Enzymatic synthesis of novel bioactive oligosaccharides via microbial transglycosidases acting on sucrose. structural characterization and bioactivity study

ENZYMATIC SYNTHESIS OF NOVEL BIOACTIVE

OLIGOSACCHARIDES VIA MICROBIAL TRANSGLYCOSIDASES ACTING ON SUCROSE. STRUCTURAL CHARACTERIZATION

AND BIOACTIVITY STUDY.

MARINA DÍEZ MUNICIO PhD Thesis

Madrid, 2015

ENZYMATIC SYNTHESIS OF NOVEL BIOACTIVE

OLIGOSACCHARIDES VIA MICROBIAL TRANSGLYCOSIDASES ACTING ON SUCROSE. STRUCTURAL CHARACTERIZATION

AND BIOACTIVITY STUDY.

MARINA DÍEZ MUNICIO

Thesis submitted in fulfilment of the requirements for the degree of doctor at Autónoma University of Madrid (UAM)

to be defended in public on Wednesday 15 July 2015 at Institute of Food Science Research,

CIAL (CSIC-UAM).

Thesis supervisors (Spanish Council of Scientific Research, CSIC):

Dr. F. Javier Moreno Andújar Dr. Miguel Herrero Calleja

Thesis tutor at Autónoma University of Madrid (UAM):

Dr. Guillermo Reglero Rada

This research was mainly conducted at Institute of Food Science Research, CIAL – CSIC-UAM (Madrid, Spain).

M. Díez-Municio was supported from 2011 to 2015 by CSIC through

JAE-Pre Programme co-funded by European Social Fund (ESF).

C/ Nicolás Cabrera, 9.

Campus de la Universidad Autónoma de Madrid 28049 Madrid

F. Javier Moreno Andújar and Miguel Herrero Calleja, Tenured Scientists of the Spanish Council of Scientific Research (CSIC) at the Institute of Food Science Research (CIAL),

CERTIFY:

That Marina Díez Municio, has performed, under their supervision, the research work entitled: “Enzymatic synthesis of novel bioactive oligosaccharides via microbial transglycosidases acting on sucrose. Structural characterization and bioactivity study.”

This work memory is submitted in fulfilment of the requirements for the degree of doctor at Autónoma University of Madrid (UAM).

Madrid, 9 June 2015.

Fdo. D. F. Javier Moreno Andújar Fdo. D. Miguel Herrero Calleja

For Lourdes & Fidel Municio,

my mother & my grandfather,

« I among those who think that science has great beauty. »

Marie Curie, 1867-1934.

Preface

The Doctoral Thesis you hold in your hands brings together the research carried out during my four- year PhD on “Enzym„†fc ‟yn†hi‟f‟ ge ngvil bfg„c†fvi glfhg‟„cch„rfdi‟ vf„ mfcrgbf„l

†r„n‟hlycg‟fd„‟i‟ „c†fnh gn ‟ucrg‟i. S†ruc†ur„l ch„r„c†irfz„†fgn „nd bfg„c†fvf†y ‟†udy”. It was submitted in 2015 at Autónoma University of Madrid – UAM (Madrid, Spain) to obtain the degree of doctor with international accreditation under the “Biology and Fggd Scfinci” PhD Programme (RD 1393/2007).

The great opportunity to undertake a PhD Thesis came up to me in October 2011 through a grant supported by the Spanish Council of Scientific Research (CSIC) co-funded by European Social Fund (ESF) (JAEPre 2011-01374). The laboratories where most of the work was performed belong to the Department of Bioactivity and Food Analysis of the Institute of Food Science Research (CIAL-CSIC), located at UAM campus (Madrid, Spain). However, part of the work was conducted in close collaboration with the Department of Biotechnology and Microbiology of CIAL (Functional Biology of Lactic Bacteria group, BFBL) and other CSIC institutes such as Cin†ri egr Orh„nfc Chimf‟†ry “Lgr„-T„m„yg” - CENQUIOR (NMR service), Institute of Food Science, Technology and Nutrition – ICTAN (Bacterial Biotechnology group) and Institute of Agrochemical and Food Technology – IATA (to perform in vivo studies). Furthermore, I had the opportunity to broaden my knowledge in an international environment at the University of Reading – UoR (Reading, United Kingdom), which is why this PhD Thesis aims to have the international accreditation (Council of Government of December 15, 2011, for UAM doctoral studies). In this regard, I would like to acknowledge Dr. M. Victoria Moreno, the director of CIAL, on behalf of all the members of the institute, for their support all these years. Likewise, I greatly thank all the time, effort and expertise that contributor authors have devoted to this PhD Thesis.

The work was accomplished under the supervision of Dr. F. Javier Moreno and Dr. Miguel Herrero (Tenured Scientists at CIAL) and tutored by Dr. Guillermo Reglero (Professor at UAM).

I am sincerely grateful for all the professional advices and numerous lessons learned from their

excellent work experience, as well as for the support I have received at all times. There can be no

doubt that my supervisors had provided me a great assistance and source of inspiration through their

constantly positive encouraging and proactive approach.

"Enzymatic synthesis of new bioactive oligosaccharides by using glycosyltransferases produced by lactic-acid bacteria. Structural and functional characterization", supported by the Spanish Ministry of Economy and Competitiveness (MINECO) co-funded by European Regional Development Fund (FEDER) (AGL2011-27884/ALI). Moreover, the granting of another research project within the CSIC international program for the promotion of scientific collaboration with foreign institutions (i-LINK+2013-0827), allowed me conducting the short-term scientific stay in Reading besides another in Haifa, at the Israel Institute of Technology –Technion.

If you take a look to the table of contents that is on the next page, you will see that this Thesis dissertation mainly includes a general introduction section, followed by the aim, the work plan and an overview of the research, a series of chapters that gather the experimental results of this research, a general discussion and a conclusions section. The general introduction deals with functional foods, gut microbiota and food-grade oligosaccharides (production, properties, analysis, structural characterization and purification, among others aspects). Following, the core of this Thesis is constituted by ten chapters grouped in two parts, the first is about synthesis and characterization of novel oligosaccharides and the second about the bioactivity studies. Six of these chapters have already resulted in scientific publications published in international journals of high impact factor included on the Science Citation Index (SCI), while the other four are undergoing the process of peer review. The general discussion section is devoted to give the reader an overview of the study done along this PhD Thesis work and to provide some insights into the possible directions that this research may take in the future. Likewise, this section emphasizes the significance of finding novel bioactive oligosaccharides, the importance of producing them by efficient processes, and some aspects of their vulnerability on the road to the market. Finally, a general conclusions section compiles the most remarkable findings achieved during this PhD Thesis work.

With the belief that these results can be made known both at national and international level, this PhD Thesis dissertation is written in English. Nevertheless, the summary and conclusions are also presented in Spanish to comply with regulatory requirements of UAM.

I hope you will enjoy reading this PhD Thesis dissertation.

Marina Díez Municio.

Madrid, June 2015.

Contents

Summary

‘Resumen’

1 3

General Introduction

Functional foods and modulation of the human gut microbiota Overview of functional foods

The human gut microbiota

Functional food ingredients for gastrointestinal health Food-grade oligosaccharides as functional ingredients Production of food bioactive oligosaccharides

Definition and properties of non-digestible oligosaccharides Enzymatic synthesis of food-grade oligosaccharides

Analysis, structural characterization and purification of oligosaccharides

Analytical techniques Fractionation techniques

6 6 9 15 25 25 30 33 42

42 52

Aim, Work Plan and Overview of the Research 55

Part I.

Enzymatic synthesis and structural characterization of novel oligosaccharides

Chapter 1 - Synthesis and characterization of a potential prebiotic trisaccharide from cheese whey permeate and sucrose by Leuconostoc mesenteroides dextransucrase Chapter 2 - A sustainable biotechnological process for the efficient synthesis of kojibiose

Chapter 3 - Efficient synthesis and characterization of lactulosucrose by Leuconostoc mesenteroides B-512F dextransucrase

Chapter 4 - Enzymatic synthesis and characterization of fructo-oligosaccharides and novel maltosyl-fructosides by inulosucrase from Lactobacillus gasseri DSM 20604 Chapter 5 - Novel bi-enzymatic system for the efficient synthesis of lactosyl-fructosides structurally characterized by NMR spectroscopy

Chapter 6 - Synthesis and structural characterization of raffinosyl-fructosides upon transfructosylation by Lactobacillus gasseri DSM 20604 inulosucrase

63

79

93

107

127

141

Part II.

Bioactivity study of the novel oligosaccharides synthesized

Chapter 7 - Selective fermentation of potential prebiotic lactose-derived oligosaccharides by probiotic bacteria Chapter 8 - In vitro faecal fermentation of novel oligosaccharides enzymatically synthesized using microbial transglycosidases acting on sucrose

Chapter 9 - Structural differences of prebiotic oligosaccharides influence their capability to enhance iron absorption in deficient rats

Chapter 10 - Kojibiose ameliorates arachidic acid-induced liver alterations in hyperglycemic rats

157

167

183

195

General Discussion

In the search of new bioactive oligosaccharides

Enzyme catalysis as an efficient tool for producing bioactive oligosaccharides

Road to the market: there is still a very long way to go

208 211

214

General Conclusions

‘Conclusiones Generales’

217 219

Annexes Annex A - Supplemental Material Chapter 1 –

Full set of NMR spectra of 2-α-D-glucopyranosyl-lactose, also denominated 4’-galactosyl-kojibiose

Annex B - Supplemental Material Chapter 3 – Full set of NMR spectra of lactulosucrose Annex C - Supplemental Material Chapter 4 – Full set of NMR spectra of maltosyl-fructosides Annex D - Supplemental Material Chapter 5 – Full set of NMR spectra of lactosyl-fructosides.

Annex E - Supplemental Material Chapter 6 – Full set of NMR spectra of raffinosyl-fructosides Annex F - Spanish Patent Application –

“Procedimiento de síntesis de kojibiosa y su aplicación en la elaboración de composiciones alimentarias y farmacéuticas”

Annex G - Spanish Patent Application –

“Procedimiento bi-enzimático de síntesis eficiente de oligosacáridos fructosilados derivados de lactosacarosa, productos obtenidos y su uso en la mejora de la salud gastrointestinal”

223

227

233

243

253

263

275

1

Summary

The production of new bioactive oligosaccharides today raises a great interest due to their potential use as functional components in the food and pharmaceutical industry. Among the various strategies employed for the production of these oligosaccharides, enzymatic processes have great potential, as they usually have a high substrate specificity as well as regio- and stereo- selectivity. In this PhD Thesis, the production, via enzymatic synthesis, of various oligosaccharides that could present a potential industrial interest, particularly regarding their prebiotic capacity, has been studied. In general, these oligosaccharides are synthesized by transglycosidase enzymes (EC 2.4.1) produced by lactic acid bacteria capable of catalyzing reactions of transferring the glucose or fructose unit of sucrose (donor) to a wide range of carbohydrate acceptors, giving rise to high yields of synthesis. Specifically, in this PhD Thesis, the optimized synthesis of the trisaccharide 2-α-glucosyl-lactose, also called 4'-galactosyl- kojibiose, the disaccharide kojibiose, the trisaccharide lactulosucrose and sets of fructosyl oligosaccharides derived from maltose, lactosucrose, and raffinose is described. Besides, the structural characterization (glycosidic linkage, monosaccharide composition and degree of polymerization) of novel synthesized oligosaccharides, carried out by nuclear magnetic resonance (NMR) or mass spectrometry (MS) is also discussed. Both the synthesis and characterization of these oligosaccharides are presented in the first six chapters, encompassing the first part of this thesis. Moreover, once the synthesis was optimized, the purification of compounds of interest was carried out to be used in the bioactivity studies in order to determine the prebiotic potential of these oligosaccharides. These studies are presented in the second part of this thesis, Chapters 7 to 10. Depending on the viability of the purification process, which is usually one of the most expensive in the process of obtaining oligosaccharides, different types of tests were conducted both in vitro and in vivo using animal models. Both the structural characterization of these oligosaccharides as well as bioactivity studies conducted, will further allow deepen in the knowledge of the structure-function relationship, essential for the development and eventual commercialization of these new ingredients with potential functionality.

3

‘Resumen’

La producción de nuevos oligosacáridos bioactivos suscita en la actualidad un gran interés debido a su posible uso como ingredientes funcionales en la industria alimentaria y farmacéutica. Entre las diversas estrategias empleadas para la producción de estos oligosacáridos, los procesos enzimáticos tienen un gran potencial, ya que normalmente presentan una alta especificidad por el sustrato, así como regio- y estéreo-especificidad. En esta Tesis Doctoral se ha estudiado la obtención, vía síntesis enzimática, de varios carbohidratos que podrían presentar un potencial interés industrial, particularmente, en cuanto a su capacidad prebiótica. En general, estos oligosacáridos se han sintetizado mediante enzimas transglicosidasas (EC 2.4.1) producidas por bacterias lácticas con capacidad de catalizar reacciones de transferencia de la unidad de glucosa o fructosa de la sacarosa (donante) a una amplia gama de carbohidratos aceptores, dando lugar a rendimientos de síntesis elevados.

Concretamente, en el presente trabajo de tesis, se describe la producción optimizada del trisacárido 2-α-glucosil-lactosa, también denominado 4’-galactosil-kojibiosa, el disacárido kojibiosa, el trisacárido lactulosacarosa y oligosacáridos fructosilados derivados de maltosa, lactosacarosa, y rafinosa. Asimismo, también se discute la caracterización estructural (tipo de enlace glicosídico, composición en monosacáridos y grado de polimerización) de los nuevos oligosacáridos sintetizados, llevada a cabo mediante resonancia magnética nuclear (NMR) o espectrometría de masas (MS). Tanto el proceso de síntesis como la caracterización de estos oligosacáridos se presentan en los primeros seis capítulos, englobando la primera parte de esta tesis (“Chapters 1-6, Part I”). Por otra parte, una vez optimizada su síntesis, se procedió a la purificación de los compuestos de interés para posteriormente llevar a cabo estudios de bioactividad que permitan determinar fundamentalmente el potencial prebiótico de estos oligosacáridos. Estos estudios se presentan recogidos en la segunda parte de esta tesis, del capítulo 7 al 10 (“Chapters 7-10, Part II”). En función de la viabilidad de este proceso de purificación, que normalmente es uno de los más costosos en el proceso de obtención de carbohidratos, se realizaron distintos tipos de ensayos, tanto in vitro como in vivo utilizando modelos animales. Tanto el conocimiento de la estructura de estos oligosacáridos como los estudios de bioactividad llevados a cabo, permitirán profundizar en un futuro en el conocimiento de la relación estructura-función, fundamental para el desarrollo y posible comercialización de estos nuevos ingredientes con potencial funcionalidad.

5

General Introduction

Functional foods and modulation of the human gut microbiota Overview of functional foods

The human gut microbiota

Microbial diversity in the human gastrointestinal tract Techniques used to characterize the gut microbiota The role of the gut microbiota in human health Functional food ingredients for gastrointestinal health

Probiotics Prebiotics

Prebiotic concept

Mechanisms of action of prebiotics Bioactivity assessment of prebiotic effect Food-grade oligosaccharides as functional ingredients Production of food bioactive oligosaccharides

Definition and properties of non-digestible oligosaccharides Enzymatic synthesis of food-grade oligosaccharides

Microbial enzymes as biocatalysts for the synthesis of food-grade oligosaccharides Classification of microbial enzymes involved in the synthesis of food-grade oligosaccharides

Structural and catalytic insights to glycansucrases Transglycosylation acceptor reactions by glycansucrases

Analysis, structural characterization and purification of oligosaccharides Analytical techniques

High performance liquid chromatography (HPLC)

Hydrophilic-interaction liquid chromatography (HILIC) Anion exchange chromatography (AEC)

Gas chromatography (GC) Mass spectometry (MS)

Nuclear magnetic resonance (NMR) spectroscopy Fractionation techniques

FUNCTIONAL FOODS AND MODULATION OF THE HUMAN GUT MICROBIOTA

OVERVIEW OF FUNCTIONAL FOODS

The main role of food is to provide enough nutrients and energy to ensure the metabolic requirements of each individual, in addition to pursue consumer satisfaction through appetite and hedonic attributes such as taste. However, beyond this widely accepted nutritional value, diet can also provide positive physiological and psychological effects that can contribute to promote consumer‟s health, in terms of either improving physical and mental well-being and/or reducing the risk of disease. In fact, in today‟s Western world, the concept of nutrition is shifting from a traditional perspective that only sought the prevention of nutritional deficiencies, to a modern conception that seeks the prevention of chronic diseases. Moreover, consumers‟ interest in healthy eating is evolving into a balanced diet towards the consumption of foods that may provide additional potential health benefits. This has led in recent times to a growing interest towards the so-called “functional foods”. The specific bioactive food components capable of producing these additional benefits are called “functional ingredients”.

The increasing demand for such functional foods can be also explained by the increasing cost of health care due to the steady increase in life expectancy.¹

The term functional food was firstly coined in Japan in the early 1980s, and a regulatory framework known as FOSHU (Foods Of Specific Health Use) was established in 1991 to favor the development of these foods. The concept quickly spread through Europe and the United States but that is not the case for legislation governing these foods, which varies worldwide and has always lagged behind. In the European Union (EU), the scientific study of functional foods was first stimulated in 1999 through the European Commission (EC) Concerted Action FUFOSE (Functional Food Science in Europe), although at present, these products are still a

“virtual category” in terms of food law and its definition is still an ongoing issue. FUFOSE was coordinated by the International Life Sciences Institute (ILSI) Europe (http://europe.ilsi.org/) and its aim was to develop and establish a science-based approach for concepts in functional food science. As a culmination of this EU Concerted Action, recommendations and key points raised were summarized in a consensus document published in the „British Journal of Nutrition‟.² According to this document, a functional food can be an unmodified natural food, traditionally considered as a “healthy food”, or a conventional food product to which its chemical composition is modified to give a beneficial effect to one or more functions of the body. Thus, the different strategies that may be followed for the development of functional foods involve the addition or removal of a food component, the increase on the concentration or bioavailability of a functional component, or any combination of these possibilities. In that document it was also specified that a functional food must remain food, that is, maintaining a physical structure equivalent to its non-functional analog, and it must demonstrate its effects in the amounts commonly consumed in the diet as part of a normal food pattern. Examples of functional foods are shown in Table 1.

1 European Commission – Research area of Food, Agriculture& Fisheries & Biotechnology. Functional Foods. 2010.

Available in: ftp://ftp.cordis.europa.eu/pub/fp7/kbbe/docs/functional-foods_en.pdf

2 Diplock AT, Aggett PJ, Ashwell M, Bornet F, Fern EB, Roberfroid MB. Scientific concepts of functional foods in Europe: consensus document. Br. J. Nutr., 1999, 81: S1-S27.

Table 1. Examples of functional foods, naturally occurring or developed by different strategies.

An unmodified food that contain a naturally occurring bioactive compound Tomato (source of lycopene); carrots (rich in β-carotene); fish (rich in omega-3).

A food to which a new bioactive component has been added

Prebiotic and probiotic foods to enhance gastrointestinal function and regulate the immune system by improving the intestinal microbial balance; margarine with plant sterol and stanol esters to regulate blood cholesterol levels.

A food from which a component known to cause a deleterious effect has been removed or reduced Free or low-lactose milks; gluten-free foods and infant formula with hydrolyzed protein to deal with food intolerances or allergies.

A food in which the bioavailability or concentration of a functional component has been increased Skimmed milks with vitamin D to enhance calcium absorption; calcium-enriched milks; fruit juices fortified with vitamin C; omega-3 fatty acids-enriched eggs achieved by altered chicken feed.

In the same line, the nutraceutical concept was introduced for those products containing purified preparations of biological active food components that beneficially affect one or more functions of the body but do not maintain the presentation and structure of a conventional food.

They can be ingested in greater amounts than those frequently consumed being marketed as dietary supplements in tablets, capsules and other pharmaceutical presentations. Examples of nutraceuticals could be those containing omega-3 fatty acids that may be protective against cardiac death, sudden death, and myocardial infarction in people who have a history of cardiovascular disease, and β-glucan from oat bran to lower blood cholesterol levels.

One of the most promising areas for the development of functional foods lies in the use of ingredients designed to manipulate the composition and metabolic activity of the human gut microbiota and hence, to improve gastrointestinal health among others benefits. These are represented by the so-called probiotics and prebiotics which represent two of the five categories in which functional food ingredients are commonly classified along with phytochemicals, polyunsaturated fatty acids and bioactive proteins and peptides³ (Figure 1).

The concepts of probiotics and prebiotics will be defined below.

Figure 1. Representation of the five categories in which functional food ingredients are commonly classified. Most prominent among them are prebiotics for being of particular importance in this PhD Thesis.

3 Xu, Y. Perspectives on the 21st century development of functional foods: bridging Chinese medicated diet and functional foods. Int. J. Food Sci. Technol., 2001, 36: 229-242.

Bioactive Proteins /

Peptides

Phytochemicals

Probiotics

Polyunsaturated fatty acids

Prebio

tics

Figure 2 shows a representation introducing the concept of prebiotic which will be looked at in depth throughout this PhD Thesis dissertation.

Figure 2. Representation introducing the concept of prebiotic.

Foods that fall under functional foods concept for containing several components that may deliver benefits beyond basic nutrition should demonstrate their positive health effects based on scientifically-sustained information. A follow-on project from FUFOSE known as PASSCLAIM (Process for the Assessment of Scientific Support for Claims on Foods), explored how health claims might be substantiated, including the evidence needed to support them. Thus, harmonization on nutrition and health claims made on foods was achieved in 2006 with Regulation EC No. 1924/2006 of the European Parliament and of the Council, which complements the general principles for labeling and advertising of foodstuffs laid down in Directive 2000/13/EC. Under this legislation, health claims made in the presentation of foods (including food supplements) can only be used when approved by the European Food Safety Authority (EFSA). EFSA, established with Regulation EC No. 178/2002 laying down the general principles and requirements of food law, is entrusted with the assessment of the scientific data that demonstrate clear cause-and-effect relationships between intake of a compound or food and a health benefit.

In this context, the Regulation EC No. 285/97 concerning novel foods and novel food ingredients lays down an authorization procedure for all foods or ingredients that have no history of significant consumption in EU before May 15, 1997. According to its regulation, a novel food or ingredient should be subjected to a safety assessment prior to acceptance to the EU market. A simplified notification of “substantial equivalence” procedure applies if novel foods have similar composition, nutritional value, metabolism, intended use and level of undesirable substances as existing products. This regulation from 1997 is currently under revision and a proposed update was published in December 2013. Potential changes in the update may cover traditional foods from 3rd countries, as well as the submission and evaluation route (i.e., directly to EFSA rather than being conducted by competent authorities in Member States).⁴

4 European Commission. Proposal for a regulation of the European Parliament and of the Council on novel foods.

Brussels, 18/12/2013. COM(2013) 894 final. 2013/0435(COD). 2013.

Fermented by intestinal colonic microbiota

Selective stimulation of the growth and/or activity of health-promoting bacteria Prebiotics (non-digestible

food ingredients)

The current legislative framework governing introduction of novel probiotics and prebiotics products in the EU, United States, Canada and Japan, has recently been reviewed by a group of experts of the International Scientific Association for Probiotics and Prebiotics (ISAPP).⁵ In the case of bacteria added into foods, which could also be considered novel, there is a periodically updated list of microbes (at the species level) intentionally added to foods which are considered safe for foods and feeds by the EFSA (QPS, Qualified Presumption of Safety).⁶ Similarly, in the United States, the Food and Drug Administration (FDA) maintains a list of ingredients that are GRAS (Generally Recognized As Safe) for use in foods.^7,8 In relation to this, it is also worth mentioning that, at European level, there is also a positive list of authorized enzymes which perform a technological function in foods (Regulation EC No.

1332/2008).

THE HUMAN GUT MICROBIOTA

MICROBIAL DIVERSITY IN THE HUMAN GASTROINTESTINAL TRACT

The human gastrointestinal tract (GIT) harbors a complex and diverse microbial community, mostly bacterial, commonly referred to as “gut flora” or, more appropriately, “gut microbiota”.

It has been estimated that the number of intestinal bacteria is at least 10 times greater than the total number of human somatic cells in the body and suggested that they encode about 100-fold more genes than those found in the human genome.⁹ In this context, the Human Microbiome Project (HMP) Consortium (http://hmpdacc.org/) publishes the most extensive catalogue yet of organisms and genes pertaining to our so-called second genome (or the metagenome of the human gut microbiome). The metabolic activity performed by these bacteria is equal to that of a virtual organ, leading to gut microbiota being often termed as a “forgotten” organ.¹⁰ Fungi, in particular yeasts, and members of the Archaea kingdom (Methanobrevibacter smithii and Methanobrevibacter stadtmanae) also make up a part of the gut microbiota, but in a much more limited number than bacteria.

A fraction estimated at < 30% of the gut microbiota has been cultured to date; nevertheless, since the 1990s, contemporary culture-independent techniques (shown in detail in the next section) have revolutionized our knowledge of the gut microbiota, providing an estimation of the microbial composition and biodiversity of this niche.

5 Kumar H, Salminen S, Verhagen H, Rowland I, Heimbach J, Bañares S, Young T, Nomoto K, Lalonde M. Novel probiotics and prebiotics: road to the market. Curr. Opin. Biotechnol., 2014, 32: 99-103.

6 EFSA Panel on Biological Hazards (BIOHAZ). Statement on the update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA 1: Suitability of taxonomic units notified to EFSA until October 2014. EFSA J., 2014, 12: 3938, 41 pp.

7 Hoadley JE, Rowlands JC. FDA perspectives on food label claims in the United States. In Bagchi D. Nutraceutical and Functional Food Regulations in the United States and Around the World. Elsevier: San Diego, USA. 2014; pp.

121-140.

8 Agarwal S, Hordvik S, Morar S. Nutrition and Health-Related Labeling Claims for Functional Foods and Dietary Supplements in the United States. In Bagchi D. Nutraceutical and Functional Food Regulations in the United States and Around the World. Elsevier: San Diego, USA. 2014; pp. 141-150.

9 Qin J, Li R, Raes J, Arumugam M, Burgdorf K S, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, Mende D R, Li J, Xu J, Li S, Li D, Cao J, Wang B, Liang H, Zheng H, Xie Y, Tap J, Lepage P, Bertalan M, Batto J-M, Hansen T, Le Paslier D, Linneberg A, Nielsen H B, Pelletier E, Renault P. A human gut microbial gene catalogue established by metagenomic sequencing. Nature, 2010, 464: 59-65.

10 O‟Hara AM, Shanahan F. The gut flora as a forgotten organ. EMBO Reports, 2006, 7: 688-693.

Thus, it was found out that the enormous population of bacteria harboring the human intestine is dominated by relatively few phyla (7 of the 55 known, and of these, 4 are rare). However, these are highly diverse at the strain / subspecies level (more than 800 species and possibly over 7000 strains). The phyla contributing to the majority of human gut microbiota are Bacteroidetes (e.g., the genera Bacteroides and Prevotella) and Firmicutes (e.g., the genera Clostridium, Eubacterium, Lactobacillus, Roseburia, and Ruminococcus). Actinobacteria (including Bifidobacterium genus) is also numerically important within the gut microbial community. Other members, present in minor proportions in the human gut, are distributed between Proteobacteria, Verrucomicrobia, Fusobacteria and Cyanobacteria phyla.¹¹

The gut microbiota is highly dynamic and exhibits variations along the length of the gut, both in terms of composition and density. This is due to the different physicochemical features characterizing each of the anatomical regions of the GIT, such as transit rates of the luminal content, local pH, redox potential, availability of diet-derived compounds, and host secretions (e.g., hydrochloric acid, digestive enzymes, bile, and mucus). Thus, the number of bacteria (colony forming units (cfu) per mL) generally increases going down the GIT, ranging from 100-1000 cfu mL^-1 in the highly acidic environment of the stomach to around 10⁵ cfu mL^-1 in the upper small intestine, and up to 10¹² cfu mL^-1 in the colon. In this latter region, conditions are more favorable to bacterial colonization and growth due to a higher exposure to nutrients, slow transit time, near neutral pH, and low-redox potential^12,13 (Figure 3). Furthermore, the intestinal microbial composition is responsive and will highly depend on host genetics, age, diet, lifestyle and other environmental factors (Figures 4 and 5). The fact that colon bacteria contributes to 60% of fecal dry mass makes feces an ideal source to study the gut microbiota by extracting the nucleic acid from fecal specimens, generating bacterial 16S rRNA gene sequences with bacterial primers or using oligonucleotide probes (DNA probes) that target single strand rRNA molecules localized within the ribosomes.

Figure 3. Anatomy of the human gastrointestinal tract and factors affecting microbial density along its length.

Abbreviations: O2, oxygen; MMC, migrating motor complexes; H⁺, medium acidity; cfu, colony forming units.

11 TapJ, MondotS, LevenezF, Pelletier E, Caron C, Furet JP, Ugarte E, Muñoz-TamayoR, Paslier DLE, NalinR, Dore J, Leclerc M. Towards the human intestinal microbiota phylogenetic core. Environ. Microbiol., 2009, 11: 2574-2584.

12 Kovatcheva-Datchary P, Tremaroli V, Bäckhed F. The gut microbiota. In Rosenberg E. The Prokaryotes – Human Microbiology. Springer-Verlag:Heidelberg, Germany. 2013; pp. 3-24.

13 Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nat. Rev. Immunol., 2014, 14: 667-685.

Oral cavity (mouth) Pharynx

Esophagus Stomach

Large intestine

▪ Cecum

▪ Colon

▪ Rectum Small intestine

▪ Duodenum

▪ Jejunum

▪ Ileum

Anus

10¹² O₂ MMC

H⁺

Microbial load (cfu mL^-1)

10²

Ascending Transverse Descending

Figure 4. Factors involved in microbiota establishment from newborn to adult. Figure adapted from Villanueva-Millán et al.¹⁴

Figure 5. Variation in bacterial diversity within the colonic microbiota of three healthy humans. These phylogenetic trees are based on the 16S rRNA bacterial sequence data set (n = 11831) and alignment of Eckburg et al.¹⁵ (A), (B), and (C) show the portion of the whole tree that is contributed by individuals A, B, and C from the study. Each tree represents the whole data set. Within a tree, colored portions (red, blue, yellow) represent diversity unique to the individual, white branches indicate portions of the tree that are shared with another individual, and black branches indicate diversity that was not encountered in a given individual. Individual B had the highest levels of bacterial diversity, individual C had intermediate bacterial diversity and individual A the lowest bacterial diversity. Division names are indicated. The scale bar indicates degree of sequence divergence. Note that 16S rRNA sequenced-based surveys do not always recover identical communities, even from subsamples of the same material; rather, statistically related communities may be recovered. Therefore, while the exact sequences may not be resampled, related sequences from the same phylogenetic fan may be retrieved. Figure reported from Ley et al.¹⁶ Reproduced with kind permission from Elsevier.

14 Villanueva-Millán MJ, Pérez-Matute P, Oteo JA. Gut microbiota: a key player in health and disease. A review focused on obesity. J. Physiol. Biochem., 2015, 1-17.

15 Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA.

Diversity of the human intestinal microbial flora. Science, 2005, 308: 1635-1638.

16 Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature, 2006, 444: 1022-1023.

Mother gestational age Sanitary conditions Exposure to antibiotics

Mode of delivery

Microbiota establishment

> Diversity & > Stability < Diversity

Age Diet

Breast Milk

Infant Formula

At birth Neonate

6 months

2.5 years Adult

Eldery

Vaginal C-section

Microbiota similarto mother’s

vagina > Firmicutes and Bacteroides

Microbiota similar to mother’s skin

Introduction of solid foods and complete

colonization

Similar adult microbiota

> Actinobacteria andFirmicutes

> Actinobacteria and Proteobacteria

Diet Health status Geographical location

Exposure to antibiotics, probiotics, prebiotics Stress

A B C

Because of the low oxygen concentration, anaerobic organisms (i.e., Bacteroides, Bifidobacterium, Eubacterium, Clostridium) dominate the gut by 100- to 1000-fold greater amount than aerobes (i.e., Escherichia, Enterococcus, Streptococcus). Molecular analysis has shown that the present aerobic species reach relatively high cell densities and metabolic activity in the human cecum. In fact, 50% of total bacterial rRNA was found to correspond to these species in this region of the gut. This is in contrast to feces in which only 7% of the total bacterial rRNA from these aerobic species is found.¹⁷ The most common anaerobic and aerobic genera found in different parts of the human GIT are listed in Table 2.

Table 2. Main gut microbiota components present in the different parts of the human gastrointestinal tract. Table adapted from Iannitti & Palmieri.¹⁸

Oral cavity (saliva)

Streptococcus, Veillonella, Lactobacillus, Bifidobacterium, Fusobacterium, Staphylococcus, Bacteroides, Corynebacterium, Neisseria, Yeasts.

Stomach & Duodenum

Streprococcus, Lactobacillus, Bifidobacterium, Bacteroides, Enterobacteriaceae, Yeasts.

Ileum

Streptococcus, Lactobacillus, Bifidobacterium, Bacteroides, Clostridium, Enterobacteriaceae, Yeasts.

Colon

Bacteroides, Eubacterium, Ruminococcus, Coprococcus, Peptostreptococcus, Bifidobacterium Streptococcus, Enterobacteriaceae, Lactobacillus, Clostridium.

In a healthy GIT, all these organisms live in a natural balance called “symbiosis” where many classes of bacteria such as symbionts (health promoting organisms), commensals (organisms which not provide benefit but neither detriment to the host) and pathobionts (potentially pathogenic microorganisms) coexist in equilibrium. When this balance is altered leading to an unnatural shift in the composition of the microbiota (either a reduction in the numbers of symbionts and/or an increase in the numbers of pathobionts), “dysbiosis” occurs, and can involve many human diseases such as inflammatory bowel disease, obesity, diabetes, and celiac disease orsimply digestive discomfort suchas bloating, flatulence, andabdominal pain.¹⁹

TECHNIQUES USED TO CHARACTERIZE THE GUT MICROBIOTA

The techniques currently available to chracterize the gut microbiota cover from the classical cultural microbiological methods to sequencing, the gold standard for taxonomic identification to species level. These techniques are able to demonstrate (i) the microbial diversity of the gut microbiota, (ii) qualitative and quantitative information on bacterial species, and (iii) changes inthegutmicrobiota inrelation todisease. When designing astudy to assess the gut microbiota, the required cost and the depth of analysis must be considered for deciding which technique to use. Table 3 provides a brief description of each of the techniques currently available, along with some of their advantages and disadvantages and Figure 6 gives an overview of them. As shown, the majority of the so-called culture-independent techniques are based on sequence divergences of the small subunit ribosomal RNA (16S rRNA). 16S rRNA genes are highly conserved between bacterial species, but vary in a manner that allows species identification.²⁰

17 Wells A, Saulnier D, Gibson G. Gastrointestinal microflora and interactions with gut mucosa. In Gibson G &

Roberfroid M. Handbook of Prebiotics. CRC Press: Boca Raton, USA. 2008; pp. 13-38.

18 Iannitti T, Palmieri B. Therapeutical use of probiotic formulations in clinical practice. Clin. Nutr., 2010, 29:701-725.

19 Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat.

Rev. Immunol., 2009, 9: 313-323.

20 Fraher MH, O‟Toole PW, Quigley EM. Techniques used to characterize the gut microbiota: a guide for the clinician.

Nat. Rev. Gastroenterol. Hepatol., 2012, 9: 312-322.

Table 3. Techniques employed to characterize the gut microbiota with some of their advantages and disadvantages.

Table adapted from Fraher et al.²⁰

Technique Description Advantages Disadvantages

Culture Isolation of bacteria on selective media.

Semi-quantitative, cheap.

Labor-intensive, limited to culturable bacteria.

Quantitative polymerase chain reaction (qPCR)

Amplification and quantification of 16S rRNA. Reaction mixture contains a compound that fluoresces when it binds to double-stranded DNA.

Phylogenetic identification, quantitative, fast.

PCR bias, unable to identify unknown species.

Denaturing and temperature gradient gel electrophoresis (D/TGGE)

Gel separation of 16S rRNA amplicons in bands using denaturant /

temperature. Obtaining a molecular fingerprint. Each band represents a species.

Bands can be excised for further analysis, semi-quantitative, fast.

No phylogenetic identification, PCR bias.

Terminal restriction fragment length polymorphism (T-RFLP)

Fluorescently labelled primers are amplified and then restriction enzymes are used to digest the 16S rRNA amplicon. Digested fragments separated by gel electrophoresis.

Semi-quantitative, cheap, fast.

No phylogenetic identification, PCR bias, low resolution.

Fluorescence in situ hybridization (FISH)

Fluorescently labelled oligonucleotide probes hybridize complementary target 16S rRNA sequences. When

hybridization occurs, fluorescence can be enumerated using fluorescence microscopy or flow cytometry.

Phylogenetic identification, probes can be designed to target specific phyla or species, no PCR bias, semi-quantitative.

Dependent on probe sequences, unable to identify unknown species.

DNA microarrays Fluorescently labelled oligonucleotide probes hybridize with complementary nucleotide sequences. Fluorescence detected with a laser.

Phylogenetic identification, semi-quantitative, fast.

Cross-hybridization, PCR bias, species present in low levels can be difficult to detect.

Cloned 16S rRNA gene sequencing

Cloning of full-length 16S rRNA amplicon, Sanger sequencing and capillary electrophoresis.

Phylogenetic identification, quantitative.

PCR and cloning bias, laborious, expensive.

Direct sequencing of 16S rRNA amplicons

Massive parallel sequencing of partial 16S rRNA amplicons (e.g. 454 Pyrosequencing^®).

Phylogenetic identification, quantitative, fast, identification of unknown bacteria.

PCR bias, laborious, expensive.

Microbiome shotgun sequencing

Massive parallel sequencing of the whole metagenome (e.g. 454 Pyrosequencing^® or Illumina^®).

Phylogenetic identification, quantitative.

Expensive, analysis of data computationally intense.

Figure 6. Overview of techniques used to characterize the gut microbiota. Figure adapted from Fraher et al.²⁰

THE ROLE OF THE GUT MICROBIOTA IN HUMAN HEALTH

Extensive research has been dedicated to identify microbiota aberrations that are associated with diseases and to study the effect that mutualistic microorganisms (symbionts) exert on human health. Figure 7 provides an overview of the fundamental role played by a “healthy”

gastrointestinal microbiota in human subjects.¹²

Figure 7. Beneficial contributions of intestinal microbiota to human health.

Culture

Total bacterial DNA extracted (human, viral and protozoal)

FISH

Quantitative PCR

DNA microarrays Microbiome

shotgun sequencing

Direct sequencing of partial 16S rRNA amplicons

Separation of 16S rRNA (gel electrophoresis) e.g. DGGE, TGGE, T-RFLP Sequencing of

cloned 16S rRNA amplicons

Amplification of 16S rRNA genes by PCR Stool sample

Renewal of intestinal

epithelial layer

Regulation of intestinal

barrier integrity

Recovery of intestinal

epithelial injury

Intestinal angiogenesis

Improved bowel motility Development

of the nervous system

Regulation of appetite and

behavior

Maintenance of gut homeostasis

Maturation and education of the immune

system Protection

against pathogens (colonization

resistance) Improved

energy harvest through digestion

of complex dietary fibers Production

of nutrients (SCFA and amino acids) Production of vitamins (vitamin K, B12,

and folic acid)

Metabolism of xenobiotics and procarcinogens

As shown, gut microbiota crucially influences on human physiology, metabolism and nutrition, homeostasis and immunity through structural, metabolic and protective functions. Most of these benefits have been associated with the presence of a high number of species belonging to Bifidobacterium and Lactobacillus genera within the gastrointestinal tract, both traditionally classified as beneficial for health (Figure 8).

Figure 8. Generalized scheme of the composition of predominant human fecal bacteria. The bacteria are generally split into those groups that have harmful or pathogenic influences on human health, those that have beneficial effects, and those that may have both. Figure adapted from Gibson & Roberfroid.²¹

FUNCTIONAL FOOD INGREDIENTS FOR GASTROINTESTINAL HEALTH

As it was already noted, there are two main approaches towards improving gastrointestinal health: probiotics and prebiotics. In addition, the combination of pro- and prebiotics to obtain synergistic benefits has been termed symbiotics. The efficacy of probiotics, prebiotics, and symbiotics to improve host health and nutrition is dependent on the changes they elicit in the composition and metabolic activities of the assemblages of gut microorganisms.²²

21 Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr., 1995, 125: 1401-1412.

22 Buddington R. Using probiotics and prebiotics to manage the gastrointestinal tract ecosystem. In Charalampopoulos D & Rastall RA. Prebiotics and Probiotics Science and Technology. Springer: New York, USA. 2009; pp. 1-31.

P. Aeruginosa Proteus sp.

Staphylococci Clostridia Veillonellae

Bifidobacteria Lactobacilli 10⁴

Enterococci E. coli

10²

10¹¹ 10⁸ Streptococci

Bacteroides

Harmful effects Desirable effects

Number / g feces

PROBIOTICS

Probiotics, meaning “for life”, is currently a useful and accepted term used to name bacteria associated with beneficial effects for humans and animals. The firsts to make suggestions concerning the positive role played by some selected bacteria were Elie Metchnikoff (a Russian scientist, Nobel laureate) and Henry Tissier (a French paediatrician studying infantile diarrhea), who created great scientific interest working at the Pasteur Institute in Paris during the early years of the 1900s. However, the obtained results were not always positive and many of the observations were subjective, so the probiotic concept was regarded as scientifically unproven and it received minor interest for subsequent decades. Thus, the term “probiotic” was first introduced in 1965 by Lilly & Stillwell who in contrast to antibiotics, defined probiotics as

„microbially derived factors that stimulate the growth of other organisms‟, although it was not until 1989 when the word was popularized by Roy Fuller (1989) who emphasized the requirement of viability for probiotics and introduced the idea that they would have a beneficial effect on the host. Nowadays, the most widely adopted definition of probiotics was proposed at an Expert Consultation meeting arranged by the Food and Agriculture Organization of the United Nations and the World Health Organization.²³ In a recently published expert consensus report of the ISAPP,²⁴ the FAO/WHO definition was endorsed with a minor grammatical correction as „live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.‟ Microbes from several different bacterial genera, particularly Bifidobacterium and lactic acid bacteria (LAB) such as Lactobacillus, are currently being utilized as common components of probiotic preparations in different foodstuffs such as yoghurts and dairy drinks. Most of these health-promoting bacterial strains are normal residents of, or common transients through, the human digestive system and as such do not display infectivity or toxicity. Furthermore, LAB are widely accepted as probiotics because of their historical association with food fermentation and preservation. Table 4 shows the main species of probiotic microorganisms with QPS status by the EFSA. The additional health benefits claimed by probiotic products related to the gastrointestinal function include improving digestion and immunity and managing digestive disorders such as irritable bowel syndrome and diarrhea.

Table 4. Most important strains of probiotic microorganisms. Table in accordance with published data by Felis et al.²⁵

Genus Principal probiotic strains

Bifidobacterium B. adolescentis, B. animalis, B. bifidum, B. breve, B. longum.

Lactobacillus L. acidophilus, L. casei, L. crispatus, L. delbrueckii, L. johnsonii, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius.

Others Pediococcus acidilactici, Pediococcus pentosaceus, Streptococcus salivarius thermophilus, Propionibacterium freudenreichii, Bacillus clausii, Bacillus coagulans, Saccharomyces cerevisiae boulardi.

23 Food and Agricultural Organization of the United Nations and World Health Organization (FAO/WHO). Report of an expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. 2001; Córdoba, Argentina.

24 Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat. Rev. Gastroenterol. Hepatol., 2014, 11: 506-514.

25 Felis GE, Dellaglio F, Torriani S. Taxonomy of probiotic microorganisms. In Charalampopoulos D & Rastall RA.

Prebiotics and Probiotics Science and Technology. Springer: New York, USA. 2009; pp. 591-637.

PREBIOTICS Prebiotic concept

The concept of prebiotics essentially has the same aim as probiotics, which is to improve host health via modulation of the intestinal microbiota; however, in this case, a different mechanism is employed in order to overcome the major drawback of probiotics which is to ensure a high viability in the product and to have robust survival properties in the gut. Thus, in this case, the target bacteria are already present in the host, so that prebiotics stimulate its growth; however, it should be noted that if the organisms required to promote health are not already present in the gut, due to disease for example, the prebiotic might not manifest useful effects.²⁶ A prebiotic was first defined in 1995 by Gibson & Roberfroid²¹ as „a non-digestive food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health‟;

nevertheless, already in the 1950s, Petuely²⁷ described the bifidogenic activity of lactulose in infants when added to the diet. Later on, Japanese researchers^28,29 demonstrated that specific non-digestible oligosaccharides (NDOs) (especially fructo-oligosaccharides, FOS) were selectively fermented by bifidobacteria and had the capacity, upon feeding, to stimulate their growth in human feces. Subsequently, this definition has been slightly refined and validated on several occasions to keep the concept updated and to expand the original idea (see Table 5).

Table 5. Developing definitions of the prebiotic concept.

‘A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health.’

Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr., 1995, 125: 1401-1412.

‘A prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host wellbeing and health.’

Gibson GR, Probert HM, Van Loo JAE, Rastall RA, Roberfroid MB. Dietary modulation of the human colonic microbiota: updating the concept of prebiotics. Nutr. Res. Rev., 2004, 17: 259-275.

‘A dietary prebiotic is a selectively fermented ingredient that results in specific changes, in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health.’

6th Meeting of the International Scientific Association of Probiotics and Prebiotics (ISAPP), London, Ontario, 2008.

Gibson GR, Scott KP, Rastall RA, Tuohy KM, Hotchkiss A, Dubert-Ferrandon A, Gareau M, Murphy E F, Saulnier D, Loh G, Macfarlane S, Delzenne N, Ringel Y, Kozianowski G, Dickmann R, Lenoir-Wijnkoop I, Walker C, Buddington R. Dietary prebiotics: current status and new definition. Food Sci. Technol. Bull.- Funct. Foods, 2010, 7: 1-19.

‘The prebiotic effect is the selective stimulation of growth and/or activity(ies) of one or a limited number of microbial genus(era) / species in the gut microbiota that confer(s) health benefits to the host.’

Roberfroid MB, Gibson GR, Hoyles L, McCartney AL, Rastall RA, Rowland I, Wolvers D, Watzl B, Szajewska H, Stahl B, Guarner F, Respondek F, Whelan K, Coxam V, Davicco MJ, Léotoing L, Wittrant Y, Delzenne NM, Cani PD, Neyrinck AM, Meheust A. Prebiotic effects: metabolic and health benefits. Br. J. Nutr., 2010, 104, S1-S63.

‘Prebiotics are dietary substances (mostly consisting of oligosaccharides poorly digested by human enzymes and non-starch polysaccharides) that nurture a selected group of microorganisms living in the gut. They favor the growth of beneficial bacteria over that of harmful ones.’

World Gastroenterology Organization (WGO). Global Guidelines Probiotics and Prebiotics. 2011.

26 Steed H, Macfarlane S. Mechanisms of prebiotic impact on health. In Charalampopoulos D & Rastall RA. Prebiotics and Probiotics Science and Technology. Springer: New York, USA. 2009; pp. 135-161.

27 Petuely F. Lactobacillus bifidus flora produced in artificially-fed infants by bifidogenic substances (bifidus factor).

Z. Kinderheilkd, 1957, 79: 174-179.

28 Yazawa K, Imai K, Tamura Z. Oligosaccharides and polysaccharides specifically utilizable by bifidobacteria. Chem.

Pharm. Bull. (Tokyo), 1978, 26: 3306-3311.

29 Mitsuoka T, Hidaka H, EidaT. Effect of fructooligosaccharides on intestinal microflora. Nahrung,1987,31:427-436.