Introduction

INTRODUCTION

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Amla (Emblica officinalis)

Amla (Emblica officinalis Gaertn) also known as Indian gooseberry is a medicinal

plant used in ayurvedic medicine for thousands of years. It belongs to family
Euphorbiaceae (Shukla et al., 2022). It is commercially cultivated within the state of
Uttar Pradesh in India. It is also grown in Tamil Nadu, Rajasthan, and Madhya
Pradesh. It is a small to medium sized deciduous tree with a crooked trunk and
spreading branches. Amla is the medium measurement deciduous plant. It grows to
the peak of 8-18 m with thin, gentle gray bark. Its flower is yellow-greenish in color.
The fruit is spherical faded yellow with six vertical furrows enclosing six trigonous
seeds in two seeded three crustaceous cocci (Jain et al.,2016). The traditional
weight of the fruit is 20-25 g. It has a gray bark and reddish wood. Its leaves are
feathery, linear rectangular in form, and scent-like lemon. Its wood is difficult in
texture.

It is also known as the king of all medicinal plants. At present, approximately 70% of
the arena population is depending on medicinal herbs. Medicinal plants contain so
many chemical substances which can be the principal supply of therapeutic sellers
to healing human diseases (Maurya and Srivastava, 2011). Amla is one of the
excellent sources of vitamin C containing about 20 and 160 times more vitamin C as
compared to orange and apple, respectively. The nutritional and medicinal
properties of amla make it a well known fruit. It is a rich source of vitamin C,
phytochemicals and minerals. This fruit is rich in quercetin, phyllemblic compounds,
gallic acid, tannins, flavonoids, pectin, and vitamin C and also contains various
polyphenolic compounds. A wide range of phytochemical components including
terpenoids, alkaloids, flavonoids, carbohydrates, and tannins have been shown to
possess useful biological activities. Many pharmacological studies have

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demonstrated the ability of amla as antioxidant, anti-carcinogenic, anti-tumor, anti-
genotoxic, anti-inflammatory activities, anti-cancer, anti HIV-reverse transcriptase,
anti-diabetic, inhibitory effects, anti-depressant, anti-ulcerogenic, hair growth tonic,
wound healing activities, cardiovascular diseases, neurodegenerative diseases,
cancer, and many other traditional uses of the plant. Because of all these qualities it
is also known as “Wonder fruit for health”.

Amla builds immune system to fight against all kind of viruses like Hepatitis, AIDS,
Influenza and many others. This fruit is highly valued among indigenous medicines.
It is acrid, cooling, refregerant, diuretic and laxative (Govind et al., 2011). Dried fruits
have been reported to be useful in treating hemorrhages, diarrhea, dysentery,
anemia, jaundice, dyspepsia and cough. Trifala and Chyavanprash are well known
indigenous medicines in ayurvedic system using amla fruit.

Many fruits and vegetables are season oriented. Likewise, amla is also one of the
winter season fruit. It contains higher percentage of ascorbic acid as compared to
other fruits and vegetables. As amla fruit is highly perishable in nature, its storage is
very limited. Also it is a seasonal fruit. Application of fresh amla is restricted due to
its sour and strong astringent in taste and short shelf life. Therefore, it is consumed
in various processed forms out of which dehydrated powder (Karla 1988; Aslam et
al., 2022).

Amla is in very high demand. Appropriate storage and processing methods can
curtail the post harvest losses to 30% and make the fruit available for longer period
(kohli et al., 2023). A few post harvest technologies that exist are complex and are
unaffordable to the marginal and small farmers at the farm level. Extension of
storage life may be possible by checking the rate of transpiration, respiration and
microbial infection. Plant growth regulators, certain chemicals and fungicides play a
great part in increasing the storage life. Processing into value added product will not
only reduces the post harvest losses but also provides higher returns to the growers.
Drying is one of the oldest and most widely used techniques in the food industry to
prolong the shelf life of fruits and vegetables. It extends the shelf life of food
products by reducing the water activity to the level at which microbial growth and

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deterioration reactions are minimum. In addition, drying also saves packaging,
storage and transportation costs by substantially reducing the volume. Attention
towards nutritionally superior food with higher shelf life has increased in the 21st
century after problems related to basic food needs of human beings such as health
and food security came to the fore.

Practices of postharvest technologies can reduce the quantitative and qualitative

losses of fresh fruits also maintained the product quality up to final consumption.
Attaining the hygienic agricultural produce should be focused on the varieties of
higher postharvest longevity (Wasala et al., 2014). Postharvest quality and shelf life
of the fruits related with the cultivation practices, varieties of the cultivar and
environmental aspects. The soil and climatic characteristics and integrated
management practices also affect the postharvest losses and postharvest storage
duration (singh and vishal, 2014).

Postharvest quality and shelf life of the fruits are related with the cultivation
practices, varieties of the cultivar and environmental aspects. The soil and climatic
characteristics and integrated management practices also affect the postharvest
losses and postharvest storage duration. Due to high water activity, fruits and
vegetables are considered more perishable and nearly 33% of total produced fruits
and vegetables have been spoiled during harvesting to marketing (Ziv and Carmit,
2021).

Biochemical and nutritional composition of amla fruit

1. Chemical composition

The fruit is reported to contain nearly 20 times as much Vitamin C as orange. Every
100 g edible fruit provides 470-680 mg of Vitamin C. Fruit contains moisture, protein,
fat, minerals, fibers and carbohydrate. Its mineral and vitamin contents include
calcium, phosphorous, iron carotene, carbohydrate, thiamine, riboflavin besides
Vitamin C. A recent study on amla attributes its strong antioxidant properties to its
small molecular weight tannoid complexes (Ghimire et al., 2022).

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2. Nutritive and medicinal value

The fruits of amla are highly nutritious, with the rich sources of vitamin C and having
many medicinal uses which varies from 500-1200 mg of ascorbic acid per 100 g of
pulp which is much more than the vitamin C content of guava, citrus and tomato
fruits. Its other constituents are phenols and tannins containing Gallic acid, elegiac
acid and glucose which prevent oxidation of vitamin C. It has an important
therapeutic role from time immemorial and is frequently recommended for its
synergistic effects in ayurvedic and unani systems of medicine (Agarwal and
Chopra, 2004).
Vitam
in C Moist
Iron
600m ure Carbo
1.2m
Phos g/100 81.2 hydra
g/
phoro g %
100g te
us
Calciu 14.1
0.02
F%iber
m
%
miner 3.4%
0.05
al
% Fat Prote matte
0.1% in r
0.5% 0.7%
3. Amla as a rich source of ascorbic acid

The ascorbic acid content of amla is reported to be the highest among fruits and
vegetables. Compared to edible portions, it has 20 times vitamin C of grape fruit and
15 times that of lemon (Nisha et al., 2004). More importantly, the vitamin C
contained in the amla fruit is stabilized by the presence of tannins, which help amla
to maintain its vitamin content even through processing. Consumption of only 10 g
(one average size fruit) will meet the recommended daily allowances (RDA) of
vitamin C. The ascorbic acid contents of small and large size fruits of amla found to
be 412 and 900 mg per 100 g edible portion, respectively. Vitamin C is a water
soluble vitamin and it is not stored in the body. It is important to consume it on
everyday basis in diet. Vitamin C is part of the cellular chemistry that provides

5
energy, it is essential for sperm production and for making the collagen protein
involved in the building and health of cartilage, joints, skin and blood vessels.
Vitamin C helps in maintaining a healthy immune system, it aids in neutralizing
pollutants, is needed for antibody production, acts to increase the absorption of
nutrients (including iron) in the gut and thins the blood. These are the most important
functions of Vitamin C.

Varieties/cultivar

A full mature tree bears 125-200 kg fruit tree-1; however, the fruits yield various
variety to variety. Cultivars like Banarasi, Francis (Hathi Jhool), Chakaiya, Banarasi
NA-4 (Kanchan) and NA- 5 (Krishna), NA-6 (Amrit), NA-7 (Neelum), NA-9 and NA-
10 are well known for commercial cultivation. Banarasi and Chakaiya cultivars fruits
are generally used for making murabba (preserve), candy, etc.
Some other varieties of amla on the basis of colour are-:

a. Green tinged: fairly large, nearly green in color, best for pickles and murabba.

b. Red tinged: medium size fruit having white streaks.

c. Hathiful: commercial variety found in Uttar Pradesh, India fairly large tree, fruit
size good, with shining, conspicuous glands.

Maturity indices and harvesting

The budded plants take less time and produce crop in 4-5 years, while seedlings
required a long time 8-10 years to come in the full bearing stage. Fruits are ripening
during November-December. It is also vary cultivar to cultivar, such as Banarasi and
Chakaiya cultivar mature in the November, whereas frainsic cultivar fruits are mature
in November to first week of December. Fruits should be harvested at proper
maturity when the colour of fruits turn dull green or greenish yellow or sometimes
brick red colour can also be seen. Change in seed colour from creamy white to
brown. For harvesting of amla growing degree days is also taking in consideration
(5000- 5200º days) (Dhillon et al., 2013). Fruits are harvested individually plucked by

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climbing on the tree with the help of bamboo ladders. Fruit plucking should be done
at morning or evening hours.

Postharvest disease of Indian gooseberry

Indian gooseberry has a low shelf-life of 5–6 days. The fruit is susceptible to number
of Post harvest fungal pathogens among them most prevalent are blue and black
mold. Thom et al.,1930 reported annual yield loss up to 50% due to fruit rot of Indian
gooseberry caused by blue mold. The fungal diseases deteriorate the fruit quality
and lower its marketability which affects the fruit production severely. Taking into
consideration its enormous health benefits as well as economic importance, it is
necessary that the post-harvest losses of Indian gooseberry be curtailed.
Some of the postharvest diseases in Indian gooseberry are-:
1. Anthracnose by Colletotrichum gloeosporioides.
2. Fruit rot by Penicillium digitatum
3. Soft rot by Phomopsis phyllanthus

4. Fruit rot by Aspergillus niger.

Edible Films and coatings

Edible films and coatings are produced from edible biopolymers and food-grade
additives. Film-forming biopolymers can be proteins, polysaccharides
(carbohydrates and gums), lipids, or a mixture of these. Plasticizers and other
additives are combined with the film-forming biopolymers to modify the physical
properties or other functionality of the edible films (Han and Jung, 2014).
Biopolymers have multiple film-forming mechanisms, including intermolecular forces
such as covalent bonds and electrostatic, hydrophobic, or ionic interactions. For the
resulting films or coatings to be edible, the film-forming mechanism involved in
fabrication should be an appropriate food process namely, pH modification, salt
addition, heating, enzymatic modification, drying, use of food-grade solvents, or
reactions with other food-grade chemicals.

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Uses of amla

1. Food use: Amla is used for making different products such as pickles and dry
candy. Also different amla jam, sauces etc. can be prepared from amla. It is useful
during summer and provides cool to the consumer (Ghimire et al., 2022).

2. Uses of seed: The seed of amla too has some benefits. Infusion of seed is
recommended as a drink in fever and diabetes; seeds are also used as a remedy for
bilious infections and nausea. An ointment prepared by burning seeds with oil is
prescribed for scabies and itch. The root leafs; bark and fruit are also for the cure of
snake bite (Sharma et al., 2021).

3. Pharmacological use: The pharmacological properties of the amla are numerous.

Not only is it a wonderful antioxidant, but it has proven anti-fungal, anti-bacterial,
anti-viral, anti-mutagenic, yeast inhibiting, anti-inflammatory, hypolipidemic, and
hypotensive relieving properties, it also acts as an antacid and anti-tumorganic
agent. (Singh et al., 2019).

4. Medicinal use: Amla or Emblica Officinalis is a natural, efficacious, an antioxidant

with the richest natural source of Vitamin C. The fruit contains the highest amount of
Vitamin C in natural form and cytokine like substances identified as zeatin, z.
riboside, and z. nucleotide. Its fruit is acrid, cooling, refrigerant, diuretic and laxative.
The dried fruit is useful in hemorrhage, diarrhea and dysentery. It is anti-bacterial
and its astringent properties prevent infection and help in the healing of ulcers. It is
used as a laxative to relieve constipation in piles.

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OBJECTIVES

The main objectives of this study are:

1. To study the effect of edible coatings on shelf-life extension of amla fruits.

2. To elucidate the effect of the edible coatings on quality parameters in amla

fruits.
 Qualitative – disease incidence and weight loss
 Quantitative – biochemical estimation (carbohydrate, protein, flavonoid,
phenol, catalase, SOD, chlorophyll test)

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Review of Literature

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REVIEW OF LITERATURE
According to Indian ancient history, amla is believed to be the first tree to be grown
in this universe. E. officinalis is widely distributed in tropical and subtropical
countries like China, India, Indonesia and Southeast Asia. From the ancient time, the
plant has been utilized for management of various disorders like diabetes,
hyperlipidemia, CNS disorders, ophthalmic diseases and various other life style
illnesses. Emblica officinalis is a small to moderate sized deciduous tree (Variya and
Bhavesh, 2016).

Phytochemical constituents

Emblica officinalis, well known for its nutrient qualities, contains a variety of chemical
constituents including tannins, mucic acid, amino acids, alkaloids, flavone
glycosides, phenolic glycosides, flavonol glycosides, phenolic acids,
sesquiterpenoids, norsesquiterpenoids and carbohydrates (Saini et al., 2022).

Potential therapeutic application

Every part of Emblica officinalis is useful owing to its medicinal and pharmaceutical
properties. The plant has been reported to have anti-oxidant, anti-inflammatory, anti-
cancer, adaptogenic, anti-diabetic, non-tropic, anti-microbial and immunomodulatory
potential. Besides having beneficial actions in various disorders, Emblica officinalis
also prevents hyperlipidemia, osteoporosis and several other ailments (patel et al.,
2012).

Dermato-protective

From ancient time, herbs have been used in medicines and cosmetics due to their
potential to treat skin ailments and improve skin appearance. Clinical trials and
laboratory outcomes have identified the favorable action of natural ingredients for
skin. Antioxidants, flavonoids and phenolic compounds present in herbs play critical
roles to counteract free radicals, the main cause of various unfavorable skin
changes (Fowler et al., 2010).

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Morphology

Amla tree is a small to medium sized deciduous tree with an average height of 8-18
m, with thin light grey bark exfoliating in small thin irregular flakes, exposing the
fresh surface of a different color underneath the older bark. The average girth of the
main stem is 70 cm. In most cases, the main trunk is divided into 2 to 7 scaffolds
very near to the base (Pareek et al., 2011). Leaves are 10 -13 mm long, 3 mm wide,
closely set in pinnate faishon3 which makes the branches feathery in general
appearance. After setting of the fruits leaves develop. Flowers are unisexual, 4 to 5
mm in length, pale green in color, borne in leaf axils in clusters of 6 to 10. Fruits are
fleshy, almost depressed to globose shape, 2.1-2.4 cm in diameter, 5.3-5.7 g in
weight, 4.5-5.0 mL in volume. The stone of the fruit is 6 ribbed, splitting into three
segments each containing usually two seeds; seeds are 4-5 mm long and 2-3 mm
wide, each weighing 572 to 590 mg.

Post-harvest treatments

Post-harvest treatment of amla is performed immediately after harvesting. This

procedure includes cooling, cleaning, sorting and packaging. The instant a crop is
removed from the ground, or separated from its parent plant, it begins to deteriorate
so, these amla fruits are treated with different types of edible coatings to increase its
shelf-life and maintain quality.

Following are the steps included in post-harvest treatment:

1. Sorting and grading

For efficient marketing, fruits are generally sorted and graded. Grading can be done
on the basis of size and quality. On the basis of size fruits are graded into three
grades which are large size fruits (>4 cm in diameter), small size fruits (<4 cm in
diameter) and last is defective fruits (Dhillon et al., 2013).

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2. Drying

Untreated and pretreated grated amla samples were subjected to drying for 8 hours
using cabinet tray dryer at a temperature of 55 ± 2 °C. The sample was spread as a
thin layer of about 2 mm thickness in all the trays. The dryer consisted of 24
aluminum trays of size (800 × 400 × 30 mm) with temperature control and was fitted
with a centrifugal fan for air circulation at velocity 1.2 m/s speed inside the drying
chamber. Preliminary drying studies had shown that 8 h of drying yields optimally
dried gratings (data not presented). At the end of 8 h, the trays were removed,
samples were allowed to cool to room temperature and then packed in HDPE
pouches before storing at 4 °C.

3. Packaging

There is a linear increase in quality parameters (such as specific gravity, TSS,

acidity, the (TSS: acid) ratio, fiber content, etc.) from 35 days old to fully matured
fruit (120 Days) of amla, and hence it is ideal to harvest fruits at 120 days after set
(Sivakumar et al., 2010). Selection of the correct packaging material and packing
method are equally important. At present proper packaging is inadequate in case of
amla. Generally, fruits are packed in wooden crates with polythene or newspaper
liner (to minimize bruising) for distant market. Fruits can also be packed gunny bags
and jute bags. The corrugated fiber boxes are better as this provides appropriate
ventilation inside the box and can also re-usable. Packaging of fruits, in corrugated
fiber board boxes which are lined with newspaper causes minimum spoilage (16.0%)
in fruits (Ruvini et al., 2018).

4. Storage

The fruit availability period of amla is very short; hardly 2 to 3 months. Storage
conditions should be appropriate to reduce post harvest losses in amla. Therefore,
storage of fruits at an appropriate temperature is essential to extend the availability
period and to stabilize the price in the market. Storage and processing methods can
curtail the post harvest losses upto 30% and increase the shelf-life of fruits which

13
can make the fruit available for longer period. For better and long shelf-life storage,
fruits should be stored at ambient temperature and relative humidity. Storage
facilities such as cold storage and Controlled/modified atmosphere packing are very
expensive and not within the direct reach of poor farmers (Kumar et al., 1993).

Harvesting

Infield handling

sorting

Trimming

Cleaning/washing

Drying/rinsing

Grading

packaging

Bulk packaging Retail packaging

Storage

Edible coatings

Edible coatings are thin layers of edible material applied to the product surface in
addition to or as a replacement for natural protective waxy coatings and to provide a
barrier to moisture, oxygen, and solute movement for the food they are applied
directly on the food surface by dipping, spraying, or brushing to create a modified
atmosphere. Because They will be consumed, the material used for the preparation
of edible films and coatings should be generally regarded as Safe (GRAS) approved
by FDA and must conform to the regulations that apply to the food product

14
concerned (Chavan et al., 2023). An ideal coating is defined as one that can extend
storage life of fresh fruits and vegetables without causing anaerobiosis and reduces
decay without affecting their quality. Previously, edible coatings have been used to
reduce water loss, but recent developments of formulated edible coatings with a
wider range of permeability characteristics has extended the potential for fresh
produce application. Fruit-based coatings provide enhanced nutrition to products,
which increases their market value. Edible and biodegradable coatings must meet a
number of special functional requirements, for example, moisture barrier, solute or
gas barrier, water/lipid solubility, color and Appearance, mechanical characteristics,
non-toxicity, etc. The effect of coatings on fruits and vegetables depends greatly on
Temperature, alkalinity, thickness and type of coating, and the Variety and condition
of fruit and vegetable (Milani and Jafar, 2022).

Table 1: Various gums used as edible coatings for fruits and vegetables:

S.No. Coatings Fruits Main benefits References

1. Gellan Reduction in the ethylene production, (Yousuf et al.,
gum, Apple microbiological deterioration, 2018)
sodium extension of the shelf life, anti-
alginate browning effect, maintenance of the
initial firmness, retention of the color.
2. Alginate Apple Increasing shelf-life and quality of (Solis-contriras
apple. et al.,
2021)
3. Alginate Apple Edible coatings with alginate can (Salehi et al.,
and gellan reduce weight loss, preserve quality 2020)
of fruits and prolong its shelf-life.
4. Chitosan apricot Increasing quality of apricot during (Kovač and
storage. Renata, 2022)
5. Chitosan, Coatings based on Chitosan were ( Elsabee and
modified banana much superior in increasing and Abdou, 2013)
Starch and improving the shelf life and quality of

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 cellulose fruit.
6. Arabic banana Their results suggest the using 10% (Maqbool et
gum arabic gum with 0.4% cinnamon oil al., 2011)
as a biofungicide for controlling
postharvest anthracnose.
7. Aloe Vera papaya Improving of storage life and quality (Misir et al.,
maintenance of fruit. 2014)
8. Methylcell Cherry Decreasing in the water loss. (Shakir et al.,
ulose tomato 2022)
9. Alginate, They found that gum-based edible (Magri and
pectin and melon coatings prevented the dehydration Anna, 2023)
gellan of fresh cut melon and in addition
inhibited the ethylene producing and
triggered the accumulation of total
phenol with antioxidant
characteristics.
10. Chitosan The addition of 0.361 g dry (Kharchoufi et
and locust oranges pomegranate peel extract/mL, al., 2018)
bean gum Chitosan and locust bean gum
coatings reduced disease incidence
by 49 and 28%, respectively

Historical view of edible coatings

The use of wax coating of fruits by dipping is one of the age-old methods that were
in vogue in the early 12thcentury (Dhall et al., 2013). This was practiced in China,
essentially to retard water transpiration loss in lemons and oranges. Although
Chinese did not realize its full potential and reported that wax-coated fruits could be
stored longer than non-waxed fruits. In the 1930’s hot-melt paraffin waxes became
commercially available as edible coatings for fresh fruits Such as apples and pears.
Later fat coating on food products. Specifically called “larding” was in vogue in

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England. “Sausage Casing” used very commonly nowadays is nothing but a material
derived from a protein source (gelatin). Usually a film thickness of 2.5 mm is
employed and coating is done by several methods. (Nisperos-Carriedo et al. 1990)
and (Baldwin et al. 1995) Observed that oils, waxes, or cellulose had similar effects
of pre-Venting spoilage and retaining fresh-picked quality of fruits and vegetables.
Several attempts have been made to develop other materials that could be used to
coat, produce, and modify internal gas composition of fruit/vegetables for short-term
storage. In 1982, Lowings and Cutts reported an edible coating material that is non-
phytotoxic, tasteless, odorless, and is effective in increasing shelf-life of fruits and
vegetables. This coating material is a mixture of sucrose fatty acid esters (SFAE),
sodium carboxymethyl cellulose, and mono- and di-glycerides. SFAE Was originally
developed as an emulsifier. However, coating of SFAE on fruits retards fruits’
ripening.

Action of edible coatings

Fruits and vegetables continue to respire even after harvest and use up all the
oxygen within the produce, which is not Replaced as quickly as by edible coating
and produces carbon Dioxide, which accumulates within the produce because it
cannot escape as easily through coating. Eventually the fruit and vegetable will shift
to partial anaerobic respiration that requires less oxygen (1–3%) (Dhall et al., 2013).
With less oxygen, the production of ethylene (which accelerates ripening process) is
disrupted and physiological loss of water is minimized. Thus, the fruits and
vegetables remain firm, fresh, and nutritious for longer period and their shelf life
almost doubles. The natural barrier on fruit and vegetable, and the type and amount
of coating will Influence the extent to which the internal atmosphere (oxygen and
carbon dioxide) are modified and the level of reduction in weight loss.

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Properties of edible coatings

The properties of edible coating depend primarily on molecular structure rather than
molecular size and chemical constitution. Specific requirements for edible films and
coatings are:

• The coating should be water-resistant so that it remains intact and covers a product
adequately, when applied.

• It should not deplete oxygen or build up excessive carbon Dioxide. A minimum of

1–3% oxygen is required around a Commodity to avoid a shift from aerobic to
anaerobic respiration.

• It should reduce water vapor permeability.

• It should improve appearance, maintain structural integrity, Improve mechanical

handling properties, carry active agents (antioxidants, vitamins, etc.) and retain
volatile flavor com-pounds.

• It should melt above 40◦C without decomposition.

• It should be easily emulsifiable, non-sticky or should not be tacky, and have

efficient drying performance.

• It should never interfere with the quality of fresh fruit or vegetable and not impart
undesirable order.

• It should have low viscosity and be economical.

• It should be translucent to opaque but not like glass and capable to tolerate slight
pressure.

Composition of edible coatings

Edible coatings can be produced from materials with film forming ability. During
manufacturing, film materials must be dispersed and dissolved in a solvent such as
water, alcohol, Mixture of water and alcohol, or a mixture of other solvents.

18
Plasticizers, anti-microbial agents, minerals, vitamins, colors, or flavors can be
added in this process. Adjusting the pH and/or heating the solutions may be done for
the specific polymer to facilitate dispersion. Film solution is then casted and dried at
a desired temperature and relative humidity to obtain free-Standing films. The film
solutions could be applied to food by several methods such as dipping, spraying,
brushing, and panning followed by drying. Edible coatings may be composed of
polysaccharides, proteins, lipids, and composites (Suhag, Rajat et al., 2020). Their
presence and abundance determine the barrier properties of material with regard to
water vapor, oxygen, carbon dioxide, and lipid trans-Fer in food systems. However,
none of the three constituents can provide the needed protection by themselves and
so are usually used in a combination for best results (McHugh et al., 1994; xing,
yage et al., 2019).

Types of edible coatings

Polysaccharides

Polysaccharides used for edible films or coatings Include cellulose, starch

derivatives, pectin Derivatives, seaweed extracts, exudate gums, Microbial
fermentation gums and Chitosan. Polysaccharides are generally very hydrophilic
resulting in poor water vapor and gas barrier properties. Although coatings by
polysaccharide polymers may not provide a Good water vapor barrier, these
coatings can act as sacrificing agents retarding moisture loss from food Products.

Protein films

In their native states, proteins generally exist as either fibrous proteins, which are
water insoluble and serve as the main structural materials of animal tissues, or
globular proteins, which are soluble in water or aqueous solutions of acids, Bases or
salts and function widely in living systems. Fibrous proteins are fully extended and
associated closely with each other in parallel structures, generally through hydrogen
bonding, to form fibers. The globular proteins fold into complicated spherical
structures held together by a combination of hydrogen, ionic, hydrophobic and
covalent (disulfide) bonds. The Chemical and physical properties of these proteins

19
Depend on the relative amounts of the component amino acid residues and their
placement along the protein polymer chain. Several globular proteins, Including
wheat gluten, corn zein, soy protein, and whey protein, have been investigated for
their Film properties. Protein films are generally formed from solutions or dispersions
of the protein as the Solvent/carrier evaporates. The solvent/carrier is generally
limited to water, ethanol or ethanol-water mixtures (Sim and Yong Ji, 2021).
Generally, Proteins must be denatured by heat, acid, base, and/or solvent in order to
form the more extended Structures that are required for film formation. Once
extended, protein chains can associate through hydrogen, ionic, hydrophobic and
covalent bonding. The chain-to-chain interaction that produces cohesive films is
affected by the degree of chain extension and the nature and sequence of amino
acid residues. Uniform distribution of Polar, hydrophobic, and/or thiol groups along
the polymer chain increase the likelihood of the Respective interactions. Increased
polymer chain-to-chain interactions result in films that are stronger but less flexible
and less permeable to gases, vapors and liquids. Polymers containing groups that
can associate through Hydrogen or ionic bonding result in films that are excellent
oxygen barriers but are susceptible to Moisture. Thus, protein films are expected to
be good oxygen barriers at low relative humidities. (Gennadois et al., 1993; Miserez
et al., 2023).

Lipid films

Lipid compounds utilized as protective coating Consist of acetylated

monoglycerides, natural wax, and surfactants. The most effective lipid substances
are paraffin wax and beeswax. The primarily Function of a lipid coating is to block
transport of moisture due to their relative low polarity. In Contrast, the hydrophobic
characteristic of lipid Forms thicker and more brittle films. Consequently, they must
be associated with film forming agents Such as proteins or cellulose derivatives
(Bizymis et al., 2022). Generally, water vapor permeability Decrease when the
concentration of hydrophobicity phase increases. Lipid-based films are often
supported on a polymer structure matrix, usually a polysaccharide, to provide
mechanical strength.

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Composite films

Edible films and coatings may be heterogeneous in nature, consisting of a blend of

polysaccharides, Protein, and/or lipids. This approach enables one to utilize the
distinct functional characteristics of each class of film former. The combination
between polymers to form films could be from proteins and carbohydrates, Proteins
and lipids, carbohydrates and lipids or synthetic polymers and natural polymers. The
main objective of producing composite films is to prove the permeability or
mechanical properties as dictated by the need of a specific application. These
heterogeneous films are applied either in the form of an emulsion, suspension, or
dispersion of the non-miscible constituents, or in successive layers (multilayer
coating or films), or in the form of a solution in a common solvent.

Chitosan

Chitosan is a natural linear biopolysaccharide. It is derived by alkaline deacetylation

of chitin, which is the major component of protective cuticles of various crustaceans
like crabs, shrimps, prawns, lobsters and cell walls of some fungi such as
Aspergillus and mucor. Chitosan is cheap, biodegradable and nontoxic to mammals.
Freestanding Films have been prepared from Chitosan and its derivatives. Chitosan
can form Semi-permeable coatings, which can modify the internal atmosphere,
thereby delaying Ripening and decreasing transpiration rates in fruits and
vegetables. Cross linked Chitosan films offer greater strength and resistance for
handling. Films from aqueous Chitosan are clear, tough, flexible, and good oxygen
barriers.

21
Chitosan is weak base and insoluble in water and organic solvent. However it is
soluble in dilute aqueous acidic medium (pH < 6.5). It get precipitated in alkaline
solution or with the polyanions and forms gel at low pH. Nowadays starch
conjugated Chitosan microparticles are used in sustained/controlled release system.
These microparticle prepared by a reductive alkylation cross-linking method. It has
been studied in broad range of biomedical application, because of its
biocompatibility and biodegredation properties Chitosan and starch are employed for
microencapsulation of bioactive material and as carrier in drug delivery systems.
Chitosan nanoparticles are generally prepared by ionotropic gelation, self-
assembling or microemulsion methods (Yazdi et al., 2020).

Ionotropic gelation offers a mild preparation method in the aqueous environment,

without the introduction of chemical groups into Chitosan molecules.
Nanotechnology is an emerging technology that holds great promises for the future.
Chitosan nanoparticles have been extensively explored for pharmaceutical
applications, as carriers for drug, gene and vaccine delivery (Dev et al., 2010; Yang,
Yuan, Cai, Wang, & Zong, 2009; Zheng et al., 2007). Furthermore, Chitosan
nanoparticles have been shown to have effective anti-tumor activity and
hypochelosterolemic activity.

Production of Chitosan

Conversion of chitin to chitosan is achieved by treatment with concentrated sodium

or potassium hydroxide solution (40%) generally at 100°C or higher to remove some
or all of the acetyl groups from the polymer.N-acetyl groups cannot be removed by
acidic reagents without hydrolysis of the polysaccharide; thus, alkaline methods
must be employed for N-deacetylation. According to Rigby (1936), factors affecting
the extent of deacetylation include: concentration of the alkali, temperatur of
deacetylation, time of reaction, previous treatment of the chitin, particle size, and
density of the chitin. The latter two factors affect penetration rate of the alkali. During
deacetylation, conditions are necessary that will, in a reasonable time, sufficiently
deacetylate the chitin to yield a Chitosan product soluble in dilute acetic acid, but
one which is not significantly degraded.

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Chitosan edible coating application on minimally Processed Fruits:

The integrity of fresh fruits is altered by minimal processing, including peeling and
cutting, which presents some undesirable physiological changes to the fruit (Ghidelli
et al.,2018). Surface damage occurs, causing the increase of respiration, along with
other biochemical deterioration such as browning, the development of off-flavor, and
texture breakdown. Additionally, there may be some microbiological spoilage that
leads to decay of the flesh of the fruit reducing the shelf life, and also a possible
pathogenic contamination risk presented to the consumers. As fresh-cut Fruit are
being stored, there is even some further deterioration that takes Place on the cut.
Therefore, it would be beneficial for the processors to Retard the deterioration of
fruits, starting at the beginning of the process. In the presence of oxygen, browning
enzymes (e.g. polyphenyl Oxidase) reacts readily with its substrate (e.g. chlorogenic
acid) and Produces brown pigments in fruits. This process is called oxidation. When
fruits are oxidized, they turned brown, and may not be marketable. Therefore,
oxidation should be prevented immediately after the fruit is peeled or sliced, since it
occurs within Minutes after the introduction of oxygen to phenolic compounds by
Polyphenol oxidase. The cutting of the fruit should been done under controlled
condition to prevent enzymatic browning and extend the shelf life of fruits. Chitosan
is favored for its physico-chemical and biological Properties in food coating
formulation because it significantly improves the shelf life of antimicrobial, anti-
oxidant, gelling property, and it is also a health contributor of being functional fiber.

23
Chitosan is known to be safe and non-toxic, comparable to sugar, and even safer
than salt (contributor of being functional fiber).

Not only is chitosan known to display natural antimicrobial and anti-fungal properties,
chitosan film coating also possess excellent film-Forming properties with good
mechanical property and favorable selective gas permeability towards oxygen and
carbon dioxide and, which is favorable for a fruit coating, since it prevents anaerobic
respiration. From the study, it was concluded that chitosan coating would work well
on most fruits and vegetables (Wang and Hongxia 2018).

Materials for edible films and coatings

Edible films and coatings are used as packaging material to reduce the effect of
conventional non-biodegradable packaging material on the environment. Consumer
demand also shows an increasing trend towards renewable and eco-friendly
packaging Materials. Edible films are made by casting, and the extrusion process
and coating of the edible solution are done by dipping and spraying. The significant
difference is that in edible film, solid edible laminate is wrapped around the food
products. In contrast, the edible coating forming solution is applied to the food
product. These packaging materials should be edible, and they should have film and
coating forming capability. Polysaccharides, proteins, and lipids are such materials
and can form continuous films and coatings (Saklani et al. 2019). In the casting
process, edible film-forming content is dissolved in a solvent such as water or
ethanol. Plasticizers can be added to improve the fexibility of the film. A continuous
films casting and drawdown bar process is used at the commercial level. The
combination of polysaccharides and proteins is known as hydrocolloids.
Hydrocolloids are hydrophilic, and they are long-chain polymers. When they are
mixed with the solvent water, they can form a gel-like structure. These films and
coatings are transparent, whereas lipids are opaque. Polysaccharides can develop a
strong hydrogen bond with other active additives including coloring agents or
favoring agents. Hydrocolloid films and coatings have a good oxygen barrier, but
they have a poor water vapor barrier due to their hydrophilic nature. Due to their

24
hydrophobic nature, lipids exhibit good water barrier properties (Mohamed et al.
2020).

Ocimum sanctum (tulsi)

Ocimum sanctum L. is one such medicinal plant having numerous medicinal

properties.O. sanctum L. (syn O. tenuiflorum L., Mint family: Lamiaceae), commonly
known as “Holy Basil” in English and “Tulsi” in hindi and sanskrit, is a bushy plant
with a unique fragrance found in the semitropical and tropical regions of the world
(Naquvi JK et al.,2012).In ancient hindu scriptures, tulsi occupies the supreme
position among the herbs, so much so that it is referred to as “Mother.” The ancient
works of Padmapurana and tulsi Kavacham describes tulsi as a protector of life,
accompanying human beings from birth to death. The ancient sages or rishis
ensured its integration into daily life by incorporating it in religious ritual (Watson et
al., 2011). Tulsi has been used for thousands of years for its diverse healing
properties and is regarded in ayurveda as the “elixir of life” that promotes longevity..

(a) Krishna Tulsi (b) Rama Tulsi

Morphological types

Commonly, there are three types of tulsi, one with a purple-colored leaf or dark
variety, commonly known as the Shyama or Krishna tulsi and the second type with a
green-colored leaf or light variety known as Rama tulsi or Sri tulsi. Rama tulsi is
regularly used for worshiping and is more common of the three types. A third type,
commonly known as Vana tulsi (or forest Tulsi), is O. gratissimum (Naquvi et al.,
2012). It is mainly found in two main varieties black and Green, having a same
chemical composition and medicinal properties. This plant is known for its significant

25
aromatic odour due to the presence of Essential volatile oils. This volatile oil is
mainly composed of terpenes, phenols, aldehydes and is extracted from the seeds.
Other than the oil.the plant also contains glycosides, saponins, tannons and
alkaloids whereas the leaves contain ascorbic acid and carotene.

Different type Tulsi leaf of Ocimum Sanctum

Pharmacological uses and health benefit

In the ancient Ayurvedic text, the Charaka Samhita. tulsi has been documented to
be of Immense use in the treatment of headaches rhinitis, stomach disorders.
Inflammation, Heart diseases, various forms of poisoning and malaria.

Each part of the plant has proven to offer protection against various diseases, the
Aqueous and alcoholic extract from the leaves have various pharmacological
activities such anti-inflammatory, antipyretic analgesic, antasthmatic anti-emetic,
anti-diabetic Hepatoprotective, hypotensive, hypolipidemic, and antistress agents.
Further, distilation of the leaves yields oil of the plant which is known to possess
antibacterial, anti-oxidant, and anti-inflammatory properties and is used extensively
in the pharmaceutical industry mainly for skin cream preparations (Watson et al.
2011).

1. Antimicrobial activity: Tulsi is known to possess antimicrobial activity against

various bacteria, the most common being Candida albicans, Staphylococcus aureus,
Escherichia coli by its phytoconstituents isolated from various parts.

26
2. Immunomodulatory activity: Tulsi strengthens the immune response by
enhancing both cellular and humoral immunity by boosting the call mediated immune
Responsiveness and gamma amino butyric acid (GABA) pathways.

3. Antidiabetic activity: Oral administration of Ocimum sanctum extract led to a

Marked lowering of blood sugar in normal. It has an aldose reductase activity, which
May help in reducing the complications of diabetes such as cataract, retinopathy,
etc.

4. Antifungal activity: Tulsi extract has been effective against filamentous fungi
which Include Aspergillus niger, A. fumigatus. A. Aavus. Rhizopus stolonifera and
penicillum Digitatum The fungicidal activity is said to be due to the action of
secondary metabolites Which are present in tulsi including alkaloids, glycosides,
saponins, tannins ascorbic Acids eugenol and various other metabolites (Prakash et
al 2015).

Antioxidant Extracts from Medicinal Plants in Oral Health: A Clinical Trial

Perspective

The anti-gingivitic and anti-plaque effect of fluoridated dentifrice and 4% Ocimum

Sanctum extract was studied in a triple blinded randomized clinical trial (RCT)
among 14–15-Year-old school children, and reduction in dental plaque (p = 0.01)
and gingivitis (p = 0.001)Was observed maximum in 4% tulsi extracts in comparison
with the fluoridated dentifrice group (Nadar et al 2020). In triple blinded RCT, the
effect of phenolic mouth wash and Salvadora persica oral rinse was compared
among girls 18–22-year-old for six months and were found to be equally effective as
no statistically significant difference was observed in all the examination phases
between the mean gingival and plaque scores of two groups (Malik et al., 2021). The
effect of Eucalyptus globulus extract added as an ingredient in herbal product (tooth
and Gums tonic) was compared with chlorhexidine M gel in double-blind RCT and
showed a decrease in mean gingival and plaque value at different intervals. It was
observed to be equally effective in comparison to chlorohexidine with no statistically
significant difference (p = 0.001) (Sukhabogi et al 2017).

27
Alginate

Alginate is a naturally occurring anionic polymer typically obtained from brown

seaweed, and has been extensively investigated and used for many biomedical
applications, due to its biocompatibility, low toxicity, relatively low cost, and mild
gelation by addition of divalent cations such as Ca2+. Alginate hydrogels can be
prepared by various cross-linking methods, and their structural similarity to
extracellular matrices of living tissues allows wide applications in wound healing,
delivery of bioactive agents such as small chemical drugs and proteins, and cell
transplantation. Alginate wound dressings maintain a physiologically moist
microenvironment, minimize bacterial infection at the wound site, and facilitate
wound healing. Drug molecules, from small chemical drugs to macromolecular
proteins, can be released from alginate gels in a controlled manner, depending on
the cross-linker types and cross-linking methods. In addition, alginate gels can be
orally administrated or injected into the body in a minimally invasive manner, which
allows extensive applications in the pharmaceutical arena. Alginate gels are also
promising for cell transplantation in tissue engineering.

Alginate: general properties

Commercially available alginate is typically extracted from brown algae

(Phaeophyceae), including Laminaria hyperborea, Laminaria digitata, Laminaria
japonica, Ascophyllum nodosum, and Macrocystis pyrifera. By treatment with
aqueous alkali solutions, typically with NaOH. The extract is filtered, and either
sodium or calcium chloride is added to the filtrate in order to precipitate alginate.
This alginate salt can be transformed into alginic acid by treatment with dilute HCl.
After further purification and conversion, water-soluble sodium alginate power is
produced. On a dry weight basis, the alginate contents are 22–30% for Ascophyllum
nodosum and 25–44% for Laminaria digitata (Peteiro et al., 2018).

Bacterial biosynthesis may provide alginate with more defined chemical structures
and physical properties than can be obtained from seaweed-derived alginate.
Bacterial alginate can be produced from Azotobacter and Pseudomonas. The

28
pathway of alginate biosynthesis is generally divided into (i) synthesis of precursor
substrate, (ii) polymerization and cytoplasmic membrane transfer, (iii) periplasmic
transfer and modification, and (iv) export through the outer membrane.

The molecular structure of alginates is composed of unbranched, linear binary

copolymers of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues linked
by 1–4 glycosidic bonds. An algal alginate structure could be separated into three
fractions (three uronic acid blocks): These are homopolymeric regions of M and G
blocks, and Alternating MG blocks containing both polyuronic acid. The source of the
alginate affects the ratio of M and G residues, which have an impact on the physical
and chemical properties of the alginate, as well as the viscosity of the Coating
solution and thickness on the product. Alginates (i.e., sodium alginate, potassium
alginate, ammonium alginate, and calcium alginate) are monovalent salts of alginic
acid. Alginic acid and calcium alginate are insoluble in water while sodium alginate,
Potassium alginate, and ammonium alginate are water-soluble polymers. They have
a limited solubility at low pH values. The solubility of different types of Alginates in
numerous solvents and solutions were listed by Kimica Corporation (Kimica
Corporation 2018).

Alginate is widely used in various industries such as food, beverage, textile, printing,
And pharmaceutical as a thickening agent, stabilizer, emulsifier, chelating agent,
Encapsulation, swelling, a suspending agent, or used to form gels, films, and
membrane (Hay et.al. 2013). Sodium alginate is the most common salt of alginate

Green tea

29
The green tea is obtained from the tea plant Camellia sinensis (L.) Kuntze (Common
names: green tea extract, Chinese tea) which belongs to the family theaceae. It is a
widely used medicinal plant by the tribals throughout India, China and popular in
various indigenous system of medicine like ayurveda, Unani and homoeopathy etc.
Following the various traditional claims on utility of this plant in curing number of
diseases, Considerable efforts have been made by researchers to varify its utility
through scientific pharmacological screenings. In traditional Chinese and Indian
medicine, practitioners used green tea as a stimulant, diuretic (to promote the
excretion of Urine), astringent (to control bleeding and help heal wounds), and to
improve heart health. Other traditional uses of green tea Include treating flatulence
(gas), regulating body temperature and blood sugar, promoting digestion, and
improving mental Processes.

Green, tea derived from the leaves of the Camellia sinensis plant. Originally
cultivated in East Asia, this plant grows as Large as a shrub or tree. Today, Camellia
sinensis grows throughout Asia and parts of the Middle East and Africa. Green tea is
prepared from unfermented leaves, the Leaves of oolong tea are partially fermented,
and black tea is fully fermented (Chen and Liang, 2012).

Botanical evidence indicates that India and China were among the first countries to
cultivate tea. There are three main varieties of tea- green, black, and Oolong. The
difference between the teas is in their processing. Green tea is made from
unfermented leaves and reportedly contains the highest concentration of powerful
Antioxidants called polyphenols. Antioxidants are substances that scavenge free
radicals – damaging compounds in the Body that alter cells, tamper with DNA
(genetic material), and even cause cell death. Free radicals occur naturally in the
body, but environmental toxins (including ultraviolet rays from the sun, radiation,
cigarette smoke, and air pollution) also give rise to these damaging particles.
Scientists believe that free radicals contribute to the aging process as well as the
development of a number of health problems including Cancer and heart disease.

30
Essential oil

Essential oils as defined by the European pharmacopeia 7 th edition (Asbahani et al.,

2015) defines essential oils as “aromatic products with a mixture of compounds
derived from plant raw material, either separated by steam, dry distillation, or by a
suitable mechanical technique without heating.” Essential oil is separated from liquid
phase without changing its chemical composition by physical method. Herbal plants
are very important as most of the population depends on products of these plants
(essential oils). The products of these plants are used in food, cosmetic items and
medical field etc. (Kant and Kumar, 2021). Various extraction techniques are used
for essential oil extraction from several parts of medicinal plants, such as barks,
peels, leaves, buds, seeds, flowers etc. (Tongnuanchan and Benjakul, 2014). The
methods used to extract essential oil from these plants are; steam distillation (SD),
solvent-assisted extraction, hydro distillation (HD), ultrasonic-assisted extraction,
supercritical fluid extraction and solvent-free microwave extraction (Belhachat et al.,
2018). Conventional methods of essential oil extraction have lower yield and
efficiency and higher extraction time than non-conventional methods such as
microwave-assisted extraction and ultrasonic-assisted extraction. However, they are
still used for essential oil extraction nowadays. Non-conventional methods produce
higher yield, require low cost and have relatively short extraction time (Kusuma and
Mahfud, 2017, 2018).

Essential oils extracted from medicinal plants are also used for health benefits

Various studies have been carried out on the methods of oil extraction from aromatic
and medicinal plants. A hybrid solar distillery to extract oil from peppermint leaves
and eucalyptus was developed. A secondary biomass system was also attached to
meet energy demand during rainy season and night hours (Afzal et al., 2017). Then
they extracted essential oils from aromatic plants by solar steam distillation system
(SSDS) (Munir et al., 2014). Phineas Masango used SD to extract essential oil from
vegetable raw material. Results show that steam flow rate affects oil yield. Nazem et
al. applied hydro distillation to extract essential oils from Mentha (peppermint)
species and evaluated essential oil yield, phenolic content and essential oil

31
composition (Nazem et al., 2019). Razzaghi et al. designed and developed a
condenser to extract herbaceous oil. Condenser performance was evaluated in this
study by utilizing two refrigerants named R12 and R134a. Refrigerants were used to
separate oil and water from mentha leaves by vapour and air mixture (Razzaghi et
al., 2019). Sowbhagya et al. extracted oil from cumin seed by hydro distillation
process. Flaking process was used for size reduction of cumin seed in place of
conventional method. This study evaluated the effect of flaking on quality of oil and
yield. Kusuma et al. used solvent free microwave extraction (SFME) for basil oil
extraction and compared it with microwave hydro-distillation (MHD). Results showed
that SFME has higher yield and shorter extraction time than MHD (Kusuma and
Mahfud, 2017).

Essential oils are very important as they are used as anti-cancer agents, anti-viral
agents, anti-spasmodic effects, anti-bacterial agents, anti-diabetic activity, and anti-
lipid peroxidation etc.

Cinnamon oil

Cinnamon is a common spice that has been used for several centuries by different
Cultures around the world. It is obtained from different parts of a tropical evergreen
tree belonging to the genus cinnamomum. Various reports have dealt with the
numerous properties of cinnamon and its major components not only for human
health but also for agriculture applications. In this chapter, important aspects of trees
from the Cinnamomum genus, and their products, such as botany, pharmacology,
toxicology, and some end uses, with a special focus on the pesticidal potential for
agriculture and indoor Uses, are covered.

The genus Cinnamomum (Lauraceae) includes more than 250 aromatic evergreen
trees and Shrubs of up to 10–20 m, primarily distributed in Southeast Asia, China,
and Australia (Barceloux 2009). Investigations conducted at the beginning of the
1980s have shown that this genus has a center of diversity in south India. However,
although formerly thought to be a purely Asiatic genus, Cinnamomum has been
enriched with species such as phoebe, transferred from neotropical genera based

32
on studies and investiga-Tions carried out by taxonomists such as Kostermans. The
characteristics for the Cinnamomum species identification, leading the genus to
include not only the Asiatic species but also New World ones. A very detailed
Botanical characterization of different species of the genus Cinnamomum can be
found in The monograph on cinnamon and cassia written by (Ravindran et al.,2003).

Basil

Ocimum basilicum L., commonly referred to as basil, is a plant that belongs to the
Lamiaceae Family (PushpangadanP et al., 2012). It has an aromatic odor and is an
annual or perennial herb or shrub. Most of them are in the tropics and temperate
regions of the old hemisphere. Basil is distributed throughout the tropical Asia, the
northern Africa and America. Of these, China especially has abundant resources of
basil (Kadan et al.,2016). Basil has a fragrance and taste and is well-known as a
traditional Chinese medicine with homology of medicine and food. Basil has many
biological activities pharmacological applications, such as their anti-cancer activity, 5
antioxidant activity, anti-aging activity, immunity enhancement effect, hypolipidemic
and anti-atherosclerotic effects, antibacterial effect, treatment of diabetes mellitus
(Elansary et al.,2015). And so on. The abundant biological activities have been
attributed to the presence of multiple components, such as polysaccharides,
naphtha, steroids, flavone, coumarins, vitamins, and so on. Basil polysaccharides
are believed to be one of the most important active compounds of basil (S.V. Popov
et al., 2014). Substances derived from plant ingredients, especially essential oils,
have been extensively used in many industries including cosmetics, food and
pharmaceuticals (Da silva Gundel S, et al., 2018). In fresh form, basil leaf is often
used as a daily spice and food ingredient. In traditiona Medicine, thanks to the anti-
inflammatory, anti-oxidant and antimicrobial properties of the plant, Basil is also
used to promote digestion, stimulate respiratory circulation, relieve cold symptoms
and alleviate digestion issues (Izadiyan P et al ,2016).

33
Materials and
Methods

34
Selection of samples and prepare it for coating
Fresh fruits were collected from local market of Lucknow and sorted on the basis of
shape, (round fruits), and absence of visual defects. Washed with distilled water to
remove adhering dust, then heat treatment was giving to each fruit by dipping the
fruits in hot water bath at 65°C for 90 seconds. Now put them on the blotting paper
from removing excess water. After that, put the fruits in laminar for 15 minutes under
UV light for sterilization. When the fruit is completely dried then each fruit was
coated by swabbing method with different type of coatings.

Plastic wares
The plastic wares such as microfuge tubes, centrifuge tube, micro tips, sterile
pipettes and filters were purchased from Greiner (Germany), Corning (U.S.A), Nunc
(Denmark). Plastic wares used in experiments were sterilized by autoclaving.

Glassware’s
The glassware‘s used in the experiment were purchased from borosil (India). The
glassware‘s used were sterilized in hot air oven at 180°C for at least 2 hours.
• Test tubes, boiling tubes, measuring cylinders, conical flasks.
• Beaker
• Pipette
• Petri plates
• Disposable syringes
• Slides and coverslip
Equipment’s
• Magnetic stirrer-(Remi)
• Spectrophotometer (Labtronics) 38 34

35
• Centrifuges (Remi)
• Vortex shaker (KC)
• Deep freezer (-20°C)
• Oven (Science tech India)
• Autoclave (Science tech India)
• Laminar (Science tech India)

Chemicals
• Acetic acid

• Methanol

• Chitosan

• Ethanol

• Sodium tripolyphosphate (TPP)

• Sodium hydroxide

• Anthrone

• Sulfuric acid (H₂SO₄)

• Acetone

• Potassium acetate

Preparation of Chitosan
Chitosan was dissolved at 0.5% (w/v) aqueous solutions of acetic acid at 1% (w/v).
The pH into of solution was adjusted to 6.0 using 1% NaOH. After coating, fruits
were air-dried.

36
1. Hot water Treatments alone
This group of amla fruits was subjected to hot water Treatments for surface cleaning.
Amla were dipped in hot water which was heated up to 65℃ c for 1 min for reduce
the microflora. Subsequently they were allowed to remove water by placing them
blotting sheet. After that they were packed in a poly sheet with small pores for air
exchange.
Preparation of Tulsi leaf extract
10% fresh leaves of O. sanctum were collected and washed three times with distilled
water to remove dust particles. The leaves were dried on blotting paper to remove
the excess water then chopped them and 35ml distilled water was added by using
36 measuring cylinder and boiled at 65°C for 15 minute to get leaf extract. After
boiling, the mixture was cooled and filtered with Whatman paper No. 1. Filtrate was
collected and was stored at 4°C in refrigerator.

Preparation of Green Tea leaf extract

10% fresh leaves of green tea were collected and washed three times with distilled
water to remove dust particles. The leaves were dried on blotting paper to remove
the excess water then chopped them and 35ml distilled water was added by using
measuring cylinder and boiled at 65°C for 15 minute to get leaf extract. After boiling,
the mixture was cooled and filtered with Whatman paper No. 1. Filtrate was collected
and was stored at 4°C in refrigerator.

Preparation of Treatments coating solutions

All the amla fruits were washed in running water, air dried, categorized into ten
groups which are treated with different solutions like control (without Treatments),
chitosan, tulsi extract, green tea extract. The fruits (except control) were pretreated
with hot water (65℃ for 90 sec). Coating of fruits with naturally derived compounds
as chitosan with plant extract forming nanoparticles can reduce the post-harvest loss
of fresh fruits and vegetables by preventing it from contamination with microbes
especially fungi, which is main issue for post-harvest loss of fruits. Each group fruits
were left for 20 days and after that the disease incidence and quality of fruits were
evaluated. The disease incidence was calculated as percent inhibition on the basis

37
of area infected as observed visibly in Treatments as compared to control [(Value of
Control - Value of Treated) ×100/ Value of Control].

Biochemical tests
Phytochemical analysis of the plant extracts was undertaken using standard
qualitative methods has been performed as described by various authors. The plant
extracts were screened for the presence of biologically active compounds such as
alkaloids, flavonoids, carbohydrates, proteins, phenolics, tannins and saponins.
For the quantitative estimation of primary metabolites different protocols were used.
Coating of fruits homogenize were prepared for estimation. Each treatment had
three replicates. Percentage of decayed and infected fruits was calculated by visual
infection in infected and uninfected fruits (Fallik et al.,, 2000). The fruits were
weighed. Overnight grown culture of yeast and lactobacillus culture was centrifuged
at 10,000 rpm for 5 minutes. The pellet was dissolved in 50 ml of distilled water and
then suspended in to the bottom of centrifuge tube and the supernatant is discarded.
In laminar, under sterilized condition, the suspension is dissolved in 10 ml of
sterilized distilled water. Each replicate of tomato is dipped in the bacterial
suspension and coated. Each tomato were kept and dried in the laminar and dried.
After drying replicates, they are placed in beaker containing cotton bed sheets with
few drops of Streptomycin and penicillin. The beaker were packed with autoclaved
silver foil and marked with required designation. The experiment was observed daily
for 2 weeks.

Estimation of Carbohydrate:

Preparation of Reagents:-
 Anthrone's Reagent- 1gm of anthrone was dissolved and volume
raised upto 500ml of 85% H2SO4 (chilled condition should be
maintained).
 Glucose Solution-

38
  Stock solution: 50mg of glucose dissolved and raised upto 50ml with
distilled H2O(1mg/ml).
 Standard Solution: 10ml of stock solution was raised upto 100ml with
distilled H2O (100μg/l).
Procedure:-
1. 7 tubes were taken and arranged in a series from 1-7.
2. Aliquots of 0.05ml glucose solution were taken to get a corresponding range
up to 0.3ml glucose solution.
3. The volume in each tube was raised up to 1ml with distilled water.
4. Then 6ml of anthrones's reagent was added in each of the test tube.
5. Tubes were kept in hot water bath for 10mins then cooled and optical
densities were measured at 620nm.

Estimation of Protein:

Preparation of Reagents:-
1:- Biuret reagent- 750 mg of CuSO4 and 3 gm of Rochette salt (Sodium Potassium
tartarate) was dissolved in 250ml of H2O. 150 ml of 10% NaOH was added to this
mixture and volume was raised to 500 ml.
2:-10% NaOH- 25 gm of NaOH was dissolved in 250 ml of H2O.
3:- 0.1 N NaOH:- 1gm NaOH was dissolved in 250 ml of H2O.
Standard protein solution- 1gm protein was dissolved in 100 ml of 0.1N NaOH
(10mg/ml).
Procedure:-
1. Arranged test tubes in a series from 1 to 7.
2. Aliquots of 0.1 ml protein solution were taken to get a corresponding range
upto 0.6 ml of protein.
3. The volume in each tube was raised up to 1 ml by 0.1N NaOH.
4. Then 4ml of Biuret reagent was added in each of the test tube which was
kept at 37°C for 30 mins.
5. O.D measured at 540nm by spectrophotometer.

39
Estimation of chlorophyll:
The estimation of chlorophyll was done by using dimethyl sulphoxide (DMSO)
extraction procedure. Plant samples were collected at random and were chopped
into fine pieces. 50 mg sample from these chopped material were added in
replicated tubes each containing 10 ml dimethyl sulphoxide (DMSO). The tubes
containing plant pieces and DMSO were incubated at 650C for 3 h in an oven by
providing gentle shake twice. After complete extraction, clear supernatants were
used for measuring the absorbance with the help of a spectrophotometer against
DMSO blank. The absorbance was recorded at wavelength of 663 nm for chlorophyll
‗a and 645 nm for chlorophyll ‗b. The optical density was measured and the
chlorophyll contents in the original extract was estimated using the formula (Hiscox
and Isralesham, 1979).
Total chlorophyll (mg/L) = 20.20A645 + 08.02 A663
Chlorophyll ‗a‘ (mg/L) = 12.70A663 – 02.69 A645
Chlorophyll ‗b‘ (mg/L) = 22.90A645 – 04.68 A663
These can be converted to chlorophyll content in mg/g dry weight as follows:
Chlorophyll 'a' (mg/g)
V= (12.3×A663 – 0.86×A645) 1000 × W

Chlorophyll 'b' (mg/g)

V= (19.3×A645 – 3.6×A663) 1000 × W
Total Chlorophyll = a + b
Here,
A = Absorbance or O.D. (Optical density).
V = Final volume of chlorophyll extract in 80% acetone.
W = Dry weight of plant material.

Estimation of Total Phenols:

Phenolics

40
A 35% of saturated sodium carbonate solution was prepared by dissolving 35 gm of
anhydrous Na2CO3 in 100ml of pure water. Heat and stirring were added overnight
in order to facilitate complete dissociation. Additional water was added to prevent
super saturation. Once solution was prepared it was left in low heat during analysis
to prevent precipitation as solution was used. Analysis was performed by adding
3.5ml of deionized water, 50of sample extract and folin reagent and 300μl of sodium
carbonate to cuvette. The reaction was left for 15 min. and then the absorbance was
measured in 730nm using a UV/VIS spectrophotometer. A standard curve was
fashioned using Tannic acid at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0mg/mL
diluted in ethanol. Total phenolic concentration was expressed as mg of tannic acid
equivalent via the standard curve.
Total phenols in samples were determined by the Folin– Ciocalteu method given by
(McDonald et al., 2001). All extracts were dissolved in 1 mg/ml conc. The assay
mixture (5 ml) contained 0.5ml of extract or standard (Gallic acid), 2ml aqueous (1N)
Na2CO3 and 2.5ml of (2 N) Folin–Ciocalteu reagent (Sigma-Aldrich, Germany).
Samples were incubated at room temperature for 15 minutes and absorbance at 765
nm was measured by using spectrophotometer. The standard curve was prepared
using 1, 10,100 and 200 mg/ml solution of Gallic acid in methanol. The blank
contained 500 μl of double distilled water, Na2CO3 and the Folin–Ciocalteu reagent.
Total phenol values are expressed in terms of Gallic acid equivalent (mg/g of dry
mass).

Estimation of % Total Sugar:

The phenol sulphuric acid method to estimate total carbohydrates is described
below.
In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This forms a
green colored product with phenol and has absorption maximum at 490 nm.
Reagents-
Phenol 5%: Redistilled (reagent grade) phenol (50 g) dissolved in water and diluted
to one liter.
Sulphuric acid 96% reagent grade.

41
Standard glucose: Stock—100 mg in 100 mL of water. Working standard—10 ml of
stock diluted to 100 ml with distilled water.
Procedure:-
1. followed the steps 1 to 4 as given in anthrone method for sample preparation.
2. 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard into a series of test tubes.
3. 0.1 and 0.2 mL of the sample solution in two separate test tubes. Make up the
volume in each tube to 1 mL with water.
4. The blank was with 1 mL of water.
5. Added 1 mL of phenol solution to each tube.
6. Added 5 mL of 96% sulphuric acid to each tube and shake well.
7. After 10 min shacked the contents in the tubes and place in water bath at 25–
30°C for 20 min.
8. The color was at 490 nm.
9. Calculated the amount of total carbohydrate present in the sample solution using
the Standard graph.
Calculation:
Absorbance corresponds to 0.1 mL of the test = x mg of glucose
100 mL of the sample solution contains =0.1
x × 100 mg of glucose
= % of total carbohydrate present.
Percent over control was calculated as
= {[Value in Treated- Value in Control] / Value in Control } x 100

Phytochemical Evaluation:

Estimation of Flavonoids:
The total flavonoids content of Amla fruit extract was determined use a colorimetric
method. A 0.5ml of aliquot of appropriately diluted sample solution was mixed with
2mL of distilled water and subsequently with 0.15mL of a 5% NaNO2 solution. After
6 minutes, 0.15Ml of a 10% AlCL3 solution was added and allowed to stand for 6
minutes, then 2mL of 4% NaOH solution was added to the mixture. Immediately,

42
water was added to bring the final volume to 5Ml, then the mixture was thoroughly
mixed and allowed to stand for another 15 minutes. Absorbance of the mixture was
determined at 510nm. Rutin was used as a standard compound for the identification
of total flavonoids. All values were expresses as milligrams if ruti equiv. per 100gm
of fresh peel.
Quantitative: The quantitative estimation was performed spectrophotometrically by
the aluminum chloride method based on the formation of complex flavonoid-
aluminum. All sample extracts were dissolved in 1 mg/ml conc. And 0.5 ml of each
sample was mixed with 1.5 ml of methanol, 0.1 ml of 10 % Aluminum Chloride, 0.1
ml of 1M Potassium acetate and 2.8 ml of distilled water. It remained at room
temperature for 30 minutes. The absorbance of the reaction mixture was measured
at 415 nm. The calibration curve was prepared by preparing quercetin solutions at
concentrations 12.5 to 100 μg/ml in methanol.

Estimation of SOD (superoxide dismutase):

The determination of SOD activity was based on the well-known spectrophotometric
assay introduced by McCord and Fridovich (1969) J.Biol.
Principle-
The principle of SOD is based on the ability of O 2 to interact with NBT (Nitroblue
tetrazolium) reducing the yellow tetrazolium within the gel to a blue precipitate.
Areas where SOD is active develop a clear area (achromatic bands) competing with
NBT for the O2.
Reagents-
Methionine – 100mM
NBT – 1mM
EDTA – 1mM
Enzyme extract – 100 microliter
Po4 buffer (Ph=7.8) – 50mM
Riboflavin - 1mM
Procedure:-
1. weigh 0.05gm fruit sample and homogenized in phosphate buffer (Ph=7.8).

43
 2. centrifuge it at 10,000 rpm for 10min.
3. take 100 microliter of supernatant.
4. Put it in light for (20-25 minutes).
5. O.d were measured at 560nm at 5 and 15 minutes.
Calculation:
Concentration = (v-1/Vv)*df
Where,
V= absorbance at 15 minutes
v= absorbance at 5 minutes
df= dilution factor

Catalase (CAT)
The activity of catalase was determined according to the method of Aebi (1983).
Principle-
Catalase catalyzes the decomposition of H2O2 to give H2O and O2. Catalase
2H2O2 2H2O + O2 Catalase activity can be measured by following either the
decomposition of H2O2 or the liberation of O2. The method of choice for biological
material is the UV-spectrophotometric method. In the ultraviolet range, H2O2 shows
a continual increase in absorption with decreasing wavelength. The decomposition
of H2O2 can be followed directly by the decrease in extinction per unit time at 240
nm. The difference in extinction per unit time is a measure of catalase activity.
Reagents-
Phosphate buffer – 100 mM, pH 7.0
Hydrogen peroxide – 150 mM
Procedure:-
The rate of decomposition of H2O2 was followed by decrease in absorbance at 240
nm in a reaction mixture containing 1.5 ml phosphate buffer, 1.2 ml of hydrogen
peroxide and 300 μl of enzyme extract.
Calculations:

44
One unit of the enzyme activity is calculated as the amount of enzyme required to
liberate half the peroxide oxygen from H2O2 and calculated from the following
equation:
Unit activity (Units/min/gFW) = Change in abs./min x Total volume (ml)
Ext. coefficient X Vol. of sample taken (ml)
Where, Extinction coefficient = 6.93 x 10-3 mM-1cm-1
Specific activity (mol UA/mg protein) = Unit Activity (units/min/gFW)
Protein content (mg/g FW)

45
 Results
And
Discussion

46
Results

47
Conclusion

48
Conclusion

49
Reference

50
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