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BIOACTIVE FOOD

BIOACTIVE FOOD

AS DIETARY

INTERVENTIONS

FOR LIVER AND

GASTROINTESTINAL

DISEASE

ACKNOWLEDGMENTS FOR BIOACTIVE FOODS IN

CHRONIC DISEASE STATES

The work of editorial assistant, Bethany L. Stevens and the Oxford-based Elsevier staff in communicating with authors, working with the manuscripts and the publisher was critical to the successful completion of the book and is much appreciated. Their daily responses to queries, and collection of manuscripts and documents were extremely helpful. Partial support for Ms Stevens work, graciously provided by the National Health Research Institute as part of its mission to communicate to scientists about bioactive foods and dietary supplements, was vital (http://www.naturalhealthresearch.org). This was part of their efforts to educate scientists and the lay public on the health and economic benefits of nutrients in the diet as well as supplements. Mari Stoddard and Annabelle Nunez of the Arizona Health Sciences library were instrumental in finding the authors and their addresses in the early stages of the books preparation.

BIOACTIVE FOOD

AS DIETARY

INTERVENTIONS

FOR LIVER AND

GASTROINTESTINAL

DISEASE

Edited by

RONALD ROSS WATSON AND

VICTOR R. PREEDY

Academic Press

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PREFACE: LIVER AND GASTROINTESTINAL HEALTH: LIVER AND GASTROINTESTINAL HEALTH

Optimum functioning of the liver and gastrointestinal systems is critical for health. They are critical for the digestion and absorption of nutrients and foods to produce growth. Nutrient and non-nutrients are important modulators of the liver function. The symp-toms related to liver dysfunction include both physical signs and symptoms of abnormal absorption of fat, changes in blood sugar, and altered metabolism. This book provides evidence that foods and their compounds can modify some of these diseases. Expert reviews are provided on liver function as people mature and mechanisms of fatty liver as modified wild and bioactive foods for hepato-protection and digestion. The data supporting actions of bioactive, and especially Chinese foods, to prevent and treat liver diseases are defined by experts. Specific individual foods and herbs have shown specific liver disease benefits including: betal leaf, selected Indian herbs, gooseberries, and curcu -min.Non-botanical materials in reviews show promise, including probiotics. In defining mechanisms including antioxidant capacity of antocyanins, extracts of pomegranate and medicinal plants as well as specifically their carotenoids show benefits in modifying liver function in reviews. Phytochemicals involvement in liver and gastrointestinal health is concisely defined.

More diverse information is provided about bioactive foods in the therapy of gastro-intestinal diseases and functions, which are many and important in health. In this book the gastrointestinal focuses on the stomach and intestine. It releases hormones that help reg-ulate the digestive process and is subject to many diseases and problems. An overview reviews functional assessment of gastrointestinal tract function and alkaline in digestive health. Reviews generally define the protective effects of bioactive botanical foods. The human microbiome diseases are defined in a metagenomic approach. Specific classes and types of foods are reviewed for selected gastrointestinal diseases. For example, a chap-ter defines the role of milk bacteria in gastrointestinal allergies. Then selected reviews of prebiotics and probiotics documented their value in irritable bowel syndrome, mucosal immunity, and viral infections. Their lactic acid and its stimulation of folate production are reviewed as mechanisms of probiotic gastrointestinal health. The actions of non-bioactive fiber on bowel health are reviewed. Several additional reviews focus on poly-saccharides from soy sauce and fiber from apples, sources readily available to the public. Dietary fibers and cholelithiasis are shown to be important in lipid lowering. Specific small molecules and defined substances are important in gastrointestinal health. Omega 3 fatty acids are shown to be an interesting story of biotechnology leading to health. One review describes fatty acids in inflammatory bowel diseases. Black plum has a long research history, which is summarized on its phytochemicals in health, as do bioactive polyphenols on other mucosal diseases of the lung. Indian plants have a historical appli-cation to health such as spices in treatment of ulcerative colitis. Ginger and basil are reviewed as an ancient remedy, while another expert gives an overview of medicinal plants in gastrointestinal diseases. Finally not all bioactive materials are safe. Therefore the dangers of herbal weight loss supplements and alcohol on gastrointestinal functions are reviewed. Bioactive foods however, as reviewed, appear to have a role in preventing the epidemic on non-communicable diseases. Clearly bioactive herbs, foods and their extracts can play key roles in liver function and gastrointestinal health.

CONTRIBUTERS

A. Aguirre

Universidad Nacional de Crdoba, Crdoba, Argentina

A. Alva

Father Muller Medical College, Mangalore, Karnataka, India

R. Arora

University of South Carolina, Columbia, SC, USA; Chief Controller Research and Development (Life Sciences and International Cooperation), New Delhi, India; Institute of Nuclear Medicine and Allied Sciences, Delhi, India

H. Asakura

Koukann Clinics, Kawasaki, Kanagawa, Japan

A. Azmidah

Father Muller Medical College, Kankanady, Mangalore, Karnataka, India

M.S. Baliga

Father Muller Medical College, Kankanady, Mangalore, Karnataka, India; Institute of Nuclear Medicine and Allied Sciences, Delhi, India

M.P. Baliga-Rao

Manipal College of Pharmaceutical Sciences, Manipal, Karnataka, India

S.B. Bhardwaj

Punjab University, Chandigarh, India

H.P. Bhat

Maharani Lakshmi Ammani Womens College, Bangalore, Karnataka, India

M.E. Bibas Bonet

Universidad Nacional de Tucumn, Tucumn, Argentina

H.K. Biesalski

Universitt Hohenheim, Stuttgart, Germany

S. Biswas

Dr. Ambedkar College, Nagpur, Maharashtra, India

R. Borneo

Universidad Nacional de Crdoba, Crdoba, Argentina

P.C. Calder

University of Southampton, Southampton, UK

M.am

Erciyes University, Kayseri, Turkey

A. etin


K. Chapman

The College of Richard Collyer, Horsham, UK

G. Cherian

Oregon State University, Corvallis, OR, USA

M.C. Collado

Institute of Agrochemistry and Food Science (IATA-CSIC), Valencia, Spain; Univeristy of Turku, Turku, Finland; Institute of Agrochemistry and Food Technology, Spanish National Research Council (IATA-CSIC), Valencia, Spain

M. Comalada

Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain; University of Granada, Granada, Spain

G. DAuria

Joint Unit of Research in Genomics and Health Centre for Public Health Research (CSISP), Valencia, Spain

G.S. de Giori

Centro de Referencia para Lactobacilos (CERELA CONICET), Tucumn, Argentina; Universidad nacional de Tucumn, Tucumn, Argentina

A. de Moreno de LeBlanc

Centro de Referencia para Lactobacilos (CERELA-CONICET), Tucumn, Argentina

M. Dey

South Dakota State University, Brookings, SD, USA

C.A. Dogi

Universidad Nacional de Rio Cuarto, Rio Cuarto-Crdoba, Argentina

B. Duncan

The University of Arizona, Mel and Enid Zuckerman College of Public Health, Tucson, AZ,

USA

G. Durmaz

Inonu University, Malatya, Turkey

F. Emma

University of South Carolina, Columbia, SC, USA

J.R. Endres

VMN Research, Enumclaw, WA, USA

H.E. Everitt

University of South Florida, Tampa, FL, USA

R. Fayad

University of South Carolina, Columbia, SC, USA; Father Muller Medical College, Mangalore, Karnataka, India

F. Fazal


C. Ferreri

Consiglio Nazionale delle Ricerche, Bologna, Italy

M.P. Francino

Joint Unit of Research in Genomics and Health Centre for Public Health Research (CSISP), Valencia, Spain; University of California, Merced, CA, USA

A. Gonzlez-Stuart

University of Texas at El Paso, El Paso, TX, USA

. Grzekowiak

University of Turku, Turku, Finland

D. Gyamfi

University of Westminster, London, UK

R. Haniadka


F. He

Takanashi Milk Products Co., Ltd., Yokohama, Kanagawa, Japan

R.A. Hegazi

Abbott Nutrition, Columbus, OH, USA

K. Hegde

Srinivas College of Pharmacy, Mangalore, Karnataka, India

R. Jaffe

Health Studies Collegium, Ashburn, VA, USA

I.R.A.P. Jati


R. Jimmy


N. Joseph


T. Kitahora

International University of Health and Welfare, Atami, Shizuoka, Japan

M. Kobayashi

Higashimaru Shoyu Co., Ltd., Hyogo, Japan

V. Kumar

International Centre for Genetic Engineering and Biotechnology, New Delhi, India

J.E. Laio

Centro de Referencia para Lactobacilos (CERELA CONICET), Tucumn, Argentina

J.G. LeBlanc

Centro de Referencia para Lactobacilos (CERELA CONICET), Tucumn, Argentina

C. Maldonado Galdeano

Centro de Referencia para Lactobacilos (CERELA-CONICET), Tucumn, Argentina; Universidad Nacional de Tucumn, Tucumn, Argentina

P. Malhotra

Institute of Nuclear Medicine and Allied Sciences, Delhi, India

P.P. Mane


N. Mathew


A. Mira

Joint Unit of Research in Genomics and Health Centre for Public Health Research (CSISP), Valencia, Spain

M. Mizuno

Kobe University, Kobe, Japan

V. Monedero

Instituto de Agroqumica y Tecnologa de Alimentos (IATA-CSIC), Valencia, Spain

J. Nandhini


Y. Nishitani

Kobe University, Kobe, Japan

R.J. Pai


P.L. Palatty


V.B. Patel

University of Westminster, London, UK

G. Perdign

Centro de Referencia para Lactobacilos (CERELA-CONICET), Tucumn, Argentina; Universidad Nacional de Tucumn, Tucumn, Argentina

M.M. Periera-Colaco


A.N. Prabhu


I. Qureshi

VMN Research, Enumclaw, WA, USA

I. Rahman

University of Rochester Medical Center, Rochester, NY, USA

M.P. Rai

Father Muller Medical College, Mangalore, Karnataka, India; University of Delhi South Campus,

New Delhi, India

Mishra Rashmi Priya

Tata Memorial Centre (TMC), Navi Mumbai, Maharastra, India

V.S. Ratnu

University of Delhi South Campus, New Delhi, India

A.A. Robson

Universit de Bretagne Occidentale, Plouzan, France

J. Rodrguez-Daz

Instituto de Agroqumica y Tecnologa de Alimentos (IATA-CSIC), Valencia, Spain

S. Salminen

Institute of Agrochemistry and Food Science (IATA-CSIC), Valencia, Spain

A. Seth

Abbott Nutrition, Columbus, OH, USA

R. Sharma

Amity Institute of Nanotechnology, Amity University, Noida, India; Pushpawati Singhania Institute of Liver, and Biliary Diseases, New Delhi, India

Q.-H. Sheng

Inner Mongolia Mengniu Dairy (Group) Co. Ltd., Huhhot, China

A.R. Shivashankara


S.K. Shukla

International Centre for Genetic Engineering and Biotechnology, New Delhi, India

M. Sugiura

National Institute of Fruit Tree Science, Shizuoka, Japan

S. Sundriyal

Institute of Nuclear Medicine and Allied Sciences, Delhi, India

V. Sunitha


R.K. Tandon

Amity Institute of Nanotechnology, Amity University, Noida, India; Pushpawati Singhania Instititute of Liver, and Biliary Diseases, New Delhi, India; Pushpawati Singhania Research Institute of Liver, Gall Bladder Diseases, New Delhi, India

K.R. Thilakchand


M. Thomas

South Dakota State University, Brookings, SD, USA

V. Vadivel


B. Valder


M.V. Venkataranganna

Connexios Life Sciences, Bangalore, Karnataka, India

P. Venkatesh

TotipotentSc Scientific Product, Gurgaon, India

J. Xaus

University of Granada, Granada, Spain

H.S. Yashavanth

Father Muller Medical College, Mangalore, India

H. Yetim


CHAPTER 1

The Alkaline Way in Digestive Health

R. Jaffe

Health Studies Collegium, Ashburn, VA, USA

The biochemical consequences of diet are the greatest influence on overall metabolism for most patients. Food choices clearly affect the course of common pathophysiological errors such as insulin resistance, metabolic syndrome, and their sequella. However, these dynamics can also be considered a leverage point an opportunity to reverse immune reactivity through practical interventions that patients can implement in their daily lives.

1. DIETARY FACTORS IN METABOLISM

The intestinal tract plays a key part in nutrient absorption, immune defense against for-eign invaders, physiologic repair from wear and tear, growth, neurohormone regulation and stress management. Disorders anywhere in the gastrointestinal system can affect the function of the entire body and overall health. Digestive competence tends to predict survival and the capacity to thrive years to decades later.

1.1. Profile: Metabolic Acidosis as a Major Cause of Chronic Disease

Toxin accumulation in the body can result from a diet that promotes metabolic acidosis (net acid excess after metabolism) as shown by low levels of buffering minerals such as potassium and magnesium. A number of large research studies involving thousands of participants have reported about the association between metabolic acidosis and insulin resistance (Jaffe and Mani, 2006; Souto et al., 2011), type 2 diabetes (Jaffe and Mani, 2006; Schulze et al., 2003), cardiometabolic risk (Murakami et al., 2008), coronary heart disease (Liu et al., 2000), and osteoporosis (Jaffe and Brown, 2000; Jehle et al., 2006), as well as cancer (Tavani et al., 2000). A typical American diet provides insufficient minerals and fiber to counter or buffer the buildup of metabolic acids and to help displacement of toxic wastes. As a result, alkaline cellular reserves within the body reduce and deplete as the intracellular environment becomes progressively acidic, mineral depleted and proton rich (Lim, 2007; Zeidel and Seifter, 1986).

1.1.1. Associated signs and symptoms

The symptoms associated with metabolic acidosis include malaise and fatigue, metabolic syndrome and diabetes, osteopenia and osteoporosis, and depression. Metabolic acidosis is associated with a broad range of clinical conditions in the body because of the biochemical reduction of the proton gradient, upon which cell energy depends. The ratio of ATP: ADP is a measure of cell energy. A ratio of 100:1 is healthy. A ratio less than 80 begins to shift cells from an elective protective, proactive, and prevention mode to a sur-vival mode.

1.1.1.1. Fatigue

Low energy is the major complaint that patients report to their primary care physician. Energy production and the ability to remove toxins safely are compromised when even minor increases in acidity occur. Metabolic acidosis has also been linked to chronic fa-tigue immune dysfunction syndrome (Jaffe and Brown, 2000). Fibromyalgia and chronic muscle pain that is unresponsive to pain medication have been documented to produce acidic end products that directly irritate and inflame nerve muscle end plates (Deuster and Jaffe, 1998). We observe restoration of vitality and quality of life when metabolic acidosis is corrected comprehensively using predictive tests compared to best outcome reference ranges thus incorporating personalized biochemical individuality into primary care.

1.1.1.2. Osteopenia and osteoporosis

Excess acid within the cells is also a key factor in osteoporosis (Maurer et al., 2003). One of the best examples of this metabolic sensitivity is the influence of acidalkali balance on skeletal structure, health, and integrity. Skeletal muscles are the largest storehouse of available minerals in the body and are thus exquisitely sensitive to small changes in pH. Even a 10% reduction in pH increases osteoclastic activity while inhibiting osteo-blastic function, inducing amplified bone mineral loss (Jehle et al., 2006). For the past 20 years, we have consistently observed 210% new bone growth confirmed by DEXA scores after just 2 years.

1.1.2. Relevant evaluations

One of the most useful assessments in the management of metabolic acidosis is self-testing for pH, which can be performed simply by the patient in their home. After 6 h of rest, we find the urine pH is equilibrated with the urinary tract cells. Costing pennies per day, this is a useful self-care test that motivates better compliance with healthier choices. Another assessment involves laboratory testing for reactive food antigens. In tandem, these tests can be pivotal in correcting metabolic acidosis and repair deficits often called inflamma-tion and their myriad sequellae.

1.1.2.1. Self-evaluation: Testing for pH

The hazard of metabolic acidosis is that it requires additional minerals to buffer and remove excess acids from the body, stripping out needed minerals with potential harm to the kidneys and urinary tract. The role of metabolic acidosis in chronic kidney disease has been extensively documented (Sahni et al., 2010).

Figure 1.1 Picture of pH strips.

Figure 1.2 Interpretation of first, morning-urine measurements.

A pH assessment of the first morning urine provides a clinically useful measure of met-abolic acidosis risk. The urine pH is a predictive indicator of the bodys mineral reserves, as well as acid/alkaline status (Whiting and Bell, 2002). Typically pH balance is restored during sleep and rest when excess acids are excreted (Shafiee et al., 2002). This capacity varies widely based on the specific toxic load and the individuals ability to make energy, deactivate toxins, and excrete those toxins as reported by Bazhin (2007) (see Figure 1.1, pH strips and Figure 1.2, reference range for urine measurement).

A value of 7.0 indicates a neutral state, a balance of acid, and alkaline elements. The first morning urine pH goal of 6.57.5 shows healthy mineral balance. Neutral or low-level acid excess reflected in lower pH values indicates that metabolic chemistry is appropriately alkaline and that the small amounts of metabolic acids built up from daily metabolism have been easily concentrated and excreted. Cell cytoplasm or cell juice functions in an exquisitely narrow, slightly alkaline optimum functional pH range (De Young, 1994; Zeidel and Seifter, 1986).

(NUBERING)Laboratory evaluation: Reducing immune reactivity

Immune responses directly and indirectly generate substantial amounts of acidic products. For the at-risk individual with impaired dietary buffering capacity, it is especially im-portant to avoid immune reactions due to antigen reactivity or other causes that can contribute to additional cell acidity in the system (Jaffe et al., 2006). A lymphocyte re-sponse assay (LRA) can identify delayed allergic reactivity. Substitution of immune re-active substances lowers acid loads.

1.1.3 Clinical interventions: the alkaline way (ALIGNMENT)Reduction of hyperacidity in the body can be achieved through a nutrient-rich alkaline diet, targeted supplementation with alkaline nutrients, and the inclusion of buffered fats.

1.1.3.1 Alkaline diet

The Alkaline Way diet is a health-promoting, fiber-rich diet that consists primarily of whole foods based on individual food tolerances and sensitivities. Preference is given to locally, vine-ripened, organic, or biodynamic sources of foods. Mineral-rich water is the preferred beverage. Reducing the net excess cell acidity supports a range of health benefits.

1.1.3.1.1 Enhancing immune defenses Alkalinizing foods improve immune de-fense and repair functions (Lee and Shen, 2008) by reducing host hospitality to chronic infections. This reduced infectious challenge results in lower levels of inflammation, more resources for anticancer surveillance, and enhanced repair capacity. Clinical strat-egies that accompany an alkaline diet include a rotation or a substitution diet to reduce exposure to reactive foods coupled with health-promoting food choices, fresh fruits and vegetables, pulses and grasses, whole grains, minimal animal protein, and a program of individualized nutritional supplements to fully meet biochemical needs.

1.1.3.1.2 Buffering cellular chemistry(FONT SIZE) A metabolically alkaline diet means that food has a buffering or cell acid neutralizing effect on in vivo cellular chemistry, in vivo (Budde and Crenshaw, 2003). The effects of specific food responses within the body can differ from that foods test tube chemistry (Gonick et al., 1968). For example, citrus fruits are alkalinizing in the body because citrate, malate, succinate, and fumarate all pro-mote the generation of more than twice as much bicarbonate as the acid contributed from the total amount of food metabolized (Brown and Trivieri, 2006). This means that citrus fruits and similar foods are acidic in a test tube environment, yet alkaline forming in the body.Figure 1.3 reflects this real-time perspective on metabolism assessing nutrition for in vivo efficacy rather than merely evaluating the ash residue of the food as has been historically performed in nutrient assays. The foods listed here are categorized based

Figure 1.3 Food and chemical effects on acidic/alkaline body chemical balance

on an empirical formula calculated from the actual composition of the foods total pro-tein, fat, carbohydrates, minerals, cofactors, and fiber contents (Jaffe, 1987).

1.1.3.2 Alkaline nutrients

A diet high in acidic foods tends to be less-nutrient-dense and fiber-rich than an alkaline forming, whole foods, immune tolerant diet. Once mineral depletion occurs, cells be-come progressively more acidic and less energetic. The cell cytoplasm proton gradient is required for the cellular power centers, mitochondria, to work effectively. When the cell becomes acidic, the proton gradient is reduced and cells become dependent on anaerobic survival metabolism. This is a less efficient form of energy production. Lower energy production shifts cells into minimal function survival mode until adequate mineral buffers are restored.

1.1.3.2.1 Buffering minerals Minerals are required to activate enzyme catalysts within cells; lack of specific minerals has been linked to numerous specific types of enzyme deficits. Supplementation at maintenance levels includes a healthy balance of cal-cium and magnesium, as well as copper and zinc, and all of the divalent cations that per-form essential buffering minerals needed for healthy function. These minerals are required supplements for individuals suffering from metabolic acidosis (also known as net acid excess) because buffered minerals neutralize metabolic acids to maintain healthy pH homeostasis inside the cell.

1.1.3.2.2 Buffering fats Short-chain and medium-chain fatty acids with less than 16 carbons such as octanoate and decanoate are alkalinizing. Found in palm kernel oil, coconut oil, and ghee (clarified butter), these short and medium chain fatty acids can accept acetate molecules.

1.1.4 Individual essential nutritional supplementation

Additional functional strategies in clinical management include the reduction of oxida-tive stress, support of detoxification processes (through healthy methylation), and reduc-tion of risks such as homocysteine. We find a healthier, least risk goal value for homocysteine to be 300000 new cases per year, which result in 41000 deaths (Jegatheesan et al., 2006). It is estimated that NTDs represent about a tenth of all congenital conditions and is the third most important after congenital cardiac diseases and Down syndrome. In countries where habitants do not consume folate-rich diets, the incidence of NTDs is elevated with an inverse relation to the economic situation. However, in countries with high incomes, an elevated risk of NTDs is associated with poor maternal education.

3.2. Folate and Anemia

Besides the NTDs, another important and frequent manifestation of folate deficiency is megaloblastic anemia. This pathology is the result of the lack of DNA synthesis as a con-sequence of an insufficient amount of plasmatic folate. This decreased DNA replication leads to a lower production of hemoglobin during erythropoiesis, which is manifested by the presence of abnormally enlarged erythrocytes (megalocytes) that have a lower con-centration of hemoglobin and other hematological alterations. Megaloblastic anemia is caused not only by folate deficiency but also by the deficiency of vitamin B12, as both have similar clinical manifestations.

3.3. Folate and Cardiovascular Disease

Folate deficiency has also been implicated in the increasing plasma homocysteine con-centrations that in turn can elevate the risk of cardiovascular diseases. However, fortifi-cation with folic acid has been shown to be ineffective in decreasing the risk of cardiovascular diseases and mortality in healthy adults. The American Heart Association does not recommend B-vitamin supplementation; it suggests that folate levels should be obtained from a balanced diet to reduce the incidence of heart disease and stroke (Kelly and Anne, 2010).

3.4. Folate and Other Diseases

Besides insufficient intake, folate deficiency can also be due to other causes, such as certain cancer treatments where antifolate drugs (such as methotrexate) are used. Now-adays, another multitargeted antifolate drug (ALIMTA) is used, which targets various folate-dependent enzymes such as thymidylate synthase and dihydrofolate reductase. As a part of current treatment protocols, folic acid is coadministered with the antifolate drugs to prolong the treatment.

Another cause of folate deficiency is malabsorption. The mechanism of folate absorp-tion in the jejunum has been recently described; certain pathologies (such as celiac disease and tropical sprue) can affect absorption and thus cause folate deficiency. Surgical removal of the upper intestinal tract (such as partial gastrectomy or jejunal resection), cer-tain inflammatory diseases (such as Crohns disease), the use of sulfasalazine (a noncompetitive inhibitor of the reduced folate carrier), or alcohol abuse can also affect folate absorption.

3.5. Folate and Cancer

Since folate plays a key role in DNA synthesis and repair by acting as a precursor in purine synthesis and maintains DNA stability by donating one-carbon groups, it is not surprising that this vitamin is involved in certain types of cancers. Several in vitro studies from animals and humans showed that folate deficiency can result in the demethylation of cytosine, global DNA hypomethylation, proto-oncogene activation, and chromosomal instability (Duthie, 2011). It was also demonstrated that a low intake of dietary folates can cause an insufficient incorporation of uracil, breakup of DNA strands with chromosomal breakage, and malignant transformations (Duthie, 2011).

In animal studies, it was shown that low-folate diets are associated with an elevated risk of colorectal cancer and that folic acid suppresses the growth of the cancer (Giovannucci, 2002).

Since folate levels can affect DNA methylation, folate has the potential to act in the carcinogenesis of colon cells by affecting not only their severity and duration but also their genes, tissues, and malignant transformations. This is the reason why an increased intake of dietary folate and high levels of folate in blood are generally associated with decreased risks of certain kinds of malignancies, although a complete protector role for this vitamin against carcinogenesis has been questioned by many researchers. Empiric evidence does not seem to support the hypothesis that the incomplete DNA methylation of the entire genome as a direct consequence of low folate levels can increase the risk of colon cancer in humans. Even with the evidence that folate deficiency causes genomic instability by inducing DNA damage, inhibiting DNA repair, and increasing malignant transformations, definitive evidence that shows a causal relationship between the bio-markers of genomic stability and cancer risk does not exist (Duthie, 2011). Some exper-imental evidence has suggested that folate deficiency would promote initial stages of carcinogenesis, while high doses of folic acid could increase the growth of cancerous cells. As a consequence of fortification with folic acid in the United States, an important increase in nonmetabolized folic acid and circulating folate concentration has been ob-served. These results are creating concerns regarding the safety of folic acid fortification programs, especially with respect to the risk of developing cancer (Smith et al., 2008).

Various explanations have been elaborated to explain how folic acid could contribute to cancer development, such as the possible presence of nonidentified preneoplastic injuries with the potential to develop cancer in high-risk populations and the inability of the body to metabolize increased doses of B vitamins (Carroll et al., 2010). Another event that could also be related to the development of many kinds of human cancers is the overexpression of the epidermal growth factor receptor, transferrin receptor, and folate receptor. The folate receptor has been widely used as a ligand to deliver therapeutic agents to cancerous cells due to its high-affinity union (Zhang et al., 2010).

On the other hand, large discrepancies exist between the results of studies related to populations with a history of adenomas, in which some researchers demonstrated a clear reduction of the risk of recurrence of adenomas, while others did not observe the same effects (Carroll et al., 2010). All these conflicting results have raised concerns about the supplementation with folic acid and the risk of developing cancers, especially respect to the new fortification policies of many countries (Ulrich, 2008). This demonstrates that it is important to establish the riskbenefit relationship of folate and folic acid in regard to cancer incidence and their chemopreventive effect.

4. FOLIC ACID FORTIFICATION AND SUPPLEMENTATION

The increased occurrence of folic acid deficiency in the world and the gravity of the pathologies it causes (such as neural tube malformations, cardiac diseases, and megaloblastic anemia) have obliged many governments to adopt different folic acid fortification policies. Besides the above-mentioned causes of folate deficiencies, some population groups require additional vitamin uptakes. It has been reported that at least a third of pregnant women and infants do not reach their folate requirements through their conventional diet alone. For all these reasons, fortification of flours and other foods with folic acid has now been imple-mented in over 57 countries. Thus, in countries such as Canada and the United States, flour fortification with folic acid is obligatory since 1998; other countries followed their lead, such as Argentina in 2002. However, the effectiveness of these fortification programs depends largely on the eating habits of the proposed consumers. For example, flour fortification would be ineffective in some Asian and African countries where many fam-ilies, especially in the poorest regions that are most at risk of developing vitamin deficien-cies, do not regularly consume commercial foods prepared with flour. On the other hand, many countries have not adopted a National Fortification Program with folic acid because of its potential undesirable side effects. The main concerns are based on the fact that folic acid is aggregated at concentrations that allow persons with low folate intake to reach the RDA, to prevent pathologies associated with folate deficiencies. At these levels of fortifi-cation, those with normal or elevated folate ingestions would be exposed to an excessive folic acid intake, which in turn can mask the early hematological manifestations of vitamin B12 deficiency. This is important since it has been estimated that 1030% of people older than 50 years have a reduced ability to naturally absorb vitaminB12, and consequently,20% of the general population in industrialized countries is potentially deficient in this vitamin (Asrar and OConnor, 2005).

Since folate fortification levels are based on the requirements of the general popula-tion, some groups could be exposed to extremely high levels of folic acid, such as chil-dren, whose vitamin requirements are lower than those of adults. It has even been suggested that the fetus could be exposed to excessive amounts of folic acid due to sup-plementation of the mother during pregnancy, in addition to eating fortified foods, and this could favor the selection of methylentetrahydrofolate polymorphism that is associated with a group of debilitating diseases.

With respect to supplementation, several different alternatives to folic acid have been studied, such as the administration of (6S) 5-MTHF, which is more effective to increase the concentration of folate in erythrocytes in potentially childbearing women. Since 5-MTHF does not mask B12 deficiency, this folate form would be a more efficient and secure alternative than supplementation with folic acid (Lamers et al., 2006). Al-though milk has not been traditionally considered an important source of folates com-pared to folate-rich food products, in vitro studies using a dynamic gastrointestinal model demonstrated that 5-MTHF in milk was easily released from the milk matrix and highly available for absorption (6070%) (Verwei et al., 2003). It was shown that synthetic folic acid is absorbed and transported to the liver where it is reduced and a por-tion is methylated (Wright et al., 2003). In contrast, natural folates (such as 5-MTHF present in foods and produced by microorganisms) are reduced and methylated before being absorbed, making them more bioavailable than folic acid (Asrar and OConnor, 2005).

Other foods that have been proposed as vehicles for folic acid supplementation include rice. Since rice is a poor source of essential micronutrients, including folates, met-abolic engineering strategies have been developed to increase their concentrations. By overexpressing two Arabidopsis thaliana genes of the pterin and the pABA branches of the folate biosynthetic pathway, folate concentrations were 100 times higher com-pared to the values of wild-type rice. Consumption of 100 g of polished raw grains is sufficient to meet a fourfold intake of the adult daily folate requirement (Storozhenko et al., 2007). However, people living in lower socioeconomic conditions are less willing to accept genetically modified (GM) rice compared to higher-income populations, a clear setback in fortification programs that are to be directed to these higher-risk populations whose folate intake is inadequate.

Another way to increase folate consumption is to increase its levels in foods that are consumed by large population groups; the use of folate-producing microorganisms is thus an interesting alternative to fortification with folic acid by producing fermented foods with elevated concentrations of natural forms of this essential vitamin.

5. FOLATE BIOSYNTHESIS AND LACTIC ACID BACTERIA

Lactic acid bacteria (LAB) are a group of microorganisms that are normally used as starter cultures in the manufacture of a large variety of fermented foods. Besides their important fermentative capacities, LAB can also increase the safety, shelf life, nutritional value, flavor, and overall quality of fermented products. Certain strains of LAB are able to produce, release, and/or increase specific beneficial compounds in foods. These func-tional ingredients are sometimes referred to as nutraceuticals that can be defined as any substance considered as food or a part of food that can confer medical or health ben-efits including the prevention and/or treatment of disease. These ingredients can be mac-ronutrients (such as unsaturated fatty acids present in some oils), micronutrients (such as vitamins), or nonnutritive compounds (such as hydrolytic enzymes and flavonoids) and can be naturally present in foods (such as omega-3 fatty acids in fish or vitamin C in citrus fruits) or added (such as milks fortified with calcium and vitamin D and cereals fortified with folic acid) (Hugenholtz et al., 2002). The selection of strains delivering health-promoting compounds (nutraceuticals) is now the main objective of several research groups. Most bacteria are auxotrophic for several vitamins, but it has been shown that certain strains have the capacity to synthesize B-group vitamins as demonstrated by the fact that some fermented foods contain elevated levels of B-group vitamins as a result of microbial biosynthesis.

5.1. Folate Biosynthesis from Lactic Acid Bacteria

Numerous studies have shown that industrial lactic acid bacteria such as Lactococcus (Lc.) lactis and Streptococcus (St.) thermophilus have the ability to synthesize folate. This explains why some fermented dairy products, including yogurt, contain higher amounts of folate than nonfermented milk products. However, some works have shown that the ability of microbial cultures to produce or utilize folate varies considerably and is a strain-dependent trait. The amount of folic acid found in cows milk ranges from 20 to 60 g l-1, whereas its concentration in yogurt may be increased, depending on the starter cultures used and on the storage conditions, to values above 200 g l-1(Wouters et al., 2002). This level depends on the strain of St. thermophilus and Lactobacillus (L.) delbrueckii subsp. bulgaricus used because the latter organism utilizes folates for its growth. It is now known that not only do yogurt starter cultures and Lc. lactis have the ability to produce folates but other LABs also have this important property. Lactoba-cillus acidophilus is reported as being able to produce folate in chemically defined medium as can L. plantarum (LeBlanc et al., 2010a). Other LAB such as Leuconostoc lactis and Bifidobacterium (B.) longum were also reported as folate producers. Some Propionibacterium (P.) strains, well-known producers of vitamin B12, can produce high quantities of folates, so these LAB could potentially increase folate levels in milk.

It was observed that a combination of St. thermophilus/bifidobacteria/Enterococcus (E.) faecium increased folate levels, and a combination of St. thermophilus and Bifidobacterium animalis could increase folate levels by sixfold, representing 15% of RDA (Crittenden et al., 2003). It is well established that St. thermophilus strains are dominant producers of folates in milk, principally producing 5-MTHF, giving rise to yogurts with more than six times 5-MTHF content compared with the control after 12 h of fermentation (Holasova et al., 2004). In the case of B. longum, some strains were recognized as mod-erate producers with a maximum increase of 73% of 5-MTHF after 12 h of fermentation (Holasova et al., 2004). On the other hand, Propionibacterium freundenreichii subsp. Sherma-nii strains did not influence 5-MTHF levels during fermentation. In all cases, the max-imum concentration of 5-MTHF was highest between 6 and 12 h of fermentation, and then a decrease in the 5-MTHF content was observed (Holasova et al., 2004).

In addition to obtaining fermented milk products using adequately selected starter cultures that can increase vitamin concentrations, it is possible to increase the folate level naturally through the addition of some fruit component (Holasova et al., 2005). There-fore, a good flavor of fermented melon juice or melon concentrate that possess high levels of folate and vitamin B12 could be the beginning of a line of products with a long shelf life that can be directed at populations with B vitamin deficiencies.

Besides fermented dairy products, microorganisms are able to increase folate content in a wide variety of other foods. For example, fermentation of rye dough to produce bread is frequently accompanied by increase in folate content (Kariluoto et al., 2006). In these studies, the increase of this vitamin during fermentation was mainly due to folate synthesis by yeasts, whereas LAB did not produce folate but rather consumed it. The adequate selection of strains, for example, by replacing folate consumers with folate-producing LAB, could significantly increase folate content in these breads.

Also it has been reported that it is possible to select starter cultures of LAB that pro-duce significant amounts of 5-MTHF (almost twice the basal concentration) during vegetable fermentation (Jagerstad et al., 2004). To optimize the entire process, it is im-portant to carefully check the folate concentration in raw vegetables. Folate losses during processing must be limited as much as possible and optimizing the conditions to favor the microbiological biosynthesis of folates is essential to increase folate levels in the final product.

Another example of use of LAB to improve folate level in fermented products is in the fermentation of corn flour, where an increase of folate of almost threefold after 4 days of fermentation at 30 C has been obtained (Murdock and Fields, 1984).

Other studies performed with the aim to determine if the exogenous vitamin can affect folate synthesis by bacteria have shown that production is strain dependent; some bifidobacteria did not produce folate when this vitamin was already present, whereas others produced it regardless of the vitamin concentration. It has been suggested that in some strains, folate biosynthesis might not be regulated; this is confirmed by the fact that the final concentration of this vitamin was at least 50-fold higher than the require-ment of all strains (Pompei et al., 2007).

It has been shown that different forms of folates are produced by LAB, some even produce folates with more than 3 glutamyl residues. An example is Lc. lactis, where up to 90% of the total produced folate remained in the cell and was identified as being 5,10-methenyl-THF and presumably 10-formyl-THF, both with four, five, or six glutamate residues (Sybesma et al., 2003b). In St. thermophilus, much less of the total produced folate remained in the cell and was identified as being 5-formyl-THF and 5,10-methenyl-THF, both with three glutamate residues. These differences in distribu-tion can probably be explained by the different length of the polyglutamyl tail of the two microorganisms. One of the main functions of the polyglutamyl tail is thought to be the retention of folate within the cell. It can be assumed that cell retention of folate is mainly a result of the negative charge of the carboxyl groups of (polyglutamyl) folate (pKa of 4.6). Moreover, in St. thermophilus, the intra- and extracellular folate distribution was influ-enced by the pH. Cells that were grown at low pH had a larger extracellular folate frac-tion than cells that were cultured at high pH. Consequently, at low intracellular pH, a higher concentration of the folate is protonated and electrically neutral, enhancing transport across the membrane. In Lc. lactis, pH did not seem to affect the intra- and extracellular folate distribution (Sybesma et al., 2003b).

The application of biofortification of daily products using vitamin-producing micro-organisms is an interesting alternative to the use of synthetic folic acid in fermented foods. The careful selection of folate-producing strains and the optimization of their production are essential and could lead to natural enrichment of folate in different products (Holasova et al., 2004; Sybesma et al., 2003b).

5.2. Folate and Probiotics

Because of the numerous beneficial properties that have been attributed to LAB, these are the most commonly used probiotic microorganisms and can be defined as live micro-organisms which when administered in adequate amounts confer a health benefit on the host (FAO/WHO, 2001). It was shown that St. thermophilus possesses certain pro-biotic characteristics such as providing resistance to biological barriers (gastric juice and bile salts), improving intestinal microflora and lactose digestion in lactose-intolerant individuals, stimulating the gut immune system, producing a high quantity of folate ex-tracellularly, and alleviating the risk of certain cancers, ulcer, and inflammation. To clas-sify a bacterium as a probiotic, it has to satisfy a series of requirements that vary depending on the research group, but basically it should possess a generally recognized as safe (GRAS) status to be able to survive through the gastrointestinal tract (GIT) and adhere to the human intestinal cells in addition to exerting health benefits in the host. Since some LAB can produce significant amounts of folate and certain strains are able to survive in the GIT, these beneficial microorganisms could be used as efficient probiotics to produce or liberate folate in the GIT. Some research have shown that the amount of folate synthe-sized in the human GIT is significant and could be clinically important if it is available (Rong et al., 1991). There is direct evidence in vivo that folate synthesized by bacteria could be absorbed throughout the intact large intestine and incorporated into tissues (Rong et al., 1991).

Recent reports have shown that some probiotic microorganisms (such as bifidobac-teria and propionibacteria) have the ability to synthesize folates (Holasova et al., 2004; Pompei et al., 2007). The oral administration of folate-producing probiotic strains may confer a more efficient protection against inflammation and cancer by exerting the beneficial effects of providing folate and by delivering it to colonic rectal cells (Pompei et al., 2007). In humans, folate is also produced by the microbiota in the small intestine and is assimilated by the host (Camilo et al., 1996). Although microbial folate synthesis is believed to supply only a minor source of the total absorbed folate in humans (Bates, 1993), the contribution of the microbiota to the folate requirements of the high cell turnover intestinal epithelium is unknown. A mechanism for luminal folate absorp-tion by cells in the human colon has been reported (Dudeja et al., 1997), which suggests that folate produced in situ by the colonic microbiota may be utilized by cells in the co-lonic epithelium. Asrar and OConnor (2005) also showed that bacterially synthesized folate is absorbed across the large intestine and incorporated into the liver and kidneys of piglets. They predicted that approximately 18% of the dietary folate requirement in piglets could be met by folate absorption across the large intestine. Another important finding is that increased intestinal bifidobacteria populations have been correlated with an enhanced folate status in rats (Krause et al., 1996). It is therefore possible that probiotic bacteria active in the intestinal tract may be able to contribute to the folate requirement of colonic epithelial cells. However, further research is required to determine if these bacteria produce folate in the intestinal environment, the form in which this folate oc-curs, the availability of this folate for transport and utilization by colonocytes from the lumen, and the contribution of the intestinal microbiota to the total folate requirement of colonic epithelial cells (LeBlanc et al., 2010a).

Other probiotic organisms including E. faecium and Saccharomyces cerevisiae boulardii have potential to be used in probiotic products. Yogurt is the most important delivery vehicle for probiotic organisms. Cheddar cheese, dips, and spreads are becoming popular as alternative products for incorporation of probiotics.

5.3. Folate Production Using Genetically Modified Lactic Acid Bacteria

The folate biosynthesis genes have been identified in Lc. lactis, L. plantarum (Kleerebezem et al., 2003), and L. delbrueckii subsp. bulgaricus (van de Guchte et al., 2006). This new information opened the doors to numerous studies and allowed the development of many metabolic engineering techniques that are necessary not only to understand the complex metabolic pathways but also for the genetic modification of LAB to produce biological compounds. Lc. lactis is by far the most extensively studied lactic acid bacte-rium, and over the last decades, a number of elegant and efficient genetic tools have been developed for this starter bacterium. These tools are of critical importance in metabolic engineering strategies that aim to inactivate undesired genes and/or (control) the overexpression of existing or novel ones. In this respect, especially, the nisin-controlled expression (NICE) system for controlled heterologous and homologous gene expression in Lc. lactis has proven to be valuable. The design of rational approaches to metabolic engineering requires a proper understanding of the pathways that are manip-ulated and the genes involved, preferably combined with knowledge about the fluxes and control factors. Metabolic engineering of more complicated pathways involved in sec-ondary metabolism has only recently begun with the engineering of exopolysaccharide production in Lc. lactis (Levander et al., 2002) and continued with other complicated pathways such as the biosynthesis of folate (Green et al., 1996). This biosynthesis includes parts of glycolysis, the pentose phosphate pathway, and the shikimate pathway for the production of the folate building block pABA, while the biosynthesis of purines is re-quired for the production of the building block GTP. In addition, a number of specific enzymatic steps are involved in the final assembly of folate and for production of the var-ious folate derivatives (Sybesma et al., 2003a).

It is well known that some LAB cannot synthesize folate because some of the genes involved in folate biosynthesis are not present in their genome; this is the case for L. gasseri (Wegkamp et al., 2004), L. salivarius (Claesson et al., 2006), L. acidophilus, and L. johnsonii (van de Guchte et al., 2006).

It has been shown that metabolic engineering can be used to increase folate levels (Table 16.1) in Lc. lactis (Sybesma et al., 2003a; Wegkamp et al., 2007), L. gasseri (Wegkamp et al., 2004), and L. reuteri (Santos et al., 2008).

In cells, folate is present predominantly in the polyglutamyl form because many folate-dependent enzymes have increased affinity for polyglutamyl folates than for mono-glutamyl folates. The enzyme responsible for polyglutamyl folate synthesis and the cor-responding elongation of the chain is polyglutamyl synthetase (EC 6.3.2.17), which is encoded by the folC gene in Lc. lactis. Until now, all sequenced microbial genomes pos-sess folC or a similar gene (Sybesma et al., 2003a).

The controlled overexpression of folKE genes in Lc. lactis that codes for 6-hydroxy-methyl- dihydropterin pyrophosphokinase ( folk) and GTP cyclohydrolase ( folE) pro-duced a tenfold increased in the production of extracellular folate and a threefold increase in the production of total folates; meanwhile, overexpression of folA that codes for dihydrofolate reductase decreased (by 50%) the production of total folates. Also it was observed that the combined overexpression of folKE and folC favored the accumulation of intracellular folate (Sybesma et al., 2003a). Furthermore, the overexpression of the first enzyme of the biosynthetic pathway (GTP cyclohydrolase I) showed a big potential as a strategy to increase the flux through the folate biosynthesis pathway. This presumption is based on the fact that this enzyme in Bacillus subtilis has a low turnover and is not regulated by negative feedback (De Saizieu et al., 1995).

Even though inducible systems are useful, in food fermentations, it is preferable to use constitutive promoters. Cloning the folKE gene next to a constitutive promoter resulted

Table 16.1 Total Folate Produced By Microorganisms Grown In Chemically Defined Folate-Free Medium

Microbial speciesFolate (g l-1) Reference

Lactococcus

Lactococcus lactis subsp. cremoris92116Sybesma et al. (2003b)

1213Gangadharan et al. (2010)

Lactococcus lactis subsp. lactis57291Sybesma et al. (2003b)

1314Gangadharan et al. (2010)

Lactococcus lactis subsp. lactis biovar diacetylactis79100Sybesma et al. (2003b)

Lactobacillus

Lactobacillus plantarum45Sybesma et al. (2003b)

Lactobacillus helveticus289Sybesma et al. (2003b)

Lactobacillus acidophilus1Sybesma et al. (2003b)

Lactobacillus casei32Sybesma et al. (2003b)

Lactobacillus casei subsp. rhamnosus34Sybesma et al. (2003b)

Lactobacillus delbrueckii subsp. bulgaricus54Sybesma et al. (2003b)

Propionibacterium

Propionibacterium thoenii36Hugenholtz and Smid (2002)

Propionibacterium acidipropionici36Hugenholtz and Smid (2002)

Propionibacterium jensenii40Hugenholtz and Smid (2002)

Propionibacterium freudenreichii subsp. shermanii1778Hugenholtz and Smid (2002)

Propionibacterium sp.929Hugenholtz and Smid (2002)

Bifidobacterium

Bifidobacterium adolescentis70110Pompei et al. (2007)

Bifidobacterium animalis026Pompei et al. (2007)

Bifidobacterium bifidum01Pompei et al. (2007)

Bifidobacterium breve02Pompei et al. (2007)

Bifidobacterium catenulatum3Pompei et al. (2007)

Bifidobacterium cuniculiPompei et al. (2007)

Bifidobacterium dentium29Pompei et al. (2007)

Bifidobacterium globosumPompei et al. (2007)

Bifidobacterium infantis027Pompei et al. (2007)

Bifidobacterium lactisPompei et al. (2007)

Bifidobacterium longum02Pompei et al. (2007)

Bifidobacterium magnumPompei et al. (2007)

Bifidobacterium pseudocatenulatum7590Pompei et al. (2007)

Bifidobacterium suisPompei et al. (2007)

Bifidobacterium thermophilusPompei et al. (2007)

Streptococcus thermophilus29202Sybesma et al. (2003b)

Leuconostoc

Leuconostoc lactis45Sybesma et al. (2003b)

Leuconostoc paramesenteroides44Sybesma et al. (2003b)

a Folate concentration was measured by a microbiological assay method.

in the same increase in folate production that was observed using the NICE system. Combining the overexpression of folKE with the increased or decreased expression of other folate biosynthesis genes, folate production could be significantly increased (Sybesma et al., 2003a). Other studies were performed on the overexpression of the pABA gene cluster on three different vectors, two nisin-inducible vectors, and one con-stitutive vector. The overproduction of pABA did not lead to elevated folate pools. Hence, by overexpressing the pABA and the folate biosynthesis gene clusters simulta-neously, high folate levels were reached independent of the pABA supplementation (Wegkamp et al., 2007).

The overproduction of pABA leads to relatively low intracellular folate pools and a relatively high secretion of folate.

There exists a very tight relation between folate and pABA biosynthesis. (i) The deletion of the pABA genes in Lc. lactis eliminated its ability to synthesize folate, causing a complete inability to grow in the absence of purine nucleobases/nucleosides. In the presence of purine nucleobases/nucleosides, folate is not required for growth and (ii) the combined overexpression of folate and pABA biosynthesis pathways led to a strain that produces a high folate concentration and that does not rely on the supplementation of precursors in the medium (Wegkamp et al., 2007). These studies were realized in LAB that have the ability to produce folates. In other trials, L. gasseri (ATCC 33323) was con-verted from being a folate consumer into a highly efficient folate-producing strain. In this bacterium, the folate biosynthesis genes are not present, except for folA and folC, which are involved in the regeneration and retention of reduced folates absorbed from the me-dium. L. gasseri was transformed using a plasmid that contains the complete folate gene cluster (folA, folB, folKE, folP, and ylgG and folC) from Lc. lactis MG1363 converting it into a folate-producing strain (Wegkamp et al., 2004).

It was also demonstrated that engineered L. lactis was able to improve the folate status in deficient rats (LeBlanc et al., 2010b). Supplementation with Lc. lactis overexpressing the folC, folKE, or folC+folKE genes significantly improved the folate status in deficient rats. The biosafety assessment of these genetically modified LAB (GM-LAB) was per-formed, and it was shown that these were just as safe as the native strains from which they were derived (LeBlanc et al., 2010c).

6. CONCLUSIONS

In this review, it has been shown that folate biosynthesis by LAB could increase natural folate concentrations in certain foods such as yogurts and fermented milks through careful and specific selection of the microbial species and cultivation conditions. These folates would not cause dangerous side effects, such as masking of vitamin B12 deficiency, as is the case with folic acid. On the basis of its ability to produce B-group vitamins, LAB could be useful to design new functional foods that could prevent vitamin deficiencies by improving the nutritional values of foods. The food industry should use this information for selecting folate-producing strains as part of their starter cultures to produce fermented products with elevated levels of this essential vitamin. Many benefits will be obtained from the use of these strains, such as economic advantages to food man-ufacturers by providing a value-added effect without increasing production costs. Also consumers would increase their folate intakes by including these novel fermented foods as part of their normal diet and lifestyle. In addition, selected specific folate-producing strains could also provide health benefits for consumers.

ACKNOWLEDGMENTS

The authors would like to thank the Consejo Nacional de Investigaciones Cientficas y Tcnicas (CONI-CET), Agencia Nacional de Promocin Cientfica y Tecnolgica (ANPCyT), and the Consejo de Inves-tigaciones de la Universidad Nacional de Tucumn (CIUNT) for their financial support.

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van de Guchte, M., Penaud, S., Grimaldi, C., et al., 2006. The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proceedings of the National Academy of Sciences of the United States of America 103, 92749279.

Verwei, M., Arkbage, K., Havenaar, R., et al., 2003. Folic acid and 5-methyltetrahydrofolate in fortified milk are bioaccessible as determined in a dynamic in vitro gastrointestinal model. The Journal of Nutri-tion 133, 23772383.

Wegkamp, A., Starrenburg, M., de Vos, W.M., Hugenholtz, J., Sybesma, W., 2004. Transformation of fo-late- consuming Lactobacillus gasseri into a folate producer. Applied and Environmental Microbiology 70, 31463148.

Wegkamp, A., van Oorschot, W., de Vos, W.M., Smid, E.J., 2007. Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis. Applied and Environmental Mi-crobiology 73, 26732681.

Wouters, J.T.M., Ayad, E.H.E., Hugenholtz, J., Smit, G., 2002. Microbes from raw milk for fermented dairy products. International Dairy Journal 12, 91109.

Wright, A.J., Finglas, P.M., Dainty, J.R., et al., 2003. Single oral doses of 13C forms of pteroylmonoglutamic acid and 5-formyltetrahydrofolic acid elicit differences in short-term kinetics of labelled and unlabelled folates in plasma: potential problems in interpretation of folate bioavailability studies. The British Journal of Nutrition 90, 363371.

Zhang, C., Zhao, L., Dong, Y., et al., 2010. Folate-mediated poly(3-hydroxybutyrate-co-3-hydroxyoc-tanoate) nanoparticles for targeting drug delivery. European Journal of Pharmaceutics and Biopharma-ceutics 76, 1016.

FURTHER READING

LeBlanc, J.G., Rutten, G., Bruinenberg, P., et al., 2006. A novel dairy product fermented with Propionibac-terium freudenreichii improves the riboflavin status of deficient rats. Nutrition 22 (6), 645651.

LeBlanc, J.G., Burgess, C., Sesma, F., Savoy de Giori, G., van Sinderen, D., 2005. Ingestion of milk fer-mented by genetically modified Lactococcus lactis improves the riboflavin status of deficient rats. Journal of Dairy Science 88 (10), 34353442.

LeBlanc, J.G., Burgess, C., Sesma, F., Savoy de Giori, G., van Sinderen, D., 2005. Lactococcus lactis is capable of improving the riboflavin status in deficient rats. The British Journal of Nutrition 94 (2), 262267.

LeBlanc, J.G., Savoy de Giori, G., Smid, E.J., Hugenholtz, J., Sesma, F., 2007. Folate production by lactic acid bacteria and other food-grade microorganisms. In: Mndez-Vilas, A. (Ed.), Communicating Cur-rent Research and Educational Topics and Trends in Applied Microbiology, vol. 1. Formatex Research Center, Badajoz, Spain 978-84-611-9422-3, pp. 329339.

Sybesma, W., 2003. Metabolic Engineering of Folate Production in Lactic Acid Bacteria. In: de Vos, W.M., de Vos, W.M. (Eds.), Wageningen University, The Netherlands 90-5808-935-5.

Burgess, C., Oconnell-Motherway, M., Sybesma, W., Hugenholtz, J., van Sinderen, D., 2004. Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods. Applied and Environmental Microbiology 70, 57695777.

Sybesma, W., Burgess, C., Starrenburg, M., van Sinderen, D., Hugenholtz, J., 2004. Multivitamin produc-tion in Lactococcus lactis using metabolic engineering. Metabolic Engineering 6, 109115.

Sybesma, W., Van Den Born, E., Starrenburg, M., et al., 2003. Controlled modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis. Applied and Environmental Microbiology 69, 71017107.

CHAPTER 17

Probiotics against Digestive Tract Viral

Infections

J. Rodrguez-Daz, V. Monedero

Instituto de Agroqumica y Tecnologa de Alimentos (IATA-CSIC), Valencia, SpainABBREVIATIONS

AdV Enteric adenovirus

AV Astrovirus

EV Enterovirus

NV Norovirus

ROS Reactive oxygen species

RV Rotavirus

TLR Toll-like receptor

1. INTRODUCTION

Gastrointestinal viral pathogens have great social and economic impact in both developed and developing nations. Gastrointestinal viruses are shed with human fecal wastes and are transmitted through the oro-fecal route by direct contact with an infected person and by consumption of, or contact with, contaminated water or food (Buesa and Rodrguez- Daz, 2006; Knipe and Howley, 2007). Intestinal viral infections usually result in diarrhea of varying degrees, and oral or parenteral rehydration therapy is the most common treat-ment. However, there is a growing interest in the use of probiotic antiviral therapies due to their positive influence on human health. Probiotics are defined as live microorganisms that, upon ingestion in certain quantities, exert beneficial effects on the host. The most common probiotics are members of the lactic acid bacteria (Lactobacillus, Streptococcus, Enterococcus, etc.), Bifidobacteria, and some yeast strains. These microorganisms are gener-ally recognized as safe, have a long history of use in food production, and are normal in-habitants of the gastrointestinal tract. This ecosystem is colonized by a diverse microbiota, which at some locations can reach up to 1012 microorganisms per gram of content, and it is constituted by approximately 1000 different species, making it one of the most dense and complex microbial ecosystems. The intestinal microbiota plays an important role in the organisms physiology and helps maintain host health (Collado et al., 2009). Thus, imbalances in its composition (dysbiosis) underlie some pathologies (e.g., inflammatory bowel diseases). The gut-associated immune system displays a hyporesponsiveness to this resident microbiota, but it is sensitive to products derived from it, the so-called microbial-associated molecular patterns (DNA, components of the cell wall, proteins, etc.), which are sensed by a complex family of receptors present in epithelial and immu-nocompetent cells (e.g., the Toll-like receptors TLRs). A cross-talk is established be-tween the microbiota and epithelial/immune cells, which influences cell proliferation and maturation and helps maintain the immune homeostasis and gut barrier functions (Gill and Prasad, 2008). The epithelium and the intestinal microbiota constitute a syn-ergic physical and chemical defense line against pathogens. These defense functions are susceptible to being modulated by the use of probiotics.

There are many health benefits attributed to probiotics (prevention and treatment of intestinal infections, prevention and management of allergic diseases, enhancement of im-mune function, anticancer effects, cholesterol lowering, etc.), but the accumulated clinical data point to the treatment and prevention of infectious diarrhea as one of the health effects supported by sound scientific evidence. Besides their antibacterial activities, many studies have demonstrated that specific probiotics reduce the risk and shorten the duration of di-arrheas associated with viral infections, especially in infants and children.

2. VIRUSES THAT INFECT THE GASTROINTESTINAL TRACT

Several types of viruses are able to replicate in the intestinal epithelium, but not all of them cause gastroenteritis (Buesa and Rodrguez-Daz, 2006; Knipe and Howley, 2007). In the following sections, the most important viral groups responsible for gastro-intestinal infections worldwide are described.

2.1. Noroviruses

Noroviruses (NVs) aremembers of the Caliciviridae family that infect the small intestine and cause the majority of foodborne and waterborne outbreaks of acute gastroenteritis world-wide. NVs contain a linear positive-sense single-stranded RNA genome. They have a high genetic variability, being classified in five genogroups (GIGV) that are subdivided into ge-notypes. The major NVs infecting humans belong to the GI and GII genogroups, with the GII4 genotype emerging as themain genotype causing gastroenteritis outbreaks worldwide. The incubation time ranges from 15 to 48 h, leading to gastroenteritis for 1260 h from the beginning of the symptoms. NV infection usually courses as a self-limited diarrhea and is characterized by vomiting, but in special cases, it can lead to severe dehydration and death.

2.2. Rotaviruses

Rotaviruses (RVs) are the main etiological cause of severe gastroenteritis and infantile morbidity worldwide in children under 5 years, the age when most of the population is seroconverted and thus less susceptible to RV infection. RVs also lead to high child-hood mortality in developing countries, causing (500000 deaths per year as a result of dehydration and deficient medical care. In developed countries, RV diarrheas are respon-sible for a large number of hospitalizations. Although some RV vaccines have been developed during the last few years, more economic alternatives would be desirable, especially in developing countries. RVs are 70-nm icosahedral viruses that belong to the family Reoviridae and infect mature enterocytes. Seven RV serogroups (AG) have been described based on the antigenicity of the capsid VP6 protein. Most human path-ogens belong to groups A, B, and C. RVs of group A are the most important from a public health standpoint. The virus is composed of three protein shells, an outer capsid, an inner capsid, and an internal core that surround 11 segments of double-stranded RNA. Three major structural and nonstructural proteins are of interest in epidemiological studies and vaccine development against group A RV: NSP4 (genotypes AF), VP7 (G genotypes 115), and VP4 (P genotypes from 1 to 14). VP7 and VP4 are of special interest because they are able to elicit neutralizing antibodies.

2.3. Astroviruses

Astroviruses (AVs) are nonenveloped viruses with a positive-sense, single-stranded RNA genome. AV infections occur worldwide and their incidence ranges from 2% to 9% in both developed and developing countries. Outbreaks of AVs have been associated with consumption of sewage-polluted shellfish and ingestion of water from contaminated sources.

2.4. Enteric Adenoviruses

Enteric adenoviruses (AdVs) are nonenveloped, double-stranded DNA icosahedral viruses measuring 7090 nm in diameter. AdVs are divided into two genera: Mastadeno-virus, which includes viruses that infect mammals, and Aviadenovirus, which contains viruses that infect birds. In some countries, enteric AdVs (subtypes 40 and 41) are placed as the second etiologic agents of infantile gastroenteritis.

2.5. Enteroviruses

Enteroviruses (EVs) are named after their site of replication but rarely cause gastroenter-itis, and the resulting infection is frequently asymptomatic or targets other organs. EVs belong to the family Picornaviridae and possess a positive-sense RNA genome. The two main representative EVs are polioviruses, causing poliomyelitis, and kuboviruses. Aichi virus, a member of the genus kubovirus, is responsible for gastroenteritis outbreaks usually caused by the consumption of contaminated oysters.

3. POSSIBLE MECHANISMS OF PROBIOTICS ACTION AGAINST INTESTINAL VIRUSES

The mechanisms for the antagonistic capacity of probiotics against microbial pathogens have been exhaustively investigated for microorganisms important in gastrointestinal in-fections, such as Clostridium difficile, Helicobacter pylori, Salmonella, or pathogenic Escherichia coli, where numerous in vitro and some clinical studies exist. The positive effects are attributed to multiple mechanisms (Servin, 2004), some of which can also be extended to viruses (summarized in Figure 17.1).

Probiotics are able to induce host cellular defenses against pathogenic bacteria, such as -defensins synthesized by Paneth cells, and they also produce well-characterized anti-bacterial molecules such as organic acids, H2O2, or antimicrobial peptides (bacteriocins). Some authors have postulated that certain probiotics can produce antiviral substances (Botic et al., 2007; Seo et al., 2010), although their nature is unknown and in vitro viral

Figure 17.1 Proposed mechanisms for the antiviral effect of probiotics in gastrointestinal infections. Probiotics putatively interfere with viral replication at different levels, by blocking viral attachment, synthesizing antiviral compounds by itself, or inducing their synthesis by epithelial cells. The cross-talk established between probiotics and epithelial/immune cells enhances barrier functions and innate as well as adaptive immune responses.

inhibition with bacterial supernatants might be simply explained by the presence of or-ganic acids.

One of the first viral infection mechanisms that can be targeted by robotics is viral binding to host cells. Exclusion of pathogens by direct binding, attachment inhibition, or displacement has been thoroughly studied for bacterial pathogens in in vitro and in vivo studies using robotics, but data on exclusion of viruses are scarce. Viruses can use ol-igosaccharides present as glycoconjugates on cellular surfaces as receptors for attachment and entry. RVs recognize sialic acid (N-acetylneuraminic acid) residues as a first step for cellular entry, whereas NVs display binding specificities toward 1,2-fucosylated carbo-hydrates and 2,3-sialylated carbohydrates, which form part of the histoblood group anti-gens expressed at mucosal surfaces. Many Lactobacillus and Bifidobacterium strains display lectin-like activities on their surfaces. Surface components from these bacteria have been characterized that bind to the highly glycosylated intestinal mucus and extracellular ma-trix proteins or are responsible for attachment to cultured enterocyte lines (e.g., Caco-2, HT-29, or T84 cell lines) (Lebeer et al., 2008). The surface layer proteins (SlpA) from Lactobacillus have been implicated in attachment to cellular surfaces and pathogen dis-placement, and other surface proteins with no evident secretion signals (e.g., chaperones and glycolytic enzymes) decorate the surface of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus reuteri, or Lactobacillus johnsonii strains and behave as sticky factors playing a role in adhesion. Lactobacillus species adapted to the gastrointestinal niche (L. plantarum, Lactobacillus acidophilus, Lactobacillus gasseri, L. johnsonii, and L. reuteri) possess surface-specialized proteins involved in mucin binding in a mannose-sensitive manner (lectin-like) or specific mucin-binding pili, as is the case for Lactobacillus rhamnosus GG. Other molecules of nonprotein nature present at the bacterial surface and reported to be involved in binding are lipoteichoic acids and exopolysaccharides. These types of molecules allow probiotics to attach to the intestinal mucosal surface and might be re-sponsible for their persistence in this niche and, in addition, participate in viral exclusion and displacement from the surface of target cells. Besides, probiotic strains are able to directly bind viruses, which would promote their elimination in feces. This implies that some surface molecules (glycosylated proteins or other components of the cell wall) from probiotics could be mimicking viral receptors. Interestingly, the two strains of L. rham-nosus GG and Bifidobacterium lactis Bb-12 with a better-documented efficacy in infectious diarrhea exhibited the best binding ability to RV particles (Salminen et al., 2010).

Probiotics can modulate specific host pathways. They can induce the synthesis of molecules that interfere with some step of the viral cycle, increase the mucosal barrier function, or act as immunomodulators that enhance both innate and adaptive immune response.

Some studies have addressed the synthesis of reactive oxygen species (ROS) by cultured epithelial cells in the presence of probiotics. ROS can play defensive roles in the organism, and a correlation between ROS release induction and viral protection for specific pairs of cultured cell lines/probiotic strains has been described (Maragkoudakis et al., 2010).

L. rhamnosus GG and L. plantarum 299v stimulate mucin secretion by upregulation of MUC-2 and MUC-3 genes in Caco-2 and HT-29 cells, respectively. Increased mucin secretion, which forms part of the epithelial mucus protective layer, may participate in viral exclusion by binding and entrapping viruses through specific mucinviral interact-tions, promoting their shedding from the intestine and acting as a physical barrier that limits access to the epithelium.

Viral diarrheas involve varied mechanisms that result in deficient nutrient absorption or increased secretion of water and electrolytes. During infection, paracellular epithelial permeability can be increased and epithelial damage and apoptosis occur. L. rhamnosus GG and other members of the L. casei/rhamnosus group secrete to the culture medium-specific proteins (p40 and p75) that enhance barrier functions through mech-anisms involving Akt and the PI-3K kinase and protect the intestinal epithelium from injury and apoptosis caused by inflammatory cytokines (tumor necrosis factor (TNF-) and interferon gamma (IFN-)) or oxidative damage, maintaining the structure of the tight junctions and increasing the expression of specific proteins (e.g., zonula occlu-dens-1, claudin, and occludin). Low-molecular-weight peptides produced by L. rhamno-sus GG activate mitogen-activated protein kinases and induce cytoprotective heat-shock proteins HSP25 and HSP72 in intestinal cells. In general, probiotic strains maintain epithelial integrity and reduce the decrease in transepithelial resistance in cultures follow-ing pathogen infection. Thus, they may help to keep the intestinal barrier integrity which is compromised during viral infection.

In vitro and in vivo experiments have established that probiotics can modulate the syn-thesis of an array of cytokines, for example, interleukin (IL)-1, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-, and TNF-. This leads to a range of modulatory effects on immune cells: increased cytotoxic and phagocytic capacity of NK cells or macrophages and immune cell (T and B lymphocytes) proliferation and differentiation, which can result in increased antibody responses (Gill and Prasad, 2008). The consumption of fermented milk contain-ing certain probiotics increased specific IgG and IgA titers when individuals were vac-cinated against Salmonella, hepatitis B, influenza, or poliovirus. In this sense, L. rhamnosus GG was effective in promoting specific IgA-secreting cells and higher plasma IgA titers after RV infection (Kaila et al., 1992) and showed an adjuvant effect in RV vaccination.

4. LABORATORY EVIDENCE OF PROBIOTICS-CONFERRED RESISTANCE TO GASTROINTESTINAL VIRAL INFECTIONS

Most of the data on the effect of probiotics on viral gastrointestinal infections using in vitro and in vivo models have been obtained with RVs. This derives from the fact that, in

Table 17.1 Examples of the Efficacy of Probiotics against Gastrointestinal Viruses in Different In Vitro and In Vivo Models

VirusStrainsModelEffectsReference

VSVB. breve DSM 20091 B. Longum Q46 L. paracasei A14 L. paracasei F19 L. paracasei Q85 L. plantarum M1.1 L. reuteri DSM 12246IPEC-J12 cell lineReduced in vitro infectionBotic et al. (2007)

VSVL. paracasei Q85 L. paracasei A14 L. paracasei F19 B. longum Q463D4/2 macrophage cell lineIncreased antiviral response and decreased viral infectionIvec et al. (2007)

RVL. acidophilus NCFM L. rhamnosus GGIPEC-J12 cell lineProtection and enhancement of innate immunityLiu et al. (2010)

RV and TGEVL. rhamnosus GG L. casei Shirota E. faecium PCK38 L. fermentum ACADC179 L. pentosus PCA227 L. plantarum PCA236 etc.Six different cell lines (from human, pig, and goat)Reduced in vitro infectionMaragkoudakis et al. (2010)

RVL. reuteri Probio-16TF-104 cell lineReduced in vitro infectionSeo et al. (2010)

RVL. plantarum 299vBovine intestinal epithelial cell lineReduced infection and enhancement of innate immunityThompson et al. (2010)

RVB. bifidumMiceDiarrhea reductio

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