CHAPTER ONE

1.0 INTRODUCTION

Pectin is high molecular weight acid polysaccharide, primarily made up of α-(1-4) linked

Dgalacturonic acid residues with a small number of rhamnose residues in the main chain and

arabinose, galactose and xylose on its side chain (Galiotou-Panayotou et al., 2013). Pectin is

found in the plant cell wall where it contributes to cell wall rigidity (Steven et al., 2000). Three

major pecticpolyssacharide groups are recognized with all containing D-galacturonic acid to a

greater or a lesser extent (Abe et al., 2008). They are homogalacturonan (HG) which is a linear

polymer formed by D-galacturonic acid which can be acetylated and/or methyl esterified.

Rhamnogalacturonan I (RG I) is composed of the repeating disaccharide rhamnose-galacturonic

acid. Rhamnogalacturonan II (RGII) is a homogalacturonan chain with complex side chains

attached to the galacturonic residues. Industrially extracted pectin is used for pectinase

production using Aspergillus species. Pectinase is a generic name for a family of enzymes that

catalyze hydrolysis of the glycosidic bonds in the pectic polymers (Yogesh et al., 2009).

Pectinases are classified into Pectin Methyl Esterases (PME) or pectinesterases,

Polymethylgalacturonases (PMG), Polygalacturonases (PG), Pectatelyases (PGL) and Pectin

Lyases (PL) (McCready, 2010). Pectinase is sourced from different genera of bacteria, yeasts and

moulds but Erwinia, Bacillus, Saccharomyces, Kluyveromyces, Aspergillus, Penicillium,

Fusarium and Rhizopusare the genera most frequently used over the years, with strains of

Aspergillus, Penicillium and Erwiniamainly used for enzyme production studies (Aschoff et al.,

2015). The choice for microbial source for pectinase production depends on the type of culture

required for their production, (solid-state or submerged fermentation), number and type of the

produced pectinases (esterases, hydrolytic depolymerases and eliminative depolymerases), pH

1
and thermal stability of the enzymes, and genotypic characteristic of the strain (wild type,

mutagenized strain, and homologous or heterologous recombination) (Chadha et al., 2005).

Pectinases are applied in several conventional industrial processes, such as textile, plant fibre

processing, oil extraction, treatment of industrial waste water, containing pectinacious material

etc. Pectinases have also been reported to work on purification of viruses and in making of paper

(Reena et al., 2005). The frequent use of fruits such as mango, orange and pineapple for

production of juices, nectars, concentrates, jams, jelly powders and flakes generate lots of wastes

in the form of peel wastes and seed kernels which could bring about environmental pollution

(Bali, 2003). The current study is focused on harnessing the wastes generated from fruits juice

industry in the form of peels into pectin. The pectins extracted from those peals as the only

carbon source for Aspergillus fumigates and Aspergillus niger in submerged fermentation system

for pectinase production. The optimal pH and temperature for for pectinase industrial activity

where also determined.

1.1 Aim of the study

This study is aim to determine the effect of pH and temperature on the activity of pectinase

obtained from submerged fermentation of orange pectin by Aspergillus niger.

1.2 Specific objective of the study

 To isolate pectinolytic fungus, using microbial culture.

 To identify the fungal species using its microscopic and microscopic features.

 To produce pectinase from submerged fermentation of orange pectin by the fungus

(Aspergillus niger).

 To purify the crude pectinase via G200 gel filtration chromatography

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 To determine the effect of pH on pectinase activity, using industrial citrus pectin as

substrate.

 To determine the effect of temperature on pectinase activity, using industrial pectin as a

carbon source.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Orange fruits

All citrus trees belong to the single genus Citrus and remain almost entirely interfertile. This

includes grapefruits, lemons, limes, oranges, and various other types and hybrids. As the

interfertility of oranges and other citrus has produced numerous hybrids and cultivars, and bud

mutations have also been selected, citrus taxonomy is fairly controversial, confusing or

inconsistent (Aguilar and Huirton, 2010). The fruit of any citrus tree is considered a hesperidium,

a kind of modified berry; it is covered by a rind originated by a rugged thickening of the ovary

wall. Different names have been given to the many varieties of the species. Orange applies

primarily to the sweet orange – Citrus sinensis (L.) Osbeck. The orange tree is an evergreen,

flowering tree, with an average height of 9 to 10 m (30 to 33 ft), although some very old

specimens can reach 15 m (49 ft). Sweet oranges grow in a range of different sizes, and shapes

varying from spherical to oblong. Inside and attached to the rind is a porous white tissue, the

white, bitter mesocarp or albedo (pith). The orange contains a number of distinct carpels

(segments) inside, typically about ten, each delimited by a membrane, and containing many

juice-filled vesicles and usually a few seeds (pips) (Sinclair et al., 2005). When unripe, the fruit

is green. The grainy irregular rind of the ripe fruit can range from bright orange to yellow-

orange, but frequently retains green patches or, under warm climate conditions, remains entirely

green. Like all other citrus fruits, the sweet orange is non-climacteric. The Citrus sinensis group

is subdivided into four classes with distinct characteristics: common oranges, blood or pigmented

oranges, navel oranges, and acidless oranges.

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Other citrus groups also known as oranges are:

 Mandarin orange (Citrus reticulata) is an original species of citrus, and is a progenitor of

the common orange.

 Bitter orange (Citrus aurantium), also known as Seville orange, sour orange (especially

when used as rootstock for a sweet orange tree), bigarade orange and marmalade orange.

Like the sweet orange, it is a pomelo x mandarin hybrid, but arose from a distinct

hybridization event.

 Bergamot orange (Citrus bergamia Risso), grown mainly in Italy for its peel, producing a

primary essence for perfumes, also used to flavor Earl Grey tea. It is a hybrid of bitter

orange x lemon.

 Trifoliate orange (Poncirus trifoliata), sometimes included in the genus (classified as

Citrus trifoliata). It often serves as a rootstock for sweet orange trees and other Citrus

cultivars (Philip et al., 2013).

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Plate 1: Orange fruit. (Bai et al., 2016)

2.1.1 Products made from oranges

Orange juice is obtained by squeezing the fruit on a special tool (a juicer or squeezer) and

collecting the juice in a tray underneath. This can be made at home or, on a much larger scale,

industrially. Brazil is the largest producer of orange juice in the world, followed by the United

States, where it is one of the commodities traded on the New York Board of Trade (Widmer et

al., 2009). Frozen orange juice concentrate is made from freshly squeezed and filtered orange

juice

2.1.2 Uses of orange

Sweet orange oil is a by-product of the juice industry produced by pressing the peel (Bai et al.,

2014). It is used for flavoring food and drinks and also in the perfume industry and aromatherapy

for its fragrance. Sweet orange oil consists of approximately 90% D-limonene, a solvent used in

various household chemicals, such as wood conditioners for furniture and along with other citrus

6
oils detergents and hand cleansers (McCready, 2010). It is an efficient cleaning agent with a

pleasant smell, promoted for being environmentally friendly and therefore, preferable to

petrochemicals. D-limonene is, however, classified as irritating to the skin and as very toxic to

aquatic life in different countries. Marmalade preserves are traditionally made with Seville

oranges, which are less sweet. All parts of the fruit are used: the pith and pips (separated and

placed in a muslin bag) are boiled in a mixture of juice, slivered peel, sliced-up flesh, sugar, and

water to extract their pectin, which helps the conserve to set. Gardeners use orange peel as a slug

repellent (Albersberg et al., 2003).

2.4 Nutrition One medium orange weighing 131g

 61.6 calories

 0.16 g of fat

 237 milligrams of potassium

 15.4 g of carbohydrate

 12.2 g of sugar

 1.23 g of protein (Frair et al., 2007)

The same orange provides the following percentages of a person’s daily requirement of several

essential vitamins and minerals, according to United States Department of Agriculture

guidelines:

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Nutrient Percentage of daily requirement for adults

Vitamin C 92.93% for females and 77.44% for males

Thiamin 10.36%

Folate 9.83%

Fiber At least 9.34%, depending on age and sex

Calcium Between 4.36% and 5.24%, depending on age

Potassium 5.04% (Albersberg et al., 2009).

Oranges also contain choline and zeaxanthin.

Choline is an important nutrient in oranges that helps with sleep, muscle movement, learning,

and memory. Choline also aids the transmission of nerve impulses, assists in the absorption of

fat, and reduces chronic inflammation. Zeaxanthin is a type of carotenoid antioxidant that can

reduce inflammation (Reena et al., 2005). it can positively benefit heart, liver, skin, and eye

health.

2.2 Pectin

The plant cell wall is a complex macromolecular structure that surrounds and protects the cell,

and is a distinguishing characteristic of plants essential to their survival (Alkorta et al., 2008). As

a consequence of limited mobility, plants are plastic in their ability to withstand a variety of

harsh environmental conditions and to survive attack by pathogens and herbivores. The structure

formed by the polysaccharides, proteins, aromatic, and aliphatic compounds of the cell wall

8
enables plants to flourish in diverse environmental niches. Cell wall structure is continually

modified to accommodate the developmental stage and the environmental condition. The plant

cell lays down the middle lamella and the primary wall during initial growth and expansion of

the cell (Albersberg, et al., 2003). In many cells, the wall is thickened and further strengthened

by the addition of a secondary wall. The primary wall is thought to contribute to wall structural

integrity, cell adhesion, and signal transduction (Perez-Cacho and Rouseff, 2008). The structural

constituents of a young plant cell wall are cellulose, hemicellulose and pectic substances (Alonso

et al., 2003). The cellulose micro fibrils provide strength to the cell wall, while hemicelluloses

and pectic substances act as the cementing substance for the cellulose network. Pectins or pectic

substances contribute to complex physiological processes like cell growth and cell differentiation

and so determine the integrity and rigidity of plant tissue. Pectic substances are polysaccharides

of high molecular weight, with a negative charge, appearing mostly in the middle lamella and the

primary cell wall of higher plant (Penniston et al., 2008). They are formed by a central chain

containing a variable amount although in high proportion of galacturonic acid residues linked

through α-(1-4) glycosidic bonds partially esterified with methyl groups. This molecule is known

as pectin, while the demethylated molecule is known as polygalacturonic acid or pectic acid.

Pectin was discovered in 1790 by Vauquelin and later in 1825 crudely characterized by

Braconnot (Amorim and Amorim, 2007). Compared with young, actively growing tissues,

lignified tissues have a low content of pectic substances. The content of the pectic substances is

very low in higher plants usually less than 1%. They are mainly found in fruits and vegetables,

constitute a large part of some algal biomass (up to 30%) and occur in low concentration in

forestry or agricultural residues (McCready, 2010).

2.2.1 Sources of pectin

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Pectin is naturally found in a number of plants namely: lemon peel, orange peel, apple pomace,

carrots, sunflower-heads, guava, mangoes and papaya (Arenas-Ocampo et al., 2003). The

European countries, Switzerland and USA largely produce pectin either from apple pomace or

peels of citrus fruits. Evaluation and standardization of pectin is based on its ‘Gelly-Grade’ that

is, its setting capacity by the addition of sugar. Usually, pectin having ‘gelly grade’ of 100, 150

and 200 are recommended for medicinal and food usuages (Galiotou-Panayotou and Kapantai,

2013).

2.2.1.1 Biological Sources Pectin: is the purified admixture of polysaccharides, obtained by

carrying out the hydrolysis in an acidic medium of the inner part of the rind of citrus peels, for

instance: Citrus limon (or Lemon) and Citrus aurantium belonging to the family Rutaceae, or

from apple pomace Malus sylvestris Mill (Syn: Pyrus malus Linn, family: Rosaceae).

2.2.1.2 Geographical Source: Lemon and oranges are mostly grown in India, Africa and other

tropical countries. Apple is grown in the Himalayas, California, many European countries and

the countries located in the Mediterranean climatic zone. One of the richest sources of pectin is

lemon or orange rind which contains about 30% of this polysaccharide (Aschoff et al., 2015).

2.2.2 Structure of pectin

Pectins are a family of covalently associated galacturonic acid-rich plant cell wall

polysaccharides. About 70% of all pectin contains galacturonic acid and all pectin

polysaccharides contain galacturonic acid linked at the O-1 and O-4 positions of the polymer.

The structural elements of pectin are classified into two families; galacturonans and

rhamnogalacturonan. Galactorunans consist of a backbone of α-(1,4)-linked D-galacturonic acid

residues (El Hadi et al., 2013). The branched can either be branched or unbranched. The

10
backbone in rhamnogalacturonans, however, contains diglycosyl repeating units of α-L-

rhamnose-(1,4)-α-D-galacturonic acid. The rhamnose residues are ramified at the O-4 and O-3

positions with polymeric side chains that include arabinose and galactose residues at other

positions. In pectin, four different types of polymeric side chains might exist; arabinans,

galactans, type I arabinogalactans, and type II arabinogalactans. The chemical structure of pectin

is extremely complicated as it contains as many as 18 different monosaccharides linked together

by twenty different linkages (Chadha et al., 2005). The overall structure of pectin is explained in

terms of smooth and hairy regions. The smooth regions contain linear chains of homo or

heteropolymer, whereas the hairy region contains simple or complex side chains (Bai et al.,

2016).

2.2.2.1 Galacturonans: The galacturonans found in pectin can either be homogalacturonans or

hetero galacturonans. Homogalacturonans form the smooth region of pectin, that is unbranched

chains of α-(1,4)-linked D-galacturonic acid residues that might be methyl or acetyl-esterified.

Homogalacturonans accounts form about 60% of all pectin found in different living beings. In

the case of heterogalacturonans, the homopolysaccharide chain is more or less heavily

substituted at O-2 and O-3 by monomers or dimers of xylose, resulting in xylogalacturonan. If

the polymer is substituted with complex side chains like rhamnose, it forms

rhamnogalacturonans.

2.2.2.2 Rhamnogalacturonans: Some of the pectins might exist as rhamnogalacturonan

consisting of a long chain of alternating L-rhamnose and D-galacturonic acid residues (Albers et

al., 2004). In some cases, the rhamnose residues might even be replaced by a variety of L-

arabinosyl and D-galactosyl-containing side chains. A small number of glucuronic acid and 4-O-

11
methyl glucuronic acid residues might be present. Rhamnogalacturonans account for about 20-

35% of the total pectin content in nature, but the amount is as high as 75% in the soybean plant.

Figure 1: Chemical structure of pectin (Aschoff et al., 2015).

2.2.3 Classification of pectin

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

classified pectic substances into four main types as:

2.2.3.1 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 (Neill, 2001).

2.2.3.2 Pectin (polymethylgalacturonate): It is polymeric material in which, at least, 75% of

the carboxyl groups of the galacturonate units are esterified.

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2.2.3.3 Pectic Acids: These are the galacturonans that contain negligible amounts of methoxyl

groups. Normal or acid salts of pectic acid are called pectates.

2.23.4 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 (Chadha

et al., 2005).

2.2.4 Properties of pectin

Appearance: Coarse or fine- powder

Colour: Yellowish white

Odour: Practically odourless

Taste: Mucilaginous taste

Solubility: 1. Completely soluble in 20 parts of water forming a solution containing negatively

charged and very much hydrated particles. 2. Dissolves more swiftly in water, if previously

moistened with sugar syrup, alcohol, glycerol or if first mixed with 3 or more parts of sucrose.

Chemical Constituents Pectin occurs naturally as the partial methyl ester of a (1→4) linked (+)

– polygalacturonate sequences interrupted with (1–2) – (–) – rhamnose residues. The neutral

sugars that essentially form the side chains on the pectin molecules are namely: (+) – galactose,

(–) –arabinose, (+) – xylose, and (–) – fructose. (Bai et al., 2014)

2.2.5 Production of pectin

The specific method of preparation of pectin is solely guided by the source of raw material i.e.,

lemon/orange rind or apple pomace; besides the attempt to prepare either low methoxy group or

high methoxy group pectins. In general, the preserved or freshly obtained lemon peels are gently

boiled with approximately 20 times its weight of fresh water maintained duly at 90ºC for a

13
duration of 30 minutes. The effective pH (3.5 to 4.0) must be maintained with food grade lactic

acid/citric acid/tartaric acid to achieve maximum extraction. Once the boiling is completed the

peels are mildly squeezed to obtain the liquid portion which is then subjected to centrifugation to

result into a clear solution. From this resulting solution both proteins and starch contents are

suitably removed by enzymatic hydrolysis. The remaining solution is warmed to deactivate the

added enzymes. The slightly coloured solution is effectively decolourized with activated carbon

or bone charcoal. Finally, the pectin in its purest form is obtained by precipitation with water-

miscible organic solvents (e.g., methanol, ethanol, acetone), washed with small quantities of

solvent and dried in a vaccum oven and stored in air-tight containers or polybags (Reena et al.,

2005).

2.2.6 Applications of Pectins

It is employed mostly as an intestinal demulscent. It is believed that the unchanged molecules of

polygalacturonic acids may exert an adsorbent action in the internal layers of the intestine,

thereby producing a protective action along with Kaolin to prevent and control diarrhoea. As a

pharmaceutical aid pectin is used frequently as an emulsifying agent and also as a gelling agent

preferably in an acidic medium (Penniston et al., 2008). It is employed extensively in the

preparation of jellies and similar food products e.g., jams, sauces, ketchups. Pectin in the form of

pastes exerts a bacteriostatic activity and hence, is used frequently in the treatment of indolent

ulcers and deep wounds. A combination of pectin and gelatin find its application as an

encapsulating agent in various pharmaceutical formulations to afford sustained-release

characteristics.

2.2 Aspergillus niger

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2.2.1 Feature of aspergillus niger

The Conidiophores are 400-3000um long, they are smooth and hyaline.The conidiophore

becomes dark at the apex and terminating in a globose vesicle which is 30-75um in diameter.

The metulae and phialides cover the vesicle. The phialides produce conidia that have a rough

texture, are dark brown colored, and have a diameter of 4-5um. (\Galiotou-Panayotou and

Kapantai, 2013).

2.3.2 Role of A. niger 

 A. niger is used within the biotechnology industry in production of magnetic isotope-

containing variants biological  macromolecules  for  NMR  analysis.

 Aspergillus niger is also cultured for the extraction of the enzyme, glucose

oxidase (P13006), used in the design of glucose biosensors, due to its high affinity for β-

D-glucose.

 The fungus also plays a role in the solubilization of heavy-metal sulfides (Huang et al.,

2008).

 A. niger has also been shown to be able to remediate acid mine drainage through

biosorption of copper and manganese (Bali, 2003).

2.2.2 Classification of Aspergillus niger

Kingdom: Fungi
Division: Ascomycota
Class: Eurotiomycetes
Order: Eurotiales
Family: Trichocomaceae
Genus: Aspergillus

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Species: A. niger (Arenas-Ocampo et al., 2003).

2.3 Pectinase

Pectinases is a group of enzymes (at least seven different enzymes) involved in the breakdown of

pectin obtained from various sources. Due to the diverse group of pectin that is found in different

living organisms, pectinases are also diverse. Most common and industrially important

pectinases are divided into different groups on the basis of the differences in their substrate,

structure, and reaction mechanism (Sinclair, 2015). Some of the common pectinolytic enzymes

include pectinesterases, polygalacturonases, pectin lyases, and pectin depolymerases. Pectinases

have important industrial applications as they are involved in the extraction and clarification of

juice and maceration of plant tissues. Pectinases are one of the major enzymes that take in the

global carbon cycle, assisting in natural waste recycling (Walton et al., 2005). Pectinases are

even termed carbon recycling agents in nature as they degrade pectin substances into saturated

and unsaturated galacturonans, which can then be catabolized to form either pyruvate or 3-

phosphoglyceraldehyde (Amorim and Amorim, 2007). Along with these applications, pectinases

are also used in degumming fiber crops, as enzyme complex for the generation of animal feed,

purifying plant viruses, and in the extraction of oils. Some pectinases act as virulence factors as

these enzymes help in the degradation of pectin found in the cell of plants (El Hadi et al., 2013).

Pectinases might differ in structure and mechanism on the basis of their source, like the pectinase

from fungi might be different from that of bacteria. Bacterial pectinases tend to be alkaline,

whereas fungal pectinases are acidic in nature (Alonso et al., 2003).

2.4.1 Sources of pectinase

16
Pectinases can be extracted from fungi such as Aspergillus niger. The fungus produces these

enzymes to break down the middle lamella in plants so that it can extract nutrients from the plant

tissues and insert fungal hyphae. Pectinase can be produced by a variety of microorganisms.

These enzymes act on pectin, which is the major component of middle lamella in plant cell wall

(Penniston et al., 2008). Pectinolytic enzymes are classified according to their mode of attack on

the galacturonan part of the pectin molecules such as protopectinases, esterases and

depolymerases. As we know that microbial enzymes work depends upon the type of enzymes

application, temperature, concentration, and pH and so on, therefore, pectinase enzyme also

differentiated according to their physical and chemical factors too. The biochemical structures of

pectinases include members of all the major classes and the structure–function relationship,

studies of a few available complexes of pectinases with substrate/analogs could be considered as

prototypes for related family member and the molecular characterization of pectinolytic enzymes

is also well documented. Furthermore, it provides a bird’s eye view of the possible application of

these enzymes in commercial sector pectin.

2.4.2 Classification of pectinase

Depending on the source of the enzymes, their substrates, and the reaction mechanism,

pectinases are classified into different groups;

2.4.2.1 Polygalacturonase (EC 3.2.1.15)

Polygalacturonase is a group of enzymes that hydrolyze O-glycosyl bonds in the

homogalacturonan to form monomeric units (Ramsey, 2005). These enzymes act on the 1-4-α-D-

galactosyluronic linkages between the galacturonic residues. Most of the polygalacturonases are

endo-enzymes that act on the linkages randomly to depolymerize the chain or reduce the length

17
of the polymer. The natural substrate of endo-polygalacturonase is homogalacturonan; however,

other compounds like oligogalacturonides might also act as a substrate depending on the nature

of the substrate. A class of exo-polygalacturonase is also known where they break down the

polygalacturonates into di- and mono-galacturonates. The activity of the enzyme is determined

by the measurement of reducing sugars formed as a result of hydrolysis or by the viscous

reduction method (Akita et al., 2000).

2.4.2.2 Pectinesterase (EC 3.1.1.11): Pectinesterases are a group of enzymes that catalyzes the

hydrolysis of methylated carboxylic ester in pectin to form pectic acid and methanol (Berld et

al., 2014). The natural substrate of pectinesterase is pectin; however, other compounds like

methyl pectate and methylated oligogalacturonides also work as substrate. The activity of

pectinesterase is enhanced or induced by (NH 4)2SO4, Mg2+, and NaCl (Chadha et al., 2005). It is

inhibited by the presence of Cu2+ and Hg2+. Most of the well-studied pectinesterase is produced

from plants; however, recently pectinesterase of bacterial and fungal origin have also been

discovered. Most pectinases are specific towards esterified pectic substances and thud, might not

show any activity towards pectates.

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2.4.2.3 Pectin lyases (EC 4.2.2.10): Pectin lyases degrade pectic substances in a random

fashion, yielding a 4:5 ratio of unsaturated oligomethylgalacturonates (Perez-Cacho and Rouseff,

2008). These enzymes cleave glycosidic linkages, preferentially on polygalacturonic acid

through transelimination reaction. Pectin lyases have an absolute requirement of Ca 2+ ions and

thus, are inhibited by chelating agents like EDTA (Amorim and Amorim, 2007). Exo-pectin

lyases catalyze the cleavage of the substrate from the non-reducing end of the polymer.

2.4.2.4 Pectatelyase (EC 4.2.2.2): Pectatelyase (PGL) cleaves glycosidic linkages preferentially

on polygalacturonic acid forming unsaturated product through transelimination reaction. PGL

has an absolute requirement of Ca2+ ions. Hence it is strongly inhibited by chelating agents as

EDTA (Aguila and Huirton, 2010).

2.4.3 Production of 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 (Steven et

al., 2000). There were two fermentation techniques we can use for pectinases production, as

many other enzymes. These techniques are Solid State Fermentation (SSF) and submerged

fermentation (SmF). 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 (McCready, 2010). In contrast, in submerged

fermentation (SmF) the nutrients and microorganisms are both submerged in water.

19
Approximately 90% of all industrial enzymes are produced in SmF, frequently using specifically

optimized, genetically manipulated microorganisms (Douf, 2011). 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 (Sinclair et al., 2005). 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 (Albersberg et

al., 2003).

Microbial production of pectinases has been studied during recent years (Bali, 2003) and fungi.

However, almost all the commercial preparations of pectinases are produced from fungal sources

(Reena et al., 2005).

Pectolytic enzymes have been reported to be induced by several substances. In many cases pectin

itself has been used. Many investigators had used complex media such as beet sugar, wheat bran,

ground nut meal, citrus fruit peels etc (Bai et al., 2014). Higher cost of the production is perhaps

the major constraint in commercialization of new sources of enzymes. Though, using high

yielding strains, optimal fermentation conditions and cheap raw materials as a carbon source can

reduce the cost of enzyme production for subsequent applications in industrial processes (Chadha

et al., 2005). There are many studies that have been conducted related to the characterization of

different microbial pectic enzymes concerning their mechanisms of action and biochemical

properties. The optimal pHs that these enzymes may act range between 3.5-11, while the optimal

temperatures vary between 40-75 °C (El Hadi et al., 2013).

2.4.4 Gel filtration chromatography

20
Gel-filtration chromatography is a form of partition chromatography used to separate molecules

of different molecular sizes. This technique has also frequently been referred to by various other

names, including gel-permeation, gel-exclusion, size- exclusion, and molecular- sieve

chromatography (Arenas-Ocampo et al., 2003). The basic principle of gel filtration is quite

straightforward. Molecules are partitioned between a mobile phase and a stationary phase

(comprising a porous matrix of defined porosity) as a function of their relative sizes. A column

constructed of such a matrix, typically in bead form, will have two measurable liquid volumes,

the external volume, consisting of the liquid between the beads, and the internal volume,

consisting of the liquid within the beads (Galiotou-Panayotou et al., 2013). The external volume

is usually referred to as the void volume (V 0), while the sum of the external and internal volumes

is the total volume (V t). Following sample application, molecules larger than the pores of the

stationary phase matrix will be excluded from the internal volume within the beads and will,

therefore, migrate quite rapidly through the column, emerging at V 0, while molecules both

smaller than the matrix pores, as well as those intermediate in size, will equilibrate with both the

external and internal liquid volumes, causing them to migrate much more slowly and emerge at a

volume (V e) greater than V 0. Molecules are, therefore, eluted in order of decreasing molecular

size. The elution volume, V e, of a particular molecule depends on the fraction of the stationary

phaseavailable to it for diffusion (Alonso et al., 2003). This can be represented by the constant K

d or K av (also referred to as the partition coefficient). Therefore:

Ve=V0+Kav(Vt−V0)

Rearranging this equation gives:

Kav=(Ve−V0)/(Vt−V0)

21
2.4.5 Factors affecting pectinase activity

2.4.5.1 Effect of pH: The potential of hydrogen (pH) is the best measurement for determining

the concentration of hydrogen ion (H+)in a solution. It also determines whether the liquid is

acidic, basic or neutral. Generally, all liquids with a pH below 7 are called acids, whereas liquids

with a pH above 7 are called bases or alkalines. Liquids with pH 7 are neutral and equal the

acidity of pure water at 25 C°. You can determine pH of any solution using the pH indicators

Pectinsae are protein substances that contain acidic carboxylic groups (COOH –) and basic amino

groups (NH2). So, the enzymes are affected by changing the pH value. pectinase has a pH value

that it works at with maximum efficiency called the optimal pH. If the pH is lower or higher than

the optimal pH, the pectinase activity decreases until it stops working (Sinclair et al., 2005).

2.4.5.2 Concentration of Substrate: In the presence of a given amount of pectinase, the rate of

enzymatic reaction increases as the substrate concentration increases until a limiting rate is

reached, after which further increase in the substrate concentration produces no significant

change in the reaction rate. In other words, the pectinase molecules are saturated with substrate.

The excess substrate molecules cannot react until the substrate already bound to the pectinse has

reacted and been released (or been released without reacting) (Abe et al., 2008).

2.4.5.3 Effect of Temperature: The protein nature of the pectinase makes them extremely

sensitive to thermal changes. Pectinase activity occurs within a narrow range of temperatures

compared to ordinary chemical reactions. Pectinase has a certain temperature at which it is more

active. This point is called the optimal temperature, which ranges between 37 to 40C°. The

pectinase activity gradually lowers as the temperature rises more than the optimal temperature

until it reaches a certain temperature at which the pectinase activity stops completely due to the

22
change of its natural composition. On the other hand, if the temperature lowers below the

optimal temperature, the pectinase activity lowers until the enzyme reaches a minimum

temperature at which the enzyme activity is the least. The pectinase activity stops completely at

0C°, but if the temperature rises again, then it gets reactivated once more (Bai et al., 2016).

2.4.5.4 Concentration of Enzyme: As the concentration of the pectinase is increased, the

velocity of the reaction proportionately increases. The concentration of the enzyme is important

in chemical reaction as it is needed to react with the substrate. Often a small amount of enzyme

can consume a large amount of substrate (Reena et al., 2015). ". However, with the increase of

enzyme concentration, the effectiveness of the active sites also increases, so these active sites

will convert the substrate molecules into products. This basically means that if the concentration

of the enzyme is to be increased, there needs to be an excess of substrate, in other words, which

means that the reaction must be independent of the concentration of the substrate (Tietel et al.,

2011). ".

2.4.6 Application of pectinase

2.4.6.1 Oil extraction: Oils from grape seed, coconut germ, sunflower seed, palm kernel and

olives are traditionally produced by extraction with organic solvents. Hexane, a potential

carcinogen is the most commonly used solvent. Recently, the plant cell-wall-degrading enzyme

preparation has begun to be used in olive oil preparation. During grinding of the olives, the

enzymes are added, thereby releasing the oil easily (Albersberg et al., 2003).

2.4.6.2 Chromaticity and Stability of Red Wines: Pectinolytic enzymes added to macerated

fruits before the addition of wine yeast in the process of producing red wine resulted in improved

visual characteristics (color and turbidity) as compared to the untreated wines. Red wines which

23
are enzymatically treated showed chromatic characteristics better than the control wines. These

wines also showed greater stability as compared to the control (Aschoff et al., 2015).

2.4.6.3 The chemical degumming treatment is toxic, polluting and non-biodegradable: Eco-

friendly and economic alternative to the above problem is the biotechnological degumming using

pectinases in combination with xylanases. Pectinases are involved in the retting and degumming

of jute, flax, hemp, ramie, kenaff (Hibiscus sativa), and coir from coconut husks (Arenas-

Ocampo et al., 2003). Retting is a fermentation process, in which certain bacteria (e.g.,

Clostridium sp., Bacillus sp.) and certain fungi (e.g., Aspergillus sp., Penicillium sp.) decompose

the pectin of the bark and release fiber.

2.4.6.4 Waste water treatment: For treatment of wastewater from citrus processing industries

various processes have been investigated, which include: physical dewatering, spray irrigation,

chemical coagulation, direct activated sludge treatment and chemical hydrolysis followed by

methane fermentation (Penniston et al., 2008). These processes have low efficiency due to

chemical resistance of the pectic substances, high treatment cost, long treatment periods and

complexity of the process. Vegetable processing industries release pectin, containing

wastewaters as by product. Pretreatment of these wastewaters with Pectinolytic enzymes

facilitates removal of pectinaceous material and renders it suitable for decomposition by

activated sludge treatment (Chadha et al., 2005). The waste water from the citrus-processing

industry contains pectinaceous materials that are barely decomposed by microbes during the

activated-sludge treatment. Pectin containing waste waters is released by vegetable food

processing industries as by-product. These wastewaters are pretreated with Pectinolytic enzymes

which facilitate the removal of pectinaceous material and render it suitable for decomposition by

activated sludge treatment.

24
2.4.6.5 Coffee and tea fermentation: Pectinase treatment accelerates tea fermentation and also

destroys the foam forming property of instant tea powders by destroying pectin. In coffee

fermentation, it is used to remove mucilaginous coat from coffee beans. Enzymatic treatment

accelerates tea fermentation, wherein the enzyme dose is carefully adjusted to avoid damage to

the tea leaf (McCready, 2010).

2.4.6.6 Paper and pulp industry: Pulp and paper mills are beginning to use enzymes to solve

problems in their manufacturing processes. Pectinase produced by Bacillus sp.and Erwinia

carotovora sp. due to its strong macerating activity, has been used for retting of Mitsumata bast.

During papermaking; Pectinase depolymerize polymers of galacturonic acids, and lowersthe

cationic demand of pectin solutions and the filtrate from peroxide bleaching (Alonso et al.,

2003).

2.4.6.7 Textile processing and bio-scouring of cotton fibers: Pectinases have been used in

conjunction with amylases, lipases, cellulases and hemicellulases to remove sizing agents from

cotton in a safe and ecofriendly manner, replacing toxic caustic soda used for the purpose earlier.

Bio-scouring is a novel process for removal of non-cellulosic impurities from the fiber with

specific enzymes (El Hadi et al., 2013). Degumming/retting 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.

25
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Materials

3.1.1 Reagents

3, 5-dinitrosalicylic acid (DNS):

D-(+)-Galacturonic acid monohydrate: Sigma-Aldrich (USA)

Bovine serum albumin (BSA), Bio Rad Laboratories (India)

Folin-Ciocalteau phenol reagent:

Agar-Agar: Hopkins and Wichans (England)

Ammonium sulphate ((NH4)2SO4): British Drug House Chemicals Limited Poole (England)

Dipotassium hydrogen phosphate (K2HPO4): British Drug House Chemicals limited Poole

(England)

Sodium hexametaphosphate: British Drug House Chemicals limited Poole (England)

Hydrochloric acid (HCl): Reagente Puro Erba (RPE)

Magnesium tetraoxosulphate VI heptahydrate (MgSO4.7H2O): British Drug House Chemicals

limited Poole (England)

Sodium potassium tartarate (Rochelle salt): Merck (Germany)

Sodium acetate:

Sodium Hydroxide: British Drug House Chemicals limited Poole (England)

Ammonium nitrate (NH4NO3): Merck (Germany)

Sodium nitrate (NaNO3): Merck (Germany)

Iron II tetraosulphate VI heptahydrate (FeSO4.7H2O): British Drug House Chemicals

Limited Poole (England)

26
Zinc tetraoxosulphate VI heptahydrate (ZnSO4.7H2O): British Drug House Chemicals

Limited Poole (England)

Potato destrox agar (PDA): Lab M Limited Topley House, 52 Wash Lane, Bury,Lancashire BL9

6AS (United Kingdom)

Lacto phenol cotton staining blue: Kermel Chemicals (China)

Ammonium chloride (NH4Cl): Merck (Germany)

Ammonium dihydrogen phosphate (NH4H2PO4): British Drug House Chemicals

Limited Poole (England)

Sodium trioxocarbonate IV (Na2CO3): British Drug House Chemicals limited Poole (England)

Sodium sulphite (Na2SO3): British Drug House Chemicals limited Poole (England)

Note: All other chemicals used in this work were of analytical grade unless otherwise stated.

3.1.2 Apparatus

Weighing Balance: Ohaus Dial-O-Gram, Ohaus Cooperation, N.J. USA.

Water bath: Model DK.

Magnetic stirrer: AM-3250B Surgi Friend Medicals (England).

Milling machine: Thomas Willey laboratory Mill Model 4, Anthor H (Thoma Company,

Philadelphia, USA).

Autoclave: UDAY BURDON’s Patent Autoclave (India).

Incubator: B and T Trimline incubator.

Oven Gallenkamp Hotbox (England).

pH meter: Ecosan pH meter (Singapore).

Sensitive weighing balance: B2404-5 mettler Toledo (Switzerland).

UV/visible spectrophotometer: Jenway 6405

27
Microscope: WESO microscope (USA)

Glass wares (Pyrex) (USA)

3.1.3 Collection of Pectin

Orange pectin was collected from Enzymology Unit, Department of Biochemistry, University of

Nigeria Nsukka Enugu, Nigeria.

3.1.4 Collection of Microorganism

Pectinase producing fungus was collected on Potato Dextrose Agar (PDA) slopes from

Enzymology Unit, Department of Biochemistry, University of Nigeria Nsukka Enugu, Nigeria.

3.2 Methods

3.2. Microbial Culture

3.2.1.1 Preparation of Solid Culture Medium

Potato Dextrose Agar (PDA) (gelling agent) solid media were prepared, following

manufacturer’s description and the media autoclaved at 121oC for 15min. The sterilized media

were poured aseptically into Petri dishes and allowed to gel. The plates were then incubated 30 oC

overnight to check for sterility before.

3.2.1.2 Inoculation of Plates and Sub-culturing

The fungal specie was inoculated onto the PDA culture media, using inoculation needle and

under the flame of Bunsen burner. The plates were thereafter incubated at 30 oC till visible

colonies of fungus were observed. Fungus was isolated from the morphological contrasting

colonies and purified by repeated streaking and sub-culturing on separate plates. This process

was continued till pure fungal isolate was obtained.

28
3.2.1.3 Storage of Pure Fungal Isolates

Pure fungal isolate was maintained on Potato Dextrose Agar (PDA) slopes or slants and stored at

4oC as stock cultures.

3.2.1.4 Microscopic Features of the Isolated Fungus

Three-day old pure culture was examined. The culture was sent to the Microbiology section of

Brain Phosphorelationship Laboratory Ogui Enugu, for identification. The color, texture, nature

of mycelia or spores and growth patterns were also observed. Photographs of the cultures were

also taken.

3.2.1.5. Fungal Identification

The three-day old pure culture was used in preparing microscopic slides. A little bit of the

mycelia was dropped on the slide and a drop of lacto-phenol blue was added to it. A cover slip

was placed over it and examination performed under the light microscope at X400 magnification.

Identification was carried out by relating features and the micrographs to “Atlas of mycology” by

(Barnett and Hunter, 1972). Species identification was by examining both macroscopic and

microscopic features of a three day old pure culture. Colour, texture, nature of mycelia and/or

spores produced, growth pattern in addition to microscopic features such as separation, spore

shapes and so on were examined and confirmed Aspergillus niger.

3.2.2 Fermentation Experiments

Submerged fermentation (SmF) technique was used to produce 500ml of the enzyme, using a

modification of the method described by (Nsude et al., 2019). This involved ten 250ml

Erlenmeyer flasks containing 50ml each of sterile cultivation media, made up of 0.1% NH4NO3,

0.1% NH4H2PO4, 0.1% MgS04.7H2O, 0.5% orange pectin and adjusted to pH 5.0 by using 1.0N

29
HCl/1.0N NaOH. The flasks were stopped with aluminium foil and autoclaved at 121 oC, 15psi

for 15min.

Six days old pectin agar cultures were used to inoculate the flasks. In every sterile flask, three

discs of fungal hyphae from edge of actively growing pectin agar cultures were added

respectively using a flamed and cooled cork borer of diameter 10mm and then plugged properly.

The culture was incubated for 8 days at 30oC and then harvested. The filtrates were stored at 4 oC

and used as the crude enzyme for further studies.

3.2.3 Pectinase Assay

Pectinase activity was evaluated by assaying for polgalacturonase (Pg) activity of the enzyme.

This was achieved by measuring the release of reducing groups from African star cherry pectin

using a modification of the 3,5 dinitrosalicylic acid (DNS) reagent assay method described by

(Miller, 1959) as contained in (Nsude et al., 2019) with little modifications.

The reaction mixture containing 0.5ml of 0.5% orange pectin in 0.05M sodium acetate buffer of

pH 5.0 and 0.5ml of enzyme solution were incubated for 30min. 1ml of DNS reagent was added

and the reaction was stopped by boiling the mixture in a boiling water bath for 10mins. The

mixture volume was made up to 4ml with 1ml of Rochelle salt solution and 1ml of distilled

water. The reaction mixture was allowed to cool and then absorbance read at 540nm. One unit of

enzyme activity was defined as the amount of enzyme that catalyzes the release of one

micromole of galacturonic acid per minute.

3.2.4 Protein Determination

Protein content of the enzyme was determined by the method of (Lowry et al., 1951), using

Bovine Serum Albumin as standard.

30
3.2.4.1 Procedure for Protein Determination

For protein standard curve, the reaction mixture contained 0.0-1.0ml of protein stock solution

(2mg/ml BSA) in test tubes arranged in triplicates. The volume was made up to 1ml with

distilled water. But for the test mixture, 0.1ml of sample enzyme was mixed with 0.9ml of

distilled water. In either case, 5ml of solution E was added and allowed to stand at room

temperature for about 10min. 0.5ml of solution C (dilute Folin-Ciocalteau phenol reagent) was

added with rapid mixing. After standing at room temperature for 30min, absorbance was read at

750nm using spectrophotometer. Absorbance values were converted to protein concentration by

extrapolation from the standard curve.

3.2.5 Purification of Pectinase

3.2.5.1 Centrifugation

Crude enzyme filtrate was centrifuged at 4000rpm and the supernatant kept under cold condition

for further studies.

3.2.5.2 Gel filtration

Ten (10) ml of the enzyme supernatant were gradually injected into sephadex G-200 column

(1.6 × 59cm) previously equilibrated with sodium acetate buffer (pH 5.0, 0.05M) and eluted with

the same buffer at a flow rate of 1 ml.min -1. The fractions with high activities were collected,

pooled and used for characterization and protein determination.

3.2.3. Effect of Temperature Change on Pectinase Activity

The optimum temperature was determined by incubating the enzyme with pectin solution at 30 -

70oC interval of 5oC for 30min and pH 5.0. The activity was then assayed, using the method

described by (Miller, 1959) as contained in (Wang et al., 1997) with little modifications.

31
3.2.4 Effect of pH Change on Pectinase Activity

The optimum pH for enzyme activity was determined using 0.05M sodium acetate buffer pH 3.5-

5.5 and phosphate buffer pH 6.0-8.0 at intervals of 0.5. 0.5% pectin solution was prepared by

dissolving 0.5g pectin in 100ml of 0.05M of the respective buffers. Also purified enzymes were

dispersed in the various buffers and 0.5ml of the enzyme mixed with 0.5ml pectin solution at the

corresponding pHs for pectinase assays using the method described by (Miller, 1959) as

contained in (Nsude et al., 2019) with little modifications.

32
 CHAPTER FOUR

4.0 Results

4.1 Microorganism

The fungal isolate was collected on Potato Dextrose Agar (PDA) slopes from Enzymology Unit,

Department of Biochemistry, University of Nigeria Nsukka Enugu, Nigeria. The pure isolate was

identified as Aspergillus niger (Figure 1) after macroscopic and microscopic examination of a

four day old pure culture.

Figure 2: Pure culture of Aspergillus niger

33
4.2 Production of Pectinase by Aspergillus niger

Enzyme activity and protein concentration of mass-produced pectinase of Aspergillus niger were

found to be 225.38µmole/min and 0.622mg/ml, respectively

4.3 Purification of Aspergillusniger Pectinase

The elution profiles after Sephadex G-200 Gel filtration chromatography is shown in Figure 3.
Absorbance at 540nm

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35 40

Fraction number

Figure 3: The elution profiles after Sephadex G-200 Gel filtration chromatography

4.3.1 Purification step of Aspergillusniger Pectinase

Aspergillusniger pectinase was purified with a fold of 1.73, a yield of 4.61% and specific activity

of 0.64U/mg of protein (Table 1).

34
Table 1: Purification step of Aspergillusniger Pectinase

Purification Volume Total Total Specific Fold Yield

step (ml) Protein activity activity (%)

(mg) (U) (U/mg)

Crude 100 63..06 23.19 0.37 1 100

Gel 25 4.56 2.91 0.64 1.73 4.61

filtration

4.4 Effect of pH on Aspergillusniger Pectinase Activity

Aspergillusniger pectinase has an optimum pH at 5.5 with optimum activity of 163.74µmole/min

(Figure 5) and decreased significantly below and above this value, using orange pectin as

substrate for enzyme reaction.

180
160
Activity (µmole/min)

140
120
100
80
60
40
20
0
3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
pH

Figure 4: Effect of pH on the activity of pectinase

35
4.5 Effect of Temperature on AspergillusnigerPectinase Activity

Aspergillusnigerpectinase activity was optimum at 45°C as shown in Figure 6. At the optimum

temperature,the enzyme had optimum activity of 160.14µmole/min.

Activity (µmole/min)

180
160
140
120
100
80
60
40
20
0
25 30 35 40 45 50 55 60 65 70 75
Temperature (oC)

Figure 5: Effect of temperature on the activity of pectinase

36
 CHAPTER FIVE

In this research work, industrial pectin

was used. Fungal strain was collected from Dextrose Agar

(PDA) slopes from Enzymology Unit, Department of Biochemistry, University of Nigeria

Nsukka Enugu, Nigeria. Based on macroscopic and microscopic characteristics of the

fungal isolate, Aspergillus niger was confirmed (Figure 1). The industrial pectin was used

to induce pectinase production in Aspergillus niger which had more pectinase activity under

submerged fermentation. The entire fermentation process was carried out at room temperature

(300C). The accumulation of maximal enzyme activity and protein concentration of mass-

produced pectinase of Aspergillus niger were found be 225.38µmole/min and 0.622mg/ml,

respectively after 4 days of fermentation. Similar observation was also obtained during pectinase

production from mango peels by Aspergillus tamari in solid state fermentation in which

polygalacturonase production was highest at day 3 of fermentation (Amande et al., 2013)

and in 3 days for the production of pectinase from Tamarind kernel powder by submerged

fermentation using Aspergillus spp (Viswanathan and JagadeshBabu, 2008). Banu et al., (2010)

reported maximum polygalacturonase activity on the 5th day of fermentation with Penicillium

chrysogenum. A lot of factors influence the production of pectinases. These include;

concentration of nutrients, pH and temperature. Sephadex G-200 Gel filtration chromatography

was use to purify the extracted Aspergillus niger pectinase (Figure 3). It was purified with a fold

of 1.73, a yield of 4.61% and specific activity of 0.64U/mg of protein (Table 1). The optimum

pH for Aspergillus niger pectinase were 5.5 with optimum activity of 163.74µmole/min (Figure

4) and decreased significantly below and above this value, using industrial pectin as substrate for

enzyme reaction. Optimum temperature of the enzyme was at 45°C as shown in (Figure 5). At

the optimum temperature, the enzyme had optmium activity of 160.14µmole/min. (Favela-Torres

37
et al., 2006) reported that an acidic pH of 4.0-4.5 and temperature range of 40-45oC support high

pectinase activity. The pH optima for 30 fungal pectinases reported by Niture et al., (2004)

ranged from 2.5 to 6.0. According to (Jayani et al., 2005), most microbial pectinases have

optimal pH range of 3.5-5.5 and optimal temperature range of 30-50oC. Hence, the optimal pH

and temperature obtained in this work are in agreement with those in literature. According to

(Abbott and Boraston, 2005), pectin degradation is facilitated by a battery of pectinases

including polygalacturonase, pectin lyase and pectin methylesterase. Pectin lyase breaks down

the glycosidic linkages of pectin at C-4 and simultaneously eliminates H from C-5, producing

unsaturated products and reducing groups called oligogalaturonates (Yadav et al., 2009).

5.1 Conclusion

This investigation suggests that industrial pectin gotten from orange peel could be an attractive

and promising substrate for the production of pectinases by Aspergillus niger. In addition, this

work will act as first line information to researchers who want to explore the possibilities of

converting waste to wealth and value addition. Since orange peels utilized in this process are

readily accessible as waste with little or no cost and also contain an appreciable amount of

pectin, they can be regarded as a low-cost substrate for efficient and economical production of

pectinases using Aspergillus niger. This will not only lead to the reduction in the production cost

of pectinases but also help to decrease the pollution load resulting from these wastes.

38
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