INFLUENCE OF AN EDIBLE COATING MADE OF SODIUM

ALGINATE, CALCIUM, BEESWAX, GLYCEROL, SUCROESTER

AND CHITOSAN ON THE SHELF LIFE OF THE ANDEAN
BLACKBERRY
Misael Cortés-Rodríguez a*, Rosa Amanda Alegría Macías a, Lina María

Cardona a and Rodrigo Ortega-Torob*

a Faculty of Agricultural Sciences, Universidad Nacional de Colombia, Medellín.
bUniversidad de Cartagena, Food Engineering Department, Food Packaging and Shelf
Life Research Group (FP&SL) and Complex Fluids Engineering and Food Rheology
Research Group (IFCRA), Avenida del Consulado Calle 30 No. 48 – 152, Cartagena de
Indias D.T. y C., Colombia.

* Corresponding author: mcortesro@unal.edu.co; rortegap1@unicartagena.edu.co

ABSTRACT

The aim of the research is to evaluate the influence an EC made of sodium alginate (SA),
beeswax (BW), glycerol (G), sucroester (SE), chitosan (Ch), and calcium (EC+Ca) has
on the shelf life of the Andean blackberry. In addition to the microbiological properties,
the dependent variables for the blackberries, which were stored at 4, 8 and 12°C for 0, 3,
6, 9, and 12 days, were: moisture (Xw), water activity (aw), weight loss (WL), acidity,
pH, °Brix, colour (L*, a*, b*), Surface Breaking Strength (SBF), Total Phenols (TP), and
antioxidant capacity (DDPH and ABTS methods). The blackberries with EC+Ca stored
at 4°C proved to have the best attributes. The EC+Ca that was applied to the Andean
blackberry better conserves its physicochemical, physical, and microbiological
properties, which allows the shelf life of the product to be extended. This is a 140%
increase compared to fresh fruit that do not have EC.

Keywords: Rubus glaucus Benth, fruit preservation, shelf life, edible coatings

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 13

 Abbreviations

Name Abbreviation
Edible coating EC
Chitosan Ch
Sodium alginate SA
Beeswax BW
Glycerol G
Sucrose esters SE
Edible coating + Calcium EC+Ca
Moisture Xw
Water activity aw
Weight loss WL
Total phenols TP
Surface breaking force SBF
Lightness L*
Chromaticity red green a*
Chromaticity yellow blue b*

INTRODUCTION

Fresh food consumption has inspired the food industry to seek new techniques to increase
quality during storage as well as to improve microbiological safety, i.e., increasing shelf
life (Maringgal et al., 2020; Mellinas et al., 2015). The challenge of developing new
products is being able to achieve novel processes or conservation strategies which keep
foods safe and guarantee and maintain the nutritional properties that contribute to health
(Riva et al., 2020, Hasan et al., 2019; Yousuf et al., 2018).

The Andean blackberry (Rubus glaucus Benth) is a non-climacteric fruit and is

characterized by being very fragile and susceptible to attack by fungi. When no adequate
treatments are employed, the fruit may experience weight loss, colour change, loss of
firmness, etc., which affect its optimal characteristics and nutritional qualities (Zhang et
al., 2019). Moreover, the presence of fungi such as Botrytis causes grey mould, which is
one of the most serious infectious diseases and causes great losses in fruit yield and
quality. It affects more than two hundred species, most of which are economically
significant (Horvitz et al., 2017).

For blackberries and for fruits in general, long storage periods can produce changes in
firmness, which increase their susceptibility to attack and growth of some
microorganisms. The post-harvest lifespan of fruits is determined by their susceptibility
to water loss, softening, mechanical injuries, and especially the presence of pathogenic
microorganisms (Tezotto-uliana et al., 2014; Horvitz et al., 2017; Vittori et al., 2018).
Research has suggested strategies to control the growth of these microorganisms during
post-harvest to preserve fruit quality; these include modified atmospheres, forced air
cooling, thermal shocks, osmotic treatments, irradiation, and edible coatings (EC) (Ma et
al., 2017).
[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 14
 EC are of great interest because of their ability to reduce respiration and transpiration
rates during storage periods as well as their ability to retain fruit firmness (Velickova et
al., 2013; Novaes et al., 2014; Tezotto-uliana et al., 2014). EC also provide good
mechanical properties, are non-toxic, do not cause contamination, and their application is
low cost (Hasan, Ferrentino and Scampicchio, 2019). These are generally composed of
hydrocolloids, lipids, proteins, and mixtures of the previous (Mellinas et al., 2015; Aloui
and Khwaldia, 2016). Among the hydrocolloids, alginates stand out because of their
excellent functional properties such as stabilizers, thickeners, film formations and gelling
agents (Dhanapal et al., 2012). Alginates in EC are favoured by the presence of calcium
ions, inducing EC jellification, which improve surface mechanical resistance when dried.
Some authors have efficiently used calcium to prolong the shelf life of fruit because of its
interaction with the pectic components of cell walls, strengthening firmness, cell
turgidity, mechanical resistance, and cross-linking of polysaccharides (Martins et al.,
2018). The blackberry has high calcium absorption properties, which reduces the
transpiration rate, ethylene production, firmness, and post-harvest shelf life (Tyagi et al.,
2017).

Adding antimicrobial agents to EC could improve food functionality; this is due to the
protection it offers the food in terms of microbial proliferation, which results in an
increase in post-harvest quality and shelf life (Antunes et al., 2012; Gomes et al., 2016).
Chitosan (Ch) has been shown to have antimicrobial properties to combat various
microorganisms, mainly bacteria; however, it also acts against moulds and yeasts
(Velickova et al., 2013; Tezotto-uliana et al., 2014; Kerch, 2015). The aim of this research
is to evaluate the influence that an EC made form sodium alginate (SA), beeswax (BW),
glycerol (G), sucroester (SE), and Ch and calcium (EC+Ca) has on the shelf life of the
Andean blackberry.

MATERIAL AND METHODS

In this research, we used the Andean blackberry from the municipality of Granada,
department of Antioquia, Colombia, which had a state of maturity 5 and an "extra"
category according to NTC 4106 (ICONTEC, 1997). According to the methodology
described by Cortés-Rodríguez et al. (2019), EC+Ca is created using SA (2.85%), BW
(1.00%), SE (0.18%), SA/G (2.00), Ch (0.5%), and CaCl2 (1.396%). The EC+Ca was
applied on the Andean blackberry after it was washed with potable water and sanitized
with NaClO at 50 ppm for 1 min. The fruits were submerged in the EC+Ca for 1 min,
they were drained for 30 s, and, finally, 2.4 kg loads were put in a tunnel dryer (Centricol,
Series 0803) at 30°C, 1600 rpm for 1 h.

A storage study was performed on the EC+Ca blackberries using a completely

randomized 2n factorial design. The independent variables were: time (0, 3, 6, 9, and 12
days) and temperature (4, 8 and 12°C). The dependent variables were: moisture (Xw)
using the gravimetric method 930.15, (AOAC, 2012); weight loss (WL) by weight

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 15

 difference between time 0 and time t; ºBrix, pH, and acidity according to NTC 4106
(ICONTEC, 1997); water activity (aw) using a hygrometer dew point at 25ºC (Aqualab
series 3TE, Decagon, Devices, Pullman, WA, USA); the content of total phenols (TP)
and the antioxidant activity of the fruit determined from the methods DPPH (2,2-
Diphenyl-1-Picrylhydrazil); and ABTS (azinobis 3-ethylbenzothiazolin-6-sulfonic acid),
based on the methodology described in Cortés-Rodríguez et al. (2019). The colour was
determined according to the methodology in Pérez-Gallardo et al. (2015), which used
CIE-L*a*b* coordinates with X-Rite SP62 spectrophotometer, D65 illuminant and 10°
observer. The texture was determined by puncture tests using TA.XT2 texturometer
(Stable Micro System), P/5 probe, speed 2 mm/s (it had a penetration length of 10 mm),
following the methodology described by Mannozzi et al. (2016) and expressed as the
surface rupture force (SBF). The permeability of the film or water vapour transmission
was gravimetrically determined according to the standard ASTM, (1995) method
E96/E96M, which was modified (in a climatic chamber controlled to relative humidity of
65% and T=25ºC). Finally, the microbiology was carried out through determining moulds
and yeasts by plate count as well as total and faecal coliform according to AOAC, 1998
method.

The samples were packed in perforated polypropylene boxes, and three boxes with fruit
were used for each storage condition. Three repetitions were performed for each
dependent variable (one repetition/box). Samples for some time-temperature
combinations were stored until the physical presence of microorganisms on the surface
of the fruit could be seen. The results were analysed from ANOVAS with a 95%
confidence level using Statgraphics Centurion XVI.II software.

RESULTS AND DISCUSSION

Figure 1 shows the evolution of the dependent variables for blackberries: EC+Ca is a
function of storage time and temperature. Table 1 records the ANOVA results (p-value)
for each dependent variable. The samples stored at 12ºC showed the presence of
microorganisms on the surface of the fruit during the control time of ten days; therefore,
results are only reported until day nine. There was similar behaviour at 4 and 8ºC, and
results were reported until day 12.

When the storage time was longer, there was a decrease in the Xw of blackberries with
EC+Ca, which became smaller at lower temperatures. This is because of the increased
weight loss; the lowest values were reached at 4 and 8ºC: 13.6±0.5% and 23.4±0.5%,
respectively. This behaviour is attributed to several phenomena: on the one hand, the
EC+Ca forms a layer on the fruit with hydrophobic and hydrophilic components that
affect the WL speed, which delays the ripening and senescence processes of the coated
fruits (Galus and Lenart, 2013). On the other hand, at higher temperatures, water
experiences greater molecular movement and favours mass transfer; there is higher
vapour pressure that encourages a higher evaporative rate on the fruit surface. This is

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 16

 limited by the EC+Ca, the permeability of which, to film water vapour, was 1.8x10-5 g
day/cm3 at 25ºC. Additionally, a decrease in Xw over time produces a decrease in food
structures’ (such as fruits) aw, which is evident in the graph. Temperature was not a critical
factor in food’s aw changes: the average value at day 12 was 0.953±0.001. This condition
favours deterioration reactions.

86 4 °C 25
84 8 °C
20
12 °C
82

WL (%)
15
Xw (%)

80
78 10
76
5
74
72 0
0 3 6 9 12 0 3 6 9 12
Time (days) Time (days)

0,970 12,0

0,965 11,0
10,0
°Brix
aw

0,960
9,0
0,955
8,0
0,950 7,0
0 3 6 9 12 0 3 6 9 12
Time (days) Time (days)

3,00 350
TP (mg GAE/100 g)

2,90 300
250
2,80
200
2,70 150
pH

100
2,60
50
2,50 0
0 3 6 9 12 0 3 6 9 12
Time (days) Time (days)

1000 1100
ABTS mg Trolox/100 g

1000
DPPH mg Trolox/100 g

900
900
800 800
700
700
600
600 500
0 3 6 9 12 0 3 6 9 12
Time (days) Time (days)

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 17

 24 10
22 8
20 6

a*
L*

18
4
16
14 2
12 0
0 3 6 9 12 0 3 6 9 12
Time (days) Time (days)

5 14
12
4
10

SBF (N)
3 8
2 6
b*

4
1
2
0 0
0 3 6 9 12 0 3 6 9 12
Time (days) Time (days)

Figure 1. Stability of the physicochemical and physical properties of blackberries with

EC+Ca during storage

The WL values at 4ºC are considered acceptable compared to those reported by other
authors using different food matrices: Dong et al. (2004) reported on litchi (Litchi
chinensis) with Ch-based EC for six days at 1°C: (6 and 9% without EC);Tokath and
Demirdöven (2020) reported on sweet cherry (Prunus avium L.) with Ch-based EC for
25 days at 4°C: (22.5 and 16.18% without EC), ( Oliveira et al. (2019) reported on apples
with calcium-based EC for 25 days at 5°C: (values between 3.7% - 4.2%); Jiao et al.
(2019) reported on peaches with EC using Ch and chlorogenic acid for eight days at 20ºC:
(a decrease of 18.7% with respect to the control); Thakur et al. (2019a) reported on
bananas with EC using starch for 14 days at 20°C: (2.24% compared to 3.78% for the
control); and Nawab et al. (2017) reported a reduction in WL for starch-based EC
tomatoes over 20 days at 20°C.

The °Brix presented an increasing trend with respect to time. The effect of the temperature
at 12ºC was not consistent with the WL; a soluble solids content, equivalent to 8.5%, was
reached after nine days. At 4 and 8ºC on day 12, the °Brix were higher than at 12ºC: 9.5
and 10.5%, respectively. This behaviour could be the result of several phenomenon: the
ripening of the fruit allows them to increase in size because of the conversion of starches
present in sugars as well as the dilution and metabolization of acids in this process. Also,
with the senescence of the fruit, there is a decrease in the amount of sugars that become

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 18

 alcohols and organic acids, and, as the breathing process continues, acids are taken and
converted into CO2 and H2O (Sora et al., 2006).

Table 1. ANOVA for blackberries with EC+Ca during storage

Variable Temperature Time

WL (%) 0.000 0.000
pH 0.317 0.000
Acidity (%) 0.000 0.000
Xw (%) 0.170 0.000
aw 0.696 0.000
°Brix 0.000 0.000
TP (mg GAE/100g) 0.000 0.000
ABTS (mgTrólox/100g) 0.000 0.000
DPPH mgTrólox/100g 0.000 0.000
a* 0.237 0.636
b* 0.251 0.810
L* 0.000 0.000
SBF (N) 0.000 0.000

The increasing of ºBrix over time has been observed by some authors researching
blackberries with other ECs (Sánchez et al., 2014) and with other food matrices: sweet
cherries (Tokath and Demirdöven, 2020), red guava (Formiga et al., (2019), cashew
apples (De Oliveira et al., (2019), papaya (Tabassum et al., 2019), chopped mangoes
(Chien et al., 2007), strawberries (Hernández et al., 2008), and carrots (Vargas et al.,
2009).

The pH showed an increase over time (from 2.7 to 3.0); it was higher during the first three
days of storage and then tends to become asymptotic. These changes are considered
acceptable within the framework of the phenomena mentioned above. Some authors have
reported similar changes in the parameters associated with pH, such as acidity: in
blackberries with EC using cassava starch, Ch, and kefir that were stored for 12 days at
102ºC (Oliveira, Kwiatkowski and Rosa, 2012), and in blackberries with EC using
sodium alginate that were stored for 11 days at 3ºC (Sánchez et al., 2014).

The Andean blackberry is a non-climacteric fruit rich in antioxidant properties and

polyphenols including anthocyanins, flavonols, ellagitannins, and procyanidins (Kaume
et al., 2012). The values of TP contents are highlighted for fresh blackberries: 93.5±14.5
mg gallic acid equivalent (GAE)/100g and DPPH values: 299.9±12.3 mg Trólox/100g
and ABTS: 863.2±31.7 mg Trólox/100g; which denotes a high variability: TP (15.5%),
DPPH (4.1%) and ABTS (3.6%), which is affected by the state of maturity, aspects of
cultivation, climate, incidence of light during growth, among others (Horvitz et al., 2017).

At the beginning of storage, the blackberries with EC+Ca showed the following levels of
TP: 105.3±4.5 mg GAE/100g; DPPH values of: 319.3±9.4 mg Trólox/100g; and the

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 19

 ABTS was: 962.7±15.1 mg Trólox/100g. This denotes the EC+Ca had an influence on
these parameters increasing, which could mainly be attributed to BW and Ch (Jiao et al.,
2019; Hernández et al., 2008). The TP content of the blackberries with EC increased with
storage time and temperature, which is mainly attributed to the increase in the
concentration of phenolic compounds due to the WL the fruit experienced. Horvitz et al.,
(2017) reported an increase in PT over time for Andean blackberries that were stored at
8°C (nine days) and 18°C (three days).

The antioxidant activity measured in terms of DPPH did not present a well-defined trend
with respect to storage time; it was higher at higher temperatures. At 4 and 8ºC a sharp
decrease was observed from the beginning until day three (299.9±12.3 118.2±11.2 and
299.9±12.3 253.0±11.2 mg Trólox/100g respectively); there was then progressive growth
until similar values were reached: approximately 351.0±18.6mg Trólox/100g at 12 days.
At 12ºC the behaviour of the DPPH values showed an increase until they reached 387.37.1
mg Trólox/100g on day nine. Furthermore, the ABTS values also presented undefined
behaviour as fluctuations were observed over time and with different temperatures; there
were generally more at higher temperatures. Antioxidant capacities are methods used to
determine the antioxidant potential of plant tissue, which is a function of the type and
concentration of bioactive compounds present. These fluctuate during the fruit’s
physiological processes during storage (Robles-Sánchez et al., 2013). Other authors have
had similar results using Andean blackberries during storage at 8 and 18°C; they found
that the content of ascorbic acid decreases, while the concentration of anthocyanins
increases, which explains why the values in antioxidant activity do not mark a trend over
time (Horvitz et al., 2017).

Colour is one of the desirable characteristics that could determine consumer acceptance
(Mannozzi et al., 2016). The blackberries’ colour parameters (L*, a*, and b*) with EC at
4 and 8ºC did not present significant differences with respect to time (L*, a*, and b* <
5): no changes could be seen with the human eye.

The effect that storage conditions had on colour was more evident at 12ºC: a decrease in
L* (from 22.5±0.7 to 14.7±0.9) was observed, which reflected a darkening mainly due to
surface EC+C. These brown and opaque pigments were because of the changes
experienced by the anthocyanins and phenolic compounds present (Sánchez, Real and
Perez, 2014). Moreover, a decrease in chromaticity a* over time (from 8.0 to 2.0) can
also be observed. There is a lower intensity of red pigment present, mainly attributable to
oxidation or phenolic compounds condensation reactions, which produce the loss of
anthocyanins in the fruit (Mannozzi et al., 2016). Similar behaviour has been observed
by some authors with blackberries with EC using cassava starch and kefir grains
(Oliveira, et al., 2012); EC using sodium alginate (Sánchez et al., 2014); and in
blackberries packed in an active modified atmosphere with the following concentrations:
CO2 (25%); O2 (5%), N2 (70%), CO2 (20%); O2 (10%); and N2 (70%) in low-density
polyethylene packages (Sora et al., 2006). Similar behaviour has also been observed in

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 20

 other fruits: blueberries with EC using alginate and citrus pectin (Mannozzi et al., 2016),
“cripps pink” apple with starch-based EC (Thakur et al., (2019b), and strawberries with
EC using starch, carrageenan, and Ch (Ribeiro and Vicente, 2007).

The SBF of blackberries with EC decreased with time and with increasing temperature;
the losses of firmness or surface mechanical resistance were 50, 79, and 88% at 4 and 8ºC
(12 days) and 12ºC (nine days), respectively. Firmness directly influences the quality and
lifetime of the blackberry and can be affected by several physiological and biochemical
processes including the conversion of starch into sugars during ripening and changes in
metabolism and cell wall structure (Oliveira et al., 2013). In addition, the migration of
water vapor towards the fruit surface and through the EC during storage favours the
growth of fungi and yeasts, which generates structural damages that soften the blackberry
(Pérez-Gallardo et al., 2012).

Greater losses of firmness in fresh blackberries during storage have been reported in the
literature: 42% for 21 days at 2ºC (Joo et al., 2011). However, the results have been better
for EC using sodium alginate for 18 days at 0ºC: 20% (Meneguel et al., 2008). Other
authors have reported losses of 44% for fresh blackberries with EC using aloe stalk
mucilage gel (Ramírez et al., 2013). This loss of firmness has also been reported by
Thakur et al. (2019) in Cavendish bananas with EC using rice starch and sucrose ester
that were stored at 20±2°C for six days (8.1%). The results were compared to those
obtained from bananas that did not use EC (< 6.9 N).

According to the results obtained from the physicochemical and physical properties at the
three temperatures evaluated (4, 8 and 12ºC), a microbiological evaluation (total
coliforms, faecal coliforms, moulds, and yeasts) was performed for the blackberries with
EC+Ca that had been stored at 4°C for 12 days (Table 2).

It can be observed that in the first storage times (0, 3, and 6 days) the total coliform
parameters, moulds, and yeasts did not comply with Colombian regulations (Ministry of
Social Protection, 2013) (150 and 3000 CFU/g respectively). At longer times, however,
(9 and 12 days) these values decreased, which then complied with the regulation. This
situation can mainly be attributed to the effect of the Ch contained in the EC+Ca. Its role
as a bactericide or antimicrobial is based on the generation of a change in the permeability
of the cell wall, which generates a leakage of proteins and other intracellular compounds
of microorganisms (Tokatli, and Demirdöven, 2020; No and Meyers, 2007).

Vieira et al. (2016) used Ch in an EC with aloe vera to be applied to blueberries that
reduced the microbiology in the fruits by a high percentage with respect to the control.
We consider that the results could have been affected by environmental contamination;
therefore, we suggest intensifying the disinfection of fresh fruit with NaClO (100 ppm
per immersion for 1 min), or another disinfectant could be used. Therefore, we consider
that the microbiological results can be corrected and that the product could achieve 12

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 21

 days of useful life at 4ºC: higher than the length reported at the same temperature for fresh
blackberries (4 - 5 days) (Sora et al., 2006).

Table 2. Microbiological evaluation of blackberries with EC+Ca that had been stored at
4°C for 12 days.
Faecal
Total coliforms Yeasts Moulds
Time(days) coliforms
(CFU/g) (CFU/g) (CFU/g)
(CFU/g)
30 Cladosporium
0 220 - 180 < 10 300 – 300
30 Colletotrichum
500 – 1300 Cladosporium
3 400 - 6900 < 10 2000 - 3500
900 Botrytis

6 < 10 - < 10 < 10 2000 - 4000 < 10 –30 Colletotrichum

9 30 – 30 < 10 300 – 300 < 10 - < 10

30 Cladosporium
12 < 10 – 200 < 10 270 – 300
20 Colletotrichum

CONCLUSIONS

The EC+Ca formulated from SA, BW, G, S, Ch, and CaCl2, had a positive impact on the
physicochemical, physical, and microbiological properties of the Andean blackberry. Its
shelf life was prolonged to 12 days in 4°C storage conditions; this is a 140% increase in
shelf life compared to what has been reported for fresh blackberries or those without EC.
In general, blackberries with EC+Ca have an important TP content and antioxidant
activity; in addition, the EC could represent a vehicle for the blackberry to be fortified.
The product’s critical variables during storage were weight loss and firmness, which
reached values of 13.6 and 50.0% at 4ºC, respectively. The addition of Ch showed a
significant effect on the microbiology of blackberries with EC+Ca: there was a reduction
in total coliforms, moulds, and yeasts to permissible levels, and, as such, the product was
identified as being of acceptable quality.

In conclusion, the EC SA, BW, G, S, Ch, and CaCl2, and CaCl2, has potential as post-
harvest treatment and provides promising results as protective layer to increase the shelf
life of blackberry and other berries, to maintain the physicochemical, nutritional and
microbiological properties during the storage and transport period.

Conflicts of Interest

The manuscript was prepared and revised by all authors, who declare the absence of any
conflict which can put the validity of the presented results in risk.

[SYLWAN., 164(2)]. ISI Indexed,Feb 2020 22

 Acknowledgments

The authors thank the Colciencias for the financing of the research project, as well as to
Jaimie Brzezinsk (London Metropolitan University) for the critical review of the
manuscript in english.

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