2018

Estudio del alga verde Ulva para su uso en alimentación

funcional en acuicultura:
Desarrollo de procesos biotecnológicos para determinar la
bioactividad del
polisacárido sulfatado ulvan

Study of the green alga Ulva for its use as functional food in
aquaculture: Development of biotechnologic processes to
determine the bioactivity of the sulfated polysaccharide ulvan

Olivier B.N. COSTE

DIRECTORES:
DRA. CATALINA FERNÁNDEZ DÍAZ
DR. ERIK-JAN MALTA

TESIS PARA ACCEDER AL TÍTULO DE DOCTOR EN CIENCIAS

DOCTORADO EN RECURSOS MARINOS
ESCUELA INTERNACIONAL DE DOCTORADO EN ESTUDIOS DEL MAR (EIDEMAR

OLIVIER
Study of the green alga Ulva for its use as functional food in
aquaculture: Development of biotechnologic processes to
determine the bioactivity of the sulfated polysaccharide ulvan

Estudio del alga verde Ulva para su uso en alimentación funcional

en acuicultura: Desarrollo de procesos biotecnológicos para
determinar la bioactividad del polisacárido sulfatado ulvan.

Memoria presentada por D. Olivier Bernard Noël Coste para Optar

al grado de Doctor en Ciencias por la Universidad de Cádiz.

Fdo. Olivier Bernard Noël Coste

Los Directores:

Fdo. Dra. Catalina Fernández Díaz Fdo. Dr. Erik-jan Malta

Investigadora Titular IFAPA Investigador IFAPA
Los Doctores Dña. Catalina Fernández Díaz y D. Erik-jan Malta,
Investigadores del instituto de Investigación y Formación Agraria y
Pesquera de Andalucía (IFAPA) perteneciente a la Consejería de
Agricultura, Pesca y Desarrollo Rural de la Junta de Andalucía,
HACEN CONSTAR
Que la presente Tesis Doctoral titulada “Estudio del alga verde Ulva
para su uso en alimentación funcional en acuicultura: Desarrollo de
procesos biotecnológicos para determinar la bioactividad del
polisacárido sulfatado ulvan” que presenta D. Olivier Bernard Noël
Coste para optar al grado de Doctor en Ciencias, ha sido realizada
bajo su dirección, y autorizan su presentación y defensa.
Y para que conste a los efectos oportunos, firmamos la presente en
Puerto de Santa María a 23 de Marzo de 2018.

Fdo. Dra. Catalina Fernández Díaz Fdo. Dr. Erik-jan Malta

Investigadora Titular IFAPA Investigador IFAPA
Título: “Estudio del alga verde Ulva para su uso en alimentación funcional en
acuicultura: Desarrollo de procesos biotecnológicos para determinar la
bioactividad del polisacárido sulfatado ulvan.”
Año de publicación: 2018
Estudiante: Olivier B.N. COSTE
Tesis para acceder al Título de Doctor en Ciencias por la Universidad de Cádiz
Doctorado en Recursos Marinos
Directores: Tutor Académico:
Dra. Catalina Fernández Díaz Dr. Antonio Medina Guerrero
Dr. Erik-jan Malta
Centro de desarrollo:
Instituto de Investigación y Formación Agraria y Pesquera de Andalucía,
Centro El Toruño, El Puerto de Santa María, Cádiz, España
Escuela doctoral: Escuela Internacional de Doctorado en Estudios del
Mar (EIDEMAR)
Universidad: Universidad de Cádiz
Programa: Programa Recursos Marinos
Financiación: La presente Tesis Doctoral ha sido realizada en el marco
de los proyectos Interreg ECOAQUA (Fondos Feder); “Salty Gold/Het
Zoute Goud”, Stichting Innovatie Alliantie (SIA), Países Bajos; INIA
RTA2011-00032-00-0 y EI.AVA.AVA201301.5: Nuevas tecnologías para la
diversificación y la mejora de la Acuicultura en Andalucía; Agradecer la
financiación recibida sin los cuales no se podrían haber podido realizar
los trabajos experimentales.
Olivier Coste ha sido beneficiario de una beca-contrato pre-doctoral
perteneciente a un Programa de Formación de personal investigador
gestionado por el Instituto de Investigación Agraria y Pesquera de
Andalucía (IFAPA) y financiado por el Fondo Social Europeo.

I
II
Lista de Comunicaciones

Artículos Publicados:

 Olivier Coste, Erik-jan Malta, José Callejo López, Catalina Fernández Díaz.
Production of sulfated oligosaccharides from the seaweed Ulva sp. using a new
ulvan-degrading enzymatic bacterial crude extract. Algal Research 10 (2015) pp
224-231

 Catalina Fernández-Díaz, Olivier Coste, Erik-jan Malta. Polymer Chitosan

nanoparticles functionalized with Ulva ohnoi extracts boost in vitro ulvan
immunostimulant effect in Solea Senegalensis. Algal Research 26 (2017)
pp.135-142

Artículos Pendientes de envío:

 Olivier Coste, Erik-jan Malta, M. Teresa Jiménez Peral, Catalina Fernández Díaz:
Seasonal variation of proximate composition, ulvan content and sugar
composition of Ulva ohnoi (Chlorophyta, Ulvales) in relation to growth and
environmental parameters

 Olivier Coste, Erik-jan Malta, Catalina Fernández Díaz, Improvement of

antioxidant compounds extraction in the macroalgae Ulva ohnoi with hot acidic
methanol extraction method

Presentaciones:

 Olivier Coste. Active Compunds in Seaweeds and their application in Fish

Aquaculture Enhancing microalgae production for cosmetic industry. Growing
systems, harvesting and processing. Algaecom El Puerto de Santa María
September 9th-13th 2013

 Olivier Coste. Compuestos Funcionales a partir de macroalgas. Jornadas de

Transferencia en Acuicultura. 24 y 25 de Octubre del 2013, El Puerto de Santa
María (Cádiz)

III
  Olivier Coste, Erik-jan Malta, Catalina Fernández Díaz. Ulvan nanoparticles from
ulvan-lyase degraded polysaccharides and their activity in fish in vitro
immunomodulation. Alg’n’chem 2014. 31 March-3 April 2014- Montpellier-
France.

 Olivier Coste, Erik-jan Malta, Catalina Fernández Díaz. Valoración del

alga Ulva como fuente de componentes funcionales para la acuicultura.
Primeras Jornadas Doctorales Escuela EIDEMAR. 01 de Diciembre del 2015,
Cádiz

Comunicaciones a Congresos:

 Olivier Coste. Effects of elevated CO2 Levels on Growth and biochemical

composition of two seaweed species. 5th European Phycological Congress
September 4-9th 2011 Rhodes Island, Greece

 Olivier Coste, Erik-jan Malta, Catalina Fernández Díaz. Effects of Light Culture
Intensity on Growth, Polysaccharides Composition and Sulfate Contents of Ulva
Rotundata. Plant and Seaweed Polysaccharides Workshop. July 17-20 2012
Nantes- France

 Olivier Coste, Erik-jan Malta, Catalina Fernández Díaz. Valorisation of Green

Seaweed Ulva sp. Compounds as Functional Ingredient For Aquaculture Feed.
Aquaculture Europe. October 14-17th 2014 San Sebastián, Spain

Actividades formativas como formador:

 Olivier Coste. Cultivos Integrados: El papel de las macroalgas y su valorización.

Jornadas Ecoaqua: Fundamentos Tecnológicos y biológicos para una
acuicultura sostenible. 21 de Junio 2011, Punta Umbría, Huelva

 Olivier Coste: Las algas están por todos lados… Pero dónde? Fundación
Descubre: Café Con ciencia. 14 de Noviembre 2013. El Puerto de Santa María

IV
Estancias en centros de investigación:

 Préparation et purification d’oligosaccharides à partir de polymères d’Ulva.

Laboratoire des polysaccharides Microbiens et Végétaux, IUT Amiens,
Université de Picardie Jules Verne. Del 11 de Marzo 2013 al 7 de Junio 2013.
Supervisor : Pr. Josiane Courtois

 Alimentos funcionales para la Acuicultura: potencial de macroalgas

procedentes de sistemas integrados. Nutrition and Immunobiology Group.
Centro Interdisciplinar de Investigação Marinha e Ambiental de Porto,
Portugal. Del 8 de Octubre al 14 de Noviembre 2014. Supervisor: PhD.
Benjamín Costas

V
 AGRADECIMIENTOS

Quisiera agradecer en primer lugar a Catalina y Erik-jan, gracias por la paciencia

que habéis tenido conmigo. Gracias Lina por tu creatividad, tus consejos y tu

orientación. Gracias Erik por tu pasión por las algas y la ciencia, tu orientación y

tu apoyo incondicional. Es gracias a vosotros que estoy aquí.

Gracias a todo el IFAPA y especialmente los miembros del laboratorio de

nutrición del Toruño. Euge, te echo de menos a mi lado además de una gran

profesional eres una gran persona. María del Mar siempre nos hemos reído

mucho contigo y hoy me falta el toque “Gadita” que le ponías a mi cotidiano. Sir

Mac Edu, ha sido un placer y un honor estar a su lado, un gran caballero listo para

conquistar Escocia. Anina e Ismael habéis sido mis hermanos mayores, siempre

ahí cuando os necesitada. Javi, si llegas a leer estas palabras: eres un cra’ de

verda’! También dar las gracias a Manolo M., Manolo A., Pedro, Stef, José Luis (allá

dónde estés), Aniela, Teresa y el personal técnico, todos han contribuido a esta

tesis.

Gracias a todas las personas con las que he compartido pipetas en otros

laboratorios de Francia y Portugal aunque probablemente no lo lean. Gracias a

Benjamín, Emmanuel, Josiane, Ali, Marina, Rita y Mahmud.

VI
Blanca, eres sin duda la persona que más ha hecho para que esta tesis sea posible,

gracias por aguantarme cuando me desespero, gracias por acompañarme en mis

locuras y a veces sufrir conmigo por ellas, gracias por ser la maravillosa madre de

nuestra niña Maïa y cuidarla mientras termino esta tesis en el cuarto de arriba,

gracias por ser tú. Eres una luchadora, puedes conseguir lo que te propongas.

Gracias al irrepetible grupo de Meandro 24, estos años a vuestros lados fueron

sin duda una etapa inolvidable de mi vida. No me lo habría podido pasar mejor.

Gracias a Antonio por el apoyo con la RMN.

Maman, un grand merci pour m’encourager autant, je sais que tu seras fière de

moi mais maintenant c’est à moi de t’encourager pour que tu te récupères vite et

tu viennes vite nous voir. Papa toi aussi tu ne rates pas une occasion de

m’encourager et de m’appuyer. C’est grâce à vous deux que j’ai toujours pu faire

ce dont je rêvais. Merci!

Finally, thanks to Fall Creek especially Hans and Scott who encouraged me to

finish this thesis. I really appreciate the freedom you gave me those days and the

comprehension you have shown.

Merci à Tous!

VII
VIII
 “Cuando emprendas tu viaje a Ítaca

pide que el camino sea largo,

lleno de aventuras, lleno de experiencias.”

Extracto del Poema “Itaca”

Constantine Peter Cavafy

IX
X
Aux quatre personnes les plus importantes de ma vie:

Maïa, Blanca, Maman & Papa

XI
XII
 Resumen de la tesis

El objetivo de ésta Tesis Doctoral ha sido realizar un estudio sobre el alga

verde Ulva y evaluar su uso potencial como fuente de ingredientes funcionales
en acuicultura. Principalmente ha consistido en determinar el potencial de
polisacáridos sulfatados procedentes de la macroalga Ulva y desarrollar procesos
biotecnológicos que permitan determinar su bioactividad. El trabajo se ha
desarrollado mayoritariamente en las instalaciones de IFAPA centro El Toruño
tanto en las zonas de esteros como en las salas de cultivo y laboratorios.
Se ha identificado la especie de Ulva presente en la zona y con la que se
ha realizado los estudios de la presente tesis, que resultó ser la especie Ulva ohnoi
M. Hiraoka & S. Shimada. Esta alga se puso en cultivo presentando una aceptable
tasa de crecimiento y una composición basal similar a la descrita en la
bibliografía.
Se ha realizado un trabajo de seguimiento del alga en zona de esteros que
permitio avanzar en los conocimientos sobre la respuesta de ésta especie a
cambios físico-quimicos del medio y condiciones ambientales durante un ciclo
estacional. De forma general los factores climáticos de temperatura y de
irradiación solar tuvieron efectos sobre el crecimiento de las algas y su
composición proximal. Se encontraron diferencias significativas entre la
composición del extracto acuoso según el punto de muestreo. En un punto el
alga recibió un aporte de nitrógeno exclusivamente amoniacal que se relacionó
con un nivel constante de ramnosa y xilosa mientras que el punto de muestreo
con un aporte nitrógeno nítrico mostró variaciones más importantes de estos
azúcares. Estos dos azúcares son componentes importantes del polisacárido
ulvan. Los datos sugieren un papel del ulvan como una molécula con funciones
de reserva de cationes para el alga.
Para estudiar las propiedades del alga Ulva ohnoi y, de forma especial, los
polisacáridos sulfatados que presenta dicha alga se desarrollaron procesos
biotecnológicos que han permitido avanzar en el conocimiento del polisacárido
ulvan. La extracción de ulvan y el fraccionamiento de polisacáridos permitió

XIII
obtener oligasacáridos aplicando diferentes procesos de despolimerización,
química y enzimática. Para la despolimerización enzimática aislamos e
identificamos una bacteria procedente del intestino de un anfípodo capaz de
degradar específicamente los enlaces glucosídicos que caracterizan el ulvan. Los
oligosacáridos obtenidos se purificaron y se caracterizaron.
Entre los procesos biotecnológicos aplicados en esta Tesis destacar
también la utilización de nanotecnología para poder dar aplicación a los
productos extraidos y purificados del alga. El polisacárido ulvan y sus
oligosacáridos tienen capacidad para formar parte de nanopartículas. Se
elaboraron nanopartículas poliméricas de quitosano y tripolifosfato sódico que
incorporan eficientemente tanto el polisacárido ulvan como los oligosacáridos.
Se obtuvieron nanopartículas estables y con tamaños inferiores a 300 nm. Se
estudió el efecto del polisacárido ulvan y de sus oligosacáridos tanto
encapsulados como no encapsulado sobre el estallido oxidativo de macrófagos
de Solea senegalensis. Los resultados confirman el poder inmunoestimulante del
polisacárido ulvan. Los oligosacáridos obtenidos en cambio no ejercen un efecto
estimulante en las condiciones ensayadas. La encapsulación del ulvan no
despolimerizado tuvo un efecto potenciador sobre el estallido oxidativo de los
macrófagos. La máxima actividad inmunoestimulante de las células se alcanzo
cuando el polisacárido se encontraba incorporado en una nanoparticula.
El complejo quitosano-ulvan ejerce un efecto potenciador de las
propiedades de ulvan sobre el sistema de defensa del lenguado Solea
senegalensis. La utilización de estas nanoparticulas de quitosano y ulvan con alto
peso molecular puede ser una herramienta útil en acuicultura para la prevención
de enfermedades.
Los polisacáridos sulfatados procedentes de la macroalga Ulva sp.
presentan potencial para poder ser usados como ingredientes funcionales o
nutracéuticos en acuicultura.
La metodología aplicada en la extracción de compuestos del alga es
determinante para poder conocer la carga real de compuestos de interés y su
utilización como ingredientes funcionales en acuicultura. Se propusó la hipótesis
de que los valores de antioxidantes a partir de Ulva podrían estar infraestimados.

XIV
Se describe el uso de un método alternativo de extracción de polifenoles (unos
antioxidantes mayoritarios) a partir del alga Ulva ohnoi que permite extraer
compuestos que no son extraíbles con el tradicional método de metanol-
acetona. La extracción mediante metanolisis durante 5 horas a 100 ºC permitió
alcanzar niveles más altos de extracción, del orden de 3-4 veces superior a los
obtenidos cuando se emplea la metodología habitual para la determinación de
estos compuestos en algas.

Abstract

The objective of this PhD. Thesis was to realize a study of the green alga
Ulva and evaluate its potential as a source of functional ingredients in
aquaculture. It mainly consisted of determining the potential of sulfated
polysaccharides proceeding from the green alga Ulva and develop
biotechnological processes that allowed the determination of its bioactivity. The
work has been realized principally in the facilities of the IFAPA centro el Toruño
in a zone with earthern ponds and saltmarshes as well as in the algae culture
chamber and in the laboratory.
The specie of the local Ulva that has been cultivated and for which studies
have been made has been identified as Ulva ohnoi M. Hiraoka & S. Shimada. This
alga was cultivated showing adequate growth rate for experimental purposes and
a proximal composition similar to those encountered in the literature.
A monitoring study has been carried out in the earthen pond/experimental
fish farm area, which improved the knowledge of the response in growth rate and
biochemical composition of this species to physico-chemical changes in
environmental parameters during a seasonal cycle. In general, climatic factors
temperature and solar irradiation have had a significant effect on growth and
proximal composition of the algae. Significant differences were found between
the water extract compositions depending on the sampling point. In one
sampling point the algae received almost exclusively ammonium nitrogen which
has been related to a constant level of rhamnose and xylose. Meanwhile the
sampling point where some percentage of nitrate nitrogen was measured showed

XV
more important variations in the contents of those sugars. Rhamnose and xylose
are important components of the polysaccharide ulvan.
To study the properties of the alga Ulva ohnoi and especially of its sulfated
polysaccharides, biotechnological processes were developed and that allowed us
to increase our knowledge of the ulvan polysaccharide. The extraction of the
ulvan and its oligomerisation allowed for obtaining oligosaccharides through
chemical and enzymatic processes. For the enzymatic depolymerization we
isolated and identified a bacteria proceeding from the intestine of an amphipod
that was able to degrade specifically the glucosidic links that are specific for ulvan.
The oligosaccharides that were obtained were purified and characterized.
Amongst the biotechnological processes applied in this thesis it is
noteworthy to mention that the use of a nanotechnologic tool has been
employed for the application of the products extracted and purified from the
alga. Ulvan polysaccharides and its oligosaccharides are able to form part of
nanoparticles. Polymeric nanoparticles of chitosan and sodium tripolyphosphate
were elaborated into which were incorporated successfully both the poly and the
oligosaccharides of ulvan. Stable nanoparticles with a size inferior to 300 nm were
obtained.
The effect of the encapsulated and the free form of the polysaccharide and
oligosaccharides on the oxidative burst of Solea senegalensis macrophages has
been studied. The maximum activity has been measured when the complete
polysaccharide was encapsulated in a nanoparticle.
The chitosan-ulvan complex had a synergestic effect boosting the
properties of ulvan on the immune defense system of Solea senegalensis. The use
of those chitosan nanoparticles and ulvan with a high molecular weight could be
a useful tool for its use in preventing diseases in aquaculture.
The sulfated polysaccharides proceeding from Ulva sp. present a potential
to be used as a source of functional ingredients or as nutraceuticals in
aquaculture.
The methodology applied in the extraction of algal compounds is a
determining factor to know the real content of this molecule and its potential use
as a functional ingredient in aquaculture. It was hypothesized that the antioxidant

XVI
content obtained from Ulva might have been underestimated. An alternative
methodology for the extraction of Ulva ohnoi polyphenols (major antioxidant
compounds) not extractable with the traditional methanol-acetone method is
described. A methanolysis step of 5 hours at 100°C allowed to reach the highest
levels of antioxidant molecules with values of 3-4 fold those obtained with the
methodology usually employed for the determination of those compounds in
algae.

XVII
Índice

ÍNDICE
1. INTRODUCCIÓN ........................................................................................ 1
1.1. LAS ALGAS .................................................................................................... 2
1.2. LOS POLISACÁRIDOS DE ALGAS ....................................................................... 11
1.3. EL ULVAN ................................................................................................... 22
1.4. BIBLIOGRAFÍA ................................................................................................. 34

2. HIPÓTESIS Y OBJETIVOS ............................................................................ 47

2.1 HIPÓTESIS.................................................................................................... 48
2.2 OBJETIVO GENERAL ....................................................................................... 48
2.3 OBJETIVOS ESPECÍFICOS ................................................................................. 49

3. SEASONAL VARIATION OF PROXIMATE COMPOSITION, ULVAN CONTENT AND SUGAR

COMPOSITION OF ULVA OHNOI (CHLOROPHYTA, ULVALES) IN RELATION TO GROWTH AND
ENVIRONMENTAL PARAMETERS ......................................................................... 51
3.1. INTRODUCTION............................................................................................ 53
3.2. MATERIALS AND METHODS ............................................................................ 54
3.3. RESULTS ..................................................................................................... 60
3.4. DISCUSSION ................................................................................................ 70
3.5. CONCLUSIONS ............................................................................................. 76
3.6. REFERENCES................................................................................................ 77

4. PRODUCTION OF SULFATED OLIGOSACCHARIDES FROM SEAWEED ULVA SP. USING A

NEW ULVAN -DEGRADING ENZYMATIC BACTERIAL CRUDE EXTRACT ............................. 85
4.1. INTRODUCTION............................................................................................ 87
4.2. MATERIALS AND METHODS ........................................................................... 90
4.3. RESULTS ..................................................................................................... 95
4.4. DISCUSSION .............................................................................................. 101
4.5. CONCLUDING REMARKS .............................................................................. 105
4.6. REFERENCES.............................................................................................. 105
Índice

5. POLYMER CHITOSAN NANOPARTICLES FUNCTIONALIZED WITH ULVA OHNOI EXTRACTS

BOOST IN VITRO ULVAN IMMUNOSTIMULANT EFFECT IN SOLEA SENEGALENSIS
MACROPHAGES ............................................................................................115
5.1. INTRODUCTION.......................................................................................... 117
5.2. MATERIALS AND METHODS .......................................................................... 120
5.3. RESULTS ................................................................................................... 127
5.4. DISCUSSION .............................................................................................. 133
5.5. CONCLUSION............................................................................................. 138
5.6. REFERENCES.............................................................................................. 139

6. IMPROVEMENT OF ANTIOXIDANT COMPOUNDS EXTRACTION IN THE MACROALGAE

ULVA OHNOI WITH HOT ACIDIC METHANOL EXTRACTION METHOD ............................145
6.1. INTRODUCTION.......................................................................................... 147
6.2. MATERIALS AND METHODS ......................................................................... 149
6.3. RESULTS ................................................................................................... 152
6.4. DISCUSSION .............................................................................................. 154
6.5. CONCLUSIONS ........................................................................................... 157
6.6. REFERENCES.............................................................................................. 157

7. DISCUSIÓN GENERAL ...............................................................................163

7.1. BIBLIOGRAFÍA ............................................................................................ 181

8. CONCLUSIONES GENERALES ......................................................................189

9. ANEXOS...............................................................................................193
Tabla de Abreviaturas

TABLA DE LAS ABREVIATURAS

°C: grados centígrados HPLC: High Pressure Liquid

µm: micrometros Chromatography
Ac.Ac.: Ácido acético HPTLC: High Performance Thin Layer
ADN: Ácido desoxyribonucléico Chromatography
ADP: Adenosina difosfato IL: Interleuquina
ANOVA: Analysis of Variable ISI: Intracellular Stimulation Index
ARN: Ácido ribonucléico ITS: Inter Transcibe Spacer
ATP: Adenosina trifosfato kDa: kilo Dalton
BDDE: 1,4-butanediol diglicedil éter M: Ácido manurónico
BLAST: Basic Local Alignment Search MD: Maryland
Tool MeOH: Metanol
CA: cultivated algae meq : miliequivalente
CS: Quitosano/Chitosan mg : miligramo
CytC: Citocromo C MHz : Megahercio
D2O: Deuterio min : minuto
DAD: Diode Array Detector ml : mililitro
dn/dc: Differential refractive index mM : milimolar
dNTP: desoxyribonucleotides mix MS/MS: masa / masa
DP: Degree of Polymerization MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-
DPPH: 2,2-diphenyl-1-picrylhydrazyl diphenyltetrazolium
DU : Depolymerized ulvan mV: milivoltio
EtOH : Etanol Mw: Molecular Weight
FBS : Fœtal Bovine Serum MW: Molecular Weight
FID: Flame Ionization Detector NADP: nicotinamida adenina
G : Ácido gulurónico dinucleótido fosfato
GAGs: Glicosaminoglicanos NBT: Nitro Blue Tetrazolium
GC: Gas Chromatography NF-κ B: Nuclear Factor-κ B
H1-NMR: Resonancia magnetica ng: nanogramo
nuclear del proton nm: nanometro
HBSS: Hanks’ Balanced Salt Solution NMR: Nuclear magnetic resonance
HOD: Deuterio NOD: Nucleotide-binding
oligomerization domain
Tabla de Abreviaturas

NPs: nanopartículas UL: Ulvan

P: Percentil ULE: Ulvan cortado enzimaticamente
P/DAMP: Pathogen/ Danger ULQ: Ulvan cortado quimicamente
Associated Molecular Pattern UV: Ultravioleta
PAD: Pulsed Amperometric Detector v/v: Volumen por volumen
PAR: Photosyntetically Active vis: visible
Radiation w/w: weight per weight
PC : Phenolic Coumpounds μmol: micromol
PCR : Polymerase Chain Reaction
PL/ Polysaccharide Lyase
PLS : Partial Least Square regression
PMA : Phorbol Myristate Acetato
ppm : part per million
PRR : Pattern recognition receptor
PVA: Poly vinil alcohol
rbcL: 1,5-biphosphate carboxylase
gene
rDNA: ribosomic Desoxyribonucleic
acid
Ref.: Refer to
Rf: Retention factor
ROS: Reactive Oxygen Species
rpm: rotaciones por minuto
s: segundo
S/P: Streptomicina / Penicilina
sec: Segundo
SEC-MALLS: Size Exclusion
Chromatography Multi angle Laser
Light Scattering
SLS: Static Light Scattering
TLC: Thin Layer Chromatography
TLR: Toll-Like Recptor
TPC: Total Phenolic Compounds
TPP: Sodium Tripolyphosphate
UGMA: Ulvan glicidil metacrilato
22
1. Introducción

1
 1. Introducción

1.1. Las Algas

1.1.1. Biología, Ecología y Diversidad

1.1.1.1. ORIGEN EVOLUTIVO DE LAS ALGAS

A pesar de su aparente simplicidad las algas son organismos ancestrales

que desde tiempos inmemoriales han evolucionado de forma continua
adaptando su fisiología con éxito a los nichos ecológicos que hoy ocupan. Las
estrategias de supervivencia, de reproducción y de colonización de estos
organismos son tan diversas como inexploradas si las comparamos con sus
descendientes las plantas terrestres. Para introducir las algas y comprender de
donde viene la verdadera complejidad que entrañan y por qué el hombre aún
domina poco este inmenso grupo polifilético es necesario mirarlas a la luz de la
historia de la vida en nuestro planeta.
Se estima que el primer rastro de vida en la tierra apareció hace 4.6 mil
millones de años presumiblemente en un medio líquido en el que la propia
1
entropía favorecía el movimiento de moléculas orgánicas y posibilitó
innumerables combinaciones entre ellas para formar nuevos compuestos con
inéditas propiedades. La vida primitiva tomó la forma de varias moléculas que se
organizaron para replicarse entre ellas usando como substrato las moléculas
orgánicas simples que las rodeaban en este medio.
Es probable que estos organismos primitivos usaran la hidrólisis de
moléculas orgánicas libres en el medio - como el ATP - cómo fuente de energía
para expulsar los protones de la célula y mantener el pH intracelular estable. La
primera gran crisis energética del planeta llegó cuando se agotaron las reservas
de ATP abiótico en el medio y las bacterias fermentadoras consumieron las
moléculas orgánicas simples más rápidamente que la velocidad a la que se

1
Moléculas simples de síntesis abiótica

2
 1. Introducción

producían por síntesis abiótica. En este momento un organismo que fuera capaz
de producir sus propias moléculas orgánicas precursoras saldría con una gran
ventaja evolutiva [1].
Se piensa que los primeros organismos en usar electrones como
combustible para alimentar la bomba de protones fueron bacterias que tenían
pigmentos para protegerse de las radiaciones solares y que convirtieron este
sistema de protección en una cadena de transporte de electrones capaz de
producir la fosforilación del ADP. Otras bacterias procariotas usaban la luz para
transferir electrones del H2S al NADP+ lo que servía a reducir el CO2 y fijarlo
produciendo moléculas orgánicas. Con la evolución, algunas bacterias
fotosintéticas consiguieron extraer estos electrones del H2O en vez del H2S
creando O2 como deshecho metabólico y cambiando para siempre la atmósfera
terrestre. Se sabe hoy que las responsables de este cambio fueron las
cianobacterias. [2]
Las cianobacterias se han llamado durante mucho tiempo las algas
cianófitas o algas verdeazuladas ya que son los únicos procariotas capaces de
producir oxígeno por fotosíntesis mediante la clorofila y poseen ficobilinas que
son las responsables del color turquesa de estas bacterias. Las cianobacterias
“modernas” se han demostrado que son las descendientes de las bacterias
endosimbióticas que se asociaron a las bacterias provistas de un núcleo para
formar los eucariotas fotoautótrofos. Este orgánulo capaz de realizar la
fotosíntesis se denomina plástido. Cuando se descubrió la diferencia entre
eucariotas y procariotas las cianobacterias se clasificaron en el reino de los
moneras a diferencia de las algas que se clasifican en el reino de los protistas al
no considerarse las algas como plantas, hongos o menos aún animales.
Estudios moleculares han revelado que la primera endosimbiosis entre un
eucariota y un procariota fotoautotrofo puede haber ocurrido en un rango de
aproximadamente 1900 a 900 millones de años atrás [3]. No está claro si esta
endosimbiosis primaria fue un evento único o si ocurrió en varios linajes de
eucariotas con varios tipos de cianobacterias. Más tarde, otros eucariotas - no
relacionados con los primeros - experimentaron un evento de endosimbiósis
segundaria en el que el endosimbiota fueron algas verdes o rojas. A raíz de la

3
 1. Introducción

incertidumbre y de su origen múltiple las algas son hoy un amplio grupo de

organismos definidos de forma poco confinada como “eucariotas autótrofos que
no sean plantas” y se ubicaron originalmente en el reino de los protistas.

1.1.1.2. CLASIFICACIÓN Y DIVERSIDAD DE LAS ALGAS

Naturalmente en la ausencia de las nuevas tecnologías usadas en

filogenética las primeras clasificaciones que se hicieron de las algas se basaron
en la morfología del organismo y/o de la célula. Debido a un alto grado de
polimorfismo y ciclos de vida complejos estas clasificaciones no permitieron
hacer consenso entre los científicos. Repasando la literatura aún permanece difícil
encontrar dos clasificaciones similares dentro de las algas probablemente
parcialmente debido a la complejidad del genoma que ha experimentado
numerosos intercambios transversales de genes entre huésped y endosimbiota a
lo largo de la evolución sembrando aún más dudas sobre orígenes comunes,
distancia filogenética y etapas de separación específica.
Los trabajos más recientes emplean una combinación de análisis
ultraestructurales, moleculares y bioquímicos para determinar la posición
filogenética de cada grupo de algas. El análisis de la ultraestructura de las
membranas de los plástidos permite revelar si ha ocurrido uno o varios eventos
de endosimbiosis entre un eucariota y un organismo fotosintético a lo largo de
la historia. Así por ejemplo el cloroplasto de las algas verdes posee una doble
membrana y tiene tilacoides organizados en lamelas. El análisis del ADN
ribosómico del plástido y del huésped sirven para determinar si hubo divergencia
evolutiva anterior o posterior a estos eventos.
Basados en estas características algunos autores dividen los organismos
que emergieron de una endosimbiosis primaria y una endosimbiosis secundaria.
Esta clasificación rompe con el esquema tradicional de reino presentado más
arriba pero presenta una explicación basada en la cronología de los eventos
evolutivos. Así, por ejemplo, encontraremos una distancia evolutiva mucho

4
 1. Introducción

menor entre las algas verdes y las plantas terrestres que entre las algas verdes y
las diatomeas.
El linaje de los archaeplastidae son organismos que han conocido una
única endosimbiosis a lo largo de su evolución y agrupa a las algas rojas, las
glaucocystofitas y las plantas verdes [4].
Los otros grupos entre los cuales encontramos algas derivan de una
endosimbiosis secundaria o terciaria. Esto significa que estos grupos aunque no
deriven de un huésped filogenéticamente próximo a los archaeplastidae han
formado una endosimbiosis con una de ellas. Estas endosimbiosis segundaria o
terciaria han ocurrido entre un organismo eucariótico y algas verdes o algas rojas.
Estos eventos posteriores a la endosimbiosis primaria han dado lugar a una
variedad de grupos llamados: criptofitos, haptofitos, alveolatos, stramenopiles,
rhizaria y excavatas. Conocidas macroalgas como las laminarias pertenecen al
grupo de la stramenopiles por ejemplo [5].
La complejidad de la filogenia de las algas refleja la larga historia evolutiva
que acompaña este heterogéneo grupo. Para una mejor comprensión,
adoptaremos en esta tesis la nomenclatura más popular basada en el tipo de
pigmentación accesoria del fotosistema y que distingue tres grandes grupos de
algas: las clorofitas, las rodofitas y las feofitas. El primer grupo se conoce más
familiarmente como las algas verdes y se caracterizan por tener en abundancia
las clorofilas a y b. Las rodofitas o algas rojas tienen clorofila a pero usan varios
pigmentos llamados cianinas, ficoeritrina, aloficocianinas que le dan su color rojo.
El último grupo se conoce como las feofitas pero están más comúnmente
llamadas algas pardas. En realidad este grupo se descompone en cuatro grupos:
las glaucocystófitas, las euglenófitas (excavata), las criptófitas, las stramenopiles
y los dinoflagelados (haptofitas). Tienen el punto en común de tener pigmentos
de clorofila a, c y xantófilos como pigmentos accesorios.
Es importante para el objeto de esta tesis saber que en realidad las algas
verdes pertenecen a un grupo llamado viridiplantae situado dentro de las
archaeplastidas junto con las rodofitas y las glaucofitas. Las viridiplantae se
caracterizan por su pigmentación verde rica en clorofila. Este grupo se divide a
su vez en clorofitas y streptofitas. El grupo de las clorofitas son exclusivamente

5
 1. Introducción

algas mientras que el grupo de las streptofitas contienen plantas terrestres y

organismos considerados como algas verdes como las caroficeas.
Las clorofitas cuentan numerosas clases entre el cuál se encuentra la clase
de las ulvoficeas que a su vez se divide en ordenes de los cuales se encuentra el
de las Ulvales. Dentro de éste orden la familia de las Ulváceas cuenta con varios
géneros como Enteromorpha, Ulva, Ulvaria o Umbraulva entre otros. Estos
géneros cuentan con especies que presentan similitudes morfológicas
importantes hasta tal punto que Ulva y Enteromorpha han finalmente sido
declarados como siendo el mismo género [6,7]. La falta de criterios morfológicos
distintivos entre especies del genero Ulva y la gran plasticidad fenotípica hace su
identificación complicada. Es necesario el empleo de herramientas moleculares
precisas para poder determinar la especie basándose en la alineación de
secuencias del gen rbcL2del plastido y el gen nuclear3 ITS son requeridas [8].

Aunque las estimaciones estén todavía confusa, las cifras en la web de

referencia algaebase.org apuntan a un número de especies de 72,500 con
actualmente 129 especies pertenecientes al género Ulva [9].
Esta gran diversidad representa una oportunidad para el hombre de poder
explorar las recónditas y únicas propiedades de estos organismos. Por otro lado
la complejidad que entraña entender los múltiples mecanismos fisiológicos de
las algas es un inevitable reto que se debe superar para poder controlar su
producción. Esta tesis pretende estudiar tanto la composición y las propiedades
de compuestos extraídos de algas como la fisiología subyacente al control de su
cultivo. El marco elegido para enfocar este estudio es el de la utilización de las
algas como una fuente de ingredientes funcionales para ser empleado en
acuicultura.

2
1,5-biphosphate carboxylase gene
3
Inter Transcribe Spacer

6
 1. Introducción

1.1.2. Importancia económica de las algas

Aunque el hombre ya explota las algas a nivel comercial, se estima que el

potencial de estos organismos es todavía mucho mayor. En el mundo, las algas
ocupan el tercer puesto en término de volumen para productos de la acuicultura
detrás de los peces de agua dulce y los moluscos. En 2015, 30.5 millones de
toneladas de algas fueron producidas en el mundo de cuya cantidad 29.4
millones se cultivaron en granjas acuícolas. Las laminarias son las que más se
producen en el mundo con un volumen de 5 millones de toneladas. En España,
el volumen de algas recogidas del medio natural esta en aumento y alcanzó en
2015 las 2386 toneladas pero los volúmenes producidos en 2010 fueron de unas
modestas 124 toneladas frente a las 388,520 toneladas de Chile o las 246,620
toneladas de China [10].
En Asia, las algas se consumen en la alimentación a diario por lo que la
producción mundial está concentrada en esta región del mundo. El Nori
(Porphyra spp.), el Wakame (Undaria pinnatifida) y el Kombi (Laminaria japónica)
están cultivadas de formas extensas para el consumo humano pero existen más
especies comercializadas. En el resto del mundo el uso de las algas en la
alimentación bajo su forma natural no esta tan generalizado [3]. Sin embargo, en
las regiones en las cuales la comida procesada es parte del cotidiano de sus
habitantes, las algas están presentes en muchos alimentos de producción
industrial como aditivos llamados ficocoloides. Estas moléculas son polisacáridos
y están dispersas en un medio con el objetivo de modificar su textura. Finalmente,
una tercera gran aplicación de las algas es su uso en biorefinería. Este término se
usa para nombrar una industria cuyo fin es convertir una biomasa en carburantes,
energía o productos químicos de origen natural. Esta actividad encuentra un gran
interés en los últimos años porque a diferencia de la producción de ficocoloides,
este negocio implica pequeñas cantidades de productos con alto valor añadido
[11]. Muchas moléculas presentan un gran interés en farmacéutica, en cosmética
o para compuestos funcionales de origen natural.
Comparativamente, a nivel económico, las algas se compran frescas 50 €
por tonelada cuando se trata de comercializarla para el mercado de los coloides,

7
 1. Introducción

sin embargo para el uso en alimentación o para la extracción de moléculas están

valoradas en 500€ la tonelada [12]. A pesar del importante valor añadido que le
da a las algas la extracción de moléculas de interés y el enorme potencial que
representa esta actividad en los países occidentales, todavía se sabe
relativamente poco sobre el metabolismo primario y segundario de estos
organismos y las posibles bioactividades de estos compuestos en otros seres
vivos.
En nutrición, las algas se conocen sobre todo por su alto contenido en
fibras, en minerales, en vitaminas y en algunos casos en proteínas [13]. Sin
embargo contienen otras moléculas como polisacáridos, lípidos y polifenoles que
pueden tener bioactividades beneficiosas sobre la salud. Estos efectos son
múltiples pero los que más se investigan son principalmente las actividades
antimicrobianas, antiviral, anticancerígenas, hipotensoras, antioxidantes y contra
la diabete. Fisiológicamente se pueden distinguir dos tipos de elementos en las
macroalgas que pueden tener efecto: las macromoléculas no absorbidas como
son las fibras alimentarias y las moléculas que pueden ser absorbidas y tener un
efecto directo sobre la homeostasis [11].

1.1.3. Las algas verdes como biofiltros

Al revisar la bibliografía sobre las actividades de moléculas aisladas de

algas, se observa que la inmensa mayoría de los artículos se dedica al estudio de
algas pardas o rojas, siendo minoría los estudios relacionados con las algas
verdes. Este hecho se puede explicar por dos razones principales: la primera es
que las algas verdes se asocian a episodios de contaminación de las aguas por
elevadas cargas de nutrientes, y por otra parte que las algas rojas y pardas están
cultivadas en zonas más extensas. Sin embargo, las algas verdes tienen varias
características que hacen de ellas un interesante tema de estudio con un
verdadero potencial a nivel de explotación. En primer lugar, las algas verdes
suelen tener tasas de crecimiento importantes que pueden alcanzar en el caso
del género Ulva hasta un 50% de su peso al día, en estas condiciones forman un
“bloom”. Este fenómeno se debe al hecho que las algas del genero Ulva tienen

8
 1. Introducción

una gran capacidad para absorber los nutrientes que se encuentran solubilizados
en el medio [14].
A lo largo de los 40 últimos años en el sur de la península ibérica se han
desarrollado un gran número de granjas marinas de peces de agua salada como
la dorada o la lubina. Los efluentes de estas explotaciones llevan altas cargas de
nutrientes que son liberados en el medio ambiente [15] y pueden tener un efecto
contaminante favoreciendo la aparición de especies oportunistas. Cuando las
algas mueren sedimentan al fondo, esto provoca un crecimiento de la actividad
bacteriana que tiene por efecto el aumento del consumo de oxígeno resultando
en una anoxia en las zonas bénticas. Estos ecosistemas albergan una fauna que
está en la base de la cadena alimentaria y áreas de desove de especies de peces
pelágicos. Largos periodos de anoxia pueden romper el equilibrio de estos
sistemas resultando no solo en una gran pérdida de la biodiversidad pero
también en pérdidas económicas. Por ejemplo se calcula en el mar Báltico que si
las “zonas muertas” debido a la anoxia fueran recuperadas, este mar sería de 30
a 50% más productivo [16,17].
La habilidad de absorción de los nutrientes de las algas verdes las convierte
en una candidata idónea para poder ser empleadas como biofiltros
consiguiéndose como resultado aguas con baja carga de nutrientes al medio y la
obtención de una gran cantidad de biomasa de algas. Asimismo las Ulváceas
presentan un interés para desempeñar este papel en la cadena de la acuicultura
multitrófica, su crecimiento es rápido y no presenta riesgo de introducir esta
especie en el medio ambiente ya que se encuentra naturalmente presente en la
mayoría de los ecosistemas costeros del planeta. En las costas andaluzas y las
zonas de esteros las especies del género Ulva se encuentran con facilidad a lo
largo del año.

1.1.4. El reto de la valorización de las algas verdes

Un reto clave para la consecución de semejante sistema es que además de

cumplir la función de depurar el agua pueda generar algún tipo de riqueza y que
además de medioambientalmente sostenible lo sea también económicamente.

9
 1. Introducción

Un aspecto relevante y de gran interés para la valorización de las algas esta

relacionado con la extracción de moléculas con actividad biológica para su uso
en los sectores de la nutrición, la farmacéutica, la nutracéutica4, en química o para
cosméticos.
Una de las principales puesta en valor que se han conseguido con las algas
marinas es la extracción de sus polisacáridos. Generalmente se utilizan para
modular la textura de alimentos pues tienen la capacidad de modificar su
viscosidad de forma predecible. Algunos son capaces de formar geles en
presencia de iones específicos. En los últimos años, la investigación sobre estas
moléculas ha permitido desvelar numerosas actividades biológicas que
encuentran aplicaciones tanto en farmacia, en cosmética o para concebir
alimentos funcionales. En la sección 1.2.3. y 1.2.4. se describen los principales
polisacáridos extraídos de las macroalgas y sus principales actividades biológicas.

1.1.5. Las algas como fuentes de alimentos funcionales para la acuicultura

En acuicultura el uso de las microalgas está muy extendido en la nutrición

de los alevines. También son fuentes de moléculas nutricionales y funcionales
que permiten añadir propiedades a la alimentación de los animales [18]. En
comparación existen en alimentación animal pocos estudios sobre el uso de
componentes de las macroalgas en alimentación animal.
El empleo de las macroalgas en la acuicultura de peces se ha enfocado
generalmente a reemplazar parcialmente los ingredientes procedentes de la
pesca de especies salvajes y a estudiar el impacto sobre variables del crecimiento
[19–22]. Aunque existan macroalgas con niveles altos de lípidos o proteínas
parece que su uso como ingrediente nutricional en especies de nivel trófico alto

4
El termino nutracéutica deriva de la contracción de los términos
“nutrición” y “farmacéutica” que en otras palabras se podría definir como
alimentos con propiedades biológicas

10
 1. Introducción

sea aún por desarrollar [23]. Algunos de estos estudios sin embargo describen
algunos efectos no directamente relacionados con el crecimiento pero cuyas
consecuencias parecen ser beneficiosas para el animal.
Así, Nakagawa [19] describe que la adición de 5% de harina de Porphyra
yezoensis en la dieta de Pagrus mayor tiene un efecto sobre la resistencia al estrés
de los alevines en situaciones de hipoxia. Satoh [24] ha reportado la mejora de
las cualidades inmunitarias, como la actividad fagocítica, de Pagrus mayor
cuando recibía una dieta suplementada con un 5% de Ulva.
El Aquavac® ErgosanTM es un producto comercial usado en la formulación
de alimentos para larvas y adultos de peces que contiene alginato de Laminaria
digitata y una harina de Ascophyllum nodosum. Tiene efectos positivos sobre el
sistema inmune de las truchas Oncorhynchus mykiss pero también mejora la tasa
de crecimiento específica y disminuye la ratio de conversión del alimento [25].
Los polisacáridos de algas presentan un gran potenical de uso y son de
especial interés para la inmunoestimulación en acuicultura [26].
Se ha puesto de manifiesto la efectividad de los extractos de diversas algas
como producto con efecto antivírico o antibacteriano. La actividad de extractos
metanólicos de Monostroma nitidum y Ulva pertusa quedó fueron entre las más
activas contra dos virus en Pseudopleuronectes sp.[27].
Se llevó a cabo un barrido de la actividad antibacteriana de extractos
alcoholicos de 26 algas diferentes contra varios patógenos del salmón. El extracto
de Asparagopsis armata resultó tener una actividad de inhibición bacteriana muy
fuerte sobre las 4 bacterias [28].

1.2. Los Polisacáridos de Algas

1.2.1. La pared celular de las algas

Una característica común de las algas con respecto a las plantas vasculares
es una diferenciación celular limitada pero a pesar de esto se encuentra una gran

11
 1. Introducción

diversidad de organización de las células que determina la morfología del

organismo. La primera dicotomía morfológica es la diferenciación entre algas
unicelulares y algas pluricelulares.
Las algas unicelulares, a menudo llamadas microalgas pueden tomar
diversas formas y tener estructuras como flagelos o una pared celular formada
de sílice llamada frústula. Pueden vivir libres en el medio, ancladas a un sustrato
o formar colonias entre ellas en las cuales los intercambios entre células son
limitados. Se estima que las microalgas forman el 75% de la producción primaria
del medio acuático.
Las algas multi- o pluricelulares también llamada macroalgas pueden
formar estructuras más complejas en las que las células están rodeadas de una
pared celular que une las células entre ellas y define la morfología. Las
macroalgas pueden ser filamentosas con una sola fila de células, pueden ser
filamentosas ramificadas, formar estructuras foliáceas de una o varias capas de
células, formar estructuras ramificadas de varias capas celulares e incluso formar
tejidos diferenciados que pueden formar tallos, hojas, órganos de intercambio de
gases o agarraderos para su fijación en un soporte. Muchas algas comportan en
su ciclo de vida diferentes fenotipos y pueden pasar por fases unicelulares
flagelada en la que la pared celular es inexistente.
Aunque exista lejanía taxonómica entre los diferentes grupos de algas
existen importantes similitudes entre las paredes celulares de las diferentes
macroalgas y las plantas terrestres. Aunque todos estos organismos vengan de
eventos de endosimbiosis con cianobacterias que poseen paredes celulares, no
parece ser que éstas tengan las mismas características moleculares al tener un
contenido muy inferior en polisacáridos que sus descendientes. El origen de estas
estructuras aún no es completamente conocido pero la conservación de los
genes implicados en su biosíntesis y su continua evolución ponen de manifiesto
su importancia funcional y estructural [29].

12
 1. Introducción

Figura 1.1: Esquema de la

organización ultraestructural de las
paredes celulares de las macroalgas
a: Esquelto de polisacáridos
cristalinos; b: Matriz de polisacáridos
amorfos; c: membrana plasmática; d:
citoplasma

Las paredes celulares de las algas marinas están hechas de dos fases
diferentes: la fase esquelética hecha de polisacáridos cristalinos generalmente
neutros y la fase matricial compuesta por polisacáridos aniónicos amorfos que
rellenan los huecos dejados por el entramado esquelético (Figura 1.1). Los
polisacáridos matriciales presentan un interés especial dado la originalidad de su
estructura.
Las plantas terrestres poseen células cuyas paredes están compuestas por
una gran parte de microfibrillas de celulosa y los polisacáridos matriciales ocupan
un lugar poco importante en comparación con las macroalgas. Como en las
plantas, las paredes celulares de las algas de agua dulce están hecha de
polisacáridos neutros. En el medio marino, muchas algas y plantas vasculares
poseen paredes celulares compuestas por polisacáridos sulfatados lo que sugiere
funciones en relación con el medio marino. Se postula que estas moléculas
cumplen funciones estructurales y de regulación osmótica e iónica [30].

1.2.2. Generalidades estructurales sobre los polisacáridos

Los polisacáridos son con diferencia los polímeros más abundantes en la

superficie de la tierra. Los polisacáridos son cadenas de azucares simples
(monosacáridos) ligados por enlaces glucosídicos. Se pueden caracterizar por
numerosos aspectos de su estructura.

13
 1. Introducción

1.2.2.1. DIVERSIDAD DE AZUCARES

Los polisacáridos son macromoléculas cuya unidad estructural es el

monosacárido. En los polisacáridos los monosacáridos están en forma cíclica y
cada uno puede contar de 5 a 6 carbonos denominándose furanosa o pyranosa
respectivamente. Cada uno de ellos existe en dos isómeros quirales, la forma
levógira (L-) y la forma dextrógira (D-). Dependiendo de la conformación
epimérica del carbono anomérico 5 , el monosacárido puede presentar una
conformación α- o β- que determina el tipo de enlace que se va a formar entre
dos monosacáridos (Figura 1.2).

1.2.2.2. ENLACES GLUCOSÍDICOS

Figura 1.2: Las 4 formas cíclicas de la D-glucosa 1: α-D-glucofuranosa; 2: β-D-glucofuranosa;

3: α-D-glucopiranosa; 4: β-D-glucopiranosa

El enlace se realiza entre el carbono 1 de la primera subunidad y cualquiera

de los otros carbonos de la siguiente, en el caso de que un azúcar β se enlazara
por ejemplo al tercer carbono de otro monosacárido este se apuntaría β-1,3. Estos

5
El carbono anomérico de un monosacárido es un carbono
stereoisomérico derivado del carbono de la función carboxílica del azúcar en
conformación abierta

14
 1. Introducción

enlaces determinan el impedimento estérico y a consecuencia la solidez y

flexibilidad de los enlaces y, por extensión, de la molécula entera.

1.2.2.3. SECUENCIA DE AZUCARES Y RAMIFICACIÓN

A la hora de determinar la funcionalidad final de las moléculas no solo es

determinante conocer la diversidad de monosacáridos que coexisten en éstas
sino que también es importante la secuencia de éstos, con la dificultad añadida
de que los polisacáridos si bien pueden tener una sola cadena y ser lineales
también pueden comportar ramificaciones de varias longitudes así como varios
niveles de ramificación.

1.2.2.4. FUNCIONALIZACIÓN

Dentro de los polisacáridos los propios monosacáridos pueden estar

“decorados” por grupos químicos o grupos funcionales en uno o varios carbonos
como esteres O-sulfatos, esteres N-sulfatos, sulfhidrilos, O-fosfatos, aminas,
acetilos, ácido carboxílico, hidroxilos... En definitiva, estos parámetros determinan
la estructura tridimensional de la molécula en el medio y la posibilidad de
interacción con otra molécula.

1.2.2.5. TAMAÑO

Finalmente, los polisacáridos pueden tener desde dos (disacárido) hasta

varios cientos de azúcares. Generalmente las moléculas de longitud intermedia
desde una decena hasta algunas decenas de monosacáridos son llamadas
oligosacáridos.

1.2.2.6. COMPLEJIDAD Y RETOS

Todos estos factores tienen una influencia sobre las propiedades de las
moléculas. Si bien abre numerosas posibilidades a nivel de propiedades
fisicoquímicas e interacciones biológicas, también conlleva el reto de afinar los
modos de extracción y purificación, de elucidar las estructuras químicas, de
descubrir y comprender la naturaleza de las interacciones biológicas y físicas así
como desvelar los engranajes de su aparentemente aleatoria biosíntesis.

15
 1. Introducción

1.2.3. Diversidad de polisacáridos de algas

Las algas son una fuente de polisacáridos con múltiples propiedades

biológicas y físicas. Se diferencian entre ellos por sus características estructurales
y físico-químicas. A continuación, se describirán los polisacáridos aislados de
macroalgas marinas que más atención científica e industrial han recibido hasta
hoy.

1.2.3.1. AGAR

El agar es uno de los extractos de algas más conocido en gran parte por el
éxito que tiene como matriz de cultivo en microbiología y su uso bajo el nombre
de E406 como agente texturizante en la comida procesada. Procede de algas rojas
y abunda especialmente en las especies del género Gracilaria spp. o Gelidium
spp.[31].
El agar está compuesto por una mezcla de agarosa y agaropectina siendo
la primera la parte mayoritaria alcanzando del 50% al 90% de la mezcla y siendo
responsable del fenómeno de gelación. La unidad constitutiva de la agarosa es
un disacárido de galactosa nombrado agarobiosa que está constituida por un β-
D-galactopyranosyl y un 3,6 anhydro-α-L-galactopyranosyl enlazado en el
carbono 4 [32].
Se ha demostrado que la estructura del agar puede variar en función de la
estación del año en la que se recolectan las algas. La fuerza de las interacciones
que ocurren entre las moléculas del agar dentro de un gel depende
esencialmente de la estructura y como consecuencia la firmeza de este gel puede
variar [33]. Este cambio de calidad repercuta en el precio final del agar y tiene
cierta importancia para la sostenibilidad de este negocio.

1.2.3.2. ALGINATOS

El alginato es un tipo de coloide procedente de las algas pardas que

presentan múltiples usos en productos del entorno doméstico. El alginato de
sodio, la forma salina del alginato, se encuentra con frecuencia bajo el código
E401 en la comida procesada, en los dentífricos y productos cosméticos y

16
 1. Introducción

farmacéuticos. Sectores tan diversos como la industria sanitaria, la industria

papelera o los fabricantes de cervezas usan este producto por sus interesantes
propiedades estabilizadoras.
Se extrae de las algas pardas faeofitas de los géneros Ascophyllum,
Laminaria y Macrocystis que se encuentran principalmente en aguas frías. Las
algas pardas creciendo en aguas más templadas dan un alginato de menor
calidad [34].
Una vez extraído con una solución acuosa, el alginato puede aislarse por
ultrafiltración y precipitación con alcohol o directamente con calcio. Un proceso
alcalino aplicado al alginato nativo permite obtener una sal de alginato de sodio
que tiene la forma de polvo una vez secada.
Su éxito a nivel industrial se debe a su simple e ingeniosa capacidad de gelación
en presencia de cationes divalentes como por ejemplo el calcio Ca2+ el elemento
usado con más frecuencia para este propósito por su relativa inercia. El polímero
está constituido de dos azucares ácidos principales, el ácido gulurónico (G) y el
ácido mannurónico (M). Estos azucares se repiten varias veces de manera seguida
en la cadena formando bloques G y bloques M.
El alginato presenta diferentes grados de calidad refiriéndose a la dureza
del gel que se obtiene a partir de éste. A nivel químico se considera generalmente
que un ratio elevado de G/M, o en otras palabras una mayor abundancia de ácido
gulurónico, favorece una viscosidad elevada. El tamaño molecular también es un
factor que puede influenciar la viscosidad del gel, a mayor tamaño molecular
mayor viscosidad [35]. Los fabricantes Europeos han aprovechado estas
características para diferenciarse de los productores asiáticos con productos de
calidad superior y viables para usos en farmacéutica y biotecnología. El precio
medio del alginato es de 12$ el kilo [31].

1.2.3.3. CARRAGENANOS

Los carragenanos son polisacáridos extraídos del alga Chondrus crispus.

Aunque no tenga ninguna propiedad nutricional están muy usados como aditivos
en productos de gran distribución por sus propiedades gelificantes,
emulsificantes y espesante. Se encuentra presente bajo el código E-407 y E-407a,

17
 1. Introducción

este último teniendo en su composición gran parte de celulosa. A nivel químico

los carragenanos son polisacáridos lineales constituidos por D-galactopiranosa
alternándose los enlaces α-13 y β-14. Además la galactosa enlazada α 13
posee un puente 3,6-anhydro como nivel de complejidad superior [36]. Pueden
estar sulfatados hasta más del 15 al 40% y presentan un peso molecular superior
a los 100 kDa. Existen distintas clases de carragenanos las cuales fueron atribuidas
en referencia a la diferencia de solubilidad que existía entre ellas en una solución
de cloruro de potasio. En realidad esta diferencia de solubilidad es debida en
parte a la diferencia del grado de sulfatación por subunidad disacarídica. Estas
clases están descritas por letras del alfabeto griego: el carragenano β (0 sulfato),
los carragenanos γ, α y κ (1 sulfato), los carragenanos δ, ι, θ, y μ (2 sulfatos) y los
carragenanos λ y ν (3 sulfatos). Entre ellos se diferencian por la posición de los
sulfatos o la presencia del puente 3,6-anhydro en la galactosa α 13. Estas
diferencias además de la conformación del azúcar en la cadena confiere distintas
propiedades a estos distintos grupos, por ejemplo los carragenanos kappa y iota
forman geles en presencia de iones calcios o potasio mientras que la forma
lambda no lo hace [37].

1.2.3.4. FUCANOS

Los fucanos son polisacáridos que como lo indica su nombre contienen

principalmente unidades azucaradas de fucosa. Se encuentran en dos tipos de
organismos marinos: las algas pardas y en invertebrados como los
equinodermos. Algas del orden de las fucales, las laminarias, las dictiotales entre
otras contienen fucanos también llamados fucoides o fucoidanos.
Estos polisacáridos se conocen por su amplio espectro de actividades
biológicas de las cuáles la más conocida es su actividad anticoagulante y
antitrómbica. Esta propiedad se debe en parte al patrón de sulfatación de las
subunidades de fucosa así que el tamaño molecular que le permiten interactuar
específicamente con factores proteínicos implicados en la coagulación de la
sangre.
Otro efecto interesante de la fucosa es su propiedad de ligando de las

18
 1. Introducción

selectinas, un tipo de lectina6 situada sobre los leucocitos e interviniendo en el

proceso de reclutamiento de éstos durante la inflamación 7 . Esta propiedad
encuentra interesantes aplicaciones en biotecnologías como por ejemplo como
agentes atractantes para reclutar células madre en la medula ósea o para
controlar el proceso de inflamación.
También se observan efectos de inhibición sobre crecimiento celular,
migración y adhesión de éstas, propiedad muy deseable para evitar la
proliferación de los tumores. Los fucanos son inhibidores de la adhesión del
esperma al oviducto, esto se debe a que los receptores de la zona pelúcida 8 del
ovulo están fucosilados. Adicionalmente, este polisacárido sulfatado presenta
una actividad antiviral y antiparasitaria [38]. Algunos autores distinguen de los
fucanos un tipo de polisacárido conteniendo fucosa sulfatada pero que presenta
importantes variaciones a nivel de efecto biológico con éstos, el ascofilán, el cual
se extrae de las algas del género Ascophyllum spp.[39].
Los fucanos son moléculas que contienen una columna vertebral lineal
compuesta por fucosa con enlaces α-13 y α-12 con esteres sulfatos en los
carbonos 3 o 4 y pueden tener ramificaciones cada 3 o 4 monosacáridos.
Los fucanos se extraen generalmente con solvente acuosos como buffer o
con ácido a temperatura de entre 70 °C y 100 °C, sin embargo el método que
mejor respeta la estructura de la molécula evitando al máximo la coextracción
con otros polisacáridos se realiza con un buffer de ácido cítrico y fosfato a 60 °C
[40].

6
Las lectinas son proteínas o dominios proteinícos que se enlazan con
carbohidratos con una gran especificidad
7
Este proceso se llama el “leukocytes homing”
8
Capa de glicoproteínas situada sobre la superficie del ovulo

19
 1. Introducción

1.2.3.5. LAMINARINA

Las algas pardas poseen un polisacárido de reserva llamado laminarina (o

laminarano) y representa hasta el 35% del peso seco del alga. Se trata de un
polímero linear de 25 a 50 unidades de glucosas enlazadas por enlaces β-1,3 y a
veces β-1,6 con alguna ramificación en posición 6. Pueden diferir a nivel de las
unidades terminales que pueden ser glucosa, laminaran de tipo G, o mannitol,
laminaran de tipo M. No tienen ninguna propiedad gelificante pero es una fuente
de manitol.

1.2.3.6. GALACTANS

Estos polisacáridos sulfatados están descritos a la vez como homo y

heteropolisacáridos dependiendo de la especie que los genera. Pueden
componerse de galactosa a menudo sulfatada en los carbonos 2 y 4. En el caso
de que sean heteropolisacáridos pueden contener residuos de arabinosa y de
glucosa. Se encuentra en la pared celular de algas rojas y verdes [41].

1.2.4. Actividad biológica de los polisacáridos de algas

Más allá de la diversidad estructural de los polisacáridos de algas y de sus

propiedades como ficocoloides se les puede relacionar con diversas actividades
biológicas. Entre ellas algunas revelan cierto interés para ser candidatos para su
uso nutracéutico, farmacéutico o medicinal. En muchos de los casos, la actividad
biológica está fuertemente relacionada con la estructura de las moléculas, por
esta razón el análisis de la actividad de una determinada molécula no es completa
si no está acompañada de una determinación estructural detallada. Como hemos
visto la estructura de un determinado polisacárido puede presentar variaciones
incluso en la misma especie de alga siendo el factor medioambiental el que
determina estas variaciones. En general el efecto biológico obtenido con un
producto esta correlacionado con una estructura molecular precisa. Medir el
efecto biológico de un extracto controlando la estructura de las moléculas que

20
 1. Introducción

contiene, su abundancia y la presencia de impurezas permite optimizar la

reproductibilidad de los resultados [34,42–44].
Entre las actividades descritas para polisacáridos de algas se encuentra
frecuentemente los efectos anticoagulante, inmunomodulatorio, antitrómbico,
antitumoral, antiviral o antimicrobial. Por su gran parecido con estructuras
naturalmente presentes en el cuerpo humano como los glicosaminoglicanos
(GAGs) o bien como las glicosilaciones sufridas por las proteínas a modo de
modificaciones post-translacional, los polisacáridos sulfatados demuestran
generalmente más actividad que los otros polisacáridos. Es interesante destacar
que las algas marinas son, con excepción de alguna fanerógama marina, la única
fuente de polisacáridos sulfatados producido por organismos fotosintéticos. Es
un dato relevante si se considera su uso como sustituto de algunos fármacos o
adyuvantes 9 de origen animal, evitando así posibles contaminaciones entre
especies animales como por ejemplo los priones bovinos o porcinos.

9
Los adyuvantes son sustancias farmacológicas con efecto inmunológico
usados en conjunto con las vacunas para potenciar su efecto

21
 1. Introducción

1.3. El ulvan

1.3.1. Generalidades

Como anteriormente se mencionó, la familia de las ulváceas, y más

generalmente el orden de las Ulvales, posee varios géneros en los cuales
encontramos especies de morfologías similares. Una característica de estas
especies es la organización en dos capas celulares formando talos tubulares o
planos. Estas dos capas celulares están mantenidas por una pared celular formada
por diferentes polisacáridos más o menos organizados (Figura 1.3).
La pared celular constituye una parte importante de la biomasa producida
por este organismo y puede representar entre el 38% y el 54% del peso seco del
alga. Está compuesta por 4 polisacáridos diferentes: celulosa insoluble, el ulvan
soluble en agua, el glucuronano y el xiloglucano soluble en solución alcalina. Los
dos primeros representando la gran mayoría del peso. Dependiendo del método
de extracción el ulvan puede representar entre el 8% y el 29% del peso seco del
alga. Los azúcares que forman esta molécula son principalmente la ramnosa, el

A B

100µm

Figura 1.3: A. Fotografía por microscopía óptica del corte transversal de un talo de Ulva sp. en la que
se puede apreciar la doble capa celular con la pared que las rodea. B. esquema de la repartición de
los polisacáridos en el alga según Lahaye, 2007.

22
 1. Introducción

ácido glucurónico, la glucosa, la xilosa, el ácido idurónico y contiene grupos

sulfatos.
Las características estructurales del ulvan son variables lo que puede tener
una influencia sobre las propiedades de este material. Diferentes estudios de
estructuras sobre diferentes especies del género Ulva han demostrado que el
factor genético tiene una importancia fundamental en la concentración de sus
azúcares, el grado de la sulfatación, el peso molecular, la secuencia y la repetición
de las subunidades estructurales [45].
Un estudio llevado sobre dos especies diferentes Ulva armoricana y Ulva
rotundata [46] ha demostrado que independientemente del método de
extracción y de las condiciones ambientales existía variaciones estructurales entre
ellas que se atribuyen al factor genético. También se demuestra que existen
variaciones estructurales en las algas de la misma especie relacionadas con
cambios estacionales. Estos cambios se manifiestan por rendimiento de
extracción, peso molecular, composición en azucares y secuencia de azucares.
La tasa de crecimiento máxima de estas dos especies se ha observado
durante los meses de primavera y los meses de otoño. Estos periodos
corresponden a un aumento de los nitratos de origen agrícola en las aguas
costeras arrastrados por las lluvias que caen durante estas temporadas. Se piensa
que durante estas fases de rápido crecimiento el ulvan producido se encuentra
enlazado de forma aún débil con los otros componentes de la pared celular lo
que le hace ser más disponible para su extracción. Los rendimientos son
especialmente altos en las fases finales de los periodos de crecimiento activo. En
estos periodos también se observó la extracción de moléculas de ulvan de un
peso molecular superior a los periodos de crecimiento lento en los que la
repartición del peso molecular del ulvan extraído se desplazaba hacías tamaños
inferiores [46].
La variación estacional del perfil de azucares fue limitada para las dos
especies excepto en el contenido en xilosa de U. rotundata que fue inferior en los
meses de rápido crecimiento. De forma general el contenido en ácido
glucurónico fue superior para las dos algas durante los periodos de alto

23
 1. Introducción

crecimiento mostrando una proporción de ácido idurónico menor que durante

los otros periodos10.
Los análisis que se hicieron con el fin de analizar la variación de la
proporción de los oligosacáridos obtenidos por degradación con una enzima
específica del ulvan han demostrado tener resultados desiguales entre las dos
especies. Los oligosacáridos obtenidos mostraron diferencias estacionales en
abundancia significantes en U. rotundata pero no mostraron diferencia en U.
armoricana. Esta diferencia podría estar asociada a modos de vida diferentes,
Ulva rotundata se encuentra en zonas poco profundas de zonas costeras rocosas
mientras que Ulva armoricana es una especie que vive libremente en la columna
de agua bajo condiciones más estables [46].
La información sobre la influencia de las condiciones externas y los factores
genéticos son de gran importancia para poder anticipar rendimientos y calidad
del extracto final, aspectos fundamentales para su futuro uso.

1.3.2. Composición y estructura

1.3.2.1. COMPOSICIÓN SACARÍDICA

El ulvan es un heteropolisacárido sulfatado ligeramente ramificado lo que

significa que contiene varios azucares diferentes los cuales pueden estar
sulfatados. La molécula es lineal con algunas ramificaciones cortas.
Esta molécula está formada por dos tipos de azúcares, azucares neutros y
azucares ácidos. Los azucares neutros son la ramnosa, la xilosa y la glucosa y los
azucares ácidos son los ácidos urónicos llamados ácido glucurónico y ácido
idurónico. Ocasionalmente se pueden encontrar pequeñas cantidades de ribosa,
manosa y galactosa [45].

10
Esto sugiere que la epimerasa responsable de la conversión del ácido
glucurónico en ácido idurónico tiene una cinética lenta

24
 1. Introducción

Definir precisamente la composición sacarídica del ulvan no es algo

sencillo. El análisis de la composición se hace normalmente mediante
comatografía (HPLC o GC) lo que requiere separar los azucares los unos de los
otros. Esta tarea sin embargo es más complicada que para otros poliscáridos
puesto que el ulvan contiene algunos enlaces resistentes a la hidrólisis con ácidos
débiles. Algunos autores [47–49] emplearon un método secuencial de hidrólisis
ácida con una hidrólisis enzimática sin embargo este método requiere tener las
enzimas específicas para estos enlaces lo cual no está disponible en el mercado.
La hidrólisis con ácido fuerte que si bien parece que rompe eficientemente los
enlaces también podría destruir parte de los azúcares liberados llevando a un
resultado de análisis sesgado. El método más empleado y que al parecer rompe
eficientemente la molécula a la vez que conserva los azúcares es la metanólisis
ácida11. A raíz de diferencias en la metodología de extracción y análisis, de la
complicada determinación de la especie, de las diferencias entre especies así
como a causa las variaciones derivadas de las condiciones medioambientales,
existen datos dispares sobre la composición del ulvan.
Las proporciones en las que se han descrito los azucares hasta ahora son
de 16.8-45.0% de ramnosa, 2.1-12.0% de xilosa, 0.5-6.4% de glucosa, 6.5-19.0%
de ácidos urónicos de los cuales 1.1-1.9% se encuentra en forma de ácido
idúronico, el resto siendo ácido glucúronico. El sulfato representa del 16.0-23.2%
del peso del extracto seco. En U. armoricana el porcentaje de azucares sulfatados
fue del 64.9% [50].
La ramnosa se encuentra bajo su forma α piranosa “L-“ , la xilosa en su
forma β piranosa “D-“, el ácido glucurónico en forma β piranosa “D-“ el ácido
idurónico en su forma α “L-” y la glucosa bajo fu forma α “D-“.

Los ácidos urónicos son azucares que tienen la particularidad de haber sido
oxidados en el grupo hidroxilo terminal por un atomo de oxígeno dando lugar a

11
Este paso es esencial para la derivatización de los azúcares para su
análisis en GC por el método de los TMS.

25
 1. Introducción

una función ácido carboxílico. El pKa de este azúcar es de 3.28. La ramnosa está
siempre funcionalizada con un grupo sulfato en el carbono 3. La xilosa también
puede llevar un grupo sulfato en el carbono 2. Tanto los ácidos urónicos como
los grupos sulfatos le dan a esta molécula un carácter fuertemente aniónico con
una repartición homogénea de las cargas que representa de 2.8 a 3.77meq
carga/g. Esta característica es de gran importancia para el comportamiento
fisicoquímico y biológico de esta molécula.

1.3.2.2. SECUENCIA

Otro aspecto que tiene grandes implicaciones en las propiedades de la

molécula es la secuencia de los monosacáridos previamente mencionados.
Mediante técnicas de hidrolisis enzimáticas y químicas se puede conseguir la
degradación del polisacárido en oligosacáridos. Una vez obtenidos estos
oligosacáridos se separan y cuantifican por cromatografía y su estructura se
estudia mediante varios métodos de resonancia magnética nuclear [51,52].
En estos estudios queda claro que existe repetición de algunas secuencias
disacarídicas particulares, de las cuales dos destacan particularmente y que se
denominan ácido ulvanobiourónico de tipo A y de tipo B. En el caso del tipo A se
trata de una secuencia de ácido glucurónico enlazado por enlace β-14 a una
ramnosa sulfatada en el carbono 3. En el ácido ulvanobiourónico de tipo B el
ácido idurónico reemplaza al ácido glucúronico convirtiendo el enlace en un
enlace α 14. Para más facilidad estas dos secuencias se anotan respectivamente
A3S y B3S (figura 1.4).
La estructura de otras secuencias oligosacarídicas también se han
encontrado después de hidrólisis enzimáticas: la ulvanobiosa 3 sulfato que se
compone de un residuo de xilosa enlazado con una ramnosa por un enlace β1-4
y se escribe U3S. La ulvanobiosa 2’,3 sulfato es la misma secuencia pero la xilosa
esta sulfata en el carbono 2, se escribe U2’3S.

26
 1. Introducción

La secuencia A3S puede llevar igualmente en el carbono 2 de la ramnosa

un ácido glucurónico enlazado por β 12. Esta secuencia se escribe A2g3S. La
estructura asignada a este último oligosacárido implica que el ulvan es un
polisacárido ligeramente ramificado.
Como se puede observar en la Figura 1.4 el polisacárido no se ha roto por
el enlace del ácido ulvanobiourónico sino por enlaces diferentes. Este enlace es
resistente a la hidrólisis con ácido débil y su degradación debe hacerse con
enzimas específicas. Las enzimas capaces de degradar este enlace se denominan
ulvan-lyasas pero existe aún pocas que se hayan descubierto y no se encuentran
a nivel comercial. El análisis por resonancia magnética de los fragmentos
obtenidos con este tipo de enzimas refuerzan los datos de secuencias. Los
oligosacáridos obtenidos presentan la especificidad de tener un enlace
insaturado en la extremidad no reductora efecto del proceso de corte por la
enzima. El corte del polisacárido en oligosacárido mediante hydrolysis enzimática
con una β-glucuronidasa parece indicar que estas secuencias pueden estar
interrumpidas por ácido glucurónico.
Patrones de repetición de las secuencias previamente descritas se pueden

Figura 1.4: Representación de

las subunidades discararídicas
estructurales del ulvan
A3S ácido ulvanobiourónico de
tipo A, B3S ácido
ulvanobiourónico de tipo B.
Extracto de [51]

27
 1. Introducción

observar en los oligosacáridos aislados. Algunas secuencias son comunes entre

diferentes algas de diferentes orígenes por ejemplo es –A3S-A3S- ; -A3S-U3S- ; -A3S-
U2’3S- pero otras secuencias son propia de cada tipo de alga. Aún no se sabe si
estas diferencias se deben a un origen genético diferente o si depende de las
condiciones fisiológicas del alga [45].

1.3.3.3. PESO MOLECULAR

El extracto acuoso de Ulva sp. puede contener moléculas con una gran
dispersión de tamaños sin embargo esta dispersión se puede deber a otras
moléculas que están coextraidas con el ulvan. Los pesos moleculares que se han
encontrado descrito hasta ahora se encuentran entre 1.7x105 g/mol hasta 8.2x106
g/mol. Los valores publicados parecen presentar una gran disparidad. La
temperatura de extracción es un factor importante siendo mayor el tamaño
molecular de los extractos a 80-90 °C que a 30-40 °C lo que indica que las altas
temperaturas son necesarias para extraer las moléculas de mayor tamaño [53]. El
origen del alga, el periodo de recolección, el método analítico y el solvente
usados para la extracción son otros factores que pueden impactar la estabilidad
de los resultados entre estudios.
Algunos autores tienen la teoría de que el motivo de la gran dispersión de
tamaño molecular se debe a la conformación del ulvan en soluciones acuosas. Al
no ser completamente soluble en este medio el ulvan adopta una forma de
partícula en suspensión en vez de una molécula completamente desplegada y los
agregados de distintos tamaños de estas partículas podrían ser el origen de la
disparidad de pesos moleculares [54].

1.3.3.4. CONFORMACIÓN

La composición de la molécula y su tamaño molecular no consiguen por si

solos explicar todas las propiedades de los polisacáridos pero sí son la base de
las diferentes interacciones intramoleculares que definen la conformación de la
molécula.

28
 1. Introducción

La capacidad de las macromoléculas de formar geles y tener interacciones

con otras moléculas biológicas reside en la formación de zonas de junción entre
fragmentos moleculares de conformación estructurada. El ulvan presenta una
secuencia variable que resulta en una conformación general aparentemente
desordenada. Las secuencias A3S, B3S, U3S, U2’3S y A2g3S sin embargo tienen enlaces
glucosídicos adecuados que les permite formar estructuras organizadas. Se
encuentra localmente dentro de la molécula estas repeticiones de estos dímeros
idénticos las cuales forman estructuras helicoidales que se estabilizan
internamente por enlaces hidrógenos. Los dímeros A 3S y B3S han demostrado
formar hélices internamente a partir de dos repeticiones de dímeros. La
conformación del hélice producido por B3S depende de la propia configuración
del ácido idurónico. Las interacciones electrostáticas y el pH del medio en el que
se encuentra el ulvan determinan si el ácido idurónico se encuentra en forma de
“silla” o en forma de “bote torcido”. Esta característica implica que el polímero
puede cambiar de un estado ordenado a un estado desordenado cuando cambia
la conformación de este azúcar [50]. Es probable que otros azúcares como la
ramnosa del A2g3S pueda presentar la misma flexibilidad dependiendo de las
condiciones y de su entorno electrostático. Esta flexibilidad juega un papel
primordial en las propiedades de otros polisacáridos favoreciendo las
interacciones biológicas [55]. Estas hélices son probablemente los sitios de
interacción entre moléculas que le permiten tener diversas propiedades [52].

1.3.4. Bioactividad y Usos

Existe una amplia y diversa literatura científica sobre el estudio de la

utilización de extractos de algas verdes del género Ulva. Esta sección pretende
hacer énfasis en resumir y recopilar los artículos encontrados sobre el uso del
ulvan como componente de biomateriales y sus efectos sobre el sistema inmune,
dos aspectos estrechamente relacionados con el presente trabajo.

29
 1. Introducción

1.3.4.1. PROPIEDADES FISICOQUÍMICAS

Como hemos visto el ulvan presenta características estructurales que

hacen de él una molécula de interés por sus potenciales interacciones con otras
moléculas. Una de las primeras propiedades que despertó la atención sobre este
polímero es su capacidad a formar geles.
Probablemente debido a su composición monosacarídica el ulvan tiene
una solubilidad baja en medio acuoso. Se piensa [54] que el grupo metilo de la
ramnosa es responsable del carácter hidrófobo y los grupos cargados
negativamente por su carácter hidrófilo. A consecuencia el ulvan forma
naturalmente en el agua una suspensión de partículas con interacciones limitadas
entre las moléculas, esto resulta en una viscosidad baja (95-285 ml/g) comparado
con otros polisacáridos de algas.
A consecuencia de esta conformación en el medio acuoso los geles
formados resultan en una red de partículas enlazadas entre ellas por filamentos
compuestos por ulvan, proteínas u otros poliscáridos contaminantes. La
estabilización de esta red se hace mediante cationes divalentes, ácido bórico y
un pH entre 7,5 y 8. No se conoce completamente el mecanismo de gelación
aunque se piensa que el ácido bórico forma esteres de borato que pueden
coordinar el catión divalente para formar un enlace iónico. Probablemente
debido a los escasos sitios de interacción entre moléculas las propiedades de los
geles obtenidos mediante técnicas de gelación físicas no han mostrado ser lo
suficientemente fuertes para poder ser usados en la elaboración de biomateriales
como tal. Existe sin embargo un verdadero interés para conseguir geles
biocompatibles de origen no sintético, no animal, biodegradables y que puedan
ofrecer las propiedades biológicas de los polisacáridos sulfatados a la vez que
almacenan y liberan otras moléculas farmacéuticas de forma controlada [56].
La estabilidad de los geles de ulvan ha sido aumentada por
funcionalización con anhídrido metacrilico y con glicidil metacrilato. Esto dio
lugar a moléculas fotopolimerizables que forman enlaces covalentes entre ellas
dando lugar a geles más estables [57]. Otros geles fotopolimerizables de ulvan

30
 1. Introducción

son termorreversibles a 30-31°C [58].

Sin embargo las estructuras no parecieron citocompatibles en un
experimento de cultivo celular. El autor explica este problema a que el ulvan por
tener un alto contenido en cargas negativas monopolizó el agua dentro de este
andamio molecular [59]. Para remediar a este problema se consiguó mineralizar
enzimáticamente el andamio molecular formando un material apto para el cultivo
de células óseas [60].
Los geles formados por gelación ionotrópica del ulvan y quitosano
permiten evitar también la monopolización del agua y mostraron buenas
propiedades como matriz 3D de cultivos celulares de fibroblastos de ratón [59].
La compatibilidad de los geles de ulvan y quitosano con el cultivo celular
ha sido demostrada en otras ocasiones. En un estudio realizado en paralelo se
cultivaron células óseas en membranas de quitosano y ulvan que mostraron tener
una organización semejante a la matriz extracelular de éstas células in vivo [61].
El quitosano ya ha demostrado su capacidad a formar estructuras estables
con el ulvan. La inclusión de partículas de quitosano y ulvan en un andamio
molecular de poly-D-L-ácido láctico han demostrado ser un buen candidato para
liberar moléculas con efecto biológico como la dexametasona [62]. En otro
estudio este grupo propuso realizar la gelación del ulvan mediante reacción con
el 1,4-butanediol diglicedil éter (BDDE), un agente reticulante frecuentemente
utilizado en la formación de enlaces covalentes entre polisacáridos. Las
membranas obtenidas con esta técnica son buenas candidatas para un nuevo
tipo de apósitos capaz de liberar sustancias de forma sostenida [63].
Con la misma idea de aplicación en biomateriales con capacidad de
retención de moléculas se fabricaron nanofibras de ulvan con PVA (poly-vinil
alcohol) en presencia de iones de calcio y ácido bórico. La técnica empleada fue
el electrospinning. El ratio ulvan/PVA 50:50 dio fibras perfectamente uniformes
con un diámetro de 105nm [64].

31
 1. Introducción

1.3.4.2. PROPIEDADES BIOLÓGICAS

La composición y la estructura del ulvan son únicas en los organismos

fotosintéticos. Los ácidos glucurónicos y especialmente el ácido idurónico son
unos de los mayores componentes de los glicosaminoglicanos (GAGs), unos
mucopolisacáridos con múltiples funciones que son producidos en los tejidos
conjuntivos de los animales como la heparina, el condroitin sulfato, el dermatan
sulfato, el ácido hialurónico, el heparan sulfato y el queratan sulfato. Hasta la
fecha el ácido idurónico no se ha encontrado en ningún otro vegetal. Más allá del
simple hecho que el ulvan podría ser una fuente de bajo coste de azúcares raros,
despierta la curiosidad sobre su potencial como compuesto biomimético para
actividades similares a los GAGs.
Las paredes celulares de otros organismos como las bacterias gram-
negativa pueden tener altos contenidos en ramnosa. La cascada de reacciones de
defensa de los animales contra las bacterias implica el reconocimiento por los
receptores “Toll-like” de las estructuras polisacarídicas. Este reconocimiento lleva
por ejemplo al estallido oxidativo de los macrófagos. Este mecanismo provoca la
liberación por los macrófagos de una importante de cantidad de iones
superóxidos con el fin de contratacar en caso de detección de un patógeno.
En un estudio preliminar, se estudió las reacciones de macrófagos de
rodaballo (Psetta maxima) expuestos in vitro e in vivo a extractos acuosos de U.
rigida naturales y con una desulfatación. Demostraron que este polisacárido es
capaz de desencadenar una reacción inmune tal como la activación de la
interleukina 1β (IL-1β) y el estallido oxidativo. Demuestran también que el grupo
sulfato así como el patrón de sulfatación son factores determinantes para que
ésto ocurra [65]. Estos resultados se repitieron en macrófagos de ratones
induciendo además la transcripción de una serie de genes implicados en la
respuesta inflamatoria. Estas propiedades se perdieron una vez desulfatados los
polisacáridos [66]. Para demostrar la efectividad de este compuesto como aditivo
inmunoestimulante en la dieta de los peces, se incluyeron diferentes
concentraciones de ulvan al pienso de Mugil cephalus que se alimentaron de ello

32
 1. Introducción

durante 8 semanas. Después de esto los peces se incubaron con un patógeno

letal Photobacterium damselae. El control tuvo una mortalidad acumulada de
79±1.5% después de 10 días de incubación con la bacteria, el porcentaje más bajo
de acumulación de mortandad se consiguió con la concentración de 10 mg de
ulvan por kilo de pienso en un 16±0.8%. Estos resultados están apoyados por los
resultados de niveles de lisozima y actividad fagocítica observados en los grupos
que recibieron el ulvan. Este estudio también demuestra que los peces que
recibieron esta dieta desarrollaron una capacidad antioxidante total superior a
los peces que no recibieron ulvan. El autor resalta el hecho que estos resultados
no se han encontrado para otras especies con pienso que incluían polvo de Ulva
sp. o por inmersiones puntuales en ulvan. El efecto podría depender de la especie
animal estudiada, la dosis de ulvan, el tipo de extracto y la forma de administrar
el producto [67] o bien por la acción de otros componentes presentes en el polvo
de Ulva que pueden enmascarar el efecto beneficioso del ulvan.
Resultados de inclusión de ulvan purificado también apuntan a la
activación del sistema inmune de Penaeus monodon, el langostino tigre [68].
Optimizar la supervivencia de los animales evitando que padezcan
enfermedades se ha convertido en los últimos años en una medida profiláctica
que los productores en acuicultura valoran pues el uso de antibióticos está cada
vez más restringido si no está ya prohibido [69].
Recientemente se ha demostrado que el ulvan es capaz de activar la
producción de ARN mensajero de citoquinas en un cultivo in vitro de células
epiteliales del intestino de cerdo. El reconocimiento del ulvan y la activación de
una cascada inmunógena en células del intestino permiten pensar que el
intestino puede avisar al sistema inmune mediante mensajes bioquímicos de la
presencia de algún patógeno para que esté preparado a enfrentarlo. En este
estudio descubrieron que se activaba la transcripción del gen de la citoquina IL-
8 a través del enlace del ulvan con el receptor “Toll-Like” 4 (TLR4) en células
embrionarias renales modificadas in vitro. El extracto no tuvo efecto de activación
con otros TLR lo que indica una gran especificidad [70].
La activación de las defensas por el ulvan no está exclusivamente reservada
al reino animal. El estudio de uso de ulvan purificado sobre Medicago truncatula,

33
 1. Introducción

un tipo de trébol usado como modelo vegetal, indujo la transcripción de unos

genes similares a la aplicación de una hormona vegetal, el metil jasmonato. Estos
genes están todos relacionados con la defensa de la planta contra patógenos
[71]. Estos resultados fueron confirmados por utilización de estos extractos de
U. armoricana sobre plantas de pepino, vides y guisantes incubadas con
diferentes especies de oídio. Las plantas tratadas mantuvieron el porcentaje de
hojas afectadas en niveles 80% inferiores a los controles para todas las especies
vegetales a una concentración de 6 g.l-1 [72].
La actividad inmunoestimulante del ulvan está cada vez mejor descrita y
mejor contrastada con estudios realizados por grupos de investigadores a nivel
mundial. Es probablemente la propiedad biológica que más se ha estudiado hasta
ahora y demuestra el potencial más prometedor para desarrollar aplicaciones en
productos inmunoestimulantes o adyuvantes en vacunas siendo objeto de varias
patentes.
Además de la imunoestimulación el ulvan ha demostrado tener efectos en
actividad hepatoprotectora [73], actividad anti-hyperlipidemica y
antihypercolesterolémica [74–77], función antioxidante [78–81], como anti-
adhesivo bacteriano [82], antiviral [83] y antitumoral [81].

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from Renewable Resources, in: R. Pignatello (Ed.), Biomater. Phys. Chem., InTech,
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of synthetic and natural origin for biomedical and environmental applications,
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al., Biofunctionalization of ulvan scaffolds for bone tissue engineering., ACS Appl.
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Roussis, et al., ulvan and ulvan /chitosan polyelectrolyte nanofibrous membranes
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[62] A. Alves, A.R.C. Duarte, J.F. Mano, R. a. Sousa, R.L. Reis, PDLLA
enriched with ulvan particles as a novel 3D porous scaffold targeted for bone
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42
 1. Introducción

drug delivery applications., Int. J. Pharm. 426 (2012) 76–81.

doi:10.1016/j.ijpharm.2012.01.021.

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V. Roussis, Nanofibers based on polysaccharides from the green seaweed Ulva
Rigida, Carbohydr. Polym. 84 (2011) 1093–1102.
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Stimulation of turbot phagocytes by Ulva rigida C. Agardh polysaccharides,
Aquaculture. 254 (2006) 9–20. doi:10.1016/j.aquaculture.2005.10.012.

[66] J.M. Leiro, R. Castro, J. a Arranz, J. Lamas, Immunomodulating

activities of acidic sulphated polysaccharides obtained from the seaweed Ulva
rigida C. Agardh., Int. Immunopharmacol. 7 (2007) 879–88.
doi:10.1016/j.intimp.2007.02.007.

[67] P. Akbary, Z. Aminikhoei, Effect of water-soluble polysaccharide

extract from the green alga Ulva rigida on growth performance, antioxidant
enzyme activity, and immune stimulation of grey mullet Mugil cephalus, J. Appl.
Phycol. (2017) 1–9. doi:10.1007/s10811-017-1299-8.

[68] R.S. Declarador, A.E. Serrano Jr., V.L. Corre Jr., Ulvan extract acts
as immunostimulant against white spot syndrome virus ( WSSV ) in juvenile black
tiger shrimp Penaeus monodon, Int. J. Bioflux Soc. 7 (2014) 153–161.

[69] J. Galindo-Villegas, H. Hosokawa, Immunostimulants : Towards

Temporary Prevention of Diseases in Marine Fish, in: Memorias VII Simp. Int. Nutr.
Acuícola, Hermosillo, 2004: pp. 279–319.

[70] M. Berri, M. Olivier, S. Holbert, J. Dupont, H. Demais, M. Le Goff,

et al., Ulvan from Ulva armoricana (Chlorophyta) activates the PI3K/Akt signalling

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pathway via TLR4 to induce intestinal cytokine production, Algal Res. 28 (2017)
39–47. doi:10.1016/j.algal.2017.10.008.

[71] V. Jaulneau, C. Lafitte, C. Jacquet, S. Fournier, S. Salamagne, X.

Briand, et al., Ulvan, a sulfated polysaccharide from green algae, activates plant
immunity through the jasmonic acid signaling pathway., J. Biomed. Biotechnol.
2010 (2010) 525291. doi:10.1155/2010/525291.

[72] V. Jaulneau, C. Lafitte, M.-F. Corio-Costet, M.J. Stadnik, S.

Salamagne, X. Briand, et al., An Ulva armoricana extract protects plants against
three powdery mildew pathogens, Eur. J. Plant Pathol. 131 (2011) 393–401.
doi:10.1007/s10658-011-9816-0.

[73] A. Sathivel, Balavinayagamani, B.R. Hanumantha Rao, T. Devaki,

Sulfated polysaccharide isolated from Ulva lactuca attenuates d -galactosamine
induced DNA fragmentation and necrosis during liver damage in rats, Pharm. Biol.
52 (2014) 498–505. doi:10.3109/13880209.2013.846915.

[74] Y. Pengzhan, L. Ning, L. Xiguang, Z. Gefei, Z. Quanbin, L.

Pengcheng, Antihyperlipidemic effects of different molecular weight sulfated
polysaccharides from Ulva pertusa (Chlorophyta), Pharmacol. Res. 48 (2003) 543–
549. doi:10.1016/S1043-6618(03)00215-9.

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Polysaccharides from Ulva pertusa ( Chlorophyta ) and preliminary studies on
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[76] X. Liu, X. Wang, J. Zhang, Y. Duan, H. Qi, Q. Zhang, Synthesis and

antihyperlipidemic activity of acetylated derivative of ulvan from Ulva pertusa, Int.
J. Biol. Macromol. 50 (2012) 270–272. doi:10.1016/j.ijbiomac.2011.11.006.

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[77] M. Godard, K. Décordé, E. Ventura, G. Soteras, J. Baccou, J. Cristol,

et al., Polysaccharides from the green alga Ulva rigida improve the antioxidant
status and prevent fatty streak lesions in the high cholesterol fed hamster , an
animal model of nutritionally-induced atherosclerosis, Food Chem. 115 (2009)
176–180. doi:10.1016/j.foodchem.2008.11.084.

[78] H. Qi, T. Zhao, Q. Zhang, Z. Li, Z. Zhao, R. Xing, Antioxidant activity

of different molecular weight sulfated polysaccharides from Ulva pertusa Kjellm
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Antioxidant activity of different sulfate content derivatives of polysaccharide
extracted from Ulva pertusa (Chlorophyta) in vitro., Int. J. Biol. Macromol. 37
(2005) 195–9. doi:10.1016/j.ijbiomac.2005.10.008.

[80] H. Qi, X. Liu, J. Ma, Q. Zhang, Z. Li, In vitro antioxidant activity of

acetylated and benzoylated derivatives of polysaccharide extracted from Ulva
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doi:10.1016/j.bmcl.2006.01.076.

[81] P. Shao, Y. Pei, Z. Fang, P. Sun, Effects of partial desulfation on

antioxidant and inhibition of DLD cancer cell of Ulva fasciata polysaccharide., Int.
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[82] V. Gadenne, L. Lebrun, T. Jouenne, P. Thebault, Antiadhesive

activity of ulvan polysaccharides covalently immobilized onto titanium surface.,
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P. Zapata-Benavides, E. Mendoza-gamboa, et al., Sulphated Polysaccharides from

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 1. Introducción

Ulva clathrata and Cladosiphon okamuranus Seaweeds both Inhibit Viral

Attachmente/Entry and Cell-Cell Fusions, in NDV Infection, Mar. Drugs. 13 (2015)
697–712. doi:10.3390/md13020697.

46
2. Hipótesis y Objetivos

47
 2. Hipótesis y Objetivos

2.1 Hipótesis

 Las condiciones ambientales y físico-químicas del medio en que se

encuentre el alga Ulva influye en la concentración, estructura y
propiedades de sus compuestos.

 La utilización de extractos enzimáticos procedentes de bacterias marinas

proporcionan fracciones homogéneas del polisacárido ulvan y
contribuyen a potenciar sus propiedades biológicas.

 Los polisacáridos extraídos del alga Ulva y sus derivados pueden ser
utilizados en la elaboración de nanopartículas de quitosano. El complejo
quitosano- ulvan ejerce un efecto potenciador de las propiedades de
ulvan sobre el sistema de defensa del lenguado Solea senegalensis.

 Los métodos de extracción clásicos empleados para identificar los

compuestos fenólicos pueden estar subestimando el contenido total de
dichos compuestos en el alga Ulva sp..

2.2 Objetivo General

Determinar el potencial de polisacáridos sulfatados procedentes de la macroalga

Ulva sp. como fuente de ingredientes funcionales en acuicultura.

48
 2. Hipótesis y Objetivos

2.3 Objetivos específicos

1. Determinar la influencia de parámetros ambientales y físico-químicos en la

proporción y composición de polisacáridos procedentes del alga Ulva sp.

2. Seleccionar e identificar una cepa de Ulva sp. procedente del litoral

suratlántico andaluz para su caracterización y puesta en cultivo

3. Disponer de una metodología que permita obtener oligosacáridos con una

estructura homogénea a partir del polisacárido ulvan empleando técnicas
enzimáticas y químicas.

4. Evaluar el efecto inmunoestimulante y antioxidante de polisacáridos y

oligosacáridos procedentes del alga Ulva en células de Solea senegalensis.

5. Elaborar nanopartículas complejas de quitosano- ulvan para su potencial

aplicación en acuicultura

6. Evaluar la eficiencia de técnicas alternativas de extracción de compuestos

fenólicos antioxidantes

49
 2. Hipótesis y Objetivos

La memoria de ésta tesis se ha organizado según el siguiente esquema:

 En el capítulo 3 se presenta un trabajo de estudio sobre la variación anual

de los componentes proximales del alga Ulva ohnoi en dos puntos de
muestreo de una zona de esteros respondiendo a los objetivos
específicos 1 y 2.

 En el capítulo 4 se describe la extracción y purificación del ulvan y se

describe la producción de oligosacáridos a partir de una enzima aislada
de una bacteria intestinal del anfípodo Gammarus insensibilis que da
respuesta al objetivo específico 3.

 En el capítulo 5 se describe la obtención de nanopartículas a partir de

quitosano incorporando ulvan. Se compara el efecto inmunoestimulante
de estas nanopartículas que incorporan oligosacáridos o polisacáridos
de ulvan frente a los productos de ulvan sin encapsular sobre macrófagos
de Solea senegalensis. Este capítulo responde a los objetivos específicos
4 y 5.

 En el capítulo 6 se describe un método para mejorar la extracción de

moléculas antioxidantes de Ulva ohnoi. Ésta macroalga fue recolectada,
identificada (anexo I) y puesta en cultivo respondiendo a los objetivos
específicos 2 y 6.

 En el capitulo 7 se discuten de forma general los resultados de los

diferentes trabajos realizados en la presente tesis así como los retos y
perspectivas

 En el capítulo 8 las conclusiones generales de la presente Tesis Doctoral

se presentan en español y en inglés.

50
 Seasonal variation

3 . S eas onal va ria tion of proxima te

composition, ulva n content a nd
s ugar composition of Ulva ohnoi
(Chlorophy ta, Ulvales ) in rela tion
to growth a nd environmental
pa ra meters

51
 3. Seasonal variation

Título del Artículo:

Seasonal variation of proximate composition, ulvan content and sugar

composition of Ulva ohnoi (Chlorophyta: Ulvales) in relation to growth and
environmental parameters

Autores:

Olivier Coste a, Erik-jan Maltab, M. Teresa Jiménez Perala, and Catalina Fernández-
Díaza

Publicado en:
No publicado

a
IFAPA, Centro El Toruño, Camino del Tiro Pichón s/n, 11500 El Puerto de Santa
María, Cádiz, Spain
b
Instituto de Investigación y Formación Agraria y Pesquera de Andalucía (IFAPA)
Centro Agua del Pino, Ctra. Cartaya-Punta Umbría Km 4, 21450 Cartaya, Huelva,
Spain

Abstract:
Growth parameters, composition and ulvan-rich water soluble extract
composition of Ulva ohnoi naturally present in two sampling stations of salt
marshes were measured over the 4 seasons. Important variations were found to
occur over the seasons. The main differences between the sampling stations were
nitrate input and temperature amplitude where nitrate nitrogen was almost
inexistent in one of the stations. It is concluded that temperature and light are
the main drivers of the variation in growth and biochemical composition, but that
local factors, such as nitrogen concentration and salinity might also play an
important role.

52
 3. Seasonal variation

3.1. Introduction

Macroalgae are key elements of coastal marine ecosystems, providing a

wide range of ecosystem functions [1]. They also have a long history of uses by
humans for commercial purposes, such as human and animal food and feed and
possess a wide variety of components that are interesting for nutraceutical,
pharmaceutical, cosmetic and other applications [2]. Indeed, in volume, seaweed
cultivation is the largest form of mariculture [3].
For seaweed farmers or cultivators, which might be either interested in just
total biomass yield or in specific components, it is important to obtain knowledge
on factors affecting growth and composition to develop optimal cultivation
protocols. On the other hand, this is relevant for ecologists as well as these factors
determine nutrient (C, N and P) allocation that in turn affect ecosystem
functioning.
Thus far, most field studies in applied phycology have mainly been
focusing on one or a limited number of components, centering on spatial and/or
seasonal variation (e.g. [4-6]). However, these studies rarely measure growth rate
or production and are usually not taking into account environmental variables.
Ecological studies on the contrary, typically focus on growth and/or production
in relation to variation in environmental parameters (e.g. [7-9]). However, in these
studies, tissue composition analyses are usually limited to specific parameters
related to production (such as pigment contents) or that might indicate limiting
conditions, such as tissue nutrient contents. In addition, many studies exist that
focus on specific components related to oxidative stress, UV-protection, etc. (e.g.
[10-12]).
Integrated studies, taking into account the relevant environmental
parameters, growth rates and biochemical composition, including tissue nutrient
contents, proximate composition (proteins, carbohydrates, lipids) and pigment
contents are rare.

53
 3. Seasonal variation

Species of the chlorophyte genus Ulva are key examples of the developing
use of seaweeds as a biomass resource as they can be produced across a diversity
of production systems both at sea and on land. They are highly adaptable across
a broad range of environments and can be efficiently integrated for the
bioremediation of nutrients from animal production systems such as fish farms
[13-15]. In addition, these seaweeds contain considerable amounts of the
polysaccharide ulvan that show high potential for a wide range of applications as
feed supplements, for pharmaceutics, plant growth stimulators, cosmetics, etc.
(this thesis, [16]).
This study was defined to fill the gap between ecological and applied
studies, with an integrated study on Ulva ohnoi growing in the context of an
experimental finfish cultivation system in a former saltmarsh. Seasonal variation
of Ulva growth rates were determined, together with environmental variables,
and seaweed tissue nutrient and proximate composition in order to reveal
general patterns between these parameters.

3.2. Materials and methods

3.2.1. Area description and sampling stations

Algal collections and incubations were carried out in earthen ponds of the
experimental fish farm of the research station IFAPA “El Toruño” in El Puerto de
Santa María, Cádiz province, south Spain (36° 34’ 50.34’’ N; 6° 12’ 32.47’’ W). Two
stations were selected following initial surveys in which they were found to host
important populations of Ulva ohnoi (see identification in Annex I), representing
different water bodies and that were consequently suspected of having different
nutrient regimes. Station 1 was situated in a large water storage basin, acting as
a reserve when no water can be taken in from the tidal river due to - for instance
- strong freshwater discharges following rainy periods or neap tides. Seaweed
diversity was relatively high in this station, hosting several foliose and filamentose

54
 3. Seasonal variation

Ulva ohnoi and various other filamentous red and green seaweeds. Water
exchange of this area was generally low, except for the rainy period of winter.
Station 2 was situated in a small and shallow outlet channel, receiving waste water
of outdoor (extensive) tank cultures. Vegetation consisted almost exclusively of
Ulva ohnoi, benthic diatoms and cyanophytes. Average depth of stations 1 was
approximately 1 m, whereas for station 2 this was approximately 20 cm, although
variation occurred depending on circulation regime in the fish cultivation plant.

3.2.2. Algal collection and growth measurements

For each season (March, July, and November 2011 and January 2012),
foliose Ulva ohnoi were collected for determination of growth rate and
biochemical analyses. Healthy thalli were collected the Monday of the first week
of each month from the two stations around 10.00 in the morning and
transported to the lab where they were briefly rinsed with filtered seawater and
kept separated by station of origin. Growth rate for each station was determined
by incubating thalli from that station in cilindrical (9 cm diameter), 30 cm long
mesh cages (1 x 1 cm mesh size). Discs (3 cm diameter) were punched from the
thalli, carefully blotted dry between tissues, weighed individually and put in the
cages. Five discs per cage were used and three cages per station were incubated
at the same depth of the free-floating algae, about 10 – 15 cm for both stations.
Cages were retrieved after one week, after which the discs were collected, rinsed
with seawater, blotted dry and weighed again. Relative growth rate (RGR, d-1) was
determined as (ln Wt – ln W0)/t where W0 and Wt are initial and final weight
respectively and t is time in number of days. Following this, discs were dried for
72 hours at 70 °C and weighed to determine fresh weight/dry weight ratio
(FW/DW), after which the discs were stored dry for determination of ash content.
Collections of algae for proximate composition and sugar analysis were
made at the same day as the retrieval of the cages. Thalli were stored in the dark
in ice boxes and transported to the lab within 10 minutes after collection for
further procession. Approximately 500 mg subsamples were gently blotted and

55
 3. Seasonal variation

dried or 72 hours at 70 °C and stored dry for analysis of tissue nutrient (carbon,
nitrogen and phosphorus) contents. Another approximate 5 g of fresh thalli were
rinsed with distilled water as well, blotted dry and stored in marked plastic bags
at -20 °C for analysis of proximate (total carbohydrates, lipids and protein
contents). Triplicate samples per station were stored for all parameters. In
addition approximately 20 g of algae were collected for ulvan extraction and
analysis. In every sampling station approximately 40 grams (wet weight) of algae
were collected, stored in the dark in ice boxes and transported to the lab within
10 minutes after collection. The algae were then cleaned with tap water and
epiphytes organisms were removed. The algae were then dried in duplicate for
48h in a stove at 70 °C for further conservation and analysis. The conservation
was made in the dark in a dry place and adding 25% of algal dry weight of silicate
gel to preserve dry algae against moisture.

3.2.3. Environmental data

Data on daily solar radiation and precipitation were downloaded from the
agroclimatological stations website of the IFAPA
(https://www.juntadeandalucia.es/agriculturaypesca/ifapa/ria). For July and
November 2011 and January 2012 data were obtained from the El Puerto de
Santa María meteorological station (situated at approximately 6.5 km from the
sampling site). As this station was not recording before this date, for March 2011
data were obtained from the then nearest station, the IFAPA Chipiona
meteorological station at approximately 25.5 km from the sampling site. Water
temperature during the week-long cage incubations was recorded at each station
at 30 min intervals using Thermochron iButton DS 1921G temperature loggers
(Maxim, USA). Duplicate water samples of each station were taken both at the
beginning and at the end of the incubations using 1 L PE bottles. Dissolved
oxygen, salinity, pH and temperature were measured on the site using the 550A
Dissolved Oxygen Instrument (YSI, USA) and the WTW-CF330 multiparameter
sonde (WTW, Germany) respectively. Subsequently, 10 mL samples were taken

56
 3. Seasonal variation

using syringes, were filtered over cellulose acetate disposable filters (0.45 µm
diameter, Chromafil, Machery-Nagel), stored in PE tubes and frozen at -20 °C for
analyses of inorganic nutrients. The remainder of the sample was transported to
the lab in coolboxes for determination of turbidity, using a HI 93703 turbidity
meter (Hanna Instruments, USA). Dissolved inorganic nutrients (ammonium,
ortho-phosphate, nitrite, nitrate and silicate) in the water were analyzed on a
segmented flow autoanalyzer (QuAAtro, Seal Analytical, Germany) using standard
fluorimetric (ammonium) and photospectrometric methods.

3.2.4. Algal nutrient contents and biochemical composition

Ash contents were determined on the algal discs used for growth
measurements after overnight combustion at 500 °C. For tissue nutrient analyses,
the dried algae were ground to powder in an MM400 mixer-mill (Retsch,
Germany) and the sample was split in two. Samples for tissue C and N contents
were send to the University of Málaga for analysis on a CHN elemental analyzer.
Tissue P was determined as dissolved ortho-phosphate on a nutrient-analyzer
(see above) after acid persulfate digestion of the algae [17]. Frozen samples
stored for proximate composition were lyophilized for 72 hours and ground to
powder in a mixer mill (Retsch MM-400, Haan, Germany). Total carbohydrate
content was determined using the anthrone method [18, 19]. Briefly, 1 – 2 mg dry
weight (DW) of powdered algae were weighed on an analytical balance and
inserted in a reaction tube to which 2 mL of a 3 M KOH solution was added.
Samples were then homogenized during 30 s in a sonicator, hydrolized during 1
h in a hot water bath (90 °C) and subsequent overnight continuous agitation at
room temperature. 6 mL of 2 M H2SO4 was added for neutralization after which
the samples were agitated and centrifuged at 3,000 rpm (1,690 x g) for 5 minutes.
Triplicate samples of 1 mL supernatant were cooled on ice and 5 mL anthrone
were added. The samples were put in a hot water bath (90 °C) for 16 min to
complete the color reaction after which samples were allowed to cool down to
room temperature and absorbance was read on a photospectrometer (UV-1800,

57
 3. Seasonal variation

Shimadzu, Kyoto, Japan) at 625 nm. The concentration was calibrated against a
dilution series of a D (+) glucose solution treated the same way as the samples.
Crude protein content was determined according to Lowry [20] using the DCTM
protein assay kit (Biorad, Hercules, USA). Bovine serum albumin (Sigma) was used
as a standard and absorbance was read at 750 nm in a micro-plate reader
(Sunrise, Tecan, Männedorf, Switzerland). Total lipid contents were determined
gravimetrically [21].
To perform ulvan extraction, dried algae were previously milled into a fine
powder with a Jetta Kitchen Blender. The extraction was realized in duplicate for
each sample. An aqueous extraction 1% algal powder w/v in 0.05 M oxalate was
realized at 80 °C during 2 hours then centrifuged. Aqueous phase was collected
and solid phase was extracted again in the same way. The second water phase
was put together with the first for further purification; the solid phase was
discarded. Water extract was then filtered with a 0.45 µm filter then diafiltrated
and concentrated by ultrafiltration with a 10 kDa cut-off membrane. The
concentrated aqueous extract was then frozen and freeze-dried. The resulting
material was then stored in a desiccator in the dark at ambient temperature.

Ash content of the extract has been determined by organic material

consumption at 550 °C during 12 h. Total carbohydrate content of the extract has
been determined by the phenol-sulfuric method described by Dubois [22].
Protein content was determined by the method of cupric ion described by Lowry
[20]. Sulfate content was determined by the Barium Chloride assay on a
polysaccharide degraded by sulfuric acid described by Dodgson and Price (1962)
[23]. Sugar profile has been determined by GC-FID after methanolysis and
derivatization with Trimethylsylil.

58
 3. Seasonal variation

3.2.5. Data analyses

Differences in environmental and algal parameters between stations and

seasons were tested for significance with two-way ANOVA’s using the Rcmdr
package [24] in R version 3.4.3 [25]. Bartlett’s tests for heterogeneity prior to
ANOVA, and in case of heterogeneity of variances data were ln transformed,
which solved the problem.

Month Rad (MJ m-2) Prec (mm) Tmax (°C) Tmin (°C) Tmean (°C)
March 2011 75.0 64.6 16.6 ± 1.6 9.4 ± 1.4 12.6 ± 0.7
July 2011 211.4 0 32.5 ± 2.2 17.0 ± 1.6 24.7 ± 1.2
November 2011 77.7 8.2 21.3 ± 2.3 11.5 ± 4.6 16.2 ± 3.1
January 2012 66.8 18.2 17.1 ± 2.3 5.0 ± 1.8 10.7 ± 1.7

Table 3.1: Total weekly radiation (MJ m-2), total weekly precipitation (Prec), average
weekly minimum, maximum and mean air temperature in four seasons; data from the
IFAPA meteorological station in Chiclana de la Frontera (March 2011) and El Puerto de
Santa María (other months). Temperature data are ± 1 SD.

59
 3. Seasonal variation

3.3. Results

3.3.1. Environmental variables, site

comparison

Radiation and rainfall showed the

typical annual pattern for a Mediterranean
area (Table 3.1).
Rainfall was mainly concentrated in the
winter months and was particularly heavy
during the February and March 2011 and
January 2012 incubations. The main difference
between these periods was that in March 2011
it was raining nearly every day of the week,
whereas in November 2011 and January 2012
all rainfall was recorded in one single day.
Water temperature followed the same pattern
as air temperature and radiation and showed
significant seasonal variation for both stations
(Figure 3.1; Table 3.1).
Average weekly mean temperature was
significantly higher for station 2 in July (Figure
3.1A); also, maximum temperature was always
Figure 3.1: Weekly averages of daily significantly highest for station 2 (Figure 3.1
mean (A), minimum (B, Tmin) and C), whereas minimum temperature was higher
maximum (C, Tmax) water
temperatures (°C) during sampling for station 1 in July only (Figure 3.1B).
weeks in four months at two sampling Consequently, temperature range (the
stations of an experimental fish
average of daily differences between
farm/earthen pond system at the
IFAPA-El Toruño institute in South
Spain.

60
 3. Seasonal variation

Figure 3.2 A-D: Dissolved

oxygen (A, mg L-1),
Turbidity (B), Salinity (C,
psu), and pH (D) during
sampling weeks in four
months at two sampling
stations of an experimental
fish farm/earthen pond
system at the IFAPA-El
Toruño institute in South
Spain.

minimum and maximum temperature during the incubation weeks) was always
higher for the shallow station 2.
Environmental variables dissolved oxygen and pH showed little, and
insignificant variation between seasons and stations (Figure 3.2 A and D, Table
3.2). Peak values for turbidity were detected in July, however, this was insignificant
due to generally high variation between measurements (Figure 3.2 B). Only
salinity showed significant seasonal variation with reduced salinities during the
March 2011 sampling (Figure 3.2C, Table 3.2). Figure 3.3 shows weekly average
water nutrient concentrations. Ammonium, nitrate and silicate showed
significant, but contrasting, seasonal differences (Table 3.2). Nitrate and silicate
also showed significant differences between stations. Ortho-phosphate showed
fluctuations similar to nitrate, however these were not significant due to high
variances. Ammonia, nitrate in station 1 and phosphate in station 2 peaked in
November (Figure 3.3 A-C).

61
 3. Seasonal variation

Var Factors df F ratio p value Var Factors df F ratio p value

Tmean Month 3 651.8264 <0.001 pH Month 3 0.9452 0.463
Station 1 5.3267 < 0.05 Station 1 0.1292 0.728
MxS 3 0.8758 0.462 MxS 3 0.0887 0.964
Error 40 Error 8
Tmax Month 3 161.768 <0.001 Ammonium Month 3 6.1016 < 0.05
Station 1 29.698 <0.001 Station 1 0.3688 0.561
MxS 3 2.796 0.0525 MxS 3 0.3473 0.792
Error 40 Error 8
Tmin Month 3 466.739 <0.001 Nitrate Month 3 10.4024 < 0.01
Station 1 16.2892 <0.001 Station 1 40.4728 < 0.001
MxS 3 6.8273 <0.001 MxS 3 9.7421 < 0.01
Error 40 Error 8
DO Month 3 3.5467 0.068 Ortho-P Month 3 2.4277 0.141
Station 1 0.0427 0.841 Station 1 4.2893 0.072
MxS 3 0.1172 0.947 MxS 3 0.6975 0.579
Error 8 Error 8
Turbidity Month 3 2.494 0.134 Silicate Month 3 7.3013 < 0.05
Station 1 2.91 0.126 Station 1 5.8781 < 0.05
MxS 3 0.4097 0.75 MxS 3 1.8436 0.217
Error 8 Error 8
S Month 3 11.982 < 0.01
Station 1 2.0075 0.194
MxS 3 0.4871 0.701
Error 8

Table 3.2: Results of two-way ANOVA testing the effect of the fixed factors Month and Station on
environmental variables weekly maximum, minimum and mean water temperature, dissolved oxygen
(DO) concentration, turbidity, salinity (S) and pH, in a seasonal sampling of Ulva ohnoi from two
stations in an experimental fish farm / earthen pond system.

62
 3. Seasonal variation

Figure 3.3 A-D: Dissolved

inorganic nutrients (µM)
ammonium (A, NH4+),
nitrate (B, NO3-), ortho-
phosphate (C, PO43-) and
silicate (D, SiO2) during
sampling weeks in four
months at two sampling
stations of an
experimental fish
farm/earthen pond
system at the IFAPA-El
Toruño institute in South
Spain.

It has to be noted that the heavy November rainfall occurred just the one
day before the sampling. Ammonium was generally the main component of the
total dissolved inorganic nitrogen (DIN), followed by nitrate. Nitrite generally
contributed less than 5% to DIN (data not shown). Nitrate was significantly higher
in station 1 than in station 2 in March, July, and November 2011 (Table 3.2),
whereas ammonium was slightly, but insignificantly higher in station 1. Due to
this, differences between stations in total DIN were not significant (data not
shown). Silicate was highest in station 2 during the whole year (Figure 3.3 D, Table
3.2).

63
 3. Seasonal variation

Figure 3.4: Relative growth

rates (RGR, d-1) of incubated
Ulva ohnoi during sampling
weeks in four months at two
sampling stations of an
experimental fish farm/earthen
pond system at the IFAPA-El
Toruño institute in South Spain.

3.3.2. Algal growth, nutrient contents and biochemical composition

Growth rates of U. ohnoi showed clear peaks in summer for both stations,
with high rates of over 0.35 d-1 (Figure 3.4). Seasonal fluctuations were significant
with the highest values in July 2011 and the lowest in January 2012 (Table 3.3).
Except for January, RGR was always significantly for station 1 (Table 3.3). DW/FW
and ash content of the algae showed significant seasonal variation (Figure 3.5 A-
B, Table 3.3). DW/FW was higher in January compared to the other seasons,
whereas ash content showed peaks in July and January. No significant differences
for DW/FW and ash were found between stations.
Tissue C, N, and P contents of the algae all showed significant seasonal
variation, whereas also a significant effect of sampling station was found for tissue
C and N (Figure 3.6, Table 3.3).

64
 3. Seasonal variation

Tissues fluctuations were

relatively modest, between 23 and
27% DW (Figure 3.6 A). They were
lowest in station 2 in July and highest
in station 2 in November.
Furthermore, station 1 showed
slightly, but significantly higher tissue
C contents in March 2011 and January
2012.
Tissue N content varied
between 1.7 and 3.6% and was lowest
for all stations in July (Figure 3.6 B). It
was generally higher for station 1 than
for station 2, except for the January
sampling, when it was the reverse.
Tissue P contents were significantly
higher in November and January for
both stations (Figure 3.6 C). Figure 3.7
A-C shows carbohydrate, protein, and
lipid content of the algae.
Figure 3.5: Dry weight to fresh weigh ratio
(DW/FW) and ash content of Ulva ohnoi Significant differences between
during sampling weeks in four months at sampling months were found for all
two sampling stations of an experimental
three components (Table 3.3). CH were
fish farm/earthen pond system at the
IFAPA-El Toruño institute in South Spain. highest for station 1 in March and
January, compared to the other
months and station 2 and ranged from 5 to 23% DW, although most observations
varied between 7 – 15% DW (Figure 3.7 A). Seasonal differences were less
pronounced in station 2; here, CH was slightly higher than in station 1 in July and
November, although this difference was not significant (Figure 3.7 A).
Protein contents ranged from 16% (station 2, November) to 32% (station
2, March) (Figure 3.7 B). Value for station 2 were highest in March and January

65
 3. Seasonal variation

Var Factors df F ratio p value Var Factors df F ratio p value

RGR Month 3 37.973 <0.001 Tissue P Month 3 4.1632 < 0.01
Station 1 7.1045 <0.05 Station 1 0.0033 0.956
MxS 3 1.3639 0.289 MxS 3 0.5179 0.682
Error 16 Error 8
DW/FW Month 3 10.0015 <0.001 CH Month 3 46.291 < 0.001
Station 1 0.1759 0.68 Station 1 23.044 < 0.001
MxS 3 2.6666 0.083 MxS 3 36.364 < 0.001
Error 16 Error 16
Ash Month 3 52.4723 < 0.001 Protein Month 3 6.2393 < 0.01
Station 1 0.1006 0.755 Station 1 0.0001 0.993
MxS 3 0.1529 0.926 MxS 3 6.4483 < 0.01
Error 16 Error 16
Tissue C Month 3 32.4595 < 0.001 Lipids Month 3 47.383 < 0.001
Station 1 6.802 < 0.05 Station 1 10.029 < 0.01
MxS 3 6.8521 < 0.05 MxS 3 10.964 < 0.001
Error 8 Error 16
Tissue N Month 3 90.8522 < 0.001
Station 1 5.4086 < 0.05
MxS 3 24.646 < 0.001
Error 8

Table 3.3: Results of two-way ANOVA testing the effect of the fixed factors Month and Station on
algae variables relative growth rate (RGR), dry weight to fresh weight ratio (DW/FW), ash, tissue
C, N, and P content, and carbohydrate (CH), lipid, and protein levels in a seasonal sampling of Ulva
ohnoi from two stations in an experimental fish farm / earthen pond system.

66
 3. Seasonal variation

and lowest in November, whereas for station 1

protein contents were significantly lowest in July.
Contents were higher for station 2 than for station
1 in July whereas the reverse was true for
November, explaining the significant interaction
and the absence of a significant station effect
alone. Lipid contents of U. ohnoi varied between
1.7 and 3.5% DW (Figure 3.7 C) and showed similar
seasonal patterns for both stations. Lowest
contents were found in July and highest in March
and January. Station 1 had significantly higher
lipid contents than station 2 in January, whereas
contents were higher for station 2 in March.
A variation in the yield of the water soluble
extract could be observed throughout the season
with the lowest yield being 22.3% and the highest
being 28.4% (Figure 3.8). Those two values have
been observed for the same sampling Station with
a maximum being during the month of November
and the minimum during the month of March in
the station 2 with a significative difference
between those months. In the station 2 no
significative difference could be observed in yield
Figure 3.6: Tissue Carbon (A), during the seasons with a slight variation of
Nitrogen (B) and Phosphorous (C) between 24.1% and 27.6%.
content (% DW) of Ulva ohnoi
during sampling weeks in four A variation in the yield of the freeze dried
months at two sampling stations water soluble extract could be observed
of an experimental fish
farm/earthen pond system at the throughout the season (Figure 3.8) with the lowest
IFAPA-El Toruño institute in yield being 22.3% and the highest being 28.4%
South Spain.
however no significant difference were found.
Those two values have been observed for the same

67
 3. Seasonal variation

sampling Station with a maximum being

during the month of November and the
minimum during the month of March in
the station 1. In the station 2 no significant
difference could be observed in yield
during the seasons with a slight variation
of between 24.1% and 27.6%.
Figure 3.8 shows that November is
the month for which the most
carbohydrates could be found in the
freeze dried ulvan rich water soluble
extract in both sampling stations with a
percentage of carbohydrate extracted
representing 13% of algal dry weight for
the station 1 and 15% of algal dry weight
for the station 2. The variation of
carbohydrate extracted has been varying
from 9 to 15% of algal dry weight. Figure
3.8 also highlights the fact that some
components of the freeze dried ulvan rich
water soluble extract couldn’t be
identified. This fraction is especially
pronounced for the station 1 in the month
of January.
The value of water-extracted
Figure 3.7: Tissue carbohydrates (A, CH), rhamnose percentage per algal dry weight
lipids (B) and protein (C) content of Ulva is a good indicator of the yield of ulvan as
ohnoi during sampling weeks in four
months at two sampling stations of an this is the dominant sugar in this molecule.
experimental fish farm/earthen pond Significant variations have been observed
system at the IFAPA-El Toruño institute in
South Spain. for the station 1 compared to station
where the amount of rhamnose was

68
 3. Seasonal variation

significantly lower than in March and January at station 2 (Figure 3.9 A). The
rhamnose level was consistently and significantly higher for station 2 than for
station 1 (Table 3.4).
The highest Rhamnose content was measured at Station 2 during the
month of January with a value of 6.7% of algal dry weight. Rhamnose results were
consistent with the xylose results for which the same pattern of variation has been
found (Figure 3.9 C).
Glucuronic acid (Figure 3.9 D) shows a similar pattern but did not show any
significant differences amongst station and between seasons. The glucose
variation has shown a significant increase in the month of November and January

Figure 3.8: Yield and Reconstructed Composition of Aqueous extract as percentage normalized
to algal dry weight (DW) represented for each season and each sampling station. CBHs:
Carbohydrates; N.I.: Not Identified

69
 3. Seasonal variation

for station 2 and in the month of January for station 1 (Figure 3.9 D). Glucose was
significantly affected by season and station (Table 3.4). Galactose did not show
any significant variation during the year.
The level of ribose was significantly lower during the month of November
than during the month of January for Station 2. The level of ribose was
significantly higher in the station 1 than in the station 2 for the month of
November.
Sulfate contents (Figure 3.9 G) were very stable during the year. Ashes
(Figure 3.9H) showed a significant difference between station 1 and 2 during the
month of March. A significant decrease of extracted proteins was observed for
station 1 between the month of November and January (Figure 3.9 I).

3.4. Discussion

3.4.1. Spatial and seasonal variation in environmental variables

Season was the factor responsible for the majority of significant variation
in the values of the environmental variables temperature, salinity, ammonium,
nitrate and silicate (Table 3.2). Temperature, nitrate, and silicate also showed
significant differences between sampling stations. Variation in water temperature
logically follows the seasonal pattern. Variation in salinity was characteristic for
this region [26], where the decrease in March can be explained by the heavy
rainfall preceding and during the March sampling, whereas temperature driven
evaporation are responsible for increased salinity in July. The reason for the
variations in nutrients are less obvious. The peak in ammonium in November is
most likely related to the first rains of the year in that month; these may have led
to increased ammonium rich run-off from the land. Nitrate peaks following March
and November rains, especially in station 1 are probably related to imports from
riverine water. The peak in silicate in summer is in agreement with earlier

70
 3. Seasonal variation

Var Factors df F ratio p-Value Var Factors Df F ratio p-Value

Yield M 3 1 0,44 Galactose M 3 0,29 0,82
S 1 0,08 0,79 S 1 0,45 0,52
MxS 3 1,21 0,36 MxS 3 0,51 0,68
Error 8 Error 8
Rhamnose M 3 0,44 0,73 Ribose M 3 0,85 0,50
S 1 7,03 <0,05 S 1 0,11 0,75
MxS 3 1,03 0,42 MxS 3 2,86 0,1
Error 8 Error 8
Xylose M 3 0,6 0,6 Sulfates M 3 0,037 0,95
S 1 13,11 <0,01 S 1 0,012 0,85
MxS 3 1,92 0,20 MxS 3 0,18 0,65
Error 8 Error 8
Glucose M 1 11,94 <0,01 Ashes M 3 0,67 0,59
S 3 7,1 <0,05 S 1 0,01 0,91
MxS 3 2,34 0,14 MxS 3 2,77 0,11
Error 8 Error 8
Glucur. Ac. M 3 1,43 0,3 Proteins M 3 1,13 0,39
S 1 2,36 0,16 S 1 0,33 0,58
MxS 3 0,5 0,69 MxS 3 2,28 0,15
Error 8 Error 8

Table 3.4: Results of two-way ANOVA testing the effect of the fixed factors Month and Station on
algae variables yield, rhamnose, xylose, glucose, glucuronic acid, galactose, ribose, sulfates, ashes,
proteins levels in a water extract of a seasonal sampling of Ulva ohnoi from two stations in an
experimental fish farm / earthen pond system.

71
 3. Seasonal variation

Figure 3.9: Variation of abundancy of ulvan-rich water soluble extract components throughout the year. Black: Station 1; Grey: Station 2; A:
Rhamnose; B: Glucuronic Acid; C: Xylose; D: Glucose; E: Galactose; F: Ribose; G: Sulfates; H: Ashes; I: Proteins. Letters show the group of
significance by one-Way ANOVA, when no letter appears no significant difference has been observed. P<0,05

72
 3. Seasonal variation

observations from several saltmarsh station in the Bay of Cádiz area that showed
increases in benthic silicate fluxes in summer months [27].
To explain the differences between stations, it is important to recall the
specific characteristics of each. Station 1 was located in a water storage reserve,
forming a relatively large water mass, with a medium depth of 1 m and relatively
low water exchange. Station 2 on the other hand was located in the run-off canal
of the experimental fish farm; it was shallow (0.2 to 0.3 m on average) and had
relatively high exchange, depending on the flushing regime of the fish ponds.
Most conspicuous were the differences in temperature with station 2 having
slightly higher averages and especially lower minimum and higher maximum
temperature, resulting in a significantly larger average temperature range in
station 2. The higher influence of riverine water probably explains the higher
nitrate concentrations in station 1. The reduced depth of station 2 allowed for
large diatom blooms (pers. obs. E. Malta) in this station, which are related to
increased silicate remineralization in summer [27].

3.4.2. Variation in algal growth and composition

Season (month) was a significant factor affecting all algal variables,

whereas station caused significant effects on growth, tissue C and N content and
carbohydrate and lipid content. Growth appeared to be mainly regulated by
temperature and light, in agreement with earlier observations on U. cf. rotundata
from this area [8, 15]. The generally lower growth rates found in station 2 are
most likely due to the harsher environmental conditions the shallow station
presents, as shown in the differences between stations in maximum and minimum
temperatures. Furthermore, especially for July, the higher water nitrate
concentrations might have had a positive impact on station 1 growth rates, as
tissue N indicates slight N limitation of growth during this month (see below for
detailed discussion on N limitation).
Seasonal variation in water and ash content has been observed before in
Ulva [e.g. 28, 29] with a tendency towards lower water and higher ash content in

73
 3. Seasonal variation

winter [30]. Winter accumulation of (insoluble) carbohydrates as observed here

and inorganic constituents that are no longer diluted in the algal tissue due to
low growth rates might be the main cause; nevertheless this does not explain the
peak in ash content in summer observed in this study for both stations. Seasonal
dynamics of DW/FW, tissue C and carbohydrate contents were more or less
similar, supporting the idea of winter carbohydrate accumulation. Malta and
Verschuure [9] found similar patterns of tissue C dynamics in Ulva in the
Netherlands, although considerable differences between sampling years and
sampling stations may occur [8, 31], indicating that more factors interact in their
dynamics.
Tissue N contents typically decrease with increasing growth rate due to
internal dilution and decreasing external nutrient contents and vice versa [8, 9] as
was also the case in this study, although less obvious for the station 1 sampling
in January 2012. It is important to note that, except for the July observation for
station 2, tissue N was always over 2.1% DW, which is considered the critical level
for limitation of growth, indicating that growth was never N-limited [32, 33]. This
is in agreement with the nitrogen concentrations (ammonium, nitrate and total
DIN) found in the water, which were never below 10 µM, whereas for nitrogen
limitation of growth this appears to be a threshold concentration [1, 34]. The
significantly higher tissue N concentration in station 1 algae are well in
agreement with the higher total N, caused by higher nitrate concentrations in
that station. Protein contents generally followed the same pattern as tissue N, as
can be expected, N being an essential building block for proteins [35, 36].
Tissue P contents showed similar seasonal patterns as water ortho-
phoshate levels. The relation between tissue P and growth rate is less
straightforward than for tissue N and experimental data on critical tissue P levels
are scarce and show large differences. A relatively high critical tissue P level of
0.65 % DW was experimentally found for U. lactuca from Norway [37], whereas
other authors suggest values of 0.2% DW [38, 39]. Observations from U. rotundata
growing in a fish farm in the same area as where this study has been performed,
suggest even lower values of around 0.1% DW tissue P [15]. Thus, March and July

74
 3. Seasonal variation

data suggest that algal growth might have been slightly P-limited during this
season. In experiments with six common seaweed species from New Zealand,
Ulva was the only one that showed supply driven uptake, supporting this
hypothesis [40]. On the other hand, the generally higher (although this difference
was not significant) ortho-phosphate levels in the water in station 2 were hardly
reflected in tissue P levels. Clearly, more work is needed on the relation between
water P-levels and algal growth and composition.
Lipid contents were in the range typically found for Ulva spp. [29, 41, 42].
The most conspicuous seasonal effect was the distinct decrease in summer,
concomitant with experimental results U. pertusa showing that temperature is the
main factor influencing lipid contents, with lower temperatures leading to
increases in internal lipids and vice versa [43, 44]. Salinity also has been found to
have significant effects on lipid contents and differences in salinity between
stations might have led to the difference in lipids in July in particular [45, 46].

3.4.3. Ulvan-rich water soluble extract composition

The yield of the ulvan-rich water soluble extract was relatively stable during
the whole year and for both stations. The composition of the water extract
consisted mainly of ash, rhamnose, glucuronic acid and sulfates which indicates
that this extract is mainly composed of ulvan [47]. Other compounds were also
present in this extract such as glucose or proteins. Glucose content is probably
related to co-extracted starch [48]. In station 1 the level of rhamnose decreased
only between the month of November and the month of January. At the same
time the level of glucose increased significantly between the month of July and
the Month of November. This suggests a shift of carbohydrates metabolism from
vegetative to overwintering. This is supported by the fact that at station 1 after
the nitrogen pulse of November, the algae accumulate N in tissue which is
consumed in the month of November with a very low growth rate so nitrogen
might be used as a source of nutrients for synthesizing enzymes to convert
structural sugars into starch reserve. The stimulus that triggers this shift of

75
 3. Seasonal variation

behavior does not seem to be related to nutrient deficiency but rather could be
due to a decline of the temperatures and light hours.
As it can be seen in Figure 3.9 A,D and Figure 3.6 D the station 2 does not
experiment this metabolism shift to the same extent nor at the same time than
Station 1. Instead station 2 maintains a stable level of ulvan and nitrogen and
starts to accumulate starch during the month of November. This earlier response
to winter conditions might be explained by the fact that station 2 algae are
exposed to a broader spectrum of temperatures over the day due to low volume
of water (Figure 3.1) reaching lower temperatures earlier in the year thn station
1. The impact of temperature amplitude on carbohydrates metabolism has been
shown previously [49]. This change in metabolism is also supported by a lower
growth rate at this period with a high glucose content in the soluble extract.
Table 3.3 indicates that the station has a significative influence on
rhamnose, xylose and glucose levels. Rhamnose and xylose maintain a constant
level in station which suggests that ulvan stays more constant in this station. The
abiotic factors that are influenced by the station are nitrate, T° and silicates.
However silicates levels are most likely related to local diatoms bloom dynamics
as argumented in 3.4.1. Nitrate nitrogen was practically inexistent in station 2,
thus the only source of nitrogen in this station was ammonium. The high
dependency on this cation might have promoted this behavior as maintaining the
level of ulvan could be a strategy to trap those cations inside this anionic
polysaccharide as a reserve to use in case N scarcity. Further studies are needed
to determine whether N source and cationic forms of nutrients in general can
have an influence on the concentration of ulvan in the algae.

3.5. Conclusions

We conclude that temperature and light were the principal drivers behind
the variation in growth and composition. However, differences observed between

76
 3. Seasonal variation

the stations indicate that other factors, specifically salinity and nitrate
concentrations also have a significant influence on algal growth and composition.

3.6. References

[1] F. Mineur, F. Arenas, J. Assis, A.J. Davies, A.H. Engelen, F. Fernandes, E.J.
Malta, T. Thibaut, T. Van Nguyen, F. Vaz-Pinto, S. Vranken, E.A. Serrao, O. De
Clerck, European seaweeds under pressure: Consequences for communities and
ecosystem functioning, J. Sea Res., 98 (2015) 91-108.

[2] S.L. Holdt, S. Kraan, Bioactive compounds in seaweed: functional food

applications and legislation, J. appl. Phycol., 23 (2011) 543-597.

[3] FAO, The State of World Fisheries and Aquaculture 2016. Contributing
to food security and nutrition for all, FAO, Rome, 2016, pp. 200.

[4] B.J. Gosch, N.A. Paul, R. de Nys, M. Magnusson, Spatial, seasonal, and
within-plant variation in total fatty acid content and composition in the brown
seaweeds Dictyota bartayresii and Dictyopteris australis (Dictyotales,
Phaeophyceae), J. appl. Phycol., 27 (2015) 1607-1622.

[5] R. Patarra, L. Paiva, A.I. Neto, E. Lima, J. Baptista, Nutritional value of

selected macroalgae, J. appl. Phycol., 23 (2011) 205-208.

[6] A. Robic, J.F. Sassi, P. Dion, Y. Lerat, M. Lahaye, Seasonal Variability of

Physicochemical and Rheological Properties of ulvan in Two Ulva Species
(Chlorophyta) from the Brittany Coast, J. Phycol., 45 (2009) 962-973.

77
 3. Seasonal variation

[7] M. Altamirano, A. Flores-Moya, F. Conde, F.L. Figueroa, Growth

seasonality, photosynthetic pigments, and carbon and nitrogen content in
relation to environmental factors: a field study of Ulva olivascens (Ulvales,
Chlorophyta), Phycologia, 39 (2000) 50-58.

[8] I. Hernández, G. Peralta, J.L. Pérez-Lloréns, J.J. Vergara, F.X. Niell,

Biomass and dynamics of growth of Ulva species in Palmones river estuary, J.
Phycol., 33 (1997) 764-772.

[9] E.-j. Malta, J.M. Verschuure, Effects of environmental variables on

between-year variation of Ulva growth and biomass in a eutrophic brackish lake,
J. Sea Res., 38 (1997) 71-84.

[10] K. Bischof, P.J. Janknegt, A.G.J. Buma, J.W. Rijstenbil, G. Peralta, A.M.
Breeman, Oxidative stress and enzymatic scavenging of superoxide radicals
induced by solar UV-B radiation in Ulva canopies from southern Spain, Scientia
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83
 3. Seasonal variation

84
4 . Production of s ulfa ted
oligosa ccha rides from s eaweed Ulva
s p. us ing a new ulva n -degra ding
enzy ma tic bacterial crude extrac t

85
 4. Production of sulfated oligosaccharides

Título del artículo:

Production of sulfated oligosaccharides from the seaweed Ulva sp. using a new
ulvan- degrading enzymatic bacterial crude extract

Autores:

Olivier COSTEa, Erik-jan MALTAb, José CALLEJO LÓPEZa, Catalina FERNÁNDEZ

DÍAZa*

a
Instituto de Investigación y Formación Agraria y Pesquera de Andalucía (IFAPA),
Centro El Toruño, Camino del Tiro Pichón s/n, 11500 El Puerto de Santa María,
Cádiz, Spain
b
IFAPA Centro Agua del Pino, Ctra. Cartaya-Punta Umbría Km 4, 21450 Cartaya,
Huelva, Spain

Publicado en: Algal Research Sometido: 12 de Febrero 2015

Número: 10 Acceptado: 19 de Mayo 2015
Páginas: 224-231

Abstract

Green macroalgae of the genus Ulva have complex and hardly degradable
polysaccharidic extracellular matrix. One of its main component, the ulvan, is a 3-
sulfated rhamnoglucuronan that has a wide range of properties and is a source
of rare sugars. The production of mono- and oligo-saccharides from this
polysaccharide could motivate its use to an industrial scale. Enzymatic tools to
realize this process are still scarce. Here we describe the activity of an enzyme
crude extract proceeding from a new Alteromonas species isolated from the gut
of Gammarus insensibilis, an amphipod from southern Spanish saltmarshes.

86
 4. Production of sulfated oligosaccharides

HPTLC separation and NMR spectrometry allowed to describe the formation of

low molecular weight oligosaccharides formed principally of unsaturated 3-
sulfated rhamnose and uronic acids. The control of hydrolysis kinetics allowed
preparing different molecular weight fractions down to a 5 kDa oligosaccharides
mix. These results highlight the presence of a potentially new β-lyase produced
by this hitherto undescribed bacteria. This work identifies a new source of
enzymes with potential application for the production of high added value
compounds from Ulva sp.

4.1. Introduction

Marine green macroalgae are fast growing species with an important

capacity to fix CO2 and remove excess nutrients from the water. Although they
are excellent biofilters and can be cultivated for that purpose the biomass
generated is still largely underexploited mainly because of its low value [1]. Due
to their unique composition, algal polysaccharides have been shown to exhibit a
wide range of bioactivities. Green algal cell walls, especially of species of the
genus Ulva are constituted by a long chain polymer called “ulvan” [2]. This
heteropolysaccharide is interesting because it is mainly composed of rare sugars
and is highly sulfated as are many polymers of animal origin [3]. It may contain
between 16%-23.2% of sulfate, up to 45% of rhamnose, 2.1-12% Xylose, 0.5-6%
glucose and 6.5-19% of uronic acids. The main structural subunit ulvan obiuronic
acid type A (A3S) is composed of L-rhamnose sulfated on the third carbon and
linked on its non-reducing end with a β-D-glucuronic acid with an α 1-3 linkage,
the B type ulvan obiuronic acid contains iduronic acid instead of glucuronic acid
(B3S)[4–6]. Its molecular weight depends on the algal species and the extraction
protocol; however it is generally high and can round up to 700-800 kDa [7]. This
glycosaminoglycan-like structure has anticoagulant [8], antioxidant [9–11],
immunomodulator [12, 13], antihypercholesterolemic [14], antihyperlipidemic

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 4. Production of sulfated oligosaccharides

[15,16], antiviral [17], antitumoral [18,19] and plant defense elicitor activities [20–
23]. It has been used in forming biomaterials such as nanofibers, nanofibrous
membrane [24, 25], microparticles [26], molecular sponges for cell culture [27],
antiadhesive activity [28] or as ion exchanger hydrogel [29]. L-rhamnose as well
as L-α-rhamnosyl oligosaccharides and glycoconjugates have been described to
strongly interact with specific lectin domains in several proteins and organisms
[30–38].
The importance of sugars in many biological processes has given rise to
the study of new carbohydrate-based drugs able to mimic endogenous sugars
and glycoconjugate behaviors and act as receptors antagonists. Sulfated
oligosaccharides are implicated in biological events such as protein localization
at cell surfaces [39]. These interesting features make ulvan an excellent candidate
as a renewable and cheap compound for industrial purposes. Unfortunately its
structure is difficult to define [7,40,41]. An enzymatic or chemical reduction of its
molecular weight could lead to a more definable structure and improve the range
of its activities, the efficiency and reduce the polydispersity of its molecular
weight. The physical properties of carbohydrates mainly depend upon the sugar
composition of the chain, the glucosidic linkages, the side groups and the
molecular size. Their conformation in solution determines their affinity for
receptors and their biological properties. Given that a change in molecular weight
can have a great influence on the geometrical conformation of some
carbohydrates [42], the bioactivity of carbohydrate can be enhanced by
depolymerization and making more accessible the active sequences to ligand
receptors [43,44]. In addition, Ulva and other seaweeds are viewed as promising
candidates for the production of biofuels [45], however, as industrial microbes
cannot currently realize the metabolization of the complex polysaccharide
components, an efficient depolymerization step will increase their potential for
biofuel production [46, 47].
Several methods to reduce the molecular weight of polysaccharides have
been described. Mild acid hydrolysis reduces the molecular weight but sulfate
groups are lost during the process. Furthermore, the alpha 1-4 linkage of the
aldobiuronic acid moiety is resistant to this reaction [4]. Enzymatic cleavage of

88
 4. Production of sulfated oligosaccharides

polysaccharides generally does not have these disadvantages. Intestinal

microbiota associated to animals feeding on Ulva sp. can be a source of specific
enzymes. Recently a new bacteria species Persicivirga ulvanivorans [48] was
isolated from aplysia feces and was found capable of degrading ulvan in to
disacharidic subunits with an extracellular Ulvan-lyase [49]. Such an enzyme has
already been described in Ochrobactrum tritici [50]. The genus Persicivirga has
recently been merged in the genus Nonlabens [51] and the newly named
Nonlabens uvanivorans has been described to produce a β-glucuronyl hydrolase
implicated in the degradation of ulvan [52]. Other authors have described the
degradation of ulvan by enzymatic means but with alginate-degrading
commercial enzymes or without further characterization of the bacteria used to
obtain the enzyme [7,19,53]. The main challenge of enzymatic degradation is to
maintain the basic structure of repetitive subunits with the sulfate functional
groups while controlling the kinetics of degradation to obtain oligomers with a
constant yield and molecular size.
The objective of this work was to obtain oligosaccharides from the large
polysaccharides produced by the green alga Ulva ohnoi Hiraoka et Shimada by
enzymatic means in order to assay their biological activity in further studies. In
this paper we characterize the degradation products and the kinetics of an
enzymatic degradation of ulvan. The crude enzyme mix has been extracted from
a culture of a newly identified species of bacteria belonging to the genus
Alteromonas isolated from the gut of the amphipod Gammarus insensibilis.
Amongst several animal species proceeding from different taxonomic groups
these amphipods were identified in a preliminary experiment as efficient grazers
of Ulva spp. The degradation level has been assayed by static light scattering
(SLS), thin layer chromatography (TLC) and subsequently characterized by H1-
NMR.

89
 4. Production of sulfated oligosaccharides

4.2. Materials and Methods

4.2.1. Ulva ohnoi cultivation

Ulva ohnoi Hiraoka and Shimada strain UOHN120810 was isolated from
the outlet channel “Caño de Agua del Pino” close to the mouth of the Rio Piedras
tidal inlet in SW Spain (37°12'57.39" N, 7°5'5.29" E) on 12 August 2010 and has
been maintained in culture since then in the aquaculture centers of the IFAPA.
Identification was confirmed by the analyses of the rbcL and ITS2 sequences
(genbank submission pending, Malta et al. in prep.). To obtain the biomass
needed for ulvan analyses, stock cultures were upscaled to 15 L cilinders under
constant light (150 μmol photons.m-2.s-1) and temperature (20 ºC) in filtered (0.2
μm) natural seawater enriched with f/2 medium. After two weeks of cultivation
sufficient biomass was obtained. Algae were harvested, rinsed with tap water,
freeze-dried and kept in a dry place until the extraction.

4.2.2. Ulvan extraction and purification

Approximately 10 g of freeze dried algae were milled to a fine powder

(around 8 µm particle diameter) using a coffee mill (Jata, Spain). Ulvan was
extracted as described by Robic [54] with some further purification steps. Briefly,
6.7 g.L-1 of alga dry weight were in hot water (80 ºC) with 0.05 M sodium oxalate
(Sigma Aldrich, USA) during 2 hours. The extract was centrifuged for 20 min at
14000 rpm (Beckman, USA) filtered at 0.45 µm and a secondary extraction was
made on the residue. Both supernatants were merged together and concentrated
by ultrafiltration using a 10 kDa membrane mounted on a Centramate
Ultrafiltration Unit (PALL, USA). Subsequently, pH was lowered to 2 by adding
phosphoric acid and centrifuged in order to precipitate possible glucuronan. The
extract was neutralized by adding NaOH, autoclaved for 21 minutes at 121 ºC and
centrifuged for 30 min at 30,000 g to remove proteins and contaminating
molecules. The supernatant was purified by diafiltration with a 10 kDa cut-off

90
 4. Production of sulfated oligosaccharides

membrane until the filtrate conductivity reached that of distilled water. Finally,
the supernatant was freeze-dried and the resultant purified ulvan extract was kept
in a dry and dark place.

4.2.3. Bacterial isolation

Approximately ten individuals of the marine amphipod Gammarus

insensibilis were collected from an earthen pond in the salt marsh ecosystem of
the Rio San Pedro tidal inlet next to the centre IFAPA El Toruño (36°34’44.6”N,
6°12’25.0”W). The amphipods were maintained on a diet of freshly collected Ulva
sp. from the same locality during 3 days in natural seawater. Animals were then
externally sterilized by submerging them during 5 sec in a 10 % H2O2 solution.
The gut was immediately extracted in sterile conditions and kept in 3 ml of
sterilized seawater at a salinity of 35 ppm. A pool of five G. insensibilis intestines
was homogenized and 5 µl were spread on a sterile ulvan gel prepared with
autoclaved 10 mg.ml-1 ulvan in 35 ppm seawater (not containing agar). After one
night of incubation the gel had been entirely liquefied and the supernatant was
frozen for 24 hours with 15 % glycerol at -80 °C to eliminate helminths. The
resulting bacterial culture was sterilely plated on a marine agar petri plate
enriched with 4 % ulvan [55]. After 24 hours several colonies had formed and
amongst them some had formed small craters. The well separated colonies were
individually picked up from the plate and newly spread on gelificated ulvan. No
growth could be observed in liquid bacterial culture. The bacteria that degraded
the gel were considered as ulvanolytic. Only one strain showed such activity and
was conserved in glycerol 15 % at -80 ºC for further uses.

4.2.4. Crude Enzyme extract isolation

50 ml ulvan gel (10 mg.ml-1 in 35 ppm sterilized natural seawater) was

incubated with 100 µl of the bacteria strain in the dark at 25 °C. When the gel was
completely liquefied the culture batch was centrifuged for 10 min at 10 000 g at
4 °C after which the supernatant was filtered twice with over 0.22 µm pore
cellulose filters (Millipore, MA, USA) to remove bacteria. Proteins were then

91
 4. Production of sulfated oligosaccharides

precipitated by salting out with to 60 % and 80 % ammonium sulfate (Panreac,

Spain). The pellet was recovered in a 100 mM tris-HCl buffer at pH=8 and 200
mM NaCl and dialyzed with a 14 kDa membrane (Sigma-Aldrich, USA) during 24
hours against the same buffer in order to remove all the salts and small molecules.
Protein was concentrated 4-fold by centrifugal frontal ultrafiltration with a 5 kDa
Amicon Ultra-4 tubes (Millipore, USA). Proteins in these crude enzymes mix were
then quantified following the Bradford method [56] with Bovine Serum Albumine
(Sigma-Aldrich, USA) as standard.

4.2.5. Bacterial identification

For the molecular identification of the bacterial isolate, DNA extraction was
performed from 40 mg of frozen pellet using the FastDNA kit for 40 s at speed
setting 5 in the Fastprep FG120 instrument (Bio101, Inc., Vista, CA). Primers used
for PCR amplification of 16S rDNA were 63f and 1387r [57]. PCR reactions were
carried out in 25 µl of reaction volume: 1 µl DNA template (20 ng) was added to
24 µl PCR mix consisting of 17.25 µl of sterile distilled water, 2.5 µl dNTP mix 10
mM, 2.5 µl of 10x buffer, 1 µl MgCl2 50 mM, 0.25 µl (1.25 units) BioTaqTM DNA
polymerase (Bioline, London, UK), and 0.5 µl of each primer (10 mM). The thermal
cycle profile was an initial denaturation step of 96 °C for 2 min was followed by
30 cycles of 96 °C for 30 s, 60 °C for 30 s, and 72 °C for 1.5 min. PCR products
were examined by electrophoresis on ethidium bromide–stained 2.0 % agarose
gel and visualized by UV transillumination. Double-stranded DNA products were
purified using a PCR product purification kit (Marlingen Bioscience, Ijamsville,
MD) and directly sequenced using the BigDye Terminator v3.1 Cycle Sequencing
kit in an 309 ABI3130 Genetic Analyzer (Applied Biosystems). The rDNA16S
sequence has been deposited on the GenBank database under the accession
number GIIUL2. Sequences were aligned by BLAST in the 16S RNA ribosomal
sequences database (www.ncbi.nlm.nih.gov) and a phylogenetic tree was
generated with the same tool [58].

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 4. Production of sulfated oligosaccharides

4.2.6. Enzyme kinetics

A 180 µl solution of 40 mg.ml-1 ulvan in 100 mM tris HCl buffer with 200
mM NaCl was incubated with a final concentration of 0.01 mg.ml -1 of the crude
enzyme mix. Bases on preliminary experiments, it was found that this amount was
sufficient to produce significant ulvan degradation within a reasonable amount
of time (Fig. S1). The enzymatic hydrolysis was made in triplicate and microtubes
were placed in a dry bath incubator at different temperature (see below). 15 µl
samples were taken at different times: 0, 2.5, 5, 7.5, 10, 12.5 , 15, 25, 50 hours after
incubation, dissolved 4-fold in distilled water and heated for 10 min at 100 ºC in
a dry heat bath in order to stop the enzyme activity. The sample was then
centrifuged for 20 min at 13200 rpm and the supernatant was kept at 4 ºC for
further TLC analysis. Three pH buffer conditions (6, 7 and 8) were first assayed for
degradation rate at 35 °C and the kinetics of the reaction were followed by
analyzing the evolution of the area under the curve of each TLC peak. The pH
condition with the highest degradation rate was used to assay the degradation
rate at four different temperatures (20, 25, 30, 35 °C) (n=3).

4.2.7. Thin Layer Chromatography

4 µl of each sample was applied on aluminum HPTLC silicagel 60 plates

(Merck, Germany) with an Automatic TLC Sampler 4 (CAMAG, Switzerland) with a
bandwidth of 5 mm. The liquid phase was composed of n-butanol:formic
acid:water (4:8:1) and the front was eluted until 5.5 cm from the plate basis. Before
the plate revelation the plate was read at 244 nm with a TLC Scanner 3 (CAMAG,
Switzerland). Carbohydrates were stained with a mix of 1 ml HCl 37.5%, 2 ml of
aniline, 10 ml of ortophosphoric acid 85 %, 100 ml of ethyl acetate and 2 g of
Diphenylamine [59]. Then the plate was dried and heated 1 min at 170 ºC. After
revelation the HPTLC plate was read at 380 nm and data were analyzed using the
winCATS software. The relative abundance of each oligosaccharide was calculated
as the area under the peak divided by the total of area under the curve. The
absorbance spectrum was acquired with the same device used in the spectrum

93
 4. Production of sulfated oligosaccharides

mode for the determined HPTLC spot.

4.2.8. Semi-Preparative degradation

To prepare more quantity of oligosaccharide for detailed analysis the

process was scaled up in a 12 ml tube during 40 hours at 35 °C. The objective of
this step was to obtain as much different oligosaccharides inferior to 10 kDa as
possible.

4.2.9. Oligosaccharides purification

The crude enzymatic hydrolyzed sample was split in a fraction with

molecular weight smaller than 10 kDa (UD < 10 kDa) and one larger than 10 kDa
(UD > 10 kDa) by ultrafiltration using a 10 kDa cut-off filter. Static light scattering
analysis (see below) was used to confirm that the average molecular weight was
actually inferior to 10kDa. After ultrafiltration, the filtrate, corresponding to the
UD < Kda fraction, was collected and concentrated with a rotavapor and freeze-
dried. The retained fraction with a molecular weight superior to 10 kDa (UD >
10kDa) was freeze-dried as well for molecular weight determination. 100 mg of
the UD < 10 kDa freeze dried powder was dissolved in 20 ml of pure water (MilliQ,
Millipore, USA) and loaded on a 20 x 3 cm activated carbon column.
Subsequently, the column was washed with 125 ml pure water, eluted with 250
ml of 80 % EtOH with a flow of 2-3 ml.min-1; 10 ml fractions were recovered. The
presence of salts in the fractions was assayed by measuring conductivity with a
probe (Crison, Spain) and presence of carbohydrates was assayed by staining (see
TLC paragraph for staining solution composition) of 4 µl of the spotted fraction
on silica plate. Salt-free carbohydrate-rich fractions were pooled and freeze dried.

4.2.10. H1-NMR

The one dimension Proton Nuclear Magnetic Resonance spectra of none-

degraded ulvan and oligoulvans inferior to 10 kDa were acquired on an Agilent-
500 MHz spectrometer at 25 °C in deuterium (D2O). Chemical shifts are expressed

94
 4. Production of sulfated oligosaccharides

in ppm in reference to the deuterium peak. Peaks were assigned by comparison

with literature [60].

4.2.11. Static Light Scattering

Molecular weight (Mw) measurements of ulvan fractions were performed

on a Zetasizer Nano-ZS90 (Malvern, UK) with Static Light Scattering Mode. The
mean scattering intensity was calculated from 20 intensities readings of 15
seconds, using 5 concentrations: 0.2, 0.4, 0.6, 0.8, 1.0 g.l -1 for ulvan -rich extract;
0.25, 0.5, 0.75, 1.0, 1.25 mg.ml-1 for hydrolyzed ulvan > 10 kDa; 6, 7, 8, 9, 10 mg.ml-
1
for hydrolyzed ulvan < 10 kDa. All measurements were made in 5 kDa
ultrafiltrated MilliQ water. The sample was filtered with a 0.22 µm Millipore
syringe filter into the quartz cell applying an equilibration time of 240 seconds at
25 °C. The dn/dc used was 0.127 ml.g-1 [6]. Intensities were then plotted against
concentration and a Debye Plot was calculated by the Zetasizer Software.

4.3. Results

4.3.1. Bacterial isolation and growth

Since the bacteria formed small aggregates, the measurement of growth

by optical density change was not suitable. The bacterial growth was assumed to
have reached its maximum when the gel was entirely liquefied, which occurred
overnight when incubated at 25 °C. No growth was observed in liquid medium.
The active enzymes cocktail precipitated with 60% ammonium sulfate. The
hydrolyzing activity was confirmed by the HPTLC analysis of ulvan after one night
of incubation with this fraction. The protein fraction precipitated with 80% of
ammonium sulfate showed no activity. After dialysis and ultrafiltration
concentration, the yield of protein in the active enzyme mix obtained with the
bacterial hydrolysis of a 50 ml ulvan gel was 0.82 mg crude protein.

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 4. Production of sulfated oligosaccharides

4.3.2. Taxonomical determination

Comparison of the 16S ribosomal DNA sequence (GIIUL2) identified the

bacteria as belonging to the genus Alteromonas, however it presented less than
95 % of homology with the registered species in a GenBank BLAST search (Figure
4.1).

Figure 4.1: Phylogenetic tree of

Alteromonas genus and position of the
newly described strain based on 16S
ribosomal sequences

4.3.3. Enzyme Kinetics

TLC allows to characterize the products present in the solution by the

retention factor (Rf) which is the migration distance of the solute over the
migration distance of the solvent (Figure 4.2). The evolution of the well-

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 4. Production of sulfated oligosaccharides

Figure 4.2: A. TLC plate of degraded ulvan w/o molecular weight separation. B.
Digitalized chromatogram at 380nm. C. Absorbance spectrum of peak R f 0.42

Figure 4.2: Evolution of the relative area of Peak Rf 0.0 and Rf 0.55 of HPTLC plate of ulvan
oligosaccharides after enzymatic degradation under different pH and temperature conditions

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 4. Production of sulfated oligosaccharides

defined Rf 0.0 and Rf 0.55 HPTLC

peaks obtained by analysis of ulvan
hydrolyzed under different pH (6, 7
and 8) and temperature (20, 25, 30
and 35 °C) conditions is shown in
Figure 4.3. The Rf 0.0 showed the
fastest degradation rate when buffer
pH was 8. With this condition the
relative peak area reached down to
21.9 % ± 0.31 of the total area. This
means that almost 80% of the
polysaccharide degradation was
achieved after 20 hours of enzymatic
treatment. The weakest
depolymerization was observed in
the slightly acidic conditions of pH 6.
This result is reflected by the area of Figure 4.3: Evolution of area under the peaks of
TLC plate in optimal degradation conditions
peak Rf 0.55 which shows a rapid A.Peaks Rf 0.25, 0.32, 0.42, 0.55, 0.67 B. Peaks Rf
formation at pH 8 while pH 6 and pH 0.00, 0.06

7 conditions showed no increase. The

maximum area of the Rf 0.55 peak was reached after 40 hours at the optimal pH
8. The degradation rate of the enzyme was faster for higher temperatures, the 35
°C condition showed the best results while low depolymerization was obtained
at 20 °C.
The selected condition for depolymerization in semi-preparative
conditions were then 35 °C and pH 8 during 20 hours. Figure 4.3 shows the
evolution of HPTLC peaks under the optimal conditions. It is worthwhile noting
that Rf 0.42 and Rf 0.55 peaks are still growing after 40 hours while the other
peaks are stable after 20 hours. The Rf 0.06 peak starts growing but decreases
after 10 hours. It suggests that peaks near the Rf 0.0 are large oligosaccharides
that are still not fully degraded and peaks with higher R f are smaller
oligosaccharides that have reached a maximum of degradation.

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 4. Production of sulfated oligosaccharides

Figure 4.4: TLC plate of Ulvan (U),

degraded ulvan with molecular weight
greater than 10 kDa (DU > 10 kDa) and
degraded ulvan with molecular weight
inferior to 10 kDa (DU < 10 kDa)

4.3.4. TLC separation

The non-degraded ulvan, the DU > 10 kDa and the DU < 10 kDa fractions
showed different separation patterns (Figure 4.4). No band displacement was
shown in the non-degraded ulvan. DU > 10 kDa showed slight migration of bands
principally at low Rf whereas the fraction DU < 10 kDa fraction almost entirely
migrated. Figure 4. shows a HPTLC separation of a degraded ulvan before
ultrafiltration (Figure 4. A), the digitalized chromatogram (Figure 4. B) and the
absorbance spectrum of the Rf 0.42 peak (Figure 4. C). The chromatogram shows
the appearance of 8 peaks, each of them corresponding to a level of degradation
of polysaccharide and/or a specific molecular subunit. The absorbance spectrum
of degraded extracts peaks before staining shows the appearance of an
absorbance peak at 244 nm while no relevant UV absorbance was observed for
the non-degraded ulvan.

4.3.5. Yield and Molecular weight

After the preparative enzymatic hydrolysis, the DU > 10 kDa fraction

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 4. Production of sulfated oligosaccharides

yielded 36.8 % of the initial dry weight of ulvan. After the desalting of the UD <
10 kDa with activated charcoal, 39.8 % of initial ulvan dry weight was recovered.
It is assumed that the missing 23.4 % were lost during the desalting process in
the form of mono- and small size oligosaccharides. The calculated molecular
weight (MW) of the undegraded ulvan was 674 kDa ± 38, the fraction larger than
10 kDa had a MW of 81.3 kDa ± 2.01 and the oligosaccharidic fraction had an
average MW of 4.43 kDa ±0.423.

4.3.6. H1-NMR

The NMR spectrum of the none-treated ulvan is included in the electronic

supplementary material (Figure annex S1). It clearly shows that the ulvan was not
degraded before enzymatic treatment. Typical peaks for rhamnose and
glucuronic acid were observed, whereas no peaks corresponding to unsaturated
uronic acid were observed before degradation (Figure. annex S2). Figure 4.5
shows the spectrum of proton magnetic resonance obtained for the UD < 10 kDa
fraction. Chemical shift are expressed refereed to the Residual Hydrogen Oxygen
Deuterium (HOD) at 4.81ppm. Several peaks with a chemical shift around 1.15
and 1.4 ppm were observed, typical of the rhamnose methyl group. The strong
displacement of the 6.05 ppm and 5.53 ppm peaks are attributed to chemical
shift of hydrogens of an unsaturated uronic acid sugar ring. The peaks situated
between 3.3 and 4.8 ppm are attributed to rhamnose 3-sulfate, xylose and uronic
acids. This shows that the oligosaccharides have conserved the basic saccharidic
structure of ulvan with the appearance of an unsaturated bond.

100
 4. Production of sulfated oligosaccharides

Figure 4.5: H1-NMR spectrum of enzymatically degraded ulvan with molecular weight
inferior to 10 kDa (DU < 10 kDa). Δ: refers to unsaturated uronic acid; R: refers to
rhamnose; Rα/β: refers to the reducing end rhamnose anomers; I: refers to Iduronic
acid; G/G’: refers to glucuronic acid; X: refers to xylose. Numbers refers to the proton of
the corresponding carbon

4.4. Discussion

Depolymerization is considered an important step to enhance the general

activity of algal polysaccharides and obtain products with more reproducible
characteristics [43]. Here we have shown that a crude enzyme extract of a thus far
unknown bacteria produced very good results in the depolymerization of ulvan,
the main polysaccharide of the marine macroalga Ulva sp. The bacteria were
isolated from Gammarus insensibilis individuals, a herbivorous amphipod feeding
on Ulva. Phylogenetic analysis placed these hitherto unknown bacteria in the
genus Alteromonas close to A. hispanica which is a moderately halophilic species
that has been described from a saline lagoon 140 km from our location [61]. This
genus is well known in relation to carbohydrates biotechnology and several

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 4. Production of sulfated oligosaccharides

patents have been published both for their degrading capacity as well as for their
exopolysaccharide biosynthesis activity [62-64]. It was not possible to culture the
bacteria in liquid media. An explanation may be that the bacteria is possibly film
forming as has been described for several Alteromonas species [65]. During the
bacterial growth the formation of an insoluble substance has been observed.
Such a product has been characterized for Alteromonas macleodii. It was
described as a polysaccharide made of glucose, galactose, glucuronic acid,
galacturonic acid and pyruvate mannose combined in a repetitive hexasaccharide
unit [66]. A Black Sea strain of this species has been described to synthesize a
great number of hydrolytic enzymes including α-L-rhamnosidase [67]; these
bacteria are highly opportunistic and their genome and consequent properties
can present high variation inside the same species depending on the substrate
where the strain is grown [68]. These enzymes can be extracellular or intracellular.
In our case, the bacterial extract was isolated from the supernatant of the gel
hydrolysate without any cell lysis step. Thus it is likely that several extracellular
carbohydrate active proteins are present in this crude extract.
As high (≥ 40 mg.ml-1) concentrations of ulvan are required to obtain a
sufficient quantity of oligosaccharide for the analysis, we were unable to properly
follow the variation of absorbance at 232 nm to assay the enzyme activity in a
normal benchtop spectrophotometer, this is the reason why the TLC method was
preferred. Moreover this versatile technique allows a semi-quantitative
monitoring of the different oligosaccharidic fraction release over time and a UV-
vis spectrum of each TLC spot can be acquired in order to get more information
on each of those fractions. The kinetic study of the enzyme depolymerization
show that the first band (ref. Rf 0.0) reaches a minimum value after 20 hours,
however the maximum value of other bands like Rf 0.55 is obtained after 40 hours.
This means that the enzyme mix is still functional after 40 hours, however not on
the larger polysaccharides. A possible explanation of this phenomenon can be
that the Rf 0.0 spot correspond to large sugar sequences that would have reached
their maximal degradation. That could mean that this enzyme cocktail is capable
of producing oligosaccharides but is not able to cleave all linkages types that are
present in this polysaccharide. Alternatively, this might indicate the presence of

102
 4. Production of sulfated oligosaccharides

two types of enzymes in the mix, one that has an activity on major molecular
weight polysaccharides that loses its activity after 20 hours and one active on
smaller weight polysaccharides that maintains its activity up to 40 hours. As the
final yield of oligosaccharides shows, this interruption of degrading activity on
higher molecular weight polysaccharides has an effect on the efficiency of the
production, being still superior to 10 kDa 36.8 % of the initial polysaccharide.
Regarding the yields of the degraded and the purified products, only 39.8
% of the initial weight was recovered in the form as oligosaccharides < 10 kDa
which means that approximately 23.4 % of the oligosaccharides were lost during
the elution of salts on the activated charcoal column. This requires an
improvement of the purification method by changing the eluents volumes used
on the activated charcoal or using a more efficient gel column or ultrafiltration
with a smaller cut-off filtration range.
Considering the MW of the main disaccharide subunit, the aldobiuronic
acid, it would be reasonable to conclude that the average polymerization degree
of oligosaccharides is in the order of 20 monomers for the DU < 10 kDa fraction.
The Figure 4.4 suggests that oligosaccharides inferior to 10 kDa are able to
migrate more easily on the silica plate than fractions of higher molecular weight.
Assuming that HPTLC spots with a higher Rf have a lower molecular weight [56],
it is possible that the smallest fragment (Ref. 0.67 Rf) could have only two sugars
residues. Further studies are necessary to determine the exact composition and
degree of polymerization (DP) of each HPTLC band. The objective of this work
was to obtain oligosaccharides from a large polysaccharide in order to assay their
activity in further studies. No attempt or assay was made to reach the degradation
to monosaccharides for fine chemical production or fermentation to biofuel. Most
of sugars of Ulva sp. have been shown to be fermentable for the production of
biofuel however acidic or alkaline treatment coupled to an enzymatic treatment
are necessary for the release of all sugars [69]. Further studies are required to
determine if this bacteria would be able to achieve a complete degradation of
the polysaccharide, which would be interesting for the aforementioned purposes.
The proton NMR spectrum clearly shows that basic ulvan structure was
conserved after enzymatic degradation. Strong signals of the sixth carbon of the

103
 4. Production of sulfated oligosaccharides

rhamnose ring are characteristically present in the 1.15-1.4 ppm zone of the ulvan
spectrum. Moreover peaks corresponding to protons of the anomeric carbon of
an internal 3-sulfated rhamnose are present around 4.72-4.93 ppm, also showing
that rhamnose is not only present at the reducing ends of the oligosaccharide. A
peak indicates the presence of iduronic acid in 5.12 ppm. The appearance of two
strongly deshielded protons indicates that a new type of linkage has been
generated. The chemical shifts of 6.05 (doublet) and 5.53 ppm are typically
produced by protons situated on the carbon 4 and 1 of an unsaturated uronic
acid respectively [60].
The absorbance in the UV range of the degraded ulvan (which did not
occur in intact ulvan) and the H1-NMR signal demonstrate that the
oligosaccharides have an unsaturated link on the sugar ring [49]. This suggests
that the enzyme cocktail has a polysaccharide lyase (PL) activity. The lyases are
known to catalyze a β-elimination and produce an unsaturated sugar ring on the
non-reducing end side of the linkage [70]. This is in agreement with the fact that
several Alteromonas species have been described to produce both internal and
external carbohydrate degrading enzymes towards algal polysaccharides [71, 72].
This type of enzyme is highly desirable for the processing of raw algal
material in order to efficiently release molecules from the extracellular matrix
using a mild extraction method. It is able to selectively cut the polysaccharide,
thereby obtaining alternative saccharidic products with a modulated bioactivity.
Further studies should be directed towards the elucidation of the exact structure
of each oligosaccharide fraction and the characterization of the enzymes present
in the crude mix. In this respect, an effective research strategy would include a
complete sequencing of the bacteria in combination with a detailed bioinformatic
analysis to provide the list of potential enzymes. Furthermore it would be
interesting to study the efficiency of the enzyme extract on raw algal material for
the release of other molecules for biorefinery, biofuel or other purposes.

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 4. Production of sulfated oligosaccharides

4.5. Concluding remarks

This study identifies a new bacterial ulvan -lyase producer with an

interesting enzymatic cleaving activity on the cheap and abundant ulvan
polysaccharide. Based on its 16S ribosomal sequence, it has been identified as
belonging to the genus Alteromonas. This is the first time that this gram-negative
bacteria genus has been described to have the capacity to grow with ulvan as
sole source of carbon. An enzymatic degradation of ulvan polysaccharide allowed
obtaining small size oligosaccharides conserving the original subunit pattern.
This work contributes to obtaining efficient tools to produce compounds with
potential biological activities and high-added value from a renewable biomass
source.

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5 . Poly mer chitosa n na noparticles
func tionalized with Ulva ohnoi
extracts boost in vitro ulva n
immunostimula nt effect in Solea
senegalensis macropha ges

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 5. Polymer chitosan nanoparticles

Título del artículo:

Polymer chitosan nanoparticles functionalized with Ulva ohnoi extracts boost in

vitro ulvan immunostimulant effect in Solea senegalensis macrophages

Autores:

Catalina Fernández-Díaza , Olivier Costea , Erik-jan Maltab

a
Instituto de Investigación y Formación Agraria y Pesquera de Andalucía (IFAPA),
Centro El Toruño, Camino del Tiro Pichón s/n, 11500 El Puerto de Santa María,
Cádiz, Spain
b
IFAPA Centro Agua del Pino, Ctra. Cartaya-Punta Umbría Km 4, 21450 Cartaya,
Huelva, Spain

Publicado en: Algal Research

Número: 26
Páginas: 135-142
Sometido: 7 de Junio 2017
Acceptado: 6 de Julio 2017

Abstract

The potential of chitosan-based nanoparticles to incorporate ulvan was

investigated and the immunostimulant properties of ulvan polysaccharide as
nanocarrier in Solea senegalensis macrophage were evaluated. ulvan native
extract (UL) and ulvan fractionated by chemical (ULQ) or enzymatic (ULE) methods
were obtained from Ulva ohnoi macroalgae in culture. These different ulvan types
were tested for their ability to form polymeric nanoparticles. Nanoparticles
including either unaltered or fractionated ulvan were prepared by ionotropic
gelation and characterized. Activity of the ulvan -loaded nanoparticles was tested
in vitro on fish macrophages against free unaltered or fractioned ulvan extract.

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 5. Polymer chitosan nanoparticles

Intracellular and extracellular reactive oxygen species (ROS) production from the
different ulvan products was determined in S.senegalensis macrophages using
oxidative burst assay. The native ulvan extract (UL) as well as the fractioned form
(ULQ and ULE) successfully yielded nanoparticles with a size of 250–300 nm and
with a Z-potential of 30–40 mV. Highest nanoparticle production was achieved
using ulvan native extract (NPsUL). Our results indicate that the configuration of
the structure of ulvan molecules influence the immune response; in this way,
unaltered ulvan is required for successful stimulation of Solea macrophages by
Ulva ohnoi polysaccharides. This immune response significantly increases when
unaltered ulvan is nanoencapsulated. We conclude that the here developed
hybrid polysaccharide nanoparticles composed of chitosan and ulvan are
functional. This might open the way for production of nanocarriers that can be
used for oral administration of active compounds in aquaculture.

5.1. Introduction

Nanoparticles possess several features that are already applied in a wide

range of disciplines such as food technology, pharmaceuticals or cosmetics.
Generally, the main objective of using nanoparticles is to enhance the
bioavailability of molecules by both improving intestine epithelium absorption
and protecting compounds against degradation. The entrapped biochemical,
generally drugs or rare nutrients, significantly increased their bioavailability and
release [1]. The field of nanotechnology has undergone a strong development
over the last years, however studies on nanotechnology applications in
aquaculture are still lagging behind. Nevertheless, despite its early stage, the
potential to play an important role in answering the questions related to animal
health, production, and reproduction, as well as disease prevention and treatment
is apparent. Nanoparticles have been recently shown to be good candidates as
aquaculture feed additives or as oral vaccine vehicles [2,3].
Beyond the purely structural role, constitutive materials of the nanoparticle

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 5. Polymer chitosan nanoparticles

can offer some functional features. Polysaccharides from natural origin are
especially indicated to fulfil all these requirements. One of the most widely used
natural polymers for oral delivery of many bioactive agents is chitosan, which
encompasses a number of desirable properties for this goal, including its
biocompatibility, biodegradability and no-toxicity [4].
A straightforward method for the manufacturing of nanoparticles is the
ionotropic gelation between molecules of opposite charges. Recently, Bugnicourt
and Ladavière [5] reviewed ionical cross-linking of chitosan nanoparticles with
tripolyphosphate. This is especially feasible for biomedical applications due to a
number of inherent, desirable properties (notoxicity, anti-microbiological activity,
mucoadhesivity, haemocompatibility) and also the possibility to incorporate
several bioactive compounds of interest into these nanoparticles. In addition,
current developments foresee the linking of chitosan with anionic
polysaccharides to form a new generation of nanoparticles (NPs) that can
combine the promising properties of chitosan with outstanding
biopharmaceutical properties of other polysaccharides [6].
In recent years there has been a surge in the interest to tap underexploited
marine resources to develop novel materials. Polysaccharides of algal origin have
exhibited exceptional biological properties and researchers have been focusing
on application in the health and food sector principally [7].
There is an increasing interest in finding new sources of marine bioactive
products with immunostimulant properties with applications in fish aquaculture.
ulvan is a complex and biochemically variable heteropolysaccharide from Ulva sp.
that is naturally abundant (8%–29%) in these fast growing and opportunistic
green macroalgae [8]. It is formed of neutral and acidic sugars and contains up
to 16% of O-sulphate groups. Specific ulvan composition varies extremely, but
most frequently consists of the following sugars: rhamnose, glucuronic acid,
glucose, iduronic acid and the xylose. Sulphate groups are generally
functionalizing the rhamnose on the C-3 bond. As uronic acids and sulphate
groups have a negative charge in solution, ulvan can be viewed as an anionic
chain. Conformation of the ulvan biopolymer is essentially disordered due to its
heterogeneous chemical composition and the various forms of ulvan greatly

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 5. Polymer chitosan nanoparticles

differ in molecular weight and intrinsic viscosity [9]. Ulvan characteristics and
properties mainly depend on extraction conditions and remain unknown thus far
[10]. Bioactivities attributed to ulvan include antioxidant activity, antilipidemic
effect, antiviral or immunomodulator [11,12,9].
Chemical composition, charge density and the molecular weight are largely
responsible for these properties [13]. Although promising, thus far the number of
studies on the use of ulvan for biomedical or food applications is scarce [9].
Considering reported results in the scientific literature, a potential application of
sulphated polysaccharides obtained from marine algae may be related to
immunostimulation and in controlling activity of macrophages [9]. The structural
characteristics of ulvan also suggest a potential immunomodulation property
resulting in one of the most interesting properties of ulvan. Sulphated groups
and the sulphation pattern of ulvan are essential for the activation of turbot
(Psetta maxima L.) macrophages, while inducing a potent stimulating effect on
macrophage oxidative burst [14,15]. Ulvan had the capacity to upregulate the
transcriptional activity of several genes whose products are related with
inflammation response and innate immunity on RAW 264.7 cell line murine
macrophages [16]. Clearly, both complete and fractioned seaweed extracts
possess immunomodulatory capacities on the immune system of fish that
deserve more attention than received thus far [15].
Apart from this, the polyelectrolyte properties of ulvan allow for the
establishment of interactions with cations, interaction of the latter, in turn with
drugs and/or polymers has already led to the development of applications in
different fields [17]. Other anionic sulphated polysaccharides such as fucoidan,
carragenate or alginate have been associated with chitosan and tripolyphosphate
(TPP) to form NPs [18–20]. However, ulvan has been scarcely studied in this
respect and no information focusing on its role in the formation of NPs is
available to date.
Developing and applying technologies to improve the incorporation of
ulvan into NPs can contribute to a better understanding of the functionality of
this compound. Fish response to hybrid nanoparticles, including either ulvan, can
be useful in aquaculture. This study had the objective of obtaining nanoparticles

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 5. Polymer chitosan nanoparticles

from ulvan and comparing their activity on the oxidative burst in Solea
senegalensis macrophages against free ulvan molecules in an attempt to
determine an optimum ulvan format to be used in fish aquaculture. For that
purpose we produced several types of ulvan modifying the extraction conditions
and poly or oligosaccharides were obtained. These poly or oligosaccharides were
included into nanoparticles made by ionotropic gelation. Nanoparticles were
characterized and tested to know whether encapsulation of ulvan in chitosan/TPP
nanoparticles improves ulvan functionality.

5.2. Materials and methods

A schematic summary of the whole experiment is represented in Figure 5.1

and every step is detailed in the following points.

5.2.1. Ulva ohnoi culture

In order to produce ulvan under controlled conditions, Ulva ohnoi algae

Figure 5.1: Schematic representation of the different steps of the experiment. CS: chitosan; NPs:
nanoparticles without ulvan form added; TPP: tripolyphosphate; UL: enriched ulvan extract;ULE:
oligoulvans obtained by enzymatic depolymerization; ULQ: oligoulvans obtained by chemical
depolymerization.

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 5. Polymer chitosan nanoparticles

were grown in natural 0.2 μm filtered seawater brought to constant salinity of 30

ppm with tap water until sufficient biomass for polysaccharide extraction was
reached. The water was enriched with a modified F/2 medium, temperature was
maintained at 20 °C and 1% CO2-enriched air bubbling allowed permanent water
movement and avoided an excessive pH increase. The light intensity was fixed at
200 μmol photons·m−2 s−1. When the alga reached a self-shading density (5 g·l−1)
the excess biomass was harvested and rinsed with tap water before freeze-drying.

5.2.2. Ulvan extraction and depolymerization

5.2.2.1. EXTRACTION AND PURIFICATION OF ULVAN

Ulvan polysaccharides were extracted following the method described by

Robic [21] with some further modifications. The freeze-dried algae were milled
until reaching a particle size of around 8 μm diameter. The ground algae were
extracted in hot distilled water (80 °C) with 0.05 M sodium oxalate for 2 h. The
extract was centrifuged for 20 min at 30,000g (Beckman, USA), filtered at 0.45 μm
and a secondary extraction was made on the residue. Both supernatants were
combined and filtered through 0.45 μm filters. The co-extracted glucuronic acid
was precipitated by lowering pH down to a value of 2 with orthophosphoric acid.
The solution was centrifuged and the supernatant was neutralized with 0.1 M
NaOH and diafiltrated to 10 kDa until conductivity of filtrate reached the one of
distilled water. This extract was concentrated and dialyzed by ultrafiltration to 10
kDa. Eventually the solutions were frozen and freeze dried to obtain the enriched
ulvan extract (UL).

5.2.2.2. CHARACTERIZATION OF ULVAN

Sulphate content was determined by the barium chloride method of

Dodgson [22]. The molecular weight was determined by Static Light Scattering
(SLS) with a dn/dc of 0.145 with concentration of 1, 2, 3, 4 and 5 mg·ml−1 for
higher molecular weight and 11, 12, 13, 14 and 15 mg·ml −1 for smaller
oligosaccharides on a Zetasizer NanoS (Malvern, UK). The presence of the
different sugars was assayed by 1 H NMR.

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 5. Polymer chitosan nanoparticles

5.2.3. Enzymatic depolymerization of ulvan

Enzymatic depolymerization was performed as described in Coste [10].

Briefly, a crude enzyme mix was isolated from an Alteromonas sp. strain. Enriched
ulvan extract (UL) was solubilized at 40 mg·ml−1 in a 0.1 M Tris-HCl buffer with
0.2 M NaCl at pH 8 to which 0.25 mg protein equivalent·ml−1 was added and the
degradation was performed during 48 h at 35 °C after which degradation was
stopped by heating to 100 °C during 10 min. The hydrolyzate was then
ultrafiltrated at 10 kDa. The retentate was then diafiltrated to remove all salts and
the filtrate was ultrafiltrated and diafiltrated against ultrapure water with a
membrane cut-off of 1 kDa. The resulting solutions were then freeze-dried. The
fraction lower than 10 kDa was named ULE.

5.2.4. Free radical depolymerization of ulvan

800 mg of enriched ulvan extract (UL) was dissolved in 500 ml of distilled

water and heated to 60 °C under agitation at 200 rpm. To this 30 ml of copper
acetate were added. Then 12 ml of H2O2 10% was added dropwise during a period
of 15 min to obtain a final ratio of polysaccharide/H2O2 of 1.5. This exothermic
degradation was performed in a bioreactor that allowed maintaining pH 7 and
60 °C constant temperature. An automated addition of NaOH 1 M regulated pH.
These conditions were maintained during 1 h after the addition of the hydrogen
peroxide. Afterwards the reaction was stopped by adding 1 g of NaBH4 and
cooling to 20 °C during 2 h. The copper ions were removed by washing the
hydrolyzate on a chelex 100 (Bio-Rad, USA) column before the ultrafiltration of
the obtained oligosaccharide with a membrane of 1 kDa cut-off. Eventually the
degraded polysaccharide was freeze-dried to obtain a fine white powder and
referred to as ULQ in the rest of this paper.

5.2.5. Nanoparticles methodology

5.2.5.1. NANOPARTICLES PREPARATION

Based on the methods of Wen Fan [23] and Jiménez-Fernández [2] with

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 5. Polymer chitosan nanoparticles

some modifications, nanoparticles (NPs) loaded with ulvan (UL) or both

oligoulvan types (ULE or ULQ) were prepared.
Briefly, low molecular weight chitosan (CS) (Brookfield viscosity 20,000 cps)
(SigmaAldrich, USA) was dissolved in 0.4% glacial acetic acid and stirred
overnight; pH was adjusted to 4.7 with NaOH. Chitosan final concentration was 1
mg ml−1. The solution was filtered through a 0.2 μm cellulose sterile filter and
kept at 4 °C. UL, ULE and ULQ fractions were dissolved in MilliQ water and passed
through a syringe filter (pore size 0.22 μm Millipore, USA). Tripolyphosphate (TPP)
was dissolved in ultrapure water at a concentration of 1 mg.ml−1 and also passed
through a syringe filter (pore size 0.22 μm Millipore, USA). Six milliliters of CS was
heated to 55 °C with agitation of 300 rpm, then 2 ml of the negatively charged
ion polymers (TPP and/or ulvan fractions) was rapidly added at 4 °C. The formed
NPs were allowed to stabilize during 15 min at 4 °C with an agitation of 300 rpm.
A small aliquot (≈2 ml) of nanoparticles suspension was kept at 4 °C for further
characterization. 4.5 ml of the particles suspension was centrifuged at 195,500g
during 6 min with a small amount (≤0.5 μl) of glycerol. The resulting pellet was
then freeze-dried and thawed to obtain a white powder. Nanoparticle powder
was conserved at −20 °C to avoid any possible alteration.

5.2.5.2. CHARACTERIZATION OF NANOPARTICLES

NPs suspension was diluted ten times in a phosphate saline buffer at pH

7.4. Size and polydispersity index were then calculated by dynamic light
scattering on a ZetaSizer Nano-ZS90 (Malvern, UK). For determination of Z-
potential, the NPs suspension was diluted ten times in a 1 mM KCl buffer and
calculated on the same device by Laser Doppler Micro-Electrophoresis. The yield
of nanoparticles was calculated as the weight of freeze-dried quantity divided by
the weight of the initially dissolved matter.
A qualitative assay for the presence of ulvan was done using Alcian Blue.
Briefly, the initial nanoparticles suspension was centrifuged at 195,500g for 6 min
to obtain the equivalent of 1 mg of dry nanoparticles and washed two times with
MilliQ Water. The pellet was dissolved in 1 ml of Alcian Blue 8 GX (Panreac, Spain)
dissolved in 3% acetic acid and water (v/v, pH 2.5), incubated for 30 min at room

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 5. Polymer chitosan nanoparticles

temperature and centrifuged. The supernatant was then discarded and the pellet
suspended in ultrapure water. As a control, nanoparticles of CS and TPP without
ulvan added (NPs) were treated the same way.

5.2.6. Oxidative burst assay

5.2.6.1. MACROPHAGE EXTRACTION AND CULTURE

The method followed is the one described by Secombes & Fletcher [24].
Five individuals of Solea senegalensis between 40 and 100 g in weight were
sacrificed after anaesthesia with 1 ml.l−1 of 2-phenoxyethanol in seawater (v/v).
Head kidney was subsequently aseptically removed and placed in an L15 medium
containing 2% of fetal bovine serum (FBS), heparin (1000 UI·ml−1), streptomycin
and penicillin (S/ P). Macrophages were released by dispersing the head-kidney
in a petri dish with the same medium and the suspension was passed through a
100 μm mesh. Alternatively, cells of each fish were maintained separated all along
the cell extraction process. The cell suspension was loaded on a 34%/51%
discontinuous Percoll gradient (GE Healthcare, Sweden) prepared with modified
Hanks' balanced salt solution (HBSS) without phenol red (SAFC, Switzerland). The
lymphocytes were collected on the interface of the two Percoll concentrations
after 40 min of centrifugation at 400 g in a swinging bracket centrifuge
programmed without brake. The cells were then washed three times by adding
L15 with 0.1% FBS and S/P and centrifuged 5 min at 400 g. Viable cells were
counted with the trypan blue method. A suspension of 4.106 cells.ml−1 was
prepared and 100 μl were incubated in each well of a 96-well plate during one
night at 20 °C. Wells were washed with HBSS containing S/P on the second day
to remove non-adherent cells. The remaining adherent cells were incubated for
one hour with 100 μl of the samples dissolved in L15 containing 5% FBS.

5.2.6.2. INTRACELLULAR REACTIVE OXYGEN SPECIES (ROS) PRODUCTION WITH NITROBLUE

TETRAZOLIUM (NBT)

Immediately after macrophage incubation, the samples were removed and

the cells were incubated with NBT dissolved in HBSS at 1 mg·ml −1 for 1 h. The

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 5. Polymer chitosan nanoparticles

supernatant was removed, the cells were washed again with HBSS after which 100
μl of 70% MeOH was added for 3 min to fix the cells. Then the cells were washed
with 100% MeOH and immediately removed. The wells were allowed to air-dry
during 5 min. 46 μl of KOH and 54 μl of dimethyl sulphoxide were added to the
wells to solubilize the produced formazan. The absorbance was read at 640 nm
on a multiwell plate reader. All samples had a concentration of 1 mg·ml −1 and
were added in 8 wells each. As controls, 8 wells were incubated with only L15 5%
FBS. Phorbol myristate acetate (PMA), as positive control, was added to the NBT
solution at 1 μg·ml−1 in an untreated column of wells. Each plate was repeated 3
times. The different multiwell plates were normalized by quantifying proteins in
the wells with the Bradford (1976) method [25] in 16 untreated wells and lysed
with a 10 mM citric acid and 0.5% Tween 20 Buffer during 15 min.
The results were expressed as intracellular stimulation index and calculated
as:
Intracellular stimulation Index (ISI) = (X Sample – X Blank)/ (X Control-X Blank)
where X is arithmetic means of absorbance data.

5.2.6.3. EXTRACELLULAR REACTIVE OXYGEN SPECIES (ROS) PRODUCTION WITH CYTOCHROME

C (CYTC)

In this assay, isolated macrophages of each fish were kept separately and
the assay was performed 24 h after the extraction of the cells. After that period,
cells were washed and immediately incubated with a CytC solution (1 mg·ml −1 )
with different amounts (10 and 100 μg) of nanoparticles (NPsUL) or free
polysaccharides (UL). Cells were incubated with PMA as a positive control. A blank
was performed with CytC in HBSS and a negative control was performed by PMA
with superoxide dismutase in CytC solution. Absorbance was read at 550 nm at
60 min after incubation. As CytC was oxidized by extracellular ROS absorbance
was increased. Each treatment was repeated with a minimum of five individuals.
The results were expressed as extracellular stimulation index and calculated as:
Extracellular stimulation Index = (X Sample – X Blank)/ (X Positive Control-X
Negative Control)
where X is arithmetic means of absorbance data.

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 5. Polymer chitosan nanoparticles

5.2.7. Statistical analysis

The 96-well experiment was repeated three times with different fish pools
each time. Differences in the intracellular stimulation response between different
ulvan extracts (native or depolymerized) and the effect of encapsulation of ulvan
were tested by two-way ANOVA followed by Tukey pairwise comparisons test
when significant differences were found at p < 0.05. A two-way ANOVA test
(significance, p < 0.05) followed by Tukey's pairwise comparison was also applied
to test the main effect and the interaction among ulvan product concentration
and the ulvan nanoencapsulation effect using extracellular stimulation index. The
results were expressed as mean and standard error. Statistical analyses were
conducted using the software Stat graphic Centurion.

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5.3. Results

5.3.1. Characterization of
ulvan fractions

Depolymerization greatly
reduced the molecular weight of the
ulvan extract as is shown in Table 5.1.
Decrease was roughly 200-fold for
the ULQ and 100-fold for the ULE
compared to the intact UL extract.
Main structure was preserved in all
extracts as shown by 1H NMR
analyses (Figure 5.2). ULQ showed
two peaks with a shift of 5.35 and
5.85 ppm, indicating the presence of
strongly deshielded protons, which
correspond to the protons of the
Figure 5.2: Image relative to the different ulvan
fourth and first carbon of the
fraction. H1-NMR of enriched ulvan extract (ULE),
Chemically depolymerized ulvan (ULQ) and unsaturated uronic acid, respectively.
enzymatically depolymerized ulvan (UL<10).

Table 5.1: Molecular weight of different ulvan fractions. Intact ulvan (UL); enzymatically
depolymerized ulvan (ULE); chemically depolymerized ulvan (ULQ).

Fraction Molecular Weight (kDa) % Sulphation

UL 698±33.2 15.07±0.64

ULE 5.92±2.25 14.08±0.49

ULQ 2.28±0.44 15.70±2.23

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 5. Polymer chitosan nanoparticles

A B

Figure 5.3: Characterization of nanoparticles (NPs). A) Representative picture of a

suspension of Alcian Blue tainted NPs with and without enriched ulvan extract. B)
Microscopic view of ulvan nanoparticles.

5.3.2. Nanoparticles

Several tests were used to set up the method and composition ratios that
allowed obtaining nanoparticles with characteristics adequate of size, Z-potential,
dispersion and yield. The Tyndall effect was observed between the chitosan and
all the different ulvan fractions in the presence of TPP. Attempts to incorporate
cyclodextrin with ulvan were not successful as the formation of aggregates was
instantaneous. The characterization of nanoparticles elaborated is depicted in
Figure 5.3 and Figure 5.4.
Alcian Blue assay gave a qualitative result for the presence of ulvan. All the
particles that were made with some fraction of ulvan showed a blue colour,
whereas the control particles CS/TPP didn't show any incorporation of the
pigment (Figure 5.3A).
Z-potential, size, polydispersion index values and the yields obtained are
indicated in Table 2. As it is generally admitted that the nanoparticles have a good
stability in solution when the Z-potential value ranges 25–40 mV, particles with
these characteristics were selected for the following bioactivity tests. Particles
with ratios CS/TPP/UL of 2/0.25/1 had particularly good features with good z-
potential and excellent yield.

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Figure 5.4: Ulvan nanoparticles characterized using Nano Zsizer equipment. A) Repartition of sizes
of nanoparticles; B) Z-potential repartition of ulvan NPs

The NPs including ulvan in all the formats had a diameter < 300 nm.
Control NPs of CS/TPP had the smallest size of 123.9 nm. Particle size and
encapsulation yield positively correlated with increase in ulvan MW.

Table 5.2: Composition, characteristic (Z-potential and particle size) and process efficiency (yield) of different type
of nanoparticles. PI: polydispersity index; CS: chitosan; TPP: tripolyphosphate;UL: enriched ulvan extract; ULE:
enzymatically depolymerized ulvan; ULQ: chemically depolymerized ulvan

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5.3.3. Oxidative burst assay

5.3.3.1. NITROBLUE TETRAZOLIUM FOR INTRACELLULAR REACTIVE OXYGEN SPECIES

The formation of oxygen radicals inside the adhered macrophages after

incubation with different ulvan formats and nanoparticles including ulvan were
assayed with the NBT test and the results of estimated intracellular stimulation
index (ISI) of the different samples are exposed in the Figure 5.5. In general, cells
incubated with ulvan both in free form and as nanoparticles produced an effect
on the oxidative burst. Increased ROS level was significantly higher (ANOVA test;
p < 0.05) in cells treated with UL than in cells treated with ULQ and ULE.

Figure 5.5: Stimulation of Solea senegalensis macrophages after incubation with ulvan fromUlva
ohnoi macroalgae. Internal reactive oxygen species (ROS) cells production. Different format of
ulvan: enriched ulvan extract (ulvan native) or enzymatically depolymerized ulvan. Different
letters indicate significant differences (p < 0.05) between ulvan products assayed. Asterisk
indicates differences (p < 0.05) between encapsulated or not encapsulated ulvan.

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It seems that the method of depolymerization did not result in a significant

difference (p > 0.05 between ULE and ULQ). Ulvan native loaded nanoparticles
exhibited significantly higher intracellular stimulation index than free ulvan and
depolymerized ulvan loaded nanoparticles.
A significant interaction polymerization ∗ nanoencapsulation was found
(two-way ANOVA; p < 0.05). Thus, a seven-fold increase in the internal
immunostimulation index was detected when cells were incubated with UL NPs
versus free UL. Incubation of macrophage cells with either free or
nanoencapsulated depolymerized ulvan induced lowest ROS production (p <
0.05; Figure. 5.5). When cells were incubated with non-loaded ulvan nanoparticles
minimum response was detected (ISI = 0.16 ± 0.14).

5.3.3.2. CYTOCHROME C FOR EXTRACELLULAR REACTIVE OXYGEN SPECIES

Figure 5.6 shows the effect of different concentrations of ulvan native

extracts and the nanoencapsulation on ROS cells production. Significantly
increased ROS (p < 0.05) was detected when cells were incubated with higher
product dose (100 μg·ml−1). Ulvan extracts promoted the highest ROS production

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 5. Polymer chitosan nanoparticles

(p < 0.05) when included in NPs at concentrations above 100 μg·ml−1. A

significant interaction between the supplied ulvan concentration and
nanoencapsulation was detected (Two-way ANOVA; p < 0.05). Whereas cells
incubated with high dose of ulvan nanoencapsulated exhibited higher
extracellular stimulation index, no differences between cells incubated in similar
condition with free ulvan were observed.

Figure 5.6: Stimulation of Solea senegalensis macrophages after incubation with ulvan from Ulva
ohnoi macroalgae. External reactive oxygen species (ROS) cells production. Free or
nanoencapsulated ulvan (UL and NPsUL) extract at different concentrations. Different letters
indicate significant differences between ulvan products assayed. Asterisk indicates differences (p
< 0.05) between encapsulated or not encapsulated ulvan.

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 5. Polymer chitosan nanoparticles

5.4. Discussion

5.4.1. Polysaccharide depolymerization

To define the impact of ulvan extraction and purification on its

conformation and capacity to make NPs, ulvan was depolymerized by different
methods using chemical and enzymatic processes and were then
nanoencapsulated. NPs could be successfully produced applying all ulvan types.
Unsaturated uronic acid presence at the reducing end of the oligosaccharide,
when the ulvan molecule was enzymatically degraded as indicated by the
spectroscopy results, makes it likely that the rough enzyme mix isolated from a
new strain of Alteromonas sp. contained an ulvan lyase [10]. This class of enzymes
cut the polysaccharides by β-elimination yielding an unsaturated link at the
reducing-end. Clearly, depolymerization successfully reduced molecular weight
without affecting sulphation.
Extraction conditions of ulvan may favour the modification of the
polysaccharide conformation and properties as concluded by Robic [21]. Particle
formation can be exhibited by polysaccharides associated ionic interactions with
cationic surfactants, depending on their molecular weight and the structure and
number of charges along the polysaccharide chain [17]. In addition, as the results
from the present study show, extraction conditions may favour the capacity of
effective ulvan integration into nanoparticles.

5.4.2. Nanoparticle characteristics

We tried to obtain NPs from UL and CS without any additional cross linker.
Even if the characteristics of those particles were acceptable, the yields were still
low (15.8%). The use of TPP as multi anionic cross linker allowed greatly improved
nanoparticle yields, even when added in small amounts. Native ulvan extract or
depolymerized ulvan were nanoencapsulated in CS/TPP NPs.
In all cases polymer interaction was produced and complex CS/TPP/ ulvan

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nanoparticles were successfully obtained. The size of the NPs obtained with ratios
of CS/TPP/UL of 2/0.25/1 gave fairly good Tyndall effect. The mean size of the
particles containing ulvan was greater than the control particle made of CS/TPP
without any ulvan added. The > 20 mV Z-potential of the solution indicates its
stability [26]. This value is positively correlated to the number of chitosan amino
groups on the particle surface [27]. The smaller Z-potential shown by NPs made
of lower MW polysaccharides than that of the unfractionated UL NPs could be
due to the more efficient neutralization of protonated amino groups by ulvan
anions.
Existence of nanoparticles probably resulted from electrostatic attraction
and showed that the interaction of native ulvan was stronger than that for
oligosaccharides. The ulvan of greater molecular weight allowed better yields
with nearly 90% of the dissolved molecules converted into NPs. This result
suggests that high molecular weight polymer chains could lead to better
incorporation into the mix and would be more easily trapped during nanoparticle
formation [5]. This coincides with the observations of several research groups
studying other polymers of chitosan indicating that there is a linear increase of
size and nanoparticle yield with increasing chitosan MW [28,27].
Surface charge plays a major role in stabilizing particles in suspension,
through the electrostatic repulsion effect between particles, and performance [2].
The repulsive interactions between particles will increase with increase in Z-
potential, leading to the formation of more stable particles with a more uniform
size distribution [29]. In the present study, no change in size and Z-potential, and
no aggregation or precipitation, could be observed in NPs suspensions during
several months preservation at 4 °C. Consequently, all NPs developed here can
be considered stable and optimal for further study.
Ionotropic gelation of chitosan with TPP and ulvan is a suitable,
straightforward and cheap method to obtain ulvan nanoparticles. The
physicochemical properties of ulvan enable its interaction with a cationic polymer
such as chitosan, obtaining nanoparticles that can be used as activators of the
defense system or as carriers of interesting bioactive compounds. Jiménez-
Fernández [2] demonstrated that CS NPs with similar size (< 300 nm) could be

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internalized by zebrafish hepatocytes. The size of the NPs elaborated in the

present work was considered adequate to be used in Solea senegalensis
macrophages for in vitro experimentation.

5.4.3. Biological effect

In this study we have investigated the in vitro bio-functionality as new

immunostimulant candidates for aquaculture of new ulvan formats, comparing
the effects of both nanoencapsulated and native polysaccharides. The close
relation between innate immune system response and macrophages is known
and hence their role in the defense of the host against pathogens, by the release
of cytokine, is essential [30]. Reports of the stimulation of macrophage cells by
sulphated polysaccharides obtained from green seaweeds have been published
before [31,32]. According to Cunha and Grenha [9], these sulphated
polysaccharides can potentially be used in stimulation of the immune system or
in controlling macrophage activity. To date there are no previous studies on the
functionality of polysaccharide ulvan in macrophages of sole.
In vitro models are being used with the idea to extend and transfer these
to fish and the management of their production in aquaculture. The central idea
is using in vitro models for the evaluation of immune responses, with the aim of
projecting it to the fish and its productive management in aquaculture. Here, the
quantification of ROS provides evidence of the stimulating effect of ulvan
products on macrophage activity in culture, as shown in their increasing
destructive activity by phagocyte oxidase. Stimulation of polymeric ulvan from
Ulva rigida of macrophages and thereby contributing to disease resistance in fish
was observed by Castro [15].
Our study reveals that native ulvan extracted from Ulva ohnoi macroalgae
can be considered a macrophage activator. Both intracellular and extracellular
ROS production was enhanced in S. senegalensis macrophages after exposure to
ulvan. Among all the ulvan fractions tested, native ulvan extracts with high
molecular weight induced maximum stimulatory activity. When this ulvan format
was incorporated into chitosan nanoparticles a potential effect was detected and

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the maximum ROS production obtained. Better immune-stimulation properties

of the soluble polysaccharides ulvan NPs was evidenced by enhancement of ROS
production versus free ulvan fractions. An effect of product concentration and
molecular weight was evidenced. S. senegalensis macrophage cells showed a
positive dose-response after exposure to the dilutions of ulvan NPs. Hence, the
highest dose assayed 100 μg ml−1 was much more effective compared to lower
doses. Macrophages were incubated with same quantity of product, but
considering that nanoparticles were made with a ratio of 2:0.25:1 to CS/TPP/UL,
only a third part of the nanoparticle is constituted of ulvan. Obviously, 100 μg
ml−1 extract contains more ulvan than 100 μg ml−1 of nanoparticles but
surprisingly the ROS production is increased by 5–6 fold in this later case. The
CS/TPP control nanoparticles (NPs) showed that it had no effect at all on the ROS
production. Other polymers of chitosan showed similar properties as ulvan and
an increased effect was evidenced when they were encapsulated [5]. Blending of
two biopolymers such as chitosan and ulvan by ionic gelation provides a synergic
effect in the immune-stimulant system of S. senegalensis macrophages. The
synergic properties of these NPs may enhance ulvan bioactivity. It has been
shown that the degree of polysaccharide polymerization is a key issue for their
secondary structure in solution and hence for their interaction with cell receptors.
Laminarihexaose (DP6), for instance, inhibits the production of ROS by murine
macrophages, however, laminariheptaose (DP7) stimulated their production
[33,34]. The ulvan fractions obtained both enzymatically and by free radical
degradation are crude mixes of oligosaccharides with some diversity of receptors.
However our results show that low molecular weight ulvan s loose the immune-
stimulatory activity.
We also showed that the nonreducing end seems to have no relevance for
stimulation of receptors that induce ROS production in the macrophage. These
two observations support the hypothesis that ROS production is triggered by
large ulvan sequences rather than by an extremity of the polysaccharide. It has
already been shown that desulphated ulvan was unable to activate the
production of ROS [15]. In mice this activation of macrophages is associated with
the release of several pro-inflammatory cytokines [15]. It seems that ulvan

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 5. Polymer chitosan nanoparticles

triggers innate immune response acting as Pathogen/ Danger Associated

Molecular Pattern (P/DAMP). It has been hypothesized that TOLL-receptors (TLR)
were probably involved, but inhibition assays showed that it's unlikely that this
activation is made by lectin domains [16]. As lectins sugar-binding domains are
specific to the terminal sugars of the polysaccharides, our observations seem to
confirm that the macrophage activation by ulvan is not triggered through this
binding.
Sulphated fucans have been shown to activate the production of NF-κB in
vitro via the specific binding to TLRs 2 and 4 in human kidney cell-lines [34].
Earlier work from our group showed that chitosan NPs are efficiently endocyted
by zebrafish hepatic cell line in vitro [2]. We also described the ability of ascorbic
acid-loaded chitosan/cyclodextrin NPs to penetrate the Solea senegalensis
intestine epithelium [2]. It should be further determined whether this new type of
nanoparticle is able to penetrate the cells. A recent study demonstrated the
targeting effect of heparin-chitosan nanoparticles towards dendritic cells in mice
lymph nodes but relatively few of these NPs were endocyted [35,36]. Nasti [37],
based on tetrazolium dye (MTT) assays with macrophages and fibroblasts
exposed to chitosan nanoparticles, suggested that macrophages have a high
internalization rate of nanoparticles. Cationic NPs are especially good for the
transport of molecules because, unlike anionic NPs, they escape the lysosome
system and, hence, are more available in the cytosol. This offers particles the
opportunity to interact with intracellular receptors [38]. Nucleotide-binding
oligomerization domain (NOD) like receptor (NLR) is a class of cytosolic Pathogen
Recognition Receptors (PRR) that is implied in the innate immune response by
recognizing P/DAMPs [38]. Interestingly, it has recently been shown that
encapsulation of NOD1/2 ligand in nanoparticles enhanced the production of
proinflammatory cytokines compared with the free ligand. The authors
hypothesized that the endocytosis of the nanoparticle could favour the
endoplasmic delivery of the ligand of the cytosolic PRR [39]. In channel catfish
the upregulation and downregulation of those molecular factors have been
reported to be tissue specific after challenge with the pathogenic gram-negative
bacteria Edwardsiella ictaluri [40]. It would be interesting to study whether ulvan

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 5. Polymer chitosan nanoparticles

is able to act as a ligand for cytosolic PRRs when presented in NP form and trigger
an innate immune response. This property is especially interesting to increase the
immunogenicity of antigen.
Furthermore, even if low molecular weight oligosaccharides containing
particles did not show any immunostimulant activity, they could still be useful in
applications where this phenomenon is undesirable and where oligosaccharides
could have a biological effect. For this purpose it is worth following the study of
bioactivity of different ulvan fractions. Further works are needed to evaluate the
activity of those particles in vivo. Our study demonstrated that the properties of
ulvan were improved in the chitosan ulvan nanoparticles. Therefore, the
production of ROS is boosted when the macrophages are incubated with high
molecular weight ulvan NPs.
Currently, our laboratory is undertaking studies testing the efficacy of
water-soluble ulvan fraction as a feed additive for practical application in fish
aquaculture. With these perspectives ulvan nanoparticles have a great potential
as carriers with adjuvant properties for oral delivery of nutrients and vaccines in
aquaculture. In addition, this study contributes to enhance ulvan knowledge and
the valorisation of Ulva ohnoi biomass.

5.5. Conclusion

The incorporation of ulvan or oligoulvan into nanoparticles was confirmed

both by particle size, Z potential and by Alcian Blue assay. Ulvans are able to
create gels in the presence of phosphate ions and cationic polysaccharides such
as chitosan. Nanoparticles prepared by ionic gelation allow the inclusion of
different ulvan derivate forms. High MW ulvan favoured nanoparticles formation,
and a higher response in sole macrophages was detected when ulvan native NPs
were utilized. This paper shows that ulvan can produce a stimulant effect in S.
senegalensis macrophages that can be boosted when delivered through
nanoparticles. Ulvan nanoparticles can be considered as a macrophage activator

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and present a potential as immune-stimulant ingredient in marine fish feed and

adjuvants.

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6 . Improvement of a ntioxidant
compounds extra ction in the
macroa lgae Ulva ohnoi with hot
a cidic methanol extra ction method

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 6. Improvement of antixiodant compounds extraction

Título: Improvement of antioxidant compounds extraction in the macroalgae

Ulva ohnoi with hot acidic methanol extraction method

Autores: Olivier Costea, Erik-jan Maltab, Catalina Fernández Díaza

a
Instituto de Formación Agraria y Pesquera de Andalucía “el Toruño” Camino del
tiro Pichón s/n 11500 El Puerto de Santa María, Cádiz, SPAIN
b
Instituto de Formación Agraria y Pesquera de Andalucía “Agua del Pino” Crtra.
El Rompido-Punta Umbría km 4 21450 Cartaya, Huelva, SPAIN

Publicación: No publicado

Abstract

Phenolic compounds (PC) can be freely present or being covalently linked to

other molecules like protein, polysaccharides or lipids. As a consequence an
underestimation of the PC content could be caused by extraction methods using
solvents without cleaving capacity of these linkages. Green algae are organisms
rich in soluble fibers and containing apparently low levels of PC. Here we briefly
report on the development of a method of hot methanolysis of raw Ulva material
that allows to significantly improve the reducing capacity of the extract.
Separation of the compounds with thin layer chromatography (TLC) and DPPH
revelation allowed for the detection of the presence of previously undetected
antioxidant compounds. The compounds were characterized by their retention
factor (Rf) value and UV-vis absorbance spectrum. Almost all the compounds
showed a peak around 269-280 nm. Only phloroglucinol and gallic acid could be
unequivocally assigned. Comparison with other commercial standards failed to
identify the compounds present in the hydrolysate. In conclusion, these data
indicate the presence of hitherto non-detected compounds in Ulva with a
potential as natural antioxidants. This work opens a new perspective on the
determination of antioxidants in green macroalgae through the rupture of their
resistant extracellular matrix.

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 6. Improvement of antixiodant compounds extraction

6.1. Introduction

Phenolic compounds (PC) are molecules constituted of at least one phenyl

ring mono- or poly-substituted with an alcohol group. Because of the numerous
side-chains and functional groups that they can bear this is a highly diversified
group of molecules. PC are generally abundant in nature and specifically in the
plants kingdom where they are present as secondary metabolites. As they are
potent bioactive and antioxidant molecules their extraction, purification,
characterization as well as their activities have been extensively studied with the
aim to replace synthetic molecules in the food industry, cosmetics,
pharmaceuticals and nutraceuticals [1, 2]. Their biological role is not fully
understood but it is often associated to protection against oxidative stress and
defense against herbivores, pathogens and fouling organisms [3]. Oxidative stress
occurs when an imbalance between production of Reactive Oxygen Species (ROS)
and scavenging of the former is achieved. Excess of ROS can initiate oxidative
chain reaction that can cause harmful damages to biological molecules such as
cell membrane lipids, proteins, DNA and even lead to cell-death. [4].
In autotroph organisms there is a wide network of antioxidant compounds
acting to control this balance. Principally there are two types: preventive
antioxidants that impair the formation of radicals, and radical scavengers that
stop radical chain reaction sacrificially [2]. PC are able to donate H• with the effect
of terminating the chain reaction and become themselves a radical in which the
electron is stabilized by delocalization and resonance on the phenyl ring with
formation of a quinone [5]. Polyphenols are also known to be enzyme regulators
and metals chelators so that they can also regulate the formation of hydroxyl
radicals by the Fenton effect [6]. On the other side the oxidized form of phenolic
compounds, the phenoxyl forms, have been shown to be potential pro-oxidant if
not stabilized. For instance, this mechanisms occurs in the growing parts of the
plants and seems to be a way to locally generate superoxide anions and disrupt
glycosydic linkages of the cell wall polysaccharides [7]. When the phenoxyl form

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 6. Improvement of antixiodant compounds extraction

is not stabilized by a metal ion, it can be regenerated by non-enzymatic reaction

with ascorbate or by enzymatic means with monodehydroascorbate-reductase
[8].
A review was made on these molecules for red and brown algae [1].
Phaeophytes are known to accumulate especially large amounts of phlorotannins.
These macromolecules are polymers of the phenolic subunit phloroglucinol and
constitute up to 25% of dry weight [9]. Many marine macroalgae inhabit the
intertidal and higher subtidal zones, where they are constantly experiencing
changing conditions of light, temperature, and salinity. In the Ulva genus, such
events can produce oxidative stress and stimulate the organism’s antioxidant
defenses [10–15].
Despite its harsh conditions of life, antioxidant capacity and PC levels of
Ulva are apparently low in comparison to brown macroalgae [1, 3, 9]. Traditionally
organic-aqueous solvents extraction methods have been employed to extract
phenols however it has been shown [16] that a significant amount of PC remains
in the solid residue after this step. This author describes the presence of ester of
phenolic acids covalently bound to cell wall carbohydrates in plants, in particular
in species rich in fibers. It is generally recognized that the colonic fermentation
of natural bran allows the release of phenolic compounds (PC) with beneficial
effect for colorectal cancer [17,18]. In vegetables and fruits, significant amounts
of PC have been shown [19] to be undetected due to their covalent linkages to
other molecules and the inefficacy of the traditional aqueous-organic solvent
extraction. These molecules are likely present in the organisms in the form of
polyesters of PC or as carbohydrate esters. The Ulva species are known [20] to
possess an elevated content of fibers such as cellulose, hemicellulose, glucuronan
and rhamnoglucruronan (ulvan) that have been shown to be hardly degraded
both by enzymatic or chemical means.
Taking these considerations into account, it is hypothesized that a
significant amount of non-extractable PC is present in Ulva spp. and that the total
content of PC is underestimated in Ulva spp. Here, we evaluate the use of a

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transesterification step by methanolysis to break the possible covalent linkages

and release the PC in solution.

6.2. Materials and Methods

The first experiment consists of the comparison of antioxidant capacity of

the products of the traditional methanol: acetone extraction and the product of
the hot acidic methanol extraction method. In a second experiment the
conditions in which the hot acidic methanol extraction is performed are studied.
Three extraction time and two temperatures have been done in triplicate. Finally
the products obtained by different extractions methods are separated on a TLC
plate and characterized by their absorbance spectrum.

6.2.1. Algal material

6.2.1.1. ALGAL CULTURE

The experiment was carried out with Ulva ohnoi M. Hiraoka & S. Shimada
isolated from an outflow channel next to the IFAPA Centre Agua del Pino in
Cartaya, Spain (see annex I). Algae were grown in 5 liter bottles under continuous
light conditions (100 µmoles photons.m-².s-1) of photosyntetically active radiation
(PAR) at 20 °C and a salinity of 30 ppm. In order to avoid change of pH a constant
bubbling with 1% CO2 enriched air was provided. The water was enriched with
nutrient with a modified f/2 solution with nutrient concentrations of 200 µM
nitrate and 20 µM ortho-phosphate. Medium was changed weekly.

6.2.1.2. PC EXTRACTION

When a sufficient amount of algae was reached, algae were harvested,

blotted dry between two layers of tissue and stored frozen. Before the analysis,
algae were freeze-dried for 72 hours in the dark. The freeze-dried algae were
ground into a fine powder (media of 8 µm diameter) using a mill. Protocol of

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Arranz et al. [19] for the extraction of the so-called “non-extractable polyphenols”
was followed with some modifications. Briefly, 500 mg of algal powder were put
into a 20 ml screw-cap glass tube and extracted at room temperature during 1
hour under vigorous agitation with MeOH:water (50:50) adjusted at pH 2 with HCl
2M prior to centrifugation for 30 min at 1500 rpm after which the supernatant
was stored.
A second extraction was made with 20 ml acetone and water (70:30) at
room temperature during 1 hour under strong agitation. After centrifugation
both supernatants were merged and stored at -20 °C until analysis. The residues
were freeze-dried and weighted. 100 mg of the freeze-dried residue were put into
an 8 ml screw cap tube and 5 ml of 10% H2S04 in MeOH was added, this first
methanlosysi was performed at 80°C for 20 hours.
In order to know which were the best conditions a second set of
methanolysis was performed on the residues of methanol and acetone extracts.
Three time 5 h, 10 h and 20 h and two temperatures 80 °C and 100 °C were tested.
After that time, samples were allowed to cool during 10 minutes and then
were centrifuged 30 min to 4000 rpm. The supernatant was then neutralized with
KHCO3 in a 20 ml screw cap tube allowing the formed CO2 to escape. The volume
was adjusted to 10 ml with methanol and 1.5 ml was dried with using a vacuum
evaporator. PC were then solubilized in MeOH and then in ethyl acetate to avoid
the presence of salts, passed to a new tube and evaporated in a speed vacuum
evaporator. The solid brown pellet was then stored at -20 °C until analysis. Each
extraction was made in triplicate.

6.2.2. Reduction Capacity

Reduction capacity assay was performed with the Folin-Ciocalteu method

also known as total phenolic content method [22]. Solid extracts were dissolved
in 200 µl of methanol and diluted 10 fold with ultrapure water. 20 µl of these
extracts were placed in the wells of a transparent 96-well microplate, then 100
µml of Folin-Ciocalteu reagent was added and mixed with the sample. After five

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 6. Improvement of antixiodant compounds extraction

minutes 80 µl of sodium carbonate 7.5% was added. The plate was shaken in the
dark during 2 hours at room temperature. The plate was read at 750 nm in a
microplate reader. A calibration curve was made with phloroglucinol. Results are
expressed in mg of phloroglucinol equivalent per gram of algal dry weight. All
reactants and standards were purchased from Sigma (USA).

6.2.3. Thin Layer Chromatography

In first place, to determine whether the extract had phenolic compounds

or not we used TLC aluminum thin layer F264 plates. A mix of solvent
CH3Cl:MeOH:H20:Ac.Ac. (65:25:4:3) was used to perform the separation. Then each
lane of the plates was read on a TLC-scanner (CAMAG, CH) at 280 nm and the
UV-vis spectra was acquired for each relevant peak. Standards of phloroglucinol,
gallic acid, coumaric acid, protocatechuic acid, chlorogenic acid, resorcinol,
ferrulic acid, pyrogallic acid, hydroxybenzoic acid, vanillic acid, syringic acid,
caffeic acid and rutine were separated on TLC after following the same
methanolysis treatment in order to identify algae products by R f and spectrum.
Following this, the plate was submerged in DPPH 0.1 gr.l-1 and heated for 10
minutes to 160 °C for the formation of white spots on the violet background.
Image of the plate was treated to enhance contrasts and to better appreciate the
presence of several spots [21].

6.2.4. Treatments of Data

Data have been treated with the software Statgraphics Centurion XVI with a one-
way ANOVA and statistical significance when p<0,05.

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 6. Improvement of antixiodant compounds extraction

6.3. Results

In a first part of the experiment, we

compare the reducing capacity of the joined
extracts of methanol and acetone with the
extract obtained by the hot methanolysis on
the residue in preliminary standard
conditions (20 h at 80 °C). Figure 6.1 shows
that the amount of reducing compounds was
significantly increased by the methanolysis
step. While the methanol/acetone residual
pellet was still green after multiple solvent
extraction the methanolysis residue had Figure 6.1: Reducing activity of
almost completely lost its color after a few merged Methanol and Acetone
extract and Methanolysed extract.
minutes.
PGE: phloroglucinol equivalent
The results from the optimization test
at two temperatures and three time durations
showed that the optimal quantity of reducing compounds was obtained after 5

Figure 6.2: Reducing capacity of extracts performed in different

time and temperature conditions

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 6. Improvement of antixiodant compounds extraction

hours at a temperature of 100 °C. The methanolysis of 80 °C during 5 h showed

the lowest results but still extracted reducing compounds (Figure 6.1).
TLC separation of the methanol and acetone extracts showed a slight signal
at Rf 0.0 but no other clear peak could be detected (Figure 6.3 A). Figure 6.3 B
shows the separation of compounds proceeding for the hot methanolysis
extraction on a TLC plate, it can be observed that a significantly larger amount of
molecules are separated than for the traditional extraction. Figure 6.4. shows a
typical chromatogram obtained after methanolysis and separation by TLC.
Approximately 10 peaks could be counted. As reflected in the Figure 6.4
chromatograms of the methanolysed extracts at 80 °C and 5 h showed a lower
amount of peaks and with an inferior resolution of peaks. Gallic acid and
phloroglucinol were the only standards that could be unequivocally identified
based on their rentention factor and spectrum (not shown) in 0.6 Rf and 0.8 Rf
respectively.
The UV-vis spectrum of each spot was acquired to try to identify the
compounds. Amongst the absorption spectrum of spots, three types of shapes

Figure 6.3: TLC plates stained with DPPH. A: Methanol and Acetone extracted B: Replicate
of hydrolysates with different times and temperatures

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 6. Improvement of antixiodant compounds extraction

were observed. Representative

spectra are shown in Figure 6.5.
The spot with a Rf of 0.09
showed an absorption peak at
269 nm. The 0.6 Rf spot showed
a double peak of absorption at
227 nm and 280 nm. The 0.7 Rf
spot has a maximum of
absorption at 280 nm. These
Figure 6.4: Chromatogram of a representative TLC lane peaks are representative for the
scanned for absorption at 269nm before staining with rest of the absorption spectra of
DPPH.
the other peaks that had all one
of these three shapes. Intense
absorption at 200 nm is
attributed to the background
absorption of the residual
solvents.

6.4. Discussion

To the best of our

knowledge the extractability of
Figure 6.5: Representative Absorption spectra of TLC
spots
secondary metabolites
compounds in Ulva has been
rarely studied. The dark green colour of the residue following standard organic
solvent extraction steps suggestion that extraction was incomplete and unable to
remove for instance all chlorophyll. Hence we concluded that solvents generally
used for extraction are not able to efficiently access the cells and dissolve the
lipid layer and the chloroplast membrane. This could greatly impair the extraction

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 6. Improvement of antixiodant compounds extraction

of several compounds including antioxidants. One of the possible reason is that

even if this algae is composed basically of a bilayer of cell, they are entrapped in
a very dense matrix of polysaccharides and proteins that is difficult to degrade
[20]. These polysaccharides are insoluble in organic solvents and could form an
impermeable barrier between the external medium and the cells. Moreover, these
molecules like ulvan or glucuronan are resistant to acidic pH [20] and could
impair the total extraction of intracellular compounds and/or cell lysis.
On another hand, these compounds are usually quantified via the Folin-
Ciocalteu method. Unless it is thought that this assay measures the reducing
capacity of an extract and not only the polyphenols it is commonly named Total
Polyphenol Content (TPC) [2]. In plants the compounds that have the major
reducing activity are generally the PCs because of their ability to donate electrons
[5].
Extracts contents can be systematically underestimated because of a poor
extraction yield. Hassan and Ghareib [22] have identified 8 phenolic compounds
in Ulva lactuca organic solvent extracts by HPLC. Vanillin and p-coumaric acid
were the most abundant but also smaller amounts of protocatechuic acid, caffeic
acid, pyrogallic acid, chlorogenic acid, resorcinol, salicylic acid, and ferulic acid.
Recently, Farvin and Jacobsen [23] made water and ethanolic extracts and
determined the abundance of several phenolic compounds in several seaweeds
of the Danish coast by HPLC and detected the presence of gentisic acid,
protocatechuic acid, gallic acid, hydroxybenzoic acid, vanilic acid syringic acid,
caffeic acid and chlorogenic acid. Ulva has been shown also to have a peroxidase
III, an enzyme implicated in the lignin polymerization. It was active on sinapyl
alcohol or syringaldazine, however authors were unable to unequivocally link it
to the presence of lignin [24].
All these substances were used as standards but except gallic acid and
phloroglucinol, we were unable to exactly identify the other products by TLC Rf
and spectrum correspondance. This mode of extraction shows that thus far
unknown phenolic secondary metabolites are present in Ulva. Several important
cues indicate that they are phenolic compounds. The results obtained by TPC

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 6. Improvement of antixiodant compounds extraction

obtain with the Folin-Ciocalteu reagents show that we are in presence of strongly
reducing compounds. In plant, the phenolic compounds are generally the
strongest reducing compounds. Moreover the eluent used in our method shows
similar Rf to standards with slight differences. That means that some little
structural differences occur in the composition when compared to these
standards. This hypothesis is supported by the fact that the absorbance spectrum
recorded on the CAMAG TLC Scanner showed spectrum of absorbance very
similar to these compounds with maximum of absorbance from 270 nm to 280
nm which is typical of aromatic compounds. In the absence of known standard a
liquid MS/MS spectrometry should be performed to determine the fine structure
of each compound.
Methanolysis is a chemical method that allows to perform a
transesterification reaction. It is likely that these compounds are present in the
algae under the form of ester of some other larger molecules which are impairing
their extraction employing the normal solvents like methanol, water or acetone.
This process convert the so-called “condensed tannins” to their soluble methyl
esters [25]. In land plants raw material like maize bran or wheat, hemicellulose is
covalently crosslinked by ferullic acid esters where it can be present up to 3%
w/w. An interesting fact is that some polysaccharides of the Ulva are resistant to
degradation and a way to perform an analysis of their monosaccharide
composition is to break them by acid hydrolysis. The most efficient hydrolysis is
the sulfuric acid methanolysis [26]. This is an aggressive treatment that has been
shown to degrade even the sugar monomers after 4 hours, however the phenolic
acids are more resistant to this treatment. The results showed that treatment at
100 °C during 20 h gives highly variable results, this could indicate that the
compounds present are beginning to undergo a degradation.
To our knowledge, this is the first time that a methanolysis is performed
on this marine algae to analyze the condensed phenolic compounds. It reveals
the presence of several unidentified aromatic compounds with an enhanced
antioxidant activity compared to the classical extraction method. Further studies
are needed to find out the exact structure of these compounds. However, authors

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 6. Improvement of antixiodant compounds extraction

of this paper recommend to use this technique in the future to evaluate the
content of phenolic compounds and antioxidant capacity of this algae as the
classical method could be greatly underestimating its content.

6.5. Conclusions

In this research work we adapted an acidic metanolysis extraction of non-

extractable polyphenols to the alga Ulva ohnoi. This extraction method showed
a higher polyphenol content than with traditional methanol acetone method. The
extraction 5 hours at 100 °C showed the highest and most reproductible result in
TPC. Gallic acid and phloroglucinol were the only molecules unequivocally
identified. Complete composition of the extract remain to be identified.

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162
7 . Dis cusió n general

163
 7. Discusión general

En este capítulo, se mencionan diferentes aspectos relacionados con la

metodología utilizada y con los resultados que pueden influir en la determinación
del potencial del polisacárido ulvan como fuente de compuestos funcionales en
acuicultura. Además, se introducen ideas y perspectivas para seguir desarrollando
e incrementando el interés por ésta molécula.

Uno de los retos a los que se enfrenta la acuicultura es poder disponer de

alimentos para peces con una formulación base a los que la incorporación de
compuestos funcionales proporcione equilibrio nutricional y salud. Dichos
aspectos son fundamentales para asegurar rentabilidad y calidad a los
productores. Una de las ideas subyacentes en esta investigación consiste en la
contribución al avance de la acuicultura y a la puesta en valor de un recurso
marino, como son las algas, para su utilización como producto funcional. La
obtención de dichos compuestos requiere de un conocimiento previo exhaustivo
acerca del origen de los mismos, garantizando una productividad óptima de los
mismos a la vez que se mantiene la estabilidad estructural.

En la presente Tesis doctoral se exponen los avances realizados en el

conocimiento de la especie de alga verde Ulva ohnoi. Ésta fue empleada en el
trabajo experimental y es importante destacar que apenas había estudios
realizados de su presencia en el litoral suratlántico español. Esta especie, es
probablemente originaria de Japón, aunque es conocida como una especie de
origen “criptogénico” [1]. Se ha encontrado de forma repetitiva en el entorno de
la Bahía de Cádiz, dónde se efectuaron los muestreos para el capítulo 3, y en la
costa onubense, de dónde procede el espécimen que se puso en cultivo para los
experimentos siguientes. Se observó una gran abundancia de esta especie en las
zonas de esteros creciendo estacionalmente de forma abundante y rápida. Hay
referencias de cultivos intensivos en el exterior de Ulva ohnoi con elevadas tasas
de crecimiento [2]. Aunque éstas fueron irregular durante los seis meses que duró
el experimento descrito por el autor, se comprobó que el rendimiento de ulvan
fue constante sin que se pudiera observar variación significativa.

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 7.Discusión general

Los polisacáridos y los polifenoles son dos tipos de compuestos de interés

procedentes de algas para su inclusión como compuestos funcionales en
acuicultura por su potencial para inmunoestimulación y como antioxidantes
respectivamente. Para optimizar el rendimiento de extracción de estos productos
y determinar su bioactividad, es preciso disponer de métodos que permitan
extraer y purificar éstos compuestos de la forma más eficiente posible. A la vez
para determinar el rendimiento, la pureza y el efecto es imprescindible adquirir
la metodología analítica la más adecuada posible. A continuación se contrastan
los resultados obtenidos en la extracción del polisacárido ulvan de Ulva ohnoi y
de sus polifenoles.

Existen numerosas publicaciones que describen la extracción y la

purificación del ulvan. La primera etapa de esta tesis fue adquirir estas técnicas
así como la metodología de determinación estructural. Este conjunto constituye
la base con la cual se ha desarrollado este trabajo. Se describen a continuación
todos los aspectos técnicos que se deben tener en cuenta para optimizar estos
procesos.
Según la literatura, el polisacárido sulfatado ulvan extraído de la macroalga
Ulva puede representar de 8% a 29% del peso seco del alga [3]. Los datos
obtenidos en nuestro experimento de ciclo anual están en el rango encontrado
en la literatura indicando que Ulva ohnoi crecida en zonas de esteros contiene
entre un 22.3% y un 28.4% de ulvan en relación a su peso en seco. A escala
industrial la estabilidad del rendimiento en la extracción del compuesto es un
factor importante ya que puede resultar en una gran variación de los costes de
producción.
La variación en el rendimiento de extracción del polisacárido puede
deberse principalmente al método de extracción, al método de purificación, a la
especie de alga o el estado fenológico del alga.
Tanto la fisiología de las algas como su ciclo reproductivo aún están lejos
de estar completamente controlados. Comprender las fases de crecimiento de las
algas con sus correspondientes requerimientos nutricionales puede ayudar a
obtener cultivos de algas más controlables. Diseñar sistemas de cultivo eficientes

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 7. Discusión general

que se adapten tanto al medio natural como a la propia alga es imprescindible

para maximizar el aprovechamiento de estos efluentes a la vez que se controla el
crecimiento.
Dentro del diseño de estos sistemas de cultivo se deberá incluir el factor
genético. Se considera necesario un mayor esfuerzo investigador para avanzar a
largo plazo para la mejora genética de las algas buscando carácteres fenotípicos
mejorados como la regularidad de crecimiento, la adaptabilidad a sistemas de
cultivo, la susceptibilidad a enfermedades y plagas, la productividad de
compuestos o la idoneidad para su consumo humano. Estos avances permitirán
optimizar el cultivo contribuyendo a mejorar la rentabilidad y diversificar los usos.
Para ello sería de interés la constitución de una colección de individuos y su
diferenciación molecular, el estudio de la variabilidad de carácteres genéticos y
el control del ciclo reproductivo [4-7].
Hasta ahora, los rendimientos de ulvan obtenidos tanto en nuestras
investigaciones como en la literatura se encontraron en un rango situado entre
el 8% y el 29%, lo que se puede considerar como rendimientos variables. En
realidad esto incluye a todos los grados de pureza y de calidad y hace difícil su
comparación. Por ello, la metodología utilizada para el proceso de extracción y
purificación es clave y se debe ajustar a las características requeridas para un uso
determinado. Es importante pues conocer los requerimientos de pureza y calidad
para cada uso con el fin de optimizar el proceso. Para la industria este concepto
tiene gran importancia ya que la variabilidad de rendimiento puede tener un
impacto importante sobre los costes de producción.
El proceso de extracción del polisacárido es el primer paso y de gran
importancia ya que puede influir en las carcteristicas del producto final. El
método empleado en el trabajo experimental de ésta tesis para efectuar la
extracción de ulvan consistió en una extracción acuosa en presencia de oxalato.
Existen otras técnicas alternativas de extracción realizadas a pH ácido con ácido
clorhídrico siendo más eficiente que la que hemos utilizado en los trabajos
realizados en la tesis. El punto en contra de esta técnica es que disminuye
drásticamente el peso molecular del ulvan [8] y nuestro objetivo era poder

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disponer de fracciones de ulvan con diferentes pesos moleculares para

determinar su funcionalidad. La técnica de extracción ácida consiste en realizar
una extracción en caliente del alga descolorida y secada en una proporción peso
por volumen de 1% en una solución de HCl al 0,05M a 85°C durante una hora. El
extracto se neutraliza con NaOH una vez la extracción terminada [9]. Se ha
demostrado que a pH cercano al pKa del ácido glucurónico (3.28) el ulvan - que
tiene una forma más o menos agregada en solución - se desagrega, lo que le
hace ser más soluble y menos propenso a la agregación [10].
Para adaptar esta metodología sería preciso tener en cuenta el uso que se
pretende hacer con el producto extraído. El objetivo es maximizar la extracción y
a la vez respetar el tamaño de la molécula el cuál es un requisito imprescindible
para el efecto inmunoestimulante. Podría ser interesante comprobar la eficacia
de ambos métodos de extracción evaluando la capacidad inmunoestimulante
que ejercen dichos extractos de ulvan en peces.
Aunque las despolimerizaciones llevadas a cabo en esta tesis obtuvieron
cadenas de azúcares con tamaños muy inferiores a lo que se obtendría con una
extracción ácida, hasta ahora nuestros resultados han demostrado que el peso
molecular del ulvan es importante para su efecto in vitro sobre macrófagos de
peces. Ni los oligosacáridos, ni las fracciones > 10 kDa - < 50 kDa (dato no
publicados) de ulvan fueron capaces de inducir estallido oxidativo intracelular en
el test del NBT.
El extracto que contiene la fracción soluble en agua si bien tiene un alto
contenido en polisacáridos también está compuesto por moléculas
contaminantes libres en el medio o ligadas al ulvan.
Existen diferentes procedimientos para separar estos diferentes
compuestos. Los compuestos co-extraídos pueden ser proteínas, sales, almidón
u otros polisacáridos. Un método que se ha ido usando en muchos de los
artículos relativos al ulvan es la precipitación del polisacárido añadiendo un gran
volumen de alcohol isopropílico a baja temperatura. Este método no resultó ser
eficiente en este trabajo obteniendo rendimientos muy débiles (dato no
mostrado). Durante la extracción acuosa es posible que algún compuesto soluble

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 7. Discusión general

en alcohol se haya extraído con el ulvan uniéndose con él de alguna forma para
hacerlo soluble en alcohol e impidiendo su precipitación de forma eficiente.
Queda indeterminada si la causa por la cual fue imposible extraer los pigmentos
fue por metodología o por peculiaridad de nuestra cepa de alga. Algunos autores
inician la extracción del polisacarido con una primera fase de decoloración del
alga extrayendo los pigmentos con etanol, para evitar impurezas, en nuestro caso
fue imposible eliminar toda la coloración del alga por lo que se decidió prescindir
de este paso y purificar después.
La metodología empleada para la purificación del ulvan fue la
ultrafiltración con membrana de corte molecular de 10 kDa. Esta técnica deja
pasar todas las moléculas inferiores al tamaño de poro reteniendo las moléculas
de tamaño superior. Esto permite retener todas las moléculas deseadas de
polisacárido eliminando sales y moléculas de tamaño pequeño pero retiene
también las moléculas no deseadas como las proteínas u otros polisacáridos de
gran tamaño. Los datos obtenidos en el experimento de campo revelan un
contenido en proteína del extracto acuoso rico en ulvan de 0.9% a 1.5% del peso
seco del alga, lo que son datos relativamente altos. Asimismo el contenido en
glucosa varió de 0.7% a 2.5% del peso del alga variando de forma estacional lo
que parece indicar la coextracción de almidón.
En experimentos ulteriores se añadieron pasos de eliminación del
glucuronano por acidificación con ácido fosfórico a pH 2 y centrifugación.
También se añadió un paso de eliminación de proteínas por desnaturalización
termal a 121 °C y centrifugado. El efecto fue una reducción de aproximadamente
un 30-40% en el contenido de ácido glucuronico al aplicar los cambios
mencionado en el protocolo de extracción aunque no se redujo el porcentaje de
proteínas del extracto. Los niveles mas bajos de glucosa, alrededor del 0.9% (peso
seco del alga), sugiere una hidrólisis del almidón durante el tratamiento termal
con subsecuente eliminación de los azucares simples por ultrafiltración
(resultados publicados en tesis de máster realizada en nuestro laboratorio). Es
importante señalar que estos resultados fueron obtenidos con un alga cultivada

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 7.Discusión general

en condiciones de nitrógeno alto lo que puede haber incidido en la variabilidad

encontrada.
Una alternativa para solucionar el problema de las impurezas - al menos
en parte- es por tratamiento enzimático de α-amilasa y proteinasa k tal como
descrito por Costa et al. [11]. El rendimiento obtenido en estos estudios fue alto
extrayendo el 88% de la ramnosa contenida en el alga y con valores de proteína
y glucosa del 1.3% y de 1.9% respectivamente. Este método contiene varios pasos
suplementarios con respecto a otros protocolos lo que le pueden hacer más
costoso pero podrían también ser de gran utilidad según el uso al que vaya
destinado el ulvan y si requiere gran pureza. En esta publicación el autor no habla
de eliminación del glucuronano en el extracto, sin embargo esto podría explicar
el nivel inusualmente alto de ácido glucurónico que obtiene. Es probable que se
pueda conseguir un ulvan libre de proteínas, de almidón y de ácido glucurónico
por combinación de una purificación enzimática con precipitación de
glucuronano.
La determinación de la estructura de los carbohidratos complejos es un
tema muy extenso que sobrepasa las fronteras de esta tesis. Existen muchas
metodologías distintas para caracterizar los diferentes aspectos de estas
moléculas.
El perfil de azucares es uno de los mayores retos técnicos ya que puede
existir una gran variabilidad de resultados entre los diferentes métodos de
análisis. Uno de los puntos críticos es la separación de los diferentes azucares en
monosacáridos. Ciertos enlaces se muestran resistentes a la hidrólisis por ácido
trifluoroacético generalmente usado para hidrolizar otros polisacáridos. Una
alternativa viable para el ulvan es la metanólisis ácida que lleva a la liberación
estable de metilglicosidos [12]. Se decidió trabajar con metanólisis ácida de ulvan
en HCl 3M en metanol durante 4 h a 100 °C que demostró ser un buen
compromiso entre liberación de monosacáridos y estabilidad de resultados. Un
estudio demuestra la eficiencia de éste método sin embargo también resaltan
que puede permanecer alguna cantidad pequeña de discáridos de ulvan (< 2%)
sin separar [11]. La aparición de nuevas enzimas capaces de degradar

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 7. Discusión general

específicamente el ulvan van a permitir una caracterización más completa y más

segura [13].
Una vez tiene lugar la degradación es necesario identificar y cuantificar los
azucares, para ello existen varios métodos cromatográficos con varios métodos
de detección. Un importante reto es encontrar estándares especialmente el ácido
idurónico que es extremadamente raro y costoso. Sin estdándar, no pudimos
identificar este azúcar – minoritario pero de gran interés - de forma segura en
cromatografía pero sí por RMN pero sin poder cuantificarlo.
La cromatografía en fase líquida es la más fácil porque no requiere ninguna
derivatización avanzada de los compuestos y las muestras gozan por lo tanto de
una muy buena estabilidad. La columna más frecuentemente encontrada en la
literatura es la de intercambio de aniones. Para ello es necesario tener un detector
adaptado como el detector de masas, un diode array detector (DAD), un de
detector de índice de refracción (IR) o un pulsed amperometric detector (PAD)
acoplados con un detector de UV.
En cromatografía en fase gaseosa es necesario operar una derivatización
sobre los monosacáridos para convertirlos en compuestos volátiles. La
derivatización por el método de los trimetilsilanos es un método fácil que permite
una detección de señal con un detector de ionización de llamas (FID) corriente.
El aspecto negativo de esta derivatización es que la disolución de los compuestos
da lugar a diferentes tautómeros que resultan en múltiples picos por cada azúcar.
En el caso de heteropolisacáridos con una gran variedad de azucares el
cromatograma se puede volver un tanto complicado [14]. El método de los
acetatos de alditol consiste en una apertura del ciclo glucídico con la ayuda de
tetrahidrato de boro en medio básico, lo que crea una cadena carbonada
polialcolisada. Estas funciones alcohol se transforman a continuación en acetatos
de alditol mediante anhídrido acético y metilimidazole. La ventaja que presenta
este método es la aparición de un único pico por azúcar sin embargo es tedioso
y requiere numerosos pasos.
En ausencia de detectores apropiados para la fase líquida optamos por
trabajar en cromatografía en fase gaseosa. Los resultados preliminares obtenidos

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 7.Discusión general

con el método de los acetatos de alditol no fueron satisfactorios probablemente

porque la hidrólisis ácida que debe acompañar este método no es capaz de
liberar todos los azúcares. En las pruebas realizadas con el método de los
trimetilsilanos los patrones mostraron una buena correlación entre el área bajo
las señales del cromatograma y la cantidad del patrón que se había usado (Figura
S ). Este método presenta la ventaja de ser relativamente fácil de ejecutar sin
embargo los compuestos derivados no permanecen estables durante mucho
tiempo a causa de su gran sensibilidad a la humedad y la temperatura siendo
necesario realizar los análisis de forma rápida después de la derivatización. El
tiempo de análisis de cada muestra en el cromatógrafo es de aproximadamente
90 minutos, la preparación de varias muestras a la vez y su disposición en el tren
portamuestras del aparto se hace muy complicado. Dependiendo de las
condiciones ambientales el tiempo que pase entre las primeras y las últimas
muestras puede ser suficiente como para que las últimas se destruyan antes de
llegar a ser inyectadas en el aparato. Este método es bueno pero no ideal y
mejorar este aspecto podría mejorar enormemente la simplicidad y la rapidez con
la que se realizan este tipo de análisis.
Aunque se han obtenido resultados muy válidos y fiables, una alternativa
para simplificar los cromatogramas y sería añadir un paso de oximación antes de
la sililación con el fin de reducir el número máximo de picos a 2 por azúcar. Es
recomendable también extremar las condiciones de estabilidad en la que se
encuentran las muestras para evitar su destrucción antes de análisis, los
trimetilsilanos son entre otras cosas muy sensibles a la humedad y la temperatura
[14].
La metodología relativa a la cuantificación de los grupamientos sulfatos es
bastante directa y fácil de realizar. A pesar de esto la interpretación de los datos
depende, como en todos los elementos cuantificados, de la cantidad de
impurezas que lleva el extracto. Por lo tanto las variaciones que se pueden
observar a veces pueden ser fruto de una variación de las impurezas más que de
una variación natural de la sulfatación. Analizando los datos obtenidos en este
trabajo, los contenidos en sulfatos de los extractos acuosos ricos en ulvan

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 7. Discusión general

presentaron una variación de entre 3% y 4% con una correlación negativa con la

glucosa de r=-0.89 (dato no mostrado) lo que puede significar la gran influencia
de una impureza como el almidón sobre el contenido aparente en sulfato. Este
hecho es un buen ejemplo de la necesidad de implementar aún más los pasos de
purificación del ulvan.
Se ha demostrado que para un análisis rápido de los mayores
constituyentes del ulvan una combinación de FT-IR con PLS 12 puede dar
resultados razonablemente fiables. Este análisis permite predecir de forma rápida
el contenido en sulfatos, ramnosa, ácido glucurónico y xilosa pero carece de
precisión en la determinación de los componentes menores como la glucosa, la
galactosa o las proteínas. Este análisis constituye una buena herramienta para
controlar la calidad del ulvan en la industria [15].

Los pesos moleculares para el ulvan que se describen en la literatura

presentan valores de entre 500 y 800 kDa con índices de dispersión relativamente
altos. Este hecho se podría explicar en parte por la mala solubilidad del ulvan, en
solución acuosa esta molécula adopta una conformación condensada que forma
agregados de partículas en forma de frambuesa. Este comportamiento de la
molécula puede ser el origen de la variabilidad de resultados así como de su alto
índice de polidispersión [16]. A pH inferior al pKa del ácido glucuronico los
agregados de partículas se disocian dando lugar a partículas libre en el medio. Es
probable que las interacciones que llevan a la agregación de las moléculas de
ulvan tengan lugar entre los ácidos carboxílicos que una vez protonados dejan
de interactuar. Sin embargo los grupos sulfatos se quedan cargados y las
moléculas se mantienen solubles en el agua.
Nuestros resultados con SLS nos han permitido encontrar un peso
molecular de 674 kDa, este método resultó ser bastante rápido y relativamente
fiable. El valor se verificó mediante SEC-MALLS dando lugar a un pico mayor de
elución a 800 kDa con un “hombro” entre 200 y 300 kDa correspondiendo a otro

12
PLS : Partial Least Square regression

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 7.Discusión general

tipo de molécula. El índice de polidispersión obtenido fue relativamente elevado

lo que indica una gran variedad de tamaños moleculares probablemente debido
a la presencia de impurezas y al efecto de agregación del ulvan en medio acuoso.
Sería preciso desarrollar un método que permite la medición del tamaño
molecular del ulvan en ambiente ácido por debajo del pka del ácido glucurónico
para minimizar el nivel de polidispersión. Mientras que en cromatografía el tipo
de columna de exclusión de tamaño puede limitar el uso de eluentes con pH
bajo, el SLS permite elegir el solvente que uno decida. El SLS es una metodología
rápida y que permite tener un valor de tamaño medio de las moléculas en el
medio sin embargo no permite distinguir entre varias poblaciones de moléculas.
Es probable que la optimización de la purificación del compuesto consiga con
ambas metodología resultados más estables y con índices de polidispersion
inferiores.

La resonancia magnética nuclear es una herramienta muy versátil que

permite realizar desde un análisis superficial de la composición sacarídica hasta
la conformación fina de amplios fragmentos de la molécula. Una de las ventajas
de esta herramienta es que es no destructiva. En esta tesis esta herramienta fue
usada a nivel relativamente básico para estudiar como las despolimerizaciones
habían podido cambiar la estructura y la conformación de la molécula. Existe en
la literatura numerosos trabajos que describen los espectros de resonancia del
protón y del carbono del ulvan entero o en forma de oligosacáridos [3, 10, 12].
Esta herramienta es especialmente útil en el desarrollo del potencial de productos
en el cual se aportan modificaciones a la molécula con el objetivo de modular
sus propiedades. Nos ha permitido caracterizar la actividad de la enzima capaz
de degradar el ulvan y conocer la diferencia estructural que el oligosacárido
obtenido por esta vía presentaba respecto a los oligosacáridos obtenidos por vía
de despolimerización química.
Los polisacáridos son moléculas que tienen un número alto de carbonos y
de hidrógeno y en los casos de los heteropolisacáridos los espectros suelen ser
especialmente complicados. Con el fin de obtener un espectro de calidad y

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 7. Discusión general

reducir el ruido provocado por las otras moléculas es importante asegurarse que
el compuesto tiene una pureza máxima.
El control de la extracción y de la purificación así como la determinación
estructural son los pasos previos e imprescindibles para estudiar la actividad
relacionada con los compuestos. Esta tesis doctoral ha permitido adquirir y afinar
éstas técnicas. También ha servido para saber dónde están las mayores
dificultades en estos procesos y conocer cuáles son los puntos que se pueden
seguir mejorando. La finalidad es poder contar con un sistema fiable y tan estable
como sea posible para llegar a tener un producto base con una pureza óptima y
constante. Esta base nos sirvió para empezar a estudiar los usos del ulvan con la
confianza de tener la menor variabilidad posible dentro de los extractos
conseguidos.

Existe actualmente interés en la obtención de inmunoestimulantes de

origen natural, no patogénico y que procedan de fuentes sostenibles. Aunque se
atribuyen numerosas propiedades a los polisacáridos sulfatados extraídos de la
macroalga Ulva, en esta tesis hemos focalizado el estudio en las propiedades
inmunoestimulantes de ulvan debido al impacto positivo que puede ocasionar
en acuicultura. En el capítulo de introducción se hizo mención a los estudios que
demostraban el poder inmunoestimulante in vitro del ulvan en macrófagos de
rodaballo Psetta máxima y en macrófagos de ratón [17,18]. Estos resultados se
han confirmado recientemente in vivo en lisas (Mugil cephalus) y en cultivos in
vitro de células intestinales de cerdo [19,20]. La acuicultura del lenguado es una
actividad reciente que suscita mucho interés a nivel comercial. Con la prohibición
del uso de antibióticos el uso de inmunoestimulantes es una medida preventiva
necesaria para evitar enfermedades y sus consecuentes pérdidas económicas.
Nuestro objetivo aquí fue testar el efecto in vitro de ulvan sobre macrófagos del
lenguado S. senegalensis e investigar formas de suministrar ulvan que pudieran
maximizar la actividad.
Algunos carbohidratos como los mananoligosacáridos son usados en su
forma de oligosacáridos para estimular el sistema inmune de peces marinos [21].

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 7.Discusión general

La particularid de los oligosacáridos con respecto a los polisacáridos de misma

composición y misma cantidad de azúcares, es que pueden aumentar el efecto
biológico por multiplicación de los sitios de interacción con moléculas dianas
[22]. Esto nos llevó a investigar el poder de los oligosacáridos de ulvan como
inmunoestimulante para peces.
Los enlaces de la molécula de ulvan presentan una gran resistencia a la
hidrólisis ácida y por ello la obtención de oligosacáridos de esta molécula se tuvo
que hacer por medios alternativos. La degradación de polisacáridos mediante
ataque por radicales libres es una opción fácil, directa y rápida sin embargo
presenta la desventaja de realizar una ruptura totalmente aleatoria del
polisacárido destruyendo gran parte de éste con un impacto sobre el rendimiento
final sin que se obtenga una patrón estructural determinado. La migración de los
oligoulvans obtenidos por degradación con radicales libres tuvieron un tamaño
molecular medio de 2.28 kDa lo que demuestra la gran eficacia de este método.
Los análisis estructurales demostraron también que la estructura principal fue
mantenida. A parte del rendimiento, este método presenta el inconveniente de
ser un método muy agresivo, los productos usados en este método como el
peróxido de hidrogeno y el ion cúprico produjeron importantes daños a las
membranas de ultrafiltración que resultaron inutilizables tan solo después de
algunas horas de filtración. Precauciones adicionales para neutralizar estos
productos son necesarias.
La degradación enzimática representa un método alternativo suave. Sin
embargo no existen enzimas comercialmente disponibles capaces de degradar el
enlace del ácido ulvanobiourónico del ulvan. Las enzimas que hasta ahora se han
descrito para romper el ulvan son la β-glucuronidasa que rompe el polisacárido
fuera de las secuencias de ácido aldobiourónicos y las endo-1-3(4) glucanasas
cuya actividad no ha sido descrita [12,23].
Para paliar la escasez de disponibilidad de enzimas específicas diseñamos
una nueva estrategia que consistió en identificar organismos que vivieran sobre
la Ulva alimentándose de ella para poder conseguir aislar bacterias de su tracto
digestivo. Así aislamos, de un anfípodo, una bacteria capaz de degradar el ulvan.

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 7. Discusión general

Se realizó el aislamiento de la bacteria y la purificación de un cocktail de enzimas

así como la subsecuente caracterización de los oligosacáridos obtenidos.
Se identificó la bacteria como perteneciente al género Alteromonas sin
embargo el estudio de la bacteria fue superficial para no desviarnos del objetivo,
el cual era diferente. El desarrollo del uso de esta bacteria es una posible vía de
exploración para aumentar el potencial del polisacárido a través de la obtención
de oligosacáridos pero también podría servir de herramienta para dislocar la
pared celular y aumentar la extracción de otros compuestos [23]. Este interés está
reflejado por un número crecientes de publicaciones y patentes sobre este tema
[24–28]. Los efectos de los oligosacáridos de la Ulva a pesar de presentar
intereses en cosmética o en agricultura [29,30] están aún por explorar. A parte
del efecto inmunoestimulante los oligoulvans tienen posibilidad de poder ser
empleados como ingredientes funcionales por sus propiedades probioticas,
antivirales, antioxidantes, antibacterianos o antiinflamatorios [23]. Estas
funcionalidades presentan un gran interés en nutraceutica pero necesitan más
investigación.
Otra forma de potenciar la actividad de un compuesto es favorecer su
absorción y aumentar su efecto mediante incorporación en nanopartículas.
Gracias a trabajos anteriores que describen una novedosa forma de encapsular
compuestos para su uso en acuicultura [31] se pudo adaptar un protocolo de
trabajo a la encapsulación del ulvan y de los diferentes oligosacáridos. Se
obtuvieron nanopartículas con características estables para todos los tipos de
molécula con tamaños de partículas de entre 200 y 300 nm. Estos resultados
demuestran la gran eficiencia del ulvan para formar enlaces iónicos con
polímeros catiónicos.
Aunque esta idea ha sido sugerida recientemente por diferentes autores
[20,32] hemos sido los primeros en conseguir, describir el uso del ulvan para
formar nanopartículas. Esto abre nuevas posibilidades para la exploración de las
especificidades y funcionalidades de estas nanopartículas, no solo en acuicultura
sino en muchas otras áreas también [33]. Notablemente el uso de compuestos

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 7.Discusión general

para hacer diana con receptores del sistema inmune - y especialmente los TLR -
es altamente demandado en la industria farmacéutica [34].

Siguiendo nuestro objetivo de promover el uso del ulvan y los productos

derivados para la inmunoestimulación de peces, se puso a punto un plan de
estudio de los diferentes compuestos en este campo. Con la finalidad de trasladar
los resultados obtenidos a estudios in vivo se ha realizado en esta tesis la fase
previa que consiste en seleccionar in vitro cuál de los compuestos o
construcciones moleculares presenta el efecto más potente.
El protocolo estándar para efectuar un cribado de la actividad
inmunoestimulante de un compuesto se divide en dos partes: la primera parte
consiste en poner en cultivo celular macrófagos extraídos del riñón cortical de
peces y la segunda en medir la producción de ROS de éstos en presencia de un
determinado componente. La segunda fase es conocida como estallido oxidativo
y el protocolo completo esta descrito por Secombes [35].
La consecución de cultivos celulares de macrófagos de lenguados, aunque
no sean estables a largo plazo, permite realizar ensayos en placas multipocillos
lo que desmultiplica el número posible de muestras por ensayo. Como
consecuencia de esto, podemos encontrar un rendimiento variable de células
entre individuos pudiendo este hecho influir sobre los resultados obtenidos
dependiendo del estado inmunológico de los peces. En defecto de la existencia
de una línea de células estables in vitro este método es recomendable dada su
versatilidad y las posibilidades que ofrece.
Un método alternativo es la inyección del inmunoestimulante por vía
intraperitoneal al pez con el objetivo de medir la respuesta de los macrófagos
por diferentes vías después de algunos días.
La detección de las especies reactivas de oxígeno producidas por las
células puede realizarse de forma intracelular o extracelular mediante la
oxidación de sondas colorimétricas (NBT y citocromo C respectivamente).
El método de producción de ROS en intracelular mediante oxidación del
NBT para formar un compuesto azul llamado formazan permite cuantificar la

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 7. Discusión general

respuesta de las células. Este ensayo se hizo con pool de células de 5 peces
diferentes. Este método nos dio resultados satisfactorios aunque la amplitud de
la respuesta obtenida por la inmunoestimulación fue variable entre placas.
Además de la posible variabilidad del estado inmunológico de los peces otros
factores entran en juego. El protocolo requiere un paso previo a la disolución del
formazan que consiste en fijar la célula con una limpieza con metanol, este paso
imprescindible tiene por efecto arrastrar la sales de formazan dando
probablemente lugar a la variabilidad entre replicados de muestras durante la
lectura en el espectrofotómetro. El formazan además es muy poco soluble y es
necesario homogeneizar fuertemente el medio para obtener una correcta lectura.
A pesar de esto se pudo observar después de vaciar las placas que algunos
pocillos presentaban residuos de formazan no disueltos. El método de medición
de los ROS extracelulares se basa en el cambio de absorbancia del citocromo C
cuando esta oxidado. Esta molécula no es liposoluble y permanece fuera de las
células. Este método presenta los mismos inconvenientes a nivel de variabilidad
de resultados. Para minimizar esta variabilidad decidimos hacer un análisis sin
juntar macrófagos de diferentes peces. Este método permite descartar un efecto
de variabilidad por inmunogenicidad por parte de células ajenas. Sin embargo
no permite realizar un gran número de pruebas ya que el número de células que
se obtiene es muy limitado y varía entre individuos.
Los datos obtenidos en estos dos tests demostraron claramente que el
ulvan tiene un efecto inmunoestimulante in vitro alcanzando los niveles de
respuesta obtenidos con el control positivo. El ulvan encapsulado provocó una
respuesta aún mayor incluso en dosis bajas lo que demuestra que esta forma de
suministro desmultiplica el efecto producido especialmente en intracelular
sugiriendo que las partículas y el ulvan entran en el citoplasma e interactúan con
receptores. No se sabe si el ulvan y las nanopartículas siguen el mismo camino
pero sería interesante conocer los receptores implicados en los dos casos. Un
estudio de expresión de genes sobre células incubadas con nanopartículas y con
moléculas de ulvan podría ser de gran interés para determinar si existe alguna
diferencia. Esto confirma el gran potencial de las nanopartículas de quitosano y

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 7.Discusión general

ulvan para hacer diana con células del sistema inmune y transportar fármacos
relacionados o vacunas. También es necesario estudiar el efecto de la molécula y
de la nanopartícula in vivo para saber si refuerzan las defensas de los peces contra
patógenos.
Los oligosacáridos no demostraron tener efecto alguno sobre la
producción de ROS libre o en nanopartículas. Sin embargo su acción antiviral [28]
y su posible efecto como agentes profilácticos antibacterianos son
funcionalidades dignas de atención para su uso como alimento funcional en
acuicultura [36].

Estos resultados en conjunto confirman el poder inmunoestimulante del

ulvan y la potenciación de su efecto a través de su inclusión en una nanopartícula.
Además de confirmar el buen potencial del ulvan como inmunoestimulante para
la acuicultura se incrementó el potencial de esta molécula lo que aumenta el
interés por ella. Abre también un horizonte con diversas líneas de investigación
para el futuro tanto para el sector de la acuicultura como para otros sectores
relacionados con la nutrición y la salud.

Los antioxidantes son aditivos alimentarios que reciben mucho interés por
sus cualidades de preservación. Por su modo de vida en ambiente salino, con
fuerte exposición al sol, al oxígeno y con un metabolismo eficiente las algas
verdes poseen un sistema antioxidante de alto rendimiento.
Al inicio de la presente tesis, la metodología empleada para la
determinación de polifenoles en algas fue la existente en la bibliografía, no
obstante ésta especie de Ulva resultó contener niveles muy bajos de polifenoles
en comparación con otras algas (Fucus sp.). Estos resultados preliminarios
coincidían con los descritos en la literatura [37–39] y parecían confirmar el bajo
contenido de la Ulva en éstos compuestos. Los polifenoles pueden estar
asociados en las paredes celulares a fibras y a proteínas e impedir su correcta
extracción al utilizar el método clásico de extracción de polifenoles [40]. En esta
tesis se ha descrito de forma exhaustiva las peculiaridades de las paredes

179
 7. Discusión general

celulares de la Ulva y por ello decidimos probar un método de extracción

diferente adaptado a las peculiaridades de esta especie. Un elemento que nos
pareció ser un indicador de la incompleta extracción de algunos elementos en
esta alga fue que no conseguimos extraer la clorofila del alga hasta hacerle
perder su coloración por completo. Esta decoloración únicamente sucedía de
forma muy llamativa cuando se realizaba la metanólisis (o transesterificación) del
alga para el análisis composicional de los azucares totales. El método de análisis
de la fracción de polifenoles no extraíbles es precisamente la metanólisis ácida
con ácido sulfúrico. Nos planteamos realizar pruebas de metanólisis ácida
consiguiendo resultados significativamente más altos en un factor de 3 a 4 veces
con respecto a la extracción tradicional con acetona o metanol. La separación de
esto compuestos en cromatografía de capa fina parece indicar que hay una gran
variedad de compuestos que participan en este efecto y sería de gran interés
poder aislarlos e identificarlos.
El método que hemos utilizado dio resultados reproducibles, sin embargo
el método de purificación es bastante largo y somete el extracto a unas
condiciones oxidantes. Esto puede ser una fuente de variación y podría dar una
subestimación del poder antioxidante. Al aplicar éste método se producen
además esteres metílicos de polifenoles lo que también podría modificar su
comportamiento. Existe un número creciente de trabajos tratando de conseguir
métodos que permitan una máxima extracción de antioxidantes [41] pero sería
adecuado utilizar métodos más suaves de degradación.
La extracción de los “polifenoles no extraíbles” se ha realizado en plantas
terrestres pero nunca en algas marinas. Esto abre la puerta a numerosas pistas de
investigación como la elucidación de la estructura de estas moléculas, sus
propiedades biológicas, su relación con los polisacáridos y su papel en la
estabilización de la pared celular. El estudio realizado en la presente tesis supone
un avance en la metodología para la extracción de compuestos antioxidantes de
la macroalga Ulva. Sin embargo, esto solo representa un primer paso de un largo
desarrollo para establecer el potencial real de estas moléculas antioxidantes y su
funcionalidad en acuicultura.

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 7.Discusión general

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188
8 . Conclusiones g enera les

189
 8. Conclusiones generales

1. En base a un análisis de marcadores genéticos, se concluye que la cepa

de Ulva recogida en zonas intermareales de la provincia de Cádiz y
Huelva que se ha puesto en cultivo en las instalaciones del IFAPA el
Toruño pertenece a la especie Ulva ohnoi M. Hirakoa & S. Shimada. /
Based on genetic markers, It is concluded that sthe strain of Ulva isolated
from the interdial zone of the provinces of Cádiz and Huelva that was
cultivated in the facilities of the IFAPA el Toruño belongs to the species
Ulva ohnoi M. Hirakoa & S. Shimada.

2. La temperatura y la luz son las principales causas de la variación en el

crecimiento y la composición en carbohidratos, lípidos y proteínas del
alga Ulva ohnoi creciendo en los esteros de la Bahía de Cádiz, mientras
que otros factores, particularmente la concentración en nitratos, también
tienen influencia. /Temperature and light are the principal variables
determining the variation in growth and biochemical composition in
carbohydrates, lipids and protein levels of the alga Ulva ohnoi growing
in the earthen ponds in the Bahía de Cádiz área, whereas other factors,
in particular nitrate concentrations also affect growth and composition.

3. La composición del extracto soluble de Ulva ohnoi procedente de esteros

presenta un alto contenido en ulvan. / The composition of the ulvan-rich
water soluble extract from Ulva ohnoi collected in saltmarshes has a high
content in ulvan.

4. Una bacteria procedente del intestino de Gammarus insensibilis y

perteneciente al género Alteromonas e identificada bajo el código
Alteromonas sp. GIIUL2 en la base de datos GenBank es capaz de
degradar un gel de ulvan. / A bacteria isolated from the gut of Gammarus
insensibilis and that was identified as belonging to the genus
Alteromonas and was identified under the code Alteromonas sp. GIIUL2
in th eGenBank database had the capacity to degrade an ulvan gel.

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 8. Conclusiones generales

5. El cóctel de enzimas aislado de la bacteria Altermonoas sp. GIIUL2 es

capaz de degradar ulvan en oligosacáridos. / An enzyme cocktail isolated
from the bacterial strain Altermonoas sp. GIIUL2 is able to degrade ulvan
to oligosaccharides.

6. Oligosacáridos obtenidos por despolimerización enzimática contienen

una extremidad no saturada y se ha podido confirmar que al menos una
de las enzimas contenida en el cóctel es una ulvan -liasa. Se describe por
primera vez una actividad de ulvan -liasa por parte de una bacteria del
género Alteromonas. / Oligosaccharides obtained by enzymatic
depolymerization contained an unsatured end where at least one of the
enzymes contained in the cocktail is an ulvan -lyase. This is the first time
that the ulvan -lyase activity is described from the genus Alteromonas.

7. El método de nanoencapsulación por gelación ionotrópica de quitosano

y tripolifosfato es adecuado para vehicular polisacáridos y/u
oligosacáridos extraídos de la macroalga Ulva ohnoi en nanopartículas.
Las nanopartículas que incluyen ulvan mediante la metodología descrita
en el presente estudio son estables, con un tamaño menor de 300 nm y
con potencial Z de +35-40mV. / The Chitosan and tripolyphosphate
Ionotropic gelation nanoencapsulation method is suitable to introduce
polysaccharides and/or oligosaccharides extract from the macroalgae
Ulva ohnoi into nanoparticles. The nanoparticles that include ulvan
through the methodology describe din this study were stable with a size
inferior to 300nm and a Z-potential of +35-40mV.

8. Los oligosacáridos obtenidos a partir de la macroalga Ulva ohnoi tanto

en forma libre como nanoencapsulados no presentan actividad
inmunoestimulante en macrófagos de Solea senegalensis. / The
oligosaccharides obtained from the macrolagae Ulva ohnoi do not
present any immunostimulatory activity under a free or

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 8. Conclusiones generales

nanoencapsulated form for the macrophages of Solea senegalensis.

9. El polisacárido sulfatado ulvan extraído del alga Ulva ohnoi bien en

forma libre o nanoencapsulado proporciona efecto inmunoestimulante
in vitro en macrófagos de Solea senegalensis. / The sulfated
polysaccharide ulvan extracted from Ulva ohnoi does trigger an
immunostimulant effect on Solea senegalensis macrophages both under
free and nanoencapsulated form.

10. La encapsulación del polisacárido ulvan en una nanopartícula de

quitosano/TPP mejora el efecto inmunoestimulante del polisacárido libre
en macrófagos de Solea senegalensis. / The chitosan/TPP nanoparticle-
encapsulated form of ulvan improves the immunostimulant effect of the
crude polysaccharide on Solea senegalensis macrophages.

11. El método de extracción de antioxidantes basado en una metanólisis

ácida con calor permite conseguir un extracto algal para Ulva ohnoi con
una actividad antioxidante cuatro veces más elevada que con la técnica
tradicional de extracción con metanol y acetona/ The antioxidant
extraction method based on hot acidic methanolysis allows for obtaining
an algal extract with a 4-fold higher antioxidant activity than with the
traditional methanol acetone extraction technique.

192
8. Co

9 . A nexos

193
 9. Anexos

Annex 1:

Identification of a species of the Ulva genus proceeding from the

saltmarshes of the Southern Atlantic Spanish Coast
The Ulva strain used in the experimental chapters of this thesis (Chapters
4 – 6) was isolated as a single thallus from a cannel named “Caño de Agua del
Pino” close to the mouth of the Rio Piedras tidal inlet in SW Spain (37°12′ 57.39″
N, 7°5′ 5.29″ E) on the 12th of August 2010. Vegetative clones of this fragment
were maintained in culture in filtered seawater enriched with f/2 medium since
then in the IFAPA centers Agua del Pino in Cartaya (Huelva, Spain) and El Toruño
in El Puerto de Santa María (Cádiz, Spain). Morphology was studied under the
light microscope. For molecular identification, sequences of the chloroplast-
encoded RuBisCo gene (rbcL) were compared with GenBankTM sequences
archived by NCBI (https://www.ncbi.nlm.nih.gov/genbank/). The same was done
for nine samples collected from different locations and in the four different
seasons in the El Toruño experimental fish farm described in the field study in
Chapter 3. Although morphological characteristics, especially of field samples,
were variable, GenBank’s BLAST search identified all samples as 100% agreement
with Ulva ohnoi sequences in the database.
Ulva ohnoi M.Hiraoka & S.Shimada is a species most likely native from
Japan, but currently shows a cosmopolitan distribution and is specifically
mentioned as an example of a ‘cryptogenic’ species [1]: “species whose alien or
native status may be difficult to demonstrate, due to the ancient character of such
introductions, and the likely high connectivity, with bidirectional exchanges,
between native and introduced populations” [2]. U. ohnoi has been found before
in Cádiz in 2005 and 2006 [1], furthermore, the species has been found in
neighbouring waters such as the Ria Formosa lagoon in South Portugal [3], and
in ship ballast water and on ship hulls in Mediterranean harbours [4, 5].

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 9. Anexos

[1] F. Mineur, C.A. Maggs, Macroalgal cryptogenesis: where are we?, Eur. J.
Phycol., 46 Suppl. 1 (2011) Abstracts of the Fifth European Phycological Congress,
Rhodes, Greece, 4-9 September 2011, 98.

[2] F. Mineur, F. Arenas, J. Assis, A.J. Davies, A.H. Engelen, F. Fernandes, E.J.
Malta, T. Thibaut, T. Van Nguyen, F. Vaz-Pinto, S. Vranken, E.A. Serrao, O. De
Clerck, European seaweeds under pressure: Consequences for communities and
ecosystem functioning, J. Sea Res., 98 (2015) 91-108.

[3] T. Alsufyani, A.H. Engelen, O.E. Diekmann, S. Kuegler, T. Wichard,

Prevalence and mechanism of polyunsaturated aldehydes production in the
green tide forming macroalgal genus Ulva (Ulvales, Chlorophyta), Chemistry and
Physics of Lipids, 183 (2014) 100-109.

[4] M.M. Flagella, M. Verlaque, A. Soria, M.C. Buia, Macroalgal survival in

ballast water tanks, Mar. Pollut. Bull., 54 (2007) 1395-1401.

[5] F. Mineur, M.P. Johnson, C.A. Maggs, H. Stegenga, Hull fouling on

commercial ships as a vector of macroalgal introduction, Mar. Biol., 151 (2007)
1299-1307.

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 9. Anexos

Figure S 1: H1-NMR spectrum of ulvan before enzymatic degradation. R: refers to rhamnose; Rα/β:
refers to the reducing end rhamnose anomers; G/G’: refers to glucuronic acid; X: refers to xylose.
Numbers refers to the proton of the corresponding carbon

Figure S 2: Area of the TLC peak 0.55 Rf of the produced oligosaccharide after 40
hours in relation to protein concentration of crude enzyme extract

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Figura S 3: Rectas de calibrado de patrones por GC por el método del trimetilsilil

197