to measure the water vapour permeability (WVP) of film
samples. Film solubility was determined according to a previously re-
ported method (Saberi et al., 2016). WVP was measured using the ASTM
E96-92 method described by Martins et al. (2012) and calculated as:
WVP =
m T
A t P (1)
2
Food Packaging and Shelf Life 22 (2019) 100409
Where m / t = weight of moisture gain per unit time (gs−1) and can be
calculated by the slope of the graph; A= area of the exposed film surface
(m2); T = thickness of the film (mm); P = represents the water vapour
pressure difference inside and outside of the film (Pa).
2.3.2. Mechanical properties
2.3.2.1. Tensile strength (TS) and elongation at break (EAB). Tensile
strength (TS) and elongation at break (EAB) were determined with the
Texture Analyzer (LLOYD Instrument LTD, Fareham, UK) using the
ASTM standard method - D 882-91 (Martins et al., 2012). Pre-
conditioned (60% RH) films (15 x 40 mm) were placed between the
tensile grips with 40 mm separation and 1 mm/s crosshead speed
respectively. 10 film samples of each type of films were replicated. TS
was expressed in Nm2 and EAB was expressed in mm.
2.4. Experimental design and statistical analysis
2.4.1. Box-Behnken experimental design (BBD)
Optimization of film solution requires many experiments which are
time-consuming, often leading to poor optimization efficiency.
Therefore, RSM was applied to study the effect of variables and their
responses using the minimum number of experiments and experimental
conditions. RSM is a combination of statistical and mathematical
methods which are useful for developing, improving and optimizing
processes (Agbovi & Wilson, 2017; Thakur, Saberi et al., 2017). A Box-
Behnken design was employed which comprised of four independent
variables, viz., starch (1–3% w/w), carrageenan (0.5–2% w/w), sucrose
esters (0.5–2 w/w) and glycerol (0.3–2 w/v) were selected to study and
optimize the influence of process variables. The levels of independent
variables were selected based on preliminary experiments (data not
shown). The designed model was used to predict the relationship be-
tween the response studied (thickness, WVP, solubility, tensile strength
and elongation and break) and the selected experimental variables. The
design recommended 27 experimental runs which were randomized to
maximize the effects of unexplained variability in the observed re-
sponses. In this regard, the postulated quadratic polynomial model is
represented by equation 1. JMP software (Version 22, SAS, Cary, NC,
USA) was used to study the regression model and statistical analysis.
k k 1 k k
Y= o i Xi + i=1 ij Xi Xj + ii Xi2
i=1
i<j
i=2 i=1
(2)
Xi = independent variables; 0= intercept; i, ii, ij = regres-
sion coefficients for intercept, linear, quadratic, and interaction terms;
k = number of variables
Statistical analysis was performed using SPSS 23.0.0 statistical soft-
ware for Windows (IBM SPSS statistics version 24.0 Inst., Cary, North
Carolina, USA). All measurements were performed in triplicate and the
results reported mean values. To evaluate the fit of the model, an analysis
of the variance (ANOVA) was undertaken. Mean values were evaluated
using the Duncan test, with p < 0.05 deemed statistically significant.
Multiple correlation coefficients (R2) and adjusted R2 were used as an
indicator to evaluate the fitness of the second order polynomial equation.
3-D plots were prepared to demonstrate the relationship and interactions
between the independent variables and responses. The final optimal
formulation for the film was obtained by solving the regression equation
and also by analyzing the surface plots. The fitted model was used to
study the relative sensitivity of the responses to the variables and to
determine the optimum ingredient concentration.
2.5. Fruit coating experimental design
A randomized experimental design, comprising 84 homogeneous
lots (same size) was established. Four lots were used to measure the
fruit properties at day 0 and the remaining 80 lots were divided into 2
groups (40 lots each) for control and coating treatments. The starch
coating (0.5 mL) was applied over the fruit surface in a circular motion
R. Thakur, et al.
using gloved hands ensuring the whole fruit was coated uniformly. The
freshly coated fruit was then dried using a hair dryer (controlled tem-
perature 35 °C for 15 s) then left to stand at room temperature (20 °C)
for 1 h for complete drying. Control and coated fruit were further di-
vided into two groups for a storage study at 20 °C (RH 55 ± 2%) and
5 °C (RH 90 ± 2%) + 1 day at 20 °C. Four lots (four replications) from
each treatment were assessed on the sampling day to gauge the effect of
coating treatment on fruit physiological and nutritional properties.
Fruit stored at 20 °C was assessed at weeks 1, 2, 3, 4 and 5; while fruit
stored at 5 °C + 1 day at 20 °C was assessed at weeks 2, 4, 6, 8 and 10.
2.5.1. Determination of weight loss percentage and respiration rate
Weight loss of the fruit was determined as the difference between the
initial and final fruit weight with the results expressed as a percentage
loss of the initial weight. Fruit respiration rate was measured using the
method reported by Thakur et al. (2018a); Thakur et al., 2018b. Briefly,
apples were transferred to a sealed, 1500 mL glass jar at 20 °C for 1 h,
after which a gas sample (1 mL) was collected by syringe and the carbon
dioxide content was analysed. Carbon dioxide concentration was mea-
sured to within 0.1% using an ICA40 series low volume gas analysis
system (International controlled Atmosphere Ltd., Kent, UK).
2.5.2. Determination of total soluble solids (TSS) and titratable acidity
(TA)
For the assessment of total soluble solids (TSS) and titratable acidity
(TA), fruit samples were chopped into small pieces and blended in a juicer
mixer. TSS, expressed as °Brix, was measured from the pressed juice of
fruit with a digital refractometer (ATAGO Inc., Bellevue, WA, USA). TA
was determined by titrating 5 mL of juice with 0.1 N NaOH standard
solution to pH 8.2 using an automatic titrator (Mettler Toledo T50,
Switzerland) and the result was expressed as a percentage of malic acid.
2.5.3. Fruit firmness, greasiness and background colour
Fruit firmness (n = 7) per replication was measured at the peeled
sides with a firmness tester (Penetrometer) equipped with an 8 mm
probe. The average of 2 reading points from each side of the fruit was
taken. The firmness results were expressed in N/m. Apple fruit greasiness
was recorded subjectively by rubbing fruit against the hand and scoring
the degree of greasiness as, (1) none or very low, (2) slight, (3) moderate
(4) high (5) and severe (Dadzie, Banks, Hewett, & Cleland, 1995). For the
skin background colour, the scoring index was (1) deep green, (2) green
(3) more green than yellow (4) more yellow than green.
2.5.4. Polyphenol determination
2.5.4.1. Total polyphenolic content (TPC) and DPPH radical scavenging
activity. A modified Folin-Ciocalteu method as described by Thakur
et al. (2018a); Thakur et al., 2018b was used for the determination of
total polyphenolic content (TPC). Briefly, diluted juice sample (1 mL of
fruit juice in 50 mL of water) was mixed with 5 mL of Folin-Ciocalteu
reagent and 4 mL of 7.5% Na2CO3 solution. The mixture was left at
room temperature for 1 h to complete the reaction. The optical
absorbance was measured at 765 nm using a UV-spectrophotometer
(Varian Australia Pty. Ltd., Victoria, Australia). A calibration curve
(R2 = 0.998) was constructed with ten points using gallic acid as a pure
standard. The results were expressed as gallic acid equivalents (GAE)
mg GAE mL−1 of fresh fruit juice sample. The DPPH (1,1-diphenyl-2-
2picrylhydrazyl) radical scavenging activity was determined using the
assays described previously (Bhuyan et al., 2015). Trolox was used as
standard (R2 value for standard curve = 0.997) and results expressed as
millimole Trolox equivalent (mg TE mL−1 juice sample).
2.6. Statistical analysis
JMP software v. 11 (SAS, Cary, NC, USA) was used to establish the
model equation. The adequacy of the RSM second-order polynomial
model was determined based on the lack of fit and coefficient of
3
Food Packaging and Shelf Life 22 (2019) 100409
determination (R2). Analysis of variance (ANOVA) was performed on the
test data (films and fruit samples) by using the SPSS software package, v.
24.0 for Windows (SPSS, Inc., Chicago, IL). Analyses of films samples
were carried out in triplicate. For the fruit quality assessment, fruit
samples with four replications including seven fruit per replications were
used. Multiple Ranges Duncan’s test was used to determine whether
differences among treatments were significant at p < 0.05.
3. Results and discussion
3.1. Statistical analysis and model fitting of Box-Behnken design
Different concentrations of the ingredients were studied. Data from
the targeted responses were obtained from the different film formulations
using BBD (Table 1). The optimization procedure comprises of designing
experiments, estimating the coefficients by fitting the experimental data
to the response functions, predicting the responses of the fitted model and
the goodness of fit of the model (Agbovi & Wilson, 2017). ANOVA was
used to conclude the suitability of the response functions and the sig-
nificance of the effect of the independent variables. Results obtained from
the analysis of variance of the design for establishing the model fit is
demonstrated in Table 2. In the ANOVA table second-order polynomial
equation represents the actual relationship between the dependent and
independent variables. The lack of fit and analysis of variance were used
to evaluate the suitability of response variables. RSM mathematical model
was calculated for thickness, WVP, solubility, tensile strength and elon-
gation at break. The p-values for the aforementioned process variables
were 0.0004*, 0.008*, 0.002*, 0.0001*, and 0.002*, respectively. The p-
values were less than 0.05, verifying the reliability of the model. The
higher F value (Table 2) for these film attributes showed that the math-
ematical model was a successful predictor of thickness, WVP, solubility,
tensile strength and elongation at break properties of rice starch compo-
site films. The goodness of fit by the regression model was evaluated by
the coefficient of R2 and adjusted R2 values. In fact, the better model was
obtained for the higher values of R2. Different process variables were
estimated satisfactorily with R2 values of 0.91 (T), 0.83 (WVP), 0.87 (Sol),
0.96 (TS), and 0.88 (EAB) (Table 2), specifying a close correlation be-
tween the predicted values and experimental values. These results de-
monstrated that the present model was significant and reliable for pre-
diction of physical, barrier and mechanical properties of the composite
films as a function of tested variables (rice starch, ι-carrageenan, FAEs
and, glycerol). The adjusted R2 statistic is more suitable for comparing the
model with independent variables. The adjusted R2 values for the model
were 0.80 (T) 0.64 (WVP), 0.72 (Sol), 0.91 (TS), and 0.73 (EAB), in-
dicating the reliability of model in predicting the properties of edible
films. 3-D response surface plots were used to visualise the relationship
between the variables and responses. The response surface is a graphical
representation of the regression equation that shows the relationship
between the responses and experimental levels of each variable (Agbovi &
Wilson, 2017). The 3-D response surface plots were obtained from the
model-predicted responses by keeping two independent variables con-
stant at the optimal level while varying the other two terms within the
experimental conditions.
3.2. Second order polynomial mathematical models
Equations 3, 4, 5, 6 and 7, which represent the second order
quadratic equations for model responses, were analyzed with multiple
regression analysis to obtain a good fit. These mathematical models
were applied to demonstrate the relationship between independent
variables and the responses on the film physical, mechanical and bar-
rier properties. The values are derived from Table 3 and the final
equations in term of coded factors are given below:
Thickness = 0.083 + 0.034X1+ 0.023X2+0.0084X3+0.018X4+
0.0075X1X2+0.001X1X3+0.001X2X3-0.005X1X4-0.005X2X4-
R. Thakur, et al. Food Packaging and Shelf Life 22 (2019) 100409

Table 1
Box-Behnken design matrix experimental data for the optimization of starch films.
Independent variables (Factors) Dependent variables (Factors)

Runs Starch X1(%) Carrageenan X2(%) FAEs X3(%) Glycerol X4(%) Thickness (mm) WVP x 10−10 (gs−1 m−1 Pa−1) Sol (S) TS (N/m2) EAB (mm)

1 1 0.5 1.25 1.15 0.03 2.09 33.03 46.56 11.9

2 2 0.5 1.25 0.3 0.04 5.95 42.69 70.57 7.34
3 2 0.5 0.5 1.15 0.05 5.33 55.39 30.88 4.27
4 2 0.5 2 1.15 0.07 2.3 34.36 80.35 1.08
5 2 0.5 1.25 2 0.08 3.54 37.25 55.45 8.22
6 3 0.5 1.25 1.15 0.09 5.23 57.23 112.46 2.66
7 1 1.25 1.25 0.3 0.05 5.03 53.58 49.84 21.69
8 1 1.25 0.5 1.15 0.07 4.94 44.95 27.98 30.7
9 1 1.25 2 1.15 0.07 2.89 39.37 90.73 20.7
10 1 1.25 1.25 2 0.05 4.61 47.65 55.21 27.5
11 2 1.25 0.5 0.3 0.03 5.23 61.96 60.25 11
12 2 1.25 2 0.3 0.06 3.8 41.8 145.7 11.33
13 2 1.25 1.25 1.15 0.09 4.88 61.35 75.66 22.2
14 2 1.25 1.25 1.15 0.09 2.84 53.48 82.55 17.53
15 2 1.25 1.25 1.15 0.1 4.06 50.29 85.95 25.76
16 2 1.25 0.5 2 0.09 5.29 71.02 78.33 22.19
17 2 1.25 2 2 0.08 3.05 46.88 99.76 13.96
18 3 1.25 1.25 0.3 0.14 5.66 55.26 127.38 22.58
19 3 1.25 0.5 1.15 0.13 7.55 75.36 111.36 22.02
20 3 1.25 2 1.15 0.14 4.02 53.25 185.21 12.45
21 3 1.25 1.25 2 0.12 5.2 56 155.25 24.94
22 1 2 1.25 1.15 0.09 4.1 55.32 65.11 23.45
23 2 2 1.25 0.3 0.08 5.2 54.07 124.36 9.00
24 2 2 0.5 1.15 0.09 6.02 61.35 84.36 35.05
25 2 2 2 1.15 0.12 4.49 49.31 121.73 21.32
26 2 2 1.25 2 0.1 5.51 56.73 86.31 29.28
27 3 2 1.25 1.15 0.18 7.23 61.24 188.56 29.53

0.004X3X4+0.012X12+0.004X 2 2
2 +0.001X3 +0.017X4
2
(3) of the final solution(Saberi et al., 2016; Whistler & Daniel, 2000). Effect
of solution viscosity has an impact on water evaporation as reported by
WVP = 4.9367 + 0. 1.2318X1+ 0.3758X2-0.6413X3-0.1438X4-
Rodríguez, Osés, Ziani and Maté (2006). Moreover, the higher viscosity
0.0025X1X2-0.1727X1X3+0.1750X2X3-0.0100X1X4 + 0.6800X2X4-
of the film matrix solution increases the thickness of the final film due
0.0945X3X4+0.5946X12+0.3996X 22+0.0304X32+0.5346X42 (4)
to lower water evaporation. The thickness of the edible film was af-
Solubility = 62.0299 + 10.3427X1+ 4.7078X2-4.0692X3+1.3102X4- fected by the concentration of ingredients as observed in this study.
4.5700X1X2-1.9285X1X3+1.0488X2X3+1.6675X1X4 + 2.0250X2X4- These results are supported by previous studies as reported by Han, Seo,
0.4643X3X4+0.3671X12+4.6508X 22+0.0048X32+1.2858X42 (5) Park, Kim and Lee (2006) and Sanyang, Sapuan, Jawaid, Ishak and
Sahari (2016). These authors indicated that ingredient concentrations
Tensile strength = 62.5541 + 43.1792X1+ 25.2667X2+9.1255X3+ and hydrophilic nature of partaking ingredients influenced the thick-
8.8215X4+14.3875X1X2+1.2950X1X3-1.4117X2X3+5.6250X1X4 ness of prepared films. In contrast, incorporation of plasticizers showed
-5.7325X2X4-7.4690X3X4+17.3588X12-1.26008X no significant effect on the measured property, signifying that inter-
2 2 2
2 +1.0851X3 +3.9538X4 (6) actions between plasticizers (glycerol and FAEs) and biopolymers im-
EAB = 22.978 -1.899X1+ 11.455X2+0.386X3+ 5.308X4+3.830X1X2- proved the film flexibility without affecting the film thickness.
0.0050X1X3-1.230X2X3-0.863X1X4 +4.850X2X4-0.999X3X4+2.677X12-
5.701X 22-0.616 X32-2.460X42 (7) 3.3.2. Film water vapour permeability (WVP)
The linear correlations between predicted and experimental values for
WVP is presented in Fig. 1. Water vapour permeability was significantly
3.3. Effect of independent variables on the responses influenced by starch and FAEs concentrations (p < 0.05). The results
showed that film WVP decreased with the increased amount of sucrose
3.3.1. Film thickness fatty acid esters, and increased with the biopolymer concentrations. These
The thickness of starch composite films was influenced by starch results indicated that WVP was influenced by the hydrophilic and hy-
(1–3%), carrageenan (0.5–2%) and glycerol content (0.3–2%) (Table 1). drophobic nature of starch and FAEs components. Similar observations
The hydrophilic nature of starch, carrageenan and glycerol, im- were made by Escamilla-García et al. (2017) who indicated that an in-
mobilized water within the film matrix, thereby increasing the viscosity crease in starch content significantly increased the WVP of the resulting

Table 2
The independent variables, levels, the experimental design and the observed responses.
Parameters Thickness (mm) WVP (gs-1m-1Pa-1) Solubility (%) Tensile strength (N/m2) EAB (mm)

Lack of fit 9.05 0.55 0.90 6.79 1.42

R2 0.91 0.83 0.87 0.96 0.88
Adjusted R2 0.80 0.64 0.72 0.91 0.73
F-ratio of the model 8.38 4.23 5.69 19.80 6.04
Prob > F 0.0004* 0.008* 0.002* 0.0001* 0.002*

WVP = Water vapor permeability, EAB = Elongation at break * significant difference with p < 0.05.

4
R. Thakur, et al. Food Packaging and Shelf Life 22 (2019) 100409

Table 3
: The analysis of variance for the experimental results.
Thickness WVP Solubility Tensile strength EAB

s Term Estimate Prob > |t| Estimate Prob > |t| Estimate Prob > |t| Estimate Prob > |t| Estimate Prob > |t|

βo Intercept 0.0835 < .0001* 4.9367 < .0001* 62.0299 < .0001* 62.5541 < .0001* 22.978 < .0001*
X1 Starch (%) 0.0347 0.0009* 1.2318 0.0095* 10.3427 0.0023* 43.1792 < .0001* −1.899 0.438
X2 Carrageenan (%) 0.0230 0.0131* 0.3758 0.3659 4.7078 0.1053 25.2667 0.0016* 11.455 0.000*
X3 Ester (%) 0.0084 0.1604 −0.6413 0.0433* −4.0692 0.0543 9.1255 0.0618 0.386 0.822
X4 Glycerol (%) 0.0180 0.0419* −0.1438 0.7254 1.3102 0.6346 8.8215 0.1829 5.308 0.045
X1X2 Starch*Carrageenan 0.0075 0.3678 −0.0025 0.9952 −4.5700 0.1192 14.3875 0.0421* 3.830 0.136
X1X3 Starch*Ester 0.0012 0.7605 −0.1727 0.3794 −1.9285 0.1551 1.2950 0.6687 0.050 0.965
X2X3 Carrageenan*Ester 0.0012 0.7605 0.1750 0.3732 1.0488 0.4254 −1.4117 0.6411 −1.230 0.293
X1X4 Starch*Glycerol −0.0050 0.5444 −0.0100 0.9807 1.6675 0.5518 5.6250 0.3914 −0.863 0.725
X2X4 Carrageenan*Glycerol −0.0050 0.5444 0.6800 0.1193 2.0250 0.4715 −5.7325 0.3827 4.850 0.066
X3X4 Ester*Glycerol −0.0047 0.2359 −0.0945 0.6264 −0.4643 0.7212 −7.4690 0.0264* −0.999 0.390
X12 Starch*Starch 0.0129 0.0874 0.5946 0.1161 −0.3671 0.8789 17.3588 0.0081* 2.677 0.222
X22 Carrageenan*Carrageenan −0.0046 0.5215 0.3996 0.2773 −4.6508 0.0721 −1.2600 0.8220 −5.701 0.018
X32 Ester*Ester −0.0015 0.3276 0.0304 0.6979 −0.0048 0.9927 1.0851 0.3810 −0.616 0.198
X42 Glycerol*Glycerol −0.0171 0.0300* 0.5346 0.1537 −1.2858 0.5957 3.9538 0.4843 −2.460 0.259

WVP = Water vapor permeability, EAB = Elongation at break *significant at 5% level.

film. Moreover, sucrose fatty acid ester improves the permeability prop-
erties of the starch film as reported previously by Thakur et al. (2016).
Starch exhibits higher hydrophilicity because the absorption and deso-
rption of water molecules are caused by the modifications of starch
structure in the presence of water (gelatinization process). Water increases
the molecular mobility and increases the diffusion coefficients for gas or
water. In a similar study by Bertuzzi, Castro Vidaurre, Armada and
Gottifredi (2007) revealed a strong correlation between starch and plas-
ticizer system. More information on the interactions between the in-
gredients affecting the WVP value could be obtained from the plots. The
WVP statistical model revealed that the linear term (X1, X3) were sig-
nificant for the WVP of rice starch composite film, whereas interactions
between other variables were not significant (p > 0.05).
3.3.3. Water solubility (WS)
WS is a key parameter for edible films where potential applications
require water resistance for starch biodegradable packaging (Saberi
et al., 2016). The effect of independent variables on solubility content is
presented in Fig. 2. The solubility content was affected by varying
concentrations of rice starch and FAEs. Solubility decreases as the
concentration of FAEs and starch increase in the formulation which is
probably due to the stronger FAEs-starch (Zeng, Gao, & Li, 2013) and
starch-carrageenan interactions (Huc et al., 2014; Lascombes et al.,
2017). The interactions might have decreased the water affinity of the
matrix and hence impeded water absorption capacity.
3.3.4. Tensile strength (TS) and elongation at break (EAB)
Film mechanical properties of starch composite films were studied.
As shown in Fig. 2, starch and carrageenan significantly affected the
tensile strength of the edible film. Tensile strength of edible films showed
a positive correlation with experimental variables. The interactions be-
tween starch and carrageenan are well reported in the literature (Huc
et al., 2014; Lascombes et al., 2017). As the concentration of starch and ι-
carrageenan in the formulation increase, the molecular associations

Fig. 1. Correlations between predicted and experimental thickness, water vapour permeability (WVP), solubility, tensile strength, elongation at break (EAB).

5
R. Thakur, et al. Food Packaging and Shelf Life 22 (2019) 100409

Fig. 2. Response surface plots showing the interaction effects of process variables on thickness, water vapour permeability (WVP), solubility, tensile strength,
elongation at break (EAB).

among these ingredients become dominant, leading to improved me- mechanical properties of starch composite films were positively influ-
chanical film properties (Thakur et al., 2018a, 2018b). Interactions be- enced by starch and ι-carrageenan content. EAB is an indication of film
tween the independent variables - starch x ι-carrageenan, and starch x flexibility and expansion capacity. It is also of interest that ι-carrageenan
starch also had a significant impact on tensile strength (p < 0.05). The and glycerol also had a significant impact on the film flexibility

Fig. 3. Effects of starch-based edible coating on weight loss (a and b) at 20 °C and 5 °C + 1day at 20 °C and respiration rate (c and d) at 20 °C and 5 °C + 1day at 20 °C
of coated and uncoated apple fruit. n = 28 (7 fruit x 4 replications). In the Fig, the colour green and black represents control and coated samples respectively.
Different letters on the bars indicate significant difference at P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)

6
R. Thakur, et al. Food Packaging and Shelf Life 22 (2019) 100409

Table 4
Effect of starch coating on the TPC, DPPH and firmness of apple fruit stored at 20 °C and 5 °C + 1 day at 20 °C. Different letters in the same row represent the
significant differences in the control and coated fruit (p < 0.05).
TPC (mg GAE mL−1 juice)

20 °C 5 °C + 1 day at 20 °C

Storage time (weeks) Control Coated Storage time (weeks) Control Coated

a a a
0 0.039 ± 0.007 0.039 ± 0.007 0 0.039 ± 0.007 0.039 ± 0.007a
1 0.014 ± 0.000a 0.024 ± 0.000a 2 0.031 ± 0.001a 0.029 ± 0.003a
2 0.020 ± 0.002a 0.023 ± 0.004a 4 0.034 ± 0.003a 0.032 ± 0.002a
3 0.025 ± 0.002a 0.030 ± 0.003a 6 0.042 ± 0.007a 0.036 ± 0.003a
4 0.026 ± 0.005a 0.027 ± 0.004a 8 0.038 ± 0.004a 0.030 ± 0.004a
5 0.024 ± 0.002a 0.023 ± 0.003a 10 0.047 ± 0.008a 0.036 ± 0.002b

DPPH (mg TE mL−1 juice)

20 °C 5 °C + 1 day at 20 °C

Storage time (weeks) Control Coated Storage time (weeks) Control Coated

b b a
0 0.122 ± 0.003 0.122 ± 0.003 0 0.122 ± 0.003 0.122 ± 0.003a
1 0.161 ± 0.001a 0.158 ± 0.000a 2 0.143 ± 0.000a 0.148 ± 0.003a
2 0.160 ± 0.002a 0.157 ± 0.003a 4 0.142 ± 0.001a 0.145 ± 0.002a
3 0.100 ± 0.019a 0.122 ± 0.007a 6 0.135 ± 0.006a 0.145 ± 0.003b
4 0.130 ± 0.007a 0.152 ± 0.002b 8 0.122 ± 0.012a 0.148 ± 0.003b
5 0.147 ± 0.001a 0.140 ± 0.009b 10 0.124 ± 0.005a 0.143 ± 0.002b

Firmness (N/m)

20 °C 5 °C + 1 day at 20 °C

Storage time (weeks) Control Coated Storage time (weeks) Control Coated

a a a
0 44.1 ± 0.55 44.1 ± 0.551 0 44.1 ± 0.551 44.1 ± 0.551a
1 42.9 ± 0.979a 42.1 ± 0.726a 2 43.6 ± 1.357a 44.1 ± 2.028b
2 42.2 ± 0.260a 42.8 ± 0.891a 4 42.2 ± 0.391a 42.8 ± 1.743b
3 42.0 ± 1.563a 42.5 ± 0.929a 6 40.7 ± 0.839a 42.3 ± 0.974b
4 40.9 ± 1.789a 40.4 ± 1.116a 8 38.2 ± 0.659a 40.1 ± 0.959a
5 41.3 ± 0.448a 41.7 ± 0.285a 10 38.8 ± 1.112a 39.6 ± 0.457a

(p < 0.05) (Fig. 2), which could be a product of favourable cross-linking
interactions (Shah, Naqash, Gani, & Masoodi, 2016). Plasticizers such as
glycerol restrict individual polymeric chain hydrogen bonding, thereby
reducing order and crystallinity of the network, leading to the creation of
a higher entropy film structure. Interactions between the variables ι-
carrageenan x ι-carrageenan had a significant impact on the film flex-
ibility (p < 0.05), while starch x starch, FAEs x FAEs, glycerol x glycerol
had no significant impact on EAB.
The aim of this step was to obtain a film formulation developed from
starch, ι-carrageenan, FAEs and glycerol with minimum thickness,
minimum permeability, high solubility and strong mechanical properties.
Based on the mathematical models and functioning by JMP software, we
selected the optimal formulation, which met the requirement of the film
with minimum thickness, minimum permeability, high solubility and
strong mechanical properties. Optimal formulation was starch (2.5%), ι-
carrageenan (1.5%), FAEs (2%) and glycerol (1.5%) respectively.
3.4. Coating applications- weight loss (WL) and respiration rate
Fruit weight loss showed gradual increasing tendencies in both con-
trol and coated fruit stored at two different temperatures (20 °C and 5 °C
+ 1 day at 20 °C). Results presented in Fig. 3a and b shows that the
lowest weight loss was manifested in RS-ι-car coated fruit irrespective of
storage temperature. Apple fruit stored at 20 °C showed higher weight
loss compared to the fruit stored at 5 °C. At the end of the storage period,
weight losses for control and coated fruit stored at 20 °C and 5 °C were
2.02% and 1.85% and 1.11% and 1.0%, respectively. A significant
weight reduction was observed in coated apple fruit after week 3 (20 °C)
and week 8 (5 °C) of storage (p < 0.05). These results indicated that the
RS-ι-car coating was effective with regard to weight loss irrespective of
storage temperatures. WL reduction in coated fruit could be due to the
uniform thin film layer created over the fruit surface as a result of
complexation between FAEs-starch and ι-carrageenan molecules (Thakur
et al., 2018a, 2018b; Zeng et al., 2013). This complex thin layer structure
restricts the movement of water vapour through the membrane. Re-
spiration rate (O2/CO2) for coated and control fruit at different storage
temperature is presented in Fig. 3c and d. For control and coated fruit
respiration rate varied between 1.82 to 2.15 mLCO2 kg−1 h−1 and 1.82
to 1.86 mLCO2 kg−1 h−1 at 20 °C and 1.82 to 2.46 mLCO2 kg−1 h−1 and
1.82 to 2.23 mLCO2 kg−1 h−1 at 5 °C, respectively. Starch coated fruit
showed a lower respiration rate. However, significant differences were
observed in the control fruit as the storage period progresses. These re-
sults are in line with previously reported data by Garcia, Pereira, Luca
Sarantópoulos, and Hubinger, 2012) and Chiumarelli, Pereira, Ferrari,
Sarantópoulos and Hubinger (2010) who explained the efficiency of
starch composite coating in delaying the WL and respiration rate of
strawberry and mango fruit during postharvest storage. In summary,
starch coating in combination with low temperature was found to be
more efficient in controlling the moisture loss from the fruit surface
hence resulting in lower weight loss at 5 °C + 1 day at 20 °C than 20 °C.
3.4.1. Fruit firmness, gloss, skin greasiness and change in skin background
colour
Firmness is an important quality attribute associated with overall
consumer acceptability. Firmer fruit tends to be juicier, crisper,
crunchier and less mealy than control fruit (Johnston, Hewett, &
Hertog, 2002). Table 4 shows the firmness changes in control and
coated fruit stored at 20 °C and 5 °C + 1 day at 20 °C. For control and
coated fruit, firmness varied between 44.1 to 41.3 N/m (20 °C), 44.1 to
38.8 N/m (5 °C) and 44.1 to 41.7 N/m (20 °C), 44.1 to 39.6 N/m (5 °C),

7
R. Thakur, et al. Food Packaging and Shelf Life 22 (2019) 100409

Fig. 4. Evaluation of skin quality by skin background colour (a and b) and greasiness (c and d) score in control and coated apple fruit stored at 20 °C and 5 °C + 1day
20 °C. Skin background colour was visually recorded and skin greasiness was assessed subjectively by rubbing fruit against the hand. In the Fig, the green and black
colour represents control and coated samples respectively. Each value represents the mean ± SE of four replicates. Different letters on the bars indicate
significant difference at P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Effect of starch coating on TSS (a and b) and TA (c and d) values of control and coated apple fruit stored at 20 °C and 5 °C + 1day 20 °C. In the Fig, the green
and black colour represents control and coated samples respectively. Each value represents the mean ± SE of four replicates. Different letters on the bars indicate
significant difference at P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

8
R. Thakur, et al.
respectively. Fimness for coated fruit was higher than control fruit at
low-temperature storage, probably due to the controlled moisture loss
from the fruit surface (Link, Drake, & Thiede, 2004). No significant
differences were observed in control and coated fruit stored at 20 °C
(p > 0.05) compared to fruit stored at 5 °C + 1 day at 20 °C
(p < 0.05), which indicates that surface water loss (Fig. 3a and b) was
not sufficient to affect the apple firmness. The possible firmness dif-
ferences at 5 °C + 1 day at 20 °C could be due to the intracellular air-
spaces created during cold storage. However, the study needs more
investigation as insufficient information is available in the literature.
Fruit greasiness is a sign of ripening and related to fruit maturity
(Dadzie et al., 1995). Fig. 4c and d shows the skin greasiness of control
and coated fruit stored at 20 °C and 5 °C+ 1 day at 20 °C. Fruit greasy
feeling noted as a greasiness score, varied between 2 to 4.5 at 20 °C and
2 to 0.3 at 5 °C + 1 day at 20 °C, respectively (p < 0.05). We judged
that surface of fruit at the start of the experiment was slightly greasy,
which indicated the maturity of fruit. For the control fruit stored at
20 °C and 5 °C+, 1 day at 20 °C, greasiness increased with fruit ripening
and reached to severe level at the end of week 2. By contrast, apple fruit
treated with rice starch composite coating showed a negligible or low
level of greasiness during different temperature regimes. According to
Embuscado and Huber (2009) edible coatings either are functioned by
creating a thin layer over the fruit surface or by blocking the skin pores.
Spraying of coating suspension might have covered the fruit natural
wax covering and formed a thin dry coating layer over the fruit surface
masking the natural greasiness hence resulting in lower greasiness va-
lues. Fruit with a greener background colour, are regarded by most
consumers as fresher than those that have gone yellow (Harker,
Gunson, & Jaeger, 2003). There was an important difference in the rate
of yellowing of background colour of control and coated fruit stored at a
different temperature. Fruit skin colour was green at the start of the
experiment with a score of 2 on the fruit skin background colour
(Fig. 4a and b). The control fruit, stored at 20 °C and 5 °C+ 1 day at the
20 °C start losing the green colour as the storage period progresses. Loss
of green colour was significant for fruit stored at 20 °C and 5 °C+ 1 day
at 20 °C (p < 0.05) after week 2 however, the background colour
change was faster at ambient temperature. The change in skin colour is
associated with the progressed chlorophyll breakdown reactions
causing underlying yellow pigment to be unmasked (KNEE, 1980).
Starch coating delayed the change of colour from green to yellow ir-
respective of storage regimes probably due to the slower activity of
degradation enzymes as a function of the applied coating.
3.4.2. Total soluble sugars (TSS), titratable acidity (TA)
Sugars and acids content are two major determinants of flavour
characteristics of fruit including apple (Cohen et al., 2014). Fig. 5(a–d)
shows the TSS and TA values of control and coated fruit stored at 20 °C
and 5 °C + 1day at 20 °C. The results from 20 °C storage showed a
gradual increase in fruit TSS value for control and coated fruit
(p < 0.05), however, the increase was comparatively slower at 5 °C
than 20 °C. These results could be correlated with the water loss of the
fruit samples. The loss of water during storage increased the fruit TSS as
reported previously (De León-Zapata et al., 2018). For the TA data
(Fig. 5c and d), a non-uniform trend was observed for the fruit stored at
a different temperature. TA values were significantly higher for coated
fruit during week 2–3 (20 °C) and week 4–6 (5 °C + 1day at 20 °C),
respectively (p < 0.05), indicating the slow acid degradation rate of
the apple fruit, however, no significant differences were observed as the
storage progresses. The cessation of acid degradation in apple fruit
during storage could be attributed to the lower utilization of acids in
the metabolic process. Overall, fruit stored at 5 °C + 1day at 20 °C
shows higher TA values than 20 °C at the end of the storage period. The
higher TA during cold storage is in agreement with the findings of a
previous study on apple fruit (Kweon et al., 2013).
9
Food Packaging and Shelf Life 22 (2019) 100409
3.4.3. Effect of coatings on bioactive compounds
Bioactive compounds play an important role in the nutritional and
sensory properties of fresh produce (Guerreiro, Gago, Faleiro, Miguel, &
Antunes, 2017). Chlorogenic, p-coumaric and caffeic acids, quercietin,
epicatechin, catechin, rutin, phlorizin are the main phenolic com-
pounds in apple fruit (Socha, Łakoma, & Fortuna, 2017). The variations
in the content of bioactive compounds (total phenolic compounds and
DPPH compounds) in the control and coated fruit stored at 20 °C and
5 °C + 1 Day at 20 °C is presented in Table 4. At 20 °C, TPC content for
control and coated fruit at the end of storage period was 0.024 and
0.023 mg mL−1 juice. However, the TPC content for control and coated
fruit was higher for apple fruit stored at low temperature (5 °C + 1 Day
at 20 °C). At low-temperature storage, the TPC content for control fruit
was 0.011 mg mL−1 and TPC was higher at the end of storage period
than coated fruit (p > 0.05). These observations showed that the ed-
ible coating did not affect the synthesis of total phenolic content since
no significant differences were observed among control and coated fruit
at both storage temperatures. However, low-temperature storage was
more suitable for apples long term storage w.r.t maintaining the level of
phenolic compounds.
The antioxidant potential status of fruit and vegetables is linked
with the amount of bioactive compounds presents in the produce
(Moreira, Tomadoni, Martín-Belloso, & Soliva-Fortuny, 2015). Table 4
shows the changes in the DPPH radical scavenging activity of apple
fruit subject to starch edible coatings. From the data shown, it is clear
that significant differences were observed between control and coated
fruit, however, there was no uniform trend in the DPPH content at
different storage period. As the storage period progresses free radical
scvangeging activity was increased initially irrespective of applied
treatment and storage temperature. At 20 °C DPPH content was higher
in coated fruit after week 4 (p < 0.05) as compared to control fruit.
The trend was similar in fruit stored at 5 °C where coated fruit showed
higher DPPH content after week 4 during storage. The variations in the
antioxidant activity of the fruit during storage could be due to the
postharvest physiological activities as reported previously (Valero &
Serrano, 2013).
4. Conclusion
Rice starch-ι-car coating formulation was optimized for minimum
thickness, water vapour permeability and strong mechanical properties.
The study demonstrated that interactions between the ingredients are
critical factors in the design of coating material. Apple fruit coated with
starch optimised formulation showed a reduction in weight loss, re-
spiration rate, firmness, skin colour change and fruit greasiness without
affecting the flavour (TSS and TA) and phytochemical profile during
postharvest storage. The effect of the coating was more prominent at
5 °C than room temperature (20 °C) indicating that starch coating re-
tains its integrity at high humidity environment. However, future re-
search is required to understand the behaviour and compatibility of
coating material to the fruit surface.
Acknowledgments
This work was supported by the University of Newcastle, Australian
Research Council (ARC) Training Centre for Food and Beverage Supply
Chain and Optimisation (IC140100032). NSW Department of Primary
Industries is a partner organisation in the training centre.
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