Epilogue: Concluding Thoughtson Food BioactiveOligosaccharidesRobert A. RastallDepartment of Food and Nutritional Sciences, University of Reading, Reading, UK

The level of interest in the biological activities of carbohydrates displayed by the scientific community is increasing.

Scientists in areas as diverse as food, pharmaceuticals, bioenergy and environmental science are looking to the vast

and diverse world of carbohydrates for interesting and exploitable activities. This is not, perhaps surprising given that

carbohydrates are the largest resource of organicmaterial on the planet and can give rise to far greater structural diversity

than other organic molecules. This interest is spread across a wide range of activities making glycobiology a rewarding

multidisciplinary world in which to work.

Of all bioactivities found within carbohydrates, perhaps the one subject to the most intense scrutiny at the present

time is their ability to modulate the activities of the gut microbiota. We are currently undergoing an explosion of under-

standing of the ecology of the mammalian gut and of the impact that this ecology and its metabolites has on human and

animal health. The term “prebiotic” was first applied to carbohydrates in 1995 in recognition that dietary carbohydrates

are a very easy way to induce changes in the gut microecology to improve health. Early research tended to focus on

the widespread changes in population of a small number of microbial groups with known activities, health attributes

and pathogenic or toxigenic potential. Developments in molecular microbiology and DNA sequencing technology have,

however, broadened our view considerably and it is now normal to look at changes in diversity of the ecosystem on a

very broad scale. Our thinking of what constitutes a prebiotic has also evolved somewhat since 1995. Current research

focusses much more on the systemic metabolic consequences of prebiotic dietary intervention via improvements in

metabonomic techniques. These new techniques enable a true systems biology approach to gut microbiota management

and are opening up rich seams for future prebiotic development.

There is now a sizable industry supplying prebiotics for food use. This is based on extraction and hydrolysis of inulin

from chicory, synthesis of fructooligosaccharides from sucrose and synthesis of β-galactooligosaccharides from lactose.

These all share the characteristic of being derived from readily available and inexpensive raw materials. Research is

identifying a rapidly expanding set of candidate prebiotic molecules but each of these will have to compete economi-

cally with these established market leaders. This is likely to demand a clearly defined performance advantage or novel

biological activity.

Where will such novel prebiotics come from? Polysaccharides from plants andmicrobial sources have a lot of potential

for generating bioactive oligosaccharides. Advances in analytical chemistry techniques, particularly multidimensional

NMR and mass spectrometric techniques, are revealing the complexity of these molecules and they represent a rich

resource of available complex carbohydrates that can be modified to generate bioactivity. Wherever one finds complex

carbohydrates, there are likely to be somewhere in the environment enzymes that can degrade them. A search of the

databases reveals hundreds of identified glycosidases with many putative glycosidases identified in sequencing studies.

Food Oligosaccharides: Production, Analysis and Bioactivity, First Edition. Edited by Dr. F. Javier Moreno and Dr. Marıa Luz Sanz.

© 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

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Isolation and characterization of these enzymes is, however a sizeable task but will give us the tools to degrade complex

polysaccharides to oligosaccharides efficiently and with control over the product spectrum.

Alternatively, novel oligosaccharides can be synthesized from sucrose and lactose using either glycosidases (which are

actually transferases that usually usewater as an acceptor) or glycosyltransferases. Our ability tomanipulate the activity of

such enzymes by a combination of genetic engineering and reaction engineering is increasing steadily. Modified glycosyl

transferases can be used to design specific oligosaccharide structures, which have particular properties whenmetabolized

by the gut microbiota.

A seductive biological activity often ascribed to complex oligosaccharides is their ability to inhibit the adhesion of

pathogens and there is significant activity in this area within the scientific community. Whilst it is clear that certain

oligosaccharides can inhibit pathogen binding in vitro, we have essentially no information on how they act in a complex

microbiota. It is not clear at the present time that antiadhesive oligosaccharides have the required selectivity to inhibit

pathogens and not desirable commensals or prophylactic probiotics. This is due to our profound lack of knowledge of

the molecular receptors of commensal bacteria, with virtually all of our knowledge coming from studies on pathogens.

Prebiotics may help fight off infectious organisms by other mechanisms, however. Many of the health-positive species

that reside in the gut can produce powerful antimicrobial agents and stimulating their activity would seem to be a desir-

able aim. Establishing that this mechanism actually operates in the gut environment is, however, challenging and we

need more functional ecology research to establish such mechanisms. Prebiotics can also potentially have an impact on

the immune system. This could either be by changing the balance of bacteria in the gut or due to direct interactions with

immune cells. Such interactions have been demonstrated in vitro but, once again, we need to establish that such interac-

tions occur in the gut environment in vivo. What has been shown repeatedly is that prebiotic consumption does induce

changes in markers of immune function. These are typically cytokines, chemokines, and antibody responses, and shifts

in such markers have been seen several times in well designed human studies. Unfortunately there is no solid consensus

that changes in specific immune function markers necessarily correlate with an improvement in host health. In order

to make scientifically sound claims on improvement of immunity, studies with hard health outcomes are needed. Good

data has been obtained with probiotics with respiratory infections and immunization, cases where an immune challenge

can be predicted to some degree or induced. Such studies are currently thin on the ground for prebiotics and this is

clearly an area deserving of more study.

Prebiotics can, however, bring about large-scale changes in the diversity and activity of the colonic microbiota and this

can potentially have important metabolic consequences for the host. Research in this area is in its infancy but prebiotics

are currently being investigated for their potential to impact on some of the most important diseases of society: obesity

and metabolic syndrome. Early research in human volunteers and animals is encouraging and a thorough evaluation of

the ability to manage these conditions in humans would be timely.

In research of this nature it is common to have small quantities of oligosaccharides for initial biological testing. This

demands that we develop good, reproducible and validated models of the human and animal situation in order to assess

the potential for scale up and further in vivo studies. This is, however, a considerable challenge. In the context of pre-

biotic activity, there are models of the human gut ranging from relatively simple pH-controlled batch culture systems

to very sophisticated multivessel systems that model fermentation and absorption from the gut. Animal models are less

useful for investigating fermentation processes but are essential for physiological studies to give the mechanistic under-

standing to prebiotic function. All such models, however, use evacuated human or animal feces as an inoculum and

there is constant debate about how representative this is of the in vivo gut. The problem is even worse when investi-

gating antiadhesive activities of oligosaccharides. Realistically, such activity can only be investigated using tissue cul-

ture cell lines or ex vivo tissue samples in culture. In most cases it is not at all clear that this activity actually takes

place in vivo.

It is clear that we are rapidly making the goal of designer oligosaccharides a reality in the laboratory. Commercial

exploitation of such technology is, however, lagging behind the discovery research. Manufacture of bioactive oligosac-

charides on a commercial scale demands economically viable processing technology and this is currently the limiting

factor in oligosaccharide manufacture. Isolation and purification of oligosaccharides is, in most cases, a laborious and

time-consuming activity.

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Development of novel prebiotics with interesting and useful biological activities will only be of use to society if they are

formulated into foods and consumed by consumers. This is an area where there is still a lot to be done. Most discovery

research in this field (at least that which is published in scientific journals) has been limited to the evaluation of the

effects of such molecules on the colonic microbiota. There is a marked lack of information on the physicochemical and

organoleptic properties of candidate prebiotics in the context of real food products.

Without doubt, at the present time, the biggest obstacles to the development and application of novel bioactive

oligosaccharides are the regulatory authorities. This is particularly acute in the EU as the European Food Safety Authority

(EFSA), the body tasked with substantiating health claims, has raised the bar very high in terms of supporting evidence

required. The EFSA is applying pharmaceutical standards to the evaluation of functional food products and at the cur-

rent time, it has not passed any positive opinions on prebiotic activity. It seems clear that the food industry will have to

invest substantially in multiple large human studies with clear health outcomes if it is to get approved health claims – not

easy in the healthy population that is the usual target of functional foods.

What, then, does the future hold for bioactive carbohydrates as functional food ingredients? Whilst the level of sci-

entific activity in this area continually increases, particularly at the discovery level, commercial development is lag-

ging behind. In order for them to reach their potential, increased attention needs to be given to the economic and

large-scale manufacturing of oligosaccharides and to the establishment of real health benefits in healthy humans. Our

increased understanding of the role that the gut microbiota plays in human and animal health is stimulating scientists

to look at carbohydrate functional foods. There is, however, a lot more work to be done in communicating the benefits

to consumers.