(LUẬN văn THẠC sĩ) isolation, selection and identification of aspergillus oryzae producing high salt tolerant neutral protease

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

VU THI LAN

ISOLATION, SELECTION AND IDENTIFICATION OF
ASPERGILLUS ORYZAE PRODUCING HIGH SALT
TOLERANT NEUTRAL PROTEASE

Major:

Food technology

Code:

24.18.05.54

Supervisor:

Dr. Nguyen Hoang Anh

AGRICULTURAL UNIVERSITY PRESS - 2017

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DECLARATION
I hereby declare that the thesis entitled “Isolation, selection and identification of
Aspergillus oryzae producing high salt tolerant neutral protease” is the result of the
research work carried out by me under the guidance of Dr. Nguyen Hoang Anh in the
Central Laboratory of Food Science and Technology, the faculty of Food Science and
Technology, Vietnam National University of Agriculture.

I certify that the work presented in this thesis has not been submitted to any
other universities. Any help received in preparing this thesis and all sources used have
been specifically acknowledged.
Hanoi, May 10th, 2017
Master candidate

Vu Thi Lan

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ACKNOWLEDGEMENT
I would like to express my deep gratitude and appreciation to my supervisor, Dr.
Nguyen Hoang Anh, Vice Dean as well as Head of Central Laboratory of the faculty of
Food Science and Technology whose encouragement and guidance supported me to do
this thesis. His patience, motivation, enthusiasm, and immense knowledge helped me
during the time of my research and thesis writing.
I am grateful to Research and Teaching Higher Education Academy-Committee
on Development Cooperation (ARES-CDD) for generous financial support for the
course work and research work.
I sincerely thank all the teachers in the Department of Food Safety and Quality
management, Faculty of Food Science and Technology, who gave me many valuable
suggestions and ideas for my thesis.
Finally, I would like to acknowledge my family and friends for their love and
encouragement during the completion of the thesis.
Hanoi, May 10th, 2017
Master candidate

Vu Thi Lan

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TABLE OF CONTENT
Declaration .................................................................................................. i
Acknowledgement...................................................................................... ii
Table Of Content ....................................................................................... iii
List Of Abbreviations ................................................................................. v
List Of Tables ........................................................................................... vi
List Of Figures ......................................................................................... vii
PART I. INTRODUCTION ..................................................................... 1
1.1.
Introduction ................................................................................ 1
1.2.
Objectives of study ..................................................................... 2
PART II. LITERATURE REVIEW........................................................ 3
2.1.
Enzyme protease......................................................................... 3
2.1.1.
2.1.2.
2.1.3.
2.1.4.
2.2.
2.2.1.
2.2.2.
2.2.3.

Enzyme protease......................................................................... 3
Classification of proteases .......................................................... 4
Application of proteases in industries ......................................... 6
Sources of proteases ................................................................... 8
Aspergillus group ....................................................................... 9
General characteristics of Aspergillus oryzae ........................... 10
Use of Aspergillus oryzae ......................................................... 13
Enzyme production of A. oryzae ............................................... 14

PART III. MATERIAL AND METHOD ............................................. 16
3.1.
Material .................................................................................... 16
3.1.1.
3.1.2.
3.1.3.

Sample collection ..................................................................... 16
Reference fungi ........................................................................ 18
Fungal media and buffers ......................................................... 18

3.2.
3.2.1.

Methods.................................................................................... 20
Isolation of Aspergillus oryzae from natural substrates ............. 20

3.2.2.
3.2.3.

Primary identification of Aspergillus oryzae ............................. 21
Determination of protease activity by well diffusion and
enzymatic assay ........................................................................ 21
Effect of pH on activity and stability of protease ...................... 23

3.2.3.

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3.2.4.
3.2.5.

Effect of NaCl concentrations on activity and stability of
protease .................................................................................... 23
Identification of Aspergillus oryzae by molecular biological
method ..................................................................................... 23

PART IV. RESULTS AND DISCUSSION ........................................... 25
4.1.
Isolation and primary identification of Aspergillus oryzae ........ 25
4.1.1.
Isolation of Aspergillus oryzae from the natural sources ........... 25
4.1.2.
4.2.

Primary identification of the isolated fungal isolates ................ 27
Determination of protease activity produced from isolated

A. oryzae................................................................................... 29

4.2.1.

Determination of protease activity produced from the
isolates………..... ..................................................................... 30
Growth rate of the fungi on the different media ........................ 32

4.2.2.
4.3.

Effect of Sodium chloride (NaCl) on protease activity and
stability..................................................................................... 33

4.3.1.
4.3.2.
4.4.
4.4.1.
4.4.2.
4.5.
4.5.1.
4.5.2.

Effect of NaCl on protease activity ........................................... 33
Effect of salt on protease stability ............................................. 35
Effect of pH on the protease activity and stability..................... 36
Effect of pH on the protease activity......................................... 36
Effect of pH on the protease stability ........................................ 37
Identification of the fungi by molecular biological method....... 38
DNA extraction and PCR ......................................................... 38

BLAST search .......................................................................... 39

PART V. CONCLUSION AND RECOMMENDATION .................... 41
5.1.
Conclusion ............................................................................... 41
5.2.
Recommendation ...................................................................... 41
REFERENCES......................................................................................... 42

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LIST OF ABBREVIATIONS
Acronym

Abbreviations

A.flavus

Aspergillus flavus

A. oryzae

Aspergillus oryzae

A. sojae

Aspergillus sojae

A. nomius
A. parasiticus

Aspergillus nomius
Aspergillus parasiticus

ITS

Internal Transcribed Spacer

PCR

Polymerase Chain Reaction

BLAST

Basic Local Alignment Search Tool

bp

Base pair

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LIST OF TABLES
Table 2.1. Characteristics of types of proteases.................................................... 5

Table 3.1. Characteristics of the collected samples ............................................ 16
Table 3.2. The enzymatic assay procedure of protease ....................................... 22
Table 4.1. Natural sources of Aspergillus oryzae and isolation results ............... 25
Table 4.2. Morphological characteristics of four isolates on PDA...................... 28
Table 4.3. Diameter of clear zones of protease produced from isolates .............. 30
Table 4.4. Diameter (mm) of colony on PDA and CYA..................................... 32

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LIST OF FIGURES
Figure 2.1.

Crystal structure of protease from Aspergillus oryzae .................... 3

Figure 2.2.

Aspergillus oryzae morphology. ................................................... 11

Figure 2.3.

Conidial head of A. oryzae............................................................ 12

Figure 2.4.

Conidial head of A. flavus............................................................. 12

Figure 3.1.

The hyphae on the surface of soybeans (Hung Yen) and rices
(Nam Dinh) .................................................................................. 16

Figure 3.2.

Aspergillus oryzae from Institute of Microbiology and
Biotechnology .............................................................................. 18

Figure 4.1.

Morphological characterization of strain TB1............................... 29

Figure 4.2.

Aspergillus oryzae in 4-day PD broth culture ............................... 30

Figure 4.3.

The clear distinct zones of proteases on the casein agar plates
flooded with BCG reagent after 3 day incubation. ........................ 31

Figure 4.4.

The growth rate of fungus TB1 on CYA and PDA after 2 days
and 5 days. ................................................................................... 33

Figure 4.5.

Effect of NaCl concentrations on protease activity of two

isolates TB1 and G2. .................................................................... 34

Figure 4.6.

The NaCl tolerance of the protease from TB1and G2 at
16% NaCl..................................................................................... 35

Figure 4.7.

Effect of pH on protease activity from TB1 and G2 ...................... 37

Figure 4.8.

The pH stability of protease produced from A.oryzae TB1
and G2.......................................................................................... 38

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THESIS ABSTRACT
Master candidate: Vu Thi Lan
Thesis title: Isolation, selection and identification of Aspergilus oryzae producing
high salt tolerance neutral protease
Major: Food technology

Code: 24180554

Education organization: Vietnam National University of Agriculture (VNUA)

This study was to isolate, select and identify Aspergillus oryzae producing high salt
tolerant neutral protease. Four isolates (TB1, TB2, G2 and M1) in 12 isolates were
primarily assumed to be A. oryzae by morphological characterization. TB1 and G2
revealed the highest protease activity with 49.26 u/l and 29.10 u/l, respectively. The
protease was labile in the sodium chloride solution alternated from 0% to 20%. The
protease activity of TB1 and G2 behaved high salt tolerance in 16% NaCl and retained
49.2% and 34.8%, respectively, of initial activity after 9 hours. The optimum pH for
activity of the extracellular protease of both isolates TB1 and G2 were shown to be 7.0.
The protease was more stable in the neutral condition than in acid or alkaline
environments. After incubation at 37oC for 12 hours at pH 7.0, the enzyme activity left
were detected only 37% for TB1 and 41% for G2. TB1 was determined to be
Aspergillus oryzae by the molecular method.
Key words: Aspergillus oryzae (A.oryzae), protease, salt tolerance.

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PART I. INTRODUCTION
1.1. INTRODUCTION
Proteases are multifunctional enzymes and represent a fundamental group
of enzymes due to diversity of their physiological roles and biotechnological
applications (Silva et al., 2011). These enzymes are extremely important in the
pharmaceutical, medical, food, and biotechnology industries, accounting for
nearly 60% of the whole enzyme market (Ramakrishna, Rajasekhar et al., 2010).
It has been estimated that microbial proteases represent approximately 40% of
the total worldwide enzyme sales (Rao et al., 1998).
Proteases are ubiquitous but to get high salt tolerant neutral proteases is
still receiving considerable attention. Proteases can be classified into three types

based on their optimum functional pH. Neutral protease is more important for
food industry because it can hydrolyze the proteins of the raw materials
thoroughly and reduce the bitterness. It is mainly used in the industry of food
fermentation, brewing and feed additives etc. In addition, some kinds of food are
unique due to its high concentration of sodium chloride. The higher sodium
chloride content provided a lower degree of protein degradation. The salt stable
proteases are used in fermented food production, antifouling coating preparation
and waste treatment, especially at marine habitat (Gao et al., 2016). The protease
activity and stability decreased sharply when the materials is mixed with sodium
chloride at high concentration, which is used for inhibiting spoilage bacteria,
selectively retaining the slow growth of osmotolerant yeast and lactic acid
bacteria as well as prolonging the preservation time. Consequently, a protease
which could tolerate high concentration of sodium chloride is important in order
to improve food quality, to shorten the time for the maturation process and to
improve the efficiency of raw material utilization (Wang et al., 2013).
Since proteases are physiologically necessary for living organisms, being
found in a wide diversity of sources such as plants, animals, but commercial
proteases are produced exclusively from microorganisms. Fungi of the genera
Aspergillus, Penicillium and Rhizopus are especially useful for producing
proteases, as several species of these genera are generally regarded as safe, of
which, Aspergillus oryzae (A.oryzae) is mentioned (Chutmanop, Chuichulcherm
et al. 2008). This fungus is also a potential source of proteases due to their high

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proteolytic activity, broad biochemical diversity, their susceptibility to genetic
manipulation, high productivity, and being extracellular and are easily

recoverable from the fermentation medium (de Castro and Sato 2014).
Many studies have characterized the proteases from A.oryzae and
disclosed their role in food processing technology. However, there have been
only a few reports on the mechanism of the protease stability under high
concentration of sodium chloride. In order to enhance the performance of the
enzyme in shortening the production cycle and conversion rate of raw materials,
studies on the protease properties under high concentration of sodium chloride
are necessary. Besides morphological and physical chemical characteristics,
identification of the accurate A. oryzae by methods of biochemistry and
molecular biology is extremely necessary. In this context, the study "Isolation,
selection and identification of Aspergillus oryzae producing high salt tolerant
neutral protease" is conducted.
1.2. OBJECTIVES OF STUDY
 General objective
The aim of this study is to isolate, select and identify the Aspergillus
oryzae producing high salt tolerant neutral protease from some Vietnam natural
sources.
 Specific objectives
-

Isolate A. oryzae from some Vietnam natural sources and primarily
identify by morphological method;

-

Select strains producing protease by well diffusion and enzymatic assay;

-

Determine the high salt tolerance of protease activity and stability;

-

Determine the effect of pH on protease activity and stability;

-

Identify isolated strains using molecular biology method.

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PART II. LITERATURE REVIEW
2.1. ENZYME PROTEASE
2.1.1. Enzyme protease
Protease or peptidase is one of member in hydrolysis enzyme group that is
capable of cutting the peptide link of polypeptide molecules, proteins and some
other similar substrates into free amino acids and low molecular peptides.

Figure 2.1. Crystal structure of protease from Aspergillus oryzae
(Kamitori, et al., 2003)
The characters of this enzyme are common with respect to optimum pH,
temperature and stability. The biochemical characterization showed that the
enzyme was most active over the pH range 5.0–6.5 and was stable from pH 4.5
to 5.5. The optimum temperature range for activity was 55–60°C, and the
enzyme was stable at temperatures below 45°C (Vishwanatha, 2009). Majority of
these enzymes show low thermostability and lose their activities and structure at
high temperature (Rao et at., 1998).

In the body, proteins in food are digested in the digestive tract by proteindegrading enzymes, first pepsin in gastric juice and then secretions in the
pancreas and from mucosal cells, intestine. Amino acids are absorbed into the
liver and then involved in the metabolism. Protein hydrolysis plays an important
role in the production of many foods. This process can be accomplished by the
protease of the food itself or the microbial protease introduced into the food
processing process.

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Protease is one of the most important commercial enzymes, and is used in
food processing, detergents, diary industry and leather making. Proteases occur
widely in plants and animals, but commercial proteases are produced exclusively
from microorganism. Molds of the genera Aspergillus, Penicillium and
Rhizopusare especially useful for producing proteases, as several species of these
genera are generally regarded as safe (Chutmanop, Chuichulcherm et al. 2008).
2.1.2. Classification of proteases
As reported by Pushpam, proteases are classified into six types based on
the functional groups in their active sites. They are aspartic, cysteine, glutamic,
metallo, serine, and threonine proteases. They are also classified as exopeptidases and endo-peptidases, based on the position of the peptide bond
cleavage. Proteases are also classified as acidic, neutral or alkaline proteases
based on their pH optima.
Exopeptidases: The exopeptidases act only near the end of polypeptide
chains. Based on their site of action at the N or C terminus, they are classified as
aminopeptidases and carboxypeptidases, respectively (Barrett, 1994). The former
act at a free N terminus of the polypeptide chain and liberate a single amino acid
residue, a dipeptide, or a tripeptide while the later act at C terminals of the
polypeptide chain and liberate a single amino acid or a dipeptide.

Endopeptidases: The peculiar characteristics of endopeptidases are by
their preferential action at the peptide bonds in the inner regions of the
polypeptide chain away from the N and C termini. Endopeptidases are
categorized into four subgroups based on their catalytic mechanism, (i) serine
proteases, (ii) aspartic proteases, (iii) cysteine proteases, and (iv)
metalloproteases.
The serine and cysteine proteases act directly as nucleophiles to attack
the substrate, generate covalent acyl enzyme intermediates. The aspartyl and
metallo proteases activate water molecules as the direct attacking species on the
peptide bond.
General features of four types of proteases are described by Vishwanatha,
2009, as follows:

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Table 2.1. Characteristics of types of proteases
Types of Molecular
pH
Tem.
Active site
o
proteases weight
optimum optimum ( C) residues

Major inhibitors

Aspartic

30 - 45

3 -5

40 -55

Aspartic
acid

Pepstatin

Cysteine
or thiol

34 -35

2 -3

40 -45

Aspartate
or cysteine

Iodoacetamide,
p-CMB

Metallo

19 -37

5-7

65 - 85

PhenylChelating
alamine or such as
leucine
EGTA

Serine

18 - 35

6 -11

50 - 70

Serine,
histidine
and
aspartate

agents
EDTA,

PMSF,
DIFP,
EDTA,
soybean,

trypsin
inhibitor,
phosphate buffers,
indole,
phenol,
triamino acetic acid

Major sources

References

Aspergillus,
Mucor,
Endothia,
Rhizopus,
Penicillium, Neurospora,
Animal tissue (stomach)
Aspergillus, stem of
pineapple,
latex
of
Figureure tree, papaya,
Streptococus
Bacillus,
Aspergillus,
Penicilium,
Pseudomonas,
Streptococus
Bacillus,
Aspergillus,

animal
tissue
(gut),
Tritirachium album

North, 1982; Rao et
al., 1998; Kovaleva et
al., 1972

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Keay and wildi, 1970;
Keay et al, 1972;
Gripon et al., 1980
Aunstrup, 1980

Boyer,
1970;
Nakagawa, 1970


2.1.3. Application of proteases in industries
Generally proteases have a large variety of applications, in various
industries. These include food industries, detergent, pharmaceutical industries...
The application of these enzymes varies considerably.
Detergents industry:
In 1913, pancreatic extract was reported to be used for the first time in the
enzyme-detergent preparation (Rao et al., 1998). Then, after four decades, a

microbial enzyme was used commercially in the detergents under the trade name
of BIO-40 (Kumar et al., 2008). Detergent industry represent the largest
industrial application of enzymes amounting to 25–30 % of the total sales of
enzymes and expected to grow faster at a CAGR of about 11.5 % from 2015 to
2020 (Singh et al., 2016). Protease digests on stains due to food, blood and other
body secretions. Proteases are used as one of key constituent in detergents
formulations to improve washing performance for use in domestic laundering to
solution for cleaning contact lenses or dentures (Baweja et al., 2016). The
application of enzymes in detergents has the advantages of removing spots in
eco-friendly manner with shorter period of soaking and agitation (Saba et al.,
2012). The enzymes used as detergent additives should be effective in very small
amount over a broad range of pH and temperature with longer shelf life. Most
often, the proteases used in detergent formulations are serine proteases produced
by Bacillus strains. Alkaline proteases from fungal sources are also gaining
interest due to ease in downstream processing. In many formulations, cocktail of
different enzymes including protease, amylase, cellulase and lipase are also used
for improved washing effect for household purposes (Cherif et al., 2011).
Peptide synthesis:
Peptide synthesis through chemical methods has disadvantages, such as,
low yield, racemization issues and health and environmental concern due to toxic
nature of solvents and reagents used in the processes (Gill et al., 1996; Kumar,
2005). Whereas the enzyme mediated peptide synthesis offers several advantages
like enantioselectivity, racemization free, environmental friendly reaction
conditions etc. Besides, no or minimal requirement of pricey protective groups,
solvents, reagents in enzyme based synthesis are cost effective in comparison to
chemical synthesis. Enzymatic synthesis of peptides has attracted a great deal of
attention in recent years. Proteases from bacterial, fungal, plant and animal

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sources have been successfully applied to the synthesis of several small peptides,
mainly dipeptides and tripeptides. Peptide bonds can be synthesized using
proteases in either a thermodynamically controlled or a kinetically controlled
manner. Proteases from microbial sources have been used satisfactorily for
synthesis of peptide bonds as well as hydrolysis of peptide bonds. Organic
solvent tolerant alkaline proteases from the species of Aspergillus, Bacillus and
Pseudomonas have shown promising potential in the synthesis of peptide.
Proteases from microbial sources have also established their potential for
synthesis of peptide in minimal water system. Small peptides such as di or
tripeptide synthesized through enzyme mediated processes are used for nutrition
and in pharmaceuticals (Guzman et al., 2007).
Leather Industry:
The conventional methods for leather processing involve toxic and
hazardous chemicals that generate environmental pollution and consequently a
detrimental effect on living organisms. The enzyme mediated leather processing
has proved, successfully, to overcome the issues generated by chemical methods.
The application of enzymes in leather processing has improved leather quality
and reduction of environmental pollution (Jaouadi et al., 2013). Proteases are
used to degrade noncollagenous constituents of the skin and elimination of
nonfibrillar proteins. Microbial alkaline proteases are used to ensure faster
absorption of water, which reduce the soaking time. Application of alkaline
proteases coupled with hydrated lime and sodium chloride during dehairing and
dewooling reduce waste disposal. The protease mediated leather processing is an
efficient alternative in an environmental friendly manner to improve the quality
of leather, help to shrink waste and, save time and energy (Zambere et al., 2011).
Food Industry:
Proteases are used in food industry for a wide range of applications. These

enzymes are efficiently involved in the modification of properties of food
proteins to improve nutritional value, solubility, digestibility, flavour, palatability
and minimizing allergenic compounds. Besides, their basic function, they are
also used to modify functional properties, such as coagulation, emulsification,
foaming, gel strength, fat binding etc. of food proteins (Pardo et al., 2000). The
catalytic function of proteases is used in the preparation of protein hydrolysate of
high nutritional value, which is used in infant food products, medicinal dietary
products, fortification of fruit juice and soft drinks (Ward, 2011). In dairy

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industry, proteases are primarily used in cheese manufacturing to hydrolyze
specific peptide bonds to produce casein and macropeptides. The ability of
proteases to hydrolyze connective tissues and muscle fibre proteins is used for
tenderization of meat. The alkaline proteases play an important role in the
production of soy sauce and other soy products. In baking industry, they are
added to ensure dough uniformity, reduce dough consistency, maintain gluten
strength in bread and, improve flavor and texture in bread. These hydrolytic
enzymes are utilized for degradation of the turbidity complex resulting from
protein in fruit juices & alcohol based liquors; in gelatin hydrolysis and recovery
of meat proteins (Souza et al., 2015).
Other applications:
Since ancient time proteases have been included in the preparation of
sauce and other products from soy that help in the degradation of high protein
content grains. Proteolytic modification by fungal alkaline and neutral proteases
in soy processing improves their functional properties (Inacio et al., 2015).
2.1.4. Sources of proteases

Proteases are widely distributed in most of biological (plants and animals)
and microbial sources.
1- Plant proteases: Papain, bromelain and ficin represent some of the well
known proteases of plant origin. Papain is a traditional plant protease and
isolated from the latex of Carica papaya fruits. This enzyme is active between pH
5- 9 and is stable up to 90C. Bromelain is extracted from the stem and juice of
pineapples. The enzyme is also called as cysteine protease which is less stable
than that of papain. A neutral protease is also purified from Raphanus sativus
leaves. An aspartic protease is also present in potato leaves with different
physiological roles and Thiol protease is also purified from Pineapple crown leaf.
Serine protease was also found in artificially senescing parsley leaves whose
proteolytic activity was found low in young leaves and increased from the start of
senescence lead to reduction in the protein content of the leaves. Endoproteases
were also isolated from alfalfa; oat and barley senesced leaved which are
involved in the process of protein degradation during foliar senescence
(Gonzalez et al., 2011).
2- Animal proteases: The most common proteases of animal origin are
pancreatic trypsin, chymotrypsin, pepsin and rennins. Trypsin is the main

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intestinal digestive enzyme which is responsible for the hydrolysis of food
proteins. It is a type of serine protease and hydrolyzes peptide bonds in which
carbonxyl groups are contributed by the lysine and arginine residues. It is
specific for the hydrolysis of peptide bonds in which the carboxyl groups are
provided by one of the three aromatic amino acids, i.e., phenylalanine, tyrosine,
or tryptophan. It is used extensively in the de-allergenizing of milk protein

hydrolysates. Pepsin is an acidic protease and present in the stomachs of
vertebrates. Rennet is a pepsin-like protease (rennin, chymosin) which is present
in its inactive precursor, pro-rennin, in the stomachs of all ruminants. It is used
exclusively in the dairy industry to produce food flavored curd (Rao et al., 1998).
3- Microbial proteases: Microorganisms regarded as an important source
of proteases because they can be obtained in large quantities using cultural
techniques within a shortest possible time by established fermentation methods,
and they produce a regular and abundant supply of the desired product.
Furthermore, microbial proteins have a longer shelf life and can be stored under
less than ideal conditions for weeks without significant loss of activity (Gupta,
2002). Microbial proteases generally have been pointed as to be extracellular in
nature and directly express in the fermentation medium. This help in simplicity
of downstream processing of the enzymes relative to their plants and animal
counterparts. The appropriate producers of these enzymes for commercial
exploitation are non-toxic and non pathogenic that are designated as safe.
Bacteria are known to produce alkaline proteases with genus Bacillus as the
prominent source. Different exotic environment has been the sources of different
Bacillus species with alkaline protease production abilities. A large number of
microbes belonging to bacteria, fungi, yeast and actinomycetes are known to
produce alkaline proteases of the serine type (Kumar et al., 1999).
2.2. ASPERGILLUS GROUP
The genus Aspergillus represents a grouping of a very large number of
asexual fungi whose taxonomy is based on morphological features. The genus
has been divided into groups based on attributes of the spores, conidiophores,
and sclerotia. Because this separation of individual species into groups is based
on morphological or physiological characteristics, it has resulted in somewhat
tenuous and overlapping classification (Bennett, 2010). Aspergillus oryzae is a
member of the A. flavus group of aspergillus species. The A. flavus group, which
also includes A. sojae, A. nomius and A. parasiticus are defined by the production

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of spore chains in radiating heads which range in color from yellow-green to
olive brown. A.flavus and A. parasiticus are known to produce the potent
carcinogen aflatoxin. A.oryzae and A. soji have been used for producing food
grade amylase and fermentation of oriental foods for centuries (Gunawardhane et
al, 2004).
The genus Aspergillus is a diverse group of common molds and the
approximately 175 species are inhabitants of virtually all terrestrial environments,
when conditions in indoor situations are favorable for fungal growth. Most
species have relatively low moisture requirements and some are extremely
xerophilic (dry tolerant), allowing them to colonize areas that cannot support
other fungi and where only minimal or intermittent moisture is available. Their
rapid growth and production of large numbers of small, dry, easily aerosolized
spores makes them a significant contaminant concerning Indoor, air quality and
potential human exposure-related illnesses (Abbott, 2002).
2.2.1. General characteristics of Aspergillus oryzae
2.2.1.1. Morphological characteristics
Identification of the hyphomycetes is primarily based on microscopic
morphology including conidial morphology, especially septation, shape, size,
color and cell wall texture, the arrangement of conidia as they are borne on the
conidiogenous cells, e.g., solitary, arthrocatenate, blastocatenate, basocatenate or
gloiosporae, the type conidiogenous cell, e.g., non-specialized or hypha-like,
phialide, annellide or sympodial and other additional features such as the
presence of sporodochia or synnemata. For identification, PDA and cornmeal
agar are two of the most suitable media to use and exposure to daylight is
recommended to maximize culture color characteristics. Aspergillus colonies are

usually fast growing, white, yellow, yellow-brown, brown to black or shades of
green, and they mostly consist of a dense felt of erect conidiophores.
Conidiophores terminate in a vesicle covered with either a single palisade- like
layer of phialides (uniseriate) or a layer of subtending cells (metulae) which bear
small whorls of phialides (the so-called biseriate structure). The vesicle,
phialides, metulae (if present) and conidia form the conidial head. Conidia are
one-celled, smooth- or rough-walled, hyaline or pigmented and are
basocatenate, forming long dry chains which may be divergent (radiate) or
aggregated in compact columns (columnar). Some species may produce Hulle
cells or sclerotia (Fayyad, 2008).

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Scientific classification:
Kingdom

Fungi

Division

Deuteromycota

Class

Eurotiomycetes

Order

Aspergillals

Family

Aspergilluceae

Genus

Aspergillus

Species

A. oryzae

Figure 2.2. Aspergillus oryzae morphology. A–C. Colonies incubated at 25 °C
for 7d, A. CYA, B. MEA, C. Sclerotia, D–I. Conidiophores and conidia
( />
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Figure 2.3. Conidial head of A. oryzae

Figure 2.4. Conidial head of A. flavus

(www.emlab.com/m/media/Aspergillua.jpg) ( />lab/publications/publications.html)
A. oryzae (figure 2.3) is a member of the A.flavus group of Aspergillus
species. The conidiophores are roughened and colorless. The spores themselves

have conspicuous ridges and echinulations (spines). A.oryzae/flavus species have
never been connected to a sexual or teleomorphic stage. However, the
telemorphic stages of other Aspergillus species have been domonstrated by the
formation of cleistothecia (Raper, 1997; Chang, 2006). The fruiting body (or
conidial head) of A. flavus is shown in figure 2.4. In nature, selection places
limits on conidial size as may be critical to dispersal or survival conidia of
domesticated yellow-green Aspergilli from strains of A. oryzae (Ahlburg) Cohn
and A. sojae are used in the preparation of koji inoculums, germinate
approximately 3h sooner than conidia produced by related wild species
(Wicklow, 1984).
Although the details of the genetic relationship between A. oryzae and
A.flavus remain unclear, the two species are so closely related that all strains of
A. oryzae are regarded by some as natural variants of A. flavus modified through
years of selection for fermenting of foods. A. oryzae is regarded as not being
pathogenic for plants or animals, though there are a handful of reports of
isolation of A. oryzae from patients. There are also several reports of products of

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A. oryzae fermentations, e.g. amylase, that seem to be associated with allergic
responses in certain occupations with high exposure to those materials. A. oryzae
can produce a variety of mycotoxins when fermentation is extended beyond the
usual time needed for production of these foods. While wild A. flavus isolates
readily produce a flatoxins and other mycotoxins, A. oryzae has not been shown
to be capable of aflatoxin production (Raper, 1997).
Because A. oryzae has GRAS status for use in the food industry, efforts
have been made to develop molecular methods to unambiguously distinguish A.

oryzae from A. flavus. These methods include restriction fragment length
polymorphism, amplified fragment length polymorphism, electrophoretic
karyotyping, isozyme profiling and analysis of ribosomal DNA internal
transcribed spacer regions. Generally, these methods have not been successful in
unambiguously separating A. oryzae as a distinct species (Chang, 2006).
2.2.1.2. Biochemical characteristics
Under laboratory conditions, optimal growth of A. oryzae occurs within a
temperature range of 32oC to 36oC and a pH range between 2 and 8, and it
requires ions of the trace elements Fe and Zn (Domsch et al., 1980) for growth.
The protease was most active over the pH range 5.0–5.5 and was stable
from pH 4.5 to 5.5. The optimum temperature range for activity was 55–60°C,
and the enzyme was stable at temperatures below 45°C (de Castro, 2014).
2.2.2. Use of Aspergillus oryzae
A.oryzae is a filamentous fungus, which has an ability to secrete large
amounts of hydrolytic enzymes such as xenlulase, pectinase, xylanase,
hemixenlulase,... It is widely used in the manufacture of traditional fermented soy
sauce in Asia. The extracellular proteins in soybean koji inoculated with A.oryzae
contain different protein profiles including neutral and alkaline protease, amylase,
glutaminase and metallopeptidase. Moreover, A.oryzae is genomically well
characterized and considered to be a safe organism for producing of food enzymes
because it lacks expressed sequence tags for the genes responsible for aflatoxin
production (Chancharoonpong, Hsieh et al. 2012).
A. oryzae has been used for centuries in the production of many different
oriental foods such as soy sauce, sake and miso. As a "koji" mold, A. oryzae has
been used safely in the food industry for several hundred years. It is also used to

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produce livestock probiotic feed supplements. The koji mold enzymes were
among the first to be isolated and commercialized nearly 100 years ago. In koji
preparation, A. oryzae also produces a low-molecular-weight iron- chelating
compound, termed deferriferrichrysin, a type of siderophore (Morita, 2007;
Yamada, 2003 and Hocking, 2006).
A. oryzae is currently used in the production of organic compounds such
as glutamic acid, and several enzymes that are of potential use commercially, for
example, amylase, protease, ß -galactosidase, lipase, and cellulase. While
these enzymes could be used as Toxic Substances Control Act (TSCA)
products, several of them have been more often used in food processing. In
1989, Environmental Protection Agency (EPA) reviewed a pre manufacture
notice (PMN) for a strain of A. oryzae modified for enhanced production of a
lipase enzyme to be used primarily in detergent formulations for the removal of
fat-containing stains. In 1994, EPA reviewed a PMN for a similar strain of A.
oryzae modified for enhanced production of a cellulase gene for use in
detergents as a color-brightening agent.
Submerged fungal fermentations are widely used in the production of
enzymes, antibiotics and organic acids, which have many applications in the
food, medicine, pharmaceutical, chemical and textile industry. However, their
filamentous growth characteristic creates a number of process engineering
problems attributed to the morphological change accounted during the
fermentation process in large scales. It is well documented that the fungal
culture exhibits two major morphologies observed as pellet or mycelia, which
are very much determined by several environmental and genetic factors These
are; type of the strain, pH and composition of the media, inoculation ratio,
type of the inoculums, agitation speed, aeration rate, feeding rate, and genetic
factors of the culture (Amanullah, 2000; Gogus, 2006).
2.2.3. Enzyme production of A. oryzae

Protease: is one of member in hydrolysis enzyme group that is capable of
cutting the peptide link of polypeptide molecules, proteins and some other
similar substrates into free amino acids and low molecular peptides. Proteases
are multifunctional enzymes and represent a fundamental group of enzymes due
to diversity of their physiological roles and biotechnological applications (de
Castro, 2014).

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α –amylase: is composed of a group of ubiquitous endoglycosidases that
hydrolyze 1,4-glucosidic linkages in polysaccharides containing three or more
α- 1,4-linked D-glucose units yielding a mixture of maltose and glucose
(Hocking, 2006).
Cellulase: refers to a group of enzymes that act together to hydrolyze cellulose
to glucose. Although cellulases are distributed throughout the biosphere, they are
manifest in fungi and microbial sources (Quirce et al., 1992).
D-galactosidases: such as α-galactosidases, it hydrolyses variety of simple
oligosaccharides and more complex polysaccharides (Shankar, 2007).
Lactase: a disaccharidase enzyme produced by A. oryzae and A. niger, is
used extensively in the food and drug industries (Bernstein, 1999).
Glutaminase: is generally regarded as a key enzyme that controls the delicious
taste of fermented foods such as soy sauce. This unique taste called umami,
elicited by meat, fish and vegetable stocks, has been confirmed as the fifth basic
taste beside sweet, acid, salty and bitter (Weingand Ziade et al., 2003).
Acid phosphatase: A. oryzae produce three types of acid phosphatase (ACP-I,
ACP-II, and ACP-III) in a submerged culture by using only phytic acid as the
phosphorous substrate (Fujita et al., 2003).

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PART III – MATERIAL AND METHOD
3.1. MATERIAL
3.1.1. Sample collection
In this study, protein containing substrates have been considered to isolate
Aspergillus oryzae. The collected substrates were described in Table 3.1.

Figure 3.1. The hyphae on the surface of soybeans (Hung Yen)
and rices (Nam Dinh)
Table 3.1. Characteristics of the collected samples
Samples

No. samples

Collected places

Soysauces

3

Ban town, Hung Yen

Pictures of samples

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