Saxby Hawii 0085A 10844

THE POTENTIAL OF TARO (COLOCASIA ESCULENTA) AS A DIETARY
PREBIOTIC SOURCE FOR THE PREVENTION OF

COLORECTAL CANCER
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE

UNIVERSITY OF HAWAIʻI AT MĀNOA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
NUTRITIONAL SCIENCES
DECEMBER 2020
By
Solange Majewska Saxby
Dissertation Committee:
Yong Li, Chairperson

Carol J Boushey
Rachel Novotny
Marie Kainoa Fialkowski Revilla
Chin Nyean Lee (University Representative)
Keywords: Taro, Colocasia esculenta, probiotic, prebiotic, gut microbiota, colorectal cancer
DEDICATION
I dedicate this dissertation to my husband, Nathan; my parents, Wojciech and Xiomara Majewski;
my sister, Toto; my parent-in-laws, Kevin and Mei Saxby, and loyal dog, Kuma, for their constant
support and love.
I
ACKNOWLEDGMENTS
Thank you to Jari S.K. Sugimoto for helping collect taro samples from Waimānalo Research Station;
Jessie Kai and Nathan Saxby for the additional set of hands during the in vitro fecal fermentation
assay;
Dr. Jia Wei for running short-chain fatty acid analysis through the Metabolomics Shared Resource
gas chromatographer;
Dr. Kiana Frank for helping guide the gut microbial analysis;
my dissertation committee: Dr. Marie Kainoa Fialkowski Revilla, Dr. Rachel Novotny, and Dr. Chin
Nyean Lee for your advice, critique and sharing your expertise, knowledge, and time;
Dr. Carol J Boushey for your mentorship during my time as a GA in the UH Cancer Center
Multiethnic Cohort;
Dr. Yong Li, my advisor, without your time, guidance, mentorship, support, and friendship, this
dissertation would not be possible.
II
ABSTRACT
Taro (Colocasia esculenta) is a high dietary fiber tuber that holds great cultural and agricultural
importance in the Pacific. Dietary fiber is the portion of food that is indigestible by the human
gastrointestinal tract. Some dietary fibers are prebiotics since they can promote the growth of probiotic
bacteria in the gut and their production of healthful short-chain fatty acids (SCFA). Maintaining a
homeostatic gut microbiota through dietary modifications with the inclusion of high fiber foods has
been shown to reduce the risk of colorectal cancer (CRC). CRC development is highly influenced by
diet, with high fiber diets showing preventative properties. Thus, consumption of taro could
potentially promote healthy gut microbiota and SCFA production and reduce CRC risk.
This dissertation aimed to investigate the potential of taro as a prebiotic and explore its
preventative characteristics against CRC through biochemical and epidemiological means. Through
the biochemical methodology, five taro varieties were analyzed for the following objectives: 1)
Determine the nutrient, physicochemical, and functional properties of taro varieties; 2) Determine the
prebiotic fiber contents of taro varieties and their prebiotic activity scores after they were digested and
absorbed in vitro; and 3) Understand the microbial changes that occur in the gut microbiome due to
the presence of taro via in vitro fecal fermentation. Through the epidemiological methodology, the
inclusion of taro as a high dietary fiber source was explored for the following objectives: 4) Determine
the influence of taro on the risk of CRC through the analysis of the Multi Ethnic Cohort (MEC) Study
and 5) Determine the association of dietary patterns, that include taro and taro products in the food
groups, with CRC risk, using the MEC data.
The outcomes of this dissertation contribute to increased knowledge of the biochemical and
epidemiological aspects of taro’s beneficial properties for CRC prevention. Evidence of the nutrient
composition and dietary prebiotic properties of taro, and its association with the activity of gut
microbiota and the risk of CRC may help formulate effective prevention strategies for CRC.
III
TABLE OF CONTENTS
CHAPTER 1 DISSERTATION OVERVIEW ..................................................................... 1

1.1 INTRODUCTION ............................................................................................................................................ 1
1.1.1 History of Taro (Colocasia esculenta).............................................................................. 1
1.1.2 Food Processing .......................................................................................................... 2
1.2 PROBLEM STATEMENT .............................................................................................................................. 2
1.2.1 Rationals and Significance ............................................................................................ 4
1.3 OBJECTIVES...................................................................................................................................................... 4
CHAPTER 2 LITERATURE REVIEW ............................................................................... 7
2.1 INTRODUCTION ............................................................................................................................................ 7
2.1.1 Taro (Colocasia esculenta) ............................................................................................ 7
2.1.2 Functional Foods........................................................................................................ 7
2.1.3 Taro’s Medicinal Use .................................................................................................. 7
2.1.4 Colorectal Cancer ....................................................................................................... 8
2.2 OBJECTIVES...................................................................................................................................................... 8
2.3 BIOACTIVE AND NUTRIENT COMPONENTS OF TARO ........................................................... 8
2.3.1 Minerals ................................................................................................................... 8
2.3.2 Fat .......................................................................................................................... 9
2.3.3 Carbohydrates.......................................................................................................... 10
2.3.4 Probiotics ................................................................................................................ 12
2.3.5 Prebiotics ................................................................................................................ 13
2.3.6 Amino Acids and Proteins ......................................................................................... 14
2.3.7 Phytochemicals ......................................................................................................... 16
2.4 TARO NUTRIENT ABSORPTION .......................................................................................................... 17
2.5 CANCER PREVENTION OF TARO ....................................................................................................... 17
2.6 OTHER HEALTH BENEFITS ................................................................................................................... 18
2.6.1 Food Allergies ......................................................................................................... 18
2.6.2 Complementary Food................................................................................................. 19
2.6.3 Tooth Decay ............................................................................................................ 19
2.6.4 Wound Healing ....................................................................................................... 20
2.7 CONCLUSIONS .............................................................................................................................................. 20
CHAPTER 3 NUTRITIONAL, PHYSICOCHEMICAL AND FUNCTIONAL
PROPERTIES OF FIVE VARIETIES OF TARO (COLOCASIA ESCULENTA) ............. 22
3.1 ABSTRACT........................................................................................................................................................ 22
3.2 INTRODUCTION .......................................................................................................................................... 23
3.3 MATERIALS & METHODS ........................................................................................................................ 24
3.3.1 Taro Processing ........................................................................................................ 24
3.3.2 Nutrient Analysis .................................................................................................... 24
3.3.3 Physicochemical Analysis ........................................................................................... 24
3.3.4 Functional Properties Analysis .................................................................................... 26
3.3.5 Statistical Analysis ................................................................................................... 27
3.4 RESULTS............................................................................................................................................................ 27
IV
3.4.1 Nutrient Composition ............................................................................................... 27
3.4.2 Physicochemical Properties .......................................................................................... 27
3.4.3 Functional Properties of Taro ...................................................................................... 28
3.5 DISCUSSION.................................................................................................................................................... 28
3.5.1 Nutritional Properties ............................................................................................... 29
3.5.2 Physicochemical Properties .......................................................................................... 29
3.5.3 Functional Properties................................................................................................. 31
3.6 CONCLUSION ................................................................................................................................................ 33
CHAPTER 4 PREBIOTIC ACTIVITY SCORES OF TARO (COLOCASIA
ESCULENTA) WITH DIFFERENT LACTOBACILLUS SPECIES ................................... 47
4.1 ABSTRACT........................................................................................................................................................ 47
4.2 INTRODUCTION .......................................................................................................................................... 48
4.3 METHODOLOGY ......................................................................................................................................... 49
4.3.1 Taro Sample Preparation ........................................................................................... 49
4.3.2 Total Dietary Fiber, Resistant Starch (RS), and Non-Resistant Starch .............................. 49
4.3.3 In Vitro Human Digestion ........................................................................................ 49
4.3.4 Bacterial Strains ...................................................................................................... 50
4.3.5 Prebiotic Activity Assay ............................................................................................ 50
4.4 RESULTS............................................................................................................................................................ 51
4.4.1 Dietary Fiber, Resistant Starch and Non-resistant Starch ................................................ 51
4.4.2 Percent Recovery ....................................................................................................... 51
4.4.3 Growth of Lactobacilli and E. coli on Different Carbohydrates .......................................... 51
4.4.4 Prebiotic Activity Score .............................................................................................. 52
4.4.5 Correlation Between Dietary Fiber Components and Prebiotic Activity Scores with Tested
Lactobacillus Species.................................................................................................................... 52
4.5 DISCUSSION.................................................................................................................................................... 52
4.6 CONCLUSION ................................................................................................................................................ 54
CHAPTER 5 IN VITRO FECAL FERMENTATION OF TARO (COLOCASIA
ESCULENTA) ON THE MODULATION OF GUT MICROBIOTA COMPOSITION
AND SHORT-CHAIN FATTY ACIDS PRODUCTION ....................................................... 62
5.1 ABSTRACT........................................................................................................................................................ 62
5.2 INTRODUCTION .......................................................................................................................................... 63
5.3 METHODS ........................................................................................................................................................ 64
5.3.1 Fecal Collection ........................................................................................................ 64
5.3.2 Fecal Fermentation ................................................................................................... 65
5.3.3 Short-Chain Fatty Acids Analysis .............................................................................. 65
5.3.4 Gut Microbiome ....................................................................................................... 65
5.3.5 16S rRNA Sequencing ............................................................................................. 66
5.4 RESULTS............................................................................................................................................................ 67
5.4.1 Gas Production ........................................................................................................ 67
5.4.2 pH Changes ............................................................................................................ 67
5.4.3 Short Chain Fatty Acids (SCFA) .............................................................................. 67
V
5.4.4 Gut Microbial Profile ................................................................................................ 67
5.5 DISCUSSION.................................................................................................................................................... 69
5.5.1 Limitations ............................................................................................................. 73
5.6 CONCLUSION ................................................................................................................................................ 74
5.7 SUPPLEMENT MATERIAL........................................................................................................................ 84
CHAPTER 6 INTAKE OF TARO (COLOCASIA ESCULENTA) AND RISK OF CRC:
THE MULTIETHNIC COHORT ........................................................................................... 87
6.1 ABSTRACT........................................................................................................................................................ 87
6.2 INTRODUCTION .......................................................................................................................................... 88
6.3 METHODS ........................................................................................................................................................ 88
6.3.1 Study Population ...................................................................................................... 88
6.3.2 Questionnaire and follow-up data................................................................................. 89
6.3.3 Nutritional Data ..................................................................................................... 89
6.3.4 Colorectal Cancer Cases ............................................................................................. 89
6.4 RESULTS............................................................................................................................................................ 90
6.5 DISCUSSION.................................................................................................................................................... 91
6.6 CONCLUSION ................................................................................................................................................ 92
CHAPTER 7 DIETARY PATTERNS ASSOCIATED WITH GUT MICROBIAL
HEALTH AND RISK OF COLORECTAL CANCER: THE MULTIETHNIC COHORT
STUDY…………………………………………………………………………………………..99
7.1 ABSTRACT........................................................................................................................................................ 99
7.2 INTRODUCTION ........................................................................................................................................ 100
7.3 METHODS ...................................................................................................................................................... 101
7.3.1 Study Population .................................................................................................... 101
7.3.2 Colorectal Cancer Cases ........................................................................................... 101
7.3.3 Dietary Assessment and Food Groupings .................................................................... 101
7.3.4 Statistical Analysis ................................................................................................. 102
7.4 RESULTS.......................................................................................................................................................... 103
7.5 DISCUSSION.................................................................................................................................................. 104
7.5.1 Dietary Pattern 1 ................................................................................................... 105
7.5.2 Dietary Pattern 2 ................................................................................................... 106
7.5.3 Dietary Pattern 3 ................................................................................................... 106
7.5.4 Study Strengths ...................................................................................................... 107
7.5.5 Study Weaknesses .................................................................................................. 107
7.6 CONCLUSION .............................................................................................................................................. 107
CHAPTER 8 GENERAL DISCUSSION AND FUTURE DIRECTIONS................... 117
REFERENCES ................................................................................................................................................... 121
APPENDIX .......................................................................................................................................................... 151
VI
LIST OF TABLES
Table 3.1 Chemical composition of taro corms………………………………………...….……35
Table 3.2 Total starch content of taro varieties and their starch granule size and shape…....……36
Table 3.3 Physicochemical properties of taro varieties………………………………………….37
Table 3.4 Functional properties of taro varieties………………………………………………..38
Table 3.5 Gelling properties of taro…………………………………………………………….39
Table 3.6 Correlation analysis of physicochemical and functional properties of taro varieties…..40
Table 4.1. Total Dietary Fiber, Resistant Starch, Non-resistant Starch Content of Taro varieties..56
Table 4.2. Percent Recovery of Taro After in vitro Human Digestion…………………………….57
Table 4.3. Increase in cell count (log10 cfu mL -1) of tested Lactobacillus spp. between 0 h and
24 h on various carbohydrates…………………………………………………………………..58
Table 4.4 Correlation analysis of prebiotic fiber components and prebiotic activity scores of taro
varieties with tested Lactobacillus species………………………………………………………...59
Table 5.1 Nonparametric correlation coefficients (Spearman’s rank) between combinations
of microbial taxa and SCFA………………………………………………………………….…82
Table 6.1 Baseline characteristics of the study population (n=190,985), Multiethnic Cohort, 1993-
2010………………………………………………………………………………………...…..93
Table 6.2 Taro consumption and frequency of taro intake and colorectal cancer risk in the
Multiethnic Cohort Study, 1993-2013…………………………………………………………..94
Table 6.3 Taro consumption and frequency of taro intake and colorectal cancer risk in men
and women from the Multiethnic Cohort Study, 1993-2013………………………………...…..95
Table 6.4 Taro consumption and frequency of taro intake and colorectal cancer risk in the
five ethnicities from the Multiethnic Cohort Study, 1993-2013…………………………………96
Table 6.5 Estimated dietary fiber intake from taro and incidence of CRC Multiethnic cohort,
1993-2010………………………………………………………………………………………97
Table 7.1 The twenty-two food groups derived from the food frequency questionnaire from
the Multiethnic Cohort Study used in the reduced rank regression analysis…………………...107
Table 7.2 Study participants who completed the baseline food frequency questionnaire………108
Table 7.3 Baseline characteristics of 190,985 participants by lowest and highest quintiles of
the three derived reduced rank regression dietary patterns in the Multiethnic Cohort Study…...109
Table 7.4 Derived dietary patterns from reduced rank regression and colorectal cancer risk in
the Multiethnic Cohort Study, 1993-2013…………………………………………………….110
Table 7.5. Derived dietary patterns from reduced rank regression and colorectal cancer risk in
the Multiethnic Cohort Study, 1993-2013……………………………………………………..111
Table 7.6 Derived dietary patterns and colorectal cancer risk by race/ethnicity in the
Multiethnic Cohort Study, 1993-2013………………………………………………………....112
VII
LIST OF FIGURES
Figure 1.1 Flow chart of dissertation approach…………………………………………………..5
Figure 3.1 Taro corms: Bun Long (a), Mana Ulu (b), Moi (c) Kauaʻi Lehua (d), Tahitian (e)…….41
Figure 3.2 Flow chart of taro processing………………………………………………………...42
Figure 3.3 Cross sectional view of taro corm flesh: Bun Long (a), Mana Ulu (b), Moi (c) Kauaʻi
Lehua (d), Tahitian (e)…………………………………………………………………………..43
Figure 3.4 Scanning Electron Microscopy (SEM) of starch granules from taro varieties: Bun
Long (a), Mana Ulu (b), Moi (c) Kauaʻi Lehua (d), Tahitian (e)………………………………….44
Figure 3.5 X-ray Diffraction (XRD) of taro varieties: Bun-Long, Mana Ulu, Moi, Kauaʻi Lehua,
Tahitian……………………………………………………………………………………..…...45
Figure 4.1 Prebiotic activity scores of Taro Varieties with Lactobacillus spp…………………...…..60
Figure 5.1. The gas volume of fermentation slurry over a 24-hour period in vitro fecal
fermentation………………………………………………………………………………….…74
Figure 5.2. The pH of fermentation slurry over a 24-hour period in vitro fecal fermentation……75
Figure 5.3. Total short-chain fatty acid (SCFA) concentrations at 24 hours of in vitro fecal
fermentation………………………………………………………………………………….…76
Figure 5.4 Individual short-chain fatty acid (SCFA) production at 24 hours of in vitro
fecal fermentation………………………………………………………………………………77
Figure 5.5 Heatmap of hierarchical clustering of bacterial microbiota composition
profiles represented by 16S ribosomal RNA (rRNA) amplicons per sample of ‘heavy’ and ‘
light’ gradient fractions from treatments: A) Baseline, B) Control, C) FOS, D) glucose, E)
Bun-long, F) inulin, G) Tahitian, H) Mana Ulu, I) Kauaʻi Lehua, and J) Moi………………….....78
Figure 5.6. Principal Coordinate Analysis (PcoA) of bacterial community structures
using permutational MANOVA (PERMANOVA) statistical method and unweighted
UniFrac distance………………...……………………………………………………………….79
Figure 5.7 Alpha-diversity of community ANOVA statistical method and Shannon Index……... 80
Figure 5.8 Relative abundance of phylotypes at the phylum level and at the family level
from in vitro fecal fermentation samples……………………………………………………….....81
Figure 5.9 Alpha-diversity index of bacterial community ANOVA statistical method
and (A) observed; (B) Chao1 diversity measure, p-value: 0.36535; F-value: 1.1667………..……..83
Figure 5.10 Relative abundance of phylotypes at the phylum level from in vitro fecal
fermentation samples……………………………………………………………………………84
Figure 8.1 Translation model of taro basic research, public health research, and development
of new approaches……………………………………………………………………………….120
VIII
LIST OF ABBREVIATIONS AND SYMBOLS
A1, albumin one
A2, albumin two
AI, acceptable intake
ASV, amplicon sequence variants
BD, bulk density
BSCFA, branched-chain fatty acid
Ca, calcium
CD-MGDG, γ-cyclodextrin monogalactosyl diacylglycerols
COX-1, cyclooxygenase-1
COX-2, cyclooxygenase-2
CRC, colorectal cancer
DGDG, digalactosyl diacylglycerols
DWB, dry weight basis
EA, emulsifying activity
EA, emulsifying activity
eGI, estimated glycemic index
ES, emulsifying stability
FC, foam capacity
Fe, iron
FOS, fructooligosaccharides
FS, foam stability
FUFOSE, Functional Food Science in Europe
G1, globulin one
G2, Globulin two
GH, glycoside hydrolases
GI, glycemic index
GRAS, generally recognized as safe
GS, granule size
hOSC, human lanosterol synthase
HPLC, high performance liquid chromatography
HSD, Tukey’s honestly significant difference
ISAPP, International Scientific Association for Probiotics and Prebiotics
K, potassium
LAB, lactic acid bacteria
LAK, lymphokine activated kill cells
LGC, least gelation concentration
LGG, L. rhamnosus
LPS, lipopolysaccharide
MC, moisture content
Mg, magnesium
MGDG, monogalactosyl diacylglycerols
Mn, manganese
MRS, De Man, Rogosa and Sharpe
NADPH, nicotinamide adenine dinucleotide
NRS, non-resistant starch
OAC, oil absorption capacity
IX
P, phosphorus
PGE2, prostaglandin E2
RDA, recommended daily allowance
rRNA, ribosomal RNA
RS, Resistant starch
SC, swelling capacity
SCFA, short-chin fatty acid
SEM, scanning electron microscopy
TCH, total cholesterol
TDR, total dietary fiber
TG, triglycerides
TSB, tryptic soy broth
USDA, United States Department of Agriculture
VFA, volatile fatty acids
VLDL, very-low-density lipoprotein
WAC, water absorption capacity
WAI, water absorption index
WSI, water solubility index
XOD, xanthine oxidase
XRD, x-ray diffraction
Zn, zinc
X
CHAPTER 1 DISSERTATION OVERVIEW
1.1 INTRODUCTION
1.1.1 History of Taro (Colocasia esculenta)

Taro is a root vegetable belonging to the genus Colocasia, within the sub-family Colocasioideae of
the monocotyledonous family Araceae [1]. The long history of taro’s vegetative propagation has
contributed to considerable confusion of its origins [1]. However, ethno-botanical evidence traces the
origin of taro back to South Central Asia, most likely in the India-Malaysian Peninsula [1-3]. From the
South Central Asia origin, cultivation of taro spread to the rest of the world. The westward expansion
of taro began as early as 500 B.C., and cultivation was seen in Egypt, the Arabian region and the
Mediterranean region [1, 4]. The origins of taro cultivation in the Arabian and Mediterranean regions
still remain unclear, despite the existence of many written records [5]. However, taro cultivation
continued to spread to south and West Africa [1, 4]. Western introduction of taro to the Americas and
the Caribbean is relatively recent, primarily thought to have come from Africa through the slave trade
[4, 6].
The eastward journey of taro saw similar expansion. The cultivation of taro began as early as
100 B.C. for the rest of South-East Asia, Burma, China, and Japan [1, 4]. In addition, wild forms began
growing in various parts of South-East Asia [7]. The cultivation of taro continued throughout the
Pacific Ocean, New Zealand and Polynesia [4]. Polynesian, Micronesian, and Melanesian voyagers
were bringing taro, along with other foods, in their canoes, becoming known as “canoe plants’. The
settlers across the Pacific cultivated the “canoe plants” as food staples and staffs of life [6].
Today, taro can be seen across the tropical and subtropical latitudinal band [6]. This is
reflected by various names given to taro by the various cultures cultivating it, including; kalo (Hawaiʻi),
gabi (Philippines), eddoe (Japan), arbi (India), cocoyam (Africa), and others [5]. However, the greatest
cultivation and highest contribution to the diet occurs in the Pacific Islands, especially in Hawaiʻi [6].
Early Hawaiians planted taro in marshes close to the mouths of rivers [8]. The progressive expansion
of taro led to the cultivation on slopes and along rivers in wetland farming, also known as kalo loʻi
(flooded taro patches in Hawaiian), which reached a unique level of engineering sophistication and is
still seen today [2, 9]. This great cultivation can be attributed to Native Hawaiians, growing taro as not
merely an activity of food production, but also strongly bound to their culture and beliefs [2].
In Hawaiʻi, taro is extremely important to the Native Hawaiian people, the Kānaka Maoli, who
associate it with their Gods and their story of creation [10]. In Native Hawaiian tradition,
Papahānaumoku (Earth Mother) and Wākea (Sky Father) had a daughter named Ho‘ohokukalani (the
making of the stars in heaven). Ho‘ohokukalani first pregnancy was to a baby boy named Hāloa (long
eternal breath); however, ended with a stillbirth . After burying the child, a taro plant sprouted on the
very site, which provided sustenance to the Hawaiian people [4]. In Hoʻohokukalani’s subsequent
pregnancy, she birthed a baby boy named Hāloa, in honor of his older brother, which became the first
Hawaiian man [11]. As such, taro has become a symbol of survival for the Hawaiian people [12].
Taro has become a globally significant crop and is considered an essential food crop for
millions of people worldwide [13], with evidence from the various common names and use by
different cultures.
1.1.2 Food Processing
Taro’s primary use is as a food product that is consumed primarily for its edible leaves and
corm [13, 14], though this dissertation will mainly focus on the corm when referring to taro, unless
otherwise stated. Taro has great potential to be a versatile food source that can be processed into
different consumable products. Unfortunately, taro is exposed to post-harvest losses because of the
high moisture content, sustained metabolism, and microbial attack, resulting in damages during
harvest and shortened storage time [15-17]. In addition, taro, like other plants in the Araceae family, is
considered poisonous when eaten raw because the tissues contain acrid components and calcium
oxalates [2]. Thus, converting taro to non-perishable products through food processing operations
extends the shelf life, reduces food waste, eliminates toxicity elements, and increases nutritional value
of the taro based products [15, 18, 19].
In Hawaiʻi, Native Hawaiians traditionally use taro to make poi, which is the most common
food form of taro consumed in Hawaiʻi [10]. Poi is made by steaming taro corms and then pounding
with a small amount of water into a starchy paste [10, 20]. In other cultures, various taro products are
available in the forms of taro flakes (Taiwan), frozen taro chunks (China), dried taro chips (Fiji and
Western Samoa), and frozen taro cake (Taiwan) [21]. Current products that include taro as the
ingredient include: poi, poi in the jar (baby food), dehydrated poi, deep-fried taro chips (snack), and
taro baskets (found mostly in restaurants) [21, 22]. Other products that use taro as one of the main
ingredients include: taro bread or rolls, taro pancakes, and kūlolo (a type of fudge-like dessert)
formulations [21, 22]. Nevertheless, new food products can be manufactured using taro, such as: flour,
pasta, canned products, cereal bars, and beverage powders [15, 16, 19].
In addition, taro has been utilized in non-food applications with the starch being manufactured
into biodegradable plastics [20, 22, 23]. Furthermore, it has the potential of acting as good filling agents
for biodegradable polyethylene film [20, 23]. Improvements on existing technologies can make taro
products more attractive to the consumers [21].
1.2 PROBLEM STATEMENT
Taro is a culturally important staple food of Pacific Islanders’ traditional diet. It often sits at
the core of Pacific origins, in addition to culturally significant dishes and medicinal practices. For
thousands of years, Native Hawaiians have cultivated this sacred plant and traditionally used it in
medicinal practices to treat human ailments [4]. However, colonization of the Pacific Islands has led
to a nutrition transition to a more westernized diet — high in saturated fats, cholesterol, and sugar —
that have been linked to an increase in the incidence of chronic diseases [24].
Historical evidence illustrates that prior to Western contact, Native Hawaiians had low rates
of cardiovascular disease, obesity, cancer, and other chronic illnesses [25]. This might be attributed to
their wholesome natural diet, which mainly consisted of taro, sweet potato, yams, breadfruit, seaweed,
greens (fern shoots and leaves of taro, sweet potato, and yams), fruit, fish and chicken [26]. These
foods provided a traditional diet high in dietary fibers, complex carbohydrates, and polyunsaturated
fatty acids, and low in fat and saturated fats [25]. High dietary fiber diets have been shown to remedy
the disruption of homeostasis that is caused by Western diets. Specifically, taro is a nutrient dense
food, high in carbohydrates and minerals (potassium, magnesium and calcium), making it an
exceptional dietary option [27]. In addition, the small irregularly-shaped starch granules in taro may
help increase the bioavailability of its nutrients, due to higher efficiency of digestion and absorption
[19]. Taro is high in dietary fibers, and 100 g of flesh provides an individual with 4.1 g or 11% of daily
dietary fiber [28]. Therefore, it has the potential to serve as a prebiotic food [4, 29].
2
Prebiotics are carbohydrates that are indigestible by the human digestive tract and promote
probiotic growth in the gut. The concept of prebiotics was first introduced by Glenn Gibson and
Marcel Roberfroid in 1995, and has since been defined as: “The selective stimulation of growth and/or
activity(ies) of one or a limited number of microbial genus(era)/species in the gut microbiota that
confer health benefits to the host” [30]. Prebiotics may be non-digestible dietary fibers that are found
in daily foods. A single food might contain different prebiotics that differ in their effects on the gut
microbes and gut health. However, not all dietary fibers are prebiotics. Dietary fibers require certain
characteristics and properties to be considered prebiotics. Prebiotic characteristics include improved
bowel function, removal of carcinogenic toxins, reduced risk of colon cancer, and preferential growth
of protective bacteria over pathogenic strains [31-33]. The efficacy of a prebiotic depends on its ability
to interact with different probiotic species in the gut microbiome. Thus, to improve gut health, it is
important to understand the nutritional composition and prebiotic properties of food.
Diet is important in maintaining the homeostasis of the gut microbiome. The gut microbiome
is the collective genome of microbes (composed of bacteria, bacteriophage, fungi, protozoa and
viruses) that reside in the colon, playing an important role in human health status [34]. The bacterial
constituents of a microbial population can be identified by sequencing the 16S rRNA-encoding genes
followed by comparison to known bacterial sequence databases to understand the role of the
microbiota in human homeostasis and disease pathogenesis [34]. Some established beneficial
microbiota are probiotics. Probiotics are live microorganisms that promote health by helping digest
food, producing vitamins, and outcompeting pathogens that are also present in the gut microbiota
[31, 33]. In the presence of certain prebiotics, the health benefits of probiotics may increase, as
microbes can utilize the prebiotics to produce beneficial fermentation products and increase gut
microbial diversity and gut health [33, 35]. The major fermentation products of prebiotics in the colon
by probiotic bacteria are short-chain fatty acids (SCFAs). The increased concentration of SCFAs in
the colon has been shown to have several beneficial effects, including: improved intestinal barrier
function, increased intestinal mucus synthesis, stimulated immunosuppressive cytokines, and reduced
levels of proinflammatory mediators [36, 37]. However, important SCFAs, such as butyrate,
propionate, and acetic acid, are only produced from certain prebiotics. As such, dietary factors heavily
influence the gut microbiome and human health.
Dysbiosis, the disruption of gut homeostasis, can affect the gut microbiome through altering
the composition of the gut microbiota and has been linked to the development of colorectal cancer
(CRC) [38-40]. Evidence from ecological studies, migrant studies, and secular trend studies suggests
that diet is a controllable factor influencing the development of CRC [41-45]. Western nations’
characteristic diet includes a high consumption of fat, red meat, and sugary food and drinks with low
dietary fiber intake [39, 40], which has been shown to have negative impacts on the gut microbial
composition [40, 46, 47] and may lead to CRC. High fiber diets have been shown to significantly
decrease the risk of CRC [39, 48]. Altering the development of CRC with dietary prebiotic
interventions may prove to be more beneficial in the long term. Returning back to a wholesome natural
diet, such as the traditional Native Hawaiian diet prior to Western colonization, can be a potential
CRC prevention method. As such, taro may be used as a dietary aid in preventing CRC— harkening
back to its traditional medicinal roots.
Taro has been shown to have anti-cancer and CRC prevention potential [49]. An in vitro study
showed that poi, a pounded taro corm food, induced apoptosis of colonic adenocarcinoma cells and
activated lymphocytes that can lyse cancerous cells [50]. This study suggested that poi may inhibit the
proliferation of colon cancer cells and stimulate the immune system [50]. In addition, poi has been
shown to support the growth of probiotic lactic acid bacteria (LAB), resulting in 85% of the total
microflora composition to be LAB after 24 hours [51]. This increase in probiotic bacteria is necessary
for enhanced production of beneficial bacterial fermentation products, specifically SCFAs. A study
3
looking at in vitro fermentation of tropical foods showed that SCFAs production increased as the starch
content of the tropical food samples increased and was the highest for taro [52]. Furthermore,
butyrate, a SCFA from bacterial fermentation, was shown to induce differentiation of phenotypes in
colorectal tumor cells, induce apoptosis of CRC cells, and downregulate certain CRC related genes
[31, 32, 53, 54]. As such, the high fiber content of taro may help improve gut health and reduce the
risk of CRC by promoting the growth of probiotic bacteria and the production of SCFAs.
1.2.1 Rationals and Significance

Taro has a long history in the Native Hawaiian community, tracing their genealogy to this
sacred plant. As such, taro is a crucial cultural component in the Pacific. However, colonization of the
Pacific Islands brought the introduction of Western diet — high in saturated fats, cholesterol, and
sugar [24]. Evidence exists that prior to colonization, the rates of CRC and other chronic diseases
were low [55]. However, as of 2018, CRC is the 3 rd most frequently diagnosed cancer in Hawaiʻi, with
approximately 720 newly diagnosed cases and 220 deaths each year. In Hawaiʻi, CRC is the 2nd leading
cause of cancer death in men and the 3rd leading cause of cancer death among women [56].
Unfortunately, evidence of taro’s potential health benefits is outdated or only biochemically
based. This dissertation seeks to be interdisciplinary by encompassing biochemical and
epidemiological explorations of the gut health benefits of taro. The results of this study will contribute
to increased knowledge on dietary prebiotic properties of taro. Understanding of the relationship
between diet and activity of the gut microbiota may formulate positive prevention strategies for CRC
and perhaps guide research in the direction to include taro as an effective dietary therapy.
1.3 OBJECTIVES
This study aims to investigate the prebiotic potential of common taro varieties through nutritional
composition, biochemical characterization, and epidemiological methods. Specific objectives are as
follows.
Objective 1: Determine the nutrient, physicochemical, and functional properties of taro varieties
Objective 2: Determine the prebiotic fiber components of taro and the prebiotic activity score after it
is digested and absorbed in vitro;
Objective 3: Conduct an in vitro fecal fermentation that simulates the human gut microbiome to
understand the microbial changes that occur in the gut microbiome due to the presence of taro;
Objective 4: Determine the influence of taro on the risk of colorectal cancer through the analysis of
men and women in the Multiethnic Cohort (MEC) Study;
Objective 5: Determine dietary patterns, that include taro and taro products in food groups, and their
association to colorectal cancer, using MEC data.
4
Figure 1.1 Flow Chart of Dissertation Approach
5
6
CHAPTER 2 LITERATURE REVIEW
2.1 INTRODUCTION
2.1.1 Taro (Colocasia esculenta)

Taro (Colocasia esculenta) has a long history of cultivation and cultural significance around the
world. Taro is a generic name for four related species of the family Araceae (aroids): Colocasia
esculenta (taro), Cyrtosperma chamissonis (giant swamp taro) and Xanthosoma sagittifolium, of which the
corms are eaten, and Alocasia macrorhiza (giant taro), of which the edible part is the thickened
underground stem [57]. Only the nutritional value of taro (Colocasia esculenta) corms will be discussed
in this review, unless otherwise stated.
Taro is the most widely cultivated crop in Asia, Africa, and the Pacific, as well as the Caribbean
Islands [58]. It is cultivated mainly for its tuber, an essential food for millions of people, as it is
considered the 14th most cultivated vegetable/staple around the world [59].
Taro is unique amongst root crops due to its high nutritional value. It has a broader complement
of vitamins and nutrients than other tubers [60-62]. The plant is a rich source of calcium, phosphorous,
iron, vitamin C, thiamine, riboflavin, and niacin, which are all important components of the human
diet [62]. Raw taro consumption is affected by the presence of acridity factors, specifically calcium
oxalates [2, 62]. Thus, ingestion of raw taro is toxic, causing sharp irritation and burning sensation in
the throat and mouth [2, 62]. However, acridity can be reduced through processing, such as: peeling,
grating, soaking, cooking, and fermentation [63]. Despite the acridity component, taro’s nutritional
content makes it a potential functional food.
2.1.2 Functional Foods

The most current definition of functional foods comes from the Functional Food Center,
which defines “functional foods” as: “Natural or processed foods that contain biologically active
compounds; which, in defined, effective, and non-toxic amounts, provide a clinically proven and
documented health benefit utilizing specific biomarkers for the prevention, management, or treatment
of chronic disease or its symptoms” [64, 65]. In addition, the European Union Concerted Action on
Functional Food Science in Europe (FUFOSE) definition of functional foods includes: “a functional
food must remain food and must demonstrate its effects in amounts that can normally be expected to
be consumed in the diet: it is not a pill or a capsule, but part of the normal food pattern” [66]. Thus,
taro’s nutrient dense composition makes it a potential function food, which will be discussed in further
depth.
2.1.3 Taro’s Medicinal Use

Historically, taro was not only consumed as food, but also utilized, and continues to be used,
for medicinal purposes in different cultures. In the Philippines, the Pinatubo Negritos use the binate
taro cultivar as a medicine [67]. The leaves and corms have been documented to be boiled and eaten
by women experiencing a difficult childbirth [50, 67]. In Malaysia, taro’s leaves are warmed and used
to compress a child’s head after birth if the head is too long [68]. In Chinese medicine, taro is utilized
in tonic prescriptions and used to treat gastrointestinal disorders, especially after tumor resection [1].
In India, taro corms are heated and placed on painful areas [61]. In Hawaiʻi, taro varieties with low
acridity, such as Lauloa and Haokea, are used for healing purposes [69]. Similarly, in Cuba, hospitals
have included taro into the diets of elderly patients for its high nutritional value and digestive qualities
[70]. Furthermore, certain taro varieties are used in traditional medicine to treat arterial hypertension,
liver problems, ulcers, snakebites, and rheumatism [1].
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These traditional uses of taro come from common knowledge that utilizes many parts of the
plant, corm, leaf, and petiole. Taro has been applied for several health disorders that include: diabetes
mellitus, internal hemorrhages, gastrointestinal disease, alopecia, body aches, snakebite, anemia,
inflammation, and cancer [71, 72].
2.1.4 Colorectal Cancer

Colorectal cancer (CRC) is the third most commonly diagnosed and deadly cancer in both men and
women [73]. CRC incidence is decreasing annually among older individuals benefiting from the large
scale of colonoscopy screening, in the USA. Conversely, the incidence and mortality of CRC is still
increasing in young individuals, especially in developing countries [39]. Based on analysis of
epidemiological studies around the world, an estimate of over 90% of gastric and colonic cancers can
be attributed to diet [39, 48, 74]. Altering the development of CRC with dietary interventions can
prove beneficial in the long term.
Taro has become a dietary staple and is of cultural importance in several nations around the world.
Not only is taro a dietary staple, but is also utilized for medicinal purposes. Thus, combining both
dietary and medicinal properties, taro has great potential as a functional food. Specifically, taro has
been shown to have anti-cancer and CRC preventative properties which will be outlined in this review.
Thus, taro may be used as a dietary aid in reducing risk for CRC.
2.2 OBJECTIVES
The wide spread knowledge of taro’s medicinal properties proves its great potential as a
functional food. The high nutritional content of taro is consistent with taro being promoted as a rich
dietary source of certain vitamins and minerals. In addition, taro contains several bioactive and
nutritional components that may reduce risk for CRC. Though it may not be possible to cure diseases
with medicine or food, functional foods may be serve as a source of potential relief of symptoms that
improve the quality of life [65]. Thus, this literature review aims to present bioactive and nutrient
components of taro that may contribute to its value as a functional food for the prevention of CRC.
2.3 BIOACTIVE AND NUTRIENT COMPONENTS OF TARO
2.3.1 Minerals
Taro has superior nutritional value compared to other tuber crops, such as: potato, sweet potato,
cassava, and rice [60, 61]. Studies have shown that potassium is the most abundant mineral in taro,
along with magnesium, phosphorus, and calcium [75-77].
The nutritional composition of taro corms can vary widely and is dependent on the genotype,
growing conditions, and the interaction between the genotypes and the environment [76].
Furthermore, the level of minerals, type and chemical composition of the soil, soil fertility, root-soil
interface, absorption mechanism, and translocation in the plant may also affect the nutrient
composition of taro corms [78].
Mergedus et al. [27] calculated the percentages of recommended daily intakes (RDIs) of
essential minerals, calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), phosphorus (P), and zinc
(Zn), based on an average consumption of 200 g of fresh taro corms per day in regard to children
aged 4-8 years and adult females and males of 19-50 years old. The percentages of RDIs for children
aged 4– 8 years were: 33.2–52.6% for Mg, 2.91–5.35% for Ca, 12.5-21.5% for P, 30.4–80% for Zn,
5.2–9.2% for Fe, and 66.8–120.6% for Cu. Furthermore, for males aged between 19 and 50 years,
calculated values were: below 15% for Mg and P, and below 10% for Ca and Fe [27]. In contrast, for
females aged 19 and 50 years, Mg and Fe were below 20% and 5%, respectively [27]. The values
8
calculated for Zn and Cu were notably higher and approached 35% and 46% respectively [27].
However, these values were based on the assumption of 100% absorption of individual minerals [27].
Based on the average mineral content in the central part of taro corms and the recommended daily
allowance/acceptable intake (RDA/AI) values provided by the Food and Nutrition Board of the
National Academy of Sciences, 2004, taro was found to make substantial contributions [27].
Concentrations of most minerals (P, Mg, Fe, Cu, and Zn) were found to be higher in the upper and
the central parts of a taro corm [27]. In addition, potassium was particularly accumulative in the central
part, whereas its contents in other parts were lower but not significantly different [27].
2.3.1.1 Colorectal cancer prevention of minerals from taro

So far, no known studies have shown anti-cancer potential of taro derived minerals. This may
be because specific mineral studies for cancer prevention have yet to provide credible results, as
optimal age for intervention, best dose, and duration for testing nutritional agents against cancer
prevention are largely unknown, making findings hard to interpret [79]. Furthermore, baseline
nutritional status of participants can be critical for mineral studies [79], making it also harder to
interpret intervention results. However, it is well known that essential minerals are vital for
maintenance of normal body functions, which include: maintenance of pH, osmotic pressure, muscle
contraction, transport of gasses [80]. These minerals are important components of enzymes and
hormones for normal metabolic and physiological processes [80]. Thus, taro’s high mineral content
should focus on prevention of CRC from a whole food approach, or dietary pattern, in future studies.
2.3.2 Fat
Taro has a relatively low fat composition. The fat content of raw taro was found to be 0.65%
[81], and cooked taro was reported to range from 0.3 g/50 g to 0.7g/50 g [13]. These values are lower
than those of other root and tuber crops, such as yam [82, 83] and sweet potato [84].
Fatty acid derivatives extracted from taro corm skin showed new molecules, including: a
monoglyceride, (2’S)-1-O-(9-oxo-10(E),12(E)- octadecadienoyl) glycerol, and known compounds: I-(-
)-9-hydroxydecenoic acid, I-(-)-9-hydroxydecanoic acid, (2E,4S)-4-hydroxy-2-nonenoic acid, (S)-
15,16-didehydrocoriolic acid, 1-O-(octanoyl) glycerol, 12,13-epoxyoctadec-9(Z)-enoic acid,
(9S,10E,12Z)-10,12-octadecadienoic acid methyl ester, (9S,10E,12Z)-10,12-octadecadienoic acid, and
12,13-epoxyoctadec-6(Z),9(Z)-dienoic acid [85]. Specifically, 12,13-epoxyoctadec-6(Z),9(Z)-dienoic
acid, dose-dependently inhibited melanin content with significant cell toxicity in murine melanocyte
melan-a cells and inhibited melanin biosynthesis with an effective ratio of 35.43 [86].
2.3.2.1 Colorectal cancer prevention of fat from taro

A study by Sakanoa et al. found that taro, amongst 130 vegetables tested, showed significant
inhibition on human lanosterol synthase (hOSC), a cholesterol synthesizer [87]. Taro exhibited a 55%
inhibition on hOSC at 300 mg/mL, with Aichi-wase and Yatsugashira cultivars, showing the most
potent hOSC inhibition activity [87]. Using silica gel column chromatography, ethanolic extract of
Aichi-wase was partitioned with hexane and aqueous methanol, with two major active fractions
showing inhibitory activity. Purification of the fractions by high performance liquid chromatography
(HPLC) yielded three monogalactosyl diacylglycerols (MGDG) and five digalactosyl diacylglycerols
(DGDG) as active compounds that showed 28-67% inhibitory activities at the concentration of 300
mg/mL [87]. Blocking cholesterol synthesis may prove to decrease cholesterol production, as elevated
serum cholesterol levels have been linked to a higher risk of colorectal adenoma and CRC [88].
MGDG and DGDG have a high content of polyunsaturated fatty acids, mainly omega-3s [89].
Both MGDG and DGDG have shown several biological activities including: antiviral [90], anti-
inflammatory [89], and anti-tumor and anti-proliferative activity [91-93]. MGDG possibly activates
9
the anti-inflammatory loop triggered by cyclooxygenase (COX-2) via 15ΔPGJ2 production, indicating
a possible role of COX-2 in resolution of inflammation, which was shown active in human articular
cartilage [89]. Furthermore, MGDG has shown potential to suppress the proliferation of Colon26
mouse colon cancer cells with an LD50 of 24 μg/ml in vitro [93]. Similarly, oral γ-cyclodextrin (CD)-
MGDG complex administration for the treatment of implanted solid tumors in mice reduced tumor
volume by ~60%, compared to control [93]. Furthermore, in immunohistochemical analysis, the CD-
MGDG complex group showed a decreased number of proliferating cell nuclear antigen (PCNA)-
positive cells and reduction of mitosis in the tumor cells compared with the control group [93]. In
addition, the CD-MGDG complex increased the number of terminal deoxynucleotidyl transferase
dUTP nick-end labeling (TUNEL)-positive apoptotic cells and inhibited CD31-positive tumor blood
vessel growth significantly [93]. These results provide evidence for taro’s MGDG extracts and fat
composition to be explored as potential natural products for antitumor, anti-proliferative, anti-
angiogenesis and apoptosis-inducing activity— suggesting that taro fat may have cancer-preventive
and health-promoting properties.
2.3.3 Carbohydrates
As a gluten-free food, the consumption of taro corms may be a healthier alternative
carbohydrate source, for avoiding food allergy reactions and allergy related disorders [62, 94]. Taro
carbohydrates have been reported to account for 29% on a fresh weight bases [62]. A study found
that unavailable carbohydrates in taro account for 14.7% of the dry matter, with almost 70% of the
fraction being hemicellulose, 17% pectin, and 13% cellulose [95], therefore, making taro corms a great
alternative flour source. Taro flour may be used in many food preparations, including: cookies,
noodles, paste, bread, and infant formulas [62, 94, 96]. The preparation of bread using taro flour in
combination with wheat flour was reported to have similar or better functional characteristics,
compared to breads prepared exclusively with wheat flour [96-98]. Thus, taro shows potential as an
alternative food product ingredient.
2.3.3.1 Dietary Fiber

Compared to other tubers, especially potatoes, taro provides good source of dietary fiber.
Dietary fiber is comprised principally of polysaccharides, which are subjected to varying degrees of
breakdown in transit of human digestion [99]. Compared to other tropical crops, taro reported the
highest dietary fiber content of 13.5 g/100 g for raw taro corms [100] and 3.21% for cooked taro
corms [101]. Based on the dietary fiber data of Bun-long and Lehua Hawaiian taro varieties, Huang et
al. [29] estimated the consumption of 1 lb (454 g) of taro per day by Pacific Islanders to meet the
recommended 25 g per day AI of dietary fiber.
2.3.3.1.1 Resistant Starch (RS)

Resistant starch is a form of starch that is also considered a dietary fiber because it evades
digestion in the small intestine, and enters the colon for fermentation [102, 103]. Resistant starch can
be found in foods naturally and also be formed through processing at home and/or a factory [104].
There are four types of RS: RS1, is physically inaccessible starch that is locked within the cell walls,
limiting the accessibility of digestive enzymes, such as grains, seeds, or tubers; RS 2, native granular
starch that prevents digestive enzymes from break down, such as raw potatoes, unripe bananas; RS 3,
retrograded starch, usually disrupted by heating or cooking, causing gelation, such as cooked potatoes
and corn-flakes; and RS4, chemically modified starch due to cross-linking with chemical reagents [102,
103, 105]. As a functional ingredient, resistant starch has gained increasing importance as a new source
of dietary fiber [105].
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Taro has been found to be a great source of resistant starch, having been reported as having
51.6 g/100 g [52]. One study found that taro starch, compared to several other tropical crops,
contained the highest levels of resistant starch and was found to be rapidly digested, resulting in the
highest predicted glycemic index value [52]. Srikaeo et al. [106] found that taro containing resistant
starch of 12.0 g/100 g had a low GI value. Similarly, Simsek et al. [104] were able to produce RS3 from
taro corms. The estimated glycemic index (eGI) of taro starch and taro resistant starch were 60.6 +/-
0.5 and 51.9 +/-0.9, respectively, with the decrease in eGI of taro resistant starch found to be
statistically significant (p<0.05) [104]. The lower hydrolysis rate (low eGI) of taro RS3 compared to the
taro starch makes it an alternative source for dietary fiber in product formulations that can be used
for diabetic patients and weight management [104]. Furthermore, resistant starch has been shown to
improve the functionality of food. When taro flour is added into noodles, the GI was lowered, proving
to be a potential crop to be utilized as an ingredient to improve the health of foods and food security
[106].
In the gastrointestinal tract, resistant starch is highly influential on the colonic microbiota [107,
108]. Through bacterial fermentation, colonic bacteria utilize resistant starch for the production of
secondary metabolites, specifically SCFA [109]. SCFA are saturated fatty acids that consist of one to
six carbons of which the most important are: acetate (C2), propionate (C3), and butyrate (C4).
However, the amount and type of resistant starch has dramatic effects on the composition of the
intestinal microbiota and consequently on the type and amount of SCFA produced. Thus, through
the high resistant starch content, taro has the ability to potentially mediate the gut microbiome and
gut health in humans.
2.3.3.2 Colorectal cancer prevention of carbohydrates from taro

Carbohydrates from taro have been shown to have CRC preventative properties. Park et al.
[110] extracted polysaccharides from taro corms and obtained a purified active polysaccharide
compound (Taro-4-I), with a molecular weight of 200 kDa and a polysaccharide composition of 64.4%
neutral sugars and 35.6% uronic acid [110]. Treatment of peritoneal macrophages with Taro-4-I
activated the complement system through the classical and alternative pathways, increasing the
production of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) in a dose-dependent manner,
though Il-12 production showed a maximal activity at 56 µg̸ml. In addition, splenocytes obtained from
BALB̸c mice administered Taro-4-I intravenously showed a higher toxicity to lymphoma Yac-1 cells,
compared to those obtained from untreated mice. Furthermore, administration of Taro-4-I
significantly inhibited the lung metastasis of B16-BL6 melanoma cells [110]. These data suggest
polysaccharides from taro have potential anti-cancer activity, though the exact mechanism is unknown
and merits further studies.
Dietary fiber has been found to have protective measures against CRC, though not all types
of dietary fiber are thought to be equally protective [111]. A study revealed that taro’s dietary fiber had
mutagen-biding properties, which was shown by the ability to absorb 1,8-dinitropyrene (DNP), an
environmental hydrophobic mutagen [112]. The greatest adsorption occurred with dietary fiber from
the leaf blade, followed by petiole and corm walls, although the differences were shown to not be
major [113]. Furthermore, it has been shown that dietary fiber is metabolized by gastrointestinal tract
bacteria into sodium butyrate (NaB), a known differentiation inducer, believed to increase the
expression of tumor suppression genes, such as p21, in HT29 human colonic adenocarcinoma cells,
thereby blocking cdk-cycling complexes and causing cell cycle arrest [114]. Thus, the adsorption of
carcinogenic mutagens by dietary fiber from taro may assist in protecting against the development or
progression of CRC.
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RS has been shown to stimulate the growth of specific butyric acid producing bacteria [115].
Butyrate is metabolized by colonocytes as the major source of energy for the cells [107]. Furthermore,
butyrate has been shown to induce differentiation of phenotypes in colorectal tumor cells, induce
apoptosis of CRC cells, and down regulate certain CRC related genes [31, 32, 53, 54]. It is well known
that the disruption of gut homeostasis, dysbiosis, can affect the gut microbiome through altering the
composition of the gut microbiota and has been linked to the development of CRC [38-40].
A study by Martín Bernabé et al. [52] comparing tropical starches found that taro contained a
significantly higher amount of resistant starch than the other tropical starches. Furthermore, through
in vitro fermentation, an in vitro assay that mimics the upper intestine through in vitro enzymatic
digestion methods using human feces, showed that taro corms had one of the highest production of
total SCFAs, compared to other tropical starches. Furthermore, SCFA production increased as the
resistant starch content of the samples increased, and this was especially true for taro [52]. Similarly,
Srikaeo et al. [106] showed that taro was a good source of the SCFA, specifically acetate, in an in vitro
fermentation assay. SCFAs produced from anaerobic bacterial fermentation within the colon have
been shown to be protective against colon carcinogenesis [116-118]. Thus, the high resistant starch of
taro may prove to have CRC prevention through the increased production of SCFA from gut
microbial fermentation.
In addition, taro resistant starch has been shown to have an effect on bile acids. The in vitro
binding of bile acids by taro starch and taro resistant starch relative to cholestyramine were 5.2 +/-
0.2% and 7.6 +/- 1.7%, respectively [104]. Studies have shown a significant correlation between bile
acid binding capacity and total dietary fiber, especially non-soluble dietary fiber [119, 120]. Bile acids
have been shown to increase the risk of CRC [121]; therefore, taro has potential CRC risk reduction
properties, though further studies are necessary to substantiate this evidence.
2.3.4 Probiotics
The International Scientific Association for Probiotics and Prebiotics (ISAPP) most recently
defined probiotics as: ‘live microorganisms that, when administered in adequate amounts, confer a
health benefit on the host’ [122, 123]. As such, taro has potential to be a probiotic source, as it contains
naturally occurring yeast and LAB on the surface that initiate the fermentation process without the
need of a starter culture [124, 125].
A study by Yoshioka et al. [126] looking at sick piglets fed fermented taro skins confirmed the
presence of beneficial LAB on taro skins. On the fermented taro skin, 91% of the isolated bacteria
were found to be LAB strains, with over 75% of the LAB found to be a dominant species, Leuconostoc
mesenteroides [126], with Lactococccus and Weissella genera also present on taro skins. Leuconostoc
mesenteroides [127, 128] and Lactococcus genera [129, 130] have been identified as potential probiotics,
along with select Weissella species [131, 132] having probiotic properties. As such, feeding fermented
taro skins helped the recovery of sick piglets following weaning. These results imply that taro skins
have high levels of LAB species and are an important source of probiotic species that could be
potentially used in health implications for humans as well. Further animal and human studies are
warranted to confirm the beneficial effects of taro’s naturally occurring probiotics.
One of taro’s food consumables, poi, has been identified as a potentially good source for
probiotic bacteria from the natural fermentation of taro [125]. Leaving poi at room temperature for
two days begins the fermentation process of starch to dextrin, sugars, and acids, with the help of the
LAB— giving poi a sour taste [20, 124]. During the “souring” process, the acid production has been
shown to change the pH from 6.3 to 4.5 within 24 hours and reach its lowest pH on the fourth or
fifth day of fermentation [12, 124]. The length of fermentation of poi depends on the preference of
“sour” taste flavor and method of pounding. The acid fermentation process that takes place in fresh
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poi to sour poi is similar to the fermentation of sauerkraut and yogurt, and the souring is mainly due
to the action of the LAB [124, 133].
Furthermore, an early study showed that fresh poi experimentally inoculated with pathogenic
enteric bacteria that was stored at room temperature was able to purify itself from the pathogenic
bacteria in about 3 days, which was thought to be a consequence of the fermentation process [134].
This is thought to occur because of the rapid drop in pH during the fermentation process that results
in less competition from contaminating bacteria and faster growth of L. lactis, the predominant species
in souring process and an established probiotic [124, 135, 136]. This out competition of pathogenic
bacteria characteristic is seen in probiotics. As such, taro and poi can be used as a probiotic in medical
nutrition therapy [50]; however, further research is warranted to provide substantial evidence for their
use as a probiotic and unearth additional beneficial bacteria.
2.3.4.1 Colorectal cancer prevention of probiotics from taro

An in vitro study with rats showed that poi inhibits the proliferation of colon cancer cells and
stimulates the immune system [50]. Brown et al. (2005) found that poi extract can have two distinct
inhibitory effects towards colon cancer [50]. Poi inhibited rat YYT colon cancer cells in a dose-
dependent manner [50]. In addition, it was observed that the rat YYT cells exposed to poi rounded
up and failed to thrive, which indicated cell apoptosis [50]. To exclude the possibility that poi acts as
a non-specific cytotoxic agent, poi was tested on normal splenocytes and caused enhanced
proliferation of the splenocytes [50]. Thus, these data suggest that an agent within the poi extract can
activate lymphocytes and selectively inhibit the growth of specific cells.
Other studies suggest that poi has an endogenous mitogen, that has been shown to have a
mannose-binding lectin receptor that activates lymphocytes [49, 137]. Lectins induce lymphocyte
proliferation by the production of interleukin-2 (IL-2). High doses of IL-2, when incubated with
lymphocytes for 1-2 days, induce non-specific tumoricidal activity called lymphokine that activated kill
cells (LAK). LAK cells and mitogen activated kill cells eradicate multiple types of cancer cells,
including colon cancer [138]. Thus, poi could induce LAK cells to start to form within the colon to
prevent the development of CRC polyps.
2.3.5 Prebiotics
Prebiotics also belong to other categories of “functional food ingredients”. According to the
ISAPP the current definition of “dietary prebiotics” is: “a selectively fermented ingredient that results
in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus
conferring benefit(s) upon host health” [139]. In addition, criteria used to classify prebiotics include:
1) resistance to acidic pH of stomach, hydrolyzation by mammalian enzymes, and absorption, 2)
fermentation by intestinal bacteria, and 3) selective growth and/or activity of the intestinal bacteria
and improvement of host health [139]. Under the Functional Food Center definition, further
characteristics of prebiotics include: being part of conventional or everyday foods, to be part of the
normal/usual diet, being composed of natural (as opposed to synthetic) components, sometimes in
increased concentrations or present in foods that would not normally supply them, and having a
positive effect on target functions that may enhance well-being and health and/or reduce the risk of
disease [66]. In addition, the most efficient prebiotics will also reduce numbers and activities of
potential pathogenic organisms [140].
Taro has previously been suggested as a potential prebiotic [50]. Taro has been clearly shown
to have the potential ability to inhibit pathogenic organisms. A study found that a minimum of 1%
taro medium was necessary for L. lactis supsp. lactis ATCC 11454 to produce nisin, a bacteriocin
approved by the Food and Drug Administration (FDA) for use as a preservative in certain foods, that
inhibited the growth of Micrococcus luteus ATCC 10240, which causes invasive disease in
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immunocompromised patients [125]. Similarly, an investigation on the antimutagenicity effects on the
Trp-P2-induced mutagenicity of Salmonella Typhimurium using root crops found that processed taro
showed the highest inhibition of Salmonella Typhimurium at 60%. Further antimicrobial analysis of
taro extracts from the corm showed to have MIC values of 15.6 mg/L against Edwardsiella tarada,
Escherichia coli, Flavobacterium sp., Pseudomonas aeruginosa, and Vibrio cholera; and 31.4 mg/L against
Klebsiella sp., aeromonas hydrophila and Vibrio alginolyticus; and 125 mg/L against Salmonella sp. and Vibrio
parahaemolyticus [141]. In addition, research has suggested that taro may be useful to control Salmonella
Typhimurium. A study investigated the effects of water extracts, ethanol extracts, and gummy
materials prepared from four root crops, nagaimo (Chinese yam, Dioscorea polystachya), jinenjo
(D.japonica), satoimo (taro, Colocasia esculenta), and processed taro (poi, Colocasia esculenta), on S.
Typhimurium TA 98. The gummy of processed taro showed the highest inhibition of 60% among the
root crops used [142].
2.3.5.1 Colorectal cancer prevention of prebiotics from taro

A study found that mucilage isolated from taro was arabinogalactan [143], an established
prebiotic [144]. Mucilage from taro was fed to rats at a dose of 4 mg/100 g body weight for 8 weeks
and resulted in a hypolipidemic effect, decreased lipid levels in both serum and tissues. In addition,
hepatocytes isolated from the livers of the mucilage-fed rats showed a decrease in the synthesis and
secretion of apoB-containing lipoproteins, specifically, very-low-density lipoprotein (VLDL) [143]. An
association between dyslipidemia and colorectal neoplasia has been observed with
hypertriglyceridemia [145]. The levels of total cholesterol (TCH) and triglycerides (TG) in serum and
the levels of TCH and high density lipoprotein- cholesterol in cancerous tissue in patients with
colorectal cancer have been shown to be significantly correlated with metastasis stage [146]. Thus,
disordered and abnormal levels of lipids in cancerous tissue and serum of patients with CRC have
been correlated with the occurrence and development of colorectal cancer [146].
In addition, prebiotics may prevent CRC development in humans by modifying the
composition or activity of the colorectal microflora. The gut microbiota, either as individual microbe
or as a microbial community, exerts a collective effect that has potential to mitigate CRC risk [147].
The high bacterial density in the colon and the observation of bacteremias with specific microbes, like
Streptococcus gallolyticus, have been suggested to be clinical indicators of occult colonic adenomas and
CRC, which emphasizes the importance of studying the roles of gut microbes in CRC [147].
Modulation of gut microbiota by prebiotics could positively influence the immune system and
microbiota, which would be beneficial in preventing inflammation and CRC. Thus, further research
into the modulation of taro’s prebiotic potential to modulate CRC is warranted through in vitro and in
vivo methodologies.
2.3.6 Amino Acids and Proteins

Taro corms have a low protein content between 1.4% to 3.0% on a fresh weight basis [148].
Though taro has been shown to be low in protein, essential amino acid content has been shown to be
adequate, except for sulfur-containing amino acids [95]. Analysis of amino acids for total protein from
taro corms indicated lower levels of methionine and cysteine [95, 149]. The polypeptide composition
of taro corm can change slightly among distinct cultivars, as demonstrated by electrophoresis analysis
[149, 150]. However, generally, the amino acid compositions of the corms have been comparable with,
and even better than that of potato protein [95, 151].
Taro corms have four major storage protein families: two globulins (G1 and G2) and two
albumins (A1 and A2) [137]. Storage proteins are defined as proteins whose major role is to act as
stores of nitrogen, sulfur, and carbon [152]. However, storage proteins have been shown to have
14
multiple functions, beyond their storage roles [137]. Storage proteins’ additional functions include
defense mechanisms and potential health benefit functions that have yet to be unearthed [137, 149].
The G1 globulin, named tarin [153], is an identified lectin that accounts for about 40% of the
total soluble proteins [153, 154]. Tarin has shown sequence homology with mannose-biding lectins,
including 40% identical with the snowdrop (Galanthus nivalis) lectin [137]. In addition, tarin showed
45% identity with curculin, a taste-modifying protein from fruits of Curculago latifolia (hypoxidaceae)
[137]. Tarin can bind to a wide variety of ligands, including high-mannose and complex N-glycans,
but preferentially binding to complex N-glycans over high mannose [155].
Similarly, G2 globulin has been shown to account for about 40% of the total soluble tuber
protein [154]. G2 globulin from taro has shown sequence homology to trypsin inhibitor family
members found in soybeans, winged beans, sweet potato, and barley [137, 156, 157]. Globulins are
important, not only for the storage function, but also the proteinase function. Proteinase inhibitors,
especially food-additive grade inhibitors, are in demand for protecting myofibrillar proteins from
proteolysis by endogenous proteinases to maintain the integrity of food products [158].
Though taro albumins are not the most abundant protein, they account for about 11% of the
total protein [149]. The A1 albumin, also known as colocasin, is composed of six 8.3 kDa homogenous
monomers [149]. Albumin A1 is not always present in the aqueous extract, which may be due to the
cultivar difference, extraction procedures, or corm maturation stage [60, 156]. Similarly, A2 albumin
was found to accumulate in the first two stages of corm development, and decreased in the last 3
stages [156]. Amino acid analysis of the major albumins indicated low levels of sulfur-containing amino
acids [149]. Little information is available about the identification of albumins or their biological
properties [148].
Taro contains a unique composition of protein polypeptides that have not been found in other
root crops, and exclusively found in tubers from C. esculenta [157]. Furthermore, taro differs from
sweet potato, cassava and yams, in that it contains two major types of storage protein: G1 (a mannose-
binding lectin) and G2 (a trypsin inhibitor related to sporamin) [159]. Thus, the low protein content
and gluten-free composition of taro makes for great hypoallergenic food and potential food substitutes
for individuals with food allergies [10].
2.3.6.1 Colorectal cancer prevention of amino acids and protein from taro
Taro has been shown to have anti-cancer activities through its unique protein content. The G1
globulins from taro demonstrated agglutination of erythrocytes from rabbits and were inhibited by
mannose, but not other monosaccharides [153]. A study by Kundu et al. [49] showed that active
protein components in taro appeared to have anti-metastatic activity. Active protein components
extracted from uncooked taro concluded to be: tarin, lectin, and a 12 kD storage protein [49]. All three
proteins contain similar amino acid sequences, post-translational processing, and carbohydrate binding
domain [49]. Using two highly metastatic, ER, PR, and HER-2 negative murine mammary tumor cell
lines 66.1 and 410.4, the taro extracts were shown to nearly complete ablation of metastasis in the lung
colonization model in vitro [49]. In the in vivo BALB/cByJ mouse model, taro extract treatment was
initiated, after mammary tumors were established in mice using mammary tumor cell line 66.1, and
was found to significantly inhibit the spontaneous metastases to the lung, with no further colonization
in the heart [49].
Similarly, a study by Chan et al. [160] found that tarin extract from Hong Kong small taros
showed antitumoral effects against hepatoma HepG2 cells, demonstrating that tarin inhibited the
proliferation of the tumoral cells. Furthermore, tarin demonstrates the ability to stimulate the
expression of cyctokines that are currently involved with cancer treatments to stimulate the immune
15
response against tumor cells, which includes: IL-2, TNF-⍺ and interferon- γ [160-162]. In addition,
interleukin-1β (IL-1 β) is also released, though not currently used for cancer treatments [160].
Taro’s anticancer properties can be potentially explained by the presence of tarin. Tarin
exhibits specificity towards glycan chains that make part of many cell surface antigens including cancer
cells, viruses, insect cells and also hematopoietic cells [148]. Furthermore, tarin binds specifically to
cell membrane glycans, exclusively binding the high mannose N-glycan 49 [man⍺1-3(Man⍺1-6)Man
β1-4GlcNAcβ1-4GlcNAc β] that is commonly found in human cancer tissue, and not in healthy
tissues [163]. In addition, the complex N-glycan 465 shows exact similarities to the LeY antigen (Lewis
Y/CD174-Fuc⍺1-2Gal β1-4[Fuc⍺1-3]GlcNAc β1-), a cell marker specifically associated with cancer
[164]. The LeY antigen has been shown to be up-regulated in a variety of cancer cells, including: colon,
stomach, ovary, breast, pancreas, prostate, and lung [164]. Furthermore, tarin has the ability to bind
the H2 antigen (CD173-Fuc⍺1-2Gal β1-4FlcNAc β1-) that is present at the glycan 358 expressed in
the leukemia cells linages KG1 and KG1a (pro-myeloid ells) and TF1 (pre-erithroblastoid cells) [165].
Taro can also potentially bind the CA-125 antigen, which characterizes ovary cancer cells [166]. Taro
tarin exhibits remarkable biological potential by having: anti-metastatic, mitogenic, antitumoral,
insecticidal, and antiviral activities [148]. Thus, tarin extract from taro has promising potential as a
future biopharmamceutical for cancer, with further research warranted [148].
As such, taro provides great evidence for the possibility of broad spectrum actions for
anticancer activities. Specifically, the active protein component, tarin, shows promising potential as a
natural source for controlling cancer metastasis, migration, colonization; however, in vivo and clinical
studies are necessary to uncover its full potential.
2.3.7 Phytochemicals
Taro corms have been found to be rich in various phytochemicals. A study analyzing tropical
root crops found that 24 of the taro cultivars had accumulation of four anthocyanins in the corm,
with a large diversity of other phenolic compounds including 20 flavonols, nine flavanols and two
phenolic acids [167]. Similarly, Muñoz-Cuervo et al. [168] found that taro corms contain six
carotenoids, 35 flavones/flavonols, six flavanones, two flavanols, and one indol. Alcantara et al. [81]
found that after the drying of taro powder at 60C, the phytochemical components increase by
124.89% for phenols, 89.59% for saponins, and 124.89% for flavonoids. However, when taro-powder
based food products, specifically taro noodles and taro cookies, were assessed, all the phytochemical
components reduced [81]. The decrease of phytochemicals may be due to heat, degrading certain
compounds with antioxidant properties, such as, phenols, flavonoids, tannins, and saponins; and
leaching the antioxidant compounds [169].
According to recent findings, steroidal saponins, a type of phytochemical, could be a novel
class of prebiotics to LAB and are effective candidates for treating fungal and yeast infections in
humans and animals [170]. An antifungal compound isolated from tubers of taro inoculated with black
root fungus (Ceratocystis fimbriate) identified as 9,12,13-trihydroxy-( E)-10-octadecenoic acid together
with two enzymes, lipoxygenase and lipid hydroperoxide-converting enzyme was found responsible
for the production of antifungal lipid peroxides [171]. Similarly, an isolated bioactive molecule from
taro corms identified as 2, 3-Dimethylmaleic anhydride (3, 4-Dimethyl-2, 5-furandione) proved to be
an efficient biofumigant that is highly toxic to insect pests for stored grains at very low concentrations,
with no adverse effects on seed germination [172].
Taro has been reported to contain anthocyanins, a type of phytochemical. Anthocyanins
isolated from taro corms and petioles were identified as: pelargonidin 3-glucoside, cyanidin 3-
rhamnoside, and cyanidin 3-glucoside [173]. Anthocyanins were highest in the skin of the corm, 16.0
mg/100 g, with equal amounts in both corm and petiole, 3.29 mg% [174]. Using chromatographic and
16
spectrophotometric methods, pigments identified included: pelargonidin 33-glucoside, cyanidin 3-
rhamnoside, and cyanidin 3-glucoside [174]. In addition, anthocyanogens were also present in the
corm [174]. Anthocyanins are reputed to improve circulation by decreasing capillary fragility [175],
improve eyesight, act as potent antioxidants, act as anti-inflammatory agents, and inhibit human cancer
cell growth [176-178]. As such, further in vitro clinical trials are necessary to understand the mechanism
of action.
2.3.7.1 Colorectal cancer prevention of phytochemicals from taro

Several types of phytochemicals, such as carotenoids, flavonoids, and the antioxidative
vitamins C and E are believed to reduce cancer incidence in humans [179]. Oxidative stress is
recognized to be involved in the tumor promotion stage because organic peroxides or radical-
generating agents are tumor promoters.
Taro has been reported to show significant antioxidative effects by inhibiting O 2- generation
from nictotinamide adenine dinucleotide (NADPH) oxidase and xanthine oxidase (XOD) pathways,
from in vitro TPA-stimulated in HL-60 and AS52 cells [180]. NADPH oxidase and XOD are enzymes
involved in oxidative stress; thus, inhibiting O 2- generation suggests taro’s antioxidative and cancer
preventative potential [180]. Taro corm’s anthocyanins, cyanidin 3-glucoside, pelargonidin 3-
glucoside, and cyanidin 3-rhamnoside, have been reported to have antioxidant and anti-inflammatory
properties [173, 181]. These substances may protect the intestine from carcinogens [181]. However,
further studies are necessary to glean of taro’s phytochemical potential as a colorectal cancer
preventative.
2.4 TARO NUTRIENT ABSORPTION
Taro’s functional food potential can be attributed to its easy absorption capabilities. The small
sized granule of starch in taro helps increase the bioavailability of its nutrients, due to high efficiency
of digestion and absorption. Taro starch has irregular, polygonal shapes, and small granular sizes that
promotes rapid digestibility [19].
An early study comparing the raw starches of rice, arrowroot, canna, cassava, taro, tree-fern, and
potato found that rice and taro root were considerably more digestible than arrow root and potato
starch, with taro starch being 98.9% assimilable [182]. The study concluded that there was a direct
relationship between the size of the starch granule and the digestibility [182]. Similarly, another early
study found that the starch granule of the taro variety, Kauʻuliuli, was one tenth the size of a potato
starch granule, but about the same order of magnitude as the starch granule of rice with a greater
concentration of nutrients [183]. As such, taro was proved to be a more nutritious alternative.
The high digestibility of poi also appears to be related to the relative ease with which it breaks
down. An early study reported that the easy digestibility of poi and the high absorbability of its
minerals, specifically calcium and phosphorus, appear to be related to its rapid fermentation process
[184]. Furthermore, the high digestibility and absorption was further demonstrated in a human study
where poi eaten in high quantities was found to have no measures of undigested fiber in the participant
feces [182]. Thus, most of taro nutrients are easily absorbed through the digestive tract, potentially
facilitating their beneficial properties.
2.5 CANCER PREVENTION OF TARO
Taro has been shown to have potential anticancer properties. An anticancer property taro has
is through the means of inhibiting mutagens. Mutagens are agents that directly alter a cell’s DNA
17
sequence; while antimutagens are suppressors of mutation frequency, which possess cancer prevention
capability [185]. In Japan, taro showed the highest antimutagenicity capability compared to sweet
pepper, eggplant, oriental pickling melon, pumpkin, and edible burdok [185]. Antimutagenicity on the
Trp-P2-induced mutagenicity to Salmonella Typhimurium TA98 showed the highest inhibition of
mutagenicity from processed taro of 60% [142]. Similarly, preincubated mutagenicity assay with
Salmonella Typhimurim TA1538 against the mutagenicity of the heterocyclic amine 2-amino-3-
methylimid-azo[4,5-f]quinoline (IQ), showed strong antimutagenic properties of taro leaves [186]. As
such, taro can be considered a potential starting material to identify new bio-antimutagens for cancer
prevention [185].
Furthermore, taro has shown great promise as cancer therapy in in vitro cell line studies. Taro
corms and the skin of taro showed markedly greater inhibitory effects on adult T-cell leukemia cells,
Su9T01, than genistein, a soy-derived isoflavone phytoestrogen [187]. Similarly, water-soluble extract
of taro strongly inhibits lung colonizing ability, as well as spontaneous metastasis from mammary
gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2 negative breast
cancer; with taro also showing antiproliferative activity [49]. Taro extract significantly inhibited
proliferation of in vitro cell lines of murine breast cancer cell (66.1 and 410.4), human breast
adenocarcinoma cell lines (MCF-7, MDA-MB-231), and human mammary epithelial cell line MCF10A
in a dose dependent manner [49]. Likewise, research using human breast adenocarcinoma MCF-7
cancer cells found that the corm and stem of taro extract inhibited growth of cancer cells by 30% at a
concentration of 20 and 30 μg/m, respectively [141].
Taro has also shown significant inhibition against tumor cell colonization and migration in in
vivo mouse studies. Taro significantly inhibited tumor cell colonization with evidence from mice
injected daily with taro extract resulting in a 98-99% reduction in lung tumor colony formation and
also significant inhibition of tumor colonies in the heart [49]. Similarly, mice transplanted
subcutaneously with tumor cells, significantly reduced the number (85% inhibition) of spontaneous
lung metastases; however, the treatment had no effect on the size of the locally growing tumors [49].
Taro has also shown promising capability of affecting inflammatory mediators associated with
tumorigenicity. Pharmacologic inhibition of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2
(COX-2) has shown reduced tumor cell proliferation, prostaglandin E2 (PGE2) synthesis, and tumor
growth [188, 189]. Taro extract showed significant inhibition of PGE2 synthesis, as well as
downregulated expression of COX-2 mRNA and, to a lesser extent, COX-1 in vitro [49]. Thus, taro
has potential as a tumorigenic mediator, as it is well established that COX-2 and its product PGE2 are
positively associated with increased tumorigenic and metastatic potential for aggressive cancers [189,
190].
Thus, strong evidence presented above illustrates taro’s great potential for anti-cancer activity
and warrants further studies linking the nutrient content to cancer preventing properties in vitro and
clinical trials to further understand the anti-cancer effects in vivo.
2.6 OTHER HEALTH BENEFITS
2.6.1 Food Allergies

Taro has been found to be a great substitute for allergenic foods, due to its hypoallergenic
properties from having a very low protein composition [10]. For centuries, Native Hawaiians had
knowledge of taro’s medicinal value, had not only been using taro to make nutritious foods, but also
making lāʻau lapaʻau (herbal medicines) for treating boils and other body ailments [4]. In 1939, Alverez
[191] showed that poi, a taro based food, could be a good alternative for food allergies. Subsequently,
around 1942, Feingold [192] was one of the first researchers to suggest that poi be considered a
18
substitute for soy milk in infants allergic to both soy and cow’s milk [12]. In 1961 in Hawaiʻi, Dr.
Jerome Glaser, a pediatrician and allergist, reported that many infants in Hawaiʻi with allergies and
other gastrointestinal disorders were being fed with poi, which could potentially be used as a cereal
allergy substitute [193]. As such, Glaser et al. [193] attempted to conduct a 6-month study in which
100 babies admitted to the clinic would be assigned either a poi (50 babies) or rice diet (50 babies) and
followed at 4-week intervals for a period of six months. However, the study contained several
limitations such as problems with parental compliance and inability to complete metabolic studies,
with only three babies remaining on the rice cereal diet and five on the poi diet for six months [193].
Similarly, Roth et al. conducted a study on 132 babies at Honolulu hospitals that were fed cow’s milk
substitutes, finding that only 7.27% (4/55 babies) of rice-fed babies exhibited signs of allergies and
6.85% (5/73 babies) poi-fed babies exhibited signs of allergies, while breast babies presented no signs
of allergies [194]. In addition, individuals with celiac disease can benefit from taro or poi consumption
because of the lack of gluten [10, 193]. Though no further studies have been conducted to date
comparing taro or taro based food with food allergy substitutes, taro remains a potential food allergy
substitute and warrants further investigation.
2.6.2 Complementary Food

In as early as the 1950s, physicians recognized the importance of poi as a beneficial
complementary food for infants. A mailed questionnaire survey to medical professions in Hawaiʻi
asked about the extent to which they recommend poi as a staple and/or therapeutic food for healthy
infants and children [195]. When asked when feeding poi should begin, about 64% of the pediatricians
and 55% of other specialties stated that they would recommend feeding poi to babies [195]. Thus,
emphasizing the potential benefits of taro based food. Furthermore, a case study of a failing to thrive
premature infant only weighing 1,500 grams, gaining 100 grams after 54 days fed various formulas,
saw that upon being fed poi her weight quickly responded and she was able to be discharged from
the hospital after maintaining a healthy weight to 2,500 grams [193]. Furthermore, taro corm flour,
dried and milled, contains easy digestion starches that can widely be used as infant food [196].
A recent study in Hawaiʻi following the first feeding behaviors of Native Hawaiian, Pacific
Islander, and Filipino infants (3-12 months of age) examined the dietary diversity and feasibility of a
mobile phone food record found that the most common first food introduced infant/rice cereal
(n = 19) followed by poi (mashed taro root, n = 16) [197]. Thus, taro remains a beneficial first solid
food to introduce into infant diets; though, further research is need to strengthen this statement.
2.6.3 Tooth Decay

In the early 1930s, Hawaiʻi’s Queen’s Hospital Research Department study showed that babies
in the infant feeding center of Ewa Plantation Health Project fed a diet of poi and sweet potatoes di
not develop odontoclasis, a form of tooth decay; whereas infants fed grain as their major carbohydrate
source, developed odontoclasia [195]. Furthermore, a number of cases were found to have arrested
the tooth decay process, when grain foods were omitted from the diet and replaced with taro and
sweet potato [198]. In villages in the Sepik and Fly River regions of Papua-New Guinea found that
Chinese taro provided beneficial effects in regards to dental caries, from the molybdenum, manganese,
vanadium, phosphorous and titanium content [199]. Though currently, there is no association between
taro consumption and decline in dental caries, it has been hypothesized that taro’s high degree of
alkalinity is a potential important factor in maintaining healthy teeth [198]. However, further studies
ae needed to confirm taro and poi’s potential as a dietary tooth decay preventative.
19
2.6.4 Wound Healing
Apart from its nutritional value, C. esculenta leaves and tuber were also used in the treatment
of cutaneous wounds [200]. The wound healing property of C. esculenta can be attributed to its
antioxidant activity, namely its superoxide radical scavenging potential, and the inhibition of
hyaluronidase, thus protecting skin cells from oxidative damage and accelerating the recovery of the
wound in the inflammatory state [201]. However, further investigation on the mechanisms of action
of taro’s wound healing properties are necessary to understand the potential health benefits.
2.7 CONCLUSIONS
Taro (Colocasia esculenta) has been traditionally used as a medicinal plant and is a rich source of
nutrients and bioactive compounds. Taro’s unique composition provides great potential as a
functional food for the prevention of CRC. Specifically, several bioactive components of taro show
great promise containing anti-cancer activities, such as dietary fiber, RS, probiotics, and
phytochemicals. Some of the anti-cancer actives include: inhibition of mutagens, increasing expression
of tumor suppressor genes, increasing SCFA production, inhibiting bile acids, anti-inflammatory,
antioxidant, anti-tumorigenic, and anti-metastatic. Similarly, other nutrients in taro, fat and
carbohydrates, have also shown potential as prevention for CRC. This unique nutrient composition
of taro poses great potential as a dietary functional food.
Functional foods represent one of the most promising and developing area in the food and
nutrition industry. In addition, functional foods in diets can provide a preventative measure against
the development and progression of cancer. Taro poses as a potential dietary source to reduce the risk
of CRC, with many of the traditional uses of taro as a medicinal plant scientifically corroborated [148].
Thus, taro warrants further exploration of its bioactive nutrients, specifically prebiotic and probiotic,
to fully understand the potential health benefits.
20
21
CHAPTER 3 NUTRITIONAL, PHYSICOCHEMICAL AND
FUNCTIONAL PROPERTIES OF FIVE VARIETIES OF TARO
(COLOCASIA ESCULENTA)
3.1 ABSTRACT
Taro (Colocasia esculenta) has been shown to be a good source of nutrients and may be an
alternative as a dietary carbohydrate source for food production and health implications. However,
the amount of starch as well as other nutritional characteristics may be different amongst varieties and
geographical regions. This study aimed to evaluate five taro varieties grown in Hawaiʻi, Bun-long,
Mana Ulu, Moi, Kauaʻi Lehua, and Tahitian, for nutritional, physicochemical and functional
properties. Macronutrients, minerals, water absorption capacity (WAC), oil absorption capacity
(OAC), foam capacity (FC) and stability (FS), emulsifying activity (EA) and stability (ES), swelling
capacity (SC), water absorption index (WAI), water solubility index (WSI), bulk density (BD), gelling
(GP) and boiling points (BP), least gelation concentration (LGC), and starch content, granule size and
diffraction pattern were analyzed and compared. Among the five taro varieties, Moi had the highest
concentrations of potassium, copper, and manganese at 1.75 g/100 g, 0.97 mg/100 g, and 12.46
mg/100 g, respectively. Tahitian exhibited the highest concentrations of iron and zinc at 7.74 mg/100
g and 13.68 mg/100 g, respectively. Tahitian, Bun-long, and Moi showed high total starch content of
40.8 g/100g, 38.9 g/100g, and 34.1 g/100g, respectively. In addition, total starch content of taro was
significantly correlated with its WAC, OAC, EA, WBI, and WSI. The diameter size of taro starch
granules ranged between 2.08 m and 2.93 m, with Mana Ulu having the smallest and Tahitian having
the largest values, which were significantly different. The lowest GP was observed in Moi at 62.3C,
while Bun-long having the highest at 68.3C. The BP of the taro varieties ranged from 74.7C to
79.7C, with Bun-long having the lowest and Moi having the highest values. These results indicate
that different taro varieties can be used to supplement nutritional components, and improve food
quality and human health.
22
3.2 INTRODUCTION
Taro (Colocasia esculenta) belongs to the Araceae family and is a starchy root crop with wide
leaves. The most frequently eaten part of the plant is the corms, which are formed underground by
the thickening of the stem base. Taro is the main food source for about 500 million people living in
Asia, Africa, Middle America, and the Pacific Islands [202]. Specifically in Hawaiʻi, taro is of cultural
importance, and it is used for pounding paʻiʻai and poi, a fermented sticky paste [124].
Taro is a nutritious crop that has several potential health benefits, such as high mineral content.
Minerals are vital in all body fluids and tissues and play important roles in metabolic processes such
as: maintenance of pH and osmotic pressure, muscle contraction, and transport of gases [80]. These
minerals are important components of enzymes and hormones, and crucial for bone formation and
the synthesis of vitamins [80]. In addition, raw taro has a high starch content that has been reported
to be 70-80% [203]. Starch is used in the food industry mainly as a modifier of texture, viscosity,
adhesion, moisture retention, gel formation and films [204]. Starches with desirable functional
properties play important roles in improving food quality and conferring health benefits [205, 206].
Due to the small size of its starch granules, taro is hypoallergenic and easily digested. Therefore, it
serves as a great dietary alternative for individuals allergic to cereals and milk and a great dietary option
as baby food [21]. However, taro is underutilized and sold in markets as raw vegetable and has high
pre-harvest and post-harvest losses [207, 208]. The post-harvest losses of taro are due to its high
moisture content and large size of corms [207, 209]. These losses can be minimized by converting it
into non-perishable forms by drying or cooking, which may reduce the storage space, extend the shelf
life, reduce food waste, and increase nutritional value of products [18, 19, 62, 208].
Currently, flour is covered by four conventional sources: wheat, corn, potato, and cassava
[210]. However, more nutritious alternatives are available that can provide similar food applications.
Alternatives to conventional wheat flour from local sources has become an increasingly important
objective of the Food and Agricultural Organization policy [211]. Flour and starch from tubers and
roots can be used to substitute wheat flour in certain food applications [212]. The performance of
flours as food ingredients depends upon the physicochemical properties of the root crop, which in
turn affect the functional characteristics and sensory qualities imparted on the end products [213].
Physicochemical properties of flours affect the quality factors of the subsequent products, such as
swelling capacity, water absorption index, water solubility index bulk density, and starch characteristics
[214]. Knowledge of the starch granule characteristics is of significant importance for the food
industry, which seeks to maintain and enhance the properties of food products during storage [15].
Functional properties are the fundamental physicochemical properties that reflect the complex
interaction between the composition, structure, molecular conformation and physicochemical
properties of food components together with the nature of environment in which these are associated
and measured [215]. These may include foaming, emulsification, texture, gelation, water/oil
absorption capacities, and viscosity which are influenced by proteins, carbohydrates and other
components to various extents [14]. Thus, there is a need for extensive research on alternative flour
sources to assess their nutritive, physicochemical, and functional characteristics so that possible
applications may be developed using substitutes for traditional and chemically modified flours.
Few reports have differentiated the Pacific Island taro cultivars and their functional properties
[29, 216]. With the increase in popularity of taro-based food products, such as taro chips and taro
buns, it is vital to provide the public accurate nutrient information of different taro varieties [29]. Bun-
long, also known as Bun woo, is variety that contains a white flesh and purple fiber corm, that is
frequently used for chip production because of the copious fibers [217]. Mana Ulu, also known as
Mana Owene, has a yellow flesh and fiber corm, that is named Ulu due to the resemblance of the flesh
23
of breadfruit, and is one of the more commonly cooked taro varieties, since it is more attractive than
other varieties [217]. Tahitian variety has a white flesh and yellow fiber corm, that is traces it’s origins
back to Tahiti, and is commonly used as table taro [217]. Moi variety has a light pink flesh, with yellow
fiber corm that is one of the more common varieties used for poi pounding [217]. Manufacturers of
taro-based products in Hawaiʻi use the same set of USDA taro nutrient data as a reference source that
does not account for any varietal differences [29]. As such, data on nutrient composition of different
taro varieties would be useful for taro processing and product development. It is also evident that a
significant amount of work needs to be done to characterize taro flour for it to become competitive
amongst other commercial flours. Therefore, the present study aimed to investigate the nutritional,
physicochemical, and functional properties of taro varieties from the Pacific region.
3.3 MATERIALS & METHODS
3.3.1 Taro Processing

Full-grown corms of five taro varieties (Bun-Long, Moi, Tahitian Variety, Kauaʻi Lehua, Mana
Ulu) were collected at the University of Hawaiʻi Waimānalo Research Station (Waimānalo, Hawaiʻi)
(Figure 1). The loʻi (field) was planted in September 2017 and harvested in July 2018 at maturation.
The field design was 8 lines with 9 varieties per line. The lines ran from mauka (mountain-side, north)
to makai (ocean-side, south). Varieties were planted 2 feet in between hulis (taro seed that includes
upright stem between the leaf and the corm and a piece of the corm attached where roots emerge)
and 5 feet between rows. Three corms of each cultivar were pulled. Following the protocol in Figure
2, immediately after the harvest, the corms were washed and left on a clean wooden cutting board
until the surface was dry. Subsequently, the skin was peeled using a ceramic knife to remove inedible
portions. The edible corm flesh was cut into 2 cm width cubes, weighed, and autoclaved at 121°C for
15 minutes to simulate pressure cooking. Afterwards, the cubes were frozen for 48 hours in a -80°C
freezer. The samples were then lyophilized for 48 hours at -51°C at 0.021 mBar pressure (FreeZone,
Labcono, Kanasa City, MO). After lyophilization, the samples were weighed and ground to 1 mm size
taro powder particles (Industrial Electric Peppe Grain Mill, CGOLDENWALL). The three taro corm
samples of the same variety were combined and stored under anaerobic conditions until further use.
3.3.1.1 Moisture Content (MC)

Moisture content of samples were calculated using the weight before and after lyophilization.
Samples were then calculated using the following calculation:
Moisture (%) = (W1 – W2) x 100

W1
Where: W1 = weight (g) of sample before lyophilization

W2 = weight (g) of sample after lyophilization
3.3.2 Nutrient Analysis

Macronutrients in the samples were analyzed by the Agricultural Diagnostic Service Center of
the University of Hawaiʻi. Minerals were analyzed using an inductively coupled plasma machine
(ICP/6500, Perkin-Elmer Instruments, Norwalk, CT).
3.3.3 Physicochemical Analysis
24
3.3.3.1 Swelling Capacity (SC)
Samples were weighed to 0.5 g and transferred into a 50 mL graduated cylinder centrifuge
tube. Bed volume was measured. Distilled water of 20 mL was added to samples and incubated at
room temperature. After 16 h of incubation the bed volume was recorded and expressed as mL/g dry
sample [218, 219].
3.3.3.2 Water Absorption Index (WAI) and Water Solubility Index (WSI)
Samples of 2.5 g were individually added into centrifuge tubes, and 30 mL of distilled water was added.
The samples were heated for 15 minutes at 90°C in a water bath. The cooked paste was cooled to
room temperature and centrifuged at 3000 x g for 10 minutes. The supernatant was decanted into a
tared evaporating dish to determine the solid content, and weight was recorded. The samples were
then lyophilized for 48 hours at -51°C at 0.021 mBar pressure (FreeZone, Labcono). Afterwards, the
weight of the dry solids was recorded and used to calculated WAI and WSI using the following
equations [62, 220].
WAI (g/g) = Weight of sediment

Weight of sample
WSI (g/100 g) = Weight of Dissolved Solids in Supernatant

Weight of sample
3.3.3.3 Bulk Density (BD)

A 10 mL graduated cylinder was weighed and filled with sample to the 10 mL mark. The
cylinder was continuously tapped at the bottom until there was no visible change of the sample volume
and the final weight was record. The difference of the weight and volume was determined to compute
bulk density, which was expressed as grams per unit of volume of sample (g/mL) [94, 221].
3.3.3.4 Starch Properties Analysis
3.3.3.4.1 Total Starch Content

Total starch was determined suing Megazyme Resistant Starch assay kit (Megazyme
International, Wicklow, Ireland). In brief, the samples were hydrolyzed for 16 hours using pancreatic
α- amylase and Amyloglucosidase at 37C. After exhaustive amylolysis, the samples were centrifuged
to form a pellet, which was then hydrolyzed to glucose with KOH, and the glucose content was
quantified. Starch contents were calculated following manufacturer’s instructions.
3.3.3.4.2 Granule Size

Samples were mounted with conductive carbon tape on aluminum stubs and sputter coated
with gold/palladium with a Hummer 6.2 sputter coater. Samples were viewed with a Hitachi S-4800
Field Emission Scanning Electron Microscope at an accelerating voltage of 2kV.
Size of the starch granules was estimated by measuring the diameters of 20 randomly selected
granules from the micrographs [18, 209].
3.3.3.4.3 X-Ray Diffraction (XRD) Pattern

X-ray diffraction patterns of samples were obtained with beryllium using a diffractometer (Miniflex
II, Riagaku, Japan). Diffractometer was operated at 15 mA and 30 kV, 2 range from 10 to 50.
25
3.3.4 Functional Properties Analysis
3.3.4.1 Water Absorption Capacity (WAC)

Samples were weighed to 0.5 g and hydrated in 20 mL of distilled water. After 24 h of
incubation at room temperature, samples were centrifuged at 1,500 rpm for 5 minutes at room
temperature. Excess supernatant was decanted. Pellet was weighed and expressed as grams of water
held by 1 g of sample [222-224].
WAC (g/g) = (weight of centrifuge tube & pellet -weight of centrifuge tube) – sample weight
sample weight
3.3.4.2 Oil Absorption Capacity (OAC)

OAC was determined using the above stated methodology for WAC; however, instead of 20
mL of distilled water, vegetable oil was used. Vegetable oil density was 0.93 g/cm 3. OAC was recorded
as grams of oil held by 1 g of sample [222, 223].
OAC (g/g) = weight of centrifuge tube after drawing oil – (centrifuge tube weight + sample weight)
Sample weight
3.3.4.3 Foam Capacity (FC) and Foam Stability (FS)

Samples of 1.5 g were added to 50 mL of distilled water in a 100 mL cylinder, stirred and noted
for the volume. The suspension was blended with a hand held blender (Hand held blend, Braun) at
high setting for 3 minutes to form foam. The blend was immediately transferred into a graduate
cylinder and the volume was record after whipping. The volume of the foam after 30 seconds was
recorded [225]. The foam capacity and foam stability were calculated using the following formulas [62,
226].
FC = Volume after homogenization – initial volume x 100

Initial volume
FS = foam volume changes in the graduated cylinder was recorded at an interval of 20, 40, 60, and
120 minutes of storage as a percentage of the initial foam volume
3.3.4.4 Emulsifying Activity (EA) and Emulsifying Stability (ES)

The samples (3.5 g) were homogenized for 30 seconds in 50 mL of water at high setting. Vegetable
oil (25 mL) was added and the mixture was again homogenized for 30 seconds. Then another 25 mL
of vegetable oil was added, and the mixture was homogenized for 90 seconds. The emulsion was
evenly divided into 50 mL centrifuge tubes and centrifuged at 1100 x g for 5 minutes. Emulsifying
activity (EA) was calculated by dividing the volume of the emulsified layer by the volume of the
emulsion before centrifugation and multiplied by 100.
The emulsion stability (ES) was determined using the samples prepared for the measurement
of emulsifying activity. The samples were heated for 15 minutes at 85°C, cooled and then centrifuged
at 1100 x g for 5 minutes. The emulsion stability was expressed as the percentage of emulsifying
activity remaining after heating [62, 227].
3.3.4.5 Gelling Properties Analysis
3.3.4.5.1 Gelling and Boiling Points
26
Samples of 10 g were added into 100 mL of distilled water in a 250 mL beaker. A thermometer
was added into the sample so that only the bulb was submerged. A magnetic stir bar was continuously
spinning while the suspension was heated. The samples were heated until the suspension began to gel
and then boiled, when the corresponding temperatures were recorded [228].
3.3.4.5.2 Least Gelation Concentration (LGC)

Samples were added to 5 mL of distilled water in test tubes to achieve 2, 4, 6, 8, 10, 12, 14, 16,
18, and 20 g/100 mL. The suspensions were heated for 1 hour in boiling water, followed by rapid
cooling under running water. The tubes were further cooled at 4C for 2 hours. LGC is the
concentration above which the sample did not fall down or slip when the test tube was inverted [229].
3.3.5 Statistical Analysis
Measurements of triplicate samples were analyzed to obtain means and standard deviations.
Differences among the means were analyzed for statistical significance using analysis of variance
(ANOVA) and post-hoc Tukey’s Honest Significant Difference (HSD) Test.
3.4 RESULTS
3.4.1 Nutrient Composition

Chemical compositions of the five taro varieties are show in Table 1. The nutrient contents of
the taro corms were calculated on a dry-weight basis. The main minerals found in the taro corms in
relatively high concentrations were K, P, Mg, and Ca, with the mean values of the highest variety
being, 1.75 % (0.1) in Moi, 0.23 % (0.1) in Moi, 0.15 % (0.1) in Bun-Long, and 0.23 % (0.1) in Moi,
respectively. In addition, high concentrations of trace minerals found were manganese, zinc, and iron,
with the highest mean values being 12.46 mg/100g (9.1) in Moi, 13.68 mg/100g (8.3) in Tahitian, and
7.74 mg/100g (1.2) in Tahitian, respectively. Potassium was the most abundant mineral, ranging from
1.29% in Mana Ulu to 1.75 % in Moi.
Calcium showed a range of 0.11 – 0.23% with Bun-long having the lowest and Moi having the
highest values, which were significantly (p<0.05) different. Manganese results showed a range of 5.23
– 12.46%, with Mana Ulu having the lowest and Moi having the highest values, which were
significantly (p<0.05) different. Iron results ranged from 1.67 to 7.74 mg/100 g, with Mana Ulu having
the lowest and Tahitian having the highest values, which were shown to be significantly (p<0.05)
different. In contrast, the concentrations of Molybdenum and Selenium were below the limit of
quantification.
3.4.2 Physicochemical Properties

Starch properties of taro are vital for understanding its physical and functional characteristics
(Table 2). Based on a dry weight basis the total starch ranged from 19.8 to 40.8 g/100 g, with Mana
Ulu having the lowest and Tahitian having the highest values, which were significantly (p<0.05)
different. Taro starch granules diameter size ranged between 2.08 and 2.93 m, with Mana Ulu having
the smallest and Kauaʻi Lehua having the largest values, which were significantly (p<0.05) different.
The physicochemical properties of taro are vital in understanding its structure, texture,
rheology and interfacial properties, and composition when taro undergoes processing (Table 3). The
SC of the taro varieties ranged from 1.2 to 2.1 (g/g), with Mana Ulu exhibiting the lowest SC and
Tahitian exhibiting the highest SC, showing a significant (p<0.05) difference. The WAI of the taro
varieties ranges from 4.2 to 5.8 (g/g), with Kauaʻi Lehua exhibiting the lowest WAI and Tahitian
exhibiting the highest WAI values, which were significantly (p<0.05) different. In addition, WAI was
found to be significantly correlated with total starch (dwb), OAC, bulk density, FC, EA, and ES, with
27
correlation coefficients being 0.553, 0.793, 0.883, 0.907, 0.871, and 0.868, respectively (Table 6). The
WSI of taro varieties ranged from 15.7 to 33.3 g/100 g, with Kauaʻi Lehua exhibiting the lowest WSI
and Tahitian exhibiting the highest WSI values, which were significantly (p<0.05) different. Similarly,
WSI was significantly correlated with total starch (dwb) with a correlation coefficient of 0.621
(p<0.05). The BD of the taro varieties ranged from 0.589 to 0.691 g/mL, with Mana Ulu having the
lowest BD and Moi having the highest BD, which were significantly (p<0.05) different. Though BD
was not significantly correlated with total starch, it was found to be positively and significantly
correlated with OAC, FC, EA, ES, WAI, WSI, LGC, FS-60, and FS-80 (Table 6).
3.4.3 Functional Properties of Taro

Taro’s functional properties describe behaviors during preparation and cooking that will affect
the finished food product in terms of appearance, taste, and texture (Table 4) [230]. The WAC of the
taro varieties varied from 0.93 to 3.48 g/g, with Kauaʻi Lehua having the lowest WAC and Tahitian
having the highest WAC. However, Tahitian did not exhibit significantly (p<0.05) different WAC from
Bun-long or Moi. The OAC of the taro varieties ranged from 1.2 to 3.2 g/g, with Kauaʻi Lehua and
Mana Ulu exhibiting the lowest OAC and Tahitian exhibiting the highest OAC. However, Tahitian
did not show significantly (p<0.05) different results from Bun-long and Moi varieties. The OAC of
the five taro varieties showed a significant correlation with total starch (dwb) and WAC, with
correlation coefficient being 0.689 (p<0.01) and 0.802 (p<0.001), respectively (Table 6). The FC of the
five taro varieties ranged from 4.7 to 28.3 mL/100 mL, with Mana Ulu having the lowest and Tahitian
having the highest values, respectively, which were significantly (p<0.05) different. In addition, FC
was found to be significantly (p<0.05) correlated with OAC, BD, EA, ES, WAI, WSI, LGC, SC, GP,
FS-60, FS-80, and FS-100 (Table 6). The FS for taro varieties exhibited Tahitian variety having the
longest FS lasting till 80 minutes, while Mana Ulu had the shortest FS lasting till 20 minutes. In
addition, FS-40 minutes was found to be positively correlated with total starch (dwb) and WAC with
correlation coefficients being 0.921 (p<0.001) and 0.799 (p<0.001), respectively (Table 6). The EA of
the five taro varieties ranged from 20.0 to 36.7 %, with Kauaʻi Lehua having the lowest and Tahitian
having the highest, respectively. In addition, EA was positively and significantly correlated with total
starch (dwb) with a correlation coefficient of 0.56 (p<0.05) (Table 6). The ES of the five taro varieties
ranged from 12.7 to 32.7 %, with Mana Ulu having the lowest and Tahitian having the highest values,
respectively.
Gelling properties of taro are vital in understanding its phase transition for food processing
(Table 5). The lowest GP was Moi variety with a 62.3C, while Bun-long had the highest at 68.3C,
which were significantly different (p<0.05). The BP of the taro varieties ranged from 72.33C to
79.7C, with Tahitian having the lowest and Moi having the highest, respectively, which were
significantly (p<0.05) different. Bun-long and Tahitian taro varieties both had an LGC of 10 g/100
mL whereas Kauaʻi Lehua had the highest LGC of 14 g/100 mL.
3.5 DISCUSSION
Taro (Colocasia esculenta) proves to be a good source of essential nutrients, with certain varieties
exhibiting greater nutrient concentrations than others and dietary carbohydrate source for food
production and health. The variations in nutrient concentrations amongst the studied varieties are
likely due to genetic differences because growing conditions (i.e. plot, planting distance, and planting
period) were identical. In addition, the high level of variability, not only in the mineral contents but
also in other nutrient contents, has been studied on taro germplasm to further understand nutrient
differences amongst taro varieties and has been found to have narrow genetic variation [231].
28
3.5.1 Nutritional Properties
Taro has superior nutritional value compared with other tuber crops, such as: potato, sweet
potato, and cassava [60, 61]. Studies have shown that potassium is the most abundant mineral in taro,
along with Mg, P, Ca [75-77]. This was confirmed with the results of the present study. These results
were similar to other reports on potassium content of taro varieties from: Vanuatu with a range of
1.60% to 2.24% [27], and Taiwan ranging from 2.25% to 4.14% [75], but higher than the values
reported in samples from Hawaiʻi with a range of 0.35-0.86% [29], and Ghana with a range of 0.76-
1.00% [232]. The high potassium content can be explained by the high requirement of root crops for
potassium due to being valuable sources of carbohydrates [27]. Potassium is known to influence the
metabolism of sugars, their polymerization, and the synthesis of starch [233]. From a health stand
point, potassium is one of the most important intracellular ions and is essential for the homeostatic
balance of body fluids. It is involved in the transfer of phosphate from ATP to pyruvic acid, and
probably plays a role in other enzymatic reactions [27]. In addition, Mergedus et al. [27] calculated the
percentages of recommended daily intakes (RDIs) of the essential minerals, Ca, Cu, Fe, Mg, P, and
Zn, based on an average consumption of 200 g of fresh taro corms per day in regard to children aged
4-8 years and adult females and males of 19-50 years old. Based on the average mineral content in the
central part of taro corms and the current recommended daily allowance/acceptable intake (RDA/AI)
values provided by the Food and Nutrition Board [234], for children aged 4-8 years taro was found to
make substantial contributions for Mg, Ca, P, An, Fe, and Cu, for males aged 19 to 50 taro made
substantial contributions for Mg, P, Ca, and Fe, and for females aged 19 to 50 taro made substantial
contributions for Mg and Fe [27]. Concentrations of most minerals (P, Mg, Fe, Cu, and Zn) were
found to be higher in the upper and the central parts of a taro corm [27]. Overall, taro has good
mineral content required for human health.
3.5.2 Physicochemical Properties

The physicochemical properties of taro are crucial for understanding how processing affects
the preservation of the food products, nutritional qualities, and development of organoleptic
properties [235].
SC was found to be highest in the Tahitian variety (Table 3). This can be explained when starch
molecules are heated in excess water, the semi-crystalline structure is broken, and water molecules
associate by hydrogen bonding to hydroxyl groups exposed on the amylose and amylopectin
molecules. This association causes swelling and increases granules size and solubility [236]. The
swelling capacity illustrates the interactions of the polymeric chains comprising the amorphous and
crystalline granule fractions [237]. In addition, low protein and high carbohydrate contents in taro
flour provide water easy access to starch granules and lead to better swelling ability [238]. These results
are comparable to the SC of other taro varieties in other countries, including in South Africa SC
averaging around 1 g/g at 55C and 2 to 6 g/g at 65C [239]. However, the SC values observed in the
present study were lower than those of taro varieties in: Thailand ranging 11.0-17.4 g/g dry flour [240]
and South Africa ranging from around 20 g/g to 25 g/g at 75C [239]. SC is a measure of hydration
capacity. Because the determination is a weight measure of swollen starch granules and their occluded
water [236], that may explain the difference in SC of taro in other countries. SC is a vital characteristic
as food quality is often connected with retention of water in the swollen starch granules that affects
the texture of foods [241]. In addition, previous studies have shown the dietary inclusion of fiber high
in SC and WAC was shown to promote satiety and reduced food intake [242, 243].
Taro’s high WAI and WSI can be explained by their significant (p<0.05) correlation with starch
content, which illustrates the importance of starch in the physical properties of taro (Table 6). WAI
represents the volume occupied by starch after swelling in excess water, which maintains the integrity
29
of starch in aqueous dispersion [62]. Water absorption and heating of the starch dispersion break the
hydrogen bonds responsible for granule cohesion, partially solubilizing the starch [244]. Water
penetrates the interior of the starch granule, hydrating linear fragments of amylopectin [245], leading
to irreversible swelling, and increasing the granule size and the paste viscosity. This can be seen with
other taro varieties that have reported WSI of 18.55-25.64 g/100 g for taro cultivated in Cameroon
[246] and 2.443 – 6.013 g/100 g for taro cultivated in India [62]. In addition, a study comparing the
physiochemical and functional properties of taro, rice, pigeon pea flour, and their blends, found that
taro had the greatest WSI [62]. It was also observed that with an increase in the amount of taro flour,
the WAI and WSI of blends increased [62]. The increase in WSI with the addition of taro flour is of
significance since it gives an indication that taro flour addition can be used to increase the amount of
soluble materials such as starch and amino acids which can be easily digestible [62]. Water absorption
results in swelling, and the swelling power of flour depends on the concentration of protein, starch
and fiber [247]. The high WAI and WSI of taro is important to the food industry because taro can be
a good nutrient dense flour alternative and an additive during food production to improve functional
properties.
Bulk density (BD) is the mass of bulk solid that occupies a unit of volume of a bed [248]. The
BD of taro in the present study (Table 3) was similar to those observed in earlier studies that reported
0.480–0.689 g/mL from taro cultivated in India [62], 0.57-0.71 g/mL from taro cultivated in
Cameroon [246], 0.689 g/mL from taro cultivated in India [94], 0.43-0.49 g/mL from taro cultivated
in Hawaiʻi [249], and 0.14-1.18 g/mL from taro cultivated in Nigeria [228]. BD is dependent on the
measure of heaviness of solid samples and upon the particle size of the sample [228]. The high bulk
density of taro implies potential applications in the food industry, especially for determining packaging
requirements and material handling [228]. Furthermore, taro is a potential nutrient additive to help
reduce paste thickness for convalescence, formulation of complementary foods, and early child
feeding [62].
The high starch content of taro is of great value for the food industry, diet, and human health.
Digestible starches, such as amylose and amylopectin, are broken down (hydrolyzed) by the enzymes
-amylases, glucoamylase and sucrase–isomaltase in the small intestine to yield free glucose that is
then absorbed [103]; thus, starch is a vital carbohydrate source in the human diet [250]. The results of
the present study (Table 2) are lower in starch concentration than what have been previously reported
for total starch in taro cultivated from: Turkey ranging between 58.5 and 68.8 g/100 g [251], Thailand
averaging 71.53 g/100 g [52], India averaging 67.42 g/100 flour [62], and Hawaiʻi ranging between
73.0 – 76.1 g/100 g [216]; however, it was similar to China averaging 18.8 g/100 g [252]. This may be
due to differences in genetics and growing conditions. In addition, the taro in the present study was
cooked to simulate real life application; whereas the other studies obtained starch content from raw
taro. Cooking has been shown to decrease starch content [253, 254], which may also explain the lower
starch content in this study compared to previous reports. Due to its high starch content, taro would
be a good carbohydrate alternative in the formulations for the baking industry, as starch is one of the
components responsible for the structure and properties of bakery products [210]. In addition, starch
is also used in the preparation of diverse types of pasta, in the preparation of noodles and those
intended for extrusion, and in the formulation of instant foods and fried foods [210], in which taro
could also be used as a great carbohydrate alternative.
Furthermore, taro starch granules show small irregularly polygonal shape and agglomerate into
clusters (Figure 4). The unique starch granule morphology of taro may be due to the biological origin
and physiology of the plant and the biochemistry of the amyloplast [205]. Furthermore, amylose and
amylopectin contents may play an important role in the control of the starch granule shape and size
[213]. The small size of taro starch granules in the present study corroborates previous studies
30
reporting taro starch granule size ranging: 1.1 to 4.2 m in India [209], 1.0 to 2.0 m in India [205],
2.3 to 4.0 m in Brazil [207, 255], 0.6 to 6 m in Mexico [256], 1.3 to 2.2 m in Thailand [240], 0.3 to
4.3 m in Hawaiʻi [216], 1.3 to 2.2 m in China [257], 0.5 to 5.0 m in Venezuela [18], 5 m in Nigeria
[19], and averaged 2.5 m in Turkey [104]. The small starch granule size of taro offers great benefits
to the food industry, including entrapping flavoring compounds like vanillin and as a potential fat
substitute [205]. The size of starch granules from food crops is of great importance as it affects the
behavior of the food during food processing [228]. This can be explained by small starch granules
being more resistant to rupture and loss of molecular order [91]. Moreover, small starch granule size
is useful for bread and noodle production [92]. In addition, taro can be used for cosmetic formulations,
such as face powder, and in aerosol-dispensing systems [19, 258]. Furthermore, the small granules size,
may also have industrial applications, such as being a good filling agent for biodegradable polyethylene
film [20]. Taro starch with small granule size is easily digested and has high bioavailability due to
efficiency of digestion and absorption [257]. As such, taro is a great food ingredient alternative for
baby foods, in diets of people allergic to cereals, and in children sensitive to milk [205].
In addition, all tested taro varieties illustrated a type A x-ray crystallinity pattern (Figure 5),
which is similar to those of taro cultivated in India [205, 209, 259], Malaysia [260], China [257], Hawaiʻi
[216], Mexico [258], Brazil [207], and Turkey [104]. Two crystalline structures of starch have been
identified as ‘A’ and ‘B’ type, which contain differing proportions of amylopectin [105]. A-type
starches are found in cereals, while B-type starches are found in tubers and amylose-rich starches [105].
A third type called ‘C-type’ appears to be a mixture of both A and B forms and is found in legumes
[105]. Typically, tuber starches have B or C type pattern; however, taro varieties exhibit type-A pattern,
which is characteristic of cereal starches [257]. The difference between A- and B-type crystallites is
due to the packing of double helices in the crystal unit cell and the number of water molecules
stabilizing them [257]. In A-type crystal pattern, the double helices are packed in a monoclinic unit
cell, an arrangement corresponding to densely packed structure with only four water molecules per
unit cell [261]. Benefits of A-type crystals include being more resistant to enzyme digestion and higher
melting point than to B-type starches [257, 262]. Furthermore, A-type starch is more resilient to
human digestive enzymes in the upper gut and has been associated with health benefits, such as a
slower release of glucose into the bloodstream resulting in reduced postprandial glycemic and insulin
responses [15, 263]. Thus, taro’s starch composition may play a vital role in health.
3.5.3 Functional Properties

Taro’s relatively high gelling and boiling point are vital to understanding the phase transition
during food processing (Table 5). The results of the gelling points in the present study are comparable
to those in previous studies of taro varieties from: Nigeria ranging 58.5C to 72.5C [228], and China
averaging 73.6C [257]. Gelling point is the temperature at which a food solution forms an observable
thicker consistency when heat is applied [264], which is vital for processing and cooking of foods.
Boiling point is the temperature at which the vapor pressure of the liquid equals the pressure of the
surrounding gases [265]. The relatively high boiling points of the taro varieties could be due to the
presence of other components in taro flours, such as proteins and lipids, that might obstruct the
swelling of starch granules and increase the amount of heat required to reach the final swelling [228].
In addition, pectins, starches and gums have been shown to form strong gels [230]; therefore taro’s
high starch content further alludes to stronger gelling. The results of the present study are comparable
to previous studies reporting BP of taro from: Nigeria ranging 81.5C to 88.5C [228], and Hawaiʻi
80C to 88.5C [216]. Thus, the relatively high gelling and boiling points provide vital information for
processing taro into other food products.
31
The different WAC of the five taro varieties can be attributed to the presence of varying
amounts of starch (Table 4), which is confirmed by a significant (p<0.001) correlation between WAC
and total starch content (Table 6). This can be explained by the non-starch components, such as
mucilage, that have been suggested to contribute to the WAC of taro flours [19]. Previous studies have
reported similar results of WAC with 1.34-2.45 g/g for taro cultivated in India [62], 2.2 g/g for taro
cultivated in India [94], 2.42-3.21 g/g for taro cultivated in Cameroon [246], 1.50-1.80 g/g for taro
cultivated in Hawaiʻi [249], and 2.1 g/g-3.6 g/g for taro cultivated in Nigeria [228]. In addition, the
difference in WAC of the taro varieties can be attributed to the degree of attachment of the water
molecule hydroxyl groups to form hydrogen and covalent bonds between starch chains [266]. As such,
the taro varieties with high WAC may have more hydrophilic components, such as polysaccharides
[62]. This was further shown in a study looking at different flour blends which observed that an
increase in the amount of taro in the flour blend also increased the overall WAC [62]. The high WAC
of taro suggests its great potential to be used as food additives as a thickener or gelling and viscosity
increasing agent. WAC is the ability of the starch or flour to hold water and swell, which improves the
consistency in food [267]. As such, taro may be a potential additive for food formulations that include:
soups, gravies, sausage, dough, processed cheese, and bakery products [62].
The OAC is the ability of starch or flour to bind fat by capillary attraction, which is of great
interest to the food industry as it increases flavor retention and mouthfeel of foods [62]. Previous
studies have reported similar OAC to the present study, with 1.12-1.89 g/g from taro cultivated in
India [62], 1.04-2.51 g/g from taro cultivated in India [94], 1.74-1.86 g/g for taro cultivated in
Cameroon [246], and 2.50-3.35 g/g from taro cultivated in Nigeria [228]. The high OAC of taro
suggests its great potential for structural interaction in food production for flavor retention,
improvement of palatability, and extension of shelf life [62]. This can be further explained by the
different non-polar side chains of taro that may bind the hydrocarbon side chains of the oil forming
lipids [268]. In addition, the high OAC of taro can be attributed to the intrinsic factors like amino acid
composition, protein conformation, and surface polarity of hydrophobicity [62]. As such, taro may
potentially serve as an additive in meat products, fried foods, doughnuts, mayonnaise, bakery products,
and fat filled foods [62].
Foam capability (FC) and foam stability (FS) properties of taro are determined by its ability to
rapidly absorb on the air-liquid interface during whipping or bubbling, and by its ability to form a
cohesive viscoelastic film by way of intermolecular interactions, respectively [269]. Previous studies
found similar FC results to the present study (Table 5), with 29-31 mL/100 mL of taro cultivated in
Hawaiʻi [249], 18-27 mL/100 mL of taro cultivated in Cameroon [246], 9 mL/100 mL of taro
cultivated in India [94], and 4.46-18.28 mL/100 mL of taro cultivated in Nigeria [228]. The foaming
of the flours is suggested to be due to proteins that form a continuous cohesive film around the air
bubbles in the foam, as well as a lowering of the surface tension at the water-air interface [62, 270].
Furthermore, the FC of taro flour could be due to its mucilage, a soluble glycoprotein, content [249].
Foams are used to improve texture, consistency and appearance of foods [271], which can be seen
with taro FC having positive and significant (p<0.001) correlations with other texture and flavor
improving properties, including: OAC and BD. As such, the high FC of taro may be useful as an
ingredient to improve textural and leavening characteristics of food products, such as: ice-cream, cakes
or topping and confectionery products [62]. Similarly, FS is important in the food industry for
products that require whipping agents to maintain the whip for long periods of time [62]. The present
results corroborate previous studies that found FS had 100% stability until 20 minutes from taro in
India [94], 100% stability until 4 hours at 25C from taro in Hawaiʻi [249], and 100% stability until 80
minutes from taro in India [62]. These long FS periods can be explained by the ability of the film to
form around trapped air bubbles and remain intact without draining, in which stable foams can only
32
be formed by the high surface active solutes [62]. This may be due to taro’s high starch content, as its
FS-40 was found to be positively and significantly (p<0.001) correlated with total starch content, with
a decrease correlation significance as the time increased. Taro with high FS can be a beneficial
ingredient for food processing, especially for baked and confectionery products [62].
The high EA activity results of the present study (Table 4) are confirmed by previous studies
that found EA of 40 ± 1.52 mL/100 mL and ES of 42.3 ± 0.57 mL/100 mL from taro in India [62].
Taro’s high EA and ES activity may be attributed to its high starch content, as total starch was
positively and significantly (p<0.05) correlated with EA (Table 6). This may be explained as
emulsifying properties have been reported to be greatly influenced by the presence and composition
of soluble proteins, as well as components other than proteins, such as carbohydrates [62, 270]. More
specifically, properties of taro have been attributed to the hydrophobicity, solubility, and
conformational stability of the proteins [62]. Furthermore, ES can be additionally explained because
proteins can act as surface active agents that form and stabilize the emulsion by creating electrostatic
repulsion on oil droplet surface [62]. As such, taro can be used to entrap flavoring compounds, like
vanillin, for flavor enhancement in food processing [272, 273]. Increasing EA and fat binding during
food processing can be beneficial for food products such as meat products, salad dressings, frozen
dressing, and mayonnaise [62]. Furthermore, these results indicate that taro can also be a useful
additive for the stability of fat emulsion, especially for production of sausages, soups, and cakes [62].
The results of taro LGC from the present study (Table 5) were corroborated with previous
reports of LGC at 6 g/100 mL from taro cultivated in Hawaiʻi [249], 10 g/100 mL from taro cultivated
in India [62], and 6.0 to 10 g/100 mL from taro cultivated in Nigeria [98]. In addition, flours of taro
cultivated in India formed relatively firm gels at a significantly lower (p<0.05) concentration (10 g/100
mL) than pigeon pea flour (12 g/100 mL) [62]; however, the variety of taro was unknown. The various
LGC levels can be due to the starch interaction during heat treatment and the different ratios of
nutrient components, such as proteins, carbohydrates, and lipids [62, 249]. In addition, it has been
reported that exposure of hydrophobicity and sulfhydryl of proteins greatly affects gelation [274]. As
such, the low LGC of taro has potential as a nutrient additive to increase gel forming in food products,
such as gravies and soups, at lower concentrations [62].
3.6 CONCLUSION
This study provides a comprehensive analysis of the nutritional, physicochemical and

functional properties of five taro varieties, Bun-long, Mana ulu, Moi, Kauaʻi Lehua, and Tahitian. The
five varieties showed significant differences in their nutritional, physiochemical, and functional
properties, although all hold promise for food production and health implications. All taro varieties
had high mineral content, proving to be a great food source of certain nutrients and a potential additive
to increase the nutrient contents and health benefits of food products. The high WAC of taro flour
makes it a good body providing agent and can thus be used as a thickener or gelling agent in food
production [94, 104]. In addition, taro’s small starch granule size lends itself to be a great
hypoallergenic alternative for individuals suffering food allergies or sensitivities. Not only does the
small granule size provide health benefits, but taro can also act as a good filling agent for biodegradable
polyethylene film [20, 23]. The high EA and OAC of taro are of great value during food production
as they increase flavor retention and mouthfeel of foods. Understanding the nutritional,
physicochemical, and functional properties of taro provides the foundation for the creation of new
food products, alternative flour sources, and potential health benefits. As such, results of this study
further demonstrate taro’s great potential to be used in the food industry as an alternative flour source
for improving food quality and human health.
33
ACKNOWLEDGMENTS:
Authors are grateful to Agricultural Diagnostic Services of the University of Hawaiʻi.

Supported in part by the Grants and Awards Program, Graduate Student Organization (GSO); USDA-
NIFA Hatch Grant No.HAW02034H.
34
Table 3.1. Chemical composition of taro corms
Mean and standard deviation (St. Dev.). The results are given on dry weight basis.
Nutrients Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD
Proximates (g/100g)
Moisture 69.46 (1.5) a 67.82 (1.8) ab 65.44 (2.7) ab 63.34 (2.3) b 68.52 (2.4) ab 5.87
Dry Matter 29.59 (1.5) a 23.58 (2.3) b 22.88 (2.0) b 24.58 (2.2) ab 20.25 (2.2) b 5.53
Ash 1.08 (0.2) ab 0.86 (0.1) bc 1.17 (0.2) a 0.81 (0.0) bc 0.73 (0.1) c 0.30
Crude Protein 3.33 (0.8) b 4.19 (1.2) ab 6.57 (1.1) a 5.37 (0.7) ab 3.09 (0.7) b 2.47
Crude Fat 1.49 (0.3) a 1.63 (0.1) a 1.61 (0.3) a 1.26 (0.2) a 1.54 (0.5) a 0.79
Neutral Detergent Fiber 17.28 (1.8) bc 14.67 (1.0) c 25.52 (1.1) a 20.37 (2.7) b 18.94 (1.4) bc 4.59
Acid Detergent Fiber 4.86 (1.6) a 4.1 (0.2) a 6.51 (1.4) a 4.75 (1.2) a 4.08 (0.8) a 3.08
Lignin 0.64 (0.1) b 0.82 (0.1) b 1.39 (0.3) a 0.91 (0.1) b 0.81 (0.1) b 0.38
Cellulose 3.8 (0.4) b 3.39 (0.3) b 5.31 (0.9) a 3.78 (0.2) b 3.63 (0.6) b 1.48
Minerals (%)
Phosphorus (P) 0.17 (0.1) a 0.08 (0.0) a 0.23 (0.1) a 0.36 (0.4) a 0.1 (0.0) a 0.49
Potassium (K) 1.68 (0.2) a 1.29 (0.1) a 1.75 (0.1) a 1.45 (0.2) a 1.51 (0.3) a 0.56
Calcium (Ca) 0.11 (0.0) b 0.16 (0.0) ab 0.23 (0.1) a 0.15 (0.1) ab 0.13 (0.0) b 0.10
Magnesium (Mg) 0.15 (0.1) a 0.11 (0.0) a 0.14 (0.1) a 0.09 (0.0) a 0.10 (0.0) a 0.12
Sodium (Na) 0.0 (0.0) a 0.0 (0.0) a 0.01 (0.0) a 0.01 (0.0) a 0.01 (0.0) a 0.01
Trace Minerals (mg/100 g)

Boron (B) 0.58 (0.7) a 0.54 (0.5) a 0.58 (0.7) a 0.52 (0.7) a 0.52 (0.7) a 1.84
Copper (Cu) 0.57 (1.5) b 0.34 (1.5) b 0.97 (1.5) a 0.47 (0.6) b 0.58 (1.3) b 3.58
Iron (Fe) 3.24 (2.5) d 1.67 (2.1) e 7.08 (2.6) b 5.35 (3.1) c 7.74 (1.2) a 0.64
Manganese (Mn) 7.70 (2.0) b 5.23 (3.5) c 12.46 (9.1) a 6.62 (3.7) b 7.97 (2.1) b 1.67
Molybdenum (Mo) nq nq nq nq nq N/A
Zinc (Zn) 7.40 (2.6) bc 5.23 (5.3) d 8.63 (5.2) b 6.48 (3.0) cd 13.68 (8.3) a 1.33
Selenium (Se) nq nq nq nq nq N/A
a-e
Means with different letters within the same row differed significantly by Tukey’s HSD (p<0.05),
nq = not quantifiable
N/A = not applicable
35
Table 3.2 Total starch content of taro varieties and their starch granule size and shape
Mean and standard deviation (St. Dev.). The results are given on a “dry weight basis” (dwb).
Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD

Total Starch (g/100g) 38.9 (2.7)a 19.8 (1.2)c 34.1 (1.9)b 17.7 (1.3)c 40.8 (1.3)a 4.79
content
Size (m) 2.61 (0.6)ab 2.08 (0.6)b 2.30 (0.7)b 2.93 (0.7)a 2.20 (0.6)b 0.41
Shape Small rounded, Small rounded, Small rounded, Small rounded, Small rounded,
irregular irregular irregular irregular irregular N/A
polygonal polygonal polygonal polygonal polygonal
a-d
Means with different letters within the same row differed significantly by Tukey’s HSD (p<0.05)
N/A= not applicable
36
Table 3.3 Physicochemical properties of taro varieties

Water Absorption
Index (WAI) (g/g) 4.53 (0.3) b 4.18 (0.2) b 5.68 (0.2) a 4.32 (0.1) b 5.81 (0.1) a 0.50
Water Solubility Index 19.33 (2.9) bc 17.04 (2.3) c 26.70 (5.0) ab 15.72 (2.8) c 33.30 (3.3) a 9.17
(WSI) (g/100 g)
Swelling Capacity (SC) 1.33 (0.2) b 1.63 (0.2) ab 1.67 (0.1) ab 1.20 (0.2) b 2.07 (0.2) a 0.47
(g/g)
Bulk Density (BD) 0.62 (0.01) b 0.59 (0.01) b 0.69 (0.01) a 0.62 (0.01) b 0.66 (0.02) a 0.03
(g/mL)
a-d
37
Table 3.4. Functional properties of taro varieties

2.43 (0.5) ab 2.25 (0.1) b 2.77 (0.1) ab 0.93 (0.6) c 3.48 (0.5) a 1.13
Water Absorption Capacity (g/g)
2.24 (0.1) ab 1.25 (0.4) b 2.68 (0.2) a 1.25 (0.5) b 3.15 (0.7) a 1.17
Oil Absorption Capacity (g/g)
12.33 (3.5) b 4.67 (1.2) c 23.67 (2.1) a 15.00 (2.6) b 28.33 (2.5) a 6.72
Foam Capacity (mL/100 mL)
Foam Stability (%)
100 (0.0) a 100 (0.0) a 100 (0.0) a 100 (0.0) a 100 (0.0) a 0.00
20 minutes
100 (0.0) a 99.7 (0.6) a 100 (0.0) a 99.3 (1.2) b 100 (0.0) a 0.824
40 minutes
99 (1.0) ab 97.3 (1.2) b 100 (0.0) a 100 (0.0) a 100 (0.0) a 1.558
60 minutes
95.3 (1.2) bc 93.3 (2.3)c 98 (2.0) ab 100 (0.0) a 100 (0.0) a 2.818
80 minutes
94 (2.0) a 92 (2.0) a 95 (1.0) a 99.3 (1.2) a 99.3 (1.2)a 5.037
100 minutes
92.7 (1.2) ab 89.3 (1.2) b 95 (1.0) ab 98.7 (1.2) ab 98.7 (1.2) a 5.397
120 minutes
22.00 (2.6) b 19.33 (4.2) b 32.67 (3.1)a 20.00 (4.0) b 36.67 (2.3) a 8.90
Emulsifying Activity (%)
21.33 (3.1) b 12.67 (3.1) c 26.67 (1.2) ab 20.67 (2.3) b 32.67 (3.1) a 7.07
Emulsifying Stability (%)
a-d
38
Table 3.5 Gelling properties of taro varieties

Gelling Point (°C) 68.33 (1.5) a 67.00 (1.0) ab 62.33 (2.5) b 67.67 (1.2) a 63.33 (2.5) b 4.99
Boiling Point (°C) 74.67 (3.1) ab 77.67 (1.5) ab 79.67 (2.1) a 78.33 (2.1) ab 72.33 (2.5) b 6.20
Least gelation concentration (g/100 mL)

2 No gelation No gelation No gelation No gelation No gelation N/A
8 Gel No gelation Gel No gelation Gel N/A
10 Firm Gel Gel Firm Gel No gelation Firm Gel N/A
12 Firm Gel Firm Gel Firm Gel Gel Firm Gel N/A
14 v. firm gel Firm Gel v. firm gel Firm Gel v. firm gel N/A
16 v. firm gel v. firm gel v. firm gel Firm Gel v. firm gel N/A
18 v. firm gel v. firm gel v. firm gel v. firm gel v. firm gel N/A
20 v. firm gel v. firm gel v. firm gel v. firm gel v. firm gel N/A
a-b
Means with different letters within the same row differed significantly Tukey’s HSD (p<0.05)
39
Table 3.6 Correlation analysis of physicochemical and functional properties of taro varieties
Nonparametric correlation coefficients (Spearman’s rank) between dry weight basis of TS, WAC, OAC, BD, FC, EA, ES, WAI, WSI, SC, GP, BP, GS, FS-
40, FS-60, FS-80, FS-100, and FS-120.
Variables TS WAC OAC BD FC EA ES WAI WSI SC GP BP GS FS-40 FS-60 FS-80 FS-100 FS-120
0.830 0.689 0.560 0.553 0.621 -0.616 0.921 0.724 0.595
TS 1 0.456 0.316 0.392 0.506 -0.407 -0.223 0.234 0.428
*** ** * * * * *** ** *
0.802 0.687 0.634 0.767 0.691 -0.664 0.799 0.623 0.673 0.717
WAC 1 0.402 0.417 0.481 -0.474 -0.339 0.505
*** ** * *** ** ** *** * ** **
0.709 0.752 0.830 0.753 0.793 0.811 0.589 -0.523 0.517 0.720 0.639 0.627 0.650
OAC 1 -0.430 -0.447
** *** *** *** *** *** * * * ** ** * **
0.849 0.820 0.764 0.883 0.689 -0.753 0.768 0.623
BD 1 0.414 -0.001 -0.133 0.253 0.414 0.276
*** *** *** *** ** *** *** *
0.853 0.936 0.907 0.747 0.547 -0.693 0.675 0.715 0.641
FC 1 -0.387 -0.214 0.108 0.502
*** *** *** *** * ** ** ** **
0.807 0.871 0.794 0.701 -0.750 0.777 0.702 0.547 0.567
EA 1 -0.196 -0.416 0.406
*** *** *** ** *** *** ** * *
0.868 0.779 0.556 -0.663 0.598 0.643 0.761 0.589
ES 1 -0.439 -0.215 0.155
*** *** * ** ** ** *** *
0.854 0.672 -0.788 0.752 0.855 0.624 0.566
WAI 1 -0.299 -0.318 0.411
*** ** *** *** *** ** *
0.668 -0.831 0.693 0.738 0.616 0.578
WSI 1 -0.309 -0.445 0.466
** *** ** ** ** *
0.568 0.779 0.678 0.813
SC 1 -0.481 -0.389 -0.219 0.349
* *** ** ***
-0.706 -0.570
GP 1 -0.149 0.362 -0.243 -0.328 -0.217
** *
-0.565 -0.635
BP 1 0.223 -0.173 -0.116 -0.502
* *
-0.699
GS 1 -0.507 -0.396 -0.050 -0.281
**
0.578
FS-40 1 0.510 0.127 0.420
*
0.644
FS-60 1 0.180 0.239
**
0.550 0.670
FS-80 1
* **
0.893
FS-100 1
***
FS-120 1
Spearman Correlation Coefficient * total starch (TS), water absorption capacity (WAC), oil absorption capacity (OAC), bulk density (BD), foam capacity (FC),
emulsifying activity (EA), emulsifying stability (ES), water absorption index (WAI), water solubility index (WSI), swelling
-1 -0.5 0 0.5 1 capacity (SC), gelling point (GP), boiling point (BP), granule size (GS), foam stability (FS-40, FS-60, FS-80, FS-100, and FS-120)
*P < 0.05 **P< 0.01 ***P<0.001
40
A B C
D E
Figure 3.1 Taro corms: Bun Long (A), Mana Ulu (B), Moi (C), Kauaʻi Lehua (D), Tahitian (E)
41
Figure 3.2. Flow chart of taro processing
42
Aa B C
D E
Figure 3.3 Cross sectional view of taro corm flesh: Bun Long (A), Mana Ulu (B), Moi (C), Kauaʻi Lehua (D), Tahitian (E).
43
A B C
D E
Figure 3.4. Scanning Electron Microscopy (SEM) of starch granules from taro varieties: Bun Long (A), Mana Ulu (B), Moi (C), Kauaʻi Lehua (D),
Tahitian (E).
44
Figure 3.5. X-ray Diffraction (XRD) of taro varieties: Bun-Long, Mana Ulu, Moi, Kauaʻi Lehua, Tahitian.
*Abstract artifact
45
46
CHAPTER 4 PREBIOTIC ACTIVITY SCORES OF TARO
(COLOCASIA ESCULENTA) WITH DIFFERENT
LACTOBACILLUS SPECIES
4.1 ABSTRACT
Prebiotic potential of dietary fiber is dependent on its hydrolysis and utilization by probiotic
species. Taro is rich in starch and has potential to serve as a prebiotic source. Dietary fiber and resistant
starch in this food may promote the growth and activity of specific probiotic species, such as
Lactobacillus spp., after they escape digestion in the upper gut. However, prebiotic utilization by
probiotics is species-specific, which ultimately confers health benefits to the host. The prebiotic
potential can be quantitatively determined through the prebiotic activity score calculation, which
reflects the ability of a prebiotic carbohydrate to support the growth of probiotic bacteria relative to
an enteric bacteria and relative to the growth on a non-prebiotic substrate, such as glucose. As such,
this study aimed to determine the prebiotic potential of different taro varieties. The concentrations of
dietary fiber and resistant starch in Bun-long, Mana Ulu, Moi, Kauaʻi Lehua, and Tahitian were
determined. These taro varieties also underwent in vitro human digestion simulation and then tested
individually with Lactobacillus acidophilus, L. paracasei, L. plantarum, L. rhamnosus, and Escherichia coli. Inulin
and fructooligosaccharides (FOS), two established prebiotics, were used as controls. Cell counts of
the bacteria were determined at 0 h and 24 h during incubation and used to calculate prebiotic activity
scores of each taro variety with different Lactobacillus strains. The results of this study illustrated that
the taro varieties, Tahitian, Bun-long, and Moi, exhibited significantly (p<0.05) higher total dietary
fiber and resistant starch contents than Mana Ulu and Kauaʻi Lehua. This translated to the highest
prebiotic activity scores coming from pairings of L. paracasei with Tahitian and inulin, suggesting that
the probiotic species paired with Tahitian will more likely be active in the gastrointestinal tract, similar
to the pairing with inulin, a well-established prebiotic. The present study provides evidence for taro
varieties to serve as sources of dietary prebiotics and has shown their probiotic species-specific
utilization. It lays the groundwork for further explorations of potential health benefits of taro as
prebiotics or part of synbiotics.
47
4.2 INTRODUCTION
Taro (Colocasia esculenta) is a nutrient dense root crop, potentially superior to other root crops
[29]. Specifically, taro is high in dietary fibers, with starch content of 70-80 g/100 g [203] on a dry
weight basis and dietary fiber content of 4.1% on a fresh weight basis [28]. Based on the dietary data
of Bun-long and Lehua taro, Huang et al. [29] estimated that taro consumption of 1 lb (454 g) per
meal by Pacific Islanders can fulfill the Dietary Guidelines for Americans 2015-2020 dietary fiber
recommendation of 25 g per day (dietary guidelines). Dietary fiber may promote the growth of
beneficial bacteria in the colon; thus, taro can be a potential dietary source of prebiotics.
Prebiotics are defined as non-digestible food ingredients that beneficially affect host health by
selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon
[275]. Prebiotic characteristics include improved bowel function, removal of carcinogenic toxins,
reduced risk of colon cancer, and preferential growth of protective bacteria over pathogenic strains
[31-33]. A single food may contain various prebiotics that differ in their effect on gut health. Resistant
starch (RS) has gained attention as a new source of dietary fiber and a potential prebiotic [105]. RS is
the portion of starch or starch hydrolysis products that escapes digestion in the stomach and small
intestine and enters the colon for fermentation [99]. RS also has physiological beneficial effects such
as: improving colonic health, increasing absorption of minerals, assisting in the control of diabetes,
lowering plasma triglyceride and cholesterol levels, and being a substrate of bacterial fermentation for
the production of short-chain fatty acids (SCFA) [99, 105, 276, 277]. The efficacy of a prebiotic
depends on its ability to interact with different probiotic species in the gut microbiome. Thus, to
improve gut health, it is important to understand the prebiotic properties of food, especially with
probiotics.
Probiotics, as defined by The International Scientific Association for Probiotics, are ‘live
microorganisms that, when administered in adequate amounts, confer a health benefit on the host’
[122, 123]. The Agency for Healthcare Research and Quality (AHRQ) released an NIH-sponsored
report in 2011 reviewing the safety of probiotics in general, and Lactobacillus was one of the six
organisms listed in the report which included data from 622 studies [278]. Furthermore, Lactobacillus
spp. have a reputed Generally Recognized as Safe (GRAS) status [279]. The health-promoting
properties of specific strains belonging to the genus Lactobacillus have led to their application in
products that are marketed as probiotic foods or probiotic pharmaceutical preparations [280].
Lactobacillus species such as L. paracasei, L. plantarum, L. acidophilus, and L. rhamnosus (LGG) have been
found to have beneficial properties including, but not limited to: antimicrobial activity [281], helping
treat diarrhea [281, 282], improving signs of acute gastritis [283], improving immune response [284],
and lowering triacylglycerol levels [285]. In addition, several probiotic bacteria are also used in the
food industry to ferment food products, such as: dairy foods, kimchi, and sauerkraut [286].
Ultimately, the ability of probiotics to metabolize prebiotics results in their selective
enrichment in the gastrointestinal tract and the formation of lactic, acetic, and other short-chain fatty
acids (SCFA) that may be antagonistic to their intestinal competitors [287, 288]. Thus, prebiotics alone
or combined with probiotic bacteria in the form of synbiotics are believed to influence and improve
the gastrointestinal health of humans [289]. However, not all dietary fibers are suitable substrates for
selective growth of specific probiotic strains [286]. Fermentation of prebiotic carbohydrates is
dependent on the bacterial species [286, 287]. Bacterial species, especially probiotics, may metabolize
prebiotic carbohydrates differently [286]. Studies have shown that Lactobacillus ferments prebiotic
carbohydrate in a strain and substrate specific manner [290, 291].
Yet there are certain characteristics that provide information about the substrates prebiotic
potential. One such characteristic is the prebiotic activity score, which reflects the ability of a given
substrate to support the growth of an organism relative to other organisms and relative to growth on
48
a non-prebiotic substrate, such as glucose [286]. The prebiotic activity score method assesses prebiotic
activity through the combination of a prebiotic with specific strains of accepted probiotic bacteria
[13]. Carbohydrates with a positive prebiotic activity score mean they are metabolized just as well as
glucose by probiotic strains and are selectively metabolized by probiotics but not by enteric intestinal
bacteria, such as Escherichia coli [292]. Thus, a prebiotic activity score identifies combinations of
probiotics and prebiotics that could be added into food products and provide potential health benefits
[286].
A synbiotic product beneficially affects the host by improving the survival and implantation
of live microbial dietary supplements in the gastrointestinal tract and selectively stimulating the growth
and/or metabolism of intestinal health-promoting bacteria [291, 293]. Taro has high amounts of
dietary fiber; therefore it has the potential to serve as a prebiotic food [1, 23]. In addition, taro naturally
harbors yeast and lactic acid bacteria on the surface that can initiate the fermentation process without
a starter culture [124, 125]. Thus, taro may be a potential source of synbiotics. Dietary intervention
through food or food supplements containing live beneficial microbes and prebiotics could be a
possible step to improve the gut microbiota and human health. Therefore, the objective of this study
was to determine the prebiotic potential of five varieties of taro in relation to different Lactobacillus
spp.
4.3 METHODOLOGY
4.3.1 Taro Sample Preparation

Fresh full-grown taro corm varieties (Bun-Long, Moi, Tahitian Variety, Kauaʻi Lehua, Mana
Ulu) were selected because of their popular use for food preparation as: cooking, table taro, chip
production, and poi pounding [217]. The taro varieties were collected at Waimānalo Research Station
(Waimānalo, Hawaiʻi), were processed into 2 cm width cubes, weighed, and autoclaved at 121 °C for
15 minutes to simulate pressure cooking. The taro samples were freeze-dried to remove water content
for 48 hours at -80 °C, then subsequently for 72 hours at -51 °C and 0.021 mBar pressure (FreeZone
6 Liter Benchtop Freeze Dryer, Labcono, Kansas City, MO). Afterwards, the samples were weighed
and ground to 1mm size taro powder particles (Commercial Stainless steel Industrial Electric Peppe
Grain Mill, CGOLDENWALL). The respective taro corm variety powder were blended together and
treated as an individual sample and stored under anaerobic conditions until further use.
4.3.2 Total Dietary Fiber, Resistant Starch (RS), and Non-Resistant Starch
Freeze-dried taro samples were analyzed for total Dietary Fiber using Megazyme Total Dietary
Fiber kit (Megazyme International, Wicklow, Ireland) (AACC Method 32-21.01 and AACC Method
32-06.01) and resistant starch (RS) and non-resistant starch (NRS) using Megazyme Resistant Starch
assay kit (Megazyme International, Wicklow, Ireland) (AOAC Method 2002.02; AACC Approved
Method 32-40) following the manufacturer’s instructions.
4.3.3 In Vitro Human Digestion

Freeze dried taro samples were subjected to in vitro digestion. A negative control sample, blank,
was tested to assess the impact of residual digestive enzymes on the growth of Lactobacillus strains. A
positive control glucose was used to confirm the viability of the bacterial strains. Inulin and fructo-
oligosaccharide (FOS) were used as prebiotic controls. Human salivary α-amylase (EC 3.2.1.1), porcine
pepsin (EC 3.4.23.1), porcine pancreatin (EG/EC 232-369-0), bovine bile (EG/EC 232-369-0), and
all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
49
The digestion simulation was conducted according to the procedure of Amrein et al. [294] and
Stewart [295] with one digestion vessel prepared for each treatment. Briefly, 10 g of each treatment
was suspended in phosphate-buffered saline (PBS) (500 mL, 20 mM, containing 10 mM NaCl, pH
6.9) at 37 C. The entire experiment took place under continuous agitation in a water bath at 37C.
Human salivary -amylase (0.25 mL, 20 mg/mL, in 1 mM CaCl2) was added to the treatment solution
and incubated for 15 minutes. Using 2M HCl, the pH of treatment solutions was adjusted to 2.0
followed by adding porcine pepsin (1.25 mL, 1.0 mg/mL, containing 9.0 g/L NaCl) and incubating
for 30 minutes. The pH was raised to 6.9 with 1M NaOH followed by adding porcine pancreatin in
PBS (5 mL, 102 mg/ML) and 3.4 g of bovine bile and incubating for 3 hours. Subsequently, each
treatment was placed in dialysis tubing with pore size of 3,500 DA molecular size cutoff (Fischer
Scientific, Waltham, MA) and subjected to continuous movement in distilled water for 24 hours.
Digestion residues were removed from the tubing and frozen at -80 C for 72 hours and then freeze-
dried for 72 hours at -51 °C and 0.021 mBar pressure (FreeZone 6 Liter Benchtop Freeze Dryer,
Labcono, Kansas City, MO). Percentage recovery was calculated based on the dry weight of digestion
resides and starting weight of samples and enzymes.
4.3.4 Bacterial Strains

Four test strains of Lactobacillus were selected because they are already established as probiotics
with health benefits. L. paracasei, L. acidophilus, L. plantarum, and L. rhamnosus cultures were maintained
at -80 C in MRS Broth (Difco Laboratories, Sparks, MD, USA) containing 15% (wt/vol) glycerol,
and E. coli culture was maintained at -80 °C in Tryptic Soy Broth (TSB; Difco Laboratories) containing
15% (wt/vol) glycerol.
For the prebiotic activity assay, frozen cultures were streaked onto MRS agar for the
Lactobacillus cultures or Tryptic Soy Agar (TSA), for E. coli, followed by incubation at 37 °C for 24–48
h. Then, one colony from each plate was transferred into 10 mL of MRS broth or TSB and incubated
overnight. For E. coli, an additional transfer of 1% (vol/vol) was made from a TSB overnight culture
into 10 mL of M9 Minimal Medium broth [296] and incubated overnight.
4.3.5 Prebiotic Activity Assay

The assay was performed by adding 1% (vol/vol) of an overnight culture of each probiotic
strain individually to separate tubes containing MRS broth with the carbon source substituted with
either 1% (wt/vol) glucose, prebiotic controls, or taro samples that had been treated by in vitro human
digestion. The cultures were incubated at 37 °C under ambient atmosphere conditions. At 0 and 24 h
during incubation, samples were serially diluted with 0.1% peptone water and enumerated on MRS
agar. In addition, overnight culture of E. coli was added at 1% (vol/vol) to separate tubes containing
M9 broth with the carbon source substituted with either 1% (wt/vol) glucose, prebiotic controls, or
taro samples that had been treated by in vitro human digestion. The cultures were incubated at 37 °C
ambient atmosphere, and enumerated on TSA at 0 and 24 h during incubation.
4.3.5.1 Prebiotic activity score

Prebiotic activity score was calculated with the formula below [286].
A= (probiotic log cfu mL -1 on prebiotic at 24h – probiotic log cfu mL-1 on prebiotic at 0h)
(probiotic log cfu mL-1 on glucose at 24 h – probiotic log cfu mL -1 on the glucose at 0)
B= (enteric log cfu mL -1 on prebiotic at 24 h – enteric log cfu mL 1 on prebiotic at 0 h)
(enteric log cfu mL -1 on glucose 24 h – enteric log cfu mL -1 on the glucose at 0 h)
Prebiotic Activity Score = A – B
50
All experiments in this study were repeated three times. Bacterial counts were log transformed.
All data were analyzed to obtain mean and standard deviation of different treatments. Differences
among the means were analyzed for statistical significance using analysis of variance (ANOVA) and
post-hoc Tukey’s Honest Significant Difference (HSD) test.
4.4 RESULTS
4.4.1 Dietary Fiber, Resistant Starch and Non-resistant Starch

Total dietary fiber (TDF), resistant starch (RS), and non-resistant starch (NRS) were
determined to understand the prebiotic fiber composition of the five taro varieties, which are
presented on an “as is” and “dry weight basis” (DWB) (Table 1). On the “as is” basis, Bun-long,
Tahitian, and Moi exhibited significantly (p<0.05) higher TDF concentrations than Kauaʻi Lehua and
Mana Ulu. Similarly, on the DWB, Moi, Bun-long, and Tahitian exhibited TDR concentrations of
53.69 (4.7), 52.22 (3.9), 48.62 (3.0) g/100g, respectively, which were significantly higher than those of
Kauaʻi Lehua and Mana Ulu. Moreover, The RS (As Is) concentration of Tahitian was found to be
8.37 (0.5) g/100 g, significantly (p<0.05) highest than those of Kauaʻi Lehua and Mana Ulu. However,
on the DWB, Tahitian, Bun-long and Moi were not significantly different in their RS contents. In
contrast, the NRS (As is) concentration of Tahitian was significantly (p>0.05) higher than Nun-long,
Moi and Mana Ulu. However, on a DWB, the NRS of Mana Ulu was significantly (p<0.05) lower than
other four taro varieties.
4.4.2 Percent Recovery

Percent recovery determines the quantity of sample residuals from the in vitro human digestion
(Table 2). Bun-Long exhibited a percent recovery of 59.05 (2.0)%, which was significantly higher than
glucose. The rest of the treatment groups did not show significantly difference in their percent
recovery values.
4.4.3 Growth of Lactobacilli and E. coli on Different Carbohydrates

The growth of four Lactobacillus species and E. coli from 0 to 24 h was determined as a change
in log10 (cfu mL -1) from plate counts (Table 3). Prebiotics should be metabolized by a probiotic species
as well or nearly as well as glucose is metabolized. Thus, an increase in cell count between 0 and 24 h
comparable to the glucose treatment implies the ability of tested substance to stimulate probiotic
growth [286]. In this study, the growth of L. paracasei paired with Tahitian and inulin was significantly
(p<0.05) higher than the glucose, FOS and other taro counterparts. The growth of L. plantarum paired
with glucose was not significantly (p<0.05) different from pairings with Bun-long, inulin, Moi,
Tahitian, and Kauaʻi Lehua. Similarly, the growth of L. acidophilus paired with glucose was not
significantly (p<0.05) different from Moi and inulin pairings. All the treatments but Kauaʻi Lehua
exhibited similar effects on the growth of L. rhamnosus.
The other characteristic property of a prebiotic substance is that it should be selective and not
fermented by commensal organisms [286]. Therefore, E. coli was tested in a similar manner as the
Lactobacillus species to represent the enteric portion of the commensal flora. The growth of E. coli
paired with glucose was significantly higher than all other pairings (Table 3). Actually, the bacterial
counts declined with Bun-long, Mana Ulu, Tahitian, and Kauaʻi Lehua treatments.
51
4.4.4 Prebiotic Activity Score
The prebiotic activity score illustrates the prebiotic potential of a substance in relation to a
specific bacterium. As shown in Figure 1, the prebiotic activity scores of tested substances differed
with the four Lactobacillus species even though they belong to the same genus. With L. plantarum, the
prebiotic activity score of Bun-long was significantly higher (p<0.05) than those of all other taro
varieties and prebiotic controls inulin and FOS. With L. paracasei, the prebiotic activity scores of Bun-
long, Moi, Tahitian and inulin were not significantly different. But Tahitian exhibited a significantly
higher prebiotic activity score (p<0.05) than Mana Ulu, Kauaʻi Lehua and FOS. With L. acidophilus,
the prebiotic activity scores of Bun-long, Mana Ulu, Moi, Kauaʻi Lehua, and inulin were significantly
higher (p<0.05) than those of Tahitian and FOS. With L. rhamnosus, the prebiotic activity scores of all
tested taro varieties and the two prebiotic controls were not significantly different.
4.4.5 Correlation Between Dietary Fiber Components and Prebiotic Activity Scores with Tested Lactobacillus
Species
Nonparametric correlation coefficients (Spearman’s rank) illustrates mixed relationships
between fiber components and prebiotic activity scores of tested taro varieties with different
Lactobacillus species (Table 4), further demonstrating the utilization of fiber components is bacterial
species specific.
The prebiotic activities scores with Kauaʻi Lehua was the only taro variety that illustrated a
positive and significant (p<0.01) correlation with all four types of prebiotic fiber components, RS (as
is), RS (DWB), TDF (as is), and TDF (DWB). In contrast, Tahitian illustrated a negative correlation
with all four prebiotic fibers, with significance (p<0.05) only obtained with RS (as is), RS (DWB), TDF
(DWB). Similarly, Inulin exhibited the same correlations as Tahitian.
4.5 DISCUSSION
The prebiotic potential of carbohydrates is highly dependent on their chemical structures and
utilization by specific probiotic species. Not all probiotic species have the same ability to metabolize
prebiotic carbohydrates [290, 291]. Thus, determining the prebiotic activity score of a carbohydrate is
vital to understanding its potential effect on probiotic species that might improve gut health. In the
present study, the prebiotic potential of five taro varieties was determined with four common probiotic
Lactobacillus species. The relationships between the dietary fiber and resistant starch contents of tested
taro varieties and their prebiotic activities scores with different Lactobacillus species were assessed.
Dietary fiber may promote the growth and activity of probiotics, improve digestive health, and
impact the development of chronic diseases, such as coronary heart disease, stroke, hypertension,
diabetes, obesity, and certain gastrointestinal diseases [297, 298]. The TDF (as is) was found to range
from 8.1 g/100 g in Bun-long to 5.27 g/100 g in Mana Ulu (Table 1). These results are comparable
with previously reported TDF contents of taro from Hawaiʻi with 3.6 g/100 g in Lehua and 3.8 g/100
g in Bun-long [29] and slightly lower than TDF contents of taro from Turkey ranging 12.8 – 14.0
g/100 g [251]. Thus, the high TDF of taro lends itself to be a potential dietary prebiotic source that
has the ability to meet nutrient requirements. Similarly, RS has also been identified as a potential
prebiotic source and represents the portion of starch that remains undigested passing through the
upper gastrointestinal tract [105, 115]. Apart from having prebiotic effects, RS has been shown to
provide several other health benefits, including increasing absorption of minerals, reducing plasma,
and improving insulin resistance [102, 105]. On the DWB, the high RS concentrations of Tahitian,
Bun-long, and Moi are similar to results of previous studies with taro from: Turkey ranging 33.5 –
51.4 g/100 g [251], Thailand averaging 51.60 g/100 g [52], and China 27.5 g/100 g [252]. Overall,
52
Tahitian, Moi and Bun-long have high TDF and RS contents indicating great potential to serve as
prebiotic sources.
Prebiotic carbohydrates may be fermented by different bacteria, resulting in selective growth
of certain species that benefit the host [299]. The metabolic diversity of Lactobacillus spp. might explain
different increase in cell count and thus the prebiotic activity score of a single prebiotic paired with
different probiotic strains [286]. Genome sequencing of probiotic Lactobacilli revealed versatile
carbohydrate metabolic gene repertoires dedicated to the catabolism of various oligosaccharides [300].
Even within the genus Lactobacillus, genes encoding for metabolic systems that break down prebiotics
may be present or absent in different strains, resulting in varied prebiotic activity scores [290, 301].
Specifically, in the presence of prebiotic carbohydrates, probiotic bacteria begin to express the
carbohydrate-active enzymes (CAZymes), such as glycoside hydrolases (GHs), which enable
carbohydrate utilization as carbon and energy sources [302]. Furthermore, GHs have been shown to
strongly vary within bacterial species [299]. Thus, the different cell growth results of tested Lactobacillus
spp. in this study (Table 3) confirm that carbohydrate utilization is a species-specific characteristic.
The specific use of prebiotic carbohydrates by bacterial species is exemplified by their different
prebiotic activity scores with the four Lactobacillus spp. (Figure 1). All the prebiotic activity scores were
positive; however, for the same Lactobacillus species, the utilization of prebiotic fibers also varied
greatly. Thus, the prebiotic activity score was based on each Lactobacillus species’ ability to grow on a
specific prebiotic carbohydrate. This was further exemplified by several null and negative correlations
between the prebiotic activity scores of the taro varieties and the concentrations of their prebiotic
fiber components (TDF and RS) (Table 4). Therefore, high prebiotic nutrient content does not
automatically equate to high prebiotic activity scores for particular probiotic species.
From a species specific perspective, inulin and Tahitian exhibited much higher prebiotic
activity scores with L. paracasei than other tested pairings (Figure 1). This can be explained as that L.
paracasei is a great metabolizer of FOS-type fiber [290]. Tahitian was shown to have one of the higher
dietary fiber concentrations (Table 1) of the taro varieties, providing potential to have higher FOS. In
addition, an in vitro study showed that L. paracasei W20 had the highest overall affinity for fructose
[299], which further corroborates the high prebiotic activity score of Tahitian, as taro has shown to
have 0.7g/100 g of fructose [303], though the taro variety was not specified. Furthermore, a human
isolate L. paracasei subsp. Paracasei 8700:2 was observed to break down inulin-type fructan
extracellularly, which can benefit other members of the gut microbiota [304, 305]. These gut
microbiota benefits was confirmed with Lactobacillus paracasei W20 that was found to act as a keystone
strain in the degradation of prebiotic inulin and cross-feed other probiotic species in the human gut
[299].
L. acidophilus was found to have the highest cell number increase between 0 and 24 h (Table 3)
for glucose and inulin pairings. This can be explained as that strains of L. acidophilus have the ability to
metabolize oligosaccharides [306]. This was further exemplified by inulin exhibiting the highest
prebiotic activity score among all tested carbohydrates with L. acidophilus (Figure 1); however, Bun-
lung, Mana Ulu, Moi, and Kauaʻi Lehua showed similar results. This suggests that these taro varieties
have similar prebiotic potential to inulin in relation to L. acidophilus. In addition, the pairing of L.
acidophilus and inulin has been studied extensively to illustrate its synbiotic health benefits. A study
illustrated that the pairings of inulin and/or okra flour and L. acidophilus La-5 under in vitro simulated
gastrointestinal conditions show high viability, even after 28 days of storage at 4 ºC [307]. Furthermore,
the combination of L. acidophilus and inulin exhibited health benefits in a randomized, double-blind,
placebo-controlled, parallel study that improved the irregularity in shape of red blood cells (RBC) in
hypercholesterolemic subjects [308]. Similarly, hypercholesterolemic pigs fed with a high fiber diet and
treated with synbiotics containing L. acidophilus ATCC 4962, FOS, inulin, and mannitol improved
53
plasma lipid profiles linked to obesity, decreasing plasma total cholesterol, triglycerides, and low-
density lipoprotein-cholesterol levels [309]. Thus, since in this study taro varieties exhibited similar
prebiotic activity scores to inulin when paired with L. acidophilus, taro could potentially be used develop
synbiotics to elicit health benefits.
L. plantarum exhibited cell number increase between 0 and 24 h with all of the tested
carbohydrates expect for FOS (Table 3). This was corroborated by a study that found that L. plantarum
NCDO326 was unable to grow with FOS as the sole carbon source [310]. Furthermore, Bun-long
exhibited a significantly higher (p<0.05) prebiotic activity score than other carbohydrates when paired
with L. plantarum (Figure 1). This was corroborated by a study that illustrated that L. plantarum paired
with soluble and insoluble fiber fractions from oats showed the highest growth among all the fiber
pairings [311]. Therefore, this study disclosed the strong prebiotic potential of Bun-long paired with
L. plantarum. Furthermore, the symbiotic relationships of prebiotics and L. plantarum have health
benefits. The pairing of inulin and L. plantarum LS/07 was shown to be effective against breast cancer
in a rat model through immunomodulatory mechanisms [312]. Thus, the health benefits of Bun-long
with L. plantarum warrant further investigation.
L. rhamnosus paired with all the taro varieties except Kauaʻi Lehua exhibited an increase in cell
number from 0 to 24 h similar to or higher than that with glucose (Table 3), illustrating the prebiotic
potential of taro. Moreover, all the prebiotic activity scores of taro were not significantly different
from inulin and FOS, the established prebiotics (Figure 1). This may be explained as mannitol and
sorbitol are fermentable substrates for L. rhamnosus [48], which taro varieties may be high in. Mannitol
and sorbitol are sugar alcohols that have a natural occurrence in starchy vegetables, such as potatoes.
They can also be obtained by hydrolysis or isomerization of natural raw materials, such as starch [313].
A mice study with prebiotic inulin, probiotic L. rhamnosus, and their combination (synbiotic) found
treatments with the synbiotic and prebiotic increased concentrations of total IgA, whereas the
probiotic alone had no effect [314].
Since E. coli paired with glucose showed the highest growth among pairings with all tested
carbohydrates, the E. coli culture used in this study was active and could represent enteric bacteria in
the determination of prebiotic activity scores. The prebiotic activity score is a ratio of the probiotic
grown on a specific prebiotic and glucose subtracted from the ratio of enteric growth on the same
prebiotic and glucose. Thus, a high prebiotic activity represents, in theory, a very low relative growth
of the enteric bacteria on the same prebiotic [286]. In this study, the reduction in E. coli paired with
taro varieties, with the exception of Moi, provides evidence for the taro varieties to serve as potential
prebiotics.
In the human gastrointestinal tract, commensal organisms are likely to have some ability to
utilize prebiotic carbohydrates [315]. Furthermore, a particular organism may initiate metabolism of
an oligosaccharide via extracellular hydrolysis. The products (mono- or disaccharides) that are released
in vivo may then ‘‘cross-feed’’ other organisms [286], thus, warranting further investigation on the
effects of synbiotics in the human intestine. These findings create a foundation for further evaluation
of taro, specific probiotic species, and their combinations for food applications and health.
4.6 CONCLUSION
Overall, the results of this study provide evidence for taro varieties to serve as potential sources
of dietary prebiotics and have shown their probiotic species-specific utilization. Bun-long, Tahitian,
and Moi exhibited significantly (p<0.05) higher TDR and RS concentrations than Mana Ulu and
Kauaʻi Lehua, indicating differences in their prebiotic contents. Moreover, the pairings of L. paracasei
with Tahitian and inulin exhibited the highest prebiotic activity score, suggesting that the probiotic
54
species paired with Tahitian will more likely be active in the gastrointestinal tract, similar to the pairing
with inulin, a well-established prebiotic. The present study lays the groundwork for further exploration
of potential health benefits of taro as a prebiotic or as part of synbiotics.
55
Table 4.1. Total Dietary Fiber, Resistant Starch, Non-resistant Starch Contents of Taro Varieties
Data are shown as mean and standard deviation (St. Dev.). The results are given on “as is basis”, and “dry weight basis” (DWB).
Bun-Long Kauaʻi Lehua Mana Ulu Moi Tahitian HSD
Total Dietary As Is g/100 g 8.10 (0.3)a 5.47 (0.5)b 5.27 (0.3)b 7.23 (0.3)a 7.47 (0.4)a 0.97
Fiber
DWB (g/100g) 52.22 (3.9)a 40.45 (3.7)b 14.49 (0.8)c 53.69 (4.7)a 48.62 (3.9)ab 9.79
Resistant Starch As Is (g/100 g) 7.60 (0.5)ab 1.20 (0.4)c 6.14 (1.5) b 7.10 (0.6) ab 8.37 (0.5)a 2.19
DWB (g/100 g) 23.98 (1.6)a 5.02 (0.2)c 15.08 (0.8)b 22.30 (0.9)a 25.07 (1.5)a 3.05
Non Resistant As Is (g/100 g) 3.74 (1.1)bc 4.78 (1)ab 1.64 (0.6)c 3.12 (0.5)bc 6.56 (0.8)a 2.22
Starch
DWB (g/100 g) 12.34 (1.1)a 12.69 (1.4)a 4.74 (0.4)b 11.77 (1.2)a 15.70 (2.7)a 4.17
a-c
Means with different letters within the same row differed significantly by Tukey’s HSD (p<0.05).
56
Table 4.2. Percent Recovery of Taro After in vitro Human Digestion
Data are shown as mean and standard deviation (St. Dev.).
Substrates Percent Recovery (%)

Bun-Long 59.05 (2.0) a
Mana Ulu 51.62 (4.0) ab
Moi 53.34 (3.1) ab
Kauaʻi Lehua 55.93 (4.4) ab
Tahitian 50.54 (1.9) ab
Glucose 48.17 (5.9) b
Inulin 52.37 (1.6) ab
FOS 52.63 (1.4) ab
Control 53.36 (2.0) ab
HSD 9.30
a-b
Means with different letters within the same column differed significantly by Tukey’s HSD (p<0.05)
57
Table 4.3. Increase in cell count (log10 cfu mL -1) of tested Lactobacillus spp. between 0 h and 24 h on various carbohydrates
Data are shown as mean and standard deviation (St. Dev.).
Kauaʻi
Bun-long Mana Ulu Moi Tahitian Inulin FOS Glucose HSD
Lehua
L .plantarum 1.54 (0.19) a 0.03 (0.33) bc 0.81 (0.23) ab 0.64 (0.23) ab 0.67 (0.5) ab 1.01 (0.6) ab -0.55 (0.23) b 1.24 (0.26) a 1.00
L. paracasei 0.22 (0.63) bc 0.29 (0.51) bc -0.89 (0.48) c -0.43 (0.13) c 2.67 (0.1) a 3.43 (0.39) a 0.09 (0.41) bc 1.11 (0.62) b 1.27
L. rhamnosus 1.14 (0.23) a 1.17 (0.17) a 1.51 (0.31) a -0.66 (0.09) b 1.75 (0.49) a 1.66 (0.87)a 0.99 (0.26) a 0.87 (0.19) a 1.13
L. acidophilus 1.24 (0.2) b 0.94 (0.15) bc 2.01 (0.5) a 0.64 (0.18) bc 0.52 (0.11) c 2.70 (0.2) a 0.63 (0.27) bc 2.70 (0.2) a 0.71
E. coli -0.99 (0.11) cd -1.22 (0.23) d 0.38 (0.26) b -1.19 (0.32) d -0.82 (0.33) cd -0.23 (0.35) bc -0.94 (0.37) cd 1.31 (0.38) a 0.87
a-d
Means with different letters within the same row differed significantly by Tukey’s HSD (p<0.05).
FOS, fructooligosaccharide
58
Table 4.4 Correlation analysis of prebiotic fiber components and prebiotic activity scores of taro varieties with tested Lactobacillus species
Nonparametric correlation coefficients between RS (as is), RS(DWB), TDF (as is), TDF (DWB), and prebiotic activity scores of taro varieties with L.
plantarum, L. Paracasei, L. rhamnosus, and L. acidophilus.
Variables RS (as is) RS (DWB) TDF (as is) TDF Bun-long Mana Ulu Moi Kauaʻi Tahitian Inulin FOS
(DWB) Lehua
RS (as is) 1 0.938 0.688 0.920 0.284 0.291 -0.300 0.690 -0.623 -0.767 0.142
*** ** *** ** * ***
RS (DWB) 1 0.835 0.945 0.376 0.338 -0.239 0.731 -0.521 -0.675 0.163
*** *** ** * **
TDF (as is) 1 0.702 0.402 0.256 -0.022 0.667 -0.186 -0.384 0.153
** **
TDF 1 0.252 0.407 -0.186 0.710 -0.514 -0.611 0.246
(DWB) ** * *
Bun-long 1 -0.002 0.300 0.352 0.025 -0.060 -0.157
Mana Ulu 1 0.335 0.682 -0.257 -0.115 0.706
** **
Moi 1 0.284 0.592 0.728 0.416
* **
Kauaʻi 1 -0.157 -0.304 0.395
Lehua
Tahitian 1 0.883 -0.197
***
Inulin 1 0.094
FOS 1
Spearman Correlation Coefficient
-1 -0.5 0 0.5 1
*P < 0.05 **P< 0.01 ***P<0.001

FOS, fructooligosaccharides; RS (DWB), resistant starch dry weight basis, TDF (DWB), total dietary fiber dry weight basis
59
Figure 4.1 Prebiotic Activity Scores of Taro Varieties with Lactobacillus spp.
a-c
Means with different letters within the same bacteria group differed significantly by Tukey’s HSD (p<0.05).
60
61
CHAPTER 5 IN VITRO FECAL FERMENTATION OF TARO
(COLOCASIA ESCULENTA) ON THE MODULATION OF GUT
MICROBIOTA COMPOSITION AND SHORT-CHAIN FATTY
ACID PRODUCTION
5.1 ABSTRACT
Taro (Colocasia esculenta) has been shown to contain high dietary fiber and resistant starch.
Dietary fiber and resistant starch are the non-digestible fraction of complex polysaccharides. Reaching
the large bowel, dietary fiber and resistant starch, such as inulin and fructooligosaccharides (FOS), can
function as prebiotics to modulate the bacterial community and confer health benefits to the host.
One of the health benefits is the bacterial fermentation product short-chain fatty acids (SCFAs).
However, dietary fiber, resistant starch, and nutritional characteristic may vary amongst foods, and
knowledge about different taro varieties’ ability to influence complex gut microbial communities and
secondary metabolites is unknown. Thus, this study aimed to understand how taro varieties modulate
the colon microbial community through 16S ribosomal RNA sequencing and analyze their effects on
the production of SCFAs, through an in vitro batch fecal fermentation system. Results have shown that
Bun-long taro yielded a significantly higher amount of SCFAs than prebiotic inulin and FOS. Bun-
long and Moi produced more acetic acid and propionic acid than the two well established prebiotics.
All tested taro varieties, except Kauaʻi Lehua, exhibited significantly higher concentrations of butyric
acid than inulin and FOS. Furthermore, hierarchical cluster analysis indicated that the community
structure profiles of all taro varieties were similar to inulin, with a clear delineation from control
treatments. Alpha diversity analysis exhibited diverse microbial abundance profiles for tested taro
varieties, similar to inulin, suggesting that taro has potential to modulate gut microbiota. Taro
promoted beneficial Firmicutes and Bacteroidetes and suppressed potentially pathogenic
Proteobacteria during fecal fermentation. These results indicate that the taro varieties may dynamically
change the gut microbiota and the microbial species benefited from the available nutrients to increase
SCFA production.
62
5.2 INTRODUCTION
Taro (Colocasia esculenta) is a root crop with great cultural importance cultivated throughout the
subtropical and tropical regions of the world. It is a major source of food for about 500 million people
living in Asia, Africa, Central America, and the Pacific Islands [15]. From a nutrition point of view,
taro is a good source of vitamins (B vitamins, vitamin E, and vitamin C), minerals (potassium,
magnesium, and calcium), dietary fiber, and resistant starch [20, 27, 29, 316]. Taro’s dietary fiber and
resistant starch are the most notable nutrients that have been explored for potential human health
benefits [1, 15, 20].
Beneficial effects of dietary fibers are mainly dependent on their physicochemical properties
such as particle size, cell wall architecture, solubility, degree of polymerization, distribution, and degree
of cross-linking of the polymers [317, 318]. There are several categories of dietary fiber, with some
falling under the prebiotic characterization. Prebiotics are non-digestible food components that reach
the human colon intact, are fermented by colonic bacteria, and enhance microflora species associated
with intestinal health, such as Lactobacillus and Bifidobacterium [2]. Some of the well-known prebiotics,
inulin and fructooligosaccharides (FOS), can be found in dietary sources, such as wheat, bananas,
onions, and garlic [3].
Dietary fibers and resistant starch have also shown to reduce the risk of diseases, such as heart
disease, stroke, hypertension, diabetes, obesity, gastrointestinal disorders, and certain cancers [298,
319-321]. Specifically, epidemiological and experimental studies have shown dietary sources of fiber
and resistant starch exhibit possible preventive effects on colon cancer [322-324]. This may be due in
part to dietary fiber and resistant starch’s ability to dilute potential carcinogens by stool-bulking,
acceleration of transit through the colon, and enhancement of microflora species associated with
intestinal health [325] .
Several studies have shown that diet-based interventions improve health through selective
modulation of the gut microbiota, with the three phyla Bacteroidetes (gram-negative), Firmicutes
(gram-positive), and Actinobacteria (gram-positive) being the most abundant in the intestine [326,
327]. Specifically, dietary fibers, resistant starches, and prebiotic polysaccharides, such as inulin and
FOS, have shown to stimulate the growth of certain bacterial genera under in vitro conditions such as
Bacteroides, Bifidobacterium, Eubacterium, Lactobacillus, Roseburia, and Ruminococcus [328-331]. Furthermore,
compositional changes in the large bowel microbial community have been linked to several
gastrointestinal disorders, such as: inflammatory bowel disease, obesity, allergies, diabetes, and cancer
[[332]. Consumption of such non-digestible carbohydrates may selectively stimulate the bacteria that
transform the fermentable substrates into diverse short chain fatty acids (SCFAs) [333, 334].
SCFAs are the major end products of bacterial fermentation in the human colon, in particular,
acetate, propionate, and butyrate [109, 335-337]. SCFAs are characterized by containing fewer than 6
carbons in their aliphatic chain. According to the number of carbons, SCFAs include: acetic (C2),
propionic (C3), butyric (C4), valeric (C5) and caproic (C6) acids [337]. Acetic, propionic, and butyric
acids have been shown to have the greatest influence on the host health and gut microbial composition
[99, 338]. In particular, butyrate has been shown to be important for maintaining health through
regulating the immune system [337], serving as nutrients for the colonocytes [339], and promoting
satiety after meals [340]. In addition, butyrate has been regarded as a key factor for potential protection
against colon cancer, since it can act at different levels, such as reducing tumor cell growth , inducing
cancer cell differentiation, and inhibiting apoptosis [115, 341 2000 #503]. Propionate has shown to
inhibit cholesterol synthesis [342] and be the second preferred energy source for colonocytes [343].
Acetate plays a role as an energy substrate for peripheral tissues [327], immune system response [344],
and body weight control [327, 345]. The amount and type of fiber consumed has dramatic effects on
the composition of the intestinal microbiota and consequently on the production of SCFAs [327].
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In vitro fecal fermentation models have proved to be a useful tool for studying the effects of
food components, probiotics, and pharmaceutical molecules on the gut microbiota composition [346].
Compared to in vivo models, in vitro models are cheaper, able to be performed under standardized
conditions, and easier to control and repeat [346]. The advantages and limitations of in vivo and in vitro
models as well as the developments to improve the modeling of host-microbe interactions have been
extensively reviewed [183, 346-349]
Thus, the aim of this study was to understand the potential of five varieties of taro to modulate
the gut microflora and SCFAs production in an in vitro batch fecal fermentation system, which
simulates gut microbiome in the human colon.
5.3 METHODS
5.3.1 Taro Sample Preparation

Fresh full-grown taro varieties (Bun-Long, Moi, Tahitian Variety, Kauaʻi Lehua, Mana Ulu)
collected at Waimānalo Research Station (Waimānalo, Hawaiʻi), were processed into 2 cm width
cubes, weighed, and autoclaved at 121 °C for 15 minutes to simulate pressure cooking. The taro
samples were freeze-dried for 48 hours at -80 °C and then for 72 hours at -51 °C and 0.021 mBar
pressure to remove water content (FreeZone 6 Liter Benchtop Freeze Dryer, Labcono, Kansas City,
MO). Afterwards, the samples were weighed and ground to 1mm size taro powder particles
(Commercial Stainless steel Industrial Electric Peppe Grain Mill, CGOLDENWALL).
5.3.2 In Vitro Human Digestion

Freeze dried taro samples were subjected to in vitro digestion. A negative control sample, blank,
was tested to assess the impact of residual digestive enzymes on the growth of Lactobacillus strains. A
positive control glucose was used to confirm the viability of the bacterial strains. Inulin and fructo-
oligosaccharide (FOS) were used as prebiotic controls. Human salivary α-amylase (EC 3.2.1.1), porcine
pepsin (EC 3.4.23.1), porcine pancreatin (EG/EC 232-369-0), bovine bile (EG/EC 232-369-0), and
all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The digestion simulation was conducted according to the procedure of Amrein et al. [294] and
Stewart [295] with one digestion vessel prepared for each treatment. Briefly, 10 g of each treatment
was suspended in phosphate-buffered saline (PBS) (500 mL, 20 mM, containing 10 mM NaCl, pH
6.9) at 37 C. The entire experiment took place under continuous agitation in a water bath at 37C.
Human salivary -amylase (0.25 mL, 20 mg/mL, in 1 mM CaCl2) was added to the treatment solution
and incubated for 15 minutes. Using 2M HCl, the pH of treatment solutions was adjusted to 2.0
followed by adding porcine pepsin (1.25 mL, 1.0 mg/mL, containing 9.0 g/L NaCl) and incubating
for 30 minutes. The pH was raised to 6.9 with 1M NaOH followed by adding porcine pancreatin in
PBS (5 mL, 102 mg/ML) and 3.4 g of bovine bile and incubating for 3 hours. Subsequently, each
treatment was placed in dialysis tubing with pore size of 3,500 DA molecular size cutoff (Fischer
Scientific, Waltham, MA) and subjected to continuous movement in distilled water for 24 hours.
Digestion residues were removed from the tubing and frozen at -80 C for 72 hours and then freeze-
dried for 72 hours at -51 °C and 0.021 mBar pressure (FreeZone 6 Liter Benchtop Freeze Dryer,
Labcono, Kansas City, MO). Percentage recovery was calculated based on the dry weight of digestion
resides and starting weight of samples and enzymes.
5.3.3 Fecal Collection

University of Hawai’i Institutional Review Board (IRB) approval was obtained before fecal
collection occurred. Three healthy donors were recruited using convenience sampling based on the
64
following criteria: consuming an unspecified Western diet, not taking antibiotics for 3 months prior
to collection, not pregnant, and no history of bowel conditions [45, 46]. Fecal collection procedures
were provided to participants and samples were collected under aerobic conditions using the
Fisherbrand™ Commode Specimen Collection System (Fisher Scientific, Waltham, MA). Fecal
samples were kept under sealed and used within four hours of collection.
5.3.4 Fecal Fermentation

Digestion residues and nondigested samples were fermented using an in vitro batch method.
Each treatment at all time points was examined in triplicate. Previously digested treatments were
hydrated for 12 hours in 40 mL of sterile trypticase peptone fermentation medium at 4 C before the
start of the fermentation. One liter of trypticase peptone medium contained 2 g of trypticase peptone,
0.8 g of ammonium bicarbonate, 2 g of anhydrous sodium phosphate dibasic, 1.25 g of anhydrous
potassium phosphate monobasic, 0.5 g of magnesium sulfate, 100 mg of calcium chloride, 63.5 mg of
manganous chloride, 15.5 mg of cobalt chloride, 5.2 mg of ferric chloride, and 0.01 mg of resazurin.
Bottles were warmed to 37 C in a shaking water bath 2 hours prior to inoculation.
Fresh feces were homogenized with phosphate-buffered saline in the ratio of 1:6. Two parts
reducing solution (950 mL of distilled water, 6.25 g of cysteine hydrochloride, 40 mL of 1 N NaOH,
and 6.25 g of sodium sulfide) were combined with 15 parts fecal slurry [350]. To initiate fermentation,
10 ml of the fecal inoculum was added to each bottle along with 0.8 mL of Oxyrase  (Oxyrase Inc.,
Mansfield, OH, USA) to remove oxygen.
Bottles were flushed with carbon dioxide with a headspace of around 90 mL. Subsequently,
bottles were sealed at atmospheric pressure using rubber stoppers and aluminum seals, and placed in
a 37C shaking water bath. Bottles were removed at 0, 4, 8, 12, and 24 h. Over pressurized gas was
allowed to flow into a 20 mL syringe and the volume was recorded. pH was determined using a pH
meter (Oakton WD-35613 pH Handheld Meter). Copper sulfate (1 mL of 200 g/L) was added to each
bottle to cease fermentation. A 2-mL aliquot was taken from each bottle and frozen at -20 C until
SCFAs extraction was completed.
5.3.5 Short-Chain Fatty Acid Analysis

Samples collected at 0 and 24 h were analyzed in triplicate for SCFAs. In brief, fecal
fermentation samples were homogenized with 300 μL of NaOH (1M) solution and centrifuged at
16,000 rcf at 4 °C for 20 minutes. Subsequently, 200 μL of supernatant was transferred into an
autosampler vial, and the residue was further exacted with 200 μL of cold methanol. After the second
round of homogenization and centrifugation, 167 μL of supernatant was combined with the first
supernatant in the sample vial. Samples were then analyzed by GC/TOFMS (Agilent 6890N gas
chromatography coupled with a LECO Pegasus HT time-of-flight mass spectrometer) and followed
the protocol described elsewhere [351, 352]. Raw data from GC/TOFMS analysis were exported in
NetCDF format to ChromaTOF software (v4.50, Leco Co., CA, USA) and subjected to the following
preprocessing, baseline correction, smoothing, noise reduction, deconvolution, library searching, and
area calculation. Individual compound identification was performed by comparing both MS similarity
and Kovats Afterward. Data sets were exported to a CSV file [351, 352].
5.3.6 Gut Microbiome

Microbial DNA was extracted from the fecal fermentation samples using the QIAamp
PowerFecal Pro DNA Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
The DNA extracts were kept frozen at -20C.
65
5.3.7 16S rRNA Sequencing
Libraries of the 16S rRNA gene were prepared according to the Illumina 16S Metagenomic
Sequencing Library Preparation protocol [353], with slight modifications. The V3-V4 region of the
16S rRNA gene was amplified with the following primers with gene-specific sequences of the primers
underlined [354].
16S Forward Primer:

5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’
16S Reverse Primer:

5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-
3’
The first round of PCR followed standard Illumina 16S Metagenomic PCR amplification [353].
The second round of PCR used Nextera XT v2 indexes (Illumina, San Diego, California) and included
a denaturation step at 95°C for 3 minutes, 8 cycles of 95°C for 30 seconds, 55°C for 30 seconds and
68°C for 30 seconds, followed by a final extension at 68°C for 5 minutes. After bead purification, the
indexed libraries were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen,
Thermo Fisher Scientific, Carlsbad, California), normalized, and pooled. The pooled library was run
on a Bioanalyzer High Sensitivity DNA chip (Agilent, Santa Clara, California) to determine the average
size. Sequencing was performed on an Illumina MiSeq desktop sequencers to generate paired 300-bp
reads at the Advanced Studies in Genomics, Proteomics and Bioinformatics (ASGPB) at the
University of Hawaiʻi at Mānoa.

Data collected from Illumina 16S Metagenomic PCR amplification were subjected to the
following analysis. The raw paired reads were preprocessed using the dada2 R package. Reads were
truncated at position 250/230 (forward/reverse read). A read pair was discarded if at least one of the
reads contained one or more bases with quality scores less than 2, or more than 3 expected errors.
Denoising was performed using dada2 with default parameters. Paired reads were merged into
Amplicon Sequence Variants (ASVs) and any pairs with an overlap of fewer than 10 bases, or with
more than one mismatch, were discarded. Mothur [355] along with the Silva (release 132) database
[356] were used to align the sequences. Alignments with a start or stop position outside the 5 th-95th
percentile range over all sequences were discarded. Potential chimeras were removed using Mothur’s
chimera.vsearch utility. Taxonomies were assigned using the RDP classifier [357] as implemented in
Mothur. All mitochondrial or chloroplast ASVs were removed, as well as sequences with no
annotations at the domain level. ASVs were removed with a total abundance of 2 or fewer reads and
subsampled each sample to 5,000 reads. Samples with less than 5,000 reads were discarded. The Lulu
R package was used to refine ASVs as follows. Two ASVs were merged if all of the 3 following
conditions were satisfied: 1) They co-occur in every sample, 2) One of the two ASVs has a lower
abundance than the other in every sample and 3) they share a sequence similarity of at least 97%.
Post-processing analysis was done on MicrobiomeAnalyst, a web-based program that
integrates exploratory analysis on common abundance profiles and taxonomic signatures generated
from microbiome studies [358]. The sequences were aligned against Mothur-adapted SILVA bacterial
reference database [359]. Differences in beta-diversity were determined by permutational multivariate
analysis of variance (PERMANOVA), a permutation-based multivariate analysis of variance to a
matrix of pairwise distance to partition the inter-group and intra-group distances.
66
Analysis of variance (ANOVA) with Tukey’s pair-wise test was used in all tests to determine
differences of treatment means. In addition, principal component analysis (PcoA) of bacterial
community using unweighted UniFace distance was done. Statistical significance was achieved for p-
values less than 0.05.
5.4 RESULTS
5.4.1 Gas Production

The production of gas showed evidence of active microbial activities in fecal fermentation
experiments (Figure 5.1). At 24 hours, Tahitian, Moi, and Bun-long produced higher gas volumes than
other six treatments. In contrast, the control and glucose treatments exhibited no gas production
throughout the entire 24 hour period, indicating a less occurrence of microbial activities. Inulin, an
established prebiotic [297], exhibited no significant difference in gas production from Mana Ulu and
Kauaʻi Lehua, suggesting that these two taro varieties exhibited similar microbial activities as inulin,
as gas production is a by-product of bacterial fermentation.
5.4.2 pH Changes
Throughout the 24-hour fermentation period, the pH values of all treatments dropped, with
the 24 hour time point having the lowest pH value, indicating the occurrence of microbial
fermentation (Figure 5.2). At 24 hours, Tahitian, Moi, and Bun-long, exhibited significantly (p<0.05)
lower pH values than inulin. In contrast, the control exhibited a significantly higher pH value of 7.8
(0.1) than all taro treatments, which can be explained by no carbon sources being added; therefore,
not providing starches for bacteria to ferment. Meanwhile, the pH value of the control is not
significantly different from those of inulin, FOS, and glucose. The in vitro fermentation was reflected
by the drop pH of the glucose treatment from 8.6 at 0 h to 7.6 at 24 h, indicating viability of microbes
from the fecal slurry [295].
5.4.3 Short Chain Fatty Acids (SCFA)

At 24 h, Bun-Long exhibited the highest total SCFAs of 332032.4 ng/mL (50813.35), which
was significantly higher than that of inulin (Figure 5.3). Moi, Tahitian, Mana Ulu, and Kauaʻi Lehua
varieties exhibited similar total SCFA production, which was not significantly different from inulin. In
contrast, control, glucose, and FOS illustrated significantly (p<0.05) lower total SCFA production than
the taro and inulin treatments — a result of lower microbial activities. Among all SCFAs, propionic
acid, acetic acid, and butyric acid, exhibited high percentages of the total SCFAs, which further
exemplifies microbial fermentation of prebiotic carbohydrates. Propionic, acetic, and butyric acid are
produced as metabolites from bacterial fermentation of non-digestible fibers [327].
Individually, propionic acid was produced in significantly (p<0.05) higher concentration from
Bun-long, Moi, and Tahitian (Figure 5.4). Similarly, acetic acid was produced in significantly (p<0.05)
higher concentration from Bun-long and Moi. In contrast, butyric acid was significantly (p<0.05)
higher concentration from only Bun-long, compared to the other taro varieties and controls.
5.4.4 Gut Microbial Profile

The overall bacterial community composition was analyzed using 16S rRNA gene amplicon
sequencing. For this analysis, all data were normalized to 24,911 reads per sample. A total of 116
OTUs were obtained, which could be identified to four bacterial phyla (Proteobacteria, Bacteroidetes,
Firmicutes, Actinobacteria) and 21 bacterial families.
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Heatmap of hierarchical clustering of bacterial microbiota composition profiles of the
treatments illustrated two distinct delineation hierarchical clusters (Figure 5.5). The first group was
presented between the baseline, control, FOS, and glucose treatments, while the second group was
between Bun-long, inulin, Tahitian, Mana Ulu, Kauaʻi Lehua, and Moi. Alpha-diversity measures were
calculated using Shannon Index for microbial diversity within the community. ANOVA exhibited a
F-value of 2.2206 (p=0.17914) (Figure 5.6). Treatments were grouped into baseline, control (glucose,
inulin, and FOS), and taro (Bun-long, Mana Ulu, Moi, Kuai Lehua, Tahitian) and represented by
different colors. The analysis of α-diversity indices demonstrated the highest phylogenetic diversity in
the baseline; however, only one data point was available. Apart from the baseline, the taro group
showed the second highest phylogenetic diversity. PcoA of bacterial community using unweighted
UniFrac distance (Figure 5.6) showed three distinct bacterial community groups; baseline, control, and
taro. PcoA permutational MANOVA (PERMANOVA) exhibited an F-value of 6.0143; R-squared:
0.63213; p-value < 0.005.
The relative abundance of the bacterial community from the pooled fecal slurry (Figure 5.8),
showed that the baseline sample (0 hour) was dominated by Firmicutes (61.22%), Proteobacteria
(25.44%), Bacteroidetes (12.7%), and Actinobacteria (1.18%) phyla, with the most dominant families
being Lachnospiraceae (33.90%), Enterobacteriaceae (25.08%), and Ruminococcaceae (24.52%).
After 24 hours of fecal fermentation, the bacterial composition in the control sample (without
carbon substrates) differed from that in the baseline sample. At the phylum level, there was an increase
in Proteobacteria (64.61%), while Firmicutes (32.60%), Bacteroidetes (2.27%), and Actinobacteria
(0.52%) decreased. At the family level, there was an increase in the relative abundance of
Enterobacteriaceae (60.87%), while the levels of bacterial families, such as Bacteroidaceae (1.86%),
Lachnospiraceae (19.47%), and Ruminococcaceae (10.02%) decreased.
The relative abundances of bacterial communities in the FOS and glucose samples were
similar. At a phylum level, FOS and glucose samples were dominated by Proteobacteria (68.16% and
74.49%), Firmicutes (31.33% and 25.21%), Bacteroidetes (0.10% and 0.01%), and Actinobacteria
(0.41% and 0.30%), respectively. Similarly, at the family level, FOS and glucose samples were
dominated by Enterobacteriaceae (68.12% and 74.33%) and Lachnospiraceae (18.07% and 15.07%),
respectively.
The inulin and taro (Bun-Long, Mana Ulu, Moi, Kauaʻi Lehua, Tahitian) samples exhibited
similar bacterial compositions. At the phylum level, they were dominated by Firmicutes,
Proteobacteria, Actinobacteria. However, at the family level, Enterobacteriaceae, Veillonellaceae,
Bacteroidaceae, Lachnospiraceae, and Bifidobacteriacea are dominant in the bacterial communities.
Nonparametric correlation coefficients (Figure 8) for the associations between fermentation
end products SCFAs and major bacterial families showed all the SCFAs were strongly and positively
correlated with one another (p < 0.05). Veillonellaceae was strongly and positively correlated with
isovaleric acid (0.696; p < 0.05), with positive correlations with valeric acid and butyric acid (0.617 and
0.521, respectively). Desulfovibrionaceae exhibited positive correlations with isovaleric acid, and heptanic
acid, along with strong and positive correlations to Acidaminococcaceae and Bacteroidaceae (0.888; p < 0.01
and 0.720; p < 0.05, respectively). Acidaminococcaceae was found to have positive correlations with
isovaleric acid and valeric acid. Bacteroidaceae and heptanoic acid were significantly (0.711; p < 0.05)
correlated.
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5.5 DISCUSSION
Prebiotics from dietary sources have the potential to modulate the human gut microbiome
and SCFA production. Specifically, taro (Colocasia esculenta) may be a dietary prebiotic due to its high
fiber content of 4.1% of dietary on a fresh weight basis [28]. Huang et al. [29] estimated the
consumption of 1 lb (454 g) per meal by Pacific Islanders, using dietary data obtained for Bun-long
and Lehua taro varieties, and showed that taro alone can fulfill the Dietary Guidelines for Americans
2015-2020 dietary fiber recommendation of 25 g per day [234]. Dietary fiber content has been shown
to have prebiotic characteristics, thus, making taro a potential dietary source of prebiotics. However,
not all taro varieties contain the same concentration of dietary fibers; therefore, their effects on the
gut microbial community and SCFA production may be different. Hence, understanding how taro
varieties modulate microbial communities, compared to established prebiotics, inulin and FOS, reveals
taro’s potential prebiotic activities.
Batch fermentation system using human fecal inoculum is designed to mimic the human
digestive tract [348, 360]. Though in vitro batch fermentation models do not reflect the real conditions
of the colon environment, such as certain biological conditions, they have been extensively reviewed
and have shown to provide a controlled environment that represents the colon microbial community
[183, 346-349]. Thus, they provide a non-invasive, controlled model to understand the ability of
prebiotic dietary sources to modulate microbial communities and SCFA production, in this case five
taro varieties and established prebiotics.
Changes in the gas production, especially Tahitian, Moi, and Bun-long, suggest that the five
taro varieties have fermentable carbohydrates, with steady fermentation profiles similar to that of
inulin (Figure 5.1). However, the overall gas production was lower compared to the findings by Chiu
et al. [295]. Dietary fiber and prebiotic carbohydrates have been shown to produce gas as a byproduct
from anaerobic bacterial fermentation, which includes: hydrogen, carbon dioxide, and methane [99,
318, 361]. Since hour 0 measurements showed no gas production, it can be presumed that the serum
bottles were not over pressured and at ambient pressure. Thus, subsequent gas production can be
attributed to pressure buildup from anaerobic bacterial fermentation.
Decrease in pH across the 24 hours, especially of Tahitian, Moi, and Bun-long samples, further
suggests the occurrence of anaerobic bacterial fermentation (Figure 5.2). These results are
corroborated by a study looking at wetland taro and dry-land taro that found the pH of cooked
fermented taro skins decreased to 5.1 for wetland taro and 4.4 for dry-land taro at around 34 hours,
from a starting pH of 5.8 and 6.0, respectively [126]. Tahitian, Moi, and Bun-Long samples had the
lowest pH, compared to other treatments, suggesting that these varieties had the more fermentable
nutrient compositions. Dietary nutrients have been shown to be substrates for microbial metabolisms,
such as oligosaccharides and simple sugars, which have an impact on pH by promoting acid
production through fermentation [362]. A byproduct of microbial fermentation is lactic acid, which
has shown to decrease pH value in vitro and indicate bacterial activity [363, 364]. Specifically, acidity
can selectively stimulate microbial growth and production of microbial metabolites [363]; therefore,
the decrease in pH of taro varieties, similar to inulin, implies their microbial fermentation activities
may modulate microbial community and microbial metabolites.
Five tested taro varieties showed high total SCFA production in fecal fermentation, compared
to inulin and FOS. This suggests the taro varieties have favorable prebiotic fiber composition to
promote specific bacterial fermentation products (Figure 5.3). Fermentation of carbohydrates, mainly
dietary fiber and resistant starch, by specific colonic anaerobic bacteria yield more SCFAs [109].
Though the main sources of SCFAs are carbohydrates, branched-chain amino acids obtained from
protein breakdown, such as valine, leucine, and isoleucine, can also be converted into isobutyrate,
isovalerate, and 2-methyl butyrate, which are known as branched-chain SCFAs (BSCFAs). Though
69
BSCFAs contribute little (5%) to the total SCFA production [365], the five taro varieties yielded a
higher concentration of each BSCFA than control, glucose, FOS, and inulin (Figure 4), which can be
attributed to their protein content.
Specific SCFA, acetic acid, propionic acid, and butyric acid were found to be in high
concentrations from the five taro varieties and inulin (Figure 5.4). This can be explained as more active
fermentation of DF and resistant starch occurs from human colonic bacteria [99]. Resistant starch is
considered to be the most powerful butyrogenic substrate, with in vitro as well as in vivo studies showing
it has significantly higher level of butyrate production than non-starch polysaccharides [366]. Butyrate
has shown to have several health benefits and is currently regarded as one of the most important and
studied SCFA [367]. Butyrate has anti-inflammatory properties through the inhibition of pro-
inflammatory cytokine IL-12, upregulation of anti-inflammatory cytokine IL-10, monocytes [368], and
suppression of proinflammatory molecules TNF- ɑ and IL-1β [369]. Butyrate is responsible for anti-
inflammatory effects, not only within the gut, but also systemically, affecting even the brain via the
blood-brain barrier [109]. In addition, butyrate has also demonstrated the ability to inhibit a variety of
factors that propagate the ignition, progression and growth of colon tumors [115].
While in the present study FOS exhibited a low total SCFA yield (Figure 5.5), FOS is part of
the oligosaccharides family, which are short chains of monosaccharide units. The oligosaccharide
family includes galactooligosaccharides, mannanoligosaccharides, and chitooligosaccharides, which
have also been identified as substrates for SCFA production [370]. However, resistant starch and FOS
have been shown to act synergistically in the digestive system to cause a prebiotic effect that benefits
human health [105]. This may explain the low SCFA production by FOS in the present study. Since
each substrate was individually tested in the process, FOS’s full potential as a prebiotic may have not
been exhibited.
Tested taro varieties showed prebiotic potential to modulate microbial communities.
Hierarchical cluster analysis revealed community structure profile of baseline, being separated from
control, FOS, and glucose, and further separated from inulin and the taro varieties (Figure 5.5). The
clear delineation in hierarchical clustering from baseline suggests that the nutritional composition of
taro has the ability to dynamically change the microbiota. This is further supported by the analysis of
overall β-diversity of gut microbiota signatures (Figure 5.6) in the microbial community in terms of
unweighted (qualitative) produced two distinct clusters; thus, indicating that the gut microbiota in
these two groups have different composition (unweighted). The PcoA of the bacterial community
structure (Figure 5.6) illustrated that the taro varieties exhibited the most similar bacterial profile. Thus,
these results further suggest that more bacterial species benefited from the available nutrients from
the taro varieties. Similarly, α-diversity indices (Figure 5.7) demonstrated the highest phylogenetic
diversity in the baseline; however, only one data point was available, and thus it is not conclusive.
Apart from the baseline group, the Shannon index (I biodiversity in terms of I richness, abundance,
and evenness) was higher in the taro group from the control group.
Taro’s ability to modulate the microbial community was observed at the phylum and family
levels. In the present study, Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria phyla were
found in the microbial communities (Figure 5.8). Although there are considerable variations among
the microbial compositions of different individuals, the gut microbiota of healthy adults is commonly
dominated by four major bacterial phyla: Firmicutes and Bacteroidetes (two groups of obligate
anaerobes), which constitute ~90% of the microbial ecosystem, and Proteobacteria and
Actinobacteria, which contribute to a lesser degree [371]. Therefore, the microbial phyla found here
represent a healthy adult’s gut microbiota profile.
70
5.5.1 Firmicutes Phylum
The Firmicutes phylum was the most abundant phylum amongst the taro varieties, inulin, and
baseline, and the second most abundant phylum amongst the glucose and FOS (Figure 5.8). These
results corroborate with a study comparing mice, non-human primates, and human fecal microbiota
composition that found the phylum-level analyses demonstrate higher Firmicutes–Bacteroidetes ratio
across all samples [372]. The Firmicutes phylum is a highly abundant member of the gut microbiota
and contains many groups associated with health and producers of SCFAs, specifically butyrate and
propionate [109, 373, 374]. Since the taro varieties exhibit a higher abundance of the Firmicutes
phylum, similar to the abundance of inulin, it suggests that taro varieties potentially promoted the
production of butyrate and propionate.
At a family level, Veillonellaceae, which belongs to the Firmicutes phylum, was found in highest
abundance amongst the taro varieties, specifically Kauaʻi Lehua, Tahitian, and Moi; compared to the
baseline, control, FOS, and glucose, all had the an abundance over 35%. This clear delineation between
the control group and taro varieties was verified with the hierarchical clustering heatmap of the
bacterial microbiota profiles (Figure 5.5). Veillonellaceae is known to produce SCFAs and ubiquitous
in the human gut microbiota [375]. Specifically, Veillonellaceae has been found to produce propionic
acid through the acrylate pathway lactate and is converted to propionate through the activity of the
lactoyl-CoA dehydratase and downstream enzymatic reactions [376]. In addition, Veillonellace was
found to be significantly and positively correlated (0.696, p> 0.0.5) with isovaleric acid (Figure 5.9).
This can be explained as some species of Veillonellace, such as Anaeroglobus geminatus, may produce the
metabolic end products, acetic acid, propionic acid, butyric acid, butyric acid and isovaleric acids in
the gastrointestinal tract, with isovaleric acid being produced in the highest concentrations [377, 378].
Furthermore, Dialister and Megasphaera, among other members of the Veillonellaceae family, have been
also reported to produce valeric acid as an end metabolite of the fermentation of carbohydrates and
lactate [379]. Similarly, A. geminatus has been found to be saccharolytic, being able to break down
galactose and mannose [378]. Thus, taro’s high carbohydrate, dietary fiber, and resistant starch
contents are potentially responsible for the promotion of Veillonellace, which increases the production
of SCFAs, specifically isovaleric acids.
Lachnospiraceae, part of the Firmicutes phylum, was found in relatively high abundance across
all treatments, but particularly high in the baseline, Tahitian, and control. This may be because the
family Lachnospiraceae has been shown to dominate the metabolically active gut microbiome [380, 381].
Specifically, members of this family are good digesters of starch, as well as more complex
polysaccharides, such as cellulose, hemicellulose, and pectin [382, 383]. In addition, Roseburia spp., a
part of the Lachnospiraceae family, have been found to produce a major extracellular amylase enzyme,
known as neopullulanase, that converts starch and glycogen into simple sugars [384]. The amylase
enzyme appears to be anchored to the cell surface via a sortase-mediated mechanism and may
contribute to digesting various starches that can be used by other bacterial species for fermentation
[384]. This corroborates several in vitro fermentation studies that found an increase in Roseburia spp,
upon the addition of wheat dextrin [361, 385, 386]. Furthermore, Lachnospiraceae includes butyrate-
producing species that are interspersed with butyrate non‐producing species, which will cross feed to
utilize by-products of other bacteria for their own metabolism [123, 141]. This is particularly true for
Roseburia species, which constitute a major group of butyrate‐producing Firmicutes [101], that at mildly
acidic pH are able to produce butyrate as a fermentative byproduct through the consumption of
acetate or lactate [387]. In addition, both Roseburia spp. and Coprococcu spps are some of the only
anaerobic bacterium capable of producing both butyrate and propionate during fermentation [149].
Lachnospiraceae, are also butyrate-producing species and have been found to be interspersed with
butyrate non‐producing species that will cross feed to utilize other bacterial by-products for their own
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metabolism [123, 141]. In particular, the Roseburia species constitute a major group of butyrate‐
producing Firmicutes [101], which at mildly acidic pH produce butyrate as a fermentative byproduct
through the consumption of acetate or lactate [387]. Furthermore, Roseburia spp. and Coprococcu spp.,
both parts of the Lachnospiraceae family, are some of the only anaerobic bacteria capable of producing
both butyrate and propionate during fermentation [149]. As such, this may explain the higher
concentrations of butyric acid and propionic acid with Tahitian and other taro varieties during fecal
fermentation, which may be attributed to the higher abundance of Lachnospiraceae. Thus, the metabolic
cross feeding from substrate-producing to substrate utilizing bacteria may also be a factor in the
butyrogenic effects of high dietary fiber sources [388]. Therefore, the taro varieties, which are high in
starch, dietary fiber, and resistant starch, may be contributing to the higher abundance of
Lachnospiraceae, which ultimately contributes to the higher concentrations of butyrate and propionate.
5.5.2 Bacteroidetes Phylum

The Bacteroidetes phylum was found abundant amongst the taro varieties, inulin, and baseline,
compared to the 24 hour control, FOS, and glucose (Figure 5.8). Bacteroidetes is one of the most
abundant microbial phylum in the intestine and feces, that has been associated with health benefits
that includes being primarily fermenters of various types of starch to produce organic acids, including
SCFAs, and hydrogen [389-391]. Starch utilization by Bacteroidetes is mediated through a well-
characterized starch binding and degradation system referred to as starch utilization system [391, 392].
In particular, the Bacteroidetes phylum members mainly produce acetate and propionate [393, 394],
which may further explain the high acetate and propionate production by the taro varieties.
At the family level, Bacteroidaceae, a part of the Bacteroiddetes phylum, was found in higher
abundance in Bun-long, Inulin, and Mana Ulu samples, compared to the control, FOS, and glucose
samples, which all had abundances close to zero. Bacteroidaceae has been shown to be digesters of
starch, along with complex polysaccharides that include: cellulose, hemicellulose, and pectin [382, 383].
Members of the genus Bacteroides also have the ability to metabolize dietary protein [395]. This is
corroborated with a study analyzing fiber effects in an in vitro batch fermentation system that found
dramatic increases in the fiber-digesting group, Bacteroides, which constituted 68.9 and 24.0% of the
total bacteria at 48 h in the fiber samples Psyllium husk and Benefiber, respectively [147]. Similarly, a
study using an in vitro human digestive and gut microbiota model system revealed that the relative
abundance of the OTUs for the genus Bacteroides were significantly higher in samples with Psyllium
husk [396]. In addition, in the presence of dietary fibers, Bacteroidaceae produces SCFAs, specifically
propionic acid [397]. Propionic acid production from Bacteroidetes has been found to primarily use
the succinate pathway [398], which helps reduce the overall environmental pH [130]. Clinical studies
have also shown that the decrease in pH promotes growth of beneficial bacteria such as Bacteroides
[399]. This was exemplified in a study that showed that in the presence of Benefiber and Psyllium
husk, higher levels of SCFAs were produced and were correlated with the higher relative abundance
of Bacteroides [396]. This can also be exemplified in the current study that shows lower pH values with
taro varieties. Thus, it suggests that taro varieties promote the growth of Bacteroides, which in turn leads
to the higher levels of SCFAs and low pH.
5.5.3 Proteobacteria Phylum

The taro varieties exhibited lower abundances of Proteobacteria, compared to the 24 hour
control, FOS, glucose, and inulin treatments (Figure 5.8). The phylum Proteobacteria is vast,
comprising six classes based on phylogenetic analysis of 16S rRNA; however, many of the common
human pathogens are found in this phylum [400]. This is because a common trait of Proteobacteria is
the presence of the lipopolysaccharide (LPS) in the outer membrane, which has been linked to low-
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grade inflammation, called endotoxemia, and has been well established to be connected with the
development of metabolic disorders [401, 402]. Thus, compared to FOS, glucose, inulin, and control
treatments, the lower abundance of Proteobacteria from the taro varieties suggests they potentially
reduce the presence of human pathogenic bacteria and inflammatory agents, such as LPS.
At the family level, Enterobacteriaceae, a part of the Proteobacteria phylum, was present in much
higher abundance in glucose, FOS, and control samples, compared to low abundance in the taro
varieties, with Tahitian variety having the lowest abundance. This may be because Escherichia coli is one
of the most common species in the family Enterobacteriaceae, which is also the most common species
of facultative anaerobe found in the human gastrointestinal tract and the most commonly encountered
pathogen from the Enterobacteriaceae family [403]. These results are similar to studies with dietary
interventions with fiber that have been found to decrease the abundance of Enterobacteriaceae spp. In an
in vitro human digestive and gut microbiota model system to investigate the effect of three commercial
fiber products; NutriKane™, Benefiber® and Psyllium husk (Macro) on the adult gut microbiota,
Enterobacteriaceae showed a reduction in the relative abundances upon addition of all fiber treatments
compared to the no added fiber control [396]. These studies corroborate with the present results,
which found low abundance of Enterobacteriaceae in fecal fermentation samples of taro varieties, which
are high in dietary fiber, total starch, and resistant starch. Thus, this finding suggests that taro can
inhibit the growth of Enterobacteriaceae and may be used as a dietary intervention to combat pathogenic
bacteria in this family.
5.5.4 Actinobacteria Phylum

Bifidobacteriaceae, which belongs to the Actinobacteria phylum, was found in the taro varieties
and inulin samples at 24 h; though the levels were much lower than the other bacterial families. These
results agree with other in vitro model studies that have shown an increase in the abundance of
Bifidobacteriaceae upon consumption of inulin, short chain FOS or galactooligosaccharides (GOS) [385,
404-406]. Furthermore, in the presence of dietary fiber, Bifidobacteriaceae has been also shown to
increase the production of SCFAs [396]. Bifidobacteriaceae can produce SCFAs, specifically acetate,
butyrate, and propionate, typically occurring in a 3:1:1 ratio, respectively [376]. Despite increasing the
abundance with the presence of fiber, Bifidobacteriaceae typically makes up less than 10% of the adult
human gut microbiome [407], which may be explained by the fact that the Actinobacteria phylum is
proportionally less abundant than other phyla in the human gut [408]. As such, taro may increase the
abundance of Bifidobacteriaceae, through the higher dietary fiber content, which can contribute to higher
concentrations of generated acetate, butyrate, and propionate.
Overall, these results illustrate the potential of taro as a dietary prebiotic. Diet provides the
main energy source for the gut microbiota; thus, playing a major role in dictating which bacteria thrive
in the large intestine [409]. The ability to change microbial communities also affects the metabolites
produced from the bacterial fermentation, such as SCFAs. Certain dietary constituents, specifically
dietary fiber and RS, are known to increase the bacterial production of SCFAs, such as butyrate,
propionate, and acetate [410]. This study has revealed significant effects taro has on the composition
and activity of gut microbiota and potential health benefits to the host.
5.5.5 Limitations
The amount of SCFAs excreted for each individual is relatively constant during several months
[411]. Inter-individual variations in the concentrations of SCFAs in the feces may be a potential
reflection of differences in the diets [411]. Cummings et al. [412] studied the excretion of SCFAs in
six subjects on carefully controlled diets and found no increase in the total concentrations of SCFAs
in their feces when the subjects increased their intake of dietary fiber from 17 g/day to 45 g/day [412].
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However, fecal SCFAs do not represent the complete microbial SCFA production, as there are
individual differences in absorption and fluxes of the microbial metabolites [413]. Furthermore, only
up to 5% of microbially produced SCFAs are excreted in the feces [414]. Though interdependence
has been shown between diet, the gut microbiota, and host metabolism; changes in SCFAs and the
microbiota have been shown to be associated with profound effects on host metabolism, which
cannot be assessed in an in vitro model [415]. In addition, only five taro varieties were used, which may
not be representative of the full taro diversity species. Thus, results of the present study would require
further explorations of microbial SCFA production in an in vivo investigation.
5.6 CONCLUSION
The results of this study provide evidence for taro varieties to be a potential dietary prebiotic
source that promotes a diverse gut microbiota profile and production of SCFAs, especially butyric
acid, acetic acid, and propionic acid. Based on hierarchical clustering analysis, taro varieties exhibited
similar gut microbiota profiles to inulin, an established prebiotic, and a clear delineation from the
baseline, control, glucose, and FOS treatments. Furthermore, alpha diversity analysis exhibits diverse
microbial abundance profiles for tested taro varieties, similar to inulin, suggesting that taro has
potential to modulate gut microbiota and promote diverse bacterial phylotypes. This is further
supported by significant higher concentrations of propionic acid, acetic acid, and butyric acid, which
are vital SCFAs produced from microbial fermentation, from inulin and the taro varieties. Moreover,
taro promoted beneficial Firmicutes and Bacteroidetes and suppressed potentially pathogenic
Proteobacteria during fecal fermentation. As such, this study reveals that taro may serve as a dietary
prebiotic to modulate the gut microbiota and increase production of vital SCFAs, such as propionate,
acetate, and butyrate, for human health.
ACKNOWLEDGEMENTS
The authors are grateful to Wei Jia, PhD, Lu Wang, PhD; Huizhen Zhang, MA, Cancer
Biology Program, University of Hawaiʻi Cancer Center; Jennifer Saito, University of Hawaiʻi ASGPB,
and participants who volunteered for study. Supported in part by the #MahiMicrobes2018 Grant, C-
MĀIKI; Grants and Awards Program, Graduate Student Organization (GSO); Travel and Research
Grant, Graduate Women in Science (GWIS) Hawaiʻi Chapter; USDA-NIFA Hatch Grant
No.HAW02034H.
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Figure 5.1. The gas volume of fermentation slurry over a 24-hour period in vitro fecal fermentation. Data are mean values (n=3). Values with different letters
showed significant differences among the treatments (p < 0.05).
75
Figure 5.2. The pH of fermentation slurry over a 24-hour period in vitro fecal fermentation. Data are mean values (n=3). Values with different letters
showed significant differences among the treatments (p < 0.05)
76
Figure 5.3. Total short-chain fatty acid (SCFA) concentrations at 24 hours of in vitro fecal fermentation. Data are mean values (n=3). Values with different
letters showed significant differences among the treatments (p < 0.05).
77
Figure 5.4 Individual short-chain fatty acid (SCFA) production at 24 hours of in vitro fecal fermentation. Data are
means values (n=3). Values with different letters showed significant differences among the treatments (p < 0.05).
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A B C D E E F G H I J
Figure 5.5 Heatmap of hierarchical clustering of bacterial microbiota composition profiles represented by 16S
ribosomal RNA (rRNA) amplicons per sample of ‘heavy’ and ‘light’ gradient fractions from treatments: A) Baseline,
B) Control, C) fructooligosaccharides (FOS), D) glucose, E) Bun-long, F) inulin, G) Tahitian, H) Mana Ulu, I) Kauaʻi
Lehua, and J) Moi. RNA was extracted from fecal samples before (0 Baseline) and at 24 hours of the fermentation.
The presented community profiles are results of the triplicate pooled samples for each treatment. Bacteria shown
represent the taxa with the highest mean relative abundance across all fraction samples. Heatmap color (red gradient)
displays the row scaled relative abundance of each taxon across all samples.
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Figure 5.6. Principal Coordinate Analysis (PcoA) of bacterial community structures using permutational MANOVA
(PERMANOVA) statistical method and unweighted UniFrac distance. Treatments were grouped by baseline, control
(glucose, inulin, and fructooligosaccharides (FOS)), and taro (Bun-long, Mana Ulu, Moi, Kauaʻi Lehua, and Tahitian
varieties). PcoA shows three distinct bacterial communities. F-value: 6.0143; R-squared: 0.63213; p-value < 0.005.
80
Figure 5.7 Alpha-diversity of community ANOVA statistical method and Shannon Index. F-value: 2.2206; p-value:
0.17914
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Proteobacteria
Kauaʻi Lehua
Firmicutes
Bacteroidetes
Actinobacteria
Figure 5.8 Relative abundance of phylotypes at the phylum level and at the family level from in vitro fecal
fermentation samples.
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Table 5.1 Nonparametric correlation coefficients (Spearman’s rank) between combinations of microbial taxa and SCFA.
Isobutyric Heptanoic Acidaminococcac Desulfovibrionac Enterobacteriace

Variables Acetic acid Propionic acid Butyric acid Isovaleric acid Valeric acid Bacteroidaceae Lachnospiraceae Veillonellaceae
acid acid eae eae ae
Acetic acid 1 0.994 *** 0.945 *** 0.901 *** 0.918 *** 0.897 *** 0.604 *** -0.127 -0.316 -0.052 0.299 -0.455 -0.017
Propionic acid 1 0.946 *** 0.910 *** 0.920 *** 0.913 *** 0.623 *** 0.216 -0.143 0.268 -0.015 -0.546 0.322
Isobutyric acid 1 0.974 *** 0.990 *** 0.975 *** 0.715 *** 0.335 -0.039 0.369 -0.108 -0.543 0.377
SCFAs Butyric acid 1 0.953 *** 0.978 *** 0.739 *** 0.421 -0.012 0.460 -0.259 -0.476 0.521
Isovaleric acid 1 0.966 *** 0.695 *** 0.623 0.270 0.625 -0.535 -0.376 0.696 *
Valeric acid 1 0.778 *** 0.527 0.108 0.485 -0.397 -0.412 0.617
Heptanoic acid 1 0.457 0.711 * 0.566 -0.456 -0.004 0.165
Acidaminococcaceae 1 0.711 * 0.888 ** -0.853 ** -0.385 0.886 ***
Bacteroidaceae 1 0.720 * -0.692 * -0.088 0.359
Desulfovibrionaceae 1 -0.818 ** -0.331 0.758 *

taxa Enterobacteriaceae 1 -0.124 -0.813 **
Lachnospiraceae 1 -0.302
Veillonellaceae 1
Spearman Correlation Coefficient

-1 -0.5 0 0.5 1
*P < 0.05 **P< 0.01 ***P<0.001
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5.7 SUPPLEMENT MATERIAL
(A) (B)
Figure 5.9 Alpha-diversity index of bacterial community ANOVA statistical method and (A)
observed; (B) Chao1 diversity measure, p-value: 0.36535; F-value: 1.1667.
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Tahitian
Moi
Mana Ulu
Kauai Lehua
Kauaʻi
Bun-long Actinobacteria
Bacteroidetes
Inulin Firmicutes
Proteobacteria
Glucose
FOS
Control
Baseline
0% 20% 40% 60% 80% 100%
Figure 5.10 Relative abundance of phylotypes at the phylum level from in vitro fecal fermentation
samples.
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CHAPTER 6 INTAKE OF TARO (COLOCASIA ESCULENTA) AND
RISK OF COLORECTAL CANCER (CRC): THE MULTIETHNIC
COHORT
6.1 ABSTRACT
Background/Objective: Taro (Colocasia esculenta) may be protective against colorectal cancer (CRC) due to
its higher total dietary fiber content. The current study determined the association of taro
consumption, frequency of consumption, and dietary fiber intake with CRC incidence in the
Multiethnic Cohort (MEC) study.
Methods: The study included 168,294 participants of Native Hawaiian, Japanese American, Latino,
African American, and white ancestry with 1,781 incident CRC cases after 16 years of follow-up.
Dietary intake was assessed using a validated quantitative food frequency questionnaire, and dietary
fiber from taro was estimated from self-reported consumption. Cox hazard regression, adjusted for
potential confounders, was applied to estimate hazard ratios (HR) and 95% confidence intervals (CI)
for CRC.
Results; The consumers of taro showed a lower risk of CRC, though significance was not obtained,
which was also evident in stratified groups for men, women, Native Hawaiians, Latinos, and white.
Increased frequency of consumption (<1 /month, 1-3 /month, ≥ 1 /week) also exemplified a
decreased risk. The estimated dietary fiber intake (50-100 mg/day) from taro also showed a significant
association with CRC risk (HR =0.88; 95% CI, 0.78-0.99).
Conclusion: The results of this study illustrates that intake of taro, frequency of taro consumption, and
dietary fiber from taro show a protective trend and have potential to reduce the risk of developing
CRC.
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6.2 INTRODUCTION
Taro (Colocasia esculenta) is a nutrient dense root crop that is culturally important across Asia
and the Pacific region [58]. Nutritionally, taro has broader complement of vitamins and other nutrients
than other tubers, including: potato, sweet potato, and cassava [60-62]. The consumption of 200 g of
fresh taro corm per day meets the recommended daily allowance/acceptable intake (RDA/AI) values
for calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), phosphorus (P), and zinc (Zn) [27], all of
which are important constituents of the human diet [62, 75-77]. Similarly, an estimated consumption
of 1 lb (454 g) taro per day by Pacific Islanders was found to meet the recommended RDA value of
dietary fiber of 25 g per day [29]. As a gluten free and a high energy yielding root of around 135 kcals
per 100 g [416], taro may be a healthier alternative carbohydrate source for avoiding food allergies and
allergy related disorders, serving as a potential food security crop, and offering other health benefits
[62, 94].
Taro’s unique nutrient composition has anti-cancer properties and prevention potential,
especially against colorectal cancer (CRC). Several in vitro studies have shown that certain nutrient
constituents from taro have protective characteristics against CRC, such as fat [87], protein [160, 188],
and dietary fiber [110, 112]. Specifically, taro’s high concentration of dietary fiber, 5.1 g /100 g [417],
is noteworthy for several health benefits [104, 111]. Dietary fiber has consistently been associated with
decreased risk for the development of CRC [418-420].
CRC is the third most diagnosed cancer in both men and women [73]. As of 2018, CRC is the
rd
3 most frequently diagnosed cancer in Hawaiʻi, with approximately 720 newly diagnosed cases and
220 deaths each year. In Hawaiʻi, CRC is the 2nd leading cause of cancer death in men and the 3 rd
leading cause of cancer death among women [32]. Based on the analysis of epidemiological studies
around the world, an estimate of over 90% of gastric and colonic cancers can be attributed to diet.
[39, 48, 74]. Specifically, high fiber diets have been shown to significantly decrease the risk of CRC
[39, 48]. Thus, dietary interventions, by including foods rich in dietary fiber, such as taro, may prove
to have preventative effects against CRC in the long term.
Despite several in vitro studies, epidemiological evidence of taro consumption and its effects
on CRC is currently very limited. CRC affects individuals around the world, with the incidence of
CRC at younger ages (before age 50 years) showing increased disease burden [421]. Taro has much
potential to serve as a food security crop fulfilling essential nutrient requirements, with the additional
protective benefits against CRC development. Thus, this study aimed to understand the effects of taro
consumption (g/day), frequency of consumption, and dietary fiber intake from taro on the risk of
CRC using multiethnic cohort (MEC) data, which included participants of Native Hawaiian, Japanese
American, Latino, African American, and white ancestry.
6.3 METHODS
6.3.1 Study Population

The Multiethnic Cohort (MEC) is a prospective cohort study established to investigate the
influence of lifestyle, such as diet, and genetic factors on the occurrence of cancer and other chronic
diseases [422]. The Institutional Review Boards of the University of Hawaiʻi and the University of
Southern California approved the study, with the baseline questionnaire comparing of informed
consent for participation in the investigation. The study participants were recruited through targeted
recruitment primarily of five major races/ethnicities: African American, Native Hawaiian, Japanese
American, Latino, and white. During an average 16 years, 4,333 incident colorectal cancer cases
diagnosed.
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6.3.2 Questionnaire and follow-up data
At cohort entry, a 26-page, self-administered questionnaire (QX1) [422] collected detailed
exposure information on demographics, anthropometric characteristics, lifestyle factors, medical
conditions, family history of cancer, reproductive history, and a diet history by means of a validated
quantitative food frequency questionnaire (QFFQ), which included ethnic-specific foods and was
linked to a large database containing local recipes [423]. In the QX1, one of the QFFQ food items
was taro, which provided the g/day intake and frequency of consumption.
6.3.3 Nutritional Data

The intake of taro was assessed as taro, a specific food item in the QFFQ, and taro food
products, which were a combination of food items from the QFFQ that included taro and poi.
Participants were able to select from 8 frequency categories and 3 serving sizes [422]. The frequency
of consumption and daily intake categories for taro and taro food products were analyzed separately.
Daily intake was categorized as non-consumers vs. consumer; while frequency of intake was classified
using cutoffs of <1 /month, 1-3 /month, ≥ 1 /week, which approximately represents the 50 th, 75th,
and 90th percentiles, respectively.
To asses dietary fiber intake from taro of participants, United States Department of
Agriculture (USDA) Food Data Central (FDC) was used as a reference for taro (cooked, without salt;
FDIC ID: 168486) total dietary fiber content of 5.1 g/100 g [417]. Individual levels of dietary fiber
were categorized using the cutoff of <50 mg/day, 50-100 mg/day, and >100 mg/day, which
approximately represents the 50th, 75th, and 90ths percentiles, respectively.
6.3.4 Colorectal Cancer Cases

The colorectal cancer cases were obtained from surveillance, epidemiology, and end results
program (SEER) registries in Hawaiʻi and California. Participant deaths were identified through death
certificate files in both states and the National Death Index, which were completed to December 31,
2013. Participant colorectal cancer diagnosis was 4,333 cases, throughout an average of 16 years.

For the current analysis, 168,294 cohort members were retained after excluding the following
observations: 13,987 other ethnicity, 2,251 reported previous CRC, 301 reported CRC from tumor
registry, 11 case classification error, 3 inconsistent date, 8,116 missing/invalid data point, and 22,691
missing data on covariates.
Cox regression was applied to estimate hazard ratios (HR) and 95% confidence intervals (CI)
for colorectal cancer risk for the highest vs. lowest-intake categories. The age period of observation
was the age at cohort entry to the earliest of the following ages: age at diagnosis, age at death, and age
at study close (December 31, 2013). Base models for men and women separately were adjusted for
race/ethnicity as a strata variable and age at cohort entry as a covariate.
Similarly to prior analysis [45, 424] the following multivariate models were further adjusted
using the following covariates: family history of colorectal cancer (yes/no), history of colorectal polyp
(yes/no), body mass index (<25, 25 to <30, and ≥ 30 kg/m2), multivitamin use (yes/no), nonsteroidal
anti-inflammatory drug use (yes/no), pack-years of cigarette smoking (continuous), physical activity
(hours spent in vigorous work or sports per day), menopausal status and menopausal hormone therapy
use (premenopausal, postmenopausal: never, past, current use) for women only, alcohol consumption
(g/day), dietary fiber (g/kcal/day), dietary folate (mcg/day), vitamin D (IU/day), and total energy (log
transformed kcal/day). Because sub-group analysis showed similar association patterns in men and
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women between different ethnicities, we present models combining men and women using
multivariate adjustment. All statistical tests were performed by using SAS statistical software, version
9.4 (SAS institute, Inc., Cary, NC).
6.4 RESULTS
Baseline characteristics for all the covariates of the men and women included in the present
analysis are shown by taro consumption (non-consumers and consumers) and taro frequency
(<1/month, 1-3/month, ≥ 1 /week) (Table 1). Taro non-consumers were more likely to have a higher
age at cohort entry, ever smoked, multivitamin use, family history of colorectal cancer, history of
intestinal polyps, red meat intake, and ever used menopausal hormone therapy (MHT), compared to
taro consumers. Consumers of taro frequency <1 /month were more likely to be at a higher age at
cohort entry, use multivitamins, nonsteroidal anti-inflammatory drugs (NSAID) users, have family
history of CRC, history of intestinal polyps, alcohol intake, red meat, and MHT users. In contrast,
consumers of taro frequency ≥ 1/week were more likely to have a higher body mass index, energy
intake, dietary fiber intake, calcium intake, dietary folate intake, and vitamin D intake.
Results by daily intake amounts and frequency of intake showed an inverse relationship
between taro consumption and taro frequency and CRC risk (Table 2). Though no significance was
attained, the HR for the multivariate model for taro consumers was 0.98 (95% CI, 0.86-1.10), while
the HR for frequency of 1-3 /month vs <1 /month was 0.99 (95% CI, 0.88-1.13). Despite the low
taro frequency intake, more frequent (≥ 1 /week vs <1 /month) was related to lower CRC risk
(HR=0.76; 95% CI, 0.47-1.21).
In sex-specific analyses, daily taro intake amounts and frequency of intake also showed an inverse
relationship (Table 3). The HR for taro consumers among men was 0.99 (95% CI, 0.84-1.17) and for
women was (0.92 (95% CI, 0.79-1.09). The HR for men showed that 1-3 /month vs <1 /month of
taro was 1.01 (95% CI, 0.85-1.19), though more frequent intake (≥ 1 /week vs <1 /month) was related
to a lower risk of CRC ( HR= 0.88; 95% CI, 0.47-1.165). Similarly, the HR for women showed that
1-3 /month vs <1 /month of taro was 0.99 (95% CI, 0.82-1.20), with more frequent intake (≥ 1 /week
vs <1 /month) being related to a greater reduction in risk of CRC (HR= 0.66; 95% CI, 0.33-1.34).
In race/ethnicity-specific analyses with men and women combined, taro consumption and
frequency of taro intake were inversely related to CRC. The HR for Native Hawaiians, Latino, and
white populations were 0.83 (95% CI, 0.64-1.08), 0.59 (95% CI, 0.27-1.33), and 0.865 (95% CI, 0.640-
1.17), respectively (Table 4). In contrast, African Americans and Japanese taro consumers showed an
increased risk of CRC. Similarly, frequency of taro consumption of 1-3 /month vs <1 /month for
Native Hawaiians, Latino, and White populations showed a decreased risk of CRC with HR being
0.87 (95% CI, 0.66-1.15), 0.653 (95% CI, 0.292-1.46), and 0.91 (95% CI, 0.67-1.23), respectively. In
contrast, African American and Japanese American showed an increased risk of CRC for frequency
of taro consumption of 1-3 /month vs <1 /month.
The estimate of dietary fiber intake from taro was significantly inversely related to CRC risk
(P for trend = 0.046) (Table 5). Those in the 50-100 mg/day category had a significantly lower HR of
0.88 (95% CI, 0.78-0.99) than the lowest intake. Furthermore, the highest category >100 mg/day
showed a protective HR of 0.86 (95% CI, 0.73-1.02) as compared to the lowest intake, though
significance was not obtained. In sex specific models, men had a protective HR of 0.86 (95% CI, 0.73-
1.02) for the 50-100 mg/day category compared to the lowest intake, though significance was not
obtained. Similarly, women had a protective HR of 0.89 (95% CI, 0.75-1.06) with 50-100 mg/day
category compared to the lowest intake, and an even further protective HR of 0.86 (95% CI, 0.67-
1.10) with >100 mg/day, though significance was not obtained. In ethnic-specific models, across all
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ethnicities a protective HR trend was seen with 50 – 100 mg/day, though no significance was obtained.
In contrast, the highest category of >100 mg/day only had protective HR trends from Native
Hawaiian, Japanese American, Latino, and white groups, though no significance was obtained.
6.5 DISCUSSION
A decreased risk trend for CRC was shown among taro consumers and those with high
frequency of consumption, with significance only attained from estimated taro dietary fiber and an
inverse relationship with CRC. These results suggest that dietary taro consumption and frequency of
consumption have potential to decrease the risk of CRC, and provide a foundation for future
explorations of taro’s dietary disease prevention.
Potential mechanisms for the protective association between taro and CRC may be the unique
nutritional constituents of taro. The fat extract from taro, monogalactosyl diacylglycerols (MGDG),
has shown to have inhibitory effects against human lanosterol synthase (hOSC) and showed great
promise to suppress cholesterol biosynthesis [87]. Furthermore, spinach MGDG extract has shown
to suppress proliferation of colon cancer cells, in vitro, by selectively inhibiting the activities of
mammalian replicative DNA polymerases (α, δ and ε) [93]. Thus, taro MGDG extract could potential
suppress the proliferation of CRC and warrants further exploration. Dietary fiber of taro was shown
to have mutagen-biding properties and specifically absorb 1,8-dinitropyrene (DNP), an environmental
hydrophobic mutagen that can contribute towards the progression of CRC [112]. The protein tarin, a
globulin 1, was shown to have anti-metastatic [49] and anti-tumoral effects [160] against cancer cells.
From a whole foods perspective, poi, a Hawaiian fermented taro corm food, induced apoptosis of
colonic adenocarcinoma cells and activated lymphocytes that can lyse cancerous cells. A study
suggested that poi may inhibit the proliferation of colon cancer cells and stimulate the immune system
[50]. In addition, poi has been shown to support the growth of probiotic lactic acid bacteria (LAB),
resulting in 85% of the total microflora composition to be LAB after 24 hours [51]. This increase in
probiotic bacteria is necessary for enhanced production of beneficial bacterial fermentation products,
specifically short chain fatty acids (SCFAs). A study looking at in vitro fermentation of tropical foods
showed that SCFAs, specifically butyrate, production increased as the starch content of the tropical
food samples increased and was the highest for taro [52]. Butyrate has shown to induce differentiation
of phenotypes in colorectal tumor cells, induce apoptosis of CRC cells, and downregulate certain CRC
related genes [31, 32, 53, 54]. Thus, these studies provide strong evidence for using taro as a dietary
preventative measure against CRC.
Taro’s high dietary fiber content is also a strong variable for its preventative characteristics
seen against CRC. Dietary fiber has been shown to decrease the risk of CRC development [425]. These
results have been corroborated by other population studies. Results of the Prostate, Lung, Colorectal,
and Ovarian Cancer Screening Trial illustrated that elevated total dietary fiber intake was associated
with a significantly reduced risk of incident distal colorectal adenoma (OR highest vs. lowest tertile of intake: 0.76;
95% CI: 0.63-0.91; P-trend = 0.003) [323]. In the Japan Collaborative Cohort Study, participants were
found to have a decreasing trend in risk of CRC with increasing intake of total dietary fiber; the
multivariate-adjusted rate ratio (RR) across quartiles were 1.00, 0.96 (95% CI, 0.72-1.27), 0.72 (95%
CI, 0.53-0.99), and 0.73 (95% CI, 0.51-1.03; Ptrend = 0.028) [419]. The European Prospective
Investigation into Cancer and Nutrition (EPIC) cohort study with over 400,000 European participants
reported that dietary fiber protected against colorectal cancer incidence [420]. A previous analyses of
the MEC after an average 7.3 years of follow-up with 2,110 colorectal cancer cases found a 38%
lowered risk (95% CI for HR: 0.48–0.79) in the highest intake quintile among men, but a 12%
reduction (95% CI for HR: 0.67–1.14) among women (compared to 25%, 95% CI: 0.61–0.92, before
91
adjustment for other lifestyle factors) [426]. These results are further corroborated by a meta-analysis
of 25 prospective studies showing that the risk of CRC decreased by 10% (95% CI, 6%–14%) per 10-
g/day increase in dietary fiber intake [425].
Strengths of the current analysis include the prospective cohort design, large participant size,
diverse racial/ethnic backgrounds, a long follow-up period, and comprehensive information on a wide
range of potential confounding factors. In addition, diet assessed at baseline uses a validated QFFQ
designed to capture nutritional intakes of the five different ethnic groups [423]. The long follow-up
accommodates CRC’s long latency period, estimated to be over 10 years [427, 428]. Limitations of the
current analysis include a lack of validation for taro varieties in the FFQ that may have introduced
misclassification bias [429]. Without the inclusion of other food sources of dietary fiber, it is not
possible to examine potential differences due to taro-derived dietary fibers and total dietary fiber
intake. However, analysis of the EPIC cohort with multivariable adjustments, similar to the present
studies, confirmed significant inverse trends between consumption of total dietary fibers from all food
sources and CRC risk [418]. In addition, dietary measurements are based on a self-administered
QFFQ, which is subject to measurement error. However, this error is unlikely to be correlated with
disease risk in a cohort study, and thus should result in attenuation of the risk estimates [427].
Although the overall sample size was large, the subgroup analyses for taro consumption had limited
statistical power [45] that was seen with the small number of cases in the racial/ethnic group analysis,
such as Native Hawaiians. Lastly, dietary habits may have changed during the follow-up period [45].
6.6 CONCLUSION
The results of this study provide evidence that taro consumption has potential preventative
properties to decrease the risk of CRC development. This was exemplified by dietary fiber from taro
showing a significant inverse relationship with CRC. In future studies, a better assessment of taro
varieties would allow for a more accurate assessment of dietary fiber intake and elucidate the role of
dietary fiber in CRC disease development. Thus, this study suggests that taro has nutritive
constituents, such as dietary fiber, that potentially hold preventative properties against CRC
development. Therefore, it lays the groundwork to explore dietary taro varieties, dietary fiber from
taro, and potential health benefits of taro in chronic disease development.
ACKNOWLEDGMENTS:
The authors are grateful to Minji Kang, PhD, Gertraud Maskarinec, PhD, and Carol J Boushey,
PhD, Cancer Epidemiology Program, University of Hawaiʻi Cancer Center, and the MEC participants.
Supported in part by the National Cancer Institute at the National Institutes of Health grants
P01CA168530, P30 CA071789 and U01CA164973.
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Table 6.1 Baseline characteristics of the study population (n=190,985), Multiethnic Cohort, 1993-2010
Taro Consumption Frequency of Taro Consumption
None Consumers Consumers <1 /month 1-3 /month ≥4 /week
Sex, n (%)
Men 79,530 (44.91) 6,379 (45.85) 72541 (45.34) 5879 (46.7) 484 (37.69)
Women 97,543 (55.09) 7,533 (54.15) 87440 (54.66) 6711 (53.3) 800 (62.31)
Race/Ethnicity, n (%)a
African American 32,556 (18.39) 5216 (1.55) 27863 (17.42) 174 (1.38) 35 (2.73)
Native Hawaiian 8,920 (5.04) 4,827 (34.7) 8298 (5.19) 4011 (31.86) 803 (62.54)
Japanese American 48,589 (27.44) 5,383 (38.69) 46422 (29.02) 5188 (41.21) 189 (14.72)
Latino 42,922 (24.24) 572 (4.11) 35722 (22.33) 506 (4.02) 61 (4.75)
Non-Hispanic White 44,086 (24.9) 2,914 (20.95) 41676 (26.05) 2711 (21.53) 196 (15.26)
Age at Cohort Entry, y, mean (SD) 60.0 (8.8) 59.2 (9.0) 59.63 (8.83) 59.28 (9.05) 58.8 (8.97)
Body Mass Index, kg/m , mean (SD)
2 26.6 (5.1) 26.8 (5.6) 26.5 (5.07) 26.67 (5.47) 28.51 (6.67)
Ever smokers, n (%) 97,829 (56.13) 7386 (53.72) 88879 (56.2) 6636 (53.31) 725 (57.31)
Physical activity, h/d, mean (SD)b 1.17 (1.4) 1.5 (1.6) 1.2 (1.35) 26.67 (5.47) 1.63 (1.88)
Multivitamin Use, n (%) 88,867 (51.37) 6,544 (48.15) 80332 (50.91) 5938 (48.19 585 (47.52)
NSAID use, n (%) 91,405 (52.93) 6,576 (48.23) 81976 (51.87) 5926 (47.94) 630 (50.97)
Family History of Colorectal Cancer, n (%) 13,972 (7.89) 1,203 (8.65) 12889 (8.06) 1108 (8.8) 93 (7.24)
History of Intestinal Polyps, n (%) 9,622 (5.43) 790 (5.68) 8869 (5.54) 727 (5.77) 62 (4.83)
Energy Intake, kcal/d, mean (SD) 2142.5 (1044.7) 2,655.2 (1,233.6) 2147.08 (1028.38) 2585 (1173.25) 3344.96 (1547.92)
Alcohol Intake , g/d, mean (SD) 9.0 (25.1) 8.5 (25.5) 9.19 (25.13) 8.59 (25.67) 7.38 (23.52)
Red Meat, g/kcal/day, mean (SD) 18.5 (12.7) 17.7 (10.7) 18.38 (12.41) 17.9 (10.68) 16.19 (11.18)
Dietary Fiber, g/kcal/day, mean (SD) 11.8 (4.3) 11.8 (4.2) 11.64 (4.21) 11.62 (4.08) 13.69 (4.34)
Calcium, mg/day, mean (SD) 850.9 (524.4) 977.7 (582.1) 845.76 (514.47) 946.21 (553.05) 1286.56 (745.07)
Dietary Folate, mcg/day, mean (SD) 692.0 (433.5) 821.0 (490.3) 689.54 (426.86) 797.86 (469.83) 1047.97 (611.34)
Vitamin D, IU/day, mean (SD) 143.9 (108.5) 184.6 (138.9) 143.7 (107.5) 178.49 (132.91) 244.93 (176.6)
MHT Ever Use Among Postmenopausal
43,018 (55.36) 3,074 (52.77) 39235 (56.06) 2795 (53.72) 269 (44.46)
Women
MHT, menopausal hormone therapy; NSAID, nonsteroidal anti-inflammatory drug.
aColumn percentages.
bHours spent in vigorous work or sports per day.
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Table 6.2 Taro consumption and frequency of taro intake and colorectal cancer risk in the Multiethnic Cohort Study, 1993-2013
Overall (n=168,294)
Basic modela Multivariate modelb
Casesc HR (95% CI) HR (95% CI)
Taro Consumption
Non Consumers 155,851 1.00 (ref.) 1.00 (ref.)
Consumers 12,443 0.97 (0.86-1.09) 0.98 (0.86-1.10)
P for trendd 0.6199 0.6924
Taro Frequency
<1 /month 159,981 1.00 (ref.) 1.00 (ref.)
1-3 /month 12,590 0.99 (0.88-1.12) 0.99 (0.88-1.13)
≥1 /week 1,284 0.75 (0.47-1.19) 0.76 (0.47-1.21)
P for trendd 0.5468 0.5755
aAdjusted in Cox regression model of colorectal cancer for age at cohort entry, ethnicity, and sex
bAdditionally adjusted for family history of colorectal cancer, history of colorectal polyp, body mass index, pack-years of cigarette smoking, multivitamin use,
nonsteroidal anti-inflammatory drug use, vigorous physical activity, menopausal hormone therapy use for women only, ethanol, dietary fiber, dietary folate, vitamin D,
and totally energy
cExcluding participants with missing data on any of the covariates
dTrend variables were assigned median values for quintiles
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Table 6.3 Taro consumption and frequency of taro intake and colorectal cancer risk in men and women from the Multiethnic Cohort
Study, 1993-2013
Men (n= 77,951) Women (n= 90,343) P for

Casesa HR (95% CI)b Casesa HR (95% CI)b Heterogeneityc
Taro Consumption
Non Consumers 72,096 1.00 (ref.) 83,755 1.00 (ref.)
Consumers 5,855 0.99 (0.84-1.17) 6,588 0.92 (0.79-1.09)
P for trendd 0.9079 0.6798 0.3755
Frequency of Taro
<1 /month 67619 1.00 (ref.) 78,575 1.00 (ref.)
1-3 /month 5,424 1.01 (0.85-1.19) 5930 0.99 (0.82-1.20)
≥1 /week 418 0.88 (0.47-1.65) 647 0.66 (0.33-1.34)
P for trendd 0.9182 0.5115 0.5626
aExcluding participants with missing data on any of the covariates.

bAdjusted in Cox regression model of colorectal cancer for age at cohort entry, ethnicity, family history of colorectal cancer, history of colorectal polyp, body mass
index, pack-years of cigarette smoking, multivitamin use, nonsteroidal anti-inflammatory drug use, vigorous physical activity, menopausal hormone therapy use for
women only, ethanol, and total energy.
cBased on Wald test comparing associations between men and women and adjusting for covariates in the multivariate models
dTrend variables were assigned median values for quintiles.
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Table 6.4 Taro consumption and frequency of taro intake and colorectal cancer risk in the five ethnicities from the Multiethnic Cohort
Study, 1993-2013
African American Native Hawaiian Japanese American Latino White

(n=32,772) (n=13,747) (n=53,972) (n=43,494) (n=47,000) P for
heterogeneityb
Cases HR (95% CI)a Cases HR (95% CI)a Cases HR (95% CI)a Cases HR (95% CI)a Cases HR (95% CI)a
Taro Consumption
Non Consumers 26690 1.00 (ref) 8096 1.00 (ref) 44788 1.00 (ref) 35580 1.00 (ref) 40697 1.00 (ref)
Consumers 157 1.45 (0.65-3.24) 4265 0.83 (0.64-1.08) 4857 1.07 (0.91-1.27) 492 0.59 (0.27-1.33) 2672 0.865 (0.64-1.17)
P for trendc 0.3661 0.1599 0.394 0.2031 0.3486 0.5316
Frequency of taro
<1 /month
24204 1.00 (ref) 7662 1.00 (ref) 43531 1.00 (ref) 31684 1.00 (ref) 39113 1.00 (ref)
1-3 /month
129 1.84 (0.82 -4.11) 3590 0.87 (0.66-1.15) 4694 1.07 (0.91-1.26) 443 0.653 (0.292-1.46) 2498 0.91 (0.67-1.23)
≥1 /week
25 nq* 668 0.65 (0.35-1.21) 158 1.24 (0.56-2.77) 47 nq* 167 0.34 (0.05-2.40)
P for trendc 0.5749 0.1286 0.3641 0.17111 0.2899 0.6055
aAdjusted for age at cohort entry, sex, family history of colorectal cancer, history of colorectal polyp, body mass index, pack-years of cigarette smoking, multivitamin
use, nonsteroidal anti-inflammatory use, vigorous physical activity, menopausal hormone therapy use, ethanol, and total energy.
bBased on Wald Test comparing associations between race/ethnicity and adjusting for the covariates in the multivariate models
cTrend variables were assigned the median values for quintiles
nq: not quantifiable
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Table 6.5. Estimated dietary fiber intake from taro and incidence of CRC Multiethnic cohort, 1993-2010
Estimated Dietary Fiber Intake (mg/day)

Categories P for trendd
<50 50- 100 >100
Casesa HR (95% CI)b Casesa HR (95% CI)b Casesa HR (95% CI)b
Multivariate Model 9655 1.00 (ref.) 146194 0.88 (0.78-0.99) 12445 0.86 (0.73-1.02) 0.046
Sex
Men 4476 1.00 (ref.) 67619 0.86 (0.73-1.02) 5856 0.86 (0.68-1.08) 0.202
Women 5179 1.00 (ref.) 78575 0.89 (0.75-1.06) 6589 0.86 (0.67-1.10) 0.228
P for Heterogeintyc 0.6672
Ethnicity
African American 2486 1.00 (ref.) 24204 0.84 (0.68-1.04) 157 1.24 (0.54-2.83) 0.196
Native Hawaiian 433 1.00 (ref.) 7662 0.79 (0.45-1.4) 4266 0.67 (0.37-1.20) 0.108
Japanese American 1256 1.00 (ref.) 43531 0.88 (0.67-1.17) 4858 0.95 (0.69-1.31) 0.746
Latino 3896 1.00 (ref.) 31684 0.95 (0.76-1.17) 492 0.56 (0.25-1.29) 0.341
white 1584 1.00 (ref.) 39113 0.83 (0.61-1.12) 2672 0.72 (0.48-1.10) 0.131
P for heterogeintyc 0.5341

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CHAPTER 7 DIETARY PATTERNS ASSOCIATED WITH GUT
MICROBIAL HEALTH AND RISK OF COLORECTAL CANCER:
THE MULTIETHNIC COHORT STUDY
7.1 ABSTRACT
Background & Aims: Dietary patterns may reveal effective means to reduce disease burden of colorectal
cancer (CRC). This study aimed to determine dietary patterns informed by gut health principles
derived from reduced rank regression (RRR) and whether hypothetical patterns could be associated
with CRC.
Methods: Participants of the Multiethnic Cohort (MEC), a prospective cohort study, completed a
baseline food frequency questionnaire. Responses were used to derive three dietary patterns using
RRR. Dietary pattern scores were divided into quintiles and evaluated for association with CRC among
168,294 participants. During a mean follow-up of 16 years, 4,333 incident invasive cases were
identified. Cox proportional hazard models were used to calculate basic and multivariable-adjusted
hazard ratios (HR) and 95% confidence intervals (95% CI).
Results: Dietary pattern 1, which loaded on fruits and vegetables, dairy products and legumes and
exhibited an inverse association with CRC risk with the highest vs lowest quintile score having a HR
of 0.81 (95% CI: 0.73-0.91) and P for trend = <0.0001. Dietary Pattern 2 loaded on red and processed
meats was not significantly associated with CRC risk, with the highest vs lowest quintile score having
a HR of 1.06 (95% CI: 0.95-1.18) and P for trend = 0.16. Dietary Pattern 3 loaded on red meat,
processed red meats, processed poultry, and alcohol was associated with an increased CRC risk with
the highest vs lowest quintile score having a HR of 1.14 (95% CI: 0.99-1.31) and P for trend = 0.07.
Conclusion: Based on dietary patterns derived from RRR, CRC risk decreased with fruits, vegetables,
dairy, and legumes intake.
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7.2 INTRODUCTION
Colorectal cancer (CRC) is the third most frequently diagnosed form of cancer, and the fourth
leading cause of cancer related deaths in the world [430]. By 2030, the burden of CRC is predicted to
increase to more than 2.2 million new cases and cause around 1.1 million deaths per year, an estimated
60% increase [431]. The increasing incidence of CRC parallels economic development that can be
seen rapidly increasing in low-income and middle income countries and stabilizing in highly developed
countries [431, 432], although the incidence rates since the mid-1980s in the United States have
increased annually among adults younger than 55 years [73].
Evidence from ecological studies, migrant studies, and secular trend studies suggests lifestyle
risk factors are primary causal factors for CRC [42-45]. One such controllable lifestyle etiology is diet.
Studies have revealed that dietary factors affect colorectal carcinogenesis through a number of
mechanisms, including altering the composition of the gut microbiota, referred to as dysbiosis [38,
39]. Dysbiosis, the disruption of gut homeostasis, can occur with an unbalanced diet that causes the
dominant activities of gut microbiota in the colonic lumen to be protein fermentation through bile
acid deconjugation that creates damage in colon epithelial cells through proinflammatory and
preneoplastic by-products— leading to an increased risk of CRC [39].
The diet of developed nations is characterized by a high consumption of fat, red meat, and
sugary food and drinks and a high intake of alcohol and low intake of dietary fiber [39, 40], which
have been shown to have negative impacts on the composition of the gut microbiome [40, 46, 47].
Dietary sugars have been shown to affect the gut microbiome composition in humans [433-436], as
sugar is the preferred carbohydrate source of many bacteria [35, 435, 436]. Compared to
carbohydrates, fat is generally considered to be less important to the metabolism of microbes [37];
however, several studies have shown that high fat diets, such as animal based diets high in processed
and red meats, greatly alter the composition of gut microbiome and produce carcinogenic byproducts
[437-439]. Although alcohol metabolism occurs mainly in hepatocytes, several of the enzymes involve
alcohol oxidative metabolism present in the intestinal mucosa [440], with intestinal bacteria also
producing ethanol and acetaldehyde in the gastrointestinal tract [441]. Several studies have
demonstrated that alcohol promotes both dysbiosis and bacterial overgrowth that can contribute to
deleterious effects to the gut microbiome [442-444]. In contrast, dietary fiber has been shown to be
beneficial to the gut microbiome, as microbes utilize dietary fiber to produce beneficial fermentation
byproducts and to increase gut microbial diversity [35, 445, 446]. These data suggest that food
components of diets are great effectors of the composition of the gut microbiome and can serve as
indicators of the gut microbiome health status.
Food components, such as prebiotics and probiotics, have been identified to improve gut
microbial health [33, 275, 447-450]. Probiotics, according to the International Scientific Association
for Probiotics and Prebiotics (ISAPP), are defined as “live microorganisms that, when administered
in adequate amounts, confer a health benefit on the host” [449]. On the other hand, prebiotics are
indigestible in the human gastrointestinal tract and promote the growth of beneficial gut microbiota
[33]. Food sources of prebiotics include, but are not limited to: soybean, inulin, unrefined wheat and
barley, raw oats, yacon, and non-digestible oligosaccharides such as fructans, polydextrose,
fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), and
arabinooligosaccharides (AOS) [53, 446]. In the distal colon, prebiotics have been associated with
improved bowel functions, removal of carcinogenic toxins, and an overall reduced risk of colon cancer
[33, 43-45].
The combination of prebiotics and beneficial gut microbiota forms a symbiotic relationship
that helps maintain homeostasis and enhances health benefits [31, 140]. A health benefit of the
symbiosis is the production of short chain fatty acids (SCFA). These beneficial SCFAs are, acetate,
100
propionate, and butyrate, which are only produced through the fermentation of specific prebiotics,
specific dietary fibers, and resistant starches, found in foods by the gut flora [36]. Although the
production of these three SCFAs account for only around 5% of the total SCFA, they are of particular
interest resulting from their vast health benefits, e.g., energy source for colonic epithelium, regulators
of host immunity, metabolic homoeostasis, gut-brain signaling, interaction with host metabolites (bile
salts, local hormones such as glucagon-like peptide-1 and peptide YY), differentiation of phenotypes
and apoptosis in CRC and adenoma development [32, 451]. As such, the dietary components, such as
dietary fiber, found in foods greatly impact the richness and diversity of microbes that colonize and
flourish in the gut and ultimately affect the disease state of CRC [36, 439].
Foods are not consumed in isolation but are part of a total diet and few dietary guidelines have
considered the role of prebiotic and probiotic foods in gut health. Understanding the impact of certain
prebiotic and probiotic foods is important to glean information about modulating gut microbiome
homeostasis [36]. Poor quality diets affect the bacterial diversity of the gut microbiome that affects
human health disorders [452, 453]. Highlighting these symbiotes in dietary patterns may help
formulate the right preventative strategies for CRC and perhaps guide research in the direction of
more effective and less toxic therapies.
Previous studies addressing diets associated with risk of CRC found a reduced incidence with
high quality diets, however the majority of studies done to date have primarily been with non-Hispanic
white participants. Currently, few studies have been conducted with dietary patterns selected to
represent gut microbial health. As such, the use of the Multiethnic Cohort (MEC) population allowed
for an investigation of the associations between gut healthy dietary patterns and CRC risk in a racially
heterogenous population.
7.3 METHODS
7.3.1 Study Population

The Multiethnic Cohort (MEC) is a prospective cohort study established to investigate lifestyle
factors, such as diet, in the relation to cancer and other chronic disease [422]. In brief, more than
215,000 adults aged 45 to 75 enrolled in the MEC between 1993 and 1996 by completing a
comprehensive self-administered questionnaire that included a detailed food frequency dietary
assessment [422]. The study participants were recruited through targeted recruitment primarily of five
major race/ethnicities: African American, Native Hawaiian, Japanese American, Latino, and white.
For the present analysis, participants who were not from one of the five racial/ethnic groups (n=
13,987), had previous colorectal cancer identified on the baseline questionnaire (n=2,251) or from
tumor registries (n=301), data inconsistencies (n=13) or reported implausible diets based on total
energy intake or its components (n= 8,116) were excluded.
7.3.2 Colorectal Cancer Cases

Information on invasive incident colorectal cancer cases was ascertained from linkages to
surveillance, epidemiology, and end results program (SEER) registries in Hawaiʻi and California.
Deaths were identified by linkage to death certificate files in both states and the National Death Index.
Case and death ascertainment was complete through December 31, 2013. During an average of 16
years, 4,333 incident colorectal cancer cases diagnosed.
7.3.3Dietary Assessment and Food Groupings

The MEC baseline questionnaire included a quantitative food frequency questionnaire
(QFFQ) with over 180 food items, which was developed from 3-day measured dietary records from
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approximately 60 men and women in each of the five racial/ethnic groups [422]. Subsequently, a
calibration study showed satisfactory correlations for nutrients between the QFFQ and 3 repeated 24-
hour recalls for all ethnic-sex groups [429]. Daily nutrient intakes were calculated using the food
composition tables developed and maintained at the University of Hawaiʻi Cancer Center for use in
the MEC [422].
The 182 food items and food item groups from the MEC QFFQ were collapsed to 22 food
groups by macronutrient composition, food group designation (e.g. fruits), culinary usage, cultural
specificity, impact on the gut microbiome and classifications found in the literature [39, 454-456]. The
22 food groups included the following: red meat excluding processed red meat, processed red meat,
fresh poultry, processed poultry, fish excluding shellfish, shellfish, al legumes, light green vegetables,
dark green vegetables, yellow-orange vegetables, rice, potatoes and tubers, breakfast cereals, breads,
pasta, fruit juice alone, yellow-orange fruit, all dairy products, eggs, beer, wine, nuts excluding coconut.
These food groups served as the predictor variables. Unit designation for the foods comprising the
final 22 food groups was g/day. Multiple combinations of food groups were tested, with the final food
groupings shown in Table 1.

The statistical method reduced rank regression (RRR), otherwise known as the maximum
redundancy analysis, was used to derive dietary pattern scores. The use of this method to derive dietary
patterns has been described in detail elsewhere [454, 455, 457]. In brief, RRR allows for the calculation
of dietary pattern scores similar to those extracted by factor analysis. However, where factor analysis
determines dietary pattern scores by maximizing the explained variation of a set of predictor variables,
such as food groups, RRR derives dietary pattern scores from response variables that are disease
related nutrients, and predictor variables are food groups or items [457]. The percent energy from
total fat, the percent energy from alcohol, density of dietary fiber (g/1,000 kcal), and sugar intake
(g/day) were selected as the response variables because these variables have been frequently and
consistently associated with gut health [32, 39, 458-460]. Intake data from food groups determined by
the MEC QFFQ served as predictors. These food groups, the predictor variables, were classified into
distinct dietary patterns to capture the variation in the total fat, alcohol, fiber, and sugar, response
variables associated to gut health. In RRR, the number of extracted dietary patterns cannot be higher
than the number of selected responses variables. In this case, four dietary patterns were obtained; of
which three contributed enough variance for inclusion.
Factor loadings, which reflect the correlation of individual food groups within each of the
derived dietary patterns, were obtained from the RRR. To focus on food groups that significantly
contributed to the dietary pattern, values with an absolute factor loading > 0.2 are primarily displayed.
The food groups above the cut-off were used to inform the food exposures, a dietary pattern score
was calculated by summing the product of all the contributing food group intakes and scoring
coefficients. The scores for each dietary pattern were then converted into quintiles for use in further
analysis. Thus, for each dietary pattern Quintile 5 would be composed of those who conform most to
that particular pattern, while Quintile 1 would be the lowest conformers.
Cox proportional hazards models of colorectal cancer with age as the time metric were used
to calculate hazard ratios (HRs) and 95% confidence intervals (95% CI). The age period of observation
was the age at cohort entry to the earliest of the following ages: age at diagnosis, age at death, and age
at study close (December 31, 2013). The three derived patterns were categorized into quintiles based
on their distributions across the entire cohort. Additionally, trend variables for the indexes were
assigned median values for quintiles. Base models for men and women separately were adjusted for
race/ethnicity as a strata variable and age at cohort entry as a covariate. Multivariate models were
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further adjusted for family history of colorectal cancer (yes/no), history of colorectal polyp (yes/no),
body mass index (<25, 25 to <30, and ≥ 30kg/m 2), multivitamin use (yes/no), nonsteroidal anti-
inflammatory drug use (yes/no), pack-years of cigarette smoking (continuous), physical activity (hours
spent in vigorous work or sports per day), menopausal status and menopausal hormone therapy use
(premenopausal, postmenopausal: never, past, current use) for women only, alcohol consumption
(g/day), and total energy (log transformed kcal/day).
Participants with missing data on covariates (n=22,691) were excluded from the multivariate
models, resulting in n=168,294 participants. Because sub-group analysis showed similar association
patterns in men and women, we present models combining men and women using multivariate
adjustment. All statistical tests were performed by using SAS statistical software, version 9.4 (SAS
institute, Inc., Cary, NC).
7.4 RESULTS
Foods with a factor loading > |0.2|, which indicates the level of correlation to the derived
dietary patterns, are shown in Table 7.2. Based on the derived dietary patterns from RRR, Dietary
Pattern 1 exhibited factor loadings high in yellow-orange fruit, yellow-orange vegetables, dark green
vegetables, light green vegetables, fruit juice alone, all dairy products, all legumes, and low in beer. In
contrast, Dietary Pattern 2 was distinguished by high intakes of red meat, processed red meat, eggs,
breads, processed poultry, and low intakes of beer and wine. Dietary Pattern 3 predominately loaded
on breads, red meats, all dairy products, processed red meat, fruit juice, beer, eggs, and processed
poultry.
Baseline characteristics (Table 7.3) for all the covariates of the men and women included in the
present analysis are shown by extreme quintiles of the dietary patterns. Across all the dietary patterns,
individuals in the highest dietary pattern quintiles (Q5) were more likely to have a higher body mass
index, have ever smoked, and nonsteroidal anti-inflammatory drugs (NSAID) use compared with
those in the lowest quintiles (Q1). Participants in Q5 had higher energy intakes than in Q1, with the
exception of Pattern 3. Family history of colorectal cancer was more common in Q1 than Q5 across
dietary patterns.
In men and women combined (Table 7.4), Dietary Pattern 1 was significantly associated with
a decreased risk of CRC (P for trend <0.0001) with adjustment for age at cohort entry, race or
ethnicity, and sex, while Dietary Pattern 2 was not consistently associated to CRC in the basic model
and not statistically significant (P for trend 0.14). In contrast, Dietary Pattern 3 was significantly
associated with an increased risk of CRC (P for trend < 0.0001) with adjustment for age at cohort
entry, race or ethnicity, and sex. With further adjustment for CRC covariates, Dietary Pattern 1
remained having a strong statistical significance associated with a decreased risk (P for trend <0.0001).
Whereas Pattern 2 had a null association with CRC (P for trend = 0.16). Dietary Pattern 3 remained
associated with an increased risk of CRC, though not statistically significantly (P for trend = 0.07).
In sex-specific analyses (Table 7.5), only Dietary Pattern 1 exhibited statistically significant
inverse relationships to CRC for men and women (P for trend = 0.002 and P for trend = 0.03,
respectively), while the test for heterogeneity did not show statistically significant differences in the
associations between men and women (P’s for heterogeneity = 0.57). For Dietary Pattern 2, both men
and women did not show associations for CRC (P for trend = 0.58; P for trend = 0.12, respectively),
though the risk of CRC increased with every subsequent quintile in Dietary Pattern 2. For Dietary
Pattern 3, men exhibited an association with an increased risk of CRC (P for trend = 0.05); whereas,
in women, Dietary Pattern 3 showed no association for CRC (P for trend = 0.51).The test for
heterogeneity by sex for Dietary Pattern 3 showed a significant differences in the associations between
men and women (P’s for heterogeneity = 0.04).
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In the race/ethnicity-specific analyses in men and women combined (Table 7.6), Dietary
Pattern 1 was significantly associated with a decreased risk of CRC only in whites (P for trend = 0.02)
and Latinos (P for trend = 0.001) (P’s for heterogeneity = 0.34). None of the race or ethnic-specific
associations for Dietary Pattern 2 were statistically significant. However, Dietary Pattern 3 was
significantly associated with an increased risk for CRC only in whites (P for trend = 0.03), with the
test for heterogeneity across race/ethnic groups being not statistically significant (P for heterogeneity
= 0.35).
7.5 DISCUSSION
The dietary patterns hypothetically derived using the MEC data reflected different types of
eating habits. Dietary Pattern 1 positively loaded on yellow-orange fruit, yellow-orange vegetables,
dark green vegetables, light green vegetables, fruit juice alone, all dairy products, all legumes, and
negatively loaded on beer and was inversely associated with CRC risk. In contrast, Dietary Pattern 2
negatively loaded on beer and wine and positively loaded on red meat, processed red meat, eggs,
breads, processed poultry and was not associated with CRC risk. Lastly, Dietary Pattern 3 positively
loaded on breads, red meats, all dairy products, processed red meat, fruit juice, beer, eggs, and
processed poultry and was associated with a higher CRC risk. Collectively, these results can be used
to inform the development of a theoretically based dietary pattern to reflect the exposures associated
with the diverse complexity of food interactions with CRC and gut microbiome.
The use of RRR to determine dietary patterns is a relatively new approach to determining dietary
patterns in population-based studies [457]. The method has the combined strengths of a priori and a
posteriori methods that accounts for current scientific evidence and data from the study and correlate
the results with dietary components [461]. RRR has become the recommended method to use when
evaluating food group predictors, by maximizing the amount of variation in the response variables
[454, 456]. RRR seeks to capture the variation in intake with regard to certain response variance
variables [457]. The response variables selected in this study were specifically associated to a healthy
gut microbiome and include: fat, sugar, dietary fiber, and alcohol.
Although, in humans, most nutrient absorption from food occurs in the small intestine, several
nutrients can escape absorption in the small intestine and reach the distal gut, site of the gut microbiota
[462, 463]. As such, dietary intake becomes of vital importance in maintaining a healthy gut
microbiome. Evidence supports intake of dietary fiber influencing the composition of the gut
microbiota, which may impact the risk of CRC [36, 38, 44, 447]. In contrast, diets high in processed
and red meats, which are high in fat, have been proposed to increase risk of CRC through, among
other mechanisms, modulating pathogenic bacteria in the gut microbiome [39, 43-45, 47, 437, 464].
In addition, diets high in dietary fiber have been associated with a reduced risk of colorectal cancer
[33, 43-45]. Similarly, dietary sugars, especially diets high in simple carbohydrates relative to complex
carbohydrates, alters the microbial composition of the gut microbiome, increasing the risk of colon
cancer, possibly through their modifications of body mass and implications of high sugar lifestyle [433,
434, 465, 466]. Furthermore, alcohol has also been shown to change the composition of the gut
microbiome and has been directly linked to CRC [47, 467-469]. These food components have been
shown to affect the gut microbial composition, suggesting that bacterial gut composition mediate the
utilization of dietary food components and, thus, is important to highlight in dietary patterns.
Derived Dietary Pattern 1 contributed the most variance to fat, sugar, dietary fiber, and
alcohol, and was dominated by food items considered gut healthy, such as legumes, light green
vegetables, dark green vegetables, yellow-orange vegetables, breakfast cereals, and dairy products,
similar to results found in other populations [456, 470, 471]. In contrast, the Dietary Pattern 2 which
contributed the second highest variance to the responses variables, was heavily influenced by foods
104
considered detrimental to the gut and associated to colorectal cancer and included red meat, processed
red meat, processed poultry, similar to results found in other population studies [455, 456, 458, 470-
473]. Dietary Pattern 3 contributed the least to the response variables, heavily influenced by high
consumption of red meat, processed red meat, processed poultry, breads, dairy products, fruit juice,
eggs, and beer, similar to results found in other populations [467, 474, 475].
Patterns characterized by higher loadings of fruits, vegetables, fish, and poultry, with less red
meat and processed meat, have been inversely associated with CRC [455, 472, 476-478]. In contrast,
patterns characterized by higher loadings of red meat, processed meat, egg, refined grains, sugar
containing foods, and alcohol with low levels of fruit and vegetable intake have been associated with
higher risks of CRC [472, 473].
7.5.1 Dietary Pattern 1

Pattern 1, which positively loaded on yellow-orange fruit, yellow-orange vegetables, dark green
vegetables, light green vegetables, fruit juice alone, all dairy products, and all legumes and negatively
loaded on beer was associated with a decreased risk of CRC; in both men and women, and especially
in Latino, and whites. These foods are high in dietary fiber, which have been associated with a reduced
risk of CRC [39, 44, 439, 479]. In addition, Pattern 1 greatly resembled other population study dietary
patterns that have also positively loaded on vegetables and fruits [455, 472, 476-478] and corroborate
other MEC study findings [45, 480]. In addition, the latest American Institute for Cancer Research
(AICR) and the World Cancer Research Fund (WCRF) report concluded that foods containing fiber
decrease the risk of CRC [481].
7.5.1.1 Dietary Fiber and Gut Microbiome

Vegetables, such as light green vegetables, dark green vegetables and yellow-orange vegetables,
which were loaded in Pattern 1, have high amounts of inulin, oligofructose, and non-digestible
oligosaccharide ingredients, which are known prebiotic ingredients [140]. The fermentation of these
prebiotic ingredients by the gut microbiota produce SCFA, which have been shown to be potential
therapeutic tools to modulate inflammatory responses and for CRC treatment [482]. Prebiotics also
demonstrate anti-cancer properties by downregulating the expression levels of inflammatory markers
(COX-2, iNOS, and NF-kB) and gastrointestinal glutathione peroxidase through their bifidogenic
effects and immunomodulatory roles [479]. The administration of prebiotics in diets causes changes
in the gut microbiome composition that confer health benefits to the host and have potential for
prevention of CRC. [30]. One study found that diets rich in whole grains and dietary fiber were
associated with a lower growth of Fusobacterium nucleatum, which is a bacteria positively associated with
CRC progression [483]. This may explain the complex inverse association between diets high in dietary
fiber and the development of CRC, although further studies need to be conducted to identify gut
microbiota that are associated with diet and CRC development.
Prebiotics work synergistically with probiotics (symbiotic) to exert a beneficial impact on the
gut microbiota, making them a potential therapeutic strategy for CRC [479]. Probiotic bacteria,
specifically Lactobacillus spp. and Bifidobacerium spps., are common bacteria found in dairy products,
which positively loaded in Pattern 1. An in vitro study illustrated that administering Lactobacillus
plantarum to 5-FU-resistant colorectal cancer cells selectively inhibited the cells expression of certain
cancer-specific markers (CD44, 133, 166, and ALDH1) [484]. Similarly, ribosomal 16S ribosomal
RNA sequencing identified Bifidobacterium as being associated with antitumor effects with oral
administration to mice improving tumor control to the same degree as programmed cell death protein
1 ligand 1 (PD-L1)–specific antibody therapy (checkpoint blockade), and combination treatment
nearly abolished tumor outgrowth [485]. Administration of these bacteria has been studied as potential
CRC therapies for their antitumor responses [450, 485, 486].
105
Pattern 2 positively loaded on red meat, processed red meat, eggs, breads, processed poultry,
and negatively loaded on beer and wine; and was not significantly associated with risk of CRC.
Although Pattern 2 was not statistically significant, there was the suggestion that, as the intake quantity
increased, exemplified from quintile 1 to quintiles 5, so did the risk of CRC. The AICR and WCRF
report concluded that every 50 gram of processed meat consumed daily, which is equivalent to one
hot dog, is linked to a 16 percent increased risk of CRC [481]. Processed and red meats have been
associated with an increased risk of CRC in other population-based studies [472, 476-478, 487, 488].
Our results would suggest an association for processed and red meats and a higher risk for CRC.
7.5.2.1 Meat and Gut Microbiome

Red or processed meats have been associated with an increased risk of development of CRC
through mechanisms involving exposure to mutagens, such as polycyclic aromatic hydrocarbons,
heterocyclic amines, and dietary N-nitroso compounds.[47] Specifically, heterocyclic amines,
produced from meats cooked at high temperature, are chemical carcinogens to human cells [489]
[490]. Studies have suggested that meat processing, such as curing and smoking, potentially causes the
production of carcinogenic chemicals including heterocyclic aromatic amines (HAA), N-nitroso-
compounds (NOC), and polycyclic aromatic hydrocarbons (PAH), while cooked red meat, contains
suspected carcinogens, including HAA and PAH, all of which have been associated with CRC [491,
492]. As such, the International Agency for Research on Cancer (IARC) has classified the
consumption of processed meat as “carcinogenic to humans” (Group 1) on the basis of sufficient
evidence for colorectal cancer and classified the consumption of red meat as “probably carcinogenic
to humans” (Group 2a) [491]. It has been hypothesized that heterocyclic amines are CRC initiators,
while dietary fats promote the further development of CRC [493], which was one of the response
variables chosen in this study and a major component of the high red meat diets.
Besides exposure to carcinogenic compounds, a high red meat diet may play a role on the
effect of gut microbiome on the development of CRC [47]. Several studies have demonstrated that
the Bacteroides enterotype is highly associated with dairy, animal protein, and saturated fat, which is
characteristic of Western diets [494, 495]. Positive associations were found between consumption of
red meat and carcinoma-producing bacteria that includes: Bacteroides massiliensis, Alistipes finegoldii and
Bilophila wadsworthia [46]. Furthermore, red and processed meats might promote the growth of sulfate-
reducing bacteria that produce hydrogen sulfide, a genotoxic agent [459]. Specifically, B. wadsworthia, a
hydrogen sulphite-reducing bacterium, has been shown to be increase abundance and activity has been
shown to inflame intestinal tissues in both mice models fed a high-fat diet [496] and human’s fed a
high-fat animal diet [497]. As such, diet-induced changes to the gut microbiota have major implications
to contribute to the development of CRC. These factors may partially explain the association between
red and processed meats consumption and the development of CRC through the modulation of the
gut microbiome.

Pattern 3 was predominately loaded on breads, red meats, all dairy products, processed red
meat, fruit juice, beer, eggs, and processed poultry and exhibited an association with an increased risk
of CRC. Our results suggest pairing alcohol with processed and red meat, may significantly increase
risk for CRC. Slattery et al. [472], using principal components analysis, found that a “drinker” pattern,
in which alcoholic beverages and processed and red meats had the highest loadings, was associated
with an increased CRC risk. In addition, alcohol based dietary patterns have been found to be
associated with CRC risk in other studies [458, 467, 472, 498].
106
7.5.3.1 Alcohol and Gut Microbiome
Current evidence reviewed by the WCRF and the AICR concluded that there is convincing
evidence that consuming alcoholic drinks, approximately 30 grams per day (about two drinks per day),
increase the risk of CRC [499]. Though the mechanistic effects of alcohol carcinogenicity has not been
well established, some suspected mechanisms include: alcohol as a solvent for penetration of
carcinogens in cells; production of reactive oxygen species and reactive nitrogen species; alcohol
mediating estrogen and prostaglandin concentration in the body; formation of reactive and genotoxic
metabolites of alcohol (acetaldehyde); diets of high alcohol consumers being relatively low in essential
nutrients [500].
Alcohol has been proposed to affect the intestinal microbiota by reducing gastrointestinal
motility, suppressing innate and adaptive immune response, and inhibiting bactericidal protein
expression [501]. A study among hospitalized patients with chronic alcohol intake had higher levels
of aerobic and anaerobic microorganisms in jejunum aspirates, compared to hospitalized patients
without alcohol abuse [502]. In addition, colonic microbiome samples from colonic biopsies from
participants with chronic alcohol abuse showed dysbiosis with lower median abundances of
Bacteroidetes and higher Proteobacteria [442]. Similarly, a study found reduced levels of Bacteroidetes and
highly enriched Proteobacteria and Fusobacteria contents of stool samples in the alcohol-related cirrhosis
group [503]. As such, alcohol may also contribute to changes in the gut microbiome composition, and
ultimately development of CRC; however, further studies need to be conducted to strengthen the
evidence for such associations.
7.5.4 Study Strengths

Strengths of this study include its prospective design, large number of participants with diverse
racial/ethnic backgrounds, a long follow-up period and comprehensive information on a wide range
of potential confounding factors, as well as, diet assessed at baseline using a QFFQ designed to capture
nutritional intakes of 5 different ethnic groups. The long follow-up accommodates CRC’s long latency
period, estimated to be over 10 years [427, 428].
7.5.5 Study Weaknesses

Dietary measurements based on a self-administered QFFQ are subject to measurement error.
This error is unlikely to be correlated with disease risk in a cohort study, and thus should result in
attenuation of the risk estimates [427]. Although the overall sample size was large, the subgroup
analyses had limited statistical power [45]. In addition, dietary habits may have changed during the
follow-up period [45]. In addition, the gut microbiome of participants was not analysed and therefore,
the interaction between the gut microbiome and their diet is also unknown. Since the healthy gut
dietary pattern derived is study population-specific, the associations between these food groups and a
healthy gut microbiome needs to be confirmed in other populations.
7.6 CONCLUSION
In conclusion, in a multi-ethnic population, a healthful dietary pattern derived using RRR

loaded positively on intakes of yellow-orange fruit, yellow-orange vegetables, dark green vegetables,
light green vegetables, fruit juice alone, all dairy products, all legumes, and negatively on intake of beer,
which associated with a lower risk of CRC. A second dietary pattern with positive loadings of red
meat, processed red meat, eggs, breads, processed poultry, and negative loadings of beer and wine was
not significantly associated with CRC risk. Finally, a third dietary pattern which predominately loaded
on breads, red meats, all dairy products, processed red meat, fruit juice, beer, eggs, and processed
107
poultry, increased the risk of CRC. These results support that derived dietary patterns informed by
gut health principles have potential prevention for CRC.
ACKNOWLEDGMENTS:
The authors are grateful to Song-Yi Park, PhD, Lynne R Wilkens, PhD, Minji Kang, PhD Loïc
Le Marchand, PhD Carol J Boushey, PhD, Cancer Epidemiology Program, University of Hawaiʻi
Cancer Center, and the MEC participants. Supported in part by the National Cancer Institute at the
National Institutes of Health grants P01CA168530, P30 CA071789 and U01CA164973.
108
109
Table 7.1 The twenty-two food groups derived from the quantitative food frequency questionnaire
from the Multiethnic Cohort Study used in the reduced rank regression analysis
Food Group Foods
Red Meat Excluding
All preparation (beef, veal, pork, and other meat, organ meats)
Processed Meat
All preparation (luncheon meats, bacon, ham, hot dog, sausage, chorizo, spam, bologna,
Processed Red Meat
salami, pastrami, Vienna polish)
Fresh Poultry All preparation (chicken, turkey)
Processed Poultry All preparation (ham, turkey bacon)
Raw, fried, baked, and canned (tuna, ahi, kamaboko, snapper, cod, sea bass, aku, salmon,
Fish Excluding Shellfish
mackerel, sardines)
Raw, fried, baked, and canned (shrimp, cuttlefish, iriko, taegu, bacalao, crab, octopus,
Shellfish
squid, oysters)
All Legumes All types (legumes, soybean, soybean products)
Raw, Mixed, Canned, Cooked (celery, zucchini, green peppers, asparagus, cucumbers,
Light Green Vegetables
sprouts)
Raw, Mixed, Canned, Cooked (Broccoli, spinach, collard greens, watercress, mustard
Dark Green Vegetables
cabbage, choi sum, chard, green beans, peas,)
Raw, Mixed, Canned, Cooked; (Carrots, yellow-orange winter squash, yams, pumpkin,
Yellow-Orange Vegetables
corn)
Rice All types of rice (Spanish/Mexican, brown, white, sushi, fried rice,)
Boiled, baked, mashed, fried (potatoes, taro, poi, purple sweet potatoes, french-fried,
Potatoes and Tubers
hash-browned)
All types (bran or High fiber Cereal, raisin bran, bran flakes, cold cereals, corn flakes,
Breakfast Cereals cheerios, cooked cereals, oatmeal, corn grits, cream of wheat, granola, cereal bars,
fortified beverages/bars)
All types (white, whole wheat, rye, mixed grain, raisin, oat bran, buns, rolls, bagels,
Breads
English muffins, flour tortilla, muffins)
All types of pasta and noodles (Ramen or Saimin, Fried Noodle Dish, Pasta with
Pasta Tomato Sauce, Pasta with Cheese Cream Sauce, Noodle Casseroles, Pasta or Somen
Salad)
Fruit Juice Alone Blended Fruit Juice, orange, grapefruit, cranberry juice, apple juice, passion-orange
Fresh, Canned, Dried (Oranges, grapefruit, Pomelo, Papaya, Pineapple, Peaches,
Yellow-Orange Fruit
Apricots, Bananas, Cantaloupe, Mangoes)
All types of milk and milk products (yogurt, pudding, whipped cream, custard, ice
All Dairy Products
cream, ),All types of cheese (low-fat and regular cheese)
Eggs Eggs
Beer Beer
Wine Wine
Nuts Excluding Coconut All types of nuts, seeds, and peanut butter
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Table 7.2 Study participants who completed the baseline food frequency questionnaire
Reduced Rank Regression Derived Patterns % of variance
Food Group
Pattern 1 Pattern 2 Pattern 3 explained
Red Meat Excluding Processed Meat 0.368 0.335 47.5
Processed Red Meat 0.336 0.318 43.1
Processed Poultry 0.226 0.202 17.3
Fresh Poultry 6.6
Fish Excluding Shellfish 7.3
Shellfish 11.3
All Legumes 0.225 14.7
Light Green Vegetables 0.330 27.7
Dark Green Vegetables 0.335 27.3
Yellow-Orange Vegetables 0.389 35.9
Rice 2.5
Potatoes and Tubers 0.216 14.9
Breakfast Cereals 0.316 23.0
Breads 0.245 0.346 44.2
Pasta 10.3
Fruit Juice Alone 0.283 0.286 38.5
Yellow-Orange Fruit 0.418 47.5
All Dairy Products 0.233 0.332 40.6
Eggs 0.247 0.265 26.1
Beer -0.255 -0.531 0.267 68.8
Wine -0.344 26.6
Nuts Excluding Coconut 12.7
% Variance Explained 22.9 16.3 11.6 =50.8
a Factor loadings < |0.2| are not shown
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Table 7.3 Baseline characteristics of 190,985 participants by lowest and highest quintiles of the three derived reduced rank regression
dietary patterns in the Multiethnic Cohort Study
Pattern 1 Pattern 2 Pattern 3
Quintile 1 Quintile 5 Quintile 1 Quintile 5 Quintile 1 Quintile 5
Sex, n (%)
Men 12,958 (33.9) 20,677 (54.1) 12,597 (33.0) 24,611 (64.4) 12,958 (33.9) 20,677 (54.1)
Women 25,239 (66.1) 17,520 (45.9) 25,600 (67.0) 13,586 (35.6) 15,827 (41.4) 22,051 (57.7)
Race/Ethnicity, n (%) a
African American 9,582 (25.1) 5,556 (14.6) 5,635 (14.8) 7,465 (19.5) 1,535 (4.0) 10,518 (27.5)
Native Hawaiian 2,071 (5.4) 4,046 (10.6) 2,120 (5.6) 4,163 (10.9) 5,825 (15.3) 1,061 (2.8)
Japanese American 10,387 (27.2) 7,054 (18.5) 11,549 (30.2) 8,447 (22.1) 22,966 (60.1) 2,889 (7.6)
Latino 6,628 (17.4) 14,709 (38.5) 10,530 (27.6) 8,719 (22.8) 3,266 (8.6) 14,838 (38.9)
Non-Hispanic White 9,529 (25.0) 6,832 (17.9) 8,363 (21.9) 9,403 (24.6) 4,605 (12.1) 8,891 (23.3)
Age at Cohort Entry, y, mean (SD) 60.1 ± 9.0 59.5 ± 8.6 62.3 ± 8.4 57.2 ± 8.6 59.0 ± 9.0 60.5 ± 8.5
Body Mass Index, kg/m , mean (SD)
2 26.3 ± 5.1 27.6 ± 5.4 25.6 ± 4.7 28.0 ± 5.6 26.1 ± 4.9 27.6 ± 5.4
Ever smokers, n (%) 21,042 (55.9) 21,176 (56.6) 16,604 (44.4) 25,745 (68.2) 21,766 (57.6) 22,000 (58.9)
Physical activity, h/d, mean (SD)b 0.2 ± 0.6 0.6 ± 1.0 0.4 ± 0.8 0.5 ± 1.0 0.5 ± 0.9 0.4 ± 0.9
Multivitamin Use, n (%) 17,714 (47.9) 19,996 (53.5) 21,824 (58.7) 16,685 (44.5) 17,820 (47.5) 19,643 (53.0)
NSAID use, n (%) 19,047 (51.5) 21,237 (57.0) 18,766 (50.7) 20,778 (55.5) 16,273 (43.4) 21,824 (59.1)
Family History of Colorectal Cancer, n (%) 3,083 (8.1) 2,667 (7.0) 3,073 (8.1) 2,892 (7.6) 3,303 (8.7) 2,733 (7.2)
History of Intestinal Polyps, n (%) 1,862 (4.9) 1,865 (4.9) 2,063 (5.4) 1,993 (5.2) 2,612 (6.8) 1,678 (4.4)
Energy Intake, kcal/d, mean (SD) 1135.6 ± 339.8 3710.4 ± 1081.5 2262.9 ± 1067.3 2970.5 ± 1204.8 2647.8 ± 1108.2 2395.9 ± 1242.1
Alcohol Intake , g/d, mean (SD) 7.3 ± 21.6 10.6 ± 29.3 5.2 ± 18.9 13.8 ± 30.7 13.4 ± 39.2 8.2 ± 21.2
MHT Ever Use Among Postmenopausal
10,738 (54.4) 7,119 (50.7) 11,870 (55.3) 5,076 (51.3) 6,832 (56.7) 9,361 (52.1)
Women
MHT, menopausal hormone therapy; NSAID, nonsteroidal anti-inflammatory drug.
aColumn percentages.
bHours spent in vigorous work or sports per day.
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Table 7.4 Derived dietary patterns from reduced rank regression and colorectal cancer risk in the Multiethnic Cohort Study, 1993-2013
Overall (n=168,294)
Basic modela Multivariate model
Casesc HR (95% CI) HR (95% CI)
Dietary Pattern 1
Quintile 1 1022 1.00 (ref.) 1.00 (ref.)
Quintile 2 867 0.81 (0.74-0.89) 0.88 (0.80-0.97)
Quintile 3 820 0.73 (0.67-0.81) 0.82 (0.75-0.91)
Quintile 4 828 0.72 (0.65-0.79) 0.82 (0.74-0.91)
Quintile 5 796 0.70 (0.63-0.77) 0.81 (0.73-0.91)
P for trendd <0.0001 <0.0001
Dietary Pattern 2
Quintile 1 964 1.00 (ref.) 1.00 (ref.)
Quintile 2 845 0.90 (0.83-1.00) 0.95 (0.87-1.05)
Quintile 3 845 0.97 (0.88-1.06) 1.00 (0.90-1.10)
Quintile 4 841 1.00 (0.91-1.10) 1.02 (0.92-1.12)
Quintile 5 838 1.04 (0.95-1.15) 1.06 (0.95-1.18)
P for trendd 0.14 0.16
Dietary Pattern 3
Quintile 1 861 1.00 (ref.) 1.00 (ref.)
Quintile 2 853 0.96 (0.88-1.06) 0.98 (0.89-1.09)
Quintile 3 853 0.97 (0.88-1.07) 1.01 (0.91-1.12)
Quintile 4 865 1.00 (0.90-1.10) 1.04 (0.93-1.17)
Quintile 5 901 1.11 (1.01-1.21) 1.14 (0.99-1.31)
P for trendd <0.0001 0.07
Dietary Pattern 1 loaded positively yellow-orange fruit, yellow-orange vegetables, dark green vegetables, light green vegetables, fruit juice alone, all dairy products,
all legumes, and negatively on intake of beer. Dietary Pattern 2 loaded positively on red meat, processed red meat, eggs, breads, processed poultry, and negative
loadings of beer and wine. Dietary Pattern 3 loaded positively on breads, red meats, all dairy products, processed red meat, fruit juice, beer, eggs, and processed
poultry.
aAdjusted in Cox regression model of colorectal cancer for age at cohort entry, ethnicity, and sex
bAdditionally adjusted for family history of colorectal cancer, history of colorectal polyp, body mass index, pack-years of cigarette smoking, multivitamin use,
nonsteroidal anti-inflammatory drug use, vigorous physical activity, menopausal hormone therapy use for women only, ethanol, and totally energy
cExcluding participants with missing data on any of the covariates
dTrend variables were assigned median values for quintiles
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Table 7.5. Derived dietary patterns from reduced rank regression and colorectal cancer risk in the Multiethnic Cohort Study, 1993-2013
Men (n=77,951) Women (n=90,343) P for
Casesa HR (95% CI)b Casesa HR (95% CI)b Heterogeneityc
Dietary Pattern 1
Quintile 1 767 1.00 (ref.) 255 1.00 (ref.)
Quintile 2 456 0.86 (0.76-0.97) 411 0.92 (0.79-1.09)
Quintile 3 384 0.81 (0.71-0.92) 436 0.85 (0.73-1.00)
Quintile 4 372 0.84 (0.74-0.96) 456 0.82 (0.70-0.97)
Quintile 5 329 0.81 (0.70-0.93) 467 0.85 (0.72-1.01)
P for trendd 0.002 0.03 0.57
Dietary Pattern 2
Quintile 1 583 1.00 (ref.) 381 1.00 (ref.)
Quintile 2 387 1.00 (0.88-1.15) 458 0.90 (0.79-1.04)
Quintile 3 370 1.00 (0.87-1.14) 475 0.99 (0.86-1.14)
Quintile 4 434 1.01 (0.89-1.16) 407 1.03 (0.89-1.20)
Quintile 5 534 1.04 (0.91-1.19) 304 1.09 (0.92-1.29)
P for trendd 0.58 0.12 0.82
Dietary Pattern 3
Quintile 1 302 1.00 (ref.) 559 1.00 (ref.)
Quintile 2 406 1.06 (0.91-1.24) 447 0.93 (0.82-1.06)
Quintile 3 432 1.01 (0.86-1.18) 421 1.03 (0.89-1.19)
Quintile 4 508 1.07 (0.91-1.26) 357 1.06 (0.90-1.24)
Quintile 5 660 1.25 (1.03-1.51) 241 1.00 (0.81-1.23)
P for trendd 0.05 0.51 0.04
poultry.
114
Table 7.6 Derived dietary patterns and colorectal cancer risk by race/ethnicity in the Multiethnic Cohort Study, 1993-2013
African American Native Hawaiian Japanese American Latino White
(n=26,847) (n=12,361) (n=49,645) (n=36,072) (n=43,369) P for
Case heterogeneityb
HR (95% CI)a Cases HR (95% CI)a Cases HR (95% CI)a Cases HR (95% CI)a Cases HR (95% CI)a
s
Dietary Pattern 1
Quintile 1 159 1.00 (ref) 95 1.00 (ref) 406 1.00 (ref) 143 1.00 (ref) 219 1.00 (ref)
Quintile 2 173 0.85 (0.68-1.06) 52 0.98 (0.69-1.39) 302 0.94 (0.80-1.10) 164 0.88 (0.69-1.11) 176 0.81 (0.66-1.00)
Quintile 3 172 0.89 (0.71-1.12) 42 0.89 (0.61-1.31) 283 0.90 (0.77-1.07) 146 0.68 (0.53-0.87) 177 0.76 (0.62-0.94)
Quintile 4 162 0.95 (0.75-1.20) 38 0.76 (0.51-1.14) 290 0.88 (0.74-1.04) 166 0.71 (0.56-0.91) 172 0.76 (0.61-0.95)
Quintile 5 137 1.02 (0.79-1.32) 49 0.71 (0.47-1.05) 274 0.87 (0.73-1.04) 190 0.68 (0.53-0.88) 172 0.76 (0.60-0.97)
P for trendc 0.64 0.05 0.10 0.001 0.02 0.34
Dietary Pattern 2
Quintile 2 125 0.94 (0.70-1.26) 48 1.03 (0.69-1.54) 400 1.03 (0.90-1.19) 118 0.86 (0.66-1.13) 154 0.86 (0.70-1.07)
Quintile 3 192 1.08 (0.82-1.42) 42 0.97 (0.64-1.47) 286 1.01 (0.87-1.18) 171 1.04 (0.81-1.34) 154 0.85 (0.69-1.06)
Quintile 4 198 1.05 (0.80-1.38) 46 0.94 (0.63-1.42) 237 1.08 (0.92-1.27) 178 0.98 (0.77-1.26) 182 0.97 (0.79-1.19)
Quintile 5 206 1.07 (0.81-1.42) 79 1.28 (0.88-1.87) 160 1.03 (0.85-1.25) 221 1.04 (0.81-1.34) 172 1.02 (0.82-1.28)
P for trendc 0.38 0.32 0.52 0.42 0.72 0.99
Dietary Pattern 3
Quintile 2 170 1.03 (0.83-1.28) 27 0.72 (0.42-1.23) 368 0.99 (0.85-1.15) 132 0.90 (0.70-1.15) 156 1.13 (0.88-1.46)
Quintile 3 151 1.01 (0.79-1.29) 58 1.20 (0.73-1.96) 311 0.94 (0.80-1.11) 149 0.97 (0.76-1.25) 184 1.19 (0.92-1.54)
Quintile 4 144 1.09 (0.83-1.44) 65 1.05 (0.62-1.77) 276 0.97 (0.81-1.17) 159 0.93 (0.71-1.22) 221 1.31 (1.00-1.71)
Quintile 5 133 1.07 (0.76-1.50) 99 1.02 (0.56-1.87) 211 1.11 (0.88-1.40) 229 1.09 (0.79-1.49) 229 1.38 (1.01-1.88)
P for trendc 0.63 0.57 0.78 0.62 0.03 0.35
poultry.
aAdjusted for age at cohort entry, sex, family history of colorectal cancer, history of colorectal polyp, body mass index, pack-years of cigarette smoking, multivitamin
use, nonsteroidal anti-inflammatory use, vigorous physical activity, menopausal hormone therapy use, ethanol, and total energy.
bBased on Wald Test comparing associations between race/ethnicity and adjusting for the covariates in the multivariate models
cTrend variables were assigned the median values for quintiles
115
116
CHAPTER 8 General Discussion and Future Directions
Taro (Colocasia esculenta) is a significant food in Hawaiʻi that has been valued for its cultural,
health, and medicinal importance. Prior to colonization, the rates of CRC and other chronic diseases
were low in Hawaiʻi [55]. The low rates can be attributed to Native Hawaiians wholesome traditional
diet, which was high in dietary fibers, complex carbohydrates, and polyunsaturated fatty acids, and low
in fat and saturated fats [25]. However, Western dietary influence has caused Hawaiʻi, as of 2018, to
have CRC as the 2nd leading cause of cancer death in men and the 3rd leading cause of cancer death
among women [56]. Diet is a vital contributor to the overall health status of an individual and greatly
affects the gut microbiota. Disruption of the homeostasis of the gut microbial community through
dietary means, which has been shown with Western diets, results in dysbiosis of the gut microbiota
that has been linked to the development of CRC. Therefore, returning to traditional diets could reverse
chronic disease trends.
Thus, to better understand taro’s health and medicinal potential, this dissertation explored two
avenues: 1) the biochemical analysis of taro’s nutrients (Chapter 3), prebiotic potential (Chapter 4),
and effects on the gut microbiota (Chapter 5) to provide evidence of its CRC prevention properties
through maintenance of a healthy gut microbial community; and 2) the epidemiological analysis of
food intake in a human population to understand the relationship between eating taro and the risk of
developing CRC (Chapter 6) and the association of dietary patterns with the CRC risk (Chapter 7).
Taro’s nutrient composition (Chapter 3) showed high total starch and essential minerals, which has
superior nutritional value compared with other tuber crops, such as: potato, sweet potato, and cassava
[60, 61]. This nutritional composition, especially the high total starch and low protein content, lends
itself for favorable food processing characteristics, such as physical and functional properties, that
enable taro to be utilized in different food processing conditions. Furthermore, the low protein
content makes taro hypoallergenic and a great food ingredient alternative for baby foods, diets of
people allergic to cereals and children sensitive to milk [205]. Similarly, the small starch granules are
efficiently digested and absorbed, making it a great food ingredient with high nutrient bioavailability
[257]. Thus, these results illustrate taro’s nutrient composition would make for a great nutritional
alternative ingredient for the food industry and for health purposes.
A specific health benefit from taro focused on understanding its prebiotic potential (Chapter
4). Prebiotics are a selectively fermented ingredient that results in specific changes in the composition
and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health [139].
Usually, prebiotics come in the form of various fibers; thus, total dietary fiber and resistant starch (RS)
contents of taro were determined. In addition, prebiotics must evade digestion in the human digestive
tract; thus, an in vitro digestion simulation was conducted to represent those conditions. Findings
showed that taro varieties, Tahitian, Bun-long and Moi, exhibited high total dietary fiber and RS
concentrations. After in vitro human digestion, prebiotic activity scores of taro residuals were
determined. L. paracasei parings with inulin and Tahitian illustrated the highest scores. Results of this
test indicate that prebiotic activity of taro is species specific, as no two prebiotic carbohydrates were
utilized similarly by different probiotic species, despite being in the same Lactobacillus genus.
Correlation analysis illustrates weak relationships between fiber components and prebiotic activity
scores of tested taro varieties with different Lactobacillus species, further demonstrating the utilization
of fiber components is bacterial species specific.
The CRC preventative properties of taro may rely on its modulation of gut microbial
community and increased production of SCFA (Chapter 5). This is because epidemiological and
117
experimental studies have shown dietary sources of fiber and RS exhibit possible preventive effects
on colon cancer [322-324], which may be due in part to dietary fiber and RS’s ability to enhance
microflora species associated with intestinal health and SCFA production [325]. Thus, previously in
vitro human digested taro samples were subjected to in vitro fecal fermentation, to simulate the gut
microbiome of humans. Results illustrated that taro varieties and inulin had distinctly different
microbial communities from the baseline. Furthermore, microbial relative abundance results
illustrated that taro varieties promoted the proliferation of healthy microbial communities, such as
Enterobacteriaceae, Veillonellaceae, Lachnospiraceae, and Bifidobacteriacea, and decreased pathogen-associated
microbial communities, such as Enterobacteriaceae. In addition, Bun-long yielded a significantly higher
amount of SCFAs than prebiotic inulin and fructooligosaccharides. All tested taro varieties but Kauaʻi
Lehua exhibited significantly highly concentrations of butyric acid than the two prebiotic controls.
Thus, these results suggest that taro can serve as a dietary source to promote the modulation of gut
microbial communities towards a healthier composition and boost metabolism.
To understand taro’s ability to lessen CRC risk, epidemiological analysis was conducted using
the Multiethnic Cohort (MEC) study (Chapter 6) to determine the association of taro consumption,
frequency of consumption, and dietary fiber intake with CRC incidence. The MEC is a prospective
cohort study established to investigate the influence of lifestyle, such as diet, and genetic factors on
the occurrence of cancer and other chronic diseases [422], with participants from five major
races/ethnicities: African American, Native Hawaiian, Japanese American, Latino, and white. Dietary
intake was assessed using a validated quantitative food frequency questionnaire, and dietary fiber from
taro was estimated from self-reported consumption. Cox hazard regression was applied to estimate
hazard ratios (HR) and 95% confidence intervals (CI) for CRC. Results illustrated that taro consumers
had a decreased risk of CRC, though the trend was not significant. In addition, as the frequency of
taro consumption increased, a greater reduction in risk of CRC was observed. The dietary fiber intake
of 50-100 mg/day from taro also showed a significant association with CRC risk (HR =0.88; 95% CI,
0.78-0.99). Therefore, these results suggest that consumption of taro potentially decreases the risk of
CRC.
Despite taro’s CRC prevention potential, food items are not eaten in individual manners; but
rather, in combination with other food items. Thus, understanding how taro in a gut microbial-driven
dietary pattern affects CRC risk, provides better understanding of overall potential disease outcome,
on a population basis (Chapter 7). Using the MEC cohort, dietary patterns were derived from RRR
that was driven by gut microbiome components. Three dietary patterns were derived, with dietary
pattern 1 loaded high on fruits and vegetables, dairy products and legumes exhibiting an inverse
association with CRC risk; dietary pattern 2 loaded on red and processed meats that was not
significantly associated with CRC risk; and dietary pattern 3 loaded on red meat, processed red meats,
processed poultry, all dairy products, beer, eggs, and potatoes and tubers that was associated with an
increased CRC risk. These outcomes illustrate that diet high in fruits and vegetables may result in a
decreased CRC risk. In this case, taro was grouped in the potato and tuber group, which showed to
be associated with CRC risk increase. The grouping of taro in the potato and tuber group may have
masked taro’s beneficial properties. Taro is a socio-culturally important food for people of the Asia
and Pacific region [63] that may have had lesser participant consumption compared to potato and
other tubers in the group. Thus, future studies should explore re-grouping food items to further
include criteria of similar dietary fiber content. This addition could help prevent masking ethnic/race
specific foods that may have limited consumption in participants of different ethnic/race groups.
Findings from this dissertation provide evidence to bridge the gap between biochemical and
epidemiological research about taro’s beneficial properties for potential CRC prevention—it provides
evidence for basic research of taro and it’s implication in human health (Figure 8.1). It demonstrates
118
basic research of taro’s nutrition, physiochemical, and functional content (Chapter 3), holds prebiotic
potential after being subjected to an in vitro human digestion (Chapter 4), can modulate gut microbial
communities and increase production of SCFA (Chapter 5), serves as a potential dietary source for
public health research to reduce CRC risk (Chapter 6), and is part of the gut healthy dietary pattern
that reduces CRC risk (Chapter 7). Thus, this study provides empirical evidence of taro’s potential
health benefits, specifically for CRC prevention. The results of this study also contribute to increased
knowledge of the prebiotic properties of taro that may serve as a dietary source for CRC prevention
that may be used for development of new approaches of nutritional strategies to minimize the risk of
CRC (Figure 8.1).
Future research may seek to explore more of taro’s health potentials. Other taro varieties can
be analyzed for their nutrient and dietary prebiotic fibers to fully understand health benefits taro may
confer. In addition, analysis of taro’s impact on the gut microbiome through an in vivo model can
further help elucidate microbial composition changes that may occur from dietary taro and potential
CRC prevention. This is because the gut microbiota has been closely linked to CRC development, as
well as considered a platform for studying CRC interactions of host and environment [503]. In
addition, this study opens an avenue for further research to investigate if combinations of taro with
other food items or taro in specific dietary patterns could prove to be better representatives of dietary
selections for the prevention of CRC. Furthermore, populations that specifically eat taro can be
recruited to study the benefits of taro and the impacts on the gut microbiome and the implications on
health outcomes. This would allow taro to be highlighted in a diet that normally consumes it, along of
potential interactions that may occur from consumption with other foods. It would be further
interesting to observe the effects these applications on the gut microbial community on a longer
timeline to better understand how diet affects the gut microbiota and human health. Understanding
the relationship between diet and activity of the gut microbiota may formulate the right prevention
strategies for CRC and perhaps guide research in the direction to include taro as an effective dietary
therapy.
119
Figure 8.1 Translating basic and public health research to develop new approaches for examining
taro’s contribution to overall health
120
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THE POTENTIAL OF TARO (COLOCASIA ESCULENTA) AS A DIETARY

PREBIOTIC SOURCE FOR THE PREVENTION OF

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE

Yong Li, Chairperson

CHAPTER 1 DISSERTATION OVERVIEW ..................................................................... 1

1.1.1 History of Taro (Colocasia esculenta)

1.2 PROBLEM STATEMENT

1.2.1 Rationals and Significance

2.1.1 Taro (Colocasia esculenta)

2.1.2 Functional Foods

2.1.3 Taro’s Medicinal Use

2.1.4 Colorectal Cancer

2.3 BIOACTIVE AND NUTRIENT COMPONENTS OF TARO

2.3.1.1 Colorectal cancer prevention of minerals from taro

2.3.2.1 Colorectal cancer prevention of fat from taro

2.3.3.1 Dietary Fiber

2.3.3.1.1 Resistant Starch (RS)

2.3.3.2 Colorectal cancer prevention of carbohydrates from taro

2.3.4.1 Colorectal cancer prevention of probiotics from taro

2.3.5.1 Colorectal cancer prevention of prebiotics from taro

2.3.6 Amino Acids and Proteins

2.3.7.1 Colorectal cancer prevention of phytochemicals from taro

2.4 TARO NUTRIENT ABSORPTION

2.5 CANCER PREVENTION OF TARO

2.6 OTHER HEALTH BENEFITS

2.6.1 Food Allergies

2.6.2 Complementary Food

2.6.3 Tooth Decay

3.3 MATERIALS & METHODS

3.3.1 Taro Processing

3.3.1.1 Moisture Content (MC)

Moisture (%) = (W1 – W2) x 100

Where: W1 = weight (g) of sample before lyophilization

3.3.2 Nutrient Analysis

3.3.3 Physicochemical Analysis

WAI (g/g) = Weight of sediment

WSI (g/100 g) = Weight of Dissolved Solids in Supernatant

3.3.3.3 Bulk Density (BD)

3.3.3.4 Starch Properties Analysis

3.3.3.4.1 Total Starch Content

3.3.3.4.2 Granule Size

3.3.3.4.3 X-Ray Diffraction (XRD) Pattern

3.3.4.1 Water Absorption Capacity (WAC)

3.3.4.2 Oil Absorption Capacity (OAC)

3.3.4.3 Foam Capacity (FC) and Foam Stability (FS)

FC = Volume after homogenization – initial volume x 100

3.3.4.4 Emulsifying Activity (EA) and Emulsifying Stability (ES)

3.3.4.5 Gelling Properties Analysis

3.3.4.5.1 Gelling and Boiling Points

3.3.4.5.2 Least Gelation Concentration (LGC)

3.4.1 Nutrient Composition

3.4.2 Physicochemical Properties

3.4.3 Functional Properties of Taro

3.5.2 Physicochemical Properties

3.5.3 Functional Properties

This study provides a comprehensive analysis of the nutritional, physicochemical and

Authors are grateful to Agricultural Diagnostic Services of the University of Hawaiʻi.

Trace Minerals (mg/100 g)

Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD

Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD

Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD

Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD

Least gelation concentration (g/100 mL)

P < 0.05 P< 0.01 P<0.001

P < 0.05 P< 0.01 P<0.001

Acidaminococcaceae 1 0.711 * 0.888 -0.853 -0.385 0.886 ***

P < 0.05 P< 0.01 P<0.001