Effects of packaging materials and disinfectants on quality changes of ceylon spinach (Basella alba L.) during storage
ISSN 2588-1299
VJAS 2019; 2(2): 397-408
Vietnam Journal
of Agricultural
Sciences
Effects of Packaging Materials and
Disinfectants on Quality Changes of Ceylon
Spinach (Basella alba L.) during Storage
Tong Thi Anh Ngoc & Phan Thi Thanh Que
Food Technology Department, Faculty of Agriculture, Can Tho University, Can Tho,
94115, Vietnam
Abstract
The objectives of the study were to determine the effective washing
disinfectant (chlorine dioxide and r peracetic acid) in reducing total
aerobic counts, E. coli and coliform, and then to test different
packaging materials, namely polypropylene (PP), high density
polyethylene (HDPE), and low density polyethylene (LDPE), on the
quality and shelf-life of Ceylon spinach. The results showed that
washing Ceylon spinach with 100 ppm peracetic acid solution for
five minutes significantly reduced E. coli and coliform counts
(reduction of 1.1 and >2 log CFU g-1, respectively). Ceylon spinach
packed in HDPE material might optimally prolong the shelf-life up
to 7 days whereas spinach packed in LDPE was recommended if
used within 6 days at 10 ± 1oC.
Keywords
Ceylon spinach, microbial counts, packaging materials, peracetic
acid, quality, storage
Introduction
Basella alba L. is an important green leafy vegetable commonly
found in the tropical regions of the world (Deshmukh & Gaikwad,
2014; Varalakshmi, 2016). In Vietnam, there are more than 100
industrial sectors of vegetable production with a total capacity of
300,000 tons each year (MARD, 2018). Fresh vegetables are sold in
local markets, supermarkets, and retailers, etc. in Vietnam. Most of
the vegetables marketed are produced as primary production and are
rinsed and stored under ambient temperatures in local markets or
refrigerated conditions in supermarkets. For convenience and safety
reasons, it is necessary to produce ready-to-use vegetable products
as minimally processed vegetables, especially Basella alba (Ragaert
et al., 2007; Kakade et al., 2015).
Received: March 11, 2019
Accepted: July 3, 2019
Minimally processed vegetables, which includes vegetables
being selected, washed and dried, and packaged with sealed. These
steps may not be efficient to eliminate contamination
Correspondence to
397
Effect of packaging materials and disinfectants on quality changes of Ceylon spinach during storage
on minimally processed vegetables (Graça et
al., 2017). Hence, washing vegetables with
disinfectants could potentially reduce or
eliminate the microbial loads on vegetable
products as much as possible (Tong et al., 2015;
Tong et al., 2018). Chlorine (the most common
disinfectant) and peracetic acid (an alternative
disinfectant) have been studied intensively on
vegetables (Arturo-Schaan et al., 1996; Li et al.,
2001; Allende et al., 2004; Jin & Lee, 2007).
Though the use of chlorine is reasonable
regarding cost and efficiency, the chlorination
process has some negative effects, such as the
formation of carcinogenic by-products, skin
irritation, and negative respiratory tract effects
(Tong, 2015). Unlike chlorine, peracetic acid is
harmless and produces environmentally friendly
residues; it is a promising and widespread
substitute for chlorine.
Materials and Methods
Experimental design
Decontamination efficacy
Ceylon spinach (Basella alba) was grown
on a farm-scale in Chau Thanh A district, Hau
Giang province, Vietnam and was harvested
after 30-35 days of cultivation. The Ceylon
spinach plants (35-50cm in length) were
harvested from January to April 2018 and
transported to the laboratory at the Food
Technology Department, Can Tho University.
Preparation and disinfection were carried out in
a cleanroom at 28-30oC. Three separate
experiments were repeated independently. The
white rust, downy mildew, and yellow leaves
were removed prior to washing the plants in tap
water (for 60 seconds) to remove the soil, sand,
and foreign matters. The excess water on the
surface of the Ceylon spinach was drained
manually using a salad spinner (Japan) for one
minute. Thereafter, the Ceylon spinach was
dipped in chlorine dioxide (5%, Thailand; pH
adjusted to 6.5 via acetic acid) or peracetic acid
(12%, Korea) at a concentration of 100ppm for
five minutes with the ratio of 1:10 of fresh-cut
spinach and the solution. Then, the Ceylon
spinach was rinsed with potable water
(commercial drinking water produced by Can
Tho University) with the same ratio used with
the disinfectant for one minute to remove any
disinfectant residues and centrifuged for another
minute.
In addition to using sanitizing treatments as
chemical interventions in the reduction of
microbial loads on vegetables, packing
vegetables in plastic containers as a physical
intervention could be inacted to extend the
shelf-life of minimally processed vegetables.
The storage of the minimally processed
vegetables can be combined with refrigeration
in order to reduce the respiration rate and inhibit
microbial growth (Barbosa et al., 2011).
However, the types of packaging materials and
packaging methods also partly affect the quality
changes and the shelf-life of products during
storage (Suryawanshi, 2008; Costa et al., 2011;
Muizniece-Brasava et al., 2013), particularly for
vegetables (Piagentini & Güemes, 2002;
Jacobsson et al., 2004), meat (Kotzekidou &
Bloukas, 1996), and fish products (Ly & Tong,
2017).
In each experiment, the decontaminated
Ceylon spinach was compared to the unwashed
and water washed samples. Effective
decontamination was evaluated based on the
reduction of microbial counts, i.e. total
mesophilic counts, E. coli and coliform counts,
and organoleptic quality.
The objectives of the present study were to
determine the most effective disinfectant in
reducing the total aerobic counts of Escherichia
coli (E. coli) and coliform on Ceylon spinach
vegetables (Basella alba) in preliminary
treatments and identify the proper packaging
materials that can minimize the changes in
quality and organoleptic properties of Ceylon
spinach vegetables during storage.
Storage of the Ceylon spinach
Similar to the procedure described above,
the Ceylon spinach plants were washed in the
selected disinfectants and were packed in
different
packaging
materials:
PP
(polypropylene, thickness of 25µm), HDPE
(high density polyethylene 67%, thickness of
35µm), or LDPE (low density polyethylene,
thickness of 40µm) with package sizes of
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Vietnam Journal of Agricultural Sciences
Tong Thi Anh Ngoc & Phan Thi Thanh Que (2019)
18x21cm. Each independent experiment,
twenty-four sealed bags of the Ceylon spinach
plants packed on three above materials
weighing 50g each were placed in a refrigerator
(Sanaky, Vietnam) at 10 ± 1oC. The control
samples were placed in plastic disks but were
not sealed, and then stored in the same
refrigerator. The three different storage
experiments were repeated independently.
(TMC), total yeasts and molds, and total
coliform and E. coli counts were determined by
pour plating the decimal dilutions onto Plate
Count Agar (PCA, India, Himedia), Yeast
Extract Glucose Chloramphenicol Agar (YGC,
Merck,
Darmstadt,
Germany),
and
Chromocult® Coliform Agar ES (Merck,
Darmstadt, Germany), respectively. The pour
plates were incubated for 2-3 days at 37oC to
determine the TMC and total yeasts and molds,
and for 24h to determine the total coliform and
E. coli. Microbial counts were reported as log
CFU g-1 of tissue.
Analytical methodology
Physical and chemical qualities of the Ceylon
spinach
The initial weight and storage weight of
each sample were determined on the sampling
day prior to analysis using a balance (OHAUS,
China). The extent of spoilage was calculated
by weighing the amount of spoiled leaves from
each stored sample.
Sensory evaluation
After the spinach samples were washed
with one of the two disinfectants mentioned
above at a concentration of 100ppm for 5min
(the preliminary treatment), the sensory quality
of the spinach samples, general appearance,
color, odor, and texture, were evaluated by 18-
19 panelists who participated in triangle tests.
After being washed with a disinfectant, the
samples were rinsed in tap water for one minute
before the sensory tests. In all cases, a set of
three samples of the vegetable (each sample ca.
50g), randomly coded with 3-digit numbers,
was presented to each panelist. The panelists
were told that two of the samples were similar
and were asked to identify the odd sample. The
number of correct answers was compared to the
number expected by using a statistical table to
achieve a significant answer (5% level)
according to the BS ISO 4120:2004.
The pH of the Ceylon spinach was
determined by an electronic pH meter (Vernier,
USA).
The vitamin C content (mg/100g dry
weight) of the Asian spinach was analyzed by
titration methods with 2.6-dichlorophenol-
indophenol reagents.
The color of the whole leaves was measured
using a spectrophotometer-colorimeter (model
NR60CP, China). The overall color difference
(ΔE) in color spaces L*, a*, and b* was calculated
according to the following equation (1):
∆퐸 = (퐿∗ − 퐿)2 + (푎∗ − 푎)2 + (푏∗ − 푏)2 (1)
√
The sensory evaluation of the Ceylon
spinach during storage was implemented by
scoring methods. The spinach was scored on a
scale from 0 to 5, with 0 being the lowest and 5
being the highest. A descriptive table with the
lowest and highest quality attributes was
provided. Each assessment was carried out by
ten panelists and they were also asked to state
whether or not the spinach was acceptable. This
was used to determine the shelf-life of the
products.
where L*, a*, b* and L, a, b are the
measured values of the samples on day 0 and
day of sampling, respectively.
Microbiological analysis
A 15g composite sample collected from
different parts of each spinach sample was
transferred aseptically to a Stomacher bag by
means of sterile scalpels and tweezers. One
hundred thirty-five (135) mL of sterile
Maximum Recovery Diluent (MRD; Merck,
Darmstadt, Germany) was added and the
mixture was homogenized for 1min. A tenfold
serial dilution was made from the sample in the
MRD. The total aerobic mesophilic counts
Statistical analysis
Data were subjected to one-way analysis of
variance (ANOVA) using the Statgraphics
Centurion 15.1 program (U.S.A.). LSD (Least
399
Effect of packaging materials and disinfectants on quality changes of Ceylon spinach during storage
Significant Difference) test was applied for
pairwise comparisons of means at P ≤0.05. All
results were reported as the mean values ±
standard deviations.
reported that cucumbers washed in chlorine
dioxide water resulted in less than 1 log unit of
various microbes (i.e. total aerobic counts, lactic
acid bacteria, yeast and molds) on product
surfaces. Additionally, the microbial reductions
for aerobic bacteria coliform, yeast and molds
on fresh-cut celery, cabbage, and potatoes
washed in 80ppm of peracetic acid were less
than 1.5 log units (Joshi et al., 2013).
Results and Discussion
Effects of the chlorine dioxide and peracetic
acid disinfectants on the microbial counts of
the spinach
The sensory qualities of the Ceylon spinach
samples after being washed with chlorine
dioxide or peracetic acid were evaluated using
triangle tests. The results in Figure 2 show that
the Ceylon spinach samples washed with the
two solutions were not significantly different
compared to those washed with tap water. The
number of correct responses was lower than the
required number of correct responses to achieve
a significant difference. Based on these results,
peracetic acid is recommended as a suitable
sanitizer to wash Ceylon spinach for further
storage.
Chlorine dioxide and peracetic acid are
considered alternative sanitizers because of their
strong oxidation capacities and lower risk of
forming harmful by-products (Monarca et al.,
2002; Silveira et al., 2008). Both disinfectants
are safe and create environmentally friendly
residues (Demirci & Ngadi, 2012).
The antimicrobial effects of chlorine
dioxide and peracetic acid (PAA) were
evaluated on the Ceylon spinach (Figure 1).
The initial total mesophilic counts (TMC),
coliform counts, and E. coli counts on unwashed
Ceylon spinach were 7.1 ± 0.12, 4.9 ± 1.23, and
2.9 ± 1.02 log CFU g-1, respectively. The TMC
on the Asian spinach washed in tap water,
chlorine dioxide, and PAA were 6.9 ± 0.41, 6.4
± 0.81, and 6.5 ± 0.06 log CFU g-1, respectively.
The coliform counts on the Ceylon spinach
washed in tap water, chlorine dioxide, and PAA
were 4.7 ± 0.7, 4.1 ± 1.14, and 3.8 ± 0.3 log
CFU g-1, respectively. The E. coli counts on the
Ceylon spinach washed in tap water, chlorine
dioxide, and PAA were 1.6 ± 1.13, 1.1 ± 0.15,
and <1 log CFU g-1, respectively. These data
indicate that the microbial counts on Ceylon
spinach washed in tap water (control samples)
were not reduced significantly. The chlorine
dioxide and peracetic acid solutions were not
effective in reducing TMC but significantly
reduced coliform and E. coli counts on the
Ceylon spinach. Surprisingly, a significant
reduction of E. coli was observed (detection
limits, <1 log CFU g-1) when the Ceylon
spinach was washed with peracetic acid. This
result was probably because peracetic acid has a
stronger oxidation capacity and is less affected
by organic matter than chlorine dioxide (Kitis,
2004). The obtained results are in agreement
with the previous study of Sapers (2001) who
Physical and chemical changes of the spinach
during storage
Weight loss and rate of spoilage during storage
The weight loss and rate of spoilage of the
spinach were measured by the change in weight
of the samples over the storage period at 10 ±
1oC. The percent of weight loss and rate of
spoilage of the Ceylon spinach are shown in
Figure 3 and Figure 4, respectively. The
highest weight loss (17.28%) and almost
complete spoilage (99.24%) were observed in
the control samples (not packed) after 3 days.
The direct contact of the Ceylon spinach with
cool air resulted in a rapid loss of moisture
during storage. After 7 days of storage, the
weight loss (6.89%) and rate of spoilage
(44.21%) of the Ceylon spinach packed in
LDPE were higher than the samples packed in
PP (2.56% and 22.53%, respectively) and
HDPE (4.27% and 12.53%, respectively). It is
well known that the rate of water vapor
transmission of LDPE is higher than the other
packaging materials, thus, the samples packed
in LDPE had a higher loss of moisture and
higher spoilage rate (Piagentini & Güemes,
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Vietnam Journal of Agricultural Sciences
Tong Thi Anh Ngoc & Phan Thi Thanh Que (2019)
2002). In addition, the loss of moisture can
cause undesirable changes in color, soft
structure, and loss of nutritional quality (Nagar
et al., 2012).
color difference (ΔE = 2.94) of the control
samples was not different with the samples
contained in the PP and HDPE packages (ΔE =
1.95 and 1.94, respectively) at day 3. The
statistical results also showed that all three
packaging materials used did not have an impact
on the overall color difference of the Ceylon
spinach during storage at 10 ± 1oC.
Color and pH changes of the spinach during
storage
The color changes of the spinach during
storage shown in Table 1 reveal that the overall
9
8
7
6
5
4
3
2
1
<
0
TMC
Coliform
Raw materials (after cutting)
E. coli
Microbial counts
Water (5 min)
Chlorine dioxide (100 ppm, 5 min)
Peracetic acid (100 ppm, 5 min)
Figure 1. Microbial counts on the Ceylon spinach after being washed with water, chlorine dioxide, or peracetic acid (PAA)
20
18
16
14
12
10
8
6
4
2
0
Chlorine dioxide
Peracetic acid
Figure 2. Sensory test of Ceylon spinach treated with chlorine dioxide or peracetic acid water at 100ppm for 5 minutes. number
of panelists choosing the wrong sample as the odd sample, number of panelists choosing the correct sample as the odd sample.
The line (‒) shows the minimum number of correct answers needed to have a significant difference in sensory qualities between
series (α = 0.05).
401
Effect of packaging materials and disinfectants on quality changes of Ceylon spinach during storage
50
40
30
20
10
0
0
3
6
7
days
-10
Control
PP
HDPE
LDPE
Packaging materials
Figure 3. Weight loss of the Ceylon spinach during storage
100
80
60
40
20
0
0
3
6
7
days
-20
Control
PP
HDPE
LDPE
Packaging materials
Figure 4. Rate of spoilage of the Ceylon spinach during storage
Vitamin C changes of the spinach during storage
The effects of the packaging materials on
the pH value of the spinach during storage at 10
± 1oC are shown in Figure 5. The initial pH
value of the spinach was 6.4 ± 0.14. The pH
values ranged from 6.36 to 6.5 during storage.
The type of packaging material did not
influence the pH value of the Ceylon spinach
during storage.
Vitamin C, including ascorbic acid and
dehydroascorbic acid, is one of the most
important nutritional quality factors in many
horticultural crops and has many biological
activities in the human body. More than 90% of
vitamin C in the human diet is supplied by fruits
and vegetables, especially green leafy
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Vietnam Journal of Agricultural Sciences
Tong Thi Anh Ngoc & Phan Thi Thanh Que (2019)
vegetables (Lee & Kader, 2000; Citak &
Sonmez, 2010). In this experiment, changes in
vitamin C contents of the Ceylon spinach were
also determined during storage (Table 2).
Microbial quality of the spinach during
storage
Total aerobic mesophilic counts
The results of the total aerobic mesophilic
counts (TMC) of the spinach during storage at
10 ± 1oC (with a relative humidity of 44-60%)
in different packaging materials are shown in
Figure 6. The initial total mesophilic counts of
the Ceylon spinach were from 7.0 to 7.3 log
CFU g-1. This is in agreement with previous
studies that showed initial TMC ranged from 2
to 8 log CFU g-1 (Lee et al., 2013; Ottesen et al.,
2013; Gu et al., 2018). Samples washed with
peracetic acid (100ppm, 5min) had initial TMC
values (at day 0) of 5.3 ± 0.4 log CFU g-1. As
recommended by Debevere et al. (2006), the
microbiological criteria of TMC for fresh
vegetables was targeted at 5 log CFU g-1 at the
end of shelf-life, and an acceptable limit of 8 log
CFU g-1. Therefore, the initial microbiological
Generally, a decrease in the vitamin C
content of the Ceylon spinach was observed in
all the packed samples after 7 days at 10 ± 1oC
(Table 2). The vitamin C content of the
unpackaged samples reduced rapidly after 3
days (loss of 82%). Although the packaging
materials delayed the loss of vitamin C of the
Ceylon spinach during storage, the vitamin C
content was not significantly different among
the three packaging materials. At day 7, the loss
of vitamin C of the Ceylon spinach was 78-86%
compared to day 0. Gil et al. (1999) found that
the decrease of vitamin C of fresh-cut spinach
both in air and MAP packaging was up to 50%
after 3 days of storage at 10oC.
Table 1. Effects of the packaging materials on the overall color difference (ΔE) of the Ceylon spinach during storage at 10 ± 1oC
Overall color difference (ΔE)
Day
Control
PP
HDPE
0a
LDPE
0a
0
3
6
7
0a
2.94b ± 0.23
NA
0a
1.95ab ± 0.49
2.25a ± 1.59
1.72a ± 0.13
1.94ab ± 1.14
1.22a ± 0.53
0.99a ± 0.56
1.72a ± 0.13
1.52a ± 0.60
1.01a ± 0.21
NA
Note: Unpackaged samples served as the control; Values with the same letter within the same row are not significantly different at P
<0.05; NA: Not analyzed.
100
80
60
40
20
0
0
3
6
7 days
-20
Control
PP
HDPE
LDPE
Packaging materials
Figure 5. Changes in the pH of the Ceylon spinach during storage
403
Effect of packaging materials and disinfectants on quality changes of Ceylon spinach during storage
Table 2. Changes in vitamin C contents of the Ceylon spinach in different packaging materials during storage at 10 ± 1oC
Changes in vitamin C (mg/100g dry weight)
Day
Control
30.79a ± 2.77
5.53a ± 3.36
NA
PP
HDPE
LDPE
0
3
6
7
30.79a ± 12.77
22.55a ± 14.70
8.07a ± 1.06
6.42a ± 2.27
30.79a ± 12.77
16.05a ± 10.30
5.93a ± 2.76
6.54a ± 1.28
30.79a ± 12.77
15.36a ± 12.46
6.21a ± 2.17
4.06a ± 2.38
NA
Note: Unpackaged samples served as the control; Values with the same letter within the same row are not significantly different at P
<0.05; NA: Not analyzed.
quality of the Asian spinach was acceptable. No
stored samples exceeded 8 log CFU g-1 after 6
days of storage. The shelf-life of the samples in
the PP and HDPE packages can be extended to day
7 with TMC counts of 7.8 and 7.7 log CFU g-1,
respectively.
The spoilage of vegetables may result from
the degradation of physiological and sensory
characteristics (color, texture, and odor) as well
as from microbiological degradation (Kakade et
al., 2015). In this study, the sensory quality had
a high correlation with the chemical and
microbiological quality. The samples stored in
the PP and HDPE packages (both had an overall
score of 4) were better than those packed in
LDPE (overall score of 3). According to the
results of this study, packaging the Ceylon
spinach in LDPE at 10 ± 1oC is recommended if
the spinach is used within 6 days, while
packaging the Ceylon spinach in the PP or
HDPE packages extends the shelf-life up to 7
days. More importantly, after 7 days of storage,
the Ceylon spinach in the HDPE package had
better quality compared to those packed in PP
based on the microbiological quality and
spoilage rate. Therefore, the HDPE package is
suggested to prolong the quality of the Ceylon
spinach during storage.
Total yeasts and molds
Generally, yeast and mold counts of the
samples in all the packaging materials increased
gradually during storage at 10 ± 1oC (Figure 7).
The initial counts of yeasts and molds were 3.6
± 0.7 log CFU g-1. This was in line with the
study of Debevere et al. (2006) who reported a
tolerance limit of 4 log CFU g-1. During storage,
all the samples showed rapid growth of yeasts
and molds after day 3. Particularly, the total
yeasts and molds of the samples in the PP
package increased rapidly during storage. At
day 7, the yeast and mold counts in the PP were
5.2 ± 0.1 log CFU g-1, whereas the counts in the
HDPE and LDPE packaging were 4.5 ± 0.4 and
4.4 ± 0.6 log CFU g-1, respectively. The
recommended acceptable limit of yeasts and
molds is 5 log CFU g-1 (Debevere et al., 2006).
The Ceylon spinach stored in LDPE as well as
HDPE can be maintained up to 7 days at 10 ±
1oC before becoming unacceptable for human
consumption.
Conclusions
Ceylon
spinach
should
first
be
decontaminated with peracetic acid (100ppm for
5 minutes) before being packed then stored at
10 ± 1oC. The results indicated that the use of
PP or HDPE packages might optimally prolong
the shelf-life of the Ceylon spinach up to 7 days.
The Ceylon spinach in the LDPE packaging is
recommended if the produce is used within 6
days, while the Ceylon spinach in HDPE had
better quality compared to those packed in PP
based on the microbiological quality and
spoilage rate. Therefore, the HDPE packaging
material is recommended for storage of Ceylon
spinach at 10 ± 1oC.
Sensory evaluation
Sensory evaluation was implemented on the
characteristics of the Asian spinach to evaluate
the color, odor, texture, and overall changes
during storage (Figure 8). At day 0, the color,
odor, and texture of the Asian spinach showed
very high acceptability with scores of 4.6-4.8.
Rapid changes in terms of the sensory aspects
were observed in the control samples during
storage (scores of 2.1-3.4 at day 3).
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Vietnam Journal of Agricultural Sciences
Tong Thi Anh Ngoc & Phan Thi Thanh Que (2019)
9
8
7
6
5
4
0
3
6
7
days
Control
PP
HDPE
LDPE
Packaging materials
Figure 6. Total aerobic mesophilic counts (TMC) on the spinach during storage
6
5
4
3
2
0
3
6
7
days
Control
PP
HDPE
LDPE
Packaging materials
Figure 7. Total yeast and mold counts of the Ceylon spinach during storage
(a)
5
4
3
2
1
0
0
3
6
7
days
Control
PP
HDPE
LDPE
405
Effect of packaging materials and disinfectants on quality changes of Ceylon spinach during storage
(b)
5
4
3
2
1
0
days
7
0
3
6
Control
PP
HDPE
LDPE
(c)
5
4
3
2
1
0
days
7
0
3
6
Control
PP
HDPE
LDPE
(d)
5
4
3
2
1
0
days
0
3
6
7
Control
PP
HDPE
LDPE
Figure 8. Sensory changes of (a) color, (b) odor, (c) texture, and (d) overall attributes of the Ceylon spinach during storage
406
Vietnam Journal of Agricultural Sciences
Tong Thi Anh Ngoc & Phan Thi Thanh Que (2019)
(2018). Shifts in spinach microbial communities after
chlorine washing and storage at compliant and
abusive temperatures. Food Microbiology. 73: 73-84.
Acknowledgements
We would like to thank Ms. Pham Hong
Nhi and Mr. Dinh Van Khanh Linh for their
assistance in this study.
Jacobsson A., Nielsen T. & Sjöholm I. (2004). Effects of
type of packaging material on shelf-life of fresh
broccoli by means of changes in weight, colour and
texture. European Food Research and Technology.
218: 157-163.
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