LISETE SOUSA PAIVA

LISETE SOUSA PAIVA

Nutritional evaluation and research and characterization

of peptides with inhibitory activity of angiotensin I-converting

enzyme (ACE) in macroalgae of the Azores.

UNIVERSIDADE DOS AÇORES

FACULDADE DE CIÊNCIAS E TECNOLOGIA

PONTA DELGADA, 2016

LISETE SOUSA PAIVA

Nutritional evaluation and research and characterization

of peptides with inhibitory activity of angiotensin I-converting

enzyme (ACE) in macroalgae of the Azores.

Dissertation for PhD degree in Biology

presented to the University of the Azores

Supervisors: Professora Elisabete Maria de Castro Lima

Professor José António Bettencourt Baptista

Professora Ana Isabel Azevedo Neto

UNIVERSIDADE DOS AÇORES

FACULDADE DE CIÊNCIAS E TECNOLOGIA

PONTA DELGADA, 2016

This research was partially supported by CIRN (Centro de Investigação de Recursos Naturais,

University of the Azores), CIIMAR (Interdisciplinary Centre of Marine and Environmental

Research, Porto, Portugal) and CITA-A (Centro de Investigação e Tecnologias Agrárias dos

Açores). It was also partly supported by the European Regional Development Fund (ERDF)

through the COMPETE - Operational Competitiveness Programme and national funds

through FCT - Foundation for Science and Technology, under the project "PEst-

C/MAR/LA0015/2013, by the Strategic Funding UID/Multi/04423/2013 through national

funds provided by FCT - Foundation for Science and Technology and European Regional

Development Fund (ERDF), in the framework of the programme PT2020 and by cE3c funding

(Ref: UID/BIA/00329/2013).

Furthermore, the FRC (Fundo Regional da Ciência) supported my Doctoral scholarships ref.

M.3.1.2/F/014/2011, which is co-financed by the European Social Fund through the action-

type 4.2.2 from Pro-Emprego.

“Mestre é aquele que estende a mão, inicia o diálogo e encaminha para a aventura da vida.

Não é só o que ensina fórmulas, regras, raciocínios, mas o que questiona e desperta para a

realidade. Àqueles que nos ensinam muito mais que teorias e que nos preparam também

para a vida.”

Autor desconhecido

ACKNOWLEDGMENTS

To my supervisors, Professors Elisabete Lima, Ana Neto and José Baptista for their

orientation, unconditional support for their constant guidances, motivation and

concerns to transmit knowledge, for their friendliness, friendship and commitment

that helped me in the preparation and completion of this thesis. For helping me

figure out what to do better and thus make it better. Thanks for leaving messages

that will never be erased from my mind. And it's not always through the words that

we learn, but also from the attitudes and qualities that are seen in the actions, which

were an example and inspiration. Thank you for devoting themselves to your work

with such enthusiasm. I will never be able to convey my appreciation fully.

To the group of Marine Biology of Azores University for the help in the collection and

identification of algae and for the friendship.

To Department of Technological Sciences and Development and to the Azores

University for the facilities to perform and accomplish all the experimental work.

To the doctoral coordinating committee in Biology.

To Professor Monteiro from Guelph University, Canada, for the help in peptides

amino acid sequencing determinations.

To FRC (Fundo Regional da Ciência) for supported my Doctoral scholarships.

To my parents for their guidance and for the teachings and sense of responsibility

instilled in me and for the spiritual support, values that have governed my life.

To all the friends who in one way or another contributed to the performance and

accomplishment of all work.

I

TABLE OF CONTENTS

LIST OF ORIGINAL MANUSCRIPTS……………………………………………………………………………………..XIII

RESUMO……………………………………………………………………………………………………………………………XV

ABSTRACT……………………………………………………………………………………………………………………….XVII

FRAMEWORK AND THESIS OBJECTIVES…………………………………………………………………………….XIX

THESIS OUTLINE……………………………………………………………………………………………………………..XXIII

CHAPTER 1 – GENERAL INTRODUCTION………………………………………………………………………………3

1. Macroalgae and their utility for therapeutic and industrial applications…………………………3

2. Nutritional potential……………………………………………………………………………………………………….4

2.1. Proteins and Amino acids……………………………………………………………………………………….….6

2.2. Lipids and Fatty acids…………………………………………………………………………………………………6

2.3. Fibers…………………………………………………………………………………………………………………………7

2.4. Minerals…………………………………………………………………………………………………………………….8

2.5 Vitamins and related compounds……………………………………………………………………………….9

3. Antioxidant potential……………………………………………………………………………………………………10

4. Therapeutic potential for hypertension disease…………………………………………………………….11

4.1. Hypertension and the renin-angiotensin system (RAS)……………………………………………. 11

4.2. Bioactive peptides…………………………………………………………………………………………………….12

4.2.1. Mechanism of hypotensive action of bioactive peptides…………………………………..14

4.2.2. Mechanism of transport and absorption of bioactive peptides ........................... 16

4.2.3. Methods for bioactive peptides purification and identification ........................... 17

4.2.3.1. Enzymatic hydrolysis………………………………………………………………………………17

4.2.3.2. Purification…………………………………………………………………………………………….19

4.2.3.3. Molecular mass and amino acid composition and sequence………………….22

4.2.4. Studies with in vitro and in vivo bioactive peptides .............................................. 22

4.3. Angiotensin I-converting enzyme (ACE)……………………………………………………………………24

4.3.1. Physical and chemical structure of ACE ................................................................ 24

4.3.2. Determination of ACE-inhibitory activity…………………………………………………………..25

5. The studied macroalgae………………………………………………………………………………………………. 26

References………………………………………………………………………………………………………………………..28

II

CHAPTER 2 – Edible Azorean macroalgae as source of rich nutrients with impact on human

health……………………………………………………………………………………………………………………………….43

ABSTRACT…………………………………………………………………………………………………………………………46

1. Introduction………………………………………………………………………………………………………………….47

2. Material and methods…………………………………………………………………………………………………..48

2.1. Chemicals and reagents……………………………………………………………………………………………48

2.2. Macroalgal collection……………………………………………………………………………………………….49

2.3. Sample preparation………………………………………………………………………………………………….49

2.4. Nutrient analysis………………………………………………………………………………………………………49

2.4.1. Dry weight and moisture content determination ................................................ 49

2.4.2. Determination of crude proteins .......................................................................... 50

2.4.3. Extraction of crude lipids, total lipid determination and fatty acids profile ........ 50

2.4.4. Determination of ash ............................................................................................ 51

2.4.5. Determination of soluble carbohydrates content ................................................ 51

2.4.6. Mineral analysis .................................................................................................... 51

2.4.7. Amino acids analysis (composition) ...................................................................... 52

2.4.8. Antioxidant properties .......................................................................................... 52

2.4.9. Fat soluble vitamins content ................................................................................. 53

2.5. Statistical analysis…………………………………………………………………………………………………….53

3. Results and discussion…………………………………………………………………………………………………..54

3.1. Proteins……………………………………………………………………………………………………………………54

3.2. Lipids ……………………………………………………………………………………………………………………….54

3.3. Ash…………………………………………………………………………………………………………………………..55

3.4. Soluble carbohydrates……………………………………………………………………………………………..55

3.5. Minerals…………………………………………………………………………………………………………………..56

3.6. Amino acids……………………………………………………………………………………………………………..57

3.7. Fatty acids (FA)…………………………………………………………………………………………………………59

3.8. Antioxidants……………………………………………………………………………………………………………. 62

3.9. Fat soluble vitamins………………………………………………………………………………………………… 62

4. Conclusions………………………………………………………………………………………………………………….. 64

Acknowledgements…………………………………………………………………………………………………………..65

References………………………………………………………………………………………………………………………..65

III

CHAPTER 3 – Health-promoting ingredients from four selected Azorean macroalgae……….71

ABSTRACT………………………………………………………………………………………………………………………….74

1. Introduction…………………………………………………………………………………………………………………..75

2. Material and methods……………………………………………………………………………………………………76

2.1. Chemicals and reagents……………………………………………………………………………………………76

2.2. Macroalgae collection………………………………………………………………………………………………77

2.3. Macroalgae samples preparation……………………………………………………………………………..77


2.4.1. Total lipid determination and fatty acids (FA) profile ........................................... 78

2.4.2. Mineral determination .......................................................................................... 78

2.4.3. Amino acids (AA) determination ........................................................................... 79

2.4.4. Coenzyme Q10 determination ............................................................................... 79

2.4.5. Vitamin E (α-tocopherol) determination............................................................... 79

2.4.6. Total phenolic content (TPC) determination ......................................................... 80

2.4.7. Energy value determination .................................................................................. 80

2.5. Biological activity assay…………………………………………………………………………………………….80

2.5.1. Free radical scavenging activity (FRSA) determination ......................................... 80

2.6. Statistical analysis…………………………………………………………………………………………………….81

3. Results and discussion…………………………………………………………………………………………………..81


3.1.1. Crude lipid content and fatty acids (FA) profile…………………………………………………81

3.1.2. Mineral composition ............................................................................................. 84

3.1.3. Selected amino acids (AA) with specific functional properties ............................. 85

3.1.3.1. The algae with anti-ageing AA………………………………………………………………..85

3.1.3.2. Selected algae for patients with phenylketonuria disorder…………………….85

3.1.4. Coenzyme Q10 content .......................................................................................... 86

3.1.5. Vitamin E (α-tocopherol) content ......................................................................... 87

3.1.6. Total phenolic content (TPC) ................................................................................. 88

3.1.7. Calculated energy value ........................................................................................ 88

3.2. Biological activity assay…………………………………………………………………………………………….89

3.2.1. DPPH free radical scavenging activity (FRSA) ........................................................ 89

4. Conclusions……………………………………………………………………………………………………………………90

IV

Acknowledgements…………………………………………………………………………………………………………..90

References………………………………………………………………………………………………………………………..90

CHAPTER 4 – Nutritional and functional bioactivity value of selected Azorean macroalgae….

………………………………………………………………………………………………………………………………………….97

ABSTRACT………………………………………………………………………………………………………………………. 100

1. Introduction………………………………………………………………………………………………………………. 101

2. Materials and methods……………………………………………………………………………………………… 102

2.1. Chemicals and reagents………………………………………………………………………………………… 102

2.2. Macroalgae collection…………………………………………………………………………………………… 103

2.3. Macroalgae sample preparation…………………………………………………………………………….103

2.4. Nutrient analysis…………………………………………………………………………………………………….104

2.4.1. Dry weight and moisture content determination ............................................... 104

2.4.2. Crude protein determination .............................................................................. 104

2.4.3. In vitro algae protein digestibility evaluation ..................................................... 104

2.4.4. Amino acids (AA) composition ............................................................................ 104

2.4.5. Crude lipid extraction and total lipid and fatty acid (FA) groups determination……

…………………………………………………………………………………………………………………………………105

2.4.6. Soluble carbohydrate content determination .................................................... 105

2.4.7. Total, soluble and insoluble dietary fiber (TDF, SDF and IDF) determination..... 105

2.4.8. Ash determination ............................................................................................... 105

2.5. Biological activity assay………………………………………………………………………………………….106

2.5.1. Algae enzymatic hydrolysates preparation and fractionation………………………….106

2.5.2. ACE inhibitory activity determination................................................................. 106

2.6. Statistical analysis…………………………………………………………………………………………………. 107

3. Results and discussion…………………………………………………………………………………………………107

3.1. Nutrient analysis…………………………………………………………………………………………………….107

3.1.1. Moisture .............................................................................................................. 107

3.1.2. Protein ................................................................................................................. 108

3.1.3. Amino acids composition .................................................................................... 109

3.1.4. Protein digestibility.............................................................................................. 110

3.1.5. Lipids .................................................................................................................... 111

3.1.6. Fatty acid (FA) groups .......................................................................................... 112

V

3.1.7. Soluble carbohydrates ......................................................................................... 113

3.1.8. Total, soluble and insoluble dietary fiber (TDF, SDF and IDF) ............................. 113

3.1.9. Ash ........................................................................................................................ 114

3.2. Biological activity assay………………………………………………………………………………………….114

3.2.1. Angiotensin-converting enzyme (ACE) inhibitory activity………………………………..114

4. Conclusion………………………………………………………………………………………………………………….115

Acknowledgements…………………………………………………………………………………………………………116

References………………………………………………………………………………………………………………………116

CHAPTER 5 – Isolation and characterization of angiotensin I-converting enzyme (ACE)

inhibitory peptides from Ulva rigida C. Agardh protein hydrolysate………………………………..123

ABSTRACT……………………………………………………………………………………………………………………….126

1. Introduction………………………………………………………………………………………………………………..127

2. Material and methods…………………………………………………………………………………………………128

2.1. Collection and preparation of U. rigida sample………………………………………………………128

2.2. Chemicals and reagents………………………………………………………………………………………….128

2.3. Enzyme screening for ACE-inhibitory peptides production from U. rigida protein….129

2.4. Preparation of U. rigida enzymatic hydrolysate……………………………………………………..130

2.5. Purification of ACE-inhibitory peptides…………………………………………………………………..131

2.6. Determination of ACE-inhibitory activity………………………………………………………………..132

2.7. Determination of amino acid composition of the purified ACE-inhibitory peptides..133

2.8. Identification of molecular mass and amino acid sequence of the purified ACE-

inhibitory peptides………………………………………………………………………………………………………….134

2.9. Stability of the purified ACE-inhibitory peptides…………………………………………………….134

2.10. Determination of the inhibition pattern on ACE of the purified peptides……………..135

2.11. Statistical analysis……………………………………………………………………………………………….. 135


3.1. Enzyme screening for ACE-inhibitory peptides production from U. rigida protein….135

3.2. Preparation of U. rigida protein hydrolysate (URPH) and purification of their ACE-

inhibitory peptides………………………………………………………………………………………………………….137

3.3. Amino acid composition and sequence of the purified ACE-inhibitory peptides…….140

3.4. Stability of the purified ACE-inhibitory peptides…………………………………………………….143

3.5. Determination of the inhibition pattern on ACE of the purified peptides……………….144

VI

4. Conclusion…………………………………………………………………………………………………………………..145


References………………………………………………………………………………………………………………………146

CHAPTER 6 – Angiotensin I-converting enzyme (ACE) inhibitory activity of Fucus spiralis

macroalgae and influence of the extracts storage temperature – A short report…………….153

ABSTRACT……………………………………………………………………………………………………………………….156

1. Introduction………………………………………………………………………………………………………………..157

2. Material and methods…………………………………………………………………………………………………158


2.2. Collection, preparation, extraction and fractionation of F. spiralis (Fs) sample………158

2.3. ACE-inhibitory activity determination of Fs methanol extract/fractions…………………159

2.4. Total phenolic content (TPC) determination of Fs methanol extract/fractions……….160

2.5. Chromatographic and spectrophotometric analysis of Fs methanol extract/fractions

……………………………………………………………………………………………………………………………………….160

2.6. Statistical analysis………………………………………………………………………………………………….161

3. Results and discussion………………………………………………………………………………………………..161

3.1. HPLC ACE-inhibitory activity assay…………………………………………………………………………161

3.2. ACE-inhibitory activity, TPC, and chromatographic and spectrophotometric analysis of

Fs methanol extract/fractions…………………………………………………………………………………………162

3.3. Effect of the Fs-DME storage temperature on ACE inhibition…………………………………164

4. Conclusion…………………………………………………………………………………………………………………..165


References………………………………………………………………………………………………………………………166

CHAPTER 7 – Angiotensin I-converting enzyme (ACE) inhibitory activity, antioxidant

properties and total phenolic content of Fucus spiralis L. protein hydrolysate fractions…171

ABSTRACT……………………………………………………………………………………………………………………….174

1. Introduction………………………………………………………………………………………………………………..175

2. Material and methods………………………………………………………………………………………………...176


2.2. Collection and preparation of F. spiralis sample……………………………………………………..177

2.3. Extraction of protein from F. spiralis………………………………………………………………………177

2.4. In vitro protein digestibility evaluation…………………………………………………………………..178

VII

2.5. Preparation of protein hydrolysate………………………………………………………………………..179

2.6. Purification of protein hydrolysate…………………………………………………………………………179

2.7. Protein and peptide contents analysis in FSPH fractions………………………………………..179

2.8. Amino acids composition in FSPH fractions……………………………………………………………179

2.9. ACE-inhibitory activity determination of FSPH fractions…………………………………………180

2.10. Antioxidant activity assays on FSPH fractions……………………………………………………….181

2.10.1. Free radical scavenging activity (FRSA) determination ..................................... 181

2.10.2. Reducing power assay ....................................................................................... 181

2.10.3. Metal chelating capability ................................................................................. 182

2.11. Total phenolic content (TPC) determination in FSPH fractions……………………………..182

2.12. Statistical analysis………………………………………………………………………………………………..183


3.1. Protein content and in vitro digestibility evaluation……………………………………………….183

3.2. F. spiralis protein hydrolysate (FSPH), purification by ultrafitration and ACE-inhibitory

activity of FSPH fractions…………………………………………………………………………………………………184

3.3. Protein and peptide contents of FSPH fractions……………………………………………………..184

3.4. Amino acids composition of FSPH fractions……………………………………………………………185

3.5. Antioxidant activities of FSPH fractions………………………………………………………………….186

3.5.1. Free radical scavenging activity (FRSA) assay ..................................................... 186

3.5.2. Reducing power assay ......................................................................................... 187

3.5.3. Metal chelating capability assay ......................................................................... 188

3.6. Total phenolic content (TPC) of FSPH fractions……………………………………………………...189

3.7. Pearson correlation between parameters………………………………………………………………190

4. Conclusion…………………………………………………………………………………………………………………..191


References………………………………………………………………………………………………………………………192

CHAPTER 8 – FINAL CONSIDERATIONS AND REMARKS…………………………………………………….199

References………………………………………………………………………………………………………………………203

ANNEX I …………………………………………………………………………………………………………………….......207

ANNEX II………………………………………………………………………………………………………………………….209

ANNEX III…………………………………………………………………………………………………………………………211

ANNEX IV………………………………………………………………………………………………………………………..213

VIII

ANNEX V ………………………………………………………………………………………………………………………..215

ANNEX VI ……………………………………………………………………………………………………………………….217

IX

LIST OF TABLES

Table 1. Classification of angiotensin I-converting enzyme inhibitory peptides by inhibition

mode. ………………………………………………………………………………………………………………………………….16

Table 2. Composition (moisture, protein, lipid, ash, and soluble carbohydrates) of the edible

Azorean macroalgae (EAM). ………………………………………………………………………………………………..55

Table 3. Mineral content of edible Azorean macroalgae compared to some whole foods. ….57

Table 4. Amino acid profiles of edible Azorean macroalgae and some traditional foods.

.........................................................................................................................................……….59

Table 5. Gas chromatography determination of fatty acid composition of edible Azorean

macroalgae (EAM).………………………………………………………………………………………………………………61

Table 6. Fat soluble vitamins content of edible Azorean macroalgae compared to some

whole foods. ……………………………………………………………………………………………………………………….64

Table 7. Total lipid content and gas chromatography determination of fatty acid composition

of the studied macroalgae. ………………………………………………………………………………………………….83

Table 8. Mineral content in the studied macroalgae compared to some common whole

foods, and Na/K and Ca/Mg ratios. . ……………………………………………………………………………………84

Table 9. Selected amino acids content of the studied macroalgae and content of essential

amino acids ………………………………………………………………………………………………………………………..86

Table 10. Coenzyme Q10 and vitamin E (α-tocopherol) contents in the studied macroalgae

compared to some common whole foods.…………………………………………………………………………..87

Table 11. Free radical scavenging activity (FRSA) and total phenolic content of the studied

macroalgae methanolic dry extracts. …………………………………………………………………………………..88

Table 12. Composition (moisture, protein, lipids, soluble carbohydrates, dietary fiber and

ash) of the studied macroalgae…………………………………………………………………………………………108

Table 13. Amino acid profiles of the studied macroalgae compared to some traditional high-

protein foods and protein standards…………………………………………………………………………………110

Table 14. Gas chromatography determination of fatty acid groups of the studied

macroalgae……………………………………………………………………………………………………………………....112

Table 15. Angiotensin-converting enzyme (ACE) inhibition of the studied macroalgae

enzymatic hydrolysates……………………………………………………………………………………………………..115

X

Table 16. Angiotensin-I converting enzyme (ACE) inhibitory activity, yield, protein and

peptide content of pepsin-bromelain URPH and their fractions obtained by ultrafiltration cell

dialyser system. …………………………………………………………………………………………………………………138

Table 17. Summary of the purification of ACE inhibitory peptides from URPH-III fraction by

sequential chromatographic techniques. …………………………………………………………………………..140

Table 18. Simulated gastrointestinal digestion of the purified peptides.……………………………144

Table 19. Angiotensin-I converting enzyme (ACE) inhibitory activity, recovery yield and total

phenolic content (TPC) of the fractions obtained by ultrafiltration from the Fs-DME (Fucus

spiralis dry powder methanol extract) . …………………………………………………………………………….163

Table 20. Angiotensin-I converting enzyme (ACE) inhibitory activity, recovery yield, protein

and peptide contents of the fractions obtained by ultrafiltration from the F. spiralis protein

hydrolysate. ………………………………………………………………………………………………………………………185

Table 21. Amino acid profiles of the fractions obtained by ultrafiltration from the F. spiralis

protein hydrolysate. ………………………………………………………………………………………………………….186

Table 22. Correlation matrix of the studied parameters (Pearson correlations coefficients).

………………………………………………………………………………………………………………………………………….191

XI

LIST OF FIGURES

Figure 1. Renin-angiotensin-aldosterone system ............................................................. 12

Figure 2. Steps for purification and identification of marine organisms derived bioactive

peptides ............................................................................................................................. 18

Figure 3. Azorean macroalgae - A: F. spiralis; B: U. rigida; C: U. compressa; D: Porphyra

sp.; E: O. pinnatifida; F: P. capillacea; G: G. microdon ...................................................... 26

Figure 4. Comparison of free radical scavenging activity (FRSA) of methanolic extract

from edible Azorean macroalgae (EAM) ........................................................................... 63

Figure 5. Calculated energy value of the studied macroalgae .. ....................................... 89

Figure 6. Relative percentage of protein digestibility to sodium caseinate normalized at

100% in the studied macroalgae. .................................................................................... 111

Figure 7. (A) Angiotensin-I converting enzyme (ACE) inhibitory activity and IC50 value and

(B) yield of U. rigida protein hydrolysed by various proteases ....................................... 136

Figure 8. Purification of angiotensin-I converting enzyme (ACE) inhibitory peptides from

U. rigida protein hydrolysate (URPH) digested with pepsin plus bromelain. ................. 139

Figure 9. Determination of molecular mass of the purified peptides (IP and AFL) from

URPH-III fraction (MW < 1 kDa). ...................................................................................... 141

Figure 10. Lineweaver–Burk plots of angiotensin-I converting enzyme (ACE) inhibition by

the two purified peptide from URPH-III fraction: IP and AFL (B), and by the FL ............. 145

Figure 11. Comparison of angiotensin-I converting enzyme (ACE) inhibitory activities of

Fucus spiralis dry powder methanol extract (Fs-DME) ................................................... 162

Figure 12. Representative HPLC chromatograms of angiotensin-I converting enzyme

(ACE) reaction mixture using the inhibitory Fucus spiralis dry powder methanol extract

(Fs-DME)…………………………………………………………………………………………………………………….165

Figure 13. Free radical scavenging activity (FRSA) of the fractions obtained by

ultrafiltration from the F. spiralis protein hydrolysate ................................................... 187

Figure 14. Reducing power of the fractions obtained by ultrafiltration from the F. spiralis

protein hydrolysate (FSPH). ............................................................................................. 188

Figure 15. Ferrous ions (Fe2+) chelating (FIC) activities of the fractions obtained by

ultrafiltration from the F. spiralis protein hydrolysate ................................................... 188

XII

Figure 16. Total phenolic contents (TPC) of the fractions obtained by ultrafiltration from

the F. spiralis protein hydrolysate ................................................................................... 189

XIII

LIST OF ORIGINAL MANUSCRIPTS

This thesis is supported by the following published and submitted manuscripts that are

referred in the text by their roman numerals.

I. Paiva L., Lima E., Patarra R. F., Neto A. I., Baptista J. (2014). Edible Azorean

macroalgae as source of rich nutrients with impact on human health. Food Chemistry,

164, 128–135. [IF: 3.391] (Annex I)

II. Paiva L., Lima E., Neto A. I., Massimo M., Baptista J. (2016). Health-promoting

ingredients from four selected Azorean macroalgae. Food Research International,

http://dx.doi.org/10.1016/j.foodres.2016.08.007. [IF2015: 3.182] (Annex II)

III. Paiva L., Lima E., Neto A. I., Massimo M. J. Baptista J. (2016). Nutritional and

functional bioactivity value of selected Azorean macroalgae. Submitted to European

Journal of Phycology. (Annex III)

IV. Paiva L., Lima E., Neto A. I., Baptista J. (2016). Isolation and characterization of

angiotensin I-converting enzyme (ACE) inhibitory peptides from Ulva rigida C. Agardh

protein hydrolysate. Journal of Functional Foods, 26, 65–76. [IF2015: 3.973] (Annex

IV)

V. Paiva L., Lima E., Neto A. I., Baptista J. (2016). Angiotensin I-converting enzyme (ACE)

inhibitory activity of Fucus spiralis macroalgae and influence of the extracts storage

temperature – A short report. Journal of Pharmaceutical and Biomedical Analysis,

131, 503-507. [IF2015: 3.169] (Annex V)

VI. Paiva L., Lima E., Neto A. I., Baptista J. (2016). Angiotensin I-converting enzyme (ACE)

inhibitory activity, antioxidant properties and total phenolic content of Fucus spiralis

L. protein hydrolysate fractions. Submitted to Marine Biotechnology. (Annex VI)

XV

RESUMO

As macroalgas Fucus spiralis, Ulva rigida, U. compressa, Porphyra sp. e Osmundea

pinnatifida são consumidas como alimento em algumas das ilhas dos Açores, enquanto que

Gelidium microdon e Pterocladiella capillacea são coletadas para a produção de agar. No

entanto, pouca informação está disponível sobre o seu valor como fonte natural de

compostos nutricionais e funcionais.

Esta tese investigou principalmente a composição nutricional e os aspetos promotores

de saúde destas macroalgas, bem como o seu potencial como fonte de frações peptídicas

e/ou de péptidos purificados inibidores da ECA.

Em relação aos aspetos nutricionais das macroalgas, esta tese investigou, pela primeira

vez, as proteínas, o perfil de aminoácidos, a digestibilidade das proteínas in vitro, os lípidos,

o perfil de ácidos gordos (saturados, monoinsaturados, polinsaturados e a razão n6/n3 e

h/H), os hidratos de carbono solúveis, a fibra alimentar, as cinzas, os minerais e a razão Na/K

e Ca/Mg, as vitaminas, a coenzima Q10, a humidade, o teor de fenólicos totais, as atividades

antioxidante e inibitória da ECA in vitro e o valor energético. Os resultados sugerem que um

consumo regular destas algas, quer diretamente ou através de suplementos alimentares,

pode melhorar a saúde humana ou pode ter um efeito protetor sobre algumas doenças

degenerativas e, consequentemente, sobre o processo de envelhecimento.

As macroalgas podem também ser utilizadas para a produção de produtos

farmacêuticos com potencial valor económico. Como é do conhecimento geral, a enzima

conversora da angiotensina I (ECA) tornou-se um importante alvo para o controlo da pressão

arterial, uma vez que catalisa a conversão de angiotensina I num potente vasoconstritor a

angiotensina II. Recentemente, cada vez mais atenção tem sido dada às algas marinhas

como fontes naturais de novos inibidores da ECA. Neste trabalho foram isolados e

caracterizados alguns péptidos em U. rigida e os resultados revelaram, pela primeira vez,

que péptidos inibidores da ECA podem ser eficientemente obtidos a partir das proteínas

hidrolisadas pelas enzimas pepsina-bromelaína. Dois péptidos inibidores da ECA (IP e AFL)

foram isolados e purificados com sucesso a partir deste hidrolisado. Os seus modos de

inibição e estabilidade a diferentes temperaturas e os estudos in vitro do efeito das

proteases gastrointestinais na atividade destes péptidos foram também caracterizados pela

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primeira vez. Além disso, o tripéptido AFL foi hidrolisado por peptidases da mucosa

intestinal dando origem a um dipéptido FL, que apresentou uma maior inibição da ECA

relativamente ao seu percursor.

Esta tese também apresenta, pela primeira vez, as atividades inibitória da ECA e

antioxidante in vitro por frações proteicas hidrolisadas de F. spiralis, bem como o perfil de

aminoácidos e o conteúdo em fenólicos totais destas frações. Os resultados revelaram que

não só os péptidos ativos, mas também os compostos fenólicos contribuem para

as elevadas atividades inibitória da ECA e antioxidante das fracções proteicas

hidrolisadas de F. spiralis. Avaliou-se, ainda, pela primeira vez, o efeito inibitório da ECA

por extratos/frações metanólicas de F. spiralis, o respetivo conteúdo em fenólicos totais e o

efeito da temperatura de armazenamento do extrato metanólico seco de F. spiralis na

inibição da ECA. Os resultados sugerem que esta macroalga é muito rica em florotaninos, os

polifenóis mais abundantes em algas castanhas e que têm sido referidos como

sendo uma fonte potencial de compostos inibidores da ECA.

Em conclusão, as macroalgas estudadas podem ser usadas como alimentos funcionais

e são uma fonte natural e potencial de frações/péptidos inibidores da ECA que podem ser

utilizados para a produção de nutracêuticos e farmacêuticos para prevenir e/ou tratar a

hipertensão. A sua utilização seria uma terapia natural alternativa, económica e mais segura,

aos medicamentos comerciais sintéticos e atuaria como um incentivo para a conservação da

biodiversidade e para a manutenção de um ambiente limpo nos Açores.

Palavras-chave: Macroalgas; Inibidores naturais da ECA; Péptidos bioativos; Alimentos

nutricionais e funcionais; Composição bioquímica; HPLC.

XVII

ABSTRACT

The macroalgae Fucus spiralis, Ulva rigida, U. compressa, Porphyra sp. and Osmundea

pinnatifida are consumed as food in some of the Azorean Islands, whereas Gelidium

microdon and Pterocladiella capillacea are collected for agar production. Little information

is, however, available on their value as natural sources of nutritional and/or functional

metabolites content.

This thesis investigated mainly the nutritional composition and health-promoting

aspects of those selected macroalgae as well as their potential as source of protein-derived

ACE-inhibitory peptide fractions and/or purified peptides.

Regarding the macroalgal nutritional aspects, this thesis evaluated, for the first time,

the proteins, amino acid profiles, in vitro protein digestibility, lipids, fatty acid profiles (SFA,

MUFA, PUFA and n6/n3 and h/H ratios), soluble carbohydrate, dietary fiber, ash, minerals

and Na/K and Ca/Mg ratios, vitamins, coenzyme Q10, moisture, total phenolic content, in

vitro antioxidant and ACE inhibition activities and energy values. The results suggested that a

regular consumption of these macroalgae, either directly or through food supplements, may

improve human health or may have a protective effect on some of the degenerative diseases

and consequently on the ageing process.

Macroalgae can also be used for the production of pharmaceuticals with potential

economic value. It is well known that angiotensin-I converting enzyme (ACE) became a major

target control for high blood pressure, since catalyzes the conversion of angiotensin I to a

potent vasoconstrictor angiotensin II. Recently, increasing attention has been paid to the

marine algae as natural sources of novel ACE-inhibitors. In this thesis the isolation and

characterization of some peptides from U. rigida were achieved and the results revealed, for

the first time, that ACE-inhibitory purified peptides could be efficiently generated from U.

rigida protein hydrolyzed by the pepsin-bromelain enzymes. Two ACE-inhibitory peptides (IP

and AFL) were successfully isolated and purified from this hydrolysate. Their inhibition

patterns and stability at different temperatures and the in vitro study of the gastrointestinal

proteases effect on the activity of these peptides were firstly characterized. Furthermore,

AFL was hydrolyzed by intestinal mucosa peptidases to a dipeptide FL with a more potent

ACE inhibition relatively to its percurssor.

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This thesis also reports, for the first time, the in vitro ACE-inhibitory and antioxidant

activities by F. spiralis protein hydrolysate fractions and their amino acid profiles and total

phenolic content. Results suggest that not only the active peptides, but also the phenolic

compounds contribute to the strong ACE-inhibitory and antioxidant activities of F. spiralis

protein hydrolysate fractions. Furthermore, this is the first study reporting the ACE inhibition

by methanol extract/fractions from F. spiralis, their total phenolic content and the effect of

the storage temperature of F. spiralis dry powder methanol extract on ACE inhibition.

Results suggest that this macroalgae is very rich in phlorotannins, the most abundant

polyphenols in brown algae that have been reported to be a potential source of powerful

ACE-inhibitory compounds.

In conclusion, the studied Azorean macroalgae can be used for functional foods

supplementation and are a natural potential source of ACE-inhibitory fractions/peptides that

may be used for the production of nutraceuticals and pharmaceuticals to prevent and/or to

treat hypertension. Their use would be economical, safer and a natural alternative therapy

to commercial synthetic drugs, and would make an incentive for the biodiversity

conservation and for the maintenance of a clean environment in the Azores.

Keywords: Macroalgae; Natural ACE-inhibitors; Bioactive peptides; Nutritional and

Functional foods; Biochemical composition; HPLC.

XIX

FRAMEWORK AND THESIS OBJECTIVES

The angiotensin I-converting enzyme (ACE) inhibition has become a major target to

control hypertension, which is a main risk factor for the development of cardiovascular

diseases that are a significant public health problem worldwide. Natural products have been

investigated in order to prevent or treat high blood pressure as an alternative potentially

safer than the use of synthetic drugs that can have some adverse effects. Indeed, searching

for ACE-inhibitors from natural resources, such as marine organisms including macroalgae,

has become one of the major areas of research in the field of nutraceutical and

pharmaceutical industries, and some antihypertensive products such as peptides and

phenolic compounds have already successfully been isolated. Recently, some researchers

have reported that the ACE-inhibitory compounds also present other significant bioactivities,

including antioxidant properties.

Marine macroalgae are undoubtedly a valuable sustainable resource of “unique”

bioactive phytochemical structures as well as sources of nutritious food components that

have a high potential for producing health-care products for the pharmaceutical, medical,

cosmetic and food industries. However, little information is available on the potential of

macroalgae from Azores Islands (Portugal) as natural sources of functional metabolites.

The main aim of this thesis was to evaluate the nutritional and health-promoting

aspects of selected Azorean marine macroalgae species, and to investigate their potential as

sources of protein-derived ACE-inhibitory peptide fractions and purified peptides. The goal

was mainly to gather knowledge on the important nutrients contents, and on the structure

and activity of the ACE-inhibitory peptides, having in mind the possibility of producing, in the

future, new health-promoting products for nutraceutical, pharmaceutical and cosmeceutical

marine biotechnology markets.

More specifically, the objectives of this thesis were:

To investigate the biochemical composition and the health-promoting aspects of the

selected macroalgae in terms of moisture, proteins, lipids, amino acids and fatty acids

profiles, soluble carbohydrates, dietary fiber, vitamins, coenzyme Q10, ash, minerals and

total phenolic contents, and also the energy value, the in vitro protein digestibility, and

XX

the in vitro antioxidant and ACE inhibition properties. In this study seven common

species of macroalgae from the Azorean littoral zone were investigated: Fucus spiralis

(Ochrophyta, Phaeophyceae), Ulva rigida, Ulva compressa (Chlorophyta) and Porphyra

sp., Osmundea pinnatifida, Pterocladiella capillacea and Gelidium microdon

(Rhodophyta).

To investigate and to evaluate the potential of the selected edible macroalgae U. rigida

as a source of ACE-inhibitory peptide fractions and purified peptides, namely:

- to optimize the protein extraction process, to screen for the most effective enzymes

and to find the enzymatic hydrolysis reaction conditions in order to maximize the

number of ACE-inhibitory peptides in the macroalgae protein hydrolysates;

- to isolate the ACE-inhibitory peptide fractions from the selected enzymatic protein

hydrolysate for further purification in order to obtain the purified bioactive peptides;

- to determine the yield, protein and peptide content of the selected enzymatic

protein hydrolysate and also from the ultrafiltration fractions;

- to evaluate in vitro the ACE-inhibitory activity of the macroalgae hydrolysate fractions

and the purified peptides;

- to determine the amino acids composition and sequences of the purified ACE-

inhibitory peptides;

- to synthetize the most active ACE-inhibitory peptides to compare their activities with

the isolated natural ones and also to the synthetic antihypertensive drugs usually

prescribed by physicians;

- to investigate the purified ACE-inhibitory peptides in terms of yield and in vitro

properties, e.g. inhibition patterns and stability against temperature and

gastrointestinal enzymes (pepsin, trypsin, chymotrypsin and intestinal mucosa

peptidases).

To investigate the ACE inhibition by methanol extract/fractions from F. spiralis, their

total phenolic content and the effect of the storage temperature of F. spiralis dry

powder methanol extract on ACE inhibition.

XXI

To investigate and to evaluate the ACE-inhibitory and antioxidant activities of protein

hydrolysate fractions from the selected edible macroalgae F. spiralis, namely:

- to optimize the protein extraction process and the enzymatic hydrolysis reaction

conditions to obtain higher yield of the macroalgae protein hydrolysates for further

fractionation by ultrafiltration;

- to determine the yield, protein, peptide, total phenolic content and amino acids

profile of the macroalgae protein hydrolysate fractions;

- to evaluate the in vitro ACE-inhibitory activity of the macroalgae protein hydrolysate

fractions and also their in vitro antioxidant activities;

- to determine the correlation between the various parameters (ACE inhibition,

antioxidant activity and total phenolic content).

XXIII

THESIS OUTLINE

This thesis is composed of eight chapters.

Chapter one is a general introduction that includes information on nutritional and

health-promoting aspects of macroalgae species and on their potential as sources of

antihypertensive compounds considering the relevant published literature. Chapter starts

with the principal information of therapeutic and industrial applications of macroalgae,

followed by the evaluation of their nutritional composition (proteins and amino acids, lipids

and fatty acids, fibers, minerals, and vitamins and related compounds) and antioxidant

potential. Then, it follows with the approach of hypertension disease and the explanation of

the hypotensive action mechanism of bioactive peptides and their mechanism of transport

and absorption. Chapter continues with more exhaustive explanation of the methods used in

the bioactive peptides purification and identification in order to support the methods

mentioned in chapter’s five. It is also referred the studies with in vitro and in vivo bioactive

peptides and a description of physical and chemical structure of the angiotensin I-converting

enzyme (ACE) as well as its activity determination. Chapter ends with a description of the

selected Azorean macroalgae.

Chapter’s two to four (corresponding to manuscripts I to III) detail the information

regarding the analytical methods used to determine the nutritional content of macroalgae.

Chapter two provides, for the first time, full information on the biochemical and nutritional

composition of the three most consumed edible Azorean macroalgae (Osmundea

pinnatifida, Fucus spiralis and Porphyra sp.) in order to determine their nutritional value for

human consumption and their potential impact on human health. Chapters three and four

report, for the first time, the nutritional aspects, the health promoting ingredients content

and the in vitro antioxidant and ACE-inhibitory activities of the Azorean macroalgae Ulva

compressa, U. rigida, Gelidium microdon and Pterocladiella capillacea, in order to evaluate

their use as functional foods and/or for producing health-care products for the

pharmaceutical, medical, cosmetic and food industries.

Chapter five describes, for the first time, the investigation of the edible U. rigida

protein hydrolysate as a source of ACE-inhibitory peptide fractions and their purified

XXIV

peptides in order to evaluate its potential use in the preparation of antihypertensive drugs

or functional foods. Chapter details the following steps: (a) screening for effective

proteolytic enzymes and investigating the enzymatic hydrolysis conditions for producing U.

rigida protein hydrolysates with the higher ACE-inhibitory activity, (b) isolating and

identifying ACE-inhibitory peptides from U. rigida protein hydrolysed with pepsin plus

bromelain enzymes, and (c) investigating in vitro the purified peptides in terms of ACE-

inhibitory activity, inhibition pattern, and stability against temperature and gastrointestinal

proteases.

Chapter six reports, for the first time, the studies of in vitro ACE-inhibitory activity of

crude and size-fractionated methanol extracts from F. spiralis, detailing the following steps:

(a) determining their total phenolic content (TPC), (b) analyzing them by chromatography

(TLC and HPLC-DAD) and spectrophotometry (UV and IR) methodologies, and (c)

investigating the effect of F. spiralis dry powder methanol extracts storage temperature on

the ACE inhibition, in order to maximize its potential use in the preparation of

antihypertensive drugs or functional foods.

Chapter seven describes, for the first time, the investigation of F. spiralis protein

hydrolysate as a source of ACE-inhibitory and antioxidant peptide fractions, and also their

protein, peptide, phenolics content and amino acids composition in order to evaluate its

potential to be incorporated as multifunctional ingredients into foods as alternatives to

conventional antihypertensive drugs or synthetic antioxidants.

Finally, chapter eight presents the final considerations and remarks, the limitations of

the work and discusses future perspectives.

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LISETE SOUSA PAIVA