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1 INTRODUCTION 1.1 Background Materials used for the packing of foods and
dry goods today consists of a variety of petroleum-derived plastic polymer,
metals, glass, paper, and board, or combinations. Among those packing
materials, plastic was accessible to be the best due to its long shelf life properties.

Thus, its use is also increasing every day by day. Nevertheless, plastics cannot be
degraded by natural processes in a short period of time; so, they are left as
plastic surplus, affecting environmental problems. Methods normally used to
terminate other types of waste such as burning and burying are not good for
plastic demolition.

In response to those problems related to plastic waste, there has been

considerable interest in the development and synthesis of degradable bioplastic
plastics (Ezeoha & Ezenwanne, 2013). Packaging plays an important role in recent
years as over 67 million tons of packaging waste which resulting in environmental
concerns. That packaging had many different processing, for instance, the use of
additives for example fillers and plasticizers to produce polymers as packaging
applications.

Environmental, economic furthermore safety challenges have triggered many

scientists to investigate degradable bioplastic polymers as a substitute for
petrochemical-based polymers. This degradable polymer or bio-plastics shows
that more eco-friendly environment than the conventional plastics which lead to
environmental issues. An option was proposed for low cost and the renewable
substrate so that it would be used in agriculture waste (AW).

Besides to that of performance and plentiful sources, starch from agriculture

waste is the solution for an alternative. Starch as the biodegradable polymer is
proper to use it as a material for the production of bioplastics because of its low
cost (Maulida, Kartika, Harahap, & Ginting, 2018). The word polymer is derived
from the Greek, poly, and meros meaning many and parts respectively.

Scientists also use the word macromolecule meaning huge molecule instead of
polymer. The long chains of atoms that comprise polymers and the bonds within
and between the chains determine plastics ?properties. Polymers only cannot be
plastics. Plastics are polymers or macromolecules which have been modified with
additives and mechanically strong plastic structure that approve a dimensionally
stable form.

In the last 60 years, synthetic polymers have practiced advanced growth,

constituting an important area in polymer science. Biodegradable polymers,
obviously from renewable resources, are being inspected for replacement of
synthetic plastics. The most common ASTM description of biodegradable
polymers is states that a degradable polymer is one in which degradation results
from the action of naturally happening microorganisms such as bacteria, fungi,
and algae. Biodegradable polymers may be transformed back into carbon dioxide
after they can be used.

With this ability, polymers which produced by renewable resources may create a
“carbon neutral” life cycle of in which the total amount of carbon dioxide
unconfined into the environment leftovers relatively constant. 1.2 Statement of
the Problem Researches that have done over the past few years showed that
degradable bioplastic which synthesized from starch (MSK) alone is thermally
unstable and is not mechanically strong enough due to the lack of reinforcing
materials and hence the produced bioplastic could not give any longer service.

The other issue is the increase in demand for packaging materials. In order to
improve the thermo-mechanical properties of the film in addition to extending its
shelf life and service, natural pottery clay is the best solution to overcome such
shortcomings (Maulida et al., 2018).

The unseen protection given by reinforcing natural pottery clay and plasticizer
can result in materials, which lead to new products for even more demanding
situations. Packaging plays a significant part in recent years as over 67 million
tonnes of packaging waste accumulation which results in a big environmental
concern (Obasi, Egeolu, & Oparaji, 2015).

The clay layers also induce changes in the molecular mobility of the polymer
matrix, which may enhance its thermal and mechanical properties as well as
oxidation stability, among other advantages (Alvarez, n.d.). 1.2.1 Justification The
use of starch in the manufacturing of degradable bioplastics will reduce the levels
of environmental pollution since it takes only 0.8

metric tons of carbon dioxide to create bio-plastics which is 3.2 metric tons less
than normal plastics (Moore C.J, 2014). Bioplastics which are biodegradable and
can be made from starch have a potential solution to the problem,
environmentally and economically. The depletion of petroleum resources coupled
with an awareness of global environmental problems provides the alternatives for
new green materials that are compatible with the environment and their
development is independent of petroleum-based resources.

There are a number of alternatives by which one can avoid the impact of waste
plastic on the environment and can reduce the lack of packaging. Among these
developments of natural films (agricultural and industrial by-product), reinforced
biodegradable plastic promotes the use of environmentally friendly materials
(Ave´rous et al., 2001). 1.3 Objectives of the Research 1.3.1

General Objective The general objective is to develop and characterize

degradable bioplastic from mango seed kernel starch reinforced with micro-
pottery clay as a filler. 1.3.2 Specific objectives Ø To extract and characterize
starch from mango seed kernels Ø To evaluate the parameter effects of time and
mass liquor ratio on the yield of starch Ø To develop and characterize degradable
film reinforced with micro-pottery clay Ø To investigate individual and interaction
effects of pottery clay and glycerol concentration on the films overall properties.

Ø To determine and investigate the films physico-mechanical properties Ø To

analyze the thermal properties of the films using thermogravimetry analysis 1.4
Scope of the Study This research focusses on the synthesis of degradable
bioplastic through the extraction of starch from the mango seed kernel. This
includes the collection of mango seed kernels, extraction, production of the
degradable bioplastic, testing the properties, gathering, and analysis of data and
finally, arriving at the conclusions. This was taken three weeks to finish the
extraction of starch since there are parts where drying is desired.
The experimentation has done up to laboratory scale. The materials used in this
study were locally available. Mango seed kernel is the major raw material used in
the study, can be collected in Bahirdar City local juice traders. These mango seed
kernels are the by-product of the mango juice house.

The experimental variations involved in this study are types of solvent,

plasticizers, fillers, ratio of a material to a reagent, Concentration and molar ratio
of reagents. 1.5 Significance of the Study Successful achievement of the research
will able to play a great role by providing a remedy for the bottlenecked
problems of environmental pollution due to the discharge of the waste plastics.

Besides to that it also reduces packaging problems of food with prolonged shelf
life due to reinforcement with micro-pottery clay. With this research, the
conventional petroleum-based commercial plastics will soon be replaced by
these degradable bioplastics made from mango seed kernel starch. An advantage
of this is that they will not fill up the landfills because they are biodegradable and
just for months, disposed of bioplastics are completely gone, unlike petroleum-
based plastics which takes about many centuries.

This research will also be significant to the whole scientific community since it
would provide added information about how to make a good, environmentally-
friendly, inexpensive and toxic-free biolistic from mango wastes. 2 LITERATURE
REVIEW 2.1 Mango Seed Kernel Mangifera indica (Mango) it is a insistent crop of
the family Anacardiaceae. It is grownup in the whole tropical and sub-tropical
areas of the world.

Its fruit is oval -shaped with smooth, leathery skin and the color can be ranges
from light or dark green to pure yellow when it is ripe. Theseed kernel of the
mango fruit can be used as fresh as a dessert or can be processed into drinkable
juices, jams and other varieties of products despite the fact, the seeds are
discarded which frequently causes a critical environmental pollution (Journal,
Science, & Vol, 2017).

According to Maulida eta'l (2018) studies on the title of 'Utilization of waste

mango seed kernel as a source of starch for the development of films reinforced
with micro particle clay using glycerol as plasticizer. Use of organic waste like
mango seeds for the development of starch-based film can help diminish the
environmental critical issues that are mainly caused by conventional petroleum
basednplastics. Best films an be found by improving their properties with the
addition of micro pottery clays. Table 2.1

Percent distribution of mango fruit parts Components Percentage Peels 15-20

Pulp 45-65 Seed Coat 10-15 Kernel 10-20 Source: central food technological
research institute, 1985 Mango seed kernel is a good source of starch and fat. It
has been reported that about 75 % of the fruits are knocked off, right from the
flowering stage till ripening.

The losses, however, it can be diminished to a great extent by utilizing the

dropped fruits. Fortunately, mango is one of the few fruits, which can be utilized
in all stages of maturity. The aim of this work is to study the properties of films
from cultivation waste such as mango seed kernel starch.

The effect of filler and plasticizer on this film is also examined. Mango seed kernel
(MSK) is a worthy source of starch (58-80%) (Diarra, 2014). According to some
studies, the yield of mango seeds is about 21% of starch. Amylase treatments
prove that the extracted product from mango seed is starch (Saadany, 1980).
Table 2.2

Chemical composition of mango kernel, on a dry basis Components Amount

Starch 58-80% Reducing sugars 2.9% Proteins 5.7% Pectin 0.8% Fat 9.3% Tannins
1.1% Moisture 15.2% 2.1.1 Native starch and It's Composition Starch is a
polysaccharide which mainly consisting of two fractions such as amylose (10-
20%) and amylopectin (80-90%).

Amylose forms a colloidal suspension in water whereas amylopectin is insoluble

(Sonthalia, 2015). It is a natural polymer made up of hundreds of thousands of
glucose units connected together by glycosidic bonds. The molecular formula of
starch is (C6H10O5)n. It is the most common carbohydrate in human diets and is
contained in large amounts in such as yam, cassava, wheat, corn, rice, potatoes,
mango, mushroom, etc.

Starches can be used for industrial packaging applications, adhesives, non-food

fillers, textile stiffening agents are extracted for edible plant sources regardless of
their final-use (Alobi, Sunday, Magu, Oloko, & Nyong, 2017). Several studies have
been carried out on starch-based films obtained by melt processing or casting
from a solution or gel with the addition of a plasticizer.

The addition of water or other plasticizers such as glycerol considerably improves

mechanical properties. Even so, starch films have poor mechanical properties
when compared to those of synthetic polymers. This is due to their hydrophilic
nature and thus their sensitivity to moisture content, a factor that is difficult to
control.

Starch is made of long chains of glucose molecules united. Strictly, it contains

two polymers: amylose, which is straight-chained, and amylopectin, which is
branched. When starch is dried from an aqueous solution it forms a film as an
outcome of hydrogen bonding between the chains. However, the amylopectin
hinders the formation of the film (Sonthalia & Sikdar, 2015).

The physicochemical properties of starch differ from source to source of raw

material (Ginting, Hasibuan, Lubis, Iqbal, & Dicky, 2016). Starch composition
mainly consists of branched and linear chains of glucose molecules, namely
amylopectin and amylose, respectively. Figure 2.1 Structures of starch amylose (A)
& amylopectin (B) (Ginting et al.,

2016) However, the wide application of starch film is limited by its mechanical
properties and efficient barrier against low polarity compounds. In order to
overcome its shortcomings, blend the starch with different reinforcing additives
to improve its thermo-mechanical characteristics. Higher starch water content can
affect a shorter starch saving time because it would faster be contaminated by
the microorganisms.

The presence of endogenous lipids in starch may have a positive effect on the
swelling of individual granules by repelling the water (Ginting et al., 2016). Figure
2.2 ?-1, 4 and ?-1, 6 glycosidic bonds of starch (Ginting et al., 2016) 2.1.2 Physico-
chemical properties of starch The length of the ?-glucan chains, amylose-
amylopectin ratio and branching degree of amylopectin define the size, structure
and particular utility of starch granules in each plant species.

Other characteristics associated with the granule such as form, surface type, and
phosphate groups influence the starch's properties and uses (Alcázar-alay,
Angela, & Meireles, 2015). Starch exhibits some similar properties as polymers
when subjected to hydrolysis. There exist two types of molecules in starch
explicitly unbranched molecules which consist of glucose and branched
molecules which consist of amylose and amylopectin.

The shapes, as well as the size of the granules of the starch, are important factors
to be considered in the course of processing concerning their properties. For
example, mango seeds, potato, and cassava starch have low protein content, but
high phosphate content as compared to wheat starch and according to a recent
study. The length of the ?-glucan chains, amylose-amylopectin ratio and
branching degree of amylopectin define the size, structure and particular utility of
starch granules in each plant species. (Smith, 2001). 2.1.3

Starch functional properties i) Gelatinization and retrogradation properties of

starch: When starch heated in the presence of excess water, undergoes a
transition phase known as gelatinization, and there are characteristic temperature
intervals for gelatinization corresponding to each of the starch species.
Gelatinization happens when water disseminates into the granule, which then
swells considerably due to the hydration of the amorphous phase causing loss of
crystallinity and molecular order (Alcázar-alay et al., 2015).

Gelatinization happens initially in the amorphous region, which favored by the

weak hydrogen bonds present in this region. The process then extends to the
crystalline region. Amylose presence reduces the fusion point in the crystalline
area and the amount of energy necessary to initiate the gelatinization process
(Alcázar-alay et al.,

2015). Gelatinization is necessary for particular processes, e.g., textile and

hydrolyzed starch industries. Gelatinization affects the rheological properties and
viscosity of the paste, making the starch granule more accessible to enzymatic
action. When starch granules swell and its components are in solution, the
medium properties change from a simple starch granules suspension to a starch
paste.

Amylose and amylopectin separate phases are occurred because of

thermodynamic immiscibility. Actually, starch is typically in combination with
other polymeric constituents, such as proteins and other polysaccharides,
forming different phases. Many processes cause gelatinization of granules or the
manufacturing of products from raw materials based on starch, especially cereals.

Gelatinization progress along the granule is determined by the physicochemical

characteristics of the starch, the presence of other constituents, the availability of
water and process parameters applied (i.e., temperature, time and mechanical
energy) (Alcázar-alay et al., 2015). Solubilized starch polymers and the remaining
insoluble granular fragments have a tendency to reassociate after heating. This
reassociation is called retrogradation.

One definition of starch retrogradation is also followed (1): Starch retrogradation

is a process which occurs when starch chains start to reassociate in an ordered
structure. In its initial phases, two or more starch chains may form a simple
interval point, which then may develop, into more extensively ordered areas.
Ultimately, under favorable conditions, a crystalline order appears.
Retrogradation is especially evident when amylose-containing starches are
cooled.

Upon cooling, less energy is available to keep the solubilized starch molecules
apart. Retrogradation results in the formation of crystalline aggregates and a
gelled like texture. Although amylopectin can retrograde upon cooling, linear
amylose molecules have a greater tendency to reassociate and form hydrogen
bonds than the larger, “tumbleweed-like” amylopectin molecules. As the
retrogradation process occurs, the starch paste becomes increasingly opaque and
forms a cuttable gel.

With time, this gel becomes rubbery and has a tendency to release water (Atwell,
1999). The molecular interaction produced after gelatinization and cooling of the
paste is also known as retrogradation. During retrogradation, amylose molecules
associate with other glucose units to form a double helix, while amylopectin
molecules re-crystallize through the association of its small chains.

Initially, the amylose content exercises a strong influence over the retrogradation
process; a large amount of amylose is associated with a strong tendency for
retrogradation. Starch retrogradation is intensified by repeated freezing and
defrosting of paste. In general, retrogradation in starch pastes, as well as foods
containing starch, is unfavorable in terms of food quality, causing syneresis of
gels or hardness.

Currently, retrograded starch is classified as a form of resistant starch (RS) and is

used in industry for different purposes described in other sections of this review.
Starch granules when heating in excess water above their gelatinization
temperature, the linear structure cross-bond and increases starch stability. It
strengthens the hydrogen bonding of starch to-starch chain with covalence
chemical bonding.

During cooling (storage) starch pastes may become cloudy and eventually
deposit an insoluble white precipitate. This is caused by the recrystallization of
starch molecules; initially, the amylose forms double-helical chain segments
followed by helix aggregation (Alcázar-alay et al., 2015). The starch
transformation at different stages of processing is as shown in Figure 2.5.
Amylose is considered primarily responsible for the short-term retrogradation
process because of the fact that the dissolved amylose molecules reorient in
parallel alignment. The long-term retrogradation is represented by the slow
recrystallization of the external branches of amylopectin. The recrystallized
amylopectin in the retrograded gel can be melted at 55?C, whereas for the
recrystallized amylose the melting temperature rises to 130?C.

The rate and the extent of retrogradation increase with an increase in amylose
content (Atwell, 1999). Figure 2.3 Gelatinization transformation stages of starch
(Atwell, 1999) As it can be seen in the above figure the first stage (A) represents
the raw mango seed starch granules that mainly consists of the linear stracture
(Amylose) and the branched chain (Amylopectin). The second stage (B) represents
startup heating of starch solution.

During heating, water is primarily absorbed in the amorphous regions of amylose

due to its low numerous hydrogen bonding. Here the intra and intermolecular
bonds of the starch start to break and allowing H-bonding sites to engage more
water so that granules become more swelled. The third transformation stage (C)
stands for the gradual infiltration of water via the amorphous regions to the
tightly bonded areas of amylopectin.

At ambient conditions, they do not allow water to enter rather than, they settled
down. Continuous heating causes such regions start to diffuse, the amylose part
completely dissolved and the size of crystalline region becomes decrease. The
final stage (D) stands for the amylose part completed leached out into the
surrounding water and gelation is happened upon cooling.

Researchers have tried to accurately defined starch gelatinization for many years.
Some claim that gelatinization starts when granular birefringence is lost; others
believe it begins when an increase in viscosity occurs. Words such as “cooking,”
“solubilizing,” and “thickening,” although not exactly accurate from a starch
chemist's point of view, are quite often used to characterize the gelatinization
process.

On the basis of the results of this survey, the following definition of gelatinization
was proposed: Starch gelatinization is the breakdown or the disruption of
molecular orders within the starch granule manifested in irreversible changes in
properties like granular swelling, native crystalline melting, loss of birefringence,
and starch solubilization (Atwell, 1999).
Granular starch is essentially insoluble in cold water, and even when it is added to
water at room temperature, little happens until heat is applied. A combination of
heat and water, however, causes uncooked granules to undertake unique and
irreversible changes, the most dramatic of which are the disruption of the semi-
crystalline structure, as evidenced by a loss of birefringence; and an increase in
granule size, although not all granules within a given population swell at the
same rate or to the same extent. As these changes are taking place, there is an
attendant increase in the viscosity of the medium in which the starch is heated.

When a majority of the granules has undergone this progression, the starch is
considered“pasted” or “cooked-out.” In most cases, it is this pasting (i.e.
Viscosity-forming) ability that makes starch so well-designed as a food
ingredient. The temperature at which starch begins to undergo these changes is
called gelatinization temperature.

In actuality, because not all the granules of a given starch begin to gelatinize at
the exact same temperature, the gelatinization temperature is more appropriately
defined as a relatively narrow temperature range than one specific temperature.
Temperatures also vary depending on the type and source of the starch. From a
mechanistic perspective, heating of starch in water causes disruption of the
hydrogen (H) bonds between polymer chains, thereby weakening the granule.

It is believed that the initial swelling takes place in the amorphous sections of the
granule where hydrogen bonds are less numerous and the polymers are more
susceptible to dissolution. As the structure begins to weaken, the granule absorbs
water and swells. Because not all the granules gelatinize simultaneously, different
degrees of structural disruption and swelling exist.

ii) Pasting properties of starches: As heating continues and more and more
granules become swollen, the viscosity of the medium increases. The paste
viscosity reaches a maximum when the largest percentage of swollen, intact
granules are present; this is referred to as the peak viscosity. At this point, the
starch is considered to be fully pasted.

For native starches, continued heating eventually results in a decrease in viscosity

as granules dissolve and polymers are solubilized. The following proposed
definition of the pasting process differentiates it from gelatinization (1): Pasting is
the phenomenon following gelatinization in the dissolution of starch. It involves
granular swelling, exudation of molecular components from the granule, and
eventually, total disruption of the granules.

It is important to note, however, that pasting is not exactly separate from

gelatinization, but rather an overlapping occurrence perhaps best described as a
continuation of gelatinization. Although there is no definitive point at which
gelatinization ends and pasting begins, pasting is usually related to the
development of viscosity. However, this link between gelatinization and pasting
often results in these two terms being used interchangeably to describe the same
process.

iii) The viscosity of starch pastes: Further heating in water above gelatinization
temperature causes greater swelling of starch granules. The swollen granules
gradually disperse and form a viscous paste. The viscosity of the starch paste
increases with the temperature until the "peak viscosity" is reached.

This increase in viscosity is caused by the swollen granules being sufficiently close
together to have their movement restricted. Figure 2.4 Graphic summary of films
due to amylose content (Guti et al., 2018) After the viscosity reaches its peak, the
cohesive forces in the original granular structure become excessively weakened
and the viscosity of the paste gradually breaks down as the integrity of the
granules is lost. (Chen, 1990).

Some researches showed that films derived from starches with a lower amylose
content show a lower plasticizer-polymer compatibility. This allows the plasticizer
to migrate toward the surface of the film, thereby increasing the number of Lewis
sites and reducing the water contact angle (Alvarez, n.d.). A diagrammatic
summary of the surface properties of thermoplastic starch films associated with
their amylose content effects is provided in Fig 2.7. 2.2

The Natural Pottery Clay as a Filler Based on the studies of Kartika eta'l (2018) the
influence of the addition of clay causes more hydrogen bonds in the
biocomposite so that the chemical bonds are tougher and harder to break
because they need a large amount of energy to break the bond. Whereas the
percentage elongation is inversely proportional to the addition of natural micro
clay.

Where the addition of clay particles, causing percentage elongation reduced. This
is because of the decreasing intermolecular bonding distance between the
chains. The clay particle has a micron-size surface area that is large enough to be
able to interact effectively with the film matrix at low concentrations (5-8%). As a
result, polymer clay composite showed that an increase in modulus, thermal
stability and barrier properties without an increase in polymer density and loss of
its optical properties. In addition, the high mass of natural clay as filler can
contribute to a slowdown in the interaction between molecules of starch
polymer.

This scenarios leads to the development of bioplastic structures becoming more

heterogeneous and the results being discontinuities. According to Nuridin eta'l
(2018) studies: An increase in interaction process of poor adhesion forces
between the chains caused the decrease in polymer tensile strength. The particle
size has a relationship to the surface area per gram of filling material.

A very small size of particles produces a large surface area on the film matrix and
fillers to enhance the strength of such polymeric materials. This is consistent with
the results of the study [7], which stated that the surface area can be increased by
the presence of a surface that is a pivot or void space on the filling surface.
Polymers can enter the surface that is shafted during the continuous mixing
process.

Homogeneously scattered particles can increase the interaction through

absorption of the polymer over the surface of the filler material. On the other
hand, particles that are not scattered homogeneously may be produce bumps in
the polymer matrix. The presence of lumps in the polymer matrix reduces the
surface area then causes interaction between fillers, the matrix weakens and
results in a decline in the physical properties of polymeric materials (Zikri Noer
eta'l, 2018) Although clays are naturally compatible with starch, they can be
chemically modified to obtain composites with improved properties. To
overcome high permeability caused by the plasticizer, micro-pottery clay as an
additive was used.

In this area, the synthesis of micro-composites films has proven to be a promising

option, since polymer composites are increasingly gaining importance as a
substitute for materials due to their greater tensile properties, making them
especially suited for transportation and packaging applications. Mineral clays are
technologically vital and are mainly composed of hydrated aluminosilicate with
neutral or negative charged layers.

Clay is a potential filler; itself a naturally plentiful mineral that is toxin-free and
can be used as one of the components for food, medical, cosmetic and
healthcare products. Moreover, clay is environmentally sociable and inexpensive.
Clay/starch composites have been the most frequently studied, demonstrating a
potential for an advance of tensile strength, Young's modulus, water-resistance
and decrease of the water vapor permeability of starches from many different
sources (Souza et al., 2012).

The influence of the addition of pottery clay causes more hydrogen bonds in the
biocomposite so that the chemical bonds are stronger and harder to break
because they require a large amount of energy to break the bond. While the
percentage elongation is inversely proportional to the addition of natural clay.
Where the natural addition of clay, causing the percentage of elongation
decreased. results is due to the decreasing intermolecular bonding distance.

Clay has a micron-size surface area large enough to be able to interact effectively
with the polymer matrix at low concentrations (5-8%). As a result, polymer clay
showed an increase in modulus, thermal stability, and barrier properties without
an increase in density and loss of optical properties. In addition, the high mass of
clay filler can contribute to a slowdown in the interaction between molecules of
starch bioplastic.

The pottery clay mainly composed of 32% silica, 38% alumina, 5% iron (III) oxide,
up to 1% calcium oxide and magnesium oxide combined. Fillers act as a
discontinuous phase in the composite, they are usually scattered and distributed
within the matrix, playing the role of enhancing or reinforcing. When fillers are
incorporated into the matrix phase, a complex structure of interphase is formed
whereby the configuration and interaction between the fillers and the matrix
determine the properties of a composite. Both the filler and the matrix phases
can complement one another, thus producing a composite with enhanced
properties.

For examples, the tensile and barrier properties of the neat starch composite
improved after incorporating CaCO3 as reinforcing filler alginate-based
composite films filled with silver nanoparticles exhibited improvement in the
mechanical, barrier, and other additional properties such as thermal and
antimicrobial. There are myriad types of organic and inorganic fillers; however,
this review emphasized the most predominantly used fillers in polysaccharide
composite films to enhance the thermo-mechanical and water barrier properties.

Organic fillers are derived from living organisms that usually contain carbon-
hydrogen, carbon-carbon, as well as covalent bonds between carbon, hydrogen,
and nitrogen while inorganic fillers are obtained or derived from nonliving things
such as salt, metal and elemental compound that do not contain carbon-
hydrogen bonds (Khalil et al., 2019). Mineral or metallic fillers are considered as
inorganic fillers. Clay, nano-clay i.e.,

montmorillonite, silver nanoparticles (AgNPs), and calcium carbonate (CaCO3) are

among the common inorganic reinforcement materials that can be used in
polysaccharide-based composites. Among the inorganic fillers, clay is the most
frequently used fillers in the composite field since pre-historic civilization because
of its extraordinary natural abundance. Clay minerals are phyllosilicate minerals,
which are usually intercalation property and it, can swell with the absorption of
water.

Clay can be classified into various groups including smectite (pottery clay),
kaolinite, montmorillonite, illite, chlorite, fibrous silicate (e.g., sepiolite and
palygorskite) which sometimes known as attapulgite (Khalil et al., 2019). 2.3
Glycerol as a Plasticizer The glycerol (propane-1, 2, 3-triol) is acting as a
plasticizer.

Plasticizers are used in commercial products to alter the properties of the

polymer, just as the propane-1, 2, 3-triol have been used to change the
properties of the bioplastic. The propane-1, 2, 3-triol gets in between the
polymer chains and averts them from lining up in rows to form a crystalline
structure. When the polymer becomes crystalline, it also becomes brittle and
inflexible.

It can be considered plasticizer as a small molecule that gets between the

polymer chains and aids them to slide easily over each other so that the polymer
behaves like plastic. A plasticizer is a major component, required to overcome
film brittleness and helping to evade chipping and cracking of films during
subsequent handling and storage.

The plasticizer decreases intermolecular forces and increases polymeric chain

mobility. Moreover, the plasticizer reduces the glass transition temperature of the
thermoplastic. Plasticizing agents commonly used for thermoplastic starch
production include water and glycerol, polyethylene glycol and other polyols,
such as sorbitol, mannitol, and sugars (Gail, C, & G, 2016).

The physical properties of plasticizer (glycerol) and starch are presented below
the table (Mohsen Esmaeili, Gholamreza Pircheraghi*, 2017) Table 2.3 Physical
properties of starch and glycerol. Material Starch Glycerol Chemical formula
(C6H10O5)n C3H8O3 Molecular mass (g/mol) Variable 92.10 Melting point (oC)
Decomposes 18 Boiling point (oC) ---- 290 Density (g/cm3) 1.5 1.26 According to
Harahap eta'l (2018) investigations: The addition of glycerol as a plasticizer and
its molecules in the film can be located between biocomposite bonding chain
and can be able to interact by forming hydrogen bonds in the bond between the
film chains, causing interactions between the molecules of biocomposite film
have become less and may cause a decline in film stiffness. This is because of the
increased speed of the film's viscoelastic response and its molecular chain
mobility.

The incorporation of glycerol as serves as a conduit on the elastic properties of

the films, so that more glycerol addition provided will increase the values of film
extension. The glycerol can reduce the internal hydrogen bonds of molecules and
lead to the decrease in intermolecular attractive forces adjacent to the film
chains, thereby reducing the tensile breaking.

According to Sarbon eta'l (2017) findings on the title of “Effects of plasticizer

concentrations on functional properties of chicken skin gelatin films” They have
observed the effects of glycerol on the transparency of the film as follows. The
increased glycerol concentration resulted in a decrease in light transmission.
Lower UV light transmission is probably due to the different molecular weight,
composition and size of glycerol concentration that impedes the light
transmission properties of those films. The results indicated that the transparency
value of the films was decreased as the percentage of glycerol concentration
increased.

The lower the transparency value means that the higher the opacity of the films.
Figure 2.5 Graphic summary of films due to glycerol effect (Guti et al., 2018) 2.4
Petro-Based Plastics and Their Adverse Effects The development and progress of
the chemical industry in the mid-19th century are directly correlated to the
discoveries of fossil reserves which provide the raw material for the synthesis of
all petrochemical products.

Overall, 90% of the raw material for the chemical industry is presently produced
by fossil feedstocks in the form of petroleum and gas. This puts the chemical
industry in the third position as a manipulator of oil and gas, while energy
generation and transportation hold the first and second positions, respectively.
As to the chemical industry, the highest priority region for the utilization of fossil
feedstocks is for polymer fabrication.
The annual plastic production will be increased by up to 300 million tonnes by
the year 2010 (Gadhave, Das, Mahanwar, & Gadekar, 2018). This is because of the
success of synthetic polymer to produce plastics for the manufacturing of a range
of household and industrial products over the last 50 years. Petro-based plastics
are playing a crucial role in modern society due to their versatile nature.

The Petro-based plastics possess a range of divergent properties e.g. they can be
rigid or elastic, breakable or resilient, transparent or intensely colored, and can
have many added advantageous properties (e.g. cheap, recyclable, insulators)
(Gadhave et al., 2018).

The adverse effect of Petro based plastics are as follows: A) Utilization of

petroleum and energy resources Plastic production is directly affecting petroleum
consumption because tonnes of plastics are fabricated from petroleum products
every year. Several types of plastics e.g. ethylene, propylene, and styrene are
directly extracted from crude oil enhancing crude oil consumption.

In the year 2009, the total world's petroleum depletion was 98.3 million barrels
per day. If this rate of consumption persists, the known oil reserves which are
almost 1.24 trillion barrels will last for 41 years (Gadhave et al., 2018). The amount
of oil used to accomplish plastics products is 4% of the absolute oil exploitation.

Thus, by keeping in view the bulk petroleum utilization of the world, 4% results in
a large amount of oil used for plastic production. The European contribution to
the World's plastic production is 25%, resulting in 60 million tons of plastics per
year. A significant amount of energy is necessary for the synthesis of Petro-based
plastics and plastic-based products.

About 6% of all the energy used by American industries is utilized by the plastic
industry. This results in a 1.3% heat loss and it, in turn, makes the addition of
0.5% global warming (Gadhave et al., 2018). B) Emissions of heat and greenhouse
gas Besides the release of heat from the plastics industry, also CO2 is released
from the plastic industry further subsidizing to global warming. The release of
CO2 from the plastic industry to the environment was increased by 160% from
1971-2004 resulting in a release of 1.0 Gt/year.

As the manufacturing of plastics and plastic products is growing day by day it is

obvious that CO2 production will likewise be increasing. Plastic production and
manufacturing produce heat that contributes to global warming (Gadhave et al.,
2018). C) Health hazards Increased use of Petro-based plastics has created many
health hazards.

The key health hazards that are associated with Petro-based plastics are mainly
result of monomers present in the plastics. These monomers are trapped in the
polymer matrix during the process of fabrication and then, under certain
conditions e.g. heating, may leak out. For instance, styrene, a monomer can leak
out from its polymer polystyrene when subjected to heating and it is assumed to
be involved in endocrine sicknesses and cancer (Gadhave et al., 2018).

D) Plastics disposal problems and the inability of degradation All over the world,
a considerable quantity of leftover streams is produced from the manufacturing
and utilization of Petro-based plastics. Municipal waste products in the US
encompass almost 10% of plastic. The huge amount of plastics that are discarded
every year end up in landfills and water.

These dumps of plastics are polluting almost every ecosystem including marine,
freshwater, and terrestrial and deserts posing numerous environmental
complications. Plastics are generally resistant to microbial degradation making
them even more hazardous to the ecosystems. Further, the presence of plastics
everywhere is a threat to the survival of wildlife.

The health and environmental hazards of Petro-based plastics have resulted in

the prevention of the use of plastic bags in a large part of the European Union
(EU) countries, Australia, China, the city of San Francisco and the sanction was
also tried in California (Gadhave et al., 2018). Major impacts of plastics under
disposal problems are - (i) spoiling of plastic waste causes hindering of the
sewage systems which leads to scattering of water-borne diseases and increasing
the cost of sewage maintenance, (ii) plastic remains in soil for years deprived of
degradation and negatively affects the soil fertility, (iii) improperly disposed
plastic food bags, when consumed by animals, causes death of animals because
of suffocation, and stomach and intestine related diseases, (iv) plastic wastes in
water bodies carriage severe risk to the marine life, i.e.,

fish, sea birds, otters, and other marine species encounter premature death upon
swallowing these plastic wastes, and (v) disposal of chemicals used in the
manufacturing of plastic material in addition to its by-products into close to
water channels and open spaces by plastic manufacturing industries, thereby
causing health hazards as well as environmental pollution. 2.5 Degradable
Bioplastics Degradable bioplastics are an alternative to petroleum-based plastics.
Biodegradable plastics are plastics that will totally decompose to carbon dioxide,
methane, water, biomass and inorganic compounds underneath aerobic or
anaerobic conditions and the action of living organisms. Biodegradation or biotic
degradation is a specific property of certain plastic materials - namely, of the
polymers, these materials are made of.

A polymer material decomposes under the effect of biotic components (living

organisms) by a process. Microorganisms (bacteria, fungi, algae) recognize
polymers as a foundation of organic compounds (e.g. simple monosaccharides,
amino acids, etc.) and energy that sustains them. In other words, biodegradable
polymers are their nutrition.

Under the effect of intracellular and extracellular enzymes (endo- and

exoenzymes) the polymer undertakes chemical reactions and the polymer
degrades by the process of scission of the polymer chain, oxidation, etc. The
result of this process that can be influenced by a great number of different
enzymes is increasingly smaller molecules, which go into cellular metabolic
processes (such as the Krebs cycle), generating energy and turning into water,
carbon dioxide, biomass, and additional basic products of biotic decomposition.

These products are non-toxic and occur normally in nature and in living
organisms. This process turns artificial materials, such as plastics, into natural
components. A process, in which an organic substance, such as a polymer, is
changed to an inorganic substance, such as carbon dioxide, is called
mineralization. This process can be influenced by non-living (e.g. ultraviolet light,
heat, water) or living factors (enzymes, organisms).

Decomposition generally activates with fragmentation, i.e. the material that is

insecure to living or non-living factors undergoes a chemical decomposition of
the polymer and consequently decomposes mechanically (fragments). In the next
phase, the products of this decomposition are mineralized by microorganisms.

This second phase is a necessary step that characterizes this process as

biodegradation for the reason that the partially degraded polymers (fragments)
are hereby metabolized into end products. There are other cases (oxo-
degradable materials) where the material undergoes a fast fragmentation under
the influence of heat and UV light but the mineralization stage is very sluggish,
which means that the relatively inert micro-particles of the plastic material remain
that have a poor susceptibility to biodegradation.
The last stage of biodegradation is determined by the mineralization level.
Because organic carbon is transformed into carbon dioxide in the process of
aerobic metabolism, the most widely used method of monitoring this stage is by
measuring the amount of carbon dioxide made in a closed system.

To certify proper results, sufficient conditions must be maintained in the closed

system (humidity, temperature, pH, nonappearance of toxic substances) for the
existence of the microorganism culture. The method comprises determining the
amount of carbon in a polymer with a known structure and known mass. This is
followed by precise measurements to establish the amount of carbon that was
converted to carbon dioxide during biodegradation.

At its core, this process is similar to human metabolism, where food is converted
to energy and exhaled as carbon dioxide (Gadhave et al., 2018). Degradation at
high temperatures, such as in pyrolysis (burning) tends to cause emission of toxic
fumes. Plastic accumulation in the environment thus makes tremendous
problems for the world, presently and in the future.

Environmental problems caused by plastics comprise changes to the carbon

dioxide cycle, problems in composting, and increased toxic emissions. Stimulated
by environmental concerns, scientists are currently concentrating on ways to
develop plastics that will be used more efficiently. Two simple strategies are to
“recycle” (reuse), or to produce plastics that will degrade when no longer
essential.

Degradable plastics are grouped by the American Society for Testing and
Materials as (a) Photodegradable plastics: a degradable plastic in which the
degradation results from the action of natural light of day; (b) Oxidatively
degradable plastics - a degradable plastic in which the degradation results from
oxidation; (c) Hydrolytically degradable plastics: a degradable plastic in which the
degradation results from hydrolysis; and (d) Biodegradable plastics - a
degradable plastic in which the degradation results from the action of naturally
occurring microorganisms for example bacteria, fungi and algae.

As the plastics defined in categories (a), (b) and (c) require extra inputs, such as
light (UV) or oxygen for degradation, the biodegradable plastics (d) offer the only
products which are “naturally” degradable. There has been a widespread interest
in films prepared from renewable and natural polymers which can degrade
naturally and more rapidly than petroleum-based plastics.
Among all biopolymers, starch is being investigated as a potential material for
biodegradable films (Ezeoha & Ezenwanne, 2013). 2.5.1 Degradability and
Compostability Properties Biodegradable materials are materials that can be
broken down by microorganisms like bacteria or fungi into the water, naturally
happening gases like carbon dioxide (CO2) and methane (CH4) and biomass.

Biodegradability depends powerfully on the environmental conditions:

temperature, presence of microorganisms, and availability of oxygen and water.
Compostable materials are materials that break down at composting conditions.
Industrial composting conditions require raised up the temperature (55?C-60?C)
joined with high relative humidity and the presence of oxygen, and they are in
fact the most optimal when compared against other degradation conditions like
in soil, surface water, and marine water.

Compliance with EN 13432 is considered a good measure for industrial

Compostability of packaging materials. According to the EN13432 standard,
plastic packaging can only be called compostable (Gadhave et al., 2018). The
chemical properties of plastics can be used as standards for differentiating them
into degradable and non-degradable polymers.

Non-biodegradable plastics, frequently known as synthetic plastics, are derived

from petrochemicals. They have a lot of repetitions of small monomer units;
make them a very high molecular weight. In comparison, biodegradable plastics
are made from renewable resources that completely biodegrade in their natural
forms, such as components of living plants, animals and algae as a foundation of
cellulose, starches, protein and algal materials. They can also be produced by a
range of microorganisms.

Biodegradable plastics usually break down upon interaction with UV, water,
enzymes and gradual fluctuations in pH. There are four types of degradable
plastics: Photodegradable bioplastics, compostable bioplastics, bio-based
bioplastics and biodegradable bioplastics (Alshehrei, 2017). Photodegradable
bioplastic has light-sensitive collections connected directly into the backbone of
the polymer.

Ultraviolet Radiation exposure for an extended time can disintegrate their

polymeric structure, rendering them open to further bacterial degradation.
Landfills, however, typically lack sunlight, thus keeping these plastics non-
degraded (Alshehrei, 2017). Bio-based bioplastics are defined as “plastics” in
which 100% of the carbon is derived from renewable agricultural and forestry
resources, such as corn starch, soybean protein, and cellulose.

Compostable bioplastics are decomposed biologically in a composting process

that occurs at a related rate to other compostable materials, without leaving
visible toxic remainders. In order to designate a plastic as bio-compostable, its
whole biodegradability, its disintegration degree and the possible ecological
toxicity of its degraded materials must be determined by standardized tests.
Biodegradable bioplastics are fully degraded by microorganisms, without
departure visible toxic remainders.

The term “biodegradable” refers to materials that can disintegrate or break down
naturally into biogases and biomass (mostly carbon dioxide and water) as a result
of existence exposed to a microbial environment and humidity.
Polyhydroxyalkanoic acids (PHAs) are a significant type of biodegradable plastics
since they possess properties similar to conventional plastics.

They are completely biodegradable but may be melted and modeled, making
them ideal for use in consumer products (Alshehrei, 2017). 2.6 Polymer
Biodegradation Steps and Mechanisms Microorganisms break down the
compounds into a simpler form through biochemical transformation.
Biodegradation of polymer is described as any alteration of the polymer
properties such as digestion by microbial enzymes, reduction in molecular
weight, and loss of mechanical strength and surface properties, in other words,
the breakdown of material into fragments via microbial digestion.

Degraded particles are redistributed and probably non-toxic to the environment.

In nature, microorganisms form catalytic enzymes for biodegradation. This
approach is proficient for environmental waste management, and
microorganisms involved in this process for oxidation serve as a tangible
alternative mode to maintain a healthy environment. The degradation process is
accomplished by microorganisms via different enzymatic activities and bond
cleavage.

This degradation occurs in sequential steps, bio-deterioration (altering the

chemical and physical properties of the polymer), bio-fragmentation (polymer
breakdown in a simpler form via enzymatic cleavage) and assimilation (uptake of
molecules by microorganisms) and mineralization (production of oxidized
metabolites (CO2, CH4, H2O) after degradation). Mineralization of polymers takes
place in both aerobic and anaerobic conditions.
In the aerobic condition, CO2 and H2O are formed, while under anaerobic
conditions, CH4, CO2 and H2O are produced (V. M. Pathak, 2017). The
biodegradation procedure of a few polymers is known. Most of the microbial
communities are able to utilize polyester and polyurethane at a slower rate.
Starch- or cellulose-based plastics are biodegradable; they degrade easily
through composting, which can reduce landfilling and solve the waste
management problem.

Biodegradation with the help of microorganisms is an approachable way to clean

up such plastic waste. Table 2.4 Typical microorganisms related with polymer
degradation Types of polymer Microorganisms References Polyethylene
revibacillus borstelensis, Comamonas acidovorans TB-35, Pseudomonas
chlororaphis, P. aeruginosa, P. fluorescens, Rhodococcus erythropolis, R. rubber,
R. rhodochrous, Staphylococcus cohnii, S.

Ghiglione 2014; Shah et al. 2008a; Kale et al. 2015a, b; Grover et al. 2015;
Restrepo-Flórez et al. 2014; Bhardwaj et al.2012a) Polyvinyl chloride
Pseudomonas fluorescens B-22, P. putida AJ, P. chlororaphis, Ochrobactrum TD,
Aspergillus niger Dussud 2014; Shah et al. 2008a; Shah et al. 2008a; Kale et al.
2015a, Polycaprolactone Bacillus brevis, Clostridium botulinum, C.
acetobutylicum, Amycolatopsis sp., Dussud and Ghiglione 2014; Shah et al.

2008a; Bhardwaj Polylactic acid Penicillium roquefort, Amycolatopsis sp., Bacillus

brevis, Rhizopus delemar (Shah et al. 2008a) There are various misconceptions of
the term “Bio-plastic.” Bio-plastic, however, comprises bio-based plastics or
biodegradable plastics. Bio-based bioplastic has some or all its content or carbon
produced from a renewable source (animals, plants, or micro-organisms).

Biodegradable plastics degrade into methane (CH4), carbon dioxide (CO2), water
(H2O), and biomass in a defined timescale and in defined environments - soil and
marine environments, anaerobic digestion, and composting - through biological
action. A common misinterpretation exists between “bio-based” and
“biodegradable” polymers as they are inappropriately always linked, there is a
fine difference as they are not, given that a biodegradable bioplastic may not be
bio-based [e.g.

polybutylene adipate-co-terephthalate and polycaprolactone] and a bioplastic

that is bio-based may not essentially be biodegradable (e.g. polyethylene-
terephthalate and bio polyethylene). Biodegradation takes place in two steps. The
initial step is the disintegration (disintegration is a somatic process, while
biodegradation is a chemical process.

But, both must occur together for complete decomposition to occur) of the
polymers under suitable conditions into lower-molecular weight varieties by
means of either biotic reactions, i.e. photodegradation, hydrolysis or oxidation.
Closely followed by mineralization of the polymer remains by microbes i.e.
degradations by microbes or abiotic reactions (Africa, 2018). Plastics can degrade
via different mechanisms: thermal, chemical, photo and biological degradation.

The degradation of plastics is a physical or chemical alteration in polymers that

occurs as a result of environmental factors, like light, heat, moisture, chemical
conditions or biological activity. Biodegradation is a biochemical process that
refers to the degradation and assimilation of polymers by living microorganisms,
to produce degradation products.

Biodegradation is defined as any physical or chemical change in a material

caused by biological activity. Microorganisms such as bacteria, fungi, and
actinomycetes are involved in the degradation of both natural and synthetic
plastics. Bioplastics are usually biodegraded aerobically in nature, anaerobically in
sediments and landfills and partly aerobically in compost and soil.

Carbon dioxide and water are produced during aerobic biodegradation, while
anaerobic biodegradation produces carbon dioxide, water, and methane
(Alshehrei, 2017). The following figure shows the mechanism of polymer
biodegradation under aerobic and anaerobic conditions. Figure 2.6 Mechanisms
of polymer biodegradation (Alshehrei, 2017) A) Aerobic biodegradation Aerobic
biodegradation is an important part of the natural attenuation of contaminants in
many risky waste sites. Aerobic microbes use oxygen as an electron acceptor and
break down organic chemicals into minor organic compounds.

CO2 and water are the by-products of this process (Alshehrei, 2017). B) Anaerobic
Biodegradation Anaerobic biodegradation is the breakdown of organic pollutants
by microorganisms when oxygen is not present. It is also an important
component of the natural attenuation of contaminants at hazardous waste sites.

Some anaerobic bacteria consume nitrate, sulfate, iron, manganese and carbon
dioxide as their electron acceptors, to break down organic chemicals into smaller
compounds. Microorganisms are unable to transport the polymers directly
through their outer cell membranes, into the cells where most of the biochemical
processes take place since polymer molecules are long and not water-soluble.
In order to use such materials as a carbon and energy source, microbes
developed a strategy in which they excrete extracellular enzymes that
depolymerize the polymers outside the cells. Anaerobic and aerobic
biodegradation mechanism pathways are given in Figure 2.10. Extracellular and
intracellular depolymerize enzymes are actively involved in the biological
degradation of polymers.

During degradation, microbial exoenzymes break down complex polymers,

providing short chains or smaller molecules similar to oligomers, dimers, and
monomers. These molecules are small sufficient to be water-soluble and can pass
through the semi-permeable outer bacterial membranes to be used as carbon
and energy bases. This preliminary process of breaking down polymers is called
depolymerization; and when the final products are inorganic species (e.g.,

CO2, H2O, or CH4), the degradation is named mineralization (Alshehrei, 2017).

Mechanism of biodegradation: Biodegradation of polymers encompasses the
following steps: Attachment of the microorganism to the surface of the polymer,
growth of the microorganism, using the polymer as a carbon source, final
degradation of the polymer.

Microorganisms are able to assign to a polymer's surface, as long as the latter is

hydrophilic. Once the organism is attached to the surface, it is able to grow using
the polymer as its carbon source. In the primary degradation stage, the
extracellular enzymes secreted by the organism causing the main chain to leave,
leading to the formation of low-molecular-weight fragments, like oligomers,
dimers or monomers. These low molecular weight compounds are further used
by the microbes as carbon and energy sources.

Small oligomers may also diffuse into the organism and get assimilated in its
internal environment (Ibrahim & Ahmed, n.d.). 2.6.1 Factors affecting the
biodegradation of plastics The biodegradability of a polymer is essentially
determined by the following physical and chemical characteristics: The availability
of functional groups that rise hydrophobicity (hydrophilic degradation is faster
than hydrophobic), the exposure conditions, the enzyme characteristics, the
polymer properties such as the molecular weight and density of the polymer
(lower degrades faster than higher), the morphology: amount of crystalline and
amorphous areas (amorphous degrades faster than crystalline), structural
complexity such as linearity or the presence of branching in the polymer,
presence of easily breakable bonds such as ester or amide bonds. Chain coupling
(ester > ether > amide > urethane), molecular composition (blend), nature and
physical form of the polymer (e.g.,

films, pellets, powder or fibers), stiffness (Tg) (soft polymers degrade faster than
hard ones) (Alshehrei, 2017). 2.7 Advantages of Degradable Bioplastics A) Eco-
friendly Long-established plastics are petroleum-based plastics that depend on
fossil fuels which is an untenable source. Also, acquiring fossil fuels does a lot of
damage to the natural environment.

Bio-plastics on the additional hand, are crafted from biomass like trees,
vegetables, even waste which is completely bio-degradable. So bioplastics are
prepared from completely renewable sources. Even during the engineering of
plastics, a lot of pollution follows, such as, during fabrication, PVC plants can
release dioxins, known carcinogens that bio-accumulate in humans and wildlife
and are linked with the reproductive and immune system maladies (S. Pathak,
Sneha, & Mathew, 2014).

B) Need less time to degrade Conventional plastics take thousands of years to

break down and degrade, these plastics stopover in the environment, most
markedly on the ocean beds where they do the maximum damage for years.
These plastics obstruct the development and destroy natural habitats. On the
other hand, bioplastics involve considerably less time to biodegrade.

This degradation can be carried out at home for some bioplastics and even for
the bioplastics which need definite conditions, the time needed to degrade
completely is significantly less. This reduces the vast pressure on our prevailing
landfills (S. Pathak et al., 2014). C) Poisonous-free Some of the plastics degrade
speedily in the oceans liberating very harmful chemicals into the water bodies,
thus spoiling the animals, plants and also harming the humans by inflowing the
food chain. Biodegradable plastics are entirely safe and do not have any
chemicals or pollutants.

This plastic innocuously breaks down and gets absorbed into the earth. Such
advantages of bioplastics are of great importance, as the toxic plastic load on the
earth is maturing and at this rate will trigger a complete span of problems for
future generations (S. Pathak et al., 2014).

D) Consume less energy Companies still use fossil fuels for the production of
bioplastics; however, several bioplastics use considerably less fuel for their
construction. For example, Polylactic acid production encompasses much lesser
energy consumption than the other plastics. E) Environmental protection
Incinerating fossil resources heightens the share of CO2 in the atmosphere, which
results in an increase of the average temperature (greenhouse effect). Scientists
see a distinct association between CO2 increase in the atmosphere and the
increase of the number of thunderstorms, floods, and aridity.

Climate protection is presently is an essential part of environmental policy, due to

the fact that climate alteration can generate extensive negative aftermaths.
Governments and organizations work against this menace with focused measures
(S. Pathak et al., 2014). 2.8 Biodegradable Plastics from Starch Starch is a low-cost,
annually reloaded material that occurs widely as an energy store in the form of
carbohydrate in most plants, predominantly in the seeds, roots, fruits, shoots, and
leaves.

Starches obtained from various sources contain amylopectin and amylose, at

proportions that differ with the source. This disparity gives a distinctive
instrument to varying starch material properties. Starch is one of the most make
use of and researched biodegradable substances for bio-plastic production.

Starch begins to show plastic properties when mixed with a plasticizer. Table 2.5
Different sources of amylose & amylopectin content in starch Starch Amylose (%)
Amylopectin (%) Mango 18-20 80-92 Wheat 30 70 Corn 28 72 Potato 24 76 2.9
Reviewed Articles concerning to the Effect of Glycerol and clay on Film properties
The incorporation of clays has been reported as a good approach to improve
polysaccharide-based film properties. Kaolinite, smectite, and sepiolite are the
most frequently used clays in polymer composite studies.

These clays are environmentally friendly, naturally abundant, and economical.

Smectites, in particular, are often chosen for the preparation of polymer-based
composites. The parallel layers are stacked together by weak electrostatic forces
leading to a regular van der Waals gap between the layers called the interlayer or
gallery. As the forces that hold the stacks together are relatively weak, the
intercalation of small molecules between the layers is straightforward.

Thus, in addition to their low cost, high surface area, and large aspect ratio, the
rich intercalation chemistry of these clays enables them to be easily chemically
modified. In order to obtain composites with improved final properties, the clay
layers must be highly dispersed in the polymer matrix (intercalated or exfoliated)
as opposed to being aggregated as factoids.
The clay layers also induce changes in the molecular mobility of the polymer
matrix, which may enhance its thermal and mechanical properties as well as
oxidation stability, among other advantages. It is important to mention that all
these improvements can be achieved with low amounts of below 10 wt% (Nawab,
Alam, Haq, & Hasnain, 2016).

According to (Khairunnisa & Rostini, 2018) the addition of glycerol with different
concentrations significantly affected thickness, tensile strength, percent
elongation, and transparency of films. Based on (Souza et al., 2012) cassava
starch-based biodegradable films: Influence of glycerol and micro-clay particles
content on tensile and barrier properties and glass transition temperature.

Films prepared with lower glycerol content would have better tensile and barrier
properties than films with higher content. Films were then prepared with the
addition of micro-clay particles and their tensile and barrier properties and glass
transition temperature were measured. This indicated that both glycerol and
micro-clay particles influenced significantly the tensile and barrier properties,
diminishing film water vapor permeability.

The presence of micro-clay particles did not influence glass transition

temperature. According to (Basiak, Lenart, & Debeaufort, 2017)the
amylose/amylopectin ratio in starch films play a significant role in the physical
and chemical properties. This ratio strongly influences the microstructure and
thus the viscosity of the film-forming suspensions that affect the retraction of the
network during the film drying and then the final thickness. The addition of
glycerol concentration in the development of packaging films also resulted in an
increase in film thickness.

This observation is consistent with Sudaryati et al. (2010) that improvement of

thickness due to the addition of glycerol is because the glycerol molecules will
occupy voids in the matrix and interact with the film-forming polymer, which
causes the distance between the polymer increases thus improving the film
thickness.

Bourtoom (2007) has reported that the plasticizer added to the manufacture of
the film can bind with starch to form starch plasticizer polymer so that the starch-
starch bond is replaced by the starch-glycerol-starch bond which led to an
improvement of the film thickness. Nemet et al. (2010) have reported that the
film-forming solution with higher concentrations of glycerol has a higher dry
matter content resulting in a thicker film.
Furthermore, high glycerol concentration can increase the ability to absorb
moisture to some extent and can cause an increase in film thickness due to the
swelling process (Vieira et al., 2011; Ahmadi et al., 2012). 3 MATERIALS AND
METHODS 3.1 Materials Mango seed kernels were obtained from local juice
traders in Bahir-Dar city.

The pottery clay locally available in south Gondar, particularly in Adiszemen

which is 90km far away from Bahir-Dar city. Addis Zemen (Amharic "New Era";
also known as Addis Abraham) is a town in northern-central Ethiopia. Located in
the south Gondar Zone of the Amhara Region, on the road connecting Gondar
and Bahir Dar, Addis Zemen has a latitude and longitude of 12°07?N 37°47?E and
an elevation of 1975 meters above sea level. Almost all 99% of glycerol, was also
obtained from the nearby local market. 3.1.1

Chemicals Chemicals that mainly used during the experiment were distilled water,
glycerol as a plasticizer (Almost all 99% of glycerol was also obtained from the
nearby local market), liquid sulfur dioxide, and micro-pottery clay as a filler.
Sodium hydroxide, methanol, ethanol, iodine solution, KI, methanol, acetic acid,
hydrochloric acid, sulfuric acid and sodium chloride. 3.1.2

Equipment's The laboratory equipment's for conducting mechanical, chemical

and physical properties were: analytical weighing balance (JF-2004), peeler,
grinder, sieve, centrifuge (642E), thermometer, beakers, water bath (TBS451PA)
shaker, measuring cylinder, burettes, micropipettes, filter paper, magnetic stirrer,
heating oven (DHG-9023A 20L), different-sized standard measuring flasks, UV
spectrophotometer (UV-2700), tensile tester (UTM 100ST). 3.2 Experiments 3.2.1

Methods of starch extraction from mango seed kernels Mango seed kernels were
obtained from the mangoes, which were then used for extracting starch from the
kernels. Starch was isolated from the ripe mango seed kernel (Nayak & Rayaguru,
2018). Mango seeds were obtained from the waste container at local juice traders
were washed with clean water and peeled and then the kernel shredded to small
pieces and exposed to the sun dry for six days. The shredded mango seed kernels
were fed to the jaw crusher and the obtained fine powder of seed kernel was
sieved using 0.125µm and soaked in liquid sulfur-dioxide (as extracting agent)
with a mass liquor ratio (mango seed powder to liquid sulfur-dioxide were 1:05,
1:10, 1:15 and 1:20 for four days). Here the effects of parameters (time and mass
liquor ratio) on the yield of starch were investigated.
After four days, each starch slurry was filtered and then later placed in the tank
for settling that took at least 30 minutes. Then after each starch sediment was
separated from the slurry using nylon cloth and washed again with distilled water.
After the second settling, each starch sediment was dried using an oven with
temperature 100ºC for moisture removal.

Finally, the dried starch was ground and sieved within the range of 0.5µm -
0.125µm for better homogeneous size and the actual yield was calculated
(Maulida et al., 2018). Figure 3.1 Flow chart for extraction of starch from the
mango seed kernel 3.3 Characterization of Mango Seed Starch 3.3.1
Determination of starch overall properties A) pH determination The PH can be
determined according to the method of (Alobi et al., 2017).

The electrode of the pH meter will be rinsed with distilled water and inserted in
the starch sample slurry. The pH value of the sample will be recorded. 5g of
starch is will be taken in 20ml of distilled water and is mixed thoroughly for 5
minutes. B) Moisture content determination The moisture content of the starch
was determined based on (A. O. A. C., 1975).

Three grams of mango seed starch was weighed into a crucible and placed in an
oven with a temperature of 105oC and dried for 24 hours to constant weight.
(AACC, 2005). Moisture content in the dried starch was determined by keeping
the weighed quantity of sample in a thermostat-controlled oven at 105ºC for 24
hours. The dry weight of each sample was taken on a weighing balance (A. O. A.
C., 1975).

The percentage of the moisture content and dry matter was then calculated by
the formula as presented below: C) Amylose content determination One hundred
milligrams of amylose was dispersed using 1 mL absolute ethanol, and 9 mL 1M
NaOH was added to the solution. The solution was positioned in a boiling water
bath for 15 min with frequent shaking to obtain a clear solution; then the entire
volume was adjusted to 100 mL using distilled water to obtain the stock solution.
To generate absorption spectra, 4 mL amylose stock solution or 2.6

mL amylopectin stock solution was added to 30 mL distilled water and the pH

was adjusted to 3.6 using 0.5M HCl. Then, 0.5mL potassium iodide-iodine (I2-KI)
solution (2g/L I2, 20g/L KI) was added, and the total volume was adjusted to
50mL using distilled water. This reaction solution was mixed and placed at room
temperature for 30min, after which it was scanned using a spectrophotometer at
the 600nm wavelengths.
To generate the amylose standard curve, the amylose stock solution was diluted
to obtain reaction solutions with final concentrations of 10, 20, 30, 40, 50, 60 and
70 ?g/mL, respectively (Ma & Deng, 2017). Based on this amylose standard
calibration curve, the maximum values of the amylose that is found in mango
seed starch were determined using the following formula.

This was determined by using the method of (Hassan et al., 2013). High amylose
content of the starch sample indicates a decrease in crystallinity and gel
tackiness; increased amorphous regions, gel hardness, higher pasting
temperature and amylose aggregation/spoilage tendencies. High amylose
starches have an increased tendency for water absorption.

Amylopectin content increased with an increase in amylose, this shows that one is
a function of the other and both properties are important in food preparation
and development. Amount of amylose and amylopectin present in starch affects
the mechanical property of the starch polymer. High amylose starch presents
higher stiffness and hardness than high amylopectin starch due to the
comparatively less branching structures (Alobi et al., 2017).

D) Ash content determination Ash content was determined by using the AOAC
method. Total ash content was determined by igniting a previously dried starch
sample in a muffle furnace at 540C for 2 hours. On cooling, the resulting ash was
weighed and the percentage ash was calculated as: E) Density determination The
bulk density was determined by dividing the mass per unit volume and the true
density is determined by the water displacement (Omar, 2016).

Four gram of the starch sample was mixed and shacked with 12 ml of distilled
water. The density of the starch was investigated by the standard of ASTM D792-
91. The true density can be calculated as follows: F) Swelling power and solubility
The method of Nadia et al. (Hassan et al., 2013) was used in the determination of
swelling power and solubility. Briefly, 0.400g of the starch sample in a centrifuge
tube was treated with 40 cm3 of distilled water. The slurry was heated at 85oC in
a water bath for 30 min.

After cooling to room temperature, the solution was centrifuged at 3000 rpm for
15 min. The supernatant was carefully recovered, and the swollen starch sediment
was weighed. An aliquot of the supernatant was then evaporated overnight
(110oC) and swelling power and solubility were calculated as follows (Series &
Science, 2018): G) Effects of parameters (Mass liquor ratio) on the yields of starch
The yield of starch extraction mainly depends on the types of raw materials used,
the types of extracting agent and extraction time.

The yield of starch was determined as the quantity of starch isolated from the
fresh seed kernel as well as dried seed kernel, expressed in percentage (Nayak &
Rayaguru, 2018). Here temperature and mass of samples were kept constant. The
yield of starch was calculated as follows (Ratnayake, Wassinger, & Jackson, 2007):
3.3.2 Preparation of micro-pottery clay Six grams of unmodified pottery clay were
suspended in 100ml of distilled water and stirred for 24h using a water bath at
room temperature. Five hundred milliliters of a solution of sodium hydroxide was
prepared.

The suspension was added to the sodium hydroxide solution and the final
product was stirred for 30h in a water bath at 90°C. The precipitate was washed
with 200ml of 25% ethanol solution. After the micro-pottery clay had been dried
at 100°C for 24hrs, it was grounded in a mill with a sieve size of (0.125µm)
(Krump, Luyt, & Hudec, 2006). 3.3.3

Experimental design The research using a Randomized blocks design with a

factorial experiment. The first factor was micro-pottery clay to starch ratio of 0%,
5%, 7% and 10% w/w. The second factor was glycerol to starch ratio with
concentration varied from 0%, 25%, 30% and 35% w/v.

The individual and interaction effects of both glycerol and micro-pottery clay on
the films overall properties were studded. 3.4 Development of Biodegradable
Films Initially, five gram of starch with 100 ml of distilled water was prepared and
by varying the gelatinization temperature such as 55oC, 65oC, 75oC and 85oC at
a heating rate of 10ºC/min with a continuous handshaking which results a
gelatinized starch solution and then after the micro-pottery clay (0, 5, 7 and 10% )
and glycerol concentration (0, 25, 30 and 35 %) were added and mixed based on
the experimental design and the film-forming solutions were left to cooled down
to 50oC prior to casting in glass Petri-dishes (so as to remove air bubbles)
(Maulida et al., 2018).

After mixing, the solution (50ml) was cast onto the flat Petri dishes (15cm*15cm).
The glass Petri-dishes serving as casting surface enables the film to have a
smooth and flat surface. The films were then dried for 6hrs in an oven at about
33°C to allow evaporation until they reached a constant weight.

After 6hrs the plates were removed from the oven, films were peeled off
(Gooding, 2016). Figure 3.2 Film development process 3.4.1 Effects of
Gelatinization Temperature against on the Viscosity The prepared solution was
heated using a boiling water bath at different temperature ranges from 55 ºC to
85ºC at a heating rate of 10ºC/min. During the process, the heating and cooling
cycle was used. The heating rate and the stirring speed were kept constant.

At the end of the gelatinization temperature (85ºC), the gelatinized solution was
cool down to a temperature of 50ºC at 15ºC/min, and then held at 50ºC for one
minute and the viscosity was determined up to 85ºC using a viscometer (Nilani et
al., 2010). The effect of gelatinization temperature in each interval of 10ºC during
the process of film formation was practically observed. 3.5

Characterizations of Degradable Film 3.5.1 Measurement of the film's overall

properties A) Measurement of film's solubility in water The solubility of films in
water was measured following the method of Jouki, Yazdi, Mortazavi, and
Koocheki (2013). Film samples were dried at 85oC for 24hrs in a laboratory oven
and then weighed to determine initial solid content.

Pre-weighed film samples (1 cm × 3 cm) were immersed under constant agitation

in 50ml of distilled water for 6hours at 25oC. Then after the remaining pieces of
films were filtered and dried at 100oC till constant weight obtained (final dry
weight). The solubility was reported as the difference between the initial and final
dry matter with respect to the initial dry matter (Khazaei, Esmaiili, Emam, &
Ghasemlou, 2014). B) Measurement of film density. The density of the best film
was examined by the standard of ASTM D792-91.

The mass of the best was measured using an analytical balance. The film sample
was prepared with a size of 2x2cm and weighted (g) before immersing it into
distilled water. The convex mica measuring cylinder was filled with 30ml of
distilled water. The amount of liquid displaced after immersing film into the liquid
was recorded as V.

Eq. (3.9*) was utilized to calculate the density (?). The test was performed in
triplicate. Density was calculated as follows: C) Measurement of film thickness The
films thickness were measured using digital micrometer and measurements were
made in at least, three random locations for each film and an average value was
carried out. D) Measurement of film transmittance and transparency.

The materials used in this study were UV spectrophotometer. The transmittance

of films was determined at 620nm as described by Bourtoom and Chinnah (2009).
The film samples were cut into rectangles and placed in the internal side of the
spectrophotometer cell at a wavelength of 620nm (Nemet, Šošo, & Lazi, 2010b).
The micro-composite films were cut into strips (1*3cm2) and placed directly in
the spectrophotometer test cell. Air was used as a reference.

Transparency was expressed as transmittance units per thickness unit (Liu et al.,
2016). All samples were carried out in triplicate. The transparency of the films was
calculated according to equation (9): Where T620 is the transmittance at 620nm
and X is the thickness of the film (mm). E) Measurement of films moisture uptake
Films Moisture/water uptake were studied by cutting off each individual film with
a size of nearly 3x3 cm and then weighed the masses. The pieces of such films
were oven-dried at 105°C for 24 h (till equilibrium weight was obtained).

The weight differences were determined after drying, and moisture uptake by the
films was obtained using the following equation: F) Measurement of tensile
strength The tensile strength of the resultant bio-plastic sheets was determined
according to the ASTM D-882-9 using the universal testing machine. A dumbbell-
shaped specimen was used for the tensile strength and % elongation.

Initial grip separation was set at 20 mm; while cross-head speed is also set at 100
mm/min. The thickness and width of the samples were measured using a caliper
micrometer. Tensile strength is then calculated by dividing the maximum load
developed during the test by an initial sheet cross-sectional area.

The tensile strength can be obtained from the observed data. G) Measurement of
films biodegradability The biodegradable behavior of micro-pottery clay
reinforced bioplastic was determined using soil burial decomposition test. The
film was cut into 5*5 mm2, then, buried in the ground at 100mm depth for two
weeks, prior to burial, the initial weight (W1) was determined and the final
weight, W2 (weight after decomposition) of the film was measured. The
decomposition of each sample was determined by equation 3.16 for weight loss.

Percentage weight loss, (%W) can be calculated from the following equation 3.16
(Wahyuningtiyas & Suryanto, 2018). 3.5.2 Individual and interaction effects of
glycerol and micro-pottery clay on the film over all properties. Individual and
interaction effects of both glycerol and micro-pottery clay on the film's overall
properties were studded by using design experts.

Here what was used for parameter interaction is a design expert that incorporates
response surface methodology. Response surface methodology (RSM) is a
collection of mathematical and statistical techniques for empirical model
building. The analysis was carried out with a design order (quadratic) and a
design model (polynomial).

Analysis of variance was also applied to the linear regression and general linear
models that quantified the relationship between the dependent variables (the
responses) and the independent variables (the fixed factors). 3.5.3
Thermogravimetric analysis (TGA) The thermal stability of the sample was studied
by using Thermogravimetry analysis (TGA) and were performed on the Shimadzu
TGA-50 equipment under the nitrogen atmosphere, the flow rate of 50 mL/min, a
heating rate of 10°C/min and temperature range of 20ºC to 200°C.

Approximately 2mg was placed on the platinum pan. Here the thermal properties
of the best film (which reinforced with micro-pottery clay and has optimum
values of tensile strength) and the non-best film (which is not reinforced with
micro-pottery clay but plasticized with glycerol) were studied. 4 RESULTS AND
DISCUSSION 4.1

Characterization of Mango Seed Starch The pH of the starch solution was

measured using pH meter which is found to be 6.2±0.231 which really agrees
with Uchendu eta'l (2013) findings which are 5.5. In any food industry, the pH test
can be used to identify the presence of molds and other unnecessary impurities.

In the different industries, the starch of low acid factor should be chosen except
when starch is needed for dextrin formation (Uchendu, 2013a) findings5.5. The
swelling power of starch was found to be 0.233±0.23%. Swelling power is the
capacity to uptake water by starch granules as a heating temperature increases.
This may be endorsed to the disruption of starch granules at higher temperatures
and subsequent release of all the amylose from the amylopectin network.

Based on this experimental result, the amount of moisture and solubility found to
be 10.33±0.21% and 1.65±0.33 respectively which is nearly consistent with
(Hassan et al., 2013) findings. According to Hassaeta'l (2013) studies, they found
8.33±0.58% and 0.135±0.002 solubility and moisture content respectively.
Concerning the present swelling power and the previous one, it has been seen a
great difference (0.233±0.23%, 19.40±0.30% respectively) this might be due to
the hydrophilic nature of starch.

The relatively small moisture amount of starches makes them easy to store at
room temperature and less prone to colonization by micro-organisms
degradation for mango seed starch making it amenable for utilization in
packaging industries. The ash content of mango seed starch is an indicator of the
total mineral/inorganic content. According to this study, the residual ash content
of starch was 0.41±0.13%, which is a little bit greater than the result obtained by
Kalpana eta'l (2018) which is around (0.28±0.04%).

This is mainly because of some presence of impurities in the starch, which did not
burn off completely (Uchendu, 2013b). Based on the standard calibration curve,
the obtained amylose content was 19.278% which is consistent with (Sonthalia,
2015) findings. 4.1 Standard Amylose Calibration Curve A calibration curve is a
mathematical tool that provides a set of reference points that an unknown
concentration of samples can be compared too.

Once the curve is generated and all other values that pertain to techniques like
linear regression (for example, the correlation coefficient (R2) were determined,
the curve can also be used to determine the concentration of the unknown
sample. Figure 4.1 Standard amylose calibration curve The amylose was
calibrated using spectrophotometry that the absorbance of the standard sample
was read at 620nm wavelength and the unknown sample (starch) concentration
was determined which is around 0.315±0.01 (using the slope and the intercept
from the standard calibration equation) was also determined which is within the
range of the standard absorbance and it implies that the unknown solution
certainly contains amylose which was about 19.28% based on equation (3.2)
which is also presented in appendix 4.2 table 4.2

Effects of Parameters on the Yield of Starch For the analysis of the yield: four days
with four different samples of mass liquor ratios (mango seed kernel powder to
liquid sulfur-dioxide i.e. 1:5, 1:10, 1:15 and 1:20) were taken. The maximum yield
of starch was determined based on the equation (3.8). Starch is a nutrient for
many organisms and since water is present in the final structure so that starch is
readily consumed.

Water can easily be absorbed by starch resulting in the disintegration of the

material by partial solubility. Partially solubilized starch is even more readily
consumed by enzymes principally from microorganisms (Shanks & Kong, 2012).
Figure 4.2 Effect of parameters (mass ratio and extraction time) on the yield of
starch As we can see from the above graph (Figure 4.2) the maximum starch yield
was obtained on the third day at the mass liquor ratio of 1:10 as presented in
Appendix 4.1 which is around to 19.97±0.021% this is less than by 1.03% than the
theoretical yield.
As days increases the percentage yield of starch was decreased due to the mold
(cream like) formation on its surface. This mold might contain micro-organisms
that used starch as their source of food. Gradually the micro-organisms
multiplied rapidly and their consumption to starch increases.

Due to the lack of starch, they release toxic excreta and hence the percentage
yield of starch was gradually reduced at three days letter (Sciences & Medan,
2018). The yield of starch extracted from dried mango seed kernel
was19.97±0.021%, which is lower than the result obtained by Rayaguru eta'l
(2018) on the title of 'studies on extraction of starch from dried and fresh mango
seed kernel' which has 64.41±5.51 and 47.37± 2.56% respectively. The yield
difference might be due to the effect of starch extraction techniques (alkalization.

Sedimentation etc), the type of chemicals used during extraction, purities and the
type of mango varieties (Nayak & Rayaguru, 2018). 4.3 Effects of Gelatinization
Temperature against on the Viscosity Gelatinization happens when water
disseminates into the granule, which then swells considerably due to the
hydration of the amorphous phase causing loss of crystallinity and molecular
order. Figure 4.3

Effect of gelatinization temperature against the viscosity As was depicted in the

graph, there was observed an increase and decrease the phenomena of viscosity
during gelatinization. The increase in viscosity (from 1.491±0.271 to 1.867±0.213)
in the early heating stages from 55-65oC is due mainly the release of amylose
while, in later stages 65-75oC, the continued viscosity increase (1.867±0.213 to
4.033±0.541) is due to the interaction of extra granular material and swelling of
the granules. The peak viscosity was observed at a temperature of 75oC.

The granules swell and contact each other thus increasing the viscosity of the
paste. There was observed a sharp decline of viscosity (4.033±0.541 to
2.485±0.132) as a temperature increases from 75-85oC. This sharp decrease in
viscosity is because it was reached a certain critical limit and the loss of starch
granular integrity (Taylor, Lund, Lorenz, & Lund, 2009).

Finally, it was observed that once again the viscosity raised from 2.485±0.132 to
3.368±0.371 while the gelatinized solution cooled down from 85-50 oC. This is
mainly because of gel formation so that starch granules are reordered in an
advanced structure form. The heated gelatinized solution was cooled down from
85-50 oC in order to remove air bubbles.
It is believed that the initial swelling takes place in the amorphous regions of the
amylose where hydrogen bonds are less numerous and the polymers are more
susceptible to dissolution. As the structure begins to weaken, the granule absorbs
more water and became swelled as a result of the amylose leach out and finally,
gel formation was observed.

Because not all the granules gelatinize simultaneously, different degrees of

structural disruption and swelling exist (Alcázar-alay et al., 2015). Crystallinity is
mainly influenced by the chain length of amylopectin. The smaller the starch
granule, the higher the gelatinization temperature would be (Primadona, 2018).
4.4

Density of Reinforced Film The density of the best film was examined by the
standard of ASTM D792-91. Initially, the mass of the film was determined using
an analytical balance which is around 0.801g and later on the piece of the film
which displaced the distilled water was recorded (0.5ml).

The density of the best-reinforced film was performed using equation (3.9*)
which is 1.58±0.233 g/cm3. Similar findings were reported by Ginting et al (2018)
which is around 1.315g/cm3. This little difference might be due to the types and
varieties of raw materials and additives. Table 4.1

Composition of the developed films Run Micro-pottery clay grounds (%w/w)

Glycerol (%w/v) Starch ground (g) Distilled water (ml) 1 0 0 5 100 2 5 25 5 100 3 7
30 5 100 4 10 35 5 100 5 0 0 5 100 6 5 25 5 100 7 7 30 5 100 8 10 35 5 100 9 0 0 5
100 10 5 25 5 100 11 7 30 5 100 12 10 35 5 100 13 0 0 5 100 14 5 25 5 100 15 7
30 5 100 16 10 35 5 100 4.5

Individual Effects of Glycerol and micro-pottery clay on the Film's overall

Properties A) Effect of glycerol and micro-pottery clay on the film's solubility
Solubility in water is an important property of films, since potential applications
may require water insolubility to enhance product integrity and water resistance
(Nemet, Šošo, & Lazi, 2010a).

Solubility in water is defined as the percentage of the dry matter of each film,
which solubilized after 24hrs soaking (Farahnaky, Saberi, & Majzoobi, 2013).
Solubility influenced by hydrophilic and hydrophobic components of the
ingredients. Glycerol is an organic compound which has a three-carbon molecule
that has three hydrophilic hydroxyl group responsible for its solubility in water
and its hygroscopic nature. Glycerol and water is an ideal example of a
homogeneous mixture. Figure 4.4

Effect of glycerol and pottery clay on the film solubility The increment of glycerol
concentration from 0%w/v to 35%w/v led to an increase in film solubility of
18.75±0.214 due to the strong affinity of glycerol towards the water. The higher
the value of a hydrophilic material then the solubility would be higher, and the
reverse holds true for hydrophobic material.

As it has been observed from figure 4.4 as the concentration of glycerol

increases, the solubility of the films was increased from 3.85±0.134 to
18.75±0.214 for all treatments because the hydroxyl group which presents in
glycerol is responsible for its solubility in water. The highest value of solubility
was observed at glycerol concentration of 35% w/v which is around 18.75±0.214.

This showed that the concentration of glycerol has a direct relationship with the
solubility of the film. For the respective of sixteen treatments, it was observed a
sharp increment of solubility as the glycerol concentration increases. Evaluation
of the water solubility of the films is an important factor in determining
biodegradability potential since polymers are more susceptible to degradation at
higher solubility because components are easier to hydrolyze and consequently
become smaller molecules.

The higher the film solubility, the higher the degradability of the material b/c the
film could not resist the penetration of water. The value of low solubility in the
biodegradable film very good used as a material packaging (Indrianingsih &
Rosyida, 2018). These results suggested that the increase in solubility of the films
with glycerol is partly due to a certain hydrophilic tendency (Sifuentes-nieves et
al., 2015).

The addition of micro-pottery clay has an antagonistic effect with glycerol on the
film properties. Increment of micro-pottery clay in all treatments reduces film
solubility in water. This pottery clay interacts with water and makes a good
network by hydrogen bonds, increasing the cohesiveness of the starch matrix and
reducing its solubility in water (Of, On, Of, Starch, & Films, 2013). The increase in
glycerol concentration resulted in increasing the film solubility.

Improvement of the film solubility due to the influence of increasing

concentrations of glycerol caused by hydrophilic and hygroscopic properties of
glycerol (Agricultural & Pangkep, 2016). Bourtoom (2008) has reported that the
increase of the solubility of the film with increasing plasticizer concentrations can
be explained by the hydrophilic properties of plasticizer, where the dry matter
solubilized in water is likely formed by the plasticizer.

Therefore, the increase in the glycerol content of film would increase the content
of dry matter solubilized in water, thereby increasing the film solubility. Ramos et
al. (2013) suggested that improve film solubility because of the increased
concentration of glycerol is caused by hygroscopic properties of glycerol which
attract and bind water molecules, thus supporting the film surface wetting and
moisture absorption.

The high solubility of the film with glycerol is because the glycerol has a strong
affinity towards water molecules, as well as low molecular weight facilitates the
entry of glycerol between polymer chains, thereby increasing the free space
volume between the chains (Sanyang et al., 2015). Film solubility in water is an
important property in selecting suitable food packaging plastics.

For most food applications, films with good water insolubility are essential to
provide water resistance and boost the shelf-life of food products. Plasticization
of mango seed starch films significantly increases film solubility in water as
presented in Fig. 4.4. Regardless of the plasticizer type, the solubility of
plasticized films in water increased with an increase in glycerol concentration.

A similar result was reported in previous studies, regarding the effect of

plasticizer concentration on biodegradable film solubility in water. Due to the
hydrophilic nature of glycerol, it has an essential role in weakening the interaction
between films molecular chains, increasing the free space volumes between the
chains.

This, in turn, promoted water diffusion into the film matrix and, consequently, led
to an increase in solubility of plasticized films (Sanyang, Sapuan, Jawaid, Ishak, &
Sahari, 2015). B) Effect of glycerol and micro-pottery clay on the film's moisture
uptake Figure 4.5 Effect of pottery clay and glycerol on the films moisture uptake
Based on the above figure 4.5

initially, it has been observed that moisture uptake by the films was increased as
the concentration of glycerol increases from 0% to 35%. Moisture content is
another packaging film property of great importance in foodstuff packaging
applications because it helps to retain moisture levels within packaged products.
Here what observed from the graph is the moisture content of films is directly
proportional to the glycerol concentration as presented in appendix 4.3, table 4.
The moisture content of the film which made from 35%w/v glycerol
concentration was the highest value (19.31±0.17).

The first four treatments of the films were prepared without glycerol. Films with
no glycerol content had the lowest moisture uptake (8.21±0.03, 4.35±0.13,
2.98±0.23 and 1.77±0.08) for the first four respective runs). The hygroscopic
properties of glycerol itself migrated to the surface of the film may be
contributed to the absorption of moisture and increases film moisture uptake
(Nor, Nazmi, & Sarbon, 2017).

For all plasticized mango seed starch films, the moisture uptake increased
significantly as glycerol concentration increases from 0 to 35%w/v. Generally,
starch-based films turn out to be more hydrophilic with an increase in glycerol
concentration. Thus, several investigations reported that the moisture content of
starch-based films increases by adding more glycerol (Sanyang et al.,

2015), Similar findings were reported by Aguirre et al. (2013). Usually, films are
highly hygroscopic above their critical/compatibility limit of glycerol
concentration and therefore film moisture content is affected by ambient
conditions. The best film was obtained at its exact compatibility limit of the
ingredients concentration aspect ratio.

Higher levels of glycerol concentration increase the film's moisture affinity and
these results could be attributed to the hydrophilicity of the glycerol, with
accessible -OH groups capable to interact with water by hydrogen bonds. Based
on the result obtained, whole, starch-glycerol films had shown a higher ability to
uptake water at all concentrations (Farahnaky et al., 2013).

Micro-pottery clay has an antagonistic effect on the film response as compared

with glycerol concentration. As was observed in the above figure 4.5, film
moisture uptake was decreased as the micro-pottery clay content increases. This
is because clay has less tendency towards water uptake as compared with the
remaining two ingredients.

Therefore, it can be possibly said that the larger the micro-pottery clay content,
the lower the moisture uptake by the films would be. From 0%w/w -10%w/v
micro-clay content, the moisture content of the films was decreased from
19.31±0.17 to 8.21±0.03. The moisture uptake of the film which made from 10%
w/w micro-pottery clay content was the lowest value (8.21±0.03).
The first four possible combinations of the films were prepared with no micro-
pottery clay. Films with no micro-pottery clay content had the highest moisture
uptake (19.31±0.17, 17.81±0.26, 15.15±0.11 and 8.21±0.03) (Zeng, Yu, Lu, & Paul,
2005) as compared with reinforced films due to the combined hydrophilicity
nature of glycerol and MSK starch.

For more information look appendix 4.3, table 4. The larger the glycerol amount,
the greater the polarity of the surface, which favors hydrophilicity and moisture
uptake. Properties of starch-based films strongly depend on water-glycerol and
micro-pottery clay contents.

Altering the plasticizer content allows an increase or decrease of selected

physical, chemical and functional parameters (Glycerol & Films, 2018). C) Effect of
glycerol and micro-pottery clay on the film's thickness Figure 4.6 Effect of
glycerol and pottery clay on the film thickness The film's thickness was measured
using a manual digital micrometer in at least, three random locations for each
film and the average values of the replicate were taken as shown in appendix 4.3
table 6.

As the micro-pottery clay content increases during film formation process, the
values of the thickness of the films in each possible treatments were decreased
(0.43±0.002, 0.37±0.042, 0.30±0.032, 0.35±0.006, 0.32±0.022, 0.31±0.010,
0.27±0.019, 0.18±0.001, 0.15±0.001) this is because of the formation of some
micro-void spaces which need to be filled by the plasticizer. For more information
take a look at appendix 4.3.

However, glycerol has antagonistic effects on the thickness of the films as

compared with the clay effect. As the glycerol concentration increases, the values
of films thickness were proportionally increased this is because glycerol causes
the films to favored more swelling properties (Jafarzadeh, Alias, Ariffin, &
Mahmud, 2018) consequently, increased films thickness and also the micro-voids
are expected to be filled by the glycerol and as result, the films had become more
thicker so that inter-particle compactness was increased.

The other reason is because of glycerol is a clear compound, simply dissolves in

water, enhances the viscosity of the solution and easily binds with water. So, the
higher the addition of glycerol content, the lower the water evaporation rate
would be because some of the water in the film's solution is bounded by glycerol
so that it significantly affected the thickness of developed films (Khairunnisa &
Rostini, 2018).

Transmittance is mainly depended on the film's thickness and hence based on

equation 3.10 as film thickness increases, transparency of films would be
decreased (Mazur, Domaradzki, Wojcieszak, & Song, 2010). The largest film
thickness was obtained at the higher glycerol concentration of 35%w/v which is
around 0.43±0.002 as listed in Appendix 4.3 (López et al., 2015).

The figure clearly had shown that, as the glycerol concentration increases, almost
all the thickness of the films was increased (Khairunnisa & Rostini, 2018). Samples
with a higher content of pottery clay had a lower film thickness this is due to the
formation of porous media on the films which prevent polymer molecular chains
from coming close and compact together. The addition of glycerol content
during the development of packaging film also resulted in increasing film
thickness.

This observation is consistent with Sudaryati et al. (2010) that improvement of

thickness due to the addition of glycerol is because the glycerol molecules will
occupy the micro-voids in the matrix and strongly interact with the film-forming
polymer, which causes the distance between the polymer increases thus
improving the film thickness.

Bourtoom (2007) has reported that the plasticizer (glycerol) added to the
developing of packaging film can bind with starch to form starch plasticizer
polymer so that the starch-starch bond is substituted by the starch-glycerol-
starch bond which led to the improvement of the film thickness. Nemet et al.
(2010) have reported that the film-forming solution with higher concentrations of
glycerol has a higher dry matter content resulting in a thicker film.

Moreover, high glycerol concentration can increase the ability to absorb moisture
to some extent and can cause an increase in film thickness due to the swelling
process (Vieira et al., 2011; Ahmadi et al., 2012). D) Effects of glycerol and micro-
pottery clay on the film's transparency Figure 4.7 Effects of glycerol and pottery
clay on the film transparency As it was observed in fig 4.7

the maximum transparency value was observed at 10%w/w micro-pottery clay

content which is around 4.93±0.178. As the clay content increases from 0%, 5%,
7% and 10% , the pattern of light passing through the film (%T) was increased
from 0.51±0.140, 1.36±0.210, 1.54±0.109, 2.01±0.035, 3.11±0.321, 4.93±0.178.
Based on the observed results glycerol has an antagonistic effect on the film's
response as compared with that of micro-pottery clay. The increased glycerol
concentration resulted in a decrease in light transmission. Lower UV light
transmission is probably due to the different molecular weight, composition and
size of glycerol concentration that impedes the light transmission properties of
those (Nor, M. H. M., Nazmi, N. N. M. and *Sarbon, 2017). A similar tendency was
reported by (Nor et al., 2017). According to figure 4.7

initially, it was observed that film transparency slightly decreased as glycerol

concentration increases this is because of the increase in glycerol concentration
resulted in an increase in film swelling properties and hence more denser films
that prevent the light from being passing though. Transparency values represent
the level of clarity of the films produced.

Films translucency can be affected by various factors including the biopolymer

type, additives, and thickness. The experimental results with respect to film
transparency ranged between 4.93±0.178 to 0.51±0.140 as presented in appendix
4.3. The film's transparency value was reduced when its glycerol concentration
was increased this is due to the increase in film thickness.

The average results of the analysis in each treatment are presented in Appendix
4.3 which had shown that the treatment of glycerol concentration had a
significant effect on the film's response which implies that as glycerol content
increases, the transparency of the films was decreased (Sad, 2014). The
transparency values of each film produced in this study experienced decreased in
the range of 4.93±0.178 to 0.51±0.140 as the glycerol concentration increased
from 0 to 35%w/v. Transparency of films highly affected by film thickness.

The higher the thickness values of the films would have resulted in an increase in
diffusion of light so that the film object would appear as more turbid and hence
the brightness was observed much lower than its original appearance. The thicker
the film will give a color that is not transparent and looks less attractive. In
addition, the rise of viscosity would affect the increase in the thickness of the
films.

This is due to the increase in film thickness which in turn reduces its
translucency/clearness so that the transparency value is reduced (Khairunnisa &
Rostini, 2018). The higher glycerol concentration caused the color of the
developed film to become blurred so that the brightness level that passing
through into the film were decreased this is due to the increase in film thickness
which preventing the light passing through the film and makes the film to
become more blurred(Khairunnisa & Rostini, 2018).

E) Effect of glycerol and micro-pottery clay on the film's tensile strength

Mechanical properties reflect the durability of films and their ability to enhance
the mechanical integrity of foods. Tensile strength is the supreme tensile force
that can be held by the films. Films with high tensile strength are desired for food
packaging that helps to protect foodstuffs during handling, transporting, and
marketing (Khairunnisa & Rostini, 2018). Figure 4.8 Effect of glycerol and pottery
clay on films tensile strength As we can see in figure 4.8

addition of glycerol concentration over 25%, molecules in starch-based films

situated between the films bonding chain and be able to interact by forming
hydrogen bonds in the bond between the film chains, causing interactions
between the molecules of films have become less and caused a decline in films
tensile strength (from 7.66±0.012 to 0.95±0.012) as presented in Appendix 4.3
table 4.

This is due to the increased speed of viscoelastic response and molecular mobility
of the polymer chains (Series & Science, 2018). The best film was obtained at its
compatibility limit with ingredients aspect ratios of which five grams of starch
with 5% micro-pottery clay content and 25% glycerol concentration results in an
optimum tensile strength of 7.667±0.012 Mpa as it could be found in Appendix
4.3 table 6.

Based on this result it is absolutely possible to say that the film was highly
resistant between the chains as compared with other treatments due to strong
intermolecular hydrogen bonding to the group which caused molecular bonds
between amylose to be more compact between the chains as compared with
other treatments (Sciences & Medan, 2018). The tensile strength of the films
gradually decreases from 7.66Mpa to 3.82Mpa) as glycerol concentration
increases over 25%w/v to 35% due to over plasticization above its critical limit led
to films had become more flexible but, less resistant.

In fact films, tensile strength was decreased as glycerol concentration increases

after the second four treatments. For more information, it is also found in
Appendix 4.3 table 6. This is because the higher the concentration of glycerol
results in the higher the elongation value, but it could not have expected any
resistant.
According to (Khairunnisa & Rostini, 2018) the use of glycerol tends to reduce the
tensile strength and increase the percentage elongation of the films because
plasticizers can reduce intermolecular forces and increases the mobility of
biopolymer chains. The glycerol can also reduce internal hydrogen bonds of
molecules and lead to the weakening of the intermolecular attractive forces
adjacent to polymer chains, thereby reducing the tensile breaking.

From the analysis of the result, it has been observed that film tensile strength was
decreased over 5% micro-pottery clay content because of the less binding
capacity of the natural pottery clay as compared with glycerol. Based on the
results in figure 4.10, above 25%w/v addition of glycerol has an antagonistic
effect with that of pottery clay.

The above shows that with increasing glycerol content greater than 25% cause
the tensile strength to be decreased because glycerol can increase the distance
between the molecules (or glycerol reduces the intermolecular interaction forces
between molecules) that make the films become more flexible but less resistant.
With more pottery clay (5- 8%w/w according to the literature) added to the film,
then the elongation will be dropped, but the tensile strength is increased
(Manshor et al., 2018).

An increase in tensile strength till its compatibility limit (5% pottery clay, 25%
glycerol) was observed because there is a strong interaction between
compositions of the film that is starch molecules with micro-pottery clay and
glycerol. This bonds happened between starch molecules with pottery clay is
more tight and compact so that the film is more strong enough and difficult to
stretchable and elongated, it will reduce the percentage of film elongation.

The addition of plasticizer in low concentration to medium concentration (1-

20%) facilitated the formation of a crystal in a starch film, which is direct to anti-
plasticizer behavior. This is due to movement from polymer chain which causes
water molecules, and glycerol disappears slowly, it impacted the amylose and
amylopectin from strong hydrogen bonding, so it happens recrystallization or
retrogradation (Manshor et al., 2018). According to Harahap et'al.

(2014) which reported that micro-clay reinforced films in matrices are uniformly
dispersed and the uniform distribution of micro-clay as a filler in matrices plays
an important role in improving film mechanical properties. F) Effects of glycerol
and micro-pottery clay on the film's degradability The increase in glycerol from
0% to 35% content resulted in an increased in moisture content which can in turn
influenced film biodegradation in various ways due to the essential requirement
of water for growth and multiplication of microbes.

Hence, film degradation speed is enhanced in the presence of sufficient moisture

due to rapid microbial action. In addition, moisture-rich conditions support the
process of hydrolysis by generating more chain scission reactions. The
breakdown of the films mainly depends on its chemical composition, which
assists the growth of microorganisms in the form of nutrient sources.

The starch-based packaging film is favorable for the microbial attack, and
hydrolytic enzymes act on the film matrix to reduce their weight (V. M. Pathak,
2017). Figure 4.9 Effects of glycerol and pottery clay addition against on
degradability The biodegradability test was done based on the soil burial method
for two weeks. The simplest quantitative methods to characterize the occurrence
of biodegradation of a polymer is to determine the mass loss of the polymer
material (Bp, 2009).

Mass loss was determined by weighing the mass of the films before and after
biodegradation process for two weeks. The greater the mass loss means the
faster the degradation of the films would have resulted. As the micro-clay content
increases from 0 to 35%, the degradation rate (the mass loss) of the films were
decreased from 74.38±0.012, 59.93±0.141, 58.37±0.105, 46.62±0.031,
44.76±0.221 …9.91±0.109 as it can be observed in Appendix 4.3 this is due to the
micro clay results strong compactness within and among polymer chains.

However, glycerol has an antagonistic effect on the film's degradation rate as

compared with the addition of the clay. The mass loss was increased with
increasing concentrations of glycerol because high glycerol concentration caused
films to be hydrophilic with an active hydroxyl -OH group which can initiate the
hydrolysis reaction (which leads growth of micro-organism), so that the films
easily degraded into small pieces (Manshor et al.,

2018). The increase in glycerol concentration also led to an increase in film

moisture content which can affect film biodegradation rate in different ways due
to the vital requirement of water for the growth and multiplication of microbes.

Hence, film degradation rapidity is enhanced in the presence of sufficient

moisture due to swift microbial action. Furthermore, moisture-rich conditions
support the process of hydrolysis by generating more film chain scission
reactions (Ahmed et al., 2018). Based on the obtained result in figure 4.9
degradability of the films was increased from 9.91±0.109, 15.13±0.122.
16.36±0.208, 58.37±0.105, 59.93±0.141, 74.38±0.012 as the glycerol
concentration increased from 0%, 25%, 30% and 35%w/v. According to figure 4.9,
the maximum degradability of the film was observed at glycerol concentration of
35% which found to be 74.38±0.012.

After two weeks, the samples were weighed and the percentage weight loss
(degradability) was calculated using (equation 3.14) (Bp, 2009). Glycerol is the
basic additives for the film-forming polymers. It reduces intermolecular forces,
which increase the molecular spacing and mobility of biopolymer chains.

Researches that have done earlier shows that films made without plasticizer are
extremely brittle and shattered upon handling. Polar groups (-OH) along
plasticizer chains are believed to develop polymer-plastic hydrogen bonds that
replace the polymer-polymer interactions in the biopolymer films. Due to its
small size and high polarity, glycerol is most commonly used as a plasticizing
agent (Alshehrei, 2017).

Generally, starch-based films had shown that greater biodegradability as

compared to other synthetic polymers. In addition to degrading starch-based
films, microorganisms also play an important role in the degradation of
petroleum-based polymers, even if it takes time to degrade it. Glycerol as an
additive plays an important role in the chemical process during photo-
degradation.

Such alterations affect their thermal sensitivity and UV-absorbing capacities.

Chemically sensitive films have a better biodegradability rate as compared with
other petroleum-based polymers. Likewise, thermal exposure is also involved in
the breakdown of films into simpler forms that increase the accessibility of
microorganisms (V. M. Pathak, 2017).

Table 4.2 Differences in mango seed starch and reinforced film properties Films
properties Mango-seed starch bioplastic Micro-clay reinforced bioplastic 5%w/w ,
25%w/v Solubility (%) 12.2±0.343 10.87±0.012 Transparency (%) 0.81±0.140
2.01±0.035 Moisture (%) 8.21±0.03 11.27±0.01 Thickness (MM) 0.32±0.022
0.24±0.014 Tensile strength (Mpa) 3.33±0.008 7.66±0.012 Degradability (%)
27.38±0.132 29.56±0.188 Table 4.3

Validation of the best film as compared with the previous studies Film
characterization Present study Values Previous studies Values References Density
(g/cm3) 1.58±0.233 1.315 Ginting eta'l (2018) Solubility (%) 14.5±0.051 32.76
Jawaid eta'l (2015) Moisture content (%) 15.15±0.11 32.282 Maulida eta'l (2018)
Transparency (%T) 1.38±0.012 2.98 Vera L eta'l (2010) Thickness (mm) 0.43±0.002
0.25 Ariska eta'l (2015) Tensile strength (Mpa) 7.66±0.012 5.657 Kartika eta'l
(2018) Degradability (%) 74.38±0.012 21.317 Pamungkas eta'l (2019) 4.6

Interaction Effects of both Glycerol and Micro-pottery clay on the Films overall
Properties The parameter interaction in terms of the 3D surface was studied by
using a design expert that incorporates response surface methodology. Response
surface methodology (RSM) is a collection of mathematical and statistical
techniques for empirical model building.

The analysis was carried out with a design order (quadratic) and a design model
(polynomial). I. Interaction effects of both glycerol and micro-pottery clay on the
film's solubility Figure 4.10 Interaction effects of glycerol and pottery clay on
film's solubility As it can be seen in the above figure 4.10, as the concentration of
both glycerol and micro-pottery clay increases, the response towards the center
point was increased for both independent variables.

In fact, the graph tends to more likely in the direction of glycerol axis (ruby color)
and the reverse holds true regarding the effect of pottery clay on the response
(the blurred color). The maximum value of solubility was observed at glycerol
concentration of 35%w/v which has 18.75±0.214. The ruby color indicated that
the highest values of films solubility which corresponding to the largest glycerol
concentration (35%) values this is because of its low molecular weight which led
to the free space volume between the chains and resulting strong affinity towards
water (Sanyang et al., 2015).

According to the results of ANOVA analysis in appendix 4.4 indicated that the
two independent variables had a highly significant interaction effect on the
response (p<0.05). The increment of glycerol content up to its compatibility limit,
led to an increase in film solubility whereas increment of micro-pottery clay
content had an antagonistic effect against the response as compared with
glycerol concentration increment.

This is caused by the increased amounts of insoluble fine particles from the
pottery-clay and the increasing number of bonds between molecules in a
solution of the films (Agricultural & Pangkep, 2016). The best of-fit of the model
was tested by the determination coefficient (R2) of 96.6%. The correlation and
adjusted correlation values of the response model were 0.966 and 0.935
respectively which indicated that the model is suitable to define the possible
combined effects of pottery clay and glycerol concentration on the response.

The "model f-value" of 29.811 implies that the actual association between the
response and model process variables can be navigated. In this case, X1, X2, X1X2
and X12, X22 are significant model terms. Based on the final coded equation
independent variables X1, X1X2 and X12 had a significant positive effect, as it has
a positive coefficient, on the other hand, X2 and X22 had antagonistic effects on
the response. On the basis of this correlation coefficient in (equation 4.1), it was
evident that the response (Ro) affected by the two independent variables.

As shown in the final equation in terms of a coded factor, the response (film
solubility) influenced by the linear terms (X1, X2), linear interaction terms (X1X2)
and quadratic terms (X12, X22). The final equation in terms of coded factors: Ro
=4.18 + 0.28*X1 - 0.663*X2 + 3.209*X1*X2 + 7.3*X12 - 0.341*X22 (4.1) Where X1,
X2, and Ro represent micro-pottery clay, glycerol and response respectively. II.

Interaction effects of both glycerol and micro-pottery clay on the film's moister
uptake Figure 4.11 Interaction effects of glycerol and pottery on films moisture
uptake The 3D response surface plot is a graphical representation of the
regression coded factor equation. It is plotted to know the interaction effects
between the response and the independent variables.

The maximum value of moisture uptake was observed at glycerol concentration

of 35%w/v which has a maximum value of 19.31±0.17. Each response surface
plotted for degradable bioplastic synthesis represented the possible
combinations of two test variables one at a time. The convex response surfaces
suggested that there was observed the optimal value of the response at 35%w/v
glycerol concentration (Lima, Coelho, & Contiero, 2010).

The best of-fit of the model was tested by the determination coefficient (R2) of
98.9%. In this case, the R2 value (0.989) for Eq.4.2 showed that the sample
variation for the film of 99.9 % was attributed to the independent variables. The
adjusted determination coefficient (adj. R2=0.979) was also satisfactory for
confirming the significance of the model (Lima et al., 2010). Based on figure 4.13
lower moisture value (2.98±0.23) of the film was observed at micro-pottery clay
content of 10%w/w, on the other hand, the highest moisture value (19.31±0.17)
was obtained at glycerol concentration of 35% w/v.

The more glycerol concentration results in the more uptake of moisture content
of by the film due to its accessible hydroxyl groups capable to interact with water
by hydrogen bonds and more hydrophilic nature (Zeng et al., 2005). The values of
probability(p=0.003) implies that the actual relation between the response and
the fixed model variables were highly significant (p<0.05).

In this case, X1, X2, X1X2 and X12, X22 are significant model terms. The results
show that the independent variable (X1), X1X2 and X12 had a significant positive
effect, as it had a positive coefficient, on the other hand, the linear model term
(X2) and the quadratic model term (X22 ) had an antagonistic effect on the
response. On the basis of this correlation coefficient in (equation 4.2), it was
evident that moisture content increased with increasing glycerol but decreased
with increasing micro-pottery clay (Food, 2018).

As shown in the final equation in terms of a coded factor, the response positively
and negatively influenced by the linear terms (X1, X2), quadratic terms (X12, X22)
and linear interactive terms (X1X2). The final equation in terms of coded factors:
R1 =3.02 + 2.60*X1 - 0.85*X2 + 4.19*X1*X2 + 1.49*X12 - 3.32*X22 (4.2) Where X1,
X2, and R1 represent glycerol, micro-pottery clay and response respectively. III.

Interaction effects of both glycerol and micro-pottery clay on the film's

transparency Figure 4.12 Interaction effects of glycerol and pottery clay on film's
transparency The analysis of variance (p = 0.003) indicated that the variation of
both independent fixed variables significantly affected the response (p<0.05).

The variation of one variable with respect to the other would have an
antagonistic effect against on the film response as compared with each other. In
this case, X1, X2, X1X2 and X12, X22 are significant model terms that can
negatively and positively affect the response. As shown in the final equation (4.3)
in terms of a coded factor, the response is influenced by the positive and
negative significant model terms such as the linear terms (X1, X2), linear
interactive terms (X1X2) and quadratic terms (X12, X22).

The final equation in terms of coded factors: The fit model analysis of the
response of the film gives a quadratic model with a coefficient of correlation and
adjusted correlation values of 0.878 and 0.764 respectively. On the basis of this
correlation coefficient in (equation 4.3) it was evident that the response was
increased from the center point towards in the glycerol axis this is due to the film
became thicker/denser which in turn resulted in less transparent films. The final
equation in terms of coded factors: R4 =0.45 + 0.219*X1 - 0.314*X2 +
2.271*X1*X2 - 6.21*X12 - 0.81*X22 (4.3) where X1, X2, and R4 represent glycerol,
micro-pottery clay and Response respectively. IV.

Interaction effects of both glycerol and pottery clay on the film's thickness Figure
4.13 Interaction effects of glycerol and pottery clay on film's thickness As it can
be observed from figure 4.13 the red, yellow and green colors represented the
maximum, the medium and the minimum values of the response respectively.

The increase in glycerol concentration from 0% to 35% have a great significant

effect on the response found to be increased from 0.15±0.001mm - 0.43±0.002,
but the increase in micro-pottery clay contents from 0% to 10% had antagonistic
effect on the response as compared with an increment of glycerol concentration
(Odewole, Sunmonu, Adeyinka-ajiboye, Adako, & Oyeniyi, 2017). The correlation
and adjusted correlation values of the response model were 0.868 and 0.932
respectively which indicated that the model is suitable to describe the treatment
effects of pottery clay and glycerol concentration to the film thickness (Food,
2018). On the basis of this correlation coefficient in (equation 4.4), it was evident
that the response was increased with an increase in glycerol concentration but
decreased with an increase in pottery clay content due to the formation of micro-
voids. These phenomena happened at a time.

In this case, the response can be affected by the linear model terms (X1, X2),
linear interactive terms (X1*X2) and quadratic (X12, X22) terms. The independent
variables X1, X2, X1*X2 and X12, X22 are significant model terms that can
negatively and positively affect the response. The fit model analysis of the
response of the film gives a quadratic model with a coefficient of correlation and
adjusted correlation values of 0.868 and 0.932 respectively.

The final equation in terms of coded factor is: R5 =0.171 + 0.382*X1- 0.302*X2 +
0.461*X1*X2 + 0.94**X12 - 4.21*X22 (4.4) Where X1, X2, and R5 represent
glycerol, micro-pottery clay and thickness respectively. V. Interaction effects both
glycerol and micro-pottery clay on the film's tensile strength The "model f-value"
of 2.388 implies that the model cannot be used to navigate the design space as it
is not greater than 4.0. There is a 71.08 % chance that a "model f-value" this large
could occur due to noise. The values of "Probability less than 0.050 indicates that
model terms are significant.

In this case, X1, X2, X1-X2 and X12, X22 are significant model terms. The response
can be affected by such linear model terms (X1, X2), linear interactive terms
(X1*X2) and quadratic (X12, X22) terms. The fit model analysis of the response
provides a quadratic model with a coefficient of correlation and adjusted
correlation values of 0.995 and 0.990 respectively.

On the basis of this correlation coefficient in (equation 4.5), it was evident that
films tensile strength increases towards in the micro-pottery clay direction up to
its compatibility limit (5%). Figure 4.14 Interaction effects of glycerol and pottery
clay on film's tensile strength Based on figure 4.14 increasing both glycerol and
micro-pottery clay content above their critical limit caused lower tensile strength
values due to the interaction effects among the ingredients of is not so good
hence the clay particles led an agglomeration as a result films become less
resistant (Wahyuningtiyas & Suryanto, 2018).

When glycerol and pottery clay are incorporated over their critical limit (5%w/w,
25%w/v), competition for the formation of hydrogen bonding started. As a result,
direct interactions between starch chains were partly reduced due to hydrogen
bond formation with glycerol, allowing the film chains to have more freedom of
motion (Victo, Grossmann, Alves, & Bele, 2007) so that films become less
resistant.

The above surface plot showed that the increase in micro-pottery clay and
glycerol content caused the values of the response to increase and later on the
tensile strength dropdown at the highest glycerol concentration (Odewole et al.,
2017). Tensile strength varied between 0.95±0.012 to 7.66±0.012 Mpa. This is due
to the formation of intermolecular hydrogen bonding between glycerol and -OH
groups of clay-starch which increases with the increase in concentration.

The results are in line with the previous study reported by Chinma et al. (2015)
which showed that tensile properties of starch-based films decreased with an
increase in plasticizer concentration which. in turn led to an increase in film
humidity (Thakur et al., 2017). The final equation in terms of codded factor: R6 =
0.87 + 0.70*X1 + 1.74*X2 - 0.99*X1*X2 + 0.15*X12 + 6.42*X22 (4.5) Where X1, X2,
and R6 represent glycerol, micro-pottery clay and tensile strength respectively. VI.

Interaction effects of both glycerol and micro-pottery clay on the film's

degradability Figure 4.15 Interaction effects of glycerol and pottery clay on films
degradability Based on the obtained result in figure 4.15 the increase in
ingredients amount over their critical limit resulted in an increased in film
degradability from 9.91±0.109 to 74.38±0.012 towards the glycerol axis.

This is due to the fact that starch-based films can absorb more moisture from the
surrounding so that films became more comfortable for the growth and spread
of micro-organisms during the soil burial method so that films highly consumed
by micro-organisms and disintegrated upon oxidation and radiation (Odewole et
al., 2017). The maximum degradation of the film was observed at glycerol
concentration of 35% which was around 74.38±0.012 because glycerol is a
hydrophilic molecule that can promote the growth of micro-organisms which can
use the starch as their source of food (Farahnaky et al., 2013).

Both glycerol and micro-pottery have had a significant interaction effect on the
response (p<0.05) Values of "Probability less than 0.05 indicates that model terms
are significant. In this case, X1, X2, X1-X2 and X12, X22 are significant model
terms that can negatively and positively affect the response. As it can be seen in
the final equation (4.6) in terms of a coded factor, the response is influenced by
the positive and negative significant model terms such as the linear terms (X1,
X2), linear interactive terms (X1X2) and quadratic terms (X12, X22).

The final equation in terms of coded factors: The fit model analysis of the
response of the film gives a quadratic model with a coefficient of correlation and
adjusted correlation values of 0.992 and 0.898 respectively. On the basis of this
correlation coefficient in equation 4.6, it was evident that film degradability
increases towards in the glycerol direction. The final equation in terms of coded
factors: R7 = 10.26 + 0.041*X1 + 0.063*X20.13*X1*X2 + 043* X12 + 0.14*X22 (4.6)
Where X1, X2, and R7 represent glycerol, micro-pottery clay and response
respectively. 4.7

Thermal Analysis of Films Using TGA A) TGA analysis for the best-reinforced film
Thermal analysis is a branch of materials science
<http://en.wikipedia.org/wiki/Materials_science> where the properties of
materials are studied as they change with temperature
<http://en.wikipedia.org/wiki/Temperature>. The glass transition of the material
is a change, in which the material turns from the glassy to the rubbery state
during heating (Victo et al., 2007).

Thermogravimetry is a technique measuring the variation in the mass of a sample

as a function of temperature when it undergoes temperature scanning in a
controlled, inert or oxidative atmosphere (Bp, 2009). Figure 4.16 TGA analysis for
the best-reinforced film As shown the TGA curve exhibit four mass loss stages.
The first stage: The horizontal portion of the TGA curve below 60oC showed that
there was only a little weight-loss about 2.15% was observed.

This implied that the film is thermally stable below 60oC The initial mass loss
below 60°C is due to the gradual evaporation of absorbed moisture, volatile
components (glycerol), monomers and unbound water (free water), began at
room temperature because of the dry purge gas flowing over the film. Here chain
segments are frozen in fixed positions; atoms underwent only low-amplitude
vibratory motion at those range of temperatures (Kim, Yang, Kim, & Park, 2004).

The second stage: As the temperature raised from 57.5-115.23oC in the second
stage, this portion of the TGA curve stands for the derivative weight loss curve
which told us the point at which a change of weight loss was observed around
38.25% which was most apparent. More weight loss change was observed within
this temperature range as compared to other stages.

In this stage dehydration of bound water taken place. In the crystalline portion of
the film, the changes at a glass transition temperature are less drastic. This is
because these changes are restricted mainly to the amorphous regions while the
crystalline zones remain relatively unaffected. In the third stage, there was
observed a change of weight loss of about 20.26% as the temperature shifted
from around 115.23-140oC.

Here the film chemical bonds started to break up and hence resulting in
decomposition of the sample to taken place. The decomposition of such
materials might be due to the presence of glycerol and micro-pottery clay. In this
stage, the micro-pottery clay as a filler remains as a stable residue. The rate of
decomposition of the film mainly depends on the heating rate.

The higher the heating rate, the higher the decomposition temperature would be.
The thermo-oxidative decomposition of the film happened due to the presence
of oxygen. The pyrolysis (the pure thermal decomposition) could be achieved by
using inert purge gases such as nitrogen. (Mohsin, Iannucci, & Greenhalgh, 2019).
The final stage is the film material is starting to melt and flow as the temperature
shifted from 140-175oC which had a corresponding change of weight loss of
3.32%.

In this stage, all the crystalline portions of the material are expected to be melted
at a temperature of 170.31oC. At this temperature, a total weight loss of around
94.5% was observed. The remaining percent considered as residues. B) TGA
analysis for a plasticized film which is not reinforced with pottery clay The
molecular motion in a polymer sample is encouraged by its thermal energy and
opposed by the cohesive forces between structural segments (groups of atoms)
along with the chain and between neighboring chains.
These cohesive forces and, consequently, thermal transitions in polymers depend
on the structure of the polymer (Mohsin et al., 2019). Figure 4.17 TGA analysis for
a plasticized film which is not reinforced with clay First stage: Initially, from a
temperature range of 20-45.25 oC there was not observed any significant change
of weight loss. This showed that the film is hard had brittle as a result it is highly
thermally stable.

In this glassy region, evaporation of volatile components such as plasticizer,

solvents, monomers and unbound water (free water), started at room
temperature due to overflowing of dry purge gas on the film material. Second
stage: An apparent weight loss change of 56.75% was observed at a temperature
range of around 45.25-78.24 oC.

This stage is known as the transition stage in which the film material transits from
the hard and brittle glass into a softer, rubbery state that occurred over that
narrow temperature range referred to as the glass transition temperature.
Another key consequence of a glass transition in film structures is an increase in
molecular mobility due to the existence of glycerol which may lead to a decrease
in transition temperature as compared with the best-reinforced film in the
previous TGA curve (Khazaei et al., 2014).

The presence of this glycerol as a plasticizer probably made the polymer

molecules more moveable (Khazaei et al., 2014). The weight loss in this region
related to complex processes including the dehydration of the polymer rings,
depolymerization, and glycerol evaporation. The decrease in transition and
melting temperature on the overall process accompanied by the presence of
glycerol content and had been observed in other studies too (Khazaei et al.,
2014).

The formation of crosslinking of in the rubbery plateau caused significant

improvement of film thermal stability as it has been observed in the glassy state
(Shayan, Azizi, Ghasemi, & Karrabi, 2019). The third stage is a rubbery state, here
almost all the mass loss is nearly constant at which temperatures ranging from
78.24-102.15oC, here more chains entangled and flexible.

From the results and those from the literature, the type of starch does not
significantly influence the thermal properties of films (Basiak et al., 2017). In the
case of a partially crystalline polymer, solid-to-liquid phase transformation occurs
only in the amorphous regions. The crystalline zones remain unchanged and act
as reinforcing elements thus making the sample hard and tough.

The equilibrium crystalline melting point, for polymers, corresponds to the

temperature at which the last crystallite started to melt. The formation of
crosslinking in the rubbery plateau caused significant improvement of film
thermal stability as it has been observed in the glassy state (Shayan et al., 2019).
The last region is the viscous flow region; in this region, a higher change of
weight loss around 57.9% was observed within a temperature range of 102.15-
140 oC. Here almost all amorphous and crystalline portions are expected to be
converted into a viscous liquid (140.53oC).

In this region, nearly 90% of the sample weight was lost as the operating
temperature reached its equilibrium melting point (140.53oC). The decrease in
the melting point (as compared to the previous TGA curve ) may be caused by a
decrease in the number of hydroxyl groups in the polymer molecules which can
form strong hydrogen bonds among polymer chains (Khazaei et al., 2014). Here
the total weight loss of a sample was around 98.75%.

The negligible weight loss of about 1.25% should have accounted for ashes and
filler glass fibers. Hence, the carbon is virtually unaffected at 200oC (Mohsin et al.,
2019). 5 CONCLUSION AND RECOMMENDATION 5.1 Conclusion From the study
carried out the development and characterization of degradable bioplastics from
mango seed kernel starch reinforced with micro-pottery clay as a filler, the
following conclusions pointed out.

Mango seed kernel is generated as a waste during mango fruit processing can be
effectively treated as a by-product to extract starch. Starch extracted from this
mango seed kernel used as a source of raw material for the development of
degradable films that finds an application in many food packaging industries and
can be readily converted into useful products.

According to the results obtained from the analysis of variance for a multivariate
response, the statistical models showed that all data points well fitted a curve or
line. Response surface methodologies involving central composite designs have
been successfully applied to evaluate the interaction effects of both glycerol and
micro-pottery clay on the film's overall properties. The mango seed kernels
starch/micro-pottery clay results in an increased in film tensile strength from
3.33±0.008MPa for the pure starch film to a starch reinforced with micro-pottery
clay could have an optimum tensile strength of 7.66±0.012MPa.
The improvement of films with micro-pottery clay could be attributed to the
strong hydrogen bond between hydroxyl groups of the interface of both micro-
pottery clay and starch matrix. The film thicknesses were increased (from
0.15±0.001 to 0.43±0.002 mm) as the glycerol concentration increased from
0%w/v to 35%w/w. The film with the highest tensile strength (which was found to
be at micro-pottery clay content of 5% w/w and glycerol concentration of 25%
w/v) anticipated having better characteristics than the other films. The highest
film degradability was obtained at glycerol concentration of 35%w/v which is
around 74.38±0.012.

This result has been shown that as the plasticizer concentration increases, film
degradability was also increased. It mainly implied that the higher the glycerol
content, the higher the degradability of film was observed. That is films would
not take more time to degrade by any. The influence of glycerol content and
micro-pottery clay on the film's overall properties have been studied.

Generally altering the glycerol concentration and pottery clay allows an increase
or decrease in the physical, chemical and functional properties of the developed
films. This work has shown that how glycerol and micro-pottery clay can strongly
affect the functional properties of the mango seed kernel starch/clay-based
packaging films.

The higher the glycerol content, the lower the film thermal stability. The TGA
results clearly indicated that thermal degradation of the starch films is relevant
features in order to convert this biopolymer into materials with useful and
desirable properties which can be important for different packaging applications.

The TGA analysis was conducted under a flow rate of 50ml/min and a heating
rate of 10°C with a sample amount of 2mg approaches with an operating
temperature range of 200°C. This analysis is also able to forecast and estimate
the shelf life of the materials. Thermal analysis was performed for plasticized and
reinforced films. during the analysis, it was observed that the plasticized film was
less stable and decomposed at low temperature, in addition, it has a low
transition and melting temperature as compared with that of reinforced film. 5.2

Recommendation From this paper, it was clear that degradable bioplastic could
be produced using mango seed kernel starch reinforced with micro-pottery clay
as a filler and glycerol as a plasticizer. Even though degradable bioplastic is
produced, further investigations of the following points can be suggested. The
present study was done by treating the pottery clay, but further research works
should be importantly focused on the composition of the clay using composition
analyzer devices such as gas chromatography.

To investigate the effects of various gelatinization temperatures during the

gelatinization process, further research works necessarily advice to use vacuum
aspiration so as to remove air bubbles which resulted from above its optimum
gelatinization temperature. It should be also noted that the extrusion process is
necessarily advised in order to produce homogenous/evenly distributed
suspensions to control the weight of each individual film.

The extruder is an excellent mixing device and particularly suitable for the
processing of highly viscous fluids that can produce a more consistent quality of
the products. The present study has been done for none edible film for
packaging application, it is also advised importantly focusing on the synthesis of
edible film for food packaging exclusively micro-pottery clay and characterizing
all the properties as it was in the present study and including the barrier
properties (water vapor permeability, Oxygen permeability, and Carbon dioxide
permeability).

Based on the present study the developed degradable bioplastic can be used for
food packaging at a small scale so that future study researchers shall importantly
focus on the feasibility study for food packaging on a large scale. The present
study was done at the micro level so that the future study will be a concern on
Nanoscale production and characterization of the films to improve the quality,
mechanical and physicochemical properties of the reinforced film so as to meet
the desired properties of the films.

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