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Addition of glycerol and sodium chloride into garcinia atroviridis chitosan film,
and its application for wrapping of chicken meat

Article in Malaysian Journal of Analytical Science · January 2021

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 Malaysian Journal of Analytical Sciences, Vol 25 No 3 (2021): 399 - 414

ADDITION OF GLYCEROL AND SODIUM CHLORIDE INTO Garcinia

atroviridis CHITOSAN FILM, AND ITS APPLICATION FOR WRAPPING OF
CHICKEN MEAT

(Penambahan Gliserol dan Natrium Klorida Pada Garcinia atroviridis Filem Kitosan dan
Aplikasinya Pada Pembungkusan Daging Ayam)

S-Navin Sivanasvaran, Ianne Kong, Hui-Ling Tan, Liew-Phing Pui*

Department of Food Science and Nutrition, Faculty of Applied Sciences,

UCSI University, 56000 Cheras, Kuala Lumpur, Malaysia

*Corresponding author: puilp@ucsiuniversity.edu.my

Received: 28 February 2021; Accepted: 22 May 2021; Published: 27 June 2021

Abstract
Glycerol and sodium chloride (NaCl) have a strengthening effect that can be incorporated into edible films to enhance their
mechanical properties. This study evaluates the effects of glycerol (0.5-2.0% v/v) and NaCl (5.0-10.5 mM w/v) on the physical,
mechanical, and antimicrobial properties of chitosan film incorporated with Garcinia atroviridis. Storage tests were conducted on
chicken meat that were wrapped with and without the film. The moisture content, water solubility, and elongation at break of film
with 1.5% (v/v) glycerol and 10 mM (w/v) NaCl was the highest. The increment of glycerol and NaCl reduced thickness, tensile
strength, and Young’s modulus. The total color difference of the film with 0.5% (v/v) glycerol and 0 mM (w/v) NaCl was highest
and showed the greatest inhibition against Pseudomonas aeruginosa and Staphylococcus aureus. The pH of the control increased
whereas the pH of chicken meat wrapped with the film decreased as the number of storage days increased. In conclusion, film with
1.5% (v/v) glycerol and 10 mM (w/v) NaCl might be a suitable film because it demonstrated the highest elongation at break and a
prolonged shelf life for the chicken meat of at least 15 days, which was longer than of the control.

Keywords: glycerol, sodium chloride, edible film, chitosan, Garcinia atroviridis

Abstrak
Gliserol dan natrium klorida (NaCl), yang mempunyai kesan pengukuhan, dapat dimasukkan ke dalam filem yang boleh dimakan,
untuk meningkatkan sifat mekanikal filem yang boleh dimakan. Tujuan kajian ini adalah untuk menilai kesan gliserol (0.5-2.0%
v/v), dan NaCl (5.0-10.5 mM w/v) terhadap sifat fizikal, mekanikal, dan antimikrob filem kitosan yang diperbadankan dengan
Garcinia atroviridis. Ujian penyimpanan dilakukan pada daging ayam yang dibungkus dengan dan tanpa filem. Kandungan
kelembapan, kelarutan air, dan pemanjangan pada pemecahan filem dengan gliserol 1.5% (v/v), NaCl 10 mM (w/v) adalah yang
tertinggi. Peningkatan gliserol dan NaCl mengurangkan ketebalan, kekuatan tegangan, dan pekali Young. Perbezaan warna
keseluruhan filem dengan 0.5% (v/v) gliserol dan 0 mM (w/v) NaCl adalah tertinggi dan ia menunjukkan penghambatan terbesar
terhadap Pseudomonas aeruginosa dan Staphylococcus aureus. Pengawalan pH meningkat sedangkan pH daging ayam yang
dibungkus dengan filem menurun ketika hari penyimpanan meningkat. Kesimpulannya, filem dengan gliserol 1.5% (v/v) dan NaCl

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Sivanasvaran et al: ADDITION OF GLYCEROL AND SODIUM CHLORIDE INTO Garcinia atroviridis
CHITOSAN FILM, AND ITS APPLICATION FOR WRAPPING OF CHICKEN MEAT

10 mM (w/v) mungkin merupakan filem yang sesuai kerana ia mempunyai pemanjangan tertinggi pada pemecahan dan jangka
hayat daging ayam yang berpanjangan sekurang-kurangnya 15 hari yang lebih lama daripada kawalan.

Kata kunci: gliserol, natrium klorida, filem boleh dimakan, kitosan, Garcinia atroviridis

Introduction According to Basri et al. [13], Garcinia atroviridis

In Malaysia, chicken meat is one of the most consumed extracts inhibited gram-positive bacteria
foods. Statistics show the poultry consumption per (Staphylococcus aureus, Staphylococcus epidermis,
person in Malaysia was approximately 49 kg in 2019 Bacillus subtilis) and gram-negative bacteria
(https://www.statista.com/statistics/756920/malaysia- (Salmonella enterica, Salmonella typhimurium,
meat-consumption-per-capita-by-type/). Chicken meat Escherichia coli and Pseudomonas aeruginosa).
had a shelf life of 6 days when in refrigerated storage at Chitosan film incorporated with Garcinia atroviridis
4 °C. Common pathogens that were reported in chicken extracts showed promising characteristics for use as
meat are Campylobacter, Salmonella, Staphylococcus food packaging [6]. From their study, it was found that
aureus, and Pseudomonas aeruginosa [1]. Food the film increased the shelf life of Indian mackerel
packaging was invented to preserve foods from external during storage and inhibited the microbial growth of
contamination [2]. Edible packaging is gaining greater Pseudomonas aeruginosa, Bacillus subtilis, and
interest due to the disadvantages associated with Staphylococcus aureus. However, the tensile strength
petroleum-based food packaging and how they deplete and elongation at the break of film were low.
non-renewable natural resources [3]. This study focuses
on consumable films or coatings that are applied onto The mechanical properties of packaging materials are
food products [4]. Edible packaging provides important to ensure product safety and security until it
mechanical protection and extends shelf life of foods reaches the consumer. Glycerol improves the elongation
[5]. at the break of edible film [14, 15]. Another study, Choi
et al. [16] reported that addition of NaCl increased the
Chitosan is widely used to produce edible films because elongation at break of pea starch films by adjusting
it possesses good antimicrobial and gas barrier formation and strength of intermolecular hydrogen
properties that give it a film forming ability [6]. Several bonds of starch molecules. Further, increasing the NaCl
researchers have found that chitosan has a proven concentration enhanced the hydration effect of chitosan
antibacterial effect towards gram-positive and gram- films. More hydrated chloride ions impeded the
negative bacteria [7-8]. The incorporation of 20% (v/v) hydrophobic interactions between chitosan molecules
Piper nigrum leaf extract increased the inhibition zone and disrupted hydrogen bonding on the chitosan film
of chitosan and polyvinyl alcohol blended films against [17].
Escherichia coli [9]. On the other hand, chitosan film
incorporated with grape seed extract extended the shelf Since studies on enhancing the mechanical properties of
life of chicken breast fillets and inhibited lipid oxidation chitosan film incorporated with Garcinia atroviridis are
[10]. Additionally, chitosan film incorporated with scarce, the study of addition levels of glycerol and NaCl
cinnamon oil inhibited lipid oxidation and reduced on chitosan films with Garcinia atroviridis can provide
microbial growth on chicken breast fillets [11]. further insights into the application of film for chicken
meat. This research evaluates the effects of physical,
Garcinia atroviridis is known as ‘asam keping’ or ‘asam mechanical, and antimicrobial analysis of chitosan film
gelugur’. It is commonly used to make pickles, in tea, incorporated with Garcinia atroviridis by optimizing
and as a seasoning in curries. Al-Mansoub et al. [12] the additional levels of glycerol and NaCl. The
found that Garcinia atroviridis extract had antioxidant, optimized film shall be applied to chicken meat for
antimicrobial, and antitumor-promoting properties. storage tests. The chicken meat slices will be wrapped

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 Malaysian Journal of Analytical Sciences, Vol 25 No 3 (2021): 399 - 414

with the optimized chitosan film incorporated with Garcinia atroviridis extracts were poured into a
Garcinia atroviridis and stored in a chiller at 4 °C for 15 universal bottle and kept at 4 °C until future use.
days for further analysis.
Preparation of film
Materials and Methods Film formation was conducted following the casting
Materials procedure of Zaman et al. [6]. To prepare the film
Garcinia atroviridis and chicken breast meat (Gallus solution, 1.5 g of chitosan (CH) powder was added in
gallus domesticus) were purchased from a local market 100 mL of 1% (v/v) acetic acid to form (1.5%, w/v) CH
(Petaling Jaya, Malaysia). Chitosan analytical grade, solution. The CH solution was magnetically stirred for
glycerol (≥99.5%), and sodium chloride analytical grade 24 h at room temperature. It was then filtered using a
were purchased from Sigma-Aldrich (M) Sdn Bhd, vacuum filter with filter paper to filter out insoluble
Petaling Jaya, Malaysia. Glacial acetic acid (≥99.99%) residue.
and Tween-80 analytical grade were purchased from
LGC Scientific Sdn Bhd, Malaysia. Ethanol (≥99.8%) For film formation, 25 mL of the CH solution was
(Synertec Enterprise Sdn Bhd, Malaysia), barium poured into a 50 mL beaker. After that, 5% (v/v)
chloride (≥99.99%) (Sigma Aldrich, USA), and Garcinia atroviridis extract and 0.01% (v/v) Tween-80
sulphuric acid (≥96%) (Fisher Scientific, Malaysia), were added to the solution with additional levels of
Mueller-Hinton agar (Synertec Enterprise Sdn Bhd, glycerol (0.5, 1.0, 1.5, 2.0%, v/v) and NaCl (5, 10, 10.5
Malaysia), plate count agar (Chemolab, Malaysia), mM, w/v) as shown in Table 1. The solutions were
peptone water (Synertec Enterprise Sdn Bhd, Malaysia), homogenized using a homogenizer (HG-15D, Daihan
stomacher bags (LGC Scientific Sdn Bhd, Malaysia), Scientific, South Korea) at 9000 rpm for 4 min. After
and petri dishes (90 mm × 15 mm) (Synertec Enterprise homogenization, the solution was transferred to a petri
Sdn Bhd, Malaysia) were purchased. dish and placed into the oven for 24 h at 50°C. Once the
films were dried, they were peeled and transferred into
Preparation of Garcinia atroviridis extracts a zip-lock plastic bag and stored in a desiccator for
The Garcinia atroviridis was cut into smaller pieces and further analysis.
dried in an oven (1350 FX, Sheldon Manufacturing,
Oregon, USA) overnight at 60 °C. After drying, it was Physical properties of film: Moisture content
ground into powder with a grinder (8011G, Waring, The film moisture content was analyzed according to the
USA) to increase the total surface area of the Garcinia method described by Lee et al. [3]. Films with a size of
Atrovidis to obtain a higher yield during extraction. The 2 × 2 cm were placed in a desiccator overnight. Then,
powder was then placed into a sealed pack and kept in a the initial mass of the film was weighed (𝑀𝑖 ). The film
chiller at 4 °C for further analysis. was then placed into an oven at 90°C until there were no
changes in the final mass (𝑀𝑓 ). Moisture content was
The extraction was conducted as previously described determined using the equation below:
by Zaman et al. [6] in which 80 g of Garcinia atroviridis
powder was added to 800 mL of 99.8% (v/v) ethanol in 𝑀𝑖 − 𝑀𝑓
Moisture content = × 100% (1)
a 1 L beaker. The mixture was then mixed for 24 h with 𝑀𝑖

a magnetic stirrer (MS-H280-Pro, DLAB Scientific,

Beijing, China) at 1500 rpm at room temperature. After where Mi and Mf refer to the mass of the initial dried film
2 h, a vacuum filter (WP6222050, Milipore, MA, USA) and final dried film (g), respectively.
and filter paper (Whatman No. 3, Thermoline, NSW,
Australia) were used to filter out the residue in the Water solubility
mixture. The filtrate then underwent evaporation using The film water solubility was measured following
a rotary evaporator (R-000, BÜCHI, Flawil, procedures from Chan et al. [18] with some
Switzerland) at 60 °C under vacuum condition and the modifications. The final mass of the dried films from the

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Sivanasvaran et al: ADDITION OF GLYCEROL AND SODIUM CHLORIDE INTO Garcinia atroviridis
CHITOSAN FILM, AND ITS APPLICATION FOR WRAPPING OF CHICKEN MEAT

moisture content determination were taken as the initial crosshead was adjusted to 20 mm/min. The values from
mass (𝑊1 ). The dried films were placed into a boiling the machine called peak load and peak extension were
tube filled with 30 mL of distilled water for 3 h. After taken as tensile strength (MPa) and elongation at break
immersion, they were taken out and underwent drying (%), respectively. Young’s modulus was determined
again in an oven for 24 h at 60°C. The final dry mass of using the following equation:
the film was measured (𝑊2 ). The water solubility was
𝑆𝑡𝑟𝑒𝑠𝑠
determined using the following equation: 𝑌𝑜𝑢𝑛𝑔′ 𝑠 𝑚𝑜𝑑𝑢𝑙𝑢𝑠 𝑀𝑃𝑎 = (4)
𝑆𝑡𝑟𝑎𝑖𝑛

𝑊1 − 𝑊2
Water solubility = × 100% (2) where stress is determined as the tensile force (F) / cross-
𝑊1
sectional area (A) while strain is the length of film
where W1 and W2 refer to the mass of the initial dried extension (e) / original film length (lo).
film and final dried film (g), respectively.
Antimicrobial properties
Color profile The film antimicrobial properties were analyzed using
The film color profile was measured according to the the agar disk diffusion method described by Chan et al.
method described by Chan et al. [18]. A colorimeter [18]. Antimicrobial activities of the film were examined
(ColourFlex EZ, Hunterlab, Virginia, USA) was used to against two bacteria (Pseudomonas aeruginosa and
determine the color. The values, L* (luminosity), a* Staphylococcus aureus). The bacterial suspension was
(green-red) and b* (blue-yellow) were used to express prepared with reference to the 0.5 McFarland standard.
the film color. The total color difference (𝛥𝐸) of the film Then, 100 µL of the suspension was inoculated on a
was determined using the following equation: Mueller-Hinton Agar and spread aseptically with a glass
hockey stick.
ΔE = √(L − 𝐿∗ )2 + (a − 𝑎 ∗ )2 + (b − 𝑏 ∗ )2 (3)
Films were shaped into 6 mm diameter discs and
aseptically placed onto the Mueller-Hinton Agar plates
where the color parameters of the white plate are
that were previously inoculated. The positive control for
expressed by L, a, and b and the color of the Garcinia
Pseudomonas aeruginosa was Streptomycin (10
atroviridis films are expressed as L*, a* and b*.
mg/mL). For the Staphylococcus aureus, penicillin (10
mg/mL) was used as the positive control. Ethanol was
Thickness
used as the negative control. The plates were then put
A manual micrometer (QB420100, JY, China) was used
into the incubator at 37°C for 24 h. The inhibition zone
to measure the film thickness as described by the method
diameter including the edible film disk (6 mm) was
from Kuan et al. [4]. Measurements were made at five
determined with a vernier caliper.
random places of the film for which an average value
was determined.
Storage test of chicken meat
The chicken meat for storage test was prepared
Mechanical properties
according to the method described by Higueras et al.
The measurements of tensile strength and elongation at
[20]. After purchasing the chicken meat from the
break were conducted according to the procedures
market, it was placed in an icebox and brought to the
demonstrated by Lim et al. [19] with some
laboratory. It was cleaned with distilled water and
modifications. A universal testing machine (CS4921,
lightly tamped with tissue paper. The chicken meat was
Lloyd Instrument, UK) was pre-set with the
cut into 2 cm × 2 cm size, 10 ± 1 g, and placed in the
specifications for speed, gauge length, thickness, width,
chiller at 4°C for further use.
units, and tension mode. Film strips of 20 × 50 mm were
held parallel to the grip of the machine. The separation
of the grip was adjusted to 30 mm. The speed of the

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 Malaysian Journal of Analytical Sciences, Vol 25 No 3 (2021): 399 - 414

The slices of chicken meat were then wrapped with the modifications. The films were peeled off the chicken
optimized film , which was based on the results of a meat and the chicken meat were put into a stomacher
previous analysis. They were then placed into a zip-lock bag. Then, 90 mL of peptone water was poured into the
bag. The chicken meat without film was also placed into stomacher bag, which underwent homogenization for 2
a zip-lock bag and served as the control in this test. The min using a stomacher. The homogenized chicken
chicken meat wrapped with film and the control were mixture underwent serial dilutions from 10-1 up until 10-
6
placed in the chiller at 4°C until analysis. They were using peptone water. After homogenization, 100 µL of
stored for 15 days and the analysis on the chicken meats each serial dilution was inoculated on a plate count agar
were conducted on day 0, 3, 6, 9, 12, and 15 of storage. and a glass hockey stick was used to spread the
inoculations evenly. The plates were incubated for 24 h
pH changes of chicken meat at 37°C. After incubation, the colonies were counted
The pH of the chicken meat wrapped with film and with the results expressed as log10 (CFU/mL).
control were measured following procedures described
in Tantasuttikul et al. [21]. The films were peeled off the Statistical analysis
chicken meat and the chicken meat were put into a Results were expressed as mean ± standard deviation of
stomacher bag. Then, the stomacher bag was filled with three determinations. The results were statistically
100 mL of distilled water. Then it underwent analyzed using Minitab version 17 (p ≤ 0.05) (Minitab,
homogenization for 2 min using a stomacher (BagMixer USA). The comparison of means was performed by one-
400, Interscience, France). The pH reading was way analysis of variance followed by Tukey’s test. The
measured by dipping the pH meter (pH700, EUTech Paired T-test was conducted to analyze results of total
Instruments, Singapore) into the mixture. colony count and pH reading between the chicken meat
wrapped with film and chicken meat without film
Microbial changes of chicken meat (control).
The total colony count was analyzed according to the
procedures outlined in Remya et al. [46] with slight

Table 1. Formulation of chitosan film incorporated with Garcinia atroviridis extract

Film Formulation Glycerol Concentration Sodium Chloride Concentration
(%) (mM)
A 0.0 0.0
B 0.5 0.0
C 1.0 5.0
D 1.5 10.0
E 2.0 10.5

Results and Discussion

Physical properties of films The increment of glycerol and NaCl increased the
Moisture content moisture content of the film from 15.42% to 27.29%.
Dehghani et al. [22] showed that low moisture content This might be due to the glycerol’s hygroscopicity
is usually favorable for edible films as it reduces the risk whereby it tended to absorb and retain moisture from its
of microbial growth. Table 2 presents the moisture surrounding [23]. Additionally, as a humectant, the
content of each chitosan film with Garcinia atroviridis hydroxyl groups in glycerol formed hydrogen bonds in
extract with different addition levels of glycerol and the water [24]. The addition of glycerol also elevated the
NaCl. viscosity and allowed for film matrix interaction with

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Sivanasvaran et al: ADDITION OF GLYCEROL AND SODIUM CHLORIDE INTO Garcinia atroviridis
CHITOSAN FILM, AND ITS APPLICATION FOR WRAPPING OF CHICKEN MEAT

water to increase [25]. These results are also similar to glycerol and 0 mM (w/v) NaCl was highest. However,
Peng et al. [26] and Dick et al. [27] in which the there was no significant difference between the color
increment of glycerol caused the moisture content of a profile of the films. The ΔE of the film reduced when
film to increase. the addition level of glycerol and NaCl increased. This
could be attributed to the fact that glycerol and NaCl was
Water solubility colorless and it did not have the color imparted into the
Water solubility is an important attribute because it film. Peng et al. [26] and Dick et al. [27] observed the
determines biodegradability, integrity, and water increment of glycerol had not changed the ΔE of film in
resistance [28]. Table 2 shows that the increment of combination with tea polyphenols. Another factor to
additional levels of glycerol and NaCl increased the consider is the concentration of chitosan used that
water solubility of the films from 46.40% to 59.30%. influences the color of the edible film. However, in this
The increment in water solubility might be due to the study, the concentration of chitosan was kept constant.
hydrophilic behavior of glycerol. The incorporation of Therefore, it did not influence the color of the films.
glycerol increased the affinity of the film matrix to bind
to water thus increasing the solubility of the film [29]. Thickness
Thickness is a key characteristic in an edible film
Additionally, Yang et al. [30] suggested that glycerol because thicker films provide more security for the food
increased the hydrogen bonds in the chitosan film matrix product. Figure 2 shows the results of the film thickness
and leads to a higher availability for it to interact with analysis. The thickness of the films ranged from 0.097–
water molecules. Furthermore, it could be explained that 0.122 mm. The thickness value of film with 2% (v/v)
glycerol disrupted the chitosan network interaction glycerol and 10.5 mM (w/v) NaCl was the lowest. The
density. The presence of a polar molecule, glycerol concentration of NaCl in the film with 2% (v/v) glycerol
would increase the interaction with water. It caused a and 10.5 mM (w/v) allowed the permeation of hydrated
decrease in intermolecular forces by interacting with the chloride ions (Cl-) into the chitosan films, which
functional groups of the chitosan, causing an increase in disrupted the hydrogen bonds and hydrophobic
solubility. This led to the elevation of the water interactions. This led to the loss of cohesion capacity of
solubility of the film [23]. This trend of increasing water chitosan films. As a result, water molecules were
solubility was also observed by Cerqueira et al. [28], squeezed out from hydrated ions and reduced the film’s
whereby the water solubility of chitosan films increased thickness [17]. Similar results were reported for chia
from 51.86% to 69.94% when the glycerol was mucilage films [27].
increased from 0.5% (v/v) to 2.0% (v/v).
Mechanical properties of film
Color profile Tensile strength measures the amount of tensile stress
Color plays a significant part in the consumer’s the film can withstand until it breaks. Conventional
perception of food as it is one of the first characteristics standards state that food packaging should have a tensile
that the customer will evaluate [4]. Figure 1 shows the strength greater than 3.5 MPa to be deemed as
appearance of the films. As seen in the figure, the films satisfactory [31]. Table 4 presents the tensile strength of
were yellowish-brown in color. According to Zaman et the films was in the range of 4.03–9.24 MPa. Hence,
al. [6], the yellowish-brown film color was mainly due based on this standard, all the film formulations in this
to the presence of pigments in Garcinia atroviridis study fulfilled this criterion.
called xanthonoids.
Further, the tensile strength reduced as the additional
The overall color profile of the films is best described levels of glycerol and NaCl increased. The increment in
using the total color difference (ΔE) (Table 3). The the additional levels of glycerol in the film caused a
range of the ΔE of the films was from 0.00–1.37. The plasticizing effect for which the chitosan network chains
results showed that the ΔE of the film with 0.5% (v/v) became weaker [31]. Furthermore, more mobile regions

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 Malaysian Journal of Analytical Sciences, Vol 25 No 3 (2021): 399 - 414

were created due to the interference caused by glycerol. electroviscous effect and renders the chitosan molecules
High additional levels of glycerol elevated the film more flexible with a random, coiled shape. Coiled
water content. This could be attributed to the molecules will result in less inter-molecular
hygroscopic behavior of glycerol that led to the entanglements. The ability of the film to stretch was
reduction in the interactions between adjacent increased, increasing the elongation of the film. The
macromolecules [29]. results of this study are similar to Liu et al. [36] in that
the elongation of chitosan-starch blended films had an
Several studies showed that the increment in the incorporation of 2.5-10% (v/v) glycerol increased from
additional levels of glycerol reduced the tensile strength 13.5-18.0%, accordingly.
of the film [33, 34]. Apart from that, the results from this
study showed an improvement in tensile strength when However, the elongation of the film decreased from
compared to the results of Zaman et al. [6]. The authors 123.35% to 70.45% when the additional levels was
detected that at 5% (v/v) addition level of Garcinia increased to 2% (v/v) glycerol and 10.5 mM (w/v) NaCl.
atroviridis extract, the tensile strength was 3.28 MPa, This was probably associated to the anti-plasticizing
which was lower than for films in this study. This effect exhibited by glycerol at high additional levels
indicated that the addition of glycerol and NaCl [36]. Souza et al. [38] found that at high additional
improved the tensile strength of the film. levels, plasticizers exhibited strong interactions with
biopolymers. This led to the decrement in the
Elongation at break is the film’s capability to resist macromolecular mobility and elongation at break of the
changes in shape without breaking. For edible films, a film. This explained why, at higher plasticizer
high elongation is favorable as it enhances the ability of concentrations, such as the anti-plasticization
the film to wrap and package food. Table 4 shows the phenomena were associated with stronger interactions
range of the elongation at break was from 59.32– between plasticizer and chitosan molecules, which
123.35%. The film with 1.5% (v/v) glycerol and 10 mM impedes macromolecular mobility. When the amount of
(w/v) NaCl had the highest elongation at break. plasticizer molecules in a plasticized chitosan-based
Additionally, the increment in additional levels of film exceeded a critical value, anti-plasticization
glycerol and NaCl in the film showed increments on the occurred. As a result, the film's elongation at the break
elongation at break except for the film with 2% (v/v) was reduced [47].
glycerol and 10.5 mM (w/v) NaCl.
Young’s modulus measures the ability of a film to
The increment in elongation of the film can be due to the withstand elastic deformation when under elongation or
interactions of the glycerol with the chitosan chains. The compression. Table 4 shows that the range of the values
plasticizing effect of glycerol reduced the acting forces of Young’s modulus was from 0.046–0.146 MPa. The
between the molecules leading to the sliding of the increment in additional levels of glycerol and NaCl
polymer chain. Thus, the flexibility and ductility of the decreased the Young’s modulus of the film. The
film were increased [33]. Apart from that, the addition decreasing trend of Young’s modulus might be due to
of NaCl enhanced elongation of the film. The NaCl ions the effect of glycerol in increasing the elasticity of the
may cause an increase of electrostatic repulsion among film and reducing the interactions among the chitosan
chitosan chains leading to the formation of stretched chain. This resulted in the film becoming less stiff [32].
chitosan chains from coiled chitosan chains [35]. Similarly, Kusumaningtyas et al. [39] detected that the
Prateepchanachai et al. [48] demonstrated that salt increment in glycerol lowered the Young’s modulus of
produces a counter-ion effect that depresses the third films as well.

Antimicrobial properties of film showed that film with 0.5% (v/v) glycerol and 0 mM
Figures 3 and 4 show the inhibition zone of the different (w/v) NaCl, which showed the highest inhibition zone
film formulations on selected pathogens. The findings

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Sivanasvaran et al: ADDITION OF GLYCEROL AND SODIUM CHLORIDE INTO Garcinia atroviridis
CHITOSAN FILM, AND ITS APPLICATION FOR WRAPPING OF CHICKEN MEAT

against Pseudomonas aeruginosa (10.50 mm) and of the bacterial cell. This led to the disruption of the cell
Staphylococcus aureus (11.00 mm). membrane, followed by the intracellular leakage that led
to cell death [8].
The inhibition zone of the films ranged from 9.67–10.50
mm for Pseudomonas aeruginosa and 10.17–11.00 mm The extracts from Garcinia atroviridis are known to
for Staphylococcus aureus. The results indicated that the exhibit antibacterial properties against various bacteria
films exhibited antimicrobial properties towards these due to flavonoids and other phenolic compounds [13].
pathogens. The antimicrobial properties of the film Zhang et al. [40] demonstrated that chitosan film with
could be attributed to the chitosan film and the Garcinia 10% (w/v) mangosteen rind powder was able to inhibit
atroviridis extract. Chitosan has been widely known to Salmonella and Staphylococcus aureus. Additionally,
exhibit antimicrobial properties because it possessed Zaman et al. [6] observed that chitosan film incorporated
charged groups and has ionic interactions with bacterial with 5% (v/v) Garcinia atroviridis extract was able to
cell walls. The antibacterial action of chitosan was done inhibit Pseudomonas aeruginosa and Bacillus subtilis.
via the binding of the amino group with the membrane

Table 2. Moisture content (%) and water solubility (%) of the different formulations of chitosan films incorporated
with Garcinia atroviridis extract
Film Formulation Moisture Content, (%) Water Solubility (%)
A 15.42 ± 1.84a 46.40 ± 1.31a
B 18.64 ± 2.10b 49.60 ± 1.94ab
C 22.31 ± 1.96c 52.13 ± 2.43b
D 24.15 ± 1.64c 55.77 ± 1.64c
E 27.29 ± 1.37d 59.30 ± 2.04d
Results are presented as mean value ± standard deviation of three replicates.
Different letters superscripted denote significant difference (p0.05) between samples for each parameter in the same column.

A C D

Figure 1. The appearance of the films made from formulation A, C and D

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 Malaysian Journal of Analytical Sciences, Vol 25 No 3 (2021): 399 - 414

Table 3. Color parameters of the different formulations of chitosan film incorporated with
Garcinia atroviridis extract
Film Formulation L* a* b* ΔE
A 8.50 ± 1.03ab 0.39 ± 0.65a 8.70 ± 0.62ab 0.00 ± 0.00a
B 8.30 ± 0.64a -1.43 ± 0.40b 8.37 ± 1.46a 1.37 ± 0.93b
C 9.19 ± 0.72ab 0.77 ± 0.94a 8.40 ± 0.67a 0.78 ± 0.61a
D 8.46 ± 0.21ab 1.20 ± 0.38a 9.16 ± 0.51b 0.27 ± 0.91a
E 9.31 ± 1.07b 1.12 ± 0.25a 8.81 ± 0.06ab 0.63 ± 0.78a
Results are presented as mean value ± standard deviation of three replicates.
Different letters superscripted denote significant difference (p0.05) between samples for each parameter in the
same column

0.16

b
0.14
b
ab ab a
0.12
Thickness (mm)

0.10

0.08

0.06

0.04

0.02

0.00
A B C D E

Figure 2. The thickness (mm) of the different film formulations of chitosan film incorporated with Garcinia
atroviridis extract. A represents film containing 0% glycerol and 0 mM NaCl; B represents film containing
0.5% glycerol and 0 mM NaCl; C represents film containing 1.0% glycerol and 5 mM NaCl; D represents
film containing 1.5% glycerol and 10 mM NaCl; and E represents film containing 2.0% glycerol and 10.5
mM NaCl. Error bars indicate mean ± standard deviation of three replicates. Bars with different letters
denote significant difference (p≤0.05) between samples.

Table 4. Mechanical properties of the different film formulations of chitosan film incorporated with
Garcinia atroviridis extract
Film Formulation Tensile Strength Elongation at Break Young’s Modulus
(MPa) (%) (MPa)
A 9.24 ± 0.77d 59.32 ± 2.50a 0.156 ± 0.011c
B 7.44 ± 0.55c 93.77 ± 2.85c 0.080 ± 0.005b
C 6.17 ± 0.72b 109.63 ± 3.36d 0.056 ± 0.006a
b e
D 5.66 ± 0.64 123.35 ± 3.73 0.046 ± 0.054a
a b
E 4.03 ± 0.62 70.45 ± 1.69 0.057 ± 0.009a
Results are presented as mean value ± standard deviation of three replicates

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Sivanasvaran et al: ADDITION OF GLYCEROL AND SODIUM CHLORIDE INTO Garcinia atroviridis
CHITOSAN FILM, AND ITS APPLICATION FOR WRAPPING OF CHICKEN MEAT

14

b
12
a ab a
a
10
Inhibition zone (mm)

0
A B C D E

Figure 3. The antibacterial activity of the different film formulations of Garcinia atroviridis extract incorporated into
chitosan film on Pseudomonas aeruginosa. A represents film containing 0% glycerol and 0 mM NaCl; B
represents film containing 0.5% glycerol and 0 mM NaCl; C represents film containing 1.0% glycerol and
5 mM NaCl; D represents film containing 1.5% glycerol and 10 mM NaCl; and E represents film containing
2.0% glycerol and 10.5 mM NaCl. Error bars indicate mean ± standard deviation of three replicates

14

12 a b
a a
a
10
Inhibition zone (mm)

0
A B C D E

Figure 4. The antibacterial activity of the different film formulations of Garcinia atroviridis extract incorporated into
chitosan film on Staphylococcus aureus. A represents film containing 0% glycerol and 0 mM NaCl; B
represents film containing 0.5% glycerol and 0 mM NaCl; C represents film containing 1.0% glycerol and
5 mM NaCl; D represents film containing 1.5% glycerol and 10 mM NaCl; and E represents film containing
2.0% glycerol and 10.5 mM NaCl. Error bars indicate mean ± standard deviation of three replicates

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 Malaysian Journal of Analytical Sciences, Vol 25 No 3 (2021): 399 - 414

Storage test of chicken meat: Optimized film Microbial changes of chicken meat
The film formulation that was used for the storage test The total colony count of the chicken meat wrapped with
was chosen through the evaluation of physical, film and control during storage was calculated and
mechanical, and antimicrobial properties of the films. expressed in log (CFU/mL) as shown in Figure 6. The
Each film formulation showed antimicrobial properties. black dotted lines in the line graph represents the safe
Hence, films with optimum additional levels of glycerol microbial limits for consumption of chicken meat,
and NaCl were selected based on its mechanical which was less than log 6.0 [10]. The chicken meat was
properties. The elongation at break of the film with 1.5% considered to be unsafe for consumption when its total
(v/v) glycerol and 10 mM (w/v) NaCl was the highest colony count exceeded log 6.0. The total colony count
(123.35%) and had an acceptable tensile strength (5.66 of the chicken breast meat at beginning of the storage
MPa) and Young’s Modulus (0.046 MPa). The film with test was log 3.48.
incorporation of 1.5% (v/v) glycerol and 10 mM (w/v)
NaCl was applied to the chicken meat for the storage The total colony count increased as the number of
tests. storage days increased for the controls and the chicken
meats wrapped with film. The total colony count for the
pH changes of chicken meat controls reached a value of log 8.17 after 15 days of
Figure 5 presents the pH values of the chicken meat storage. Conversely, the chicken meats wrapped with
wrapped with the film and chicken meat without film the film reached a total colony count of log 6.10 after 15
(control) during storage. The pH of the chicken meat at days. The total colony count for the chicken meats
the beginning of the storage test was 5.86. The pH of the wrapped with the film was lower than the control for all
control increased as the number of storage days days of the storage period.
increased whereas the pH of the chicken meat wrapped
with film decreased as the number of storage days In addition, from Figure 6, the colony-forming unit
increased (Figure 5). The increment in the pH value of (CFU) for the control exceeded log 6.0 on the 6th day of
the control might be caused by the accumulation of storage; whereas, the chicken meats wrapped with the
amines and ammonia due to the growth of bacteria. The film only exceeded log 6.0 on the 15th day of storage.
bacteria broke down amino acids, which produced an Hence, we deduced that the control was safe for
accumulation of ammonia that increased the pH value of consumption up to day 5 of storage while the chicken
the chicken meat [41]. The studies on chicken breast meats wrapped with the film were safe for consumption
meat storage have shown that pH and bacterial growth up to day 14 of storage. It can be concluded that the
had a positive correlation [42]. Further, it was suggested chitosan film incorporated with 5% (v/v) Garcinia
by Bazargani-Gilani et al. [43] that the rise in pH value atroviridis extract can extend the shelf life of chicken
of chicken meat during storage because of a rise in meat from 5 days to 14 days when stored at 4°C. The
volatile bases caused by the activity of endogenous ability of the film to prolong the shelf life of the chicken
enzymes such as protease and lipase. meat might be due to the presence of antimicrobial
properties in the film. Figures 3 and 4 show that the films
Krishnan et al. [41] showed that the pH of raw chicken inhibited the growth of Pseudomonas aeruginosa and
meat increased after 15 days of storage at 4 °C, which Staphylococcus aureus.
agrees with our findings. On the other hand, the decrease
of the pH value of the chicken meats wrapped with film Basri et al. [13] showed that Garcinia atroviridis extract
may be caused by the reduction of bacterial activity and can inhibit the growth other pathogenic bacteria such as
the production of volatile base compounds in the Bacillus subtilis, Salmonella enteritidis and Escherichia
chicken meats [44]. coli. Hence, the films can reduce the total colony count
of the chicken meat and consequently extended its shelf
life. Several studies have similarly reported on the
ability of edible films to increase the shelf life of food

409
Sivanasvaran et al: ADDITION OF GLYCEROL AND SODIUM CHLORIDE INTO Garcinia atroviridis
CHITOSAN FILM, AND ITS APPLICATION FOR WRAPPING OF CHICKEN MEAT

products. Chitosan film incorporated with clove fish was wrapped with chitosan film incorporated with
essential oil and nisin increased the shelf life of pork 5% (v/v) Garcinia atroviridis [6].
patties from 6 days to 12 days [45]. In another study, the
shelf life of Indian mackerel fish was extended when the

6
pH Value

Control
3
With Film

2
0 3 6 9 12 15
Storage (day)

Figure 5. Changes in pH value throughout the storage period

8
Total plate count (log cfu/g)

5
With film

4 Control

3
0 3 6 9 12 15
Storage (day)

Figure 6. Changes in total plate count in log (CFU/mL) throughout the storage period

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 Malaysian Journal of Analytical Sciences, Vol 25 No 3 (2021): 399 - 414

Conclusion 6. Zaman, N. B. K., Lin, N. K. and Phing, P. L. (2018).

This study has demonstrated the incorporation of 1.5% Chitosan film incorporated with Garcinia
(v/v) glycerol and 10 mM (w/v) NaCl into chitosan film atroviridis for the packaging of Indian mackerel
incorporated with Garcinia atroviridis extract had the (Rastrelliger kanagurta). Ciência e
highest elongation at break and tensile strength that Agrotecnologia, 42(6): 666-675.
fulfilled the conventional standard. Therefore, glycerol 7. Ahmed, S., Ahmad, M. and Ikram, S. (2014).
and NaCl improved the mechanical properties of film. Chitosan: A natural antimicrobial agent-a review.
Storage tests showed that that the chicken meat wrapped Journal of Applicable Chemistry, 3(2): 493-503.
with the film had a shelf life less than 14 days whereas 8. Tan, H., Ma, R., Lin, C., Liu, Z. and Tang, T.
the controls had a shelf life of 5 days. Additionally, the (2013). Quaternized chitosan as an antimicrobial
results from the storage test indicated that applying the agent: antimicrobial activity, mechanism of action
film onto meat products such as chicken meat can extend and biomedical applications in orthopedics.
its shelf life. Hence, edible films with better mechanical International Journal of Molecular Sciences, 14(1):
properties are able to extend shelf life of food products 1854-1869.
and were developed in this study. 9. Kasai, D., Chougale, R., Masti, S., Chalannavar, R.,
Malabadi, R. B., Gani, R. and Gouripur, G. (2019).
Acknowledgement An investigation into the influence of filler Piper
This work was supported by UCSI University Pioneer nigrum leaves extract on physicochemical and
Scientist Incentive Fund (Proj-In-FAS-056) from UCSI antimicrobial properties of chitosan/poly (vinyl
University and UCSI Fundamental Research Grant alcohol) blend films. Journal of Polymers and the
Scheme (FRGS/1/2020/STG01/UCSI/02/3) from the Environment, 27(3): 472-488.
Ministry of Education Malaysia. 10. Sogut, E. and Seydim, A. C. (2018). The effects of
chitosan and grape seed extract-based edible films
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