The Protective Effects of Anthocyanin-Rich Potato Meals against the Adverse Effects of ...

89 Pages·2017·1.3 MB·English

The Protective Effects of Anthocyanin-Rich Potato Meals against the
Adverse Effects of Polychlorinated Biphenyls (PCBs) in the Human
Simulated Gut Digestion Model

Mo Yu

School of Dietetics and Human Nutrition
McGill University
Montreal, Quebec, Canada

March 2017

A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Master of Science

© Mo Yu, 2017
TABLE OF CONTENTS
Title Page ................................................................................................................................... i
Table of Contents ..................................................................................................................... ii
Abstract .................................................................................................................................... iv
Résumé ..................................................................................................................................... vi
Acknowledgements ............................................................................................................... viii
Contribution of Authors ......................................................................................................... ix
List of Tables ............................................................................................................................. x
List of Figures .......................................................................................................................... xi
List of Abbreviations .............................................................................................................. xii

Chapter 1- Introduction and Objectives ................................................................................ 1
1.1 Rationale and Statement of Purpose ..................................................................... 2
1.2 Thesis Objectives ................................................................................................. 5
Chapter 2- Literature Review ................................................................................................. 6
2.1 Background of Polychlorinated biphenyls ........................................................... 7
2.2 The Adverse Effects of PCBs on Health .............................................................. 9
2.2.1 Central Nervous System ............................................................................. 9
2.2.2 Immune System ........................................................................................ 10
2.2.3 Intestinal Function .................................................................................... 11
2.2.4 Reproductive System ................................................................................ 12
2.2.5 Thyroid Function ...................................................................................... 12
2.2.6 Metabolic Syndrome, Diabetes and Cardiovascular Diseases .................. 13
2.2.7 Other Adverse Effects of PCBs ................................................................ 15
2.3 Health and Gut Microbiota ................................................................................. 15
2.4 PCBs and the Gut Microbiome .......................................................................... 16
2.5 Health Influence of Altered Gut Microbiota ...................................................... 17
2.6 Potato and Health ............................................................................................... 19
2.7 Prebiotic Effect of Dietary Polyphenols ............................................................. 20
2.8 Protective Effects of Polyphenols on PCB Toxicities ........................................ 21
2.9 Health Benefits of SCFAs .................................................................................. 22
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2.10 Effects of Polyphenols on Colonic SCFAs ...................................................... 23
2.11 Biotransformation of Anthocyanins from Fruits and Vegetables in Simulated
Human Digestion Models ..................................................................................................... 23
2.12 Effect of Digestion of Polyphenols on Antioxidant Capacity .......................... 25
Chapter 3- Manuscript .......................................................................................................... 26
3.1 Introduction ........................................................................................................ 28
3.2 Materials and Methods ....................................................................................... 29
3.2.1 Chemicals .................................................................................................. 29
3.2.2 Sample Preparation ................................................................................... 29
3.2.3 In-vitro Enzymatic Digestion .................................................................... 30
3.2.4 Fecal Sample Preparation ......................................................................... 30
3.2.5 Batch-culture Fermentation ...................................................................... 31
3.2.6 Lactobacillus Enumeration ....................................................................... 31
3.2.7 Ferric-reducing Antioxidant Power (FRAP) Assay .................................. 32
3.2.8 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay ......................................... 32
3.2.9 SCFA Analysis.......................................................................................... 32
3.2.10 Solid Phase Extraction Procedure ........................................................... 33
3.2.11 LC-ESI-TOF-MS Analysis ..................................................................... 33
3.2.12 Statistical Analysis .................................................................................. 34
3.3 Results ................................................................................................................ 34
3.3.1 Lactobacillus Populations ......................................................................... 34
3.3.2 Total Antioxidant Capacity by the FRAP Assay ...................................... 34
3.3.3 Total Antioxidant Capacity by the DPPH Assay ...................................... 35
3.3.4 Analysis of SCFAs using Gas Chromatography ....................................... 35
3.3.5 Anthocyanins in Digested ALL Blue Potatoes ......................................... 35
3.4 Discussion .......................................................................................................... 36
Chapter 4- Final Conclusion ................................................................................................. 49
4.1 General Summary and Conclusions ................................................................... 50

References ............................................................................................................................... 53

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ABSTRACT

Background: Polychlorinated biphenyls (PCBs) are persistent environmental organic
pollutants that have been widely used by various industries since 1929. Although the
production of PCBs was banned in the late 1970s, they are still found in significant
concentrations in the environment due to their slow degradation. PCBs have low acute toxicity
but they are of public health concern due to their persistence in the environment and
bioaccumulation in human tissues, which has been associated with chronic or delayed toxicities
in multiple organs and tissues. The toxicities of PCBs are related to their chlorination pattern.
Highly chlorinated PCB 126 and PCB 153 congeners are more toxic than other PCB congeners
and have a greater bioaccumulation in human tissues. More recently, PCB exposure has been
associated with metabolic disorders such as type 2 diabetes, cardiovascular disorders and
obesity. Altered gut microbiota have been implicated in metabolic diseases and PCB exposure
has been linked with dysbiosis in mouse models including decreased numbers of colony
forming units (cfu) of Lactobacillus spp. Purple-fleshed potatoes are rich in anthocyanins,
which have prebiotic effects and can promote gut health-promoting short chain fatty acids
(SCFA) and antioxidant capacity. It is therefore possible that intake of anthocyanin-rich purple-
fleshed potatoes such as the cultivar All Blue may protect against adverse effects of PCBs on
gut microflora. Conversely, gut microbial PCB exposure could inhibit these prebiotic effects
of anthocyanins; depressing gut microbial metabolism of anthocyanins and resulting in reduced
SCFA generation and antioxidant capacity.

Overall objectives: The objectives were to use a human simulated gut digestion model to
study whether: i) digestion of a meal of anthocyanin-rich purple-fleshed potatoes can generate
microbial anthocyanin metabolites with associated increase in antioxidant capacity, SCFAs,
and Lactobacillus spp. cfu; ii) PCB exposure can inhibit the beneficial effects of anthocyanin-
rich meals on colonic Lactobacillus spp. cfu, SCFAs, antioxidant capacity, and microbial
anthocyanin metabolites; and iii) the potential prebiotic effects of purple-fleshed potatoes can
protect against possible adverse effects of PCBs on colonic Lactobacilli spp. cfu.

Methods: A total of four colonic vessel reactors treated with different substrates were exposed
to in-vitro enzyme digestion and a 48-h batch-culture fermentation that included: (a) control
(no treatment), (b) a mixture of PCB 126 and PCB 153 congeners, (c) a portion of cooked All-
Blue potatoes (ABP); and (d) a combination of ABP and the PCB mixture. Samples were
collected at four time points (0, 12, 24, 48 h) during the batch fermentation for Lactobacillus
spp. cfu enumeration, assessment of total antioxidant capacity (ferric-reducing antioxidant
power; FRAP) assay and 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) assay, and SCFA
analysis using gas chromatography. Samples from the ABP-containing vessels were collected
for anthocyanin metabolite profile analysis by liquid chromatography-mass spectrometry (LC-
MS).
Results: PCB exposure (b) led to a significantly (P<0.05) lower number of Lactobacillus cfu
counts compared with control (a). Treatment with ABP (c) significantly (P<0.05) increased
Lactobacillus cfu, antioxidant capacity, and SCFA levels. Combined APB and PCB (d)
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suggested that Lactobacillus were protected since cfu were unaffected. Additionally,
generation of anthocyanin metabolites, SCFAs, and antioxidant capacity were unaffected when
PCBs were fed along with ABP and in the same meal.

Significance: Intake of purple fleshed anthocyanin-rich potato meals may: (a) enhance gut
antioxidant capacity and SCFAs, which can promote gut health; and (b) protect against the
adverse effects of dietary PCBs on human gut microflora. On the other hand, dietary PCB
exposure does not seem to adversely affect the gut health promoting effects of anthocyanin-
rich meals.

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RÉSUMÉ

Contexte: Les biphényles polychlorés (PCB) sont des polluants organiques environnementaux
persistants largement utilisés par diverses industries depuis 1929. Bien que la production de
PCB ait été interdite à la fin des années 1970, elles se retrouvent encore dans des concentrations
importantes dans l'environnement en raison de leur lente dégradation. Les PCB ont une faible
toxicité aiguë, mais ils sont préoccupants pour la santé publique en raison de leur persistance
dans l'environnement et de leur bioaccumulation dans les tissus humains, qui a été associée à
des toxicités chroniques ou retardées chez de multiples organes et tissus. Les toxicités des BPC
sont liées à leur taux de chloration. Les congénères hautement chlorés contenant des PCB 126
et PCB 153 sont plus toxiques que d'autres congénères de PCB et ont une plus grande
bioaccumulation dans les tissus humains. Plus récemment, l'exposition aux PCB a été associée
à des troubles métaboliques tels que le diabète de type 2, les troubles cardiovasculaires et
l'obésité. Le microbiota de l'intestin modifié a été impliqué dans les maladies métaboliques et
l'exposition aux PCB a été liée à la dysbiose chez les modèles de souris, y compris un nombre
réduit d'unités formant des colonies (cfu) de Lactobacillus spp. Les pommes de terre à tête vive
sont riches en anthocyanines, qui ont des effets prébiotiques et peuvent favoriser les acides gras
à chaîne courte favorisant la santé intestinale (SCFA) et la capacité antioxydante. Il est donc
possible que l'apport de pommes de terre à chair violet riches en anthocyanèse, telles que le
cultivar All Blue, puisse protéger contre les effets néfastes des PCB sur la microflore intestinale.
À l'inverse, l'exposition intestinale microbienne aux PCB pourrait inhiber ces effets pré
biotiques des anthocyanines; Déprime le métabolisme microbien intestinal des anthocyanines
et entraîne une génération réduite de SCFA et une capacité antioxydante.

Objectifs généraux: les objectifs étaient d'utiliser un modèle de digestion intestinale simulée
pour
Étudier si: i) la digestion d'un repas de pommes de terre à chair violet riches en anthocyanèse
peut générer des métabolites anthocyaniques microbiens avec une augmentation associée de la
capacité antioxydante, des SCFA et de Lactobacillus spp. Cfu; Ii) L'exposition aux PCB peut
inhiber les effets bénéfiques des repas riches en anthocyanines sur le Lactobacillus spp colique.
Cfu, SCFA, capacité antioxydante et métabolites anthocyaniques microbiens; Et iii) les effets
prébiotiques potentiels des pommes de terre à chair violet peuvent se protéger des effets
néfastes potentiels des PCB sur le Lactobacilli spp colique. Cfu.

Méthodes: Au total, quatre réacteurs de navires coliques traités avec des substrats différents
ont été exposés à une digestion enzymatique in vitro et à une fermentation de culture
discontinue de 48 h qui comprenait: (a) le contrôle (sans traitement), (b) un mélange de PCB
126 Et PCB 153 congénères, (c) une partie des pommes de terre All-Blue cuites (ABP); Et (d)
une combinaison d'ABP et du mélange de PCB. Les échantillons ont été prélevés à quatre temps
(0, 12, 24, 48 h) lors de la fermentation discontinue pour Lactobacillus spp. L'énumération cfu,
l'évaluation de la capacité antioxydante totale (dosage de l'antioxydant réducteur de ferrique,
FRAP) et le dosage de radicaux libres de 2,2-diphényl-1-picrylhydrazyle (DPPH) et l'analyse
SCFA en utilisant une chromatographie en phase gazeuse. Des échantillons provenant des
vaisseaux contenant de l'ABP ont été collectés pour l'analyse du profil métabolique anthocyané
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par chromatographie liquide-spectrométrie de masse (LC-MS).

Résultats: l'exposition aux PCB (b) a conduit à un nombre significatif (P <0,05) de
Lactobacillus cfu comparé au contrôle (a). Le traitement par l'ABP (c) a significativement
augmenté (P <0,05) l'augmentation du Lactobacillus cfu, de la capacité antioxydante et des
niveaux de SCFA. L'APB combiné et le PCB (d) ont suggéré que les Lactobacillus étaient
protégés puisque l'UFC n'était pas affectée. En outre, la génération de métabolites
anthocyaniques, de SCFA et de capacité antioxydante n'a pas été affectée lorsque les PCB ont
été nourris avec de l'ABP et dans le même repas.

Importance: L'apport de plombs riches en anthocyanines à la vapeur peut: (a) améliorer la
capacité antioxydante intestinale et les SCFA, ce qui peut favoriser la santé intestinale; Et (b)
se protéger contre les effets néfastes des PCB alimentaires sur la microflore intestinale humaine.
D'autre part, l'exposition alimentaire aux PCB ne semble pas affecter négativement les effets
favorables à la santé intestinale des repas riches en anthocyanés.

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ACKNOWLEDGEMENTS
Two years ago, I was struggling to apply for my master degree program. In order to obtain a
thesis-based position in nutrition, I sent more than 100 emails to inquire about the chances. At
my most difficult time, I was so lucky to be accepted by Dr. Kubow as one of his students. He
gave me a valuable chance to start my new career in nutritional research. I would like to thank
Dr. Kubow for all his effort and being such a good role model, giving me new ideas through
the whole research process and teaching me how to think differently. I wish to thank my
committee members Dr. Stephane Bayen, Dr. Danielle Donnelly and Dr. Jeff Xia for their
helpful instructions and advice.

I would also like to thank Hamid Abdolmalek for his support for building the batch-culture
fermentation model in our laboratory. He has been working on the model since August 2015
almost every single day. Hamid, the whole experiment cannot be started without your help and
I want to thank you for your huge contribution to the model, especially at the troubleshooting
time.

A special thank-you to my colleague Salam Habib. She truly showed me that a student
majoring nutrition can be all-around. As a PhD human nutrition, she is not only good at
nutrition, but also good at electronic engineering and computer science. Salam, I want to thank
you for helping me with my research preparation, especially the installation of electric circuit
and programming for the batch-culture fermentation model.

I want to thank Dr. Kebba Sebally for teaching me a lot of analytical chemistry skills for
analyzing my samples and methods for processing LC-MS data. I see Kebba as my mentor and
my brother who is always willing to help me under any circumstances. I wish to thank Dr.
Michele Iskandar for contributing her ideas on my protocols and helping me with my
calculation problems and also Behnam Azadi for showing me the plating skill for microbiology
parts in my research.

My father, Dr. Bin Yu, I truly appreciate for your understanding and both financial and
mental support through my master study at McGill. You always want me to believe knowledge
is the power that can change my life. You gave me a lot encouragement and taught me how to
stand up as a man when in difficulties. My mother long way from China was always concerned
about the health condition and could not hold back her tears when making distant calls to me.
Her care truly motivated me and pushed me forward. The periods I spent in Montreal were
mixed with delight and sorrow. I was happy to study knowledge from different aspects and
work with a great research team. I waited too long for my research to proceed due to some
objective reasons and delayed my graduation and I lost the one I loved. No matter what
happened to me, I always believed in a Chinese proverb: When God is about to place a great
responsibility on a great man, it always first frustrates his spirit and will, exhausts his muscles
and bones, exposes him to starvation and poverty, harasses him by troubles and setbacks so as
to stimulate his spirit, toughen his nature and enhance his abilities.

My new life starts from here and I am on my way to chase my dreams
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CONTRIBUTION OF AUTHORS

Mo Yu (Candidate) was mainly responsible for building batch-culture fermentation model,
developing protocols, conducting experiments (batch-culture fermentation, Lactobacillus
counting by plating method, FRAP and DPPH assays, quantification of short chain fatty acids
by GC, preparation of samples for LC-MS injection) and analyzing experimental data. The
candidate wrote thesis and prepared all the figures and tables.

Dr. Stan Kubow (Supervisor of Candidate, Associate Professor, School of Dietetics and
Human Nutrition, McGill University): Provided the research direction, guidance and
feedback for this study and edited this thesis.

Dr. Danielle Donnelly (Committee member; Department of Plant Science, McGill
University): Provided ideas for potato sample selection and thesis presentation and helped edit
the thesis.

Dr. Stéphane Bayen (Committee member; Department of Food Science and Agricultural
Chemistry, McGill University): Guided PCBs calculation, study design and thesis
presentation and helped edit the thesis

Dr. Jeff Xia (Committee member; Institute of Parasitology, McGill University): Provided
ideas for data analysis and thesis presentation and helped edit the thesis.

Dr. Michele Iskandar (Postdoctoral Fellow; School of Dietetics and Human Nutrition,
McGill University): Guided protocol development, study design and reactor volume
calculation.

Dr. Kebba Sabally (Research Associate; School of Dietetics and Human Nutrition, McGill
University): Provided training with FRAP, DPPH assays, GC and LC-MS.

Mr. Behnam Azadi (Research Assistant; School of Dietetics and Human Nutrition, McGill
University): Provided training with Lactabacillus plating.

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LIST OF TABLES

Table 3.1 Log number of bacteria per 1 ml of medium with treatment of DMSO (control),
10
PCBs, All Blue potatoes (ABP), and mixtures of All Blue potatoes (ABP) and PCBs ........... 41

Table 3.2 Proportion of each SCFA with treatment of DMSO (control), PCBs, All Blue
potatoes (ABP), and mixtures of All Blue potatoes (ABP) and PCBs .................................... 42

Table 3.3 Proposed identification of anthocyanin peaks in vessel with All Blue potatoes and
mixtures of All Blue potatoes (ABP) and PCBs ...................................................................... 43

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