COVER STORY Agro FOOD Industry Hi Tech customer's publication

Reducing blood glucose with Actilight®, an easy to use soluble fibre

DECREASED GLYCAEMIC RESPONSE The health benefits of the usage of Actilight® fibre (in partial or total replacement of sugars) to reduce glycemia The glycæmic response is the increase in the blood glucose has been demonstrated in 2 major clinical studies. These level after eating and/or drinking. It is an indication of the studies, published in peer-reviewed journals, demonstrate amount of glucose available to produce energy within that recipes using FOS for sugars replacement could lower the body. It could be particularly beneficial to reduce the blood glucose rise compared to the reference recipes postprandial glycæmia for glucose-intolerant or diabetic (Lecerf et al., 2015; Respondek et al., 2014). This effect was subjects (Wiebe et al. 2011, EFSA, 2011). Indeed, some studies, observed without any increase of insulin blood level. This but not all, showed reducing postprandial blood glucose condition proves the health benefit, as recommended by and insulin levels could be beneficial to prevent type 2 the EFSA experts. diabetes (Blaak et al. 2012). Knowing that the prevalence of type 2 diabetes is expected to increase by more than 50% between 2010 and 2030, it is particularly important to be able ACTILIGHT®: WHICH TYPE OF FIBRE IS IT? to formulate food that may contribute to regulate glucose metabolism, even if the current recommendations for Actilight® is composed of fructo- those people are the same as the ones for healthy people. oligosaccharides (FOS), which are soluble fibres obtained from sugar beet. They are not absorbed, nor digested in the small THE EU HEALTH CLAIM ON GLYCEMIA REDUCTION BY NON- intestine. Selectively fermented by the intestinal microflora, DIGESTIBLE CARBOHYDRATES IS APPLICABLE TO ACTILIGHT® FIBRE. FOS stimulate the development of some beneficial such as bifidobacteria, and are thus considered as The European commission has officially approved a new health fibres. Some recent scientific studies confirm an improvement claim that is fully authorized since June 20th 2016. It states of intestinal comfort and glucose metabolism (Paineau et al., that the consumption of foods and drinks containing non- 2008; Respondek et al., 2013). digestible carbohydrates instead of sugars induces a lower blood glucose rise after their consumption compared to sugar- Actilight® fibre is the most active soluble fibre to containing foods and drinks. Non-digestible carbohydrates re-balance the nutritional profile of your foodstuffs are neither digested nor absorbed in the small intestine. For without any compromise on the sensory properties the health claim to be applicable, the food and drinks should of your finished products. contain at least 30% less sugars compared to the reference containing sugars. Actilight® has a nutritional interest for fibre supplementation, With this new health but also for sugar and calorie reduction. From a technological claim officially point of view, FOS are very soluble fibres whose rheological approved, food properties are very similar to the ones of sugar, which manufacturers using facilitates their use in a wide range of recipes. Actilight® Actilight® fibre to fibre tastes good. Its sweetness is about 30% compared reduce sugars by with sugar’s, and its organoleptic profile is similar. It has a 30% in their recipes, flavour enhancer effect on the aroma intensity and masks can use health the aftertaste of intense sweeteners. In order to maintain claims such as the sensory properties of food, and notably the sweet taste, “stabilises glycemia” Tereos is able to provide its expertise and know-how, together or “regulates blood with a wide range of sweetening ingredients including fibres, glucose”. polyols and stevia extracts.

Monographic special issue: PRE/ - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 Tereos Syral Z.I. et portuaire - BP 32 F-67390 - Marckolsheim France www.tereos-starchsweeteners.com

Nutrition claims according to European regulation: • Sugars: Reduced in sugars: -30% compared with a reference product. No sugars: less than 0.5g/100g • Fibre intake: - Source of fibres: 3g/100g - Rich in fibres: 6g/100g

CHOCOLATE DAIRY DESSERT REDUCED IN SUGARS WITH ACTILIGHT®

A recent study showed that the intake of Actilight® FOS for partial replacement of D-glucose in the formulation of a dairy dessert, would significantly reduce the glycæmic response compared with the reference dessert. This study, conducted in 24 healthy subjects, also confirmed this reduction was not due to the increased insulin secretion, as the insulin response was itself reduced compared with the reference food. This effect is easily explained by the non-digestible nature of FOS. (Lecerf et al. 2015, presentation).

THE MUST-HAVE INGREDIENT FOR SWEET BAKERY

Actilight® fibre can be used for the replacement of 30% sugar in various sweet bakery products, such as cookies, short bread, pound cakes and sponge cakes. • Cookies: texture and sweetness are preserved, while sensory analysis shows that cookies containing Actilight® are globally more appreciated than the reference product. • Short bread: sugar-reduced biscuits display excellent cohesion. The typical short bread texture, sweetness, butter aroma and global satisfaction of reduced short bread with Actilight® are preserved • Pound cakes: Sensory analysis shows no difference between sugar reduced products and sugar-based products in terms of colour, softness, stickiness in mouth, sweetness and global satisfaction. • Sponge cakes: Compared to oligofructose from inulin, Actilight® allows to preserve colour. Specific volume is increased with Actilight® compared to the sugar-based and oligofructose/ inulin based reference.. Softness is also increased compared to the references, which is particularly appreciated in sponge cakes.

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 I CONTENTS Agro FOOD Industry Hi Tech is a peer-reviewed journal devoted to functional food and addressed to a readership belonging to the industry. The 2015 impact factor is 0.225, according to the Thomson Reuters Journal PREBIOTICS Citation Reports. PROBIOTICS www.actilight.com TEKNO SCIENZE SRL Viale Brianza, 22, 20127 Milano (Italy) Tel +39-02-26809375/28381260 Fax +39-02-2847226 e-mail: [email protected] web: www.teknoscienze.com

Agro FOOD Industry Hi Tech Editor in Chief vol. 27(5) - Sept/Oct 2016 Carla Scesa - [email protected] Editorial Director Silvana Maini - [email protected] COVER STORY Reducing blood glucose with Actilight®, Associate Editors an easy to use soluble fibre Gayle De Maria - [email protected] Tereos Syral Carlo Buonamico - [email protected] Marketing & Sales III Shining a light on prebiotics: their role in human health Giulio Fezzardini - [email protected] S. Saville, B. A. Saville Silvia Baldina - [email protected] VII Market insights on probiotics Simona Rivarollo - [email protected] G. Paraskevakos Marketing & Events Paola Passadore - [email protected] XI The cost and benefit of use Production Manager & Editorial Coordinator A. C. Ouwehand Elisa Novaresi - [email protected] XIV Probiotics to prevent upper respiratory tract infections Production Assistants & IT department C. Gerritsen, G. Ormel Luis A. Alburqueque Diaz - [email protected] Matteo Cattane - [email protected] XVIII Host microbiota interactions in gut health Administrative & Account An update on current understanding and future perspectives S. C. M. Fong, S. P. Claus Alba Aprea - [email protected]

XXII Use of the Simulator of the Human Intestinal Microbial Ecosystem PRINTING OFFICE: ® (SHIME ) to study the fate of food ingredients and actives Arti Grafiche Colombo (Muggiò - MB, Italia) D. Gottardi, P. van den Abbeele, M. Marzorati Authorization from Tribunale di Milano n. 514 of XXV Cereal bound antioxidants July 27th 2006 (Autorizzazione del Tribunale di E. D. Cömert, V. Gökmen Milano n. 514 del 27 Luglio 2006).

XXIX Application of Arthrospira platensis (Spirulina) biomass SCIENTIFIC ADVISORY BOARD: to improve properties of probiotic doogh Production of sinbiotic doogh Anton Alldrick (CCFRA) A. Eslami-Moshkenani, V. F. Noghani, K. Khosravi-Darani Sybille Buchwald-Werner (Vital Solutions GmbH) Undurti N Das (UND Life Sciences) Inga Koehler (Pfizer Consumer Health Care) Find out more... in our past monographic supplement series! Rossella Koning (Evonik) Visit our website www.teknoscienze.com Ray A. Matulka (Burdock Group) Vicente Micol (University Miguel Hernández) Arthur Ouwehand (Danisco Health & Nutrition) Demosthenes B. Panagiotakos (Harokopio University) Denis Poncelet (ONIRIS and Capsulae sarl) Igor Pravst (Nutrition Institute – Slovenia) Joe Romano (Waters) Yang Zhu (Eurasia Agrofood Biotech)

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Associated to USPI - Unione della Stampa Periodica Italiana.

II Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 SANDRA SAVILLE1, BRADLEY A. SAVILLE1,2* *Corresponding Author 1. Prenexus Health, 4231 CA-86 #9 Brawley, CA 92227 USA 2. University of Toronto, Department of Chemical Engineering and Applied Chemistry, 200 College Street, Toronto, ON M5S 3E5, Canada

Bradley A. Saville

Shining a light on prebiotics: their role in human health

KEYWORDS: Prebiotics, non-digestible oligosaccharides, bifidobacteria, microbiome, fiber.

Prebiotics are gaining recognition for their potential health benefits, particularly for their role in feeding Abstract the beneficial bacteria within the GI tract. However, there is much to be learned about the role of prebiotics, particularly differences between prebiotics that relate to structure and degree of polymerization, which impact their function and ability to modulate the gut microbiome. This article provides some history regarding prebiotics, a discussion of sources and different types of prebiotics, their health impacts, and distinguishing features.

INTRODUCTION information (or consensus) regarding daily consumption of prebiotic fiber. Suggested intakes have ranged from There is growing consumer awareness about probiotics 5 to 20g per day (2), although it is likely that this will also and the role that they may play in health promotion, but depend upon the type of prebiotic and the dose required consumer awareness regarding prebiotics is still somewhat for efficacy. The International Scientific Association for limited. Prebiotics can play an important synergistic role Probiotics and Prebiotics suggests prebiotic consumption with probiotics, as so-called “synbiotics”, but also have of at least 5g per day (3). It is also apparent that it is an important independent role in feeding the microbiota difficult to consume enough prebiotic-containing foods already present within us. In this article, we discuss the to elicit a health benefit. For example, based upon a health benefits and sources of various prebiotics, while also targeted inulin/FOS intake of 15g/day, a person would outlining differentiating factors. need to consume about 600 grams of wheat/grain While humans have consumed prebiotics for millennia, as products (i.e., a whole loaf of bread), 400 grams of onions, part of their regular diets, the important role of prebiotics about 100 grams of Jersualem artichokes, 600 grams of in human health has only just begun to be elucidated. asparagus, or over 20 bananas. Food and beverage Prebiotics are non-digestible substrates that promote the fortification and consumption of supplements will likely growth of beneficial bacteria, particularly in the distal play an important role if the health benefits of prebiotics part of the colon, ultimately leading to health benefits. are to be achieved. These prebiotic compounds are also referred to as “non- digestible oligosaccharides”, or “microbiota accessible carbohydrates” ( MACs). For many of us, our first exposure DIFFERENCES BETWEEN INSOLUBLE DIETARY FIBER AND PREBIOTICS to prebiotics is from breast milk, which serves to promote the growth of beneficial gastrointestinal bacteria in Dietary fiber is a complex carbohydrate found in plants. infants, aiding digestion and immunity. According to a Historically, it had been recognized as insoluble “roughage” USDA study (1), the most significant source of inulin and that passed through the colon intact, without being digested. oligofructose prebiotics in the U.S. diet is via wheat and More recently, the attributes of soluble fiber fractions that similar grain products – not because these are particular are partially digestible have also been recognized. Insoluble rich sources, but rather, because a lot of grain products dietary fiber is often assumed to be, or promoted as, a are consumed in the Western diet. prebiotic; however, insoluble fiber cannot be classified as While general recommendations are available regarding a prebiotic unless it can promote the growth of beneficial daily consumption of dietary fiber, there is limited bacteria that lead to a health benefit (2).

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 III In contrast, most prebiotics can be classified as dietary transglycosylation using lactose as the substrate. fiber, although regulatory regimes in different countries The GOS produced enzymatically tends to have a low may limit this classification based upon the degree of degree of polymerization, and includes unreacted polymerization (DP) of the carbohydrate prebiotic. lactose, galactose and glucose; For example, the US FDA recently updated its regulations • arabinoxylanoligosaccharides (AXOS), also produced regarding classification of fiber, to include oligomers that from the xylan fraction in plant fiber, except that the are comprised of 3 to 10 carbohydrate units (4). Several xylan fraction includes arabinan side chains as part of prebiotics would meet this requirement; however, some their structure; commercial products contain a high percentage of dimers • isomalto-oligosaccharides (IMOS), produced by (DP2), which may not satisfy the FDA regulation for fiber. enzyme-catalyzed hydrolysis of starch, followed by removal of glucose using yeast. The alpha-1,4 glycoside Compounds commonly considered as prebiotics include: linkages in the maltooligosaccharide intermediate are then converted enzymatically into alpha-1,6 linkages • fructo-oligosaccharides (FOS), made up of fructose using a transglycosidase enzyme; subunits connected by beta linkages. FOS often • resistant starch, i.e., starch that is not readily hydrolyzed contains 2 – 6 fructose subunits, and is readily soluble in by amylase or pullulanase. It may take one of four water; forms. The first form, RS1, is the starch within milled • oligofructose, also mainly made up of fructose subunits, grains and seeds that is physically inaccessible to tends to have a greater degree of polymerization. It is enzymes. RS2 is a tightly packed, compact polymer often produced from inulin using a final hydrolysis step. that limits access to water and enzymes. RS3 is the most Oligofructose derived from chicory may contain resistant form, essentially retrograded amylose fructose chains alone, and fructose chains with produced after gelatinized starch has been cooled. terminal glucose units; RS4 is a chemically crosslinked or enzymatically • inulin, also made up of fructose subunits, has an modified form of starch. Crosslinking agents such as average DP often 20 or greater, with 11 to 60 subunits phosphates, acetate, or adipic acid are commonly typical in most inulin fractions. The higher DP fractions used for RS4. Corn and rice are typical sources for would tend to be insoluble. Inulin is often produced by these starches. direct extraction from inulin-rich plants such as chicory roots or Jerusalem artichokes; • xylooligosaccharides (XOS), produced by converting IMPACT OF PREBIOTICS ON GASTROINTESTINAL BACTERIA the insoluble xylan fraction in plant fiber into shorter- chain fractions that are soluble and more readily Typically, prebiotics stimulate beneficial bacteria within digestible by the microbiota. XOS products may have the gastrointestinal tract, and the most prominent benefit different degrees of polymerization, depending upon is to improve digestive health. Typically, the Firmicutes, whether or not an enzymatic hydrolysis step was used Bacteriodes, , Prevotella and Xylanobacter in manufacturing; genera are more prevalent in populations that consume • galactooligosaccharides (GOS), produced from the more fiber, including prebiotic fiber. These genera are also galactan fraction in plant fiber, or more adept at fermenting xylans. commonly, produced enzymatically by As shown in Figure 1, there is a cascade of impacts associated with consumption of prebiotics (5, 6). Typically, prebiotics stimulate the growth of lactobacilli and bifidobacteria, producing key metabolites, such as lactate and short chain fatty acids (SCFAs), that have direct and indirect health benefits. Collectively, these SCFAs reduce the pH in the colon, which inhibits growth of certain pathogenic species that tend to grow at higher pH. The lower pH also promotes the absorption of key minerals such as calcium and , which are more soluble under lower pH conditions, Figure 1. Role of Prebiotics in Gut Health. and thus more readily absorbed.

IV Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 Propionate, one of the SCFAs, plays a role in lipid and of insoluble fiber, contributing to dietary fiber intake in a cholesterol synthesis in the liver, and also helps to regulate similar manner as non-digestible plant fibers. blood glucose (7). Butyrate, another SCFA, is generally Cluster A and B bifidobacteria, which includes B. breve and regarded as a key compound that inhibits colonic B. bifidum, lack the fructofuranosidase needed to process oxidative stress, inflammation and carcinogenesis (8). inulin (17). Cluster B bifidobacteria can use short chain FOS, Although bifidobacteria typically do not produce butyrate, but not long chain FOS, whereas Cluster C bifidobacteria they produce lactate and other metabolites that cross- can use virtually all types of fructans, while producing feed butyrate producers. The ability of certain bacteria to fructose as a metabolite. However, fructose utilization by break down insoluble and long chain arabinoxylan and non-bifidobacterial species has been hypothesized as a arabinogalactan into soluble oligomers and monomers trigger for intestinal inflammation and discomfort. also facilitates cross-feeding of beneficial bacteria. Bifidobacteria are among the few intestinal bacteria that There is also considerable evidence that reduced have the intracellular enzymes that can use five-carbon consumption of key carbohydrates, including sources of sugars such as xylooligosaccharides. In the presence of soluble and insoluble prebiotic fibers, can contribute to xylooligosaccharides (XOS), Prevatella spp. also express dysbiosis (9, 10). Individuals who must limit consumption of genes that encode xylanases and xylosidases. This feature grain products or gluten also reduce their intake of inulin is key to the selectivity of xylooligosaccharides, which are and other prebiotics from these food sources, and may be not otherwise used by Clostridia, E. coli, or other bacteria at risk of decreased intestinal levels of bifidobacteria spp. considered to be pathogenic. In contrast, FOS and GOS and F. prausnitzii, and the corresponding key metabolites (both six-carbon sugars) are readily used by E. coli and that impact health. Low FODMAP diets also reduce a variety of pathogenic bacteria, and thus, while they exposure to prebiotics that feed the good bacteria in promote the growth of beneficial bacteria, they are not the gut; the long term consequences of these dietary particularly selective. interventions are just beginning to be investigated. Makelainen et al. from Danisco (18) conducted a comprehensive study to assess the in vitro growth of various bacteria on a range of carbohydrate sources, STRUCTURE/FUNCTION RELATIONSHIPS including glucose, FOS, GOS, xylan, xylobiose, XOS, and polydextrose (PDX). They observed that most B. lactis The impact of prebiotics on bacterial profiles in the strains were able to use XOS to a high degree, while gut is dictated to a large extent by chemical structure other bifidobacteria and lactobacilli used XOS to a lesser and degree of polymerization (DP). These features also degree. FOS and GOS are more broadly utilized, while influence the selectivity of the prebiotic for good bacteria polydextrose was fermented only to a limited extent. versus bad bacteria. Many bacteria are able to use six- While the broader utilization of FOS and GOS may seem carbon sugars for growth, whereas far fewer have the beneficial, they also observed that pathogenic bacteria ability to use five-carbon sugars. Oligomers and polymers such as E. coli, Clostridia, Salmonella, Staphylococcus, with alpha linkages (such as those based upon starch) are Bacteroides fragilis and Bacteroides vulgatus, grow well on more readily utilized than those with beta linkages (such FOS and GOS, whereas there is very limited growth on XOS. as those based upon cellulose or xylan). Furthermore, Thus, XOS is more selective, by specifically feeding “good” shorter chain soluble oligomers tend to be more readily bacteria, while FOS and GOS feed “good” and “bad” utilized than longer chain oligomers, although longer bacteria. The propensity of FOS and inulin to promote chain oligomers also tend to persist into the distal part the growth of Clostridia and unfriendly bacteria may of the colon. The ability of bacteria to utilize different contribute to the unpleasant side effects associated with types of carbohydrates is dependent upon enzymes that consumption of these prebiotics. are encoded within the bacterium. Fructofuranosidases, amylases, pullulanases, endoglucanases, xylanases, and beta xylosidases are examples of key enzymes that must DOSAGES REQUIRED FOR HEALTH BENEFITS be present in GI bacteria in order to utilize the prebiotic. Some bacteria contain genes that encode a “fructan A large number of clinical trials have been conducted utilization locus”, which helps them to ferment fructans of to evaluate health impacts of prebiotic consumption, various chain lengths (11, 12). with a variety of endpoints. The dose of FOS required to Similarly, Hamaker et al. (13) described the complexity significantly increase counts in the human of dietary fiber structures, and the impact of various gut is typically in the range of 10 to 20 g/d, and the inulin polysaccharide utilization loci within bacteria that influence dose is typically 15g/d or higher (9, 16). In contrast, a digestibility and selectivity of different types of fiber. bifidogenic effect has been observed with XOS at doses as Surprisingly, one of the most widely recognized prebiotics, low as 1.5 g/d (19), and some studies with XOS have also inulin, is poorly utilized by bifidobacteria; an in vitro study shown beneficial impacts upon lipid profiles and blood by Rossi et al. (14) indicated that only 8 of 55 bifidobacteria glucose at similar doses (20). The duration of intake also strains were able to utilize inulin, while all were able to affects dose-response, with longer-term intake leading to utilize short chain fructo-oligosaccharides (FOS). A separate a lower daily dose requirement. Doses needed to trigger study by Scott et al. (15) also showed that inulin with a DP other health effects, such as impacts upon cholesterol and greater than ~25 were poorly utilized by bifidobacteria. blood glucose, sometimes differ from the doses needed to These studies may account for the fact that a very high elicit an impact upon bifidobacteria and lactobacilli. dose of inulin, typically 15 – 20 g/day or higher, is required For example, Alles et al. were unable to detect an impact in order to see a bifidogenic effect in vivo (9, 16). of 15g/d FOS upon levels of blood glucose and lipid profiles Nonetheless, inulin has been well received as a source in a clinical trial with 20 subjects (21).

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 V Differences in dose requirements also have implications Comp. Rev. Food Sci. Food Safety, 10, 2-16, 2011. on consumer acceptance and tolerance, and ease of 6. Gullon, P., et al., Production and Bioactivity from Biomass formulation into function foods and beverages. Hemicelluloses, in Food Oligosaccharides: Production, Analysis and Bioactivity, First Edition. Edited by Dr. F. Javier Moreno and Dr. Marıa Luz Sanz, John Wiley & Sons, Ltd., 2014. 7. Hosseini, E., et al., Propionate as a health-promoting microbial SUMMARY metabolite in the human gut, Nutr Rev., vol 69, 245-258, 2011. 8. Hamer, H.M. et al., The Role of Butyrate on Colonic Function, Prebiotics are increasingly gaining recognition as an Aliment Pharmacol Ther., 27(2):104-19, 2008. important part of the human diet, with diverse and 9. Whelan, K., Mechanisms and effectiveness of Prebiotics in significant impacts beyond gut health. Several types of Modifying the Gastrointestinal Microbiota for the prebiotics are commercially available and others are Management of Digestive Disorders, Proc. Nutr. Soc., 72, 288- under development. Prebiotics contribute to dietary fiber 298, 2013. intake, but have a distinctly different role compared to 10. Scott, K., et al., The Influence of Diet on the , insoluble fiber that acts as roughage and regulates fecal Pharm. Res., 69, 52-60, 2013. 11. Sonnenberg, E.D., et al., Specificity of polysaccharide use in bulk and moisture. The different structures of prebiotics, intestinal bacteroides species determines diet-induced including 5-carbon vs 6-carbon subunits, different types microbiota alterations, Cell, 141(7), 1241-1252, 2010. of bonds between subunits, degree of polymerization, 12. Martens, E.C., et al., The devil lies in the details: how variations and solubility, all affect utilization by different bacteria in polysaccharide fine-structure impact the physiology and within the gut microbiome, and production of certain evolution of gut microbes, J Mol. Biol., 426(23), 3851-3865, 2014. metabolites. Consequently, there are differences in 13. Hamaker, B.R., et al., A perspective on the complexity of selectivity, dose, and health impacts among the various dietary fiber structures and their potential effect on the gut prebiotics. As knowledge of the microbiome increases, microbiota, J. Mol. Biol., 426(23), 3838, 2014. and structure-function relationships are better understood, 14. Rossi, M. et al., Fermentation of Fructooligosaccharides and human health benefits may be achieved by targeted Inulin by Bifidobacteria: a Comparative Study of Pure and Fecal Cultures, Appl. Env Microbiol, 71(10), 6150 - 6158, 2005. intake of specific prebiotic fibers to selectively enhance 15. Scott, K., et al., Prebiotic stimulation of human colonic the growth of healthy gut microbes. butyrate-producing bacteria and bifidobacteria, in vitro, FEMS Microbiol Ecol 87, 30-40, 2014. 16. Bouhnik, Y., et al., Short-chain fructo-oligosaccharide REFERENCES administration dose-dependently increases fecal bifidobacteria in healthy humans, J. Nutr., 129, 113-116, 1999. 1. Moshfegh, A., et al., Presence of Inulin and Oligofructose in 17. Sarbini, S, et al., Prebiotics: Metabolism, Structure and the Diets of Americans, J. Nutr. 129: 1407S–1411S, 1999. Function, Funct. Food Rev., 3, 93-106, 2011. 2. Binns, N., International Life Sciences Institute Europe Concise 18. Makelainen, H., et al., Xylo-oligosaccharides and lactitol Monograph Series, Probiotics, Prebiotics and the Gut promote the growth of Bifidobacterium Microbiota, 2013. http://ilsi.wpengine.com/europe/ lactis and species in pure cultures, Beneficial wp-content/uploads/sites/3/2016/05/Prebiotics-Probiotics.pdf Microbes, 1(2), 139-148, 2010. 3. Scott, K., International Scientific Association for Probiotics and 19. Finegold, S.M. et al., Xylooligosaccharide increases Prebiotics, http://isappscience.org/prebiotics/ bifidobacteria but not lactobacilli in human gut microbiota, 4. US Food and Drug Administration, 21 CFR Part 101, “Food Food Funct., 5, 436 - 445, 2014. Labelling: Revision of the Nutrition and Supplement Facts 20. Na, M.H., and Kim, W.K., Effects of Xylooligosaccharide Intake Labels”, https://www.federalregister.gov/ on Fecal Bifidobacteria, Lactic Acid, and Lipid Metabolism in articles/2016/05/27/2016-11867/food-labeling-revision-of-the- Korean Young Women, Korean J. Nutr., 40, 154-161, 2007. nutrition-and-supplement-facts-labels, 2016. 21. Alles, M.S., et al., Consumption of fructooligosaccharides does 5. Aachary, A.A., et al., Xylooligosaccharides (XOS) as an not favorably affect blood glucose and serum lipid Emerging Prebiotic: Microbial Synthesis, Utilization, Structural concentrations in patients with type II diabetes, Am. Clin. Characterization, Bioactive Properties, and Applications, Nutr., 69, 64-69, 1999.

VI Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 GEORGE PARASKEVAKOS International Probiotics Association (IPA)

George Paraskevakos

Market insights on probiotics

KEYWORDS: Global, demand, probiotics, ingredients, market, analysis, consumers, trends, microbial.

OVERVIEW part to preventing and/or treating many chronic diseases and disorders. This remarkable expansion of duties or The probiotics market is experiencing unprecedented year- rather “discovered capabilities” will be a force behind over-year growth. Industry analysts estimate the probiotics increased adoption of their applications. market at $33 billion in 2015. That number is predicted to • Demographics: Aging populations across many reach over $50 billion by 2021, as usage expands globally. continents, most notably North America and Western Three sectors are included in the growth: foods containing Europe, are also a driver for increased consumption. As natural probiotics with yogurt and kimchi as market people age, health needs escalate and interest in health leaders; foods fortified with probiotics such as sports bars generally increases as well. and beverages; supplements which are undergoing rapid • Dwindling healthcare resources: Rising healthcare costs change in delivery systems. These probiotics are for human are propelling enhanced attention to prevention across application; the animal probiotics segment is growing also as the sphere of health care. Probiotics are part of the probiotics are increasingly added to feed in conjunction with strategy for many consumers and health practitioners. or as a replacement for antibiotics. Some countries rely more on indigenous forms of fermented foods—think kimchi in South Korea or yogurt in Uzbekistan—for REGIONAL PROBIOTIC MARKETS their daily dose of beneficial microbes. These countries have seen flatter growth in supplement usage. As diets change The following regional snapshots characterize trends and however to include more packaged and processed foods, changes impelling the market of probiotics across the globe. demand for supplements may change. Countries which had These regions in most cases do not include a complete list of not embraced probiotic-rich foods traditionally are seeing countries; rather, the countries represent the largest or most rapid growth of the sector. For example, kefir and kombucha accessible markets in which to derive data. The material are entering the popular lexicon in North America. according to Euromonitor International (1), which was the source for much of the following country-specific information, should not serve as guidance or a comprehensive view of DRIVERS OF GROWTH the market.

• Awareness: Growth is being driven by increased North America awareness across global platforms, driven by North America leads the world in per household expenditure technological expediency via social media like Facebook in 2015, spending $13 on probiotic supplements. and YouTube. But public health programs and traditional Market size estimates currently stand at slightly over US$2,0 billion media are also highlighting the role of probiotics in health. and are expected to grow at an estimated CAGR of 9.8%. • Broadened capability: It is noteworthy that probiotics are no longer relegated to digestive health categories on the United States shelf. In the past decade, large research initiatives and Consumers are increasingly searching for ways to improve resources, most notably the NIH Human Microbiome diets and decrease unhealthy diets. The trend is driving Project, have spurred discovery that the microbiota is increased sales of probiotics foods and supplements. Total integral to the immune system, metabolic systems, and retail value in the United States exceeded US$1,8 billion respiratory system and eventually may play an integral according to analysts.

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 VII Canada Italy A much smaller but aging population coupled with Wellness through natural health products is a growing unhealthy diets and stressful lifestyles boosted sales of interest in Italy. Industry sources report that physicians are probiotics and fiber rich foods as well supplements to increasingly recommending dietary supplements along with counteract constipation and increase regularity. traditional prescription medicine for minor health problems. Probiotics have long had a favoured place as a prescription Australasia alongside antibiotic therapy in this country. Absolute growth from 2015 to 2020 is estimated to be US$8 million. This translates to 1.8% CAGR for the 5 year period. Sweden Probiotics in the form of supplements and packaged foods New Zealand with added probiotics are being marketing strongly to Probiotic supplement sales grew at a fast pace in 2015: 10% Swedish consumers. Sales of both categories are rising. Food growth in addition to being the second highest performer in with either intrinsic or added probiotic value has proven non-herbal/traditional dietary supplements market category. increasingly popular in Sweden. Growth in awareness of the Probiotics which used delivery systems that did not require microbial impact on health is driving the market. refrigeration experienced a strong introduction. United Kingdom Australia Foods and beverages with probiotic fortification continued The population is generally aware of factors fueling good to gain market share in 2015. The pediatric subset market health and the lifestyle ingredients which sustain it. An continues to grow. Sources believe a strong market may exist aging population will spur use of probiotic supplements. In for dietary supplements combining probiotics and protein, as addition, people are working later in life and will add dietary both components are spurring widening interest. supplements to regimens. Eastern Europe and Russia Western Europe Absolute growth from 2015 to 2020 is estimated to be Absolute growth from 2015 to 2020 is estimated to be US$82.4million. This translates to 4.2% CAGR for the 5 year US$111.5 million. This translates to 2.5% CAGR for the 5 year period. period. Belarus Finland Advertising as well as public health initiatives promote While not as brisk as in past years, product launches in the use of dietary supplements in the goal of reducing established brands continued in 2015. Some were innovative, disease and disorders. Probiotic advice takes a back seat but most were extensions of popular brands. to initiatives on reducing consumption of cigarettes and alcohol. France Market data put the probiotic supplement group in the Russia number two spot in value capitalization in 2015 within dietary Probiotic supplements are becoming increasingly popular as supplements in France. traditional diets, rich in yogurt and other fermented foods, Not only gut health and its attendance were the drivers in growth. One product which claimed to be important for health of the immune system proved especially popular. Thus, the aging population will be a big factor in the growth of probiotic usage, say market analysts.

Germany Probiotic foods and supplements saw some growth in 2015, driven by positive media attention and growth in the aging population. New products with emphasis on long- acting and viability broadened the market demand. Probiotic Figure 1. Total sales for top countries and drivers of growth. supplement sales grew 1% Source: © EUROMONITOR INTERNATIONAL – on behalf of IPA in 2015.

VIII Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 are replaced by processed, packaged foods as well as fast foods from retailers. Probiotic supplements claimed 28% value share of the category in 2015. Probiotic supplements are advised by the medical community in conjunction with antibiotics, an obligatory practice not as common in western cultures. In addition, probiotic supplements are routinely suggested for digestive complaints. Both adults and children are included in the protocol. Prebiotics include inulin are often included with the probiotic supplement.

Uzbekistan Increased consumer awareness campaigns are boosting sales of probiotic supplements according to market data.

Latin America Absolute growth from 2015 to 2020 is estimated to be US$8.8million. This translates to 1.6% CAGR for the 5 year period.

Mexico Probiotic supplements were increasingly popular in 2015. Analysts believe the stressful lifestyle has led to more stomach ailments and gastritis for which probiotics are seen as a solution. On the retail level, sales of probiotic supplements increased by 11% in 2015.

Asia Pacific Absolute growth from 2015 to 2020 is estimated to be US$86.8 million. This translates to 2.8% CAGR for the 5 year period. Rising disposable incomes will increase demand.

South Korea By regularly consuming probiotics, consumers are preventing constipation and keeping their bowels healthy. Consumers are buying more yogurt; devices to make yogurt at home are selling at a faster clip. Consumption of yogurt in South Korea is rivalling that of milk.

Indonesia Fermented milk and yogurt products are competing with digestive remedies in this market. When probiotics are included in their marketing claims, products are shown to be more popular.

Malaysia The probiotic supplement market here grew 17% in 2015. Consumers with busy lifestyles consumed probiotic supplements to maintain digestive health.

Pakistan Medical practitioners educate consumers about dietary supplements including probiotics.

Middle East and Africa The Middle East and Africa represent small markets with plenty of room to grow. Absolute growth from 2015 to 2020 is estimated to be US$55.4 million. This translates to 17.7% CAGR for the 5 year period.

Cameroon Probiotic supplement sales have minimal presence in Cameroon. Egypt not easily answered by current evidence-based science: which Supplements may gain traction with middle and upper class strain, which dose and when to take them. The confusion may Egyptians. Some analysts expect that probiotic supplements lead to inertia in some and refusal in others. The plethora of on- will dominate the and category in coming going research, will in time provide much needed answers. years. Meanwhile a young population and poor incomes will impede a broad market demand for probiotic supplements. Inconsistent regulation in the global arena Government regulation of probiotics, like the multitudes of Israel countries across the globe, is unfortunately all over the map. The 7% surge in dietary supplement sales in 2015 followed a 6% George Paraskevakos, Executive Director of International rise the prior year. Probiotic supplement sales appear to be the Probiotics Association (IPA) states that it will require much work biggest factor in growth. Widespread awareness of benefits of and education for governments to regulate probiotic s in the probiotics, particularly for babies suffering with colic, was raised same manner. “We are at the cusp of our journey in trying to due to media coverage in the country. profess regulatory equality but some of early successes has allowed for the IPA to receive traction when reaching out to Nigeria the different regulators,” said Paraskevakos. “Just recently we Probiotic supplements exhibited strong growth in Nigeria in 2015. had 5 government’s representatives come and present at our IPA world congress meeting in Chicago which is very positive South Africa and proves that there is a need for discussion.” IPA has been An emphasis on prevention rather than treatment is moving able to engage in discussions and is some cases had positive the South African dietary supplements market. Combination outcomes with countries such as Canada, Brazil, Mexico, and supplements which are viewed as more economical are hopefully soon the USA. popular. Negative media coverage United Arab Emirates (UAE) Sensational headlines, especially on the internet, serve as Consumers are becoming more aware of how diet impacts “click-bait” to garner attention. Paraskevakos reports that IPA health. Healthcare professionals, industry and advertising has dug into the facts behind what can seem as doomsday campaigns are raising awareness and driving sales of probiotic scenarios for the industry and has found the actual science foods and supplements. Analysts report that consumers are does not align with the headline. At times, IPA has reached out more receptive to foods beyond the standards. Probiotics fit in to authors of such articles and received no response in return. this category. With high per capita income for the most part in But he adds that “… in this very fast paced world we exist in the the UAE, sales in the probiotic category should continue on a three seconds of fame is all people tend to look at, which in positive trajectory. turn drives the medias actions.”

Saturation of processed food category PROBIOTIC MARKET CHALLENGES Many players are entering the probiotic market, whether through supplements or ingredients added to a food or Global demand for probiotics and ingredients is growing at beverage. It remains a question whether consumers will a fast pace. While heightened awareness and increased continue to welcome the abundance of probiotics in various consumer ability to pay, especially in Asian markets, are fueling products including coffee, tea and chocolate bars. the growth, several factors must be addressed as challenges to the probiotic market. CONCLUSION Confusion Probiotics are not drugs and therefore are commercially sold In conclusion, market analysis reports a robust growth market as dietary supplements or complementary and alternative for probiotics in all categories in the years to come. Widening medicine products. In most countries, probiotics are not awareness by the consumer secondary to advertising and subjected to rigorous oversight as in the pharmaceutical public health campaigns is driving increased consumption industry. Evidence for efficacy can be limited and is further across a global market. In addition, evidence-based research fragmented by various research designs and strain variability. is expanding the array of diseases and disorders which respond to probiotics in either a preventive or therapeutic capacity. Peer-reviewed data from randomized, double-blind, placebo- While all of these market drivers point to increased microbial controlled clinical trials support probiotic usage in the following: health worldwide, challenges must be addressed. treating and preventing acute diarrhea and antibiotic-induced diarrhea; preventing cow milk-induced food allergies. Less demanding studies point to effectiveness in: traveler’s diarrhea, REFERENCES relapsing Clostridium difficile-induced colitis, and urinary tract infections. Other areas of promise include: dental carries, 1. Some of the information in this letter is of a statistical nature and, obesity, depression, respiratory infections, irritable bowel while every attempt has been made to ensure accuracy and syndrome, inflammatory bowel disease, and asthma. reliability, Euromonitor International cannot be held responsible for omissions or errors. Analyses may not totally reflect the companies’ However, the broad array of targets leads to confusion among opinions, viewer discretion is advised. both practitioners and consumers. Safety, especially in critically 2. The compound annual growth rate (CAGR) measures growth over ill patients who may rely on ventilators, is being studied in multiple time periods. It is the growth rate that spans from the initial multicenter studies. However, even in less lethal scenarios, such investment value to the ending investment value if one assumes as the yogurt aisle, consumers are asking questions which are that the investment has been compounding over the time period.

X Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 ARTHUR C. OUWEHAND DuPont Nutrition & Health, Active Nutrition, Sokeritehtaantie 20, 02460 Kantvik, Finland

Arthur C. Ouwehand

The cost and benefit of probiotic use

KEYWORDS: Probiotics, lactobacillus, bifidobacterium, health economics.

There is a substantial body of literature showing that probiotic use can have beneficial effects on health. Abstract In general, probiotics tend to reduce risk or severity of disease. Here, an overview is given of studies that have calculated to cost-benefit of probiotic use. All studies indicate a positive financial effect of probiotic use. However, most studies take into account only the effect of probiotic use on the direct costs. Furthermore, many of the reviewed studies base the size of the probiotic effect on a single study. Only few studies aimed to calculate both direct and indirect costs and base the effect size on a systematic review/meta-analysis. Thus, although an overall beneficial economic effect is predicted for the use of probiotics on various health conditions, more high quality studies are required in this area.

INTRODUCTION Dietrich and co-workers (5) calculated that for their particular hospital setting AAD was responsible for an additional annual As the readership of this journal knows, probiotics are defined cost of 77800 EUR for isolation of the patients; probiotic use as live microorganisms that, when administered in adequate would reduce these specific costs by 63400 EUR. Taking into amounts, confer a health benefit on the host (1). account the additional cost for the probiotic (4600 EUR /year) That probiotics indeed do have health benefits is documented the net savings would be 58800 EUR /year. Unfortunately, by numerous randomised, double blind, placebo controlled, the calculation does not express costs per patient; making it trials and also by the many systematic reviews and meta- difficult to compare the savings to other studies. analyses that have been published. In general, probiotics do Clostridium difficle associated diarrhoea (CDAD) is a sub- not prevent disease but rather reduce risk. So, the ‘success class of AAD. C. difficile is reported to be responsible for rate’ needs to be taken into account. Assuming a certain approximately 25% of all AAD cases and is responsible for effect size and efficacy of the consumption of probiotics one the more severe cases of AAD (6). Although probiotics can calculate the costs of the probiotic consumption and have been reported to be efficacious also in reducing the estimated potential savings that are associated with this CDAD, Mergenhagen and co-workers (7) came to the consumption. This way it is possible to estimate the economic conclusion that the economic effect of probiotics could impact of the consumption of probiotics. This does, of course, not be estimated for this type of diarrhoea. Nevertheless, not take into account any influence the probiotics may have they state that probiotics have the potential to provide an on quality of life. Main factors that influence the potential inexpensive method for reducing CDAD burden if utilized in economic benefit of probiotic use are mentioned in Table 1. the appropriate patient population. This way of calculating cost-benefit; health economics, has However, Kamdeu Fansi and colleagues (8) modelled the long been used for the economic evaluation of health care costs/benefits for two different doses of probiotics in CDAD. strategies; it has only recently been applied to nutrition (2). Below, will be described what has been published to date on the topic for probiotics.

PROBIOTICS AND ANTIBIOTIC ASSOCIATED DIARRHOEA

Antibiotic associated diarrhoea (AAD) is one of the most common side effects of antibiotic use. Meta-analyses indicate that probiotics have a significant impact on the risk, duration and severity of AAD (3, 4). A number of studies have made attempts to calculate what the economic impact of probiotic use is on AAD associated costs.

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XI The model assumed that all patients with AAD would be The mean cost for families of children in the placebo group tested for CDAD and CDAD patients would be treated with was 238 EUR and 237 EUR for society. Savings were thus 192 either vancomycin or metronidazole, and used treatment EUR in total; main drivers for this cost reduction were reduced success for these antibiotics as reported in the literature. loss in work days, less use of medication and fewer visits for Hospitalisation due to CDAD was assumed as reported in medical care. the literature. Also reinfections were modelled according to the literature. Prophylactic use of probiotics by atrisk patients would lead to savings, per patient, of 1968 USD PROBIOTICS AND INFECTIOUS DIARRHOEA for a single dose and 2661 USD for double dose probiotics compared to placebo. Cost savings were mainly due to Infectious gastroenteritis is common in children and may reduced CDAD risk and reduced risk for reinfection. have various aetiologies. The most common treatment is oral Also Lenoir-Wijnkoop and colleagues calculated the rehydration. Probiotic supplementation has been shown to potential savings of the use of a fermented probiotic dairy shorten the duration of diarrhoea in children (11). Vandenplas product, Actimel, on AAD and CDAD in elderly subjects (9). and colleagues (12) calculated that costs for medication in They calculated that on average 243 GBP would be a paediatric population with diarrhoea would be 1.13 EUR saved on average per case treated with antibiotics by with probiotic vs. 4.04 EUR without. However, the additional preventing AAD, while 96 GBP would be saved per case cost for the probiotic was calculated to be 7.15 EUR; making treated with antibiotics through the prevention of CDAD. probiotic treatment more expensive. This is set off by fewer The Cost impact for CDAD is greater, however, since CDAD visits to a practitioner. The overall health care cost compare represents 15% of the total AAD cases, the total cost savings then favourable for probiotics; 10.74 EUR vs. 14.41 EUR for for CDAD are lower than for AAD. The overall savings probiotic or non-probiotic respectively. This calculation does for CDAD and AAD combined with the use of probiotics not take into account indirect costs; children staying home was calculated to be 555 GBP per patient. The cost for one day less will results in less loss of work days by parents and probiotic use was estimated to be 216 per patient. The use thus reduced loss of productivity. of probiotics may therefore lead to a potential cost saving of 339 GBP per hospitalized patient older than 65 years treated with antibiotics; regardless whether s/he develops PROBIOTICS AND ALLERGY AAD or not. There is quite some discrepancy between the estimated Estimates for the additional dietary costs of childhood eczema savings by Kamdeu Fansi and colleagues (8) and Lenoir- vary from 0 to 81 and 360 AUD annually for mild, moderate and Wijnkoop and colleagues (9). This is not so much related to severe eczema, respectively in 1997 (13). A more recent study the effect size assumed for the probiotics as well as the way (2006) estimated the additional dietary costs at 117 to 452 EUR of calculating the costs and the magnitude of the expenses annually for moderate and severe eczema respectively (14). included in the calculations. These calculations do not take Comparing daily dietary costs between infants with and into account additional costs for longer sick leave and without food allergy did not suggest a difference (15). However, thereby loss of productivity. Nor does the estimate consider these only considered the costs for the family; they did not a potential ‘herd-effect’; with fewer cases of AAD, infection consider society’s cost in the nutrition; reimbursement of costs risk may also be reduced; leading potentially to an overall for hydrolysed formula, estimated at 4.86-8.67 EUR/day, or reduced incidence. the costs associated with a larger burden to the health care system; not estimated. Taking this into account, it is likely that the additional cost of the probiotics, estimated at 0.50-0.70 EUR, PROBIOTICS AND COLIC would lead to an overall economic benefit. The benefit would probably be highest if probiotics were used in at-risk populations Colic is a common but rather undefined condition in infants; where substantial risk reductions have been reported; overall characterised by excessive crying without any know underlying risk ratio 0.78 (16), though this does not take into account condition; though a gastrointestinal origin is suspected. subjects that still have allergy but with milder symptoms. Thus, Indrio and colleagues calculated what probiotics; L. reuteri, 22% of the costs could potentially be avoided; in the example use in the case of colic would mean for costs associated mentioned above, this would be between 1 and 2 EUR while with this condition (10). To that end, they identified four cost the added cost would 0.50-0.70 EUR . In the at-risk population, drivers; emergency paediatric medical examination, los of incidence of eczema is around 50% (17); this is likely to work days, medication and probiotic treatment. provide an economic benefit in this population. In the general The authors used the effect size as observed in their study population, however, eczema incidence is only 7% (15). It on randomly is therefore chosen infant; not likely that primary consumption prevention; not of probiotics in at-risk children. by all infants The mean cost in the general for the families population of children who would provide received the an economic probiotic was 150 benefit with EUR and 133 EUR respect to for society. allergy.

XII Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 PROBIOTICS AND RESPIRATORY TRACT INFECTION CONCLUSIONS

In general, the cost of common cold is relatively small, Various probiotics have shown potential for reducing the but because it is so wide spread; affecting everybody risk or severity of disease. Besides an improved quality in the population, and recurring; the overall costs are, of life for the patient, substantial savings for society are nevertheless, substantial. These costs tend to be country possible. The size of these savings varies depending on the specific as different health care systems provide different health target. Most studies cited here calculated savings compensations and may have different cultures in how to only on the direct costs of disease and did not take into deal with common cold. account the costs for society. Such calculations can thus Probiotics are relatively inexpensive. However, for an be expected to underestimate to potential savings. On effect on the common cold, they would require prolonged the other hand, most calculations are based on a single consumption; at least during the winter season; adding up successful study. Authors of exceptionally successful the cost. This cost will have to be carried by the individual studies may be more motivated to perform cost-benefit as the national health care system will not compensate calculations than authors of less successful studies. A more their use. reliable approach is therefore to use the effect size of In a recent publication, Lenoir-Wijnkoop and colleagues (18) probiotics as determined in a broad meta-analyses; as made an attempt to calculate the economic impact of done in the study by Lenoir-Wijnkoop and colleagues (18). probiotic use on common cold for the French population. This study also takes into account indirect costs and gives To this end, a model French population was constructed therefore a more in-depth view of the financial benefits on a 1/1000 scale, taking into account age distribution of probiotics. However, despite the short comings in the (small children are more likely to get ill), smoking status calculations of some of the studies, they do indicate that (smokers are more likely to contract common cold) and there is an economic benefit to be gained from the use ‘community setting’ (being at school, day care or open of probiotics; in some cases for the general population; office exposes to more contagious interactions then when such as in respiratory tract infections, or in specific sub- working individually). For the success rate, two meta- populations as e.g. in the case of allergy. analyses were used; by Hao and co-workers (19) and by King and co-workers (20). The incidence of common cold infections was retrieved REFERENCES AND NOTES from a database; likewise costs for medication, visits to general practitioners, loss of income, compensations by 1. Hill C, Guarner F, et al. Nature reviews Gastroenterology & the health care system, etc. were calculated based on hepatology. 2014. Epub 2014/06/11. publicly available data for France. Likewise, the costs for 2. Koponen A, Sandell M, et al. Microbial ecology in health and disease. probiotics were estimated. 2012;23. Epub 2012/01/01. Using the data from King and co-workers, the French 3. Hempel S, Newberry SJ, et al. JAMA. 2012;307(18):1959-69. Epub population would have 2.383 million fewer sick days of 2012/05/10. which 581 000 days of sick leave; resulting in savings 4. Ouwehand AC, DongLian C, et al. Vaccine. 2014;32(4):458-63. Epub of 84.4 million EUR, 14.6 million EUR and 16.2 million EUR, 2013/12/03. 5. Dietrich CG, Kottmann T, Alavi M. World J Gastroenterol. for the society, the national health care system and the 2014;20(42):15837-44. Epub 2014/11/18. family, respectively. Using the data from Hao and co- 6. Larcombe S, Hutton ML, Lyras DTrends in microbiology. 2016. Epub workers suggested a reduction of 6.639 million fewer sick 2016/02/22. days of which 1 453 000 days of sick leave, resulting in 7. Mergenhagen KA, Wojciechowski AL, Paladino JA. calculated savings of 253.6 million EUR, 37.7 million EUR PharmacoEconomics. 2014;32(7):639-50. Epub 2014/05/09. and 131.1 million EUR, respectively. Biggest savings could 8. Kamdeu Fansi AA, Guertin JR, LeLorier J. Journal of medical actually be generated by targeting young children as economics. 2012;15(1):53-60. Epub 2011/10/26. they have the highest incidence of common cold and 9. Lenoir-Wijnkoop I, Nuijten MJ, et al. Frontiers in pharmacology. associated costs. 2014;5:13. Epub 2014/03/07. There are of course a few remarks to be made with this 10. Indrio F, Di Mauro A, Riezzo G. JAMA pediatrics. 2014;168(8):778. Epub 2014/08/05. calculation. Not all common cold infections are reported 11. Ahmadi E, Alizadeh-Navaei R, Rezai MS. Caspian journal of internal to general practitioners and thus do not end up in the medicine. 2015;6(4):187-95. Epub 2015/12/09. statistics, suggesting that incidence and thereby also 12. Vandenplas Y, De Hert S. Beneficial microbes. 2012;3(3):189-94. Epub the benefit are under estimated. The cold and flu season 2012/07/28. of 2012, that was used to calculate the incidence, was 13. Su JC, Kemp AS, Varigos GA, Nolan TM. 1997;76(2):159-62. Epub actually rather mild, thus on average a bigger effect 1997/02/01. could be possible. On the other hand, part of the French 14. Ricci G, Bendandi B, et al. Journal of pediatric health care : official population is already consuming probiotics and would publication of National Association of Pediatric Nurse Associates & thus already benefit from them; the effect size could Practitioners. 2006;20(5):311-5. Epub 2006/09/12. thus be overestimated. The additional cost of probiotics 15. Alanne S. Microbial ecology in health and disease. 2012;23. Epub 2012/01/01. could be smaller if e.g. a plain yogurt was replaced by 16. Zhang GQ, Hu HJ, et al. Medicine. 2016;95(8):e2562. Epub 2016/03/05. a probiotic one, or bigger if dietary supplements are 17. Thomsen SF. 2015;2. Epub 2015/11/12. purchased or e.g. probiotic yogurts not replacing plain 18. Lenoir-Wijnkoop I, Gerlier L, et al. PloS one. 2015;10(4):e0122765. Epub yogurt. Finally, the study does not take into account 2015/04/11. a ‘herd effect’; when everybody is more resistant to 19. Hao Q, Lu Z, Dong BR, et al. Cochrane Database Syst Rev. common cold, it spreads less and the risk of exposure is 2011(9):CD006895. Epub 2011/09/09. reduced. 20. King S, Glanville J, et al. Br J Nutr. 2014;112(1):41-54. Epub 2014/05/02.

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XIII COLINE GERRITSEN*, GELINE ORMEL *Corresponding author Winclove Probiotics, Hulstweg 11, 1032 LB Amsterdam, The Netherlands

Coline Gerritsen Probiotics to prevent upper respiratory tract infections

KEYWORDS: Microbiota, probiotics, upper respiratory tract infections.

Upper respiratory tract infections (URTIs) are illnesses caused by an infection of mucosal surfaces in the Abstract nose, sinuses, pharynx and/or larynx. These infections include the common cold, rhinosinusitis, tonsillitis and otitis media and are very common, especially among infants, children and elderly. URTIs are among the most common reasons for people to visit their doctor and they account for a large part of antibiotic use in high-income countries. Although antibiotics are very effective in clearing infections, they come with negative side-effects such as antibiotic resistance. This has prompted researchers to look into more natural alternatives, such as probiotics, for treatment and/or prevention of URTIs. Mechanisms by which probiotics may be effective are thought to be related to restoration of the microbial balance in the upper respiratory tract and stimulation of the immune response. Based on these scientific insights the probiotic formulation, Winclove 381 Respiratory, to prevent URTIs was developed.

INTRODUCTION PROBIOTICS

The importance of the microbial communities living in our According to the latest definition by the World Health intestinal tract for our health is well known. Due to extensive Organization, probiotics are live-micro-organisms that, when research we have learned that establishment of a symbiotic administered in adequate amounts, confer a health benefit relation between the host and the gut microbiota in early to the host (4). Probiotic interventions are aimed to restore life is crucial for optimal health later in life. However, only the microbial balance and/or prevent disturbances in this recently we have started to appreciate the role of the balance. Their action can take place at different levels, microbial communities that inhabit the upper respiratory tract. they; reduce the risk of outgrowth of potential pathogenic Nevertheless, the upper respiratory tract, just like our intestines, bacteria, restore lost bacterial or metabolic activities, or needs a variety of bacteria to maintain human health. stimulate a specific immune response (5,6). Probiotics have The upper respiratory tract is an important site of pathogen shown extensive beneficial effects in the gastro-intestinal tract colonization. In order to colonize and/or outgrow in the upper and have established positive effects in the treatment of e.g. respiratory tract, bacterial pathogens must compete with diarrhea and functional gastro-intestinal disorders (7,8). each other and with commensal members. If the commensal Moreover, their effects seem to reach beyond the gut. microbiota is compromised, for example by use of antibiotics, Human clinical trials have shown that probiotics could also be antimicrobial nasal sprays, or a weakened immune system, effective in cases of allergies, mental disorders, periodontitis, pathogens may be able to cause upper respiratory tract vaginal infections and also in respiratory infections (9,10). infections (URTIs). Recently, studies have been published addressing the microbial communities that inhabit the upper respiratory tract (1,2). These studies show quite a difference UPPER RESPIRATORY TRACT INFECTIONS between the key bacterial players in the respiratory tract compared to the ones in the gastro-intestinal tract. Data indicate Upper respiratory tract infections (URTIs) are illnesses caused that, just like in the gastro-intestinal tract, imbalances in the by an infection of mucosal surfaces in the nose, sinuses, microbiota seem to play an important role in the susceptibility to pharynx and/or larynx. These infections include non-allergic infections. A healthy upper respiratory tract microbiota provides rhinitis (the common cold), rhinosinusitis, pharyngitis, tonsillitis us with local colonization resistance. A lack of commensals and otitis media. URTIs are a very common problem, might lead to outgrowth of potential pathogenic bacteria, especially among infants, children and elderly, and they consequently resulting in respiratory illness (3). account for 9% of all consultations in general practice (11).

XIV Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 URTIs can be caused by viruses or bacteria. cavity that are able to reach the more inaccessible The most common viruses causing URTIs are rhinoviruses, niches such as the sinuses and middle ear and cause coronaviruses, parainfluenza viruses and influenza virus (11). infection (18-20). When administering a probiotic food The most common bacterial pathogens causing URTIs are supplement the bacterial strains can temporarily colonize Group A streptococci, Mycoplasma pneumonia, Chlamydia the upper respiratory tract, thereby inhibiting and pneumonia, Corynebacterium diphtheria, Staphylococcus excluding pathogens in the upper respiratory tract and aureus and Streptococcus pneumonia (11). For example preventing their shedding into other niches. This microbiota pharyngitis in children is in 15-40% of the cases caused management can not only prevent the healthy microbiota by a viral infection and 38-40% of the cases caused by a from being disturbed, it also is able restore the microbiota. bacterial infection. In adults these numbers are respectively The systemic pathway is through immune-modulation. 30-60% and 5-10% (12). Probiotic strains are able to elicit a strong immune response Usually an infection starts by direct invasion of the mucosa against invaders of the respiratory tract. This is done via the of the upper airways by the pathogen. The pathogens innate immune system (enhancing phagocytic activity), have to fight the commensal healthy microbiota present in via acquired immunity (inducing specific immunoglobulins) the airways and overcome the local and systemic immune and by enhancing local immunity (increasing the system. Usually respiratory infections are self-limiting and production of TH1 cells and cytokines). It is now thought will resolve spontaneously. Common symptoms of URTI that these combined strengths are responsible for the includes nasal congestion, sore or scratchy throat, fever beneficial effect of probiotics in URTIs. For example in and coughing. However, sometimes an infection left common cold infections, the symptoms like a stuffy nose untreated can impair breathing and swallowing in such a and sore throat are the body’s inflammatory response way that hospitalization is necessary, especially when fever toward a virus, not a direct action of the virus itself. and diarrhea are symptoms as well. Treatment depends on Probiotic microorganisms may soften the immune system’s the cause of the infection, however most treatments are reaction by reducing the body’s inflammatory response. aimed at alleviating the symptoms and not at treating the cause. Nevertheless, in some case antibiotics or antivirals are administered. Because antibiotics are associated with WINCLOVE 381 RESPIRATORY many side-effects and can promote bacterial resistance and secondary infections, they will have to be used To offer a natural alternative for the prevention of URTIs cautiously (13,14). Alternative therapies that support the Winclove has developed Winclove 381 Respiratory. Winclove commensal microbiota in the upper respiratory tract are 381 Respiratory consists of the following probiotic strains; highly desired. Streptococcus oralis 89a, Lactobacillus rhamnosus LB21, Lactobacillus rhamnosus WGG, Bifidobacterium animalis subsp. lactis W12, Bifidobacterium animalis subsp. lactis W51, PROBIOTICS AND UPPER RESPIRATORY TRACT INFECTIONS and Lactococcus lactis W19, blended on a carrier matrix consisting of rice starch, maltodextrin, inulin and vitamin Several studies and meta-analyses have shown that C. The total cell count of the formulation is 1,0 x 109 colony probiotics can be effective in preventing URTIs. In addition, forming units (cfu)/gram. The bacterial strains in Winclove recent studies suggest that probiotics may reduce the 381 Respiratory have been selected based on their in vitro risk of various symptoms of URTIs. A recent Cochrane capacity to inhibit URTI related pathogens, and to positively meta-analysis of 13 clinical studies including 3720 infants, influence the immune system. Winclove 381 Respiratory is children and adults, showed that probiotics were more developed as preventative therapy for URTIs. beneficial in preventing URTIs compared to placebo. Two strains in the formulation, S. oralis 89a and L. rhamnosus This review concluded that; despite the heterogeneity of LB21, have been added to the formulation for their in the RCTs, probiotics were found to reduce the number vitro capacity to inhibit URTI related pathogens such of people experiencing episodes of acute URTI by 47%. as S. pneumoniae, H. influenzae, M. catarrhalis, and S. Furthermore, overall the duration of an episode of URTI pyogenes. In addition, they have shown positive effects in was reduced almost two days. Both these positive results preventing ear- and throat infections in several clinical trials may further reduce the use of antibiotics and cold-related (21-26). S. oralis is a natural member of the microbiota of school absence (15). A second review included 14 the upper respiratory tract. RCTs and concluded that probiotics in children Furthermore, the bacterial strains L. rhamnosus have a modest effect both in diminishing the WGG and B. animalis subsp. lactis W12 (genetically incidence of URTIs as well as the severity of the identical to respectively L. rhamnosus LGG and B. symptoms (16). animalis subsp. lactis BB12), have been added to the Several possible mechanisms have been formulation because they have shown in previous proposed to explain the beneficial effects clinical trials to prevent common cold (27). observed in clinical studies (17). In general these The last two strains in the formulation, B. animalis mechanisms can be divided into local and subsp. lactis W51 and Lc. lactis W19, have systemic effects. The first is based upon been added to further improve the competitive exclusion of the pathogens immune boosting function of the by the probiotic bacteria. It has been formulation. The capacity of these hypothesized that especially recurrent two strains on modulating the immune infections are caused by the constant system has been measured in vitro by shedding of pathogens from biofilms TH1 cell induction and the production within the nasal and nasopharyngeal of cytokines.

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XV SELECTION OF STRAINS BASED ON IMMUNOMODULATORY PROPERTIES

In healthy conditions commensal microorganisms colonize the mucosa upper respiratory tract without inducing inflammation. Pathogens disturbing the commensal microbiota and responsible for (acute) infections often have developed mechanisms to evade an immune response by the host. One of the mechanisms by which probiotics can help fight infections, including URTIs, is by communicating with the immune system to elicit an appropriate response against invaders. Probiotics have shown to be able to regulate the adaptive immune response against pathogens mediated via T cell activation, see figure 1. Specific bacterial (probiotic) strains can provide signals from the lumen via dendritic cells across the epithelial barrier to naïve T cells, followed by differentiation of naïve CD4+ T cells Figure 2. The induction of T-bet gene expression by diverse probiotic strains, including Lc. Lactis W19, which is indicative for the induction of TH1 cells. into T helper (TH) cells. These TH cells play a central regulatory role in immune and autoimmune responses. They can regulate the activity of other immune cells by releasing cytokines. Proper differentiation of naïve CD4+

T cells into TH cells is critical for a T-dependent immune response. Of the subtypes of TH cells that are known to date, the TH1 cells play a major role in the initial attack of invaders.

More in depth, TH1 cells are a lineage of CD4+ T cells that promote cell- mediated immune responses and are required for the host defense Figure 3A. In vitro production of TNF-α by PBMCs Figure 3B. In vitro production of IFN-γ by PBMCs after 24 h co-culture with Lc. Lactis W19 or B. after 24 h co-culture with Lc. Lactis W19 or B. against intracellular viral and bacterial animalis subsp. lactis W51 compared to control. animalis subsp. lactis W51 compared to control. pathogens. TH1 cells mainly secrete IFN-γ, TNF-α and IL-2, cytokines that promote macrophage activation, nitric oxide production, An in vitro screening of a subset of Winclove’s probiotic strain and cytotoxic T lymphocyte proliferation, leading to the collection was performed in collaboration with the Wilhelmina phagocytosis and destruction of microbial pathogens. Children’s Hospital Utrecht, the Netherlands, to investigate the capacity of the bacterial strains to trigger differentiation

of a naïve T cell into either TH1, TH2, TH17 or Treg cells (28). After co-culture of peripheral blood mononuclear cells (PBMCs) with + H probiotic bacteria, the induction of CD4 THH HHh cell subsets was analyzed based on gene expression levels of signature transcription factors using reverse transcriptase PCR. For Winclove 381 Respiratory strains were selected for their capacity

to induce TH1 cells, which was measured by the induction of T-bet gene expression. The results of the screening are found in figure 2. As can be seen from figure 2,Lc. lactis W19 is a very

good inducer of the differentiation of naïve T cells into TH1 cells.

Another property for which a subset of Winclove’s probiotic Figure 1. Schematic representation of the induction of naïve CD4+ strain collection was screened was the in vitro ability to T cells differentiation by probiotic bacteria. Characteristic cytokines modulate the production of cytokines by mononuclear and signature transcription factors for TH cell subsets (TH1, TH2, TH17 and regulatory T cells) are indicated. cells. Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy adult donors.

XVI Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 The effects of the probiotic strains on cytokine production 7. Allen S.J., Martinez, E.G., et al., Probiotics for treating acute by antigen-stimulated peripheral blood mononuclear infectious diarrhea. Cochrane Database Syst Rev, 10(11), (2010). cell (PBMC) were evaluated. Cell-free supernatants were 8. Girardin, M. and Seidman, E.G., Indications for the use of collected of 24 h co-cultures of PBMCs with individual probiotics in gastrointestinal disease. Dig Dis, 29(6), 574-87 (2011). 9. MacPhee R.A., Hummelen R., et al., Probiotic strategies for the probiotic strains. Cytokine profiles were measured by the treatment and preventions of bacterial vaginosis. Expert Opin multiplex immunoassay of Luminex as described by De Jager Pharmacother, 11(18), 2985-9 (2010). et al. (29). Production of IFN-γ and TNF-α, which are cytokines 10. Ya W., Reifer C., et al., Efficacy of vaginal probiotic capsules mainly produced by TH1 cells, was measured and compared for recurrent bacterial vaginosis: a double-blind, randomized, to control, see figure 3A and 3B. Data are mean ±SEM values placebo-controlled study. Am J Obstet Gynecol, 203(2),1-6 (2010). of three or four healthy donors. As can be seen from figure 11. Bourke J.P., Lecture Notes: Respratory Medicine. Oxford, UK: 3A and 3B, B. animalis subsp. lactis W51 and Lc. lactis W19 Blackwell Publishing (2007). are able to induce the production of IFN-γ and TNF-α. These 12. Pichichero, M., Clinical Management of Streptococcal Pharyngitis. West Islip, NY: Professional Communications, Inc. (2007). cytokines will strengthen the TH1 immune response against pathogens in the respiratory tract. 13. Brook I., Gober A.E., Antimicrobial resistance in the nasopharyngeal flora of children with acute otitis media and otitis media recurring after amoxicillin therapy, J. Med. Microbiol. 54(1), 83–5 (2005). 14. Nord C.E., Sillerström E., et al., Effect of tigecycline on normal CONCLUSIONS AND FUTURE DIRECTIONS oropharyngeal and intestinal microflora,Antimicrob. Agents Chemother, 50(10), 3375–3380 (2006). With this article we aimed to stress the importance of the 15. Hao Q., Dong B.R., et al., Probiotics for preventing acute upper microbiota for the prevention of upper respiratory tract respiratory tract infections (review). Cochrane Database Syst Rev. infections. Living in a time where the drawbacks and the risks of 3(2), (2015). antibiotic treatment are being more recognized, alternatives 16. Ozen M., Kocabas Sandel G., et al., Probiotics for the prevention treatment options such as probiotics are highly desirable of pediatric upper respiratory tract infections: a systemic review. for doctors. A large majority of clinical trials conducted on Expert Opin Biol Ther, 15(1), 9-10 (2015). prevention and treatment of URTIs with probiotics have shown 17. Hatakka K., Probiotics in the prevention of clinical manifestations of common infectious diseases in children and in the elderly, Ph.D. positive outcomes (15,16). Probiotics such as Winclove 381 Thesis, Helsinki, University of Helsinki (2007). Respiratory could offer an alternative treatment solutions. An 18. Chao Y, Marks L.R., et al. Streptococcus pneumoniae biofilm important aspect that should be addressed before developing formation and dispersion during colonization and disease. Front a probiotic product is safety. All the probiotic strains in Winclove Cell Infect Microbiol, 4:194 (2014). 381 have the Qualified Presumption of Safety status (QPS) or 19. Duell, B.L., Su, Y.C. et al. Host-pathogen interactions of an extensive safety file and have been tested independently nontypeable Haemophilus influenzae: from commensal to for their efficacy on pathogen inhibition and/or immune pathogen. FEBS Lett (2016). parameters. Since probiotic strains can show synergistic 20. Marks, L.R., Davidson, B.A., et al. Interkingdom signaling induces effects but also competition, testing of the final formulation for Streptococcus pneumoniae biofilm dispersion and transition from effectivity, preferably in vivo, is desired. asymptomatic colonization to disease. MBio 4 (2013). Besides the food supplement Winclove 381 Respiratory, 21. Roos K., Grahn E., et al., Interfering alpha-streptococci as a protection against recurrent streptococcal tonsillitis in children. Int Winclove is further investigating to develop more products that J Pediatr Otorhinolaryngol, 25(1-3),141–8 (1993). could alleviate or prevent upper respiratory tract infections. 22. Roos K., Holm S.E., et al., Alpha-streptococci as supplementary At the moment a medical device, aimed at reducing the risk treatment of recurrent streptococcal tonsillitis: a randomized of recurrent ear and throat infections is being developed. placebo-controlled study. Scand J Infect Dis, 25(1), 31–5 (1993). Further research is planned to show effectiveness of these 23. Roos K., Holm S.E., et al., Recolonization with selected alpha- formulations in vivo. streptococci for prophylaxis of recurrent streptococcal pharyngotonsillitis--a randomized placebo-controlled multicentre study. Scand J Infect Dis, 28(5), 459–62 (1996). REFERENCES 24. Falck G., Grahn E., et al., L. Tolerance and efficacy of interfering alpha-streptococci in recurrence of streptococcal 1. Lima S.F., Teixeira A.G, et al., The upper respiratory tract pharyngotonsillitis: a placebo-controlled study. Acta Otolaryngol, microbiome and its potential role in bovine respiratory disease 119(8), 944–8 (1999). and otitis media. Sci Rep, 1(6), (2016). 25. Roos K., Grahn E., et al., Effect of recolonisation with “interfering” 2. de Steenhuijsen Piters W.A., Sanders E.A., et al., The role of the alpha streptococci on recurrences of acute and secretory otitis local microbial ecosystem in respiratory health and disease. Philos media in children: randomised placebo controlled trial. BMJ, Trans R Soc Lond B Biol Sci, 19(370), (2015). 322(7280), 210–2 (2001). 3. Philos Trans R Soc Lond B Biol Sci. 2015 Aug 19;370(1675). pii: 26. Skovbjerg S., Roos K., et al., Spray bacteriotherapy decreases 20140294. doi: 10.1098/rstb.2014.0294. The role of the local middle ear fluid in children with secretory otitis media.Arch Dis microbial ecosystem in respiratory health and disease. de Child, 94(2), 92–8 (2009). Steenhuijsen Piters WA1, Sanders EA, Bogaert D. 27. Smith T.J., Rigassio-Radler D., et al,. Effect of Lactobacillus 4. Report of a Joint FAO/WHO expert consultation on Evaluation of rhamnosus LGGw and Bifidobacterium animalis ssp. Lactis BB-12w health and nutritional properties of probiotics in food including in health-related quality of life in college students affected by powder milk with live lactic acid bacteria. Cordoba: FAO/WHO upper respiratory infections. Brit. J. Nutr, 109(11), 1999-2007 (2013). (2001). 28. de Roock S., van Elk M., et al., Gut derived lactic acid bacteria 5. Ouwehand, A.C., Salminen, S., et al., Probiotics: an overview of induce strain specific CD4(+) T cell responses in human PBMC. Clin beneficial effects.Antonie Van Leeuwenhoek, 82(1-4), 279-289 Nutr, 30(6), 845-51 (2011). (2002). 29. De Jager W. te Velthuis H., et al., Simultaneous detection of 15 6. Kalliomaki, M. and Isolauri, E., Role of intestinal flora in the human cytokines in a single sample of stimulated peripheral development of allergy. Curr Opin Allergy Clin Immunol, 3(1), 15-20. blood mononuclear cells. Clin Diagn Lab Immunol, 10(1),133-9 (2003) (2003).

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XVII STEVEN CHUNG MING FONG1, SANDRINE P. CLAUS2,* *Corresponding author 1. Gastroenterology Clinical Research Fellow, IBD Centre, Guy’s and St Thomas NHS Foundation Trust, London, SE17EH, United Kingdom 2. Department of Food and Nutritional Sciences, University of Reading, Reading, United Kingdom

Sandrine P. Claus

Host microbiota interactions in gut health An update on current understanding and future perspectives

KEYWORDS: Microbiota, metabonomics, metagenomics, gastrointestinal health.

The knowledge landscape of the gut microbiota is evolving rapidly, in part driven by the advances in Abstract analytical methodologies like metagenomics and metabonomics. Increasingly, the importance of diet, food-based interventions and host microbiota interactions in normal and diseased gastrointestinal states is being recognised. Links between the gut microbiota and diseases such as inflammatory bowel disease, irritable bowel syndrome, metabolic disorders such as type 2 diabetes and colorectal cancer have been identified. Future research efforts may translate into practical biomarkers for predicting disease evolution and new therapeutic strategies including the manipulation of the gut microbiota to improve drug efficacy and safety.

INTRODUCTION the gut microbiota has now become a shining beacon for research into gastrointestinal health. This fascination with The latest estimate of the total number of bacteria living on the microorganisms that inhabit our bowels is partly fuelled and in our body revealed that we are made of approximately by the promise of new therapeutic agents and strategies 50% microbes (1), representing a staggering genetic pool 360 to target various diseases. Much has been learnt about the times larger than the human genome (2). The composition complex interplay between the microbiome and us as their of the gut microbiota can be classified according to the host, yet many questions remain. Do we really know what a Linnaean system of taxonomics, in which bacteria are healthy gut microbiome should look like? How can we best divided into different hierarchies called phyla. While there manipulate it to improve our overall health and ameliorate are at least 10 phyla that have been identified in the human disease? Do certain bugs predispose us to gastrointestinal gut microbiota community, the two main phyla are by far disease? This minireview serves to update readers on the the Firmicutes and Bacteroidetes (Figure 1). Some studies current understanding of the gut microbiota in gastrointestinal have suggested that the ratio of these two phyla might diseases and elaborate on the future directions that research be a strong regulator of energy metabolism, although the into this once ‘forgotten organ’ may go. exact biological mechanisms remain unclear (3, 4). Debate also continues over whether specific species truly cause or propagate gastrointestinal diseases. Nevertheless, there is general consensus that the gut microbiome is involved in a multitude of complex interactions with the host, such as shaping of the immune system, synthesising , metabolising certain endogenous molecules such as bile acids and xenobiotics, including many drugs, and fermenting indigestible carbohydrates derived from our diet. This latter has been an extensively studied function as it leads to the production of large amounts of short chain fatty acids (mostly acetate, propionate and butyrate) that account for an important source of energy to the host and have been associated with a range of health benefits including the maintenance of a healthy intestinal epithelium (5). Figure 1. Average gut microbial composition in control (left) and IBD (right) individuals. Source: data were derived from Frank et al. 2007. Therefore, although once ignored and underestimated,

XVIII Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 ASSESSING THE GUT MICROBIOTA DIVERSE FUNCTIONS: probiotic, VSL#3, is commonly used in the treatment of METAGENOMICS AND METABONOMICS pouchitis, which is an inflammation of the pouch that may occur following surgery on patients with To better understand how our knowledge of the gut microbiota (15). When patients with Crohn’s disease take the antibiotic has developed, it is important to be aware of the different metronidazole after a surgical resection, they have a methods used in this research field. Amongst the various decreased rate of endoscopic and clinical recurrence (16). technologies used, genetic analysis and metabonomics are at Faecal microbiota transplants have been trialled in IBD the forefront. Genetic analysis techniques can range from early patients, but information is too scarce to conclude whether 16S ribosomal DNA gene sequencing to cutting edge next these treatments are truly efficacious. Indeed, the two most generation sequencing metagenomics. The latter terminology recent randomised clinical trials have produced conflicting refers to extraction of all the DNA within a specific environment, results, with one trial showing no difference in remission giving us an ‘all-inclusive’ look at an entire community of between the two groups (17), and the other showing better microbes. rates of remission with faecal transplant (18). One of the The gut microbiota produces numerous metabolites as a main differences between these two trials was the mode of result of various metabolic processes, which can be measured delivery, as the first study administered the faecal transplants to inform on the metabolic state of a system (e.g. bacterial through the upper gastrointestinal route, and the second community, organ, human body etc.). Metabonomics was was via faecal enemas. This factor could be an important defined by Nicholsonet al. (6) as ‘the quantitative measurement determinant in the efficacy of this therapeutic approach and of the dynamic multiparametric metabolic response of living calls for further studies. systems to pathophysiological stimuli or genetic modification’. Immunomodulator drugs such as azathioprine are useful in This approach is ideally suited to study the metabolic activity of IBD, but patients commonly have side effects and are unable the colonic microbiota in the context of disease development to continue long-term treatments. There is therefore an urgent because it uses the complete set of measureable metabolites need to find alternative therapies. Altogether, the evidence (so called metabolic profile) to gain insights into the complex is mounting for a central role of the gut microbiota in the biological interactions resulting to pathogenesis. When this pathogenesis of IBD, which may provide the next generation approach is used to monitor a response to diet, it can be of treatments. referred to as ‘nutrimetabonomics’ (7). The untargeted quantification of the metabolome is therefore at the core of the metabonomics approach, which is dominated THE GUT MICROBIOTA IN IRRITABLE BOWEL SYNDROME by two technologies, namely Nuclear Magnetic Resonance spectroscopy (NMR) and hyphenated methods using Mass Irritable bowel syndrome (IBS) is characterised by bloating Spectrometry (MS) as a detector. and stomach cramps associated with diarrhoea or Together, these two combined high throughput ‘omic’ constipation. It is extremely common and those with methodologies provide a wealth of information about the symptoms can find the quality of their lives significantly composition and metabolic activity of our gut microbial affected. Yet, although it is estimated that up to 20% of ecosystem that can be used to understand their influence on the UK population suffers from IBS, only a third of people the pathophysiology of gut disorders. experiencing these symptoms consult a specialist (19). The current treatment options for severe IBS are limited. Therefore, an innovative therapeutic strategy in this field THE GUT MICROBIOTA IN INFLAMMATORY BOWEL DISEASE would have a tremendous impact on patients and is an area of ample opportunity for industry and academic The exact cause of ulcerative colitis and Crohn’s disease, researchers. collectively known as inflammatory bowel diseases (IBD), is not From a microbiota perspective, the concentration of known. Early studies have sought to implicate microorganisms Bifidobacteria appears to be reduced in people with IBS (20). such as Mycobacterium avium subsp paratuberculosis but the To compensate this lack of lactic acid bacteria, probiotics data have been inconclusive (8, 9). The emerging evidence and prebiotics have been used but the data so far have does suggests, however, that the gut microbiota in IBD is been unequivocal. In a recent systematic review of altered, a phenomenon referred to as ‘dysbiosis’. Although probiotics in IBS, there was only a marginal benefit seen this is not systematically observed in every IBD patients, in with probiotics (21). For prebiotics, a recent randomised most patients it seems that the Bacteroidetes phylum is controlled trial showed some improvement in symptoms reduced (10, 11) (Figure 1). There was also considerable but lacked an intention to treat analysis which limits the interest generated when Sokol et al. found that one particular interpretation of the results (22). species, F. prausnitzii, was reduced in faecal samples from Dietary manipulation for IBS, in the form of the low FODMAP patients with Crohn’s disease(12). This suggested that this diet (fermentable oligosaccharides, disaccharides, particular species may have a protective effect on the gut monosaccharides and polyols), has become more inflammatory response, a hypothesis that was then further popular in recent years (23). This is based on the rationale supported by additional evidence that F. prausnitzii was a that the lack of complete hydrolysis or absorption of butyrate producer in human colon, which is a metabolite these compounds aggravates IBS symptoms. However, a known to be a potent anti-inflammatory compound (13). study has shown that a low FODMAP diet also reduced In light of this link between IBD and the gut microbiota, Bifidobacteria levels (24). With this in mind, there is ongoing probiotics and antibiotics have been used for several years in research looking at the effect of combining the low the treatment of IBD. In one study, the probiotic E. coli Nissle FODMAP diet with prebiotics or probiotics in hope that this 1917 was as efficacious as standard mesalazine therapy could maintain Bifidobacteria concentrations at the same in preventing relapses of ulcerative colitis (14). Another time as improving gastrointestinal symptoms.

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XIX THE GUT MICROBIOTA IN COLORECTAL CANCER FUTURE PERSPECTIVES: USING THE GUT MICROBIOTA TO PREDICT DRUG METABOLISM The role of the microbiome in the development of colorectal cancer is not clearly defined, but recent metagenomic The role of the gut microbiome in gastrointestinal health studies have shown that there may be an individualised and disease has become increasingly defined through the microbiomic signature found on tumour sites (25). There use of modern methodologies such as metagenomics and are different theories about how microbial pathogens may metabonomics. predispose us to colorectal cancer. One theory is the ‘driver- The potential opportunities for clinicians, academia and passenger’ model in which a particular bacterial ‘driver’ industry to work together to formulate better microbiota pathogen may lead to DNA damage, and the subsequent modulating therapies has never been more attractive. tumour formation leads to another set of ‘passenger’ The next direction for researchers is to move away from bacteria that propagate the cancer development (26). establishing mere associations and seek to prove causation From a dietary perspective, the finding that there are fewer through well thought and constructed studies. These butyrate producers in patients with colorectal cancer endeavours can hopefully translate into new biomarkers to suggests fibre metabolism may have a part to play in predict gastrointestinal disease progression and therapeutic pathogenesis (27). One recent fascinating study looked strategies based on a more personalised medicine. at the effects of a 2 week high fat, high protein, low fibre, or a so called Westernised diet compared to a high fibre diet in African Americans, who have a higher incidence of REFERENCES colorectal cancers than rural South Africans (28). The study found that the Westernised diet cohort was associated with 1. R. Sender, S. Fuchs, and R. Milo, “Are We Really Vastly Outnumbered? reversible changes in mucosal biomarkers of cancer risk Revisiting the Ratio of Bacterial to Host Cells in Humans,” Cell, vol. 164, such as mucosal Ki67 expression. In addition, the microbial no. 3, pp. 337–340, Jan. 2016. landscape of individuals following the high fibre diet 2. C. Huttenhower, D. Gevers, R. Knight, and S. Abubucker, “Structure, function and diversity of the healthy human microbiome,” Nature, showed an increase in bacteria involved in saccharolytic 2012. fermentation, butyrogenesis, and suppressed secondary 3. R. E. Ley, F. Backhed, P. Turnbaugh, C. A. Lozupone, R. D. Knight, and bile acid synthesis , indicating that high fibre diets may J. I. Gordon, “Obesity alters gut microbial ecology.,” Proc. Natl. Acad. help to keep a healthy gut environment less favourable to Sci. U.S.A., vol. 102, no. 31, pp. 11070–11075, Aug. 2005. the development of colorectal cancers. A detailed review 4. R. Ley, P. Turnbaugh, and S. Klein, “Microbial ecology: human gut summarising the current knowledge of the links between gut microbes associated with obesity,” Nature, 2006. microbial metabolites and colorectal cancer was published 5. A. Koh, F. De Vadder, P. Kovatcheva-Datchary, and F. Backhed, by Petra Louis in 2014 (35). “From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites,” Cell, vol. 165, no. 6, pp. 1332–1345, Jun. 2016. 6. J. Nicholson, J. Lindon, and E. Holmes, “‘Metabonomics’: understanding the metabolic responses of living systems to THE GUT MICROBIOTA IN OBESITY pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data,” Xenobiotica, vol. 29, no. 11, pp. Finally, obesity is often associated with functional 1181–1189, 1999. gastrointestinal disorders, although the evidence is limited to 7. S. P. Claus and J. R. Swann, “Nutrimetabonomics: Applications determine whether it is a causative factor (29, 30). A great for Nutritional Sciences, with Specific Reference to Gut Microbial amount of attention is being focussed on understanding the Interactions.,” Annu Rev Food Sci Technol, vol. 4, pp. 381–399, Jan. metabolic link between the gut microbiota and development 2013. of obesity. A number of studies have demonstrated that 8. J. D. Sanderson, M. T. Moss, M. L. Tizard, and J. Hermon-Taylor, obesity was associated with gut microbiota dysbiosis, often “Mycobacterium paratuberculosis DNA in Crohn’s disease tissue.,” characterised by a reduction in the relative abundance of Gut, vol. 33, no. 7, pp. 890–896, Jul. 1992. 9. D. Mishina, P. Katsel, S. T. Brown, E. C. Gilberts, and R. J. Greenstein, Bacteroidetes (4). Research by Le Chatelier et al. divided “On the etiology of Crohn disease.,” Proc. Natl. Acad. Sci. U.S.A., vol. obese individuals between those associated with a gut 93, no. 18, pp. 9816–9820, Sep. 1996. microbiota with a High Gene Count or Low Gene Count 10. A. W. Walker, J. D. Sanderson, C. Churcher, G. C. Parkes, B. N. (i.e. high or low microbial diversity) (31). Those with High Hudspith, N. Rayment, J. Brostoff, J. Parkhill, G. Dougan, and L. Gene Counts had higher counts of bacterial species with Petrovska, “High-throughput clone library analysis of the mucosa- anti-inflammatory features such asF. prausnitzii. Those with associated microbiota reveals dysbiosis and differences between Low Gene Counts had higher numbers of pro-inflammatory inflamed and non-inflamed regions of the intestine in inflammatory bacteria and tended to develop insulin resistance. bowel disease.,” BMC Microbiology 2009 9:123, vol. 11, p. 7, 2011. Therefore it seems that a higher degree of diversity was 11. D. N. Frank, A. L. St Amand, R. A. Feldman, E. C. Boedeker, N. associated with an improved metabolic status. Some of the Harpaz, and N. R. Pace, “Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel biological mechanisms that may be involved in this interplay diseases.,” Proc. Natl. Acad. Sci. U.S.A., vol. 104, no. 34, pp. 13780– include regulation of fatty acid transport and modulation of 13785, Aug. 2007. bile acid metabolism (32, 33). This latter is a key regulator of 12. H. Sokol, P. Seksik, J.-P. Furet, O. Firmesse, I. Nion-Larmurier, L. lipid absorption and bile acids are also known to be strong Beaugerie, J. Cosnes, G. Corthier, P. Marteau, and J. Doré, “Low activators of energy metabolism through their interaction with counts of Faecalibacterium prausnitzii in colitis microbiota.,” Inflamm. the nuclear receptor Farnesoid X Receptor (FXR) (34). Yet, Bowel Dis., vol. 15, no. 8, pp. 1183–1189, Aug. 2009. even if the role of the gut microbiota in energy homeostasis 13. M. T. Khan, S. H. Duncan, A. J. M. Stams, J. M. van Dijl, H. J. Flint, and and metabolic dysfunction is slowly becoming clearer, there H. J. M. Harmsen, “The gut anaerobe Faecalibacterium prausnitzii are still a lot of unanswered questions in order to translate uses an extracellular electron shuttle to grow at oxic–anoxic these findings into a therapeutic option. interphases,” The ISME Journal, vol. 6, no. 8, pp. 1578–1585, Feb. 2012. 14. W. Kruis, P. Frič, J. Pokrotnieks, M. Lukáš, B. Fixa, M. Kascák, M.

XX Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 A. Kamm, J. Weismueller, C. Beglinger, M. Stolte, C. Wolff, and trial: the effects of a trans-galactooligosaccharide prebiotic on faecal J. ScHULZE, “Maintaining remission of ulcerative colitis with the microbiota and symptoms in irritable bowel syndrome.,” Aliment probiotic Escherichia coli Nissle 1917 is as effective as with standard Pharmacol Ther, vol. 29, no. 5, pp. 508–518, Mar. 2009. mesalazine.,” Gut, vol. 53, no. 11, pp. 1617–1623, Nov. 2004. 23. H. M. Staudacher and K. Whelan, “Altered gastrointestinal microbiota 15. P. Gionchetti, F. Rizzello, U. Helwig, A. Venturi, K. M. Lammers, P. in irritable bowel syndrome and its modification by diet: probiotics, Brigidi, B. Vitali, G. Poggioli, M. Miglioli, and M. Campieri, “Prophylaxis prebiotics and the low FODMAP diet.,” Proceedings of the Nutrition of pouchitis onset with probiotic therapy: a double-blind, placebo- Society, vol. 75, no. 3, pp. 306–318, Aug. 2016. controlled trial.,” Gastroenterology, vol. 124, no. 5, pp. 1202–1209, May 24. H. M. Staudacher, M. C. E. Lomer, J. L. Anderson, J. S. Barrett, J. G. Muir, 2003. P. M. Irving, and K. Whelan, “Fermentable carbohydrate restriction 16. P. Rutgeerts, M. Hiele, K. Geboes, M. Peeters, F. Penninckx, R. Aerts, reduces luminal bifidobacteria and gastrointestinal symptoms in and R. Kerremans, “Controlled trial of metronidazole treatment patients with irritable bowel syndrome.,” Journal of Nutrition, vol. 142, for prevention of Crohn’s recurrence after ileal resection.,” no. 8, pp. 1510–1518, Aug. 2012. Gastroenterology, vol. 108, no. 6, pp. 1617–1621, Jun. 1995. 25. J. R. Marchesi, B. E. Dutilh, N. Hall, W. H. M. Peters, R. Roelofs, A. 17. N. G. Rossen, S. Fuentes, M. J. van der Spek, J. G. Tijssen, J. H. A. Boleij, and H. Tjalsma, “Towards the Human Colorectal Cancer Hartman, A. Duflou, M. Löwenberg, G. R. van den Brink, E. M. H. Microbiome,” PLoS ONE, vol. 6, no. 5, p. e20447, May 2011. Mathus-Vliegen, W. M. de Vos, E. G. Zoetendal, G. R. D’Haens, and 26. H. Tjalsma, A. Boleij, J. R. Marchesi, and B. E. Dutilh, “A bacterial driver- C. Y. Ponsioen, “Findings From a Randomized Controlled Trial of Fecal passenger model for colorectal cancer: beyond the usual suspects.,” Transplantation for Patients With Ulcerative Colitis.,” Gastroenterology, Nature Reviews Microbiology, vol. 10, no. 8, pp. 575–582, Aug. 2012. vol. 149, no. 1, pp. 110–118.e4, Jul. 2015. 27. N. Wu, X. Yang, R. Zhang, J. Li, X. Xiao, Y. Hu, Y. Chen, F. Yang, N. Lu, 18. P. Moayyedi, M. G. Surette, P. T. Kim, J. Libertucci, M. Wolfe, C. Onischi, Z. Wang, C. Luan, Y. Liu, B. Wang, C. Xiang, Y. Wang, F. Zhao, G. F. D. Armstrong, J. K. Marshall, Z. Kassam, W. Reinisch, and C. H. Lee, Gao, S. Wang, L. Li, H. Zhang, and B. Zhu, “Dysbiosis signature of fecal “Fecal Microbiota Transplantation Induces Remission in Patients microbiota in colorectal cancer patients.,” Microb. Ecol., vol. 66, no. 2, With Active Ulcerative Colitis in a Randomized Controlled Trial.,” pp. 462–470, Aug. 2013. Gastroenterology, vol. 149, no. 1, pp. 102–109.e6, Jul. 2015. 28. S. J. D. O’Keefe, J. V. Li, L. Lahti, J. Ou, F. Carbonero, K. Mohammed, 19. J. W. T. C. Caroline Canavan, “The epidemiology of irritable bowel J. M. Posma, J. Kinross, E. Wahl, E. Ruder, K. Vipperla, V. Naidoo, L. syndrome,” Clinical Epidemiology, vol. 6, pp. 71–80, 2014. Mtshali, S. Tims, P. G. B. Puylaert, J. DeLany, A. Krasinskas, A. C. Benefiel, 20. G. C. Parkes, N. B. Rayment, B. N. Hudspith, L. Petrovska, M. C. H. O. Kaseb, K. Newton, J. K. Nicholson, W. M. de Vos, H. R. Gaskins, Lomer, J. Brostoff, K. Whelan, and J. D. Sanderson, “Distinct microbial and E. G. Zoetendal, “Fat, fibre and cancer risk in African Americans populations exist in the mucosa-associated microbiota of sub-groups and rural Africans.,” Nat Comms, vol. 6, p. 6342, 2015. of irritable bowel syndrome.,” Neurogastroenterol. Motil., vol. 24, no. 1, 29. W. Ho and B. Spiegel, “The relationship between obesity and pp. 31–39, Jan. 2012. functional gastrointestinal disorders: causation, association, or 21. A. C. Ford, E. M. M. Quigley, B. E. Lacy, A. J. Lembo, Y. A. Saito, L. neither?,” Gastroenterol Hepatol (N Y), 2008. R. Schiller, E. E. Soffer, B. M. R. Spiegel, and P. Moayyedi, “Efficacy 30. U. P. Phatak and D. S. Pashankar, “Prevalence of functional of prebiotics, probiotics, and synbiotics in irritable bowel syndrome gastrointestinal disorders in obese and overweight children.,” Int J and chronic idiopathic constipation: systematic review and meta- Obes (Lond), vol. 38, no. 10, pp. 1324–1327, Oct. 2014. analysis.,” Am. J. Gastroenterol., vol. 109, no. 10, pp. 1547–61– quiz 1546– 1562, Oct. 2014. Readers interested in a full list of references are invited to visit our 22. D. B. A. Silk, A. Davis, J. Vulevic, G. Tzortzis, and G. R. Gibson, “Clinical website at www.teknoscienze.com DAVIDE GOTTARDI1,*, PIETER VAN DEN ABBEELE1, MASSIMO MARZORATI2 *Corresponding author 1. ProDigest BVBA, Technologiepark 4, B-9052 Ghent, Belgium 2. Center for Microbial Ecology and Technology (CMET), Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

Davide Gottardi Use of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) to study the fate of food ingredients and actives

KEYWORDS: SHIME, gastrointestinal tract, in vitro model, food ingredients.

The development of novel food ingredients, supplements or drugs demands consistent studies to validate Abstract their mechanism of release, absorption and action. Human in vivo trials are the gold standard to test one or few compounds, but extensive screening of novel products is often unfeasible. As a result, in vitro models represent a suitable complementary tool to test the fate of novel ingredients and molecules in the gastrointestinal tract (GIT). In this review, we describe the main parameters of the harmonized in vitro static model and we further provide practical examples of the use of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) technology for testing the fate of compounds delivered in the GIT.

INTRODUCTION vitro digestion method, as a result of the differences in the experimental facilities. In this review we report the principal The market for food ingredients with biological activities guidelines of the consensus method and describe examples (“functional foods”) is projected to reach $2.5 billion by where the computer-controlled Simulator of Human Intestinal 2020 (1). Over the last two decades, this market attracted Microbial Ecosystem (SHIME®) platform was applied. This food, pharmaceutical and retail businesses, aiming at validated system can simulate the full GI tract, from the mouth earning higher returns and a competitive edge (2). Novel to the different colon compartments. Several parameters compounds are being developed to prevent chronic can be adapted to simulate specific physiological conditions illnesses (such as cardiovascular disease, Alzheimer’s, and and guidelines from the consensus method can be applied osteoporosis), to increase energy, boosting immune system and improved. In this respect, the SHIME® becomes an and general wellbeing (2). To exert their functionality in the important dynamic tool that can be applied for digestibility, body, these compounds must be successfully released from bioavailability, bioaccessibility, and bioactivity studies. the food matrix/delivering system (i.e. bioaccessibility) and being absorbed in the GIT (i.e. bioavailability). The first step is crucial for digestibility and allergenicity of the product (3), SIMULATION OF THE HUMAN UPPER GIT while the second impacts its bioactivity. As a result, studying the activities of novel food, ingredients and pharmaceuticals The main goal of the digestion in the upper GIT is to break during the passage through the GIT is essential (4). Human down food to their basic constituents (bioacessibility) and then studies are indispensable, prior to product commercialization. absorb them (bioavailability). Mechanical performance of However, ethical and time restrictions, as well as the high in vitro models used to simulate mastication, insalivation and costs involved, are some of their drawbacks. On the contrary, bolus formation have been already reviewed (6). in vitro models represent promising tools to pre-screen such Moreover, the parameters of in vitro static models focused on products, because of the larger number of samples that can bioavailability and bioactivity tests have been described (5). be measured simultaneously. In vitro experiments may lack Although the consensus method standardizes the key biological feedback mechanisms, however, they allow for strict parameters for the digestion, some practical limitations have parameter control and sampling accessibility. In this way, they arisen. For instance, highly viscous food-saliva boluses cannot can be used for mechanistic and hypothesis-building studies. be mixed and swallowed in static models as they might be in A consensus protocol has been proposed to harmonize in vivo. Thus, the food needs to be mixed with artificial saliva to vitro systems, which simulate static digestive processes (5). obtain sufficient liquid input for the subsequent gastric step. This protocol defines key parameters to simulate oral, gastric In vivo, partial hydrolysis of starch happens already in the and small intestine conditions. However, the authors also mouth and the use of α-amylase inhibitors is frequently warn about potential limitations of the harmonised static, in considered as therapeutic functional food (7).

XXII Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 The mouth step simulation in the SHIME® technology platform aggregates more than pea and whey protein. This aggregation has been used to assess the efficacy of a novel treatment, is crucial for the protein metabolism and its entrance in the small which was shown to decrease the amount of maltose release intestine. In fact, whey protein rapidly enters in the small intestine as compared to the control test (Figure 1). and has a faster bioavailability (9). Using the SHIME® technology the digestibility of two proteins with different proteolytic profiles was investigated (Figure 2).

Figure 1. Inhibition of α-amylase, through quantification of maltose release, using the SHIME setup simulating the in vitro mouth incubation. * = p < 0.05 according to a Student T test (n = 3)

Once the bolus reaches the stomach, the presence of low pH and gastric enzyme (pepsin) allows proteins and peptides Figure 2. Digestion of protein A and B, expressed in percentage, in the stomach and small intestine of the SHIME system. Protein hydrolysis was digestion. This digestion continues in the duodenum, the first evaluated over time. A dialysis step was coupled to the small intestine segment of the small intestine. Here the pancreas helps the simulation to quantify the absorbed and the not absorbed digested fractions. ST: stomach; SI: small intestine. By means of the computer, process by secreting other proteases (mainly trypsin and the physiological conditions of the small intestine are modified in time chymotrypsin), amylases and lypases. Considering in vivo to become representative for duodenum, jejunum and ileum during parameters, Minekus et al. (5) proposed to use pH 3 and 2 h of the first, second and third hour of incubation, respectively. incubation, as representative values for a general meal in the stomach. In particular, these parameters were selected to avoid Protein A and B were digested under similar physiological complete peptide digestion after prolonged incubation at low conditions in the stomach but they showed different profiles pH. Moreover, a defined amount of pepsin (2000U/ml) was in the small intestine. In particular, hydrolysis of protein A was added (5). The small intestine step is performed at pH 7 for 2 h faster at the beginning of the small intestine (i.e. duodenum) and pancreatic enzymes are added in the form of pancreatin or compared to protein B. This aspect can be crucial for the individual enzymes, and bile salts. The amount of enzymes needs final bioaccessibility of the derived peptides and aminoacids. to be based on trypsin activity (100 U/ml), while the final amount Bioaccessibility of nutrients (such as peptides, aminoacids, of bile salts should be 10 mM. Nevertheless, the authors of the vitamins, sugars) can be measured using solubility assays. The consensus method indicated that the specificity of the protocol results reported in Figure 2 refer to samples incubated under may entail some practical limitations. For instance, gastric pH is standard simulated healthy conditions. However, fasted/fed, highly dynamic and dependent on the presence of food (8). This pancreatitis, consumption of protein pumps inhibitors, short aspect can be easily simulated using the SHIME® technology. bowel syndrome, etc… can also be simulated using the SHIME. In fact, a sigmoidal pH profile can be set with this system. This is For instance, fasted (i.e. empty stomach) or fed (i.e. full stomach) to say, a higher pH (5.5-6) - once the food has been ingested - conditions can impact the final activity of food ingredients and gradually decreasing to a lower pH (1.8-2) when hydrochloric supplements. By means of this approach it is therefore possible acid and enzymes are added. Moreover, lipases were omitted to evaluate the efficacy of different targeted delivery strategies. from the stomach incubation in the standardized method, Marzorati et al. (10) showed that the performance of two because they have an optimum pH between 4 and 6, while different capsules impacted the final bioactivity and viability of the system is fixed to 3, and the gastric lipolytic activity takes the encapsulated enzymes and probiotics, respectively. In fact, place only partially (5-40%) (5). On the contrary, the use of a higher enzymatic activity and viable cells were detected in the decreasing sigmoidal pH profile allows to test gastric lipases and sample delivered with DRcaps®. In general, the protective effect proteases at the same time, if needed. In addition, pepsin can seemed to be stronger in the absence of a food matrix (fasted be slowly delivered over time, reproducing in vivo conditions. conditions) as compared to fed conditions (10). The possibility to adapt all these parameters, makes the SHIME® Although not present in their method, another aspect highlighted technology the perfect tool for studying digestion kinetics, and by Minekus et al. (5) is the need of an adsorption step to the release and production of bioactive molecules. evaluate the bioavailability of micro and macronutrients. This can The extent in which digestion occurs in stomach and small be assessed using different strategies. For instance, a dialysis step intestine is not only dependent on the presence of HCl and associated with the simulation of the small intestine. In this case, enzymes, but also on the structure of the original proteins. the compound in the dialyzed fraction can be quantified with As structure and composition strongly differ between proteins, ad hoc analytical techniques (11). Looking at the example in differences may therefore occur in the digestibility of proteins Figure 2, protein A released more peptides and aminoacids - as originating from different sources. For instance, casein compared to protein B - capable of diffusing through the dialysis.

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XXIII This fraction would be easily available for investigating probiotics colonization in vivo transport and thus for absorption. and their performance in the colon. Another approach consists in the use Moreover, the extent of probiotic of human epithelial cell lines derived adhesion can be determined using from human colonic adenocarcinoma the mucosal-SHIME® (M-SHIME) (16). (Caco-2 cells). In this way, for instance, the absorption of the digested fraction of proteins (Figure 3) can be measured. CONCLUSIONS

This methodology allows to consider Recently, in the frame of the COST how the molecular physicochemical Action INFOGEST, a harmonized Figure 3. Transport of digested protein to the characteristics (such as lipophilicity) may basolateral side of a Caco-2 cell monolayer. The protocol has been developed and ultimately impact their permeability. basolateral content was collected after 15, 30 and 60 validated to simulate the main In fact, as shown in Figure 3 as an min of incubation and the concentration of the total digestive steps in the upper GIT. In this peptides was determined. example, although all the three proteins review, we showed that the were digested after the small intestine controlled in vitro conditions of the incubation, only protein A and B simulator of the full GIT (i.e. computer- generated peptides that were easily controlled SHIME®) can be used to transferred to the basolateral side. overcome some of the limitations identified in the harmonized method. In this respect, SHIME® represents a FURTHER ON IN THE GIT useful tool for the development of novel food ingredients, supplements The main function of the upper GIT in or drugs. Moreover, validation of their terms of digestion is to break down bioaccessibility, bioavailability and food to their basic constituents and ultimate performance, can be readily then absorb them. However, there implemented. are specific compounds that need an additional stage to become bioactive or Figure 4. Conversion of IX (gray) into 8-PN (red) in samples taken from ascending (square), transverse bioavailable. For instance, polyphenols (triangle) and descending (circle) colon of the SHIME ACKNOWLEDGMENTS are only partially absorbed in the small system (Adapted from Possemiers et al., 2006). intestine (5-10%) and then transferred Davide Gottardi has received into the colon. As a result, they have low bioavailability in the funding from the People Programme (Marie Curie Actions) upper GIT, and the real bioactivity has to be estimated in the of the European Union’s Seventh Framework Programme , where they are subjected to the enzymatic FP7/2007-2013/ under REA grant agreement n° 606713 activation by the gut microbiota. Here, bacteria transform BIBAFOODS. the original molecules so that the resulting polyphenols, rather than the original compounds found in foods, provide beneficial effects on health (12). The main advantage of REFRENCES using the SHIME® technology is the possibility to have a full simulation of the GIT from the mouth to the colon to study these 1. Markets and Markets: http://www.marketsandmarkets.com/ processes. For instance, Possemiers et al. (13) showed that the PressReleases/functional-food-ingredients.asp hop prenylflavonoid isoxanthohumol (IX) can pass unaltered 2. Khan R.S., Grigor J., et al., Trends food sci tech, 30(1), 27-37 (2013). through the stomach and small intestine until the colon. Here, 3. Schulten V., Lauer I., et al., Mol Nutr Food Res, 55(10), 1484-1491 IX is converted (up to 80%) into 8-prenylnaringenin (8-PN), a (2011). potent phytoestrogen. The use of the SHIME system including 4. Rein M.J., Renouf M., et al., Br J Clin Pharmacol, 75(3), 588-602 the three different regions of the colon showed that this (2013). conversion mainly occurred in the distal colon (Figure 4). 5. Minekus M., Alminger M., et al., Food funct, 5(6), 1113-1124 (2014). 6. Morell P., Hernando I., et al., Trends Food Sci Tech, 35(1), 18-31 (2014). 7. Sales P. M., Souza P. M., et al., J Pharm Pharm Sci, 15(1), 141-183 In another example, production from flax (lignans) has (2012). been investigated in the SHIME (14). The initial precursor SECO- 8. Tyssandier, V., Reboul, E., et al., Am J Physiol Gastrointest Liver diglucoside (SDG) needs to be released from flax matrix in the Physiol, 284(6), G913-G923 (2003). upper intestine. Further on, sugar chain is removed from SDG in 9. Overduin J., Guérin-Deremaux L., et al., Food nutr res, 59, 25622- the ascending colon and SECO is formed. SECO is fermented 25622 (2014). into enterodiol (END), which can lead to enterolactone (ENL) 10. Marzorati M., Possemiers S., et al., LWT Food Sci Tech, 60(1), 544- in the transverse and descending colon. Interestingly, the 551 (2015). production of END and ENL depends on the metabolic activity 11. Etcheverry P., Grusak M.A., et al., Front Physiol, 3 (2012). of the colonic microbiota and, therefore, on the interindividual 12. Cardona F., Andrés-Lacueva C., et al., J nutr biochem, 24(8), 1415-1422 (2013). variability observed. 13. Possemiers S., Bolca S., et al., J nutr, 136(7), 1862-1867 (2006). Finally, several studies reported that probiotics can become 14. Decroos K., Eeckhaut E., et al., J nutr, 136(4), 946-952 (2006). viable but not cultivable after stomach and small intestine and 15. Possemiers, S., Marzorati, M., et al., Int J Food Microbiol, 141(1-2), their fitness to the colonic environment may vary in response to 97-103 (2010). the stress encountered in the upper GIT (15). As a result, the use 16. Van den Abbeele P., Roos S., et al., Microbial biotech, 5(1), 106- of an in vitro simulator covering the full GIT is fundamental for 115 (2012).

XXIV Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 EZGİ DOĞAN CÖMERT, VURAL GÖKMEN* *Corresponding Author Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering, Hacettepe University, 06800 Beytepe Campus, Ankara, Turkey

Cereal dietary fiber bound antioxidants

KEYWORDS: Cereal grains, bound antioxidants, dietary fibers, QUENCHER, digestion behavior of bound antioxidants.

Cereals have potential role in human health due to their high amounts of antioxidant compounds Abstract especially in insoluble bound forms. Bound antioxidants are crucial for living organisms as they exert their antioxidant effects much longer than soluble antioxidants. They reach the colon without being digested through the small intestine and contribute to the formation of reduced environment in the colon, while soluble antioxidants ingested with diet cause a rapid and short-term increase in plasma antioxidant capacity upon absorption in the small intestine. From this point of view, cereal dietary fiber- antioxidant compounds complexes have come into prominence in recent years and researches have focused on their health benefits, digestion behavior and bioavailability in gastrointestinal system and determination of their antioxidant capacity both in vivo and in vitro. This review aimed to collect important knowledge related to cereal antioxidant compounds and give a general perspective on their potential antioxidant effects.

INTRODUCTION the high amount of antioxidant compounds in addition to dietary fiber (20-26). Antioxidant compounds bound to Researchers have emphasized that most of chronic diseases, dietary fibers exert their antioxidant effects much longer in particular obesity, diabetes, some cancers, are linked with than soluble antioxidants in living organisms (27). The human diet (1-3). Therefore, dietary guidelines encourage continuous presence of a relatively low concentration of people to consume more healthful foods and beverages antioxidant compounds may have a higher potential benefit leading to achieve and maintain a healthy weight, promote for body defense than the peak of plasma antioxidant health, and prevent diet related diseases like obesity, type 2 concentration observed immediately after the ingestion of diabetes, certain cancers and cardiovascular diseases (4-7). a free antioxidant compound-rich food (27). From this point Moreover, consumption of the functional foods containing of view, there is a growing scientific interest for antioxidant biologically active compounds increase as they provide compounds in addition to dietary fibers and antioxidant health benefits and reduce the risk of mentioned diseases (8). potential of cereal grains. Researchers have specifically Antioxidant compounds and dietary fibers have come into focused on their health benefits, digestion behavior and prominence due to their functional properties. bioavailability in gastrointestinal system and determination Antioxidants counteract the oxidative stress, which cause of their antioxidant capacity both in vivo and in vitro. This chronic and degenerative diseases, and prevent oxidation review aimed to collect important knowledge related to of oxidizable substrates in foods (9). Most common antioxidant compounds in addition to dietary fibers and dietary antioxidant compounds in foods are vitamins (C give a general perspective on the structure of cereal grains and E), carotenoids, chlorophylls and a wide variety of and their antioxidant effects,, the digestion behavior of phytochemicals such as phenolic compounds, flavonoids, antioxidant compounds bound to dietary fibers and their and complex polyphenols (10-12). They may generally be importance. found in free and bound forms in foods (13). According to most previous literature, free soluble antioxidant compounds in fruits and vegetables were outstanding sources of STRUCTURE OF CEREAL GRAINS antioxidants, while cereals were underestimated by the reason of their low levels of antioxidant capacity (14). Cereal grains consist of 3 main anatomical parts: an embryo However, researchers have revealed that it is compulsory (germ), an endosperm, and a bran. The inner layer of to bear in mind the contribution of bound antioxidant grains, endosperm, is rich in starch and protein, while bran, compounds to total antioxidant capacity of cereals since the outer layer protecting the grain, is rich in dietary fiber, cereal grains include most of antioxidant compounds in minerals, vitamin B6, thiamine, and and bound forms (15-19). It was emphasized that cereal grains some phytochemicals, in particular antioxidants such as have potential benefits for human health as they include phenolic compounds (28,29).

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XXV As phenolic compounds are concentrated in the outermost layers, the bran fractions may be used as a natural source of antioxidants and as value- added products in the preparation of functional food ingredients or for enrichment of certain products (30). The health effect of cereals is mainly attributed to the fiber and antioxidant compounds in the bran fraction of the cereals (20,31,32). This part gives the fundamental information about dietary fiber and antioxidant compounds in cereal grains.

Dietary fiber Dietary fiber is generally defined as: ‘‘Dietary fiber consists of the remnants of edible plant cell, polysaccharides, lignin, and associated substances Figure 1. Primary cell wall structure of plant materials and cross-linking between resistant to digestion and absorption through small structural components and phenolic compounds (A) Cellulose (B) Hemicellulose (C) Structural proteins (D) Pectin (E) Phenolic acids (F) Lignin (49). intestine with complete or partial fermentation in the large intestine’’ (33). Dietary fibers are divided into two categories based Bound antioxidant compounds on their solubility in water: soluble and insoluble dietary According to the previous literature (before 1990’s), free fibers. Soluble dietary fiber delays the gastric emptying by soluble antioxidant compounds in fruits and vegetables attracting with water and forming a gel, which slow down were considered as excellent sources of antioxidants, digestion (34). Slowing the gastric emptying decreases whereas cereal grains were underestimated due absorption of macronutrients and cause not only control to their relatively low levels of antioxidant capacity the blood sugar level but also control the weight (35). In (14,15,18,45,46). However, in recent years, researchers addition, they regulate the gastrointestinal hormones that have revealed that cereals have significantly higher serve as incretions to control the insulin sensitivity, leading antioxidant capacity due to their insoluble bound to control diabetes (36). Soluble fibers also reduce the LDL phenolic compounds (18,19). Fruits and vegetables blood cholesterol level via preventing the absorption of contain mostly free and conjugated soluble phenolic dietary cholesterol (37). Insoluble fibers pass through the compounds while approximately 24% of the total phenolic gastrointestinal tract relatively intact, and speed up the compounds are in bound forms in these food matrices passage of food and waste through the gut. In addition, (47, 48). Conversely, most of the phenolic compounds they add bulk to faeces and prevent constipation (38). associated with whole cereal grains are in the insoluble Insoluble fibers such as cellulose, hemicellulose, lignins bound forms (49). Apple (Malus domestica), orange are mainly found in cereal grains especially in their (Citrus sinensis) and onion (Allium cepa) contain 6.50%, bran fractions. Maize and wheat have predominantly 24.30% and 9.70% insoluble bound phenolic compounds insoluble dietary fiber in their bran fractions while oat has a (47, 50), while about 85, 75, and 62% of the total phenolic considerable amount of soluble dietary fiber. compounds are found in the insoluble bound forms in corn, wheat, and rice, respectively (48, 49). Antioxidant compounds Bound antioxidant compounds such as phenolic Antioxidant compounds have a crucial role in human compounds are generally located in the bran fraction body by means of counteracting the oxidative stress which and covalently bound to cell wall materials such as plays a major role in the development of chronic and cellulose, hemicellulose (arabinoxylan units) and proteins degenerative ailments such as cancer, cardiovascular as shown in Figure 1 (48). Phenolic acids, especially diseases, arthritis, aging, autoimmune disorders and hydroxycinnamic acids (ferulic acid, sinapic acid, cafeic neurodegenerative diseases (39-43). In addition, acid etc.) form ether linkages with lignin through their antioxidant compounds delay or inhibit the oxidation of hydroxyl groups and ester linkages with proteins and oxidizable substrates in foods and prevent formation of carbohydrates through their carboxyl groups (19,51,52). off-flavor and toxic compounds or destruction of essential As the most consumed cereal, wheat, particularly its nutrients by oxidative damage. They inhibit the oxidation non-endosperming tissues, contain high concentration by different mechanisms such as scavenging free radicals, of arabinoxylan units as an insoluble dietary fiber. chelating prooxidative metals, quenching singlet oxygen Arabinoxylan units consist of α-(1,4)-linked D-xylopyranosyl and photosensitizers, and inhibition of lipoxygenase residues with α-L-arabinofuranose side chains, potentially activity (44). substituted with ferulic acid as an antioxidant compounds Antioxidants may be generally found in two forms in foods which accounts for up to 60% of wheat bran antioxidant as free (I) and bound forms (II, III, IV). More specifically, capacity (53,54). Ferulic acids in addition to arabinoxylan their location in foods may be divided into five groups: unit are able to cross-link to other arabinoxylan units via (I) free from chemical or physical interaction with diferulates (55, 56). Dimerization of ferulates occurs by other macromolecules; (II) chemically bound to other phytochemical coupling reactions or radical coupling macromolecules that are insoluble antioxidant material reactions (55, 56) and allows the cell wall to build (usually of high molecular weight); (III) ionically bound to more stable structure (56). Therefore, the solubility of food matrix; (IV) physically entrapped in food matrice and; arabinoxylan units decreases, leading to become more (V) physically entrapped into different cellular structure (13). resistant to digestive enzyme.

XXVI Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 MEASUREMENT OF ANTIOXIDANT CAPACITY: ‘QUENCHER fecal bulk and shortened transit times (72), and affects METHOD’ indirectly by lowering colon pH via short chain fatty acid (SCFA) production (73). Therefore, it prevents carcinogenesis There are several antioxidant capacity measurement methods exposure of colon mucosa. In addition, ferulic acid bound that have similar principles. In these methods, radicals to insoluble dietary fiber shows antioxidant effect by are generated and the antioxidant capacity of a sample scavenging free radicals (74-76), inhibits carcinogenesis against radicals is measured. All of these assays assume in animal models (77, 78), protects against the formation that all the antioxidant compounds present in foods are of nitroso compounds (79) and prevents DNA damaged completely extracted by different extraction procedures (13). in cultured cells (80). Furthermore, ferulic acid units in However, complex structure of foods together with location addition to dietary fiber exert their antioxidant effects much and solubility of antioxidant compounds affect extraction longer than free ferulic acid by quenching the radicals efficiency. Most foods contain both lipophilic and hydrophilic continuously formed in intestinal tract and they maintain antioxidant compounds that are soluble or insoluble forms reducing environment in colon. In the colon, fermentable (57, 58). Therefore, there is no unique solvent or mixture to fibers induce health-promoting bacterial growth, such as solve all of the antioxidant compounds in food structure lactobacilli and bifidobacteria, by acting as prebiotics (81). and the resulting extracts are not representative of the Fogliano et al. investigated the prebiotic activity of insoluble exact antioxidant capacity. As these classical methods are cocoa fiber and also demonstrated the importance of insufficient to measure the real antioxidant capacity of food, antioxidant compounds in addition to dietary fiber in the ‘QUENCHER’ method has been introduced to overcome the colon. They showed that insoluble cocoa fraction cause drawbacks of extraction methods. In this method, there is no increase in lactobacilli and bifidobacteria numbers, which need for extraction and hydrolysis processes. It is based on is related not only to insoluble fiber content but also to the the direct measurement of solid samples by mixing them with flavanol compounds (82). the free radicals followed by a subsequent spectrometric In view of these beneficial health effects of bound measurement (13). The principle of this method is the direct antioxidant compounds, increasing the amount of bound interaction between solid and radical solution such as antioxidant compounds has come into prominence ABTS•+ radical solution (Figure 2). In this assay, not only the (49, 83). In a recent study conducted by our group, the soluble antioxidant compounds interact with radical solution in vitro bound antioxidant capacity of insoluble wheat according to the liquid-liquid type reaction but also insoluble bran increased to remarkably high level exceeding 100 antioxidant compounds come into contact with radical mmol TE∙kg−1 from the level less than 10 mmol TE∙kg−1 solution by means of the surface solid-liquid type reaction by means of its treatment with free polyphenols under (13, 59). In summary, QUENCHER method provides direct alkaline conditions (84). This study revealed that increased measurement of the antioxidant capacities of both insoluble concentration of bound antioxidants enhanced their and soluble compounds without an extraction procedure. This positive health effects and the findings might be used to method is important especially for cereals, as their antioxidant develop functional dietary fibers rich in bound antioxidant activities are largely dependent on insoluble parts. Chemical capacity. mechanism behind other classical methods such as DPPH, FRAP, CUPRAC, ORAC and Folin-Ciocalteu are adapted to direct QUENCHER procedure (60-64). DIGESTION BEHAVIOR OF ANTIOXIDANTS BOUND TO DIETARY FIBER

There are some doubts about in vivo antioxidant activity of antioxidant compounds bound to dietary fiber, due to the fact that dietary fibers may affect the release and absorption of some molecules, including phenolic compounds, and bioavailability of the antioxidants (85). Therefore, it is necessary to understand the digestion behavior of antioxidant compounds associated with insoluble dietary fibers. Figure 2. Principle of the QUENCHER procedure for the measurement of dietary fiber bound antioxidant capacity as exemplified for ABTS•+ Perez-Jimenez et al. reported that the antioxidant activity radical solution (black symbols: macromolecules binding antioxidant of cereals in the gastrointestinal digestion system might compounds, orange symbols: bound antioxidant compounds, red symbols: bound antioxidant radical forms, dark blue symbols: ABTS•+ probably be higher than expected from literature data radicals, light blue symbols: quenched ABTS molecules). on measurements of chemical extract (86). Some of the phenolic compounds may become bioavailable and their antioxidant activities significantly enhance following THE IMPORTANCE OF BOUND ANTIOXIDANT IN HUMAN HEALTH the gastrointestinal digestion (87). However, phenolic compounds strongly bound to cell wall materials are not Epidemiological studies have shown that consumption affected by the acid conditions in gastric phase or small of cereal grain reduce the risk of colorectal and gastric intestinal enzymes. Dietary fibers reduce the absorption cancers (65-68). The reason for this beneficial effect is of phenolic compounds as a primary effect in small most probably the content of insoluble dietary fiber and intestine. There are two main reasons for that: (i) phenolic antioxidant compounds (69, 70). The mechanism under compounds may be entrapped with dietary fibers or (ii) protection against carcinogenesis may be direct removal of phenolic compounds may not reach the intestinal wall as a the negative effects of this carcinogenesis such as binding consequence of increased viscosity in gastric phase by the carcinogens (71), absorbing water resulting in increased insoluble dietary fibers (88).

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XXVII Therefore, a significant part of the antioxidant compounds 161, 1542–48, (2001). bound to dietary fiber reach the colon without being 3. Calle E. E., Rodriguez C., et al., N. Engl. J. Med., 348, 1625–38, digested through the small intestine (89) and contribute to (2003). the formation of reduced environment in the colon where 4. WHO and FAO, Diet, Nutrition, and the Prevention of Chronic Diseases: Report of a Joint WHO/FAO Expert Consultation, Report they can be fermented by the microflora, yielding different 916, Geneva: WHO, (2003). compounds that may be metabolized (86). Some part of 5. Bacon C. G., Mittleman M. A., et al., Ann. Intern. Med., 139, 161– the bound antioxidants is converted to free form by the 68, (2003). colon microbiota, and is released slowly and continuously 6. Fox K. R., Public Health Nutr., 2, 411–18, (1999). (90). It provides an increase in the basal plasma antioxidant 7. Willett W. C., Koplan J. P., et al., Prevention of Chronic Disease by capacity via absorption through the system (91). Thus, Means of Diet and Lifestyle Changes, Chapter 44, in Disease antioxidants bound to dietary fibers exert their effects much Control Priorities in Developing Countries, Edited by Jamison D. T., longer than soluble antioxidants in living organisms. Kroon Breman J. G., et al., 2nd edition, Oxford University Press, New York, et al. showed that over 95% of the total feruloyl groups USA, (2006). take place in colonic fermentation, whereas only 2.6% of 8. Urala N., Lahteenmaki L., Food Qual. Prefer., 18, 1-12, (2007). 9. Rahman K., Clin. Interventions Aging, 2, 219-36, (2007). total feruloyl groups in the fiber are released after gastric 10. Kris-Etherton P. M., Hecker K. D., et al., Am. J. Med., 113 (9B), and intestinal enzymatic treatment (90). According to the 71-88, (2002). study performed on rats, free ferulic acid ingested with diet 11. Pellegrini N., Serafini M., et al., J. Nutr., 133(9), 2812-19, (2003). cause rapid increase in plasma antioxidant activity but 12. Stanner S. A., Hughes J., et al., Public Helath Nutr., 7(3), 407-22, this effect fade away within 4 hours. However, upon wheat (2004). bran consumption, a constant concentration of ferulic acid 13. Gokmen V., Serpen A., et al., Trends Food Sci. Technol., 20, 278- in the plasma was observed even 24 hours after meal (92). 88, (2009). To sum up, the continuous presence of a relatively low 14. Miller H. E., Rigelhof F., et al., J. Am. Nutr., 19(3), 312-19, (2000). concentration of phenolic compounds may have a higher 15. Jimenez-Escrig A., Rincón M., et al.,J. Agric. Food Chem., 49, potential benefit for body defense than the peak of plasma 5489-5493, (2001). 16. Saura-Calixto F., J. Agric. Food Chem., 46, 4303- 4306, (1998). antioxidant concentration observed immediately after the 17. Martinez-Tome M., Murcia M. A., J. Agric. Food Chem., 52, 4690-9, ingestion of a free phenolic compound-rich food (27). (2004). From this point of view, cereal phenolic compounds bound 18. Chandrasekara A., Shahidi F., J. Agric. Food Chem., 58, 6706- 14, to dietary fiber gain importance and are particularly (2010). beneficial for human health. 19. Liyana-Pathirana C. M., Shahidi F., J. Agric. Food Chem., 54, 1256- 64, (2006). 20. Fardet A., Nutr. Res. Rev., 23, 65-134, (2010). CONCLUSION 21. Belobrajdic D. P., Bird A. R., Nutr. J., 12, 62, (2013). 22. Vitaglione P., Mennella I., Am. J. Clin. Nutr., 101, 251-61, (2015). It is well known that soluble antioxidant compounds inhibit 23. Slavin J., Tucker M., et al., Cereal Foods World, 58, 191-8, (2013). 24. Cho S. S., Qi L., et al., Am. J. Clin. Nutr., 98, 594-619, (2013). oxidation by counteracting with oxidative stress and 25. Lefevre M., Jonnalagadda S., Nutr. Rev., 70, 387-96, (2012). modulate cell signaling pathways. On the other hand, 26. Ye E. Q., Chacko S. A., et al., J. Nutr., 142, 1304-13, (2012). the importance of soluble antioxidant compounds as 27. Vitaglione P., Napolitano A., et al., Trends Food Sci. Technol., 19, radical and oxidant scavengers in vivo might be minor, 451-63, (2008). based on their limited concentrations achieved in plasma 28. Shewry P. R., Nutrition Bulletin, 34, 225-231, (2009). and tissues for a few hours (93). However, fiber-bound 29. Stevenson L., Phillips F., et al., Int. J. Food Sci. Nutr., 63, 1001-13, antioxidants may reach the colon without being digested, (2012). and contribute to the formation of a reduced environment. 30. Liyana-Pathirana C., Chandrika D., et al., J. Agric. Food Chem., They are also converted to simpler phenolic compounds by 54, 6177-84, (2006). the colon microflora and released slowly and continuously. This partly explains what the cereal grain consumption role Readers interested in a full list of references are invited to visit our website at www.teknoscienze.com is in the prevention of colon cancer and other diseases. From another perspective, fiber-bound antioxidants may exert their inhibitory effects on lipid oxidation much longer than soluble antioxidants due to the continuous and slow releasing. However, further studies about the effects of bound antioxidants on the lipid oxidation during food processing or storage should be performed to validate this hypothesis. In addition, it is necessary to further confirm about in vivo antioxidant activity of bound antioxidants. Dietary fiber rich in bound antioxidant capacity should also be used for developing and commercializing a functional ingredient used in breakfast cereal like products.

REFERENCES

1. Swinburn B. A., Caterson I., et al., Public Health Nutr., 7,123–46, (2004). 2. Hu F. B., Leitzmann M. F., et al., Arch. Intern. Med.,

XXVIII Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 ATEFEH ESLAMI-MOSHKENANI1, VAJIHEH FADAEI NOGHANI1,*, KIANOUSH KHOSRAVI-DARANI2 *Corresponding author 1. Department of Food Science and Technology, Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran 2. Research Department of Food Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, P.O. Box: 19395-4741, Tehran, Iran Kianoush Khosravi-Darani

Application of Arthrospira platensis (Spirulina) biomass to improve properties of probiotic doogh Production of sinbiotic doogh

KEYWORDS: Doogh, probiotics, spirulina platensis, lactobacillus acidophilus (La5).

The plenty of biologically important compounds in the algae, provide a new opportunity for producing Abstract functional dairy products. The main purpose of this study was to determine the effect of some incorporation variables on the survival of Lactobacillus acidophilus (La-5). So, Spirulina platensis powder (0, 0.3, 0.5 and 0.8 % w/v) was incorporated to doogh containing 0.5 and 1% w/v mint powder. Chemical properties (e.g. , protein, pH and acidity) as well as sensorial characteristics of the product were investigated. The results showed that viability of L. acidophilus remained at the standard amount in all samples containing Spirulina during 21 days of storage (p< 0.05). Incorporation of microalgae increased the iron and protein content of the samples. The increase in the concentration of Spirulina platensis induced a significant decrease in the sensory acceptability. According to chemical and sensorial analyzes and economical characteristics samples with 0.3%, w/v Spirulina were chosen as the best treatment.

INTRODUCTION their incorporation to food may lead to a possible change in the food’s organoleptic characteristics. So additives Spirulina (S.) platensis is the best known genus of can be applied to mask sensorial properties. In the case of cyanobacteria found in our environment with a high doogh, herbal additives, especially Mentha, are common for nutritional value as food (1-5). It consists of essential amino improving its flavoring and medicinal properties. acids, natural pigments, fatty acids, enzymes, minerals (e.g. The objective of this research was to study the impact of 2+ 2+ Ca and Fe ), and Vitamins A, B2, B6, B12, E and K (1,5-8). S. platensis incorporation (0, 0.3, 0.5 and 0.8 % w/w) on the Spirulina has been used as a food supplement in juices, viability of L. acidophilus (La-5) and some chemical and cereals and bakery products, desserts, cakes, soups, salad sensorial properties of probiotic-heated doogh containing 0.5 dressings, and dairy products such as ice cream, fermented and 1% mint during cold storage (21 days at 4°C). milk, yogurt, and dairy drinks (9). Doogh is a traditional Iranian drink with special sensorial and nutritional characteristics. Incorporation of probiotics and MATERIALS AND METHODS natural prebiotics to doogh may lead to more beneficial health impacts for the host. To obtain the claimed probiotic Starter culture and additives benefits, survival of bacteria in processing and shelf life is the L. delbrueckii ssp. bulgaricus and S. thermophilus (YF-3331) as most important criterion (10). One of the most famous species well as L. acidophilus (La-5) were supplied by Chr. Hansens of probiotic microorganisms is Lactobacillus (3). (Horsholm, Denmark). Starters were used in freeze dried direct The effect of S. platensis on the viability of starter and probiotic vat set and maintained at -8°C. Spirulina platensis was used bacteria has been investigated in fermented milk (7-12), in a powdered form obtained from Gheshm Sina Microalgae yogurt (5,13,14), probiotic yoghurt containing spinach (15), UF Co., Tehran, Iran. Menthe longifolia L. (mint) was used in a feta cheese (16), Doogh (17), cookies (18), and media (3, 19). powdered obtained from Goar, Markazi, Iran. However, to our knowledge, the influence of microalgae on the viability of Lactobacillus (L.) acidophilus in doogh has not Preparation of doogh samples been studied yet. Cow milk (1.2% fat, 2.9% protein, 9.6 % total solid, 8.21% non-fat The use of cyanobacteria in the manufacture of cultured solids, pH 6.63 and titratable acidity 14.5°D) was pasteurized, dairy foods can be extended to doogh enrichment due to its cooled and inoculated with starter culture (0.1% w/v). widespread consumption in Iran. Specific cellular compounds During the fermentation, pH drop and titratable acidity were of microalga stimulate the viability of probiotic (3,7,11), but monitored until pH reached to 4.5±0.02 (Figure 1).

Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 XXIX Produced yogurt was diluted by water (50% v/v & and NaCl The maximum viability of L. acidophilus was related to the 0.7% w/v) to produce Doogh. Doogh samples were heated samples containing 0.8% S. platensis and the minimum to 85ºC (15 min) and inoculated with 0.2% w/v L. acidophilus viability belonged to the control samples at the end of and divided into 2 groups (M1 and M2 containing 0.5 and shelf life. The number of L. acidophilus in the day 14th was 1% w/v mint, respectively), and enriched with 0, 0.3, 0.5 and reduced in compare to 7th day which may be due to 0.8% w/v S. platensis (A1, A2, A3 and A4, respectively). Then competing of bacteria to obtain nutritional resource and they were filled into 250-ml PET bottles and stored at 4ºC for production of secondary metabolites.The reduction was 21 days. more significant in the control, and was rarely observed when S. platensis was added (due to buffering property Microbiological analysis and nutritional enrichment by free amino acids, peptone, MRS-bile agar medium was used for determining the hypoxanthine, adenine, etc (13,19). Similar results are viability of L. acidophilus (La5) (MRS agar by Merck, reported on acidophilus milk (13), acidophilus milk Darmstadt, Germany and bile by Sigma-Aldrich, Inc., containing Bifidobacterium, milk (7), and yoghurt (14). The Reyde, USA). One mL of each sample was diluted with 9 viability of L. acidophilus was influenced by increasedS. mL of sterile 0.1% w/v peptone water (Oxoid, Hampshire, platensis content and the storage time. The percentage UK), and mixed uniformly with a vortex mixer. Subsequent of mint powder used had no significant antimicrobial serial dilutions were made. The counts of the viability of L. effect. May be little concentrations had used that had no acidophilus (La5) were taken after incubating the plates antibacterial effect. Simsek et al. (23) indicated that mint’s anaerobically at 37°C for 72 h (20). essential oil had no significant effect on the viability of yoghurt’s traditional bacteria. Chemical analysis Total nitrogen content was determined by Kjeldahl method in the middle of storage period and multiplied by 6.38 to obtain protein content (21). The Fe content was measured on days 1,14 and 21 by atomic absorption (Youngline 8020, Korea). Homogenized sample (5- 10 g) was dried at 450°C for 5-6 h, charred, and then ashed in a muffle furnace to obtain a whitish or greyish ash. The ash was treated with concentrated hydrochloric acid and made up to 50 ml in a volumetric flask (22).

Sensory analysis A panel composed of 5 trained panelists from Pakban (Dairy Company, Iran) was used to determine the Doogh score with 5 points hedonic scale for external appearance (color), Table 1. Viability of Lactobacillus acidophilus La-5 (cfu.ml-1) in flavor, taste and texture using standard questionnaire. The Doogh samples containing different concentrations of S. platensis scores (1 = dislike very much, 2= dislike a little, 3= neither like and 0.5% Mint (M1A1 0%, M1A2 0.3%, M1A3 0.5%, M1A4 0.8%) and nor dislike, 4= like a little, 5 means like very much) were given 1% Mint (M2A1 0%, M2A2 0.3%, M2A3 0.5%, M2A4 0.8%) during storage at 4°C*. by the expert panelists for each of the mentioned properties. *Each value in the table is the mean±SD of eight replications. a, b, The samples were poured into special 100-mL coded paper c,…Means in the same row with different lowercase superscripts cup and transferred into the booths of the evaluating team. differ (p<0.05). A,B,C ,… Means in the same column with different Analysis of variance (ANOVA) was applied using SAS 9.1 uppercase superscripts differ (p<0.05). software. According to this study, S. platensis-incorporated doogh Statistical analysis possesses more than 105 CFU ml-1 (initial cell concentration Each experiment was independently replicated three times before treatment was approximately 107 CFU ml-1) probiotics in a completely randomized design. Data are expressed in shelf life during the storage. as means±SD. Analyses were performed using the SAS software (version 9.1). Differences between the means were As illustrated in Figure 1, the samples containg 0.8% S. platensis evaluated using one-way ANOVA. The differences between had a higher amount of protein content (p<0.05). It confirms the mean values were compared at the significant level of other reports (4,7,8). Dried algae contain 46-63% protein (14). p < 0.05. Mint had no effect on protein content.

RESULTS AND DISCUSSION

The viability of probiotic bacteria in different samples during 21 days is shown in Table.1. As indicated, the viable count of L. acidophilus (La5) changes in different growth phases. By incorporation of S. platensis, the viability of L. acidophilus increases significantly in compare to the control, which is due to nutritional enrichment of yoghurt. At the beginning of the Figure 1. Protein in probiotic Doogh samples containing different concentrations of S. platensis and 0.5 % and 1% mint in the middle fermentation period, an increase in the number of L. acidophilus of storage period at 4°C. depended significantly on the concentration ofS. platensis.

XXX Monographic special issue: PRE/PROBIOTICS - Agro FOOD Industry Hi Tech - vol. 27(5) - September/October 2016 Figure 2. Changes of the iron (ppm) in probiotic Doogh samples containing different concentrations of S. platensis and 0.5% Mint (M1A1 0%, M1A2 0.3%, M1A3 0.5%, M1A4 0.8%) and 1% Mint (M2A1 0%, M2A2 0.3%, M2A3 0.5%, M2A4 0.8%) during storage at 4°C.

Figure 2, indicated that Fe content increases significantly by adding S. platensis microalgae as well as by increasing the amount of mint (p<0.05), which was due to the existence of iron in this herb. However, it did not change during the storage time (1, 14 and 21 days). Similar results were reported by Guldas and Irkin (13).

Samples containing different amount of algae showed different sensorial evaluation (by color, taste, smell, texture and overall acceptance) (p<0.05). However, different amounts of mint because of the low amount (w/v) and more flavor of microalgae and time exhibited no significant effect on sensory evaluation. Figure 3, shows that by increasing the content of algae, sensorial properties of the samples are reduced. There was no significant difference between the sample with 0.3% and the control in this regard. The different amounts of mint (0.5 or 1%) had no impact on sensorial properties. The best scores were related to the control (like very much), and the samples containing 0.3% algae (like very much), 0.5% algae (like a little), and 0.8% algae (neither like nor dislike) respectively. Anyway, all samples were acceptable. These results are similar to those reported on yoghurt containing Chlorella (24), probiotic yoghurt and acidophilus milk (13), fermented milk (8), and probiotic yogurts (5) which indicate that the lower percentage of microalgae leads to the highest sensory evaluation.

Figure 3. Overall acceptability (Sensory analysis) in probiotic Doogh samples containing different concentrations of S. platensis and 0.5% Mint (M1A1 0%, M1A2 0.3%, M1A3 0.5%, M1A4 0.8%) and 1% Mint (M2A1 0%, M2A2 0.3%, M2A3 0.5%, M2A4 0.8%) during storage at 4°C, using score methodology with 5 point hedonic scale (from 1 means dislike very much to 5 means like very much).

CONCLUSION

The impact of incorporation of S. platensis to the probiotic heated doogh on the viability of L. acidophilus, as well as on the nutritional and sensorial properties of the product was investigated. S. platensis increased the viability of L. acidophilus during the 21 days of storage; it further increased the product’s iron and protein content (p<0.05). Also, microalgal incorporation increased protein content. The score of probiotic doogh containing 0.3% microalga was et al. "The potential of probiotics". Food Technol. Biotechnol., 48, similar to that of the control in sensory evaluation. 413-434 (2010). Due to its high content of vitamins, proteins, essential 11. Gyenis, B., Szigeti, J., Molnar, N., Varga, L. "Use of dried fatty acids and trace elements, S. platensis provides new microalgal biomasses to stimulate acid production and growth of Lactobacillus plantarum and Enterococcus faecium in milk". Acta opportunities in the production of functional products. Agraria Kaposváriensis, 9: 53-59 (2005) Incorporation of S. platensis microalgae to Doogh can be 12. Beheshtipour, H., Mortazavian, A. M., Mohammadi, R., helpful to overcome the iron deficiency in Iran. Nevertheless, Sohrabvandi, S., et al. "Supplementation of Spirulina platensis and the examined mint does not seem to have a significant Chlorella vulgaris algae into probiotic fermented milks", Compr impact on masking the changes of sensorial properties. Rev Food Sci ,12, 144-154 (2013). A possible solution would be to incorporate other herbal 13. Guldas, M., Irkin, R. "Influence of Spirulina platensis powder on additives to probiotic Doogh containing L. acidophilus and the microflora of yoghurt and acidophilus milk In: Microflora of S. platensis as prebiotics. yoghurt and acidophilus milk", Mljekarstvo, 60, 237-243 (2010). 14. Beheshtipour, H., Mortazavian, A.M., Haratian, P., Khosravi-Darani, K. "Effect of Chlorella vulgaris and Arthrospira platensis addition on viability of probiotic bacteria in yogurt and its biochemical ACKNOWLEDGEMENT properties". Eur. Food Technol., 235, 719-728 (2012). 15. Fadaei, V., Mohamadi-Alasti, F., Khosravi-Darani, K. "Influence The authors are grateful to Afshin Afshar (Director of the of Spirulina platensis powder on the starter culture viability in Department of Food Science, Alborz Medical University) for his probiotic yoghurt containing spinach during cold storage". Eur. J. valuable support during this study. Exper. Biol., 3, 389-393 (2013). 16. Fadaei, V., Mazinani, S., Khosravi-Darani, K. "Influence of Spirulina platensis powder on viability of Lactococcus strain in probiotic REFERENCES UF feta cheese containing Mentha longifolia L". Int, J. Biol, Biotech.,10, 475-478 (2013). 1. Hosseini, S.M., Khosravi-Darani, K. Mozafari, M.R. "Nutritional and 17. Fadaei, V., Eslami-Moshkenani, A., Khosravi-Darani, K. "Effects Medical Applications of Spirulina Microalgae", Mini-Rev. Med. of powdered Spirulina platensis biomass on pH and titratable Chem., 13, 1231-1237 (2013). acidity of probiotic Doogh containing powdered mint during cold 2. Hosseini, S.M., Shahbazizadeh, S. Khosravi-Darani, K., Mozafari, storage". Int. J. Biol. Biotech., 10, 631-635 (2013). M.R. "Spirulina platensis Food and Function", Curr. Nutr. Food Sci., 18. Shahbazizadeh, S. Khosravi Darani, K., Sohrabvandi, S. 9, 189-193 (2013). "Fortification of Iranian traditional cookies with Spirulina platensis". 3. Parada, J.L., Ceire, G.Z.D., Mule, M.C.Z., Cano, M.M.S. "Lactic acid Ann. Rev. Res. Biol., 7, 144-154 (2015). bacteria growth promoters from Spirulina platensis". Int. J. Food 19. Bhowmik, D., Dubey, J., Mehra, S. "Probiotic efficiency of Spirulina Microbiol., 45, 225-228 (1998). platensis- stimulating growth of lactic acid bacteria". World. J. 4. Molnar, N., Gyenis, B., Varga, L. "Influence of a powdered Spirulina Dairy. Food. Sci., 4 (2), 160-163 (2009). platensis biomass on acid production of lactococci in milk", 20. SOHRABVANDI, S., MORTAZAVIAN, A.M., DOLATKHAHNEJAD, Milchwissenschaft, 60, 380-382 (2005). M.R., MONFARED, A.B. "Suitability of MRS-bile agar for the 5. Akalin, A.S., Ünal, G., Dalay, M.C. "Influence of Spirulina platensis selective enumeration of mixed probiotic bacteria in presence on microbiological viability in traditional and probiotic yoghurts of mesophilic lactic acid cultures and yoghurt bacteria". Iran J during refrigerated storage", Ital. J. Food Sci., 21, 357-362 (2009). Biotechnol., 10, 16-21 (2012). 6. Soheili, M., Khosravi-Darani, K. "The potential health benefits 21. A.O.A.C. Association of Official Analytical Chemists. Official of algae and micro algae in medicine: a review on Spirulina methods of analysis. 18th ed., Arlington, USA, Method The total platensis". Curr. Nutr. Food Sci., 7(4), 279-285 (2011). nitrogen (N) content was measured by the Kjeldahl method No. 7. Caire, G.Z., Parada, J.L., Zaccaro, M.C., De Cano, M.M.S. "Effect 991.20 (2005). of Spirulina platensis biomass on the growth of lactic acid 22. SIONG, T. CHOO, K.. and SHAHID, S. 1986. Determination of Iron bacteria in milk". World J. Microbiol. Biotechnol., 16, 563-565 in Foods by the Atomic Absorption Spectrophotometric and (2000). Colorimetric Methods. Pertakina; 12(3):313-322. 8. Varga, L., Szigeti, J., Kovacs, R., Foldes, T., et al. "Influence of a 23. SIMSEK, B., SAGDIC, O., OZCELIK, S. "Survival of Escherichia coli Spirulina platensis biomass on the microflora of fermented ABT milk O157:H7 during the storage of Ayran produced with different during storage". J. Dairy Sci., 85, 1031-1038 (2002). spices". J. Food Eng., 79, 679-680 (2007). 9. Spolaore, P., Joannis, C., Duran, E., Isambert, A. "Commercial 24. SUNG, Y., CHO, J.R., OH, N.S., KIM, D.C., IN, M.J. "Preparation and applications of microalgae". J. Biosci. Bioeng., 101, 87-96 (2006). quality characteristics of curd yogurt added with chlorella". Appl. 10. Soccol, C.R., Vandenberghe, L.P.S., Spier, M., Medeios, A.B.P., Biol. Chem., 48, 60-64 (2005).

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