Probiotics and Bioactive Carbohydrates in Colon Cancer Management Probiotics and Bioactive Carbohydrates in Colon Cancer Management

Maya Raman Padma Ambalam Mukesh Doble Probiotics and Bioactive Carbohydrates in Colon Cancer Management Probiotics and Bioactive Carbohydrates in Colon Cancer Management

Maya Raman • Padma Ambalam Mukesh Doble

Probiotics and Bioactive Carbohydrates in Colon Cancer Management Maya Raman Padma Ambalam Bioengineering and Drug Design Lab Department of Biotechnology Department of Biotechnology Christ College, Saurashtra University Indian Institute of Technology Madras, Rajkot, Gujarat, India Chennai, TN, India

Mukesh Doble Bioengineering and Drug Design Lab Department of Biotechnology Indian Institute of Technology Madras, Chennai, TN, India

ISBN 978-81-322-2585-0 ISBN 978-81-322-2586-7 (eBook) DOI 10.1007/978-81-322-2586-7

Library of Congress Control Number: 2015945580

Springer New Delhi Heidelberg New York Dordrecht London © Springer India 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

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Springer (India) Pvt. Ltd. is part of Springer Science+Business Media (www.springer.com) Preface

Colorectal cancer is the third leading cause in both incidence and mortality amongst men and women. The American Cancer Society estimated that 136,830 people are affected by it and 50,314 died in 2014. Developing countries are also showing increased incidences of colorectal cancer. Genetic and environmental factors contribute to the disease, of which major contribution is due to the latter. While 10–15 % of cancers are due to familial and heredi- tary factors, diet is associated with 50–80 % cases. Animal, epidemiological, case–control and cohort studies have indicated that various nutrients and components of food aid in the development or prevention of colorectal cancer. Excess consumption of red and processed meat, roasted coffee, etc. have shown a consistent increase in the incidence of colorectal cancer, indicating that compounds formed in food containing free amino acids and sugars interact at elevated temperatures to form mutagens or carcinogens, namely, heterocyclic amines and polycyclic aromatic hydrocarbons. These mutagens can cause alterations and damage to DNA, causing colorectal cancer. The usual remedy for colorectal cancer includes surgeries and invasive chemotherapy or radiation regimen; however, the cancer grows undetected and recurs after treatment. Several lifestyle and dietary factors could prevent this ailment. This book comprises of seven chapters. The introductory chapter describes the gut system and its microbiome. Four chapters describe the dietary habits (probiotics, synbiotics and bioactive carbohydrate, namely, prebiotics and dietary fi bres) that could modify and reduce the risk of developing colorectal cancer. Probiotics, prebiotics and synbiotics have developed into a major research focus area due to their specifi c health benefi ts in human. The applications of these include functional foods, health supplements and nutraceuticals. Emphasis is also laid on the dietary fi bres that have shown signifi cant health benefi ts against colorectal cancer. Consumer interest in the relationship between diet and health has improved the dietary habits presently. This has also paved the way for functional food and nutraceuticals that supply the nutrients for benefi cial normal reactions in the body. The chapters discuss the concept, defi nition and status of the “biotics” in the current market as well as their different physiological and molecular mechanisms of action. Short-chain fatty acid, which is involved in CRC considerably and forms the molecular pathway in preventing colorectal carcinogenesis, is discussed in Chap. 6. Short-chain fatty acid is formed in the colon as a result of the microbial fermentation of undigested bioactive carbohydrates including prebiotics (oligosaccharides, inulin, lactulose, lactitol) and dietary fi bres by

v vi Preface

Bifi dobacterium and Lactobacillus. Acetate, propionate and butyrate are major SCFA products. Butyrate, specifi cally, is involved in the inhibition of histone deacetylase, resulting in histone hyperacetylation and growth inhibition in the colonic epithelial cells. Apart from these, it is also involved in minimizing infl ammation, thereby exhibiting immunomodulatory effects. Several benefi ts of probiotics, prebiotics, synbiotics and dietary fi bre are novel and have not been fully explored. Similarly, the mechanism of action of these also has not been understood with reference to colorectal cancer and other diseases. This book will be of practical and scientifi c use to academi- cians, research scholars, students, health professionals, nutritionists, etc. and could support the cause of preventing CRC by adopting smart food habits.

Chennai, Tamil Nadu, India Maya Raman Rajkot, Gujarat, India Padma Ambalam Chennai, Tamil Nadu, India Mukesh Doble Abbreviations

3-MA 3-Methyladenine 7,8-DiMeIQx 2-Amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline AAD Antibiotic-associated diarrhoea AAR Annual incidence rates ACA Anticancerous activity ACF Aberrant crypt foci AGJ Artifi cial gastric juice AIJ Artifi cial intestinal juice Akt Protein kinase-B AMA Antimicrobial activity AMP Antimicrobial peptides AOM Azoxymethane ATP Adenosine triphosphate AXOS Arabinoxylo-oligosaccharides AαC 2-Amino-9H-pyrido[2,3-b]indole B(α)P Benzo(α)pyrene CAGR Compound annual growth rate cAMP Cyclic adenosine monophosphate CD Crohn’s disease CDK Cyclic dependent kinase CFTR Cystic fi brosis transmembrane conductance cfu/g Colony-forming unit per gram

CH4 Methane CHOS Chito-oligosaccharides CIN Chromosomal instability Cl− Chloride ion CLA Conjugated linoleic acid CMC Carboxymethyl cellulose

CO2 Carbon dioxide CRC Colorectal cancer CS Caesarean section CSC Cancerous stem cell

DCR Dentritic cells DiMeIQx 2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline DNA Deoxyribonucleic acid EcN E. coli strain Nissle 1917 EGFR Epidermal growth factor receptor

vii viii Abbreviations

EPA Environmental Protection Agency ERK Extracellular signal-regulated kinase FAO Food and Agriculture Organization FOS Fructo-oligosaccharide FP Faecalibacterium prausnitzii FP7 Framework Programme for Research Fru Fructose Gal Galactose GIA Global Industry Analysts GIT Gastrointestinal tract GLP1 Glucagon-like peptide 1 GLP2 Glucagon-like peptide 2 Glu Glucose Glu-P-1 2-Amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole Glu-P-2 2-Aminodipyrido[1,2-a:3′,2′-d]imidazole GOS Galacto-oligosaccharide GPCRs G-protein-coupled receptors GRAS Generally recommended as safe GTO Gentio-oligosaccharides H+ Hydrogen ion

H2 Hydrogen

H2O2 Hydrogen peroxide

H2S Hydrogen sulphide HCA Heterocyclic amines − HCO3 Carbonate ion HDAC Histone deacetylase HepG2 Human hepatoma cell lines HTS High-throughput sequencing technologies IARC International Agency for Research on Cancer IBD Inﬂ ammatory bowel disease IBS Irritable bowel syndrome IDL Intermediate-density lipoprotein IL Interleukin IMO Isomalto-oligosaccharide IQ 2-Amino-3-methylimidazo[4,5-f]quinoline ISCs Intestinal stem cells kD Kilodalton kJ Kilojoule LAB Lactic acid bacteria LcS L. casei strain, Shirota LDL Low-density lipoprotein MAPK Mitogen-activated protein kinase MeAαC 2-Amino-3-methyl-9H-pyrido[2,3-b]indole MeIQ 2-Amino-3,4-dimethylimidazo[4,5-f]quinoline MeIQx 2-Amino-3,8-dimethylimidazo [4,5-f]quinoxaline MLN Mesenteric lymph nodes MMPs Metalloproteinases MNNG N-Methyl-N′-nitro-N-nitrosoguanidine Abbreviations ix

MOS Mannan-oligosaccharides MSI Microsatellite instability Na+ Sodium ion NF-κB Nuclear factor-κB

NH3 Ammonia + NH4 Ammonium ion NIH National Institutes of Health NK Natural killer NOC N-Nitroso compounds PAH Polycyclic aromatic hydrocarbons PARP Poly (ADP-ribose) polymerase PhIP 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine PI Prebiotic index PI3K Phosphatidylinositol 3-kinase POS Pectin-oligosaccharides PPAR-γ Peroxisome proliferator-activated receptor-gamma RD Resistant dextrin ROS Reactive oxygen species RS Resistant starch SCFA Short-chain fatty acids

SO4 Sulphate SOS Soy-oligosaccharide TGOS Trans-galacto-oligosaccharide TIM Tissue inhibitor matrix metalloproteinase TNF Tumour necrosis factor TOS Trans-oligosaccharide Trp-P-1 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole Trp-P-2 3-Amino-1-methyl-5H-pyrido[4,3-b]indole UC Ulcerative colitis USD United States Dollars USNFIA United States National Food Ingredient Association UTI Urinary tract infection VLDL Very-low-density lipoprotein WHO World Health Organization XOS Xylo-oligosaccharide

Contents

1 Introduction: Gastroinstestinal System and Colorectal Cancer ...... 1 1.1 Introduction ...... 2 1.1.1 Gastrointestinal Tract ...... 2 1.1.2 Gut Microbiota as an Organ ...... 3 1.1.3 Gut Microbiota and Health ...... 5 1.1.4 Colorectal Cancer ...... 8 References ...... 12 2 Probiotics and Colorectal Cancer ...... 15 2.1 Probiotics and Colorectal Cancer ...... 16 2.1.1 History, Defi nition, and Statistics ...... 16 2.1.2 Probiotics as Functional Food ...... 17 2.1.3 Bifi dobacteria ...... 17 2.1.4 Selection Criteria for Probiotic Organism ...... 18 2.1.5 Probiotics as Health Promoter ...... 18 2.1.6 Probiotics and Gut Microbiota ...... 21 2.1.7 Probiotics and Health ...... 22 2.1.8 Improvement of Mineral Absorption ...... 23 2.1.9 Mechanism of Action of Probiotics on Colorectal Cancer ...... 24 2.1.10 Inactivation of Mutagenic and Carcinogenic Compounds ...... 25 References ...... 29 3 Bioactive Carbohydrate: Dietary Fibers and Colorectal Cancer ...... 35 3.1 History, Defi nition, and Statistics ...... 36 3.2 Types and Components of Dietary Fiber ...... 38 3.2.1 Dietary Recommendations ...... 41 3.3 Dietary Fiber and Gut Microbiota ...... 41 3.4 Dietary Fiber and Health ...... 43 3.4.1 Anticancer and Antitumor ...... 43 3.4.2 Anti-infl ammatory ...... 45 3.4.3 Immunomodulation ...... 46

xi xii Contents

3.5 Mechanism of Action of Dietary Fiber and Its Fractions on Colorectal Cancer ...... 47 3.5.1 Physiological Mechanism...... 47 3.5.2 Cellular Mechanism ...... 48 References ...... 50 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer ..... 57 4.1 History, Defi nition, and Statistics ...... 58 4.2 Types of Prebiotics ...... 61 4.2.1 Inulin ...... 61 4.2.2 Fructo-oligosaccharides (FOS)...... 62 4.2.3 Galacto- ligosaccharides (GOS) ...... 62 4.2.4 Lactulose (Lactosucrose) ...... 64 4.2.5 Lactitol ...... 65 4.2.6 Fructans (Polydextrose) ...... 65 4.2.7 Soy-Oligosaccharides (SOS) ...... 65 4.2.8 Xylo-oligosaccharides (XOS) ...... 66 4.2.9 Isomalto- oligosaccharides (IMO)...... 66 4.2.10 Resistant Starch (RS) and Dextrin (RD) ...... 67 4.3 Prebiotics and Gut Microbiota ...... 69 4.4 Prebiotics and Health...... 70 4.4.1 Prebiotics and Cancer ...... 70 4.4.2 Prebiotics and Lactose Intolerance ...... 70 4.4.3 Prebiotics and Cholesterol ...... 71 4.4.4 Prebiotics and Osteoporosis ...... 72 4.4.5 Cancer ...... 72 4.4.6 Anti-infl ammatory ...... 73 4.5 Mechanism of Action of Prebiotics against Colorectal Cancer ...... 73 4.5.1 Stimulation of Benefi cial Gut Microbes ...... 73 4.5.2 SCFA and Lactic Acid ...... 74 4.5.3 Absorption of Micronutrients by Colon Cells ...... 75 4.5.4 Prebiotics Modulate Xenobiotic Metabolizing Enzymes ...... 75 References ...... 76 5 Synbiotics and Colorectal Cancer ...... 83 5.1 History, Defi nition, and Statistics ...... 84 5.2 Synbiotics and Gut Health ...... 85 5.2.1 Anticancer ...... 89 5.2.2 Antiinfl ammation and Immunomodulation ...... 92 5.3 Mechanism of Action ...... 93 References ...... 93 6 Short-Chain Fatty Acids ...... 97 6.1 Introduction ...... 98 6.2 Production of Short-Chain Fatty Acids ...... 100 6.2.1 SCFA Determination in Colon ...... 102 6.3 Butyrate Paradox ...... 102 Contents xiii

6.4 Short-Chain Fatty Acids, Gut Microbiota, and Health ...... 103 6.4.1 Anticancer and Apoptosis ...... 104 6.4.2 Inﬂ ammation ...... 105 6.5 Metabolic Regulation ...... 106 6.6 Mechanism and Effects of Short-Chain Fatty Acids on Health ...... 107 6.6.1 Luminal Effects of SCFA ...... 107 6.6.2 Absorption and Metabolism of SCFA by Colonocytes ...... 107 6.6.3 Effect of SCFA on Colonic Blood Flow and Muscular Activity ...... 108 6.6.4 Trophic Effects of SCFA (Butyrate and Propionate) in Maintaining Normal Colonic Cell Phenotype ...... 108 6.6.5 Molecular Mechanism of Short-Chain Fatty Acid in Preventing Colon Cancer ...... 109 References ...... 111 7 Conclusion ...... 117

Index ...... 121 Introduction: Gastroinstestinal System and Colorectal Cancer 1

Abstract The gastrointestinal (GI) system is an important part of the human body involved in digestion. Animals and humans, live in harmony with microbes and establish a symbiotic relationship with them. The human microbiota contains as many as 1014 bacterial cells, which is ten-fold higher than the number of human cells present in our body. With the development of high- throughput sequencing technique, it is possible to study the gut microbiota and its role in health and disease. It performs a number of essential protective, structural and metabolic functions for host health, including food processing, digestion of complex host-indigestible polysaccharides, pathogen displacement, and synthesis of vitamins. The gut microbiota may exert benefi cial functions beyond the gut such as controlling obesity, gastrointestinal disorders, infl ammatory bowel disease, and irritable bowel syndrome. Also, the altered microbiota has been linked to neuropsychological disorders (depression). The gut microbiota has also been implicated in the development of colorectal cancer (CRC). CRC is one of the major health problems in the world, and the risk factors associated with it are genetic as well as environmental. It evolves through a stepwise accumulation of genetic and epigenetic alterations, leading to the transformation of normal colonic mucosa into invasive cancer. In this chapter, the role of the gut microbiota in health and disease, CRC and its types, prevalence and mechanism are briefl y discussed.

Keywords Gut microbiota • CRC • Intestinal stem cells • Colon stem cell • Crypt • Metagenomics

© Springer India 2016 1 M. Raman et al., Probiotics and Bioactive Carbohydrates in Colon Cancer Management, DOI 10.1007/978-81-322-2586-7_1 2 1 Introduction: Gastroinstestinal System and Colorectal Cancer

1.1 Introduction Table 1.1 Overview of the functions of gastrointestinal organs Human system needs energy for proper meta- Organ Major function bolic activities, development and growth, which Mouth and Ingestion: food is voluntarily placed comes from the breakdown of food. Food as such other into oral cavity associated cannot be absorbed into the blood until they are Propulsion: voluntary phase of deglutition (swallowing) initiated by broken down into small soluble chemicals. This tongue propels food into the pharynx chemical digestion works with the help of Mechanical breakdown: mastication enzymes. Once the digestion is completed, the (chewing) by teeth and mixing small molecules pass through the wall of the movement by tongue stomach and small intestine by either active or Chemical digestion: salivary passive diffusion. The digestive system of the amylase in saliva, produced by salivary gland, begins chemical body is termed as gastrointestinal system. breakdown of starch Pharynx Propulsion: peristaltic waves move food bolus to stomach, thus 1.1.1 Gastrointestinal Tract accomplishing involuntary phase of deglutition Stomach Mechanical digestion and The gastrointestinal (GI) system is an important propulsion: peristaltic waves mix part of the human body, is involved in the diges- food with gastric juice and propel it tion of food, and it includes all the parts of the into the duodenum body from mouth to anus. It is uniquely con- Chemical digestion: digestion of structed to perform its specialized function of protein begun by pepsin turning food into the energy needed for the body Absorption: absorbs a few fat soluble substances (aspirin, alcohol, and packaging the residue for waste disposal. drugs) The different components and functions of the GI Small intestine Mechanical digestion and system are briefl y discussed below (Table 1.1 ) and associated propulsion: segmentation by smooth (US Department of Health and Human Services accessory muscle of the small intestine 2010 ). organs continually mixes content with digestive juices and moves the food along tract and through ileocecal Mouth It is at the beginning of the digestive tract valve at a slow rate, allowing and is the fi rst place where digestion starts with suffi cient time for digestion and intake and fi rst bite. Food is broken down into absorption pieces and further digested by salivary enzymes Large intestine Chemical digestion: some remaining food residues are digested by gut and enters into the esophagus. microbiota Absorption: absorbs most remaining Esophagus It is present in the throat near the tra- water, electrolytes and metabolites chea and delivers food to the stomach upon produced by bacteria swallowing. Propulsion: propels feces towards rectum by peristalsis, haustral churning, and mass movement Stomach The stomach is a main storage tank that Defecation: refl ex triggered by rectal produces a mixture of acid, mucus, and digestive distension; eliminates feces from enzymes to further rupture the ingested food and body then release them into the small intestine.

Small Intestine It is a 22.8-foot long tube and is ing it with digestive secretions from the pancreas made up of three segments – the duodenum, jeju- and liver. The main function of duodenum is to num, and ileum. Peristalsis also works in this further break down the digested food. The jeju- organ, namely moving the food through and mix- num and ileum are responsible for absorption of 1.1 Introduction 3 nutrients into the bloodstream. In the small intes- 1.1.2 Gut Microbiota as an Organ tine, the contents start out as semisolid and is then converted into a liquid form after passing Microbiota is the total microorganisms (includ- through the organ with the help of water, bile, ing bacteria, archaea, eukaryotes, and viruses) enzymes, and mucous. After absorption of nutri- that live in and/or on a human or a specifi c part ents, the leftover food residue liquid moves on to such as the gastrointestinal tract, oronasopharyn- the large intestine or colon. geal cavity, skin, urogenital tract (Reid et al. 2011; Sommer and Bäckhed 2013 ). The human Pancreas Its main function is to secrete diges- microbiota contains as many as 1014 bacterial tive enzymes into the duodenum that is required cells, which is tenfold higher than the number of for further digestion. It also produces insulin, a human cells present in our body (Sekirov et al. chief hormone for metabolizing sugars, which it 2010 ). This area of research has gained lots of secretes directly into the bloodstream. interest in the last 10 years and improved our knowledge and understanding about the resident Liver It is the body’s chemical “factory” and is species and their functions. In the last 5 years within the digestive system. It helps to process (since 2010), more than 4,500 publications are the nutrients absorbed from the small intestine. It available on PubMed against 635 publications in takes the raw materials absorbed by the intestine 2005–2009. The recent research on gut microbi- and helps in the synthesis of various chemicals ome has brought forth its signifi cance in health required for the whole system. The liver also and the input of high-throughput sequencing detoxifi es potentially harmful chemicals. technologies (HTS). Here, the NIH-funded Human Microbiome Project and MetaHIT proj- Gallbladder It is a small organ where bile is ect fi nanced by the European Commission under stored and concentrated before it is released into the 7th FP program are worth mentioning. the small intestine. There it helps to absorb and The mucosal surface of the human gastroin- digest fats. testinal (GI) tract is about 200–300 m2 and is colonized by 10 13 –10 14 bacteria with >500 differ- Colon (Large Intestine) It is a six-foot long mus- ent species and subspecies (Hao and Lee 2004 ). cular tube that connects the small intestine to the The gastrointestinal microfl ora is divided into rectum. It is made up of the cecum, the ascending two types: (right) colon, the transverse (across) colon, the descending (left) colon, and the sigmoidal colon, 1. Autochthonous fl ora ( indigenous fl ora ), which which connects to the rectum. The main function colonize particular habitats, i.e., physical of the colon is to process the waste obtained from spaces in the GI tract; the small intestine so that emptying of the bowels 2. Allochthonous fl ora ( transient fl ora ), which is easy and convenient. cannot colonize particular habitats except under abnormal conditions (Hao and Lee Rectum It is an 8-inch chamber that connects the 2004 ). colon to the anus, and it serves as a temporary storehouse for feces. The expansion of the rectal Most pathogens are allochthonous microor- walls causes the stretch receptors within the walls ganisms with few exceptions where autochtho- to stimulate the urge to defecate. nous may turn as opportunistic pathogens when the gut microbiota is disturbed. Anus It is the opening where the gastrointestinal The structure and composition of the gut fl ora tract ends and exits the body. It is present below refl ect the natural selection at both the microbial the rectum and is the last portion of the colon. and host levels, which promotes mutual coopera- The function of the anus is to transmit feces from tion within and functional stability of this com- the rectum to the external environment. plex ecosystem (O’Hara and Shanahan 2006 ; Ley 4 1 Introduction: Gastroinstestinal System and Colorectal Cancer

Table 1.2 Variations in microbial numbers and composition across the length of the gastrointestinal tract Organ Population density Bacterial diversity Function and pH Stomach 101 –10 2 Lactobacillus HCl secretion Streptococcus Macromolecule Helicobacter Digestion Peptostreptococcus pH 2 Duodenum 10 1 –10 3 Streptococcus Main digestion, absorption of Lactobacillus monosacchrides, amino acid, fatty acid, and water pH 4–5 Jejunum 10 3 –10 4 Bacteroides Clostridium Streptococcus Actionmychaea Ileum 10 7 –10 9 Colon 10 11 –10 12 Bacteroides Bile acids, vitamin B12, and Clostridium water absorption pH 7 Biﬁ dobacterium Enterobacteriaceace Eubacteria

et al. 2006 ; Rawls et al. 2006 ). Composition of disease-associated dysbiosis that differs among the microbes in different parts of the gut is het- women. The fetal gut is thought to be sterile and erogeneous and is infl uenced by different factors colonization begins immediately after birth, and such as acid, bile, and pancreatic secretions, it undergoes dramatic changes during the devel- which hinder the colonization in the stomach and opment. However, some evidence suggests the proximal small intestine (Ridlon et al. 2014 ; presence of bacteria in the intrauterine environ- Endesfelder et al. 2014 ). However, bacterial den- ment (Prince et al. 2014 ). The bacteria found in sity is higher in the distal small intestine and the meconium might have translocated from the increases in the large intestine to 1011 –10 12 bacte- mother’s gut via the blood stream and may infl u- ria per gram of colonic content (Table 1.2 ) ence the microbiota of the infant before birth (Whitman et al. 1998 ; Ley et al. 2006 ). Moreover, (Madan et al. 2012 ). Recently, microbiome is surface-adherent and luminal microbial popula- also discovered in preterm and full-term placen- tions are different (Carroll et al. 2011 ; Ringel tas with a unique bacterial community consisting et al. 2015 ). For example, Bacteroides , of low number of organisms and large number of Bifi dobacterium , Streptococcus, members of species (Aagaard et al. 2014 ). These are mostly Enterobacteriacea, Enterococcus , Clostridium , nonpathogenic commensals belonging to the Lactobacillus , and Ruminococcus are predomi- phyla Firmicutes, Tenericutes, Proteobacteria, nant in feces, whereas only Clostridium , Bacteroidetes, and Fusobacteria and very much Lactobacillus, and Enterococcus are identifi ed in resemble the microbiome of the oral cavity the mucus layer and epithelial crypts of the small (Aagaard et al. 2014 ). Experimental evidence intestine (Swidsinski et al. 2005 . suggests that a growing embryo and fetus does Koren et al. ( 2012) studied the dynamics of not (always) develop in a sterile world but may the gut microbiota throughout the course of preg- encounter the presence of bacteria, at least in the nancy. During the fi rst trimester, the gut micro- placenta, which needs further study to elucidate biota is similar in many aspects to that of a the role of this placenta. healthy nonpregnant male and female controls, After birth of the baby, development of micro- but by the third trimester, the structure and the biota is infl uenced by genetic, epigenetic, and composition of the community resembles a environmental factors such as delivery mode, 1.1 Introduction 5 use of antibiotics, and breastfeeding (Bermon sary for immune homeostasis. Initial coloniza- et al. 2015 ). The delivery mode at childbirth has tion by microbes in infants is very important to an impact on early microbiota composition and balance immune homeostasis (Walker 2013 ). For could subsequently infl uence on long-lasting example, initial colonization of infants is ham- metabolic effects and postnatal maturation of pered by various factors such as birth by cesarean immune system development (Dominguez-Bello section or receiving excessive perinatal antibiot- et al. 2010; Biasucci et al. 2010). Vaginally ics leading to aberrant mucosal immune function delivered children display a microbiota that (Fig. 1.1 ). As a result, later in childhood, they are shares characteristics with the vaginal origin susceptible to immunity-related diseases like (Huurre et al. 2008 ), whereas the GI tract of asthma and autoimmune diseases (e.g., celiac babies delivered by cesarean section is mainly disease). The gut microbiota performs a number colonized by environmental microfl ora (Biasucci of essential protective, structural, and metabolic et al. 2008 ). The reduced microbial stimulation functions for host health, including food process- during infancy would result in slower postnatal ing, digestion of complex host-indigestible poly- maturation of the immune system and develop- saccharides, pathogen displacement, and ment of an optimal balance between TH1 and synthesis of vitamins (Table 1.3 ) (Grenham et al. TH2-like immunity (Björkstén et al. 2001 ; 2011; Trompette et al. 2014 ; Neish 2014 ; Clarke Björkstén 2006 ). Infants born by cesarean sec- et al. 2014 ). The gut microbiota produces numer- tion (CS) show the presence of lower numbers of ous chemicals of a hormonal nature that are bifi dobacteria and Bacteroides when compared released into the bloodstream, and they act at dis- to the vaginal-born ones. This suggests that the tal sites and are implicated in many aspects of microbiota derives, at least in part, from the physiological functions of the gastrointestinal mother during the delivery and modulates the tract, including the regulation of secretion, early colonization of microbiota and immune absorption and digestion, and gut motility (Clarke development (Min and Rhee 2015 ). et al. 2014 ) (Table 1.4 ). There are signifi cant differences in the micro- Unlike other endocrine systems or organs, biota of breast-fed infants when compared to the which secrete a single or at most a small number formula-fed infants (Azad et al. 2013 ; Guaraldi of humoral agents, the gut microbiota has the and Salvatori 2012 ). Breast milk contains benefi - potential to produce hundreds of products (Clarke cial bacteria like bifi dobacteria and oligosaccha- et al. 2014 ). The gut microbiota is the most com- rides, which serves as synergistic synbiotics and plex and biochemically heterogeneous than any helps in the colonization of these bacteria in such other endocrine organ in the human being. This infants. The difference in the gut microbiome of complexity is attributed to the biochemical and a formula-fed baby when compared to the former metabolic activity of the microbial cells. They may underpin the health risks associated with contribute approximately a weight of 1–2 kg in formula feeding. Hence, the colonization process an average adult (Clarke et al. 2014 ). of the infant gut microbiome is chaotic and associated with life events, such as illnesses, dietary changes, and antibiotic treatment, suggesting that 1.1.3 Gut Microbiota and Health differences in the colonization patterns of multiple babies would most likely refl ect differences With recent advancements in research, there is in their daily lives (Koenig et al. 2011 ). enough evidence towards a possible coevolution A balanced gut microbiota is vital for the of the host and its indigenous microbiota (Sekirov development of an appropriate innate and adap- et al. 2010 ). This coevolution process helps them tive immune response (Walker 2013 ; Bermon to establish a symbiotic relationship with the host et al. 2015 ). The disruption of the normal coloni- (Prakash et al. 2011 ). The gut microbiota contrib- zation process could lead to alterations in the utes to human health by performing important important symbiotic relationship, which is neces- functions like providing protection, structural 6 1 Introduction: Gastroinstestinal System and Colorectal Cancer

Based on feeding type Based on treatment type -Breast feeding: Bifidobacterium ↑ -Pro-/pre-biotics: Bifidobacterium, Lactobacillus↑ -Formula: Enterobacteriaceae ↑ -Antibiotics: Microbial variety ↓

PRENATAL BIRTH FIRST WEEK FIRST MONTH ADULT (Conception to (0-5 days) (5-7 days) (10-30 days) (18 year birth) onwards)

Based on environmental

Based on intrauterine contamination Clostridium↑ -Mother to baby: Bifidobacterium, Lactobacillus, Enterococus↑

Based on delivery method -C-section: Staphylococcus, Corynebacterium, Propionbacterium↑ -Normal: Lactobacillus, Prevotella, Sneathia↑

Fig. 1.1 Factors inﬂ uencing the development of the intestinal microﬂ ora in the infant gut

Table 1.3 Function of commensal gut microbiota Protective functions Structural functions Metabolic functions Pathogen displacement Barrier fortiﬁ cation Control IEC differentiation and Nutrient competition Induction of IgA proliferation Receptor competition Apical tightening of tight junction Metabolize dietary carcinogens Production of antimicrobial factors, Immune system development Synthesize vitamins, e.g., biotin, e.g., bacteriocins, lactic acid, folate short-chain fatty acids Ferment nondigestible dietary residues and endogenous epithelial- derived mucus Ion absorption Salvage of energy and functional stability and various metabolic to produce various antimicrobial compounds. To functions (Prakash et al. 2011 ; Sekirov et al. colonize and proliferate in the intestine, microbes 2010 ). develop a mechanism to outcompete each other. Commensal bacteria produce antimicrobial com- Protective Effect The gut microbiota mainly ponents, including bacteriocins and protein- protects its host by serving as a physical barrier aceous toxins that speciﬁ cally inhibit members of to incoming pathogens by competitive exclusion, the same or similar bacterial species (Hammami such as occupation of attachment sites, consump- et al. 2013 ). Antimicrobial peptides (AMPs) are tion of nutrient sources, and production of anti- low molecular weight proteins produced in the microbial substances. It also stimulates the host mammalian GIT, that act by disrupting the sur- 1.1 Introduction 7

Table 1.4 Peptide hormones of the gastrointestinal tract Hormone Produced by Function Gastrin Gastric antrum Gastrin stimulates gastric juice production Duodenum and smooth muscle contraction in the stomach and small and large intestines and controls the pyloric sphincter Glucose-dependent insulinotropic Mucosa Inhibits gastric acid secretion peptide (GIP) Insulin Pancreatic β cells Anabolic hormones Motilin Mucosa Also known as the “housekeeper of the gut”; it helps in improving gastrointestinal motility or peristalsis Pancreatic polypeptide Pancreas Self-regulates endocrine and exocrine pancreatic secretion activities and affects hepatic glycogen levels and gastrointestinal secretions Neurotensin Mucosa Delays gastric emptying Cholecystokinin (CKK) Mucosa Stimulates the digestion of fat and protein Peptide tyrosine tyrosine (PYY) Mucosa Decreases gastric acid secretion Neurotensin Mucosa Delays gastric emptying Secretin Mucosa Promotes the secretion of digestive juice from pancreas with high amounts of bicarbonates and also stimulates stomach to produce pepsin (the protein digestive enzyme) and liver to produce bile Glucagon Pancreatic α cells Stimulates release of insulin and pancreatic somatostatin Cholecstokinin Mucosa Gall bladder and pancreatic secretion Somatostatin Gastric antrum, pancreatic δ Endocrine/paracrine; inhibits secretion of cells gastrin, secretin, GIP, motilin, pancreatic polypeptide, pancreatic secretion, glucagon, insulin Histamin Mast cells close to parietal Attaches to H2 receptor and sensitizes cell cells to stimuli such as gastrin Calcitonin Esophageal tumor Not normally produced in the GIT face structures of both commensal and patho- nous microbiota is also essential for the develop- genic bacteria (Sekirov et al. 2010 ). Examples of ment of the immune system. Experiments with these include defensins, cathelicidins, C-type lec- germ-free mice show underdeveloped lymphatic tins, etc. systems, with fewer Peyer’s patches and isolated Certain commensal bacterium produces short- lymphoid follicles, fewer intestinal dendritic chain fatty acids (SCFA) , which can alter the cells supporting the role of bacterial signals in B local pH to inhibit the growth of certain intestinal cell development (Prakash et al. 2011 ). The pathogens. Bifi dobacterium blocks colonization development of T-cell receptor, for example reg- of pathogenic E. coli through acidifi cation of its ulatory T, T helper type 1 and 2 cells, and T environment (Fukuda et al. 2011 ). The SCFA , helper 17 cells, is dependent on signals from butyrate, downregulates the expression of several intestinal bacteria. Capsular polysaccharide A, virulence genes, for example, those encoding the produced by Bacteroides fragilis , a commensal Type 3 secretion system (T3SS) proteins in bacteria, benefi cially infl uences the immune sys- Salmonella enteria serovar Enteritidis and tem (Mazmanian et al. 2008 ). Lactobacillus Typhimurium (Gantois et al. 2006 ). The indige- rhamnosus JB-1 has potent immunoregulatory 8 1 Introduction: Gastroinstestinal System and Colorectal Cancer effects. It changes the nerve-dependent colonic microbiota has been linked to metabolic and neu- migrating motor complexes (MMCs), enteric ropsychological disorders (depression and autism nerve function, and behavior (Al-Nedawi et al. spectrum disorder). They help in controlling obe- 2015 ). Short-chain fatty acids, such as butyrate, sity and gastrointestinal disorders, including may also exert potent immunomodulatory effects infl ammatory bowel disease (IBD) and irritable by suppressing the nuclear factor-kB activation bowel syndrome (IBS) (Cammarota et al. 2015 ; and/or by acting on G-coupled receptors. These Zhou and Foster 2015 ). effects are also demonstrated with acetate The gut microbiota is also implicated in the (Prakash et al. 2011 ). development of CRC (Candela et al. 2011 ; Compare and Nardone 2011 ; Hamer et al. Metabolic Function The combined biochemical 2012 ), by causing dysbiosis leading to IBD, which capacity of the microbiota mediates diverse ben- is considered to be a risk factor for this cancer efi cial roles, including vitamin synthesis, bile salt (Man et al. 2011 ). The complex interaction within metabolism, and xenobiotic degradation. It also and between microbiota and the host may contrib- provides biochemical pathways required for the ute to colon carcinogenesis through the induction fermentation of nondigestible substrates and of chronic infl ammation and the promotion of endogenous mucus. Through fermentation, bac- immune evasion (Turner et al. 2013 ). Analysis of terial growth is stimulated, producing short-chain colon tumor and normal tissue shows increased fatty acids (SCFA) and gases. The role of SCFA level of S. bovis , enterotoxigenic Bacteroides is discussed in more detail in Chap. 6 . fragilis , and Escherichia coli at these sites (Ellmerich et al. 2000 ; Gold et al. 2004 ). Fragilysin Structural and Histological Function The GIT producing Bacteroides spp. is associated with of a new born is structurally and functionally relapses of IBD and the induction of colon tumors immature, and its postnatal development is infl u- in Apc/Min mice (Hajishengallis et al. 2012 ). enced by a number of factors, including exposure to a developing gut microbial community (Walker 2013 ). Various animals’ models such as germ- 1.1.4 Colorectal Cancer free model and antibiotic-treated animal model provided insights on the role of the gut microbi- Colorectal cancer is the cancer of the colon or ota to provide structural stability to GIT. Butyrate rectum (large intestine) due to the abnormal produced by the gut microbiota helps to secrete growth of cells that invade or spread to other various factors such as inducing the secretion of parts of the body. Colorectal cancer evolves mucins, trefoil factors, and antimicrobial pep- through a stepwise accumulation of genetic and tides. In addition to involvement in cell and tissue epigenetic alterations, leading to the transforma- development, some bacterial communities may tion of normal colonic mucosa into invasive can- strengthen the barrier at the level of the tight cer (Arends 2013). CRC could be seen in three junctions and development of villi, crypts and different patterns based on its origin and is development of the villus microvasculature classed as inherited, familial, and sporadic (Roper (Prakash et al. 2011). Gut microbes are vital for and Hung 2013 ). Inherited and familial CRC keeping the human gut healthy. More impor- accounts for 10 and 25 %, respectively, and tantly, their functions are multifactorial and they derive, at least in part, from germ line mutations, exert benefi cial functions beyond the gut. whereas sporadic CRC derives from somatic Experiments with the germ-free model showed mutation and accounts for approximately 70 % of that a number of extraintestinal processes and CRC. The development of CRC is attributed to organ systems are dysregulated, highlighting the the loss of genomic stability leading to the acqui- contribution of the indigenous gut microbes for sition of multiple mutations (Ewing et al. 2014 ; their development and maintenance (Sekirov Munteanu and Mastalier 2014 ). The types of et al. 2010 ). Recent reports indicate that altered genomic instability include (1) chromosomal 1.1 Introduction 9 instability (CIN), (2) microsatellite instability 1,360,602 (9.7 %) new cases and 693,933 (8.5 %) (MSI), (3) aberrant DNA methylation, and (4) deaths due to CRC worldwide (Dušek et al. DNA repair defects. The genome-wide analysis 2014 ). of gene mutations shows the presence of somatic CRC is the second most common cancer in mutations in several hundred genes in CRC and Europe with 447,136 (13 %) new cases every an average of 80 mutations in any single year and 214,866 (12.2 %) deaths. Variation of CRC. This observation highlights the heteroge- CRC incidence among Asian countries are very neity of the disease (Casey et al. 2013 ). The most wide and is found higher in all developed Asian frequent and characteristic genetic changes in countries when compared to the south Asian colorectal carcinogenesis includes alterations countries (Mohandas 2011 ). The incidence of in APC, KRAS, SMAD4, TP53, and the mis- colorectal cancer has been increasing rapidly in match repair (MMR) genes, MLH1 and MSH2. recent decades, and mortality has also increased The environmental risk factors that contribute except in Japan and Singapore (Hyodo et al. to the development of CRC includes (1) lifestyle 2010). The burden of CRC has risen rapidly in factors (e.g., nutrition, tobacco use, physical some economically developed Asian countries activity), (2) naturally occurring exposures (e.g., like Japan, South Korea, and Singapore. ultraviolet light, radon gas, infectious agents), According to the National Cancer Registry (3) medical treatments (e.g., radiation and Programme conducted by ICMR in India, the medicines, including chemotherapy, hormone annual incidence rates (AARs) for colon cancer drugs, drugs that suppress the immune system), and rectal cancer in men are 4.4 and 4.1 per (4) workplace and household exposures, and 100,000, respectively (NCRP 2013 ) . The AAR (5) pollution ( www.cancer.org ; Roper and Hung for colon cancer in women is 3.9 per 100,000. 2013; Hagland and Søreide 2015). A high body Colon cancer ranks 8th for both the genders, mass index (BMI) is a clear risk factor for CRC, while rectal cancer ranks 9th among men. It is which could be reduced by physical activity. predicted that 2.4 million cases of colorectal cancer will be diagnosed annually worldwide by 1.1.4.1 Statistics 2035. The most commonly diagnosed cancers worldwide are those of the lung (13 %), breast (11 %), 1.1.4.2 Role of Mutagens/Carcinogens and CRC (9.7 %) (Ferlay et al. 2013 ). Cancer- A mutagen is a substance that induces mutation related deaths are due to lung (19.4 %), liver in cells or organisms, and a carcinogen is a sub- (9.1 %), and stomach (8.8 %). CRC is the third most stance that is responsible for causing cancer by common cancer in the world, and approximately initiating unregulated growth in cells or tissues of 95 % of it is adenocarcinomas. Other types of multicellular animals. Study shows that 157 of cancer includes mucinous carcinomas and adeno- 175 known carcinogens (approximately 90 %) squamous carcinomas. According to a recent are also mutagens (Griffi ths et al. 2000 ). The report of the American Cancer Society, there are somatic mutation theory of cancer holds that approximately 93,090 cases of colon cancer and these agents cause cancer by inducing the muta- 39,610 cases of rectal cancer to be diagnosed in tion of somatic cells. Carcinogens may have dif- US in the year 2015 (American Cancer Society ferent levels of cancer-causing potential, and the 2015 ). According to the same report, between risk associated with the development of cancer is 2007 and 2011, CRC incidence and mortality rate multifactorial and depends on how the subjects dropped by 4.3 % and 2.5 % per year, respec- are exposed to a carcinogen, the length and inten- tively, among adults above the age of 50 years. sity of the exposure, and the person’s genetic However, in adults younger than 50 years, CRC makeup (NCI, USA). According to the incidence rates have increased by 1.8 % per year. International Agency for Research on Cancer Moreover, the incidence is higher in men than in (IARC), carcinogens can be classifi ed into the women. According to another report, there are following groups based on the cancer-causing 10 1 Introduction: Gastroinstestinal System and Colorectal Cancer potential of a substance: (1) Group A: carcino- studied among more than 100 types of PAHs. genic to humans, (2) Group B: likely to be carci- B(α)P is isolated from coal tar in 1930 and shows nogenic to humans, (3) Group C: suggestive tumor-inducing capacity in mouse skin (Rubin evidence of carcinogenic potential, (4) Group D: 2001 ). Human beings are exposed to PAHs from inadequate information to assess carcinogenic dietary sources and cigarette smoke. PAHs have potential, and (5) Group E: not likely to be carci- been found particularly in charcoal-broiled, nogenic to humans. Examples of food carcino- grilled, and smoked meats (Cross and Sinha gens include heterocyclic amines (HCA), 2004). However, the infl uence of PAH from the polycyclic aromatic hydrocarbons (PAH), diet to CRC risk remains unclear because of the N-nitroso compounds (NOC), mycotoxins (afl a- diffi culties associated with quantifi cation of toxins) and acrylamide. Preclinical and clinical individual intake. studies have indicated that they are responsible for CRC, breast cancer, and prostate cancer N-Nitroso Compounds (NOC) These are among (Raman et al. 2013 . the most powerful chemical carcinogens, and so even small amounts in the human body could be Heterocyclic Amines (HCA) They are procar- hazardous (Cross and Sinha 2004 ). They are cinogens formed during the reaction between strong alkylating agents capable of causing muta- creatine or creatinine (found in muscle meats), tion and subsequently leading to cancer. They are amino acids, and sugars at high temperatures present in the diet containing a variety of cured (Sugimura et al. 2004 ). HCA are classifi ed into meats and fi sh products (Goldman and Shields two groups, based on reaction with 2 mM of 2003 ). N-nitrosamines are formed on simultane- nitrite. Group I HCA includes 3-amino-1,4- ous ingestion of nitrite or nitrogen oxides and a dimethyl- 5H-pyrido[4,3-b ]-indole (Trp-P-1); nitrosatable substrate such as a secondary amine. 3-amino-1-methyl-5 H -pyrido[4,3-b ]indole Dietary N-nitrosamines have been linked to (Trp-P- 2); 2-amino-9H -pyrido[2,3- b ]indole esophageal and other gastrointestinal cancers (Aαc); 2-amino-3-methyl-9H -pyrido[2,3- b ] (Loh et al. 2011 ; Fritschi et al. 2015 ). indole (MeAαC); 2-amino-6-methyldipyrido[1,2- a:3′,2′- d]imidazole (Glu-P-1); and Mycotoxins They are toxic for animal and 2-aminodipyrido[1,2-a :3′,2′- d ]imidazole (Glu- human health and are produced by molds as sec- P-2). They lose their mutagenicity through con- ondary metabolites (Bennett and Klich 2003 ). version of the amino to hydroxyl groups. The toxic effect of mycotoxins is referred to as However, no such changes are observed with mycotoxicosis , and its severity depends on the Group II HCA. These include 2-amino-3- toxicity of the mycotoxin, the extent of exposure, methylimidazo[4,5-f]quinolone (IQ); 2-amino- age and nutritional status of the individual, and 3,4-dimethylimidazo[4,5-f]quinoline (MeIQ); possible synergistic effects of other chemicals to 2-amino-3,4-dimethylimidazo[4,5-f]quinolone which the individual is exposed to (Peraica et al. (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f] 1999 ). Afl atoxins are acutely toxic, immunosup- quinoxaline (DiMeIQx); and 2-amino-3,7,8- pressive, mutagenic, teratogenic and carcino- trimethylimidazo[4,5-f]quinoxaline genic compounds especially responsible for liver (7,8-DiMeIQx) (Sugimura et al. 2004 ). Some carcinoma (Goldman and Shields 2003 ). HCAs can produce tumors in the colon, mammary Afl atoxins are produced by two major Aspergillus glands, and prostate leading to the development species. A. fl avus , produces only B afl atoxins, of cancer (Sugimura et al. 2004 ; Goldman and and A. parasiticus produces both B and G afl a- Shields 2003 ). toxins. Afl atoxins M1 and M2 are oxidative metabolic products of afl atoxins B1 and B2 Polycyclic Aromatic Hydrocarbons (Peraica et al. 1999 ). Afl atoxins, except afl atoxin (PAH) Benzo(α)pyrene (BαP) is one of the best M1, are classifi ed as Group 1 carcinogens. 1.1 Introduction 11

Insoluble Prebiotics fraction FOS CHAPTER 3 CHAPTER 4 Dietary GOS fibre XOS SOS Soluble Lactitol fraction Lactulose CHAPTER 5 Physiological function (bindings, reduced transit Synbiotics time, etc.) Fermentation CHAPTER 6 CHAPTER 1 Short chain fatty acids CHAPTER 2 GUT SYSTEM (Acetate, propionate, Probiotics Butyrate) (Bifidobacteria, Lactobacillus) Molecular function Colon polyps

Fig. 1.2 Schematic diagram summarizing the various topics discussed in different chapters related to colorectal carcinogenesis and strategies to prevent its development

1.1.4.3 Origin of Colorectal Cancer also contain a population of cells with a self- Intestinal stem cells (ISCs) are undifferentiated, renewal capacity, which often give rise to more multipotent, and self-renewable, and they are differentiated progeny, and are capable of driving involved in tissue homeostasis and repair. They or initiating tumor growth with highly tumori- are located at the base of mucosal invaginations genic and chemoresistant properties. Potential called crypts (Vaiopoulos et al. 2012 ). Each crypt markers of colon CSCs have been proposed, comprises three main terminally differentiated including CD133, CD166, integrin β1, and signal cell lineages (enterocytes, goblet cells, and endo- transducers CD24, CD44, ALDH1, and LGR5. crine cells) that reside in the top third of the crypt, As microenvironment plays an important role and possibly Paneth-like cells (Boman and Huang in cancer initiation, it is necessary to defi ne and 2008 ; Medema and Vermeulen 2011 ). These cells characterize regional niches within the intestine. are in continuous replacement process, as they Stem cells reside at the base of colon crypts and constantly shed into the lumen when they become are likely to target the cancer-initiating mutations senescent (Papailiou et al. 2011 ). This process is (Turner et al. 2013 ). In one study, microbial pop- regulated by stem cells and is under microenvi- ulations are identifi ed in an intestinal-crypt- ronmental infl uence (Medema and Vermeulen specifi c unique environment, adjacent to stem 2011 ). The Wnt/β-catenin, Hedgehog (Hh), cells (Pédron et al. 2012 ). Hence, a detailed Notch, the phosphatidylinositol 3-kinase (PI3K)/ investigation of intestinal microbes relevant to Akt, and the bone morphogenetic proteins colon stem cells is needed to avert cancer. To fur- (BMPs) signaling pathways play a key role for ther understand the role of the gut microbiota in the self-renewal of ISC. It is hypothesized that the initiation of CRC, it is important to identify pathways important for control of the normal and characterize those bacteria and profi le the stem cell may be equally important for loss of metabolic products generated by the microbiota control of the cancer stem cell (Davies et al. and how diet infl uences this metabolite produc- 2011 ). However, colon cancer stem cells (CSC) tion (Turner et al. 2013 ). 12 1 Introduction: Gastroinstestinal System and Colorectal Cancer

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Abstract Probiotics are living microbes that when taken in an adequate amount confers a health benefi t to the host. Predominant probiotic microorganisms include Lactobacillus spp, Bifi dobacterium spp., and Enterococcus spp. The yeast, Saccharomyces boulardii , and other bacterial species like Bacillus and Clostridium butyricum have been studied extensively. Several mechanisms have been proposed for probiosis, mainly attributed to their abilities to strengthen the intestinal barrier, to modulate the host immune system, and to produce antimicrobial compounds. The main therapeutic and health benefi ts of probiotics include prevention of diarrhea, hypercholesterolemia, and infl ammatory bowel disease and improvement in lactose utilization and mineral absorption, anti-Helicobacter pylori activity, and anticancerous activity (ACA). The ACA of probiotic is mainly accredited to the (1) inactivation of cancerogenic compounds, (2) lowering of intestinal pH, (3) modulation and enhancement of the host’s innate immunity through the secretion of anti-infl ammatory molecules, (4) alteration of the intestinal microfl ora, (5) antiproliferative effects via regulation of apoptosis and cell differentiation, and (6) inhibition of tyrosine kinase signaling pathways. Faecalibacterium prausnitzii anti-infl ammatory commensal could be used as a potential probiotic against colitis and CRC.

Keywords Antimicrobial activity • Biﬁ dobacteria • Lactobacilli • Anticancer activity • Faecalibacterium prausnitzii • Gut microbiota • Postbiotics • Probiotics

2.1 Probiotics and Colorectal Table 2.1 Timeline of probiotics history Cancer Year Contribution Reference 1908 Postulated that LAB Elie Metchnikoff 2.1.1 History, Defi nition, offered health benefi ts and Statistics capable of promoting longevity 1899 First to isolate a Henry Tissier 2.1.1.1 History Bifi dobacterium The concept of probiotics was introduced more 1917 Isolated a nonpathogenic Alfred Nissle than 100 years back by Elie Metchnikoff, a strain of Escherichia coli Russian scientist who received a Noble prize for from the feces of a First his work on phagocytosis (Metchnikoff 1907 ). World War soldier who did not develop He postulated that lactic acid bacteria (LAB) enterocolitis during a could offer health benefi ts capable of promoting severe outbreak of longevity. He developed a diet with milk fer- shigellosis mented with a bacterium, which he called as 1965 First coined the word Lilly and “Bulgarian bacillus.” In 1906 , Henry Tissier from “probiotics” and defi ned Stillwell as microbially derived Pasture Institute isolated bifi dobacteria from factors that stimulate the breast-fed infants and reported the role of this growth of other Y-shaped organism in modulating and inhibiting organisms pathogens in infants. In Japan, in early 1930s, Dr. 1974 Defi ned probiotics as Parker L. acidophilus “organisms and Shirota, a physician, isolated substances which Shirota (subsequently named L. casei Shirota) contribute to intestinal from the intestine to develop fermented milk microbial balance” (Oozeer et al. 2006). A brief summary of the his- 1989 Emphasized the Roy Fuller tory of probiotic is listed in Table 2.1 . requirement of viability for probiotics and redefi ned probiotics as “a 2.1.1.2 Defi nitions live microbial feed The term probiotic, Greek meaning “for life,” supplement which was fi rst coined by Lilly and Stillwell in 1965 . In benefi cially affects the host animal by improving 1974 , Parker defi ned the microorganisms and its intestinal microbial substances that contribute to the intestinal micro- balance” bial balance as probiotics. More than 6,000 2001 “live microorganisms FAO/WHO research papers have been published in the area which when administered of probiotics in the last decade, indicating the in adequate amounts confer a health benefi t on importance given to it by the scientifi c commu- the host” nity. An Expert Committee defi ned the term 2014 “live microorganisms International “Probiotic,” popularized by R. Fuller in 1989 , as that, when administered Scientifi c “Living microorganisms, which upon ingestion in adequate amounts, Association for in certain numbers exert healthy benefi ts beyond confer a health benefi t on Probiotics and the host” Prebiotics inherent general nutrition.” Ever since the 1990s, (ISAPP) probiotics research has become an emerging fi eld, which could be quantifi ed in terms of expo- nential increase in publications. According to the 2001, an Expert Consultation of international sci- United States National Food Ingredient entists working on behalf of the Food and Association (USNFIA), probiotics include bacte- Agriculture Organization of the United Nations ria, yeast, fungi and these are sources of direct- (FAO) and the WHO redefi ned probiotics as live fed microbial of live naturally occurring microorganisms that when administered in ade- microorganisms (Miles and Bootwalla 1991 ). In quate amounts confer a health benefi t upon the 2.1 Probiotics and Colorectal Cancer 17 host (FAO/WHO 2001 ). An expert panel was some lactobacilli are heterofermentative and convened in October 2013 by the International produce alcohol in addition to lactic acid from Scientifi c Association for Probiotics and sugars. These acid-producing bacteria can grow Prebiotics (ISAPP) to discuss the fi eld of probiot- well at low pH environments and inhibit growth ics (Hill et al. 2014 ). of other organisms. They are found in a variety of Most probiotic microorganisms include habitats, including human and animal mucosal Lactobacillus spp. (de Vrese and Schrezmeir membranes, on plants or material of plant origin, 2008 ), Bifi dobacterium spp. (Turroni et al. 2014 ), sewage, and fermented milk products. and Escherichia coli Nissle (EcN) (Franz et al. 2011 ). The yeast Saccharomyces boulardii (Dinleyici et al. 2014 ) and bacterial species like 2.1.3 Bifi dobacteria Bacillus (Cutting 2011 ) and Clostridium butyricum (Takahashi et al. 2004 ) have been studied They are nonmotile, nonsporulating, pleomor- extensively. phic rod-shaped fermentative bacteria with high G + C content belonging to the phylum Actinobacteria. Most strains are strictly anaero- 2.1.2 Probiotics as Functional Food bic. Bifi dobacteria were fi rst isolated from the feces of a breast-fed infant and then named Functional foods are designed to provide a spe- Bacillus bifi dus (Tissier 1900). Due to the close cifi c health benefi t, performance, and/or well- resemblance to properties of lactobacilli, they are being extending beyond the provision of simple classifi ed as members of the genus Lactobacillus nutrients. Most widely known functional foods during a large part of the 20th century. In the are carotenoids, dietary fi bers, fatty acids, fl avo- recent past, they have been reclassifi ed and rec- noids, isothiocyanates, minerals, phenolic acids, ognized as a different genus. They are found in plant stanols/sterols, prebiotics and probiotics, human milk and fi rst to be colonized in the gut of etc. (Hasler 2002 ). The majority of widely used a baby (Arboleya et al. 2011 ). They are a major probiotics includes species of lactobacilli, bifi do- part of the normal human gut microbiota through- bacteria, nonpathogenic Escherichia coli , bacilli, out life. They appear in the stools of a baby a few and yeasts, including Saccharomyces boulardii . days after birth and increase in number thereafter. The scientifi c and clinical evidence in support of The number of bifi dobacteria in the colon of the therapeutic potential of probiotic bacteria in adults is 1010 –10 11 cfu/g, but this number human health has been increasing steadily decreases with age. (Balakrishnan and Floch 2012 ). 2.1.3.1 Enterococcus 2.1.2.1 Lactobacilli Enterococci are Gram-positive, nonspore- Lactobacilli are Gram-positive, nonspore- forming, catalase-negative, oxidase-negative, forming, nonmotile, catalase-negative, and rod- facultative anaerobic cocci that occur singly, in shaped fermentative bacteria with low G + C pairs, or in chains and also belong to the group of content belonging to the phylum Firmicutes LAB of the phylum Firmicutes (Foulquié Moreno (Herbel et al. 2013 ). They have complex nutri- et al. 2006 ). They play an important role in the tional requirements and are strictly fermentative, ripening of cheeses, probably through proteoly- aerotolerant or anaerobic, aciduric or acidophilic sis, lipolysis, and citrate breakdown, hence con- (de Vrese and Schrezmeir 2008 ). Lactobacilli are tributing to taste and fl avor. They are also present predominant member of the LAB, encompass in other fermented foods, such as sausages and more than 25 species, and include a diverse range olives. Furthermore, the production of bacterio- of organisms. They use lactose, glucose, and cins by enterococci is well documented. However, other sugar as carbon source and produce lactate their role in these products has not been fully via homofermentative metabolism. However, elucidated. Commercial enterococcal probiotics 18 2 Probiotics and Colorectal Cancer include E. faecium SF68® (NCIMB 10415, pro- ation in food (FAO/WHO 2002 ). The functional duced by Cerbios-Pharma SA, Barbengo, requirements of probiotics should be established Switzerland) and E. faecalis Symbiofl or 1 by using in vitro methods, and the results of these (SymbioPharm, Herborn, Germany). Both strains studies should be refl ected in controlled human are produced in the form of a pharmaceutical studies. Probiotic strains should (1) preferably be preparation. However, safety of enterococci is a of human origin for human use; (2) be robust in concern, as they are known to be opportunistic terms of acid and bile tolerance; (3) adhere to the pathogens and are a prevalent cause of nosoco- epithelial surface of humans and persist in the GI mial infections that cause bacteremia, endocardi- tract; (4) survive action of toxic metabolites, pri- tis, and other infections. Several virulence factors marily phenols produced during the digestion have been mentioned for this species, and the process, as well as antibiotics and phage; grow in number of vancomycin-resistant enterococci is anaerobic and storage conditions of the food car- increasing with time (Franz et al. 2011 ). rier; (5) resist against degradation by digestive enzymes present in the intestine (e.g., lyso- 2.1.3.2 E. coli Nissle 1917 (EcN) zymes); and (6) must be safe for use. It is a nonpathogenic strain of the Enterobacteriaceae family that has probiotic properties. It was isolated from a healthy soldier 2.1.5 Probiotics as Health Promoter during a Shigella outbreak in 1917 with a hypothesis that commensal strain could protect from Postbiotics are nonviable bacterial products or presumably infectious gastroenteritis (Nissle metabolic by-products from probiotic microor- 1959). Since then, EcN has had a long history of ganisms that have biologic activity in the host use in clinical settings to treat gastrointestinal and are an important attribute of probiosis (Patel disorders. However, the molecular mechanism of and Denning 2013 ; Cicenia et al. 2014 ). Several its benefi cial activity is not well understood mechanisms have been proposed for probiosis (Schultz 2008 ). which is mainly attributed to their abilities to for- tify the intestinal barrier, to modulate the host 2.1.3.3 Faecalibacterium prausnitzii immune system, and to produce antimicrobial Faecalibacterium prausnitzii (FP) is an anti- molecules (Dobson et al. 2012). In vitro and infl ammatory commensal bacterium identifi ed by in vivo studies indicate that probiotics provide gut microbiota analysis of Crohn disease patients several benefi cial effects to host (Fig. 2.1 ) (Sokol et al. 2008 ). Its anti- infl ammatory effects (Uccello et al. 2012 ; Raman et al. 2013 ; Baboota are tested on cellular and TNBS colitis models and et al. 2013 ; Liévin-Le Moal and Servin 2014 ). are partly due to the secreted metabolites that are Each of these benefi ts will be discussed in the able to block NF-κB activation and IL-8 produc- subsequent sections. tion (Qiu et al. 2013 ; Martín et al. 2014 ). These results suggest that counterbalancing dysbiosis 2.1.5.1 Antimicrobial Activity (AMA) using FP as a probiotic is a promising strategy in Probiotic must survive and at least transiently CD treatment. They also produce butyrate, which colonize and provide protection against patho- helps in keeping colonic cells healthy, and so gens in the intestinal tract of the host (Šušković reduce the risk of disease such as CRC and IBD. et al. 2010 ). The role of AMA of probiotics against human and food spoilage organism is crucial, well documented, and critically reviewed 2.1.4 Selection Criteria for Probiotic elsewhere (Liévin-Le Moal and Servin 2014 ). Organism The ability of lactobacilli to prevent and treat the urinary tract infection (UTI) has long been To select potential and safe probiotic strains, observed and is now supported by some clinical FAO/WHO in 2002 set guidelines for their evalu- evidence (Reid et al. 2009 ). Several mechanisms 2.1 Probiotics and Colorectal Cancer 19

Fig. 2.1 Postbiotics produced by probiotic strains Bacteriocin

Modulation of tight junction Biosurfactants proteins

Postbiotic

Short chain Immunomodulation fatty acid & Lactic acid

Hydrogen peroxide

have been postulated for AMA of postbiotics, a sporicidal than as a bactericide. H2 O2 -producing which include presence of antiadhesion factors, lactobacilli might also exert control over vaginal production of hydrogen peroxide and bacterio- cancer through speciﬁ c interactions of reactive cins, and modulation of immune or signaling sys- oxygen species, such as superoxide anion, tems (Fig. 2.1 ) (Reid et al. 2011 ; Liévin-Le Moal hydroxyl radicals, and hypochlorous acid (Bauer and Servin 2014 ; Cicenia et al. 2014 ). 2001). Lactobacilli exhibit self-protection against

toxic activity of H2 O2 through the production of 2.1.5.2 Production of Acids Fe 3+ -activated extracellular peroxidase (Martín Probiotic produces short-chain fatty acids and and Suárez 2010 ). lactic acids as end products, which provide an acidic environment unfavorable for the growth 2.1.5.4 Production of Bacteriocin of many pathogenic and food spoilage microor- Bacteriocins are ribosomally synthesized antimi- ganisms. Acids are generally thought to exert crobial compounds that are produced by all major their AMA by interfering with the maintenance lineage of bacteria and archaea, including many of cell membrane potential, inhibiting an active members of the LAB, and are a heterogeneous transport, reducing intracellular pH, and inhib- group of peptides with respect to their size, struc- iting a variety of metabolic functions (Lorca ture, mode of action, antimicrobial potency, and de Valdez 2009 ; Šušković et al. 2010 ). A immunity mechanism, and target cell receptors detailed discussion of these is carried out in (Dobson et al. 2012 ). Bacteriocins are divided chapter 6. into four main classes (Fig. 2.2 ). Three classes of bacteriocins in lactobacilli have been deﬁ ned by 2.1.5.3 Hydrogen Peroxides Klaenhammer (1993 ). Later, a fourth class of AMA of hydrogen peroxide is ascribed to its “complex bacteriocins” was added to the list. strong oxidizing effect on the bacterial cell and Bacteriocins as therapeutics could be viable destroy basic molecular structure of the cellular alternatives to antibiotics because of their (1) proteins. Hydrogen peroxide could protect potency based on in vitro and in vivo assays, (2) against urogenital infections, especially in the low toxicity, (3) the availability of both broad- case of bacterial vaginosis. It is more effective as and narrow-spectrum peptides, (4) the possibility 20 2 Probiotics and Colorectal Cancer

Bacteriocins from gram- positive bacteria

Class I- Lantibiotics Class III- Class IV- (<5 kD, contain Class II-Nonlantibiotics Nonlantibiotics Nonlantibiotics lanthionine and methyl (<10 kD, heat stable) (<30 kD, heat labile (Complex with lipid and lanthionine) proteins) carbohydrate, heat stable)

Ib Ia Globular Flexible peptides with molecules no charge

IIa IIc IIb (Anti-listerial (Produced (Two peptide during single bacteriocins) secondary peptides) pathway)

Fig. 2.2 Classifi cation of bacteriocins from Gram-positive bacteria of in situ production by probiotics, and (5) the in the urogenital and gastrointestinal tracts fact that they can be bioengineered (Cotter et al. (Gudiña et al. 2010). However, detailed and 2013). Corr et al. ( 2007) showed that the bacte- extensive studies are needed to characterize them riocin Abp118 produced by Lactobacillus sali- produced from probiotic strains. varius UCC 118 was essential for protecting the host against infection by invasive Listeria mono- 2.1.5.6 Modulation of Tight Junction cytogenes. Results suggest that secretory antimi- Proteins crobial proteins other than lactic acid and Probiotics are capable of modulating the hydrogen peroxide produced by LAB exhibit a permeability of epithelial barriers, changing the broad range of activity against Gram-positive and infl ammatory potential of epithelial cells, or Gram-negative organisms (Liévin-Le Moal and directly modulating the activity of immune cells Servin 2014 ). However, the overall AMA of pro- (Ulluwishewa et al. 2011 ; Caricilli et al. 2014 ). biotics is multifactorial and is due to the synergistic action of lactic acid and proteinaceous 2.1.5.7 Immunomodulation substances (Servin 2004 ) The gut microbiota protects the host by priming the immunological defense mechanism (Weng 2.1.5.5 Biosurfactant and Walker 2013 ). The effects of few selected Biosurfactants are “surface-active” compounds LAB on the immune system are listed in Table that are synthesized by microorganisms and may 2.2 . The probiotic strains exhibit potentially use- have a role in the restoration and maintenance of ful properties, including anti-infl ammatory microbial homeostasis (Reid et al. 2011 ). effects, improvement of mucosal barrier homeo- Biosurfactants produced by probiotics can serve stasis, benefi cial effects on intestinal microbiota, as antiadhesive and antimicrobial agents and a and a reduction of visceral hypersensitivity defense weapon against other colonizing strains (Barbara et al. 2012 ). 2.1 Probiotics and Colorectal Cancer 21

Table 2.2 Immodulatory activity exerted by selected strains of probiotics Effect Strains Reference Enhancement and maintenance of tight B. infantis Ewaschuk et al. (2008 ) junction formation B. bifi dum Khailova et al. (2009 ) Probiotic mixture VSL#3 Mennigen et al. (2009 ) E. coli Nissle 1917 Ukena et al. (2007 ) L. plantarum WCFS Karczewski et al. (2010 ) Production of secretory IgA L. acidophilus Torii et al. (2007 ) L. casei de Moreno de Leblanc et al. (2011 ) Transcription of NF-κB L. acidophilus Chen et al. (2013 ) L. acidophilus , L. casei , and van Baarlen et al. (2011 ) L. rhamnosus Induction of proinfl ammatory cytokines Lactobacillus strains Maassen et al. (2000 ) L. acidophilus strain L-92 Torii et al. (2007 ) L. rhamnosus GR-1 Li et al. (2014 ) Induction of anti-infl ammatory cytokines L. reuteri and L. casei Smits et al. (2005 ) Streptococcus salivarius Kaci et al. (2014 ) Regulatory cells (Treg) B. infantis 35624 O’Mahony et al. (2008 ) Bacterial DNA induce TLR9 Probiotic mixture VSL#3 Lammers et al. (2003 ) signaling → anti-infl ammatory effect in murine colitis Elevation of serum level of IL-10 Probiotic mixture VSL#3 Hart et al. (2004 ) Induction of maturation of dendritic cells L. reuteri and L. casei Smits et al. (2005 ) (DC) Enhancement of immunoreactivity of L. casei and L. bulgaricus Perdigon et al. (1986 ) spleen cells and phagocytes and serum L. casei or L. acidophilus Perdigón et al. (1988 ) antibody response to orally and systemically administered antigens Activation of the gene for human Ganz (2003 ) beta-defensin 2 in intestinal mucosa

2.1.6 Probiotics and Gut Microbiota ome, mainly by executing any of the mechanisms of probiosis discussed earlier (Hemarajata The gut microbiota is a highly specialized organ and Versalovic 2013 ). They also upregulate the containing host-speciﬁ c assemblages of integrity of the intestinal barrier, which may microbes whereby metabolic activity directly result in the maintenance of immune tolerance, impacts human health and disease (Bocci 1992 ; modulate the intestinal immunity, decrease the O’Hara and Shanahan 2006). They live in har- translocation of bacteria across the intestinal mony with the gut of the host and help to main- mucosa, and reduce the disease phenotypes such tain the integrity of the gastrointestinal tract as GIT infections IBS and IBD (Caricilli et al. (GIT) and other organs, namely, lungs, heart, 2014 ). Different tools such as culture-dependent brain, and liver (Kau et al. 2011 ). Dysbiosis, per- methods, metagenomic sequencing, functional turbations in the composition of gut microbiota, metagenomics and metatranscriptomics, meta- may disrupt the host-microbes interaction and proteomics and metabolomics are used to study eventually may contribute to disease susceptibil- the effect of probiotics on the composition, ity. Probiotics could positively manipulate the diversity, and functioning of the gut microbiota composition and the function of the gut microbi- (Wang et al. 2015 ). 22 2 Probiotics and Colorectal Cancer

Fig. 2.3 Probiotics as health promoter. Proposed beneﬁ ts of consuming probiotic microorganisms Prevention of Diarrhea Prevention of Lactose hyperchole Intolerance sterolaemia

Probiotics as health Improvement Prevention of promoter of mineral cancer absorption

Immune enhancement IBD and UC

2.1.7 Probiotics and Health establishment of a balanced intestinal microfl ora and resistance and/or prevention of diarrhea The main therapeutic and health benefi ts of pro- (Malaguarnera et al. 2012 ). biotics are summarized in the Fig. 2.3 . 2.1.7.2 Prevention 2.1.7.1 Prevention of Diarrhea of Hypercholesterolemia Diarrhea (Greek διαρρoια = fl owing through) Hypercholesterolemia is one of the major risk means the increased liquidity or decreased con- factors for cardiovascular disease, which is the sistency of stools. It is usually associated with an leading cause of death in many countries, includ- increased frequency of stools and an increased ing India. Even a 1 % reduction in the serum fecal weight. WHO defi nes diarrhea as three or cholesterol reduces the risk of CHD by 2–3 % more watery stools on two or more consecutive (Manson et al. 1992 ). The development of new days (de Vrese and Marteau 2007 ). Although pro- dietary supplements to reduce serum cholesterol biotics have a major infl uence on the gastrointes- has gained plenty of attention. Reduction of cho- tinal fl ora composition and exhaustive research lesterol by probiotics has been demonstrated in being done on the prevention and treatment of humans, mice, and pigs (Kawase et al. 2000 ; acute gastroenteritis, antibiotic-associated diar- Haberer et al. 2003 ; Sadrzadeh-Yeganeh et al. rhea (AAD), traveler’s diarrhea in infants, the 2010 ). Cholesterol-lowering activities of probi- reports on their clear benefi ts on pediatric disor- otics are mainly attributed to the assimilation of ders remain unclear (Vandenplas et al. 2013 ). cholesterol by growing cells, binding of choles- However, data support the use of probiotics for terol to the cellular surface, incorporation of the adjunct treatment of acute viral gastroenteri- cholesterol into the cellular membrane, deconju- tis and for the prevention of gastrointestinal dis- gation of bile via bile salt hydrolase, coprecipita- eases (Weichert et al. 2012 ). tion of cholesterol with deconjugated bile, A growing number of research publications binding of bile by fi ber,, and production of short- indicate that some probiotic strains may help to chain fatty acids by oligosaccharides (Ooi and maintain the health in old people, suggesting Liong 2010 ; Bordoni et al. 2013 ). However, both health and cost-saving benefi ts in offering more clinical evidence is lacking to strengthen fermented dairy products. These benefi ts include this hypothesis. 2.1 Probiotics and Colorectal Cancer 23

2.1.7.3 Improvement in Lactose 2.1.8.1 Infl ammatory Bowel Utilization Disease (IBD) Lactose intolerance is due to the insuffi cient IBD is an idiopathic chronic condition of the GIT activity of lactase in the human gut that causes characterized by intermittent periods of infl am- various degrees of abdominal discomfort. mation and remission (Nanau and Neuman 2012 ). Probiotics, including lactobacilli and bifi dobacte- It is divided into two subtypes: ulcerative colitis ria, produce β-D -galactosidase, which auto (UC) and Crohn’s disease (CD). UC and CD dif- digests lactose and improves lactose tolerance fer based on the localization in the intestine and (de Vrese and Schrezenmeir 2008 ). features of the infl ammation. CD is often described as a prototype of T-helper 1-type dis- 2.1.7.4 Anti- Helicobacter Pylori Activity ease. It may affect a portion of the intestine in a Helicobacter pylori infects over 50 % of the segmental fashion and present a transmural population worldwide, and in developing infl ammation, extending along the entire intesti- countries 80 % of the middle-aged adults may nal wall. UC (T-helper 2-mediated condition) is a be infected (De Falco et al. 2015 ). It is associ- diffuse and a continuous infl ammation and is ated with gastritis, gastric and duodenal ulcers, confi ned to the mucosa of the colon. Microscopic and gastric malignancies. Its eradication would lesions are present only in the mucosa layer for eliminate a major worldwide cause of cancer UC, while it affects the full thickness of the intes- death (Graham 2015 ). Currently, no vaccine is tinal wall in CD (Nanau and Neuman 2012 ). The available for H. pylori and antibiotic resistance etiopathogenesis of IBD may be multifactorial, is increasing. So the need of the hour is to iden- as is the pathophysiology. It is attributed to the tify alternative treatment strategies. Results alterations in the gastrointestinal motility, vis- obtained from in vitro and preclinical studies of ceral hypersensitivity, intestinal microbiota, loss using probiotics for H. pylori reveal the feasibil- of epithelial cell barrier function, overexpression ity of this approach (Felley and Michetti of proinfl ammatory mediators in different effec- 2003; Ruggiero 2014 ). Several clinical trials tor T lymphocyte subsets (Th1 and Th17, Th2), indicate that administration of probiotics can defi cient protective and regulatory signals and/or reduce the side effects of H. pylori eradication abnormal antigen presentation, dysfunction of treatment, increase tolerability, and often the brain-gut axis or certain psychosocial factors increase the overall effi cacy. (Dai et al. 2013 ). Probiotics offer an alternative by altering the intestinal fl ora by (1) competitive exclusion, whereby probiotics compete with the 2.1.8 Improvement of Mineral microbial pathogens for a limited number of Absorption receptors present on the surface of the epithelium; (2) immunomodulation and/or stimulation Probiotics show a positive infl uence on the of an immune response of gut-associated lym- bone metabolism and bone mass density and is phoid and epithelial cells; (3) antimicrobial activ- mainly attributed to the increased mineral solu- ity and suppression of pathogen growth; (4) bility due to the production of short-chain fatty enhancement of barrier function; and (5) induc- acids, production of phytase enzyme by the tion of T cell apoptosis in the mucosal immune bacteria to overcome the effect of mineral compartment (Sheil et al. 2007 ). depressed by phytate, reduction of intestinal infl ammation followed by increase in the bone 2.1.8.2 Anticancerous Activity mass density and hydrolysis of glycoside bond CRC arises by a well-defi ned series of sequences in the food in the intestines by gut microbiota caused by mutations, activations, and deletions of (Parvaneh et al. 2014 ). However, detailed stud- oncogenes and tumor suppressor genes leading to ies are needed to validate the results from ani- histological changes (the adenoma-carcinoma mal model. sequence) (Commane et al. 2005 ). It is likely that 24 2 Probiotics and Colorectal Cancer

Anti-initiation Normal epithelium Alteration of carcinogen, metabolism & detoxification Loss of APC Enhance DNA repair Scavenge electrophiles

Hyper proliferation Anti-promotion events Alter gene expression DNA Enhance immunity hypomethylation Suppress inflammation Promote differentiation Early adenoma Suppress proliferation K-ras activation Increase apoptosis

Intemediate adenoma

DCC loss

Late adenoma

p53 loss

Carcinoma

MMP overexpression Metastasis

Fig. 2.4 Sequential steps involved in the development of CRC. Probiotics could act on various steps as shown in the boxes (Commane et al. 2005 ) different probiotic strains may exert effects at bioantimutagens , which decrease the mutation different stages of carcinogenesis (Fig. 2.4 ) rate by virtue of their involvement in the cellular (Table 2.3 ). processes. The ACA of probiotic is mainly accredited to It is well known that probiotic strains bind and (1) inactivation of cancerogenic compounds, (2) reduce the activity of the mutagens. Binding of lowering of intestinal pH, (3) immunomodula- mutagen is dependent on different factors such as tion, (4) alteration of the intestinal microﬂ ora, (5) presence of peptidoglycans, polysaccharides and regulation of apoptosis and cell differentiation, secretory glycoproteins in the strains; the growth and (6) inhibition of tyrosine kinase signaling phase of the organism; and mutagen type (Raman pathways (Uccello et al. 2012 ; Raman et al. 2013 ). et al. 2013 ). These have been discussed in detail in section 2.5. Antimutagenesis is a phenomenon in which mutation process is inhibited by the natural or 2.1.9 Mechanism of Action synthetic compounds leading to a decrease in the of Probiotics on Colorectal levels of spontaneous and induced mutations Cancer (Bhattacharya 2011 ). Antimutagens are divided into two subgroups: (1) dysmutagens , which This section focuses on the role of probiotics in affect the extracellular modiﬁ cation of the muta- preventing colon cancer. Mechanism of ACA of gens (thereby preventing DNA damage), and (2) probiotics is ascribed to the following: 2.1 Probiotics and Colorectal Cancer 25

Table 2.3 Anticancerous activity of probiotics by modulating immune response Model Effect Strains Reference Methylcholanthracene-induced Host innate immunity by L. casei strain, Shirota Takagi et al. tumor in mice stimulating the spleenic NK (LcS) (2001 ) cell activity Mouse model Improves the immune system Intrapleural injection Yasutake et al. of an inactivated strain ( 1999 ) of LcS Mouse model Potent immunomodulatory Probiotics mixture, Kwon et al. effects by upregulating or designated IRT5 ( 2010 ) potentiating the generation of Tregs by tolerogenic DCs in mesenteric lymph nodes (MLN) Mouse model Downregulated expression of Lactobacillus Chen et al. CXCR4 mRNA and MHC acidophilus ( 2012 ) class I, as well as increasing apoptosis in tumor tissue Mouse model Activation of dendritic cells Enterococcus faecalis Molina et al. and IFN-γ production CECT7121 ( 2015 )

1. Inactivation of carcinogenic compounds Lactobacillus strains could help in reducing the 2. Alteration of the intestinal microfl ora load of mutagens by exhibiting antigenotoxicity 3. Lowering of intestinal pH and activity (Table 2.4 ). 4. Immunomodulation The probiotic human strain L. rhamnosus 231 5. Regulation of apoptosis and cell differentiation (Lr 231) binds to MNNG and MeIQx, leading to 6. Inhibition of tyrosine kinase signaling their biotransformation and subsequent detoxifi - pathways cation (Ambalam et al. 2011 ). Administration of viable Lr 231 protects rats from MNNG-induced colon infl ammation (Gosai et al. 2011 ). 2.1.10 Inactivation of Mutagenic and Carcinogenic Compounds 2.1.10.1 Alteration of the Intestinal Microfl ora Potential probiotic strains bind the mutagen The GIT, particularly the colon, is densely popu- through the cell surface and peptidoglycans lated with bacteria containing commensal and (sugar and protein moieties) and exert antimuta- pathogenic bacteria. Infl ammation in GIT results genic activity and ACA (Vorobjeva and Abilev in the enrichment of certain bacterial groups with 2002; Raman et al. 2013) (Table 2.4 ). Probiotic procarcinogenic traits, including Fusobacterium bacteria, along with dietary ingredients, help in spp., Streptococcus gallolyticus subsp. gallolyti- detoxifi cation and biotransformation of procar- cus (formerly known as Streptococcus bovis cinogens and carcinogens into less toxic biotype 1), enterotoxic B. fragilis and adherent- metabolites, thus preventing tumor formation invasive E. coli (Louis et al. 2014). A diet rich in (Pool-Zobel et al. 2005). Biotransformation of animal fat and red meat stimulates the growth of mutagens/carcinogens occurs in the gut with the secondary bile salt-producing and sulfate- help of phase I and phase II type of enzymes. reducing bacteria, respectively. Gut bacteria are These regulate the toxic, mutagenic, and neoplas- important contributors to bile acid metabolism tic effects of environmental carcinogens. Phase I and thus may play a role in the biology linking of enzymes cause bioactivation, and phase II bile acids to CRC (Sears and Garrett 2014 ). enzymes cause the inactivation of mutagens/car- Commensal microbes may act by inhibiting fecal cinogens (Hammons and Lyn-Cook 2004 ). putrefactive bacteria, such as coliforms, mainly 26 2 Probiotics and Colorectal Cancer

Table 2.4 Consolidated table on antimutagenic activity of probiotics Bacteria Mechanism involved Mutagen used Reference Streptococcus cremoris Cellular fraction and cell wall Heterocyclic amines Zhang and Ohta Z-25, L. acidophilus ( 1991 ) IFO13951, and B. biﬁ dum IF014252 L. helveticus Release of peptides in the 4-nitroquinoline- N ’-oxide Matar et al. (1997 ) fermented milk Four strains of L. Binding was dependent on the Trp-P-1, Trp-P-2, Glu-P-1, Sreekumar and gasseri and B. longum mutagen, pH, bacterial strain, IQ, and MeIQ Hosono (1998a , b ) and the complex cell wall structure Bacillus subtilis- Dependent on the bacterial strain, HCA Rajendran and Ohta fermented soybean mutagen, pH, incubation time, Trp-P-1 and IQ (2001 ) product – Natto presence of metal ions, sodium chloride and alcohol, enzymes, and acetylation

L. rhamnosus GG Cell wall carbohydrate Alatoxin B1 (AFB1 ) Haskard et al. (2000 ) components’ hydrophobic and electrostatic interactions

Lactobacilli and Bacterial cell wall or components AFB 1 Peltonen et al. (2001 ) biﬁ dobacteria were found reversible L. plantarum KLAB21 Three secretory glycoproteins N-methyl- N ’-nitro-N- Rhee and Park (Kimchi, a Korean (16, 11, and 14 kD) nitrosoguanidine (MNNG), (2001a ) fermented food) NQO, 4-nitro-O- phenylenediamine (NPD)

and AFB1 Soymilk fermented Antimutagenic molecules during 3,2-dimethyl-4-amino- Hsieh and Chou with S. thermophilus , bacterial fermentation of milk biphenyl (DMABP) (2006 ) L. acidophilus , B. infantis , B. longum L. plantarum KLAB21 Culture-free supernatants MNNG, NQO, 4-nitro-O- Rhee and Park phenylenediamine (NPD), (2001b )

and AFB1 Eight LAB species Whole cell AαC, PhIP, IQ, MeIQx, Stidl et al. (2008 ) DiMeIQx Three B. longum Fermented skim milk Trp-P-1 and Trp-P-2 Sreekumar and Hosono (1998a , b ) B. longum PS+ Polysaccharides Trp-P-1 Sreekumar and Hosono ( 1998a , b ) Lactobacilli and Butyrate production HCA Lankaputhra and bifi dobacteria Shah ( 1998 ) L. plantarum 301102 Exopolysaccharides (EPS) Trp-P-1 Tsuda et al. (2008 ) LAB and B. Growth-dependent extracellular B (α) P and sodium azide Chalova et al. ( 2008 ) adolescentis ATCC bioactive compounds 15703 Lactobacillus strains Biotransformation of mutagens/ 4-NQO and MNNG Cenci et al. (2002 ); carcinogens Caldini et al. (2005 ) L. casei DN 114001 Metabolism of mutagens IQ, PhIP and MeIQx Nowak and Libudzisz (2009 ) L. rhamnosus 231 Binding and biotransformation MNNG, NQo, MeIQx Ambalam et al. ( 2011 ) L. rhamnosus IMC 501 Binding and biotransformation 4-NQO Verdenelli et al. ( 2010 ) 2.1 Probiotics and Colorectal Cancer 27 by adhering to enterocytes and producing SCFA. Th1, Th2, or even T-reg responses (Stagg et al. The enteric fl ora modifi cation in interleukin-10 2004). This desmutagenic potential of probiotics knockout mice by probiotic L. salivarius UCC118 has been recently addressed, and it is now evident results in a reduced prevalence of colon cancer that probiotic strains that induced IL-12 help in (O’Mahony et al. 2001 ). Thus, probiotics may the maturation of human monocyte-derived DCs, counteract CRC development also through a blood DCs, mouse spleenic cells, and lymph node mechanism of competition with pathogenic intes- DCs, which in turn helps in the activation of NK tinal microbiota. Faecalibacterium prausnitzii cells to produce IFN-γ (Rizzello et al. 2011). Hu (FP) is an anti- infl ammatory commensal that may et al. (2015) reported that L. plantarum can be useful as probiotic to reduce colitis and CRC. enhance the anti-tumour immune response and Probiotic strains, namely bifi dobacteria and delay tumour formation. Animal studies show lactobacilli, downregulate the expression of probiotics has the potential to prevent CRC by xenobiotic-metabolizing enzymes that mediate modulating the host immune system. carcinogenesis through various enzymes, such as β-glucuronidase, azoreductase, and nitroreduc- 2.1.10.4 Regulation of Apoptosis tase, and decrease the risk of tumor development and Cell Differentiation (Wollowski et al. 2001 ). SCFAs produced as Apoptosis is a form of genetically programmed metabolites of probiotic metabolism could inhibit cell death, which plays a key role in the regula- the generation of carcinogenic products from tion of cell numbers (Zhong et al. 2014 ). The procarcinogens by lowering the enzyme levels decreased ability to trigger apoptosis leads to (Roy et al. 2006 ). alteration in the regulation of cell proliferation and, subsequently, an important pathogenic event 2.1.10.2 Lowering of Intestinal pH associated with many cancers. However, regulat- SCFA and lactic acid produced as metabolite by ing the cell survival and death at molecular level probiotic bacteria decreases the load of pathobi- in the apoptotic process could provide an avenue onts and help in maintaining homeostasis by low- for chemoprevention and could hold therapeutic ering the intestinal pH (Roy et al. 2006 ). They potential. Probiotic could play an important role also help in lowering the solubility of bile acids in the regulation of cell apoptosis via intrinsic and ammonia absorption and increase mineral and extrinsic pathways and facilitate the preven- absorption (Cook and Sellin 1998 ). Butyrate is tion of CRC (Uccello et al. 2012 ). There are stud- the preferred energy source of colonocytes and ies supporting the evidence that probiotics may has been implicated in the control of the machin- exert anti-CRC activity through apoptosis-medi- ery regulating apoptosis and cellular differentia- ated signaling pathways (Table 2.5 ). tion (Canani et al. 2011 ; Donohoe et al. 2011 ). 2.1.10.5 Inhibition of Tyrosine Kinase 2.1.10.3 Immunomodulation Signaling Pathway Recent studies have shown that an initial coloni- Tyrosine kinase signaling pathway plays a central zation by a suffi ciently diverse microbiota is role in controlling cellular differentiation and essential for proper development and regulation proliferation and is tightly controlled and regu- of the innate and adaptive immune system (van lated. Deregulation in this pathway leads to the Baarlen et al. 2013 ). Altered gut microbiota may development of cancer. Since these effects are lead to dysbiosis and loss of metabolic and initiated by the receptor, tyrosine kinase activa- immune homeostasis. Probiotics can be used to tion, they are the key targets for inhibitors (Arora manipulate gut microbiota and maintain immune and Scholar 2005 ). p40, a Lactobacillus rhamno- homeostasis. Dendritic cells (DCs) and natural sus GG (LGG)-derived soluble protein, amelio- killer (NK) cells are important during the early rates intestinal injury and colitis, reduces defense against cancer (Fernandez et al. 1999 ). apoptosis, and preserves barrier function by Probiotics may regulate myeloid DC maturation, transactivation of the EGF receptor (EGFR) in polarizing the subsequent T-cell activity toward intestinal epithelial cells (Yan et al. 2013 ). 28 2 Probiotics and Colorectal Cancer

Table 2.5 Antiproliferative effects of probiotics via regulation of apoptosis and cell differentiation Bacterial strain Mechanism Reference In vivo model Rat Model Synbiotic combination of resistant Proapoptotic action due to Le Leu et al. ( 2005 ) starch and Bifi dobacterium lactis the carcinogen, AOM Le Leu et al. (2010 ) Mice model Lactobacillus acidophilus Severity of the colorectal Chen et al. ( 2012 ) carcinogenesis was minimized and induced apoptosis In vitro model Human cancer cell Enterococcus faecalis Induced apoptosis via Nami et al. (2014 ) lines (AGS, HeLa, secretory protein MCF-7 and HT-29) Caco-2 cells E. coli Nissle 1917 commensal Lower infl ammasome Becker et al. (2014 ) E. coli K12 activation and secretory metabolites Lactobacillus reuteri Proliferation due to Iyer et al. (2008 ) COX-2, cyclin D1 and cell survival due to Bcl-2, Bcl-xL Enhance MAPK activities Colo320 and SW480 VSL#3 Suppress the COX-2 Otte et al. (2009 ) intestinal epithelial expression cells HT-29 colon cancer Exopolysaccharides of Autophagic cell death Kim et al. (2010 ) cells L. acidophilus and L. rhamnosus activated due to induction of Beclin-1, GRP78, Bcl-2, and Bak Colorectal carcinoma L. acidophilus and L. casei Enhanced the apoptosis- Baldwin et al. (2010 ) cell line LS513 induction capacity of 5-fl uorouracil HT-29 cells Milks fermented by Lactobacillus 10–50 % decrease cell Baricault et al. (1995 ) helveticus , Bifi dobacterium, confl uency L. acidophilus or a mix of Streptococcus thermophilus , and Lactobacillus bulgaricus Human multidrug- Lactobacillus kefi ri P-IF Induction of apoptosis was Ghoneum and resistant (MDR) associated with activation Gimzewski ( 2014 ) myeloid leukemia of caspase 3, decreased (HL60/AR) cells expression of Bcl-2, and decreased polarization of mitochondrial membrane potential HT29, AGS, MCF-7, Lactobacillus plantarum 15HN Apoptosis is the main Haghshenas et al. and HeLa, as well as a and Lactococcus lactis sub sp. cytotoxic mechanism for ( 2014 ) normal human cell Lactis 44Lac secreted metabolites line CaCo-2 cells L. acidophilus and L. casei Reduced cell proliferation Soltan et al. (2015 ) and increased cell apoptosis References 29

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Abstract The health benefi ts of dietary fi bers, namely, the insoluble and soluble fractions, against colorectal cancer are focused here. The role of food and occupational-originated environmental carcinogens or mutagens (heterocyclic amines, HCA, and polycyclic aromatic hydrocarbon, PAH) and the effects of dietary fi bers against colorectal cancer (CRC) have recently gained ample interest. Epidemiological and clinical studies show that increasing the consumption of dietary fi ber decreases the incidences of CRC. They act differently from drugs, and their effects could be categorized majorly into physiological and cellular or molecular mechanisms. The benefi ts accruing through the former include increase in intestinal viscosity, detoxifi cation by binding the promutagens and mutagens, dilution and fecal bulking, pH maintenance, growth of favorable bacteria, formation of short-chain fatty acids (in particular, butyrate), and reduced transit time. The major pathway in cellular mechanism includes butyrate formation, which modulates the gene expression and assists in the elimination of damaged DNA. Immunomodulation by phase I and phase II enzymes, antitumor activity by induction of TNF-α and NK-cells, and antiproliferative activity are other cellular mechanisms that induce apoptosis and death of cells containing damaged DNA. Role of various dietary fi bers and their fractions against CRC could prove the way for reducing the incidence of the carcinogenesis.

Keywords Dietary ﬁ ber • Colorectal cancer • Short-chain fatty acids • Immunomodulation • Heterocyclic amines • Polycyclic aromatic hydrocarbons • Butyrate • Proliferation • Anticancer • Diet

© Springer India 2016 35 M. Raman et al., Probiotics and Bioactive Carbohydrates in Colon Cancer Management, DOI 10.1007/978-81-322-2586-7_3 36 3 Bioactive Carbohydrate: Dietary Fibers and Colorectal Cancer

3.1 History, Defi nition, methods to quantify these dietary fi bers and Statistics (Southgate 1978 ; Furda et al. 1979; Baker et al. 1979; Schweizer and Wursch 1979; Furda 1981 ; The benefi ts of consuming fi bers studied using Baker and Dorris 1981 ; Heckman and Lane 1981 ; coprolites have been reported approximately Asp et al. 1983 ). The focus was primarily on 10,000 years back. Though the high amounts of removing the digestible portions from the indi- fi ber was consumed in ancient times, its con- gestible portion in the food, using enzymes. The sumption decreased over the centuries as it was success of the techniques, however, was limited considered as a bothersome intestinal waste until and depended on the commercially available the scientifi c community became aware of its enzymes. By 1981, the general consensus on the benefi ts and once again started showing interests. methodology for the quantifi cation of dietary Hippocrates in 430 BC compared the laxative fi ber, as defi ned by Trowell et al. ( 1976 ) was effects of coarse and refi ned wheat and empha- achieved in the spring workshop of the sized the importance of whole grain fl our in Association of Offi cial Analytical Chemists maintaining healthy bowel system. In the 9th cen- (AOAC) in Ottawa, Canada (Prosky et al. 1985 ). tury AD, Hakim, a Persian physician, also put The enzymatic-gravimetric methodology was forth similar views. By 1920s, McCance and modifi ed due to numerous controversial views, Laurence (1929 ) developed the concept of undi- and a rough method was obtained (Prosky et al. gestible carbohydrates, which was the stepping 1985 ), which was adopted by AOAC as the fi rst stone for the modern concept of dietary fi bers as Offi cial Method of Analysis for total dietary fi ber nondigestible plant substances. Even World War (AOAC Offi cial Method 985.29, Total Dietary II saw minimum death from major ailments such Fibre in Foods-Enzymatic-Gravimetric Method) as heart diseases due to the use of unrefi ned fl our (AOAC 1995 ). Same year, AACC adopted the ( www.world-foodhistory.com ). In 1930, Kellogg, same method as AACC Approved Method 32–05 McCarrison, and Walker suggested the health (Prosky et al. 1985 ; AACC 1995 ). These meth- benefi ts of wheat bran, bread, fruits, and beans, ods extend rapidly and were widely accepted by and in 1941, dietary fi ber was reported to infl u- the research communities. Because of its wide- ence the secretory activities of the alimentary spread acceptance and use, AOAC 985.29/AACC tract (Duckworth and Godden 1941 ). 32–05 became the de facto operating defi nition Hipsley (1953 ) was the fi rst to coin the phrase for dietary fi ber and was accepted in Ottawa “dietary fi bre,” to defi ne nondigestible compo- workshop (Prosky et al. 1985 ). Due to nutritional nents, which constitute the plant cell wall. These differences in dietary fi ber fractions, this defi ni- included cellulose, hemicelluloses, lignin, and tion was modifi ed as Offi cial Method 991.42, other plant components, which resist enzymatic Insoluble Dietary Fibre in Food and Food hydrolysis in human gut. Between 1972 and Products-Enzymatic-Gravimetric Method, 1976, a group of scientists adopted Hipsley’s Phosphate Buffer (AOAC 1995 ). Through several term to develop the “dietary fi bre hypotheses” collaborative studies, Lee, Mongeau, Li, (Hipsley 1953 ; Burkitt et al. 1972 ; Painter 1975 ). Theander, and others developed and validated The hypotheses postulated an inverse relation- offi cial methods for total, insoluble, and soluble ship between the consumption of dietary fi ber dietary fi ber that was jointly approved by AOAC/ and the incidences of colon cancer and heart dis- AACC (AOAC 1995 ; AACC 1995 ). Hence, the eases. The dietary fi ber defi nition was further dietary fi ber quantifi cation defi nitions till date broadened in 1976, which included all the undi- could be summarized as following: gestible polysaccharides and storage saccharides (gums, modifi ed celluloses, mucilages, oligosac- 1. AOAC 992.16: Enzymatic-Gravimetric charides, and pectin) (Trowell et al. 1976 ). Method for Total Dietary Fibre With the growing interest and knowledge 2. AOAC 993.21: Nonenzymatic-Gravimetric about the benefi ts of dietary fi ber, numerous Method for Total Dietary Fibre in Food and researchers embarked on developing analytical Food Products with <2 % Starch 3.1 History, Defi nition, and Statistics 37

3. AOAC 994.13: Gas Chromatographic- led to further modifi cation, and following AACC Colorimetric Gravimetric Method (Uppsala prepared the analytical reference standards and Method) for Total Dietary Fibre (Determined values for total, insoluble, and soluble dietary as Neutral Sugar Residues, Uronic Acid fi ber (Caldwell and Nelsen 1999 ). Concise Residues, and Klason Lignin) accounts of various defi nitions for dietary fi ber are shown in Table 3.1 . Still, several researchers acknowledged the According to the CODEX Alimentarius, in latest defi nition as insuffi cient in quantifying cer- 2009, the dietary fi ber was defi ned as the “edible tain unique components of dietary fi bers, which carbohydrate polymers with three or more mono-

Table 3.1 Various attempts to defi ne dietary fi ber Year Defi nitions Scientists/Organization 1953 The term “dietary fi ber” was coined Hipsley 1972–1976 This describes the remnants of plant cell wall that are Trowell and coworkers resistant to hydrolysis by human alimentary enzymes 1976 Broadened the defi nition to include all digestion- Trowell and coworkers resistant polysaccharides, including gums, modifi ed celluloses, mucilages, oligosaccharides, pectins, cellulose, hemicellulose, lignin and linked waxes, cutin, and suberin 1976–1981 Developed methods to quantify the components that Asp, Schweizer, Furda, Theander, Baker, were included in the defi nition and Southgate 1979 Initial steps for developing an international Prosky consensus on defi nition and methodology for dietary fi ber 1981 Consensus on dietary fi ber defi nition and analytical AOAC Spring Workshop in Ottawa, approach Ontario, Canada 1981–1985 Validation of the consensus methodology in Prosky, Asp, Furda, Schweizer, DeVries, multinational collaborative studies and Harland 1985 Enzymatic-gravimetric method adopted to quantify AOAC Offi cial Method of Analysis 985.29 total dietary fi ber. This method that is equivalent to AACC Approved Method 32–05 becomes a working defi nition for dietary fi ber 1985–1988 Collaborative studies on developing methods for fractionation of insoluble and soluble dietary fi ber 1991 Enzymatic-gravimetric method equivalent to AACC AOAC Offi cial Method of Analysis 991.42 Approved Method 32–07 for the fractionation of insoluble dietary fi ber was fi rst adopted 1988–1994 Development, validation, and procedures for offi cial Lee, Mongeau, Li, Theander, and or approved method status for the various coworkers collaborative approaches that fi t the defi nition of dietary fi ber 1992 Reassurance on the consensus on physiological Ist Survey-International dietary fi ber defi nition 1993 Affi rms consensus on physiological dietary fi ber IInd Survey-International defi nition and complete components 1995 Reaffi rmation on the consensus on physiological AOAC International Workshop on dietary fi ber defi nition and its components Defi nition of Complex Carbohydrates and Dietary Fiber (continued) 38 3 Bioactive Carbohydrate: Dietary Fibers and Colorectal Cancer

Table 3.1 (continued) Year Defi nitions Scientists/Organization 1999 “Dietary fi ber consists of the remnants of edible plant AOAC 985.29/AACC 3205, AOAC 991.43/ cells, polysaccharides, lignin, and associated AACC32-07, and equivalent methods substances resistant to (hydrolysis) digestion by the alimentary enzymes of humans.” It identifi es a macroconstituent, including cellulose, hemicellulose, lignin, gums, modifi ed celluloses, mucilages, oligosaccharides and pectins, and associated minor substances, such as waxes, cutin, and suberin 2009 Edible carbohydrate polymers with three or more CODEX Alimentarius monomeric units that are neither digested nor absorbed in the human small intestine are naturally occurring in the food that has been obtained from food raw material by physical, enzymatic, or chemical means and has benefi cial physiological effects, and edible synthetic carbohydrate polymers, which have a benefi cial physiological effect

meric units that are neither digested nor absorbed 3.2 Types and Components in the human small intestine, are naturally occur- of Dietary Fiber ring in the food, that has been obtained from food raw material by physical, enzymatic, or chemical Dietary fi bers are primarily plant material com- means and has benefi cial physiological effects, prising of complex, nonstarch carbohydrates, lig- and edible synthetic carbohydrate polymers, nin, and analogous polysaccharides. They pass which have a benefi cial physiological effect” through the small intestine, reach intact to the (Jones 2014 ). colon where they are available for fermentation Defi ning dietary fi ber is challenging and con- by the resident bacteria resulting in new metabo- troversial, as it is not a single chemical entity but lites, thereby modulating the nutrient absorption/ a group of related compounds with more than one metabolism and preventing several diseased con- physiological health benefi ts. Currently, dietary ditions. They contribute no calories to the diet, fi bers fi nd application in various markets and are but the metabolites released by the colonic bacte- available in pure form and/or as additives. Dietary ria during fermentation are used to meet their fi bers are recommended in regular diet as they energy requirements. However, ruminants improve gastrointestinal health and associated acquire their energy from these fi bers since these bowel syndromes. TechNavio (a London-based bacteria in the rumen hydrolyze the fi bers to mol- global research fi rm) anticipate a signifi cant ecules that could be absorbed and metabolized by growth for the dietary fi ber market in North the host (Turner and Lupton 2011 ). America and is expected to reach $ 3.76 billion Dietary fi bers, based on their source, are by 2018 (CAGR). The fi rm attributes the steady broadly classifi ed into two types, namely, terres- growth of this market (in the USA) to its health trial (cereals, fruits, vegetables, and fungi) and benefi cial factors against obesity, constipation, marine dietary fi bers (marine algae). They can and chronic illnesses such as heart disease, can- also be divided into many different broad catego- cers, and gastrointestinal diseases. Some exam- ries, nonstructural or simple polysaccharides and ples of various types of dietary fi ber supplements structural polysaccharides (Fig. 3.1 ). Based on in the market are shown in Table 3.2 . their chemical, physical, and functional proper- 3.2 Types and Components of Dietary Fiber 39 bre Unifi (powdered cellulose) Metamucil (psyllium husk) Metamucil Original Metamucil Fibre Powder Drink Mixes Metamucil Fibre Wafers Metamucil Fibre Heart and Digestive Capsules Metamucil Strong Bones (300 mg calcium carbonate) Metamucil Fibre Singles Konsyl Products (psyllium hydrophilic mucilloid) Konsyl Original Konsyl Easy Mix Konsyl Fibre Easy to Swallow Capsules Metamucil Clear and Natural (inulin) Hydrocil (psyllium) Fibrecon Caplets (calcium polycabophil) FibreChoice Chewable Tablets Tablets Sugar-free Weight Management (chromium Tablets and picolinic acid) Calcium (500 mg Vitamin and 200 IU D) FibreChoice Plus Antioxidants ber product in market Citrucel Powder (sugar free available) Citrucel Soft Chews FibreChoice Citrucel Caplets FibreChoice Citrucel Fibre Shakes FibreChoice Plus Citrucel Fibre Smoothie (methylcellulose) FibreChoice (inulin) FibreChoice Citrucel Products (methylcellulose) Nonprescription fi bre Plus bre bre bre for bre Plus bre bre bre Benefi Fibre Ultra Caplets Benefi Heart Health Caplets Benefi Chewable Tablets Benefi Powder Benefi Children Benefi Calcium (300 mg) Benefi Stick Packet Brand Name (Active ingredient) Benefi Products (wheat dextrin) Table Table 3.2 40 3 Bioactive Carbohydrate: Dietary Fibers and Colorectal Cancer

pH reduction

Dilution and Rhamnose stool bulking

Lignim Fucose Adsorption

Terrestrial origin

Mannose Uronic

(Butyrate) acid Dietary fiber

Short chain fatty acid Arabinose

Marine origin

Modulation of colonic of Modulation microbial activity microbial Galactose

Xylose Binding

Glucose

intestinal epithelial cells epithelial intestinal Biological modidication of modidication Biological

Fig. 3.1 Classifi cation of dietary fi bers and their health benefi t ties, the latter can further be grouped into soluble character of red and brown algae (Kim et al. and insoluble polysaccharides. These fractions 2010 ). Brown algal sulfated polysaccharide, include neutral sugars, Klason lignin, arabinox- fucoidan (brown algal polysaccharide), has ylan, inulin, pectin, bran, cellulose, β-glucan, and recently shown to exhibit anticarcinogenic and resistant starch. antimutagenic properties in vitro (Kim et al. Terrestrial plant sources, including cereal, 2010). The binding of mutagens and carcinogens vegetable, and fruit fi bers have been widely to marine dietary fi bers is a novel area (Raman explored for their assistance against colorectal and Doble 2014 , 2015 ). cancer. Oats have high amounts of soluble dietary Very low levels of fi ber intake lead to an fi ber (Sjödin et al. 1985; Qu et al. 2005 ). Marine increase in constipation, which is the most nota- dietary fi bers are those that originate from marine ble response of the body to the diet with lesser algae, and their importance towards medical fi ber content. The mechanism of action of dietary applications has come into focus, recently. The fi bers on metabolic health is not well understood. terrestrial and marine dietary fi bers vary in their However, it is speculated that it changes the composition. Marine dietary fi bers mostly con- intestinal viscosity, nutrient absorption and rate tain predominantly a single type of glucan moi- of passage, and production of short-chain fatty ety such as galactose or fucose and other smaller acids and gut hormones (Lattimer and Haub moieties such as glucose, xylose, or arabinose. 2010). A generous dietary fi ber intake has been Presence of sulfated polysaccharides is a distinct reported to impart several health benefi ts, includ- 3.3 Dietary Fiber and Gut Microbiota 41 ing reduced cancer threat, coronary heart dis- The relationship between the dietary fi ber and eases, strokes, hypertension, diabetes, obesity, minerals is a controversial subject (Ismail-Beigi and gastrointestinal disorders, including gastro- et al. 1977 ; Hara et al. 1996 ; Greger 1999 ; esophageal refl ux disease, duodenal ulcer, diver- El-Zoghbi and Sitohy 2001 ). Dietary-fi ber- ticulitis, constipation, and hemorrhoids (Qu et al. associated substances such as phytates and poly- 2005). Dietary fi bers are reported to have anticar- phenols impair the absorption of the minerals cinogenic, antimutagenic, antitumorogenic, anti- (Ca, Zn, Mg, Mn, Cu and Fe) (Coudray et al. proliferative, antigenotoxic, anti-infl ammatory, 2003 ; Prescha et al. 2014 ). Soluble fi bers, includ- and antioxidant activity. They also improve ing pentosans, pectin gums, and mucilage serum lipid concentration, control blood glucose increase the bioavailability of minerals (Chawla in diabetes, promote bowel regularity, assist in and Patil 2010 ). Physicians recommend the con- weight loss and immunomodulation, and lower sumption of fi ber-rich foods to reduce the occur- blood pressure. Increased intake of soluble fi ber rence of obesity, cardiovascular diseases, type-2 improves glycemia and insulin sensitivity in non- diabetes, and cancers. diabetic and diabetic individuals.

3.3 Dietary Fiber and Gut 3.2.1 Dietary Recommendations Microbiota

Dietary Reference Intake value for the dietary Epidemiological and experimental studies have fi bers has been suggested based on the decreased suggested that dietary fi bers play an important risk of coronary heart disease (Turner and Lupton role in preventing colon cancer. Dietary fi bers 2011 ). The different levels of dietary fi bers that reduce the contact time of carcinogens within the are recommended for men and women for differ- intestinal lumen and promote the growth of com- ent age groups are shown in Fig. 3.2 . Females mensals in the gut would positively alter the gut have a lower recommended value than males of metabolism in a various ways. Gastrointestinal the same age group. There are no dietary intake tract is inhabited by trillions of bacterial species. recommendations for infants. No tolerable upper The bacterial gut population shifts to a healthier intake level for dietary fi bers has been suggested. composition in the presence of fermentable However, very high levels could induce and/or dietary fi ber, which forms the substrate for the reduce the absorption and mineral bioavailability bacterial fermentation (Zhu et al. 2011 ; Van Der of calcium, iron, magnesium, and zinc. Kamp et al. 2010 ). By modulating the gut micro-

Fig. 3.2 Recommended intake levels for dietary fi ber (g/day) (Turner and Lupton 2011 ). Intake of dietary fi bers in female during pregnancy and lactation are increased to 28 and 29 g/day, respectively 42 3 Bioactive Carbohydrate: Dietary Fibers and Colorectal Cancer biota landscape, dietary fi bers reduce the risk of et al. (2010 ) conducted a study to understand the type-2 diabetes mellitus, obesity, cardiovascular impact of diet in shaping the gut microbiota in diseases, and colon cancer and improve immu- the children of Europe and rural Africa and nity (Table 3.3 ). Dietary fi bers when undergoing observed that the latter had enriched with bacterial fermentation are benefi cial; however, Bacteroidetes and depleted with Firmicutes in certain dietary fi bers are subjected to concurrent comparison to the former. Fiber from oat bran, anaerobic proteolytic fermentation and cause det- pectin, and guar are highly fermentable than cel- rimental effects, resulting in a yin-yang effect lulose and wheat bran (McBurney and Thompson (Davis and Milner 2009 ). 1990 ; Lupton 2004 ). The type of dietary fi ber Bacterial fermentation is principally con- also infl uences the gut microbial composition. trolled by the amount, type, and the physiochem- For instance, inulin (a polymer of fructose mono- cial characteristics of the fi ber accessible to mers found in onions, garlic, and asparagus) bacteria in the colon (Lupton 2004 ). De Filippo stimulates the growth of bifi dobacteria but ham- pers the growth of potential pathogenic bacteria such as E. coli , Salmonella, and Listeria (Gibson Table 3.3 Soluble and insoluble dietary fi ber and its et al. 1995 ; Bosscher et al. 2009 ). Human colon health benefi ts and the mechanism of action stimulator, xylo-oligosaccharides, decreases the Dietary butyrate-producing bacteria Faecalibacterium fi ber Medical benefi t Mechanism of action prausnitzii, while total butyrate concentration is Soluble Crohn’s disease Enhancement of high in the distal vessel (Christophersen et al. fi ber short-chain fatty acid production, 2013 ). Xylo-oligosaccharides also affect the lev- and mainly acetate els of sulfate-reducing bacteria, Bacteroides fra- Celiac disease Normalization of gilis , accentuating that dietary carbohydrates intestinal modify gut microbiota and their ability to change microbiota the physiological properties of the colonic envi- Colitis Effects on epithelial ronment (Christophersen et al. 2013 ). Diet rich in permeability nonstarch polysaccharides and resistant starch Colon cancer Trophic effects on enterocytes, has intense effect on the type of fecal bacteria, short-chain fatty including Ruminococcus bromii , which contrib- acid production ute to starch degradation and short-chain fatty Metabolic Anti-infl ammatory acid (SCFA) production (Abell et al. 2008 ). syndrome effects The large colon is inhabited predominantly by Arthritis Enhancement of saccharolytic metabolic bacteria, although immune response amino-acid-fermenting bacteria and syntrophic Cardiovascular Reductions of blood diseases pressure and species are also present. Hence, dietary fi ber and reduction of LDL carbohydrate availability determines the compo- serum sition and metabolic activities of the gut micro- concentrations biota and several physiologic properties of the Insoluble Type-2 diabetes Increase insulin fi ber sensitivity, loss of microbiota, which are attributed due to the fer- weight, improved mentation of dietary fi ber and production of energy density SCFA (Cummings and Macfarlane 1991 ). Low Colon cancer Increased bulking dietary fi ber intake and low SCFA production are effect, laxation, and reported in colon-cancer-risk subjects when com- gut transit time pared to healthy individuals. These are also Cardiovascular Reduction of blood diseases pressure and accompanied by distinct fecal microbial profi les reduction of LDL between the two groups (Chen et al. 2013 ). serum Authors also observed that Clostridium , concentrations Roseburia , and Eubacterium spp. are signifi - 3.4 Dietary Fiber and Health 43 cantly less prevalent, while Enterococcus and 2002 ), G. frondosa (Hishida et al. 1988 ; Streptococcus spp. are more prevalent in the Kurashige et al. 1997; Kubo and Nanba 1997 ), colon-cancer-risk group (Chen et al. 2013 ). Low Hordeum vulgare (Cheung et al. 2002 ; Hong pH resulting from the dietary fi ber fermentation et al. 2004 ; Modak et al. 2005 ), Laminaria angus- increases the biosynthetic requirements for tata (Teas et al. 1984 ), Lentinula edodes (Nanba nitrogen- containing precursors and inhibits the et al. 1987 ), Pleurotus ostreatus (Kurashige et al. toxin accumulation in the colon (Smith and 1997 ), Saccharomyces cerevisiae (Hong et al. Macfarlane 1996 ). 2004 ), Sclerotinia sclerotiorum (Suzuki et al. Dietary fi ber consumption has signifi cant ben- 1991 ). Glucomannan from L. edodes also efi ts on health. Many of the benefi ts could be enhanced the survival rate of animals injected attributed to the fermentation of dietary fi ber in with cancer cells (Fujii et al. 1978 ). Apple and the colon and the production of SCFA, specifi - citrus pectins have also been accounted to exert cally butyrate. SCFA production and its role in anticancer effects, including reduced tumor inci- preventing colorectal cancer are discussed in dence (Watanabe et al. 1979 ; Ohkami et al. 1995 ; detail in Chap. 6 . Pienta et al. 1995 ; Hayashi et al. 2000 ; Nangia- Makker et al. 2002 ). Anticarcinogenic/antimutagenic activities of 3.4 Dietary Fiber and Health dietary fi bers are attributed to their ability to produce SCFA. Dietary fi bers may act against CRC Experimental and epidemiological studies have by binding mutagens and carcinogens/procar- emphasized the anticarcinogenic effects of cinogens and diluting them through fecal bulk- dietary fi bers against colon cancer. The general ing, SCFA formation, and reducing the pH by benefi ts of dietary fi ber are shown in Table 3.3 . supporting the colonic bacterial fermentation (Perrin et al. 2001 ). Anticarcinogenic effects have been observed in the case of prebiotics as 3.4.1 Anticancer and Antitumor well. Protective effect of dietary fi bers against dimethylhydrazine, HCA , and PAH are reported Epidemiological and clinical studies demonstrate in vitro and in vivo (Santarelli et al. 2008 ). that intake of dietary fi bers is inversely related to Consumption of cereals, pulses, fi ber-rich fruits CRC (Lattimer and Haub 2010 ). and vegetables, and marine algae has been Trametes versicolor glucans (fungal glucans) reported to reduce the prevalence of colorectal have demonstrated superior anticancer effects in adenomas and CRC (Santarelli et al. 2008 ). It is humans. Fungal polysaccharide has shown to observed that a high intake of dietary fi bers, par- increase the survival of advanced stage gastric, ticularly, cereal fi bers and whole grains, reduce colon, and colorectal cancer patients (Mitomi signifi cantly the risk of CRC (Aune et al. 2013 ). et al. 1992 ), with one study showing improved The mode of action of dietary fi bers (water insol- immune parameters (including blood NK-cell uble and water soluble fractions) on CRC risk activity, leukocyte cytotoxicity, proportion of can be summarized into physiological and cellu- helper cells and lymphocyte suppressor cells) lar/molecular mechanisms (Fig. 3.3 ). (Sun and Zhou 2014). In several animal models, Dietary fi ber intake has been claimed to infl u- a wide range of polysaccharides have shown anti- ence the physiological mechanism. It binds to tumorogenic effects. Aqueous and acid glucan procarcinogens, and carcinogens enhance fecal extracts from A. subrufescens have demonstrated bulking or reduce the system pH. The foremost anticancer activities in animal models (Kobayashi physiological effects of dietary fi bers include aid- et al. 2005 ; Murakawa et al. 2007 ). Anticancer ing in gastric emptying, reducing the intestinal effects have been reported following the intake of transit time, and helping in the production of polysaccharide in various forms from different ceco-colonic microbial fl ora by fermentation fungal sources, including G. lucidum (Lu et al. (Sjödin et al. 1985 ; Nishiyama et al. 1992 ; Lewis 44 3 Bioactive Carbohydrate: Dietary Fibers and Colorectal Cancer

Fig. 3.3 Pathway showing the mechanism of action of dietary fi bers in inducing apoptosis and preventing colon carcinogenesis and Heaton 1997 ). Processing techniques modify Fucoidan , the marine polysaccharide from the amount of soluble dietary fi bers found in brown algae, induces apoptosis in human colon cereals, fruits, and vegetables, which are fer- cancer cells, mediated via the death-receptor- mented by a wide variety of anaerobic bacteria mediated and mitochondria-mediated apoptotic resulting in an increase in the bacterial biomass, pathways. At cellular level, dietary fi ber, specifi - an increase in the fecal mass, a change in intraco- cally fucoidan, increases the levels of cleavage at lonic pH, and production of short-chain fatty caspase −3, −7, −8, and −9 (death-receptor path- acids and various gases as metabolic end prod- ways) and aids in dietary fi ber-mediated apopto- ucts (Lattimer and Haub 2010 ; Aune et al. 2011 ). sis (Yuan et al. 2006 ; Kim et al. 2010). It also On the other hand, the insoluble fi bers predomi- infl uences PI3K-Akt signaling pathway, which in nantly serve as bulking agents, which result in turn controls the Wnt signaling pathway and cell shorter transit time through the intestine and proliferation. Downregulation of ERK pathway increased fecal mass. The short-chain fatty acids by dietary fi bers, specifi cally by fucoidan, also resulting from the colonic fermentation of dietary induces apoptosis. These are also accompanied fi ber are largely absorbed via the portal blood and by changes in Bcl-2 and Bax and p38 kinase reach both the liver and the peripheral tissues (Kim et al. 2010 ). Fucoidan is also observed to (Perrin et al. 2001 ; Comalada et al. 2006 ). induce PARP cleavage. An important function of 3.4 Dietary Fiber and Health 45

PARP is to facilitate the repair of the single- vital role in leukocyte recruitment (Zapolska- strand DNA nicks. Thus, presence of cleaved- Downar and Naruszewicz 2009 ; Vinolo et al. PARP is a valuable marker for apoptosis. In p53 2009 ). Cell adhesion molecules, including selec- signaling pathway, an alteration in the Bcl-2 fam- tins, integrins, vascular cell adhesion molecule-1, ily protein contributes to mitochondrial mem- and intercellular adhesion molecule-1 are brane permeability, resulting in activation of required for adhesion and transendothelial move- caspase-9 through Bid-cleavage and ment of leukocytes (Böhmig et al. 1997 ). SCFA mitochondria- mediated pathway. Death-receptor- reduces the adherence of monocytes and lympho- and mitochondria-mediated pathways are two cytes to human umbilical vein endothelial cells, distinct yet interconnected pathways that trigger which is associated with the reduction of NF-κB caspase activation. These two are the cellular and PPARγ activities and expression of adhesion mechanisms involved in the colorectal cancer molecules (intercellular adhesion molecule-1, prevention by the dietary fi bers (Fig. 3.3 ) (Yuan ICAM-1, and vascular cell adhesion molecule-1, et al. 2006 ; Kim et al. 2010 ). VCAM-1) (Böhmig et al. 1997 ; Zapolska- Downar and Naruszewicz 2009 ). Leukocyte recruitment is altered by SCFA as it modulates 3.4.2 Anti-infl ammatory the amount and type of adhesion molecules and chemokines, which in turn reduces the chronic Infl ammation is an immediate host response of gastrointestinal (GI) tract infl ammatory response. the body to injuries of the tissue due to microbial infection or other noxious stimuli. Uncontrolled 3.4.2.2 Proinfl ammatory Mediators infl ammation and inadequate control of it may and SCFA evoke chronic conditions that could be associated Cytokines and proinfl ammatory mediators play a with cancer (Kundu and Surh 2012 ). SCFA could key role in both the extrinsic and intrinsic path- be associated with the reduction in infl ammatory ways of infl ammation-associated carcinogenesis. condition. Macrophages are the major source of infl ammatory mediators, (Kundu and Surh 2012 ) and on 3.4.2.1 Leukocyte Recruitment activation they produce considerable amounts of and SCFA tumor necrosis factor alpha (TNF-α), IL-1β, The process of infl ammation begins with the IFN-γ and IL-6, chemokines, and nitric oxide recruitment of leukocyte that migrates from the (NO) (Vinolo et al. 2011; Kundu and Surh 2012 ). bloodstream to the infl amed tissue through a Butyrate reduces the lipopolysaccharide (LPS)- multistep process involving expression and acti- and cytokine-stimulated production of proin- vation of adhesion molecules and chemokines fl ammatory mediators, including TNF-α, IL-6, (Luster et al. 2005 ). SCFA aids in the leukocyte IFN-γ, and NO, and increases the release of the recruitment (Maslowski et al. 2009 ; Vinolo et al. anti-infl ammatory cytokine IL-10 (Vinolo et al. 2009 ). Evidence indicates the directional migra- 2011 ; Zimmerman et al. 2012 ). tion of neutrophils induced by SCFA, which Histone deacetylases (HDAC) and histone depend on the activation of GPR43, a G protein- acetyltransferases modulate the degree of protein coupled receptor (Maslowski et al. 2009 ; Vinolo acetylation and gene expression and, thus, affect et al. 2011 ). The receptors GPR41 and GPR109A the expression of proinfl ammatory mediators that are related to GPR43 are activated by SCFA (Waldecker et al. 2008 ; Vinolo et al. 2011 ; (Tazoe et al. 2009 ), thus indicating the role of Zimmerman et al. 2012 ). Butyrate also augments SCFA in the migration of neutrophils to the the acetylation of nonhistone proteins such as infl ammatory sites (Vinolo et al. 2011 ). nuclear factor kappa-light-chain-enhancer of Apart from neutrophil migration, SCFA mod- activated B cells (NF-κB), tumor suppressor gene ulates the expression and secretion of cell adhe- in medulloblastoma (MyoD), and tumor protein sion molecules and chemokines, which play a p53(Vinolo et al. 2011 ). 46 3 Bioactive Carbohydrate: Dietary Fibers and Colorectal Cancer

3.4.2.3 Gastrointestinal Barriers effects of oral polysaccharides have been and Gut Microbiome observed in human subjects. The gut microbiota maintains an intact GI barrier, Arabinogalactans, galactomannans, lami- and its disruption can cause an infl ammatory pro- narin, glucomannans, and mixed polysaccharide cess (Zhu et al. 2011 ). The primary barrier is the products (Ambrotose® products) have been interaction between the microbiota and the gut shown to be metabolized by human colonic bac- epithelial cell layer, which is an active process, in teria. Orally ingested fucoidans, glucans, and which certain infl ammatory mediators are pro- mannans (or their fragments) have been detected duced. This involves ligands of toll-like receptors in numerous tissues and organs throughout the (TLRs) such as LPS and fl agellin that activate body (Sakurai et al. 1996; Irhimeh et al. 2005 ). TLR-4 and −5 to modulate different aspects of Arabinogalactans are shown to augment lympho- host metabolism and immune response (Vijay- cyte proliferation and the number of CD8+ Kumar et al. 2010 ). The secondary barrier is (transmembrane protein) lymphocytes (Nantz formed by epithelial cell secretion of mucus, et al. 2001) so as to amplify the IgG subtype which protects the intestinal epithelium from the response to pneumococcal vaccination (Udani intestinal microbiota, including invasive microbes et al. 2009 ). Panax quinquefolius (North (Salzman 2011 ). The mucus layer is composed of American ginseng) furanose extract is revealed to mucin proteins produced by goblet cells (Zhu reduce the frequency of acute respiratory illness et al. 2011 ). In the small intestine, the Paneth and symptom duration (McElhaney et al. 2006 ). cells directly sense invasive enteric bacteria Fucoidan from Undaria pinnatifi da (wakame) is through TLR activation and then release the anti- also found to stimulate as well as suppress the microbial peptides (Paolillo et al. 2009 ). immune effects in humans; increase stromal- Therefore, mucus not only forms a physical bar- derived factor-1, IFN-g, CD34+ cells, and rier and provides a nutrition source for the micro- CXCR4-expression in CD34+ cells; and decrease biota but also contains protective mediators, the blood leukocytes and lymphocytes levels including secreted antimicrobial peptides and (Irhimeh et al. 2007 ). immunoglobulin (IgA) (Salzman 2011 ; Delzenne Immune-stimulating effects are also reported et al. 2011 ). Consequently, the mucosal immune from dietary fi ber from fungal sources, including system and the homeostasis of the gut microbiota glucan products of Agaricus subrufescens are mutually dependent on each other and their (Takimoto et al. 2004 ; Chan et al. 2007 ; equilibrium maintains a stable intestinal Yuminamochi et al. 2007 ), Lentinula edodes environment. (shiitake) (Hanaue et al. 1988 ; Vetvicka et al. 2008 ), Saccharomyces cerevisiae (Tsukada et al. 2003 ), Laminaria digitata (Rice et al. 2005 ), 3.4.3 Immunomodulation Sclerotium rolfsii (Rice et al. 2004 ), Sclerotinia sclerotiorum (Suzuki et al. 1991 ), and Phellinus A large collection of literature suggests the linteus (Lim et al. 2004 ; Oh et al. 2006 ). Furanose immunomodulatory effects of dietary polysac- extract from P. quinquefolius and pectins from charides, and they have a signifi cant impact on Buplerum falcatum and Malus (apple) spp. have the gut microbes and microbial ecology (Flint also been reported to enhance immune function et al. 2007 ; Macfarlane et al. 2008 ) and, there- in healthy young animals (Sakurai et al. 1999 ; fore, infl uence the host nutrition, immune modu- Lim et al. 2003 ; Biondo et al. 2008 ). lation, resistance to pathogens, intestinal Galactomannan (guar gum) from Cyamopsis epithelial development and activity, and energy tetragonolobus or highly methoxylated pectin are metabolism (Segain et al. 2000 ; Ishizuka et al. observed to stimulate immune effects and anti- 2004 ; Mazmanian and Kasper 2006 ; Jacobs et al. body production in older animals (Yamada et al. 2009; Garrett et al. 2010 ). Immune-stimulating 2003 ). Similar reports on the effectiveness of oral 3.5 Mechanism of Action of Dietary Fiber and Its Fractions on Colorectal Cancer 47 polysaccharide against infection and immune 3.5 Mechanism of Action modulation have been demonstrated in vivo of Dietary Fiber and Its (Murphy et al. 2008 ). Similar examples include Fractions on Colorectal HSV-1 injection and feeding of Avena (oat) spp. Cancer soluble glucans (Murphy et al. 2008), feeding of E. vermiformis and Avena spp. particulate glu- 3.5.1 Physiological Mechanism cans (Yun et al. 2003 ), E. coli injections and feeding laminarin (Neyrinck et al. 2007 ), HSV Various mechanism has been demonstrated by injection and feeding with fucoidans (Hayash which dietary fi ber acts against CRC, which et al. 2008 ), Staphylococcus aureus or Candida includes binding carcinogens/procarcinogens albicans injections and feeding of S. cerevisiae and mutagens, fecal bulking, reduced transit glucans (scleroglucan) (Rice et al. 2004 ), and time, dilution, and fermentation leading to the injection of fecal solution and feeding of the formation of favorable metabolites, SCFA. aqueous extract of A. subrufescens (A. blazei Murrill) (Bernardshaw et al. 2006 ). In human 3.5.1.1 Binding Procarcinogens subjects with seasonal allergic rhinitis, S. cerevi- and Carcinogens to the Dietary siae β(1 → 3) and (1 → 6) glucan decreased the Fibers interleukin (IL-4, IL-5) and percentage of eosino- Structural polysaccharides fasten strongly to phils and increased the IL-12 in nasal fl uid 2-amino-3-methylimidazo[4,5-f ] quinoline (IQ), (Kirmaz et al. 2005 ), while a placebo-controlled 2-amino-3,4-dimethylimidazo[4,5- f ]quinoline study of patients with recurring aphthous stoma- (MeIQ), and 2-amino-3,8-dimethylimidazo[4,5- titis (canker sores) on consumption of β (1 → 3) f]quinoxaline (MeIQx), which could be due to and (1 → 6) glucans found to have an increased the presence of high amounts of Klason lignin, lymphocyte proliferation and reduced ulcer pectin, and uronic acid (Sjödin et al. 1985 ; severity scores (Koray et al. 2009 ). Nishiyama et al. 1992 ). Such binding is sug- In vivo benefi ts of dietary fi bers against gested to involve both reversible adsorption and infl ammatory bowel diseases have been reported chemical interaction of functional groups of in many cases. They include fucoidan from mutagens or carcinogens to dietary fi ber. Binding Cladosiphon okamuranus Tokida (Matsumoto is signifi cantly affected by the pH (pH 6.8) of the et al. 2004), galactomannans from Cyamopsis system. The percentage of Trp-Ps taken up by tetragonolobus (Naito et al. 2006 ), pectin from carboxymethyl cellulose (CMC) and agar in arti- Malus spp. (Lim et al. 2003), and mixed poly- fi cial gastric juice (AGJ) at a pH of 1.2 is 52–56 % saccharide supplements (Koetzner et al. 2010 ). and 58–78 %, respectively. It increases in artifi - Animals tested with ovalbumin established anti- cial intestinal juice (AIJ) at a pH of 6.8 to 97–98 % infl ammatory effects when treated with aqueous and 87–89 %, respectively. The percentage of extracts of A. subrufescens (Chan et al. 2007 ) IQ and MeIQ adsorbed by CMC is 21–27 % in and Ganoderma tsugae (Lin et al. 2006) and AGJ and 100 % in AIJ. Fungal dietary fi ber such pectin of Pyrus pyrifolia (Lee et al. 2004 ) that as Agaricus blazei β-glucan is also reported to contained soluble fi bers. Heteroglycans from have antimutagenic effects against human lym- Lycium barbarum , Lentinus lepidus , and A. sub- phocyte, human hepatoma cell lines (HepG2), rufescens have confi rmed immune-stimulating which is possibly due to its protective action effects in vivo in cancer models (Ito et al. 1997 ; against the DNA damage in the cells (Angeli Gan et al. 2004 ). Glucans from T. versicolor et al. 2006 ). The mutagenic activity of β-glucan have also shown decreased tumor growth and against 3-amino-1-methyl-5H-pyrido[4,3-b] vascular density in cancer-induced models (Ho indole (Trp-P-2) and ROS-induced DNA damage et al. 2004 ) . show dose-dependent protective effect within the 48 3 Bioactive Carbohydrate: Dietary Fibers and Colorectal Cancer range between 20 and 80 μg/ml. The protection advanced stages of cancer development, and its of DNA in the cells is also accredited to the abil- mechanism is detailed in the later section ity of glucans to bind to BαP and scavenge the (Comalada et al. 2006 ). The strong fastening free radicals produced during the metabolism. property, fecal bulking (100 %), and dilution Marine dietary fi bers, namely, alginic acid and effect of dietary fi bers, together with useful by- calcium derivatives of alginate are also reported products formed in human colon during fermen- to adsorb mutagens, and their activity is directly tation, signifi cantly aid in the physiological correlated to uronic acid content (Sjödin et al. detoxifi cation of carcinogens and mutagens. 1985 ; Nishiyama et al. 1992 ).

3.5.1.2 Laxation, Fecal Bulking, 3.5.2 Cellular Mechanism and Dilution of Procarcinogens and Carcinogens Cellular mechanism has been classed into antitu- in the Gastrointestinal System mor activity, formation of SCFA, and immuno- Dietary fi bers exhibit general hydrocolloid prop- modulation of xenobiotic enzymes that assist in erty, namely, water-holding capacity by the fi ber reducing the rate of CRC (Fig. 3.3 ). moieties. This assists in laxative effect in the large intestine where they undergo fermentation, 3.5.2.1 Antitumor Activity of Dietary enhance the microbial growth, increase the bacte- Fibers rial mass, stimulate peristalsis, and induce fecal Lignans of wheat bran exhibit antitumor activity bulking. Rapidly fermented polysaccharides, against human colon cancer cell lines (SW480) in including pectin, exhibit high activity because of dose- and time-dependent manner (Qu et al. their large hydrophobic surface area. Fecal bulk- 2005 ). Marginisporum crassissimum , Sargassum ing aids in dilution of intraluminal contents and, vulgare , and Fucus vesiculosus exhibit antitumor thereby reduces the contact time of mutagen and activity in several cell lines (mouse melanoma carcinogen to the intestinal lining, thus aiding in cell lines, B16-BL6; human colon carcinoma cell cancer prevention (Lewis and Heaton 1997 ). lines, HT-29, HCT-15, HCT 116) and in vivo These characteristic features of dietary fi ber are (transplanted with Sarcoma 180 cells and H22) similar to prebiotics, including inulin, fructo-oli- (Hyun et al. 2009 ). λ-carrageenan exhibits antitu- gosaccharide and galacto-oligosacchrides. mor activity possibly due to the induction of natural killer cells (NK) and proliferation of 3.5.1.3 Bacterial Fermentation lymphocyte and tumor necrosis factor-α (TNF-α) in the Gastrointestinal System (Yuan et al. 2006 ). The administration of Bacterial fermentation process produces SCFA, 5- fl urouracil (5-Fu) and λ-carrageenan helps in which maintains gut health and intestinal restoring the immune function in chemo-treated morphology and function. Details of the role of mice. SCFA in preventing colon cancer are discussed Treating HCT-15 cells with fucoidan from sul- in Chap. 6 . fated seaweed polysaccharide leads to apoptotic Commonly formed SCFA are acetic, propi- events, including DNA fragmentation, chromatin onic, and butyric acids at a molar ratio of condensation, and increase in the population of 60:20:20. These help in maintaining the homeo- sub-G1 hypodiploid cells (Hyun et al. 2009 ). stasis and are associated with enhanced sodium Fucoidan reduces the expression of Bcl-2 and absorption and bicarbonate excretion. Butyrate increases that of Bax in a time-dependent man- also acts as an energy source for colonocytes and ner. It also increases the activity of caspase-9, −8, is associated with the lowering of luminal pH to −7, and −3 and aids in the cleavage of poly (ADP- 5.0–5.6 (Rechkemmer et al. 1988 ; Oltmer and ribose) polymerase (PARP). Apoptosis is also Engelhardt 1994 ) and alteration of the colonic accompanied by strong activation of extracellular microfl ora. SCFA lessens and acts on the signal-regulated kinase (ERK) and p38 kinase 3.5 Mechanism of Action of Dietary Fiber and Its Fractions on Colorectal Cancer 49 and inactivation of phosphatidylinositol 3-kinase colonic epithelial cells, but the signaling pathway (PI3K)/Akt in a time-dependent manner (Hyun is still unclear. It is reported that the chemopre- et al. 2009; Kim et al. 2010 ). Similar ventive benefi ts of butyrate depends on its anticarcinogenic effects of low molecular weight amount, time of exposure with respect to the κ-carrageenan (27 and 67 kD) were observed tumorogenic process, and the type of fat in the against HCT-116 (Raman and Doble 2015 ). diet (Lupton 2004 ). Although cell culture studies Maghemite nanoparticles of ι-carageenan are support protection (Lupton 2004 ), most animal also reported to show anticarcinogenesis against studies do not. Butyrate, a fermented by-product HCT-116 emphasizing the collective preventive of dietary fi ber, maintains gene expression and action of carrageenan (red marine algal polysac- modifi es epigenetic hyperacetylation of histones charide) on colon cancer cells (Raman et al. and nonhistone proteins and consequently acts as 2015 ). a histone deacetylase inhibitor and regulates the expression of critical cell cycle (Lin et al. 2012 ), 3.5.2.2 Formation of SCFA alteration of DNA methylation that results in Due to the Fermentative enhanced accessibility of transcription factors to Hydrolysis of Dietary Fibers nucleosomal DNA (Lin et al. 2012). It also in the Gastroinstestinal System induces cell differentiation, suppresses prolifera- Soluble dietary fi bers , including arabinoxylans, tion, and enhances apoptosis to eliminate DNA- inulin, β-glucan, pectin, etc. undergo fermenta- damaged cells that might otherwise progress to tion in the small intestine and are converted to malignancy, thus reduces the risk of CRC (Le acetic, propionic, and butyric acids. The role of Leu et al. 2010 ) (Fig. 3.4 ). butyrate varies between normal and cancerous Lactate, also improves gut health and gut- cells, and this phenomenon is termed as “butyrate associated immune defense and increases the sur- paradox .” Details of the role of SCFA against face area for absorption. Propionate and acetate colon cancer are described in Chap. 6 . Butyrate induce apoptosis in human colorectal carcinoma inhibits colonic tumor cells and promotes healthy cell lines through the loss of mitochondrial trans-

Butyrate Normal colonocyte production (luminal) Mitochondria GPRs Soluble: Gradient increase in butyrate Dietary fibre Bacterial fermentation intake Colon to SCFA Nucleus Colon ↓HDAC Ac Mucosal flow Histone Insoluble: Colonic transit ↓Proliferation and ↑apoptosis Normal colonocyte (crypt base) ↑Proliferation Cancerous colonocyte

GPRs GPRs ACL/ acetyl Co-A Ac Glucose ↓HDAC ↑HAT Histone Ac Histone ↓Proliferation and ↑apoptosis

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Abstract Prebiotics are defi ned as the indigestible food components that are selectively fermented by the intestinal microbes and promote changes in the gut environment, gut microbial community structure, and their metabolism. Prebiotics have been reported to avert colorectal cancer development by altering the composition and activity of the gut microfl ora. Administration of prebiotics has been reported to reduce colon cancer and its biomarkers. Oligosaccharides such as inulin, fructo-oligosaccharide, galacto- oligosaccharide, lactulose, soy-oligosaccharide, xylo-oligosaccharide, lactitol, resistant starch, etc., are some of the commercially available prebiotics. Short-chain fructo-oligosaccharides thwart colon carcinogenesis possibly by modulating the colonic ecosystem. Galacto-oligosaccharides, lactulose, and fructo-oligosaccharides are observed to increase the intestinal concentration of lactate, stool frequency, and weight; and decrease fecal concentration of secondary bile acids, fecal pH, and nitroreductase and β-glucuronidase activities, thus preventing colon carcinogenesis. Lactulose is reported to reduce the proliferation and occurrence of adenoma. Resistant starch prevents cancer by modulating gene expression and DNA methylation. Prebiotics undergo fermentation by gut microbes leading to the formation of short-chain fatty acids and enhance the immunity of the host. Butyrate has been observed to have direct effects in preventing colon cancer. Prebiotics are observed to modulate the colonic gut environment and aid in the growth and development of Bifi dobacteria spp.

Keywords Prebiotics • Short-chain fatty acids • Oligosaccharides • Inulin • Fructo- oligosaccharides • Galacto-oligosaccharides • Lactulose • Biﬁ dogenic effects • Colon • Gut microbes

© Springer India 2016 57 M. Raman et al., Probiotics and Bioactive Carbohydrates in Colon Cancer Management, DOI 10.1007/978-81-322-2586-7_4 58 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer

4.1 History, Defi nition, the adult use of probiotics and/or prebiotics was and Statistics four times higher in 2012 than that in 2007. Oligosaccharides of low molecular weight have Prebiotics was initially introduced by Gibson and been the subject of recent interest. They form the Roberfroid (1995 ) by switching “pro” for “pre,” most accessible source of carbon for colonic bac- which means “before” or “for” and defi ned it as teria, which on ingestion reach the ileocaecal “a non-digestible food ingredient that benefi - region in a relatively unmodifi ed form (Oku et al. cially affects the host by selectively stimulating 1984 ; Nilsson et al. 1988 ). Certain oligosaccha- the growth and/or activity of one or a limited rides (Table 4.1 ) have been reported to have an number of bacteria in the colon.” These descrip- ability to support the growth of gut microorgan- tions overlap with the defi nition of dietary fi ber isms whose metabolism has positive physiologi- with the exception in selectivity for certain spe- cal consequences (Fuller and Gibson 1998 ; cies of saccharides. For instance, bifi dobacteria Gibson and Roberfroid 1999 ). showed increased growth specifi city when Today, the industry is keen on developing a ingested with fructo-oligosaccharides and inulin range of oligosaccharides that could be incorpo- (Gibson and Roberfroid 1995 ; Gibson et al. rated into the food to improve their “prebiotic 1995 ), transgalactosylated oligosaccharides effect.” This is especially true in the Western (Tanaka et al. 1983; Ito et al. 1993; Rowland and countries where their conventional daily diet has Tanaka 1993 ), and soybean oligosaccharides relatively small quantities of oligosaccharides (Hayakawa et al. 1990 ). (ca. 2–5 g per day) (Macfarlane and Cummings Gibson and Roberfroid ( 1999) also observed 1991 ). Fructo-oligosaccharides are well that these prebiotics stimulated the growth of researched prebiotics that occur naturally in the favorable and benefi cial bifi dobacteria and lacto- diet. A detailed description of the prebiotic oligo- bacilli (Gibson and Roberfroid 1999 ). Apparently, saccharides is given elsewhere in this chapter. prebiotics must endure the digestive process, the Certain prebiotics aid in the growth and meta- gut pH, and the enzymatic hydrolysis before it bolic activities of bifi dobacteria and lactobacilli reaches the colon and preferably persist through- (Rowland and Tanaka 1993 ; Fuller and Gibson out the large intestine so that its benefi ts are evi- 1998 ; Bouhnik et al. 1999 ), the benefi cial microor- dent distally (Gibson et al. 2004 ). In the colon, ganisms that exert therapeutic and prophylactic prebiotics act as the dietary bulking agent or as infl uence on the health of infants and adults the available substrate for resident colonic bacte- (Salminen et al. 1998 ). A benefi cial effect of ria, so it decreases the pH and increases the pro- Bifi dobacterium spp. in breast-fed babies against duction of short-chain fatty acids (SCFA) that childhood diseases includes the prevention of col- may reduce the number of pathogenic microor- onization of transient pathogens, which were ganisms (Morisse et al. 1993 ). reported earlier (Fuller 1991, 1992 ). Similarly, Prebiotics or oligosaccharides are normally reduced levels of bifi dobacteria may partly explain found in plants and are relatively short-chain car- the augmented vulnerability of elderly people to bohydrates. They occur widely in nature. They certain disorders and reduction in their immunity are also detected in relatively smaller quantities, (Rowland and Tanaka 1993 ; Gibson and as free sugars or glycoconjugates in human milk Roberfroid 1995 ; Buddington et al. 1996 ). The and the colostrum of various animals (Bucke and indigenous gut fl ora may be compromised due to Rastall 1990 ). They are maintained as sugar the reduction in the number of Bifi dobacterial spp. reserves in seeds and tubers and are utilized as This may reduce capacity to fi ght pathogens. growth commences. However, the effects of pre- Suitable prebiotics could increase the number of biotics or these oligosaccharides on the gastroin- bifi dobacteria or lactobacilli leading to an testinal physiology have been highlighted in improved microbial balance in the colon and colo- recent years with renewed signifi cance to human nization resistance. Although this hypothesis is health (Van Loo et al. 1999 ). In the United States, still a controversy, static batch culture fermentation 4.1 History, Defi nition, and Statistics 59

Table 4.1 Major types of prebiotics and benefi ts Components Source Microfl ora stimulated Potential benefi ts Saccharides Oligosaccharides Onion, garlic, chicory Bifi dobacterium species Increase in root, burdock, bifi dobacterium asparagus, Jerusalem Suppression of artichoke, soybean, putrefactive bacteria wheat bran, milk, Prevention of honey, leek, banana, constipation and diarrhea rye, barley, salsify (Mussatto and Mancilha 2007 ) Fructo-oligosaccharides Same as above Bifi dobacterium species Augmentation of (inulin, oligofructose) bifi dobacteria and reduction of pH Lactobacillus acidophilus Improvement of gastrointestinal health Lactobacillus casei Improvement of calcium Lactobacillus plantarum absorption Fructans Ash-free white powder Bifi dobacterium species Increase in bifi dobacteria from tubers of Jerusalem artichoke Stachyose and raffi nose Soybean extract Bifi dobacterium species Growth factor Peptides Casein macropeptide Bovine milk Bifi dobacterium species Increase in bifi dobacteria Human kappa casein and Human milk: Bifi dobacterium bifi dum Increase in bifi dobacteria derived glycomacropeptide chymotrypsin and pepsin hydrolysate Polyols Lactitol Synthetic sugar alcohol Bifi dobacterium species Increase in bifi dobacteria of lactose Lactulose Synthetic derivative of Bifi dobacterium species Increase in bifi dobacteria lactose Chart adapted from the International Food Information Council Foundation: Media Guide on Food Safety and Nutrition: 2004–2006. *Examples are not an all-inclusive list with human fecal bacteria has supported this view. transverse regions of the colon model. Similar The study indicated that fructo-oligosaccharides results were observed in rodents (Apajalahti et al. (FOS), galacto- oligosaccharides (GOS), xylo-oli- 1998 ). When inulin was fed along with the diet, gosaccharides (XOS), isomalto-oligosaccharides the caecal bacteria increased, as did the production (IMO), and lactulose alter the gut microfl ora, of SCFA, causing a reduction in the pH (Apajalahti increasing the level of bifi dobacteria and/or lacto- et al. 1998). Molecular sequencing indicated an bacilli and reducing the levels of harmful bacteria, increase in the population of bifi dobacteria and a including clostridia and bacteroides (Rycroft et al. decrease in the pathogenic bacteria, namely, clos- 2001 ). In vitro study with a simulation model for tridia and desulfovibrios. An increase in the lactic fermentation in the colon implicated that inulin acid bacteria and a decrease in the enterobacteria also aids in increasing the levels of lactobacilli but are observed in the gut of rats containing human to a lesser extent of bifi dobacteria levels, in the fl ora and fed with resistant starch (Silvi et al. proximal regions (McBain and Macfarlane 2001 ). 1999). Modulations of the fl ora are reported in These studies indicated that GOS increased both human volunteers when they are fed with prebiot- bifi dobacteria and lactobacilli in the proximal and ics. GOS and FOS are reported to augment the 60 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer fecal bifi dobacteria in a dose-dependent manner Global Industry Analysts (GIA), the prebiotic (Ito et al. 1990; Bouhnik et al. 1999). The rate of sales are approximately $ 1 billion USD in 2011 increase depended on the initial levels of the bac- (Spinner 2013 ). The US market for prebiotics is teria. Individuals with the lowest starting popula- projected to reach $ 225.31 million USD by the tions of bifi dobacteria showed maximum increase year 2015. The European market is motivated by (Rycroft et al. 2001 ). Molecular-based techniques the development of prebiotic enrichment into have confi rmed the benefi cial effects of prebiotics, new food such as, meat and snack products, and namely, FOS and lactulose, in human trials (Tuohy the US market is continuing to meet the demand et al. 2001 , 2002 ). The FOS have also been seen to for fructans, which form the largest product seg- be a highly effective prebiotic when incorporated ment in the US prebiotic market. Market analysis in a biscuit and consumed at a rate of 8 g per day shows that the prebiotic-rich infant formulae are (Tuohy et al. 2001 ). Other prebiotics, namely, inu- projected to grow at a compound annual growth lin, trans-galacto- oligosaccharide, and lactulose, rate (CAGR) of 11.3 % between 2012 and 2018 are found in wide usage in Japan. (Transparency Market Research 2015 ). The most Prebiotics fi nd application in various day-to- recent application for prebiotics has been in the day food products for humans and animals, which animal feed sector and as pet food, and it has include food and beverages, dietary supplements, become a highly lucrative market. Prebiotic and animal feed. These form a major market with demand for animal feed applications is expected tremendous potential (Table 4.2 ). Prebiotic to cross 70,000 tons by 2018 (Transparency demand for food and beverage applications is Market Research 2015 ). anticipated to attain $ 4.5 billion USD in 2018 The criteria for the classifi cation of food (Transparency Market Research 2015 ). Dietary ingredient as prebiotics are listed below supplements, a recent trend, have picked up pace (Roberfroid 2007 ): in the past few years, and they fi nd applications in the food as nutritional supplements, infant for- 1. It should resist digestion at gastric pH. mulae, and specialty nutrients. According to 2. It should not be hydrolyzed by the gut enzymes. 3. It should not be absorbed in the upper gastro- Table 4.2 Major prebiotic applications in various food intestinal tract. and the prebiotic markets 4. It is fermented by gut microbes. Major prebiotic applications Major markets 5. It should induce selective stimulation of Natural food (whole grains, onions, Europe 1 growth and activity of gut microbes that are bananas, garlic, honey, leeks, associated with health and well-being (Gibson artichokes) and Roberfroid 1995 ). Beverages Asia-Pacifi c2 Dairy products North America3 Prebiotics, the nondigestible food substances, Cereals Rest of the are accountable for the growth of the favorable world 4 bacterial species in the gut, thus benefi ting the Baked food host. The popularity of the prebiotics is rapidly Fermented meat products rising due to its wide application as a functional Dry foods food, which includes prebiotic-enriched dairy Supplements products, health drinks, nutrition bars, breakfast Dietary supplements cereals, beverages, bakery products, meat prod- Food supplements ucts, mineral supplements, weight loss products, Nutritional supplements green foods, infant food, and pet food (Table Specialty nutrients 4.3 ). This could be due to the improved consumer Infant formula awareness towards health and nutrition. Europe Animal feed has been the largest consumer of prebiotic-rich 4.2 Types of Prebiotics 61

Table 4.3 Food rich in prebiotics and the weight of pre- 4.2 Types of Prebiotics biotics in percentage Food containing prebiotics Weight (%) Prebiotics , a group of nutritional compounds, Raw chicory root 64.6 have the ability to promote the growth of specifi c Raw Jerusalem artichoke 31.5 benefi cial (probiotic) gut bacteria and suppress Raw dandelion greens 24.3 putrefactive and pathogenic bacteria. Table 4.4 Raw garlic 17.5 and Fig. 4.1 show the major types of prebiotics Raw leek 11.7 and their structures, respectively. Prebiotics Raw onion 8.6 undergo fermentation in the colon and aid in the Cooked onion 5 proliferation of benefi cial gut microbes (Fig. 4.2). Raw asparagus 5 The effect of prebiotic oligosaccharides could be Raw wheat bran 5 broadly grouped into two (Conway 2001 ): Raw banana 1 (a) Effect on the microbes: • Bifi dobacterium count increases food, owing to increased awareness of its benefi - • Lactobacillus count increases based on cial health effects. The demand for prebiotics in the type of prebiotic Europe is predicted to reach $ 1.9 billion USD in • Clostridia and enterobacteria decreases 2018. The Asia-Pacifi c is the second major con- with the type of the prebiotics sumer and is expected to be a potential market in • Reduced shedding of S. typhimurium the coming years. Asian demand for prebiotic (b) Effect on the host health and physiology: ingredients is expected to grow at a CAGR of • Reduction in blood lipids over 11 % from 2012 to 2018 (Transparency • Increase in mineral absorption Market Research 2015 ). Abbott Laboratories; • Reduced risk of cancer development BENEO-Orafti SA; Bright Food (Group) Corporation Limited; Cargill Incorporated; Cosucra Groupe Warcoing SA; Kraft Foods 4.2.1 Inulin Group, Inc.; FrieslandCampina Domo; Jarrow Formulas, Inc.; Parmalat S.P.A.; Roquette Freres; Inulin is found in around 36,000 plants, including Royal Cosun; and Yakult Honsha Co. Ltd. are herbs – chicory root, burdock root, and dandelion some of the leading companies known to manu- root; fruits – apples, bananas; sweet vegetables – facture prebiotic ingredients and prebiotic-rich onions, garlic, asparagus, leeks, and Jerusalem food (Transparency Market Research 2015 ). artichokes; and raw apple cider vinegar. Inulin- To assess the ability of prebiotic in selectively type prebiotics contain fructans of inulin type. stimulating Bifi dobacterium and Lactobacillus , Fructans are naturally occurring plant oligo- and Prebiotic Index (PI) was introduced, which can be polysaccharides with one or more fructosyl-fruc- calculated using the following formula: tose linkages forming the majority of glycosidic bonds. Inulin-type fructan has β-(2→1)-fructosyl- PI(/ Bif Total )(/ Bac Total ) fructose glycosidic bond that gives it unique structural and physiological properties. They tol- (/Lac Total )(/ Clos Total ), erate the enzymatic hydrolysis by human salivary where and small intestinal digestive enzymes. Inulin- type prebiotics include fructo-oligosaccharides Bif – Bifi dobacterium (FOS), oligofructose, and inulin. These terms Bac – Bacteroides have been interchanged in both the scientifi c lit- Lac – Lactobacillus erature and in food applications. Commercially Clos – Clostridium available inulin-type prebiotics could be extracted Total – Total bacteria from chicory root or synthesized from fundamental 62 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer

Table 4.4 Summarized table showing various prebiotics , their chemical structure, and their degree of polymerization (Cummings and Stephen 2007 ) Prebiotic Chemical structure Degree of polymerization Inulin β (2 → 1)-Fructans 2–65 Fructo-oligosaccharides β (2 → 1)-Fructans 2–9 Galacto-oligosaccharides Galactose oligomers and some glucose/ 2–5 lactose/galactose units Soy-oligosaccharides Mixture of raffi nose and stachyose 3–4 Xylo-oligosaccharides β (1 → 4)-linked xylose 2–4 Isomalto-oligosaccharides α (1 → 4)-glucose and branched 2–8 α(1 → 6)-glucose Resistant starch/dextrin Mixture of glucose oligomers Various molecule sucrose. Depending on the source and metabolites (Kolida et al. 2002 ; Sabater-Molina processing conditions, inulin-type prebiotics et al. 2009). This low molecular weight saccha- could have different chemical compositions. ride has low sweetness intensity, is calorie free, is Hence, some inulin-type prebiotics are relatively noncarcinogenic, and is considered as a soluble high in free sugars (fructose, glucose, and dietary fi ber. Additionally, FOS show prebiotic sucrose), while others have no free sugar. effect, improve mineral absorption, and decrease Mixtures consisting wholly of inulin-type oligo- the levels of serum cholesterol, triacylglycerols, saccharides, polysaccharides, or both could be and phospholipids (Kolida et al. 2002 ; Sabater- observed based on the processing conditions. Molina et al. 2009 ). FOS, similar to inulin, are Inulin, oligofructose, and FOS resist enzy- considered as a prebiotic ingredient and are matic digestion in the upper gastrointestinal tract added to food supplements and infant formulae to and reach the colon virtually intact and undergo encourage the growth of non-pathogenic gut bacterial fermentation. Inulin-type prebiotics are microfl ora (Kolida et al. 2002 ; Sabater-Molina truly bifi dogenic and stimulate the growth of et al. 2009 ). A subgroup of inulin and a prebiotic Bifi dobacteria spp. However, their effects on is regularly added to dairy foods and baked other gut organisms are less clear. A minimal goods. It improves the taste and stimulates the dose of inulin-type prebiotic is needed to induce growth of the benefi cial bacteria, bifi dobacteria. a bifi dogenic effect. However, depending on the Consumption of FOS increases fecal bolus and composition there could be variations in terms of the frequency of depositions. A dose of 4–15 g/ total number of bifi dobacteria and individual day given to healthy subjects was reported to Bifi dobacteria spp. reduce constipation in adults and newborns during their fi rst month (Sabater-Molina et al. 2009 ). It also reduces traveler’s diarrhea and combats 4.2.2 Fructo-oligosaccharides (FOS) several other diseases (Cherbut et al. 2003 ).

Fructo-oligosaccharides (FOS), the oligosaccharides occurring naturally in plants (onion, chic- 4.2.3 Galacto- ory, garlic, asparagus, banana, artichoke, etc.), ligosaccharides (GOS) are polymers of fructose linearly arranged and linked by β(2 → 1) bonds. The number of fructose GOS or trans-oligosaccharide (TOS) and trans- units range from 2 to 60 and often end in a galacto- oligosaccharide (TGOS) , a collective glucose unit (Fig. 4.1). FOS are not hydrolyzed term for the group of carbohydrates containing by small intestinal glycosidases and reach the oligo-galactose and some lactose and glucose, cecum structurally unchanged, where they are are commercially produced from lactose by metabolized by the intestinal microﬂ ora to form enzymatic hydrolysis using β-galactosidase

SCFA, L-lactate, CO2 , hydrogen, and other (Macfarlane et al. 2006 ). 4.2 Types of Prebiotics 63

Fig. 4.1 Some short-chain fructo-oligosaccharides

Oligosaccharides are the third largest compo- components of oligosaccharides are sialic acid, nent in the human milk, and high levels are found N -acetylglucosamine, L-fucose, D-glucose, and in the colostrums (24 % of total colostrums car- D-galactose, which result in a complex of over bohydrates) (Bode 2006 ). Galacto- 130 different oligosaccharides. Human milk con- oligosaccharides are naturally found in human tains large amounts of galactose with a backbone and bovine milk (Alander et al. 2001 ). Milk con- of lactose (β- D -galactopyranosyl-(1 → 4)-α-d- tains high amounts of neutral oligosaccharides glucopyranose) and external galactoses leading than acidic oligosaccharides. The principal sugar to the formation of galactosy-lactose; 1 → 3, 64 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer

et al. 1997 ; Teuri et al. 1998 ; Alles et al. 1999 ). Further clinical trials have also emphasized the benefi cial bowel functions of a mixture of GOS and FOS in infants (Fanaro et al. 2005 ). Bacterial fermentation of GOS increases biomass, leading to an increase in fecal bulk. Undigested oligosaccharides and fermentation by- products may cause an osmotic effect in the gut, thus increasing the water content of feces, which in turn stimulates peristalsis in the colon (Cummings et al. 2001). Furthermore, GOS alter the bowel function through infl uencing colonic environment. In vitro studies indicate a lowering of colonic pH because of an increase in short-chain fatty acids with bacterial fermentation (Bouhnik et al. 1997 ; Blaut 2002 ). These stimulate the growth of lactobacilli and bifi dobacteria and repress the growth of pathogens (Blaut 2002 ). SCFA may also have an effect on the intestinal motility; however, the clinical relevance and mechanisms of action remain unclear (Cherbut et al. 2003 ). The microbial fermentation of GOS may enhance the production of gases in the colon Fig. 4.2 Behavior of prebiotics in human digestive (Cummings et al. 2001 ) and may cause gastroin- system testinal symptoms, including fl atulence. Studies indicate that a maximum of 12 g of GOS daily are 1 → 4, and 1 → 6 galactosyl-lactose; and 1 → 6 well tolerated by the body (Teuri et al. 1998 ; galactosyl- lactose. These oligosaccharides con- Niittynen et al. 2007 ). tribute mainly in protecting human infants from GOS have been commercialized by Yakult gastrointestinal pathogenic bacteria (Fanaro et al. Honsha and Nissin Sugar from Japan, Corn 2005). The total concentration of lactose in Products Intl. from the United States, and Borculo human milk has been estimated to be approxi- Domo Ingredients and Clasado from Europe mately 1 g/l (Boehm et al. 2005 ). (Torres et al. 2010 ). Recently, new products have GOS and FOS show laxative effects that are come into the market, which are mixtures con- attributed to their ability to act as soluble fi bers. taining oligosaccharides of different degree of These pass undigested into the colon and undergo polymerization (CUP-oligo, Oligomate 55, TOS- bacterial fermentation (Delzenne 2003 ). It is sug- 100, Vivinal-GOS, Bimuno, Purimune, and gested that constipation could cause reduction in Promovita-GOS). Food-grade GOS are transpar- the number of bifi dobacteria and treatment with ent syrups or white powders. GOS could correct this, consequently, benefi tting bowel function (Hamilton-Miller 2004 ). Studies on human infants (Ben et al. 2004 ; Fanaro et al. 4.2.4 Lactulose (Lactosucrose) 2005 ) and adults (Ito et al. 1990 , 1993 ; Bouhnik et al. 1997 ) have emphasized the effect of GOS Lactulose, a disaccharide, comprises galactose on bifi dobacteria and the mixture of GOS and and fructose (4- O - β-D -galactopyranosyl- D - FOS on colonic fl ora. Studies have emphasized fructose) that results from heat processing of the laxative effects of GOS in the elderly (Teuri milk (Tratnik 1998 ) or alkaline isomerization of et al. 1998 ) and in adults (Ito et al. 1990 ; Deguchi lactose (Mussatto and Mancilha 2007 ). 4.2 Types of Prebiotics 65

It stimulates the growth and activity of duction (Jenkins et al. 1999 ; Gibson and Rastall Bifi dobacterium species so it has been known as 2006 ) that affects mucosal and systemic immu- bifi dus factor (Mizota et al. 1987 ; Matijevic et al. nity in the host (Hooper et al. 2002 ). Hydrolyzed 2009). It also stimulates some species of products, which include lipopolysaccharides, Lactobacillus, Clostridium , and Peptostreptococcus peptidoglycans, and lipoteichoic acids, exhibit in the large intestine in humans (Kneifel et al. 2000 ). immunomodulatory properties. Mizota et al. ( 2002 ) observed that fecal microfl ora varied with lactulose intake. Bifi dobacteria increased from 22.4 to 50.5 % during intake and 4.2.5 Lactitol then decreased to 26.2 % after the treatment. Lactulose, as a prebiotic, has been observed to act It is an emerging prebiotic, and it exists as lactitol as an energy source for bacteria and improve the monohydrate. Lactitol monohydrate quality of fermented skim milk in cocultures of the (β-galactosidosorbitol) is highly soluble in water, yogurt and probiotic cultures (L. acidophilus , L. with mild sweet taste (Riggio et al. 1990). It is a rhamnosus , and B. lactis in combination with S. disaccharide alcohol, not absorbed in the human thermophilus) (Oliveira et al. 2011 ). It also acts as an small intestine, and it reaches the colon as a osmotic active laxative characterized by shorter tran- potential substrate for the microbial fermentation sit time and accelerated bowel movement. Lactulose (Kontula et al. 1999; Probert et al. 2004 ). Intake is degraded by gut microbes into SCFA, which of high amounts of these sugar alcohols may have reduces the pH of the colon, raises the osmotic pres- adverse consequences on the host system, includ- sure, and increases the stool volume (Mizota et al. ing diarrhea. 2002 ). The amount of acetic acid rises from 30 to 35 μg/g in the feces during treatment, thus causing the fall in pH from 6.5 to 6.3 (Mizota et al. 2002 s). 4.2.6 Fructans (Polydextrose) Lactulose aids in the growth of the gut bacteria and enhances the biomass by utilizing ammonia and Polydextrose expresses prebiotic effects by nitrogen. Acidifi cation of gut inhibits ammonia pro- decreasing fecal pH, increasing residual concen- duction and reduces the residence time and passage tration of SCFA and the number of bifi dobacteria of free ammonia from intestine to blood, thus reduc- in feces (Jie et al. 2000 ; Probert et al. 2004 ). The ing the blood ammonia levels (Mizota et al. 2002 ). nature of the glycosidic bonds in polydextrose In addition to ammonia, its administration also mini- enables it to undergo only partial fermentation by mizes the concentration of putrefactive substance in gut microbes and oppose enzymatic attack the intestine, which includes skatol, indole, p -cresol, (Figdor and Rennhart 1981 ; Achour et al. 1994 ). and phenol. Other physiological functions of lactu- It is a water-soluble, randomly bonded, conden- lose include (Bouhnik et al. 2004 ; Seki et al. 2007 ) sation polymer of glucose with small amounts of the following: sorbitol and citric acid. Polydextrose is not sweet and can be used as a low-calorie bulking agent in • Improvement of blood glucose responses a wide range of food, including baked goods, • Inhibition of secondary bile acid formation, confectionery, dairy products, and functional thus preventing carcinogenesis (Challa et al. beverages. 1997 ) • Activation of immune responses • Treatment of salmonellosis 4.2.7 Soy-Oligosaccharides (SOS) • Enhancement of mineral absorption such as calcium and magnesium Though soy is commonly consumed, the prebiotic potential of its oligosaccharides (raffi nose Physiological effects of this nonabsorbable and stachyose) has been less explored. Soybean carbohydrate also involve increased SCFA pro- seed is known as a rich source of galacto- 66 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer oligosaccharides, such as raffi nose and stachy- 4.2.8 Xylo-oligosaccharides (XOS) ose. Raffi nose, a trisaccharide, contains galactose connected to glucose unit of sucrose. Stachyose Xylo- oligosaccharides have immense prebiotic is a tetrasaccharide comprising of galactose benefi t and are included into many food products. linked to the terminal galactose unit of raffi nose XOS are found in bamboo shoots, fruits, vegeta- (Kim et al. 2003). These are linked by α(1 → 6) bles, milk, corn, wheat, and honey (Vazquez et al. linkages. Noteworthy amounts of sucrose are 2000 ). It is produced on an industrial scale from observed in soy-oligosaccharides, together with lignocellulosic plant material, which is rich in the lower amounts of fructose, rhamnose, and xylan, by chemical and enzymatic methods. The arabinose. Seeds of legumes, lentils, peas, beans, latter is preferred in the food industry as it lacks chickpeas, and mustard are rich in raffi nose undesirable side reactions and by-products. The (Johansen et al. 1996 ; Sánchez-Mata et al. 1998 ). fermentation of XOS results in decrease in pH Humans do not possess α-galactosidases, and the formation of SCFA, which serves as fuel essential for hydrolyzing the α(1 → 6) linkages and regulate cellular processes. in the oligosaccharides. Hence, these prebiotics XOS are mixtures of oligosaccharides formed are not digested when consumed. Intact oligo- from xylose residues that are linked through saccharides reach colon to undergo preferential β(1 → 4) linkages (Aachary and Prapulla 2008 ). fermentation by benefi cial bifi dogenic microor- Based on the xylose residues (2–10) involved in ganisms, which contain the enzyme (Liu and Xu the XOS, it could be classifi ed into xylobiose, 2008 ). Fermentation results in production of xylotriose, and so on. Xylobiose with degree of gases (CO2 , H2 , CH4 , etc.) and SCFA that are polymerization of 2 is considered as XOS for remarkable for their prebiotic activity and asso- food applications (Vazquez et al. 2000 ). XOS ciated health benefi ts (Gibson and Roberfroid also have α- D-glucopyranosyl uronic acid or its 1995 ). High dietary levels of soy-oligosaccha- 4-O -methyl derivative. Acetyl groups or arabino- rides result in harmful effects on the intestine. furanosyl residues as side groups are also pres- The presence of soy-oligosaccharides in piglets’ ent, and these variations lead to diverse biological diet reduces the nutrient digestibility and growth properties. Their biological properties include performance and increases the incidence of diar- antioxidant activity (contributed by linked phe- rhea, since the endogenous enzymes for their nolic components), antiallergy, blood and skin- hydrolysis are not present in their gut. The prebi- related effects, antiinfection, anti-infl ammatory otic activity of soy- oligosaccharide is dose property, immunomodulatory activity, and anti- dependent. Normally, in pigs, 40–50 % of oligo- hyperlipidemic effects (Izumi et al. 2004 ). saccharides and fructans are digested in the small intestine; and are completely digested in the large intestine, resulting in an increase in the 4.2.9 Isomalto- number of lactobacilli and Bifi dobacterium and a oligosaccharides (IMO) decrease in clostridia and enterobacteria (Nemcova et al. 1999 ). Isomalto- oligosaccharide is a nondigestible low- Fermentation of SOS by lactobacilli has been calorie health sweetener, which maintains the reported in the SHIME with the inoculums of growth of colonic benefi cial bacteria in the colon, fecal bacteria inoculums. In vitro studies indicate thus acting as a prebiotic. It is composed of glu- that SOS has similar effects as other galacto- cose monomers linked by α(1 → 6) glycosidic oligosaccharides on gut bacteria. Growth of pure linkages. IMO has a mixture of isomaltose (Glu benefi cial microbes has been observed with puri- α1 → 6 Glu), isomaltotriose (Glu α1 → 6 Glu fi ed individual compounds or mixture of α1 → 6 Glu), and panose (Glu α1 → 6 Glu α1 → 4 oligosaccharides. Glu). 4.2 Types of Prebiotics 67

IMO can be easily assimilated by colonic bac- et al. 2006 ). It is heat stable, which enables its teria and selectively increase the amount of use in conventional foods. Bifi dobacterium approximately by 2–4 times. • Type 2: native high amylase – type2-RS2 is Panose, isomaltose, isomaltotriose, and isomalto- composed of native starch granules containing 900 selectively aid in the proliferation of the bifi - uncooked starch or poorly gelatinized starch. dobacteria. Human trials indicate that colonic • Type 3: retrograded type – type3-RS3 includes microfl ora fl ourish in the presence of IMO. The the substances precipitated from pap or starch minimum effective dose of IMO is 8–10 g/day gel (1.5 % amylose, 10 % amylopectin) during and Isomalto-900 is 13–15 g/day (Kohmoto et al. retrogradation. During the storage of the gel, 1991 ). The hydrolysis of IMO in human jejunum starch helix undergoes aggregation forming by the enzyme isomaltase makes them available thermally stable crystal structures, which is as substrate for colonic microbiota. resistant to amylolytic enzyme (Lafi andra IMO naturally occurs in honey, sugarcane et al. 2014). Its cooking stability enables its juice, and products derived thereof, such as trea- usage in conventional foods (Champ et al. cle or food-grade molasses (Lina et al. 2002 ). It is 2003). Storey et al. ( 2007 ) classifi ed “ retro- generally used as a low-calorie sweetener mixed graded resistant maltodextrins ” as type3-RS. with a variety of other foods and beverages for • Type 4: chemically modifi ed – type4-RS4 the purpose of sweetening. It can be used to includes chemically or physically modifi ed replace maltose syrup. Commercial Isomalto-900 starch or by a combination of these two pro- is produced by incubating α-amylase, pullula- cesses. New functional groups are added into nase, and α-glycosidase with cornstarch the starch during chemical modifi cations that (Kohmoto et al. 1988 ). prevent digestion. In physical method, during high-temperature processing or in the presence of catalyst, dextrinization occurs, releas- 4.2.10 Resistant Starch (RS) ing free glucose that binds randomly to the and Dextrin (RD) chain, resulting in resistant starch (Brown 1996 ; Onyango et al. 2006 ). Starch that escapes human digestive system and • Type 5: fatty acid complex type – type5-RS5 is transported to the lower gut for bacterial fer- is formed from high AM-starches mentation is designated as resistant starch. RS (amylomalto-starches), which involve high stimulates increased fecal bulk and low colonic gelatinization temperature and are susceptible pH, controls blood sugar level, improves bowel to retrogradation. Frohberg and Quanz ( 2008 ) health, and reduces cardiovascular risk (Slavin defi ned RS5 as water-insoluble linear poly et al. 2009). It is found in grains, cereals, tubers α-1,4-glucan that is not susceptible to degra- (especially potatoes), legumes, seeds, and some dation by alpha-amylases. They also estab- nuts (Goldring 2004 ; Fuentes-Zaragoza et al. lished that these aid in SCFA formation, 2011). RS is a linear molecule of a-1,4-D -glucan particularly butyrate, in the colon. and has a relatively low MW (120 kD) (Fuentes- Zaragoza et al. 2011 ). RS is safe for consumption and is a natural Five different types of resistant starch have component of some foods. Similar to soluble been reported, and they are as follows: fi ber, RS if consumed regularly (5–6 g/day) is proved to be benefi cial in lowering blood sugar • Type 1: physically amylase inaccessible – levels (Behall et al. 2006 ). RS has a low calorifi c type1-RS1 includes the starch in plant cells value (8 kJ/g) compared to fully digestible starch with undestroyed cell walls. It is nondigestible (15 kJ/g) (Rochfort and Panozzo 2007). It could by human gut enzymes and, together with be fermented by human gut microbes and alter plant tissue fragments, passes to the large the composition of the microbial species and bowel to undergo fermentation (Onyango their metabolic activities, and formation SCFA, 68 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer which prevent the growth of cancerous tumors totally soluble in cold water without inducing (Fuentes-Zaragoza et al. 2011). RS consumption viscosity (Lefranc-Millot 2008 ). It is produced has been reported to reduce postprandial glyce- from wheat (Nutriose FB®06) or maize starch. It mic and insulinemic responses, thereby assisting has wide applications in food and pharmaceutical in the management of diabetes and associated industries and is incorporated in fi ber-enriched cholesterol and triglycerides levels. Other bene- drinks, enteral nutrition, granulation binders, fi ts include lowering of colonic pH, increased low-calorie food, and sugar-free confectionery excretion frequency, fecal bulking, prevention of (Serpelloni 2011 ). About 15 % of this is absorbed constipation and hemorrhoids, and decreased in the small intestine and 75 % is fermented in the production of carcinogens and ammonia levels. large intestine, while the leftovers are excreted in Others also reported that RS aids in the absorp- the feces (Pasman et al. 2006). It decreases the tion of calcium, magnesium, zinc, iron, and cop- number of Clostridium perfringens in human per in rats (Lopez et al. 2001 ). However, in feces, reduces fecal pH, and increases SCFA pro- humans, RS aids in the absorption of only cal- duction (Pasman et al. 2006 ). Also, it exhibits a cium (Fuentes-Zaragoza et al. 2011 ). progressive and signifi cant impact on short-term FOS and RS may act synergistically satiety, which is time and dosage correlated (Rodríguez-Cabezas et al. 2010 ; Fuentes- (Guerin-Deremaux et al. 2011 ). It aids in a bene- Zaragoza et al. 2011 ), increasing lactobacilli and fi cial shift in the bacterial microbiome to butyro- bifi dobacteria in caecum and colon. FOS are genic genera such as Peptostreptococcus , more active in the fi rst part of the large bowel, Fusobacterium, and Bifi dobacterium (Guerin- while RS reaches the distal part of the colon. Deremaux et al. 2011 ). Administration of FOS or raw potato starches Fibersol-2 is a well-known soluble, nondigest- stimulates different bacterial populations and ible, starch-derived resistant maltodextrin and metabolites in the caecum, proximal, and distal produced from cornstarch by pyrolysis and sub- ends of the colon, and feces (Fuentes-Zaragoza sequent enzymatic treatment forming random 1, et al. 2011 ). Compared to RS, FOS double the 2- and 1, 3- α or β linkages (Ohkuma and pool of fecal fermentation products, such as lac- Wakabayashi 2001 ). It is readily dispersible in tate, while the situation is just the opposite in the water with no fl avor and odor and is highly com- distal region. These observations confi rm that patible with dry drink mix applications. It shows each prebiotic demonstrates unique properties, stability to acid and heat and could be used in which should be considered before their applica- retorted products (juices, sauces, puddings, fl uid tion for colon diseases. Furthermore, comple- milks). It also shows superior freeze-thaw stabil- mentary kinetics of different prebiotics and their ity. Studies indicate that Fibersol-2 could effec- benefi ts should be considered in promoting health tively reduce the blood glucose and insulin, blood (Younes et al. 2001 ; Fuentes-Zaragoza et al. triglycerides, and serum cholesterol. It increases 2011 ). Younes et al. (2001 ) examined the syner- the fecal bulk and reduces transit time, thus main- gistic effect of inulin and RS in male Wistar rats taining colon health and reducing the incidences and observed that these prebiotics signifi cantly of colon cancer (Ohkuma and Wakabayashi increase the mineral absorption in the intestine, 2001 ). without altering their plasma levels. Arabinogalacto-oligosaccharides , arabinoxylo- Resistant dextrin is defi ned as short-chain glu- oligosaccharides , arabino- oligosaccharides , galac- cose polymers, with no sweet taste and which turonan- oligosaccharide s, rhamnogalacturonan- show resistance to hydrolysis by human digestive oligosaccharides, and pectic- oligosaccharides are enzymes (Ohkuma and Wakabayashi 2001 ). some of the newly introduced prebiotics that Most probably, dextrinization undergoes the exhibit fermentative action in pure intestinal bac- mixed mechanism. teria, including Bifi dobacterium , Lactobacillus , Nutriose , a commercial resistant dextrin, is a Bacteroides sp., Clostridium sp., Escherichia coli , non-viscous soluble fi ber made from starch using and Klebsiella sp. (Oosterveld et al. 2002 ). Other a highly controlled dextrinization process and is oligosaccharides, which exhibit prebiotic activity, 4.3 Prebiotics and Gut Microbiota 69 include chitosan-oligosaccharide, chitin-oligosac- also display the positive capability of prebiotic- charide, gluco-oligosaccharides, and oligosaccha- enriched diet in infl uencing the gut microbial rides from melibiose, mannan-oligosaccharides, composition and the health. oligodextrans, and gentio-oligosaccharides (White Studies have indicated that metabolic equilib- et al. 2002 ). Some probiotic bacteria have been rium is maintained by gut fl ora and an altered gut reported to produce by themselves polysaccha- microfl ora could be associated with several dis- rides (but not oligosaccharide) with prebiotic eased conditions, including cancer, cardiovascular effects for their growth (Dal Bello et al. 2001 ). diseases, obesity, type-2 diabetes, antibiotic-associated diarrhea, hypercholesterolemia, and others that are very commonly prevalent in the 21st 4.3 Prebiotics and Gut century. Microbiota Prebiotics are unique in their functionalities, which selectively stimulate the growth of benefi - The gastrointestinal tract of the human embryo is cial gut bacteria and contribute to the health of virtually sterile and is colonized by microbes at the host. It is reported that changes in the compo- birth (Fuller 1991, 1992 ). The microbiome that sition of gut microbiota occur not only at phyla develops depends on the method of delivery of the but also at species levels; for instance, a lower infant (Salminen et al. 1998 ) and hygiene precau- bifi dobacteria count could be associated with tions associated with parturition (child birth) overweight and obesity. Overweight mothers (Zetterstrom et al. 1994 ). In addition to character- give birth to neonates with a decreased number of istic vaginal fl ora, lactobacilli, yeast, streptococci, bifi dobacteria suggesting that obesogenic micro- staphylococci and Escherichia coli , the neonate biota is an “inheritable” trait. may come in contact with fecal microbes and skin Bifi dobacteria serve as a perfect model for the bacteria during birth (Fuller 1991; Goldin and concept of role of prebiotics in gut management. Gorbach 1992 ). Further, inoculation from the Bifi dobacteria population (and other microorgan- general environment and other external contacts isms in the Group Firmicutes) is slightly lower in may also play a key role in determining gut micro- individuals with obesity (Ito et al. 1990 ). Similar biome (Gronlund et al. 1999 ). During acquisition observation is reported in the case of type-2 dia- period, some bacteria briefl y colonize the gut betes mellitus patients in comparison to nondia- while others become indigenous. Thus, the neona- betic patients (Mazmanian et al. 2008 ). These tal gut faces a rapid microbial succession in the conclusions recommend that bifi dobacteria play fi rst few days to months of development by lumi- an important element in the development of obe- nal redox potential (Eh) but more often by the sity and related comorbidities. Feeding prebiotics feeding regime that follows its birth (Zetterstrom like dietary fructans or inulin-type fructans to et al. 1994). Initial colonizers utilize the available mice indicates that the oligosaccharide is used as oxygen, within 48 h, and allow succession of obli- energy substrate by bacteria, which assist in its gate anaerobes belonging to bifi dobacteria, bacte- growth and reduce the levels of lipopolysaccha- roides, and clostridia groups. Hence, feeding ride, glucose tolerance, and fat mass (Ghanim method signifi cantly infl uence the gut environ- et al. 2009; Larsen et al. 2010; Basu et al. 2011 ; ment. Historically, breast-fed infants are thought Lathrop et al. 2011 ). Further, prebiotic approach to have relatively higher proportions of bifi dobac- also prevents the overexpression of several host teria (Fuller 1991, 1992 ). Breast feeding and bifi - genes related to adiposity and infl ammation. dobacteria in the gut could be linked to the lower These dietary fructans on feeding in rodents risks of gastrointestinal, respiratory and urinary increase the amount of endocrine L cells in jeju- tract infections (Kunz and Rudloff 1993 ). num and colon and promote the production and However, as the nature of commercial diet has release of the active forms of GLP1 and GLP2 in altered and enriched in recent years, the naturally the portal vein. GLP1 is reported to participate fl ourished bifi dobacterial predominance in breast- in the prebiotic-motivated appetite loss, loss of fed infants is less signifi cant. Moreover, these fat mass, and hepatic insulin resistance (Cani 70 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer et al. 2006), while GLP2 adds to the reduced which could be due to the lower proliferation in permeability of the intestinal wall and endotox- the colon at the promotion stage. Similar benefi - emia associated with obesity (Cani et al. 2009 ; cial effects of these prebiotics are observed Delzenne et al. 2011 ). against 1,2-dimethylhydrazine-induced colon cancer (Hughes and Rowland 2001 ; Pool-Zobel 2005 ). An addition of 5–15 % inulin or oligofruc- 4.4 Prebiotics and Health tose in feed is reported to lower the incidence of breast tumor in rats and mice and lung metasta- Prebiotics have been utilized in the food industry sis. High-fat diets combined with inulin and for their functional and health promoting proper- Lactobacillus acidophilus treatment signifi cantly ties. These are used in food and beverage indus- reduced formation of aberrant crypt foci (ACF) tries for their individual unique and inherent in rats (Bolognani et al. 2001 ). properties such as increase in emulsifi cation, gel Consumption of fermented dairy products has formation, low sweetness, low glycemic index, been reported to lower the prevalence of colon and modulation of viscosity. Common prebiotics cancer. Furthermore, preliminary in vivo studies that are used in improving gut health and act as have suggested that prebiotics could act as anti- supplements to food are dietary fructans, inulin, mutagens (Taper et al. 1997 ). RS suppresses the galacto-oligosaccharides, fructo-oligosaccharide, formation of colonic aberrant crypt in a dose- lactulose, lactitol, and resistant starch (Lin et al. dependent manner and may retard the growth and 2005 ; Gibson et al. 2010 ). development of neoplastic lesions in the colon. Fructo-oligosaccharides (FOS) and galacto- This suggests the usefulness of RS as a preven- oligosaccharides (GOS) are “Generally Regarded tive agent for individuals with high risk of devel- as Safe” (GRAS). Others include xylo- oping colon cancer (Liu and Xu 2008 ). oligosaccharides (XOS), isomalto- Prebiotics may directly or indirectly affect oligosaccharides (IMOS), gluco-oligosaccharides, carcinogenesis, colonization of bacteria and their pectin oligosaccharides (POS), mannan- proliferation, and infl ammation. Fermentation of oligosacharides (MOS), gentio-oligosaccharides prebiotics leads to SCFA formation, which (GTO), chito-oligosaccharides (CHOS), soybean affects colonic mucosa. Treatment of human oligosaccharides (SOS), and polydextrose, which colon carcinoma cell lines, L97 and HT29, with are not commercially available in high purity the supernatant fractions of inulin fermentation (Gibson et al. 2004 ; Rizzello et al. 2011 ). inhibited their growth and induced apoptosis The possible health benefi ts of prebiotics (Munjal et al. 2009 ). Butyrate produced during could be broadly summarized as follows: fermentation inhibits the growth of budding pre- malignant and malignant cells. Preclinical stud- • Prevention of cancer and tumor growth ies have also supported the view that butyrate • Alleviation of lactose intolerance might be a chemopreventive agent and may aid in • Lowering of serum cholesterol preventing carcinogenesis (Scheppach and • Enhancement of the immune system Weiler 2004 ) or act as guardian against colon • Prevention of vaginal infections cancer by promoting cell differentiation (Kim • Alleviation of allergic conditions et al. 1982 ) .

4.4.1 Prebiotics and Cancer 4.4.2 Prebiotics and Lactose Intolerance Femia et al. (2002 ) reported that the protective effect of probiotics on azoxymethane-induced Lactose intolerance is one of the worst problems carcinogenesis in vivo is lower in comparison to affecting the world population. Lactose acts as the effects of prebiotics (oligofructose-inulin), an osmotic and non-digestible carbohydrate. 4.4 Prebiotics and Health 71

These subjects have low amounts of lactase Yogurt containing live cultures of L acidophi- (β-galactosidase) in the intestinal mucosal cells, lus (Anderson and Gilliland 1999 ) and yogurt which help to digest lactose to glucose and galac- fermented with L acidophilus with added fructo- tose. It worsens with age. Intake of milk could be oligosaccharides (Schaafsma et al. 1998 ) are able avoided to avert this issue, but it will limit the to reduce the serum cholesterol; a reduction of availability of dietary calcium (Hamilton-Miller 4.4 % is observed with the latter. Similarly, inulin 2004 ). is also reported to lower cholesterol (Davidson Fermented dairy products (yogurt and milk et al. 1998 ). containing L. acidophilus ) have been observed In a randomized, double-blind, and crossover to be easily digested by them. This could be due study using hypercholesterolemic subjects to to the β-galactosidase activity of the bacteria that assess the prebiotic effect of inulin on blood cho- hydrolyzes lactose (Hamilton-Miller 2004 ). L lesterol level, it is observed that a daily intake of delbrueckii var bulgaricus and S salivarius var 20 g inulin signifi cantly reduced the serum tri- thermophilus are also used for fermenting milk. glycerides (Causey et al. 2000 ). Similar conclu- These organisms pass through the stomach and sions are reached in another study with 10 g of are hydrolyzed by the pancreatic secretions inulin (Letexier et al. 2003 ). In addition, the levels (Hamilton-Miller 2004 ). The role of probiotics of plasma triacylglycerol are also low (Letexier in improving lactose digestion and intolerance et al. 2003). Another randomized, double-blind, has been reported by many scientists (Mustapha placebo-controlled, and parallel design model et al. 1997 ). also indicated that prebiotics affect the lipid pro- The effects of prebiotics on lactose intolerance fi les (Brighenti et al. 1999 ). A daily consumption are uncertain. However, National Institute of of 50 g of rice-based ready-to-eat cereal (inu- Health (NIH) (2010 ) recommends that milk and lin-18 %) reduced plasma total cholesterol and milk products should not be avoided (even by lac- triacylglycerols by 7.9 and 21.2 %, respectively. tose-intolerant individuals) as they have potential Others also observed that the purifi ed diet con- benefi cial impact on the gut microbiota. Few strat- taining 10 % long-chained fructan signifi cantly egies suggested to overcome lactose intolerance reduced the blood cholesterol (29.7 %), LDL are to recondition the gut with smaller servings of (25.9 %), IDL (39.4 %), and VLDL (37.3 %) milk and increase it over a few weeks, consume (Mortensen et al. 2002 ). Other indigestible and hard cheese as it has low lactose content and con- fermentable compounds such as germinated bar- sume lactose-free milk and lactose tablets. ley, oligodextrans, lactose, hemicellulose-rich substrates, resistant starch and its derivatives, etc., have also been reported to exert hypocholesterol- 4.4.3 Prebiotics and Cholesterol emic effects (Gibson et al. 2004 ). Cholesterol- lowering effect of resistant starch is also reported The World Health Organisation (WHO 2009 ) by others (Fernandez et al. 2000 ). They also predicted that by 2030, cardiovascular diseases observed that prebiotic signifi cantly reduced will be the leading cause of mortality, affecting plasma cholesterol (27.4 %) and LDL (28.0 %). In about 23.6 million people around the world. another randomized, placebo-controlled and par- Hypercholesterolemia contributes to about 45 % allel-designed study, it is seen that feeding male of heart attacks in Western Europe and 35 % in Wistar rats with the starch from Chinese yam Central and Eastern Europe from 1999 to 2003. (Dioscorea opposita cv. Anguo) signifi cantly low- Thus, risk of heart attack is three times high in ered plasma total cholesterol, LDL-cholesterol, patients with hypercholesterolemia. and triglyceride (Wang et al. 2008 ). In another Prebiotics aid in the growth of benefi cial gut study, it is reported that daily feeding of 25 g/kg bacteria and enhance SCFA production in the of β-cyclodextrin to male Wistar rats signifi cantly gut, which has direct effects on cardiovascular reduced the plasma cholesterol and triacylglycer- diseases and lipid profi le (Wolever et al. 2002 ). ols (Favier et al. 1995 ). 72 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer

4.4.4 Prebiotics and Osteoporosis (Scholz-Ahrens and Schrezenmeir 2002 ; Scholz- Ahrens et al. 2007 ) and may contribute to the Several studies have noted that prebiotics (inu- bone mineral density. lin, oligofructose, galacto-oligosaccharides, The possible mechanisms by which prebiotics resistant starches, or lactulose) effectively stimu- enhance calcium absorption in intestine could be late calcium absorption in vivo (Scholz-Ahrens as follows: and Schrezenmeir 2002 ; Scholz-Ahrens et al. 2007 ). These studies are carried out in diseased • Prebiotic hydrolysis by the gut enzymes leads models or altered physiological status such as to SCFA production. This reduces the luminal gastrectomized, ovariectomized, cecectomized, pH and increases the solubility of calcium in and magnesium-, calcium-, or iron-deﬁ cient rats. the large intestine. It is observed that prebiotic effects of calcium • SCFA also aid in transcellular calcium absorp- absorption occur at large intestine level (Baba tion by increasing the exchange of cellular H+ et al. 1996). Brommage et al. ( 1993) reported for luminal Ca2+ . that calcium absorption in cecectomized rats • Butyrate is involved in cell growth and hence could be enhanced by stimulating them with increases absorptive surface area and increases lactulose. Griessen et al. ( 1989) reported that in mucosal calbindin D9K levels in the large healthy adult volunteers, the calcium absorption intestine. from milk (21.4 %) and lactose-free milk (26.8 %) • Increase in the metabolites, namely, poly- is similar. Lactose increased the calcium absorp- amines that lead to cell growth, increase in the tion in β-galactosidase-deﬁ cient subjects too. absorptive surface area, and so stimulate the

Increase in calcium absorption is observed with gene expression of calbindin D9K . increased concentration of lactulose in postmenopausal women (van den Heuvel et al. 2000 ). The possible mechanism of mineral absorp- Inulin, fructo-, and galacto-oligosaccharides tion in reducing the risk of osteoporosis could be are also seen to increase calcium absorption in speculated as the augmentation of bone mineral adolescents (van den Heuvel et al. 2000 ; density and assisting in bone regeneration and Cashman 2003 ). strength. Prebiotic-mineral absorption induces Further, the effect of prebiotics on calcium increased solubility of minerals due to acidic pH absorption showed a dose-dependent effect and (SCFA), incidence of SCFA-salt conjugates, aug- it depended on the amount of calcium in the mented absorption surface, increased expression diet and prebiotics in the feed. Galacto- of calcium-binding proteins, degradation of oligosaccharides stimulated the calcium absorp- phytic acid-mineral complex that liberates asso- tion in intact rats when they are fed with a diet ciated minerals, release of bone-modulating fac- containing 5 g of calcium/kg than with 0.5 g of tors, such as phytoestrogens, from food and calcium/kg (Chonan and Watanuki 1996 ). Similar stabilization of intestinal microfl ora and intesti- prebiotic effect of oligofructose on metabolic cal- nal mucus (Scholz-Ahrens and Schrezenmeir cium balance in ovariectomized rats is reported 2002 ; Coudray et al. 2003 ; Scholz-Ahrens et al. by others (Scholz-Ahrens et al. 2007 ). The effect 2007 ). is signifi cant when they are fed with 10 g of calcium/kg of diet than recommended level (5 g of calcium/kg of diet). Linear increases in the intes- 4.4.5 Cancer tinal calcium absorption in rats are observed when they are fed with different concentrations Extensive researches have been carried out and of lactulose (Brommage et al. 1993 ). are still ongoing to understand the effects of dif- Oligofructose, transgalacto-oligosaccharides, ferent prebiotics on colon cancer. Non-digestible lactulose, and resistant starch also stimulate the oligosaccharides selectively utilized by probiot- intestinal magnesium absorption in rat models ics in the colon help in reducing the risk of colon 4.5 Mechanism of Action of Prebiotics against Colorectal Cancer 73 cancer. Hsu et al. (2004 ) reported that both XOS modulate the immune parameters in gut- and FOS markedly reduced the number of aber- associated lymphoid tissue (GALT), secondary rant crypt foci in the colon of DMH-treated male lymphoid tissue, and peripheral circulation Sprague-Dawley rats. Studies indicate that GOS, (Brisbin et al. 2008 ). Infl uence of prebiotics on FOS, and lactulose boost the intestinal lactate the GALT and modulation of human immune concentration and SCFA, stool frequency, and system and colon cancer are research areas that fecal weight. They also decrease the secondary need in-depth study. Although prebiotics are bile acids, colon pH, and nitroreductase and shown to have immunomodulatory effect, more β-glucuronidase activities, signifying the appar- studies are necessary to establish the mechanistic ent role of prebiotics in CRC (Bruno-Barcena role of prebiotic-induced immunomodulation and Azcarate-Peril 2015 ) The results suggest that against cancer. dietary supplementation of prebiotics may have benefi cial impact on gastrointestinal health and prevent colon cancer. The mechanism of action 4.5 Mechanism of Action of prebiotics in preventing colon cancer is of Prebiotics detailed in the following sections. against Colorectal Cancer

The mechanism of action of anticarcinogenic 4.4.6 Anti-infl ammatory effects of prebiotics could be classifi ed as summarized below (Table 4.5 ). A schematic diagram Prebiotic consumption imparts antitumorigenic showing the potential effects of prebiotics in pre- effects through improvement of the immune venting cancer is given in Fig. 4.3 . response. Immunostimulating effects and anti- infl ammatory activities are reported for the prebiotics arabino-(glucurono) xylans and 4.5.1 Stimulation of Benefi cial Gut 4-O -methylglucuronoxylan and highly branched Microbes heteroxylan. Partially O -acetylated XOS and deacetylated XOS show mitogenic activity and Prebiotics alter gut environment and encourage development of the mitogen-induced prolifera- lactobacilli and bifi dobacteria to bind and elimi- tion of rat thymocytes, representing its nate toxins from the system. Intake of long-chain immunostimulatory potential (Nabarlatz et al. inulin-type fructans in rodents increases the bifi - 2005 ). XOS exhibit dose-dependent direct mito- dogenic effects, lowers pH and modulates immu- genic and comitogenic activities, which is similar nity, and reduces the azoxymethane with immunogenic water-soluble arabinogluc- (AOM)-induced colonic pre-neoplastic aberrant uronoxylan (Ebringerova et al. 1998 ). Feeding crypt foci (ACF) and colon tumors (Verghese modifi ed arabinoxylan to C57BL/6 and C3H et al. 2002 ). Inhibitory effects of short FOS and mice improves the activity of NK cells and their inulin are also reported in ACF-induced rat mod- binding to tumor cells indicating the induction of els (Rowland et al. 1998 ). Both XOS and FOS are host immunity (Ghoneum and Abedi 2004 ). seen to inhibit colonic ACF in dimethylhydrazine Prebiotics may show indirect immunogenic (DMH)-treated rats by lowering cecal pH and effects by manipulating the intestinal microfl ora serum triglyceride. Gain in total cecal weight, and altering the immune parameters such as NK increase in bifi dobacterial population, and reduc- cell activity, secretion of IL-10 and interferon, tion in ACF in the colon are evident (Hsu et al. and lymphocyte proliferation (Janardhana et al. 2004). GOS are also observed to considerably 2009 ). GOS is reported to decrease the incidence hamper the development of DMH- or AOM- of colitis in Smad3-defi cient mice by altering the induced colorectal tumors (Bouhnik et al. 1997 ). function and traffi cking of NK- cells Inulin, FOS, and GOS undergo fermentation in (Gopalakrishnan et al. 2012 ). Prebiotics also the large intestine, stimulate microbial growth, 74 4 Bioactive Carbohydrate: Prebiotics and Colorectal Cancer

Table 4.5 Prebiotics and their mechanism of action in various disease conditions Prebiotic Medical benefi ts Mechanism of action References Inulin Crohn’s disease Enhancement of immune Hijová et al. (2013 ) response Colitis Effect on innate immunity Macfarlane et al. (2008 ) Ramirez-Farias et al. ( 2009 ) Obesity Modifi cation of Hopping et al. ( 2009 ) microbiome and increase in bifi dobacteria Diabetes type 2 Costabile et al. (2010 ) Colon cancer Ramnani et al. (2010 ) Constipation Fructo-oligosaccharide Crohn’s disease Increase in Scholtens et al. (2006 ) bifi dobacteria Colitis Decrease in colon pH Benjamin et al. (2011 ) Obesity Reduction in lipid Aller et al. (2011 ) accumulation Constipation Secretion of anti- Cummings et al. (2001 ) infl ammatory substances Traveler’s diarrhea Local induction of reactive Arslanoglu et al. (2008 ) oxygen species Colon cancer Boutron-Ruault et al., (2005 ) Galacto-oligosaccharide Crohn’s disease Improvement of growth Saavedra and Tschernia performance and immune (2002 ) responses Macfarlane et al. (2008 ) Colitis Diminishment of intestinal Drakoularakou et al. bacterial overgrowth (2010 ) Obesity

and increase bacterial mass, fecal bulking, and tone deacetylase inhibitor, altering DNA methyl- peristalsis (Den Hond et al. 2000 ; Niittynen et al. ation resulting in improved access of transcription 2007). Bulking reduces the transit time and con- factors to nucleosomal DNA (Hammers et al. tact time to luminal lining, thus preventing 2008). Lactate has been reported to improve gut cancer. health and elevate gut-associated immune defense mechanism (Scott et al. 2013 ). Butyrate reduces colonic cell proliferation and 4.5.2 SCFA and Lactic Acid induces differentiation in colonic epithelial cells. Sodium butyrate was reported as a powerful Bacterial fermentation of prebiotics in the colon inhibitor of growth and inducer of differentiation aids in the formation of SCFA, which assists in and apoptosis, thereby possessing a beneﬁ cial maintaining gut health and intestinal morphology effect against colon cancer development. and function (Scheppach 1994 ; Roy et al. 2006 ). Fermentation by gut bacteria on a high amylose Commonly formed SCFA are acetic, propionic, starch diet produced butyrate, which increased and butyric acids. Butyrate serves as fuel for the detoxiﬁ cation of electrophilic products asso- colonocytes, lowers colon pH, and serves as his- ciated with oxidative stress (Lishaut et al. 1999 ). 4.5 Mechanism of Action of Prebiotics against Colorectal Cancer 75

Lipogenesis and controlled serum and lipids Colonocytes

Improved gut health Mineral absorption (Ca & Mg) PREBIOTICS

Fecal bulking Peristalsis induced Acidification of colon

Prebiotics Pathogens reduced Fermentation and selective growth SCFA Low bacterial metabolites Colonization resistance Lactobacillus/ Bifidobacterium Reduction of pathogens Mast cells and block adhesion Prevent colon cancer

Immunomodulatory effects Dendrite cells Anti-inflammatory Tr Suppress intestinal bowel syndrome IL-10/TGF-b Th1 X Th2 IFNg Prevention of allergy

Fig. 4.3 Potential health beneﬁ ts and mechanism of prebiotic action (Aachary 2009 )

The ability of prebiotic resistant starch type-3 4.5.4 Prebiotics Modulate Novelose 330 to reduce the incidence of colon Xenobiotic Metabolizing carcinogenesis via induction apoptosis in rats Enzymes was established, and the effect was attributed to the increased production of butyrate (Bauer- Xenobiotic metabolizing enzymes consist of Marinovic et al. 2006 ). The role of individual phase I and phase II enzymes, and they are con- SCFA in preventing colon carcinogenesis has sidered as indices of colon cancer. been discussed in detail elsewhere in the book. Phase I enzymes consist of cytochrome b5, cytochrome b5 reductase, cytochrome P450, cytochrome P450 reductase, cytochrome P450 2E1. 4.5.3 Absorption of Micronutrients Phase II enzymes consists of glutathione by Colon Cells S-transferase (GST), uridine diphospho-glucuro- nyltransferase, and DT-diaphorase. Increased mineral absorption (calcium, magne- Both phase I and phase II enzymes assist in sium, selenium), vitamin D, and antioxidants due the inactivation of procarcinogens and their elim- to the intake of short- and long-chain fructans aid ination from the system (Johnson et al. 2012 ). RS in preventing cancer risk and maintaining the is reported to induce glutathione transferase π in normal bowel structure (Scholz-Ahrens and rat colon (Wollowski et al. 2001 ). SCFA also Schrezenmei 2002 ). Inulin, oligofructose, FOS, induce glutathione transferase π as observed in GOS, SOS, RS, lactitol, and others have benefi - Caco-2 cell lines (Johnson et al. 2012 ). 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human colonic bacteria. Microbial Ecol Health Dis White LA, Newman MC, Comwell GL, Lindemann MD 14:165–173 (2002) Brewers dried yeast as a source of mannan oli- van den Heuvel EG, Schoterman MH, Muijs T (2000) Trans- gosaccharides for weaning pigs. J Anim Sci galactooligosaccharides stimulate calcium absorption 80:2619–2628 in postmenopausal women. J Nutr 130:2938–2942 WHO (2009) Cardiovascular disease. Fact sheet 317, Van Loo J, Cummings J, Delzenne N, Englyst H, Franck WHO, Geneva. http://www.who.int/mediacentre/fact- A, Hopkins M, Kok N, Macfarlane G, Newton D, sheets/fs317/en/print.html Quigley M, Roberfroid M, Van Vliet T, Van den Wolever TMS, Schrade KB, Vogt JA, Tsihlias EB, Heuvel E (1999) Functional food properties of non- McBurney MI (2002) Do colonic short chain fatty digestible oligosaccharides: a consensus report from acids contribute to the long-term adaptation of blood the ENDO project (DGXII AIRII-CT94-1095). Br lipids in subjects with type 2 diabetes consuming a J Nutr 81:121–132 high-ﬁ ber diet? Am J Clin Nutr 75:1023–1030 Vazquez MJ, Alonso JL, Domınguez H, Parajó JC (2000) Wollowski I, Rechkemmer G, Pool-Zobel BL (2001) Xylooligosaccharides: manufacture and applications. Protective role of probiotics and prebiotics in colon Trends Food Sci Technol 11(11):387–393 cancer. Am J Clin Nutr 73(2):451–455 Verghese M, Rao DR, Chawan CB, Williams LL, Younes H, Coudray C, Bellanger J, Demigné C, Shackelford L (2002) Dietary inulin suppresses Rayssiquier Y, Rémésy C (2001) Effects of two fer- azoxymethane-induced aberrant crypt foci and colon mentable carbohydrates (inulin and resistant starch) tumours at the promotion stage in young Fisher 344 and their combination on calcium and magnesium bal- rats. J Nutr 132(9):2809–2813 ance in rats. Br J Nutr 86:479–485 Wang SJ, Yu JL, Liu HY, Chen WP (2008) Characterisation Zetterstrom R, Bennett R, Nord K-E (1994) Early infant and preliminary lipid-lowering evaluation of starch feeding and micro-ecology of the gut. Acta Paediatr from Chinese Yam. Food Chem 108:176–181 Jpn 36:562–571 Synbiotics and Colorectal Cancer 5

Abstract Synbiotics are defi ned as the combination of appropriate probiotics and prebiotics, where the latter form the substrate for the growth and development of selective indigenous or introduce benefi cial bacteria in the colon. The prebiotics commonly in use are inulin, fructo-oligosaccharide, galacto-oligosaccharide, lactose, etc., and the probiotics investigated include Bifi dobacterium and Lactobacillus spp. Prebiotics form the food for the growth of probiotics. The mechanism of action of synbiotics is a collective effort of pro- and prebiotics. Administration of synbiotics could prevent the initiation or early stage of cancer and also treat the existing tumors. Synbiotic interventions bring about signifi cant alterations in the composition of the colonic microbiome leading to the altered metabolic activity of the organ. It reduces the exposure of cytotoxic agents, including mutagens and carcinogens, to the intestinal lining; decreases cell proliferation in the colonic tissue; and develops mucosal structure. It is reported that administration of synbiotics results in noteworthy changes in the fecal fl ora, with an increase in Bifi dobacterium and Lactobacillus and a reduction in the harmful pathogens such as Clostridium perfringens . These also inhibit the fecal water to cause necrosis in the colonic cells and improve epithelial barrier functions. Synbiotics are also reported to show immunomodulatory effects.

Keywords Synbiotics • Prebiotics • Probiotics • Gastrointestinal tract • Short-chain fatty acids • Microbial • Dietary supplement • Anticancer • Pathogen • Colorectal cancer

5.1 History, Defi nition, the selective function of the prebiotic compound and Statistics in supporting the growth of probiotic compound (Gibson and Roberfroid 1995 ; Fooks and Gibson The signifi cance of synbiotics, as a nutritional 2002 ). This synergistic action not only stimulates supplement, in preventing non-communicable and amplifi es the survival of desired probiotics and communicable diseases rose recently. It is a but also promotes the autochthonous-specifi c novel area of research in functional foods and strain of intestinal tract (Fotiadis et al. 2008 ). nutraceuticals that is being explored and is General health benefi ts attributed to these pro- emerging for its several hidden health benefi ts. biotics include prevention of tumors in vivo The concept of synbiotics is not well devel- (Russell et al. 2013 ); management of allergy oped and demands validation and further caused by food, including atopic eczema (Isolauri research. Roberfroid (1998 ) introduced the con- et al. 1999 ) and diarrhea in children (Isolauri cept of prebiotic in 1995, and it has become very et al. 1991 ); delay in aging (Duncan and Flint popular since then. However, the concept of syn- 2013 ); and aid in adjusting the blood lipid levels biotic has, till now, not been widely applied and (Omar et al. 2013 ). To exert these health benefi ts, is a new notion in the development of functional probiotics must be able to survive the alteration foods or nutraceuticals. Synbiotic (“Syn” means while passing through the stomach and colonize “together” and “bios” means “life”) is defi ned as the colon (Webb 2011 ). a supplement that contains both a prebiotic and a So Farnworth (2001 ) added a new point to the probiotic that work together to improve the existing defi nition of synbiotics , which says: “friendly fl ora” of the human intestine and is of “Synbiotics encourage the growth of the probi- commercial value. Two concepts were projected otic organism by providing the specifi c substrate by Kolida and Gibson (2011 ), and these are com- to the probiotic organism for its fermentation.” plementary and synergistic. Hence, the synbiotic concept was introduced as “the mixtures of probiotics and prebiotics that 1. Complementary concept: a single or a combi- benefi cially affect the host by improving the sur- nation of probiotics and prebiotics that are vival and implantation of live microbial dietary independently chosen to stimulate the benefi - supplements in the gastrointestinal tract, by cial gut microbial population. Prebiotics pro- selectively stimulating the growth and/or by acti- mote indirectly the growth and activity of the vating the metabolism of one or a limited number probiotics. of health-promoting bacteria, thus improving 2. Synergistic concept: host benefi cial probiotics host welfare” (Gibson and Roberfroid 1995 ) and the prebiotics to specially enhance the (Fig. 5.1 ). survival, growth, and activity of these probi- An ideal synergistic synbiotic supplement otic strains(s) are selected. However, the pre- would contain a suitable single or multistrain biotic may also increase the levels of benefi cial probiotic and a mixture of prebiotics. Prebiotics resident gastrointestinal microbiota. selectively favor the ingested probiotics and produce additive or synergistic effect (Kolida and The prime reason behind the use of synbiotics Gibson 2011 ; Kondepudi et al. 2012 ; Raman is that the human gastrointestinal system (GI) is et al. 2013 ). It also favors the growth of the populated with a large number and variety of bac- endogenous benefi cial bacteria and reduces the teria that have specifi c nutrient needs (bifi dobac- number of cancer-promoting bacteria or patho- teria), and hence by selecting specifi c food or genic bacteria. food ingredients, it is probable to augment the Currently available and known synbiotic numbers of these target bacteria. When probiot- supplements in the market include a combination ics and prebiotics are administered concurrently, of bifi dobacteria and fructo-oligosaccharides a synergistic activity is observed, which indicates (FOS), Lactobacillus GG and inulins, and bifi do- 5.2 Synbiotics and Gut Health 85

Fig. 5.1 Concept of synbiotics PREBIOTICS Action (Provides nutrition for intestinal microflora and host)

SYNBIOTICS ACTION

PROBIOTICS Action (Provides beneficial bacteria for the body)

Table 5.1 Examples of common synbiotics 5.2 Synbiotics and Gut Health Synbiotics Lactobacilli + lactitol The gut, though the largest organ of the human Lactobacilli + inulin system, is an important target for various kinds of Lactobacilli + fructo-oligosaccharide or inulin stresses caused by factors including sepsis, Lactobacillus rhamnosus GG + inulin trauma, burn, shock, bleeding, and infection Bifi dobacteria + fructo-oligosaccharide (Shimizu et al. 2013 ). Foreign substances includ- Bifi dobacteria + galacto-oligosaccharide ing food particles and antigens of the microbial Bifi dobacteria and lactobacilli + fructo-oligosaccharide or inulin origin that promote infectious and multiple organ dysfunction syndrome attack the gut; hence, gut microfl ora has a crucial role in health and dis- bacteria and lactobacilli and FOS or inulins eases. Imbalanced gut bacteria, deteriorated (Table 5.1 ). FOS is a potential prebiotic as they intestinal epithelium, immune system, and com- are fermented by selective benefi cial species of mensal bacteria trigger development as well as the gut (Gibson and Roberfroid 1995 ; Havenaar aggravate the infectious complications including 2011 ; DuPont and DuPont 2011 ). They are also colon cancer and others (Shimizu et al. 2013 ). used as a source of natural sweetener and syrups Studies have indicated that the altered gut micro- for persons suffering from digestive problems biota (dysbiosis) is associated with several dis- (Charalampopoulos and Rastall 2012 ). Oral treat- eases that are predominantly prevalent in the 21st ment with yacon syrup rich in FOS (50 %) mark- century. Under conditions of critical illness, it is edly accelerates colonic transit time in healthy diffi cult to maintain the normal gut fl ora. These individuals and increases defecation frequency ensue not only due to the various stresses but also and satiety sensation (Geyer et al. 2008 ; Genta due to the invasive treatment methodologies et al. 2009 ), indicating the importance of prebiot- employed. Shimizu et al. (2006 ) reported the dis- ics in enhancing the growth of benefi cial gut turbed gut fl ora is critical in stressed and ill microbes (Fig. 5.2 ). Most of the synbiotics are patients and that the gut balance could be reestab- marketed as yogurt. Synbiotic-rich yogurt, lished using synbiotics. They reported that smoothies, etc. are few commercially available patients with severe systemic infl ammatory synbiotics marketed today. response syndrome (SIRS) had 100–10,000 times 86 5 Synbiotics and Colorectal Cancer

Fig. 5.2 Different types of prebiotics commercially used as an ingredient in synbiotics 5.2 Synbiotics and Gut Health 87

Fig. 5.2 (continued) 88 5 Synbiotics and Colorectal Cancer

Fig. 5.3 Function of synbiotics in the human gut system Probiotics Synbiotics Prebiotics

Live bacteria Inulin or in the diet oligofructose in Stomach the diet

Small intestine

Total transfer to colon and Colon stimulate growth of beneficial bifidobacteria

Excretion fewer total anaerobes, including Bifi dobacterium of SCFA in the gut. The mechanisms of action of and Lactobacillus , and 100 times more patho- SCFA are detailed in Chap. 6 . genic Staphylococcus bacteria when compared to The probiotics in the sybiotics face several normal healthy human. In such cases, total challenges in the host, including competition organic acids, including acetic and butyric acids, from other indigenous pathogenic or benign were decreased considerably while the latter microorganisms, metabolic interactions between completely disappeared. The alteration in the these indigenous microbiota, pH in the upper normal gut fl ora could ultimately lead to mortal- gastrointestinal tract, and the lack of anatomical ity. Clinical studies indicated that an improved and substrate heterogeneity (to mimic the human gut fl ora is observed with the administration of proximal and distal large intestines) (Gibson and synbiotics (Shimizu et al. 2011 ). Also, the ratio Wang 1994 ) (Fig. 5.3 ). Another obstacle to probi- of Firmicutes to Bacteroidetes (F/B) determines otic survival is the high concentrations of bile the gut health (Finegold et al. 2014 ). acids (1–10 mM) in the colon (Begley et al. Combination of prebiotics with probiotics 2005 ). These constraints are overcome, and the such as FOS and Bifi dobacterium or lactitol and fl ourishing of the probiotics takes place due to Lactobacillus in vitro and in vivo has been found the utilization of the prebiotics present in the syn- to stimulate the survival and activity of the micro- biotics. Su et al. (2007 ) demonstrated a positive organism (Chakraborti 2011 ). These synbiotic effect of prebiotics on the survival and retention combinations improve the survival, implantation, of specifi c probiotic inocula in vitro and in vivo . and growth of newly added probiotic strains. The prebiotics include fructo-oligosaccharides Synbiotics comprising of Bifi dobacterium breve (FOS) , soybean oligosaccharide (SOS) , or inulin , strain Yakult and Lactobacillus casei strain and the probiotics include Lactobacillus aci- Shirota as probiotics and galacto- oligosaccharides dophilus , Bifi dobacterium lactis , or Lactobacillus as prebiotics produced considerably greater lev- casei (Table 5.1 ). They observed that prebiotics els of the benefi cial Bifi dobacterium and SOS- or FOS-containing diet stimulated the Lactobacillus and short-chain fatty acids (SCFA) growth of L. acidophilus and maintained it at its and lower the incidence of infectious complica- highest level. Soybean extract enhances the tions than the control with no synbiotics (Shimizu growth of bifi dobacteria, while SOS moderately et al. 2009 ). The benefi cial effects of these synbi- enhances the growth of these possibly due to the otics were attributed to the increased levels of the reduced percentage of polymerized sugars, two benefi cial bacteria and increased production namely, raffi nose and stachyose. FOS and inulin 5.2 Synbiotics and Gut Health 89 dietary treatment exerted similar effects on the Human milk oligosaccharides (diverse and gut B. lactis. The prebiotics, namely SOS, FOS, complex than animal milks) received lot of and inulin, enhanced the survival and retention interest in recent years due to their ability to time of L. Casei (Fig. 5.2 ). β-glucan hydrolysate augment the growth of bifi dobacteria and cell and concentrate, and fi ber gum as prebiotics are adhesion as well as aid in the growth of lactic not comparable to SOS and FOS. Similar results acid bacteria, which might be a potential are reported in randomized, double-blinded, probiotic (Kunz et al. 2000; Martín et al. 2003 ; crossover study comprising of healthy children. Collado et al. 2009). Hence, human milk is Administration of milk-based fruit juice contain- considered as a synbiotic food that can help in ing probiotic, Lactobacillus GG with galacto- the growth of benefi cial bacterial in the gut of oligosaccharide (GOS), appreciably amplifi es the breast-fed infant (Martín et al. 2003 ). Some amount of allochtonous Lactobacillus rhamno- studies have also recommended that human milk sus and autochthonous bifi dobacteria, and lacto- oligosaccharides may encourage the establish- bacilli in the feces of treated children. GOS is ment of autochthonous microbiota in children soluble dietary fi bers that pass through the colon (Heyman and Ménard 2002 ). undigested, where it is hydrolyzed and fermented Even though synbiotics are increasingly being by gut microbes. Further, the monomeric units of used for various health treatments, the potential GOS contain galactose and lactose, which are benefi ts of these to gut are limited and are the naturally found in human milk. cumulative effects of pro- and prebiotics (Figs. Xylo-oligosaccharide with Bifi dobacterium 5.4 and 5.5). Hence, new combination of synbiot- animalis subsp. lactis as synbiotic (8 g XOS + 109 ics are being experimented and developed. They cfu Bi-07/d) when administered to healthy adults exist as capsules and sachets. Pro-wel is a com- (25–65 years) for 21 days led to healthy bowel mercially available synbiotic (Patel and Patel habits and improved composition of the gut 2010 ). Usually, the probiotic intake ranges from microbiota, blood lipid concentration, and 1 to 10 billion cfu a few times a week, with vary- immune function. Nevertheless, in this double- ing prebiotic dose. blind, placebo-controlled, randomized, factorial, crossover study with the synbiotics, the symptoms such as bloating, abdominal pain, or fl atu- 5.2.1 Anticancer lence are not controlled. XOS supplementation as a synbiotic had benefi ts improving the gut micro- Colorectal cancer (CRC), though widely distrib- biota and markers of immune function and obe- uted all around the world, has the highest rate of sity (Finegold et al. 2014). Rycroft et al. ( 2001 ) mortality and occurrence in developed countries evaluated the fermentative properties of some and is gradually increasing in developing coun- prebiotics in vitro and established that XOS and tries, including India; in South America; and in lactulose produced the highest increase in bifi do- Africa. This conspicuous geographic gap is bacteria, while FOS encouraged the growth of largely due to varying food habits such as low lactobacilli. Guerra-Ordaz et al. ( 2014 ) adminis- content of insoluble and soluble vegetable dietary tered a combination of the prebiotic oligosaccha- fi ber, fondness for red meat, surplus consumption ride lactulose and the probiotic strain of of refi ned carbohydrates, and low amounts of Lactobacillus plantarum as synbiotic against protective micronutrients, in addition to obesity enterotoxigenic Escherichia coli (ETEC) and and physical idleness (Crawford and Kumar observed a reduced incidence of diarrhea. 2003 ). These types of food could generate or Inclusions of the probiotic were reported to enhance the potentiality of the carcinogens or increase the number of L. plantarum bacteria in mutagens, thereby producing DNA damage in the ileum and colon and the total lactobacilli in the crypt cells leading to mutation of genes, the colon. including adenomatous polyposis coli (APC), 90 5 Synbiotics and Colorectal Cancer

Immuno- stimulation

Cholesterol Antioxidant assimilation

SYNBIOTICS

Formation of Neutralization short chain of carcinogens/ fatty acids mutagens

Bioactive peptides, Non-adhesion oligosaccharide, of pathogens to organic acid the intestinal formation lining

Fig. 5.4 Proposed function of synbiotics in the colon

Kirsten-ras (K-ras), and p53 (protein 53 kD) activities, antigenotoxic effects, and precancer- (Brady et al. 2000; Fotiadis et al. 2008 ; Sidhu ous lesions (Burns and Rowland 2000 ). et al. 2010 ). Several researchers demonstrated the Synbiotic combination of Bifi dobacteria and detoxifying and antimutagenic properties of pro- oligofructose (FOS) has been established to hin- biotics and prebiotics and concluded that these der colon carcinogenesis in rats when compared have benefi cial protective effects on CRC to individual administration (Liong 2008 ). (Wollowski et al. 2001; Fotiadis et al. 2008 ; Administration of inulin and B. longum (6 × 109 Gupta and Garg 2009 ; Patel and Patel 2010 ). cfu/day) jointly to azoxymethane (AOM)-treated Extensive work is still pursued to demonstrate Sprague Dawley rats signifi cantly (26 %) reduced the potential preventive effects of these dietary the total aberrant crypt foci (ACF) than when interventions in combination on CRC (Liong they were ingested alone (Rowland et al. 1998 ). 2008 ). Several studies indicate that modifi cation Consumption of B. lactis and resistant starch was of the gut microfl ora through these dietary modi- also able to augment the apoptotic response to fi cations might interfere with the process of car- AOM in rats, which was probably due to the fact cinogenesis, thus minimizing the risk of that the resistant starch acted as the substrate for CRC. Epidemiological and experimental studies the optimal activity of the probiotic species. in humans and in vivo studies have indicated that Combination of oligofructose-enriched inulin, L. pro- and prebiotics can minimize the risk of car- rhamnose, and B. lactis prevents the AOM- cinogenesis by modulating the bacterial enzyme induced suppression of NK-cells (natural killer 5.2 Synbiotics and Gut Health 91

Fig. 5.5 An overview of synbiotics

cell) activity in Peyer’s patches, while adminis- observed. Combination of B. longum and lactu- tration of individual components were not effec- lose produced a 48 % inhibition of colonic ACF, tive (Roller et al. 2004 ). These studies indicate which was considerably better than either of that synbiotics have a critical role in CRC treat- these when administered alone (Challa et al. ment (Fotiadis et al. 2008 ). However, prebiotics 1997; Rowland et al. 1998). Scientists also when administered alone have shown contradic- observed that B. longum with inulin showed a tory results on carcinogen-induced ACF, which synbiotic action with a reduction in the total ACF might partly be due to the differences in the type (74 %) in rats than when B. longum (29 %) or of carcinogen/mutagen and the treatment inulin (21 %) was administered individually. regimes. Inulin (10 % in diet) had no signifi cant Also, large ACF reduced considerably (59 %) in effects on total ACF in the colon or their growth the former, while the individual treatment had no in F344 rats (Rao et al. 1998 ). Nevertheless, a effect. Liong ( 2008 ) reported fewer tumors in signifi cant decrease in ACF/cm2 of colon was cancer-induced rats when they were fed with 92 5 Synbiotics and Colorectal Cancer cereal bran. He also suggested that synbiotics are rhamnosus GG and Lactobacillus casei more benefi cial than the administration of either DN-114-001 protect and improve the epithelial probiotic or prebiotic alone. barrier and effectively suppress the Gram- Dietary synbiotics, containing L. rhamnosus negative bacteria into the gastrointestinal channel GG and Bif. lactis Bb12 as probiotics and (Parassol et al. 2005 ; Strowski and Wiedenmann oligofructose- enriched inulin as prebiotic, 2009 ; Frazier et al. 2011 ). A few probiotic strains, reduced the cancer risk factors in polypectomized including L. plantarum 299, can mitigate bacte- and colon cancer patients in a 12-week random- rial translocation (Klarin et al. 2008 ). In a pla- ized, double-blind, and placebo-controlled trial cebo-controlled trial, modifi cation of the enteric (Rafter et al. 2007 ). Administration of these also fl ora in IL-10 knockout mice by probiotic altered the composition of the fecal bacteria, Lactobacilli was associated with reduced preva- namely, increase in the population of protective lence of colon cancer and mucosal infl ammatory (benefi cial) bacteria and reduction in the num- activity (O’Mahony et al. 2005 ). bers of cancer-promoting (harmful) bacteria Synbiotics aid in producing higher amounts of (Liong 2008 ; Daniel 2010 ). They also established SCFA than probiotics and subsequently protect that certain CRC intermediate biomarkers were the onset of CRC (Le Leu et al. 2010 ; Borowicki modifi ed due to synbiotic intervention as well as et al. 2011 ). One possible explanation for this is colorectal proliferation and the fecal water to the interaction of the immunomodulating proper- induce necrosis in colonic cells decreased. ties of the probiotic bacteria with butyrate, which Additionally, the function of epithelial barrier in is produced during prebiotic fermentation result- the colon in polypectomized patients was also ing in an upregulation of apoptosis (Le Leu et al. improved. Exposure to genotoxins was also 2010 ). In addition to SCFA, probiotics are also reduced at the end of the intervention period. involved in the production of conjugated linoleic Liong (2008 ) postulated that the synbiotics modi- acids (CLA), which are shown to exert numerous fi ed the composition of the colonic bacterial eco- health benefi ts, including antiinfl ammatory and system, which subsequently altered metabolic anticarcinogenic effects (Kelley et al. 2007 ). activity of the colon and prevented the amplifi ed CLA is a mixture of positional and geometric secretion of interleukin-2 (IL-2) by peripheral isomers of octadecadienoic acid with conjugated blood mononuclear cells in the polypectomized double bonds and is a potential prophylactic patients and interferon in the cancer patients intervention for multiple infl ammatory diseases, (Rafter et al. 2007 ; Liong 2008 ). B. longum with including obesity, hyperinsulinemia, hyperten- raftiline HP (a derivative of inulin) benefi cially sion, immune response, and apoptosis altered the caecal physiology and bacterial meta- (Bassaganya-Riera et al. 2002 , 2004 ; Bassaganya- bolic activity and thereby, reduced the tumor risk Riera and Hontecillas 2010 ). In vivo studies indi- and the incidence of an AOM-induced colonic cate that CLA reduce the incidence of colonic lesions in vivo (Rowland et al. 1998 ). tumors (Liew et al. 1995 ; Kim and Park 2003 ). Conjugated linoleic acid exerts a protective effect on infl ammatory bowel disease and could be a 5.2.2 Antiinfl ammation promising chemopreventive agent against CRC and Immunomodulation in vivo. Dietary CLA ameliorate CRC activity, decrease colitis, and prevent adenocarcinoma Mice fed with a high-fat diet had an increased gut formation in part through a PPAR-γ dependent permeability and metabolic endotoxaemia mechanism (Evans et al. 2010 ). It also decreases (Safavi et al. 2013 ), which led to the leakage into the macrophage percentage in the mesenteric the body of the Gram-negative intestinal micro- lymph nodes. Ewaschuk et al. ( 2006 ) demon- biota, thus leading to the onset and progression of strated in vitro and in vivo that a mixture of the infl ammation and metabolic diseases (Cani et al. probiotic strains containing Lactobacillus aci- 2009 , 2012 ). Probiotics such as Lactobacillus dophilus , L. bulgaricus , L. casei , L. plantarum , References 93

Bifi dobacterium breve , B. infantis , B. longum , agents and, hence, the minimization of the risk of and Streptococcus thermophilus are able to pro- colorectal cancer. The bacterial fermentation by- duce CLA from linoleic acid, induce the upregu- products are mainly SCFA, specifi cally acetate, lation of PPAR-γ (a nuclear hormone receptor propionate and butyrate, and gases. Gases are expressed by monocytes, macrophages, T-cells, eliminated, and SCFA, together with nutrients dendritic cells, and gastrointestinal epithelial and growth signals, play a vital role in the pre- cells in the gut mucosa), reduce colonic tumor vention of CRC. Also, secondary bile acid is cell viability, and thus induce apoptosis (Tontonoz reduced (Mai et al. 2004 ). Femia et al. (2002 ) et al. 1998 ; Clark et al. 2000 ). Short-term synbi- observed that synbiotic combination of fructans otic treatment of ulcerative colitis, a relapsing and B. lactis (Bb12) or L. rhamnosus GG reduced infl ammatory disease of the colon resulted in a the AOM-induced colorectal adenomas and car- signifi cant improvement of the clinical appear- cinogenesis by increasing SCFA production. ance of the disease due to the changes in the This also lessens the proliferative activity and the mucosal microbiota (Furrie et al. 2005 ). expression levels of GST placental enzyme pi- type, inducible NO-synthase, cyclooxygenase-2 enzymes, which are involved in the colorectal 5.3 Mechanism of Action carcinogenesis. The details of the promising mechanism of Unlike probiotics, the possible anticarcinogenic probiotics and prebiotics are discussed in Chaps. activity of synbiotics is not tacit. As prebiotic oli- 2 and 4, respectively. The mechanism of action of gosaccharides are fermented by the probiotic SCFA in reducing cancer is discussed in Chap. 6 . bacteria and other bacteria residing in the colon, butyrate and other SCFA are formed leading to the butyrate-mediated anticarcinogenic effects References (Fig. 5.5 ) (Chakraborti 2011 ). Mechanism of action of synbiotics could be Bassaganya-Riera J, Hontecillas R (2010) Dietary CLA normally summarized as follows: and n-3 PUFA in infl ammatory bowel disease. Curr Opin Clin Nutr Metab Care 13(5):569 Synergistic action Bassaganya-Riera J, Hontecillas R, Beitz DC (2002) Prebiotics form substrate for the specifi c benefi cial Colonic anti-infl ammatory mechanisms of conjugated allochtonous and autochthonous bacteria to survive linoleic acid. Clin Nutr 21:451–459 and fl ourish Bassaganya-Riera J, Reynolds K, Martino-Catt S, Cui Y, Modifi cation of the colonic microbiome, leading to Hennighausen L, Gonzalez F, Robrer J, Benninghoff altered metabolic activity in the colon AU, Hontecillas R (2004) Activation of PPAR gamma and delta by conjugated linoleic acid mediates protection from experimental infl ammatory bowel disease. The prebiotics in the synbiotics might elevate Gastroenterology 127(3):777–791 the levels of minerals, calcium and magnesium, Begley M, Sleator RD, Gahan CG, Hill C (2005) in the colon and may enhance the growth of bifi - Contribution of three bile-associated loci, bsh, pva, and btlB, to gastrointestinal persistence and bile toler- dobacteria and lactobacilli in the large intestine. ance of Listeria monocytogenes. Infect Immun Moreover, in vitro and animal studies have shown 73(2):894–904 that these bacteria bind and inactivate carcinogen Borowicki A, Michelmann A, Stein K, Scharlau D, Scheu and hence, have the ability to directly inhibit the K, Obst U, Glei M (2011) Fermented wheat aleurone enriched with probiotic strains LGG and Bb12 modu- growth of tumor and prevent the bacterial conver- lates markers of tumour progression in human colon sion of the precarcinogens into carcinogens cells. Nutr Cancer 63(1):151–160 (Chakraborti 2011 ; http://170.107.206.70/drug_ Brady LJ, Gallaher DD, Busta FF (2000) The role of pro- info/nmdrugprofiles/nutsupdrugs/syn_0327. biotic cultures in the prevention of colon cancer. J Nutr 130(2):410S–414S shtml ). The bacterial fermentation of the dietary Burns AJ, Rowland IR (2000) Anti-carcinogenicity of components in the gut system could be associ- probiotics and prebiotics. Curr Issues Intest Microbiol ated with the production of cancer-preventive 1(1):13–24 94 5 Synbiotics and Colorectal Cancer

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Abstract Short-chain fatty acids are formed in the colon as a result of microbial fermentation (Bifi dobacterium and Lactobacillus ) of undigested bioactive carbohydrates, including prebiotics and dietary fi ber, and their signifi cant role in orchestrating colon carcinogenesis is of current interest among researchers. Acetate, propionate, and butyrate are the major by-products of SCFA formation. Propionate and butyrate are extensively involved in the cell differentiation, growth arrest, and apoptosis of cancer cells. The anticancer effects of acetate, propionate, and butyrate (molar ratio of approximately 3:1:1) is supported immensely by epidemiological, in vitro and in vivo, studies. The role of lactate has also been considered by several researchers. Diet and dietary habits alter the composition of the gut microfl ora. An inverse relationship between dietary fi ber intake and the incidence of human colon cancer has been observed. Prebiotics and dietary fi bers curtail the risk of colon cancer through diverse mechanisms. The interplay between propionate and butyrate, and gastrointestinal epithelial cells (colonocytes) in reducing colon carcinogenesis is a current topic of interest for many researchers. About 70–90 % butyrate is metabolized by the colonocytes. SCFA also modulate colonic and intracellular pH, cell volume, ion transport and regulate cell proliferation, differentiation, and gene expression. Propionate inhibits cell growth and activates apoptosis in colorectal carcinoma cells. Butyrate inhibits histone deacetylase resulting in histone hyperacetylation and growth inhibition in the colonic epithelial cells. This link between histone hyperacetylation, hyperacetylation- induced transcriptional regulation and growth inhibition has been considered as the foremost factors in preventing colon cancer. However, “butyrate paradox,” another concept, has not been confi rmed till date. The function of SCFA in the suppression of infl ammation and immunomodulation is discussed.

Keywords Anticancer • Apoptosis • Butyrate • Colon acetate • Fermentation • Inﬂ ammation • Metabolism • Propionate • Short-chain fatty acid

6.1 Introduction bacterial ecosystem, forming products that primarily comprise SCFA and gases. Carbohydrates Interest in short-chain fatty acids (SCFA) has are classifi ed into two basic groups based on their been rekindled in the last few years because of digestibility in the gut (Lattimer and Haub 2010 ). their benefi cial effects on colonic and systemic The fi rst group is simple carbohydrates (starch health. Previously, they were considered as major and simple sugars), which are enzymatically etiological factors in carbohydrate-induced diar- hydrolyzed and are absorbed in the small intes- rhea (Binder 2010 ). However, studies conducted tine, while the second group is complex carbohy- by physiologists on cattle indicated that they drates or dietary fi bers, including cellulose, derive most of their energy from cellulose and lignin, and pectin, which are resistant to diges- SCFA in the rumen, where they are easily formed tion in the small intestine and undergo bacterial and rapidly absorbed (Topping and Clifton 2001 ). fermentation in the colon (Lattimer and Haub This has led to the general conviction that SCFA 2010 ) (Fig. 6.1 ). About 95–99 % SCFA are pro- could be benefi cial to human gut health. Studies duced from resistant starch and dietary fi bers by on native East Africans indicated that they con- the bacterial fermentation (Bifi dobacterium , sumed diet rich in unrefi ned cereals and were at Ruminococcus bromii ) and are absorbed and lower risk of colorectal cancer, diverticular dis- transported via portal vein to the liver. The unab- ease, and constipation than the others (Topping sorbed fractions are distributed to other organs of and Clifton 2001 ), thus once again strengthening the body and tissues for metabolism (Topping the impact of SCFA on health. and Clifton 2001 ). Faecalibacterium prausnitzii , Prebiotics and dietary fi bers, principally com- Clostridium leptum (or clostridial cluster IV) prising of oligosaccharides and non-starchy solu- cluster and Eubacterium rectale /Roseburia spp., ble polysaccharides of low molecular weight, belonging to the Clostridium coccoides (or clos- undergo varying degrees of breakdown during tridial cluster XIVa) cluster of fi rmicute bacteria, transit through human gut (the microbiome are the two most important groups of butyrate resembles an obligate herbivores) by a complex producers (Louis and Flint 2009 ).

SMALL INTESTINE LARGE INTESTINE FECES

Proximal Distal

Starch Undigested Non-strach polysaccharide biomass- Digestion Unabsorbed secretions Carbohydrates Lignin, etc. High Low Carbohydrates, proteins, Fermentation Ingested food Fat, other nutrients

Oligosaccharide, proteins, Fat

Monosaccharides, aminoacids, Fatty acids, other nutrients

Absorption Gradient SCFA Absorption

Fig. 6.1 An overview between intake of food its digestion and fermentation in the gastrointestinal tract. Prebiotics and dietary ﬁ ber form substrate for probiotics and undergo fermentation to produce short-chain fatty acids 6.1 Introduction 99

Production of SCFA happens predominately plenty of attention in the last three decades. There in the proximal colon and is transported to distal is mounting evidence that SCFA play a notewor- regions by the fecal stream (Fig. 6.1 ). This is the thy role in maintaining gut health and in the pre- site of greatest organic disease. Hence, delivery vention and management of several of SCFA, specifi cally butyrate, to this section is noncommunicable diseases, including cancer. signifi cant. Peripheral venous SCFA are high due Soluble sugars comprising of monosaccha- to comparatively low visceral extraction and rides and oligosaccharides undergo 100 % fer- higher absorption rate during circulation. mentation in the colon. This is followed by Conversely, SCFA concentration in human pectin/psyllium (90–95 %), bran (70 %), cellu- peripheral venous blood, except for acetate, is lose (20 %), and lignin (0–5 %), with their vary- normally low, refl ecting lower SCFA production ing fermentation rates (Roberfroid 2005 ). Dietary rate and greater visceral extraction in humans. fi ber, resistant starch, and complex carbohydrates SCFA contribute to contribute largely to a high rate of SCFA production. Protein, mucus, sloughed cells, and gastro- – the normal large bowel function and intestinal secretions may also contribute to SCFA – prevent pathology through their metabolism production. Fermentation of proteins and amino by colonocytes in the lumen, colonic muscula- acids by proteolytic bacteria yields branched

ture and vasculature. SCFA, CO2 , CH4 , H2 , phenols, and amines (Roberfroid 2005 ). The primary effect of SCFA The function of SCFA, including their ion on colonic function is the result of their uptake transport property, nutrient (energy supplies) for and metabolism by colonocytes. Nonstarch poly- the colonic epithelium, modulator of intracellular saccharides, which resist digestion by intrinsic pH and cell volume, and other functions such as human intestinal digestive enzymes, do not regulators of proliferation, differentiation, and account for SCFA production and are termed as gene expression, are of keen interest for research- “ carbohydrate gap .” It is shown that consumption ers today (Fig. 6.2 ). The increased scope of SCFA of greater resistant starch is associated with and their beneﬁ ts in human health have achieved reduced risk of tumors, which may be due to

Mucosal blood flow, Absorption energy of SCFA and metabolism sodium and cell proliferation

Bacterial carbohydrate fermentation SCFA

Mucus Colitis Adaptation release, prevention to surgery cellular differentiation

Fig. 6.2 Short-chain fatty acids and their health beneﬁ t s 100 6 Short-Chain Fatty Acids

SCFA and colonocyte proliferation. However, in present in the colon (Roberfroid 2005 ), substrate epidemiological and animal studies, this was neg- source (Cook and Sellin 1998 ), and gut transit atively correlated with colorectal cancer risk. This time. A large variety of microfl oral population inconsistency may be due to the particular fea- inhabit the human colon (1010 –10 11 cfu/g wet tures of the digestive system of the rodents weight) (Hill 1995 ). SCFA production and over- (coprophagy). In vitro and in vivo studies indicate all stoichiometric reaction have been summarized that butyrate produced by resistant starch fermen- for hexose as follows (Cummings 1997 ; Topping tation may be promoting apoptosis in tumor cells. and Clifton 2001 ). The signifi cance of fermenta- It has been shown that resistant starch promotes tion by intestinal bacteria is broad. colon maintenance by alleviating infectious diar- 59 C H O + 38 H O → 60 CH COOH (acetate) rhea and promoting colonic mineral absorption. 6 12 6 2 3 + 22 CH CH COOH (propionate) High amylase maize starch, a kind of resistant 3 2 + 18 CH CH CH COOH (butyrate) starch, has been included in prebiotics as it pro- 3 2 2 + 96 CO + 268 H+ + heat + additional bacteria. motes the survival of lactic acid bacteria (LAB). 2 More than 50 genera and over 400 species of bacteria have been identifi ed in human feces. 6.2 Production of Short-Chain Bacterial numbers, fermentation, and prolifera- Fatty Acids tion are elevated in the proximal end of the colon where the carbohydrate availability is the maxi- Basic fermentative action in the human colon mum. Therefore, the principal site of colonic fer- includes hydrolysis of polysaccharides, oligosac- mentation is the cecum and proximal colon, charides, and disaccharides to their monomeric whereas the distal colon is deprived of carbohy- sugars that upon fermentation results in increased drate and water and, hence, does not participate biomass (Savage 1986 ; Topping and Clifton in colonic fermentation (except polydextrose, the 2001 ). Bacterial cell-associated and secreted second generation of prebiotics). Species such as hydrolases assist in carbohydrate hydrolysis, and Bifi dobacterium and Lactobacillus and their car- this leads to metabolizable energy for microbial bohydrate substrates have been associated with growth and maintenance, and also metabolic end an improved health, resulting in the emergence of products, including SCFA (Fig. 6.3 ). The the idea of pro- and prebiotics. The former production of SCFA is determined by several fac- involves the delivery of specifi c bacteria to the tors, including the number and type of microfl ora colon and the latter includes the administration of

Fig. 6.3 Pathway showing the production of short-chain fatty acids in the colon 6.2 Production of Short-Chain Fatty Acids 101 dietary component that promotes the growth of Total SCFA and regional differences in SCFA specifi c bacteria with defi nite metabolic concentration are of great concern in colon dis- functions. eases, especially in cancer and gastrointestinal Population survey indicated that SCFA are disorders, where disease often occurs distally produced in the order as acetate > propio- (Roediger 1980 ; Roediger and Moore 1981 ; nate ≥ butyrate (Fleming et al. 1991 ; Cummings Jenkins et al. 1999 ; Floch and Hong-Curtiss and MacFarlane 1991 ; Takahashi et al. 1993 ; 2002 ). Therefore, an increased SCFA production Segal et al. 1995 ; Muir et al. 1998 ; Topping and and their distal delivery, especially butyrate, may Clifton 2001 ). In infants fed with breast milk, have a role in preventing noncommunicable dis- acetate is predominant, propionate is very low, eases. This has led to the concept of colonic and butyrate is absent in the feces. In those fed health and development of functional foods, such with formula, butyrate may be found (Sigur et al. as “probiotics, prebiotics, and synbiotics” and 1993; Edwards et al. 1994 ; Topping and Clifton other dietary components that target the colon 2001 ). The SCFA profi le is important in gut and affect its environment, the composition of the development. In premature infants maintained in microfl ora, as well as its physiology (Bomba incubators, enhanced butyrate during the period et al. 2002 ). Dietary carbohydrates escaping between 14 and 21 days is critical as it may be digestion/absorption in the small bowel and pre- related to the development of necrotizing entero- biotics undergo fermentation in the colon and colitis (Szylit et al. 1998 ; Topping and Clifton enhance SCFA production. 2001 ). In a healthy infant, fermentation is slower SCFA are absorbed by each intestinal segment and butyrate production is established slowly but as observed in vivo and in human volunteers in 2 years, the adult SCFA profi le emerge (Binder 2010 ). Apical membrane and active (Midtvedt and Midtvedt 1992 ; Topping and transport are the modes of SCFA uptake (Fig. Clifton 2001 ). SCFA in general and specifi cally 6.4 ). The former includes nonionic diffusion, − − butyrate enhance the growth of lactobacilli and SCFA /HCO3 exchange, while the latter includes bifi dobacteria. They play a crucial role in colon SCFA transporters. The latter are monocarboxyl- physiology and metabolism (Roy et al. 2006 ). ate transporter isoform 1 (MCT1, coupled with

LUMINAL BASOLATERAL MEMBRANE

SCFA− Na+ SCFA− ATP − HCO3 K+ Cl− Cyclic AMP Butyrate Cl− SCFA Non ionic diffusion

Fig. 6.4 Cellular model showing the absorption of portation. Butyrates collected in luminal membrane undergo SCFA. Absorption is chiefl y through nonionic diffusion and recycle. Basolateral membrane K+ channels are inhibited paracellular absorption in the proximal colon. SCFA− / during SCFA absorption, and increased proton concentra- − − HCO 3 luminal membrane exchangers help in SCFA trans- tion activates Cl channels in basolateral membrane 102 6 Short-Chain Fatty Acids transmembrane H+ gradient) and SLC5A8 (Na+ (Cummings et al. 1987 ). Due to the limitations of coupled cotransporter) (Hamer et al. 2008 ; the former, the new method involving dialysis Binder 2010 ). The absorption of SCFA has a sig- sacs in gelatine capsules has been used to deter- nifi cant impact on the absorption of minerals and mine SCFA in situ in normal subjects (Wrong on electrolyte balance (Kunzelmann and Mall et al. 1965 ) with varying dietary interventions 2002 ). Butyrate exerts a powerful proabsorptive (Fredstrom et al. 1994 ). These have been adopted stimulus on NaCl transport and antisecretory in clinical ulcerative colitis, where the severity of effect towards Cl− secretion in the gut (Binder infl ammation has been correlated to butyrate and Mehta 1989 ). These effects of butyrate hap- (18.9 mM) than normal (14 mM). Continuous pen through two mechanisms: sampling is impractical in this case. Elsden et al. (1946 ) showed that there is a pro- 1. stimulation of NaCl absorption by the action gressive decline in the volatile organic acids of two coupled transport systems that involves along the large bowel of herbivorous and omniv- − − + + − Cl /HCO3 and Na /H , and Cl /butyrate and orous animal species. In pigs, depending on the Na+ /H+ and dietary profi le, total SCFA concentration in the 2. inhibition of Cl− secretion by blocking the proximal colon was ~70–140 mM, which fell to activity of the co-transporter Na-K-2Cl on the 20–70 mM in the distal colon (Marsono et al. basolateral membrane. 1993 ; Martin et al. 1998; Bird et al. 2000 ). This concentration may also vary in the distal colon In vitro studies showed that butyrate inhibits with the loss of water and digesta mass. However, Cl− secretion by inducing prostaglandin E2, chol- the availability of SCFA changes with the rate of era toxin, and phosphocholine. This inhibition digesta passage and is independent of the rates of could be due to the reduced production of intra- production. These regional differences in SCFA cellular cAMP secondary to the expression and may have implications on large bowel diseases, regulation of adenylate cyclase (Fig. 6.4 ) (Binder specifi cally colon cancer and ulcerative colitis 2010). The administration of butyrate is also (IBD). known to alleviate the symptoms associated with antibiotic use, including diarrhea (Clausen et al. 1991 ). 6.3 Butyrate Paradox Increase in SCFA results in the decrease of colonic pH, which indirectly infl uences the com- Dietary fi bers escaping digestion in the small position of the colonic microfl ora, decreases the intestine reach the large intestine in compact and solubility of bile acids, increases the absorption undergo bacterial fermentation to produce of minerals (indirectly), and reduces the ammo- SCFA, specifi cally butyrate, which has signifi - nia absorption by the protonic dissociation of cant health benefi ts. Butyrate is metabolized by ammonia and other amines (formation of the less colonocytes but inhibits colon carcinoma cells. + diffusible NH4 when compared to the diffusible Though butyrate seems to be a useful agent in

NH3 ) (Vince et al. 1978 ; Jackson 1983 ; Jenkins colon cancer therapy, its rapid metabolism and et al. 1987 ). ineffective concentration in vivo infl uence its success as an inhibitor for colon cancer. Butyric acid and propionic acid inhibit growth and pro- 6.2.1 SCFA Determination in Colon mote apoptosis of human colonic carcinoma cell lines (Fukuda et al. 2011), while antiinfl amma- Intubation has been used in humans to quantify tory properties are reported as the cumulative the intestinal digestibility of carbohydrates, effects of acetic, propionic, and butyric acids including starch. But SCFA cannot be determined (Fukuda et al. 2011; Zanten et al. 2012 ). by this method; hence, autopsy and surgery are Recently, it is reported that acetic acid produced adopted for gut content and portal venous blood by bifi dobacteria stimulates epithelial cell 6.4 Short-Chain Fatty Acids, Gut Microbiota, and Health 103 defense against infection by Escherichia coli an important mechanism in which butyrate O157:H7 (Fukuda et al. 2011 ). causes signifi cant biological effects (Comalada Butyrate is not produced by the lactic acid et al. 2006 ). bacteria; however, certain probiotics may modify the ratio of SCFA in the colon, which is considered as a likely mechanism of anticarcinogenic 6.4 Short-Chain Fatty Acids, Gut action within the colon. Butyrate also stimulates Microbiota, and Health immunogenicity of cancer cells. In a study of HCT116, colon cancer cells with wild-type p53 Carbohydrates entering the large bowel may alter or p53 mutant and treatment with drug, vorino- the colonic physiology, fi rstly, by its physical stat, indicated that 144 out of 275 miRNAs dem- presence and, secondly, by undergoing fermenta- onstrated several-fold changes in transcription tion. Undigested monosaccharides, disaccha- and most were strongly affected by vorinostat rides, and oligosaccharides induce osmotic (Shin et al. 2009 ). In colon cancer xenografts diarrhea on excess consumption (Cummings with wild-type p53, valproate has also been 1997). Fecal bulking, speed transit, and laxation established to enhance the response to radiother- are important components of the fi ber hypothesis apy (Chen et al. 2009 ). Butyrate also show simi- (Topping and Clifton 2001 ). Their effects are lar effects at molecular level (Zhou et al. divided into those occurring in lumen and those 2011 ). Nevertheless, the chemopreventive effect arise due to their uptake and metabolism by the of butyrate has not been confi rmed till date, and colonic cells. this disagreement of butyrate and colon cancer SCFA are promptly absorbed in the cecum has been termed as “butyrate paradox.” and colon with only 5–10 % excreted through the Colon bacteria that aid in producing butyrate feces. This is also associated with enhanced belongs to the clostridial clusters I, III, IV, VI, sodium absorption and bicarbonate excretion. XIVa, XV, and XVI, with cluster IV bacteria The possible mechanism of absorption has been related to Faecalibacterium prausnitzii and clus- postulated as the diffusion of protonated SCFA ter XIVa bacteria related to Eubacterium rectale (60 %) and anion exchange (Cook and Sellin and Roseburia spp. (Barcenilla et al. 2000 ). 1998). SCFA uptake is related to the transport of Butyrate plays a key role in maintaining a normal water, which is high in the distal end than in colonocyte population. It is metabolized by nor- proximal. Acetate, propionate, and butyrate are mal colonocytes for energy metabolism and pro- absorbed at comparable rates throughout the motes healthy colonic epithelial cells. However, colon (Fig. 6.5 ). Once absorbed, these are metab- it serves as an inhibitor for the proliferation and olized at three main spots: differentiation of cancer cells. Approximately, 70–90 % of butyrate is metabolized by the colonocyte (Zoran et al. 1997 ; Basson et al. 2000 ; Della Ragione et al. 2001 ). The addition of butyrate in vitro leads to the inhibition of cell proliferation, induction of apoptosis, or differentiation of tumor cells (Comalada et al. 2006 ; Hinnebusch et al. 2002 ). The anticarcinogenic effects of butyrate on normal enterocytes are not similar to that on cancer cells. Butyrate possibly stimulates the physiological pattern of proliferation in the colon basal crypt but reduces the number and the size of aberrant crypt foci (ACF), which are the initial detectable neoplastic lesions in the colon Fig. 6.5 Major short-chain fatty acids formed during bac- (Alrawi et al. 2006 ). This “butyrate paradox” is terial fermentation of bioactive carbohydrates 104 6 Short-Chain Fatty Acids

1. ceco-colonic epithelial cells use butyrate as a function, immune regulation, oxidative stress, major substrate for the maintenance of energy- intestinal motility, visceral perception, and rectal producing pathways; compliance. In extraintestinal level, it potentially 2. liver cells metabolize residual butyrate, effects insulin sensitivity, cholesterol synthesis, together with propionate, for gluconeogene- energy expenditure, ammonia scavenger, stimu- sis, while 50–70 % of acetate is taken up here; lation of β-oxidation for very long chain fatty and, ﬁ nally, acids, cystic ﬁ brosis transmembrane conductance 3. the muscle cells generate energy from the oxi- (CFTR) function, neurogenesis, and fetal hemo- dation of residual acetate. globin production (Canani et al. 2011 ). Generation of propionic and butyric acids Acetate, propionate, and butyrate are found in involves specialized bacterial groups. Acetate the gastrointestinal tract at concentrations of forms the major bulk of SCFA and is generated by about 13 mM/l in the terminal ileum, 130 mM/l many of the bacterial groups. Decarboxylation of in cecum, and 80 mM/l in the descending colon succinate and the acrylate pathway aids in the pro- (Cummings et al. 1987). Acetate is readily duction of propionic acid (Cummings 1981 ). absorbed and transported to the liver and, hence, However, the intestinal glucose uptake is minimal is not metabolized in the colon. Acetyl-CoA syn- in ruminants as the digestion and microbial fer- thetase in adipose and mammary glands use this mentation of carbohydrates take place in the acetate for lipogenesis after it enters the systemic rumen. Several biochemical pathways for propio- circulation. It is also the primary substrate for nate formation have been reported (Macfarlane cholesterol synthesis that might be absorbed and and Macfarlane 2003 ; Hosseini et al. 2011 ). The utilized by peripheral tissues (Pomare et al. succinate route is generally used by Bacteroides 1985). Further, human intestine bacteria utilize species, and the acrylate route from lactate acetate for the production of butyrate (Duncan involves fermentation by clostridial cluster IX et al. 2005 ). Other SCFA are formed by the group. Bacterium R. inulinivorans is also involved microbial degradation of lactate, formate, etha- in propionate formation from fucose (Scott et al. nol, and acetoacetate (Fig. 6.6 ), which are pro- 2006) (Fig. 6.3 ). Hooper et al. (2002 ) observed duced from other dietary components, including that SCFA account for the major source of rumi- proteins. In the intestinal level, butyrate is poten- nant energy with propionate as a primary precur- tially effective in ion absorption, cell prolifera- sor for gluconeogenesis. It has been reported that tion, cell differentiation, intestinal barrier increased propionate production may inhibit hepatic cholesterol synthesis. One of the possible determinants could be the ratio of propionate to acetate (Wolever and Bolognesi 1996 ).

6.4.1 Anticancer and Apoptosis

Colorectal cancer (CRC) is the fourth most common cause of cancer-related mortality in the world (Jemal et al. 2010 ). Many factors have been associated with CRC, which include genetic and environmental factors such as low levels of physical activity, smoking, alcohol consumption, obesity, low intake of dietary ﬁ bers, and high consumption of meat. Diet plays a sig- Fig. 6.6 Minor short-chain fatty acids formed during niﬁ cant role in the aetiology of CRC (Gill and bacterial fermentation of bioactive carbohydrates Rowland 2002 ). 6.4 Short-Chain Fatty Acids, Gut Microbiota, and Health 105

SCFA have been considered as gut-microfl ora- Rubio and Rodensjo 1996 ; Sameshima et al. 2000 ) associated biomarker for CRC. Cancer is a multi- discussed in detail in Sect. 6.6.5 . step process, including progression of Acetate stimulates the proliferation of normal carcinogenesis from hyperproliferative epithe- crypt cells in vivo (Ono et al. 2004). It improves lium to preinvasive and metastatic carcinoma ileal motility and colonic blood fl ow and plays a through ACF (Young and Gibson 1995 ). At each signifi cant role in adipogenesis and host immune step, genetic alterations occur. Most of the anti- system by interacting with G-protein-coupled carcinogenic effects of butyrate are reported in receptors (GPCR43, 41) (Brown et al. 2003 ; vitro carcinoma cell lines. Butyrate paradox has Hong et al. 2005). Acetate lessens the different effects on apoptosis in normal and lipopolysaccharide- stimulated tumor necrosis tumor cell lines. In normal cell lines, the absence factor (TNF), interleukin-6, and nuclear factor-κB of butyrate causes apoptosis within 150 min, (NF-κB) level and enhance the peripheral blood along with a fi vefold increase in the expression of antibody production in different tissues (Tedelind Bax (Hass and Wickner 1996 ), whereas it leads to et al. 2007 ). growth arrest, differentiation, and apoptosis in Propionate and acetate are also involved in tumor cell lines (Deng et al. 1992; Gamet et al. inducing apoptosis but to a lesser extent than 1992 ; Hague et al. 1995 ). Increased expression of butyrate (Hague et al. 1995 ). Propionate induces biomarkers such as brush-border glycoproteins, apoptosis in adherent cells, and butyrate induces alkaline phosphatase, and carcinoembryonic apoptosis in fl oating cells (Heerdt et al. 1994 ; antigen are observed during differentiation in Hague et al. 1995 ). Hence, the three fatty acids tumor cell lines. Normal colonic cells show exhibit differential effects in inhibiting prolifera- decreased expression of these biomarkers. Heerdt tion and inducing differentiation (Whitehead et al. ( 1994) demonstrated that alkaline phospha- et al. 1986 ; Gamet et al. 1992 ). Butyrate and ace- tase level is elevated in cancer cells treated with tate also inhibit DNA oxidative damage due to butyrate, mainly in the fl oating apoptotic cells, free radicals in vitro (Abrahamse et al. 1999 ). suggesting that apoptosis occurs following differ- Butyrate, though a trophic factor for the small entiation. One possible mechanism for differen- intestinal epithelia and important fuel for colono- tiation could be the reduction in the nuclear levels cytes (Agarwal and Schimmel 1989 ), induces of the proto-oncogene c-Myc (Collins et al. 1992 ; cell cycle arrest and apoptosis in colon carcinoma Taylor et al. 1992 ), which is important in control- cells (Freeman 1986). Butyrate inhibits NF-κB ling tumor growth. SW620 and HT-29 cells activity in the human colon cancer HT-29 cell exhibit different cell cycle factors during G0 /G1 line (Inan et al. 2000 ). and G 2 /M phases (Heerdt et al. 1994 ; Hass and Wickner 1996 ). Treatment of HT-29 tumor cells with butyrate reduces the cytoskeleton-associated 6.4.2 Infl ammation tyrosine protein kinase activity, which plays a key role in cellular responses to cytokines (trans- The emergence of adaptive immune system in forming growth factor-b1, TGF-β1) that partici- human beings indicates an advanced symbiotic pate in the growth of tumors (Barnard and relationship between SCFA and the intestinal Warwick 1993; Reimer and McBurney 1996 ). microbiota.

During G 2 /M phase, inhibition of DNA synthesis Increase of fats in the diet is observed to occurs probably through histone deacetylase amplify asthma and body weight. However, stan- inhibition. Removal of histones is a vital step in dard diet rich in dietary fi ber is observed to DNA replication (Kruh 1982 ). Apoptosis may reduce the infl ammatory diseases. This aggrava- include mutation in p53; however, apoptosis tion is attributed to the activation of infl amma- induced by butyrate does not involve the p53 some, and its reduction caused by dietary fi bers is gene (Heerdt et al. 1994 ; Hague et al. 1993, 1995 ; attributed to dendritic cells (DCR ) (Kim et al. 106 6 Short-Chain Fatty Acids

2014). Analysis of gut and lung microbial com- 6.5 Metabolic Regulation munities of mice fed with dietary fi bers indicates an increased proportion of Bacteriodaceae and Carbohydrates, including prebiotics and dietary Bifi dobacteriaceae that ferment the fi ber into fi bers, are fermented (Fig. 6.1 ) by saccharolytic SCFA. However, low-fi ber diet led to the micro- bacteria primarily in the proximal colon produc- biome with increased Firmicute (Trompette et al. ing linear SCFA (chiefl y acetate, propionate, and

2014 ). SCFA have also been linked to the genera- butyrate), CO 2, and H2 (Macfarlane and tion of Treg cells, which is essential for the control Macfarlane 2003 ), which alter the colonic physi- of adverse infl ammation in the gut. The cells of ology. Rate and degree of fermentation depend the immune system sense the metabolic by- on the type of dietary fi ber. Insoluble fi bers con- products and function of commensal microorgan- tribute mainly to the fecal bulk. ism and infl uence the balance between pro- and SCFA are organic fatty acids with 1–6 carbon antiinfl ammatory cells. atoms and are the principal anions that arise from Butyrate produced from dietary fi bers, includ- bacterial fermentation of polysaccharide, oligo- ing cellulose and pectin, facilitates the extrathy- saccharide, proteins, peptide, and glycoprotein mic generation of T reg cells in mice, which are precursors in the colon (Cummings and essential for immunological tolerance. Treg - cell Macfarlane 1991 ). SCFA arise from bacterial generation in the periphery is also potentiated by metabolism of “malabsorbed” carbohydrates that propionate. These results indicate that bacterial enter the colon, along with hydrogen gas (Cook metabolites mediate communication between the and Sellin 1998 ). This hydrogen gas is consumed commensal microbiota and the immune system by three bacterial reactions in the colon, namely (Arpaia and Rudensky 2014 ). So it could be concluded that both butyrate and propionate have (i) methanogenic bacteria that reduce CO2 to distinct immunological roles to play in the cross CH4 (50 % of population carries these talk between the bacteria and the immune system bacteria);

(Arpaia and Rudensky 2014 ). (ii) sulfate-reducing bacteria that reduce SO4 to

Butyrate has benefi cial effects on the trophic sulfi des, including H 2 S (seen in nonmethane and antiinfl ammatory effects on the gut epithelial producers); and, fi nally, cells . Butyrate transporter is downregulated in (iii) acetogenic bacteria that reduce CO2 to ace- the colonic mucosa in patients with infl ammatory tic acid by the use of hydrogen. bowel disease (IBD) (Thibault et al. 2007 ). It is also observed that in these patients, butyrate- Fermentation involves a variety of reactions producing bacteria, Bacteriodaceae and and metabolic processes, including Bifi dobacteriaceae, are considerably reduced in the gut mucosa and the fecal samples. (i) anaerobic microbial breakdown of organic Consumption of diets composed of animal or matter, yielding metabolizable energy for plant products alters microbial community struc- microbial growth and maintenance; and ture and overpowers interindividual differences (ii) other metabolic end products for host use; in microbial gene expression. Thus, gut microbi- the chief end products are SCFA together ome rapidly responds to altered diet, which with gases (CO2 , CH 4, and H 2 ) and heat explains the varied responses to food selection (Topping and Clifton 2001 ) . and ingestion in terms of health (David et al. 2014). Butyrate and its derivates have potential Lactic acid is an intermediary product of car- application in preventing and treating metabolic bohydrate fermentation (Table 6.1 ). The fore- syndromes in humans. Butyrate enemas are used most SCFA involved in mammalian physiology to treat infl ammatory bowel diseases, including are the straight chin fatty acids, including acetate Crohn’s and ulcerative colitis (Steinhart et al. (C2), propionate (C3), and butyrate (C4). Valerate 1996 ) . (C5), hexanoate (C6), isobutyrate (C4) and isova- 6.6 Mechanism and Effects of Short-Chain Fatty Acids on Health 107

Table 6.1 Metabolic fate of short-chain fatty acid 6.6.1 Luminal Effects of SCFA (SCFA) and other by-products formed during the metabolism of bioactive carbohydrates in the colon SCFA are the chief luminal anion in humans and Components Metabolic site Outcome are weak acids with pKa value ~4.8. Ingestion of Acetate Muscle, – low fermentable nonstarch polysaccharide such kidney, heart, brain as cellulose and wheat pericarp-seed coat at lev- Propionate Liver Possible gluceogenic els of 50–150 g/kg increases the cecal pH to 6.7– precursor and 8.2 (Campbell et al. 1997; Choct et al. 1998 ). suppresses However, cellulose lowers the SCFA concentra- cholesterol synthesis tion from 102 to 70 mM. A negative correlation Butyrate Colonic Regulate cell growth between the cecal SCFA and pH is established by epithelial and differentiation cells few researchers (Campbell et al. 1997 ; Choct Lactate, Colon Fermented to et al. 1998 ). In vitro incubation studies indicated succinate, short-chain fatty that lowering pH values, thence raising SCFA, pyruvate, acids and absorbed prevent the overgrowth of pH-sensitive patho- ethanol genic bacteria (Prohaszka et al. 1990 ). E. coli and Gases – Partial excretion Salmonella through breath spp. are killed by propionate and formate at high pH (pH 5). However, greater SCFA production by rapid feeding of high fermentable carbohydrates (water-soluble polysaccharides) lerate (C5) are produced in the colon in minor has been associated with the colonization of amounts (5–10 % of total SCFA) (Canani et al. Serpulina hyodysenteriae and appearance of 2011 ) (Figs. 6.5 and 6.6 ). SCFA production in diarrhea in vivo (Pluske et al. 1998). Human stud- different populations is in the order of ace- ies have been few, but SCFA can assist in the tate > propionate > butyrate in a molar ratio of management of antibiotic-induced and infectious approximately 60:20:20 or 3:1:1, respectively, in diarrhea (Ramakrishna et al. 2000 ). Fermentable the proximal and distal colon (Cummings 1981 ; carbohydrates alter the microbial ecology signiﬁ - Cummings et al. 1987 ; Topping and Clifton cantly by acting as substrates or supplying SCFA. 2001 ). The molar ratios of acetate, propionate, and butyrate vary between the right colon (1:0.38:0.36, respectively) and the left colon 6.6.2 Absorption and Metabolism (1:0.36:0.37, respectively) (Canani et al. 2011 ). of SCFA by Colonocytes The average concentration range of these in the entire intestine is 70–100 mM, with a relative Less than 5 % of bacterially derived SCFA appear ratio of 1:0.31:0.15 (Canani et al. 2011 ). Lactic in feces due to colonic uptake (McNeil et al. acid accumulates at acidic pH (<5.5) when SCFA 1978 ; Roediger and Moore 1981 ; Roediger and production is inhibited (Canani et al. 2011 ). Nance 1986 ). This is the major cause for the decline in the concentration of SCFAs in the large bowel. SCFA are absorbed from the perfused 6.6 Mechanism and Effects human large bowel in a concentration-dependent of Short-Chain Fatty Acids manner (Rupin et al. 1980 ). Most of the SCFA on Health uptake (60 %) happens due to simple diffusion (protonated SCFA) through the hydration of the

Diet has signifi cant impact on colon health. luminal CO2 . The remainder is absorbed by cel- Different mechanisms have been elaborated to lular uptake of ionized SCFA involving the discuss the action of SCFA actions in inhibiting cotransport of sodium and potassium ions colon cancer. (Fleming et al. 1991 ; Engelhardt et al. 1995 ). Its 108 6 Short-Chain Fatty Acids uptake has been associated with the transport of 6.6.3 Effect of SCFA on Colonic water that is high in distal end than proximal Blood Flow and Muscular colon (Bowling et al. 1993). SCFA stimulate Activity colonic fl uid and electrolyte transport (Clausen et al. 1991). Retention of calcium and magne- In vitro studies show that acetate and propionate sium ions is observed in animals fed with oligo- are more effective, even at low concentrations (3 saccharides (Bird et al. 2000). Studies in human mM) and dilated precontracted colonic arterioles indicate the enhanced absorption of calcium ions in human colonic segments (Mortensen et al. after the consumption of fermentable 1990). SCFA also cause greater blood fl ow (1.5– carbohydrate, including inulin and beet fi ber 5.0 fold). The mechanism of action of SCFA on (Trinidad et al. 1996 ; Choudary et al. 1997 ). blood fl ow does not involve prostaglandins and SCFA are absorbed at similar rates in various α- or β-adrenoreceptor-linked pathways regions of the large bowel (Engelhardt et al. (Mortensen et al. 1990 ). SCFA increase short- 1995 ). Acetate, propionate, and butyrate are duration contraction at low molarity (0.1–10 absorbed at comparable rates in humans and rats mM) in rats in the following order: acetate << (Umesaki et al. 1979 ; Fleming et al. 1991 ). While butyrate < propionate (Squires et al. 1992 ); and in guinea pigs, the acetate clearance is high in the this could be the combined effects of local neural proximal colon and low in the cecum and distal network and chemoreceptors. Manometric stud- colon (Engelhardt et al. 1995 ). At pH 5.5–7.5, ies in human show that ingestion of fermentable more than 50 % of SCFA are available in dissoci- carbohydrate or infusion of SCFA causes a ated form. It has been observed in vitro that puta- decrease in the gastric tone and an increase in the tive unstirred layer where reassociation of SCFA volume. SCFA possibly activate the ileocolonic may occur may affect the absorption rate. This brake in a dose-dependent manner. Modulating could be the reason for the regional difference in the upper gastrointestinal passage of food colonocyte metabolism and SCFA absorption in improves nutrient digestion. Nevertheless, rapid the colon. transit of food through the colon is believed to SCFA are the major respiratory fuels for the improve laxation. Greater blood fl ow caused by small bowel and colon (Yang et al. 1970 ; SCFA enhances tissue oxygenation and transport Windmueller and Spaeth 1977 ), and they supply of absorbed nutrients. 60–70 % of the energy needs (Roediger and McDermott 1995 ). SCFA suppress glucose (Ardawi and Newsholme 1985 ; Roediger and 6.6.4 Trophic Effects of SCFA McDermott 1995 ) and spare pyruvate metabo- (Butyrate and Propionate) lism (Butler et al. 1990). Butyrate is the major in Maintaining Normal intestinal fuel even when competing substrates Colonic Cell Phenotype such as glucose and glutamine are available (Roediger and Moore 1981 ). Butyrate is oxidized Growth of colorectal and ileal mucosal cells is more in the proximal than in the distal colon. observed in vivo , when SCFA is delivered During ulcerative colitis, a defect in butyrate colorectally or intraperitonealy (Sakata and metabolism is observed. These patients have low Yajima 1984 ; Reimer and McBurney 1996 ). This butyrate and low pH and high lactic acid levels could be due to the raise in ileal and cecal (Vernia et al. 1988 ). Intracolonic infusion of glucaogon-like peptide-1 mRNA levels. Butyrate SCFA reduces the degree of infl ammation lowers the risk of malignant transformations in (Agarwal and Schimmel 1989 ; Harig et al. 1989 ). the colon. In normal rats, butyrate enhances pro- Cell proliferation in the upper crypt, measured liferation only at the crypt base. However, this is with proliferating cell nuclear antigen (PCNA), is blocked by secondary bile acid (deoxycholic reduced by treatment with butyrate (Scheppach acid) (Velazquez et al. 1997 ). Secondary bile et al. 1999 ). acids are cytotoxic and are associated with 6.6 Mechanism and Effects of Short-Chain Fatty Acids on Health 109 greater susceptibility to the development of can- in the cell cycle inhibitor, p21 gene, which har- cer (Deschner et al. 1983 ; Lipkin et al. 1983 ; bors butyrate-responsive elements at the pro- Terpstra et al. 1987 ). Normal mucosa from moter region. Upon butyrate treatment, this gene colorectal carcinoma patients resists bile-acid- was reported to be induced due to HDAC inhibi- induced apoptosis, indicating the role of high lev- tion resulting in G1 phase arrest (Blottiere et al. els of bile acids in resisting apoptosis (Payne 2003 ) (Fig. 6.7 ). et al. 1995 ). SCFA may aid in preventing cancer Butyrate activates tissue inhibitor matrix by lowering the intracolorectal pH, as bile acids metalloproteinase (TIM) -1 and −2 and inhibits are protonated and insoluble at pH 6 and could the activity of metalloproteinases (MMPs), which not be absorbed by the colonocytes. Lower pH in turn reduce the adherence of cancer cells to the also inhibits the bacterial conversion of primary basement membrane protein laminin substrate bile acids to secondary, thus lowering the cancer through fi bronectin or type IV collagen and, thus, potentiality (Nagengast et al. 1988 ). prevent the proliferation of cancer cells. Infl ammatory cytokines (IL-4, TNF-α) aid butyrate in preventing colon carcinogenesis (Andoh 6.6.5 Molecular Mechanism et al. 2003 ). of Short-Chain Fatty Acid G-protein-coupled receptors (GPCRs) consist in Preventing Colon Cancer of proteins involved in the transduction of extracellular stimuli to intracellular signals. The fam- At a molecular level, butyrate affects the gene ily of GPCR protein is activated on binding with expression via the phosphorylation and acylation a ligand or agonist that leads to a conformational of histone proteins (Archer and Hodin 1999 ). It is change and activation of the G-protein heterodi- recognized as an inhibitor of histone deacetylase mer. Recently, acetate, propionate, and butyrate (HDAC) activity. Despite its rapid metabolism, it were reported to act as ligand for GPR41 and can have a prolonged effect on histone acetyla- GPR43 that were expressed in colonic mucosa, tion and the induction of differentiation markers suggesting their role in the normal development in colon cancer cells, including alkaline phospha- or function of the colon tissue (Tazoe et al. 2008 ). tase. It is not clear if this extended action refl ects Tang et al. revealed that exposure of GPR43 in protection on binding with HDACs or if it is an human colon cancers to SCFA causes its reduc- independent action. In NCM460 cells, butyrate is tion, thus making cells more sensitive and lead- the only HDAC inhibitor that causes a consistent ing to apoptosis by cell cycle arrest at G0/G1 increase in alkaline phosphatase activity (Lee and phase. Thus, SCFA may infl uence GPR43 expres- Workman 2007). However, this contradicts with sion and prevent colon cancer development and the results in Caco-2 cells where a variety of progression (Tang et al. 2011 ) (Fig. 6.7 ). HDAC inhibitors induces alkaline phosphatase Rapamycin (mTOR) negatively regulates activity. HDAC inhibitors in clinical trials include autophagy, and the autophosphorylation at vorinostat, phenylbutyrate and romidepsin. It is Ser2481 is considered as indicator of autophagy. postulated that inhibitors of HDAC activity cause Propionate treatment of colon cancer cells changes in the expression of genes coding for (HCT116) showed a strong time-dependent proteins and microRNAs. Similar to vorinostat, reduction in the phosphorylation state at Ser2481, sodium butyrate has also been reported to exert while no change was observed in the total mTOR antiproliferative activity on many cells types, levels. Inactivation and activation of p70S6K (at thus demonstrating the preventive effects of Thr389, Thr421/Ser424) infl uence butyrate on colon cancer and adenoma develop- mTOR. Propionate treatment reduced the phos- ment (Bornet et al. 2002 ) (Fig. 6.7 ). phorylation of p70S6K at Thr389, causing down- Histone hyperacetylation results in chromatin regulation of the mTOR signaling pathway and relaxation, which makes DNA more accessible to inducing autophagy. Propionate also mediates transcriptional factors. It also causes an increase AMPK activation causing a reduction in ATP lev- 110 6 Short-Chain Fatty Acids

Fig. 6.7 Molecular mechanism of inhibition of colon and butyrate induced ( b) cell cycle arrest and apoptosis, carcinogenesis by SCFA. (a ) Major mechanisms by and (c ) increased ROS which SCFA inhibit colon carcinogenesis. Propionate els in colon cancer cells due to the falling out of own defense mechanism (mitophagy), where mitochondrial membrane potential and releases increased mitochondrial confi scation was reactive oxygen species (ROS) (Tang et al. 2011 ). observed in lysosomes following the stimulation Excess ROS induces autophagy. of hepatocytes catabolism with glucagon (Tang Mitochondrial ATP depletes, and excess ROS et al. 2011 ). Mitophagy plays a key role during is produced when cell environment changes, cellular quality control. Mitochondrial fi ssion, causing release of proteins that promote cell fusion, and mitophagy are all important for mito- death. Fascinatingly, the cells can adapt to its chondrial homeostasis. Mitophagy has also been References 111 shown to be required for steady state turnover of References mitochondria, for the adjustment of mitochon- drion numbers to changing metabolic require- Abrahamse SL, Pool-Zobel BL, Rechkemmer G (1999) ments and during specialized developmental Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracel- stages in mammalian cells such as during red lular calcium concentration by oxidative stress in iso- blood cell differentiation (Youle and Narendra lated rat distal colon cells. Carcinogenesis 2011). HCT116 colon cancer cells treated with 20(4):629–634 propionate showed mitophagy, which selectively Agarwal VP, Schimmel EM (1989) Diversion colitis: a nutritional defi ciency syndrome? Nutr Rev targets mitochondria with a depolarized mem- 47(9):257–261 brane potential (Tang et al. 2011 ) (Fig. 6.7 ). 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Abstract Colorectal cancer (CRC) is the third most common cause of mortality in developed countries and is growing in developing countries. The International Agency for Research on Cancer and World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) have accounted that colorectal cancer (CRC) ranks third (approximately 8 % of all cancer deaths worldwide) among the list of other deadliest cancers. A large proportion (nearly 85 %) of colorectal cancers are related to environmental factors. Diet, dietary habits, and lifestyle factors are considered one of the prime environmental factors reported to be responsible for the high incidence of CRC. Red and processed meat cooked at elevated temperature, reﬁ ned starch, sugar, chocolate, coffee, and saturated and trans- fatty acids and poor ﬁ ber intake are closely linked to the increased risk of CRC. Environmental risks contain various types of heterocyclic amines and polycyclic aromatic hyrdrocarbons that are formed due to the reaction between free amines and sugars causing DNA mutation, leading to cancer. Other contributing factors include excess body mass and sedentary behav- iors. Chemotherapy, radiotherapy, antibiotics, and surgical removal of cancers or tumors are the current key modes of treating most CRCs. Nevertheless, the occurrence of cancer could not be spared by these techniques. Thus, this new concept of diet manipulating CRC has been introduced several years before. This has been reported to stimulate fermentation in the gut and encourage the production of short-chain fatty acids that have anticarcinogenic properties at molecular level.

Keywords Fusobacterium • Porphyromonas • Probiotics • Prebiotics • Synbiotics

Human gut and microfl ora inhabiting it play a symbiotic relationship between the gut microbes key role in determining the host health and func- and environmental changes interrupt metabolism, tions of the organs. An imbalance in the gut the immune system, etc. and may promote carci- microbiome may affect the health and could acti- nogenesis. Patients with colorectal cancer are vate colon carcinogenesis. This may also lower reported to have highest levels of Fusobacterium the host immunity. The role of gut microbes in and Porphyromonas bacteria . The benefi cial inhibiting carcinogenesis and the role of benefi - good bacteria that support the normal epithelial cial microbes in preventing colon cancer have cells in the colon and inhibit adenomas and carci- been reported earlier. Probiotics , prebiotics, nomas are lost with tumor growth. Manipulation dietary fi bers, and synbiotics, as functional food, of gut microbiome with antibiotics may dramati- are known to improve the gut health and act as cally decrease the number and size of tumor; anticarcinogenic agents. however, it will affect the good bacteria of the In synbiotics , the synergistic association of system too. probiotics and prebiotics causes cumulative ben- Elie Metchnikoff in 1907 established the sci- efi ts rather than individuals. Prebiotics and entifi c basis for the health benefi ts of lactic acid dietary fi bers form substrate for the probiotics bacteria suggesting that dependence of intesti- that ferment the former and aid in the formation nal microbes on the food makes it possible to of short-chain fatty acids, including acetate, pro- exchange harmful microbes with benefi cial pionate, and butyrate. Fecal bulking, laxation, microbes and could be adopted as a preventive and reduction in the contact time with the intesti- measure to improve health conditions. Hence, nal lining are the other physiological means by lactic acid fermented foods and other cultured which dietary fi bers protect the colon. Reductions milk products became an integral part of human in colon pH, improved immune system, etc. are diet and led to the development of the term other benefi ts of dietary fi ber. “ probiotics .” Probiotics are defi ned as live Microbial colonies throughout the length of microorganisms, which when administered in the digestive system vary in density and composi- adequate amount confer a health benefi t on the tion. Based on anatomical site, transit rate, host host. Probiotics alleviate lactose intolerance, secretion, environmental condition, substrate, control diarrhea and urogenital infection, reduce and the structure of gut wall, this microbiome cholesterol level, and prevent IBD and other varies. The stomach and proximal small intestine colon disorders. They also act as antioxidants support low number of microorganism, in and inhibit gut pathogens. Lactobacillus , contrast to the large intestine that favors dense Bifi dobacterium , Pediococcus , Leuconostoc , microbial community dominated by obligate Enterococcus, and yeast such as Saccharomyces anaerobes. A majority of bacteria belong to boulardii are recognized as probiotics. Scientifi c Bacteroides, Prevotella, Firmicutes, evidence has implicated a strong association Actinobacteria, Proteobacteria, and between diet and gut microfl ora composition, Verrucomicrobia. The human gut and its complex thus inhibiting colon cancer. Probiotics could be microbiome comprising of trillions of bacteria delivered in the form of capsules or tablets and contribute signifi cantly to the maintenance of the as functional food rich in probiotic bacteria. colon’s health and its proper function. Long-term Bacterial enzymes prevent the conversion of diet manipulates the structure and activity of gut procarcinogens to carcinogens. Short-chain microbes. Such diet- induced disruptive changes fatty acids (SCFA) are an additional mechanism (gut dysbiosis) to gut-associated microbial envi- in preventing colon carcinogenesis. SCFA, spe- ronment could cause chronic illness and are cifi cally butyrate, alleviate colon cancer. strongly associated with improper colon health. Increased production of interferon-γ, tumor This could be considered as a biomarker of necrosis factor-α, and interleukin-1β thwarts the colorectal carcinomas. Disturbance of the proliferation of cancer cells. 7 Conclusion 119

The mechanism of action of probiotics, probiotics. These are oligomers comprising of together with bioactive carbohydrates (prebiotics 4–10 monomeric hexose units. Common prebiot- and dietary fi bers) and the development of the ics include inulin, fructo-oligosaccharide, galacto- concept of synbiotics, has introduced a new arena oligosaccharide, xylo-oligosaccharide, lactulose, for cancer therapy. lactitol, soy-oligosaccharides, polydextrose, etc. Dietary fi bers are defi ned as the remnants of Inulin is reported to reduce the growth of potential the edible part of plants and analogous carbohy- pathogenic bacteria community, including E. coli , drates that are resistant to digestion and absorp- Salmonella , and Listeria . Prebiotics selectively tion in the human small intestine and with stimulate the growth or activity of benefi cial complete or partial fermentation in the human native bacteria or probiotics. They are nonviable. large intestine. It includes polysaccharides, As they are regularly consumed as a part of food, oligosaccharides, lignin, and associated plant sub- they are highly stable; however, safe consumption stances. Dietary fi ber exhibits one or more proper- is of great concern. Anticarcinogenic properties of ties, including laxation, fecal bulking and prebiotics are due to SCFA production and the softening, increased frequency and/or regularity, enhanced immunity. Modifi cation of gene expres- blood cholesterol attenuation, and blood glucose sion in the colon, cecum, and feces enhanced attenuation. Dietary fi ber sources include terres- micronutrient absorption, and modulation of trial and marine plant sources, such as cellulose, xenobiotic metabolizing enzymes is another wheat bran, oat bran, pectin, guar gum, sulfated means of action to prevent colon cancer. galactans, carrageenan, agaran, fucoidan, lami- Synbiotics are probiotics and prebiotics taken in narin, ulvan, etc. Marine dietary fi bers have not combination. The anticarcinogenic properties of been explored in detail as cereal, fruit, and vegeta- probiotics and prebiotics , as synbiotics, could be ble dietary fi bers. The type of dietary fi ber affects utilized due to their improved combination event the gut microbiome and fl ora. The dietary fi bers rather than from a single event. are categorized into insoluble and soluble dietary The molecular mechanism of action of probi- fi bers based on their water solubility. Soluble fi ber otics, prebiotics, synbiotics, and dietary fi bers with its high water-binding capacity contributes ultimately involves the fermented product, SCFA. to fecal bulking. The mechanism of action of Short-chain fatty acids are low molecular weight dietary fi ber in preventing colon carcinoma could fatty acids that are found in the human intestine be grouped as physiological and molecular mech- in different concentrations. Microbial fermenta- anism. The former includes binding of mutagens tion of bioactive carbohydrates by probiotics, or carcinogens and reducing their contact time Bifi dobacterium and Lactobacillus help in the with the intestinal lumen, fecal bulking, laxation, formation of SCFA. Major short-chain fatty acids mineral absorption, and decrease in colon pH. The include acetate, propionate, and butyrate, of molecular mode of cancer prevention by the which propionate and butyrate are concerned dietary fi bers could be attributed to their fermen- with cell differentiation, growth arrest, and apop- tation to SCFA, namely acetate, propionate, and tosis of cancer cells. Acetate makes up around butyrate. Other mechanism of action includes 60–75 % of total short-chain fatty acids, followed enhanced bile acid deconjugation, and modula- by propionate (15–20 %) and butyrate (15 %). tion of infl ammatory bioactive substances. Carbon dioxide and hydrogen gases are also Prebiotics are defi ned as a selectively ferment- formed along with these organic acids. Acetate able nondigestible oligosaccharide or ingredient (C2), propionate (C3), and butyrate (C4) released that brings specifi c changes, both in the composi- in the intestinal lumen are readily absorbed and tion and/or activity of the gastrointestinal micro- utilized by colonocytes and other tissues as fl ora, conferring health benefi ts. It also prevents energy sources. Acetate improves ileal motility colorectal cancer by modifying human gut micro- and immunity and increases blood fl ow in the fl ora and promotes the growth of benefi cial colon. It is also involved in adipogenesis. 120 7 Conclusion

In addition, it lessens the lipopolysaccharide- Propionate and butyrate also exhibit strong stimulated tumor necrosis factor, interleukin, and antiinﬂ ammatory properties by inhibiting the nuclear factor-κB level, thus playing a key role in production of TNF-α, NF-κB, and IL-8, IL-10, affecting host immunity. SCFA adjust the colonic IL-12 in immune and colonic epithelial cells. and intracellular pH, cell volume, and ion trans- The anticarcinogenic effects of pro-, pre-, and port and regulate cell proliferation, differentia- synbiotics and dietary ﬁ bers are an emerging area tion, and gene expression. About 70–90 % is and are of immense prospect. The functional utilized by the colonocytes, and the remaining is foods and nutraceuticals comprising of these excreted through feces. Butyrates play a note- would thwart not only cancer but also other non- worthy role in inhibiting histone deacetylase communicable diseases. The mechanism of resulting in histone hyperacetylation and growth action of these is yet to be investigated and stud- inhibition in the colonic epithelial cells. ied in detail. Index

A effects of , 102 Acetate , 106 , 107 , 119 enemas , 106 A fl atoxins , 10 gut epithelial cells , 108 Allochthonous fl ora , 3 in vitro studies , 102 Anticancerous activity, of probiotics , 23–25 tissue inhibitor matrix metalloproteinase Anticarcinogenic properties activation , 111 dietary fi bers , 43 Butyrate paradox , 49 , 104–105 prebiotics , 73 , 74 , 119 probiotics , 119 synbiotics , 119 C Anti- Helicobacter pylori activity , 23 Carbohydrate gap , 101 Antimicrobial activity (AMA), of probiotics , 18–19 Carcinogens , 9–10 Antimicrobial peptides (AMPs) , 6–7 dietary fi ber , 47 Antimutagenesis , 24 Colon cancer cells, propionate treatment , 111 Antimutagenic activity, of probiotics , 26 Colon stem cells , 11 Antiproliferative effects, of probiotics , 28 Colorectal cancer (CRC) Apoptosis description , 8 and anti-cancer , 106–108 development , 8 and autophagy , 113 dietary fi bers and cell differentiation , 27 cellular mechanism , 48 propionate and acetate , 107 physiological mechanism , 47 Arabinogalactans , 46 environmental risk factors , 9 Arabinogalacto-oligosaccharides , 68 genetic and environmental factors , 106 Arabino-oligosaccharides , 68 (see also Gut microbiota) Arabinoxylo-oligosaccharides , 68 heterocyclic amines , 10 Autochthonous fl ora , 3 incidence and mortality rates , 9 Autophagy and apoptosis , 113 mutagen/carcinogens role , 9 mycotoxins , 10 N-nitroso compounds , 10 B origin , 11 Bacillus bifi dus , 1 7 patterns , 8 Bacillus polyfermenticus , 2 9 polycyclic aromatic hydrocarbons , 10 Bacterial fermentation , 42 probiotics on galacto-oligosaccharides , 64 apoptosis and cell differentiation , 27 in gastrointestinal system , 48 immunomodulation , 27 Bacteriocins , 19 intestinal microfl ora, alteration of , 25 classifi cation , 20 intestinal pH lowering , 27 Bacteroides fragilis , 7 mutagenic and carcinogenic compounds, Bifi dobacteria , 17 , 69 inactivation of , 25 Bifi dobacterium lactis , 8 8 tyrosine kinase signaling pathway , 27 Bifi dobacterium spp., benefi cial effect , 58 Complex carbohydrates. See Dietary fi bers Bioantimutagens , 24 Conjugated linoleic acids (CLA) , 92 Biosurfactants , 20 Crohn’s disease (CD) , 23 Butyrates , 27 , 120 Crypts , 11

D esophagus , 2 Dendritic cells (DCs) , 27 , 93 gallbladder , 3 Diarrhea , 22 gut microbiota (see Gut microbiota) Dietary fi bers laxation, fecal bulking and dilution , 48 and gut microbiota , 41 liver , 3 anticancer effects , 43 mouth , 2 anticarcinogeneic/antimutagenic activity , 43 organs function , 2 anti-tumor activity , 48 pancreas , 3 classifi cation , 40 rectum , 3 colorectal cancer small intestine , 2 cellular mechanism , 48 stomach , 2 physiological mechanism , 47 Gastrointestinal tract defi nition , 36–38 , 119 fermentation,100 dietary recommendations , 41 food intake and digestion , 100 gastrointestinal barrier , 46 Genomic instability, types of , 8 gut microbiome , 46 G-protein-coupled receptors (GPCRs) , 111 history , 36 Gut bacteria , 25 hydrocolloid property , 48 Gut microbes , 8 immunomodulation , 46 benefi cial gut microbes, stimulation of , 73 infl ammatory condition, SCFA , 45 role of , 118 mechanism of action , 44 Gut microbiota , 12 physiological effects , 44 after childbirth , 4 procarcinogen and carcinogen binding , 47 breast-fed vs .formula-fed infants , 5 quantifi cation defi nitions , 36 complexity , 5 SCFA formation , 49 description , 3 sources , 119 and dietary fi ber , 41 types and components , 38 dynamics in pregnancy , 4 Dietary Reference Intake value, for dietary fi ber , 41 functions , 5 , 6 Dysmutagens , 24 human health , 5 metabolic function , 8 protective effect , 6 E structural and histological function , 8 E. coli Nissle 1917 (EcN) , 18 innate and adaptive immune response , 5 Enterococcal probiotics , 18 and probiotics , 21

F H Faecalibacterium prausnitzii (FP) , 18 , 27 HCA. See Heterocyclic amines (HCA) Familial colorectal cancer , 8 HCT116, propionate treatment , 113 Fermentation, of SOS , 66 HCT-15 cells , 48 Fibersol-2 , 68 Heterocyclic amines (HCA) , 10 , 43 Fructans , 65 Histone acetyltransferases , 45 Fructo-oligosaccharides (FOS) , 58 , 62 , 88 Histone deacetylase (HDAC) and resistant starch , 68 inhibitors , 111 Fucoidan , 44 , 48 Histone deacetylases (HDAC) , 45 Fusobacterium bacteria , 118 HT-29 tumor cells, butyrate treatment , 107 Human milk oligosaccharides , 89 Hydrogen peroxide , 19 G Hypercholesterolemia , 22 , 71 Galacto-oligosaccharides (GOS) bacterial fermentation , 64 in human and bovine milk , 63 I laxative effects , 64 Immodulatory activity, of probiotics , 21 microbial fermentation , 64 IMO. See Isomalto-oligosaccharides (IMO) Galacturonan-oligosaccharides , 68 Indigenous fl ora. See Autochthonous fl ora Gastrointestinal (GI) system Infl ammation , 107–108 anus , 3 Infl ammatory bowel disease (IBD) , 23 bacterial fermentation , 48 Inherited CRC , 8 colon , 3 Insoluble dietary fi bers , 42 , 119 Index 123

Intestinal microfl ora Prebiotics , 84 , 119 alteration of , 25 anticarcinogenic effects development, in infant gut , 5 , 6 benefi cial gut microbes, stimulation of , 73 Intestinal stem cells (ISCs) , 11 micronutrient absorption by colon cells , 75 Intubation , 104 SCFA and lactic acid , 74 Inulin , 61–62 , 90 , 119 xenobiotic metabolizing enzymes , 75 Isomalto-oligosaccharide (IMO) , 66–67 application , 60 and cancer , 70 chemical structure , 62 L and cholesterol , 71 Lactic acid , 108 in colon , 58 Lactic acid bacteria (LAB) , 16 and dietary fi bers , 98 , 118 health benefi ts of , 120 defi nition , 58 Lactitol monohydrate , 65 degree of polymerization , 62 Lactobacilli , 17 effect on microbes , 61 Lactobacillus acidophilus , 8 8 food ingredients, classifi cation of , 60 Lactobacillus casei , 8 8 food rich in , 61 Lactobacillus rhamnosus JB-1 , 7 fructans , 65 Lactose intolerance , 23 , 70–71 fructo-oligosaccharides , 62 Lactosucrose. See Lactulose and gut microbiota , 69 Lactulose , 64–65 health benefi ts , 70 health benefi ts and mechanism of action , 75 host health and physiology , 61 M in human digestive system , 64 Major SCFAs , 105–106 IMO , 66 Marine dietary fi ber , 40 inulin , 61 , 62 Metagenomics , 12 lactitol , 65 Mineral absorption, improvement of , 23 and lactose intolerance , 70 Minor SCFAs , 106 lactulose , 64 Mitophagy , 112 market analysis , 60 Mutagen , 9 and osteoporosis , 72 Mycotoxicosis , 10 resistant starch and dextrin , 67 Mycotoxins , 10 soy-oligosaccharides , 65 structures , 61 types , 61 , 86 N types and benefi ts , 59 Natural killer (NK) cells , 27 weight of , 61 N-nitroso compounds (NOC) , 10 xylo-oligosaccharides , 66 Non-prescription fi ber product , 39 Probiotics , 118 Nonstarch polysaccharides, 101 anticancerous activity , 23 Nutriose , 68 anti- H. pylori activity , 23 antiproliferative effects , 28 on colorectal cancer , 24 O apoptosis and cell differentiation , 27 Oligosaccharides , 58 , 63. See also Prebiotics immunomodulation , 27 intestinal microfl ora, alteration of , 25 intestinal pH lowering , 27 P mutagenic and carcinogenic compounds, PAH. See Polycyclic aromatic hydrocarbons (PAH) inactivation of , 25 Pectic-oligosaccharides , 68 tyrosine kinase signaling pathway , 27 Peptide hormones, of GI tract , 7 defi nition , 16 , 118 Peripheral venous SCFA , 101 diarrhea prevention , 22 Phase I enzymes , 50 , 75 functional foods Phase II enzymes , 50 , 75 bifi dobacteria , 17 Polycyclic aromatic hydrocarbons (PAH) , 10 , 43 E. coli Nissle 1917 , 18 Polydextrose. See Fructans enterococcus , 17 Porphyromonas bacteria , 118 F. prausnitzii , 18 Postbiotics , 18 , 19 lactobacilli , 17 Prebiotic Index (PI) , 61 and gut microbiota , 21 124 Index

health benefi ts , 84 luminal effects , 109 health promoter , 22 mammalian physiology , 108 acid production , 19 metabolic fate , 109 antimicrobial activity , 18 metabolic regulation , 108–109 bacteriocin production , 19 metabolism and absorption by biosurfactants , 20 colonocytes , 108–109 hydrogen peroxide , 19 production , 102–104 immunomodulation , 20 production in colon , 102 tight junction proteins, modulation of , 20 pro-infl ammatory mediators , 45 history , 16 trophic effects , 110–111 hypercholesterolemia prevention , 22 Short-chain fructo-oligosaccharides , 63 immunomodulation , 21 Simple carbohydrates , 100 infl ammatory bowel disease , 23 Soluble dietary fi bers , 42 , 49 , 121 lactose utilization , 23 Soluble sugars , 101 mineral absorption, improvement of , 23 Somatic mutation theory, of cancer , 9 organism, selection criteria for , 18 Soybean oligosaccharide (SOS) , 65 , 88 strains , 18 Sporadic CRC , 8 in synbiotics , 88 Symbiotic supplements , 84 Propionate , 120 Synbiotics , 85 , 118 Pro-wel , 89 anti-cancer , 89 anti-infl ammation and immunomodulation , 92 benefi cial effects , 88 R in colon , 90 Rapamycin (mTOR) , 111 complementary concept , 84 Resistant dextrin (RD), defi nition , 68 CRC treatment , 91 Resistant starch (RS) defi nition , 84 consumption , 67 in human gut system , 88 and FOS , 68 mechanism of action , 93 types , 67 SCFA , 92 Retrograded resistant maltodextrins , 67 synergistic concept , 84 Rhamnogalacturonan-oligosaccharides , 68

T S Terrestrial dietary fi ber , 38 Saccharomyces boulardii , 2 9 Tight junction proteins, modulation of , 20 SCFAs. See Short-chain fatty acids (SCFAs) Trans-galacto-oligosaccharide (TGOS) , 62 Secondary bile acids , 110 Transient fl ora. See Allochthonous fl ora Short-chain fatty acids (SCFAs) , 7 , 105–108 , 118 , 119 Trans-oligosaccharide (TOS). See Galacto- absorption , 103 oligosaccharides (GOS) butyrate paradox , 104–105 Tyrosine kinase signaling pathway, colon cancer prevention , 92 inhibition of , 2 7 colonic blood fl ow and muscular activity , 110 on colonic function , 101 determination in colon , 104 U formation , 49 Ulcerative colitis (UC) , 23 gut microbiota and health , 105–106 anti-cancer and apoptosis , 106–107 infl ammation , 107–108 X and health benefi ts , 101 Xenobiotic metabolizing enzymes , 50 , 75 leukocyte recruitment , 45 Xylo-oligosaccharides (XOS) , 42 , 66 , 89