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 c 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 c 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 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 c 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 Introduction 1.2 Objectives of study PART II LITERATURE REVIEW 2.1 Enzyme protease 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 Classification of proteases Application of proteases in industries Sources of proteases Aspergillus group 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 c luan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.protease luan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.protease 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 c luan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.protease luan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.protease 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 c luan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.protease luan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.proteaseluan.van.thac.si.isolation.selection.and.identification.of.aspergillus.oryzae.producing.high.salt.tolerant.neutral.protease LIST OF TABLES Table 2.1 Characteristics of types of proteases 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 c