Ascorbic acid induced degradation of beta-glucan

180 Pages·2013·6.48 MB·English

ETH Library
Hydroxyl radical mediated
degradation of cereal β-glucan
Doctoral Thesis
Author(s):
Faure, Audrey M.
Publication date:
2013
Permanent link:
https://doi.org/10.3929/ethz-a-009785350
Rights / license:
In Copyright - Non-Commercial Use Permitted
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Diss. ETH N° 21164
Hydroxyl radical mediated degradation of
cereal -glucan
β
A dissertation submitted to
ETH ZURICH
for the degree of
Doctor of Sciences
presented by
Audrey M. Faure
MSc in Biology, Chemistry and Environment,
Université de Perpignan Via Domitia
08.05.1984
citizen of France
accepted on the recommendation of
Prof. Dr. Laura Nyström, examiner
Prof. Dr. Mogens L. Andersen, co-examiner
Prof. Dr. Willem H. Koppenol, co-examiner
2013
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(Winston Churchill)

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ABSTRACT

Cereal β-(1→3),(1→4)-D-glucan can be readily subjected to oxidative cleavage in presence
of iron(II) and/or ascorbic acid (reducing agent), a denaturing process which induces
irreversible molecular weight decrease and viscosity loss. The main focus of this study was
to demonstrate the direct involvement of hydroxyl radicals in the degradation of the β-glucan
promoted by iron(II) and/or ascorbic acid and to assess the effect of various factors on the
generation of these highly reactive species. At the same time, particular attention was also
paid on elucidating the intricate mechanism behind the hydroxyl radical-mediated oxidative
cleavage of β-glucan.
The presence of ascorbic acid and iron(II) in β-glucan solutions causes the formation of a
large amount of hydroxyl radicals, which further degrades the polysaccharide, as
demonstrated by a drastic viscosity loss. The mere presence of iron(II) in β-glucan solutions
also promoted the formation of hydroxyl radicals and the related β-glucan degradation,
although to a significantly lower extent. These results demonstrated the involvement of
hydroxyl radicals in the oxidative cleavage of β-glucan induced by either iron(II) or
iron(II)/ascorbic acid. Moreover, the hydroxyl mediated-oxidative cleavage was fully inhibited
by both catalase and superoxide dismutase, which indicates that superoxide radicals and
hydrogen peroxide are intermediate species occurring during the generation of hydroxyl
radical responsible β-glucan degradation.
Thermal treatment (100° C) of a β-glucan solution containing solely iron(II) caused the same
level of hydroxyl radical mediated molecular breakdown than treatment with a reducing
agent such as ascorbic acid at 25° C. This showed that at elevated temperatures β-glucan
plays the role of a reducing agent and helps maintain the production of hydroxyl radicals.
Furthermore, at elevated temperature (85° C) β-glucan degraded in two different manners
depending on the presence or absence of oxidizing species. When treated with a hydroxyl
radical generating system, peroxyl radicals and oxidized functional groups were formed on
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the β-glucan chain which led to a quick and drastic degradation of the polysaccharide. On
the other hand, thermal treatment of the system at 85°C induced a slow hydrolysis of the
β-glucan.
Overall, the involvement of superoxide radicals, hydrogen peroxide and hydroxyl radicals in
the iron(II)-and ascorbic acid-induced degradation of β-glucan was clearly demonstrated.
Furthermore, the present study shows that the hydroxyl-mediated degradation of β-glucan
modifies its chemical structure by introducing new oxidized functional groups, which may
potentially enhance its health benefits.

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RESUME
Le β-(1→3),(1→4)-D-glucan des céréales peut subir un clivage oxydatif en présence de
fer(II) et/ou d’acide ascorbique ( agent réducteur) en solution, entrainant une diminution de
sa masse moléculaire et de la viscosité de la solution. Le but de cette étude était de
démontrer l’implication des radicaux hydroxyles dans la dégradation du β-glucan provoquée
par le fer(II) et/ou l’acide ascorbique, d’anticiper les effets de différents facteurs sur leur
génération, ainsi que d’élucider le mécanisme de clivage oxydatif du β -glucan induit par les
radicaux hydroxyle.
L’addition d’acide ascorbique et fer(II) dans une solution de β-glucan entraînait la génération
d’une grande quantité de radicaux hydroxyles et d’une rapide dégradation du
polysaccharide. Le fer(II) seul pouvait aussi induire la dégradation du β-glucan, mais de
façon moins prononcée. Ces résultats démontrent l’implication des radicaux hydroxyles dans
la dégradation de β -glucan. Cette dégradation était complètement inhibée par les enzymes
catalase et superoxyde dismutase. De ce fait, les radicaux superoxyde et le peroxyde
d'hydrogène sont des intermédiaires dans la génération des radicaux hydroxyle
responsables de la dégradation du β-glucan.
Le traitement thermal à 100°C d’une solution de β-glucan contenant du fer(II) provoquait une
formation de radicaux hydroxyles ainsi qu’une dégradation du β-glucan similaire à un
traitement avec un agent réducteur tel que l’acide ascorbique à 25° C. Ceci démontre qu’à
une température élevée, le β-glucan peut jouer le rôle d’agent réducteur. De plus, à
température élevée (85° C), le β-glucan était dégradé de deux différentes façons dépendant
de la présence ou non d’agent oxydant. Le traitement du β-glucan avec un système
générant des radicaux hydroxyles provoquait la formation de radicaux peroxydes et de
groupes fonctionnels oxydés sur sa chaîne saccharide. Ces réactions étaient en relation
directe avec la dégradation rapide et drastique du polysaccharide. D’un autre côté, un
traitement thermal à 85° C induisait seulement une lente hydrolyse du β-glucan.
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Cette étude a démontré que les radicaux superoxydes, le peroxyde d'hydrogène et les
radicaux hydroxyles étaient impliqués dans la dégradation de β-glucan provoquée par la
présence d’acide ascorbique et de fer(II). De plus, la dégradation du β-glucan induit par les
radicaux hydroxyles modifie sa structure chimique en introduisant des nouveaux groupes
fonctionnels oxydes, qui peuvent potentiellement améliorer ses effets bénéfiques sur la
santé.

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ACKNOWLEDGEMENTS

My PhD has been quite an adventure, during which I had the chance to learn a lot, not only
about research and science but also about persistence, hope, struggle, human behavior,
cultures, humility, real values and satisfaction. I definitely would not have arrived at the finish
line without the support of many people and therefore I would like to take the opportunity to
thank some of you here.
First of all, I would like to warmly thank my supervisor Laura Nyström for giving me the
opportunity to do my PhD in the Food Biochemistry Group. I am very grateful for your
constant advices, support and understanding. Your faith in my capacity really helped me to
build up confidence in my work. Thank you for creating a warm and family-like working
environment. I would also like to express my gratitude to Prof. Mogens L. Andersen, for his
valuable support and advices concerning the ESR, as well as for the fruitful collaborations.
Thanks to you, I learned a lot about ESR and I was able to enhance my knowledge. I would
like to thank Prof. Willem H. Koppenol for kindly introducing me to the inorganic chemistry
world and for the inspiring collaboration. I would like to thank Reetta Kivelä for initiating the
research on the β-glucan oxidation. Reetta, you cannot imagine how many times I went
through your thesis.
In addition I would like to thank warmly the students that I supervised A.K.A. my padawans.
First, I would like to thank Raphael who was brave enough to do both his bachelor thesis
and master thesis under my supervision. I want to thank you for trusting me twice as your
supervisor, but also for bringing a touch of masculinity and a fun atmosphere in the lab.
Thanks to Julia, who work with me on different projects and always managed to adapt really
fast. Many thanks to my demi-French student Sandrine, for her endless patience with pH
measurements and for bringing a bit of France in the lab. I want to thank Aurora for her
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