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 AGRICULTURAL UNIVERSITY

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

VU THI LAN

ISOLATION, SELECTION AND IDENTIFICATION OF ASPERGILLUS ORYZAE PRODUCING HIGH SALT

TOLERANT NEUTRAL PROTEASE

AGRICULTURAL UNIVERSITY PRESS - 2017

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

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 10 th , 2017

Master candidate

Vu Thi Lan

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 Enzyme protease 3

2.1.2 Classification of proteases 4

2.1.3 Application of proteases in industries 6

2.1.4 Sources of proteases 8

2.2 Aspergillus group 9

2.2.1 General characteristics of Aspergillus oryzae 10

2.2.2 Use of Aspergillus oryzae 13

2.2.3 Enzyme production of A oryzae 14

PART III MATERIAL AND METHOD 16

3.1 Material 16

3.1.1 Sample collection 16

3.1.2 Reference fungi 18

3.1.3 Fungal media and buffers 18

3.2 Methods 20

3.2.1 Isolation of Aspergillus oryzae from natural substrates 20

3.2.2 Primary identification of Aspergillus oryzae 21

3.2.3 Determination of protease activity by well diffusion and enzymatic assay 21

3.2.3 Effect of pH on activity and stability of protease 23

3.2.4 Effect of NaCl concentrations on activity and stability of

protease 23

3.2.5 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 Primary identification of the isolated fungal isolates 27

4.2 Determination of protease activity produced from isolated A oryzae 29

4.2.1 Determination of protease activity produced from the isolates……… 30

4.2.2 Growth rate of the fungi on the different media 32

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

4.3.1 Effect of NaCl on protease activity 33

4.3.2 Effect of salt on protease stability 35

4.4 Effect of pH on the protease activity and stability 36

4.4.1 Effect of pH on the protease activity 36

4.4.2 Effect of pH on the protease stability 37

4.5 Identification of the fungi by molecular biological method 38

4.5.1 DNA extraction and PCR 38

4.5.2 BLAST search 39

PART V CONCLUSION AND RECOMMENDATION 41

5.1 Conclusion 41

5.2 Recommendation 41

REFERENCES 42

LIST OF ABBREVIATIONS

A parasiticus Aspergillus parasiticus

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

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

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 37 o C 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

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

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

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 degrading 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

protein-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 exo-peptidases 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:

Table 2.1 Characteristics of types of proteases

Tem

optimum (oC)

Active site residues

Major inhibitors Major sources References

Aspartic 30 - 45 3 -5 40 -55 Aspartic

acid

Pepstatin Aspergillus, Mucor,

Endothia, Rhizopus, Penicillium, Neurospora, Animal tissue (stomach)

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

Aspergillus, stem of pineapple, latex of Figureure tree, papaya, Streptococus

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

Metallo 19 -37 5 - 7 65 - 85

Phenyl-alamine or leucine

Chelating agents such as EDTA, EGTA

Bacillus, Aspergillus, Penicilium,

Pseudomonas, Streptococus

Aunstrup, 1980

Serine 18 - 35 6 -11 50 - 70 Serine,

histidine and aspartate

PMSF, DIFP, EDTA, soybean, trypsin inhibitor, phosphate buffers, indole, phenol, triamino acetic acid

Bacillus, Aspergillus, animal tissue (gut), Tritirachium album

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

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

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

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

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)

40-A-C-Colonies-incubated-at-25-C-for-7-d-A-CYA-B-MEA-C)

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

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

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)

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)

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 Pictures of samples

Soysauces 3 Ban town, Hung Yen

Samples No samples Collected places Pictures of samples

Molded

soybean 3 Hanoi

Tofu dreg 3 Hanoi

Molded rice 3 Nam Dinh

Samples No samples Collected places Pictures of samples

Fermented

Three sample sites of natural sources were randomly picked up, wrapped

in plastic bags and kept at 35oC; after 2-3 days, mycelium appeared The hyphae growing on the sample surface were shown in Figure 3.1

3.1.2 Reference fungi

Aspergillus oryzae strain supplied from Institute of Microbiology and Biotechnology (IMBT) – Vietnam National University, Hanoi was used as reference

Figure 3.2 Aspergillus oryzae from Institute of Microbiology and Biotechnology (IMBT) A Colonies on PDA at 30oC for 5 days; B Sclerotia on

PDA at 30oC for 5 days 3.1.3 Fungal media and buffers

In this study, the experiments were conducted with some artificial media:

WA, PDA, PD broth, CYA and Casein agar

PDA (Potato Dextrose Agar):

Ingredients: Potatoes 200g, agar 20g, glucose 20g and distilled water Preparation: Potatoes were peeled, diced and boiled with 500 ml distilled water for 30 minutes The extract was filtered using a steel mesh Glucose and agar was added to the extract and dissolved in a microware for 3 – 5 min Water was added up to 1000 ml Medium flask was autoclaved at 1210C for 30 minutes Autoclaved medium was left to cool down 55-60OC and poured to sterilized Petri dishes in a bio-cabinet and left for 30 min for solidification and surface drying Medium dishes were wrapped with saran membrane and stored at 4 OC until use

PD broth (Potato Dextrose):

Ingredients: Potatoes 200g, glucose 20g, distilled water

Preparation: Potatoes were peeled, diced and boiled with 500 ml distilled water for 30 minutes The extract was filtered using a steel mesh Glucose was added to the extract and dissolved by stirring Water was added up to 1000 ml Medium flask was autoclaved at 1210C for 30 minutes

CYA (Czapek Yeast extract Agar)

Ingredients: Yeast extract 5g, NaNO3 0.3g, K2HPO4 1g, KCl 0,05g, MgSO4.7H2O 0,05g, agar 15g, sucrose 30g, distill water

Preparation: Adjust sucrose solution at pH 4.5, then mixed with other components in a flask Water was added up to 1000 ml Medium flask was autoclaved at 1210C for 30 minutes and then withdrawn to pour into petri dishes

WA (Water Agar):

Ingredients: Agar 20g and distilled water

Preparation: Agar was dissolved by distilled water in a microware and then sterilized and distributed in Petri dishes similarly to PDA medium

Casein agar:

Ingredients: Agar 15g, casein 10g, bromocresolgreen (BCG) 10ml and distilled water

Preparation: Agar, casein and BCG was scaled and put into a 1000ml flask

of distill water The mixture was swirled and adjusted pH 8 in order to resolve casein and make solution homogenous Medium flask was autoclaved at 1210C for 30 minutes and distributed in Petri dishes similarly to PDA medium

Potassium phosphate Buffer:

Prepare by using 11.4 mg/ml of potassium phospate dibasic, trihydrate in purified water and adjusting pH with 1M HCl This solution was placed at 37°C prior to use

Briton – Robinson Buffer:

Prepare by using 20mM sodium citrate, 20 mM sodium phosphate, and 20

Mm borate; 1M NaOH was used to adjust to the respective pH of interest

3.2 METHODS

3.2.1 Isolation of Aspergillus oryzae from natural substrates

The fungal isolation was conducted in a Laminar flow cabinet to ensure culturing work was carried out under sterilized condition Samples of sauces and seeds assumed to be contaminated with Aspergillus oryzae were collected and isolated by using isolation method of Nevalainen et al., 2014 with some modifications

Firstly, seed samples were sterilized by 70% ethanol solution, then dipped

in antibiotic solution Streptomycin 3% in 30 seconds before culturing in order to eliminate external remnants and obtain mainly endophytic strains Then the samples were aseptically cut by using sterile surgical blades and the inner tissues

of the seeds were carefully picked up and placed onto the central part of several prepared petri plates containing WA medium Theses plates were sealed and labeled by sample codes and incubated at 30oC

For sauce samples, a small amount of sauces was aseptically placed onto the central part of several prepared petri plates containing WA medium These were sealed and labeled by sample codes and incubated at 30oC

After 4 day incubation, the fungal colonies observed growing on the surface of the medium plates, colonies that differ in time of appearance, size, color, morphological shape and growth rate were recorded A loop full of each original fungal colony was picked up and sub-cultured onto PDA plates The plates were incubated again in the incubator at 30oC till the fungal colony grows to cover the whole plate surface If there was any contamination in the culture, the subculture was repeated several times until finally pure isolates were obtained For further experiments, the isolates were transferred to PDA slants and preserved at 4oC in a fridge

3.2.2 Primary identification of Aspergillus oryzae

After the incubation period, all the plates are observed for microscopic culture, such as colony diameter, colony color, conidial color, mycelia color, colony reverse, colony texture and nature of spores As description of Leck (1999), the microscopic characteristics are observed by preparing slide cultures:

- A drop of 70% alcohol was placed on a microscope slide

- A small portion of hyphae was immersed in the drop of alcohol

- One or at most two drops of lacto-phenol are added before the alcohol dries out

- The cover slip was put gently to avoid air bubbles

Accurate identification of filamentous fungi was based on the microscopic organization of sporulating parts of a colony Spores and fruiting bodies were more useful in distinguishing fungi, not their colony appearance grossly The microscopic characteristics of isolates and the reference were examined accordingly

3.2.3 Determination of protease activity by well diffusion and enzymatic assay 3.2.3.1 Preparation of crude enzyme extract

Protease production was followed the method of Fernandes et al, 2010, with some modifications After sub-cultured from isolates into PDA plates and incubated at 30oC in 4 days, some mycelia plugs (5 mm diameter) of actively growing fungi were taken out from the apical zones to inoculate and develop in Erlenmeyer flasks of 200 mL of PD broth medium These flasks were shaken in incubator at temperature 30 ±l°C After 4 day incubation, the cultures were filtered through specific filter paper (pore size of 0.45 µm) and then centrifuged

at 6000 rpm in 20 minutes The supernatants were used to determine enzyme activity and further characterization of enzyme

3.2.3.2 Determination of protease activity by well diffusion

Followed Vijayaraghavan et al., 2013, agar culture was prepared along with BCG and 1% (w/v) casein and poured in the petri dishes The plates were solidified for 30 min and wells (5mm diameter) were punched on each plate A drop of 100µl of crude extract was put into each well, then the plates were incubated at temperature 30oC for 2 – 3 days A zone of proteolysis was detected

on the casein agar plates

3.2.3.3 Determination of protease activity by enzymatic assay

Sigma's non-specific protease activity assay was used as a standardized procedure to determine the activity of proteases

In this assay, casein was used as a substrate When the protease digested casein, the amino acid tyrosine was liberated along with other amino acids and peptide fragments Folin's reagent primarily reacted with free tyrosine to produce

a blue colored chromophore The more tyrosine that was released from casein, the more the chromophores were generated and the stronger the protease activity Absorbance values generated by the protease activity were compared to a standard curve, which was generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles From the standard curve, the protease activity was determined in terms of Units, which was the amount in micromoles of tyrosine equivalents released from casein per minute

After reagents were correctly prepared, the assay was conducted as following procedure:

Table 3.2 The enzymatic assay procedure of protease

- Mixed by swirling and incubated at 370C in 30 min

- Each solution were filtered by using a 0.45µl syringe

- Pipette 2 ml of the filtrates into the new tubes