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
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.”
ACKNOWLEDGMENTS
9 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.
9 To the group of Marine Biology of Azores University for the help in the collection and identification of algae and for the friendship.
9 To Department of Technological Sciences and Development and to the Azores University for the facilities to perform and accomplish all the experimental work. 9 To the doctoral coordinating committee in Biology.
9 To Professor Monteiro from Guelph University, Canada, for the help in peptides amino acid sequencing determinations.
9 To FRC (Fundo Regional da Ciência) for supported my Doctoral scholarships.
9 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.
9 To all the friends who in one way or another contributed to the performance and accomplishment of all work.
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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
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
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. Nutrient analysis………78
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. Nutrient analysis………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
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
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. Results and discussion………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
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4. Conclusion………..145
Acknowledgements………146
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
2.1. Chemicals and reagents……….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
Acknowledgements………166
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
2.1. Chemicals and reagents……….176
2.2. Collection and preparation of F. spiralis sample………..177
2.3. Extraction of protein from F. spiralis………177
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. Results and discussion………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
Acknowledgements………191
References………192
CHAPTER 8 – FINAL CONSIDERATIONS AND REMARKS……….199
References………203
ANNEX I ………...207
ANNEX II……….209
ANNEX III………211
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ANNEX V ………..215 ANNEX VI ……….217
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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 high-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
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).
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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
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Figure 16. Total phenolic contents (TPC) of the fractions obtained by ultrafiltration from
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)
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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
XVI
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
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.
XVIII
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
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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:
9 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
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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).
9 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).
9 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.
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9 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).
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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
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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.
