DEPARTAMENTO DE QUÍMICA
DEVELOPMENT
AND CHARACTERIZATION OF SEMI-HARD GOAT’S CHEESE AND PLANT-BASED
«CHEESE» SUPPLEMENTED WITH SEAWEEDS
BRUNO MIGUEL FERNANDES CAMPOS Mestre em Ciências Gastronómicas
DOUTORAMENTO EM CIÊNCIAS DOS ALIMENTOS
Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia
DEPARTAMENTO DE QUÍMICA
DEVELOPMENT AND CHARACTERIZATION
OF SEMI-HARD GOAT’S CHEESE AND PLANT-BASED
«CHEESE» SUPPLEMENTED WITH SEAWEEDS
BRUNO MIGUEL FERNANDES CAMPOS
Mestre em Ciências Gastronómicas
Orientador: Mário Emanuel Campos de Sousa Diniz,
Professor Auxiliar, Universidade NOVA de Lisboa, Faculdade de Ciências e Tecnologia
Coorientadores: Maria Paulina Estorninho Neves da Mata,
Professora Auxiliar, Universidade NOVA de Lisboa, Faculdade de Ciências e Tecnologia
João Paulo da Costa de Noronha,
Professor Auxiliar, Universidade NOVA de Lisboa, Faculdade de Ciências e Tecnologia
Júri:
Presidente: Ana Isabel Martins Aguiar de Oliveira Ricardo,
Professora Catedrática, Universidade NOVA de Lisboa, Faculdade de Ciências e Tecnologia
Arguentes: Leonel Carlos dos Reis Tomás Pereira,
Professor Auxiliar com Agregação, Universidade de Coimbra, Faculdade de Ciências e Tecnologia
António Manuel Barros Marques,
Investigador Auxiliar, Instituto Português do Mar e da Atmosfera
Orientador: Mário Emanuel Campos de Sousa Diniz,
Professor Auxiliar, Universidade NOVA de Lisboa, Faculdade de Ciências e Tecnologia
Membros: Maria Leonor Marins Braz de Almeida Nunes
Investigadora Principal, Centro Interdisciplinar de Investigação Marinha e Ambiental
Ana Isabel Martins Aguiar de Oliveira Ricardo,
Professora Catedrática, Universidade NOVA de Lisboa, Faculdade de Ciências e Tecnologia
Ana Maria Ferreira da Costa Lourenço
Professora Auxiliar, Universidade NOVA de Lisboa, Faculdade de Ciências e Tecnologia
DOUTORAMENTO EM CIÊNCIAS DOS ALIMENTOS
Development of semi-hard goat's cheese and plant-based «cheese» supplemented with sea- weeds
Copyright © Bruno Miguel Fernandes Campos, Faculdade de Ciências e Tecnologia, Univer- sidade NOVA de Lisboa.
A Faculdade de Ciências e Tecnologia e a Universidade NOVA de Lisboa têm o direito, per- pétuo e sem limites geográficos, de arquivar e publicar esta dissertação através de exemplares impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou que venha a ser inventado, e de a divulgar através de repositórios científicos e de admitir a sua cópia e distribuição com objetivos educacionais ou de investigação, não comerciais, desde que seja dado crédito ao autor e editor.
Aos Meus Pais (pla boa hora)
À Cláudia (plo dossel y dolce sôlda)
A CKNOWLEDGEMENTS
In the first place, I want to thank my supervisors for their guidance, advising and sup- port over the last four years.
Particularly, I would like to express my deepest appreciation to Professor Mário Diniz for his commitment, encouragement, sense of pragmatism and scientific spirit, not forgetting, but underlining, the exceptional human qualities transmitted throughout the journey, among which I quote the intellectual humility, generosity, and goodness.
Words cannot express my gratitude to Professor Paulina Mata. However, I would par- ticularly like to thank for her valuable and pertinent assistance, wisdom transmitted, whether in professional achievement or in everyday life. It will never be too much to emphasize your scientific spirit, accuracy, and inspiration. My deepest thanks for all the opportunities given to me, by her generosity and dedication.
I am also grateful to Professor João Paulo Noronha for accepting me in the ALGA4Food project.
I would like to extend my sincere thanks to Professor Manuel Malfeito-Ferreira (School of Agriculture, Lisbon University), for the valuable inputs and suggestions to the work, espe- cially related to microbiological aspects.
Thanks, should also go to Professor Paulo Sousa (Federal University of Ceará, Brazil), for the Generalized Procrustes Analyis support and guidance.
I am deeply indebted to Bruno Moreira-Leite for all teachings and assistance.
I am also grateful to Maria João for all support and friendship.
I am also grateful to Abigail Salgado for her support in Flash Profile and sensory tests.
Special thanks to Edgar Ramalho for the precious help in various laboratory assays.
I would like to extend my sincere thanks to Carla Silva (Microbiology Laboratory from School of Agriculture, Lisbon University) for guidance in the microbiological analysis.
Many thanks to Mariana Carmona for the support in enzymatic assays.
I’d like to acknowledge Isa Marmelo (IPMA) for the assistance in crude protein assays.
This endeavor would not have been possible without Adolfo Henriques from Granja dos Moinhos, Maçussa. Many thanks for his support in the manufacture of cheese, teachings, good mood, energy, and friendship.
I would like to acknowledge the research units UCIBIO-REQUIMTE (Applied Molecular Biosciences Unit) and LAQV-REQUIMTE (Associated Laboratory for Green Chemistry), De- partment of Chemistry, NOVA School of Science and Technology, NOVA University of Lis- bon, for having provided the necessary facilities and equipment for research.
I would like also to acknowledge the ALGA4FOOD – «Seaweed in Gastronomy – Devel- opment of Innovative Techniques in Conservation and Utilization» (MAR-01.03.01-FEAMP- 0016) for supporting my PhD research work.
Strike a coin from every mistake.
(Ludwig Wittgenstein, 10.2.1948)
A BSTRACT
Seaweeds, also known as macroalgae, are commonly consumed by humans since early times, mainly in Asian countries. Recently, seaweeds are seen as a potential novel food capable of answering challenges posed by an expanding population, with the world set to hit 9 billion before 2050, and by the climate crisis.
In the dairy sector, several seaweeds or seaweed extracts have been added to various products to improve their functionality, nutritional and organoleptic quality, as well as en- hancing their shelf-life. Previous investigations show that seaweed can be integrated into dif- ferent types of cheese having a positive influence by optimizing its organoleptic characteris- tics.
On the other hand, plant-based alternatives to foods of animal origin are increasingly attracting consumers attention. The growth is driven by reasons such as consumer’s awareness for organic and sustainable food production, ethical reasons such as animal welfare and health related, and reduction or elimination of meat and dairy products from diets. In 2020, the COVID-19 pandemic accelerated this process as it caused consumers to rethink their lifestyles and stray towards alternative diets based on plant-based choices. Thus, the pace of develop- ment of new plant-based products has increased. These pursue to mimic the taste and texture of animal-based counterparts, including dairy products such as cheese.
In this context, the aim of this thesis was the development of new artisanal dairy and non-dairy foods products, particularly: i) a semi-hard goat’s cheese supplemented with Pal- maria palmata and Ulva sp., and ii) a cashew nut fermented cheese alternative supplemented with Chondrus crispus and Porphyra sp.; both designed with the aim of improving their quality and nutritional properties. Subsequently, the impact of these supplementation on the physi- cochemical composition, and on the microbiological and organoleptic properties of the result- ing new products was evaluated.
The results show that seaweeds and goat’s milk raw materials are viable from the point of view of general sanitary quality and, as such, viable for the manufacture of the new prod- ucts without risks to the health of consumers. In general, goat’s cheese physicochemical, color, and textural parameters were significantly affected by seaweeds addition, but, despite this, their microbial load was scarcely affected. Supplemented goat’s cheese was also characterized using more complex sensory terms, particularly for aroma and flavor. Cashew nut fermented cheese alternative shows a healthier nutritional profile and high viability as a cheese substi- tute. The physicochemical characteristics, color, texture and the sensorty profile of the supple- mented products were significantly different from the control. The microbial counts show a
lower value for Staphylococcus aureus, with LAB and AMB predominating over other microbial groups.
Overall results confirmed the potential of seaweeds to be combined with dairy and non- dairy alternative foods products, with good acceptance and global appreciation. Thus, allow- ing to contribute to increase offer for cheese consumers, and for those looking for alternatives to dairy products, as well as contributing for innovation in the dairy artisanal food and plant- based food sector.
Keywords: Seaweeds; milk; dairy products; plant-based alternatives; semi-hard goat’s cheese; cashew nut
R ESUMO
As algas marinhas, também conhecidas como macroalgas, são comumente consumidas pelo homem desde tempos imemoriais, principalmente nos países asiáticos. Atualmente, as algas marinhas são vistas como um potencial alimento passível de atender os desafios associados a uma população em constante expansão, com o mundo prestes a atingir 9 mil milhões de indivíduos antes de 2050, e à crise climática.
No setor de laticínios, algumas algas ou extratos de algas marinhas foram adicionada(o)s a vários produtos à base de leite para melhorar sua funcionalidade, qualidade nutricional e organoléptica, além de aumentarem a sua vida útil. Investigações anteriores mostraram que as algas marinhas podem ser integradas em diferentes tipos de queijo e influenciá-los positivamente, otimizando as suas características organolépticas.
Por outro lado, alternativas do tipo plant-based a alimentos de origem animal têm atraído cada vez mais a atenção do consumidor. O crescimento é impulsionado por razões como a consciencialização do consumidor relativamente à produção de alimentos biológicos e sustentáveis, razões éticas, como o bem-estar animal e redução ou eliminação de carnes e laticínios de suas dietas. Em 2020, a pandemia do COVID-19 acelerou esse processo, pois fez com que os consumidores repensassem seus estilos de vida e se desviassem para dietas alternativas baseadas em escolhas de alimentos de origem vegetal. Assim, o ritmo de desenvolvimento de novos produtos à base de plantas aumentou. Estes buscam imitar o sabor e a textura de seus homólogos de origem animal, incluindo produtos lácteos, como o queijo.
Neste contexto, o objetivo desta tese foi o desenvolvimento de novos alimentos artesanais lácteos e não lácteos, em particular: i) um queijo de cabra semi-duro suplementado com Palmaria palmata e Ulva sp., e ii) um «queijo» alternativo à base de castanha de caju enriquecido com Chondrus crispus e Porphyra sp.; ambos concebidos com o objectivo de melhorar a sua qualidade e propriedades nutricionais. Posteriormente, avaliou-se o impacto dessas suplementações na composição físico-química e nas propriedades microbiológicas e organolépticas dos novos produtos resultantes.
Os resultados mostram que as matérias-primas; as algas marinhas e o leite de cabra, são viáveis do ponto de vista da qualidade sanitária geral e, como tal, viáveis para o fabrico dos novos produtos sem risco para a saúde dos consumidores. Em geral, os parâmetros físico- químicos, de cor e textura do queijo de cabra foram significativamente afetados pela adição de algas, mas, apesar disso, a sua carga microbiana foi pouco afetada. O queijo de cabra suplementado também suscitou usos de termos sensoriais mais complexos, nomeadamente para o aroma e flavor. O «queijo» fermentado do tipo plant-based demonstra um perfil nutricional mais saudável e alta viabilidade como substituto do queijo tradicional. As suas características físico-químicas, cor, textura e perfil sensorial foram significativamente
influenciados relativamente ao controlo. As contagens microbianas mostram um valor baixo para Staphylococcus aureus, com LAB e AMB predominando sobre os demais grupos microbianos.
Os resultados gerais confirmam o potencial das algas marinhas para serem combinadas com produtos lácteos e alternativas não lácteas, com boa aceitação e valorização global. Deste modo, permitem contribuir para o aumento da oferta para os consumidores de queijo, e para quem procura alternativas aos produtos lácteos, além de contribuir para a inovação no setor de alimentos artesanais e alternativas plant-based.
Palavras-chave: Macroalgas; leite; produtos lácteos; alternativas à base de plantas; queijo de cabra semi-duro; caju
P UBLICATIONS , BOOK CHAPTERS , ORAL COM- MUNICATIONS , AND POSTERS
Scientific articles in indexed international journals (3)
Campos, B. M., Ramalho, E., Marmelo, I., Noronha, J. P., Malfeito-Ferreira, M., Mata, P., &
Diniz, M. S. (2022). Proximate composition, physicochemical, and microbiological characteri- zation of edible seaweeds available in the Portuguese market. Frontiers in Bioscienmce (Elite Edition), 14(4), 26. https://doi.org/10.31083/j.fbe1404026
Milinovic, J., Fernando, A. J., Campos, B., Leite, B., Mata, P., Diniz, M., Sardinha, J., & Noro- nha, J. P. (2021). Nutritional benefits of edible macroalgae from the central Portuguese coast:
Inclusion of low-calorie ‘sea vegetables’ in human diet. International Journal of Environmental Sciences & Natural Resources, 28(5), 1–5. 10.19080/IJESNR.2021.28.556250
Milinovic, J., Campos, B., Mata, P., Diniz, M. & Noronha, J. P. (2020). Umami free amino acids in edible green, red, and brown seaweeds from the Portuguese seashore. Journal of Applied Phycology, 32(5), 3331–3339. 10.1007/s10811-020-02169-2
Papers/abstracts in proceedings of conferences peer-reviewed (1)
Leite, B. M., Campos, B., Mata, P., Noronha, J. P., & Diniz, M. (2019). Are seaweeds the food of the future? Challenges for its conservation and introduction in the Portuguese diet. Annals of Medicine, 51 (Sup 1), 169. 10.1080/07853890.2018.1562018
Book chapter (1)
Campos, B., Noronha, J. P., Mata, P., Diniz, M., & Henriques, A. (2021). Seaweeds: An ingre- dient for a novel approach to artisanal basic dairy products. Experiencing food, designing sus- tainable and social practices (1st ed., pp. 67–72). Taylor & Francis Group, CRC Press.
10.1201/9781003046097-11
Other publications (2)
Campos, B., Noronha, J. P., Diniz, M., Henriques, A., & Mata, P. (2019). Traditional products enriched with seaweeds ― A promising strategy to introduce seaweeds in the diet of the Por- tuguese. Book of abstracts in workshop II: «Macroalgae from the Portuguese coast: An eco- nomic and nutritional approach», Porto School of Engineering, (ISEP), Porto (Portugal).
Leite, B. M., Campos, B., Gabriel, P., Diniz, M., Noronha, J. P. & Mata, P. Introducing macroal- gae from the Portuguese coast in the diet of consumers: Challenges and strategies. Menu, Jour- nal of Food and Hospitality Research, Vol. 8. Institute Paul Bocuse. Special Issue: Cook and Health Conference. ISSN 2275-5748
Oral communications (1)
Campos, B., Noronha, J. P., Mata, P., Diniz, M., & Henriques, A. (2019). Seaweeds: An ingre- dient for a novel approach to artisanal basic dairy products. International Food Design and Food Studies Conference. Experiencing Food: Designing Sustainable and Social Practices, EFOOD, School of Architecture, November 28-30th, Lisbon (Portugal).
Posters in conferences (3)
Campos, B., Diniz, M., Henriques, A., Noronha, J. P., & Mata, P. (2019). Traditional products enriched with seaweeds ― A promising strategy to introduce seaweeds in the diet of the Por- tuguese. «Macroalgae from the Portuguese Coast: An Economic and Nutritional Approach», Porto School of Engineering, (ISEP), October 24th, Porto (Portugal).
Milinovic, J., Campos, B., Leite, B. M., Diniz, M., Mata, P., & Noronha, J. P. (2019). Umami related compounds in macroalgae from the Portuguese coast. «Macroalgae from the Portu- guese Coast: An Economic and Nutritional Approach», Porto School of Engineering, (ISEP), October 24th, Porto (Portugal).
Salgado, A., Leite, B. M., Campos, B., Mata, P., Diniz, M., & Noronha, J. P. (2018). The use of ultrasound in culinary extraction processes: A study in stocks and infused oils enriched with Codium tomentosum. XIV Encontro de Química dos Alimentos (XIV EQA) – Sociedade Portu- guesa de Química (SPQ), 6-9 novembro, Viana do Castelo (Portugal).
T ABLE OF CONTENTS
Acknowledgements ... i
ABSTRAC ... v
RESUMO ... vii
Publications, book chapters, oral communications, and posters ... ix
Table of Contents ... xi
List of Figures ... xvii
List of Tables ... xix
Abbreviations ... xxi
Symbols ... xxvii
1. DAIRY CHEESE AND PLANT-BASED «CHEESE» SEAWEEDS SUPPLEMENTATION ― AN OVERVIEW ... 1
1.1 Introduction ... 2
1.2 Seaweeds ― a general view ... 3
1.2.1 Green seaweed (phylum Chlorophyta) ... 4
1.2.1.1 Ulva sp ... 4
1.2.2 Red seaweed (phylum Rodophyta) ... 5
1.2.2.1 Chondrus crispus ... 6
1.2.2.2 Palmaria palmata... 7
1.2.2.3 Porphyra sp ... 8
1.3 A new source of protein ― a current trend ... 9
1.3.1 Seaweeds supplementation in food products ... 9
1.4 Dairy industry ... 11
1.4.1 Milk ... 11
1.4.1.1 Goat’s milk ... 11
1.4.2 Dairy products supplemented with seaweeds ... 12
1.4.2.1 Cheese supplemented with seaweeds ... 13
1.5 Plant-based diets ... 14
1.5.1 Plant-based «cheese» alternatives (PBCAs) ... 15
1.5.1.1 General aspects ... 15
1.5.1.2 Non fermented PBCAs ... 16
1.5.1.3 Fermented PBCAs ― Cashew nut-based products ... 17
1.6 Overview, main aims, and scope of the thesis ... 20
1.7 Thesis layout ... 23
References ... 24
XII
2. PROXIMATE COMPOSITION, PHYSICOCHEMICAL AND MICROBIOLOGICAL
CHARACTERIZATION OF EDIBLE SEAWEEDS AVAILABLE IN THE PORTUGUESE MARKET ... 33
2.1 Introduction ... 35
2.2 Materials and methods ... 36
2.2.1 Algal biomass ... 36
2.2.2 Proximate composition ... 37
2.2.2.1 Moisture content ... 37
2.2.2.2 Ash content ... 37
2.2.2.3 Total lipids content ... 37
2.2.2.4 Crude protein content ... 37
2.2.2.5 Total carbohydrates content ... 38
2.2.3 Seaweeds chemical analysis ... 38
2.2.3.1 pH determination ... 38
2.2.3.2 Fatty acids methyl esters (FAMEs) ... 38
2.2.3.3 GC-MS analysis ... 39
2.2.3.4 Elemental analysis ... 39
2.2.4 Seaweeds microbial load ... 40
2.2.5 Statistical analysis ... 41
2.3 Results and discussion ... 41
2.3.1 Proximate composition ... 41
2.3.1.1 Moisture content ... 41
2.3.1.2 Ash content ... 41
2.3.1.3 Total lipids content ... 42
2.3.1.4 Crude protein content ... 43
2.3.1.5 Total carbohydrates content ... 43
2.3.2 Seaweeds chemical properties ... 44
2.3.2.1 Determination of pH ... 44
2.3.2.2 Fatty acid methyl esters (FAME’s) ... 45
2.3.2.3 Elemental analysis ... 47
2.3.3 Seaweeds microbial load ... 51
Conclusions ... 53
Acknowledgments ... 54
References ... 55
3. COMPARATIVE STUDY OF ENZYMES IN RAW GOAT MILK FROM INDIGENOUS AND CROSSBREED REGIONAL HERDS ... 63
3.1 Introduction ... 64
3.2 Materials and methods ... 65
XIII
3.2.1 Animals and feed ... 65
3.2.2 Samples treatment ... 65
3.2.3 Determination of enzyme activities ... 66
3.2.3.1 Alkaline phosphatase (ALP) ... 66
3.2.3.2 Catalase (CAT) ... 67
3.2.3.3 Glutathione peroxidase (GPx)... 67
3.2.3.4 Glutathione S-transferase (GST) ... 68
3.2.3.5 Lactoperoxidase (LPO) ... 68
3.2.3.6 Lysozyme (LYZ) ... 69
3.2.3.7 Superoxide dismutase (SOD) ... 69
3.2.3.8 Xanthine oxidase (XOD) ... 70
3.2.4 Total protein ... 70
3.2.5 Statistical analysis ... 70
3.3 Results and discussion ... 70
3.3.1 Alkaline phosphatase (ALP) ... 71
3.3.2 Catalase (CAT) ... 72
3.3.3 Glutathione peroxidase (GPx) ... 74
3.3.4 Glutathione S-transferase (GST) ... 75
3.3.5 Lactoperoxidase ... 75
3.3.6 Lysozyme (LYZ) ... 76
3.3.7 Superoxide dismutase ... 77
3.3.8 Xanthine oxidase (XOD) ... 77
3.3.9 Correlation between enzymes activities ... 78
3.3.10 Total protein ... 79
Conclusions ... 80
Funding ... 81
Acknowledgements ... 81
Conflicts of interest... 81
References ... 82
4.CHARACTERIZATION OF SEMI-HARD GOAT´S CHEESE SUPPLEMENTED WITH SEAWEEDS.... 89
4.1 Introduction ... 90
4.2 Materials and methods ... 92
4.2.1 Cheesemaking, seaweeds and sampling strategy ... 92
4.2.2 Cheese physicochemical analysis ... 93
4.2.2.1 Total solids and moisture content ... 93
4.2.2.2 Ash content ... 93
4.2.2.3 NaCl and pH ... 94
XIV
4.2.2.4 Total carbohydrates content ... 95
4.2.2.5 Total lipids content ... 95
4.2.2.6 Crude protein content ... 95
4.2.2.7 Elemental analysis ... 96
4.2.3 Color ... 96
4.2.4 Texture Profile Analysis (TPA) ... 96
4.2.5 Microbial load ... 97
4.2.6 Flash Profile (FP) ... 98
4.2.6.1 Panel ... 98
4.2.6.2 Procedure ... 98
4.2.7 Statistical Analysis ... 98
4.3 Results and discussion ... 99
4.3.1 Physicochemical characteristics of S-HGCs ... 99
4.3.1.1 Total solids and moisture content ... 99
4.3.1.2 Ash content ... 99
4.3.1.3 NaCl and pH ... 100
4.3.1.4 Total carbohydrates content ... 100
4.3.1.5 Total lipids content ... 101
4.3.1.6 Crude protein content ... 101
4.3.1.7 Elemental analysis ... 102
4.3.2 Color ... 104
4.3.3 Texture Profile Analysis (TPA) ... 106
4.3.4 Microbial load ... 107
4.3.5 Flash Profile (FP) ... 111
Conclusions and future perspectives ... 118
References ... 119
5.CASHEW NUT FERMENTED PLANT-BASED «CHEESE» SUPPLEMENTED WITH RED SEAWEEDS (CHONDRUS CRISPUS AND PORPHYRA SP.) ... 129
5.1 Introduction ... 130
5.2 Materials and methods ... 131
5.2.1 Manufacture of cashew nut fermented cheese alternative supplemented with seaweeds and sampling strategy ... 131
5.2.2 Physicochemical analysis ... 133
5.2.2.1 Total solids and moisture content ... 134
5.2.2.2 Ash content ... 134
5.2.2.3 NaCl and pH ... 134
5.2.2.4 Total lipids content ... 134
XV
5.2.2.5 Crude protein content ... 135
5.2.2.6 Elemental analysis ... 135
5.2.3 Color ... 135
5.2.4 Texture Profile Analysis (TPA) ... 136
5.2.5 Microbial load ... 136
5.2.6 Flash Profile (FP) ... 137
5.2.6.1 Panel ... 137
5.2.6.2 Procedure ... 137
5.2.7 Statistical analysis ... 138
5.3 Results and discussion ... 138
5.3.1 Physicochemical characterization of CNFCAs ... 138
5.3.1.1 Total solids and moisture content ... 139
5.3.1.2 Ash content ... 139
5.3.1.3 NaCl and pH ... 139
5.3.1.4 Total lipids content ... 140
5.3.1.5 Crude protein content ... 140
5.3.1.6 Elemental analysis ... 141
5.3.2 Color ... 144
5.3.3 Texture Profile Analysis (TPA) ... 145
5.3.4 Microbial load ... 147
5.3.5 Flash Profile (FP) ... 149
Conclusions and future perspectives ... 156
References ... 157
6.GENERAL CONCLUSIONS AND FUTURE PERSPECTIVES ... 165
References ... 177
Annex A ― Microbiological parameters ... 185
Annex B ― Chapter 2 ... 189
Annex C ― Chapter 3 ... 195
Annex D ― Chapter 4 ... 199
Annex E ― Chapter 5 ... 205
L IST OF FIGURES
Figure 1.1 — Species for micro- and macroalgae approved by EFSA for human consumption in the EU ... 3 Figure 1.2 — Representative image of Sea-Lettuce (Ulva sp.) ... 5 Figure 1.3 —Represenstative image of Irish Moss (Chondrus crispus) ... 6 Figure 1.4 —Representative image of Dulse (Palmaria palmata ... 7 Figure 1.5 — Representative image of Nori (Porphyra sp.) ... 8 Figure 1.6 — Application of seaweed and seaweed extracts for the development of new food products ... 10 Figure 1.7 — Goat (Capra hircus) ... 12 Figure 1.8 — Milk containing different extracts of Ascophyllum nodosum and Fucus vesiculosus ... 13 Figure 1.9 —Selected fermented cashew cheese-like product ...20 Figure 1.10 — Layout of the work developed in the thesis ... 22 Figure 2.1 — Representative images of red seaweeds (Rodophyta) and green seaweed (Clo- rophyta)... 36 Figure 3.1 — Location of the herds at Montejunto (Portugal) ... 66 Figure 3.2 — Principal Component Analysis (PCA) performed on goat’s whole milk ... 79 Figure 4.1 — Representative image of goat’s cheeses supplemented with seaweeds ... 93 Figure 4.2 — Scheme of the protocol in the production of semi-hard goat’s cheeses ... 94 Figure 4.3 — Biplot map of GPA performed on FP data and the lexicon used to describe the diverse attributes ... 117 Figure 5.1 — General appearance of the CNFCAs after 15 days of ripening ... 132 Figure 5.2 — Protocol used in the production of cashew nut fermented cheese alternatives (CNFCAs) and sampling strategy ... 133 Figure 5.3 — Biplot map of results of GPA performed on FP data and the lexicon used to describe the various attributes of the CNFCAs ... 155
L IST OF TABLES
Table 1.1 ― Median (min.–max.) values of calories and nutritional content in dairy cheese and plant-based «cheese» alternatives per 100 g ... 17 Table 1.2 — Some examples of plant-based «cheese» alternatives available on the market ... 18 Table 2.1 — Proximal composition in edible seaweeds ... 42 Table 2.2 — Fatty acid methyl esters (FAMEs) of seaweeds... 46 Table 2.3 — Macro and trace elements of seaweeds and contribution to daily dietary intake and Reference Nutrient Intake (RNI) ... 48 Table 2.4 — Microbiological level parameters of seaweeds ...52 Table 3.1 — Enzyme activities determined in goat’s whole milk phase ... 71 Table 3.2 — Enzyme activities determined in goat’s serum milk phase ... 73 Table 3.3 — Total protein determined in goat’s milk ...80 Table 4.1 — Chemical properties of S-HGCs ...100 Table 4.2 — Contents for macrominerals and trace elements of S-HGCs ... 103 Table 4.3 — Color parameters of S-HGCs ... 105 Table 4.4 — TPA parameters of S-HGCs ... 107 Table 4.5 —Microbiological load of S-HGCs ...108 Table 4.6 — Residual variance from Generalized Procrustes Analysis (GPA) of Flash Profile (FP) on S-HGCs ... 111 Table 4.7 — Residual variance values, scaling factors, and the percentage variation explained by the first two principal components of Generalized Procrustes Analysis (GPA) on S-HGCs flash profile analysis... 112 Table 4.8 — Consensus index (Rc) among panelists for each attribute of S-HGCs ... 114 Table 5.1 — Physicochemical characterization of cashew nut fermented chese alternatives (CNFCAs) ... 138 Table 5.2 — Contents for macrominerals and trace elements in cashew nut fermented chese alternatives (CNFCAs)... 142 Table 5.3 — Color parameters of cashew nut fermented chese alternatives (CNFCAs) ... 144 Table 5.4 — TPA parameters for cashew nut fermented chese alternatives (CNFCAs) ...146 Table 5.5 — Microbiological load of cashew nut fermented chese alternatives (CNFCAs) ... 148 Table 5.6 — Residual variance values for each CNFCAs from Generalyzed Procrustes Analysis (GPA) of Flash Profile (FP) ... 150
Table 5.7 — Residual variance values, scaling factors, and the percentage variation explained by the first two principal components of Generalyzed Procrustes Analysis (GPA) on CNFCAs Flash Profile (FP) analysis ... 151 Table 5.8 — Consensus index (Rc (%)) among the panelists for each attribute of CNFCAs ... 152
A BBREVIATIONS
AA Amino Acid
Abs. Absent
AC Ash content
ACE Angiotensin-I-Converting Enzyme
ALA Alfa-Linolenic Acid
ALP Alkaline Phosphatase
AMB Aerobic Mesophilic Bacteria
ANOVA Analysis of Variance
AP Acid Phosphatase
APC Allophycocyanin
ARA Arachidonic Acid
BGA Brilliant Green Agar
BP Baird-Parker
BPW Buffered Peptone Water
BSA Bovine Serum Albumine
CAGR Compound Annual Growth Rate
CAT Catalase
CDNB l-Chloro-2,4-dinitrobenzene
CFU Colony-Forming Unit
CGA Chloramphenicol Glucose Agar
CHP Cumene Hydroperoxide
CJEU Court of Justice of the European Union
CNFCA Cashew Nut Fermented Cheese Alternative
CNFCA-C Cashew Nut Fermented Cheese Alternative Control
CNFCA-CC Cashew Nut Fermented Cheese Alternative with Chondrus crispus
CNFCA-P Cashew Nut Fermented Cheese Alternative with Porphyra sp.
CO2 Carbon dioxide
CRM Certified Reference Material
DEA Diethanolamine
DF Dried Form
DFIC Dairy-Free Imitation Cheese
DHA Docosahexaenoic Acid
DPPH 2,2-Diphenyl-1-Picrylhydrazyl
DW Dry Weight
EAA Essential Amino Acid
EC European Comission
EC number Enzyme Comission number
EDTA Ethylenediaminetetraacetic Acid
EFSA European Food and Safety Authority
EPA Eicosapentaenoic Acid
ETA Eicosatetraenoic Acid
EU European Union
FA Fatty Acid
FAME Fatty Acid Methyl Ester
FAO Food and Agriculture Organization of the United Nations
FP Flash Profile
GC-MS Gas Chromatography-Mass Spectrometry
GGT y-Glutamyl Transferase
GPA Generalyzed Procrustes Analysis GPx Glutathione Peroxidase
GSH Glutathione
GST Glutathione S-Transferase
GYP Glucose-Yeast Peptone
HDL-C High-Density Lipoprotein-Cholesterol
HPA Health Protection Agency
HT Hydratation Treatment
HTST Heat Treatment Short-Time
ICP-AES Inductively Coupled Plasma-Atomic Emission Spectrometry
IMTA Integrated Multi-Trophic System
INSA Instituto Nacional de Saúde Doutor Ricardo Jorge
ISO International Organization for Standardization
LA Linolenic Acid
LAB Lactic Acid Bacteria
LPL Lipoprotein Lipase
LPO Lactoperoxidase
LYZ Lysozyme
MAC Marine Agar Counts
Max. Maximum
MFGM Milk Fat Globule Membrane
Min. Minimum
MKTTN Muller-Kauffmann Tetrathionate-Novobiocin
MRS De Man, Rogosa & Sharpe
MUFA Monounsaturated Fatty Acid
n.a. Not analyzed NaCl Sodium chloride
NADPH β-Nicotinamide Adenine Dinucleotide Phosphate n.d. Not detected
NIST National Institute for Standards and Technology
ORAC Oxygen Radical Absorbance Capacity
PA/PE Polyamide/Polyethylene
PBFA Plant Based Foods Association
PBP Phycobiliprotein
PC Phycocyanin
PCA Plate Count Agar
PCA Principal Component Analysis
PE Phycoerythrin
PFA Perfluoroalkoxy Alkanes
p-NPP p-Nitrophenyl Phosphate PUFA Polyunsaturated Fatty Acid
RH Relative Humidity
RNI Reference Nutritional Intake
R-PC R-Phycocyanin
R-PE R-Phycoerythrin
RPF Rabbit Plasma Fibrinogen
RT Retention Time
RVS Rappaport-Vassiliadis Soja
SD Standard Deviation
SFA Saturated Fatty Acid
S-HGC Semi-Hard Goat’s Cheese
S-HGC-C Semi-Hard Goat’s Cheese Control
S-HGC-PP Semi-Hard Goat’s Cheese with Palmaria palmata
S-HGC-U Semi-Hard Goat’s Cheese with Ulva sp.
SM Serum Milk
SOD Superoxide Dismutase
SOX Sulfhydryl Oxidase
TAG Tryacilglycerides
TFP Traditional Food Products
TL Total Lipids
TPA Texture Profile Analysis
UFA Unsaturated Fatty Acid
UHT Ultra-High Temperature
UK United Kingdom
USA United States of America
WM Whole Milk
WMP Whey Membrane Particle
WW Wet Weight
XLD Xylose Lysine Desoxycholate
XOD Xanthine Oxidase
S YMBOLS
a* Color parameter (red to green) b* Color parameter (yellow to blue)
B12 Cobalmin
C* Chroma
Ca Calcium
Cl Chloride
cm Centimeter
Cu Copper
ε Extinction coefficient
Fe Iron
g Grams
g Gravitational force equivalent
h Hour
I Iodine
J Joule
K Potassium
kg Kilogram
L Liter
L* Lightness
M Molar concentration
Mg Magnesium
mg Milligram
mL Millilitre
mm Millimitre
mM Millimolar
Mn Manganese
N Newton
N Nitrogen
Na Sodium
nm Nanometer
nmol Nanomole
P Phosphorus
Rc Consensus index
S Sulphur
Se Selenium
U One enzyme unit
U.mL–1 Units per millilitre
v/v Volume by volume
v/v/v Volume by volume per volume
w/v Weight by volume
w/w Weight by weight
WW Wet Weight
Zn Zinc
ω-3 Omega-3 fatty acid
ω-6 Omega-6 fatty acid
λ Wavelength
μg Microgram
μL Microlitre
μm Micrometer μmol Micromole
∆ Variance
― Unit-less
1
DAIRY CHEESE AND PLANT - BASED « CHEESE »
SEAWEEDS SUPPLEMENTATION ― AN OVERVIEW
1.1 Introduction
Seaweeds are recognised since ancient times for their importance in the diet of many countries of East Asia, particularly Japan and Korea (Bocanegra et al., 2009; Afonso et al., 2019).
Additionally, epidemiological evidence associates their regular consumption with several health benefits (Cardoso et al., 2015), including cardioprotective, neuroprotective, anti-inflam- matory (Circuncisão et al., 2018) and immunomodulatory effects, decreased blood pressure and blood sugar (Cian et al., 2015), as well as beneficial impacts on gut function and microbiota (Cian et al., 2015; Rioux et al., 2017).
While the practice of eating seaweeds remains widespread among eastern world, in the western countries’ seaweeds have found mainly increasing industrial and food applications as gelling, stabilizing, or binding agents through hydrocolloids (alginate, agar, and carragee- nan) (Pereira, 2012; Kılınç et al., 2013; Cian et al., 2015). However, since seaweeds have a bal- anced nutritional value and are an abundant source of bioactive compounds they are seen as a health- promoting food element (Afonso et al., 2019). Thus, recently seaweeds consumption has been gaining increasing attention by consumers and the food industry, in western coun- tries, due to its health benefits (Roohinejad et al., 2017), and for preventing or reducing the convalescence period of several diseases (Biris-Dorhoi et al., 2020).
Seaweeds are currently pointed as the plant-origin foods of the future, already earning a superfood status (Circuncisão et al., 2018). At the same time, their consumption is also in line with the growing consumer awareness for organic and environmentally sustainable products (Afonso et al., 2019), as they do not compete with food crops for the use of arable land and freshwater resources (Cardoso et al., 2015; Wijers et al., 2020). As consequence of this, the size of the global seaweeds market is expected to grow at a compound annual growth rate (CAGR) of 10.8% from 2021 to 2028 (Grand View Research, 2021).
In general, seaweeds differ greatly in quality, color, texture, and nutrient content (Kılınç et al., 2013). They are nutritionally very rich, being a great source of complex polysaccharides (e.g. alginic acids and fucoidans), mineral elements (e.g. Mg, P, K, Na and Fe), vitamins (e.g. A, from complex B, C, and E), and proteins (e.g. phycobiliprotein) (Afonso et al., 2019; Cardoso et al., 2014; Circuncisão et al., 2018), containing all essential amino acids (EAA) (Pereira, 2016), as well as various phycochemicals such as alkaloids, flavonoids, steroids, and terpenoids (El-Din
& El-Ahwany, 2015).
To date, more than 150 edible species of algae for human consumption have been iden- tified in Europe, of which 86% are considered macroalgae (seaweed). Of the total number of algae, only 30 species are approved as novel foods by EU legislation (Mendes et al., 2022).
Currently, diverse protein-rich seaweeds are approved by the European Food and Safety Au- thority (EFSA) for human consumption, such as Chlorophyta Ulva lactuca (29% of proteins),
Rhodophyta Chondrus crispus (20%), Palmaria palmata (19%), and Neopyropia tenera (44%); and Phaeophyceae Fucus serratus (17%) and Undaria pinnatifida (29%) (Geada et al., 2021) (see Fig.
1.1).
Figure 1.1 — Species of micro- and macroalgae approved by EFSA for human consumption in the EU as Novel Food (Regulation (EU) 2020/1820) and non-Novel Food (Regulation (EU) 2018/1023) (Source: Geada et al., 2021).
1.2 Seaweeds ― an overview
Seaweeds, also known as macroalgae, are autotrophic organisms with a simple structure with little or no cellular differentiation (Peñalver et al., 2020), whose stems exhibit a high de- gree of complexity and tissue organization (Pereira, 2016). These organisms belong to the Eu- karyota Domain and the Kingdoms Plantae (the green and red algae) and Chromista (brown algae) (Cardoso et al., 2014), and are capable photosynthesis, namely transforming light into
chemical energy, capturing CO2 to form complex organic compounds, and releasing O2 (Pe- reira, 2016). Seaweeds are classified taxonomically into several groups based on specific pig- ments, other than chlorophyll, namely: a) green seaweed (class Chlorophyceae); b) red sea- weed (class Rhodophyceae), and c) brown seaweed (class Phaeophyceae) (Biris-Dorhoi et al., 2020; Bocanegra et al. 2009; Ferrara, 2020; Peñalver et al., 2020).
1.2.1 Green seaweed (phylum Chlorophyta)
Green algae (Chlorophyta) belong to a heterogeneous group of unicellular and multicel- lular organisms (Bleakley & Hayes, 2017; Ferrara, 2020). They are photosynthetic eukaryotic organisms characterized by chloroplast pigments, chlorophyll a and b, carotenoids, stacked thylakoids, and interplastidial starch (Arora & Sahoo, 2015a), varying from greenish yellow to dark green (Peñalver et al., 2020).
Chlorophyta, and particularly, the class Ulvophyceae, are known for their ability to pro- duce marine sulfated polysaccharides, more specifically ulvans; the major constituents of their cell walls, representing 8 to 29% of the algae dry weight (Pereira, 2016; Ullah et al., 2019; Biris- Dorhoi et al., 2020).
1.2.1.1Ulva sp. (Linnaeus, 1753) (Sea Lettuce; Ulvales, Ulvaceae)
Ulva sp. (Sea Lettuce) (Fig. 1.2) is a seaweed commonly used as a food source (Kılınç et al., 2013). The taxa (genus) have around 30 described species (Arora & Sahoo, 2015b), of which U. intestinalis and U. compressa are commonly consumed (Bleakley & Hayes, 2017), being U.
lactuca the best known (Peñalver et al., 2020). According to Bocanegra et al. (2009), Ulva spp.
presents a composition (g/100g of DM) of 13.7-22.6 of ashes, 42.10 of carbohydrates, 0.6-0.7 of lipids, and 20.0-26.1 of proteins. In general, species of the genus Ulva can present essential amino acids (EAAs) at levels that can rival eggs (Hafting et al., 2015), and there are cases where all eight EAAs are present, as in U. lactuca (Ferrara, 2020). This last seaweed has a diverse chemical composition with high nutritional value due to the presence of fatty acids (FAs) with a moderate content in ω-3, complex B vitamins, mineral salts (e.g. Ca, Fe, and Mg), among others (Ferrara, 2020). Concerning U. rigida, this seaweed presents a high nutrient content, be- ing an excellent source of nitrate, sulfate, antimicrobial compounds, vitamins, and gibberellins, also containing cytostatic substances (Pereira, 2016).
Figure 1.2 — Representative image of Sea-Lettuce (Ulva sp.).
1.2.2 Red seaweed (phylum Rodophyta)
Red algae belong to the phylum Rhodophyta. Their photosynthetic pigments are chlo- rophyll a, carotenoids (e.g. carotene and zeaxanthin), and phycobiliproteins (PBPs) (Cardoso et al., 2014; Pereira, 2016). Some of these PBPs (e.g. R-phycocyanin (R-PC) and R-phycoerythrin (R-PE)) demonstrate antioxidant, anti-inflammatory, antiviral, cardiovascular, and neuropro- tective protection, among others (Cardoso et al., 2014).
The protein content of Rodophyta is higher than those of the other groups (Chlorophyta and Phaeophyceae), representing 10-50% of dry weight. In red algae, amino acids (AAs) such as tryptophan (Trp), methionine (Met), and leucine (Leu) are present in small concentrations, contrary to lysine (Lys), which is overriding and is a limited AA in terrestrial plant foods (e.g.
corn, soy, and rice) (Ferrara, 2020). The lipid content is very low (ca. 1-5%), but high in ω-3 polyunsaturated fatty acids (PUFAs), namely α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), with a balanced ratio of dietary ω-6/ω-3 (Ferrara, 2020); which is fundamental for a healthy lifestyle, since that allows the progressive brain, eyes, and heart development. At the same time, the ingestion of a dietary balanced ω-6/ω-3 reduces the risk of coronary artery heart disease (CAD) and neurodegenerative disorders (Pa- tel et al., 2022).
Among sulphated polysaccharides, red algae are an important source of galactans (aga- rose and carrageenans). These non-digestible substances are considered dietary fibres (Ferrara, 2020) and have remarkable thickening and gelling properties (Pereira, 2016). For example, E407 (carrageenan) has many applications in the food industry (e.g. puddings, chocolate milk, condensed milk, ice cream, sauces, cheeses, beer, and wine) (Pereira, 2010).
1.2.2.1Chondrus crispus (Stackhouse, 1797) (Irish Moss; Gigartinales, Gigartinaceae)
Chondrus crispus (Irish Moss or Carrageen Moss) (Fig. 1.3) is a red macroalgae with a high content of carrageenan, that has been used for several years as a thickener, emulsifier, and gelling agent in the food industry (Cardoso et al., 2014; Pereira, 2016).
Figure 1.3 —Representative image of Irish Moss (Chondrus crispus).
This seaweed is composed (g/100 g of DW) of ashes (14.2), carbohydrates (54.80), soluble dietary fibre (22.30), insoluble dietary fibre (12.0), total dietary fibre (34.3), lipids (2.6), and proteins (11.2) (Bocanegra et al., 2009), among others. It also has a high content of PUFAs min- eral salts, and vitamin A (Pereira, 2010).
1.2.2.2Palmaria palmata ((Linnaeus) F. Weber & D. Mohr, 1805) (Dulse; Palmariales, Pal- mariaceae)
Dulse (Palmaria palmata) (Fig. 1.4) is one of the most popular consumed species of sea- weeds in Western countries (Mouritsen et al., 2013) due to its pleasant taste for the western palate (Pereira, 2016). Reported values for P. palmata range from 73-89% moisture (Morgan et al., 1980), 12-37% ash, 38-74% polysaccharides, and 0.2-3.8% lipids (Rioux et al., 2017). Despite its low lipid content, it has a wide variety of FAs (Morgan et al., 1980), especially eicosapen- taenoic acid (EPA) with a relative abundance of more than 50% (Lopes et al., 2019).
Figure 1.4 —Representative image of Dulse (Palmaria palmata).
This seaweed has a particularly high protein content, varying according to the season (Bocanegra et al., 2009; Peñalver et al., 2020), ranging from 8-35% (Morgan et al., 1980). In some cases, it can represent up to 35% DW (Pereira, 2016). This seaweed has levels of AAs similar to those found in vegetables, namely isoleucine (Ile) and threonine (Thr) (Cardoso et al., 2014).
Besides these, AAs such as leucine (Leu), valine (Val), and methionine (Met) are abundant and their average contents are those generally reported for ovalbumin (Bocanegra et al., 2009). As it is also rich in glutamic acid (Glu), this seaweed can be used as a base for umami dishes (Haft-ing et al., 2015). P. palmata it has also dietary fibre (Cardoso et al., 2014), a substantial content
of vitamins B6 and B12 (Pereira, 2016), and appreciable amounts of vitamins A and C, and a higher concentration of some minerals (e.g. Cl, K, and Na). Moreover, when compared to ter- restrial fruits and vegetables, it is a good source of Fe, Mg, Ca, and I (Morgan et al., 1980).
1.2.2.3Porphyra sp. (C.A. Agardh, 1824) (Nori; Bangiales, Bangiaceae)
Among the seaweeds used for human food, Porphyra/Neoporphyra/Pyropia/Neopyropia spp. (Nori) (Fig. 1.5) is one of the most recognised (Peñalver et al., 2020), both in Asian and western countries, mainly due to its delectable flavor (Bleakley & Hayes, 2017).
Figure 1.5 — Representative image of Nori (Porphyra sp.).
Neopyropia tenera has a chemical composition (g/100 g of DW) of ashes (8.5-8.7), carbohy- drates (40.5), insoluble dietary fibre (16.8-19.2), soluble dietary fibre (14.6-17.9), total dietary fibre (33.7-34.7), lipids (0.7-1.6), and proteins (33.0-47) (Bocanegra et al., 2009).
Porphyra sp. is rich in ω-3 PUFAs (Pereira, 2016), having high concentrations of eicosa- pentaenoic acid (C20:5, ω-3, EPA), with 48.0-51.0% of total fatty acid methyl esters (FAMEs).
Adittionally, presents minor concentrations of arachidonic acid (C20:4, ω-6, ARA) and linoleic acid (C18:2, ω-6, LA), with 2.1-10.9% and 1.3-2.5% of total FAMEs, respectively (Kılınç et al., 2013).
Porphyra spp. is one of the seaweeds with the highest arginine (Arg) and glycine (Gly) concentrations (Bocanegra et al., 2009). This species is also rich in trace minerals (Pereira, 2016), presenting high concentration of iron (Fe) (Bocanegra et al., 2009). Water-soluble vitamins such as vitamin C are present at high levels in P. umbilicalis (Peñalver et al., 2020), apart from vita- mins A, B, and E (Pereira et al., 2016).
1.3 A new source of protein ― a current trend
Nowadays, the overconsumption of meat is a key factor contributing to the climate emer- gency. As such, seaweeds can be used as an alternative to animal-based proteins (Mellor et al., 2022), representing a promising and novel future protein source (Henchion et al., 2017), since traditional agriculture is no longer enough to respond to the global demand for food as the world population keeps rising (Thiviya et al., 2022). On the other hand, there is a current need for alternative sources of rich plant-based proteins to satisfy the proteins needs of vegan con- sumers and particularly vegan athletes (Raja et al., 2022), as well as to those that want to follow the general recommendation by nutritionists and organizations, such as the FAO, for a varied diet rich in plant-based proteins to prevents cardiovascular disease and diabetes (Bleakley &
Hayes, 2017). In sum, seaweeds are healthy emerging alternative protein source with some benefits over traditional high-protein crops in relation to productivity, and protein yield per unit area, having no need for arable land, freshwater and artificial fertilizer (Thiviya et al., 2022;
Wijers et al., 2020).
1.3.1 Seaweeds supplementation in food products
Seaweeds are sources of high-added value compounds that have attracted the food in- dustry sector (Peñalver et al., 2020). For this reason, they are used in countless industrial ap- plications (Kılınç et al., 2013) where conventional and fermented foods are used (Ścieszka &
Klewicka, 2018). In the western world their use was traditionally focused on the extraction of compounds used by the food industries, particularly as a source of hydrocolloids, i.e., thick- ening, and gelling agents (Wijesinghe & Jeon, 2012). The key products are agars, agaroses, algins, and carrageenans (Jiménez-Escrig & Sánchez-Muniz, 2000; Kılınç et al., 2013).
Due to their properties, seaweeds have been used as functional ingredients in food in- dustry, fortifying nutritional composition, quality, and health-related beneficial properties of different products (Roohinejad et al., 2017). There are examples of their use in products such as seafood (e.g. canned, cakes, or cutlet fish) meat (e.g. steaks, frankfurters, patties, or sau- sages), dairy products (e.g. cheese, sour cream, or yoghurt), cereal-based products (e.g. bread, noodles, pasta, or rice extrudate) (Cofrades et al., 2013; Biris-Dorhoi et al., 2020), sauces (e.g.
mustard, marinades, or pesto) and drinks (e.g. juices, gin, liquor, or ales) (Geada et al., 2021) (see Fig. 1.6).
Figure 1.6 — Application of seaweeds and seaweed extracts for the development of new food products (Source:
Roohinejad et al., 2017).
1.4 Dairy industry
1.4.1 Milk
Biologically, milk is a fluid secreted by female mammals to meet the high-level nutrition requirements of the neonate of the species (Fox & McSweeney 1998; Kelly & Larsen, 2010).
Additionally, several proteins fraction of milk, which include immunoglobulins, binding or carrier proteins, growth factors, antibacterial agents, and enzymes, among others fulfils sev- eral physiological functions (Fox & McSweeney, 1998; Claeys et al., 2014). In brief, milk con- tains different types of enzymes, which in some cases such as bovine milk, can be around 70 (Kelly & Larsen, 2010; O’Mahony & Fox, 2013). These enzymes can affect the manufacture of dairy products and their stability during storage (Moatsou, 2010).
Since the nutritional and physiological requirements of each species are subjective, the milk composition shows very marked inter-species differences (Fox & McSweeney, 1998).
Therefore, levels of milk constituents are not static but rather highly dynamic, and may even- tually fluctuate, mainly due to genetics, stage of lactation, health status, and environmental factors (e.g. feed, climate, season, or milking method) (Kelly & Larsen, 2010; Lima et al., 2018).
1.4.1.1Goat’s milk
Goat (Capra hircus) (Figure 1.7) is an important source of milk for human consumption (Lima et al., 2018), representing 2.3% of the milk produced worldwide (Claeys et al., 2014).
Goat’s milk differs from the milk of other mammals (bovine and human) in both composition and nutritional properties (Lima et al., 2018). According to Turkmen (2017), the average basic composition of goat’s milk is as follows: 69 kcal food energy, 13.2 g/100g total solids, 4.5 g/100g fat, 3.6 g/100g total protein, 4.3 g/100g lactose, and 0.8 g/100g minerals.
In comparison with human milk, goat’s and bovine milk show higher levels of ash and protein, but a lower lactose content (Yadav et al., 2016). Such as bovine, sheep, and human milk, goat’s milk fat consists of 97-98% of triglycerides, but only presents 0.5-1.5% of phospho- lipids and 0.7-1.5% of free FAs contents (Claeys et al., 2014). Concerning minerals, goat’s milk shows higher contents of Ca, Cl, K, Mg and P, and lower contents of Na and S when compared to bovine milk (Yadav et al., 2016).
Goat’s milk show some properties that confer technological advantages over bovine milk
―, for example, fat globules have a smaller size, which provides a smoother texture to the by- products; lower amounts of αS1-casein, which results in softer gel products, a higher water- binding capacity, as well as a lower viscosity (Gomes et al., 2013; Yadav et al., 2016). Due to
the health benefits of goat’s milk, it is highly recommended for infants, the elderly, and con- valescent people (Lima et al., 2018).
Figure 1.7 — Goat ((Capra hircus) Linnaeus, 1758).
1.4.2 Dairy products supplemented with seaweeds
Dairy products are nutrient-rich foods that contribute substantially to the diet with min- erals (e.g. Ca, K, Mg, P, and Zn), proteins, and vitamins (e.g. A, D and B12) (Cofrades et al., 2013).
In the dairy industry, seaweeds are added to food matrices in order to improve the nu- tritional value, organoleptic characteristics, and enhance the shelf-life of several milk-based products (e.g. cheese, cottage cheese, cream, and yoghurt) (Roohinejad et al., 2017; Biris-Dorhoi et al., 2020; Peñalver et al., 2020). In recent years, several research projects studied milk sup- plementation with seaweeds or seaweed extracts (Nuñez & Picon, 2017). For example, Del Olmo et al. (2019) showed that milk supplementation with seaweed extracts allowed attaining higher probiotic counts at the end of fermentation, most likely due to the presence of stimula- tory and inhibitory compounds present in the extracts. Another study conducted by O’Sulli- van et al. (2014) produced novel dairy products by adding seaweed extracts (0.25% and 0.5%
(w/w)) from Ascophyllum nodosum (100% H2O (AN100) or 80% ethanol: 20% H2O (AN80e)) and Fucus vesiculosus (60% ethanol: 40% H2O (FV60e)) to milk (Figure 1.8). The addition of seaweed extract showed a significant reduction in lipid oxidation and improvements in the products shelf-life characteristics.
Figure 1.8 — Milk containing different extracts of Ascophyllum nodosum and Fucus vesiculosus (Source: O'Sullivan et al., 2014).
A new set of studies on seaweed fortified yoghurts tested the same supplementation of A. nodosum and F. vesiculosus extracts revealing that yoghurts containing 0.5% of AN80e and 0.5% of FV60e exhibited lower levels of lipid oxidation. These studies also revealed that DPPH radical scavenging activity increased before and after in-vitro digestion. Despite this, parame- ters related to pH, microbiology, and whey separation were not affected by the addition of seaweed extracts (O’Sullivan et al., 2016). In both cases (milk and yoghurts) none of the prod- ucts were well accepted by panelists, even at the lowest concentrations, since they were de- scribed with negative sensory attributes such as unpleasant green/yellowish color and fishy taste (O’ Sullivan et al., 2014; O’ Sullivan et al., 2016).
1.4.2.1 Cheese supplemented with seaweeds
Supplementation of dairy products with non-dairy ingredients with th e aim of increas- ing their nutritional contents is a very common application in the dairy industry (Del Olmo et al., 2018). The nutritional richness of seaweeds allows to obtain functional dairy products (Pe- ñalver et al., 2020). Consequently, in the last few years, researchers have developed several types of cheese supplemented with seaweeds. For instance, in a study conducted by Nuñez &
Picon (2017), the supplementation with five different edible seaweeds: a) Chlorophyta (Ulva lactuca); b) Rodophyta (Porphyra umbilicalis); and Phaeophyceae (Himanthalia elongata, Saccha- rina latissima, and Undaria pinnatifida) influenced the odor, flavor, and texture sensory charac- teristics of quark cheese. The effect was dependent on concentrations and seaweed species. U.
pinnatifida at 0.5% (w/w) was the formulation that showed the highest seaweed flavor and the lowest flavor quality in quark cheese, worsening almost all their sensory aspects and making
this seaweed unattractive for quark application (Nuñez & Picon, 2017). In addition, another study concluded that the supplementation with Undaria pinnatifida or Saccharina japonica of up to 15% in cottage cheese results in higher contents of minerals (Ca, Fe, and Mg), albeit textural quality (consistency and firmness) was best with the addition of only 9% seaweed (Lalic and Berković, 2005).
The effect of adding Palmaria palmata or Saccharina longicruris to a Camembert-type cheese on both the oxygen radical absorbance capacity (ORAC) and Angiotensin I-converting enzyme (ACE)-inhibitory activity has been studied with the aim of developing a new func- tional food. The study confirmed the potential of seaweeds to be combined with a soft and surface-ripened cheese of this type, increasing the nutritional content by adding edible fibres, and by maintaining the total sodium content through minerals (Hell et al., 2017).
Del Olmo et al. (2018) developed a semi-hard cheese supplemented with each of five seaweeds: Chlorophyta (Ulva lactuca); Rodophyta (Porphyra umbilicalis); and Phaeophyceae (Himanthalia elongata, Laminaria ochroleuca, and Undaria pinnatifida). Results showed that sea- weeds addition enhance whey retention, increasing moisture and lowering pH, but it hardly influencing the antioxidant activity and cheese microbial load. The study concluded that H.
elongata offered advantages over other seaweeds when antioxidant activity and sensory char- acteristics effects were considered together.
1.5 Plant-based foods
More than 500,000 species of plants are essential for uses in medicine, fuel, clothing, and food (Krzywonos & Piwowar-Sulej, 2022). Nowadays, plants are included in a broad range of food products designed to mimic animal-based foods (e.g. meat, fish, eggs, milk and by- products) (McClements & Grossman, 2021), since people are shifting from animal-based diets to adopting plant-based dietary patterns (Fehér et al., 2020; Boukid et al., 2021).
The growing interest in plant-based foods is increasing due to health-related issues (Short et al., 2021), especially related to consumption of animal-based food products (mainly red and processed meats), which have been linked to harmful health effects such as colorectal cancer, cardiovascular disease, and diabetes (Frésan & Rippin, 2021). Contrary to these, plant- based foods have been related to lower health incidences such as cardiovascular disease, dia- betes, and cancer (Hassoun et al., 2022). Therefore, the modern food industry focuses on the development of healthier plant-based products, which are also more ethical and sustainable than animal-based foods (McClements & Grossman, 2021). For these reasons, plant-based foods quickly increased their popularity among consumers (Short et al., 2021). However, these kinds of products must be tasty, convenient, and affordable, otherwise there will be no de- mand and sales will not take place (McClements & Grossman, 2021).
In summary, a sustainable food system that shifts the consumers toward less animal- based foods and more plant-based foods is very beneficial for two reasons: i) human health;
and ii) natural resources. Diets rich in minimally processed whole grains, vegetables, fruits, nuts, and legumes have been recommended for increase human health and a sustainable en- vironment. A great variety of plant-based products have been developed to replace traditional animal-based foods namely, meat alternatives (Hassoun et al., 2022).
On the whole, plant-based food can be defined as a finished product consisting of ingre- dients derived from plants (e.g. vegetables, fruits, whole grains, nuts, seeds and/or legumes), as well fungi or algae or a combination of these. Aditionally, plant-based foods do not contain any type of animal-derived ingredients, falling into the following categories: a) tofu and tempeh; b) meat alternatives to beef, pork, chicken or fish; c) milk alternatives; d) other dairy alternatives (e.g. cheese, yogurt, ice cream, novelty and frozen desserts, butter, dips, dressings and sour cream, other beverages, and creamers); e) egg substitutes and mayo; f) meals with meat or dairy alternatives (e.g. pizza, frozen meals, and pot pies); g) baked goods; and h) pro- tein powders (PBFA, 2020)
1.5.1 Plant-based cheese alternatives (PBCAs)
1.5.1.1General aspects
With the aim of facilitating the dietary transition, i.e., reducing the consumption of pro- ducts of animal origin (Craig et al., 2022), a vast number of products imitating animal-sourced products are reaching the markets; and among them, the plant-based cheese alternatives (PBCAs) (Frésan & Rippin, 2021) based on nuts, oils, grains, soy, and other plant products.
PBCAs can be sold in various forms such as blocks, chunks, shreds, slices, spreads, and wedges (Craig et al., 2022).
Plant-based cheese alternatives are also referred to as non-dairy cheeses (Mattice & Ma- rangoni, 2020), or dairy-free imitation cheeses (DFICs) (Saraco & Blaxland, 2020), or informally as plant-based «cheeses. The denomination is a controversial issue among the distinct players in the food industry. This debate has raised questions about the nutritional composition of alternative dairy products compared to conventional ones and therefore the use of dairy tra- ditional words such as «milk», «cheese», and «yogurt» for the labeling of alternative dairy products, which might deceive the consumers (Boukid et al., 2021). According to the European Court of Justice (CJEU), in press release No 63/17, the «purely plant-based products cannot, in principle, be marketed with designations such as ‘milk’, ‘cream’, ‘butter’, ‘cheese’ or ‘yoghurt’, which are reserved by EU law for animal products» (CJEU, 2017; Boukid et al., 2021).
Concerning market size, PBCAs increased ca. 42% over 2019 sales in the USA, totalizing
$270 M, making it one of the fastest growing categories among global plant-based. The global market for PBCAs was valued at USD 2.22 billion in 2020 and is expected to grow at a com- pound annual growth rate (CAGR) of 12.4% from 2021 to 2028 (Craig et al., 2022).
Concerning flavor profile of PBCAs there are two general approaches ―, the first focus on simulating the sensory attributes of conventional dairy cheese, while the second focus on the flavors and characteristics of the plant itself. The challenge with the first approach is sub- stantial, since plant-based ingredients do not precisely mimic the sensory and physical char- acteristics of dairy-based cheese, and therefore there is limits to consumer acceptability (Short et al., 2021).
Regarding PBCAs, they usually are split into fermented and non-fermented. From the technological point of view, the PBCAs are emulsions of oil-in-water, that contain protein, sta- bilizers, emulsifiers, flavors, color agents, food preservatives, and water (Fu & Yano, 2020;
Boukid et al., 2021). These ingredients are blended to simulate dairy cheese’s texture and ap- pearance, the majority of which do not need a maturation period (Boukid et al., 2021).
The fermented type of PBCAs, are usually produced using nuts that are soaked, ground with water, and fermented (Tabanelli et al., 2018; Grasso et al., 2021). This was the type of cheese developed in the scope of this work and will be considered in chapter 5.
1.5.1.2Non fermented PBCAs
Plant-based milk substitutes can be primary ingredients in the manufacture of non fer- mented PBCAs. In general, the main ingredients of plant-based milk substitutes are divided into cereals (e.g. oats, rice, and corn), legumes (e.g. soybeans, peas, and chickpeas), nuts (e.g.
almonds), fruits (e.g. coconut), seeds (e.g. sesame and sunflower) and pseudo-cereals (e.g. qui- noa and amaranth) (Fu & Yano, 2020).
Other important ingredients in this type of PBCAs are oils (e.g. canola, coconut, sun- flower, or palm oil) and starches (e.g. corn, potato, or tapioca starch). These bases give a cheese- like fat and structure to the final product. In a certain way, they simulate the texture and melt- ability of cheese. Oil ingredients helps to simulate the meltability of cheese but not the stretch and flow, whereas starch provides some contents of stretch to the product (Mattice & Maran- goni, 2020; Harper et al., 2022).
When compared to dairy cheese, PBCAs possess higher contents of carbohydrates, fat, and saturated fatty acids (SFAs), but lower protein, salt, and sugar contents (Boukid et al., 2021). Table 1.1 shows the nutritional content of both products.
The lower protein content of PBCAs is the main limitation of these products, making them a less healthy choice (Boukid et al., 2021). The near-zero value of protein in most PBCAs
