Ultrasound-Assisted Extraction of Biopolymers from Ascophyllum nodosum Brown Seaweeds: Bioactivity and Enzyme Inhibition of Phlorotannins assessment

INTERNATIONAL DOCTORAL SCHOOL OF THE USC MAURO GISBERT VERDÚ PhD Thesis Ultrasound–Assisted Extraction of Biopolymers from Ascophyllum nodosum Brown Seaweeds: Bioactivity and Enzyme Inhibition of Phlorotannins Assessment Santiago de Compostela, 2022 Doctoral Programme in Chemical and Environmental Engineering DOCTORAL THESIS ULTRASOUND–ASSISTED EXTRACTION OF BIOPOLYMERS FROM ASCOPHYLLUM NODOSUM BROWN SEAWEEDS: BIOACTIVITY AND ENZYME INHIBITION OF PHLOROTANNINS ASSESSMENT MAURO GISBERT VERDÚ INTERNATIONAL PHD SCHOOL OF UNIVERSIDADE DE SANTIAGO DE COMPOSTELA PHD PROGRAMME IN CHEMICAL AND ENVIRONMENTAL ENGINEERING SANTIAGO DE COMPOSTELA 2022 DECLARACIÓN DEL AUTOR/A DE LA TESIS D./Dña. Mauro Gisbert Verdú Título de la tesis: Ultrasound–Assisted extraction of biopolymers from Ascophyllum nodosum brown seaweeds: Bioactivity and enzyme inhibition of phlorotannins assessment Presento mi tesis, siguiendo el procedimiento adecuado al Reglamento y declaro que: 1) La tesis abarca los resultados de la elaboración de mi trabajo. 2) De ser el caso, en la tesis se hace referencia a las colaboraciones que tuvo este trabajo. 3) Confirmo que la tesis no incurre en ningún tipo de plagio de otros autores ni de trabajos presentados por mí para la obtención de otros títulos. 4) La tesis es la versión definitiva presentada para su defensa y coincide la versión impresa con la presentada en formato electrónico. Y me comprometo a presentar el Compromiso Documental de Supervisión en el caso que el original no esté depositado en la Escuela. En Santiago de Compostela, 24 de octubre de 2022. Firma electrónica AUTORIZACIÓN DEL DIRECTOR / TUTOR DE LA TESIS Ultrasound–Assisted extraction of biopolymers from Ascophyllum nodosum brown seaweeds: Bioactivity and enzyme inhibition of phlorotannins assessment D. Ramón F. Moreira Martínez y D. Jorge Sineiro Torres En condición de Tutor y Director En condición de Director INFORMAN: Que la presente tesis, se corresponde con el trabajo realizado por D/Dª. Mauro Gisbert Verdú, bajo nuestra dirección/tutorización, y a utorizamos su presentación , considerando que reúne l os r equisitos exigidos en el R eglamento de Estudios de Doctorado de la USC, y que como directores de esta no incurre en las causas de abstención establecidas en la Ley 40/2015. De acuerdo con lo indicado en el Reglamento de Estudios de Doctorado, declaramos también que la presente tesis doctoral es idónea para ser defendida en base a la modalidad de Monográfica con reproducción de publicaciones, en los que la participación del doctorando fue decisiva para su elaboración y las publicaciones se ajustan al Plan de Investigación. En Santiago de Compostela, 24 de octubre de 2022 Fdo. Ramón F. Moreira Martínez Fdo. Jorge Sineiro Torres Als meus pares Maria José i Victor La meua inspiració, la meua força, el meu suport i per damunt de tot la meua temprança Resumo VII Unha dieta baseada en produtos sen glute implica un maior risco de sufrir problemas de diabetes, debido ao seu alto índice glicémico. Os alimentos de panificación sen glute foron deseñados inicialmente como unha necesidade imperiosa e apresurada para producir alimentos aptos para os celíacos, pero tiñan unha baixa calidade nutricional cun contido proteico baixo en relación cos de azucres e/o graxas. Por esta razón, o desenvolvemento de novos produtos sen glute converteuse nun auténtico reto, xa que debe contemplar tanto os aspectos nutricionais como sensoriais. Na actualidade, a aparición de novos produtos sen glute para satisfacer unha demanda crecente fai que este subsector do mercado alimentario sexa o que está a experimentar un maior crecemento nos últimos anos. A ausencia de glute fai obrigatorio buscar axentes estruturantes alternativos que o substitúan. Estes substitutos do glute deben emular as súas propiedades viscoelásticas e non modificar na medida do posible a aparencia e a estrutura dos produtos de panadería convencionais, sen alterar as características sensoriais. O uso de fariñas de provenientes de cereais e pseudocereais sen glute como millo, arroz, pataca, garavanzo coa adición de hidrocoloides, fibra e/ou proteínas é unha das estratexias económicas máis estendida no deseño de novos produtos sen glute. Algúns dos hidrocoloides utilizados no sector alimentario son a hidroxipropilmetilcelulosa, a goma xantana, a goma tragacanto o carraxenato, o agar e o alxinato (estes últimos obtidos de diferentes especies de algas), entre outros. A resposta glicémica postprandial está a atraer moita atención debido á súa relación directa cun alto risco de desenvolver enfermidades metabólicas coma o déficit de crecemento, anemia, deficiencias vitamínicas, enfermidades da tiroides, trastornos mentais, fatiga e/ou infertilidade. O consumo de carbohidratos ten un alto impacto nesta resposta xa que se trata de compostos esenciais para a dieta humana e son inxeridos en forma de amidón, sacarosa, glicosa, fructosa e oligosacáridos. Unha estratexia para controlar os niveis altos de glicosa en sangue é a inhibición das encimas dixestivas involucradas na descomposición do amidón, oligosacáridos e disacáridos en glicosa. Algúns fármacos antidiabéticos coma a acarbosa son inhibidores destas encimas permitindo o control da glicosa postprandial en pacientes diabéticos. Con todo, a investigación atópase máis centrada no descubrimento de inhibidores naturais das encimas α– amilasa e α–glucosidasa como os polifenois e as súas características beneficiosas para a saúde. Deste xeito proponse a reformulación deste tipo de produtos mediante o uso de aditivos alimentarios baseados en florotaninos (polifenois de algas mariñas) que poden actuar como Resumo VIII antioxidantes e tamén como axentes inhibidores de encimas dixestivos, concretamente a α– amilasa e a α–glucosidasa, e así controlar parcialmente o índice glicémico destes produtos amiláceos sen glute de nova xeración. O alto contido de polisacáridos, ácidos graxos poliinsaturados, minerais, fibra, vitaminas e moléculas bioactivas converte ás algas en candidatas ideais para seren utilizadas en diversas aplicacións médicas, farmacéuticas, cosméticas, fertilizantes, e, fundamentalmente, alimentarias. De feito, a demanda mundial de produtos derivados de algas mariñas xera beneficios socioeconómicos relevantes, estimándose que aumenten ata os 26 millóns de euros antes do 2025. Os florotaninos son metabolitos secundarios exclusivos de algas pardas. Particularmente, nesta Tese Doutoral emprégase como fonte destes aditivos o alga parda Ascophyllum nodosum presente nas costas galegas. Este tipo de polifenois son biopolímeros que derivan exclusivamente da reacción de polimerización do floroglucinol (1,3,5–trihidroxibenceno). O contido en polifenois das algas depende de diversos parámetros ambientais como as mareas, correntes, salinidade, dispoñibilidade de luz, radiación UV, entre outros. De feito, estas moléculas actúan coma defensas químicas contra os herbívoros e a radiación UV. Os florotaninos tamén teñen funcións estruturais que forman parte das paredes celulares xunto co alxinato. Estes polifenois clasifícanse en seis tipos diferentes, atendendo ás variacións da súa ensamblaxe e distribución de grupos hidroxilo, a saber, eckoles, fuhaloles, fucofloretoles, floretoles, fucoles e carmaloles. Os floretoles e fuhaloles caracterízanse por ter ligazóns aril– éter, os fucoles ligazóns aril–aril, os fucofloretoles unha mestura de ligazóns éter e fenilo. Os eckoles presentan unha unidade de 1,4–dibenzodioxina. Os carmaloles presentan unha unidade de 4–dibenzodioxina na terceira e sétima posición. Os eckoles diferéncianse dos carmaloles polo seu menor peso molecular e pola presenza dun grupo OH substituído no cuarto carbono. Os fuhaloles difiren dos floretoles pola súa secuencia regular de ligazóns para– e orto–éter, pola presenza de grupos OH adicionais en cada terceiro anel e pola falta dun ou máis grupos OH en todas as moléculas. A composición dos florotaninos segue sendo pouco coñecida, existindo moi poucos estándares comerciais dispoñibles que permitan a súa identificación, ao contrario que nos polifenois de plantas, onde o número de compostos de referencia non deixou de aumentar nos últimos anos. Numerosos métodos de illamento de florotaninos pódense encontrar na literatura, Resumo IX pero estes procesos adoitan ser complexos, custosos e ademais moi pouco eficientes polo que a identificación dos florotaninos de algas segue sendo un reto. Os florotaninos foron avaliados inicial e principalmente polas súas propiedades antioxidantes dada a súa alta capacidade para doar protóns debido aos seus múltiples grupos hidroxilo xunto coa súa capacidade para deslocalizar o radical antioxidante resultante dentro da estrutura. Nas últimas décadas, numerosas propiedades beneficiosas para a saúde destes compostos foron descubertas: antioxidantes, anticoagulantes, antitrombóticas, antidiabéticas, reguladoras da actividade encimática, bactericidas, antitumorais, antiinflamatorias, antihipertensivas e antivirais, entre outras. Tamén, encontráronse resultados prometedores no seu uso en tratamentos do Alzheimer, o Parkinson e a artrite. Os alxinatos son copolímeros binarios lineais unidos por ligazóns 1–4–glucosídicos. Os monómeros de alxinato son α–L–guluronato (G) e β–D–manuronato (M). O valor da relación M/G mide a distribución dos bloques manuronato e guluronato ao longo das moléculas de alxinato mentres que os valores de NG>1 serven para medir a lonxitude media do bloque. Os valores de M/G e NG>1 calculados mediante a norma estandarizada “Método de ensaio estándar para determinar a composición química e a secuencia en alxinato mediante espectroscopía de resonancia magnética nuclear de protóns” pódese utilizar como un índice para as propiedades físicas dos xeles de alxinato. Estas propiedades dependen da especie de alga empregada como fonte de alxinato e modificarse en función dos métodos e condicións de extracción e procesado empregadas polo que, deste xeito, pódese cubrir un amplo espectro de aplicacións industriais. A extracción asistida por ultrasóns é considerada unha técnica de extracción ecolóxica e de alta eficacia. Ofrece unha alta reproducibilidade con tempos de extracción curtos, opera a baixa temperatura, con equipos simples e de baixo custo, reduce o consumo de solventes e de enerxía e ademais é altamente efectiva na produción de extractos enriquecidos en antioxidantes. Esta técnica promove a penetración dos disolventes nos materiais celulares, mellorando a transferencia de materia e rompendo as paredes celulares vexetais, facilitando a liberación do contido celular. A selección do método de extracción e as condicións de operación son aspectos críticos que se discuten ao longo desta Tese de Doutoramento. Así mesmo, o disolvente utilizado para a extracción de compostos é un dos aspectos clave no rendemento da extracción. A auga é o disolvente máis seguro e empregado en extraccións industriais xa que ademais de ser económico respecta os principios da química verde, á conta dunha menor eficiencia de Resumo X extracción en comparación cos disolventes orgánicos. Demostrouse que as mesturas acuosas de metanol, etanol, acetona ou ácidos son disolventes máis eficaces para a extracción de florotaninos de algas pola polaridade dos mesmos e a súa afinidade por disolventes menos polares ca a auga. Con todo, estes disolventes son volátiles, inflamables e tóxicos. O uso da auga reduce as etapas posteriores de purificación e a xeración de residuos residuais. Deste xeito, nesta Tese Doutoral explorase principalmente o uso de auga como disolvente durante as extraccións de biopolímeros. O obxectivo principal desta Tese Doutoral é a obtención de biopolímeros con alta capacidade antioxidante e inhibición encimática, a partires da alga parda Ascophyllum nodosum para seren utilizados como aditivos funcionais e controladores do índice glucémico. Para cumprir cos obxectivos e metas propostas nesta Tese Doutoral, en primeiro lugar, revisouse a literatura actual sobre a extracción de florotaninos de alga Ascophyllum nodosum mediante extracción sólido–líquido convencional e asistida por ultrasóns. Revisáronse as variables principais empregadas na operación de extracción, coma o tipo de disolvente de extracción (auga-acetona, auga de mar e auga entre outros), as relacións de disolvente–alga, o tempo (t) e a temperatura (T), para avaliar o seu efecto tanto na cantidade de polifenois extraídos como na calidade destes (actividade antioxidante). Tamén se realizou unha minuciosa procura bibliografía sobre o uso de florotaninos (e polifenois) como aditivos alimentarios, así como do seu uso inhibidores encimáticos e a metodoloxía axeitada para a avaliación das súas capacidades inhibitorias. Os resultados indicaron que nos últimos 20 anos existe unha escasa información sobre o uso de polifenois de algas tanto como inhibidores encimáticos e como aditivos alimentarios. Outro problema observado foi o gran número de variables estudadas tanto na extracción como na validación dos florotaninos como inhibidores encimáticos. Durante a realización deste traballo empregáronse diferentes equipos tanto de laboratorio como escala planta piloto para levar a cabo distintas operacións como: secado, liofilización, moenda, peneirado, extracción, cromatografía e diálise, entre outras. Tamén se empregaron diversas técnicas experimentais para análises químicos (contido de: polifenois, carbohidratos, ácidos urónicos, proteínas, sales minerais, lípidos e humidade), bioactividade (actividade antioxidante determinada por ensaios de análises da capacidade antirradicalaria e/ou de doazón de electróns), inhibicións de reaccións encimáticas, análise de imaxe (microscopía electrónica Resumo XI de varrido ou SEM), métodos espectroscópicos: infravermellos por transformada de Fourier (FT–IR), resonancia magnética nuclear (NMR) e espectroscopia de masas por de desorción/ionización láser asistida por matriz e tempo de voo (MALDI–TOF–MS) que permitiron a obtención e análise dos resultados. A materia prima, a alga parda Ascophyllum nodosum, secouse por convección forzada con aire quente (temperatura de 50 ºC; humidade relativa de 30%, velocidade de aire de 2 m/s e densidade de carga de 2 kg/m2) durante 8,5 h ata que a humidade foi constante. Posteriormente, a alga seca moeuse e caracterizouse por medio de peneiras con distintas luces de malla, así como da súa capacidade para reter auga. As condicións empregadas no secado, moenda, empaquetado, selado a baleiro e conservación a 4 ºC baixo refrixeración foron axeitadas para manter as propiedades bioactivas das mostras de alga ao longo do período de realización desta Tese Doutoral. Ensaiáronse distintas condiciones experimentais para a extracción de florotaninos. Empregando un deseño experimental Box-Behnken avaliáronse os efectos da potencia de sonicación (de 70 a 90 W/cm2), tempo de sonicación (de 2 a 6 min) e relación líquido-sólido (de 20 a 40 gauga/galga) para obter mediante superficie de resposta as condición óptimas de extracción. Estas condicións foron utilizadas con outros disolventes coma auga salgada e acetona-auga (70/30 v/v). Simultaneamente, determináronse as cinéticas de extracción sen sonicación (de 2 a 60 min) empregando auga destilada e auga salgada como disolventes con fins comparativos para avaliación do efecto da sonicación. Todos os extractos caracterizáronse quimicamente determinando o seu contido en polifenois, carbohidratos, ácidos urónicos e as súas capacidades antioxidantes mediante os métodos de DPPH, ABTS, y FRAP. Os extractos con mellores propiedades foron purificados mediante cromatografía en xel e estes extractos purificados foron novamente caracterizados química, bioactivamente e a súa actividade inhibitoria fronte as ancimas dixestivas α–amilasa e α–glucosidasa. Nos extractos purificados avaliouse o efecto de diferentes tipos de oxidación (con convección natural e forzada de aire e con peróxido de hidróxeno cunha relación de 50% v/v co extracto) e a súa caracterización por tamaño despois da separación por diálise (de 2 a 20 kDa) para determinar as fraccións máis bioactiva. Así mesmo, estudiáronse as interaccións entre os polifenois das algas e o amidón de millo baixo diferentes condicións de mestura e procesos térmicos (ata 100 ºC) promovendo a xelatinización do amidón. Análises químicos, espectroscópicos e de análise de imaxe foron empregados para determinar o tipo de interacción moleculares e os cambios estruturais. Por Resumo XII último, co fin dunha valorización máis integral da alga, unha vez extraídos os polifenois, avaliouse a posibilidades de extraer por sonicación os alxinatos no refugallo sólido (secado a temperaturas < 90 ºC). As características do alxinato obtido determináronse mediante técnicas espectróscopicas (RMN y FT-IR) y viscosimétricas. A extracción acuosa e continúa asistida por ultrasóns aumentou todos os parámetros fitoquímicos dos extractos, especialmente o contido de polifenois (aprox. 1,4 veces máis alto) así como da súa actividade antioxidante (DPPH aumentou ata 2,1 veces) respecto a aqueles extractos obtidos sen sonicación. Dentro dos rangos de tempo de sonicación estudados (desde 2 a 15 min) non se produciron cambios significativos, evidenciando a alta capacidade da tecnoloxía do extracción asistida por ultrasóns para a extracción de compostos bioactivos das algas. As cinéticas de extracción dos compostos bioactivos das algas, polifenois, carbohidratos e acedos urónicos, modelizáronse satisfactoriamente mediante o emprego da ecuación proposta por Peleg. O pseudo–equilibrio alcanzouse despois de só 15 minutos independentemente do disolvente acuoso utilizado, auga destilada ou auga salgada. A auga salgada foi un disolvente adecuado co fin de obter extractos bioactivos de Ascophyllum nodosum. A modelización das cinéticas de extracción de Ascophyllum nodosum demostrou que o contido de florotaninos nos extractos dependeu principalmente da potencia de sonicación (SP) e a relación líquido–sólido (LS) e, en menor medida, do tempo de sonicación (tR). O máximo contido de florotaninos nos extractos, ademais do menor consumo específico de enerxía, acadouse nas seguintes condicións: SP de 90 W/cm2, LS de 20 g/g e tR de 2 min. Os modelos propostos permitiron establecer as condicións óptimas de extracción, que foron 15 min iniciais de SLE, axitando a mestura (170 rpm) e a continuación un curto período de operación (2 min). Estas condicións foron utilizadas ó longo da Tese Doutoral para a obtención dos diferentes extractos. Ademais o contido de polifenois nos extractos sempre foi satisfactoriamente relacionado coas diferentes actividades antioxidantes, medidas a traveso dos métodos de DPPH, ABTS e FRAP, evidenciando que a principal bioactividade dos extractos era debida o seu contido en florotaninos. Os extractos ricos en florotaninos purificáronse axeitadamente empregando cromatografía de filtración en xel de Amberlite XAD16 que permitiu a diminución do contido de carbohidratos dos extractos (ata 1,7 veces menor) sen cambios significativos do contido de florotaninos. Resumo XIII Como resultado, as capacidades antioxidantes e de inhibición encimática aumentaron polo menos 1,2 veces nos extractos purificados en comparación cos crús. Estas diferenzas atopadas entre os extractos asociáronse coa existencia de complexos florotanino–carbohidrato que coa purificación foron parcialmente eliminados. Doutra banda, tamén se concluíu que os extractos obtidos mediante extracción asistida por ultrasóns contiñan pequenas cantidades de florotaninos oxidados, principalmente quinonas, que afectaban á capacidade antioxidante e de inhibición encimática dos mesmos, pero que a purificación eliminaba parte deles. En consecuencia, a purificación é un tratamento altamente eficaz para aumentar a bioactividade xunto coa capacidade de inhibición encimática dos extractos da alga Ascophyllum nodosum. A oxidación dos extractos purificados ricos en florotaninos estudouse a través de tratamentos de distinta intensidade: por unha parte unha oxidación con convección natural de aire, aplicando aireación forzada e pola adición de peróxido de hidróxeno. Todos os tratamentos foron levados a cabo deixando a mostra 120h a temperatura ambiente. A única oxidación que significativamente afectou ás bioactividades medidas dos extractos foi o tratamento con peróxido de hidróxeno. Estes resultados puxeron en evidencia as altas capacidades antioxidantes dos extractos e a súa resistencia fronte ó emprego de condicións severas de oxidación. Paralelamente, analizáronse as diferenzas de bioactividade dos florotaninos en función do seu tamaño molecular, onde se atopou que a principal fracción bioactiva estaba no rango de 10 a 20 kDa, con contido de polifenois e valores de DPPH superiores ao 50% respecto de extractos non dializados. En canto aos extractos con menor tamaño de florotaninos (< 2 kDa) obtivéronse baixa bioactividades, que mesmo mostraron resultados similares aos extractos oxidados. Estes resultados foron corroborados empregando técnicas NMR, FT–IR e MALDI–TOF–MS. De feito, a técnica de MALDI–TOF–MS permitiu a identificación dos graos de polimerización (número de unidades de floroglucinol ou PGUs) dos florotaninos do alga (4–16 PGUs). Tamén se obtiveron os patróns de fragmentación dos florotaninos de Ascophyllum nodosum e a presenza de diversas sinais que evidenciaban a presenza de diferentes grupos de florotaninos. Cando se estudaron as interaccións e mecanismos de entrecruzamento de sistemas amidón (de millo) e fariña de alga concluíuse que a composición e as transicións de fase (xelatinización e xelificación do amidón) promovidas polas operacións de quecemento modificaban tanto as características fitoquímicas, coma a natureza e a cantidade das interaccións formadas, o cal á Resumo XIV súa vez redundou en cambios das morfoloxías transversais das mostras. O amidón mostrou diferentes retencións de polifenois dependendo do seu estado (granular ou xel). As interaccións entre os florotaninos e o amidón debéronse á fisisorción coma mecanismo principal cando as algas se puxeron en contacto con amidón. Así mesmo, a cantidade adsorbida dependeu do área superficial dispoñible, sendo superior nas mostras de xel de amidón. Con todo, a retención de polifenois aumentou notoriamente cando a xelatinización do amidón se realizou en presenza da fariña de alga, porque os florotaninos se adsorberon fisicamente na superficie do xel de amidón e tamén quedaron atrapados dentro das paredes do mesmo. Os procedementos empregados serviron para cuantificar de forma sinxela o modo de retención de moléculas bioactivas das algas en alimentos amiláceos. Os extractos acuosos de algas demostraron ser inhibidores encimáticos altamente efectivos contra os encimas dixestivos α–amilasa e α–glucosidasa, e, especialmente, con esta última. Probáronse diferentes sistemas de mesturado onde se variou a orde de adición do substrato, encima e inhibidor. Os extractos de alga foron inhibidores máis efectivos cando foron engadidos directamente ó encima antes que ó substrato. Doutra banda, a mestura de extractos cos substratos (amidón nativo ou xel, ou maltosa) antes da reacción encimática reduciu o efecto inhibitorio dos mesmos, e, particularmente na inhibición da α–amilasa. As diferenzas observadas nas capacidades de inhibición dos extractos explicáronse pola presenza de complexos alxinato–polifenois e florotaninos en estado oxidado. Os resultados obtidos comparáronse satisfactoriamente coa acarbosa, inhibidor comercial utilizado no tratamento da diabetes. A extracción secuencial de florotaninos mediante tecnoloxía asistida por ultrasón seguido da extracción de alxinatos do refugallo sólido permitiu obter dous produtos de valor comercial. Na extracción do alxinato, estudiouse o efecto da presenza dunha etapa de secado e da súa temperatura, sobre as características fisicoquímicas do alxinato de sodio. Obtivéronse alxinatos con diversas lonxitudes media de bloque (NG>1 dende 1,97 ata 2,15) e tamaño molecular viscosimétrico (Mv, desde 133,3 ata 257,3 kDa). A variabilidade das características obtidas segundo as condicións de secado supón que modulando as condicións pódense obter alxinatos á carta, é dicir coas propiedades desexadas en función do seu uso. O aproveitamento integral das algas mariñas implica claras avantaxes económicas e ambientais. De feito, este concepto de refinería baseada en algas empeza a ser unha realidade e seguindo os principios da química verde. Resumo XV Finalmente como perspectivas de futuro, proponse a adición destes florotaninos (como aditivos bioactivos) e alxinatos (como substitutos do glute) á matrices amiláceas e estudar o seu efecto sobre as características nutricionais e sensoriais, propiedades funcionais e a súa incidencia nos índices glicémicos. Outro estudo proposto sería a análise in vivo de florotaninos para determinar doses efectivas e mesmo efectos a longo prazo. Glossary XXIII YP Polyphenol sorption yield ρ Load density µgTE/mgFD Micrograms of trolox equivalents per milligram of freeze–dried extract µMTE Micromolar of Trolox equivalents 1H–NMR Hydrogen NMR 50D Alginate from UAE residue dried at 50 ºC 90D Alginate from UAE residue dried at 90 ºC Section 7 ABTS ABTS method A–region Defined between 10 and 20 min of retention time B–region Defined between 28 and 40 min of retention time CHOs Total carbohydrate content C–region Defined between 45 and 70 min of retention time D Double distilled water D20 Extraction at 20 gW/gAF with D–solvent D30 Extraction at 30 gW/gAF with D–solvent D40 Extraction at 40 gW/gAF with D–solvent DPPH DPPH method Eq. Equation FRAP Electron donor capacity gPE/L Grams of phloroglucinol equivalents per liter gGE/L Grams of glucose equivalents per liter gW/gAF Grams of water per grams of A. nodosum flour K1 related to initial extraction rate (min L/g) K2 related to equilibrium content (L/g) LS Liquid–solid LS20 Liquid–solid ratio of 20 gW/gAF LS30 Liquid–solid ratio of 30 gW/gAF LS40 Liquid–solid ratio of 40 gW/gAF mgPE/L Miligramns of phlroglucinol equivalents per liter mMTE Milimolar of Trolox equivalents RMSE Root mean squared error RP–HPLC Reverse phase high preasure liquid chromatography S Saline water S20 Extraction at 20 gW/gAF with S–solvent S30 Extraction at 30 gW/gAF with S–solvent S40 Extraction at 40 gW/gAF with S–solvent SLE Solid–liquid extraction SOL Solvent TPC Total polyphenol content UA Uronic acid content UAE Ultrasound–assisted extraction UAE20–90 Sonicated extract of LS = 20 gW/gAF and SP = 90 W/cm2 UAE20–80 Sonicated extract of LS = 20 gW/gAF and SP = 80 W/cm2 UAE20–70 Sonicated extract of LS = 20 gW/gAF and SP = 70 W/cm2 UAE30–90 Sonicated extract of LS = 30 gW/gAF and SP = 90 W/cm2 UAE40–90 Sonicated extract of LS = 40 gW/gAF and SP = 90 W/cm2 UV Ultraviolet X0 Initial content (assumed zero) Xt Content at time t (min) µMTE Micromolar of Trolox equivalents Glossary XXIV Section 8 ABTS ABTS method BB Box–Behnken CHOs Total carbohydrate content DPPH DPPH method Eq. Equation FRAP Electron donor capacity gGAE/L Grams of galic acid equivalents per liter gGE/L Grams of glucose equivalents per liter gPE/L Grams of phloroglucinol equivalents per liter gW/gAF Grams of water per grams of A. nodosum flour IC50 Concentration to achieve 50% of decayment LS Liquid–solid mgPE/L Miligramns of phlroglucinol equivalents per liter mgTE/mgDE Miligrams of Tolox equivalents per miligram of dried extract mmolDPPH Milimols of DPPH compound mMTE Milimolar of Trolox equivalents p Significance RSM Response surface model rt Room temperature (19 ± 1 ºC) S Saline water SP Sonication power tR Time residence TPC Total polyphenol content UA Uronic acid content UAE Ultrasound–assisted extraction UV Ultraviolet µMTE Micromolar of Trolox equivalents * Significance level p < 0.05 ** Significance level p < 0.01 ** Significance level p < 0.001 Section 9 DPPH 2,2–diphenyl–1–picrylhydrazyl d.b Dry basis EA UAE carried with 70% (v/v) acetone:water EW UAE using double–distilled water FD freeze–dried extracts FT–IR Fourier transform infrared GF Gluten–Free gPE/L grams of phloroglucinol equivalents per litre GRAS Generally Recognised As Safe gS/gAF Grams of solvent per gram of AF gTE/L Grams of trolox equivalents per litre GAE Galic acid equivalents GE Glucose equivalents 1H–NMR Hydrogen nuclear magnetic resonance LS Liquid–solid MALDI Matrix–Assisted Laser Desorption Ionization TOF Time–Of–Flight MS Mass Spectrometry OH Carboxylic group or alcohol group Peak 1 Peak area from chromatograms at 16.5 min of retention time; Peak 2 Peak area from chromatograms at 53.0 min of retention time; Peak 3 Peak area from chromatograms at 90.5 min of retention time PBS Podium phosphate buffer Glossary XXV PD20 PW dialysated throughout 20 kDa PD10 PW dialysated throughout 10 kDa PD3 PW fraction dialysated throughout 3.5 kDa PD2 PW dialysated throughout 2 kDa PE Phloroglucinol equivalents POA PW air oxidated (120 h rt) PON PW naturally oxidated (120 h rt) POP PW chemically oxidated with peroxide (120 h rt) PW EW purified extract RP–HPLC Reverse phase high performance liquid chromatography SLE Solid–liquid extraction spp. Species SP Sonication power tR Time residence T Temperature V volume Section 10 ABTS ABTS method CGL Gelatinized starch in presence of AF CHOs Total carbohydrate content CS Corn starch DPPH DPPH method Eq. Equation FRAP Electron donor capacity FT–IR Fourier transformed infrared GRAS Generally recognised as safe gW/gAF Grams of water per grams of AF gW/gAF Grams of water per grams of CS GL Gelatinized starch mixed with AF IC50 Concentration to achieve 50% of decayment LS Liquid–solid mgPE/gCS Miligramns of phlroglucinol equivalents per grams of CS mgPE/L Miligramns of phlroglucinol equivalents per liter NT Granular (Native) starch mixed with AF q TPC sorption value RMSE Root mean squared error SEM Scaning electron microscopy TPCAF Total polyphenol content of AF sample TPCCS Total polyphenol content of CS sample TPCAF–CS Total polyphenol content of AF–CS sample YP Polyphenol sorption yield µMTE Micromolar of Trolox equivalents Section 11 A1 Method 1 (Enzyme + Extract) of α–amylase inhibition A2 Method 2 (Sustrate + Extract) of α–amylase inhibition A3 Method 3 (Starch gelatinized with Extract) of α–amylase inhibition A–region Region comprised between 5.20 and 5.40 ppm B–region Region comprised between 5.75 and 6.40 ppm CHOs Total carbohydrate content C–region Region comprised between 6.40 and 6.55 ppm d.b Dry basis E70 A. nodosum extract sonicated at 70 W/cm2 Glossary XXVI E80 A. nodosum extract sonicated at 80 W/cm2 E90 A. nodosum extract sonicated at 90 W/cm2 FRAP Electron donor capacity FT–IR Fourier transformed infrared G1 Method 1 (Enzyme + Extract) of α–glucosidase inhibition G2 Method 2 (Sustrate + Extract) of α–glucosidase inhibition IC50 Minimal concentration value to achieve 50% of inhibition M1 Method 1 (Enzyme + Extract) inhibition M2 Method 2 (Sustrate + Extract) inhibition M3 Method 3 (Starch gelatinized with Extract) inhibition mgFD Miligrams of freeze–dried extract mgGE Miligrams of glucose equivalents mgPE Miligrams of phloroglucinol equivalents mMTE/mgFD Miliolar of trolox equivalents per miligram of freeze dried extract P90 A. nodosum extract sonicated at 90 W/cm2 and Amberlite purified RP–HPLC Reverse phase high preasure liquid chromatography SEM Scaning electron microscopy TPC Total polyphenol content 1H–NMR Hydrogen nuclear magnetic resonance µgTE/mgFD Micrograms of trolox equivalents per miligram of freeze dried extract Section 12 AF Ascophyllum nodosum flour A. nodosum Ascophyllum nodosum ASTM American Society for Testing and Materials CHNS Elemental analysis CS Comercial aglinate from Sigma FM Monad of mannuronate FG Monad of guluronate FGG Homopolymeric diad of guluronate–guluronate FGGG Homopolymeric triad of guluronate–guluronate–guluronate FGGM Homopolymeric triad of guluronate–guluronate– mannuronate FGM “or” FMG Heteropolymeric diad of guluronate– mannuronate FMGM Heteropolymeric triad of mannuronate– guluronate–mannuronate FMM Homopolymeric diad of mannuronate–mannuronate FMMM Homopolymeric triad of mannuronate–mannuronate–mannuronate FT–IR Fourier transformed infrared G Guluronate M Mannuronate M/G ratio Glucoronate – mannuronate ratio Mv Molecular viscosimetric weight ND Alginate from UAE residue not dried NG>1 average block length UAE Ultrasound–assisted extraction 1H–NMR Hydrogen nuclear magnetic resonance 50D Alginate from UAE residue dried at 50 ºC 90D Alginate from UAE residue dried at 50 ºC XXVII CONTENT XXVIII Content XXIX Acknowledgements .............................................................................................................. I Resumo ............................................................................................................................... V Glossary ........................................................................................................................ XVII Content ....................................................................................................................... XXVII 1. Introduction ................................................................................................................ 35 1.1. Generalities ............................................................................................................. 37 1.1.1. Green algae ...................................................................................................... 38 1.1.2. Red algae ......................................................................................................... 38 1.2. Brown algae ............................................................................................................ 38 1.2.1. Carbohydrates .................................................................................................. 39 1.2.2. Phlorotannins ................................................................................................... 40 1.2.3. Alginates .......................................................................................................... 43 1.3. Ascophyllum nodosum seaweed ............................................................................. 44 1.4. Market and applications ......................................................................................... 46 1.5. Antioxidant capacities ............................................................................................ 49 1.6. Antidiabetic capacities ............................................................................................ 55 1.7. State of art............................................................................................................... 60 1.7.1. Phlorotannins extraction .................................................................................. 60 1.7.2. Seaweed biopolymers in food ......................................................................... 66 1.7.3. Phlorotannins as enzyme inhibitors ................................................................. 67 2. Objectives & Hypothesis ........................................................................................... 77 3. Theoretical fundamentals ........................................................................................... 81 3.1. Extraction ............................................................................................................... 83 3.1.1. Generalities ...................................................................................................... 83 3.1.2. Ultrasound–assisted extraction ........................................................................ 86 Content 3.1.3. Fractionation–isolation of seaweed extracts ................................................... 88 3.2. Characterization of seaweed biopolymers ............................................................. 89 3.2.1. Antioxidant activity determinations ................................................................ 90 3.2.2. Chromatographic studies ................................................................................ 95 3.2.3. Fourier transformed infrared spectroscopy (FT–IR) ....................................... 97 3.2.4. Nuclear magnetic resonance (NMR) .............................................................. 98 3.2.5. Optical analysis ............................................................................................... 98 3.2.6. X–ray diffraction (XRD) ................................................................................. 99 3.3. Seaweed biopolymers in food .............................................................................. 100 3.3.1. Generalities ................................................................................................... 100 3.3.2. Starchy–based systems .................................................................................. 102 3.3.3. Phlorotannins as enzyme inhibitors .............................................................. 105 4. Experimental & Methodology ................................................................................. 107 4.1. Materials .............................................................................................................. 109 4.2. Drying & Milling & Sieving ................................................................................ 109 4.3. Particle size and water retention capacity ............................................................ 111 4.4. Biopolymers extractions ...................................................................................... 112 4.4.1. Solid–liquid extraction .................................................................................. 113 4.4.2. Ultrasound–assisted extraction ..................................................................... 114 4.4.3. Alginate extraction ........................................................................................ 117 4.4.4. Extraction yields ........................................................................................... 118 4.5. Extracts purification ............................................................................................. 118 4.6. Raw and prurified extracts characterization ........................................................ 119 4.6.1. Total polyphenol content .............................................................................. 120 4.6.2. Total carbohydrate content ........................................................................... 122 Content XXXI 4.6.3. Uronic acid content........................................................................................ 124 4.6.4. DPPH scavenging activity ............................................................................. 126 4.6.5. ABTS scavenging activity ............................................................................. 128 4.6.6. Electron donor capacity ................................................................................. 130 4.6.7. Other analyses ............................................................................................... 132 4.7. Extracts oxidation treatments ............................................................................... 132 4.8. Extracts cut–off dialysis ....................................................................................... 133 4.9. Extracts chromatography ...................................................................................... 134 4.10. Extracts fragmentation patterns .......................................................................... 135 4.11. Fourier transform infrared spectroscopy ............................................................ 136 4.12. Average viscosimetric molecular weight ........................................................... 136 4.13. Nuclear magnetic resonance ............................................................................... 137 4.13.1. Sodium alginate ........................................................................................... 137 4.13.2. Phlorotannin-eriched extracts ...................................................................... 139 4.14. Seaweed–starch interactions ............................................................................... 139 4.15. Inhibitory capacities of extracts .......................................................................... 141 4.16. Scanning electron microscopy ............................................................................ 144 4.17. Statistical analysis .............................................................................................. 144 5. Results & Discussion ............................................................................................... 145 6. Ascophyllum nodosum flour obtention .................................................................... 149 6.0. Generalities ........................................................................................................... 151 6.1. Assayed systems ................................................................................................... 153 6.2. Drying kinetics ..................................................................................................... 154 6.3. Particle size distribution ....................................................................................... 154 6.4. Water retention capacity ....................................................................................... 155 Content Highlights .................................................................................................................... 155 7. Extraction solvent and method assessment ............................................................. 157 7.1. Generalities .......................................................................................................... 159 7.2. Assayed systems .................................................................................................. 160 7.3. Solid–Liquid Extraction (SLE) ............................................................................ 162 7.3.1. Solid–Liquid extraction kinetics modelling .................................................. 165 7.4. Ultrasound–assisted extraction (UAE) ................................................................ 165 7.4.1. Analysis of composition and activity ratios .................................................. 171 7.5. Chromatography profiling: SLE vs UAE ............................................................. 175 7.5.1. Relationship HPLC areas–antioxidant capacities ......................................... 178 Highlights .................................................................................................................... 180 8. Ultrasound‑assisted extraction optimization ........................................................... 181 8.1. Generalities .......................................................................................................... 183 8.2. Assayed systems .................................................................................................. 185 8.3. Extracts chemical characterization ...................................................................... 187 8.4. Response surface modelling ................................................................................ 189 8.5. Analysis of TPC ratios ......................................................................................... 192 8.6. Antioxidant activity of extracts ............................................................................ 195 Highlights .................................................................................................................... 197 9. Influence of oxidation and dialysis on bioactivity .................................................. 199 9.1. Generalities .......................................................................................................... 201 9.2. Assayed systems .................................................................................................. 202 9.3. Bioactivity characterization ................................................................................. 204 9.4 Chromatographic characterization ........................................................................ 209 9.5. Spectroscopic characterization ............................................................................. 212 1. Introduction Page 39 of 353 Seaweeds biochemical composition is affected by botanical sources, season, geographic origin, contaminants, and their natural individual variability (Tabassum et al., 2016). Brown seaweeds are rich in nutraceuticals such as polysaccharides, polyphenols and some minerals (sodium, calcium, magnesium, potassium, chloride, sulphates, phosphorous, and fluoride). Brown seaweeds show low content of lipids (< 5% d.b) and variable content of proteins from 5 up to 24% d.b (Gómez–Ordóñez & Rupérez, 2011; Kilinç et al., 2013; Tabassum et al., 2016). These bioactive compounds have recently gained notorious relevance by their beneficial health properties that will be seen in Sections 1.4, 1.5, 1.6 and 1.7. 1.2.1. CARBOHYDRATES Seaweeds can be classified by their structural and reserve polysaccharide composition. Seaweed polysaccharides are polymeric molecules linked by glycosidic bonds, that are receiving increasing attention, together with phenolic compounds, due to their bio–functional (i.e., health benefits and biological activities) and physicochemical (e.g., emulsifiers, thickeners, or gelling agents) features (López–Hortas et al., 2021). Polysaccharide features depend on their molecular weight and conformation (Hahn et al., 2012). Hydrocolloids from seaweeds have extended industrial interest (Kim, 2011) and are used as thickener, gel formation or stabilizer ingredients. Seaweeds as a source of these hydrocolloids dates back to 1658, when the gelling properties of agar, from red seaweeds, were first discovered in Japan (Kilinç et al., 2013). Some examples of red seaweeds’ hydrocolloids are, agar, carrageenan and porphyrin, while the main hydrocolloids of brown seaweeds are laminarins, fucoidans and alginates (Holdt & Kraan, 2011). Laminarins are the main reserve of polysaccharides in brown algae, and they could be found in high quantities in Laminaria spp., Undaria spp.and Fucus spp. Laminarins are mainly dietary fiber, however, have shown promising bioactive properties (Kadam et al., 2015a). Fucoidans are sulphated polysaccharides found in the cell wall and are mainly be obtained from Cladosiphon okamuranus (ozuku) and Laminaria japonica (kombu) edible seaweeds (Gómez– Ordóñez & Rupérez, 2011). Researchers have reported promising anti–tumoral results using seaweed fucoidans (An et al., 2022). 1. Introduction Page 40 of 353 1.2.2. PHLOROTANNINS Polyphenols are secondary metabolites from plants (terrestrial and marine) and lichens (Koivikko et al., 2007). Generally, they act as cell walls structural components and shelters against environmental stress (Tierney et al., 2013a). Polyphenols are aromatic compounds with more than one hydroxylic groups (Balboa et al., 2013). Polyphenols could be divided into three categories, phenolic acids, flavonoids and non–flavonoids (Figure 1.3). Phlorotannins have achieved notorious research, industrial and medical interest due to their bioactive capacities (Figure 1.2) since have showed antioxidant, anticoagulant, antithrombotic, antidiabetic, enzymatic regulatory, bactericidal, antitumoral, anti–inflammatory, antihypertensive, antiviral, among others, properties. Besides, they have shown promising results in Alzheimer, Parkinson, and arthritis treatment (Li et al., 2011; Wang et al., 2012; Balboa et al., 2013; Harnedy et al., 2013; Kumar et al., 2020, Tavakoli et al., 2021; Cebrián– Lloret et al., 2022). Figure 1.2. Phlorotannins bioactive capacities. nti allergic nti microbial U radiations shelter euro protection nti tumor nti inflammatory 1. Introduction Page 41 of 353 Seaweeds are gaining recognition as a source of bioactive compounds, suitable to be used as health promotors and disease preventors. Phlorotannins are a group of tannins that only can be obtained from brown seaweeds (Figure 1.3) and have recently gained such a huge relevance due to their bioactive properties together with the low time– and economic– consumption that suppose the seaweed growth. Brown algae have higher contents of these compounds, with notorious content in Fucus vesiculosus and Ascophyllum nodosum species (Gómez–Ordóñez & Rupérez, 2011). Figure 1.3. Polyphenol’s classification. Phlorotannins are produced entirely by polymerization of the phloroglucinol molecule in the polyketide pathway reaction (Koivikko et al., 2007) and stored in physodes and/or cell–wall (Figure 1.4) forming complexes (Agregán et al., 2017). Phlorotannins content of seaweeds depends on environmental conditions, such as tides, salinity, light availability, UV radiation and herbivory intensity (Tabassum et al. 2016). Phlorotannins have structural roles forming part of cell walls (Figure 1.4) together with alginic molecules (Koivikko et al. 2005). These compounds are hydrophilic components with a wide range of molecular sizes ranging between 126 and 650 kDa (Ragan & Glombitza, 1986). ydroxycinnamic acids ydroxybenzoic acids Stilbenes ignans annins Ec ols Fuhalols Fucophlorethols Phlorethols Fucols armalols Flavones Flavonols Flavanones Flavanols Isoflavones nthocyanins 1. Introduction Page 42 of 353 Figure 1.4. Proposed matrix of brown seaweed cell–walls. Phlorotannins are divided into six different classes, eckols, fuhalols, fucophlorethols, phlorethols, fucols and carmalols (Figure 1.5), based upon variations in their assemblage, and distribution of hydroxyl groups (Li et al., 2011). Phlorethols and fuhalols present aryl–ether linkages, fucols aryl–aryl bonds, fucophlorethols a mixture of ether and phenyl bonds. Eckols present a 1,4–dibenzodioxin unit (Thomas & Kim, 2011). Carmalols present a 4–dibenzodioxin unit at the third and seventh position (Kumar et al., 2022). Eckols differ from carmalols by their lower molecular weight and by the presence of a OH group substituted at fourth carbon (Li et al., 2009). And fuhalols differ from phlorethols by their regular sequence of para– and ortho– ether bonds, by the presence of additional OH groups in every third ring and by the lack of one or more OH groups in the whole molecule (Ford et al 2019). ellulose microfibrils ylo fuco glucans omofucans Glycoproteic lin ages lginate networ ylo fuco glycoronans Phlorotannin complexes 1. Introduction Page 43 of 353 Figure 1.5. Phlorotannin main groups, all derived from phloroglucinol polyketide pathway reaction. 1.2.3. ALGINATES Alginate is a polyuronan isolated from some seaweeds (McHugh, 2003) or produced by some bacterial species (Govan et al., 1981) and their content can reach 55% of dry basis (d.b) in some species (Pereira, 2011). It is found in around all brown seaweeds species, but its exploitation is mainly focused on 38 species. Worldwide production of alginate comes from following species: Ascophyllum nodosum (12–16% d.b), Durvillaea potatorum (45–55% d.b), Ecklonia arborea (24–28% d.b), Laminaria hyperborea (14–21% d.b), Laminaria digitata (16– 36% d.b), Lessonia trabeculata (13–29% d.b), Macrocystis pyrifera (18–45% d.b) and Saccharina latissima (16–34% d.b) (Peteiro et al., 2018). They can reach up to 40–47% of the seaweed dried weight, varying between seasons, reaching maximums in spring (Tabassum et al., 2016). Alginate isoforms have shown antihypertensive and anti–inflammatory activities, as well as the capacity to reduce cholesterol levels (Gómez–Ordóñez & Rupérez 2011). 1. Introduction Page 44 of 353 Alginate is a structural component of the multicomponent algae matrix cell wall as can be seen in Figure 1.4 according to model proposed by Lobban & Harrison (1994). Alginates are responsible of flexibility and mechanical resistance of seaweeds (Braccini, et al., 1999; Stephen et al., 2006). In fact, alginate yield extraction is improved when seaweeds are harvested from turbulent waters. Alginate also plays a role into seaweed ion–exchange equilibrium retarding desiccation when seaweeds are air–exposed during low tide periods (Donati & Paoletti, 2009). Alginates have been widespread industrial used by their thickeners, emulsifiers, and stabilizers properties. Food industry adds alginate in sauces, jams, marmalades or mayonnaise (Jensen, 1993; Mancini et al., 2002) to improve their organoleptic qualities and the final product appealing (Paraskevopoulou et al., 2006). It is also used in ice cream production since inhibit the formation of large ice crystals producing smoother textures (Kilinç et al., 2013). It is used in dye industry, biomaterials dressing, medical and pharmaceutical sectors for microencapsulation, dental impressions, drug delivery vectors or anti–reflux therapies (Ertesvåg, et al., 1998; Ingram et al., 1998; Skaugrud et al., 1999; Mandel et al., 2000; Uludag et al., 2000). 1.3. ASCOPHYLLUM NODOSUM SEAWEED Ascophyllum nodosum (Linnaeus) Le Jolis (Figure 1.6), also known as rockweed or A. nodosum, is one of the most studied brown edible seaweeds containing 14% (polyphenol) and 28 % (d.b) of fucoidan content (Holdt & Kraan 2011; Liu & Gu, 2012; Kadam et al., 2015c; Catarino et al., 2019). Thus, together with their availability along Galicia’s coasts and the experience of the group GI–1618 with this alga (Arufe, 2017), A. nodosum was selected as main source of bioactive compounds, mainly phlorotannins and sodium alginates of the current Thesis. 1. Introduction Page 45 of 353 Figure 1.6. Ascophyllum nodosum (Linnaeus) Le Jolis seaweed (A), fronds (B) and flotation vessels (C) from Algaebase database. A. nodosum is a dark brown seaweed (Figure 1.6A) common in the North–Western coast of Europe (from Norway to Portugal), and the North–Eastern coast of North America. A. nodosum seaweed has long olive–brown fronds (Figure 1.6B) with large egg–shaped airbladders (Figure 1.6C). The fronds can reach 2 m in length and are attached by a holdfast to rocks and boulders. A. nodosum seaweed is used as a raw material for industrial production of fucoidans, alginates, ascophyllan and laminarins and as a fertilizer in the agrochemical industry (Khan et al., 2012). This seaweed is found on the northwest coast of Europe and northeastern of America (Bertness et al., 2014), Figure 1.7. Figure 1.7. Geographical distribution of A. nodosum seaweed from GBIF database, (2022). 1. Introduction Page 46 of 353 The chemical composition of this seaweed showed in Figure 1.8 of average moisture, protein, ash (mineral content), lipid, phenolic and carbohydrate content (Yuan & Macquarrie (2015). Figure 1.8. Average composition of A. nodosum brown seaweed. 1.4. MARKET AND APPLICATIONS Macroalgae, also called seaweeds, are becoming an interesting and demanded crop due to their fast growth (Kerrison et al., 2015; Wood et al., 2017) and high content in bioactive compounds. Marine organisms comprising around half of the total global biodiversity, then oceans offer a prominent resource for novel compounds (Aneiros & Garateix, 2004; Barrow & Shahidi, 2007). The benefits of macroalgae as a source of novel bioactive products are being globally revealed by the scientific community with the increasing interest on their different biological activities and the consumers are getting more attracted to marine algae–derived foods (Kim & Wijesekara, 2010; Wijesekara et al., 2010). Seaweeds provide promising and exclusive compounds, such as bioactive phenolic compounds (Isaza et al., 2013; Belanche et al., 2016), unsaturated fatty acids, fucoidans, alginate and biopolymers (Schmid et al., 2014; Angell et al., 2016) with notorious features (Ford et al., 2019; Gimpel et al., 2015a,b) and usages (Figure 1.9). ther carbohydrates 3 .5 (d.b) shes 21.5 (d.b) ipids 3.5 (d.b) thers 15. (d.b) Proteins . (d.b) 1. Introduction Page 47 of 353 Figure 1.9. Industrial potential of seaweeds. Bioactive compounds of seaweeds with relevant health benefits (Cumashi et al., 2007;) are exploited by several industrial sectors such as: biomedical (Holdt & Kraan, 2011; Valderrama, 2012), feedstocks (Craigie, 2011), biofuel production (Graham et al., 2009), wastewater treatment (Gómez–Ordóñez & Rupérez, 2011; Lawton, 2017) and food industry (Leandro et al., 2020). Hence, this rising global consumption of seaweeds and their derived products is generating notorious demand with socio–economic benefits, that can generate higher revenue streams through the processing of high value products (Ford et al., 2019), increasing up to US$ 26 million by 2025 (Ferdouse et al., 2018) that according to FAO (2018) trends to increase up to 10% in the coming years. Biomedical (medical, pharmaceutical and cosmetical): The number of cosmetic products that include seaweed compounds are increasing in recent years. It is common to see product labels with "marine extract", "extract of algae", "seaweeds extract" or similar. For example, alginate increases skin moisture retention properties of some lotions (Wijesinghe & Jeon, 2011). Seaweed–derived pastes are commonly used in thalassotherapy together with hydrotherapy to partially relief rheumatism and osteoporosis problems (Kilinç et al., 2013). arbohydrates Polyphenols Proteins Minerals, Pigments itamins ipids nti inflammatory ntithrombotic 5 nti aging Enzymatic regulatory ntitumoral 1 ntioxidant 3 actericidal 2 ntidiabetic nticoagulant 1 rthritis treatment 11 ntiviral 12 ntihypertensive thers iomedical Medical Pharmaceutical osmetical Feedstoc s griculture Fertilizers ivestoc Energy astewater treatment Food 1. Introduction Page 48 of 353 Seaweeds can provide antiaging, photoprotective, moisturizing, antioxidant, anti–melanogenic, anti–allergic, anti–inflammatory, antimicrobial, anti–acne, anti–wrinkling, whitening, low cytotoxicity and allergen content (López–Hortas et al., 2021). The use of seaweed as food supplement for the control of the glycemic index has been also investigated (Apostolidis & Lee, 2010; Kim et al 2014) and will be deeply explored in this Thesis. Feedstocks (agriculture, fertilizers and livestock): Seaweeds have been used as fertilizer since 19th century by coastal dwellers. Seaweeds enhance soil moisture retention, and their mineral content is source of trace essential elements (McHugh, 2003). Seaweeds have fast growing rates, do not require costs associated to planting, fertilization or irrigation (Ketheesan & Nirmalakhandan, 2011; Rhoades, 2012). Hence, seaweed supplemented animal feeds offer nutritionally renewable feedstocks with several positive health features for livestock (Evans & Critchley 2014). For instance, Huang et al. (2018) and Silva et al. (2018) demonstrated that pigs fed with diets supplemented with seaweeds showed a lower need of antibiotics. Additionally, Belanche et al., 2016 demonstrated that ruminants fed with seaweed diets reduced their methane emissions. Energy production: Direct combustion of algae biomass is a traditional method of generating heat or steam. However, it is not a suitable method since produce emissions together with a low efficient energy production (Milledge & Harvey, 2018). Seaweeds could be used to produce syngas throughout a gasification reaction however it is a process that requires high temperatures (Rauch et al., 2014). The production of biofuel from seaweeds can be carried out in presence of catalysts and hydrogen at high pressure and lower temperatures with hydrothermal liquefaction process (Milledge et al., 2014). Wastewater treatment: Some compounds from seaweeds have demonstrated to be efficient chelators to remove hazardous pollutants as heavy metal from industrial downstreaming (Gómez–Ordóñez & Rupérez, 2011). Food industry: Use of seaweeds as food has strong roots in Asian countries such as China, Japan, and Korea, being notoriously less consumed in occidental countries (Ali et al., 2000). Seaweeds are considered the food supplement for 21st century due to their high vitamins, minerals and dietary fibers content together with a low caloric content (Gómez–Ordóñez & Rupérez, 2011; Kilinç et al., 2013). Thus, seaweeds are being studied to be employed as food additives (Zhang et al., 2018a), to replace chemical 1. Introduction Page 55 of 353 1.6. ANTIDIABETIC CAPACITIES Celiac disease is the intolerance to gluten protein present in grains such as wheat, barley, rye and oatmeal. Celiac disease is an immune–based disease that cause the mucosal inflammation of the small intestine altering the nutrient absorption and subsequently other problems as growth deficit, anemia, vitamin deficiencies, thyroid diseases, mental disorders, fatigue and/or infertility (Patterson et al., 2009; Carstensen et al., 2014). First of celiac disease concepts were reported by Arateus of Capadocia (II century, BC) who named patients with intestinal problems as koliakos (celiacs’) that means “those who suffer from intestine”. However, it was around 1900 when Dr. Willem Karel Dicke discovered that gluten was the origin of these intestinal problems and designed the first gluten–free (GF) diet (Stoop, 1991). The estimated prevalence of the celiac disease is around 1% and it is expected that it increases (Singh et al., 2018; Cozzi et al., 2022). Celiac disease shows a variety of clinical manifestations. The incidence and different pathologies could be explained by the celiac iceberg (Figure 1.11). The well–diagnosed patients comprise the visible part of the iceberg and represent around 10% of the whole group. The last group are the silent cases, around of 75% of incidence, that suffer un–diagnosticated intestinal mucosa disorders (Maki & Collin, 1997). Finally, there is a latent group with predisposition to suffer this disease. This group is formed by childhood diagnosed patients that could recover themselves after the onset of the GF diet and were maintained in subclinical state and/or populations without previous reported gluten problems that end up developing the disease spontaneously (Nenna et al., 2013). Gluten is an amorphous protein, that represents 80% of wheat protein content. It is the responsible for the elasticity and spongy consistency of average bakery products. The unique suitable treatment for celiac disease is a GF diet during the patient's entire life. This causes a clinical and functional digestive normalization permitting the reparation of intestinal vellosities injuries (Gallagher et al., 2004). Celiac disease and their treatment in recent years, is a trending research, industrial and economical interest. The increasing consumption is correlated with the increasing incidence of gluten related disorders, and the population misconception of these products’ beneficial healthy properties. 1. Introduction Page 56 of 353 Figure 1.11. Diagram of celiac disease iceberg. Nevertheless, the adherence to GF diet frequently leads to overweight and obesity disorders (Kabbani et al., 2012). With regards to this topic, the relationship between celiac disease and diabetes mellitus has been reported by Zong et al., (2018). Diabetes mellitus is a metabolic disorder, that consists in a chronic hyperglycemia (high levels of sugar in blood), that also produces alteration in carbohydrates, lipids, proteins and mineral salts homeostasis (Khan et al., 2024). The global diabetes prevalence in 2019 was estimated around 9.3% (463 million people) and it is foreseen to reach values until 10.9% in 2050 (Saeedi et al., 2019). The fight against this disease must be priority for global health systems since it responsible for millions of death per year. Indeed, it shows an increasing trend of 3% annually in children and adolescents (Carstensen et al 2014; Patterson et al 2009). Patients with clinically overt coeliac disease Patients with undiagnosed silent coeliac disease Patients with latent coeliac disease 1. Introduction Page 57 of 353 The treatment of diabetes is limited to insulin intake by intramuscular injection or oral ingestion of agents or antidiabetic drugs, that could produce side effects like stomach disorders. Currently, the non–pharmacological interventions are limited to apply changes in dietary lifestyle promoting the consumption of low glycemic index (GI) foods. Actually, current GF products have high GI values, since they are low in proteins and high in fat and sugar content, whose consumption quickly increase glucose levels in bloodstream. Hence, consumption of these products prone to suffer diabetes disease. GF foods were initially designed as a mandatory and rushed need to produce foods suitable for celiac patients but with a low nutritional quality (Miranda et al., 2014). Then, the development of new GF products becomes a challenge, since it must be carried out attending both nutritional (GI control) and sensorial appealing (i.e., flavor, quality and sensory features) points of view (Sivaramakrishnan et al., 2004). The absence of gluten makes mandatory to seek alternative structuring agents. These gluten counterparts must be suitable to emulate its viscoelastic properties, appearance and crumb structure of conventional bakery products (Lazaridou et al., 2007) without altering products sensorial featuring. The use of gluten–free flours from alternative origin such as corn, rice, potatoes, chickpea combined with the addition of hydrocolloids (food gums), fiber and/or proteins are a suitable strategy to obtain economic, nutritional and functional GF products (Gambus et al., 2001; Behall & Hallfrisch, 2002; Novotni et al., 2012; Wolter et al., 2013;). Some of the widespread used gums in food sector are hydroxyethyl cellulose, xanthan gum, carrageenan, tragacanth gum, agar and alginate (Chenlo et al., 2009; Silva et al., 2017; Arufe et al., 2018a and b;). Some of these hydrocolloids are obtained from seaweeds, that in recent years are being in the spotlight by their high content of bioactive molecules such as polyphenols and fucoidans (Koivikko et al., 2007). To replace gluten with gums together with polyphenols from seaweed is proposed as economic, health–beneficial and novel manner of producing GF functional foods (Arufe et al., 2019a and b). Algae have become a resource for the discovery and development of ingredients and food products due to their bioactive compounds, also called nutraceuticals (Vaz et al., 2016). utraceuticals defined as “a food or a part of a food with demonstrated safety and health benefits beyond the basic nutritional functions to supplement the diet, that contain specific compounds in a quantity that exceeds those that could be obtained from normal foods” (Prasad 1. Introduction Page 58 of 353 et al., 2010). Seaweed nutraceuticals, polylactide, proteins, secondary metabolites (phlorotannins) and lipids among others, have demonstrated their beneficial health applications (Cumashi et al., 2007; Damonte et al., 2012). In addition, they have demonstrated to be promising digestive enzyme inhibitors to use them as GI regulators (Ademiluyi et al., 2012). These approaches show the potential of algae–based platforms (Scranton et al., 2015). Indeed, seaweeds are proposed as multiple resource to develop biorefineries for production of valuable compounds as unique raw material (Herrero & Ibañez 2015). European Union approved in 2017 the project MIRACLES (Multiproduct Integrated bioRefinery of Algae: from Carbon–dioxide and Light Energy to high–value Specialties) based on this concept. The multi– extraction component from seaweed has several advantages based on the 13 principles of green chemistry, since the extraction solvent could be efficiently carried out with water, the processes do not require excessive temperatures and the successive extraction of multiple components minimizes the residues generated. The current strategies to manage high glycemic levels are divided between oral ingestion of anti–diabetic agents and intramuscular injection of insulin. However, anti–diabetic drugs present undesirable side effects, such as risk of hypoglycemia, flatulence, abdominal discomfort and, sometimes, diarrhea are the main responsible for therapy discontinuation (Krentz & Bailey, 2005; Lopes et al., 2017). It is being studied the use of natural compounds as inhibitors of digestive enzymes (α–amylase and α–glucosidase) involving plant– and seaweed–derived compounds such as polyphenols due to their health beneficial features (Kan et al., 2020). Polyphenols from terrestrial origin, as catechins, quercetin, ferulic acids epicatechins, chlorogenic acids, naringenin, tannic acid, and caffeic acid have been used to control small intestinal glucose absorption via inhibition of sodium glucose co–transporters (Kobayashi et al., 2000; Johnston et al., 2005;). Johnston et al. (2003) and Dao et al. (2011) demonstrated that the polyphenols are able to mediate hyperglycemia and impaired glucose tolerance by an aided insulin response and constricted synthesis of glucagonlike polypeptide–1 (GLP–1). Waltner–Law et al. (2002) used epicatechins and catechins to moderate hyperglycemia and hepatic glucose production through regulating the expression of hepatic glucokinase and phosphoenolpyruvate carboxy–kinase. Zhou et al. (2001) and Towler & Hardie (2007) revealed that epigallocatechin gallate could trigger AMP–activated protein kinase pathway for inhibiting the expression of enzymes involved in gluconeogenesis. There are several literature that studies 1. Introduction Page 59 of 353 the effect of polyphenols on metabolism reactions, evidencing of the health beneficial properties of these compounds. Phlorotannins have demonstrated to reduce diabetes–related complications, showing that seaweeds compounds are promising anti–diabetic compound. Yang et al., (2019) reported IC50 values of phlorotannins isolated from Ishige okamurae to inhibit amylase (0.53 mM) and glucosidase (0.16 mM) enzymes. Sugiura et al. (2017) studied anti–diabetic activity of different phlorotannins isolated from Ecklonia cava (i.e., phlorofucofuroeckol–A, eckol, phloroglucinol, fucofuroec ol , diec ol, and , ’–bieckol) and reported that fucofuroeckol A and dieckol were the most effective with IC50 value of . μM. dditionally, phlorotannins have also demonstrated their capacities to inhibit several diabetic enzymes such as amylase, glucosidase, aldose reductase, protein tyrosine phosphatase and/or dipeptidyl peptidase (Gunathilaka et al., 2020). Lee & Jeon, (2013) showed that phlorotannins isolated from Ecklonia stolonifera (i.e., ec ol, diec ol, , ’–bieckol, phlorofucofuroeckol–A, phloroglucinol, and 7–phloroeckol) inhibited α–glucosidase showing IC50 values of 1 . μM. Similarly, Gotama et al. (2 1 ) found that with a dose of 300 mg/kg of phenolic compounds from Sargassum hystrix were enough to reduce blood glucose levels. There are several studies about the use of seaweeds and their derivative compounds (i.e., phlorotannins, fucoidans, laminarins, etc.) as an anti–diabetic and antioxidant agents. In the current Thesis the study was focused on the integration of seaweed derivatives into starchy based products and their subsequent capacities to inhibit diabetic enzymes. Then, it is proposed the implementation of biopolymers extracted from A. nodosum brown seaweeds in the next generation of GF products, focused on bakery products. It is a challenging task with multiple objectives, such as preventing problems derived from the short–term consumption of GF products, reduce long–term health problems of the habitual consumption of products with high GI and improving the bioactive and healthy beneficial properties of these new bakery products. The wide diversity of seaweeds put them in the forefront of research to obtain bioactive molecules with anti–diabetic properties (Barde et al., 2015). As sources of novel, structurally diverse and exclusive bioactive compounds is a promising study field (Sharifuddin et al., 2015; Lopes et al., 2017). Current researchers are focused on adding extracted, purified and fractionated seaweed bioactive compounds to food products. The use of purified fractions had the main advantage 1. Introduction Page 60 of 353 that phlorotannins added will be more efficient to increase their bioactivity features using minimal amount. Nevertheless, the production of these purified additive involves economic– and time–consuming processes. The addition of raw seaweed, as ingredient could be a quick and low–cost method. Furthermore, the use of raw algae would involve the addition of other bioactive components from algae as fucoidans and other carbohydrates improving, even more, the healthy beneficial characteristics (Koivikko et al., 2005) of these new algae–based foods (Sections 1.7.2 and 1.7.3). Therefore, as a compromise solution between these both factors, crude extracts were used in this Thesis. However, it implies the determination of chemical composition of biopolymers in the extracts and the enzymatic inhibition features related to starch digestion. Conversely, algae are prominent quelators and often retain some pollutants hazardous for humans. Thus, brown seaweeds as raw ingredient must be carefully studied taking some considerations such as chemical characterization or a detailed tracking of their origin, growth, and harvest conditions. All these aspects will be explored in Section 3.2. Then, the selection of either the raw seaweeds or the extracted/purified phlorotannin fractions is a key aspect in industrial applications and will be explored together with the optimal method to produce these fractions with acceptable antioxidant features and polyphenols content carried out with an easy– and low–cost procedure. Finally, it will be tentatively analyzed the potential anti–diabetic capacities of these phlorotannins extracted from A. nodosum brown edible seaweed, comparing the results with the reference drugs and the available literature. 1.7. STATE OF ART 1.7.1. PHLOROTANNINS EXTRACTION In the last twenty years, several researchers have studied different extraction characteristics, such as method, focusing on solid–liquid extractions (SLE) and ultrasound– assisted extractions (UAE), solvent type, liquid–solid ratio, time, and temperature as critical aspects of phlorotannin extraction from Ascophyllum nodosum brown edible seaweed (Table 1.3). 1. Introduction Page 61 of 353 Table 1.3. Overview extraction conditions (method, extractant, liquid–solid ratio, time, and temperature) to obtain phlorotannins enriched extracts from Ascophyllum nodosum. Reference Method Extractant LS (gsol/galgae) Time Temperature TPC Liu et al. (2021a) SLE Ethanol 90 30 min 30 ºC 0.7 mgPE/mgextract Sardari et al. (2021) SLE Water 100 1 h 65 ºC 7.3 mgPE/gextract Ethanol (30% v/v) 5 30 min 25 ºC 4.1 mgPE/gextract Ethanol (80% v/v) 10 20 (+ 5) h 25 (+ 65) ºC 3.4 mgPE/gextract Ford et al. (2020) SLE Acetone (70% v/v) 20 3 h rt 24.5 mgPE/gextract. Ummat et al. (2020) SLE Ethanol (50% v/v) 15 4 h 20 ºC 0.2 gPE/gextract UAE 10 30 min NS (35 kHz) 0.4 gPE/gextract Liu et al. (2019b) UAE Ethanol (40% v/v) 50 30 min 60 ºC 0.5 gPE/gextract Poole et al. (2019) SLE Water 10 24 h 4 ºC 52 mgPE/gextract Methanol (50% v/v) 77 mgPE /gextract Ethanol (75% v/v) 95.4 mgPE /gextract Dioxolane (75% v/v) 90 mgPE /gextract 1,3–propanediol 98.5 mgPE/gextract Agregán et al. (2018) UAE Ethanol (50% v/v) 10 30 min rt 46.6 mg/gextract i oš et al. (2018) SLE Methanol (70% v/v) 10 4 h rt 0.5 gGAE/gextract MAE (2.45 GHz) 15 min 110 ºC 1.4 gGAE/gextract Yuan et al. (2018) SLE Methanol (70% v/v) 10 4 h rt 0.5 mgGAE/gd.b MAE (2.45 GHz) 15 min 110 ºC 1.4 mgGAE/gd.b Moreira et al. (2017) UAE (80 W/cm2) Acetone (70% v/v) 30 4 min <35 ºC 31.8 mgPE/gd.b Kadam et al. (2015a) SLE Water 20 150 min 70 ºC 0.17 mgPE/gd.b HCl (0.1 M) 0.11 mgPE/gd.b UAE (35.6 W/cm2) Water 15 min <35 ºC 0.16 mgPE/gd.b HCl ( 0.1 M) 0.13 mgPE/gd.b continue.→ 1. Introduction Page 62 of 353 Reference Method Extractant LS (gsol/galgae) Time Temperature TPC Kadam et al. (2015b) UAE (75.8 W/cm2) + SLE HCl (0.03 M) 10 10 min + 22 h <35 ºC 135.7 mgGAE/gd.b Kadam et al. (2015c) UAE (75.8 W/cm2) HCl (0.03 M) 10 25 min <35 ºC 143.1 mgGAE/gd.b Pantidos et al. (2014) SLE Water 10 60 min 20 ºC 178.0 mgGAE/mL HCl (5 mM) 210.0 mgGAE/mL Tierney et al. (2013a) SLE Water 20 24 h rt 70.5 mgPE/gextract Ethanol (80% v/v) 10 66.3 mgPE/gextract Acetone (80% v/v) 155.9 mgPE/gextract PLE Water 2 NS 120 ºC (1500 psi) 93.4 mgPE/gextract Ethanol (80% v/v) 100 ºC (1000 psi) 101.3 mgPE/gextract Acetone (80% v/v) 60 ºC (1000 psi) 127.4 mgPE/gextract ’Sullivan et al. (2011) SLE Methanol (60% v/v) 15 3 h 40 ºC 4.5 mgGAE/gd.b Zhang et al. (2007) SLE Ethanol (50% v/v) 12.5 90 min 80 ºC 38.8 mgPE/gd.b Wang et al. (2009) SLE Water 20 24 h rt 138.0 mgPE/gextract Acetone (70% v/v) 159.0 mgPE/gextract where, galgae, is grams of algae; gd.b, is grams of algae in dry basis (d.b); gsol, is grams of solvent MAE, is microwave–assisted extraction; mgextract, is milligrams of extract; mgGAE, is milligrams of gallic acid equivalents; mgPE, is milligrams of phloroglucinol equivalents; NS, is “non– specified” in the study; rt, is room temperature; SLE, is solid–liquid extraction; UAE, is ultrasound–assisted extraction; PLE, is pressurized–liquid extraction. According to literature showed in Table 1.3 it can be clearly observed the relevance of TPC values obtained from the different extracts. TPC values ranged from 0.7 mgPE/mgextract (Liu et al., 2021a) up to 0.5 gPE/gextract (Liu et al., 2019b) both studies carried out extractions using ethanol but at different concentrations, 96% and 40% v/v, respectively. Lowest TPC values was obtained using conventional solid-liquid extraction, SLE, with one of the highest liquid–solid ratio values (LS = 90 gsolvent/gAF), during intermediate operational time (t = 30 min) and temperature (T = 30 ºC). As it was expected the highest extraction was achieved using UAE, carried out with lower LS value (50 gsolvent/gd.s), similar time (30 min) and higher temperature (60 ºC). 1. Introduction Page 63 of 353 Briefly, from these results, organo–solvents were more common employed in phlorotannin extractions, new methods as UAE, MAE or PLE increased extraction yields and low temperatures were usually employed to avoid thermal degradation of phytochemicals, except for MAE treatment. However, it is mandatory to note that expression units as well as other parameters such as temperature and/or time differ among different authors making the comparison often difficult. A suitable solution to reduce mistakes associated to data comparison could be to show in detail the experimental designs in which wide ranges of conditions were proved and their trends could be suitable used to determine the better extractive conditions. Additional publications of UAE applied to A. nodosum were found, Kadam et al. (2017) used them to extract protein from seaweed. Okolie et al. (2019) evaluated UAE influence on in vitro prebiotic assays of fucoidans enriched extracts. Garcia–Vaquero et al. (2018 & 2019) optimized the extraction of fucose and glucans using UAE technology and subsequently combining it with thermal–assisted extraction. Okolie et al. (2020) determined the impact of UAE on the structure–function relationship of sodium alginate. Several chromatographic techniques have been employed for separation, preparative isolation, purification, identification, and quantification of individual phenolic compounds from various plant materials, but still scarce studies deal with the individual phenolic compounds from brown algae (Table 1.4). These are essential research to assess and understand the phlorotannin chemical and physical structure effects on their bioactivities. Table 1.4. State of art of current phlorotannins isolated from brown seaweeds. Phlorotannin Seaweed References 2–Phloroeckol Eisenia. bicyclis Okada et al. (2004) Euphorbia stolonifera Jung et al. (2008) , ’–Bieckol Ecklonia cava Artan et al. (2008) Ishige okamurae and Euphorbia stolonifera Yoon et al. (2009) continue.→ 1. Introduction Page 64 of 353 Phlorotannin Seaweed References 7–Phloroeckol Euphorbia stolonifera Jung et al. (2008) Ascophyllum nodosum Nwosu et al. (2011) , ’–Bieckol Eisenia bicyclis, Ecklonia cava and Ecklonia kurome Shibata et al. (2008) Dieckol Eisenia bicyclis Nakamura et al. (1996) Dioxinodehydroeckol Ephorbia stolonifera Jung et al. (2008) Diphlorethohydroxycarmalol Ishige okamurae Heo et al. (2009) Eckol Ecklonia kurome Fukuyama et al. (1989) Fucodiphloroethol G Ecklonia cava Ham et al. (2007) Fucophlorethols Cystophora retroflexa Sailler & Glombitza (1999) Cupressus torulosa Glombitza et al. (1997) Sargassum spinuligerum continue.→ 1. Introduction Page 71 of 353 Seaweed spp. Extraction (Method, repetitions, LS, t, Sol and T) Inhibitor Substrate Enzyme Reaction conditions (t and T) IC50 Reference Eisenia bicyclis SLE x3 2 gsol/galgae 3 h MetOH rt Compound 1 (1 mM) Wheat starch 1% w/v α–amylase NS 15 min 37 ºC 96.2% Okada et al. (2004) Compound 2 (1 mM) 86.7% Compound 3 (1 mM) 76.0% Eisenia bicyclis SLE x3 40 gsol/galgae 3 h EtOH 25 ºC Methanolic extract n–Hexane fraction Dichloromethane fraction Ethyl acetate fraction n–Butanol fraction Water fraction Fucofuroeckol A Dioxinodehydroeckol Potato starch 1 % w/v α–amylase 0.4 mg/mL 2 h 37.5 ºC 0.5 µg/mL 3.5 µg/mL Eom et al. (2012) 0.3 µg/mL 48.1 ng/mL 0.2 µg/mL 1.9 µM 51.6 nM 93.3 nM p–NPG 3 mM α– glucosidase 0.4 mg/mL 20 min 37.5 ºC >500 µg/mL >500 µg/mL 39.98 g/mL 2.9 µg/mL 4.6 µg/mL >500 µg/mL 42.9 µM 0.47 mM Ishige foliacea SLE x3 40 gsol/galgae 3 h MetOH 80% v/v 25 ºC Octaphlorethol A p–NPG 5 mM α– glucosidase 0.7 U/mL 5min rt 0.11 mM Lee et al. (2014) continue→ 1. Introduction Page 72 of 353 Seaweed spp. Extraction (Method, repetitions, LS, t, Sol and T) Inhibitor Substrate Enzyme Reaction conditions (t and T) IC50 Reference Ishige okamurae SLE NS NS MetOH 80% v/v rt Diphlorethohydroxycarmalol p–NPG 5 mM α–amylase 0.7 U 5 min rt 0.53 mM Heo et al (2009) α– glucosidase 0.7 U/mL 0.16 mM Sargassum ringgoldianum SLE x3 NS NS MetOH 80% v/v NS Raw extract PNPG7 5 mM α–amylase 100 U 5 min rt 0.18 mg/dL Lee & Han (2012) p–NPG 5 mM α– glucosidase 0.7 U 0.12 mg/dL p–NPG 5 mM α– glucosidase NS 20 min 37 ºC 0.24 µg SF SA SS SP SD SLE x3 40 gsol/galgae 30 min EtAc 90 ºC Raw extracts Wheat starch 1% w/v) α–amylase 13 U/mL 10 min 25 ºC SF 27.0% SA 55.0% SS 30.0% SP 42.0% SD 36.0% Firdaus & Prihanto (2014) p–NPG 5 mM α– glucosidase 0.7 U 5 min 25 ºC SF 37.0% SA 65.0% SS 40.0% SP 52.0% SD 46.0% Triticum aestivum SLE x2 40 gsol/galgae 1 h EtOH 25 ºC (2–(4–(3,5– dihydroxyphenoxy)–3,5– dihydroxyphenoxy) benzene– 1,3,5–triol) Amylopectin 1% w/v α–amylase 1 µM 5 min 37 ºC 3.2 µg/mL Kawamura– Konishi et al. (2012) Maltose 100 mM α– glucosidase 1 µM 30 min 37 ºC 114.0 µg/mL Sucrose 100 mM 25.4 µg/mL where, AM, is Alaria marginata seaweed; EtOH, is ethanol; EtAc, is ethyl acetate, FD, is Fucus distichus seaweed; galgae, is grams of algae; gsol, is grams of solvent; LS, is liquid–solid ratio; Met , is methanol; S, is “non–specified” in the study; PF, is Pyropia fallax seaweed; p– NPG is p–nitrophenyl–a–D–glucopyranoside; PNPG7 is p–nitrophenyl maltoheptaoside; SA, is Sargasum aquifolium seaweed; SD, is Sargasum duplicatum seaweed; SF, is Sargasum filipendula seaweed; SG, is Saccharina groenlandica seaweed; SL, is Saccharina latissima seaweed; SLE, is solid–liquid extraction; Sol, is solvent type; SP, is Sargasum polycystum seaweed; SS, is Sargasum siliquosum seaweed; t, is time; T, is temperature; UL, is Ulva lactuta seaweed; x0, is process not repeated; x2, is process repeated twice; x3, is process repeated three times. 1. Introduction Page 73 of 353 The isolation of polyphenols from brown seaweeds has been scarcely studied until last decade, perhaps due to it is a time and cost consuming process. Then available literature of phlorotannins as digestive enzyme inhibitors is scarce, furthermore the purification and isolation of phlorotannins is another aspect with very few bibliographic sources. Additional studies where polyphenols from terrestrial plant origin (Table 1.7) isolated and identified, have been added to complement this section of polyphenols as enzyme inhibitors. Table 1.7 State of art of inhibitory capacities against α–amylase and α–glucosidase digestive enzymes of polyphenols from terrestrial plants. Polyphenol Substrate Enzyme Reaction conditions (t and T) IC50 Reference 3–O–galloylepicatechin Wheat starch NS α–amylase NS 10 min 37 ºC 739.0 µM Bhandari et al. (2008) 3–O–galloylcatechin 401.0 µM Apigenin PNPG7 NS α–amylase 0.03 mg/mL 10 min 37 ºC >0.5 mM Lo Piparo et al. (2008) Luteolin Potato starch NS α–amylase 0.2 µM 10 min rt 18.4 µM Apigenin p–NPG NS α–glucosidase NS 5 min 37 ºC 82.0 µM Proenca et al. (2017) Apigenin p–NPG 0.6 mM α–glucosidase 0.05 U/mL 15 min 37 ºC 21.9 µM Li et al. (2009b) Luteolin 17.0 µM Caffeic acid Wheat starch 0.25 mg/mL α–amylase 5.0 mg/mL 10 min 25 ºC 3.7 µM Oboh et al. (2015 a and b) Theaflavin Wheat starch 3.3 mg/mL α–amylase 0.2 mg/mL 5 min 25 ºC 0.4 mg/mL Rutin 0.04 µM Quercetin p–NPG 3.8 mM α–glucosidase 0.2 U/mL 0.04 µM Caffeic acid Wheat starch 2.5 mg/mL α–amylase 7.5 U/mL 3 min 25 ºC 6.7 mM Tan et al. (2017) p–NPG 2.0 mM α–glucosidase 0.1 U/mL 10 min 37 ºC 21202 µM continue→ 1. Introduction Page 74 of 353 Polyphenol Substrate Enzyme Reaction conditions (t and T) IC50 Reference Chlorogenic acid p–NPG 2 mM α–glucosidase 0.2 U/mL 10 min 37 ºC 4 mg/mL Venditti et al. (2015) Apigenin 0.1 mg/mL Luteolin 0.3 4 mg/mL Cyanidin–3 glucoside Corn starch 0.1 mg/mL α–amylase 0.2 mg/mL 15 min 37 ºC 0.02 mM Sui et al. (2016) (+)–catechin α–amylase 0.04 U/mL NS 37 ºC 9.3 mg/mL Cyanidin–3 glucoside Wheat starch 1.0 mg/mL α–amylase 3.0 U/mL 10 min NS 0.3 mM Akkarachiyasit et al. (2010) Cyanidin–3.5 diglucoside > 1.0 mM Daidzein Wheat starch 1.0 mg/mL α–amylase 0.1 U/mL 10 min 37 ºC > 400.0 µM Zhang et al. (2017) Genistein > 400.0 µM Luteolin >400.0 µM Epicatechin–3–gallate Rice starch 16.0 mg/mL α–amylase NS 12 min 37 ºC 1.5 mM Koh et al. (2010) p–NPG 2.0 mM α–glucosidase 0.6 mg/mL 1.4 mM Ferulic acid Potato starch 0.2 mg/mL α–amylase NS 20 min 37 ºC 0.6 mM Zheng et al. (2020) p–NPG 0.18 mM α–glucosidase 0.2 mg/mL 4.5 mM Gallic acid Wheat starch 5.0 mg/mL α–amylase 0.3 mg/mL 10 min 25 ºC 1.2 µM Adefegha et al. (2015) p–NPG 1.3 mM α–glucosidase 0.5 U/mL 7.2 µM Gallic acid p–NPG 1.3 mM α–glucosidase 0.5 U/mL 5 min 25 ºC 0.04 µM Oboh et al. (2016) Gallic acid Wheat starch NS α–amylase NS 3 min NS 57.0 mM Cirillo et al. (2010) Genistein p–NPG NS α–glucosidase NS 30 min 37 ºC 50 µM Lee & Lee (2001) Hesperidin Potato starch 5.0 mg/mL α–amylase 5.0 U/mL t30 min 37 ºC 6 µM Sahnoun et al. (2017) Naringin p–NPG NS α–glucosidase 0.04 U/mL 0.55 µM Luteolin PNPG7 NS α–amylase 0.03 mg/mL 10 min 37 ºC 0.4 mM Tadera et al. (2006) Quercetin p–NPG 1.3 mM α–glucosidase 5 µg/mL 5 min 30 ºC 7 µM continue→ 1. Introduction Page 75 of 353 Polyphenol Substrate Enzyme Reaction conditions (t and T) IC50 Reference Luteolin p–NPG 0.5 mM α–glucosidase 1.0 mM NS 37 ºC 172.0 µM Yan et al. (2014) Quercetin p–NPG 4.2 mM α–glucosidase NS 5 min 37 ºC 0.1 µM De Souza et al. (2010) Theaflavin 67.0 µM Quercetin Potato starch 2.5 mg/mL α–amylase 1.0 U/mL 3 min 25 ºC 0.9 µM Rutin 1.2 µM Theaflavin p–NPG NS α–glucosidase NS 15 min 37 ºC 54 µM Jeon et al. (2013) where, S, is “non–specified” in the study p–NPG is p–nitrophenyl–α–D–glucopyranoside; PNPG7 is p–nitrophenyl maltoheptaoside; t, is reaction time; T, is reaction temperature. To optimize and expand the use of algae and their bioproducts will be the next step to explore innovative applications in food, medical, pharmaceutical, and/or cosmetic industries. The state of art previously showed in this Thesis about extraction methods (Section 1.7.1) and the use of seaweed biopolymers as food additives in starchy–based systems (Section 1.7.2) and as digestive enzyme regulators (Section 1.7.3), evidence that seaweeds are potentially an extensive resource in the future. Page 77 of 353 2. OBJECTIVES & HYPOTHESIS 2. Objectives & hypothesis Page 79 of 353 The general objective of this Thesis is the extraction of biopolymers with high antioxidant and anti–enzymatic activities from Galician native brown seaweeds with great local socioeconomic interest. These biopolymers can be included in food formulations to improve their functional properties. Specifically, Ascophyllum nodosum (A. nodosum) was selected as a valuable and suitable source of phlorotannins (antioxidant and digestive enzyme inhibitor agent) and alginates (thickener and gelling agent) as replacements of current synthetic compounds. Antioxidant and enzyme inhibitor additives may be active controllers for the management of glycemic control over time, for the alleviation and prevention of diabetes, particularly critical in celiac people. To achieve this general objective several specific objectives were established: Reviewing in–depth bibliography on the characterization, operational conditions, ranges of interest for key variables, food application, among others, for the biopolymers extracted along the Thesis (Sections 1 up to 12). Set–up and management of experimental equipment and techniques employed for different operations such as drying, milling, sieving, compositional analysisis, in vitro enzymatic, chromatography, spectroscopic and image methods (Sections 6 up to 12). Physicochemical characterization of dried A. nodosum flour (Section 6). Comparison of conventional solid–liquid (SLE) and novel ultrasound–assisted extraction (UAE) that were discussed in Section 7. Experimental determination and modelling of polyphenol extraction kinetics from A. nodosum seaweed by means of different procedures to establish adequate extraction conditions and, on the other hand, to measure the ionic strength effect by using and salty water on aqueous extracts bioactivity (Sections 7, 8 and 9) 1 2 3 4 5 2. Objectives & hypothesis Page 80 of 353 To determine the optimal conditions of aqueous UAE of phlorotannins following a Box-Behnken experimental design to evaluate the effects of sonication power, sonication time and the liquid-solid ratio (Section 8). Characterization and purification of phlorotannins in extracts of A. nodosum for the isolation of the most bioactive (antioxidant and enzyme-inhibiting) fractions (Sections 9 and 11). To obtain phlorotannin–enriched extracts from A. nodosum UAE extracts using methods based on both size (using different cut-off dialysis membranes) and oxidation state to assess the resistance to oxidation, the bioactivity distribution and the identification of some phlorotannins (Section 9). To study the interactions between A. nodosum bioactive phlorotannins and corn starch (employed in gluten–free and non–gluten–free food formulations) by several blending methods and proportions (Section 10). To analyze and identify the in vitro inhibitory capacities of phlorotannin– enriched extracts from A. nodosum using UAE against digestive enzymes (α– amylase and α–glucosidase) by several blending methods (Section 11). Integral valorization of brown seaweeds, by means of the use the solid residue, after the UAE of phlorotannins from A. nodosum seaweed, that still contains other valuable compounds, such as alginate (Section 12). To determine the effect of the process conditions, specifically for drying operation, on physicochemical properties of alginates by means of spectroscopic and viscometry capillary techniques (Section 12) 6 7 8 9 10 11 12 3. Theoretical fundamentals Page 87 of 353 Ultrasound comprises mechanical waves that are above human hearing, ranged from 20 kHz up to 10 MHz (Picó, 2013). Ultrasound involves various phenomena including shear forces, compression pressure gradients, agitation, rarefaction, vibration, microjets, radical formation and cavitation (Tiwari, 2015; Rodrigues et al. 2015), promoting cell walls disruption and the subsequent release of compounds (Hahn et al., 2012; Agregán et al., 2018;). Cavitation is the main driving force for ultrasound extraction: it is a hydrodynamic effect that occurs when vapor cavities are created within liquid and different pressure forces are present (Picó, 2013). These processes produce expansion and implosive collapse of micro bubbles, formed due to a series of compressions and rarefactions in molecules generated by ultrasound waves improving heat and mass transfers along the system improving solvent penetration and cell walls breaking (Tiwari, 2015). The UAE method improves solvent penetration and cell walls disruption, increasing the extraction yields respect to conventional extraction methods (Soria & Villamiel, 2010; Roselló–Soto et al., 2015;). Figure 3.3 shows the general scheme of ultrasound‑assisted extraction from seaweed tissues. Figure 3.3. Ultrasound–assisted extraction process. 3. Theoretical fundamentals Page 88 of 353 UAE is a simple, cost–effective and efficient replacement for traditional technologies largely employed to extract bioactive compounds (Section 1.7.1). Indeed, UAE extraction technology is being employed in both laboratory and large–scale industrial applications (Kadam et al., 2015c) such as emulsification, homogenization, extraction, crystallization, dewatering, low–temperature pasteurization, degassing, defoaming, activation and inactivation of enzymes, among others ( i oš et al., 2 1 ). 3.1.3. FRACTIONATION–ISOLATION OF SEAWEED EXTRACTS During the extraction of phenolic compounds, other molecules are co–extracted, mainly polysaccharides and proteins. The study of phenolic compounds from seaweeds makes mandatory their separation and purification for the fractionation and/or isolation of the compounds to reduce extracts complexities. Briefly, fractionation of the extracts consists in separation based on molecular weights, charges, chemical affinities and/or solubilities (Houchi et al., 2019; Vissers et al., 2017). Adsorption–based separations are emerging due to their simplicity and the potential for scale–up and higher specificity compared to other primary fractionation techniques (Soto, et al., 2011). Separation is achieved by contact of seaweed extracts with a solid matrix with different affinities for phlorotannins and the remaining compounds. Then, phlorotannins can be recovered by separating the solid and liquid phases (McInnes, et al., 1985). Solid phase extraction is other adsorption–based technique, where sorbent is immobilized in a cartridge or column, allowing the sequential elution of compounds with gradient of solvents (Santos et al., 2019). Liquid–liquid extraction is a solubility–based separation where the broad range of polarities of phenolic compounds prone partition of the distinct compound to obtain polarity–segregated fractions enriched of phlorotannins (Santos et al., 2019). Other separations that could be performed are ultrafiltration and/or molecular–weight cut–off dialysis. Ultrafiltration and dialysis are techniques that separate compounds by their molecular weight with minor instrumental requirements, time, expertise, indeed both techniques allow separations of discrete fractions over a wide range of molecular weights with few combinations of membranes/filters (Jégou, et al., 2015; Lin, et al., 2018). 3. Theoretical fundamentals Page 89 of 353 3.2. CHARACTERIZATION OF SEAWEED BIOPOLYMERS The objective of this section was to summarize the set of techniques currently available for the characterization of extracts and biopolymers extracted from seaweeds and the information provided by each technique. Seaweeds extracts are composed by a large, diverse, and complex mixtures of compounds, whereas there are phlorotannins together with polysaccharides, proteins, and other metabolites. Chemical characterization of crude and purified compounds from seaweeds is essential to understand their bioactivities and beneficial health properties. The study of these compounds is relevant food engineering, medicine, and pharmacy areas (Munteanu & Apetrei, 2021). Nevertheless, the isolation is often difficult due to their diverse molecular weights, structural similarities and their rapid reactivities (Parys et al., 2009; Tanninou et al., 2014; Vissers et al., 2017). Initially, raw characterization is commonly carried out by spectrophotometric assays that are simple, cheap, rapid, and have a feasible comparison with other studies (Pulido et al., 2000; Munteanu & Apetrei, 2021;). Nevertheless, nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT–IR), Matrix–assisted laser desorption ionization time of flight mass spectrometry (MALDI–TOF–MS) and chromatographic (HPLC and/or UPLC) have been reported as more reliable techniques for seaweed extracts characterization (Santos et al., 2019). These techniques allow the qualitative and quantitative estimation of extracts composition. Furthermore, it is largely recommended that antioxidant activities must be tested by more than one method (Moharram & Youssed, 2014). However, the number of parameters considered in each determination hinders the comparison of results, as it was also mentioned in Section 1.7.1 for phlorotannins extractions. The current section provides a comprehensive overview of characterization techniques suitable to be applied on seaweed extracts and their isolated compounds. Some widespread techniques, also used in this Thesis, will be explained to give general background of their mechanisms, procedures and data obtained from each one (Figure 3.4). This knowledge of analytical methods available will improve the understanding of seaweed bioactive compounds and their potential uses (Sun et al., 2022). 3. Theoretical fundamentals Page 90 of 353 Figure 3.4. Summary of analytical methods often used for characterization of seaweed biopolymers whereas bold techniques were applied in this Thesis. 3.2.1. ANTIOXIDANT ACTIVITY DETERMINATIONS This section provides the background of the methods to understand the reaction mechanisms of antioxidants, together with the advantages and limitations of the different assaying tests. Spectrophotometric methods rely on the linear relationship between absorbance and concentration whereas the appearance of colors or the discoloration of the solutions at specific wavelength is measured (Ford et al., 2019). Current available methods could be divided based on transfer of hydrogen atoms ([H+]), electron ([e–]) and/or mixture of both (Figure 3.5) such as: Hydroxyl Radical Antioxidant Capacity (HORAC), Total Peroxyl Radical Trapping Antioxidant Parameter (TRAP), Total Oxyradical Scavenging Capacity (TOSC), Oxygen Radical Absorption Capacity (ORAC), 2,20–AzinoBis–(3–Thylbenzothiazoline–6–Sulfonic) acid (ABTS), 2,2–Di(4–tert–octylPhenyl)–1–PicrylHydrazyl) (DPPH), Cupric Reducing Antioxidant Power (CUPRAC), Ferric Reducing Antioxidant Power (FRAP) and finally the total polyphenol content (TPC) determinations (Prior et al., 2005; Everette et al., 2010; Apak, et al., 2018; Munteanu & Apetrei, 2021). Raw seaweed extracts , ( , 13 , q MR, R ES , M , S , 1 F, 31P, S ) R ( S , S S, PS, RF, E ), etc. on destructive tests Fast spectral acquisition Miniaturization , M , , , Sulphates, , , , etc. Most used ow sensitivity omplicated pre treatment ptical, EM, , R, SM, etc omplicated pre treatment Microstructure analysis Purified Seaweed extracts P P U P ESI QqQ MS/MS U P ESI Q F MS Suitable for quantification of low abundance phlorotannins Suitable for screaming and identification of phlorotannins Suitable for quantification of phlorotannins 3. Theoretical fundamentals Page 91 of 353 Figure 3.5. Antioxidant activity determination methods. 3.2.1.1. OXYGEN RADICAL ABSORBANCE CAPACITY (ORAC) ORAC test is used to determine the scavenging activity through the inhibition of the oxidation of peroxyl radicals. Peroxyl radicals, seen in Section 1.5, are free radicals that predominate in lipid oxidation in biological systems (Apak, et al., 2018). This test is based in the emission of peroxyl radicals by a generator (e.g., azo–compounds) that reacts with a fluorescent (e.g., fluorescein) sample that leads to loss of fluorescence due to the antioxidant effect (Becker et al., 2013). 3.2.1.2. HYDROXYL RADICAL ABSORBANCE CAPACITY (HORAC) In the HORAC method it is measured the ability of antioxidants to avoid complexation reaction between hydroxyl radical complexation and cobalt ions (Co2+). Fluorescein as fluorescence source, is incubated with the assayed antioxidant samples, then a generator of hydroxyl radicals is added (e.g., Fenton mixture). The decay of fluorescence provides a direct measurement of antioxidant capacity ( íž et al., 2 1 ; Munteanu petrei, 2 21). ydroxyl Radical ntioxidant apacity otal Peroxyl Radical rapping ntioxidant Parameter otal xyradical Scavenging apacity xygen Radical bsorption apacity upric Reducing ntioxidant Power 2,2 zino is (3 e hylbenzothiazoline Sulfonic acid 2,2 i( tert octylPhenyl) 1 Picryl ydrazyl) Ferric Reducing ntioxidant Power Folin iocalteu M 3. Theoretical fundamentals Page 92 of 353 3.2.1.3. TOTAL RADICAL-TRAPPING ANTIOXIDANT PARAMETER (TRAP) TRAP method measures the assayed compound capacity to inhibit the reaction between peroxyl radicals and target molecules by means of the oxygen consumption during the peroxidation process of 2,20 azobis(2–amidinopropane) dihydrochloride. The elapsed time for the oxygen absorption (i.e., the induction time) is used to determine the total antioxidant capacity of the samples (Apak et al., 2018). 3.2.1.4. TOTAL OXYRADICAL SCAVENGING CAPACITY (TOSC) The TOSC test is based on the inhibition of the formation of ethylene in presence of antioxidant compounds that compete with α–keto–γ–bethiolbutiric acid (KMBA) for oxidants. This test uses the relationship between area under the ethylene concentration curve (obtained by a gas chromatography) and the reaction time between KMBA and oxidants (Regoli & Winston, 1999). 3.2.1.5. CUPRIC REDUCING ANTIOXIDANT CAPACITY (CUPRAC) The CUPRAC test determine the total antioxidant capacity based on the reduction of cupper ions (Cu2+ and Cu+). Like molybdenum in the TPC method and iron in FRAP method (Bartosz, 2010) this method is based in a ligand reaction mechanism with the neocuproine (2,9– dimethyl–1,10–phenanthroline) whose color is determined at 450 nm and provides the antioxidant activity of the samples (Munteanu & Apetrei, 2021). 3. Theoretical fundamentals Page 93 of 353 3.2.1.6. FERRIC REDUCING ANTIOXIDANT POWER ( FRAP) The FRAP test is based on the reduction of iron (Fe3+ to Fe2+) as ligand by effect of antioxidants. Antioxidant activity is determined as an increase color measured at 593 nm (Antolovich et al., 2002; Prior et al., 2005). FRAP analysis is used on a large scale, providing results for a variety of purposes, including the estimation of the antioxidant content in foods and their contribution to the supply of antioxidants, to investigate the effect of storage, growth, draught, solar radiation, processing, genetic modification in foods, medicines, traditional medicines, herbs, spices, teas and wines as a standardized quality and control test (Munteanu & Apetrei, 2021). 3.2.1.7. TOTAL POLYPHENOL CONTENT (TPC) Total polyphenols content (TPC) test based on Folin–Ciocalteu method is the most common assay used with polyphenol (terrestrial and marine) enriched products (Parys et al., 2009; Tanninou et al., 2014; Vissers et al., 2017; Wootton et al., 2001). It was originally designed to analyze proteins, but it was later adopted by Singleton et al. (1999) to analyze the phenolic components in wine, after which it became a routine test for the antioxidant evaluation of food and plant extracts (Apak et al., 2018; Wright et al., 2001). TPC test detects the compounds suitable to transfer electrons from reductant compounds to molybdenum complexes of Folin-Ciocalteau reagent (Ainsworth & Gillespie, 2007; Everette et al., 2010) that promotes a color change, detected at 765 nm (Singleton & Rossi, 1965). TPC value is determined with a calibration curve carried out with a polyphenol standard compound (e.g., gallic acid or phloroglucinol). The disadvantage of this method is the possible interferences gave by non–phenolic substances such as pigments, sugars, proteins, amines, organic acids, or inorganic substances (Blasco et al., 2005; Magalhaes et al., 2008). Additionally, there is a lack of standardized methodology in what concerns the volume, incubation time and concentration of extract and reagents (Heffernan et al., 2015). Although this method has some defects it is largely applied due to the economical and convenient experimental conditions (Ignat, et al., 2011; Sun et al.,2022). 3. Theoretical fundamentals Page 94 of 353 Alternative methods to Folin–Ciocalteu method, are the Prussian blue and 2,4– dimethoxybenzaldehyde (DMBA) assays. In Prussian blue assay phenolic compounds reduce Fe(CN)63– anion (Price & Butler, 1977), and in M assay, reagent reacts with 1,3‐ and 1,3,5‐ trihydroxybenzenes. However, both methods lead to an underestimation polyphenols content (Parys et al., 2007; astro‐ lves ordenunsi 2 15; Ford et al., 2019). 3.2.1.8. DPPH METHOD DPPH● (2,2–diphenyl-1-picrylhydrazyl) is a stable radical that is soluble in different organic solvents, but not in water. (Williams, 1966). The DPPH test is based on the neutralization of DPPH● radical by the electrons donated by antioxidant assayed (Staš o et al., 2007). This reaction produces a discoloration that measured at 515 nm acts as an indicator of the scavenging activity decay of the DPPH often reported as IC50, (i.e., the efficient concentration of the antioxidant necessary to reduce the initial DPPH concentration to 50%) (Foti, 2015). The use of DPPH is widely reported to produce significant and comparable results making this method suitable to be used on several optimizing and/or standardizing protocols. Besides it is a low cost, easy, reproducible and operates at room temperature (Munteanu & Apetrei, 2021). 4.2.1.9. ABTS METHOD ABTS test is based in reaction with 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) stable radical with radical scavenging molecules, which capacity is expressed as TroloxTM Equivalent Antioxidant Content (TEAC). This test measures the antioxidant capacity to neutralize the ABTS● radical that produces a discoloration measured at 734 nm (Miller et al., 1993). ABTS test can be used over a wide pH range (Lemanska et al., 2001) and it is soluble in both, water, and organic solvents, allowing the determination of antioxidant capacity of both hydrophilic and lipophilic compounds (Cano et al., 200; Walker & Everette, 2009). It must be noted that all assays cited in this section depend on the reaction time (i.e., time contact between antioxidant and reaction mixture), intrinsic antioxidant activity, and sample concentration that often makes difficult the bibliographic comparisons. 3. Theoretical fundamentals Page 95 of 353 3.2.2. CHROMATOGRAPHIC STUDIES Liquid chromatography (LC) is the main method for the analysis of polyphenols (Sun et al., 2022). High performance liquid chromatography (HPLC) is wide used to identify the structure, linkages position, type, and size of phlorotannins (Ford et al., 2019), even purity can be evaluated with chromatographic techniques (Kerrison et al., 2015; Vissers et al., 2017). Normal phase (NP–HPLC) involves the use of a polar stationary phase, where compounds are separated based on their polarity, it leads a difficult elution of polar phlorotannins due to their high interaction with the stationary phase (Li et al., 2009a; Yoon et al., 2013;). Reverse phase HPLC (RP–HPLC) is largely used in phlorotannins analysis due to their better reproducibility and the lower retention times. RP–HPLC involves the use of apolar stationary phase. Thus, retention times for apolar molecules is longer, whereas polar molecules elute before. RP–HPLC can be a useful tool in separating compounds with very similar structures, as phlorotannins from A. nodosum, however, the rapid elution of phenols due to their high polar nature could be disadvantage (Alvarez–Rivera et al., 2019). Ultra‐performance liquid chromatography (UPLC) reduces sample and solvent usage, reducing columns size and increasing working pressures together with speed and sensitivity of analysis (Novakova et al., 2006). Size exclusion chromatography (SEC) have been used in the preparative separation of different molecular weight fractions from seaweed extract as well as to confirm the molecular size of an isolated metabolite (Kang et al., 2012b; Lee, et al., 2012;). The stationary phase or column is the key for chromatography, being octadecylsilane columns, also known as C18, with a particle size of 5 µm the most often used, but there are available with other internal diameters and lengths. Preparative columns are used to minimize problems with complex samples, avoid damage to the HPLC equipment or to remove impurities (Santos et al., 2019). The chromatographic detectors are also important. Phenolic compounds absorb ultraviolet radiation between 260 and 330 nm, and this absorption range is used to identify their presence. Thus, detectors based on visible and UV–vis are the simplest and widely applicable to these kind of molecules (Ford et al., 2019). This absorption is quite variable due to the similar structures and depends on pH and/or presence of other components (Shahidi & Naczk, et al., 2006). However, UV–VIS detectors provide ambiguous peak identification due to the 3. Theoretical fundamentals Page 96 of 353 similarities between phlorotannins. Alternatively, the use of mass spectrometers directly coupled to U/HPLC UV–vis instruments minimize this problem increasing the capacity to analyze complex extracts (Santos et al., 2019) allowing the qualitative and quantitative of hundreds of polyphenolic components (Guillarme et al., 2010). Diode array detector (DAD) used for analysis of compounds with similar molecular weights but different electronic distribution in the chromophore, leading to different UV spectra (Audibert et al., 2010; Ford et al., 2019;). HPLC combined with an UV–vis or a DAD detector and C18 column is the main system used for the separation and/or quantification of phlorotannins (Ignat et al., 2011; Motilva, et al., 2013). Mass spectrometry is an analytical method for measuring mass–charge ratio (m/z), and electrospray ionization source (ESI) is the most often used ionization procedure (Ignat et al., 2011; Devi et al., 2008). Phlorotannins throughout the ESI source is ionized in a negative ion mode to produce deprotonated molecular ions. It is worth noting that in seaweed extracts, as a complex matrix, often interferences of coeluting compounds can occur, leading to the ionization suppression or enhancement in the signals (Cladiere et al., 2018; Konermann, et al., 2013). Thus, purification is required to extract and purify the targets during sample preparation by LC– MS that could increase time and cost (Motilva, et al., 2013). In fact, the isomerization by multiple combinations of phloroglucinol units after ionization has a notorious impact on mass spectra and often it is difficult to attribute a chemical structure of the detected molecules. However, MS can easily provide the profile of degree of polymerization in a phlorotannins enriched extract (Tierney et al., 2014; Kadam et al 2015b; Lopes et al., 2017; Catarino et al., 2019; Allwood et al., 2020; Sardari et al., 2021). The main limitation of ESI is that the sample is vaporized that do not permit the analysis of higher molecular weight as well as thermally labile components (Sailler & Glombitza, 1999). Matrix–assisted laser desorption ionization (MALDI) combined with time of flight (TOF) analyser is a particularly suitable technique for the analysis of larger oligomers, with m/z above the upper limit of ESI–MS. This technique has been also used in combination with U/HPLC– ESI–MS providing information about the size and isomeric variation of phlorotannins (Wang et al., 2013; Agregán, et al., 2017; Sun et al., 2022). Other chromatography techniques have been used for the analysis of phenolic components in macroalgae, with different specificities; thin–layer chromatography (TLC) and/or Triple quadrupole (QqQ) among others (Cho et al., 2019; Santos et al., 2019). 3. Theoretical fundamentals Page 103 of 353 which is hydrolyzed in just few minutes during the first digestion stage (i.e., mastication), and its effect on the postprandial glycemia is not clear yet (Parada & Santos, 2016). An additional starch category is the resistant starch, which is not digested, and it passes to the digestion system with no effect on postprandial glycemia (Cummings & Englyst, 1995). Furthermore, starch digestion may depend on food microstructure, which controls the accessibility to the substrate and the mobility of enzymes in the food bolus throughout the digestion process, presence of substances that could alter/inhibit the action of the digestive enzymes (e.g., natural compounds as polyphenols or drugs as acarbose), the intimate interactions of starch with other bolus components preclude the direct contact between starch and enzymes (Parada & Aguilera, 2011). The profile of each consumer, including their genetic background, metabolic status, or certain diseases presence, among others, also modify the starch digestion features. Indeed, it has been found that even particle size after mastication may affect the glycemic response, showing the complexity of this process. Figure 3.7. Starch gelatinization process and their microstructure changes. Starches are being studied from multidisciplinary points of view, but the current Thesis is focused on high glycemic responses and subsequent disorders associated to the rapid digestion of gluten–free (GF) products and their derived problems to celiac patients. Glycemic response as the increasing of blood glucose levels after food consumption (Parada & Aguilera, 2011) is strongly related to starchy–based products consumption specially in traditional GF. Foods with low glycemic responses are considered favorable to health, since avoid or reduce possibilities to end up developing diabetes disease. As it was mentioned in Section 1.7 appropriate glycemic control is particularly important in celiac disease. Thus, it is mandatory to produce suitable new generation GF products to control GI responses, being the phlorotannins–enriched extracts the proposed solution in this Thesis. 3. Theoretical fundamentals Page 104 of 353 Scientific evidence refutes partially the idea that a lower starch digestibility will induce a lower glycemic response (Parada & Santos, 2016), evidencing that glycemic response is a multifactorial phenomenon. Thus, deeper research, including the interdisciplinary approach, in Figure 3.6 is necessary to increase the insight of the actual relationship between starch digestion and glycemic response. Figure 3.8 shows a simplified diagram summarizing the main steps and enzymes related with food digestion and glycemic response. Figure 3.8. Global process of starch metabolic fate. With the current advances in chemical and engineering technologies for extraction and identification of bioactive compounds from seaweeds, new food ingredients with potential functional activities in human health and nutrition is being achieved in the last years. Promising data from in vitro and animal studies have been found (Section 1.7.3), however, effects of polyphenols on glucose homeostasis in human beings are still under discussion. Further research would expand the most biologically active polyphenols screening and its derivatives and provide drug candidates for pharmaceutical purpose to the reduction or regulation of the diet–linked dysfunctions (Murugan et al., 2015). Mouth Salivary amylase iver Small intestine Intestinal glucosidase Pancreas Pancreatic amylase Stomach F Food properties affecting digestibility Starch state; global food structure; anti enzymatic compounds issues (storage and use) G U G ow glucose blood level lood I SU I igh glucose blood level Signal Response Glucose Glucose Glucose 3. Theoretical fundamentals Page 105 of 353 3.3.3. PHLOROTANNINS AS ENZYME INHIBITORS A strategy to manage the observed high levels of glucose in blood is the inhibition of α– amylase and α–glucosidase, digestive enzymes involved in the breakdown of starch and oligosaccharides (Matsui et al., 2007; Ríos et al., 2015) allowing the control of postprandial glucose in diabetic patients (Krentz & Bailey, 2005; Chen et al., 2006; Kwon et al., 2008; Zhao et al., 2021). Polyphenols are a large and heterogeneous group of phytochemicals particularly relevant as anti–enzymatic food components (Tester et al., 2004; Sajilata et al., 2006;). Polyphenols are widely known due to their ability to associate with macromolecules that could be applied on enzymes to reduce their efficacy (Parada & Aguilera, 2011). Thus, polyphenols could be used to inhibit diverse digestive enzymes, specially α–amylase and α– glucosidase, but also maltase and/or sucrase (Williamson, 2013). Gauer et al., (2018) showed that flavonoids inhibited glucose absorption in the intestine, a promising result for the selective inhibition of pathways and to develop tailor–made treatments for every patient. The ability of polyphenols to inhibit digestive enzymes is related to their well–documented interactions with some proteins and polysaccharides. The polyphenol–polysaccharide interactions are due to non–covalent (hydrogen bonds and hydrophobic interactions) or covalent interactions (Le Bourvellec & Renard, 2012). All these interactions, amount and type are largely influenced by food matrix structure and processing conditions. It is promising research that remains partially unexplored, but it can be a powerful tool to control the bioaccessibility, bioavailability, anti–enzymatic capacities of these new generation functional foods (Scazzocchio et al., 2015; Corona et al., 2017). Several in vitro studies have reported on the enzyme inhibitory activity of polyphenols from different sources (persimmon, sorghum, rowanberry, or almond seeds, among others), on α–amylase and α–glucosidase enzymes (Flores et al., 2013). Although terrestrial polyphenols are recognized to have several bioactive functions, the literature on seaweed’s polyphenols for human consumption and their disease treatment is sparse (Shibata et al., 2008) as it was previously commented (Sections 1.7.2 and 1.7.3). 3. Theoretical fundamentals Page 106 of 353 Seaweeds are natural sources of enzyme–inhibiting compounds (o enzyme inhibitors), although knowledge about them is limited (Wang et al., 2005; Nyambe–Silavwe et al., 2015; Corona et al. 2017). Lordan et al. (2013) studied the enzymatic inhibition activity of phlorotannin–enriched extracts, reporting A. nodosum extracts as the strongest α–amylase inhibitors and Fucus vesiculosus extracts the best α–glucosidase inhibitors. However, diverse characteristics must be considered, such as extract manufacture, in vitro studies conditions, substrate used and harvesting period among others (Lee & Jeon, 2015). Page 107 of 353 4. EXPERIMENTAL & METHODOLOGY 4. Experimental Page 109 of 353 4.1. MATERIALS All chemical reagents used in the treatments and characterizations were analytical grade purchased from Merck1 (Germany), Panreac2 (Spain), Megazyme3 (Ireland) or Sigma–Aldrich4 (USA). Reagents used during this Thesis were 2,2’–azino–bis(3–ethylbenzothiazoline–6– sulfonic acid) diammonium salt (ABTS4), acarbose1, Amberlite4 XAD16 resin (surface area 800 m2/g, pore diameter 1 nm), α–amylase4 (Type VI–B, EC 3.2.1.1) from porcine pancreatic (8 U/mg), chlorohydric acid1, corn starch2, deuterated water4 (D2O), deuterated dimethyl sulfoxide (DMSO–d6), 3,5–dinitrosalicylic acid, 2,2–diphenyl–1–picrylhydrazyl4 (DPPH), Folin–Ciocalteu reagent1, glacial acetic acid1, glucose4, D–glucose assay kit3 (G /P ) , α– glucosidase4 (Type I; EC 3.2.1.20) from Saccharomyces cerevisiae (11 U/mg), D(+)–guluronic acid4, 3–hydroxybiphenyl4, iron (III) chloride4, iron sulphate4, D(+)–maltose2, methanol1, phenol1, phloroglucinol4, potassium chloride4, potassium hydroxide2, resorcinol4, sodium acetate4, sodium alginate (CAS no: 9005–38–3), sodium azide4, sodium carbonate2, sodium chloride4, sodium dihydrogen phosphate2, sodium hydroxide4, sodium phosphate4, sodium tetraborate4, sulfamic acid2, sulfuric acid2, 2,4,6–tris(2–pyridyl)–s–triazine4, Trolox4, and wheat starch4. 4.2. DRYING & MILLING & SIEVING Ascophyllum nodosum (A. nodosum) seaweeds harvested from the Galicia coast (Spain) during autumn season of 2019 were supplied by Mar de Ardora S.L. (Ortigueira, Spain) with a moisture content of 73.7 ± 3.8 % d.b. Samples were washed with running tap water, dried in a hot air convective dryer, Angelantoni Challenge 250, (Figure 4.1A). Optimized conditions of A. nodosum seaweeds were previously established by Chenlo et al., (2018). Consequently, temperature (50 ºC), relative humidity (30%), air velocity (2 m/s) and of load density (2 kg/m2) were employed. A. nodosum seaweed was dried until moisture content of 4.3 ± 0.1 % d.b that was achieved after 8.5 h. Dried seaweeds were aerated for 3 days at room temperature (rt, 19 ± 1 ºC) sealed in vacuum plastic bags and conserved at 4 ºC. 4. Experimental Page 110 of 353 Then, dried samples were grounded in a blender (Figure 4.1B), milled in ultra–centrifugal mill (Figure 4.1C) and sieved using a vibratory sieve (Figure 4.1D) with standard meshes from to 5 μm. Finally, A. nodosum flour (AF) was sealed in plastic bags with a vacuum packer (Figure 4.1E) and stored at 4 °C with final moisture content of 2.9 ± 0.1 % d.b. Figure 4.1. A) Angelantoni Challenge 250 Air convective dryer (Italy), B) Laboratory blender Waring HGBTWT (USA), C) Ultra–centrifugal mill Retsch GmbH, ZM200 (Germany), D) Vibratory sieve FTL 0200, Cisa (Spain) and E) Vacuum packer Sammic V201 (Spain). Drying kinetics were determined by monitoring samples weight. Samples were weighted in an analytical balance (Figure 4.2A), every few minutes in the first stages of drying and every hour towards the end. All experiments were performed, at least in duplicate, until moisture content was close to equilibrium. Dried weight was determined after vacuum drying (Figure 4.2B), at 70ºC and 104 Pa until constant weight of samples. Moisture ratio, Eq. (4.1), was employed to evaluate drying kinetics: Moisture ratio=Xt−Xeq X0−Xeq (4.1) where Xt was the moisture content (d.b.) at any drying time, X0 was the initial moisture content (d.b.) and Xeq was the equilibrium moisture content of the sample (d.b.) evaluated from water desorption obtained from Chenlo et al., (2018). 4. Experimental Page 111 of 353 Figure 4.2. A) Vacuum oven Vacutherm VT650 (Heraeus Hanau, Germany) and B) analytical balance (Denver Instruments Spain) 4.3. PARTICLE SIZE AND WATER RETENTION CAPACITY Chenlo et al., (2018) reported that particle size of seaweed flours was critical in biopolymers extractions. Thus, the weight mean diameter, Dw, was calculated using Eq. (4.2) assuming that the particles of AF were spherical. Dw=∑ xiDpi i=n i=1 (4.2) where Dpi (µm) is the mean diameter for each fraction and xi is the weight fraction calculated as the ratio between corresponding mass of each fraction and the total mass of our employed in the sieving. Water retention capacity (WRC) measures water retained by the insoluble matrix and was determined following Robertson et al. (2000) protocol. AF was hydrated in distilled water with a liquid–solid ratio of 40 gW/gAF, for 20h in a at rt. Then, samples were centrifuged (3,000 xg. 20 min). The supernatant was decanted and conserved to be further characterized. Solids were transferred to a weighed sinter to be weighted prior to drying in a vacuum dryer. WRC was calculated by Eq. (4.3). Experimental WRC determinations were carried by triplicate. 4. Experimental Page 112 of 353 WRC =AFwet−AFdried AFdried (4.3) where AFwet is the mass hydrated and AFdried is the mass dried. 4.4. BIOPOLYMERS EXTRACTIONS Water (double distilled water) was used in all extracts as an eco–friendly and economic extraction solvent (FAO, 2018; Kadam et al., 2015a). However, to evaluate the effect of solvent type in several extraction methodologies and extraction conditions were proposed along this Thesis. To evaluate the effect of ionic strength in extractions, saline water 36 g/L NaCl, similar to seawater was studied in Section 7. Additionally, distilled and saline water were used to analyze extraction methods (solid–liquid extraction SLE and ultrasound–assisted extraction, UAE). Subsequently, using just double distilled water, the UAE and its operational conditions (sonication power, SP, time residence, tR, and liquid–solid ratio, LS) were optimized to maximize the extraction of phlorotannins (Section 8). Finally, with the UAE conditions set up (SP = 90 W/cm2, tR = 2 min and LS =20 gsolvent/galgae), 70% (v/v) acetone:water (i.e., a better extractant of phlorotannins reported by Catarino et al., 2019) was compared with water extractions. The aim was to achieve a water– based extract that was similar in bioactivity to acetone:water. Purification throughout Amberlite XAD16 permitted to achieve an aqueous extract similar to acetone:water extract in bioactivity values. Then, this purified extract was used for the study of the influence of oxidation and dialysis into phlorotannins on bioactivity and composition (Section 9). The nature, type, and number of interactions between those phlorotannins and corn starch was evaluated as preliminary approach of these new generation gluten–free foods (Section 10). Raw and purified UAE extracts were studied as digestive enzyme inhibitors (Section 11). Finally, isolated sodium alginate came from solid residue generated after UAE of phlorotannins (Section 12). Extractions were carried out in triplicate. 4. Experimental Page 119 of 353 Figure 4.5. Concentrator plus/Vacufuge plus, Eppendorf (Germany) Spectrophotometer Genesis 10S UV, Thermo Fisher Scientific (USA). 4.6. RAW AND PRURIFIED EXTRACTS CHARACTERIZATION All extract characterizations were carried out by spectrophotometry (Figure 4.5B) at least in triplicate. Seaweeds extracts characterization includes the total polyphenol content (TPC) and the evaluation of their antioxidant activity by means of scavenging activity (DPPH and ABTS methods), and electron donor capacities (FRAP) (Mesa–Vanegas et al. 2015; Dang et al. 2017b;). Total carbohydrate content (CHOs) and total uronic acid content (UA) were also determined from the extracts. All these methods are colorimetric methods (Section 3.2.1) that were used in result sections (Sections 7 up to 12). The color change observed during reaction occurred for each method is shown in Figure 4.6. Figure 4.6. Color change occurred during colorimetric assays. FR P reagent FR P reduced Folin iocalteu Reagent Reduced Folin iocalteu Reagent Seaweed extract Uronic acids Phlorotannins omplex with Phenol Sulphuric S radical S reduced PP radical PP reduced 4. Experimental Page 120 of 353 4.6.1. TOTAL POLYPHENOL CONTENT Total polyphenol content (TPC) was determined using a calibration with phloroglucinol as standard following the method proposed by Singleton & Rossi (1965). The method is based on the Folin–Ciocalteu reagent reaction with hydroxyl groups, measured spectrophotometrically at 765 nm. Aqueous seaweed extracts (0.5 mL) were mixed with 10% (v/v) Folin Ciocalteu’s reagent (2.5 mL) and 7.5% (w/w) sodium carbonate solution (2 mL). The TPC values were expressed as g of phloroglucinol equivalents per liter (gPE/L) using calibration curve showed in Figure 4.7.The blank was measured replacing seaweed extracts by double distilled water. Mixtures were incubated at 40 ºC for 15 min in completely darkness (Figure 4.8). Figure 4.7. TPC calibration curve. y 122.15x + 3. R . . 2 . . . . 1 . . .2 . . . 4. Experimental Page 121 of 353 Figure 4.8. Total polyphenol content determination methodology. Folin iocalteau solution (1 v/v) .5 (w/w) sodium carbonate nodosumextract alibration with phloroglucinol (R2 . ) avelength 5 nm P as gPE/ Resting 15 min Room temperature Static conditions lends homogenization 4. Experimental Page 122 of 353 4.6.2. TOTAL CARBOHYDRATE CONTENT The total carbohydrate content (CHOs) was evaluated using a calibration with glucose as reference spectrophotometrically at 485 nm. CHOs results were given in g of glucose equivalents per liter (gGE/L) using calibration curve showed in Figure 4.9. Aqueous extracts (1.0 mL) were mixed with 5% (v/v) phenol (0.5 mL) and H2SO4 (2.5 mL) following method proposed by Dubois et al. (1956). Mixtures were incubated at 30 ºC for 30 min (Figure 4.10). Figure 4.9. CHOs calibration curve. y 1 .31x 3.3 R . . 25. 5 . 5. 1 . 125. . .2 . . . 1. 4. Experimental Page 123 of 353 Figure 4.10. Total carbohydrate content determination methodology. alibration with glucose (R 2 . ) avelength 5 nm s as gGE/ Resting 3 min 3 Static conditions lends homogenization Sulfuric acid 5 (v/v) phenol nodosumextract 4. Experimental Page 124 of 353 4.6.3. URONIC ACID CONTENT Uronic acid content (UA) determination was carried mixing aqueous extracts (0.2 mL) with 4 M sulfamic acid (40 µL) and 12.5 mM sodium tetraborate solved in H2SO4 (1.25 mL). Mixtures were boiled at 100 ºC for 5 min and cooled for 20 min at rt. UA determinations were carried out using a calibration with glucose as reference showed in Figure 4.11 and the values are given in g of glucose equivalents per liter (gGE/L). Then, after cooling samples, 0.15% (w/v) m–hydroxybiphenyl (20 µL) was added and mixture was spectrophotometrically analyzed at 520 nm following Blumenkrantz & Asboe–Hansen, 1973 protocol (Figure 4.12). Figure 4.11. UA calibration curve. y 1 . 2x 3.2 R . . . 12 . 1 . 2 . 3 . . . . 1.2 1. 2. 4. Experimental Page 125 of 353 Figure 4.12. Uronic acids content determination methodology. alibration with rolox (R 2 . ) avelength 52 nm U as oiling 5 min 1 Static conditions lends homogenization 12.5 mM sodium tetraborate ( 2S dissolved) M sulfamic acid nodosumextract .15 (w/v) m hydroxybiphenyl 4. Experimental Page 126 of 353 4.6.4. DPPH SCAVENGING ACTIVITY DPPH scavenging activity (DPPH) measures were performed following the methodology proposed by Brand–Williams et al. (1995). Extract samples (2 μ ) were mixed with µM PP methanolic solution ( μ ). Measures were ta en after resting time of 3 min in completely darkness at rt. The scavenging activity of samples was determined measuring absorbance at 515 nm (Figure 4.14). DPPH scavenging activities were calculated using Eq. (4.7) and using calibration curve showed in Figure 4.13 with Trolox, to express results as millimolar of Trolox equivalents (mMTE). DPPH=DPPH0min−DPPH30min DPPH0min (4.7) Figure 4.13. DPPH calibration curve. y . 13x . R . . .3 .5 . 1. 1.3 . 2 . . . . 1 . 4. Experimental Page 127 of 353 Figure 4.14. DPPH scavenging activity determination methodology. µM PP methanolic solution nodosumextract alibration with rolox (R 2 . ) avelength 515 nm PP activity as µM E Resting 3 min Room temperature Static conditions lends homogenization 4. Experimental Page 128 of 353 4.6.5. ABTS SCAVENGING ACTIVITY ABTS scavenging activity (ABTS) determination was carried out following the proposed method by Re et al. (1999). ABTS reagent was prepared with 24.5 µM potassium persulfate and 15.2 mM ABTS using PBS buffer (0.14M NaCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 2.7 mM KCl and 3.1 mM sodium azide). The reagent was stirred in the dark for 16 hours at rt. ABTS solution was diluted with PBS until absorbance reach 0.70 ± 0.02 at 734 nm. Then, extracts were mixed with ABTS and incubated for 15 min at rt (Figure 4.16). ABYS scavenging activities were calculated using Eq. (4.8) and using calibration curve showed in Figure 4.15 with Trolox, to express results as millimolar of Trolox equivalents (mMTE). ABTS=ABTS0min−ABTS15min (4.8) Figure 4.15. ABTS calibration curve. y 3 .51x + .1 R . . . 12. 1 . 2 . 3 . . .1 .3 . . . min 4. Experimental Page 135 of 353 Figure 4.20. Elution gradients used during RP–HPLC analysis of A. nodosum extracts, based on gradient mode (A) and on–off mode (B). 4.10. EXTRACTS FRAGMENTATION PATTERNS The mass detectors currently used chromatographic systems have a detection limit of approximately 2000 Da and become a limiting factor working with phlorotannins that are known to be larger (Catarino et al., 2019). The analysis of oligomers, above 500 Da in the extracts was carried out with a matrix–assisted laser desorption/ionization time of flight mass spectrometry (MALDI–TOF–MS). MALDI–TOF–MS spectra were recorded for samples PW, PD20 and PD2 to assess the range of mass fragmentation. The equipment used was Bruker Ultraflex III TOF/TOF equipped with a N2 laser of 337 nm and operated in positive mode. MALDI–TOF–MS spectra data treatment was carried out with Data analysis software (Bruker, USA). Fragmentation treatments were carried out for purified (Section 4.5) and crude UAE extracts (Section 4.4.1) to evaluate the effect of phlorotannins molecular size (Section 9) on their composition (Sections 4.6), chromatographic (Section 4.9 in on–off mode) and spectroscopic (MALDI–TOF–MS, FT–IR and 1H–NMR, from Sections 4.10, 4.11 and 4.13, respectively) characteristics. 2 1 1 2 3 5 2 1 2 1 4. Experimental Page 136 of 353 4.11. FOURIER TRANSFORM INFRARED SPECTROSCOPY Fourier transform infrared (FT–IR) spectra were recorded with a Bruker FT–MIR model Vertex 70 V spectrometer. Wave number range was in the range 400 to 4000 cm–1. Samples were blended with KBr and compressed into disks. FT–IR spectra treatment was carried out with Omnic 7.1 software (Thermo Scientific, USA). FT–IR data were supplemented by elemental analyses (CHNS) carried out with a Thermo Finnigan (Flash 1112 model) elemental analyzer. FT–IR spectroscopic analyses were carried out to analyze qualitatively the complex mixtures of extracts (Section 9), interactions between corn starch and seaweed (Section 10) and as a widespread technique used for alginate characterizations (Section 12). 4.12. AVERAGE VISCOSIMETRIC MOLECULAR WEIGHT Molecular weight of extracted and commercial alginate was evaluated from kinematic viscosity value using a Ubbelohde viscometer (AVS 350, Schott–Geräte, GmbH, Germany) using an aqueous 0.1 M NaCl solution. For each sample, five measurements were taken at 25 ºC (±0.1 ºC). Then, the viscosity average molecular weight (Mv, g/mol) was determined by the Mark–Houwink equation following Dodero et al., (2020a & b) studies. Average viscosimetric molecular weight was used to analyze the effect of UAE residue drying temperature had on physicochemical properties of isolated sodium alginates (Section 12). 4. Experimental Page 137 of 353 4.13. NUCLEAR MAGNETIC RESONANCE 4.13.1. SODIUM ALGINATE Hydrogen nuclear magnetic resonance (1H–NMR) of alginate samples was carried out blended dried alginates with D2O. 1H–NMR spectroscopy was used to determine composition and mannuronic block (M) and guluronic block (G) distribution into monads (FM and FG), diads (FGG, FMM, FMG, FGM) and triads (FGGG, FMGM, FGGM), M/G ratio and block average length (NG>1) from sodium alginates samples, were also calculated Monads, diads, triads, M/G ratio and NG>1 values assessment following equations from ASTM, 2012 (Eqs. (4.9) up to (4.24)) where A, B1, B2, B3, B4 and C values are obtained peak area values form each spectra following the Figure 4.21 schematic representation. 1H–NMR is a commonly used method for sodium alginates characterization, that was used in this Thesis to analyze the effect of UAE residue drying had on the properties of isolated sodium alginates (Section 12). G=0.5(A+C+0.5 B1+B2+B3 ) (4.9) M=B4+0.5 B1+B2+B3 (4.10) GG=0.5 A+C−0.5 B1+B2+B3 (4.11) MG=GM=0.5 B1+B2+B3 (4.12) MM=B4 (4.13) GGM=MGG= B1 ·0.5 B1+B2+B3 / B1+B2 (4.14) MGM= B2 ·0.5 B1+B2+B3 / B1+B2 (4.15) GGG=GG−GGM (4.16) 4. Experimental Page 138 of 353 FG=G M+G (4.17) FM=M M+G (4.18) FGGG=GGG M+G (4.19) FMGM=MGM M+G (4.20) FGGM=FMGG=GGM M+G (4.21) NG=FG FGM (4.22) NG>1= FG−FMGM FGGM (4.23) NM=FM FMG (4.24) Figure 4.21. Average 1H–NMR spectrum shape of alginate used for quantitative analysis. 4. Experimental Page 139 of 353 4.13.2. PHLOROTANNIN-ERICHED EXTRACTS 1H–NMR spectra were collected with Bruker NEO 750 spectrophotometer operated with a 17.61 T (750 MHz resonance 1H) magnetic field strength. 1H–NMR spectra from extracts was carried out using DMSO–d6 or D2O and their treatment was carried out with MestreNova software (Mestrelab Research, Spain). NMR technique was used to analyze chemical alterations in assayed extracts due to, oxidation and dialyzation treatments (Section 9) and to evaluate UAE and purification treatments (Section 11) effects on phlorotannins features. 4.14. SEAWEED–STARCH INTERACTIONS Three methods (NT, GL, CGL) for corn starch (CS) and AF blending were tested with slight modifications respect to protocol proposed by Wang et al. (2021). NT and GL methods were assayed to study the effect of CS structural features on the interactions with bioactive compounds from AF. Conversely, CGL method was based on two stages: first, bioactive compounds extraction from AF together with CS during gelatinization step; second, a partial leaching of bioactive molecules from gel after water addition. The objective of this last method was to simulate the behavior of bioactive compounds during the heating steps during starchy products processing. So, NT consisted in the native CS and AF blending; gelled CS was blended with AF in GL, and CS was gelatinized in the presence of AF in CGL method. AF and CS control samples were also analyzed to determine chemical characteristics promoted by both powders (Figure 4.22). Starch gelatinization was performed according to the method reported by Wang et al. (2021). Aqueous CS (20%, w/w) was immersed in boiling water bath at 100 ºC for 20 min. The CGL gelatinization was carried at the same conditions for AF and CS blends. After gelatinization stage, the samples were cooled (20 min) at rt until gel temperature was lower than 35 ºC. Subsequently, GL and CGL samples were homogenized using a homogenizer (IKA Werke GmbH & Co., Germany) with 3 pulses of 5 s at 6500 rpm. Broken gel was blended with seaweeds and additional water in GL method, whereas only distilled water was added to obtain CGL samples. 4. Experimental Page 140 of 353 Figure 4.22. Process scheme summarizing methodology. NT method: AF (seaweeds flour) and corn starch (CS) blending; GL method: AF and CS blending, CGL method: CS gelatinized in presence of AF. Liquid–solid ratio was set at 100 gW/gAF. AF and CS content varied to obtain different A. nodosum flour to corn starch blending ratios (1:25, 1:2, 1:1; and 1:0.5) corresponding to 4, 50, 100 and 200 gW/gCS, respectively. Ratio 1:25 was used as average proportion studied by bakery products (O'Shea et al., 2014), meanwhile, remaining ratios were studied to determine the molecular interactions more adequately. These AF–CS mixtures in water were homogenized and rested for 15 min at rt. Elapsed time necessary to achieve a solid/liquid pseudo–equilibrium (between water and AF) was previously determined and was shorter than 15 min. This procedure agrees to the kinetics determined between potato starch and procyanidins by Qiu et al. (2016). The phytochemicals content was analyzed after samples centrifugation at 7,500 ×g for 30 s at rt followed by filtration of the supernatant through 0.45 µm microfiber filter (Meck Milipore, USA). Seaweed–starch interactions methodologies were carried out to evaluate the interactions of starch (native or gelled) with seaweed particles and their bioactive compounds. Interactions were evaluated by means of composition (Section 4.6), FT–IR (Section 4.11) and optical (Section 4.16) analyses and results are collected in the Section 10. Each proposed systems (NT, GL and CGL) and AF–CS ratio (1:0.5, 1:1, 1:2 and 1:25) was carried by triplicate. 4. Experimental Page 141 of 353 TPC values provided by AF in the AF–CS blends was calculated using Eq. (4.25): TPCLP=TPCAF−CS−TPCCS (4.25) where TPCLP is the polyphenols content in the aqueous phase from AF, TPCAF–CS is the polyphenols content measured in the liquid phase of AF–CS blends and TPCCS the corresponding polyphenols content of CS control samples. The polyphenols sorption yield at equilibrium, YP (%) was evaluated by Eq. (4.26) and the polyphenols adsorbed by CS, q (mgPE/gCS), was determined by Eq. (4.27): YP=(1−(TPC TPCAF))·100 (4.26) q= TPCAF−TPC (V mCS) (4.27) where TPCAF is the polyphenol content (gPE/L) of aqueous phase corresponding to AF control samples, V the liquid volume (L) and mCS is the final CS mass (g), evaluated by means of Eq. (4.28): mCS = mCSi – TPCCS – CHOs V (4.28) where mCSi is the initial CS mass (g) and CHOs the carbohydrate content (gGE/L) of liquid phase released from starch (adsorbent). 4.15. INHIBITORY CAPACITIES OF EXTRACTS he inhibition assay of α–amylase from porcine pancreatic was adapted from recent research protocol (Zheng et al., 2020) with minor modifications. Briefly, wheat starch solution (6.25 mg/mL) was prepared in sodium phosphate buffer (0.02 M, pH 6.9 containing 6 mM NaCl), followed by starch gelatinization in a water bath at 100 ºC for 20 min (Wang et al., 2021). Reaction systems included 50 µg of freeze–dried seaweed extracts dissolved in 20% (v/v) ethanol, 5 µ of α–amylase (50 U/mL) and 400 µL of gelatinized wheat starch. Three 4. Experimental Page 142 of 353 main methodologies were carried out to evaluate extracts inhibition activities (Figure 4.23), with minor modifications depending on the enzyme ssayed. Enzyme and polyphenol solutions were mixed and pre–incubated in an Eppendorf Thermomixer Compact at 37 ºC for 10 min at a speed of 600 rpm. Then, gelatinized starch was added, and the blend was incubated under the same conditions (A1). Gelatinized starch and polyphenolic extracts were mixed and pre–incubated at 37 ºC for 10 min before adding the enzyme, and then the mixture was incubated at 37 ºC (A2). Starch was gelatinized in the presence of the extract, and then cooled at rt for 10 min, until reaching 37 ºC. The enzyme solution was then added, and the mixture incubated at 37 ºC for 10 min (A3). To stop the reaction, 500 µL of 3,5–dinitrosalicylic acid reagent was added and the tubes placed into a boiling water bath for 10 min. Samples were diluted in distilled water (1:10), and their absorbances measured at 540 nm in a microplate reader (Epoch Biotek Instruments, Winooski, USA). he α–glucosidase from Saccharomyces cerevisiae activity was measured using maltose (10 mg/mL) dissolved in sodium phosphate buffer (0.1 M, pH 6.9). The reaction system consisted of 5 µ of seaweed extracts, 5 µ of α–glucosidase (10 U/mL) and 400 µL of maltose, but two different methodologies (G1 and G2) were tested (Figure 4.23). In G1 method, enzyme and polyphenols were initially pre–incubated at 37 ºC for 10 min and then maltose was added to initiate the enzymatic reaction at 37 ºC. For G2 methodology, maltose and seaweed extract were initially pre–incubated at 37 ºC for 10 min, the enzyme was then added. In both, G1 and G2, enzymatic reaction was stopped by boiling samples in a water bath for 10 min. Absorbance was measured at 510 nm using the GOD/POD kit. Solutions without seaweed extracts and without enzymes were analyzed as control and blank, respectively. The inhibition percentage was calculated by Eq. (4.29): Enzymatic activity decay=[1−(X+−X− C+−C−)]·100 (4.29) where, 𝑋+ was the absorbance of sample with substrate and enzyme; 𝑋− was the absorbance without enzyme; 𝐶+ was the absorbance without extracts sample; 𝐶− was the absorbance of substrate. 4. Experimental Page 143 de 353 The IC50 value is the sample concentration required for 5 inhibition of the α–amylase or α–glucosidase activity. Acarbose was used as a positive control. Inhibition of enzymatic activities (Section 11) was evaluated for purified (Section 4.5) and crude UAE (Section 4.4.1) extracts together with chemical (Section 4.6), chromatographic (Section 4.11) and 1H–NMR (Section 4.13.2) characterizations to explain the different observed activities. Each proposed method (M1, M2 and M3) and enzyme type (α–amylase and α– glucosidase) was carried by duplicate. Figure 4.23. Methodology scheme of α–amylase inhibition assays ( 1, 2 and 3) and α–glucosidase (G1 and G2) digestive enzymes against aqueous polyphenols extracts from A. nodosum seaweed. amylase Extract rt 1 min 3 1 min amylase 3 1 min Extract Gelled starch 3 1 min Gelled starch 3 1 min Granular starch Extract Granular starch rt 1 min amylase 3 1 min 3 1 min 1 2 min glucosidase Extract Maltose 3 1 min Extract glucosidase 3 1 min Maltose 3 1 min 3 1 min 1 1 min 5 2 min 4. Experimental Page 144 of 353 4.16. SCANNING ELECTRON MICROSCOPY The effect of extracts addition on samples microstructure was analyzed by scanning electron microscopy (SEM). Samples were sputtered with iridium using a vacuum metallizer/shader model Q150T S (Quorum Technologies Lewes, UK) set to obtain an upper metallic layer of 5–10 nm thickness. Samples micrographs were obtained using a scanning electron microscope (Zeiss FESEM Ultra Plus with EDX, Zeiss, Germany) at 3 Kv using a SE/InLens secondary electron detector. Cross–sectional morphologies of solid phase from seaweed–starch systems allowed the evaluation of interactions and the structural effects (Section 10). 4.17. STATISTICAL ANALYSIS Statistical analysis was carried out by IBM SPSS statistics 27 (SPSS Inc., USA) software. A one–way analysis of variance (ANOVA) was assessed based on confidence interval of 95% (p < 0.05) using a Duncan test. The experimental results were treated and plotted on Microsoft Excel (Microsoft Corporation, USA). All experimental results were expressed as 𝑥± 𝜎 of at least triplicate experiments (n=3). The root mean squared error (RMSE, Eq. (4.30)) was used to quantify differences between predicted and experimental values with Peleg model in Sections 7 and 10. RMSE=√ Xi−XModel 2 (4.30) where 𝑥𝑖 is the experimental value and XModel is the predicted value. 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 247 of 353 Figure 11.1. Assayed systems for the determination of the digestive enzyme inhibitory capacities of A. nodosum extracts and their bioactivities. Maltose Gelatinized starch Gelatinized starch in presence of extract amylase glucosidase Maltose Gelatinized starch Gelatinized starch in presence of extract amylase glucosidase Subtract omogenization Incubation Enzymatic denaturalization Subtract omogenization Enzyme Enzyme FR P P s PP P U FR P Enzymatic inhibition capacity scoph llum nodosum flour ptimal queous Ultrasound ssisted Extraction Purification Proteins Minerals Fats MR SP tR S 2 2 /cm2 min g /g F SP /cm2 /cm2 /cm2 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 248 of 353 11.3. SEAWEED EXTRACTS CHEMICAL COMPOSITION Aqueous UAE is largely effective for the extraction of polyphenols, but other compounds like carbohydrates and particularly UA were also co–extracted. Because of that proximate composition, antioxidant activities and UA content were selected to evaluate the impact of UAE and further purification. The moisture, protein, mineral, fat, CHOs, total polyphenol (TPC), UA content, and antioxidant activities (DPPH and FRAP methods) were determined from A. nodosum ultrasound–assisted crude (E90, E80 and E70), and purified (P90) extracts (Table 11.1). Sonication power did not significantly(p < 0.05) modify the proximate composition of the extracts (p > 0.05), since protein, mineral, and fat content were around 7.5 ± 0.1, 29.8 ± 0.3, and 4.1 ± 0.1 (%, dry weight, DW), respectively, CHOs (0.1 ± 0.03) and UA (0.2 ± 0.06 mgGE/mgDW) content. TPC (mgPE/mgDW), CHOs (mgGE/mgDW), and UA (mgGE/mgDW) were also invariant with sonication power with values of 0.2 ± 0.04, 0.2 ± 0.02, and 0.2 ± 0.04, respectively. Finally, moisture (%) varied in a narrow range 8.6 ± 0.3 (E80) up to 10.1 ± 0.6 (E70). No significant (p > 0.05) differences of antioxidant activities were observed between extracts obtained under different conditions, with mean values of 39.4 ± 1.0% of DPPH scavenging activity and 1.1 ± . μgTE/gDW of Fe2+ reducing power. Kadam et al. (2015a, b and c) working with A. nodosum aqueous extracts obtained from UAE (35.6 W/cm2 for 15 min) reported slightly lower TPC values (0.16 mgPE/gDW) with a DPPH decay value of 61.46%. Nevertheless, purification of the extracts significantly (p < 0.05) decreased protein (0.93– fold), mineral (0.28–fold), fat (0.73– fold), and UA (0.27–fold) content and increased CHOS (1.65–fold) and TPC (2.21–fold) content. Simultaneously, an interestingly relevant increase of DPPH decay (1.29–fold) and FRAP (1.61–fold) content was determined in the purified extract. 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 249 of 353 Table 11.1. Chemical composition of freeze dried (E90, E80 and E70) and purified (P90) extracts obtained by UAE from A. nodosum brown seaweed. Means within a raw followed with different letter are significantly (p < 0.05) different. P90 E90 E80 E70 Moisture (%, DW) 16.4 ± 0.12c 8.8 ± 0.15a 8.56 ± 0.3a 10.1 ± 0.6b Proteins (%, DW) 7.0 ± 0.30a 7.5 ± 0.02b 7.37 ± 0.1b 7.57 ± 0.1b Minerals (%,DW) 8.2 ± 0.2a 29.5 ± 0.2b 29.8 ± 0.1b 30.0 ± 0.4b Fats (%,DW) 3.0 ± 0.04a 4.2 ± 0.04b 4.0 ± 0.9b 4.2 ± 0.1b CHOs (gGE/gDW) 0.2 ± 0.04b 0.1 ± 0.02a 0.1 ± 0.01a 0.1 ± 0.02a TPC (gPE/gDW) 0.4 ± 0.04b 0.2 ± 0.03a 0.2 ± 0.03a 0.2 ± 0.03a UA (gGE/gDW) 0.05 ± 0.01a 0.2 ± 0.04b 0.2±0.02b 0.2 ± 0.04b DPPH (mMTE/gDW) 128 ± 0.05d 120 ± 0.01c 118±0.02b 107 ± 0.01a FRAP (mgTE/gDW) 1.8±0.05a 1.1 ± 0.01b 1.1 ± 0.1b 1.1 ± 0.04b According to Wang et al. (2016), polyphenols and UA could be partially forming complexes. The complexation effects on extracts bioactivity are still not elucidated. Regarding this interaction, hydrogen bond or hydrophobic interactions (Koivikko et al., 2005) and covalent bonds of ether, ester, and hemiacetal bonds (Salgado et al., 2009) have been proposed. The bonds of these complexes could affect their structural properties and promote or avoid some chemical interactions. However, the presence of these complexes was not directly discerned by common TPC and UA analyses and antioxidant activities (DPPH and FRAP; Table 11.1). Purification step reduced UA content and increased bioactivity of extracts. UA/TPC ratio was employed as a measure of cleaning of the extracts, since CHOs values varied in a narrow range (from 0.1 to 0.2 mgGE/mgDW) after purification. In fact, UA/TPC ratio decreased significantly (p < 0.05) from 0.96 ± 0.08 (E90, E80 and E70) up to 0.1 ± 0.02 (P90) after purification with Amberlite XAD16. These results indicated that this resin was an efficient adsorbent for the removal of UA in the extracts, improving bioactive features (TPC, DPPH, and FRAP) of purified extract. The reduction of UA/TPC ratio could help to explain further enzymatic inhibitory differences of tested extracts. 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 250 of 353 11.4. INHIBITION EFFECT OF EXTRACTS AGAINST DIGESTIVE ENZYMES Comparative study was conducted to determine the capability of the four different A. nodosum extracts (E , E , and E and purified P ) to inhibit α–amylase and α–glucosidase activity following three methodologies: preincubation of extract + enzyme (M1), preincubation of extract + gelatinized starch (M2), and gelatinization of starch + extract (M3). It is shown in Figure 11.2 the α–amylase and α– glucosidase inhibition induced by the different extracts, in comparison with acarbose, which was taken as standard inhibitor (Table 11.2). In M1 method (Figure 11.2), increasing the UAE sonication power significantly (p < 0.05) decreased the IC50 meaning that the inhibition capacity of the extracts increased (up to 35 and 15 for α–amylase and α–glucosidase), despite extracts showed similar TPC and DPPH values. The extract purification with Amberlite (P90) largely improved the inhibition efficiency decreasing the IC50 of α–amylase and α–glucosidase by 3.0 and 6.1 times, respectively. This result might be explained by its higher TPC (Table 11.1) compared with those measured in the extracts. Comparing the effect of tested extracts, higher extract concentrations were required to inhibit α–amylase compared to α–glucosidase. In relation to acarbose, a specific competitive inhibitor, the results were lower; IC50 values ( . and 3.2 μg/mL) of P90 were four times higher than that of acarbose on α–amylase and 2 times higher for α– glucosidase (Table 11.2). These results support the findings described by Apostolidis and Lee (2010) when analyzing an aqueous extract from A. nodosum, reporting higher inhibition activity against yeast α–glucosidase than against porcine pancreas α–amylase. Conversely, Pantidos et al. (2014) found that a tannin–rich fraction of A. nodosum obtained with different solvents was more effective inhibiting porcine pancreas α–amylase than rat intestinal α–glucosidase. Divergences might be ascribed to the different composition of the extracts, which depends on the extraction method. Zhang et al. (2007) determined an IC50 μg/mL against α– glucosidase working with water–ethanol A. nodosum extracts. Although, Liu et al. (2016) determined IC50 from . to 3 .3 μg/mL against α–glucosidase from ethanolic extracts of A. nodosum in the same range than those found in this work. Nevertheless, discussion of results based on previously published data becomes complicated due to the wide range of experimental conditions used which makes it very difficult to compare IC50 values, which are specific for the enzyme type, the substrate used, and the reaction conditions. 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 251 of 353 Table 11.2. IC50 values of seaweed extracts against α–amylase and α–glucosidase of the different analyzed methodologies: extract + enzyme (M1), extract + gelatinized starch (M2), gelatinization of starch with extract (M3). Means within a column followed with different letter were significantly (p < 0.05) different. IC50 μ α–amylase α–glucosidase A1 A2 A3 G1 G2 Acarbose 11.5 ± 0.6a 56.1 ± 3.0a 142.8 ± 12.1a 0.2 ± 0.01a 0.1 ± 0.01a P90 40.0 ± 1.1b 74.0 ± 2.6b 810.5 ± 24.3b 3.2 ± 0.1b 3.6 ± 0.2b E90 119.6 ± 0.7c 309.2 ± 4.1c 4066.3 ± 50.1c 19.5 ± 0.7c 26.2 ± 0.8c E80 128.8 ± 2.9d 342.6 ± 0.7d 4451.0 ± 49.2d 20.3 ± 0.3c 28.1 ± 0.04d E70 152.9 ± 4.3e 439.8 ± 9.8e 4777.7 ± 42.5e 29.2 ± 0.4d 30.4 ± 0.7e Given the influence of the procedure carried out to analyze enzyme inhibition, besides the sonication power during extraction and purification step, two additional methodologies (M2 and M3) were applied to identify the effect of polyphenols interaction with the enzymes on the level of inhibition. The most effective inhibition was obtained when the extracts were previously incubated with the enzyme (M1) (Figure 11.2), where the interaction between polyphenols and enzyme was favored before substrate (starch or maltose) addition for α– amylase (A1) or α–glucosidase (G1), respectively. Conversely, higher IC50 values were required with M2 when the extract was previously mixed with gelatinized starch for α–amylase (A2) (IC50 = 74.0, 309.2, 342.6, and 439.8 μg/mL for P90, E90, E80, and E70, respectively) or with maltose for α–glucosidase (G2) (IC50 = 3.6, 26.2, 28.1, and 30.4 μg/mL for P90, E90, E80, and E70, respectively) (Figure 11.2). These observations suggest that in the M2 method, the substrate hinders the polyphenol accessibility to the enzymes, increasing IC50 values. Pantidos et al. (2014) also observed lower effectiveness of a tannin–rich fraction, as porcine pancreas α– amylase inhibitor, when it was preincubated with gelatinized potato starch. Lordan et al. (2013) described porcine pancreas α–amylase inhibition (IC50 = 0.05 mg/mL) with A. nodosum extracts obtained from a three–stage process of at least 3 h duration, like the one obtained with P90 extract in M2. 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 252 of 353 Similarly, to the previously commented polyphenol–enzyme binding, phenolic compounds can bind starch by non–covalent interactions, modulating starch digestion kinetics (Giuberti et al., 2020). This lower inhibition effect when the extract was preincubated with the starch highlights the significance of the hydrolysis kinetics during in vitro analysis and subsequently to the in vivo studies, where pancreatic α–amylase and intestinal α–glucosidase both are secreted into the gut lumen, where they would meet the seaweed extract and the starch mixture. Furthermore, the inhibitory effect against α–amylase dramatically decreased when extracts were added to native starch, and the blend was subjected to high temperatures to gelatinize the starch (A3) (Figure 11.2). IC50 values varied between 810.5 (P90) and 4777.7 (E ) μgDW/mL. Nevertheless, in this case, besides the starch impediment previously mentioned for A2, polyphenols and starch interaction during the gelatinization process or polyphenols stability might be considered. Wu et al. (2011) described the existence of hydrogen bonding interaction between tea polyphenols and rice starch during gelatinization. On the other hand, high temperature can affect the stability of polyphenols and antioxidant activity (Moreira et al., 2016), therefore affecting its ability as enzyme inhibitors. Betoret & Rosell (2020) analyzed the effect of temperature (70, 80, and 90 ºC for 20 min) on phenolic compounds of Brassica napobrassica blended with maize and rice starches. These authors reported the protective role of starch with phenolic compounds in which the high apparent viscosity might contribute to protect the bioactive compounds. Authors linked the changes of bioactive compounds after thermal treatments with thermal degradation, matrix un–structuring effect and interaction with other ingredients, protecting them from degradation. Nevertheless, to better understand the interactions between A. nodosum bioactive compounds and digestive enzymes additional techniques, such as scanning electron microscopy, Fourier transform infrared spectrophotometry or X–ray diffraction, would provide valuable information. 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 253 of 353 Figure 11.2. Inhibitory capacity of purified (P90) and raw (E90, E80 and E ) seaweed extracts against α– amylase (A = A1; C = A2 and E = 3) and α–glucosidase (B = G1 and D = G2). 2 1 . . 5 .1 .15 .2 .25 P E E E 5 2 1 1 2 32 P E E E 5 2 1 . .1 .2 .3 . .5 P E E E 5 2 1 12 2 3 P E E E 5 2 1 2 1 P E E E 5 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 254 of 353 11.5. CHROMATOGRAPHY AND SPECTROSCOPIC ANALYSIS Crude (E90, E80 and E70) and purified (P90) polyphenolic extracts were chromatographically analyzed using phloroglucinol as standard. Chromatograms showed a unique peak (Figure 11.3) around 60 min of retention time, with no signals around 40 min that was the retention time of the standard used (i.e., phloroglucinol), concluding that the compounds detected had higher molecular mass. This result was expected since phloroglucinol or very small oligomers does not accumulate in the A. nodosum tissues, owing to the rapid polymerization reactions (Tierney et al., 2014; Sardari et al., 2021). Standardized phlorotannins chromatographic characterization methodology is limited since commercial standards are scarce. Very small peak signals located up 20 to 40 min of retention time show the presence of polysaccharides, in accordance with additional carbohydrates and UA standards (glucose and D (+)–guluronic acid). No significant (p < 0.05) differences on chromatographic spectra of crude extracts (E90, E80 and E70) were observed, indicating phlorotannin structure differences were not detectable with RP–HPLC technique. The increase in TPC value of P90, measured by Folin–Ciocalteu, was also confirmed by HPLC analysis, where 50 µL of 2.5 mgFD/mL of P90 presented a similar peak than 50 µL of 5.0 mgDW/mL from E90 extract. Figure 11.3. RP–HPLC profiles of purified (P90, 2.5 mgFD/mL) and crude (E90, E80, E70, 5.0 mgFD/mL) aqueous extracts obtained by UAE from A. nodosum seaweed. P E E E tR 5 . min 1. .s tR 5 . min 1. .s tR 5 . min 2 . .s tR 5 . min 2 . .s 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 255 of 353 Chromatographic results indicated that all UAE extracts contain polyphenols with high polymer size without significant (p < 0.05) presence of oligomers (expected at shorter times). Hence, differences in enzyme inhibition activities among extracts were not explained by HPLC results. Other authors have previously reported that HPLC method was ineffective separating large polymeric phlorotannins from A. nodosum (Tierney et al., 2014), and needs to be complemented with mass spectroscopy and/or NMR techniques (Koivikko et al., 2007). Figure 11.4. 1H–NMR spectra in D2O of freeze–dried extracts purified (P90) and crude (E90, E80 and E70) obtained by UAE from A. nodosum seaweed. An adequate method for identifying and quantifying purified polyphenols is 1H–NMR, but it is only qualitative in complex mixtures, since isomers appear at approximately the same chemical shifts. Nonetheless, 1H–NMR analysis could give a proximate insight of overall structure of the assayed extracts and understand inhibition results. Figure 11.4 shows 1H–NMR spectra of purified (P90) and crude UAE (E90, E80 and E70) extracts from A. nodosum seaweed. E E E P f1 (ppm) 11. Enzyme-inhibiting activity of Ascophyllum nodosum extracts Page 256 of 353 Three different ranges could be identified at 5.20–5.40 (A), 5.75–6.40 (B) and 6.40–6.55 ( ) ppm in tested samples, which agree with seaweed’s polyphenols signals reported between 5.0 and 6.5 ppm (Audibert et al., 2010; Gager et al., 2020; Susano et al., 2021). In addition, 1H– NMR spectra of crude UAE extracts showed below 5.6 ppm a notorious increasing signal associated with the presence of UA and carbohydrates (Zhang et al., 2004). Polyphenols extracted from brown seaweed are composed of phloroglucinol moieties linked by a mix of aryl–aryl and aryl–ether bonds (Choi et al., 2014). Hydrogens located near to aryl–ether bonds showed values around 6.5 ppm (C–region), meanwhile the aryl–aryl bonds signals around 5.7 to 6.3 ppm (B–region) (Choi et al., 2014; Fukuyama et al., 1990; Kim et al., 2019). This was also corroborated by phloroglucinol standard molecule spectra that showed a peak at 5.85 ppm from aryl–aryl bonds. A–region signal has been related to the presence of quinones derived from partial oxidation of polyphenols (Dobado et al., 2011). In fact, this signal dramatically decreased with sonication power, and disappeared in P90. This trend could be associated to the use of high power increased the lixiviation of less oxidated polyphenols from inner cell– structures (i.e., physodes) (Koivikko et al., 2007) and, oppositely, when low power was employed surface polyphenols from cell–wall (more exposed to oxygen) were mainly extracted. All extracts presented B–region signals and sonication power seemed to only change overall shape. Several peaks observed in this region evidenced the presence of different isomers of phlorotannins extracted during UAE treatment. Prominent signal in C–region for E70 and E80 was observed in comparison to low signal in E90, meaning that C–region signal decreased with increasing sonication power. After Amberlite purification process (P90), this signal practically disappeared. Chemical composition of extracts (Table 11.1) showed a noticeable reduction of UA/TPC ratio for P90 regarding to E90. Based on these results, it is hypothesized that signals of C–region could be indicative of the presence of some polyphenolic complexes, mainly with uronic acids. The gradual signal reduction with increasing sonication power during UAE could be related to the disruption of these complexes, that could increase polyphenol availability for enzymes inhibition. Then, NMR spectra and in vitro inhibitory activities suggested that polyphenols–UA complexes are present on the extracts. The observed trend of digestive enzyme inhibitory activities of the extracts (Table 11.1) was associated with the higher presence of “free” polyphenols (not complexed) that interacted more easily with α–amylase and α–glucosidase enzymes during in vitro inhibitory assays. 12. Alginate obtained from phlorotannins extraction solid residue Page 263 of 353 Alginates have several industrial applications (foods, dye, biomaterials, medical or pharmaceutical) by their viscosity properties (Uludag et al., 2000). However, polyphenols and other colored compounds (chlorophyl, carotenoids) from algae produce a brown discoloration, during alginate extraction, resulting in an unpleasing dark sodium alginate powder. Bleaching pre–treatments fix these colored compounds to algae tissues reducing brown discoloration, but critically affect the rheological, chemical, and structural features of alginates by modification of molecular weight and M/G ratio (Mohammed et al., 2020). Nowadays, there is an increasing interest in isolating polyphenols for their own unique bioactivity characteristics from algae (Cotas et al., 2020). A suitable strategy for the transformation of current single–product manufacturing process into biorefineries would be the sequential extraction. Polyphenols, promising bioactive molecules above mentioned in Section 1.4 up to 1.6 and, secondly, alginates. To preserve the polyphenols features and to enhance extraction yields, the use of ultrasound–assisted extraction is a promising, efficient, low–cost, and eco–friendly technology (Kadam et al., 2015a, b and c). The novelty presented in Section 12 was to contribute to the integral valorization of brown seaweeds, by means of the use the solid residue, after the ultrasound–assisted extraction (UAE) of polyphenols from AF seaweed, that still contains other active components, such as alginates. Consequently, our aim was to determine the effect of the process (existence of a drying stage) and operational conditions (drying temperature) on the physicochemical properties of alginates. Alginates were characterized by FT–IR, 1H–NMR, and capillary viscometry. 12.2. ASSAYED SYSTEMS Ultrasound–assisted extraction (UAE) of polyphenols (Section 4.4) from A. nodosum renders a solid phase, which alginates can be extracted from (Section 4.4.3). In this section alginates extracted after the UAE process, with and without an intermediate drying stage at different temperatures (50 and 90 ºC) were characterized in order to valorize UAE residue generated from all the previous Sections 6 up to 11. FT–IR (Section 4.11) and 1H–NMR (Section 4.13.1) analyses showed the high purity of alginates with features in the range of commercial alginates. Drying UAE residues at different temperatures had a notorious effect on average viscosimetric molecular size (Section 4.12) of alginate. 12. Alginate obtained from phlorotannins extraction solid residue Page 264 of 353 Tested process conditions allow the valorization of a residue to produce alginates with different properties together with the possibilities of tune their properties in order to accomplish with different characteristics depending on their future uses. The scheme of the research carried out could be seen in Figure 12.3 Figure 12.3. Scheme of process used to extract sodium alginate from UAE residues. lcoholic extraction queous Ultrasound ssisted Extraction Polyphenols enriched extracts Solid residues rying ( ) 5 (5 ) ot dried ( ) a l treatment cidic treatment l ali treatment scoph llum nodosum flour P s PP S U FR P F IR iscosimetric molecular size MR 12. Alginate obtained from phlorotannins extraction solid residue Page 265 of 353 12.3. EXTRACTION YIELD The alginates extraction yield values, calculated by Eq. (4.6), from UAE undried (ND) and dried solids (50D and 90D) were significantly (p < 0.05) different, being their values 20.3 ± 0.9%, 23.7 ± 0.5% and 18.3 ± 0.4%, respectively. Maximum yield was achieved after drying of pellet at 50 ºC and is comparable to that reported (24%) by Rioux et al. (2007) working with Ascophyllum nodosum (A. nodosum) and is also in the range (from 20 to 41%) reported for other brown seaweeds (Chee et al., 2011). The yield after drying at 90 ºC dramatically decreased due to probably alginates losses from solid (by solubilization) during processing. The low yield at rt may be justified by a dilution effect due to the presence of the water that initially accompanies the solid and that diminishes the effect of the treatments. 12.4. FOURIER TRANSFORMED INFRARED FT–IR spectra corresponding to extracted alginates from UAE undried (ND) and dried (50D and 90D) solids and CS were shown in Figure 12.4. Sodium alginates presented the characteristic bands up 4000 to 400 cm–1 range (Gómez–Ordóñeez & Rupérez, 2011). The peak between 3600 and 3200 cm–1 corresponded to stretching vibrations of O–H bonds. The 2930 cm–1 peak was stretching vibrations of C–H bonds. These peaks at 4000–2000 range are widespread reported from polysaccharides (El Atouani et al., 2016). The peak range 1680–1620 cm–1 was ascribed to asymmetric stretching vibrations of carboxylic groups. The 1415 cm–1 peak corresponded to deformation vibrations of C–OH bonds and asymmetric stretching vibrations of –C(=O)–O bonds (Mohammed et al., 2020). Around 1300 cm–1 peak appeared in the CA, but not in the A. nodosum extracted alginate. Literature refers this peak to S=O bond from sulfated polysaccharides such as fucoidans also extracted from marine brown seaweed (Gómez–Ordóñeez & Rupérez, 2011). The lack of this peak seemed to indicate the absence or low concentration of sulphated carbohydrates in the extracted alginates (Blanco–Pascual et al., 2014). Elemental analysis results (CHNS) confirmed this result, where higher sulfur content (0.09%) in CA than in 50D, 90D and ND samples (0.05, 0.01 and 0.02%, respectively) was determined. 12. Alginate obtained from phlorotannins extraction solid residue Page 266 of 353 The observed peak in the range 1091–1031 cm–1 corresponded to deformation vibrations of C–C–H and O–C–H bonds, stretching vibrations of C–O bonds, and stretching vibrations of C–O and C–C bonds, present in alginate pyranose rings. The 1030 cm–1 was promoted by stretching vibrations of C–O bonds (Gómez–Ordóñez & Rupérez, 2011). Three zones were observed in the range 950–750 cm–1: firstly, 950 to 930 cm–1, corresponding to C–O bonds stretching vibrations from uronic acid residues, secondly, 870 to 883 cm–1 corresponding to deformation vibrations of C–H bonds from mannuronic acids, and finally, 815–833 cm–1 region to stretching vibration also to C–H bonds (El Atouani et al., 2016). Figure 12.4. FT–IR spectra of commercial alginate from Sigma (CS) and extracted alginate from Ascophyllum nodosum sonication residue not dried (ND) and dried at 50 (50D) and 90 ºC (90D). 12 1 2 2 2 32 3 12. Alginate obtained from phlorotannins extraction solid residue Page 267 of 353 No significant (p < 0.05) differences were observed between 50D, 90D and ND samples and minor differences with commercial alginates CS were observed, due to probably different seaweed species origin. Alginates extracted from solid subjected previously to UAE for polyphenols extraction of AF seaweeds showed structural features like commercial alginates. It was also corroborated by alternative seaweeds F. vesiculosus and B. Bifurcata with no significant (p < 0.05) number and intensities within peaks. Further, the absence of additional peaks indicated a low level of impurities. This evidence was also supported by CHNS elemental analysis were pollutants as nitrogen or sulfur were under 1% and 0.1%, respectively. 12.5. NUCLEAR MAGNETIC RESONANCE Structural features (monads, diads and triads) of commercial (CS) and extracted alginates from A. nodosum residue after polyphenols UAE that was undried (ND), dried at 50 ºC (50D) and 90 ºC (90D) evaluated from 1H–NMR spectra (Figure 12.5) were summarized in mannuronic content (FM) of extracted alginates varied in a very narrow range (0.55 to 0.57) independently of drying conditions. Alginates from A. nodosum are characterized by their high mannuronic content in comparison to other seaweeds (Draget et al., 2006). Homopolymer diads blocks of samples presented higher FMM values around 0.30–0.33, than FGG values around 0.17– 0.20. Heteropolymer blocks (FGM or F AF) content ranged from 0.23 to 0.26. FGGG triads content ranged from 0.06 (ND) to 0.11 (AF 90D), FMGM and FGGM did not show differences among extracted samples being around 0.15 for extracted alginates. Average block size (NG>1) varied from 1.97 (90D) up to 2.15 (ND). Dried samples (50D and 90D) shower lower NG>1 values than undried (ND) sample. M/G ratio varied from 1.21 (50D) to 1.33 (90D and ND). As expected, these results indicated that drying of UAE residue did not modify significantly to monads, diads and triads distribution of the alginates, however NG>1 values seemed to decrease by increasing drying temperature. Thus, drying conditions must be carefully controlled to modulate final polymer structural features. Alginates with M/G ratios higher than one are suitable to produce elastic gels for food, cosmetical or pharmaceutical applications (Murillo– Álvarez & Hernández–Carmona, 2007). 12. Alginate obtained from phlorotannins extraction solid residue Page 268 of 353 In comparison to commercial alginate (CS), extracted A. nodosum alginates showed higher mannuronic content (around 0.56) than CS (0.48). FMM homopolymer content was similar, FMM content of CS (0.35) was notoriously higher, and FGM or FMG (0.18) lower. FGGG content was clearly higher for CS (0.30) than those determined for extracted alginates (0.09) and FMGM and FGGM showed minor differences from 0.12 to 0.15. NG>1 value (3.30) of CS was notoriously higher than those of extracted alginates (< 2.15). Finally, M/G ratio of CS was lower than one (0.91) due to glucuronic fraction was predominant above mannuronic molecules. Figure 12.5. NMR spectra in D2O of commercial alginate from Sigma (CS) and extracted alginate from A. nodosum sonication residue not dried (ND) and dried at 50 (50D) and 90 ºC (90D). The characteristics of the obtained alginates were compared with those previously reported by other authors for alginates from A. nodosum. Authors employed different methods and conditions that are necessary to specify to compare results appropriately. In fact, Yuan & Macquarrie (2015) isolated alginates involving several steps at temperatures above 70 ºC during short periods, however, the final alginates showed similar features to extracted alginates in this work. Rioux et al. (2007) characterized dialyzed alginates (>1000 Da) with high average molecular weight. This fact could explain some differences with alginates studied here. 5 S 12. Alginate obtained from phlorotannins extraction solid residue Page 269 of 353 Mannuronic content (FM) showed similar values and FMM values were slightly lower than those (from 0.38 to 0.39) reported by consulted literature (Donati & Paoletti, 2009; Yuan & Macquarrie, 2015) summarized in Table 12.1 Nevertheless, FMM values were slightly higher than value (0.28) given by Rioux et al. (2007). FGG and FGM values agreed to ranges reported by other authors (Table 12.1). Regarding triads, the values were generally in accordance with ranges previously reported: FGGG and FMGM values agreed and FGGM value was slightly higher than that (0.07) reported by Donati & Paoletti (2009). NG>1 values were lower than data (5.0) found by Donati & Paoletti (2009). Higher value of M/G ratio (1.44) was reported than the range (1.22–1.33) determined from extracted alginates in this study (Table 12.1). Average block size differences could be related to different extraction methods and conditions (temperature time) employed (Yuan & Macquarrie, 2015). Also, they can be attributed to seasonal variations and geographical origin of the seaweeds (Tabassum et al., 2016). Table 12.1. Structural features of commercial alginate (CA) and extracted alginates from and A. nodosum undried (ND) and dried at 50 (50D) and 90 ºC and (90D) and comparison with alginates of other authors. Thesis Yuan & Macquarrie (2015) Donati & Paoletti (2009) Rioux et al. (2007) Seaweed Unknown A. nodosum A. nodosum A. nodosum A. nodosum Alginate CS ND 50D 90D FM 0.48 0.57 0.55 0.57 0.59 0.59 0.46 FG 0.52 0.43 0.45 0.43 0.41 0.41 0.54 FMM 0.30 0.31 0.30 0.33 0.39 0.38 0.28 FGG 0.35 0.17 0.20 0.20 0.21 0.22 0.36 FGM 0.18 0.26 0.25 0.23 0.21 0.21 0.18 FGGG 0.30 0.06 0.10 0.11 0.13 FMGM 0.12 0.14 0.15 0.15 0.14 FGGM 0.12 0.14 0.15 0.15 0.07 NG>1 3.30 2.15 2.07 1.97 5.00 M/G 0.91 1.33 1.21 1.31 1.44 1.44 1.44 where mannuronate (M) and guluronate (G); NG>1: average block length; FM: M fraction; FG: G fraction; FMM, and FGG: homopolymer fraction diads; FGM: heteropolymer fraction diads; FGGG and FMMM homopolymer fraction triads; FMGM and FGGM: heteropolymer fraction triads. 12. Alginate obtained from phlorotannins extraction solid residue Page 270 of 353 12.6. AVERAGE VISCOSIMETRIC MOLECULAR WEIGHT Average alginate viscosimetric molecular weight (Mv) decreased significantly (p < 0.05) with the existence of a drying step and with increasing drying temperature. Mv values (kDa), obtained for extracted alginates from A. nodosum, varied in a wide range from 133.30 ± 1.20 (90D) and 427.80 ± 7.20 (ND). These results indicated clearly that molecular size of extracted alginates can be modulated by the use and conditions employed during drying conditions. No significant (p < 0.05) differences between 90D and CS (156.1 ± 1.5 kDa). Despite different seaweed origin and other aspects, it could be probably related to the industrial conditions employed during extraction process. The molecular weights obtained are within the usual ranges reported for commercial alginates (Dodero et al., 2020a). Similarly, Chen et al. (2021) determined molecular weights of several brown seaweeds (Sacharina japonica, Undaria pinnatiufida, Sargassum fusiforme and Sargassum hemiphyllum) ranging from 171.8 to 663.0 kDa. HIGHLIGHTS The integral use of seaweeds was achieved with a sequential extraction of phlorotannins by UAE, followed by a controlled drying of solid residue and the subsequent extraction of alginates. Drying temperature after ultrasound–assisted extraction of phlorotannins affected the extraction yield and rendered alginates with different average block length (from 1.97 up to 2.15) and average viscosimetric molecular weight (from 133.3 up to 257.3 kDa). Drying could be used to modulate alginate’s features producing sodium alginates with different and tuneable properties. . Page 271 of 353 13. CONCLUSIONS 14. List of figures Page 279 of 353 Figure 1.1. Seaweed color classification, main characteristics and representative species. Figure 1.2. Phlorotannins bioactive capacities. Figure 1.3. Polyphenol’s classification. Figure 1.4. Proposed matrix of brown seaweed cell–walls. Figure 1.5. Phlorotannin main groups, all derived from phloroglucinol polyketide pathway reaction Figure 1.6. Ascophyllum nodosum (Linnaeus) Le Jolis seaweed (A), fronds (B) and flotation vessels (C) from Algaebase database. Figure 1.7. Geographical distribution of A. nodosum seaweed from GBIF database, (2022). Figure 1.8. Average composition of A. nodosum brown seaweed. Figure 1.9. Industrial potential of seaweeds. Figure 1.10. Classification of antioxidants. Figure 1.11. Diagram of celiac disease iceberg. Figure 3.1. Phlorotannins extraction methods. Figure 3.2. Scheme of the coetaneous processes in SLE of compounds from seaweed cell. Figure 3.3. Ultrasound–assisted extraction process. Figure 3.4. Summary of analytical methods often used for characterization of seaweed biopolymers Figure 3.5. Antioxidant activity determination methods. Figure 3.6. Stages in the development of functional foods using seaweed as source of phlorotannins, from a multidisciplinary point of view. Figure 3.7. Starch gelatinization process and their microstructure changes. Figure 3.8. Global process of starch metabolic fate. Figure 4.1. A) Angelantoni Challenge 250 Air convective dryer (Italy), B) Laboratory blender Waring HGBTWT (USA), C) Ultra–centrifugal mill Retsch GmbH, ZM200 (Germany), D) Vibratory sieve FTL 0200, Cisa (Spain) and E) Vacuum packer Sammic V201 (Spain). Figure 4.2. A) Vacuum oven Vacutherm VT650 (Heraeus Hanau, Germany). Figure 4.3. Experimental set up for the continuous aqueous ultrasound–assisted extraction of biopolymers from A. nodosum seaweed. Figure 4.4. High–speed laboratory centrifuge Retsch GmbH, ZM200 (Germany, A) and Telstar LyoQuest lyophilizer –55, Telstar Technologies (Spain, B). Figure 4.5. Concentrator plus/Vacufuge plus, Eppendorf (Germany) 14. List of figures Page 280 of 353 Figure 4.6. Color change occurred during colorimetric assays. Figure 4.7. TPC calibration curve. Figure 4.8. Total polyphenol content determination methodology. Figure 4.9. CHOs calibration curve. Figure 4.10. Total carbohydrate content determination methodology. Figure 4.11. UA calibration curve. Figure 4.12. Uronic acids content determination methodology. Figure 4.13. DPPH calibration curve. Figure 4.14. DPPH scavenging activity determination methodology. Figure 4.15. ABTS calibration curve. Figure 4.16. ABTS scavenging activity determination methodology. Figure 4.17. FRAP calibration curve. Figure 4.18. Electron donor capacity (FRAP) determination methodology. Figure 4.19. Scheme of A. nodosum aqueous extracts dialysis. Figure 4.20. Elution gradients used during RP–HPLC analysis of A. nodosum extracts, based on gradient mode (A) and on–off mode (B). Figure 4.21. Average 1H–NMR spectrum shape of alginate used for quantitative analysis. Figure 4.22. Process scheme summarizing methodology. NT method: AF (seaweeds flour) and corn starch (CS) blending; GL method: AF and CS blending, CGL method: CS gelatinized in presence of AF. Figure 4.23. Methodology scheme of α–amylase inhibition assays ( 1, 2 and 3) and α– glucosidase (G1 and G2) digestive enzymes against aqueous polyphenols extracts from A. nodosum seaweed. Figure 5.1. General scheme of the Results & Discussion section in the current Thesis. Figure 6.1. A nodosum flour obtention, drying, grounding, milling pretreatments. Figure 6.2. Experimental kinetics of Ascophyllum nodosum seaweed drying at dry temperature of air at 50 ºC, 30% relative humidity, 2 m/s of air velocity and 2 kg/m2 of load density. Figure 6.3. Differential (A) and accumulated particle size (B) distribution curve from A. nodosum flour after drying and milling. Figure 7.1. A. nodosum extracts characterization to evaluate extraction method, solid–liquid extraction, and ultrasound–assisted extraction. 14. List of figures Page 281 of 353 Figure 7.2. Experimental data (dots) and Peleg’s model values (lines) for P ( ), s ( ) UA (C), DPPH (D), ABTS (E) and FRAP (F) of SLE kinetics using water (D) and saltwater (S) for extractions at 20, 30 and 40 gW/gAF of liquid–solid ratio. Figure 7.3. TPC (A), CHOs (B), UA (C), DPPH (D), ABTS (E) and FRAP (F) values of UAE (sonication power of 90, 80 and 70 W/cm2) and SLE using D– and S–solvents at 15 min of extraction with 20, 30 and 40 gW/gAF of LS ratios. Different superscript letters above bars mean significant (p < 0.05) differences between values. Figure 7.4. Linear relationships between TPC and antioxidant activities, DPPH (A), ABTS (B) and FRAP (C), from SLE and UAE (70, 80 and 90 W/cm2) extracts using D– and S– solvents at 15 min of extraction with 20, 30 and 40 gW/gAF LS ratios. Figure 7.5. RP–HPLC profiles (LS = 20 gW/gAF) of SLE (5 and 15 min) and UAE (15 min) at 90 W/cm2, extracts using D– (A, B and C), and S–solvents (D, E and F). Figure 7.6. Evolution of normalized area values during SLE (LS = 20 gW/gAF) from RP–HPLC profiles using D– and S–solvent, of A–, B– and C–regions. Figure 7.7. Normalized area values from the SLE (LS = 20 gW/gAF) extracts (2 to 60 min) from D– and S–solvents of A–, B–, C–regions and total correlated with measured antioxidant activities (DPPH, ABTS and FRAP). Figure 8.1. Box–Behnken experimental designing in order to optimize and set up ultrasound– assisted extraction of A. nodosum bioactive compounds. Figure 8.2. Response surface plots for A, B polyphenols (TPC, gPE/L); C, D carbohydrates (CHOs, gGE/L) and E, F uronic acids (UA, gGE/L) content vs studied variables at constant sonication power (SP, W/cm2) or time residence (tR, min): A, C SP = 70 W/cm2, E SP = 80 W/cm2, B SP = 90 W/cm2, D tR = 2 min and F tR = 4 min. Box–Behnken (black box) and additional points (white star). Figure 8.3. Linear correlations of antioxidant activities determined by DPPH (A), ABTS (B) and FRAP (C) methods, against TPC for UAE from A. nodosum. Figure 9.1. Systems experimentally obtained to evaluate the influence of oxidation and dialysis of phlorotannins on bioactivity and composition of ultrasound–assisted extracts from Ascophyllum nodosum. Figure 9.2. Phytochemical and antioxidant activity (TPC, A; CHOs, B and DPPH, C) of crude (EA and EW), purified (PW), oxidized (PON, POA and POP) and dialyzed, (PD20, PD10, 14. List of figures Page 282 of 353 PD3 and PD2) extracts. Different superscript letters indicate significant (p < 0.05) differences among values. Figure 9.3. Distribution percentage of TPC, DPPH and CHOs values among dialyzed extracts. Figure 9.4. RP–HPLC profiles of (A) standard compounds (phloroglucinol, resorcinol, glucose, glucuronic acid and BSA); (B) crude and purified (EA, EW, and PW), (C) oxidized, (PON, POA and POP) and dialyzed (PD20, PD10, PD3 and PD2) extracts. Figure 9.5. FT–IR spectra of crude (EA and EW), purified (PW), oxidized (POP) and dialyzed (PD20 and PD2) extracts. Figure 9.6. 1H–NMR (DMSO–d6) spectra of crude (EA and EW), purified (PW), oxidized (POP) and dialyzed (PD20 and PD2) extracts. Figure 9.7. MALDI–TOF–MS spectra of purified (PW) and dialyzed extracts (PD20 and PD2). Figure 10.1. Schematic process used to determine A. nodosum seaweed interactions with corn starch based on different mixing procedures. Figure 10.2. TPC values (A), sorption yields, YP (B), and CHOs values (C) of AF ( ), CS (native ( ) and gelled ( ) control samples and AF–CS blending methods of NT ( ), GL ( ) and CGL ( ) samples. Different letters indicate significant (p < 0.05) differences among samples. Figure 10.3. Adsorbed/retained polyphenols by CS, q, vs TPC of aqueous phase for AF–CS samples (NT, GL, and CGL) at 20 ºC. Lines are Halsey model (Eq. (10.1)). Figure 10.4. Antioxidant activities determined by DPPH (A), ABTS (B) and FRAP (C) against TPC in control (AF) and AF–CS samples (NT, GL, and CGL, methods). Figure 10.5. FT–IR spectra of control (AF, , and CS, ) and AF–CS (1:1 ratio) samples (NT, , GL, , and CGL, ). Figure 10.6. Cross–sectional morphologies of solid phase from AF (A), CS (B) and AF–CS (1:1 ratio) samples (NT (C), GL (D) and CGL (E)). Figure 11.1. Assayed systems for the determination of the digestive enzyme inhibitory capacities of A. nodosum extracts Figure 11.2. Inhibitory capacity of purified (P90) and raw (E90, E80 and E70) seaweed extracts against α–amylase (A = A1; C = A2 and E = 3) and α–glucosidase (B = G1 and D = G2). Figure 11.3. RP–HPLC profiles of purified (P90, 2.5 mgFD/mL) and crude (E90, E80, E70, 5.0 mgFD/mL) aqueous extracts obtained by UAE from A. nodosum seaweed. 14. List of figures Page 283 of 353 Figure 11.4. 1H–NMR spectra in D2O of freeze–dried extracts purified (P90) and crude (E90, E80 and E70) obtained by UAE from A. nodosum seaweed. Figure 12.1. Molecular structure of sodium alginate polymer (A), their monomers α–L– guluronate (G, B) and β–D–mannuronate (M, C) and diad structures (G–, M– and GM– blocks). Figure 12.2. Alginate fibers precipitation by addition of ethanol. Figure 12.3. Scheme of process used to extract sodium alginate from UAE residues. Figure 12.4. FT–IR spectra of commercial alginate from Sigma (CS) and extracted alginate from Ascophyllum nodosum sonication residue not dried (ND) and dried at 50 (50D) and 90 ºC (90D). Figure 12.5. NMR spectra in D2O of commercial alginate from Sigma (CS) and extracted alginate from A. nodosum sonication residue not dried (ND) and dried at 50 (50D) and 90 ºC (90D). Page 285 of 353 15. LIST OF TABLES 15. List of tables Page 287 of 353 Table 1.1. Reactive species. Table 1.2. Current recognized as safe and edible brown edible seaweeds (FAO, 2018). Table 1.3. Overview extraction conditions (method, extractant, liquid–solid ratio, time, and temperature) to obtain phlorotannins enriched extracts from Ascophyllum nodosum. Table 1.4. State of art of current phlorotannins isolated from brown seaweeds. Table 1.5. State of art of inhibitory capacities against α–amylase and α–glucosidase digestive enzymes of polyphenols from A. nodosum seaweed. Table 1.6. State of the art of inhibitory capacities against α–amylase and α–glucosidase digestive enzymes of polyphenols from alternative seaweeds. Table 1.7 State of art of inhibitory capacities against α–amylase and α–glucosidase digestive enzymes of polyphenols from terrestrial plants. Table 4.1. BB experimental design of UAE carried at different sonication power (SP), residence times (t) and liquid–solid ratios (LS). Table 7.1. Peleg’s model parameters ( 1 and K2) and Xeq = 1/K2 for extracts bioactive compounds content (TPC, CHOs and UA) and antioxidant activities (DPPH, ABTS and FRAP) for SLE kinetics using water (D) and saltwater (S) at 20, 30 and 40 gW/gAF. Table 7.2. Extraction yields (Eq. (4.5)) in SLE and UAE (SP = 90 W/cm2) samples of phytochemical compounds (TPC, CHOs, UA and antioxidant activities) from A. nodosum seaweed with 20, 30 and 40 gW/gAF of LS ratios using D– and S–solvents. Table 7.3. Polyphenols–carbohydrates and polyphenols–energy ratios from A. nodosum seaweed with 20, 30 and 40 gW/gAF of LS ratios using D– and S–solvents using SLE and UAE (SP = 90 W/cm2) extraction methods. Table 7.4. Ratio UAE/SLE of compound extraction (TPC, CHOs and UA) and antioxidant capacities of the extracts (DPPH, ABTS and FRAP) from A. nodosum seaweed with 20, 30 and 40 gW/gAF of LS ratios using D– and S–solvents using SLE and UAE (SP = 90 W/cm2) extraction methods. Table 7.5. Ratio D/S on compound extraction (TPC, CHOs and UA) and antioxidant capacities of the extracts (DPPH, ABTS and FRAP) from A. nodosum seaweed with 20, 30 and 40 gW/gAF of LS ratios using D– and S–solvents using SLE and UAE (SP = 90 W/cm2) extraction methods. 15. List of tables Page 288 of 353 Table 8.1. Experimental (Exp) and modeled (Mod) TPC (Eq. (8.1)), CHOs (Eq. (8.2)) and UA (Eq. (8.3)) values from aqueous extraction of A. nodosum seaweeds using UAE at different SP, tR and LS conditions following a BB design. Table 8.2. ANOVA for quadratic models of TPC, CHOs and UA content in the UAE aqueous extracts from A. nodosum seaweed. Table 8.3. TPC ratios in relation to CHOs, UA and consumed energy during for UAE of A. nodosum seaweeds at different conditions of SP, tR and LS. Table 9.1. Phloroglucinol units of dialyzed PD20 and PD2 extracts in comparison with extracts from brown seaweeds reported by bibliography. Table 9.2. List of phlorotannins identified in (Figure 9.7) and its water loss n[H2O] comparing data with Steevensz et al., (2012) & Kellogg et al. (2014). Superscript letters were used to identify different phlorotannins families within the extract Table 10.1. Polyphenol sorption yields (YP) ratios from NT, GL and CGL assayed methods. Table 10.2. Parameters of Halsey equation (Eq. (4.30)). Table 11.1. Chemical composition of freeze dried (E90, E80 and E70) and purified (P90) extracts obtained by UAE from A. nodosum brown seaweed. Table 11.2. IC50 values of seaweed extracts against α–amylase and α–glucosidase of the different analyzed methodologies: extract + enzyme (M1), extract + gelatinized starch (M2), gelatinization of starch with extract (M3). Table 12.1. Structural features of commercial alginate (CA) and extracted alginates from and A. nodosum undried (ND) and dried at 50 (50D) and 90 ºC and (90D) and comparison with alginates of other authors. 16. 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Journal Journal of Applied Phycology (2021) 33, 3297–3308; DOI: 10.1007/s10811–021– 02546–5; IF: 3.404, Q1: Marine and Freshwater Biology (18/113, 2021); Springer (ISSN: 1573-5176) Authors M. Gisbert1, M. Barcala1, C.M. Rosell2,3, J. Sineiro1, R. Moreira1 Affiliation 1. Chemical Engineering Department, Universidade de Santiago de Compostela, Campus Vida, Santiago de Compostela, Spain. 2. Institute of Agrochemistry and Food Technology, CSIC, Avenida Agustin Escardino 7, Paterna, 46980 Valencia, Spain 3. Department of Food and Human Nutritional Sciences, University of Manitoba,Winnipeg, Canada Authorship contribution: Conceptualization; Data curation; Formal analysis (characterization, response surface modelling, analysis of TPC ratios and antioxidant activity); Investigation; Methodology Software; Roles/Writing-original draft. Rights & Permissions This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your 17. Diffusion of results derived from the Thesis Page 344 of 353 intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Article (Used in Section 12) Impact of drying on the sodium alginate obtained after polyphenols ultrasound–assisted extraction from Ascophyllum nodosum seaweeds Journal Carbohydrate Polymers (2022) 272, 118455 DOI: 10.1016/j.carbpol.2021.118455 IF: 10.723, Q1: Polymer Science (3/90, 2021). ScienceDirect (ISSN: 0144-8617) Authors L. Montes, M. Gisbert, I. Hinojosa, J. Sineiro, R. Moreira Affiliation Chemical Engineering Department, Universidade de Santiago de Compostela, Campus Vida, Santiago de Compostela, Spain. 17. Diffusion of results derived from the Thesis Page 345 of 353 Contribution to the Article: Methodology, Validation, Formal analysis (extraction yields, FT-IR, 1H–NMR and average viscosimetric molecular weight), Investigation, Writing – original draft. Rights & Permissions This is an open access article distributed under the terms of the Creative Commons CCBY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article (Used in Section 10) Interactions between Ascophyllum nodosum seaweeds polyphenols and native and gelled corn starches. Journal Foods (2022) 11, 1165; DOI: 10.3390/foods11081165; IF: 5.561, Q1: Food Science & Technology (35/143, 2021). MDPI (ISSN: 2304-8158 ) Authors M. Gisbert1, A. Aleixandre2, J. Sineiro1, CM Rosell2,3, R. Moreira1 17. Diffusion of results derived from the Thesis Page 346 of 353 Affiliation 1. Chemical Engineering Department, Universidade de Santiago de Compostela, Campus Vida, Santiago de Compostela, Spain. 2. Institute of Agrochemistry and Food Technology, CSIC, Avenida Agustin Escardino 7, Paterna, 46980 Valencia, Spain 3. Department of Food and Human Nutritional Sciences, University of Manitoba,Winnipeg, Canada Contribution to the Article: Conceptualization, Data curation ; Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-original draft. Rights and permisions This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article (Used in Section 11) In vitro inhibition of starch digestive enzymes by ultrasound–assisted extracted polyphenols from Ascophyllum nodosum seaweeds Journal Journal of Food Science (2022) 87, 2405–2416; DOI: 10.1111/1750–3841.16202; IF: 3.693; Q2: Food Science & Technology (57/143, 2021); Wiley (ISSN: 1750-3841) 17. Diffusion of results derived from the Thesis Page 347 of 353 Authors A. Aleixandre1, M. Gisbert2, J. Sineiro2, R. Moreira2, CM Rosell1,3 Affiliation 1. Institute of Agrochemistry and Food Technology, CSIC, Avenida Agustin Escardino 7, Paterna, 46980 Valencia, Spain 2. Chemical Engineering Department, Universidade de Santiago de Compostela, Campus Vida, Santiago de Compostela, Spain. 3. Department of Food and Human Nutritional Sciences, University of Manitoba,Winnipeg, Canada Contribution to the Article: Data curation, formal analysis, methodology, and writing—original draft. Rights and permisions “This is an open access article under the terms of the Creative Commons AttributionNonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.” 17. Diffusion of results derived from the Thesis Page 348 of 353 Article (Used in Section 7) Polyphenols extraction kinetics from Ascophyllum nodosum seaweed employing sweet water and saltwater: Effect of ultrasound sonication. Journal Algal Research–Biomass Biofuels and Bioproducts (2022) 66, 10773; DOI: 10.1016/j.algal.2022.102773; IF: 5.276; Q2 Biotechnology & Applied Microbiology (40/158, 2021) ISSN: 2211-9264 Authors Gisbert M, Sineiro J, Moreira R. Affiliation Chemical Engineering Department, Universidade de Santiago de Compostela, Campus Vida, Santiago de Compostela, Spain. Contribution to the Article: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Software; Roles/Writing-original draft. Righsts and permisions “This is an open access article distributed under the terms of the Creative Commons CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.” 17. Diffusion of results derived from the Thesis Page 349 of 353 Article (Used in Section 9) Influence of oxidation and dialysis of phlorotannins on bioactivity and composition of ultrasound–assisted extracts from Ascophyllum nodosum. Journal Marine drugs (2022). DOI: 10.3390/md20110706 IF: 6.085; Q1 Pharmacology & Pharmacy (48/279, 2021); ISSN: 16603397. Authors Gisbert M, Sineiro J, Moreira R. Affiliation Chemical Engineering Department, Universidade de Santiago de Compostela, Campus Vida, Santiago de Compostela, Spain. Contribution to the Article: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Software; Roles/Writing–original draft Rights & permissions This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Página 351 de 353 The end I hope you have enjoyed reading this Thesis as much I did, writing it. I sincerely expect that you employ all these new knowledge in seaweeds, extractions, biopolymers, phlorotannins, alginates, enzyme inhibition and antioxidants wisely, because: “ ith great power comes great responsibilities” (Amazing Fantasy #15, 1962) Ascophyllum nodosum seaweeds was used as source of biopolymers. The ultrasound– assisted extraction was used to obtain extracts with high content of bioactive compounds with high antioxidant and enzyme inhibition activities. The physicochemical characterization of these extracts showed a high content of phlorotannins along with other substances (carbohydrates and uronic acids), being necessary subsequent purification steps. By means of gel chromatography and dialysis, extracts with an improved bioactivity were obtained. Extracts with the highest bioactivities were significant inhibitors of α–amylase and α–glucosidase digestive enzymes. Finally, alginates with modulated properties were also obtained improving the integral use of the seaweeds, after the extraction of phlorotannins. Results indicated that extracted biopolymers from Ascophyllum nodosum can be employed in gluten–free foods for celiac people and functional formulations.