Research Paper On Prebiotics

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Probiotics and Prebiotics: Current Research and Future Trends | Book

"excellent writing, and effective editing" (SIMB News)

Publisher: Caister Academic Press
Edited by: Koen Venema and Ana Paula do Carmo
Beneficial Microbes Consultancy, 6709 TN Wageningen NL, The Netheralnds and Instituto Federal do Espírito Santo, Soteco, Vila Velha ES, Brazil (respectively)
Pages: xvi + 508
Publication date: August 2015Buy book
ISBN: 978-1-910190-09-8
Price: GB £219 or US $360
Publication date: June 2015Buy ebook
ISBN: 978-1-910190-10-4
Price: GB £219 or US $360

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Composed of nearly a thousand different types of micro-organisms, some beneficial, others not, the human gut microbiota plays an important role in health and disease. This is due to the presence of probiotic or beneficial microbes, or due to the feeding of prebiotics that stimulate the endogenous beneficial microbes: these promote health by stimulating the immune system, improving the digestion and absorption of nutrients, and inhibiting the growth of pathogens. The notable health benefits of probiotic organisms have stimulated much commercial interest, which in turn has led to a plethora of research initiatives in this area; these range from studies to elucidate the efficacy of the various health benefits to analyses of the diet-microbe interaction as a means of modulating the gut microbiota composition. Research in this area is at a very exciting stage.

With state-of-the-art commentaries on all aspects of probiotics and prebiotics research, this book provides an authoritative and timely overview of the field. Written by leading international researchers, each chapter affords a critical insight to a particular topic, reviews current research, discusses future direction and aims to stimulate discussion. Topics range from the different microorganisms used as probiotics (lactobacilli, bifidobacteria, yeast, etc) and techniques and approaches used (metagenomics, etc) to the reviews of the clinical and medical aspects. The provision of extensive reference sections positively encourages readers to pursue each subject in greater detail.

Containing 33 chapters, the book is an invaluable source of information and essential reading for everyone working with probiotics, prebiotics and the gut microbiota, from the PhD student to the experienced scientist, in academia, the pharmaceutical or biotechnology industries and working in clinical environments.


"a well-developed and well researched book compiled by a broad group of expert contributors ... the book is easily read and flows smoothly from topic to topic. The book reflects the dedication and hard work of the editors as well as the contributors. With the depth of coverage, broad range of expertise of the contributors, extensive reference sections, extensive lists of Web resources, as well as the technical format of the writing, this book will be more useful for researchers, faculty, and advanced graduate students ... The breadth of the coverage, excellent writing, and effective editing allow for excellent coverage of complicated topics."fromSIMB News (2017) 67: 68

Table of contents

1. Probiotics and Prebiotics: Current Status and Future Trends

Koen Venema and Ana Paula do Carmo

Pages: 3-12.


Over the past several decades the research into the health benefits of probiotics and prebiotics has rocketed sky high. There are several new applications and diseases and disorders for these healthy dietary components that were previously unthinkable. However, the efficacy has not been scientifically substantiated for all these applications yet, and care needs to be taken that pro- and prebiotics are not considered as a cure for everything. For starters, probiotic effects are strain dependent, and hence not all strains are beneficial for all disorders. In fact, some strains may be detrimental when given to certain patients, and it may aggravate the problems that these patients have. Similarly, prebiotics are not identical, and will stimulate different microorganisms in different individuals, in some case leading to worsening of the disease. Moreover, dose-dependency has rarely been studied and in the case of probiotics, culture conditions may affect their efficacy as well. In addition, although numerous positive results have been obtained with several well-studied probiotic strains, the mechanism of action usually is still completely unclear, let alone what the molecular molecule is that is responsible for the benefit. So, despite several decades of intense research there is still much to be discovered.

2. Functional Aspects of Prebiotics and the Impact on Human Health

Vicky De Preter and Kristin Verbeke

Pages: 13-26.


Due to the recent developments in analytical techniques to analyse the composition of complex microbial ecosystems, our understanding of the intestinal microbiota has tremendously increased. Several disorders have been associated with an altered composition of the gut bacteria. As a consequence, the microbiota is increasingly recognized as a therapeutic target to improve health. Besides having a trophic and protective function, the microbiota is a metabolically very active ecosystem. Amongst the wide variety of metabolites produced, short chain fatty acids (SCFA) constitute the most relevant compounds in relation to health. As far as we know to date, administration of prebiotics selectively modifies the composition of the intestinal microbiota through several mechanisms and favours the saccharolytic fermentation resulting in increased SCFA production. These SCFA play a pivotal role in the health benefits associated with prebiotic intake as they acidify the colonic lumen, which influences metabolic pathways and inhibits pathogens, and act as signaling molecules on specific receptors. In the future, more detailed information on the exact role of each individual SCFA and on the proportion of the SCFA produced from different prebiotic substrates will be essential to further exploit the benefits of prebiotic use.

3. Lactobacilli as Probiotics: Discovering New Functional Aspects and Target Sites

Koen Venema and Marjolein Meijerink

Pages: 29-42.


Probiotic lactobacilli have been in use for several decades now. Still, we hardly know the molecular mechanisms underlying the probiotic effect. Two strains, L. rhamnosus GG and L. plantarum WCFS1 have been studied in great detail, and mutants of these strains have greatly aided in our understanding of the interaction with the host. However, several surprising results were obtained as well, and leave more questions than answers. The first part of this chapter lists the recent advances in the molecular understanding of interaction of probiotic lactobacilli with the host. Especially surface molecules are thought to play a crucial role in this interaction. In the second half of the chapter we briefly highlight some of the newest applications. Although there have not been a lot of studies with these novel approaches, the initial results are promising and require further research, not only to confirm the results found, but also to deduce the mode of action of these probiotics.

4. Bifidobacteria: Regulators of Intestinal Homeostasis

Thomas D. Leser, Caroline T. Gottlieb and Eric Johansen

Pages: 43-68.


Bifidobacteria are natural inhabitants of the gastrointestinal tract possessing genetic adaptations that enable colonization of this harsh and complex habitat. Due to their recognized benefits to human health bifidobacteria are used as probiotics; however industrial-scale production of bifidobacteria is a challenge. Bifidobacteria interact with key elements of intestinal functioning and contribute to maintaining homeostasis. Recent scientific progress has demonstrated that bifidobacteria, through strain-dependent interactions with the host may reduce mucosal antigen load, improve the intestinal barrier, and induce regulation of local and systemic immune responses. Continued research on Bifidobacterium-host interactions is expected to bring knowledge on the mechanisms involved in these health effects, and to support the identification of even more efficacious strains that will increase the variety of commercially available products.

5. Propionibacteria also have Probiotic Potential

Gabriela Zárate and Adriana Perez Chaia

Pages: 69-92.


Propionibacteria were first described by the end of the nineteenth century and named some years later by Orla-Jensen (1909) who proposed the genus Propionibacterium for referring to bacteria that produce propionic acid as their main fermentation end-product. Based on habitat of origin, they are conventionally divided into "classical or dairy" and "cutaneous" microorganisms which mainly inhabit dairy/silage environments and the skin/intestine of human and animals, respectively. Historically, the economic relevance of Propionibacterium has been related to the industrial application of classical species as dairy starters for cheeses manufacture and as biological producers of propionic acid. However, propionibacteria also display probiotic potential. Over the last two decades, the ability of these microorganisms to improve the health of humans and animals by being used as dietary microbial adjuncts has been extensively demonstrated. Both in vitro and in vivo studies revealed that propionibacteria are able to modulate in a favorable way gut physiology, microbiota composition and immunity. Much of these health benefits could be related to the ability of propionibacteria to remain in high numbers in the gastrointestinal tract by surviving the adverse environmental conditions and adhering to the intestinal mucosa. In addition, other promising properties like the production of nutraceuticals and relevant biomolecules such as vitamins B and K, conjugated linoleic acid (CLA), exopolysaccharides (EPS), trehalose, bifidogenic factors, bacteriocins, etc have been reported. In recent years, the availability of genome sequences of different propionibacteria species have allowed to deep insight into the metabolism and physiology of these microorganisms and became a useful tool for selecting appropriate strains for technological, functional or probiotic applications. In the present chapter, we review exhaustively the evidences that support the potential of propionibacteria to be used as probiotic supplements for human and animal nutrition. Besides the positive results on health obtained by us and others, the hardiness and adaptability of propionibacteria to both technological and physiological stresses encourage their usage for designing new functional foods.

6. Non-LAB Probiotics: Spore Formers

Loredana Baccigalupi, Ezio Ricca and Emilia Ghelardi

Pages: 93-104.


A growing body of evidence suggests that probiotics can be efficiently used to treat/prevent some illnesses, from gastro-intestinal or urogenital disorders to allergies, cardiovascular and autoimmune diseases and even to prevent the onset of certain cancers. Although Lactic Acid Bacteria (LAB) and bifidobacteria are the most common microbes used in probiotic preparations, yeasts and other bacteria are also widely used. This chapter focus on the use of bacterial spore formers as probiotics. Spore formers are a group of bacteria able to form an endospore (spore), an exceptionally resistant cell that contains all of the necessary genetic information needed to regenerate a new vegetative cell. Bacterial spores have been commercialized as probiotics for more than 50 years and are now extensively used in humans for the treatment of intestinal disorders and as dietary supplements, in animals as growth promoters and competitive exclusion agents and in aquaculture for enhancing the growth and disease-resistance of cultured fish and shrimps. This chapter will first describe the group of spore-forming bacteria, the sporulation process, the structure of the spore and its interactions with human intestinal and immune cells and then summarize the use of some spore former species as probiotics for human and animal use.

7. Mechanisms of Action of Probiotic Yeasts

Flaviano dos Santos Martins and Jacques Robert Nicoli

Pages: 105-114.


Some yeasts such as Saccharomyces boulardii 17 and Saccharomyces cerevisiae UFMG 905 can be used as probiotics to prevent or treat various infectious and inflammatory diseases. Similar to bacterial probiotics, beneficial effects of these yeasts are the results of simultaneous action of various mechanisms such as modulation of some aspects of local and systemic immune responses, trapping of bacterial toxin or pathogenic bacterial cells on yeast surface, and maintenance of intestinal epithelium integrity. Acting together, these mechanisms seem to be responsible for a reduction of inflammatory process, intestinal permeability and bacterial translocation observed during infectious and inflammatory diseases.

8. Yeasts as Probiotics: Established in Animals, but What About Man?

Gunnard K. Jacobson

Pages: 115-134.


Yeasts are single-celled fungi that have been associated with human activity for thousands of years. Yeast strains of Saccharomyces cerevisiae have always been a good, and traditional, source of animal and human nutrition because of their B vitamin content. The use of inactive yeasts, live yeasts, and live yeast products such as "yeast culture" is widespread in animal husbandry. The traditional use of yeast by man has been primarily in food modification (leavening and alcoholic fermentation), but also medicinally, and as a nutritional supplement. Only within the last century have the probiotic properties of some yeast strains been recognized: increased milk yields in dairy cattle and ameliorating diarrheal diseases in man, for example. Some properties may be generic, e.g., oxygen scavenging in the rumen; but other properties are not shared by all strains of Sacch. cerevisiae and the explanations for this fact are under intense scientific scrutiny. The probiotic attributes of the boulardii subgroup of strains within Sacch. cerevisiae have been attributed to effects on enteric pathogens, intestinal barrier function integrity, anti-inflammatory effects, immune stimulation, and trophic effects on the intestinal mucosa. Current research is identifying new yeast probiotics outside of the Sacch. cerevisiae ssp. boulardii cluster.

9. Escherichia coli: More Than A Pathogen?

Maïwenn Olier

Pages: 135-152.


Escherichia coli is well recognized by the wider public for its ability to cause from self-limiting to life-threatening intestinal and extra-intestinal illnesses. E. coli infections remain actually, and primarily, a global public health concern both in developed and developing countries, explaining, at first glance and understandably, the negative connotation associated with this bacterium. Consequently, E. coli employment for therapeutic purpose may be considered as an aberrant concept. We ought however to keep in mind that E. coli is not only a pathogen. Originally isolated from neonatal stools, characterized and named Bacterium coli commune by Theodor Escherich (1884, reprinted in (Escherich, 1988)), E. coli represents the predominant facultative anaerobic resident of our gut microbiota, where it behaves, at first sight, commensally. Nevertheless, clinical and preclinical beneficial effects of certain E. coli strains on the host, i.e. the probiotic E. coli strains, have also been demonstrated and they are reviewed herein. This chapter summarizes current advances in understanding geno- and phenotypic features that allow to discern the pathogen from the commensal or the probiotic E. coli. Findings in this field highlighted how complex it sometimes is to establish a clear border between probiotic and pathogenic features within E. coli populations. Consequently, ready-made guidelines for the use of probiotic E. coli strains by practicing physicians are still lacking.

10. The Paradoxical Role of Enterococcus Species in Foods

Luís Augusto Nero, Svetoslav Dimitrov Todorov and Luana Martins Peri

Pages: 153-166.


Enterococcus species present a diversity of genetic and phenotypic features, allowing them to present a high capacity to survive and grow at different conditions, explaining its constant presence in food production and processing environments and in end products. These microorganisms are able to produce a wide variety of virulence factors, highlighting their relevance as safety indicators in foods; however, they are also able to produce bacteriocins, called enterocins, and to promote specific modifications in food during fermentation. Enterococci are present in a variety of ripened cheeses, especially from the Mediterranean region, being responsible for specific aromas and flavors that determine the characteristics of these artisanal foods. Also, many Enterococcus species were characterized due to their probiotic potential, being included in commercial products to be consumed by humans and animals, aiming the promotion of health and well-being. Despite being known for possessing virulence genes, many studies demonstrated the absence of expression of such genes, especially isolates obtained from food systems, leading to studies that investigate their real relevance as pathogenic microorganisms. So, this paradoxical role of enterococci in foods must be properly discussed by food microbiologists, which is the focus of this chapter.

11. Use of recLABs: Good Bugs to Deliver Molecules of Health Interest. From Mouse To Man

Jean-Marc Chatel, Natalia Breyner, Debora Rama, Vasco Azevedo, Anderson Myioshi and Philippe Langella

Pages: 167-180.


Lactic acid bacteria (LAB) constitute a heterogeneous group of Gram-positive bacteria widely used in the food industry because of their generally regarded as safe (GRAS) status. In this regard, LAB have been widely investigated and used as live vehicles for the production and delivery of heterologous proteins or cDNA of vaccinal, medical or technological interest because of its ease for protein secretion and purification. Here, we review the expression of heterologous protein and the various delivery systems developed to target heterologous proteins to specific cell locations (cytoplasm, extracellular medium or cell wall), as well as the more recent research on LAB as DNA delivery vehicles and finale with the challenges and future trends to improve the existing strategies and develop new ones.

12. The Indigenous Microbiota and its Potential to Exhibit Probiotic Properties

Sylvie Miquel, Rebeca Martin, Muriel Thomas, Luis G. Bermudez-Humaran and Philippe Langella

Pages: 181-194.


Humans harbour a different microbiota depending on the tissue considered. Most of the microorganisms are contained in the gastro-intestinal tract (GIT) and this gut microbiota represents approximately 1014 cells that correspond to the highest bacterial density for any ecosystem. Our microbiota represents a huge diversity in term of species and functions. A healthy gut microbiota is composed of a well-balanced community of three permanent residents termed symbionts (with beneficial effects), commensals (no effect), and pathobionts (potentially induce pathologies under certain situations), but no pathogens. The term dysbiosis (microbial imbalance) has been related to many different kinds of pathologies although it is not clear whether the imbalance of such a microbiota is a cause or a consequence of the illness. Nowadays, the challenge of linking microbiota to human health and disease is being tackled by different research teams around the world with the aim to investigate the implication of potential beneficial bacteria that could be decreased in the studied microbiota of patients. From this perspective, it could be interesting to use them as potential probiotics to try to resolve dysbioses.

13. Improving the Digestive Tract Robustness of Probiotic Lactobacilli

Hermien van Bokhorst-van de Veen, Peter A. Bron and Michiel Kleerebezem

Pages: 195-204.


This chapter describes stress responses of probiotic lactobacilli, in relation to gastrointestinal (GI-)tract robustness. An overview is given of some newly developed tools to understand and improve stress responses of the model probiotic L. plantarum WCFS1 in relation to its GI persistence. These include a relative simple GI-tract assay and the development of transcriptome-trait matching that associates a trait of interest (e.g., GI-tract survival) with transcripts levels that enables the identification of genetic robustness markers, that can be modulated by pre-adaptation and/or genetic engineering to enhance robustness. Furthermore, specific additives to the in situ delivery matrix may enhance the relative tolerance of specific bacterial strains to detrimental conditions they may encounter in different regions of the GI-tract. Moreover, a methodology that allows the molecular quantification of single strains in a mixed bacterial population using engineered sequence-tags or naturally occurring discriminatory intergenic alleles in combination with next-generation sequencing offers another powerful tool for robustness evaluation. Finally, the stress responses of probiotic cultures in relation to improval of their GI persistence and some future directions for development and GI-tract research in the light of probiotic performance are discussed.

14. Biology of Reactive Oxygen Species, Oxidative Stress, and Antioxidants in Lactic Acid Bacteria

Marta C.T. Leite, Bryan Troxell, Jose M. Bruno-Bárcena and Hosni M. Hassan

Pages: 205-218.


Lactic acid bacteria (LAB) are important in food fermentations and in human health. Due to their physiological and ecological heterogeneity, they encounter a variety of stressors including acid, osmolality, heat, bile-salt, and oxygen. LAB are exposed to oxidative stress caused by the partially reduced reactive oxygen species (ROS) generated from endogenous sources as well as from the environment. Findings over the past fifty years have demonstrated that this group of organisms comprise a heterogeneous mixture of different genera, where some members of the group have the capacity to synthesis antioxidant enzymes like Mn-containing superoxide dismutase (MnSOD), non-heme catalases (i.e., Mn-containing catalase: MnKat), and heme catalases (when provided with exogenous source of heme). Furthermore, some members of the group can accumulate large intracellular concentrations of manganese to use in the detoxification of ROS. In this chapter, we discuss the natural defenses against ROS in LAB as well as the technological practices used in the food and nutraceutical industries to protect LAB from oxidative stress and loss of viability during processing and storage.

15. Functional Aspects of the Endogenous Microbiota that Benefit the Host

Koen Venema

Pages: 221-234.


Over the past 2 or 3 decades we have learned a great deal about our gut microbiota. It is well-established that the microbiota plays a role in just about any disease and disorder that human mankind suffers from. Primarily this is due to the interaction of the microbiota with the host at various levels: i) modulation of the host immune system, which has effects far beyond the local effects in the gut and may reach up to the brain, and ii) production of healthy or toxic microbial metabolites that also may have systemic effects, either directly or after co-metabolism by the host. The composition, and hence the activity of the microbiota, is determined by food and medication. Certain food-components, such as prebiotics, fibers or polyphenols, may lead to production of health-promoting metabolites, while e.g., excess protein may lead to the production or what are generally considered toxic metabolites. Antibiotics and other drugs may influence the (composition and) activity of the microbiota in such as way that the activity has deleterious effects on the host. This review discusses some of the functional aspects of the gut microbiota thought to be important for health of the host. This chapter will review recent insight in the functional role of the microbiota in health and disease.

16. Studying the Microbiota and Microbial Ecology of the Gastrointestinal Tract in the Omics Era: Tools for Stools

Kieran M. Tuohy, Francesca Fava and Nicola Segata

Pages: 235-244.


The human gut microbiota is now recognised as an important contributor to host health and disease, with diet:microbe interactions playing critical roles in immune function, energy balance and even brain development and cognitive function. Importantly, these advances in the fields of physiology and nutrition have come at a time when methodologies for studying microbial communities are providing a comprehensive tool-kit of unparalleled resolution and breadth of coverage, allowing for the first time efficacious measurement of important ecological parameters within the gut microbiome. Over the past thirty years a diverse tool kit has been developed, including molecular methods both for targeted microbial quantitation and semi-quantitative but high resolution/broad spectrum methods capable of identifying theoretically all microorganisms within an ecosystem. These methods too allow us to measure both the metabolic potential and metabolic kinetic of the gut microbiota allowing researchers to shed new light on what was until very recently the black hole of microbiota ecological function within the gut. This chapter describes a collection of molecular genetic and metabolomic approaches used to study the human gut microbiota.

17. Metagenomics of the Gut Microbiota as a Tool for Discovery of New Probiotics and Prebiotics

Hyun Ju You, Jiyeon Si and GwangPyo Ko

Pages: 245-264.


The human gut is comprised of diverse microbiota, which put substantial impact on various human diseases. The gut microbiota is linked to the maintaining gastrointestinal homeostasis and the association between metabolic and inflammatory diseases such as obesity, type 2 diabetes, low-grade chronic inflammation, and inflammatory bowel disease. Consequentially, the growing interest in gut microbiota has resulted in the development of functional foods such as probiotics and prebiotics, which have been reduce the risk of disease and improve host health. Recent advances in nucleotide sequencing technology widen the profile of the previously unknown microbial communities. International collaborative projects such as the MetaHIT initiative and the Human Microbiome Project (HMP) revealed the diversity of the gut microbiota and their metabolic functions and interactions in the host using this new technique. Current advancement of these tools eases analysis of the massive datasets, yet challenges remain for a complete construction of the microbiome. Here we discuss the development of metagenomics along with the challenges and future perspectives for metagenomic studies to find new probiotics and prebiotics.

18. Emerging Applications of Established Prebiotics: Promises Galore

Seema Patel

Pages: 265-274.


Prebiotics are non-digestible food ingredients that invigorate and boost proliferation of health-promoting bacteria in the gastro-intestinal tract, while inhibiting enteric pathogens. The term prebiotics so far encompasses fermentable carbohydrates and oligosaccharides. Prebiotics were catapulted to prominence after their stimulatory role towards bifidobacteria was recognized. They are currently being implicated as sweetener, fat replacer, starter culture formulation, for bacteriocin induction as well as for gut health maintenance, laxative and colitis prevention. However, their functionality has now ramified to multiple significant domains. The unconventional applications have been met with moderate to excellent success viz. cancer inhibition, immune-augmentation, anti-allergic, diabetes control, cholesterol reduction, cardiovascular risk mitigation, obesity management, sepsis prevention, neural protection, vaginal health restoration, bone density increment, guard for kidney ailments, addressing dermatolgical issues, as well as feed additive and antibiotic substitute in aquaculture and veterinary care. The versatile usage of prebiotics has motivated intense research in this field. The exploration of new biological roles for established prebiotics, generation of new compounds from unconventional sources (even extending beyond carbohydrates) and up-grading of existing technology have been identified as promising. This chapter presents in a nutshell, the known prebiotics and their conventional as well as evolving implications for enrichment of food, drugs, and cosmetics and use in the veterinary sector.

19. Prebiotics: Technological Aspects and Human Health

Vanessa Rios de Souza, Camila Carvalho Menezes, Luciana Rodrigues Cunha, Patricia Aparecida Pimenta Pereira and Uelinton Manoel Pinto

Pages: 275-288.


Prebiotics are known as selectively fermented ingredients that allow modifications in the composition and/or activity of the gastrointestinal microbiota conferring benefits to the host wellbeing. They are commonly described as short-chain carbohydrates containing between two and sixty monosaccharides, which are not digested by human or animal enzymes making them selectively fermented by specific beneficial colon bacteria. Besides promoting the intestinal microbiota, they also prevent the incidence of gastrointestinal infections, modulate the immune system, increase the bioavailability of minerals, regulate metabolic disorders related to obesity and diabetes and decrease the risk of cancer. In addition to the functional characteristics, the use of prebiotics by the food industry has been prompted by their favorable technological aspects. In this chapter we present the main functional and technological aspects of inulin, fructo-oligosaccharides (FOS), lactulose, galactoligosaccharides (GOS), resistant starch (RS), soybean oligosaccharides (SOS), xylooligosaccharides (XOS) and isomaltooligosaccharides (IMO).

20. New and Tailored Prebiotics: Established Applications

Shanthi G. Parkar, Paul A. Blatchford, Caroline C. Kim, Douglas I. Rosendale and Juliet Ansell

Pages: 289-314.


‘Prebiotics' have been recognised for nearly 20 years, and have recently been defined as selectively fermented ingredients that that modulate the gut microbial composition and/or activity to deliver benefits to human health, through a range of host and microbial derived mediators. Here, we discuss the emerging range of biomolecules with potential prebiotic properties beyond the well-characterised galacto- and fructo-oligosaccharides. These include carbohydrate-based foods that provide accessible glycoside substrates to the gut microbiome, polyphenols and glucosinolates, which comprise both fermentable sugar moieties and/or alternative phytochemical-based substrates. We examined the microbial and molecular responses to potential prebiotics that validate their benefits to host health in terms of the ability of the prebiotics to alter the gut microbiome and the molecular responses that mediate the link between prebiotics and established host benefits including improved gastrointestinal function.

21. Immunomodulating Effects of Prebiotics and Fibres

Hanne Frøkiær, Stine Broeng Metzdorff and Koen Venema

Pages: 315-330.


The beneficial effects of fibres have long been attributed to their bulking effect, reducing the transit time of chyme through the gastrointestinal (GI) tract, thereby e.g. reducing the contact of potential toxic compounds with the gastrointestinal epithelium and preventing constipation. Lately however, it has been recognized that fibres may have prebiotics effects by modulating the activity of the gut microbiota, and even direct effects on the immune system. This chapter highlights the effects of fibres on the immune system. Mostly direct interactions with the host are discussed, although effects of microbial fermentation products (the so-called short-chain fatty acids [SCFA]) on the immune system is also briefly mentioned.

22. Prebiotics Beyond Fibers

Delphine M. Saulnier and Michael Blaut

Pages: 331-344.


The research on the potential prebiotic properties of polyphenols, human milk oligosaccharides (HMOs), polypeptides and polyols - bioactive compounds not being strictly defined as fibers - has increased at a rapid pace. A selective stimulation of members of the microbiota traditionally associated with health benefits, such as bifidobacteria and lactobacilli, and an inhibition of potentially pathogenic species, has been demonstrated in vitro for most of these compounds. Evidence for health benefits associated with microbiota changes have started to emerge for polyphenols and HMOs in animal studies. An improvement of biomarkers for type-2 diabetes accompanied by an increase in bifidobacteria and lactobacilli occurred in a limited number of healthy subjects in response to the intake of polyphenols. Well-designed double-blind placebo controlled nutritional intervention studies should be conducted to confirm or refute a causal link between changes in microbiota composition and an improvement of type-2 diabetes or obesity. Here we present and discuss our perception of the future trends that we foresee in this field, and provide some recommendations for future research.

23. Synbiotics: More Than Just the Sum of Pro- and Prebiotics?

Koen Venema

Pages: 345-360.


This is a comprehensive but critical overview of the studies performed with synbiotics (products containing both probiotics and prebiotics) up to date. Most of the studies reviewed did not have the desired controls, i.e. arms with only the probiotic or only the prebiotic. This makes it impossible to judge whether the synbiotic (that is the combination of both probiotics and prebiotics) was required for the observed effect, or whether one of the components alone would have done the trick as well. Studies in adults, children and elderly, with and without disorders or diseases are highlighted. Only human trials are discussed, although a few in vitro studies and studies in experimental animals are reviewed to highlight potential mechanisms of action.

24. Pro- and Prebiotics: The Role of Gut Microbiota in Obesity

Marc R. Bomhof and Raylene A. Reimer

Pages: 363-380.


Obesity is a multifactorial disease that is widespread and continuing to increase in prevalence worldwide. The composition of the gut microbiota is altered in obesity and is associated with impaired gut barrier, enhanced proinflammatory response and metabolic disturbances. Manipulation of the gut microbiota as a therapeutic intervention in obesity and other chronic diseases is of major interest. Diet plays a major role in shaping the composition of the gut microbiota and prebiotics and probiotics have received much attention in this regard. This chapter examines the evidence for the effect of prebiotics and probiotics on obesity with special attention given to body weight and adiposity, appetite regulation, inflammation and gut barrier integrity and glucose and lipid metabolism.

25. The Role of the Gut Microbiota in Brain Function

Julia König, John-Peter Ganda Mall, Ignacio Rangel, Hanna Edebol and Robert-Jan Brummer

Pages: 381-390.


The intestinal microbiota forms a complex ecosystem that has an important impact on our health, and an increasing number of disorders are associated with disturbances in this ecosystem. There is growing evidence that even brain function can be affected by an aberrant gut microbiota and that the bidirectional signalling along the microbe-gut-brain axis plays a significant role in the well-being of the gut as well as the brain. In this chapter, mechanistic pathways explaining how the gut microbiota can affect brain function are described. In addition, its role is elucidated in relation to disorders such as anxiety, depression, autism spectrum disorder, Parkinson's disease and Alzheimer's disease, and possible beneficial action of prebiotics and probiotics is discussed.

26. Infant Development, Currently the Main Applications of Probiotics And Prebiotics?

Giuseppe Mazzola, Irene Aloisio and Diana Di Gioia

Pages: 391-406.


Research on probiotics and prebiotics for use in infants is very active and results on their efficacy to prevent and combat several diseases are at present available. Bifidobacteria and lactobacilli are considered beneficial bacteria for the gut, the former being the predominant group of healthy breast-fed newborns. One of the major area of probiotic research in children has been the treatment and prevention of diarrhea. Moreover, a large number of infant pathologies, both enteric (infantile colics, necrotizing enterocolitis, celiac disease) and not strictly enteric (allergies, obesity, neurologic disease) have revealed promising preventive and therapeutic effects of probiotics, although these applications need additional experimental evidences. Recent studies have shown that probiotic strain characteristics are crucial to reach a targeted therapeutic effect. One of the major aspect affecting the gut microbial composition of breast-fed neonates is the presence of oligosaccharides in breast milk. These molecules exert a prebiotic effect which is crucial for the development of a healthy gut microbiota. Research studies have been focused on the selection of fibers possessing a prebiotic role similar to human milk oligosaccharides. Galactooligosaccharides and fructoligosaccharides are abundantly used in infant formula, frequently as mixtures of the two molecules. Several studies have shown that the capability of stimulating beneficial bacteria and of shaping the gut microbiota is similar to that of breast milk. On the contrary, studies regarding the use of prebiotics in infants for the prevention of allergies showed contradictory results. Therefore, it is possible to conclude that children are a very important target, if not the main one, for probiotic and prebiotic administration and the European industry is aware of that.

27. Pro- and Prebiotics in Management of Patients with Irritable Bowel Syndrome

Ratnakar Shukla, Ujjala Ghoshal and Uday C. Ghoshal

Pages: 407-416.


Recently, several studies suggested that altered quantity and quality in gut microbiota, called dysbiosis, are important in the pathogenesis of irritable bowel syndrome (IBS). It is therefore worthwhile to evaluate whether therapeutic manipulation of the gut microbiota using probiotics would be effective and safe in management of these patients. Several studies showed that probiotics are effective in manipulation of altered gut microbiota and in improving the global IBS symptoms. Probiotics reduce visceral hypersensitivity, improve colonic transit time, enhance the intestinal epithelial barrier, modulate immune response via production anti-inflammatory and regulatory cytokines, and thus, inhibit the inflammation. Prebiotics, which are sometimes added to probiotics (symbiotics) improve the fermentation pattern, stool consistency, abdominal pain and bloating and flatulence in patients with IBS. Several meta-analyses have confirmed the efficacy of probiotics in management of IBS. Hence, it can be concluded that probiotics are useful in treatment of IBS, particularly diarrhea predominant subtypes, and most studies showed that abdominal pain, bloating and flatulence are the symptoms, which were relieved most.

28. Pro- and Prebiotics for Oral Health

Svante Twetman, Mette Rose Jørgensen and Mette Kirstine Keller

Pages: 417-428.


Recent insights in the importance of a natural microbial balance in the oral biofilm for maintaining health have brought a new concept to dentistry; to prevent ecologically disruptions rather than merely treating its consequences by restoring cavities. Consequently, an emerging interest in the use of probiotic bacteria for oral health is evident, albeit the local and systemic mechanisms of actions are still largely unknown. This chapter provides a brief background on the role of the oral biofilm in health and disease and examines the evidence for probiotic therapy within dentistry, based on clinical trials. Several studies have displayed an antagonistic role of probiotic lactobacilli and bifidobacteria against salivary Streptococcus mutans and four studies have shown reduced caries incidence and root caries arrest. Other trials have reported beneficial effects on endpoints related to gingivitis and periodontitis such as plaque index, gingival index, probing depth, subgingival microbiota and pro-inflammatory cytokine levels in gingival crevicular fluid. No adverse effects have been reported but further research is needed to confirm findings and strengthen the evidence before clinical recommendations can be advocated.

29. Cholesterol-lowering Effects of Probiotics and Prebiotics

Min-Tze Liong, Byong-H. Lee, Sy-Bing Choi, Lee-Ching Lew, Amy-Sie-Yik Lau and Eric Banan-Mwine Daliri

Pages: 429-446.


Recently, the use of probiotics and prebiotics as a cholesterol lowering agent has become increasingly popular. This chapter will highlight some of the in vitro and in vivo evidence showing the potential of probiotics and prebiotics in improving serum lipid profile. Data revealing details at molecular levels has also been included in this chapter. The proposed mechanisms for cholesterol removal by probiotics include assimilation of cholesterol by growing cells, binding of cholesterol to cellular surface and incorporation into the cellular membrane, deconjugation of bile via bile salt hydrolase, coprecipitation of cholesterol with deconjugated bile and production of short-chain fatty acids from oligosaccharides. In this chapter, we have highlighted on a few more selected cholesterol lowering mechanisms that are feasible and supported by in-depth evidence. Although cholesterol lowering abilities of probiotics has been extensively reported; recently, controversies have risen attributed to the activities of deconjugated bile acids that repress the synthesis of bile acids from cholesterol. Using a molecular docking approach, we have demonstrated that deconjugated bile acids have higher binding affinity towards some orphan nuclear receptors namely the farsenoid X receptor (FXR), leading to a suppressed transcription of the enzyme cholesterol 7-alpha hydroxylase (7AH), which is responsible for bile acid synthesis from cholesterol. Possible detrimental effects due to increased deconjugation of bile salts such as malabsorption of lipids, colon carcinogenesis, gallstones formation and altered gut microbial populations, which contribute to other varying gut diseases, are also included in this chapter. The effects of probiotics and prebiotics on other cholesterol-related disorders such as formation of abnormal erythrocytes are also discussed in this chapter. As described in the past studies, hypercholesterolemia could induce alterations in the human erythrocyte plasma membrane. Administration of probiotics and prebiotics has improved erythrocyte membrane fluidity, decreased membrane rigidity and altered membrane lipid profiles. Probiotics and prebiotics is a new feasible approach to use natural interventions for cholesterol management.

30. Perspectives on Differences Between Human and Livestock Animal Research in Probiotics and Prebiotics

Tyler E. Askelson and Tri Duong

Pages: 447-458.


Probiotics and prebiotics are used widely because of their reported benefits to digestive and immune health. While there is significant evidence to support their effectiveness in humans and livestock animals, interpretation of the results of this research is complicated by the wide differences in research performed in humans as compared to livestock animals. This chapter will explore host-specific digestive physiology, experimental constraints, and probiotic and prebiotic functionality. The insight provided by an understanding of these critically important differences will provide a context in which results of host-specific studies and their broader implications to the science can be evaluated.

31. The Use of Probiotics to Enhance Animal Performance

Juliana Teixeira de Magalhães, Luciene Lignani Bitencourt, Marta Cristina Teixeira Leite, Ana Paula do Carmo and Célia Alencar de Moraes

Pages: 459-468.


Enteric infections by pathogenic bacteria and clinical expression of disease occur frequently in young animals. Multiple environmental changes usually lead to stressful conditions and trigger transitory inflammatory responses in the gut that can contribute to anatomical and functional intestinal disorders (Berge and Wierup, 2011; Heo et al., 2013; Suda et al., 2014). These diseases have often brought about significant economic losses in animal production. In order to solve this problem, antibiotics have been included in animal feeds either at sub-therapeutic levels (acting as growth promoters-AGPs), or at therapeutic levels, to treat diseases. As growth promoters, they reduce competition for nutrients between the gastrointestinal (GI) microbiota and the host. The effects of such competition have often been at the cost of animal performance. Unfortunately, there has been considerable concern over the use of AGP's, because their long term and extensive use in animal production has resulted in selection for survival of resistant bacterial species or strains (Van der Fels-Klerx et al., 2011; Thaker, 2013). Moreover, consumer demand for organic and natural food continues to increase in the world because of an ongoing perception that organic or natural products are better than their conventional counterparts in terms of safety, taste, and increased health benefits. The general term organic foods is used to define foods that are produced without using chemical fertilizers, additives, and synthetic pesticides as well as not being processed with irradiation. Among organic foods, the overall organic meat market size is small compared with the conventional meat industries. However, according to the Organic Trade Association, the organic meat industry has grown $ 3.8 billion in 2014 (OTA, 2014). In general, with increases in organic meat products, new management approaches are needed to compensate for potential food safety concerns and animal health. Thus, the use of probiotics have been suggested as the most desirable alternative for livestock due to their beneficial effects. Probiotic bacterial species are included in the diet to promote health, protecting the intestine against pathogenic microorganisms and reducing inflammation (Ross et al., 2010; Meng et al., 2014, Cho et al., 2011; Thacker, 2013). This chapter will focus the use of probiotics as a feed additive discussing their beneficial effects in swine, poultry, aquaculture and ruminants.

32. Pharmaceutical Aspects of Probiotics and Prebiotics

Indu Pal Kaur, Parneet Kaur Deol, Simarjot Kaur Sandhu and Parveen Rishi

Pages: 469-486.


In recent times, increasing evidence of benefits of probiotics, for health restoration coupled with consumer's inclination towards safe, natural, and cost effective substitutes for drugs, has generated immense interest in exploring their therapeutic potential. Administering probiotics as such not only compromises their viability but also fails to guarantee their successful establishment in gut. In recent times emphasis has been made to take a pharmaceutical approach to probiotics which will not only maintain their viability during storage but will also assist in their successful delivery to and establishment at the site of action in a viable form. Further to above, supplementation of probiotics with prebiotics is also gaining predominance in the recent past. Numerous reports indicate that co-encapsulating prebiotics with probiotics, improves viability of the latter, apart from the independent growth promoting effect of the former on the inherent healthy gut microbiota of the host. The current chapter aims to analyze probiotics as pharmaceuticals, their encapsulation techniques, existing pharmaceutical preparations, their regulatory status in some countries, and future research needs. The current status of prebiotics in health care is also highlighted.

33. Is The Sky The Limit?

Koen Venema and Ana Paula do Carmo

Pages: 489-494.


Over the past two decades significant progress has been made in both the probiotics and the prebiotics area. Whereas earlier the primary diseases for probiotics were inflammatory bowel disease and allergy, this has now expanded to multiple other diseases and disorders too. And whereas earlier a prebiotic effect was considered to be synonymous with an increase in bifidobacteria, the research field now encompasses much more, and for instance looks at butyrate production, or levels of Faecalibacterium prausnitizii or Akkermansia muciniphila. One can therefore wonder: Are pro- and prebiotics effective for everything? This chapter hypothesizes on aspects that are beginning to be addressed, or have not even been considered yet, but might become relevant in the future as well.

How to buy this book

(EAN: 9781910190098 9781910190104 Subjects: [microbiology] [bacteriology] [medical microbiology] [molecular microbiology] [probiotics] )

Citation: Liu Y, Gibson GR, Walton GE (2016) An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly. PLoS ONE 11(9): e0162604.

Editor: Daotai Nie, Southern Illinois University School of Medicine, UNITED STATES

Received: January 29, 2016; Accepted: August 25, 2016; Published: September 9, 2016

Copyright: © 2016 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The authors received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.


Currently, there is an increase in life expectancy, thus a rapidly aging population. According to WHO, the population of adults aged 60 and over has doubled since 1980, and by 2050 this figure is forecast to reach 2 billion, outnumbering children under 14 years of age [1]. The aging population has several health issues, these may include reduced organ function and compromised immune system. Intestinal motility and transit time are slow in older people; this can lead to faecal impaction and constipation [2–4]. Slow colonic transit can also bring about increases in detrimental metabolites of proteolytic bacteria, such as ammonia and amines [5]. There are also problems associated with the diet of elderly people, for example, more limited foods, incorporating less carbohydrates and fewer nutrients [5]. This may be a result of higher thresholds for taste and smell than younger adults [6] and loss of tooth function with difficulties in masticating [7] and swallowing [8].

Elderly populations have a depleted immune defence to exogenous infectious agents but may experience increased immune response to endogenous signals caused by damage of host cells and tissues [9]. This process is loosely termed immunosenescence [10]. Increased levels of cytokines, such as interleukin-6 (IL-6), IL-1β, and tumour necrosis factor-α (TNF-α), decreased phagocytosis and natural killer (NK) cell activity have been observed in elderly populations [11–13]. During aging, the clearance of apoptotic cells is impaired and incomplete [14]. As such, abnormal immune responses including autoimmunity are observed during immunosenescence. In addition, naive B cells generated by bone marrow decrease with increasing age [15], resulting in a reduced ability to protect the host against infectious agents.

There are a great variety of microorganisms inhabiting the human intestinal tract, which is important in maintaining host health and providing a natural defence against invading pathogens [16, 17]. Due to age-related changes in the gastrointestinal tract, such as decreased transit time and increased mucosal membrane permeability [18], as well as changes in diet and immune function, microbial dysbiosis may occur in elderly populations [13]. Studies have shown decreased viable counts of Bacteroides in the elderly compared to younger adults [19, 20]. A reduction of bifidobacteria in terms of numbers and species diversity is also a notable change in elderly populations [19–25]. An increase in facultative anaerobes, such as streptococci, enterococci and enterobacteria is a confirmed age-related phenomenon [20, 21, 23, 26–28].

Overall changes in microbial numbers and species diversity may lead to a reduction in gut function which impacts on the immune response, and potentially results in greater susceptibility to gastrointestinal disorder and metabolic syndrome [24, 28]. In addition, aging is associated with declined mucin production which may lead to increased gut barrier permeability and may enable resident microbiota to more easily traverse gut epithelium cells [29]. A damaged mucosal barrier function with changes in the gut microbiota in elderly people may therefore increase translocation of pathogens and susceptibility to infection [29]. Consequently, these will lead to immune dysregulation. The triadic relationship between an impaired gastrointestinal tract, imbalanced gut microbiota and inflammation has been associated with disease risk in elderly populations, such as infections, colorectal cancer [29] and Clostridium difficile associated diarrhoea [30].

The aging population are prone to infections; therefore, there should be heightened attention to their physiological welfare. Several in vitro and in vivo approaches have shown that prebiotics and probiotics can modulate the gut microbial composition towards a potentially healthier community structure in the elderly [2, 13, 31–33]. They have also been shown to improve immune function in elderly persons [13, 34–36]. 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’ [37]. Probiotics are ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ [38, 39].

The prebiotics B-GOS and inulin have been shown to modulate microbiota composition in elderly persons [2, 13]. B-GOS has also been found to enhance immune function [13]. Compared to inulin, short chain FOS has also been shown to improve immune function in older persons [34, 40, 41]. Two synbiotics containing mixtures of Bifidobacterium bifidum BB-02, Bifidobacterium lactis BL-01 and inulin, and a mixture of Lactobacillus acidophilus and lactitol, were shown to exert positive effects on microbiota composition in healthy elderly persons [16, 42]. Bacillus coagulans GBI-30, 6086 (GanedenBC30 (BC30)) has the potential to suppress the growth of pathogens [43]. In an in vitro study, both the cell wall and the metabolite fractions of BC30 were shown to possess immune modulation properties, anti-inflammatory effects and direct induction of IL-10 [36].

Few studies have directly compared the efficacy of both probiotics and prebiotics in modulation of gut microbiota composition and immune function within the same setting. By targeting a population aged 60–75 it may be possible to target the microbiota and the immune changes in their early stages. Therefore, the aim of this study was to use an in vitro approaches with samples from donors aged 60–75 years to compare the impact of prebiotics and probiotics on the gut microbiota and selected immune markers relevant to the elderly. Common commercial prebiotic and probiotic products were used. Inulin and B-GOS were used as prebiotics; Bifidobacterium bifidum, Lactobacillus acidophilus, and Bacillus coagulans were used as probiotics. Placebos were microcrystalline cellulose and maltodextrin. Bac. thetaiotaomicron and S. typhimurium were also used to investigate the influences of commensal bacteria and a pathogen respectively on the test parameters.

Materials and Methods


Bacteriological growth medium supplements were obtained from Oxoid Ltd. (Basingstoke, Hants, U.K.). Inulin was obtained from BENEO GmbH (Mannheim, Germany) and B-GOS from Clasado Ltd (Milton Keynes, UK). All nucleotide probes used for fluorescent in situ hybridisation (FISH) were commercially synthesised and labelled with the fluorescent dye Cy3 at the 5′ end (Sigma-Aldrich Co. Ltd., Spain). Sterilisation of media and instruments was carried out by autoclaving at 121°C for 15 min.

Bacterial strains and culture preparation

Bacillus coagulans: GBI-30 (PTA-6086, GanedeBC30TM) was sourced from American Type Culture Collection (Manassas, United States) and Bacteroides thetaiotaomicron NCTC 10582 from Health Protection Agency Culture Collection (Salisbury, UK). For Bifidobacterium bifidum NCIMB 30179 (PXN23), Lactobacillus acidophilus NCIMB 30179 (PXN23), Bacteroides thetaiotaomicron NCTC 10582 and Salmonella typhimurium SL134. For each organism growth curves of optical density (OD660nm) against colony forming units (CFU) per millilitre were conducted in triplicate by regular sampling of 48 hour cultures. B. bifidum and L. acidophilus were grown in de Man—Rogosa—Sharpe (MRS) broth (10 ml) (Oxoid Ltd, Basingstoke, Hampshire, UK), at 37°C to late log phase under anaerobic (10:10:80%; H2:CO2:N2) conditions. After centrifuging at 14 000 g for 10 min, supernatants were removed. According to growth curves and standards, concentrations of cells were adjusted to 5×108 CFU/ml by addition of anaerobic phosphate buffered saline (1 M, pH 7.4). Finally, 1ml of 5×108 CFU/ml of cells was added to batch culture vessels immediately. S. typhimurium was grown in Luria Bertani (LB) broth (10 ml) (Oxoid Ltd, Basingstoke, Hampshire, UK) to late log phase in a shaking incubator at 37°C. Bac. thetaiotaomicron was grown in nutrient broth (10 ml) (Oxoid Ltd, Basingstoke, Hampshire, UK) to late log phase anaerobically (10:10:80%; H2:CO2:N2) at 37°C. They were treated in the same way as the probiotics before adding to the batch culture fermenters. 1ml of 5×108 CFU/ml of Salmonella typhimurium SL1344 and 1ml of 5×108 CFU/ml of Bacteroides thetaiotaomicron NCTC 10582 were added to corresponding batch culture vessels immediately.

Bacillus coagulans GBI-30 product contained 1×109 CFU in each capsule. Half a capsule (5×108 CFU) was suspended in 1ml phosphate buffered saline (1 M, pH 7.4). The cells were then immediately added to batch culture vessels.

Faecal sample preparation

Faecal samples were collected from three individuals (62–66 years of age). All volunteers were in good health and had not ingested antibiotics for at least 6 months before the study. Samples were collected on site on the day of the experiment and were used immediately. These were diluted 1:10 (w/v) with anaerobic phosphate buffered saline (PBS; 0.1 M; pH 7.4) and homogenised in a stomacher for 2 min (460 paddle beats/min). Resulting faecal slurries from each individual were used to inoculate batch culture vessels.

Faecal batch culture fermentation

Three separate fermentation experiments were carried out. Batch culture fermentation vessels were autoclaved and aseptically filled with 135 ml of basal nutrient medium (peptone water (2 g/l), yeast extract (2 g/l), NaCl (0.1 g/l), K2HPO4 (0.04 g/l), KH2PO4 (0.04 g/l), NaHCO3 (2 g/l), MgSO4·7H2O (0.01 g/l), CaCl2·6H2O (0.01 g/l), tween 80 (2 ml/l), hemin (50 mg/l), vitamin K1 (10 ml/l), L-cysteine (0.5 g/l), bile salts (0.5 g/l), resazurin (1 mg/l)). The vessels were gassed overnight with O2-free N2 (15 ml/min). Before addition of the faecal slurries, temperature of the basal nutrient medium was set to 37°C by use of a circulating water bath and pH was maintained at 6.8 using a pH controller (Electrolab, UK). The vessels were inoculated with 15 ml of faecal slurry (1:10, w/w), and in order to mimic conditions located in the distal region of the human large intestine the experiment was carried out under anaerobic conditions, 37°C and pH 6.8− 7.0 for a period of 48 h. During this period, samples (10 ml) were collected at six time points (0, 5, 10, 24, 30 and 48 h). Fluorescent in situ hybridisation was used for bacterial enumeration and gas chromatography (GC) for organic acid analysis.

Inoculation of substrate in the batch culture

Batch culture fermentations were conducted using a range of treatments: control (no treatment), trans-galactooligosaccharides mixture (manufactured by Clasado Ltd) called BiMuno® (B-GOS, 1.5g), standard inulin (Orafti® ST, Beneo, Tienen, Belgium; 1.5g), microcrystalline cellulose (1.5g), maltodextrin (1.5g), B. bifidum (5×108 CFU), L. acidophilus (5×108 CFU), Ba. coagulans (5×108 CFU); Bac. thetaiotaomicron (5×108 CFU, commensal bacteria); and S. typhimurium (5×108 CFU, pathogen). B-GOS and inulin are common commercial prebiotics. B. bifidum, L. acidophilus and Ba. coagulans are common commercial probiotics. Microcrystalline cellulose and maltodextrin were used as placebo treatments compared to prebiotics. Bac. thetaiotaomicron and S. typhimurium were also used to investigate the influences of a commensal bacterium and pathogen. In addition, 0.5g potato starch from Sigma-Aldrich Co. Ltd. (UK) was added to each vessel as a fermentable carbon source.

Sample processing

In preparation for FISH analysis 375 μl batch culture supernatant was taken in duplicate into two tubes of 4°C 1125 μl 4% (w/v) paraformaldehyde solution and fixed at 4°C for 4 hours. After 4 hours, the batch culture supernatant was centrifuged for 5 minutes at 11337 xg (Eppendorf centrifuge minispin, Eppendorf, UK) at room temperature. The supernatant was carefully removed and discarded. The pellet was re-suspended in 1 ml of cold 1×PBS by aspirating carefully using a pipette. Again, the sample was centrifuged for 5 minutes at 11337 xg at room temperature and the supernatant discarded. The sample was washed again in 1 ml cold PBS as above and centrifuged. All supernatant was carefully removed. Finally, the pellet was re-suspended in 150 μl cold 1×PBS and 150 μl ethanol. The sample was mixed by vortexing and then stored at -20°C.

In preparation for SCFA analysis, 1 ml of batch culture supernatant was taken in duplicate and centrifuged for 10 minutes at 11337 xg. The supernatant was stored at -20°C.

For in vitro immunoassays, 1 ml of batch culture supernatant was taken in triplicate, centrifuged for 10 minutes at 11337 xg and filtered through a 0.22 μm filter device (Millipore, Schwalbach, Germany). The cell-free supernatant was finally stored at -20°C.

Bacterial enumeration

Bacterial populations were enumerated using FISH, with oligonucleotide probes targeting specific regions of 16S rRNA. Probes were commercially synthesised and coated with the fluorescent dye Cy3. The probes used were: Ato 291 for Atopobium cluster (ATO) [44], Lab 158 for lactobacilli/enterococci (LAB) [45], Bif 164 for bifidobacteria (BIF) [46], Erec 482 for Eubacterium rectaleClostridium coccoides group (EREC) [47], Chis 150 for the Clostridium histolyticum group (CHIS) [47], Bac 303 for Bacteroides—Prevotella spp. (BAC) [48], and EUB 338 mixture consisting of EUB338, EUB338II and EUB338III for total bacteria (Total) [49]. Conditions of hybridisation and washing for individual probes are given in Table 1. Hybridisation of samples was performed as described by Daims, Stoecker [50]. Briefly, the sample was diluted for each probe. 20 μl diluted sample was added to the well of a Teflon- and poly L-lysine-coated 6-well slide (Tekdon Inc, Myakka City, FL). Slides were dried in a desktop plate incubator for 15 minutes at 46–50°C. Then, slides were dehydrated in 50, 80, 96% (v/v) ethanol series for 3 minutes in each solution and then dried for 2 minutes. For probes Lab 158 and Bif 164, 20 μl of lysozyme was added to each well before dehydration in ethanol to increase cell permeability. Then, the hybridisation mixture (0.9 M NaCl, 0.02 M Tris/HCl (pH 8.0), formamide (if required–Table 1), 10% (w/v) sodium dodecyl sulphate, 4.55 ng ml-1 probe) was added to each well, and slides placed on a tray, which was sealed and put in a hybridisation oven for 4h at probe specific hybridisation temperature (Table 1). 20 μl nucleic acid stain 4’, 6-diamidino-2- phenylindole (DAPI; 50 ng μl-1) was added to the wash buffer, once the hybridisation had completed, slides were placed into wash buffer (0.9 M NaCl, 0.02 M Tris/HCl (pH 8.0), 0.005 M ethylenediaminetetraacetic acid (EDTA) solution (pH 8.0, Table 1), H2O) and warmed at the appropriate temperature for each probe (Table 1) for 10–15 minutes. After washing, slides were dipped into ice-cold distilled water for 2–3 seconds and dried by a stream of compressed air. Finally, antifade solution (Dabco) was added to each well, a cover slip applied and slides examined using fluorescent microscopy (Nikon Eclipse E400; Nikon, Tokyo, Japan). The DAPI-stained cells were examined under ultraviolet light, and hybridised cells viewed with the use of a DM510 filter. For each slide, at least 15 random fields of view were counted. The following formula was used to calculate numbers of bacteria: (0.8 × A1 × 6732.42 × 50 × Dilution factor), where A1 is the average count of 15 fields of view, 6732.42 is area of the well divided by the area of the field of view, multiplying by 50 takes the count back to millilitre of sample. Results were expressed as Log10 (bacterial numbers per millilitre batch culture fluid).

Organic acid analysis

Organic acid production was determined by GC. Extraction and derivatisation of samples was conducted according to Richardson, Calder [51]. Briefly, samples were defrosted on ice. Each sample was vortexed and 1 ml sample or a standard solution transferred into a labelled 100 mm×16 mm glass tube (Fisher Scientific UK Ltd, Loughborough) with 50 μl of 2-ethylbutyric acid (0.1 M; internal standard). 0.5 ml concentrated HCl and 2 ml diethyl ether were added to each glass tube and samples vortexed for 1 minute. Samples were centrifuged at 2000 xg for 10 minutes (SANYO MSE Mistral 3000i; Sanyo Gallenkap PLC, Middlesex, UK). The diethyl ether (upper) layer of each sample was transferred to a labelled clean glass tube. A second extraction was conducted by adding another 1 ml diethyl ether, followed by vortexing and centrifugation. The diethyl ether layers were pooled. 400 μl of pooled ether extract and 50 μl N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) were added into a GC screw-cap vial. Samples were heated at 80°C for 20 minutes and then left at room temperature for 48 hours to allow lactic acid in the samples to completely derivatise.

A 5890 SERIES II Gas Chromatograph (Hewlett Packard, UK) using an Rtx-1 10m×0.18mm column with a 0.20μm coating (Crossbond 100% dimethyl polysiloxane; Restek, Buckinghamshire, UK) was used for analysis of SCFA. Temperatures of injector and detector were 275°C, with the column programmed from 63°C for 3 minutes to 190°C at 10°C min-1 and held at 190°C for 3 minutes. Helium was the carrier gas (flow rate 1.2 ml min-1; head pressure 90 MPa). A split ratio of 100:1 was used. The SCFA standard was run every 20 samples to update the calibration as necessary. This standard solution contained (mM): sodium formate, 10; acetic acid, 30; propionic acid, 20; isobutyric acid, 5; n-butyric acid, 20; iso-valeric acid, 5; n-valeric acid, 5; sodium lactate, 10; sodium succinate, 20. Peak areas of the standard solution, to which internal standard was added, were used to calculate response factors for each organic acid with respect to the internal standard. Response factor and peak areas within samples were calibrated and calculated using Chemstation B.03.01 (Agilent Technologies, Cheshire, UK). The response factors were calculated using Eq 1. Amount of organic acids in the samples was calculated using Eq 2.


IS = Internal Standard; SC = Specific Compound of Interest (2)

IS = Internal Standard; SC = Specific Compound of Interest; IRFSC = Internal Response Factor for Specific Compound of Interest

Preparation of peripheral blood mononuclear cells

Fasted blood samples were taken from six healthy volunteers aged 60–73 years, in sodium heparin vacutainer tubes (Greiner Bio-One Limited, Gloucestershire, United Kingdom). The study was conducted according to guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Ethics Committee of the University of Reading. The ethics approval number was UREC 14/05. Written informed consent forms were obtained from all subjects. Blood was layered over an equal volume of lympholyte (Cedarlane Laboratories Limited, Burlington, Ontario, Canada) and centrifuged at 930 xg for 15 min at room temperature. Peripheral blood mononuclear cells (PBMCs) were harvested from the interface, washed once with PBS, and then resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (containing glutamine, Autogen Bioclear Ltd., Wiltshire, UK). These steps were repeated to achieve low contamination of erythrocyte. The pellet was finally resuspended in RPMI 1640 medium and cell numbers counted using trypan blue and a cell counter (Coulter, Fullerton, CA, USA). Cells were adjusted to the required concentration.

Viability assays

To determine the appropriate supernatant concentration, PBMC viability, at different supernatant concentrations was determined using the trypan blue test. PBMCs, adjusted to 2×106 cells/ml, were incubated in twenty-four-well plates in the presence of RPMI 1640 medium, pure batch culture medium supernatant, 0h and 24h supernatant from B. bifidum treated and S. typhimurium treated vessels separately for 24 h at 37°C in an air—CO2 (19:1) atmosphere. The tested supernatant amounts of each treatment were 1%, 1.5%, 2%, 3%, 4%, 5% and 10% (v/v) of 2ml (final working volume). At the end of the incubation, cell numbers were counted using trypan blue test. According to the results, 1% (v/v) was appropriate to use for different treatment supernatants.

Cytokine stimulation and detection

PBMCs, adjusted to 2×106 cells/ml, were incubated in twenty-four-well plates in the presence of 1 mg/ml lipopolysaccharide (LPS; L4516, Sigma-Aldrich Co. Ltd. UK), 1% (v/v) pure batch culture medium, 1 mg/ml LPS with 1% (v/v) pure batch culture medium or 1 mg/ml LPS with 0h, 5h and 24h 1% (v/v) supernatants from ten vessels for 24 h at 37°C in an air—CO2 (19:1) atmosphere. At the end of the incubation, cell culture supernatants were collected and stored at -20°C for later analysis of cytokine production. Non-stimulated cultures were used as blank controls.

The production of IL-1β, IL-6, IL-8, IL-10 and TNF-α was measured using BD Cytometric Bead Array (CBA) Human Soluble Protein Master Buffer Kit (BD Biosciences, Oxford, UK) and corresponding BD Cytometric Bead Array (CBA) Human Flex Set (BD Biosciences, Oxford, UK) by BD Accuri C6 flow cytometer according to the manufacturer’s instructions. BD CBA analysis software FCAP Array v3.0.1 (BD Biosciences, Oxford, UK) was used to perform data analysis.

Statistical analysis

All statistical tests were performed with the use of SPSS version 18 (SPSS Inc, Chicago, IL). Results are presented as means (n = 3) ± SD.

For bacterial populations and SCFA concentrations, within the same treatment, differences from 0-h value were tested using paired Student’s t test. At the same time point, differences among treatments were analysed by one-way ANOVA. For cytokine production, differences from LPS value were tested using an independent t test. Within the same fermentation treatments, variations from 0-h values were tested using paired Student’s t test. At the same time point, differences among treatments in cytokine production were analysed by one-way ANOVA. Significant differences were determined by post hoc Tukey HSD (Honestly Significant Difference) test. A value of P <0.05 indicates a significant difference.


Enumeration of bacterial populations by FISH

Bacterial populations are shown in Fig 1 and S1 Fig. In the control vessel, growth of Atopobium group (p<0.05, paired Student’s t test) and total bacteria (p<0.05, paired Student’s t test) were stimulated compared to 0h. Growth of bifidobacteria was significantly stimulated by B-GOS, inulin and maltodextrin during fermentations compared to control (p<0.05, ANOVA), with higher levels following B-GOS fermentation. B. bifidum, L. acidophilus and Ba. coagulans were also shown to significantly stimulate bifidobacterial numbers compared to time 0h (p<0.05, paired Student’s t test). Numbers of lactobacilli/enterococci were significantly increased following B-GOS, inulin, L. acidophilus and Ba. coagulans at 30h and 48h compared to other treatments (p<0.05, ANOVA). Numbers of Eubacterium rectaleClostridium coccoides were increased following B-GOS fermentations at 48h compared to others (p<0.05, ANOVA). In addition, the Clostridium histolyticum group was reduced following B-GOS fermentation at 30h compared to other treatments (p<0.05, ANOVA). Following maltodextrin fermentation, levels of Bacteroides—Prevotella spp. and Clostridium histolyticum group were significantly stimulated compared to other treatments (p<0.05, ANOVA). Following the different treatments, there was no significant change in total bacterial numbers, indicating that overall bacterial numbers remained constant following prebiotic (B-GOS and inulin) and probiotic (B. bifidum, L. acidophilus and Ba. coagulans) use.

Fig 1. Bacterial populations in pH-controlled batch cultures.

Samples were collected at 0 (white), 5 (shaded), 10 (spots), 24 (fine diaganol lines), 30 (spaced diagonal lines) and 48h (black). (A) Bifidobacteria changes during batch culture fermentation. (B) Lactobacilli/enterococci changes during batch culture fermentation. (C) Eubacterium rectale—Clostridium coccoides group changes during batch culture fermentation. (D) Clostridium histolyticum group changes during batch culture fermentation. Values are mean ± SD from triplicate samples.*, significant differences from the 0h value within the same treatment, p<0.05. Significant differences (p<0.05) among treatments at the same time point are indicated with different letters from the same colour of column.

SCFA analysis

Fig 2 and S2 Fig. shows SCFA concentrations during batch culture fermentations. In the control vessel, as a carbon source, potato starch stimulated the production of all SCFAs compared to 0h (p<0.05, paired Student’s t test). Acetate production was significantly stimulated following B-GOS and maltodextrin fermentation compared to other treatments (p<0.05, ANOVA). Propionate production was significantly stimulated following maltodextrin fermentation compared to other treatments (p<0.05, ANOVA). Levels of butyrate were significantly higher in vessels with B-GOS (p<0.05, ANOVA) and inulin (p<0.05, ANOVA) compared to others. Production of branched chain fatty acids, iso-butyrate and iso-valerate, were repressed by prebiotics (B-GOS and inulin) and probiotics (B. bifidum, L. acidophilus and Ba. coagulans) (p<0.05, ANOVA). However, they were significantly higher in vessels with maltodextrin (p<0.05).

Fig 2. SCFA concentrations in pH-controlled batch cultures.

Samples were collected at 0 (white), 5 (shaded), 10 (spots), 24 (fine diaganol lines), 30 (spaced diagonal lines) and 48h (black). (A) Acetate production during batch culture fermentation. (B) Butyrate production during batch culture fermentation. Values are mean ± SD from triplicate samples. *, significant differences from the 0h value within the same treatment, p<0.05. Significant differences (p<0.05) among treatments at the same time point are indicated with different letters from the same colour of column.


After 24h incubation of PBMC with supernatants, viability of PBMC cells was measured by trypan blue. Viability was 92% with 1% (v/v) RPMI 1640 medium, 80% with 1%(v/v) pure batch culture medium, 64% and 56% with 1% (v/v) 0h and 24h supernatant from B. bifidum, 72% with both 1% (v/v) 0h and 24h supernatant from S. typhimurium. The viability of other amounts (1.5%, 2%, 3%, 4%, 5% and 10% v/v) were all lower than 40%. Differences in viability may have an impact on cytokine production; therefore 1% (v/v) supernatant was used as the most appropriate choice.

Cytokine production

Supernatants from PBMCs cultured without batch culture supernatant were used as controls (+/-). In the absence of LPS, there was no stimulation of IL-1β, IL-6, IL-8, IL-10 and TNF-α (Fig 3 and S3 Fig). Pure batch culture medium did not significantly change the production of IL-1β, IL-6, IL-8, IL-10 and TNF-α induced by LPS (p<0.05, independent t test). LPS-induced TNF-α production, observed in the positive control, was suppressed by 5h and 24h fermentation supernatants from B-GOS, inulin and maltodextrin (p<0.01, independent t test). It was also suppressed by 24h supernatants from B. bifidum, L. acidophilus and Ba. coagulans (p<0.05, independent t test). In addition, LPS-induced IL-10 production was enhanced by 5h and 24h fermentation supernatants from B-GOS, inulin, microcrystalline and maltodextrin (p<0.01, independent t test). It was also enhanced by 5h and 24h supernatants from B. bifidum, L. acidophilus, Bac. thetaiotaomicron and Ba. coagulans fermentations (p<0.05, independent t test). The LPS-induced IL-6 production was only enhanced by 24h supernatants from Ba. coagulans (p = 0.008, independent t test).

Fig 3. Effect of fermentation supernatants on cytokine production by peripheral blood mononuclear cells (PBMC).

Supernatants at 0 (white), 5 (shaded) and 24h (black). Supernatants from PBMCs cultured without batch culture supernatant were used as controls (+/-) (spaced diagonal lines). (A) TNF-α production by PBMC. (B) IL-10 production by PBMC. (C) IL-6 production by PBMC. Values are mean ± SD. PBMC from three volunteers was incubated with batch culture supernatants for 24h. #, significant differences from LPS value p<0.05. *, significant difference from 0-h value within the same fermentation treatments. At the same time point, differences among different treatments in cytokines production were analysed by one-way ANOVA. Significant differences (p<0.05) determined by post hoc Tukey HSD test were not found. In addition, cytokines in non-stimulated PBMC (blank) and in pure batch culture medium-treated PBMC (batch) were also determined. There was no significant difference between them. There was also no significant difference between LPS (LPS-stimulated PBMC) and batch+LPS (PBMC incubated with pure batch culture medium and LPS).


Prebiotics and probiotics have been shown to modulate the intestinal bacterial composition towards a potentially healthy composition in elderly populations in several studies [2, 13, 42, 43]. The current study directly compared the impact of both prebiotics and probiotics on the gut microbiota of elderly volunteers using an in vitro approach; then using an ex vivo approach monitored the potential impact on selected immune parameters.

In the current study B-GOS led to a positive microbial shift, with the potential for reduced inflammation by stimulating bifidobacteria growth, enhancing IL-10 production and inhibiting TNF-α production. Positive effects of B-GOS on colonic bacterial balance with stimulation of bifidobacteria, concurrent with reduced inflammation following intervention was observed by Vulevic et al., [13]. The reduced inflammatory potential observed in the current study was not as dramatic as that observed in the in vivo study of Vulevic, such differences could be related to the PBMC in vitro approach. The impact of B-GOS on the microbiota has been observed in different clinical settings, such as, overweight adults [52] and Irritable Bowel Syndrome patients [53]. In addition, an in vitro study looking at the porcine microbiota also confirmed the positive effects of B-GOS [54]. In the current study, the positive effect of B-GOS on beneficial bacteria at the expense of pathogenic bacteria showed that B-GOS intervention could lead to a potentially beneficial shift of microbiota composition in elderly persons [13, 55]. This is relevant when considering the changes that occur in the microbiota during ageing, this includes lower levels of bifidobacteria and increased inflammation. The results from the current study also showed a positive microbial shift following inulin with stimulation of bifidobacteria and lactobacilli which has also been supported by several in vivo and in vitro studies [2, 56–58].

The current study confirmed the bifidogenic effects of B. bifidum used as a probiotic, rather than in a synbiotic combination. In previous studies, synbiotics containing B. bifidum were also shown to induce a significant stimulatory effect of the bifidobacteria genus rather than B. bifidum alone [16, 59]. Furthermore, the stimulation of lactobacilli by probiotic L. acidophilus in this study was similar to that of a synbiotic containing L. acidophilus, observed to increase faecal lactobacilli levels in healthy elderly [42, 60]. This shows that both of these probiotics possess this functionality in the absence of a prebiotic.

Both prebiotics and probiotics may modulate the microbiota composition by targeting different beneficial bacterial groups. Consequently, gut barrier function may be improved, pathogen infections reduced and disease risk decreased. Prebiotics showed the potential to lead to greater microbiota modulation at the genus level compared to probiotics in the current in vitro study. When comparing B-GOS and inulin, B-GOS showed a greater stimulatory effect on positive bacteria and a greater inhibitory effect on harmful bacteria. This indicates that under the current conditions, B-GOS was a more effective prebiotic candidate in modulating microbiota composition.

Changes in SCFA production were associated with microbiota influences following treatment. As bifidobacteria and E. rectaleC. coccoides are producers of acetic acid [61] and butyric acid [13, 61], respectively, B-GOS and inulin showed stimulatory effects on these two acids. Alterations in SCFA and BCFA production suggest proteolytic fermentation was reduced upon fermentation of B-GOS, inulin, B. bifidum, L. acidophilus and Ba. coagulans. Proteolysis is often associated with dysbiosis and negative fermentation end-products, such as ammonia and nitrosamines [62]. As such the results indicate a potential shift in fermentation to the more beneficial saccharolysis.

Although a few studies have shown that prebiotics and probiotics could directly modulate cytokine production of elderly people in vitro [63–66], the current study is the first to directly compare their effects. In addition, the metabolites of pathogenic and commensal bacteria were considered. Cell-free supernatants contain batch culture medium, faecal water and metabolites of substrates. LPS would invoke an immune response and subsequently stimulate production of immune markers. Cell free fermentation metabolites may subsequently have anti-inflammatory effects by inhibiting production of TNF-α and enhancing production of IL-10. The SCFA production would become stable after 24 hours, therefore batch culture supernatants at 0h, 5h and 24h were collected and incubated with LPS and PBMC.

The down-regulation effects of metabolites from prebiotics and probiotics on TNF-α suggest anti-inflammatory potential. A positive impact may be directly associated with fermentation end products of prebiotics and probiotics. A few studies have shown that TNF-α production induced by stimuli in vitro could be inhibited by SCFA, especially butyrate and acetate [67–70]. This study showed fermentation supernatants from prebiotics and probiotics contained high levels of acetate and butyrate, with anti-inflammatory potential. Therefore, this study indicated the beneficial effects of prebiotics and probiotics metabolites and their beneficial effects on selected immune markers in elderly.

IL-10 is an important anti-inflammatory cytokine, which may counteract the production of proinflammatory cytokines, such as TNF-α [71, 72]. In this study, supernatants from prebiotic and probiotic fermentations enhanced production of IL-10 in vitro. There may be several fermentation metabolites associated with this impact, for example SCFA [67, 69, 72]. Similarly, enhancement of IL-10 production by Bac. thetaiotaomicron may be also linked to its fermentation end products, although the increase was not as dramatic as that produced by prebiotics and probiotics. In this study S. typhimurium has not been found to change inflammation status, although prebiotics and probiotics led to a more positive inflammatory status.

Prebiotics (B-GOS, and inulin) and probiotics (B. bifidum, L. acidophilus and Ba. coagulans) led to a change in the balance of the microbiota to a potentially positive balance, as seen by an increase in bifidobacteria, a group known to be at reduced levels in older people. Furthermore, supernatants from prebiotic and probiotic fermentations showed an anti-inflammatory effect by inhibiting production of pro-inflammatory cytokines and enhancing production of anti-inflammatory cytokines which was possibly related to SCFA concentrations. This research indicates that prebiotics and probiotics have huge potential for modulating the microbiota and inflammation status of elderly people. Furthermore, the prebiotic effect observed was more marked than that of probiotics. Such results are important when evaluating the best treatment to use in targeted interventions.

Supporting Information

S1 Fig. Mean bacterial populations in pH-controlled batch cultures at 0 (white), 5 (shaded), 10 (spots), 24 (fine diaganol lines), 30 (spaced diagonal lines) and 48h (black).

(A) Atopobium cluster changes during batch culture fermentation. (B) Bacteroides—Prevotella spp. changes during batch culture fermentation. (C) Total bacteria changes during batch culture fermentation. Values are mean ± SD from triplicate samples.*, significant differences from the 0h value within the same treatment, p<0.05. Significant differences (p<0.05) among treatments at the same time point are indicated with different letters from the same colour of column.


S2 Fig. SCFA concentrations in pH-controlled batch cultures at 0 (white), 5 (shaded), 10 (spots), 24 (fine diaganol lines), 30 (spaced diagonal lines) and 48h (black).

(A) Propionate production during batch culture fermentation. (B) iso-Butyrate production during batch culture fermentation. (C) iso-Valerate production during batch culture fermentation. Values are mean ± SD from triplicate samples. *, significant differences from the 0h value within the same treatment, p<0.05. Significant differences (p<0.05) among treatments at the same time point are indicated with different letters from the same colour of column.


S3 Fig. Effect of fermentation supernatants from batch cultures on cytokine production by peripheral blood mononuclear cells (PBMC). Supernatants at 0 (white), 5 (shaded) and 24h (black).

Supernatants from PBMCs cultured without batch culture supernatant were used as controls (+/-) (spaced diagonal lines). (A) IL-8 production by PBMC. (B) IL-1β production by PBMC. Values are mean ± SD. PBMC from three volunteers was incubated with batch culture supernatants for 24h. #, significant differences from LPS value p<0.05. *, significant difference from 0-h value within the same fermentation treatments. At the same time point, differences among different treatments in cytokines production were analysed by one-way ANOVA. Significant differences (p<0.05) determined by post hoc Tukey HSD test were not found. In addition, cytokines in non-stimulated PBMC (blank) and in pure batch culture medium-treated PBMC (batch) were also determined. There was no significant difference between them. There was also no significant difference between LPS (LPS-stimulated PBMC) and batch+LPS (PBMC incubated with pure batch culture medium and LPS).


Author Contributions

  1. Conceptualization: GG GW YL.
  2. Data curation: YL.
  3. Formal analysis: YL.
  4. Funding acquisition: GG YL.
  5. Investigation: YL.
  6. Methodology: YL GG GW.
  7. Project administration: GG GW.
  8. Resources: GG.
  9. Supervision: GG GW.
  10. Visualization: YL.
  11. Writing – original draft: YL.
  12. Writing – review & editing: GG GW YL.


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