Optimization of Pectinase Production from Organic Wastes using Aspergillus brasiliensis

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Optimization of Pectinase Production from different
Organic wastes
by
Akshita Dhingra (113014)
Ankit Deshwal (113020)
Anurag (113024)
Apoorva Joshi (113025)
Kopal Mittal (113068)
Research Project Report
Submitted to
Research Cell, National Institute of Food Technology
Entrepreneurship and Management
In partial fulfillment for the requirement of the degree of
Bachelor of Technology
in
Food Technology and Management
Under the supervision of
Dr. Chakaravarthi Saravanan
Department of Basic Applied Science
June, 2017

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DECLARATION
We the bonafide students of B.Tech in Food Technology and Management in National
Institute of Food Technology Entrepreneurship and Management, Haryana would like to
declare that the dissertation entitled ―Optimization of Pectinase enzyme from different
organic wastes” submitted by us in partial fulfilment of the requirements for the award of the
Degree of BACHELOR OF TECHNOLOGY in Food Technology and Management is our
original work.
Place: Sonepat, Haryana
Date: 21 June, 2017
Signature of the candidates
Akshita Dhingra (113014)
Ankit Deshwal (113020)
Anurag (113024)
Apoorva Joshi (113025)
Kopal Mittal (113068)

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CERTIFICATE
This is to certify that the dissertation entitled ― Optimization of Pectinase enzyme
from different organic wastes ‖is a bonafide record of group research work done by
Akshita Dhingra (113014), Ankit Deshwal (113020), Anurag (113024),
Apoorva Joshi (113025) , Kopal Mittal (113068) under my supervision and
submitted to National Institute of Food Technology Entrepreneurship and
Management (NIFTEM) in partial fulfilment for the award of the Degree of
BACHELOR OF TECHNOLOGY in Food Technology and Management.
Signature of the supervisor

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ACKNOWLEDGEMENT
We would like to express our thanks to Prof. Manjit Agarwal (Dean Research),
National Institute of Food Technology Entrepreneurship and Management,
Sonepat (Kundli) for permitting us to do our final year research project at
NIFTEM, Sonepat.
Our special thanks to Dr. Chakkaravarthi Saravanan (Guide), Department of
Basic Applied Science, for guiding us throughout the research project and
helping us completing our research project.
We express gratitude towards all other faculties, lab assistants, and other
supporting staffs who gave their extensive support throughout our research
product.
No words are sufficient to thank God, our parents and our lovely friends who
were a constant support and inspiration to us during different phases of our life.
And finally, last but by no means least, also to everyone in the impact hub. It
was great sharing laboratory with all of you during last four years.

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CONTENTS
1. ABSTRACT 12
2. INTRODUCTION 13
2.1. INTRODUCTION ABOUT SOLID STATE FERMENTATION
2.2. INTRODUCTION ABOUT PECTIN
2.3. INTRODUCTION ABOUT PECTINASE
2.4.INTRODUCTION ABOUT Aspergillus Brasiliensis
3. OBJECTIVE
4. REVIEW OF LITERATURE 21
4.1. PECTINASE ENZYME COMPOSITION
4.2.COMMERCIAL APPLICATION OF PECTINASE
4.3. PECTINASE EXTRACTION
4.4. MICROBES RESPONSIBLE FOR PECTINASE PRODUCTION
4.5. VARIOUS OPTIMIZATION PROCESSES FOR PECTINASE
PRODUCTION
4.6. ENZYME ACTIVITY ESTIMATION
5. MATERIALS 28
5.1.INGREDIENTS
5.2.AUTOCLAVE
5.3.INCUBATOR
5.4. CENTIFUGE
5.5. WEIGHING BALANCE
5.6. pH METER
5.7.HOT AIR OVEN
5.8. LAMINAR FLOW CABINET
5.9.SPECTROPHOTOMETRY

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6. METHODOLOGY 34
6.1. SAMPLE PREPARATION
6.2. REAGENT PREPARATION
6.3. FUNGAL INOCULATION PREPARATION
6.4. INOCULATION
6.5. HARVESTION OF ENZYME
6.6. ENZYME EXTRACTION
6.7. PROTEIN ASSAY
6.8. PECTINASE ASSAY
7. RESULTS AND DISCUSSIONS 42
8. CONCLUSION 58
9. BIBLIOGRAPHY 59

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LIST OF TABLES
Table 1: Pectin content in some fruits
Table 2: Preparation of trace elements
Table 3: Preparation of culture elements
Table 4: Concentration of Galacturonic acid produced using Orange peel in
ratio 1:2 at different time intervals
Table 5: Specific activity of enzyme produced using Orange Peel in ratio 1:2
(Peel: Bran)
(Peel: Bran)
(Peel: Bran)
Table 10: Concentration of Galacturonic acid produced using Kinnu peel in
Table 11: Specific activity of enzyme produced using Kinnu Peel in ratio 1:2
(Peel: Bran)

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(Peel: Bran)
(Peel: Bran)
Table 16: Concentration of Galacturonic acid produced using Mosambi peel in
Table 17: Specific activity of enzyme produced using Mosambi Peel in ratio
1:2 (Peel: Bran)
1:3 (Peel: Bran)
3:4 (Peel: Bran)
Table 22: Summary of Specific Activity of Enzyme samples

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LIST OF GRAPHS
Graph 1: Pectinolytic activity showed in different isolated strains
Graph 2: Effect of incubation period on Enzymatic activity
Graph 3: Effect of concentration of Pectin on Enzymatic activity
Graph 4: Effect of nitrogen source on Enzymatic activity
Graph 5: Effect of temperature on Enzymatic activity
Graph 6: Effect of moisture content on Enzymatic activity
Graph 7: Concentration of Glucose produced using Orange peel in
Graph 8: Concentration of Glucose (µmol/ml) vs Time(min) for the
ratio of 1:2 of Orange peel powder
Graph 13: Concentration of Glucose produced using Kinnu peel in

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ratio of 1:2 of Kinnu peel powder
Graph 19: Concentration of Glucose produced using Mosambi peel in
ratio of 1:2 of Mosambi peel powder
Graph 25: Summary of Specific Activity of Enzyme samples

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LIST OF FIGURES
Figure 1: Structure of pectin
Figure 2: Autoclave
Figure 3: Incubator
Figure 4: Centrifuge
Figure 5: Weighing Balance
Figure 6: Spectrophotometer
Figure 7: MTCC 1344 Aspergillus brasiliensis
Figure 8: Prepared culture media
Figure 9: Culture media after fungal inoculation
Figure 10: Extracted enzyme
Figure 11: Pectinase assay (DNSA Method)

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1. ABSTRACT
The use of pectinase enzyme in food industries includes clarification of fruit juices
and wines, however due to its high cost of production it is not widely produced in
India. This project focuses on the optimization of pectinase production using varying
ratios of wheat bran and citrus peels as substrate for the growth of Aspergillus, the
microbe responsible for pectinase production. The use of wheat bran and fruit peels as
substrate for microbial growth reduces the cost of production by waste utilization of
food industries. Aspergillus brasiliensis strain was used in this research project since
it shows more pectinase production as compared to other fungal strains as well as
bacteria. The growth of microbe requires certain nutrients which can be provided by
different combinations of wheat bran and citrus fruit peels. The major objective
behind the use of citrus peels is the high pectin content in them favouring the
pectinase production. The citrus fruit peels used in this study included orange,
mausambi and keenuh peels; in the ratios 1:2, 1:3 and 3:4 each with wheat bran. Other
factors favouring pectinase production maintained were, pH of around 4 to 5,
incubation days for pectinase production varying from 3 to 7 depending on the growth
and temperature of incubation for fungal growth around 25 degrees. The enzyme
activity of the pectinase extracted was determined using the dinitrosalicylic acid
method wherein the reducing sugars formed after the breakdown of pectin by
pectinase were estimated by spectrometry. The enzyme activity for the combination of
wheat bran and mausambi peels was observed to be maximum in the ratio 3:1
followed by keenu peels and then orange peels.

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2. INTRODUCTION
2.1. INTRODUCTION ABOUT PECTIN
Pectin is a structural polysaccharide found in primary cell wall and middle lamella of fruits
and vegetables. Reported molecular weights of pectin polysaccharides extracted from fruits
ranges from 25000 to 360000. Pectins have been showed to play diverse roles in cell
physiology, growth, adhesion and separation. Pectin help cement plant cells together. They
are also well known for their ability to form gels
Structural unit
The predominant structure of pectin is homopolymeric, made of a partially methylated poly-
a-(1, 4)-galacturonic acid. Sections of a-(1, 2)-L-rhamnosyl-a-(1, 4)-D-galacturonosyl
containing branch-points with L-arabinose and D-galactose can be incorporated in the main
polymeric chain. Pectin may also contain residues of D-glucuronic acid, D-apiose, D-xylose
and L-fucose attached to poly-a-(1, 4)-D-galacturonic acid sections. The molecule does not
adopt a straight conformation in solution, but is extended and curved with a large amount of
flexibility. The properties of pectin depend on the degree of esterification, which is normally
about 70%
Figure 1: Structure of pectin

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Water insoluble pectic material is referred to as protopectin and can be dissolved by weak
acids or by meta or pyrophosphates. When the solubilized material retains most of its methyl
groups and forms gels under certain conditions, it is commonly referred to as pectin. In plants
transformation of protopectin to pectin leads to fruit ripening. If all the methyl groups are
removed the remaining polymer of galacturonic acid is called pectic acid. Intermediate
substances from which methyl groups are removed are called pectinic acids.
Pectin Content in Some Fruits
Tomatoes 0.2-0.5%
Strawberries 0.6-0.7%
Raspberries 0.7-1%
Peaches 0.3-1.2%
Grapes 0.2-1%
Apples 0.5-1.6%
Pears 0.5-0.8%
Table 1
Pectin being a carbohydrate and its content varying in different fruits can present problems in
that it causes cloudiness and the pressing and filtration of juice difficult. If we consider pectin

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as a substance used in jams because of its ability to form gels it is not hard to imagine its
effect on freshly crushed fruits ready to be filtered and processed.
If the fruit processor wants to increase yield, and produce a clear product, a good Pectinase
enzyme is paramount to their operation.
2.2 INTRODUCTION ABOUT PECTINASE
Chemical Name: Poly (1, 4-a-Dgalacturonide) glycanohydrolase, poly (1, 4-a-D-
galacturonide) lyase, and pectin pectylhydrolase.
Other Names: The model enzyme is Pectinase. Among the other names for pectinase are
pectin lyase, pectin methylesterase, pectinesterase, and polygalacturonase.
Other Codes: Enzyme Commission numbers for the major components of pectinase:
 Pectin methylesterase--3.1.1.11;
 Pectin lyase: 4.2.2.10;
 Polygalacturonase: 3.2.1.15
Reaction it Catalyzes:
 Demethylation of pectin
 Hydrolysis of 1, 4-alpha-galacturonide linkages in pectin
 Eliminative cleavage of pectin to give oligosaccharides
Pectinase is a general term for enzymes that break down pectin, a polysaccharide substrate
that is found in the cell walls of plants. One of the most studied and widely used commercial
pectinases is polygalacturonase. It is useful because pectin is the jelly-like matrix which helps
cement plant cells together and in which other cell wall components, such as cellulose fibrils,
are embedded. Therefore pectinase enzymes are commonly used in processes involving the
degradation of plant materials, such as speeding up the extraction of fruit juice from fruit,
including apples and sapota. Pectinases have also been used in wine production since the
1960s. They can be extracted from fungi such as Aspergillus niger. The fungus produces

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these enzymes to break down the middle lamella in plants so that it can extract nutrients from
the plant tissues and insert fungal hyphae. If pectinase is boiled it will become denatured
(distorted) making it harder to connect with the pectin at the active site, and produce as much
juices.
Pectinases are also used for retting. Addition of chelating agents or pretreatment of the plant
material with acid enhance the effect of the enzyme. Pectinases have an optimum temperature
and pH at which they are most active. For example, a commercial pectinase might typically
be activated at 45 to 55°C and work well at a pH of 4.0 to 5.
The pectinases are required for extraction and clarification of fruit juices and wines,
extraction of oils, flavors and pigments from plant materials, preparation of cellulose fibers
for linen, jute and hemp manufacture, coffee and tea fermentations and novel applications in
the production of oligogalacturonides as functional food components. Fungal
polygalacturonases used in industrial processes for juice clarification are mainly obtained
from mesophilic aspergilli and penicillia and the range of enzyme sources is being extended
through new recombinant and non-recombinant fungal strains. Thermophilic fungi are
potential sources of various industrially important thermostable enzymes, such as lipases,
xylanases, proteases, amylases and pectinases. These enzymes have numerous applications in
the detergent, starch, food, paper and pharmaceutical industries.
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
efficient enzyme recovery procedures can reduce the cost. In addition, technical constraint
includes supply of cheap and pure raw materials and difficulties in achieving high operational
stabilities, particularly to temperature and pH. Therefore, the understanding of various
physiological and genetic aspects of pectinase is required for producing thermostable and
acid stable strains of pectinolytic fungi.
Literature highlighting the optimization, biochemical characterization, genetics and strain
improvement studies of pectinases from mesophilic fungi is available. However, the studies
on pectinases from thermophilic fungi are lacking. Considering the biotechnological
importance of thermophilic fungi in the enzyme industry, the present paper reports the
optimization of pectinase production from different organic wastes.

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2.3. INTRODUCTION ABOUT SOLID STATE FERMENTATION
Solid-state fermentation is traditionally defined as those processes in which microbial growth
and products formation occur on the surfaces of solid substrates in the near absence of free
water. Due to this low amount of water available in solid-state bioprocessing, the class of
microorganisms that are most commonly used is fungi. Several agro-industrial waste and by-
products such as orange bagasse, sugar cane bagasse, wheat bran and other food processing
waste are effective substrates for depolymerizing enzyme production by solid-state
fermentation. Recently, a large number of microorganisms, isolated from different materials,
have been screened for their ability to degrade polysaccharide present in vegetable biomass
producing pectinases on solid-state culture.
Solid-state fermentation (SSF) holds tremendous potential for the production of enzymes. It
can be of special interest in those processes where the crude fermented product may be used
directly as the enzyme source. In addition to the conventional applications in food and
fermentation industries, microbial enzymes have attained significant role in biotransformation
involving organic solvent media, mainly for bioactive compounds. This system offers
numerous advantages over submerged fermentation system, including high volumetric
productivity, relatively higher concentration of the products, less effluent generation,
requirement for simple fermentation equipment’s.
SSF processes are distinct from submerged fermentation (SmF) culturing, since microbial
growth and product formation occurs at or near the surface of the solid substrate particle
having low moisture contents. Thus, it is crucial to provide an optimized water content, and
control the water activity (aw) of the fermenting substrate—for the availability of water in
lower or higher concentrations affects microbial activity adversely. Moreover, water has
profound impact on the physio-chemical properties of the solids and this, in turn, affects the
overall process productivity.
Solid-state fermentation (SSF) processes can be defined as ―the growth of microorganisms
(mainly fungi) on moist soil materials in the absence of free-flowing water. These processes
have been used for the production of food, animal feed, and both pharmaceutical agricultural
products

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ADVANTAGE
 Comparative studies between submerged liquid fermentation (SLF) and SSF claim
higher yields and advantages for products made by SSF.
 Similar and higher yields than those obtained in the corresponding submerged
cultures.
 The low availability of water reduces the possibilities of contamination by bacteria
and yeast. This allows working in aseptic conditions in some cases.
 Similar environment conditions to those of the natural habitats for fungi, which
constitute the main group of microorganisms used in SSF.
 Higher levels of aeration, especially adequate in those processes demanding an
intensive oxidative metabolism.
 The inoculation with spores (in those processes that involve fungi) facilitates its
uniform dispersion through the medium.
 Culture media are often quite simple. The substrate usually provides all the nutrients
necessary for growth.
 Simple design reactors, with few spatial requirements can be used due to the
concentrated nature of the substrates.
 Low energetic requirements (in some cases autoclaving or vapour treatments,
mechanical agitation and aeration are not necessary).
 Small volume of polluting effluents. Fewer requirements of dissolvent are necessary
for product extraction due to their high concentration.
 The low moisture availability may favour the production of specific compounds that
may not be produced or may be poorly produced in SLF.
 In some cases, the products obtained have slightly different properties (e.g. more
thermo tolerance) when produced in SSF in comparison to SLF.
 Due to the concentrated nature of the substrate, smaller reactors in SSF with respect to
SLF can be used to hold the same amounts of substrate.
2.4. INTRODUCTION ABOUT Aspergillus Brasiliensis
Aspergillus niger (now A. brasiliensis) can be classified as a member of the
―deuteromycetes,‖ a ―class‖ reserved for organisms with no known sexual state. Although

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they are considered a deuteromycete, modern taxonomy puts them in the phlyum of
Ascomycota. Further taxonomy takes A. niger to the class of Eurotiomycetes, order of
Eurotiales, family of Trichocomaceae, and genus Aspergillus.
A. niger is a ubiquitous fungus that grows very quickly. Strains can be isolated from many
different ecological habitats such as soil, plant debris, rotting fruit, and even indoor air
environments.
Distinguishing the organism from other species of Aspergillus can be done macroscopically,
by identifying the white felt like colony turning black with conidia formation.
Microscopically one can confirm this identification by the presence of black, globose conidia
with very dark to black spores
A large number of microorganisms, including bacteria, yeast and fungi produce different
groups of enzymes. Selection of a particular strain, however, remains a tedious task,
especially when commercially competent enzyme yields are to be achieved. For example, it
has been reported that while a strain of Aspergillus niger produced 19 types of enzymes,  -
amylase was being produced by as many as 28 microbial cultures. Thus, the selection of a
suitable strain for the required purpose depends upon a number of factors, in particular upon
the nature of substrate and environmental conditions. Generally hydrolytic enzymes, e.g.
cellulases, xylanases, pectinases, etc. are produced by fungal cultures, since such enzymes are
used in nature by fungi for their growth. Trichoderma spp. and Aspergillus spp. have most
widely been used for these enzymes. Filamentous fungi too commonly produce amylolytic
enzymes and the preferred strains belong to the species of Aspergillus and Rhizopus.
Although commercial production of amylases is carried out using both fungal and bacterial
cultures, bacterial  -amylase is generally preferred for starch liquefaction due to its high
temperature stability. In order to achieve high productivity with less production cost,
apparently, genetically modified strains would hold the key to enzyme production.

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3. OBJECTIVE
The major objective of this project is optimization of ratios of citrus fruit peel powder
and wheat bran for pectinase production.
AIM
Utilization of industrial waste (citrus fruit peel waste and wheat bran) for pectinase
production.

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4. REVIEW OF LITERATURE
4.1. PECTINASE ENZYME COMPOSITION
Pectinase are a complex group of enzymes that act on the pectin substances and lead
to its breakdown into galacturonate. Pectinase are a large family of enzymes acting on
the polymers and mainly composed of pectin hydrolases, lyases and esterase (Scholar
& College, 2014). This group of enzymes includes polygalacturonase and pectolyase.
Polygalacturonase (PG) is a depolymerizing enzyme that cleaves glycosidic bonds of
pectin by means of hydrolysis. Pectinesterase (PE) is a pectolytic enzyme and is
responsible for hydrolysis of ester bonds in the pectin molecule. Action of PG and PE
together results in breakdown of pectin molecules (Martos, Vazquez, Benassi, &
Hours, 2009).
4.2. COMMERCIAL APPLICATIONS OF PECTINASE
Pectinases can be used commercially for extraction and clarification of fruit juices and
wines. Tropical fruits are usually too pulpy and pectinaceous to yield juice by simple
pressing or centrifugation. In soft fruits like guava, papaya, mangoes the extraction
process requires pectinase wherein after pulping the fruits (after peeling wherever
necessary) and warming to 60-65oC for 15 minutes to inactivate the innate enzymes,
calculated quantities of pectinase enzyme is added and mixed well. The juice is then
separated out using basket centrifuge or pressing through cheese cloth in a filter press.
Commercially pectinolytic enzymes are used for pectin degradation which is settled
down organic particles in suspension like fruit juices. The use of pectinase enzymes
resulted in higher yield and clarity of juice also preserving the nutrients, original
colour and flavour.
The application of pectinase enzyme have improved the clarification process for apple
juice, tangerine juice, pineapple juice and plum, peach, pear and apricot juice prior to
ultrafiltration. Also the juice recovery of enzymatically treated pulps increased

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significantly from 52-72% in plums, 38-63% in peach, 60-72% in pear and 50-80% in
apricot (Tapre & Jain, 2014).
4.3. PECTINASE EXTRACTION
Pectinases can be produced by submerged and solid state fermentation (SSF).
Submerged fermentation is the process of growing microorganisms on liquid broth. It
requires high volumes of water, continuous agitation and generates lot of effluents.
Solid state fermentation uses culture substrates with low water levels (reduced water
activity). The methods used to grow filamentous fungi using solid state fermentation
allow the best reproduction of their natural environment. The medium is saturated
with water but little of it is free-flowing. The solid medium comprises both the
substrate and the solid support on which the fermentation takes place. The substrate
used is generally composed of vegetal byproducts such as beet pulp or wheat
bran(Geetha, Mahalakshmi, & Reetha, 2012).
Fungi can produce both intracellular as well as extracellular enzymes. All fungi are
heterotrophic, and rely on carbon compounds synthesized by other living organisms.
Small molecules like mono or disaccharides, fatty acids and amino acids can easily
pass through the cell membrane but larger molecules cannot pass through it. For
breaking down of larger complex compounds like cellulose, hemicellulose, lignin,
starch and pectin, fungi secrete extracellular enzymes. It is well known that as
compared to intracellular enzymes, the extracellular enzymes are easier to be
extracted. Intracellular enzymes require more time and costly chemicals for
extraction.(―Chapter - 3 Fungal Extracellular Enzymes,‖ n.d.) Till date, substrates
used for solid-state fermentation are materials of plant origin like grains such as rice,
corn, root, tubers, and legumes. Apart from these, pomace, mango peels, orange waste
like peels and other fruit and vegetable industry waste are also being in much
use(Geetha et al., 2012)
4.4. MICROBES RESPONSIBLE FOR PECTINASE PRODUCTION
Several enzymes from the group of pectinases are obtained with the aid of genetically
modified moulds, for example a variety of pectinesterases (enzymes that modify

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pectin) are produced with the aid of genetically modified moulds Aspergillus,
Penicillium). However certain groups like pectatlyase can be obtained with
genetically modified bacteria (Bacillus), which is utilised only for technical industrial
purposes.
Among the moulds, Penicillium and Aspergillus strains have good prospects of
pectinase production, maximum pectinolytic activity was shown by was Aspergillus
niger, Penicillium jenseni and closely followed by Penicillium citrinum(Priya &
Sashi, 2014). Despite of the fact that pectinase can be obtained from both bacterial
and fungal cultures; the pectinases are obtained primarily through fermentation with
fungal cultures like Aspergillus, Penicillium and Trichoderma.
Graph 1
4.5. VARIOUS OPTIMIZATION PROCESSES FOR PECTNASE PRODUCTION
Aspergillus niger, ATCC 16404 was found as effective pectinase producer. Maximum
enzymatic activity (1.62 IU ml-1) was observed after 7 days incubation at 30˚C
temperature in 250 ml Erlenmeyer conical flask. In this study 1% dextrose was used
as carbon source, although citric acid as a carbon source showed better result (2.73 IU

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ml-1) but starch was not cost effective. As a substrate, combination of wheat bran and
fresh mosambi, orange and lemon peel in ratio of 9:1:1:1 showed good result (5.38 IU
ml-1) in solid state culture. Addition of 5% pectin was found to increase the enzyme
production as (3.38 IU ml-1) Pectinase production was optimum in 65% moisture thus
the wild strain Aspergillus niger ATCC16404 has outstanding pectinase producing
capability at 30◦C in 65% moisture content for 7 days of incubation in solid state
fermentation. (Khan, Sahay, & Rai, 2012)
In another experimental setup, heat tolerant filamentous fungus A. niger was used for
the optimization of pectinase production parameters in solid state fermentations and
also to clarify the specific fungal strain with the best enzyme (pectinase) production
activity. The optimization was carried out by experimental designing and surface
analysis methodology. The results indicated that enzyme activity was higher in wheat
bran and potato starch media as compared to cassava starch and rice husk(Akhter et
al., 2011). Apart from this the effect of incubation period, amount of pectin, nitrogen
sources, temperature and moisture content were compared to optimize pectinase
production. The graphs obtained were as follows:
Graph 2

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Graph 5
Graph 6
In another optimization study, orange peel solids (OPS) and orange peel extract
(OPE) were used as the substrates for pectinase production from Aspergillus niger.
The pectinase activity was found to be higher in the orange peel extract when peptone
was used as the nitrogen source. A maximum exopectinase activity of 6800 IU/g was
obtained in the submerged fermentation using orange peel extract. The citrus peel is

27
the by product from the fruit processing industry act as a valuable source for the
pectinase enzyme production. This study will act as first line information to the
researchers who are exploring the possibilities of converting waste to wealth in terms
of utilization of peels as substrates(Kharagpur, 2010).
4.6. ENZYME ACTIVITY ESTIMATION
Enzyme activity assay was based on the determination of reducing sugars produced as
a result of enzymatic hydrolysis of pectin by dinitro salicylic acid reagent (DNS)
method. The standard curve was prepared for reducing sugars with glucose. One
enzyme unit of endopolygalacturonase is the number of μM of reducing sugars
measured in terms of glucose, produced as a result of the action of 1.0 ml of enzyme
extract in 1 minute at 35°C ± 1°C.

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5. MATERIALS
5.1. Ingredients:
I. Agro- waste materials used:
a. Wheat bran
b. Fruit peel powder
i. Orange peel
ii. Mosambi peel
iii. Keenu peel
Procurement of agro-waste materials: Peels of citrus fruits, viz., Orange,
Mosambi, and Keenu were collected from a local juice shop in Sonipat
Organic 24’s Wheat bran was procured from Amazon.
II. Chemicals used:
a. Sucrose
b. Sodium Chloride
c. K2HPO4
d. Calcium Chloride
e. Urea
f. Ferrous sulphate
g. Starch
Procurement of Chemicals: The above listed chemicals were procured from
the College laboratories.
III. Fungal Culture:
a. Aspergillus brasiliensis
Procurement of culture: Fungi strain of MTCC 1344, Aspergillus Brasiliensis,
formerly known as Aspergillus Niger was supplied from
IMTECH, Chandigarh in freeze dried form.

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5.2. AUTOCLAVE
An autoclave is a pressurized device designed to heat aqueous solutions above their
boiling point to achieve sterilization. It was invented by Charles Chamberland in
1879. It is used for moist heat sterilization, which is carried out at 121°C for 30
minutes at 15 psi. Media is sterilized by autoclave under ordinary circumstances (at
standard pressure). Liquid water cannot be heated above 100 °C in an open vessel.
Further heating results in boiling, but does not raise the temperature of the liquid
water. However, when water is heated in a sealed vessel such as an autoclave, it is
possible to heat liquid water to a much higher temperature. As the container is
heated, the pressure rises due to the constant volume of the container. The boiling
point of the water is raised because the amount of energy needed to form steam
against the higher pressure is increased. This works well on solid objects; when
autoclaving hollow objects, however, it is important to ensure that all of the
trapped air inside the hollow compartments is vacuumed out.
Figure 2

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5.3. INCUBATOR
Laboratory incubator is a device which is used to grow and maintain
microbiological cultures. It is a necessary equipment for any laboratory dealing
with cell cultures or tissue culture work or study. The incubators are known to
provide a controlled, contaminant free environment for safe, reliable work with cell
and tissue cultures. In incubators, we can regulate conditions such as temperature,
humidity, CO2, and O2 in order to protect the cells from these variables.
Temperature in the incubator can be controlled via water or air jackets. Also,
constant humidity can be maintained by a humidity water trough, whereas, air
circulation is enabled by fans.
Figure 3
5.4. CENTRIFUGE
Centrifuge is the most common equipment found in laboratories, used to separate
materials into sub fractions. It is a device which rotates liquid samples at very high
speeds and thus creating a centripetal force. This causes the denser materials to

31
travel towards the bottom of the centrifuge tubes faster than they would under the
influence of gravity alone, while the low density materials rise to the top of the
tubes.
Figure 4
5.5. WEIGHING BALANCE
A weighing balance is a measuring instrument for determining the weight or mass
of an object.
Figure 5

32
5.6. pH METER
It is an instrument used to measure the hydrogen-ion activity in the water based
solutions. pH meter is an indicator to the extent of acidity or alkalinity of the water
based solution which is indicated in the unit pH. A typical pH meter consists of a
special measuring probe (a glass electrode) connected to an electronic meter that
measures and displays the pH reading. It is measured on the scale of 0 to 14. The
pH value of a substance is directly related to the ratio of the hydrogen ion [H+] and
the hydroxyl ion [OH-] concentrations. If the H+ concentration is greater than the
OH- concentration, then the solution is said to be acidic, that is the pH of the
material is displayed below 7.0 by the pH meter. But if the OH- concentration is
greater than the H+ concentration, then the pH meter shows the pH of the solution
as basic, that is, above 7.0. If equal amounts of H+ and OH- ions are present then
the solution is said to be neutral and the pH meter displays a 7.
5.7. HOT AIR OVEN
Also known as Dry heat sterilizer, these are electric devices which use dry
heat to sterilize. They were originally developed by Pasteur. It is better for
sterilizing glassware as dry heating includes good penetrability and non-
corrosive nature. It employs higher temperatures in the range of 160-180°C
and requires exposures time up to 2 hour, depending upon the temperature
employed.
5.8. LAMINAR FLOW CABINET
It is a carefully enclosed bench which is designed to prevent contamination of
biological samples or semiconductor wafers. It is used to maintain a working
area devoid of contaminants. Air is drawn through a HEPA filter which is
blown in a very smooth, laminar flow towards the user. The cabinets may have
UV-C germicidal lamp to sterilize the shell and contents when not in use.

33
5.9. SPECTROPHOTOMETER
It is a method which is used to measure the amount of analyte in the solution.
It works on the principle that materials absorb light of a certain wavelengths as
it passes through the solution.
Figure 6

34
6. METHODOLOGY
6.1. Sample Preparation
Preparation of fruit peel powder from citrus fruits peels:
1. The fruit peels were sun-dried separately for 3-4 days.
2. The dried peels were finely granulated and stored in air- tight containers.
Revival of freeze dried fungal strain
Following steps were followed in the revival of freeze dried fungal strain of A.
brasiliensis, as described in the MTCC catalogue.
1. Care was taken while opening the ampoule as the contents are in a vacuum.
2. A mark was made with a sharp file on the ampoule near the middle of the
cotton wool.
3. The surface around the mark was disinfected using alcohol.
4. Thick cotton wool was wrapped around the ampoule
5. The ampoule was then broken at the marked area.
6. The pointed top of the ampoule was then gently removed.
7. Now the cotton plug was carefully removed. About 0.4 ml of sterile water was
added to make a suspension of the culture.
8. The suspension was allowed to stand for 20 minutes.
9. A few drops of the suspension were streaked upon the Potato Dextrose Agar in
the petri plate.
10. Rest of the suspension was transferred to 5ml of Natural broth in a test tube.
11. The petri plate and the test tube containing the culture were incubated at an
appropriate temperature of about 35ºC.
12. The growth of fungus was seen within 5 days of incubation period.
13. All the remains in the original ampoule were sterilized before discarding.

35
Figure 7
6.2. Reagent Preparation:
Pectinase production was carried out in two different phases:
a. Phase 1: Preparation of trace elements
b. Phase 2: Preparation of culture materials
a) Phase 1: Preparation of trace elements
Following trace elements were used for pectinase production:-
ELEMENTS IN 150ml DISTILLED WATER
Sodium Chloride 200 mg
K2HPO4 100 mg
Calcium Chloride 50 mg
Urea 50 mg
Ferrous Sulphate 10 mg
Starch 2.0 g
Table 2

36
 All these trace elements were mixed in 150 ml distilled water.
 Mixture was heated to ensure proper mixing of the trace elements.
 The pH of the mixture was maintained at a range of 4.0 to 4.5 using 0.1N HCl.
 Distilled water was added to make up the volume to 300ml.
 The above procedure was followed for making trace elements in 10 different conical
flasks.
b) Phase 2: Preparation of culture materials
 Mixtures of fruit peel powder and wheat bran were made in 3 different ratios
Ratio (fruit peel :
Wheat bran)
1:3 1:2 3:4
Amount of peel
powder (grams)
20 27 34.5
Amount of Wheat
Bran (grams)
60 54 46
Sucrose (grams) 5 5 5
Table 3
 The above ratios of mixture were prepared for orange peel powder, mosambi peel
powder and keenu peel powder. All together 9 samples were prepared.
 A control sample was made using :
Pure Pectin: 10.2 gm
Wheat bran: 46 gm
Sucrose= 5gm was also added.
Media Preparation Procedure
o Both the prepared solutions:
o Trace elements
o Culture media

37
were mixed together to obtain a paste containing the culture medium
and the trace elements.
o The above paste was then autoclaved for 15 minutes at 121±2ºC.
Figure 8
6.3. Fungal Inoculation Preparation
i. Slants of Potato Dextrose Agar were prepared for fungal isolate preparation.
ii. The solidified media was then streaked with A. brasiliensis mother culture
iii. Slants were incubated at around 25ºC for 5 days for sporulation.
6.4 Inoculation
1. A solution of Ampicillin was prepared, dissolving 5mg in 5ml of distilled water.
2. This solution was then mixed with the spores of A.brasiliensis and further mixed with
the culture media.
** Ampicilin is an antibiotic. Thus it prevents bacterial growth in the culture media and
hence the enzyme production was solely carried out by the fungal isolate. **
3. The culture media was mixed thoroughly in the beaker.
4. The inoculum was then inoculated in the culture media; the beaker was covered with
aluminium foil in order to avoid any contact with the external environment.
5. After inoculation, the entire setup was left for 5 days in the incubator for Aspergillus
to grow in the media by Solid State Fermentation (SSF) for the production of enzyme
in the beaker.

38
Figure 9
6.5. Harvesting of Enzyme
1. 1% Sodium Benzoate was prepared in distilled water.
2. 150ml of the solution was added to the beaker containing culture media.
3. The entire setup was left for incubation for 24 hours.
** Sodium benzoate acts as an anti-fungal agent and kills the fungal spores present in the
mixture to stop further growth of fungi in the sample. **
6.6. Extraction of Enzyme
1. The media was pressed to obtain the enzyme.
2. The enzyme was then centrifuged at 14400 rpm to remove any suspended particles.
3. The cultured supernatant was used for pectinase assay.
4. The solutions (pectinase) extracted were stored in flasks at 4ºC.

39
Figure 10
6.7. Crude Protein Assay
Chemicals required:
o Protein standard (1000mg/100ml)
o Biruet Reagent
Procedure for crude protein estimation
1. A protein standard of concentration 100mg/100ml was prepared.
2. 10 test tubes were collected and following concentrations of protein standards were added:
( 0.02; 0.04; 0.06; 0.08; 0.1; 0.1; 0.2; 0.3; 0.4; 0.5)
3. The volume was made upto 2ml by addition of distilled water.
4. In another set of 10 test tubes, 0.03 ml of pectinase enzyme was added for estimation of
crude protein content.
5. 4ml Biruet reagent was then added to all these test tubes.
6. The tubes were incubated at room temperature for 30 minutes.
7. The absorbance of the samples was then checked at 550nm against the prepared standards.
8. Concentration of crude protein in various enzymes extracted was then calculated.
6.8. Pectinase assay
Chemicals required:
o Glucose
o Sodium hydroxide
o Cryo phenol
o DNS
o Sodium sulphite
o Rochelle salt

40
Preparation of chemicals for Pectinase assay:
1. Glucose standard was prepared
A glucose solution was prepared dissolving 100mg glucose in 100ml
distilled water.
The following concentrations (mg/ml) of glucose standards were
prepared using the above prepared glucose solution:
0.1; 0.2; 0.3; 0.4; and 0.5.
2. DNS Reagent:
i. 1% NaOH solution: 2.5 gm
ii. Cryo phenol: 500mg
iii. DNS : 2.5 gm
iv. Sodium Sulphite: 125mg
The reagent was prepared by dissolving each of the above chemicals in 250ml
of distilled water
3. Rochelle salt solution was prepared by adding 40 g Rochelle salt in 100ml
distilled water.
4. 1% pectin solution was prepared by adding 1g of pectin in 100 ml distilled water.
5. Citrate buffer was prepared by :
i. Making Citric acid solution: 2.1 g of Citric acid was mixed in 100ml distilled
water
ii. Making Sodium Citrate solution: 2.94 g of Sodium Citrate was mixed in 100
ml distilled water
A mixture of 8.2ml citric acid and 1.8ml sodium citrate solution was made and
the volume was made up to 100ml.
Procedure for Pectinase assay:
1. In a test tube the following were added:
i. 0.2 ml of 1% pectin solution
ii. 2 ml of Sodium citrate buffer prepared
iii. 1ml of the enzyme extracted
2. The solution was incubated for 0 minutes,15 minutes and 25 minutes
3. 1ml of this incubated solution was added to 3ml of DNS reagent

41
4. This newly prepared solution was then kept in water bath for 15minutes at 100ºC
5. After 15 minutes, 1 ml of Rochelle salt was added to this solution
The above procedure was followed for all the enzymes that were extracted from the
culture mediums of various ratios.
6. The solutions were diluted by adding 20ml distilled water.
7. Now the absorbance of these solutions was checked at 570nm.
8. The results were tabulated in a table.
Figure 11

42
7. RESULTS
The enzymes extracted after centrifugation where checked for enzymatic activity.
Pectinase activity was assayed by the Colori-metric method of (Miller, 1959).
Enzymatic hydrolysis of pectin by Dinitrosalicylic acid reagent (DNS) method was
used to determine reducing sugar produced. The absorbance was measured at 570 nm
using Spectrophotometer and standard curve was prepared using reducing sugar. One
enzyme unit of endo Polygalacturonase is the number of μM of reducing sugars
measured in terms of glucose, produced as a result of the action of 1.0 ml of enzyme
extract in 1 minute at 35°C ± 1°C.
7.1. Enzymatic activity of Orange Peel
 Enzymatic activity of Orange Peel in ratio 1:2 (Peel: Bran)
Time (in minutes) Absorbance (at 570nm) Concentration (mg/ml)
10 0.0877 0.3084
25 0.0527 0.2485
35 0.0792 0.2914
Table 4
Graph 7

43
Graph 8
O(1:2)
Slope 17.133
Concentration
of protein
(mg/ml) 0.2244
Specific Activity
(U) 76.35027
Table 5
Time (in minutes) Absorbance (at 570nm) Concentration
(mg/ml)
10 0.1125 0.3521
25 0.1386 0.3986
35 0.0733 0.2813
Table 6
0
20
40
60
80
100
120
140
160
180
200
0 5 10 15 20 25 30 35 40
Concentration (µmol/ml) vs Time (minutes)

44
Graph 9
Graph 10
O(1:3)
Slope 13.04
Concentration of
protein (mg/ml) 0.3522
Specific Activity (U) 37.02442
Table 7
0
50
100
150
200
250
0 5 10 15 20 25 30 35 40

45
(mg/ml)
10 0.0417 0.2257
25 0.0809 0.2947
35 0.0757 0.2851
Table 8
Graph 11

46
Graph 12
O(3:4)
Slope 12.538
Concentration of
Table 9
The maximum enzymatic activity was shown pectinase produced from Orange peel of ratio
1:2. The specific activity was found to be 76.350U.
0
20
40
60
80
100
120
140
160
180
0 5 10 15 20 25 30 35 40

47
7.2. Enzymatic activity of Kinnu Peel
 Enzymatic activity of Kinnu in ratio 1:2 (Peel: Bran)
(mg/ml)
10 0.1204 0.3670
25 0.1689 0.4542
35 0.1667 0.4493
Table 10
Graph 13

48
Graph 14
K(1:2)
Slope 20.388
Concentration of
Table 11
(mg/ml)
10 0.1191 0.3642
25 0.1987 0.5060
35 0.1721 0.4606
Table 12
0
50
100
150
200
250
300
0 5 10 15 20 25 30 35 40

49
Graph 15
Graph 16
0
50
100
150
200
250
300
0 5 10 15 20 25 30 35 40

50
K(1:3)
Slope 20.233
Concentration of
Table 13
(mg/ml)
10 0.0774 0.2808
25 0.2247 0.5519
35 0.2043 0.5179
Table 14
Graph 17

51
Graph 18
K(3:4)
Slope 15.672
Concentration of
Table 15
The maximum enzymatic activity was shown pectinase produced from Kinnu peel of ratio
1:2. The specific activity was found to be 138.4114 U.
0
50
100
150
200
250
300
350
0 5 10 15 20 25 30 35 40

52
7.3. Enzymatic activity of Mosambi Peel
 Enzymatic activity of Mosambi in ratio 1:2 (Peel: Bran)
(mg/ml)
10 0.0511 0.4493
25 0.1513 0.3670
35 0.1093 0.4542
Table 16
Graph 19

53
Graph 20
M(1:2)
Slope 13.555
Concentration of
Table 17
(mg/ml)
10 0.1269 0.3767
25 0.1314 0.3900
35 0.1303 0.3801
Table 18
0
50
100
150
200
250
0 5 10 15 20 25 30 35 40

54
Graph 21
Graph 22
0
50
100
150
200
250
0 5 10 15 20 25 30 35 40

55
M(1:3)
Slope 20.933
Concentration of
Table 19
(mg/ml)
10 0.1170 0.5179
25 0.1816 0.1021
35 0.1276 0.5511
Table 20
Graph 23

56
Graph 24
M(3:4)
Slope 20.005
Concentration of
Table 21
The maximum enzymatic activity was shown pectinase produced from Mosambi peel of
ratio 1:3. The specific activity was found to be 291.456 U.
Summary of Specific Activity of Enzyme Samples
Mosambi
(1:3)
Mosambi
(1:2)
Mosambi
(3:4)
Kinnu
(1:3)
Kinnu
(1:2)
Kinnu
(3:4)
Orange
(1:3)
Orange
(1:2)
Orange
(3:4)
Specific
Activity
(U)
291.54 135.27 226.30 76.52 138.41 100.33 37.02 76.35 44.66
Table 22
0
50
100
150
200
250
300
0 5 10 15 20 25 30 35 40

57
Graph 25
291.54
135.27
226.30
76.52
138.41
100.33
37.02
76.35
44.66
Mosambi
(1:3)
Mosambi
(1:2)
Mosambi
(3:4)
Kinnu (1:3) Kinnu (1:2) Kinnu (3:4) Orange
(1:3)
Orange
(1:2)
Orange
(3:4)
Specific Activity(U)

58
8. CONCLUSION
The major conclusions drawn from this study has helped to find out that the higher
pectinase activity was found in mosambi peels and wheat bran in the ratio 1:3 followed
by kinnu peels with wheat bran in the ratio 1:2 and then orange peels with wheat bran in
the ratio 1:3.
Overall it can be concluded that mosambi peels with wheat bran in ratio 1:3 shows better
pectinase activity as compared other peels.
For high yield of glucose production by break down of pectin by pectinase 15 minutes for
was found optimum time duration.

59
BIBLIOGRAPHY
WORKS CITED
Akhter, N., Morshed, M. A., Uddin, A., Begum, F., Sultan, T., & Azad, A. K. (2011).
Production of Pectinase by Aspergillus niger Cultured in Solid State Media.
International Journal of Biosciences (IJB), 1(1), 33–42.
Chapter - 3 Fungal Extracellular Enzymes. (n.d.).
Geetha, M., Mahalakshmi, S., & Reetha, D. (2012). Screening of Pectinase Producing
Bacteria and fungi for its pectinolytic activity using fruit wastes. International Journal
of Biochemistry, 30–42.
Khan, A., Sahay, S., & Rai, N. (2012). Production and optimization of Pectinase enzyme
using Aspergillus niger strains in Solid State fermentation. Research in Biotechnology,
3(3), 19–25.
Kharagpur, T. (2010). Pectinase Production from Orange Peel Extract and Dried Orange Peel
Solid as Substrates Using Aspergillus niger Pectinase Production from Orange Peel
Extract and Dried Orange Peel Solid as Substrates Using A spergillus niger, (May).
Martos, M. A., Vazquez, F. M., Benassi, F. O., & Hours, R. A. (2009). Production of
pectinases by A. Niger: Influence of fermentation conditions. Brazilian Archives of
Biology and Technology, 52(3), 567–572. https://doi.org/10.1590/S1516-
89132009000300007
Priya, V., & Sashi, V. (2014). Pectinase enzyme producing Microorganisms. International
Journal of Scientific and Research Publications, 4(1), 2250–3153. Retrieved from
www.ijsrp.org
Scholar, P. D., & College, P. S. G. R. K. (2014). Pectinase Enzyme Production by using
Agrowastes, 3(4).
Tapre, A. R., & Jain, R. K. (2014). Pectinases: Enzymes for fruit processing industry.
International Food Research Journal, 21(2), 447–453.

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