EFFECT OF CHITOSAN AND POMELO PEEL EXTRACT COATING ON THE QUALITY AND

SHELF LIFE OF GOLDEN POMPANO (Trachinotus auratus) MEAT STORED UNDER CHILLED
CONDITION USING DIFFERENT PACKAGING MATERIALS

A Thesis Proposal by

PRESNEIL S. MAGLASANG
GLYSDI CARE S. MCCARTHY

Submitted to the Department of Agricultural and Biosystems Engineering

College of Engineering and Geosciences
Caraga State University
Ampayon, Butuan City

In Partial Fulfillment of the Requirements

For the Course ABE197 - Methods of Research for ABE

ENGR. PHILIP DONALD C. SANCHEZ

Course Instructor/Professor

June 2023
 CHAPTER 1

INTRODUCTION

Rationale

Golden pompano (Trachinotus auratus) or known as “apahan” or “dawis lawin” in the Philippines
is highly regarded in international seafood markets due to its high nutritional value, delicate flavor, and
succulent qualities. Its successful aquaculture production, fast growth rates, and high market demand
contribute to its recognition as one of the most economically significant marine fish species. According
to the Bureau of Fisheries of the Ministry of Agriculture, China emerged as a primary contributor in the
production and distribution of golden pompano with an estimated substantial output of 168,000 tons on
year 2020 (Liu et al., 2021) and 243,908 tons on year 2021. While China is currently the largest
producer, other Southeast Asian countries like India, Malaysia, Vietnam, and Philippines are rapidly
expanding the development of golden pompano farming to offer consumers the highest quality and at
the most competitive prices in the global market. Based on Southeast Asian Fisheries Development
Center Aquaculture Department (SEAFDEC/AQD), pompano is relatively easy to raise and a good
alternative to milkfish wherein high recovery rate of as much as 90% have been realized by most of the
growers in the country which are found in Bolinao, Pangasinan and Igang Marine Station at Guimaras,
Island. However, this fish is highly susceptible to spoilage and bacterial growth due to its high-water
content, fragile tissues, and high microorganism loads (Zhou et al., 2023a). Hence, preservation
measures must be taken to ensure product quality (Mokrani et al., 2018; Ali et al., 2022). Currently,
several methods of fish preservation such as curing, drying, freezing, and packaging (Amit et al., 2017)
are extensively used. Among these methods, freezing is the most widely applied and effective way to
preserve fish (Altan et al., 2016).

As reported by Southeast Asian Fisheries Development Center, around 70% of the catch is
consumed fresh or chilled, while 30% is processed. Golden pompano is primarily marketed and
exported in frozen form; while freezing and refrigerating both extend the shelf life of the fish (Dawson
et al., 2018), research has shown that the morphology and distribution of ice crystals during freezing
can destroy the muscle tissues, which severely decreases fish meat quality (Zhou et al., 2023b).
Moreover, Pai (2019) reported that there is a decrease in quality and affect the sensory values during
storage. The growing need for high-quality fish products has heightened the exploration of using
additives in preservation techniques (Vijayan et al., 2021) Synthetic additives such as nitrates,
benzoates, sorbates, and parabens have been proved to be effective in reducing spoilage (Yuan et al.,
2016) and considered to prevent microbiological contamination (Yu et al., 2021). However, synthetic
additives have raised various health concerns (Saeed et al., 2019). Therefore, the demand for natural
substances as food additives has increased with the use of animal and plant derived products (Yu et
al., 2021). Natural additives can improve quality, prolong shelf-life, and inhibit the growth of
microorganisms (Teshome et al., 2022). Edible coating technology using natural substances are often
perceived as safer and healthier alternatives to synthetic additives. However, these substances can
influence color, smell, taste, and toxicity in large amounts while being effective as food preservative
(Lee & Paik, 2016). Hence, to evaluate the sensory changes and toxicity of natural additives, various
trials including combinations of other substances have been performed.

Chitosan is considered as one of the most abundant natural polysaccharides in nature that can
be obtained from marine crustacean shells such as crab, crawfish, lobster, and shrimp (Arbia et al.,
2013) and as well as in the cell walls of fungi and the exoskeletons of insects (Mohan et al., 2020). It is
a natural biopolymer which is non-toxic and exhibits antifungal and antibacterial activity. Moreover,
chitosan received greater attention because it has a wide range of applications in fields such as
agriculture, food processing, biotechnology, and medicine. Aside from its various applications, chitosan
production could also help to mitigate the disposal of crustacean shells which is under environmental
concern. While pomelo (Citrus maxima) is considered as the largest citrus fruit from the family of
Rutaceae (Kumar et al., 2019). It is a native plant and a popular fruit widely grown in Asia, which is best
cultivated in China, Southern Japan, Vietnam, Malaysia, Indonesia, Thailand and in the Philippines
because they are widely adapted to local soil and climatic conditions. And one of the most important
citrus fruits in the world revealed by Deng et al. (2022) due to its nutritional benefits discussed by Goh
et al. (2020); Zhao et al. (2019); Methacanon et al. (2014) this includes the minerals and many bioactive
compounds. Among them, naringin and hesperidin are the major compounds found in pomelo
possessing strong antioxidant capacity, creating sweetness, and flavoring (Zunli et al., 2015). According
to the study of Zain et al. (2013), pomelo peel is the largest and has the thickest peel among all citrus
fruits making up to 30% of the fresh fruit weight. However, Yumnam et al. (2022) highlighted that the
peels are generally discarded as waste. In addition, Toh et al. (2013) stated that the peel of the citrus
fruit contains a higher content of antioxidants as compared to its pulp. Thus, utilization of pomelo peel
is highly beneficial.

Numerous scientific papers have proved that developing an optimized combination of low doses
of natural substances can maintain product safety and extend shelf life while minimizing undesirable
flavor and sensory changes. For instance, research study conducted by Sabu et al. (2020), a
combination of chitosan (CH) and lemon peel extract (LPE) were coated to the yellowfin tuna and a
significant reduction in microbial counts were recorded while the sensory qualities and shelf life was
enhanced compared to LPE or CH coating alone. Additionally, Min et al. (2021) studied the different
chitosan concentration and pomegranate peel extract to produce an edible film. The findings
demonstrated the value of the CH-PPE edible film in maintaining the quality of beef and increasing its
shelf life from 4 to 7 days. A similar combination was used by Yuan et al. (2016), but in this time
melanosis and quality of Pacific white shrimp was evaluated. The results of the study revealed that on
10 days of ice storage, chitosan coating mixed with PPE prevented melanosis and color difference
changes while also enhancing the sensory quality, hardness, and springiness of Pacific white shrimp.
On the other side, a study conducted by Yumnam et al. (2022) used the extracted essential oil from
pomelo peel to test the storage stability of a few selected varieties of freshwater fish. The oil significantly
enhances the shelf stability of fish fillets, extending their freshness by 6 to 9 days compared to untreated
fillets. There have been numerous research studies conducted on the use of chitosan and combined
with other natural substances. However, there has been no inclusive research using the chitosan
combined with pomelo peel extract. Hence, this study will utilize the use of chitosan and pomelo peel
extract as edible coating to golden pompano. Test the quality of the treated fish samples in terms
physicochemical properties related to fish quality and shelf life, compare the sensory values, and predict
the cooling kinetics using different packaging materials.

Statement of the Problem

Global demand for fish and other aquatic foods is expected to double by the year 2050 (Naylor
et al., 2021), fueled by population and economic growth, globalization, urbanization in developing
countries, and changes in dietary patterns in developed countries according to the State of World
Fisheries and Aquaculture 2020. However, seafood are highly perishable although refrigeration or
freezing can prolong its shelf life, these methods may not effectively prevent lipid oxidation, rancidity,
or bacterial growth. Thus, leading to the decrease of quality and loss of nutrients. The quality of fish
holds significance for both consumers and producers as high-quality fish commands a higher price in
the market and attracts more discerning customers. Additionally, maintaining quality throughout the
supply chain is essential to minimize product losses, prevent spoilage, and ensure compliance with
food safety standards and regulations. With the promising application of edible coating, currently,
exploration of incorporating natural additives offers the advantage of improved safety by inhibiting
microbial growth and prolonging shelf life, while also satisfying consumer preferences for clean label
products and enhancing the overall sensory qualities. For instance, chitosan incorporated with pomelo
peel extract as potential edible coating for meat preservation. However, this will be subject for further
evaluation and study particularly on its efficacy to food preservation. At present, there is no study about
the application of chitosan incorporated with pomelo peel extract to improve quality and shelf life of
golden pompano. Thus, this study aims to evaluate the effect of chitosan and pomelo peel extract
coating on quality and shelf life of frozen pompano.

Objectives of the Study

The main objective of this research study is to evaluate the effect of using chitosan and pomelo
peel extract coating to the quality of golden pompano meat stored under chilling temperature using
different packaging materials. Specifically, this study aimed to:

1) Evaluate the physicochemical properties of the pompano meat under different concentrations
of chitosan and pomelo peel extracts in terms of:
a) pH Value
b) Total Volatile Basic Nitrogen (TVB-N) Value
c) Tri-Methyl Amino Nitrogen (TMA-N)
d) Peroxide Value (PV)
e) Thiobarbituric Acid Reactive Substances (TBARS)
2) Examine the sensory values of the pompano meat under different treatments of chitosan and
pomelo peel extract such as PPE1 %, CH1 %, PPE + CH1 % and PPE + CH2 %.
3) Predict the cooling kinetics of the treated pompano meat after 12 days of storage under three
different packaging materials.
a) Polyethylene Bag
b) Modified Atmosphere Packaging
c) Vacuum Packaging

Significance of the Study

This research study will evaluate the effect of chitosan incorporated with pomelo peel extract
to retain fish quality and extend shelf life of frozen pompano fish. If proven effective, such investigations
will contribute to the seafood industry or fishery sector particularly in pompano fish farming industry who
handled export and import market, food processing industry, agricultural sector, farmers, consumers,
and even researchers who are involved and concerned with the recent applications of chitosan and
pomelo peel waste. More importantly, valorizing waste by utilizing chitosan and pomelo peel extracts
can contribute to reducing food waste and promoting sustainability. Additionally, their application holds
significant potential in ensuring consumer satisfaction, while addressing the need for natural and by
incorporating these extracts into food products, a natural preservation solution can be achieved,
reducing reliance on synthetic additives and chemical preservatives. Furthermore, chitosan and pomelo
peel is economical and readily available as food waste in regions particularly in the locality.

Scope and Limitation

This research focuses only on the effect of natural additives such as chitosan and pomelo peel
extract coating on the quality and shelf life of golden pompano meat stored under chilled condition using
three different packaging materials namely: polyethylene bag, modified atmosphere packaging and
vacuum packaging. For the development of edible coating, this research is limited to the utilization and
extraction of local varieties of pomelo peel that is available in Caraga Region, Philippines. Treated
samples will be limited only to a variety of golden pompano fish weighing 250 grams for each sample.
The extraction and coating procedures will be in accordance with existing established methods in the
literature with novel modifications.
 CHAPTER 2

REVIEW OF RELATED LITERATURE

2.1 Origin of Chitosan

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is derived from chitin. Chitin
is found in cell walls of crustaceans, fungi, and insects and in some algae, microorganisms, and some
invertebrate animals (Pellis et al., 2022). According to Komi and Hamblin (2016), chitin is a natural
polysaccharide of β-(1-4)-N-acetyl-D-glucosamine monomers, first identified by the chemist Henri
Braconnot in 1811. However, chitin has limited applications because of its acetyl groups, but through
the deacetylation process chitin is converted into chitosan. During the deacetylation process, the acetyl
group present in chitin is converted into hydroxyl (-OH) and amino (-NH2) groups in the chitosan. The
modification of the reactive functional groups present in chitosan opens the possibility of broad
application in many fields (Kumari and Kishor, 2020).

2.1.1 Extraction of Chitin

The primary sources of chitin raw material are the outer coverings of various crustaceans,
primarily crabs and shrimps. Chitin is a component of the complex structure found in crustaceans,
specifically shellfish, where it forms a network with proteins. Calcium carbonate is deposited onto this
network, creating the rigid shell. The interaction between chitin and protein is close, and a small portion
of protein is also involved in a polysaccharide-protein complex. To isolate chitin from shellfish, the two
major constituents of the shell, proteins, and inorganic calcium carbonate, need to be removed. This is
achieved through deproteinization and demineralization processes, along with the removal of small
amounts of pigments and lipids typically eliminated in earlier steps. Sometimes, an additional
decolorization step is employed to eliminate residual pigments (Younes et al., 2015).

Various methods have been proposed and utilized to obtain pure chitin, but there is no
universally accepted standard method. Both chemical and enzymatic treatments can be used for
deproteinization and demineralization, and the order of these steps can be reversed for improved
results, particularly with enzymatic treatment. Microbial fermentation is another approach, wherein
deproteinization and demineralization occur simultaneously. Regardless of the chosen treatment, the
isolation of chitin begins with the careful selection of shells. The optimal quality of the final isolated
material is influenced by factors such as using shells of the same size and species. In the case of
shrimps shown in Figure 1, which have thinner shell walls, chitin isolation is relatively easier compared
to other types of shells. The selected shells are subsequently cleaned, dried, and ground into small
pieces.

Figure 1. Schematic flow chart of conventional process for chitosan production from shellfish.
Source: (Duan et al., 2022)
 Thus, the availability of crustaceans remains a key factor for the extraction of chitin (Mohammed
et al., 2013). Overall, the production of chitosan involves several steps, including the extraction of chitin
from the source material, the deacetylation of chitin to form chitosan, and the purification of chitosan to
remove impurities.

2.2 Chemical and Physical Composition of Chitosan

2.2.1 Solubility

Chitosan is produced by deacetylation of chitin, which converts some N-acetylglucosamine

moieties into glucosamine units. Its solubility depends on different factors such as polymer molecular
weight, degree of acetylation, pH, temperature, and polymer crystallinity. The lower solubility was
explained due to the increase in the polymer crystallinity after deacetylation, which offsets the effect of
the increase in glucosamine moieties (Aranaz et al., 2021).

Casadidio et al. (2019) studied the solubility of chitosan decreases when pH rises from
physiological to basic values, and with increase the ionic strength (salting-out effect). Other determining
factors that have important effects on chitosan solubility include temperature, average of DD, and DP.
The most common solvents for the solubilization of chitosan are acetic acid (1% with pH close to 4),
formic acid (0.2–100%), 1% hydrochloric acid; lactic acid; and diluted nitric acid. Therefore, the polymer
crystallinity on chitosan solubility obtained through combination of chemical and physical disruption of
the hydrogen bonds, broad solubility.

2.2.2 Molecular weight and depolymerization of chitosan

Chitosan and its derivatives were found to have physiological activities and functional properties
that depend on their molecular weight (Cota-Arriola et al., 2013). The parameters used in the
deacetylation process have been observed to have an impact on the dispersion of chitosan preparations
with various molecular weights.

There are methods for determining the molecular weight of chitosan such as light scattering
spectrophotometry, gel permeation chromatography and viscometry, and gel permeation
chromatography is the most widely used method (Niebel et al., 2014). Degradation of chitosan due to
main-chain scission leads to the opposite effect on the mechanical properties and when irradiated, both
crosslinking and degradation often occur simultaneously. The regulation of molecular weight and its
distribution is crucial in determining the technical requirements needed for an end-use since molecular
weight determines the physicochemical properties of polymers (García et al., 2015).

Therefore, the molecular weight and depolymerization has a substantial impact on chitosan's
characteristics, opening a wide range of potential uses. Because of the technical specifications of
chitosan properties of polymers are heavily influenced by molecular weight. Additionally, chitosan's
antibacterial capabilities may be impacted by its molecular weight, as high molecular weight values
provide low solubility.

2.2.3 Biological properties

2.2.3.1 Antimicrobial properties

Chitosan's broad-spectrum bacterial inhibitory abilities have been demonstrated by in vitro tests
and sophisticated food antibacterial studies (Márquez et al., 2013). Wei et al. (2020) examined how
varied chitosan concentrations affected the color and microbiological groups on the surface of chilled
duck skin. The findings found that chitosan and color-presenting chemicals may compete for
oxygenation, and that 3% chitosan could significantly reduce the number of surface colonies of duck
skin during storage. The inter-charge interactions theory is the first of three widely recognized theories
about the mechanism of chitosan inhibition. The second theory focuses on chitosan's ability to chelate
metal ions. The third theory focuses on chitosan's ability to enter the cell, attaches to DNA, and then
influences RNA transcription and protein expression. Lopez-Moya et al. (2019) pointed out that chitosan
enters the nucleus of the fungus when it passes through the cell walls of fungal pathogens that use
plant hydrolases as hosts.

Furthermore, it has been clearly demonstrated that chitosan concentration have a better
inhibiting effect in such agricultural products like meat, fruits, and vegetables. However, numerous
researchers still hold that while examining the inhibitory mechanism of chitosan, it is also important to
consider bacterial species variations, molecular weight, the degree of deacetylation, and other factors.

2.2.3.2 Antioxidant properties

Chitosan has antioxidant capabilities, which are important for meat quality. Heterocyclic amines,
generated during high-temperature processing of proteins, are carcinogenic substances. Cooking
methods, processing conditions, and the presence of antioxidant substances all influence the formation
of heterocyclic amines (Nadeem et al., 2021). Chitosan concentration and temperature are significant
variables that affect the trial findings when chitosan is applied to meat products as a food additive. Oz
et al. (2016) investigated the effect of adding different concentrations of chitosan to meatballs cooked
at different temperatures on the formation of heterocyclic aromatic amines and the quality of the
meatballs. The results showed that the heterocyclic amine content of the meatballs increased with
increasing temperature, and the heterocyclic amine content decreased with increased chitosan
concentration. Li et al. (2020) analyzed the barrier properties of chitosan–cyanidin films and concluded
that the increased hydrogen bonding between cyanidin and chitosan molecules leads to a tighter
arrangement between molecules inside the film, which improves its gas barrier properties.

Chitosan is an effective antioxidant that preserves food's commercial value by delaying ripening
and aging processes, extending its shelf life, maintaining most of the food's sensory qualities, reducing
enzymatic browning, reducing water loss, preserving the food's vibrant color, taste, and texture, and
strengthening the aroma. It also can counteract the effects of concentrations on the quality preservation
of fruits, vegetables, and meat products, as well as slow down the deterioration and aging of food,
extending the shelf life of meat products, and preventing or lessening the harm brought on by oxidation.

2.2.3.3 Enzyme activity inhibition

Foods are softened and browned because of enzymatic reactions, which are not negligible. In
some fruits and vegetables with damaged surfaces, tissue browning is unavoidable due to the activity
of polyphenol oxidase (Liu et al., 2022). Moreover, chitosan coating showed better inhibition of
enzymatic activity with increasing molecular weight, delaying banana spoilage and deterioration. Li et
al. (2020) examined the impact of a clove oil-chitosan coating on the freshness and quality of lemons
at four different temperatures (0, 4, 7, and 10 C). It was discovered by examining the variations in
peroxidase, polyphenol oxidase, and phenolic acids that the chitosan coating's ability to suppress
enzyme activity was stronger as the temperature dropped.

Enzymatic reactions are the main factor in the degrading quality of fruits, vegetables, and meat
since they make these foods extremely susceptible to deterioration. Therefore, chitosan coating is more
effective in preventing enzymatic activity so that fruits and vegetables don't turn brown and meat tissues
don't become softer. This is because it inhibits the enzymes responsible for the enzymatic reactions
that occur in food.
2.2.4 Biodegradability

Biodegradable polymers can be broken down by microorganisms, their secretions, enzyme

action, or chemical breakdown in specific conditions (Tiwari et al., 2018). Changes in chemical and
physical properties, a reduction in molecular weight, and the formation of low-molecular-weight
compounds (CO2, H2O, and CH4) are typically observed when biodegradable materials are broken
down (Liu et al., 2022). Chitosan biodegradability depends on the degree of deacetylation and
crystallinity, which reaches maximum at 0% (titin) or 100% (fully deacetylated chitosan). Croisier and
Jerome (2013) discovered that the distribution of acetyl residues along the chitosan chain influences
both the crystallinity and rate of biodegradation of chitosan. It can be deduced that smaller chitosan
chains biodegrade more efficiently than higher-molecular-weight chitosan.

As a result of its biodegradability and wide range of uses, chitosan is thought to be a biopolymer
that is exceptionally biocompatible. Chitosan also contains a biodegradable polymer that can slow down
the bacterial process of decomposition, covering and shielding food from rotting germs. Thus, chitosan
is a potent natural alternative, and it has firmly established its position as an effective substitute for food
preservation.

2.3 Benefits Acquired in Chitosan

Chitosan, a natural polysaccharide derived from chitin, is a substance that is found in the
exoskeleton of crustaceans’ shells which has been found to have several potential benefits for food
preservation.

2.3.1 Shelf-life prolongation

Chitosan coatings have been demonstrated to increase food products' shelf lives by reducing
microbial growth and oxidation, including fruits, vegetables, and meats. This may result in less food
waste and increased food safety. Chitosan is ideally suited for usage as a biodegradable antimicrobial
packaging material that can be utilized to increase the storability of perishable goods due to its inherent
antibacterial and antifungal activities and film-forming capabilities (Duan et al., 2020). Therefore, by
limiting microbial development and oxidation, chitosan can increase the shelf life of fruits, vegetables,
and meat because of its antimicrobial and antioxidant properties.

2.3.2 Safe and edible

Chitosan is a naturally occurring, biodegradable polymer that is suitable for human

consumption. Regulatory organizations like the US Food and Drug Administration (FDA) and the
European Food Safety Authority (EFSA) have approved it as a food additive. And chitosan have the
potential to improve gut health, boost the immune system, lower cholesterol levels, and other health
outcomes (Muanprasat and Chatsudthipong, 2017). As a result, chitosan is regarded as a valuable and
potentially safe food additive that can be employed in a variety of applications, including food
preservation.

2.4 Application of Chitosan as Natural Food Preservative

Chitosan is being extensively researched for use as an edible coating, which is the production
of a thin film immediately on the surface of the thing they are intended to protect. According to Inanli et
al. (2020), chitosan does not promote changes in sensory characteristics when treating meat,
vegetables, and fruits, unlike essential oil (EO). It has been reported to reduce respiration rate in many
fruit crops, leading to a delay in postharvest deterioration and weight loss of the fruits.
 Wantat et al. (2021) investigated the effects of chitosan coating on ‘Hom Thong’ banana fruit.
The results showed that chitosan coating has the highest ability to reduce respiration and ethylene
production rate, retard banana fruit ripening by decreasing weight loss, peel color change, and total
soluble solids, and keeping banana fruit firmness. Tokatl and Demirdoven (2020) studied sweet cherries
coated with four chitosans (1%) and stored at 4 °C for 25 days, and 20 °C for 15 days. The results
revealed that Chitosan-1 had the highest firmness value among the other concentrations. Gao et al.
(2018) also studied the quality and biochemical changes of navel orange (Citrus sinensis L., Osbeck)
fruits in response to cinnamaldehyde-chitosan coating during 120 days of storage at 10 ± 1 °C and 80–
90% RH. The results showed that the coating significantly reduced the decay rate and weight loss of
the navel orange fruits, delaying the decrease of the content of total soluble solids (TSS), titratable
acidity (TA) and vitamins C (Vc).

Hu et al. (2020) reported the use of chitosan nanoparticles as an edible surface coating agent
to preserve fresh-cut bell peppers at different concentrations. The experiment's findings show that fresh
sliced bell peppers coated with chitosan nanoparticles retained their weight and sensory quality at 5 °C
for 12 days while also inhibiting the growth of foodborne bacteria. Leceta et al. (2015) investigated the
effectivity of chitosan-based coatings in preserving the quality of baby carrots under modified
atmosphere packaging (MAP) and stored at 4 °C. The findings demonstrated that chitosan-based
coatings prevented microbial deterioration without degrading the qualities of baby carrots. Additionally,
studying chitosan coating may aid in reducing the danger of foodborne infections and preserving the
freshness of fruits, vegetables, and meat, enhancing their general safety, and assisting in the production
of a healthier food supply.

Huynh (2020) evaluated the effect of chitosan-based coating on the quality of fresh redfish
(Sebastes marinus) fillet during cold storage. The results revealed that chitosan-based coating
significantly increased the whiteness and limited the lipid oxidation of redfish fillets during cold storage.
Renuka et al. (2016) assessed the effectiveness of chitosan edible coating on the microbial quality of
ribbon fish (Lepturacanthus savala). The study revealed that chitosan can be used effectively to
enhance the microbial quality as well as shelf life of fishery products. Dong et al. (2020) investigated
the effects of edible chitosan (CTS) coatings (0%, 1%, 2%, and 3%) on Harbin red sausage. The results
revealed that the edible coating film containing 2% chitosan helps to enhance the storage stability of
Harbin red sausage at room temperature by preventing pH decline, stabilizing the L* value and water
migration, and reducing the growth of aerobic and lactic acid bacteria.

Therefore, chitosan coating has great potential for application in the post-harvest preservation
of fruits, vegetables, and meat products to reduce the occurrence of fruit postharvest decay, slowed
down the degradation rate of water loss, to delay the weight loss rate of fruit storage, while maintaining
high commodity value without impairing the flavor, odor, or palatability of skinned and freshly cut fruits,
vegetables, and meat product. The use of chitosan coatings could be a viable alternative for preserving
the quality and antioxidant capacity of fruits, vegetables, and meat products. For further understanding,
a list of applications of chitosan on the quality and shelf-life of fish and fisheries products were tabulated
and arranged as displayed in Table 1.

Table 1. Effects of chitosan on quality and shelf-life of fish and fisheries products.
Fish and
Mode of application Additional
fisheries Preservatives Effects References
(concentration) preservation
products
Catfish Chitosan Spraying, dipping and Stored at 4 ◦C TVB-N, TBA, pH, Bonilla et al.
(Ictalurus vacuum tumbling TVC ↓ Shelf-life (4– (2018)
puctactus) (0.5%) 8 days)
fillet
 Surimi of Chitosan Direct addition Stored at 4 ◦C TVB-N, PV, TBA, Amiza & Kang
African catfish (0.25–2%) TVC ↓ (2013)
(Clarias Shelf-life (4 days) ↑
gariepinus)
Grass carp Chitosan Wrapping (1%) Young apple pH, TVB-N, PV, Sun et al.
fillet polyphenols + TBA, TVC ↓ (2018)
stored at 4 ◦C Sensory score ↑
Pacific white Chitosan Dipping (0.5 mg/mL) Stored at 4 ◦C pH, TVB-N, TBA, K Wang et al.
shrimp Nanoparticle value, TVC ↓ (2015)
(Litopenaeus
vannamei)
Ovate Chitosan Dipping (1.5%) Citric acid or PV, TBA, pH, FFA, Qiu et al. (2016)
pompano licorice extract DL ↓
(Trachinotus + stored at – 18
ovatus) fillet ◦C
Pacific white Chitosan Dipping (1%) Pomegranate pH, TVB-N, TVC ↓ Yuan et al.
shrimp peel extract + Sensory score ↑ (2016)
stored with ice
Golden Chitosan Dipping (0.4%) Gelatin + stored pH, TVB-N, TVC, Feng et al.
pomfret at 4 ◦C Yeast and molds ↓ (2016)
(Trachinotus
blochii) fillet
Grass carp Chitosan Dipping (1%, 2%, Stored at 4 ◦C pH, TVB-N, Yu, et al. (2017)
fillet 2.5%) Biogenic amines,
TVC, HSPB, LAB,
Pseudomonas
spp.,
Enterobacteriaceae
↓ Sensory score ↑
Atlantic Chitosan Dipping (0.5%, 1.5%) Stored at − 22 Coating loss, Soares et al.
salmon ◦C Weight loss, DL, (2015)
(Salmo salar) TVBN, K value →
fillet TVC, pH ↓
Color ↑

Silver carp Chitosan and Dipping (2%) Stored at 4 ◦C pH, TVB-N, TBA, Ramezani et al.
(Hypophthalmi chitosan MBC, PBC ↓ (2015)
cthys nanoparticles Sensory score ↑
molitrix) fillet

BA - Biogenic amines, CD - Conjugated dienes, DL - Drip loss, FFA - Free fatty acid, HSPB - H2S producing bacteria, HSV -
Headspace volatiles, Hx – Hypoxanthine, LAB - Lactic acid bacteria, MBC - Mesophilic bacteria count, PBC - Psychrophilic
bacteria count, PV - Peroxide value, TBA - Thiobarbituric acid value, TMA-N - Trimethyl amine nitrogen, TVB-N - Total volatile
base nitrogen, TVC - Total viable count. Source: (Hussain et al., 2021)

2.5 Origin of pomelo

Pomelo (Citrus maxima) is considered as the largest citrus fruit from the family of Rutaceae
(Kumar et al., 2019). It is a native plant and a popular fruit widely grown in Asia, which is best cultivated
in China, Southern Japan, Vietnam, Malaysia, Indonesia, Thailand and in the Philippines because they
are widely adapted to local soil and climatic conditions. Pomelo cultivars have multiplied because of
cross-pollination with other citrus species, and they are now grown in many different countries (Tocmo
et al., 2020). Since it is widely cultivated around the world, pomelo has a history of cultivation and
consumption dating back 4,000 years ago (Puglisi et al., 2017). Roughly estimated, the total world
production of pomelo peels approached 2.8 million tons ~4.7 million tons in 2018 (Xiao et al., 2021).
Along with orange, mandarin, lemon, and grapefruit, pomelo is now one of the five citrus fruits that are
most extensively grown and consumed.
 Figure 2. Anatomy of a typical pomelo fruit. Source: (Tocmo et al., 2020)

2.6 Properties of Pomelo Fruit

2.6.1 Physical properties

Pomelos are the largest of all citrus fruits, with a diameter of up to 30 cm and can weigh up to
1 to 2 kg depending on the cultivar (Zain et al., 2013). It is generally round or slightly pear-shaped. The
outer skin is thick and tough, with a rough and bumpy texture. The color of the skin can vary, ranging
from yellow green to pink, and the flesh can be pale yellow, pink, or red (Figure 2), depending on the
fruit's maturity. The peel of pomelo is relatively thick compared to other citrus fruits. It serves as a
protective layer for the fruit and helps retain its freshness and flavor.

As the fruit ripens, the skin may become thinner and smoother. Furthermore, pomelo contains
a significant amount of juice, although it is generally less juicy compared to other citrus fruits such as
oranges or grapefruits. While the flavor of this fruit is mild and sweet, with a slightly acidic or tart
undertone. It is often described as refreshing and less tangy compared to other citrus fruits. And has a
distinct citrusy and floral aroma, with some varieties having a more pronounced fragrance than others.

2.6.2 Chemical composition

The active components that exist in citrus fruits are essential oils such as limonene, pinene,
and terpinolene (Yu et al., 2017). Essential oils found in pomelo peel contributed to the characteristic
aroma of the fruit. Wherein these essential oils consist of volatile compounds, such as limonene, α-
pinene, β-pinene, myrcene, linalool, and citral, which are responsible for the citrus scent. This fruit is
also rich in flavonoids, a class of compounds with antioxidants and potential health-promoting effects.
Flavonoids found in pomelo peel include naringin, hesperidin, rutin, and quercetin.

Pomelo fruit is low in calories and high in vitamin C, fiber, and antioxidants. They also contain
potassium, vitamin B6, and other nutrients that are essential for good health. Based on the study of Tsai
et al. (2017), the amount of total phenolic, carotenoid, vitamin C, and 𝛿-tocopherol of red pomelo juice
were notably higher than that of white pomelo juice, and the red pomelo juice exhibited higher
antioxidant which possess antimicrobial and anti-inflammatory properties reviewed and detailed by
Wanpeng et al. (2017) than white pomelo juice. However, it is important to note that the chemical
compositions and biological activity of pomelo are influenced by variety (Yin et al., 2023).

It can be concluded that pomelo offers excellent nutritional benefits. Previous studies also
revealed that vitamin C, a potent antioxidant, is abundant in pomelo. The primary antioxidants in
pomelos are naringenin and naringin that is also found in other citrus fruits. Among the three parts of
pomelo which includes the pulp, seed, and peel; peel inhibit higher antioxidant activity. This chemical
composition present on pomelo is a natural substance that may prevent or delay some types of cell
damage. Wherein, antioxidants play a major part in ensuring that the food keep their taste and color
remain edible over a longer period. Thus, the present of antioxidant in pomelo peel has a potential for
natural food preservation.

2.7 Quality Attributes of Pomelo Peel (PP)

Citrus peels are the outermost layer of citrus fruits, possess unique properties that make them
useful in various applications. Reviewed by Wedamulla et al. (2022), the waste generated in the citrus
processing industry in the form of citrus peel exhibits potential economic benefits by reentering the food
processing line as a food additive while providing sustainable and innovative solutions to food waste
utilization. In line with this, pomelo peel as part of citrus fruits also possessed properties that can
contribute to food preservation.

2.7.1 Antimicrobial activity

Contains natural compounds such as limonene, naringin, and hesperidin, which have
antimicrobial properties. These compounds can help prevent the growth of bacteria and fungi, which
can spoil food.

2.7.2 Antioxidant properties

Rich in antioxidants such as vitamin C and flavonoids. Antioxidants can help prevent or delay
the oxidation of food components, such as fats and oils, which can lead to rancidity and loss of quality.

2.7.3 Aromatics and flavor enhancement

The essential oils present in pomelo peel contribute to its characteristic aroma and flavor. These
aromatic compounds can be used to enhance the taste and fragrance of preserved foods.

2.7.4 Chelating agents

Contains natural chelating agents, such as citric acid, which can bind to metal ions that promote
oxidation and enzymatic browning.

2.7.5 Deodorizing properties

The peel has a fresh, citrusy scent that can help deodorize food. This can be particularly useful
for preserving fish or other foods with strong odors.

2.7.6 High acidity

Like other citrus fruits, pomelo peel is acidic, which can create an unfavorable environment for
the growth of spoilage-causing microorganisms.

2.7.7 Moisture-absorbing properties

It can absorb moisture, which can help prevent food from becoming too moist and spoiling.

2.7.8 Natural insect repellent

The essential oils in pomelo peel have been found to possess insect-repellent properties.
Incorporating pomelo peel or its extracts in food storage areas or packaging may help deter certain
pests and insects.

2.7.9 Natural and safe

A natural product that does not contain any harmful chemicals or additives, making it a safe
and healthy alternative to synthetic preservatives.

Summarized reviewed articles shown in Table 2 clearly emphasizes the incorporation of natural
preservatives derived from plant based could help to retain the quality and shelf-life of fish meats. Thus,
utilization of pomelo peel waste could also impact food preservation techniques. The present acidity
from its peel can help preserve the freshness of foods and inhibit microbial growth. Moreover, it can
protect against oxidative deterioration and help to improve the sensory appeal of the preserved
products. Even preserving the color, texture, and overall quality of fruits, vegetables, and juices during
preservation processes. Therefore, the combination of these properties makes pomelo peel a potential
and effective natural preservative for food.

Table 2. Effects of other natural preservatives on quality and shelf-life of fish and fisheries products.
Fish and Mode of
Preservatives Additional
fisheries application Effects References
preservation
products (Concentration)
Pompano Rosemary extract Dipping (0.2%) Nisin + stored at pH, TBA, PV, Gao et al.
(Trachinotus 4 ◦C TVB-N, TMA-N, K (2014)
ovatus) fillet value, TVC ↓
Sensory score ↑
Shelf-life (6 days)
↑
Bighead carp Pomegranate peel Dipping (2%) Stored at 4 ◦C IMP, Hx, HxR, K Zhuang et al.
(Aristichthys Extract value, BA, TVC, (2019)
nobilis) fillet HSPB,
Aeromonas,
Pseudomonas ↓
Sensory score ↑
Shelf-life (2 days)
↑
Indian mackerel Mint leaf and citrus Dipping (Mint 0.5%, Stored at 0–2 ◦C pH, TBA, PV, Viji et al. (2015)
peel extract citrus 1%) TVB-N, TMA-N,
FFA, TVC ↓
Sensory score ↑
Shelf-life (2–5
days) ↑
Anchovy Thyme, oregano, Mixing with ice Stored at 3 ◦C TVB-N, TBA, PV, Bensid et al.
(Engraulis and clove extract (0.04% thyme, MBC, PBC ↓ (2014)
encrasicholus) 0.03% oregano, Shelf-life (3 days)
0.02% clove) ↑
Banana prawn Gelatin Dipping (4.5%) Stored at 4 ◦C pH, TBA, TVB-N, Mirzapour-
shrimp hydrolysate FFA, TVC, PBC, Kouhdasht &
(Penaeus LAB ↓ Moosavi-Nasab
merguiensis) Sensory score ↑ (2020)
Shelf-life (3 days)
↑
Grass carp fillet Fish gelatin Dipping Curcumin + pH, TBA, PV, Sun et al.
β-cyclodextrin + TVB-N, TVC, (2019)
stored HSPB,
at 4 ◦C Pseudomonas
spp., Yeast and
molds, Weight
loss ↓ Sensory
score ↑ Shelf-life
(6 days) ↑
Chinese Gelatin Dipping (6%) Eugenol + pH, TBA, TVB-N, Zhou et al.
Seabass β-cyclodextrin + FFA, K value, Hx, (2019)
(Lateolabrax stored IMP, TVC, PBC,
maculatus) at − 0.9 ◦C HSPB,
Pseudomonas
spp.↓ Sensory
score ↑
Silver Carp Black cumin and Dipping (1% cumin, Stored at 4 ◦C PV, TBA, TVB-N, Eskandari et al.,
(Hypophthalmich black caraway 1% TVC, PBC ↓ (2015)
thys extract caraway) Sensory score ↑
molitrix) Shelf-life (6–9
days) ↑
 Red sea bream Sodium alginate Dipping (2%) 6-gingerol + pH, TBA, TVB-N, Cai et al.,
(Pagrosomus stored at K value, MBC, (2015)
major) 4 ◦C PBC, LAB,
fillet Enterobacteriacea
e, Pseudomonas,
Shewanella
putrefaciens ↓
Sensory score ↑
Shelf-life (10 days)
↑
BA - Biogenic amines, CD - Conjugated dienes, FFA - Free fatty acid, HSPB - H2S producing bacteria, Hx – Hypoxanthine, HxR
– Inosine, IMP - Inosine monophosphate, LAB - Lactic acid bacteria, MBC - Mesophilic bacteria count, PBC - Psychrophilic
bacteria count, PV - Peroxide value, SSO - Specific spoilage organisms, TBA - Thiobarbituric acid value, TMA-N - Trimethyl amine
nitrogen, TVB-N - Total volatile base nitrogen, TVC - Total viable count. Source: (Hussain et al., 2021)

2.8 Extraction of Antioxidant from Pomelo Peels

There have been numerous studies conducted on the extraction techniques of pomelo fruit to
develop different solutions or extracts. These solutions may be used for various purposes, such as food
additives, natural remedies, or functional ingredients. This includes hydrodistillation which is the most
used and quick and easy to apply (Ranitha et al., 2014; Stratakos and Koidis 2016); however, the
deterioration of the oil extracted in the hydrodistillation method is higher due to the instability of
components after extraction (He et al. 2019). Instead, it is suggested to use the cold percolation method
(Kumar et al., 2019). Wherein, with slight modifications, extract antioxidants from dried pomelo peel
powders using ethanol as the solvent.

In this process, 10 g of dried pomelo peel powder will be soaked in 100 ml of ethanol within a
conical flask, which is then sealed with a cotton plug. The flask will be placed in an incubator shaker at
a speed of 120 rpm, maintaining room temperature, for approximately 24 hours. Subsequently, the
extract will be centrifuged at 3000 rpm for 20 mins, and the resulting supernatant will be collected. The
supernatant will be concentrated using a rotary vacuum evaporator at 40 ᵒC for approximately 4 to 5
days. The resulting dry extract will be stored at 4 ᵒC for further analysis.

Figure 3. Graphical Representation of Extraction of Antioxidant from Pomelo Peels. Source: (Elgharbawy et al., 2019)

2.8.1 Extraction Yield

The dry extract after centrifugation will be weighed and extraction yield will be measured by the
following formula.

𝐷𝑟𝑖𝑒𝑑 𝑤𝑡. 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡

𝐸𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑌𝑖𝑒𝑙𝑑 = 𝑥 100
𝑇𝑜𝑡𝑎𝑙 𝑤𝑡. 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑒𝑒𝑙 𝑝𝑜𝑤𝑑𝑒𝑟
2.8.2 Extraction Yield (%) of Pomelo Peels

Table 3. Extraction Yield (%) of Pomelo peel extract with different organic solvents.
Source: (Kumar et al., 2019)
Organic Solvent Pomelo peel extract yield (%)
Ethanol 15.9%
Methanol 21.6%
Hexane 12.7%
Chloroform +methanol 11.1%

Presented Table 3 the percentage of pomelo peel extract obtained from dried pomelo peel
powder through refluxing with different solvents, namely ethanol, methanol, hexane, and a combination
of chloroform and methanol (2:1). The lowest extraction yield (11.1%) was obtained using chloroform +
methanol (2:1), while the highest yield (21.6%) was achieved with methanol. Ethanol extraction yielded
a percentage of 15.9%, and hexane extraction resulted in a yield of 12.7%. Comparatively, the highest
yield in this study was slightly greater with methanol and ethanol extracts, as observed in previous citrus
peel research. However, the handling or use of methanol is highly toxic and can be harmful if ingested
or absorbed through the skin. On the other hand, ethanol is generally considered safe for consumption
and has a long history of use in food and beverage applications (Ashurst and Nappe, 2022).

2.9 Current Applications of Pomelo Peel in the Preservation of Food

Utilization of pomelo peel (PP) waste has been explored for various applications in food
preservation. Research study conducted by Chollakup et al. (2020), investigated the bioactive
compounds of crude pomelo peels extract using maceration technique with 75% (v/v) ethanol and
coated onto rice straw papers at concentration of 10, 15 and 20 % (w/v). The coated papers with pomelo
peel extract showed significant antibacterial activity against food pathogenic bacteria, including
Staphylococcus aureus, Bacillus cereus and Escherichia coli. Thus, the coatings provide a protective
barrier, reducing water loss, preventing microbial growth, and extending the shelf life of fresh produce.

Additionally, since pomelo peel contains a large amount of essential oil about 0.4-1% of the
weight of the peel, based on the previous work of Yumnam et al. (2022), as they use the essential oil
from PP on the storage stability of a few selected varieties of freshwater fish. The findings of their study
highlighted the efficacy of the essential oil against pathogenic strains, offering a natural alternative to
harmful chemical antimicrobials. Moreover, the oil significantly enhances the shelf stability of fish fillets,
extending their freshness by 6-9 days compared to untreated fillets. The antimicrobial activity of the oil
synergizes with the inherent antimicrobial properties found in pomelo peel essential oil. However, future
research is required to confirm the oil's efficacy against other pathogenic strains and to conduct in vivo
studies, aiming to identify a healthy, safer, and potentially natural alternative as an antimicrobial agent.

Other than that, powdered pomelo peel was reported by Zhang et al. (2019) as novel shale
inhibitor in water-based drilling fluid. The findings indicate that when pomelo peel powder with a
concentration of 1% and particle size optimized at over 160 mesh was employed, it exhibited acidic
properties and demonstrated the ability to prevent shale collapse while reducing mud loss through
filtration. Further analysis revealed that fresh pomelo peel powder contains numerous active
substances that diminish mud filtration, enhance its rheological properties, and impede clay hydration
and expansion. Given that pomelo peel is widely available worldwide and easily obtainable, utilizing
pomelo peel powder proves to be an effective approach in alleviating ecological pressure and reducing
environmental pollution.

Pomelo peel extracts have been investigated as natural antimicrobial treatments for food
products. They can be used as dip safety and or incorporated into food matrices to control the growth
of bacteria, fungi, and other microorganisms, thereby improving food safety, and extending shelf life.
Moreover, extracts or active compounds derived from pomelo peel have been incorporated into active
packaging materials. These materials release antimicrobial compounds, such as essential oils or
phenolic compounds that can inhibit microbial growth, delay spoilage, and maintain the quality of
packaged foods. Pointed out by Chollakup et al. (2020), the benefits of natural by-products as a
potential material for active packaging. The study reported by Sowmya and Lakshmi (2023), that pomelo
peel and tea polyphenol-based composite packaging material having a 10% concentration of tea
polyphenol proved to be a good moisture barrier, and have effective anti-microbial, antioxidant and
mechanical effects. It could also be a green alternative for the packaging.

Therefore, the present antimicrobial and antioxidant properties obtained from pomelo peel can
help inhibit microbial growth, delay spoilage, and maintain the quality and safety of food items, reducing
the need for synthetic preservatives. These recent applications demonstrated the potential of pomelo
peel in food preservation as a natural, safe, and sustainable ingredient. However, further research and
development are ongoing to optimize the use of pomelo peel and its extracts in different preservation
techniques and to ensure their safety, efficacy, and sensory acceptability in specific food products.

2.10 Advantages, challenges, and future perspectives

Applications of chitosan and pomelo peel extracts to food preservation presents several notable
aspects. These natural extracts offer significant advantages in the realm of food preservation. By
incorporating these extracts into food products, a natural preservation solution can be achieved,
reducing reliance on synthetic additives and chemical preservatives (Bashir et al., 2017).

However, the review also sheds light on certain challenges associated with the use of chitosan
and pomelo peel extracts in food preservation. Formulating and applying these extracts in food products
necessitate careful optimization to ensure stability, efficacy, and sensory acceptability (Xing et al., 2016).
Moreover, navigating the regulatory landscape and obtaining necessary approvals can present hurdles,
as regulations regarding the use of these natural extracts may vary across different regions and
jurisdictions.

This review paper suggests several future perspectives to explore. Further research and
development efforts can focus on enhancing the formulations and delivery systems of chitosan and
pomelo peel extracts, with the aim of improving their effectiveness and applicability in various food
matrices. Exploring potential synergistic effects by combining these extracts with other natural
preservatives or antimicrobial agents may enhance overall preservation capabilities (Basavegowda et
al., 2021).

More importantly, valorizing waste by utilizing chitosan and pomelo peel extracts can contribute
to reducing food waste and promoting sustainability. Novel applications, such as active packaging,
edible coatings, and incorporation into innovative food products, hold promise for expanding the
utilization of these extracts in food preservation (Singh et al., 2022; Yumnam et al., 2022).

2.11 Summary and Conclusions

There have been numerous comprehensive reviews of chitosan for food preservation applied
in various perishable foods, including those of Adiletta et al. (2019), Alam et al. (2017), Cheng et al.
(2021), Eldaly et al. (2018), González (2020), Kahve and Duran (2020), Kanatt (2013), Karsli et al.
(2021), Pramonoa et al. (2018), Renuka et al. (2016), Santos et al. (2017), Tokatli and Demirdoven
(2020), Hyunh (2019), and Zhao et al. (2022). Moreover, several research studies used chitosan
combined to other substances including those of Abbas and Rahman (2020), Cao et al. (2020), Islam
et al. (2018), Kyriakidou et al. (2021), Li et al. (2020), Meherpour et al. (2020), Mehrabi et al. (2021),
Min et al. (2021), Oz et al. (2016), Riaza et al. (2021) and Sabu et al. (2020). However, there has been
no inclusive review conducted using the chitosan combined with pomelo peel extract. Thus, the primary
goal of the present study are to identify the properties, benefits and recent applications of chitosan and
pomelo peel extracts to food preservation including the methods and process of these waste material
into value added substance. An evident list of summaries were tabulated and arranged as displayed in
Table 4 regarding the recent studies on the application chitosan and pomelo peel extract to preserve
different commodities.

Table 4. Summary of recent studies on the application chitosan and pomelo peel extract to preserve different commodities.

Data Storage
Application Commodity Quality Parameter Solution Results References
Analysis Days
Chitosan Beef Sensorial SAS CH (1%, 5-8 Improved Alam et al.
properties, 1.5%, 2%) microbiologica (2017)
microbial counts l quality,
extends the
shelf life
Chitosan Beef Sensory Qualities SAS CH (0.5%) 6 Extends the Cheng et al.
+ ϵ-PL shelf life (2021)
(0.2%) + G
(0.1%)
Chitosan Harbin red Storage stability, TPA CTS (0, 1, 12 Improved the Dong et al.
sausage microbial growth 2,3%) antioxidant (2019)
capacity
Chitosan Chicken fillet Sensory LSD CH (1.0%, 12 Improved Eldaly et al.
evaluation, 1.5%, & microbiologica (2018)
microbial analyses 2.0%) l quality,
enhanced
sensory
attributes
Chitosan Orange Sensory quality SAS CH (1% & 15 Extend shelf Gao et al.
1.5%) life, (2018)
maintained
quality
Chitosan Strawberries Sensory quality, SAS CH (10%, 8 Preserved the Gonzalez et
antioxidant 20%, & quality, al. (2020)
compounds 30%) antioxidant
capacity
Chitosan Bell pepper Sensory quality SEM CH (1 % & 12 Extend the Hue et al.
3 %) shelf life (2020)
Chitosan Beef Microbiological SAS CH (1% & 7 Extend the Kahve and
quality, oxidative 2%) shelf life Duran
properties (2020)
Chitosan Meat Microbiological & SAS CH (1%) 7 Longer shelf- Kanatt et al.
chemical, sensory life 2013
characteristics.
Chitosan Catfish fillet Sensory quality, TPA CH (3%) 10 Extend shelf Karsli et al.
microbial growth and life & (2021)
SAS microbial
deterioration
Chitosan Red snapper Microbiological, SAS CH (0, 1 & 6 Prolong shelf Pramono et
sensory properties 2 % w/v) life, microbial al. (2018)
quality
Chitosan Ribbonfish Microbial quality SAS CH (1%) 16-17 Enhanced the Renuka et
microbial al. (2016)
quality and
shelf life
Chitosan Nile tilapia Physicochemical, SAS CH (1% & 12 Maintained Santos et al.
microbiological 2%) quality (2017)
quality
Chitosan Sweet Sensory quality SAS CH (1% & 15 Preserved the Tokatli and
cherries 2%) quality Demirdoven
characteristics (2020)
Chitosan Redfish fillet Physical, chemical, SAS CH (1%) 12 Increased Huynh
microbiological, shelf life & (2019)
sensory quality
characteristic
Chitosan Eggplant Sensory evaluation SAS (Nano- 15 Improved Wu et al.
chitosan quality & shelf (2021)
5%, CH life
1.5%)
Chitosan Red sea Sensory SAS CH (3%) 6-8 Improved Zhao et al.
bream fillets evaluation, quality & shelf (2022)
microbiological life
Chitosan Chicken Antimicrobial SAS CH (1%), 15 Preserved Abbas and
with breast effectiveness PPE (4%) quality Rahman
pomegranate (2020)
peel extract
Chitosan Cattle Sensory evaluation SAS CH+GPE 7 Improved Cao et al.
with Grape (2% & quality & (2020)
peel extract 0.4%) extend shelf
life
Chitosan and Banana Microbial growth SAS CH (1.0, 12 Extend the Islam et al.
Guava leaf 1.5, 2.0%), shelf life (2018)
extract & GLE
0.5%
Chitosan and Lemon Storage quality SAS CTS (d > 10 Maintained Li et al. 2020
clove oil 90%), CO qualitative
(0.10%) properties
Chitosan and Bighead fish Chemical, microbial SAS CH (1.5%), 16 Increased Meherpour
Olive Leaf properties OLE (1% & shelf life et al. (2020)
Extract 2%)
Chitosan and Chicken Sensory qualities SAS CH (2%), 12 Longer shelf Mehrabi et
Nepeta NPE (0.2% life al. (2021)
pogonosper & 0.6%)
ma extract
Chitosan and Beef Microbial properties SAS CH (1% to 4-7 Increased Min et al.
Pomegranat 2%), PPE shelf life (2021)
e peel (1.5% &
extract 4%)
Chitosan and Meatballs Quality properties SAS CH (0.25, 10 Increased Oz et al.
Heterocyclic 0.50, 0.75 & shelf life (2016)
aromatic 1%,
amines
Chitosan and Strawberry Storage quality SAS CS-APP1 6 Extend shelf Riaz and
Apple peel (0.25%, life & Aadil (2021)
0.50%, maintained
0.75%, 1%) quality
attributes
Chitosan and Yellowfin Physicochemical, SAS CH (1%) & 10-12 Enhanced the Sabu et al.
lemon peel tuna microbial, sensory LPE (1%) shelf life & (2020)
extract assessments better storage
qualities
Chitosan and Fish Microbiological SAS CH (1%) & 18 Inhibited Ucak and
Peppermint meatballs quality PEO (1%) microbial Afreen.
Essential Oil growth (2022)
Chitosan and Pacific white Sensory Qualities TPA CH (1%) & 10 Improved Yuan et al.
pomegranate shrimp and PPE (1.5%) sensory (2016)
peel extract SAS quality,
hardness,
springiness
Pomelo Peel Rice straw Fiber PPE (75%) Prevent water Chollakup et
Extract characteristics absorption al. (2020)
Pomelo peel Freshwater Antimicrobial SAS PPEO 6-9 Improved Yumnam et
essential oil fish activity (65%) storage al. (2023)
stability.
CH – Chitosan, PPE - Pomelo Peel Extract, GPE – Grapefruit Extract, GLE – Guava Leaf Extract, CO – Clove Oil, OLE – Olive
Leaf Extract, NPE – Nepeta Pogonosperma Extract, PPE – Pomegranate Peel Extract, APE – Apple Peel Extract, LPE – Lemon
Peel Extract, PEO – Peppermint Essential Oil, PPEO, Pomelo Peel Essential Oil.

The comprehensive review presented in this paper highlights the immense potential of chitosan
and pomelo peel extracts as natural food preservatives. Their remarkable properties, including
antimicrobial, antioxidant, and film-forming activities, make them effective in extending the shelf life of
various food products. By inhibiting microbial growth, reducing oxidative deterioration, and enhancing
sensory attributes, these natural extracts offer a viable solution for maintaining food quality and safety.
Their versatility in applications, such as coatings, films, and packaging materials, provides a protective
barrier against spoilage microorganisms, ensuring the preservation of food products.

The recent applications of chitosan and pomelo peel extracts in preserving meat, fish, fruits,
and vegetables have shown promising results, including the inhibition of pathogenic strains and the
reduction of microbial contamination. Furthermore, their utilization as alternatives to synthetic
preservatives aligns with the growing consumer demand for clean labels and sustainable food
preservation solutions. While the potential of these natural extracts is evident, further research is
necessary to optimize extraction methods, determine optimal concentrations, and assess the long-term
effects on food quality and safety. Such investigations will contribute to their effective and safe utilization
in the food industry.

Overall, chitosan and pomelo peel extracts represent a promising avenue for the development
of innovative and sustainable food preservation strategies. Their application holds significant potential
in ensuring consumer satisfaction, while addressing the need for natural, eco-friendly alternatives to
synthetic preservatives. By continuing to explore and enhance the capabilities of these natural extracts,
we can foster a safer, healthier, and more sustainable future for food preservation.
 CHAPTER 3

RESEARCH METHODOLOGY

3.1 Methodological Framework

The methodological framework for this study is presented in Figure 4. In general, the study
involves the following major activities namely: materials and sample preparation, oven-drying,
extraction of pomelo peel, blending and filtering of chitosan and pomelo peel extract, coating application
to the samples, packaging, chilled storage, characterization, and interpretation through data analysis.
The study will be conducted at the Caraga State University Main Campus, Ampayon, Butuan City,
particularly in the Chemistry Laboratory and Department of Agricultural and Biosystems Engineering
Laboratory. For testing, certain physicochemical properties will be analyzed at the Department of
Science and Technology Regional Office (DOST). Additional parameters will be assessed in other
universities or laboratories.

Figure 4. General framework of the study.

 The experimental research approach will be utilized to accomplish the objective of the study.
This research study was chosen because of its applicability to the problem. The collected data was
examined, and various types of scientific studies and experiments were carried out. This research
primarily aims to evaluate the effect of using natural additives such as chitosan and pomelo peel extract
coating on quality and shelf life of pompano meat and identify which packaging materials provide better
storage under chilled condition.

3.2 Sample Preparation

Sample preparation involves acquiring, gathering, and preparing the necessary materials and
ingredients. Chitosan powder will be sourced out from Galleon Company located in Makati City, Metro
Manila, Philippines. While pomelo fruit will be collected from a local pomelo producer in Cabadbaran
City, Agusan del Norte. Freshly harvested pompano fish will be purchased from Placer Fishing Port or
Wet Market in Surigao del Norte, Philippines. A total of 45 individual pompano fish (Figure 5A) with a
marketable size of 250 grams each will be chosen to obtain uniform sample sizes. Intact pompano meat
fillets (Figure 5B) of fish will be removed from guts, black film, blood clots, and any other undesirable
components. The surface water of the fillets will be gently dried with kitchen paper, and they will be
placed into different packaging materials (Figure 5C). For this study, freshly harvested pompano fish
and freshly peeled pomelo fruit will be considered to identify quality control, biological relevance,
standardization, freshness-related parameters, and real-time analysis.

Figure 5. Images of Golden Pompano (A), fillet sample (B) and packaging method (C). Source: (Zhang et al., 2018)

3.2.1 Extraction of Pomelo Peel

The pomelo fruits will be washed with flowing water and sliced to separate from its peel. The
peels will be cut into cubes and dry using convective oven dryer at 60 °C for 24 hours (Sabu et al.,
2020). After drying, the peels is subjected for grinding using a food grinder. To avoid unwanted particles
and obtain fine powder, a set of sieves will be utilized.

For the solution, a 10 g of pomelo peel powder will be soaked in 100 ml of ethanol in a conical
flask plugged with cotton and put in an orbital shaker at 120 rpm for 24 hours at room temperature.
Extract will be filtered using Whatman (No.1) filter paper and concentrate under vacuum at 40°C.

3.2.2 Preparation of coating solutions

Chitosan (1 g) will be dissolved in 1 ml acetic acid containing 100 ml of distilled water and stir
for 1 h at room temperature to obtain 1 % chitosan solution (CH1 %). Concurrently, pomelo peel extract
(PPE1 %) dipping solution will be prepared by dissolving 1 ml PPE in 100 ml distilled water. Additionally,
PPE + CH 1% and PPE + CH 2 % solutions will be prepared by combining 50 % of CH1 % with 50 %
of PPE1 % and 100 % of CH1 % with 100 % of PPE1 %, respectively.

For this study, a total of five (5) treatments will be applied for evaluation on the effect of chitosan
and pomelo peel coating to pompano meat as stipulated in Table 1. A total of forty-five (45) pompano
fish samples will be coated under different concentrations and will be stored at three (3) different
packaging materials namely, polyethylene bag (Sabu et al., 2020), modified atmosphere packaging with
70% CO2/30% N2 gas ratio (Zhang et al., 2022), and vacuum packaging (Waitrovich B., 2014).

Table 5. Factors to consider on the evaluation of chitosan and pomelo peel extract coating on golden pompano.

Modified
Polyethylene Vacuum
Treatment (T) Composition Replication Atmosphere
Bag Packaging
Packaging
1 S1 S1 S1
Control Distilled water 100mL 2 S2 S2 S2
3 S3 S3 S3
1 S1 S1 S1
PPE 10g,
T1 2 S2 S2 S2
Ethanol 100mL
3 S3 S3 S3
CH 1g, 1 S1 S1 S1
T2 Acetic acid 1mL, Distilled 2 S2 S2 S2
water100mL 3 S3 S3 S3
PPE 5 g,
Ethanol 50 mL, 1 S1 S1 S1
T3 CH 0.5 g, 2 S2 S2 S2
Acetic acid 0.5 mL, Distilled 3 S3 S3 S3
water 50 mL.
PPE 10 g,
Ethanol 100 mL, 1 S1 S1 S1
T4 CH 1 g, 2 S2 S2 S2
Acetic acid 1 mL, 3 S3 S3 S3
Distilled water 100 mL.

3.3 Coating procedure for fish samples

Fresh pompano fish meat will be brought to the laboratory under iced conditions. After washing
and cleaning, the meat will be made into fillets and randomly assigned into five groups: control C
(uncoated), PPE1 %, CH1 %, PPE + CH1 %, and PPE + CH2 % groups after washing in chilled distilled
water. The meat samples will be dipped in the prepared solutions at a pompano fish: solution ratio of
1:2 (w/v) at 4 °C for 20 min. The control meat will be dipped in chilled distilled water for 20 min. After
dipping, the fillets will be drained at ambient temperature for 3 min. The coated and uncoated samples
will then be packed in sterile polyethylene bags, modified atmosphere packaging, and vacuum
packaging and stored under chilled condition. Chilling will be applied as a practical and effective
preservation technique, where the fish will be stored at temperatures ranging from 0 to 4 °C to slow
down spoilage and maintain freshness (Sampels, 2015). According to Hassoun & Emir Coban (2017),
it is important to recognize that chilling does not completely prevent enzymatic activity or microbial
spoilage in fish due to factors such as improper handling and inappropriate packaging of the fish meat.
Immediate chilling at 0 – 4 °C of fresh whole or gutted pompano after washing/cleaning, with potable
water or clean seawater, will be conducted in accordance with hygienic practices (PNS/BAFS
206:2017). Samples representing all regions of the fillets of the respective lots (in correct quantities) will
be weighed and transferred for biochemical and microbiological analysis at every 2-day interval.
3.4 Determination of total phenolic, total flavonoid contents and antioxidant capacity of PPE

3.4.1 Total Phenolic

The total phenolic content of PPE will be assessed based on the method of Hung et al. (2020).
The total phenolic contents will be measured using a standard calibration curve, and they will be
expressed as milligrams of Gallic Acid Equivalents (GAE) per gram of PPE.

3.4.2 Total Flavonoid

The total flavonoid content of PPE will be determined using the aluminum chloride colorimetric
method, following the procedure described by Hung et al. (2020). The resulting values will be expressed
as milligrams of Quercetin Equivalents (QE) per gram of pomelo peel powder.

3.4.3 Antioxidant Capacity

The antioxidant activity of pomelo peel extracts will be assessed based on the method
described by Singh and Immanuel (2014). The DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical
scavenging activity of PPE will be used to determine the antioxidant capacity of PPE. The scavenging
of DPPH will be calculated as follows:

Absorbance sample
DPPH% = [Absorbance control − ( )] × 100
Absorbance control

3.5 Determination of changes in physical and biological properties of pompano fish meat

3.5.1 pH value

The pH will be measured using a digital pH meter. A pH meter or pH test strips will be used to
measure the pH of a pompano fish's flesh, or the liquid extracted from it. This method will allow for an
accurate measurement of the fish's pH level in the future.

3.5.2 Total Volatile Basic-Nitrogen (TVB-N) value

The TVB-N content of pompano meat will be determined using a Conway micro diffusion
method from Sabu et al., (2020). The 10 g samples will be stirred, and then 75 mL of distilled water will
be added to a reaction flask and mixed. The flask will then be left to stand for 30 min before 10 mL of
oxidase solution is added, and the mixture will be distilled using a nitrogen analyzer. The distillate will
be collected in a receiving flask to which 30 mL of 20 mg/mL boric acid solution and 3 drops of the
mixed indicator will be added, which will be generated by dissolving 0.1 g methyl red and 0.1 g
methylene blue into 100 mL of ethanol. Finally, the fractions will be titrated with 0.1 mol/L hydrochloric
acid standard solution. TVB-N values will be expressed as mg / 100 g of samples. Three replicate
experiments will be performed.

3.5.3 Tri-methyl amino nitrogen (TMA-N)

The TMA-N content of pompano meat will be determined using a Conway diffusion method
from Sabu et al., (2020). TMA-N is an important spoilage index, particularly in marine fishes and TMA-
N will be calculated and expressed as mg %.

3.5.4 Peroxide value (PV)

The fat oxidation products of pompano fish samples will be determined (Sabu et al., 2020). The
analysis will involve on extracting the fat or oil from the fish and then titrating it with a suitable reagent
to measure the peroxide content and expressed in terms of milliequivalents of peroxide per kg
(𝑚𝑒𝑞 𝑂2 ⁄𝑘𝑔). This process will allow for the measurement of the peroxide value, indicating the extent
of fat oxidation in the pompano fish samples.

3.5.5 Thiobarbituric Acid-Reactive Substances (TBARs)

TBARS will be determined according to Yerlikaya et al., (2015) to evaluate the oxidation stability
during chilled storage, and the results will be expressed as TBARS value in mg of malonaldehyde per
kg (𝑚𝑔 𝑀𝐷𝐴⁄𝑘𝑔). Briefly, 5 g of fish samples will be weighed, and 25 mL of 7.5 % trichloroacetic acid
(containing 0.1 % EDTA) will be added. The samples will be homogenized, shaken, and extracted for
30 min. The mixture will then be centrifuged for 5 min, and 5 mL of the supernatant will be taken. Then,
5 mL of 0.02 mol/L TBA solution will be added, and the solution will be heated in a boiling water bath
for 20 min. The solution will be left to turn pink and then cooled under running water. Next, 5 mL of
chloroform will be added, and the mixture will be shaken. Subsequently, it will be centrifuged for 5 min,
and the absorbance of the supernatant at 532 nm will be measured. Three replicate experiments will
be conducted.

3.5.6 Color

The color of pompano fish will be measured utilizing the CIELAB color system. Average
readings for L* (lightness), representing brightness on a scale of 0 (dark) to 100 (white), a* (redness)
ranging from negative values for green to positive values for red, and b* (yellowness) ranging from
negative values for blue to positive values for yellow, will be obtained (Min et al., 2019). Subsequently,
the chroma value (C) will be calculated using the provided formula. This procedure will enable the
quantitative assessment of the pompano fish's color characteristics.

∆𝐸 = √(𝐿 − 𝐿∗ )2 + (𝑎 − 𝑎 ∗ )2 + (𝑏 − 𝑏 ∗ )2

∆𝐶 = √(𝐿 ∗)2 + (𝑎 ∗)2 + (𝑏 ∗)2

3.5.7 Texture

The texture attributes of pompano fish meat will be assessed using a texture analyzer or a
sensory evaluation panel. The various texture tests will be performed, including hardness,
cohesiveness, springiness, chewiness, and tenderness assessment will provide valuable insights into
the texture characteristics of pompano fish meat.

3.6 Sensory evaluation of fish quality

Trained panelists will assess the sensory properties of the future fish samples using a hedonic
scale to evaluate the general appearance, color, odor, and overall acceptability. The scale will consist
of different values to indicate the panelists' reactions as follows: 1 - will indicate extreme dislike; 2 - will
indicate very much dislike; 3 - will indicate moderate dislike; 4 - will indicate slight dislike; 5 - will indicate
neutral; 6 - will indicate like slightly; 7 - will indicate like moderately; 8 - will indicate like very much, and
9 - will indicate like extremely. The average scores of the above mentioned four indices will be utilized
to determine the future shelf life of the fish. An acceptable shelf life will be determined by a sensory
score greater than 4.

3.7 Bacteriological analysis

Total aerobic plate count (TPC) will be determined based on the method described by Sabu et
al., (2020). TPC will be determined by spread plating samples on nutrient agar and incubating them at
36 °C for 48 hours. After incubation, microbial colonies will be counted, and the results will be reported
as log10 CFU/g of fresh weight.
3.7 Statistical analysis

Two major statistical analyses will be performed after data collection which include the
descriptive analysis and post hoc test. The descriptive analysis and post hoc test will be executed using
Statistical Package for the Social Sciences (SPSS). Statistical differences within treatment groups will
be established by conducting analysis of variance (ANOVA). Differences between treatments will be
assessed using the Tukey's HSD test. Significance of differences will be employed at p<0.05 level
between the means of the different treatments.
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