Luận văn bioactive peptides discovery from spirulina classification of common characteristics of

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

LUONG TAI CHIEN

Topic title:

BIOACTIVE PEPTIDES DISCOVERY

FROM SPIRULINA: CLASSIFICATION OF COMMON

CHARACTERISTICS OF ANTIHYPERTENSIVE PEPTIDES

BACHELOR THESIS

Study Mode : Full-time

Major : Biotechnology

Faculty : Biotechnology and Food Technology

Batch : 2013 – 2017

Thai Nguyen, 6/2017

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THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

LUONG TAI CHIEN

Topic title:

BIOACTIVE PEPTIDES DISCOVERY

FROM SPIRULINA: CLASSIFICATION OF COMMON

CHARACTERISTICS OF ANTIHYPERTENSIVE PEPTIDES

BACHELOR THESIS

Study Mode : Full-time

Major : Biotechnology

Faculty :Biotechnology and Food Technology

Batch : 2013 – 2017

Supervisors :Asst. Prof. Dr. Marasri Ruengjitchatchawalya

Dr. Duong Van Cuong

Supervisors 1

Asst. Prof. Dr. Marasri Ruengjitchatchawalya

Supervisor 2

Dr. Duong Van Cuong

Thai Nguyen, 6/2017

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DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Major Biotechnology

Student name Luong Tai Chien

Student ID DTN1353150004

Thesis title

Bioactive peptides discovery from Spirulina: Classification

of common characteristics of antihypertensive peptides

Supervisors

Asst. Prof. Dr. Marasri Ruengjitchatchawalya

Dr. Duong Van Cuong

Abstract:

Arthrospira (Spirulina)platensisis a spiral filamentous

cyanobacteriumcontaining high protein content about 55-70%as dry weight. The alga

has been used as human food- and animal feed supplements.Attempt to discover

bioactive peptides from A. platensis, Algal Biotechnology research group at King

Mongkut’s University of Technology Thonburi (KMUTT), Thailand,have

investigated an in-silico digestion of the algal proteome and classified the obtained

peptides regarding their bioactivities.

In this report, hands-on experience about the algal, A. platensisC1, cultivation;

cell harvesting, as well as protein extraction/ separation and a bioactivity assay, anti￾hypertensive activity (AH) of some obtained peptides has been described. In addition,

using a computational tool, Clustal Omega, to determine common characteristics of

the putative anti-hypertensive peptides showed that 10 common amino acidgroups

were classified, including 4 major groups of G, P, Y and L. The reference Novel AH

peptidewas in thegroup Y with the common amino acids―YY‖. Besides, these

peptides were mostly contained both aliphatic and aromatic amino acid (A, G, I, L,

P, V, F, W, Y) and also hydrophobic amino acid residue (A, I, L, F, V, P, G).

Keywords

Spirulina (Arthrospira) platensis C1, Bioactive peptides,

common characteristics.

Number of pages 37

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ACKNOWLEDGEMENT

This thesis was completed by the support and assistance of a number of

people whom I would like to personally thank. First and foremost, I would like

to express my gratitude to the both my supervisors Asst. Prof. Dr. Marasri

Ruengjitchatchawalya of the school of Bioresources and Technology, King

Mongkut’s University of Technology Thonburi (KMUTT), Thailand and Dr.

Duong Van Cuong from the biotechnology and food technology department of

Thai Nguyen University of Agriculture and Forestry (TUAF). Whose expertise

and understanding guided me through my internship, providing useful advice for

the improvement of this work. Some special thanks also to my lab facilitator,

PhD.Candidate,Miss Krittima Anekthanakul, for sharing her knowledge,

formaking experiments look amusing. Big thanks also go to every people on

Algal Biotechnology Laboratory (AGB research group) and Bioinformatics and

Systems Biology (BIF) Program at KMUTT for helping me fit in and feel

welcome from the moment and for the unlimited patience to explain me every

doubt I had during my internship.I would also like to acknowledge my teachers

at TUAF, Dr. Duong Van Cuong, MSc. Trinh Thi Chung, Dr. Nguyen Xuan Vu

and MSc. Duong Manh Cuong that contributed to making this work and had an

enjoyable and fulfilling experience.

Many thank you and best regards

Student

Luong Tai Chien

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CONTENTS

PART I. INTRODUCTION.................................................................................1

1.1 Background and motivation .............................................................................1

1.1.1 Arthrospira (Spirulina) platensis ...................................................................1

1.1.1.1 Classification ..............................................................................................2

1.1.1.2 Morphology of Arthrospira .........................................................................3

1.1.1.3 Ultrastructure of A. platensis......................................................................4

1.1.1.4 The life cycle of Arthrospira (Spirulina)....................................................6

1.1.2 Bioactive peptides of A. platensis C1. ..........................................................7

1.1.2.1. Antioxidant and Antiinflammatory activity. .............................................7

1.1.2.2 Antihypertension ........................................................................................8

1.1.2.3 Antimicrobial activity...............................................................................10

1.1.2.4 Antidiabetes and Antiobesity. ..................................................................10

1.1.2.5 Anticancer.................................................................................................10

1.1.3 Computational Tool: Clustal Omega...........................................................11

1.2 Objectives.......................................................................................................12

1.3 Scope of work.................................................................................................12

PART II. MATERIALS AND METHODS......................................................15

2.1. Equipment and Materials...............................................................................15

2.1.1 Types of equipment. ....................................................................................15

2.1.2. Materials. ....................................................................................................15

2.2. Methods.........................................................................................................16

2.2.1. Growing cell A. platensis C1 using Zarrouk’s medium [62]. ....................16

2.2.2. Harvesting cell A. platensis C1 by membranes filtration..........................17

2.2.3. Protein extraction from A. platensis C1. ....................................................18

2.2.4. Protein precipitation from A. platensis C1 by acetone precipitation. ........19

2.2.5. Quantitative protein of A. platensis C1 by 2-D Quant kit..........................20

2.2.6. Protein separation from A. platensis C1 by Sodium Dodecyl Sulphate

Polyacrylamide Gel Electrophoresis (SDS-PAGE). ............................................21

2.2.7. In silico digestion tools...............................................................................22

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2.2.8. ACE inhibition assay at Institute of Rangsit University. .................................23

2.2.9. Common characteristics of antihypertensive peptides using Clustal Omega. 24

PART III. RESULTS AND DISCUSSIONS....................................................25

3.1. Culture and cell growth of A. platensis C1 ...................................................25

3.2. Cell harvesting of A. platensis C1.................................................................26

3.3. Protein extraction of A. platensis C1.............................................................27

3.4. Protein precipitation from crude extract solution of A. platensis C1............27

3.5. Protein quantification of A. platensis C1 by 2D-Quant kit ...........................28

3.6. Protein separation from the crude extract of A. platensis C1.......................29

3.7 Common characteristics of antihypertensive peptides from A. platensis C1. ....30

3.8. Discussion......................................................................................................36

PART IV. CONCLUSION AND RECOMMENDATIONS...........................38

4.1. Conclusion.....................................................................................................38

4.2. Recommendations. ........................................................................................38

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LIST OF TABLE

Table 1.1 Amino acid profile of Arthrospira (Spirulina) platensis [60]................1

Table 1.2 Genome statistics of A. platensis C1......................................................7

Table 1.3 Examples of bioactive peptides from Arthrospira (Spirulina) [38].............7

Table 2.1 The equipment using for study.............................................................15

Table 2.2 The materials using for study ...............................................................15

Table 2.3 Constituents of Zarrouk’s medium.......................................................16

Table 2.4 preparation of the standard curve. ........................................................20

Table 3.1 The OD480 value of standard protein (BSA)and protein ......................28

of A. platensis C1..................................................................................................28

Table 3.2 Classification of common amino acid of candidate antihypertensive peptides,

discovered from A. platensis C1...............................................................................31

Table 3.3 Characteristics of Novel AHP based on ExactAHP_284 and

ExactAHP_460 sequences....................................................................................32

Table 3.4 Amount and percentage of 7 functional amino acid groups of

antihypertensive peptides from A. platensis C1. ..................................................34

Table 3.5 Number of amino acids found in Exactly_AHP of A. platensis C1.....35

Table 3.6 Functional amino acid pattern of Exactly_AHP of A. platensis C1.....35

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