Tài liệu 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 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 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 ii 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. iii 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 iv 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 v 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 vi 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 vii 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. viii 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 1 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. 2 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. 3 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: 4 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 5 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 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 6 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 7 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 8 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 9 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). 10 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 (https://www.researchgate.net/figure/51618627_fig17_Aspergillus-oryzae-RIB40-A-C-Colonies-incubated-at-25-C-for-7-d-A-CYA-B-MEA-C) 11