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 4.3.6 Statistical Analysis ...... 51 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 5.3.6 Statistical Analysis ...... 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 6.3.5 Statistical Analysis ...... 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 -1 Table 4.3. Increase in cell count (log10 cfu mL ) 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 3rd 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].

7 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; RS2, native granular starch that prevents digestive enzymes from break down, such as raw potatoes, unripe bananas; RS3, 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].

10 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 µgml.̸ In addition, splenocytes obtained from BALBc̸ 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.

11 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

12 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

13 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 O2 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 O2 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/cm3. 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 Mean and standard deviation (St. Dev.). The results are given on dry weight basis.

Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD 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 Means with different letters within the same row differed significantly by Tukey’s HSD (p<0.05)

37 Table 3.4. Functional properties of taro varieties Mean and standard deviation (St. Dev.). The results are given on dry weight basis.

Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD 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 Means with different letters within the same row differed significantly by Tukey’s HSD (p<0.05)

38 Table 3.5 Gelling properties of taro varieties Mean and standard deviation (St. Dev.). The results are given on dry weight basis.

Bun-Long Mana Ulu Moi Kauaʻi Lehua Tahitian HSD 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 4 No gelation No gelation No gelation No gelation No gelation N/A 6 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 4.3.6 Statistical Analysis 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 -1 in log10 (cfu mL ) 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) FOS, fructooligosaccharides

57 -1 Table 4.3. Increase in cell count (log10 cfu mL ) 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 Lehua HSD

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].

63 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.

5.3.8 Statistical Analysis 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 5th-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.

67 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.

68 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

71 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-

72 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].

73 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.

74

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). FOS, fructooligosaccharides

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) FOS, fructooligosaccharides

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). FOS, fructooligosaccharides

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). FOS, fructooligosaccharides

78 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.

79

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

81

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. FOS, fructooligosaccharides

82 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

83 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.

84 Tahitian

Moi

Mana Ulu

KauaʻiKauai LLehuaehua

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.

85

86 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.

87 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 3rd 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 [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.

88

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 50th, 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.

6.3.5 Statistical Analysis 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

89 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

90 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 (ORhighest 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.

92 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/m2, mean (SD) 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.

93 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

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

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.

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

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

96 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

c P for heterogeinty 0.5341

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.

97

98

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.

99 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.

7.3.3 Dietary 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

101 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.

7.3.4 Statistical Analysis 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

102 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/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), 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).

103 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].

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7.5.2 Dietary Pattern 2 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.

7.5.3 Dietary Pattern 3 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].

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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.

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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

110 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

111 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/m2, mean (SD) 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.

112 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

113 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 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. 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.

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 1 82 1.00 (ref) 61 1.00 (ref) 472 1.00 (ref) 121 1.00 (ref) 228 1.00 (ref) 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 1 205 1.00 (ref) 27 1.00 (ref) 389 1.00 (ref) 140 1.00 (ref) 100 1.00 (ref) 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 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 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

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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

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