CHAPTER ONE

1.0 INTRODUCTION

Pectin is an important component of the middle lamella and primary cell wall of higher

plants. Pectins are high molecular weight acidic heteropolysaccharide primarily made up of α

(1−4) linked d-galacturonic acid residues (Kavuthodi and Sebastian, 2018). Three major

pectic polysaccharides groups are recognized, all containing d-galacturonic acid to a greater

or a lesser extent. They are homogalacturonan (HG), rhamnogalacturonan I (RGI), and

rhamnogalacturonan II (RGII) (Javed et al., 2018). Pectinases are a group of enzymes that

degrade pectic substance and are classified according to their mechanism of action. For

example, methylesterases remove methoxy groups from highly or partially esterified

galacturonan. Polygalacturonases catalyze the hydrolysis of the glycosidic bonds in a random

fashion (endopolygalacturonase) or from the nonreducing end of homogalacturonan releasing

galacturonic or digalacturunic acid residues (exopoly-glacturonases) (Carassco et al., 2019).

Pectinolytic enzymes, or pectinases, are also classified according to their mode of action and

their substrate: polygalacturonases, which are sub classified as endo-polygalacturonases (E.C.

3.2.1.15) and exo-polygalacturonases (E.C. 3.2.1.67); lyases, which are sub classified into

pectatelyases (E.C. 4.2.2.9 and EC. 4.2.2.2) or pectin lyases (E.C. 4.2.2.10); and pectin

methylesterases (E.C. 3.1.1.11). It is recommended to use a combination of different kinds of

pectinases, along with other enzymes such as cellulases and hemicelullases, as multiple

enzymes can degrade different parts of the polymer, resulting in the maximal degradation of

the pectin in various raw materials such as in citrus juice processing (Cornuault et al., 2018).

Studies have reported that pectinase of microbial origin accounts for 25% of global food and

industrial enzymes sale and their market is increasing continuously (Cornuault et al., 2018).

Additionally, enzymes comprise a well-established global market projected to reach USD 6.3

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billion in 2021 (Cornuault et al., 2018). Microorganisms including fungi are promising

sources of enzymes. Fungi produces numerous extracellular enzymes that possess a special

effect in the decomposition of organic matter. These include pectinolytic enzymes which are

excreted to break down the middle lamella in plants so that it can insert fungal hyphae and

extract nutrients from the plant (Oumer and Abate, 2018). In addition to fungi, pectinolytic

enzymes are naturally produced by many other organisms like bacteria, insects, nematodes,

and protozoans (Garg et al., 2016). For the commercial production of pectinases, Aspergillus

spp., Erwinia spp., Bacillus spp., and Penicillium spp. have been extensively used (Kubra et

al., 2018). Pectinases have crucial roles in food industries. These enzymes are useful for fruit

juice extraction and wine clarification; tea, cocoa, and coffee concentration and fermentation;

vegetable oil extraction; preparation of jam and jellies; and pickling (Patidar et al., 2019).

Furthermore, these enzymes are used in paper and pulp industries, bleaching of JJ, bio-

scouring of cotton, retting and degumming of plant fibers, oil extraction, wastewater

treatment, poultry feed additives, protoplast fusion technology, and bioenergy production

(Patidar et al., 2019). Enzyme breakdown of the biomolecules depends upon the type of

microorganisms, fermentation condition such as pH, incubation time or cultivation time,

carbon and nitrogen source, types and concentration of substrate, temperature, agitation, and

use of different enzyme preparations [(Patidar et al., 2019).

1.1 Statement of the Research Problem

Recently, there has been a large increase in industrial applications of enzymes owing to their

important biotechnological prospect. Pectic enzymes are widely spread in nature and are

produced by bacteria, yeast, fungi and plants. Many extracellular enzymes are produced by

fungi which are capable of decomposing organic matter and one such enzyme is pectinolytic

enzymes. Therefore, by means of breaking down pectic substances for nutrition, microbial

pectinases play an important role in nature.

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1.2. Justification for the Study

The utilization of low-cost agro-industrial wastes as substrates has been preferable in

pectinase production. Pectinase production faced various parameters optimization constraints

such as temperature, pH and production times which are the main factors in pectinase

production. The pectinase enzyme is getting attention due to its several advantages; hence, it

needs to be explored further to take its maximum advantage in different industries.

1.3 Aim of the Study

The aim of this study is to producing pectinase producing fungi through submerged

fermentation.

1.4 Objectives of the Study

The objectives of this research are;

 To study the course of pH during fermentation

 To quantify the amount of pectinase produced by the fungal isolate

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Pectinase

Pectinases are a heterogeneous group of related enzymes that hydrolyze the pectin

substances, present mostly in plants. Pectic enzymes are widely distributed in nature and are

produced by bacteria, yeast, fungi and plants (Babu and Bayer 2017). In plants, pectic

enzymes are very important since they play a role in elongation and cellular growth as well as

in fruit ripening (Jansirani et al., 2018). Pectolytic activity of microorganisms plays a

significant role, firstly, in the pathogenesis of plants since these enzymes are the first to

attack the tissue. In addition, they are also involved in the process of symbiosis and the decay

of vegetable residues. Thus by breaking down pectin polymer for nutritional purposes,

microbial pectolytic enzymes play an important role in nature (Yadav et al., 2019). These

enzymes are inducible, produced only when needed and they contribute to the natural carbon

cycle.

2.2 Classification of Pectinases

According to the cleavage site, pectinases are divided into three groups: (1) hydrolases

consisting of polygalacturonase, PG (EC 3.2.1.15); (2) lyase/transeliminases comprising

pectinlyase, PNL (EC 4.2.2.10), and pectate lyase, PL (EC 4.2.2.2); (3) pectin esterase, PE

(EC 3.1.1.11) (Yadav et al., 2019).

2.2.1. Pectinesterase (PE)

Pectin methyl esterase or pectinesterase (EC 3.1.1.11) catalyzes deesterification of the

methoxyl group of pectin forming pectic acid and methanol. The enzyme acts preferentially

on a methyl ester group of galacturonate unit next to a non-esterified galacturonate unit. It

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acts before polygalacturonases and pectate lyases which need nonesterified substrates (Yadav

et al., 2019).

2.2.2 Polygalacturonase (EC 3.2.1.15)

Polygalacturonases (PGases) are the pectinolytic enzymes that catalyse the hydrolytic

cleavage of the polygalacturonic acid chain with the introduction of water across the oxygen

bridge (Yadav et al., 2019).

2.2.3. Pectatelyase (EC 4.2.2.2)

Pectatelyase (PGL) cleaves glycosidic linkages preferentially on polygalacturonic acid

forming unsaturated product through transelimination

 Pectin lyase (EC 4.2.2.10)

Pectin lyase catalyzes the random cleavage of pectin, preferentially high esterified pectin,

producing unsaturated methyloligogalacturonates through transelimination of glycosidic

linkages. PLs do not have an absolute requirement of Ca2+ but they are stimulated by this

and other cations (Gupta, 2015).

2.2.4 Production of Microbial Pectinase

Microorganisms are currently the primary source of industrial enzymes: 50% originate from

fungi and yeast; 35% from bacteria, while the remaining 15% are either of plant origin. The

microbial world has shown to be very heterogeneous in its ability to synthesize different

types of pectolytic enzymes with different mechanisms of action and biochemical properties

(Murad and Azzaz, 2017). There were two fermentation techniques we can use for pectinases

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production, as many other enzymes. These techniques are Solid State Fermentation (SSF) and

submerged fermentation (SmF).

2.2.4.1 Solid state fermentation

Solid state fermentation is defined as the cultivation of microorganisms on moist solid

supports, either on inert carriers or on insoluble substrates that can be used as carbon and

energy source. This process occurs in the absence or near absence of free water in the space

between substrate particles. In this system, water is present in the solid substrate whose

capacity for liquid retention varies with the type of material (Murad and Azzaz, 2017).

2.2.4.2 Submerged fermentation

In contrast, in submerged fermentation (SmF) the nutrients and microorganisms are both

submerged in water. Approximately 90% of all industrial enzymes are produced in SmF,

frequently using specifically optimized, genetically manipulated microorganisms. In this

respect SmF processing offers an insurmountable advantage over SSF. SSF has several

advantages over SmF system such as higher concentration of products, less effluent

generation, requirement for simple equipments etc. The price of commercially available

enzymes which are produced mostly by submerged fermentation is usually too high for agro-

biotechnological applications. An alternative technique of enzyme production is solid state

cultures (Murad and Azzaz, 2017).

2.3 Application of Microbial Pectinases

Application of enzymes in biotechnological process has expanded considerably in recent

years. In food and related industry, major importance was being attached to the use of

enzymes in upgrading quality, increasing yields of extractive processes, product stabilization,

and improvement of flavor and by product utilization (Murad and Azzaz, 2017). Pectinases or

pectinolytic enzymes are today one of the upcoming enzymes of the commercial sector. It has

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been reported that microbial pectinases account for 25% of the global food enzymes sales

(Gupta, 2015). On the bases of their applications, pectinases are mainly of two types: acidic

pectinases and alkaline pectinases (Murad and Azzaz, 2017).

2.3.1. Acidic Pectinases

Acidic pectic enzymes used in the fruit juice industries and wine making often come from

fungal sources, especially from Aspergillus niger (Rai et al., 2018). Potential applications of

Acidic pectinase are briefly described below.

2.3.1.1 Fruit juice clarification/extraction

Fruit juice clarification/extraction is one among the important applications of acidic

pectinases Fruit juices contain colloids that may lead to fouling problem during filtration

process and these colloids are basically polysaccharides such as pectin and starch (Rai et al.,

2018). Pre-treatment of juices with pectinases is performed to lower the amount of pectin

present and to decrease the viscosity of the juice, which in turn accelerates the subsequent

filtration process. Also, it helps to increase the clarity of the juice.

2.9.1.2 Tissue maceration

Tissue maceration is another important application of acidic pectinases in which organized

tissue is transformed into a suspension of intact cells and it is significant in the food industry

as well as in the field of biotechnology. The process can be applied for the liquefaction and

saccharification of biomass, isolation of protoplasts (Rai et al., 2018).

2.3.1.3 Wine processing

Wine processing industry also recognizes the importance of acidic pectinases (Roldán et al.,

2018), where the enzyme can be applied at different stages. The addition of pectinases during

crushing of the fruits increases the juice yield and also accelerates the release of anthocyanins

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into the juice. Pectinase treatment at the pre-fermentation or fermentation stage, settles out

suspended particles. After fermentation, enzyme is added to the wine to increase its clarity

and filtration rate (Rai et al., 2018).

2.3.2 Alkaline pectinases

Alkaline pectinases are mainly used in the degumming and retting of fibeber crops and

pretreatment of pectic wastewater from fruit juice industries. These enzymes come mostly

from bacterial sources (Rai et al., 2018). In the industrial sector, alkaline pectinases, mainly

from Bacillus spp. are applied for the following purposes.

2.3.2.1 Paper and pulp industry

During papermaking, pectinase can depolymerisepectins and subsequently lower the cationic

demand of pectin solutions and the filtrate from peroxide bleaching (Reid and Ricard, 2015).

2.3.2.2 Retting and degumming of plant bast fibers

Bast fibers are the soft fibers formed in groups outside the xylem, phloem or pericycle, e.g.

Ramie and sun hemp. The fibers contain gum, which must be removed before its use for

textile making. The chemical degumming treatment is polluting, toxic and non-

biodegradable. Biotechnological degumming using pectinases in combination with xylanases

presents an eco-friendly and economic alternative to the above problem. Pectinases have been

used in retting of flax to separate the fibers and eliminate pectins (Reid and Ricard, 2015).

2.3.2.3 Textile processing and bio-scouring of cotton fibers

Pectinases have been used in conjunction with amylases, lipases, cellulases and hemi-

cellulases to remove sizing agents from cotton in a safe and eco-friendly manner, replacing

toxic caustic soda used for the purpose earlier. Bio-scouring is a novel process for removal of

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noncellulosic impurities from the fiber with specific enzymes. Pectinases have been used for

this purpose without any negative side effect on cellulose (Reid and Ricard, 2015).

2.3.2.4 Pectic waste water treatment

The wastewater from the citrus-processing industry contains pectinaceous materials that are

barely decomposed by microbes during the activated-sludge treatment have tried to develop a

new wastewater treatment process by using an alkalophillic microorganism. Pretreatment of

these wastewaters with pectinolytic enzymes facilitates removal of pectinaceous material and

renders it suitable for decomposition by activated sludge treatment (Reid and Ricard, 2015).

2.3.2.5 Animal feed

Pectinases are used in the enzyme cocktail, used for the production of animal feeds. This

reduces the feed viscosity, which increases absorption of nutrients, liberates nutrients, either

by hydrolysis of non-biodegradable fibers or by liberating nutrients blocked by these fibers,

and reduces the amount of faeces (Gupta, 2015).

2.3.2.6 Oil extraction

Citrus oils such as lemon oil can be extracted with pectinases. They destroy the emulsifying

properties of pectin, which interferes with the collection of oils from citrus peel extracts

(Gupta, 2015).

2.3.2.7 Purification of plant viruses

Pectinases have also been reported to work on purification of viruses. But they are yet to be

commercialized. When virus particle is restricted to phloem, to release the virus from the

tissues, alkaline pectinases and cellulases are used. This gives very pure preparations of the

virus (Reid and Ricard, 2015).

2.3.2.8 Coffee and tea fermentation

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Fermentation of coffee using pectinolytic microorganisms is done to remove the mucilage

coat from the coffee beans and to enhance the tea fermentation and foam forming property of

tea. Fungal pectinases are also used in the manufacture of tea. Enzyme treatment accelerates

tea fermentation, although the enzyme dose must be adjusted carefully to avoid damage to the

tea leaf. Large-scale treatment of coffee with commercial pectinases is costly and

uneconomical, inoculated waste mucilage is used as a source of microbial pectin enzymes.

The fermentation liquid is washed, filtered and then sprayed on to the beans (Gupta, 2015).

2.4 Pectin

Pectin is a structural heteropolysaccharide contained in the primary cell walls of terrestrial

plants. It was first isolated and described in 1825 by Heneri Bracannot. Pectin, a

multifunctional constituent of cell wall is a high value functional food ingredient widely used

as gelling agent and as stabilizer (Choudhari and Bhatia, 2018). It is produced commercially

in form of white to light brown powder, mainly extracted from citrus fruits, and is used in

food as a gelling agent particularly in jams and jellies. It is also used in fillings, sweets, as a

stabilizer in fruit juices and milk drinks and as a source of dietary fiber (Pinheiro et al.,

2018). In plant cells, pectin consists of a complex set of polysaccharides that are present in

most primary cell walls and particularly abundant in the non-woody parts of nearly all

terrestrial plants. Pectin is present not only in the primary cell walls but also in the middle

lamella between plant cells where it helps to bind the cells together. The amount, structure

and chemical composition of the pectin differs between plants, within a plant over time and

in different parts of a single plant. During ripening, pectin is broken down by the enzymes

pectinase and pectin esterase, resulting in the process where the fruit becomes softer. This is

because the middle lamella which primarily consists of pectin breaks down and cells become

separated from each other. A similar process of cell separation caused by pectin breakdown

occurs in the abscission zone of the petioles of deciduous plants at the time of leaf fall

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(Pinheiro et al., 2018). Pectin is thus also a natural part of human diet, but does not

contribute significantly to nutrition. As the literature reports, the daily intake of pectin from

fruit and vegetables can be estimated to be around 5 g Srivastava et al., 2018). In human

digestion, pectin goes through the small intestine more or less intact but is acted upon by

microbial growth of large intestine. Pectin thus acts as a soluble dietary fibre. Consumption

of pectin has been shown to reduce blood cholesterol levels. The mechanism appears to be an

increase of viscosity in the intestinal tract, leading to a reduced absorption of cholesterol

from bile or food (Srivastava et al., 2018). In the large intestine and colon, microorganisms

degrade pectin and liberate shortchain fatty acids that have favorable influence on health

(also known as prebiotic effect).

2.4.1 Structure of Pectin

In terms of structure, pectin is an essentially linear polysaccharide. Like most other plant

polysaccharides, it is both polydisperse and polymolecular and its composition varies with

the source and the conditions applied during isolation. In any sample of pectin, parameters

such as the molecular weight or the contents of particular subunits differ even from molecule

to molecule. The composition and structure of pectin are still not completely understood

although pectin was discovered over 200 years ago (Choudhari and Bhatia, 2018). Through

various studies it has been brought in notice that the structure of pectin is difficult to

determine because pectin subunit composition can change during isolation from plants,

storage, and processing of plant material (Srivastava et al., 2018). At present, pectin is

thought to consist mainly of Dgalacturonic acid (GalA) units, joined in chains by means of α-

(1-4) glycosidic linkage. These uronic acids have carboxyl groups, which are naturally

present as methyl esters and others which are commercially treated with ammonia to produce

carboxamide groups (Srivastava et al., 2018). Units range in number from a few hundred to

about thousand saccharides in a chain-like configuration which corresponds to average

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molecular weights from about fifty thousand to one lack fifty thousand Dalton (Silva et al.,

2018). As the literature reports, into pectin backbone (made up of glycosides), galacturonic

acid is replaced by (1-2)-linked L-rhamnose, at some distinguishing areas. From the

rhamnose residues, side chains of various neutral sugars have been discovered to branch off.

This type of pectin is termed as rhamnogalacturonan I. Here, up to every twenty fifth

galacturonic acid in the main chain is replaced with rhamnose. The neutral sugars found in a

pectin molecule are mainly D-galactose, L-arabinose and D-xylose, whose types and

proportions vary with the origin of pectin (Silva et al., 2018).

The X-ray fibre diffraction studies have reported that the galacturonan segments in the

molecule of sodium pectate form helixes with three subunits per turn. The conformation of

Galacturonic acid units as determined by NMR spectroscopy and referred from literatures is

4C1 (Supjaroenkul et al., 2019). Calculations indicate that the helix is probably right-handed.

It was indicated that X-ray fibre diffraction patterns of sodium and calcium pectates, pectic

acids, and pectinic acids show the same helix structure, but the ways in which these helixes

were arranged relative to each other in the crystals differ to various degrees. It has been

suggested that helical pectinic acid molecules pack in a parallel arrangement, whereas the

pectates pack as corrugated sheets of antiparallel helixes (Yujaroen et al., 2019).

Another structural type of pectin is rhamnogalacturonan II, which are comparatively less

frequent complexes and a highly branched polysaccharide (Figure 2). This type of isolated

pectin has reported molecular weight of 60-130,000 g/mol, varying with origin, extraction

conditions and age of plant (Yujaroen et al., 2019). In nature, around 80% of carboxyl groups

of galacturonic acid are esterified with methanol. This proportion is although reported to

decrease more or less during pectin extraction. The ratio of esterified to non-esterified

galacturonic acid determines the behavior of pectin in food applications. On this behalf,

pectins are classified as high-ester or low-ester pectins; rather in short, HM (high-methoxy)

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versus LM (low-methoxy) pectins, with more or less than half of all the galacturonic acid

esterified (Yujaroen et al., 2019).

2.5 Sources of pectin

2.5.1 Plants Cell Wall

Pectin is a complex mixture of polysaccharides that makes up about one third of the cell wall

of dry substance of higher plants. Much smaller proportions of these substances are also

found in the cell walls of grasses. The highest concentrations of pectin are found in the

middle lamella of cell wall, with a gradual decrease as moving through the primary wall

toward the plasma membrane (Waszczynskyj and Wosiacki, 2015). Although pectin occurs

commonly in most of the plant tissues, the number of sources that may be used for the

commercial manufacture of pectin is limited. This is because; the ability of pectin to form gel

depends on the molecular size and degree of esterification (DE).

2.5.2 Fruits

Detection of a large quantity of pectin in a fruit alone is not in itself enough to qualify that

fruit as a source of commercial pectin (Waszczynskyj and Wosiacki, 2015). At present,

commercial pectins are almost exclusively derived from citrus peel or apple pomace, both of

which are by-products from juice manufacturing units. Apple pomace contains 10-15% of

pectin on a dry matter basis. Citrus peel contains relatively higher i.e. 20-30% of pectin as

compared to that of apple.

From an application point of view, citrus and apple pectins are largely equivalent (Srivastava

et al., 2018). Among the physical properties, citrus pectins are light cream or light tan in

colour whereas apple pectins are often darker. Alternative sources for pectin extraction

include sugarbeet waste obtained from sugar manufacturing, sunflower heads (seeds used for

edible oil), and mango waste (Waszczynskyj and Wosiacki, 2015).

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 Apples, quince, plums, gooseberries, oranges and other citrus fruits contain much more

pectin as compared to soft fruits like cherries, grapes and strawberries contain little pectin

(Srivastava et al., 2018). Typical levels of pectin in plants are (fresh weight): Apples, 1-1.5;

Apricot, 1; Cherries, 0.4; Oranges, 0.5-3.5; Carrots, approx. 1.4 and Citrus peels, 30%.

2.6 Classification of Pectic Substances

Based on the type of modifications of the backbone chain, The American Chemical Society

classified pectic substances into four main types as protopectin, pectic acid, pectinic acid and

pectin (Tapre and Jain, 2015).

2.6.1. Protopectin

This is a parent pectic substance and upon restricted hydrolysis yields pectin or pectinic acid.

Protopectin is occasionally a term used to describe the water-insoluble pectic substances

found in plant tissues and from which solublepectic substances are produced (Tasgin et al.,

2020).

2.6.2 Pectic Acids

These are the galacturonans that contain negligible amounts of methoxyl groups. Normal or

acid salts of pectic acid are called pectates (Tasgin et al., 2020).

2.6.3. Pectinic Acids

These are the galacturonans with various amounts of methoxyl groups. Pectinates are normal

or acid salts of pectinic acids. Pectinic acid alone has the unique property of forming a gel

with sugar. The salts of pectinic acids are either normal or acid pectinates. Under suitable

conditions, pectinic acids are capable of forming gels with sugars and acids or if suitably low

in methoxyl content with certain metallic ions (Tasgin et al., 2020).

2.6.4 Pectin (polymethylgalacturonate)

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It is polymeric material in which, at least, 75% of the carboxyl groups of the galacturonate

units are esterified with methanol. It confers rigidity on cell wall when it is bound to cellulose

in the cell wall. Pectins are the soluble polymeric materials containing pectinic acids as the

major component. They can form insoluble protopectins with other structural polysaccharides

and proteins located in the cell wall (Tasgin et al., 2020). Pectin substance consists of pectin

and pectic acid. Demethylated pectin is known as pectic acid or polygalacturonic acid.

2.7 Extraction procedures of Pectin

The most commonly used method for extraction of pectin includes direct boiling and

microwave heating (Kumar et al., 2018). Direct boiling is a conventional method of pectin

extraction, which takes approximately two hours to obtain a good yield of pectin. Due to a

relatively long period of direct heating, the extracted pectin undergoes thermal degradation

(Kumar et al., 2018). Microwave heating extraction, on the other hand, takes no more than

fifteen minutes to extract a satisfactory amount of pectin. Methods employing microwave

heating are generally more effective in term of pectin yield and give better quality products as

well. The yield of pectin also depends on the types of extraction solvents used and the use of

extra chelating agents such as EDTA and CDTA which helps in releasing pectin from cell

wall.

2.7.1 Water based extraction

The conventional water based extraction involves extracting the pectin using acidified water

(pH up to 2) at temperature not more than 70°C. The assembly is run for 2-4 h duration and

pectic substances are precipitated using ethanol or isopropyl alcohol (Metha et al., 2018).

A long list of various agents has been reported for the extraction of pectin from plant tissues.

Extraction with the hot water is the simplest and oldest method for removing the pectic

substances. The most commonly used acidifying materials are mineral acids including,

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sulfuric, hydrochloric and phosphoric acids. Many organic acids and their salts such as oxalic

acid, ammonium oxalate, tartaric acid, polyphosphates, and many others have been also used.

Meyer’s and Rouse reported the use of different types of zeolites for the extraction of pectin.

A very low yield of pectin obtained from dried orange peel was reported using Zeocarb as

extractant at 85-90°C (Metha et al., 2018). Double extraction at 85-88°C for one hour using a

cationic resin for the extraction of pectin from apple pomace has been reported to give higher

yields and better gel strength of the product. IER has been extensively used in the purification

and fractionalization of pectin.

Commercially, pectin is extracted by treating the raw material with hot dilute mineral acid at

pH about two (Metha et al., 2018). The precise length of extraction time varies with raw

material, the type of pectin desired, and from one manufacturer to another. The hot pectin

extract is separated from the solid residue as efficiently as possible. This is not easy since the

solids are by now soft and the liquid phase are viscous. The viscosity increases with pectin

concentration and molecular weight. There is a compromise between efficient extraction and

solids separation and operating cost. The pectin extract may be further clarified by filtration

through a filter aid. The clarified extract is then concentrated under vacuum. Powdered pectin

can be produced by mixing the concentrated liquid from either apple or citrus with an alcohol

(usually isopropanol). The pectin is separated as a stringy gelatinous mass, which is pressed

and washed to remove the mother liquor, dried and grounded.

2.7.2 Microwave heating extraction

Extraction of pectin from raw papaya (Carica papaya Linn.) peel, citrus peel, acerola, apple

pomace, etc. has been reported using water based extraction procedure to procure pectic

substances on large scale in industries (Srivastava et al., 2018).

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Microwave extraction has been carried out on a Milestone Ethos Microwave Lab Station.

Additional solvents used in microwave extraction included 10% ethanol, 0.05M

ethylenediamine tetra acetic acid, 1 M sodium hydroxide which was used to maintain pH up

to 2 or less. Compared with water based extraction microwave heating extraction reduces the

extraction period considerably. A fifteen minute microwave heating period is enough to

extract almost the same amount of pectin as that obtained from water based extraction with a

three hour extraction period (Metha et al., 2018).

During microwave heating considerable pressure builds up inside a material. The high

pressure then modifies the physical properties of material tissues, breaking down the cell

structure and improving the capillary porous structure of tissues. This feature allows better

penetration of extracting solvent into the tissues; improving the subsequent extraction of

pectin. Microwave extraction also gave a higher rate and amount of extraction than the

simple water based methods.

2.8 Applications of pectin

2.8.1 Mucoadhesive polymer

Different types of pectin were characterized for gastrointestinal (GI) mucoadhesion

(Sriamornsak, 2016). The mucoadhesive process involved in the formation of bioadhesive

bonds has been described in three steps: (i) wetting and swelling of polymer to permit

intimate contact with biological tissue, (ii) interpenetration of bioadhesive polymer chain and

entanglement of polymer and mucin chains and (iii) formation of weak chemical bonds

between entangles chains (Sriamornsak, 2016). Thus this property of pectin is exploited in

preparation of mucoadhesive patches in combination with other mucoadhesive polymer like

carbopol and chitosan.

2.8.2 Gelling agent, thickner and water binder

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Pectins are mainly used as gelling agents, but can also act as thickener, water binder and

stabilizer. The degree of esterification can be incompletely reduced using commercial pectin

methylesterase, leading to a higher viscosity and firmer gelling in the presence of Ca2+ ions.

Highly (2-O- and/or 3-O-galacturonic acid backbone) acetylated pectin from sugar beet is

reported to gel poorly but have considerable emulsification ability due to its more

hydrophobic nature, but this may be due to associated protein impurities. But sugar beet

pectin has different chemical features than citrus pectin, so it could find new uses, especially

in industrial products (Marshal and Chow, 2017).

As with other viscous polyanions such as carrageenan, pectin may be protective towards

milk casein colloids, enhancing the properties (foam stability, solubility, gelation and

emulsification) of whey proteins whilst utilizing them as a source of calcium. But the main

use for pectin still remains as a gelling agent, thickening agent and stabilizer in food1. The

classical application is giving the jelly-like consistency to jams or marmalades, which would

otherwise be sweet juices. For household use, pectin is an ingredient in jelling sugar where it

is diluted to the right concentration with sugar and some citric acid to adjust pH.

In some countries, pectin is also available as a solution or an extract, or as a blended powder,

for home jam making (Marshal and Chow, 2017). For conventional jams and marmalades

that contain above 60% sugar and soluble fruit solids, high-ester pectins are used. With low-

ester pectins and amidated pectins less sugar is needed, so that diet products can be made.

Pectin can also be used to stabilize acidic protein drinks, such as drinking yogurt, and as a fat

substitute in baked goods. Typical levels of pectin used as a food additive is between 0.5-1.

0% this is about the same amount of pectin as in fresh fruit.

2.8.3 Pectin in medicine and pharmaceutical industry

18
In medicine, pectin increase viscosity and volume of stool that it is used against constipation

and diarrhoea. Until 2002, it was one of the main ingredients used in Kaopectate, along with

kaolinite. Pectin is also used in throat lozenges as a demulcent. In cosmetic products, pectin

acts as stabilizer. Pectin is also used in wound healing preparations and especially in medical

adhesives, such as colostomy devices (Marshal and Chow, 2017).

2.8.4 Dietary fibre

In ruminant nutrition, depending on the extent of lignification of the cell wall, pectin is up to

90% digestible by bacterial enzymes. Ruminant nutritionists recommend that the digestibility

and energy concentration in forages can be improved by increasing pectin concentration in

the forage (Helene et al., 2015).

2.8.5 Natural prophylactic

Pectin acts as a natural prophylactic substance against poisoning with toxic cations. It has

been shown to be effective in removing lead and mercury from the gastrointestinal tract and

respiratory organs. When injected intravenously, pectin shortens the coagulation time of

drawn blood, thus being useful in controlling hemorrhage or local bleeding (Mannish et al.,

2018). Pectin and combinations of pectin with other colloids have been used extensively to

treat diarrhoea, especially in infants and children. Although a bactericidal action of pectin has

been proposed to explain the effectiveness of pectin treating diarrhoea, most experimental

results do not support this theory. However, some evidence suggests that under certain in-

vitro conditions, pectin may have a light antimicrobial action toward Escherichia coli

(Mannish et al., 2018).

2.8.6 Treatment of overeating

Pectin reduces rate of digestion by immobilizing food components in the intestine. This

results in less absorption of food. The thickness of the pectin layer influences the absorption

19
by prohibiting contact between the intestinal enzyme and the food, thus reducing the latter’s

availability (Mannish et al., 2018). Due to its large water binding capacity pectin gives a

feeling of satiety, thus reducing food consumption47. Experiments showed a prolongation of

the gastric emptying half-time from 23 to 50 minutes of a meal fortified with pectin. These

attributes of pectin are used in the treatment of disorders related to Overeacting (Mannish et

al., 2018).

2.8.7 Controlled release formulations

Pectin hydrogels have been used in tablet formulations as a binding agent and have been

used in controlled-release matrix tablet formulations (Mannish et al., 2018). Pectin has been

investigated HM-pectins for their potential value in controlled release matrix formulations

(Fassihi, 2017). The application of a binary polymer system, i.e. HM-pectin and

hydroxypropyl methylcellulose, in drug release rate modulation for oral administration was

studied. Pectin beads prepared by the ionotropic gelation method were used as a sustained

release drug delivery system (Fassihi, 2017). However, the use of these beads has some

drawbacks due to their rapid in vitro release. By changing the DE of LM-pectin, the drug

release pattern from calcium pectinate gel beads can be modified. Since pectin can react with

calcium ions, calcium pectinate has been investigated as an insoluble hydrophilic coating for

sustained release delivery by interfacial complexation process. The spherical pellets, which

contain calcium acetate, were prepared using an extrusion spheronisation method and then

coated in a pectin solution. An insoluble and uniform coating of calcium pectinate gel was

formed around the pellets. The use of pectin to develop other oral controlled release drug

delivery systems has been reported by some authors. Srivastava et al., (2018) demonstrated

the use of orange peel derived pectin as an efficient polymer to act as good binding agent

during the formulation of dosage form.

20
2.8.8 In site specific targeting

Pectin has a promising pharmaceutical uses and is presently considered as a carrier material

in colon-specific drug delivery system as indicated by the large number of studies published

over the last few years (Fassihi, 2017). The potential of pectin or its salt as a carrier for

colonic drug delivery was also studied. The rationale for this is that pectin and calcium

pectinate will be degraded by colonic pectinolytic enzymes, but will retard drug release in the

upper gastrointestinal tract due to its insolubility and because it is not degraded by gastric or

intestinal enzymes (Mannish et al., 2017). It has also been demonstrated that pectin-

degrading bacteria, Klebsiella oxytoca, could adhere to a film casted of low methoxylated

pectin. The ability of the bacteria to adhere to the films, however, was not correlated with

their ability to degrade pectin. When the dissolution of pectin matrix tablets was analyzed

with and without K. oxytoca, a significant retardation in the dissolution rate was observed in

the presence of K. oxytoca, suggesting the formation of a biofilm on the matrix or

sedimentation of insoluble pectin salts.

Thus, pectin is an interesting constituent for pharmaceutical use, e.g. as a carrier of a variety

of drugs for controlled release applications. Many techniques have been used to manufacture

the pectin-based delivery systems, especially ionotropic gelation and gel coating. These

simple techniques, together with the very safe toxicity profile, make pectin an exciting and

promising excipient for the pharmaceutical industry for present and future applications

(Mannish et al., 2017).

21
 CHAPTER THREE
3.0 MATERIAL AND METHODS
3.1 Study Area
The site chosen for experiment is Microbiology Laboratory, Kwara State University, Malete,

Kwara State. Malete is a populated place in Kwara State, Nigeria with the region font code of

Africa/Middle east. It is located at an elevation of 308 meters above sea level and its

population amounts to 102,708) (Ajayi et al., 2021).

3.2 Source of Isolates

Primarily screened isolate was gotten from Microbiology Lab, Kwara State University,

Malete, Kwara State.

3.3 Inoculum Preparation

The inoculum was prepared using the method described by Lateef and Gueguim-Kana

(2012), by transferring a loopful mycelium of the fungal strain from the plate into a sterilized

50 ml inoculum medium in a 250 ml capacity flask. The inoculum medium consisted of 0.1g

of sucrose and 0.5g of yeast extract at final pH of 5.5. 24hours old fungal strain was

introduced into the flasks and were sealed aseptically. The flask was incubated at 30±2 °C on

a shaker at 100 rpm for 24 hours to develop the inoculum (Lateef and Gueguim-Kana, 2012),

3.4 Submerged Fermentation

22
Submerged fermentation (SmF) technique was employed, using the method described by

Ezugwu et al. (2014), 10g of citrus pectin was dissolved in 1000ml of distilled water in five

250ml capacity flasks, 3g of di-Ammonium hydrogen orthophosphate (NH 4)2HPO4, 2g of

potassium di-hydrogen orthophosphate (KH2PO4), 3g of di-Potassium hydrogen phosphate

(K2HPO4), 0.1g of Magnesium Sulphate (MgSO4) was added and the pH of the medium was

adjusted to pH 5.0 using 0.4ml of Hydrochloric acid (HCL). The flask was sterilized at 121

degree Celsius for 15 minutes in the autoclave. 4ml of 24 hours old inoculum was introduced

into the flasks and was incubated at 30+2 degree Celsius on a shaker at 100 revolution per

minutes for 120 hours in order to develop the inoculum. The samples were withdrawn at

24hrs interval. At the end of fermentation, the samples were filtered through Whatman No.1

filter paper and the filtrate was used as the crude enzyme. The filtrates were analysed daily

for pectinase activity till the 5th day of fermentation (Ezugwu et al., 2014).

3.5 Pectinase Assay

Pectinase activity was evaluated using a method described by Ezugwu et al. (2014), by

preparing 1ml of 1% pectin in 01M sodium acetate buffer at pH 5.5 and was reacted with 1ml

of the crude enzyme. The reaction mixture was incubated in a water bath at 50 degree Celsius

for 30 minutes and the reaction was terminated by the addition of 1ml of 3,5-dinitro salicylic

acid (DNS) to the reaction mixture followed by heating in a boiling water bath at 100 degree

Celsius for 10 minutes. 5ml of distilled water was added after heating and allowed to cool.

The absorbance was read at 540nm using a spectrophotometer. The blank consisted of 1ml of

0.5% pectin, 1ml of sodium acetate buffer and 1ml of DNS reagent and 5ml of distilled

water. Polygalacturonic standard curve was used and one unit (U) of pectinase activity was

defined as the amount of enzyme that released 1.0 µmol reducing sugar as polygalacturonic

equivalent per minute in the reaction mixture under the specified assay conditions (Ezugwu

et al., 2014).

23
.

CHAPTER FOUR

4.0 Result

4.1 Course of pH value during enzyme production

The course of pH value of Aspergillus flavus at pH 5.5 during enzyme production are

presented in Table 1. The pH value ranged from 4.67± 0.03 to 3.0± 0.00 during 120 hours of

fermentation.

4.2 Course of pectinase activity of Aspergillus flavus during 120hours of fermentation

The course of enzyme activity of Aspergillus flavus during 120hours of fermentation are

presented in Table 2. The enzyme activity ranged from 10.0 µmol/min to 1.0 µmol/min

during 120 hours of fermentation.

24
 5

4.5

3.5

2.5
pH

1.5

0.5

0
24hours 48hours 72hours 96hours 120hours
Fermentation period (hours)

Figure 3. Course of pH value during enzyme production

25
 12

10
Pectinase activity µmol/mg/min

0
24hours 48hours 72hours 96hours 120hours

Fermenatation period (hours)

Figure 4. Course of pectinase activity of Aspergillus flavus during 120hours of

fermentation

26
 CHAPTER FIVE

5.0 Discussion

This research evaluated the amount of pectinase activity by fungal isolate using submerged

fermentation. Factors that affects enzyme production includes pH, temperature, moisture

content, and fermentation time. Primarily screened fungi was identified as Aspergillus flavus.

The pH range for enzyme production by Aspergillus flavus in this study was from 4.67± 0.03

to 3.0± 0.00 at pH 5.5. This agrees with the study carried out by Islam et al. (2013) reported a

pH range of 3-5.0 at pH 5.5. The pH affects the stability and activity of enzyme produce by

microorganisms as well as their membrane permeability and biosynthesis (Islam et al.,

2013)). During the fermentation period of 5days, daily evaluation of pectinase activity by the

organism were carried out and results used to generate daily specific activity. Highest

pectinase activity of 10 U/mg/mol/mins was obtained on the 3rd day, making the 3rd day the

maximum day of pectinase production (Figure 2). The result of this project is agreement with

that of Abdulmumini et al. (2021) who observed optimum production of polygalacturonase at

fermentation period of 72hrs, from Aspergillus flavus grown on grape fruit. Generally,

fermentation period of a microorganism depends majorly on the composition of the substrate

and biochemical characteristics of the strain like types of enzyme produced, cell division rate,

27
such as its growth rate, moisture availability, temperature, oxygen concentration and

inoculum size.

5.1 Conclusion

The study has highlighted that Aspergillus flavus isolate is a good source for producing

pectinases on cheap carbon source in short incubation period during submerged state of

fermentation process.

5.2 Recommendation

Further study should be carried out on the production, characterization and purification of

pectinases for the industrial use of this highly demanding enzyme in Nigeria as well as all

over the world. This fungal strain could be considered under genetic modification for

enormous production of pectinolytic enzymes, pectinase.

28
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32
 APPENDIX

Preparation of Polygalacturonic Acid Standard Curve

A stock solution of polygalacturonic acid was prepared by weighing 1g of polygalacturonic

acid into 100ml of sterile distilled water and then allowed to dissolve. Ten sterile test tube

were selected and labelled respectively as 1-10. One millilitre of polygalacturonic acid stock

33
solution was introduced into sterile test tube containing 9ml of distilled water. 2ml of

polygalacturonic acid solution was introduced into test tube containing 8ml of sterile distilled

water. 3ml of polygalacturonic acid stock solution was introduced into test tube containing

7ml of distilled water, 4ml of polygalacturonic acid stock solution was introduced into sterile

test tube containing 6ml of distilled water, 5ml of polygalacturonic acid stock solution was

introduced into sterile test tube containing 5ml of distilled water, 6ml of polygalacturonic

acid stock solution was introduced into sterile test tube containing 4ml of distilled water, 7ml

of polygalacturonic acid stock solution was introduced into sterile test tube containing 3ml of

distilled water, 8ml of polygalacturonic acid stock solution was introduced into sterile test

tube containing 2ml of distilled water, 9ml of polygalacturonic acid stock solution was

introduced into sterile test tube containing 1ml of distilled water, 10ml of polygalacturonic

acid stock solution was introduced into last test tube without distilled water. After this 8ml

was taken

from each test tube respectively and discarded, and all the test tubes were left 2ml contents.

Two millilitre of 3-5 dinitrosalicylic acid reagent was introduced into each test tube

respectively and then boiled at 100 degree Celsius for 10 minutes after which they were

allowed to cool. Six millilitre of sterile distilled water was added into each test tube

respectively and the absorbance were taken using a spectrophotometer at 540nm wavelength.

The amount of reducing sugar was extrapolated from polygalacturonic standard curve. One

unit of pectinase activity (UI) was defined as the amount of enzyme that released 1mg of

polygalacturonic acid in 1minute under standard assay condition (Adeleke et al., 2012).

34
 2.5

2
Absorbance reading (540nm)

1.5

1 abs
Linear (abs)

0.5

0
0 2 4 6 8 10 12
Concentration of Polygalacturonic acid (mole/ml)

Standard curve for polygalacturonic acid

Blank concentration=1.82, Unknown Absorbance= (9.61075)

35
Colonies of Aspergillus flavus on PDA plate.

36
37