Dietary prebiotics: current status and new definition

In November 2008, a group of scientists met at the 6th Meeting of the International Scientific Association of Probiotics and Prebiotics (ISAPP) in London, Ontario, Canada, to discuss the functionality of prebiotics. As a result of this, it was concluded that the prebiotic field is currently dominated by gastrointestinal events. However, in the future, it may be the case that other mixed microbial ecosystems may be modulated by a prebiotic approach, such as the oral cavity, skin and the urogenital tract. Therefore, a decision was taken to build upon the current prebiotic status and define a niche for ‘dietary prebiotics’. This review is co-authored by the working group of ISAPP scientists and sets the background for defining a dietary prebiotic as ‘‘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’’.

Bacillus coagulans as a probiotic

Probiotics are live microbial feed additions that improve human or animal health. Their activities are towards improving the composition of the gastrointestinal microbiota in a manner that reduces the risk of disorder. In some cases, probiotics are also used therapeutically. Most probiotics use lactobacilli or bifidobacteria as the main constituents. These produce lactic acid as well as other anti-pathogenic attributes. Traditionally, probiotics are incorporated in dairy products (yoghurts or fermented drinks) or in lyophilised form. Because of stability and viability factors, heated products are not usually a target for probiotic use. This is because they are temperature sensitive. However, a spore-forming genus would have the ability to overcome this limitation. Here, we discuss evidence for the spore-forming Gram-positive bacterium Bacillus coagulans as a probiotic.

Prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a randomized, controlled, double-blind, crossover intervention study

BACKGROUND: The absorption of cocoa flavanols in the small intestine is limited, and the majority of the flavanols reach the large intestine where they may be metabolized by resident microbiota. OBJECTIVE: We assessed the prebiotic potential of cocoa flavanols in a randomized, double-blind, crossover, controlled intervention study. DESIGN: Twenty-two healthy human volunteers were randomly assigned to either a high-cocoa flavanol (HCF) group (494 mg cocoa flavanols/d) or a low-cocoa flavanol (LCF) group (23 mg cocoa flavanols/d) for 4 wk. This was followed by a 4-wk washout period before volunteers crossed to the alternant arm. Fecal samples were recovered before and after each intervention, and bacterial numbers were measured by fluorescence in situ hybridization. A number of other biochemical and physiologic markers were measured. RESULTS: Compared with the consumption of the LCF drink, the daily consumption of the HCF drink for 4 wk significantly increased the bifidobacterial (P < 0.01) and lactobacilli (P < 0.001) populations but significantly decreased clostridia counts (P < 0.001). These microbial changes were paralleled by significant reductions in plasma triacylglycerol (P < 0.05) and C-reactive protein (P < 0.05) concentrations. Furthermore, changes in C-reactive protein concentrations were linked to changes in lactobacilli counts (P < 0.05, R(2) = -0.33 for the model). These in vivo changes were closely paralleled by cocoa flavanol-induced bacterial changes in mixed-batch culture experiments. CONCLUSION: This study shows, for the first time to our knowledge, that consumption of cocoa flavanols can significantly affect the growth of select gut microflora in humans, which suggests the potential prebiotic benefits associated with the dietary inclusion of flavanol-rich foods. This trial was registered at clinicaltrials.gov as NCT01091922.

Health benefits of probiotics: are mixtures more effective than single strains?

PURPOSE: Most studies on probiotics utilise single strains, sometimes incorporated into yoghurts. There are fewer studies on efficacy of mixtures of probiotic strains. This review examines the evidence that (a) probiotic mixtures are beneficial for a range of health-related outcomes and (b) mixtures are more or less effective than their component strains administered separately. RESULTS: Mixtures of probiotics had beneficial effects on the end points including irritable bowel syndrome and gut function, diarrhoea, atopic disease, immune function and respiratory tract infections, gut microbiota modulation, inflammatory bowel disease and treatment of Helicobacter pylori infection. However, only 16 studies compared the effect of a mixture with that of its component strains separately, although in 12 cases (75%), the mixture was more effective. CONCLUSION: Probiotic mixtures appear to be effective against a wide range of end points. Based on a limited number of studies, multi-strain probiotics appear to show greater efficacy than single strains, including strains that are components of the mixtures themselves. However, whether this is due to synergistic interactions between strains or a consequence of the higher probiotic dose used in some studies is at present unclear.

In vitro bioaccessibility and gut biotransformation of polyphenols present in the water-insoluble cocoa fraction

Scope: Cocoa, especially the water-insoluble cocoa fraction (WICF), is a rich source of polyphenols. In this study, sequential in vitro digestion of the WICF with gastrointestinal enzymes as well as its bacterial fermentation in a human colonic model system were carried out to investigate bioaccessibility and biotransformation of WICF polyphenols, respectively. Methods and results: The yield of each enzymatic digestion step and the total antioxidant capacity (TAC) were measured and solubilized phenols were characterized by MS/MS. Fermentation of WICF and the effect on the gut microbiota, SCFA production and metabolism of polyphenols was analyzed. In vitro digestion solubilized 38.6% of WICF with pronase and Viscozyme L treatments releasing 51% of the total phenols from the insoluble material. This release of phenols does not determine a reduction in the total antioxidant capacity of the digestion-resistant material. In the colonic model WICF significantly increased of bifidobacteria and lactobacilli as well as butyrate production. Flavanols were converted into phenolic acids by the microbiota following a concentration gradient resulting in high concentrations of 3-hydroxyphenylpropionic acid (3-HPP) in the last gut compartment. Conclusion: Data showed that WICF may exert antioxidant action through the gastrointestinal tract despite its polyphenols being still bound to macromolecules and having prebiotic activity.

Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats

The interaction between the gut microbiota and their mammalian host is known to have far-reaching consequences with respect to metabolism and health. We investigated the effects of eight days of oral antibiotic exposure (penicillin and streptomycin sulfate) on gut microbial composition and host metabolic phenotype in male Han-Wistar rats (n = 6) compared to matched controls. Early recolonization was assessed in a third group exposed to antibiotics for four days followed by four days recovery (n = 6). Fluorescence in situ hybridization analysis of the intestinal contents collected at eight days showed a significant reduction in all bacterial groups measured (control, 1010.7 cells/g feces; antibiotic-treated, 108.4). Bacterial suppression reduced the excretion of mammalian-microbial urinary cometabolites including hippurate, phenylpropionic acid, phenylacetylglycine and indoxyl-sulfate whereas taurine, glycine, citrate, 2-oxoglutarate, and fumarate excretion was elevated. While total bacterial counts remained notably lower in the recolonized animals (109.1 cells/g faeces) compared to the controls, two cage-dependent subgroups emerged with Lactobacillus/Enterococcus probe counts dominant in one subgroup. This dichotomous profile manifested in the metabolic phenotypes with subgroup differences in tricarboxylic acid cycle metabolites and indoxyl-sulfate excretion. Fecal short chain fatty acids were diminished in all treated animals. Antibiotic treatment induced a profound effect on the microbiome structure, which was reflected in the metabotype. Moreover, the recolonization process was sensitive to the microenvironment, which may impact on understanding downstream consequences of antibiotic consumption in human populations.

In vitro fermentation characteristics of whole grain wheat flakes and the effect of toasting on prebiotic potential

Population studies have shown a positive correlation between diets rich in whole grains and a reduced risk of developing metabolic diseases, like diabetes, cardiovascular disease, and certain cancers. However, little is known about the mechanisms of action, particularly the impact different fermentable components of whole grains have on the human intestinal microbiota. The modulation of microbial populations by whole grain wheat flakes and the effects of toasting on digestion and subsequent fermentation profile were evaluated. Raw, partially toasted, and toasted wheat flakes were digested using simulated gastric and small intestinal conditions and then fermented using 24-hour, pH-controlled, anaerobic batch cultures inoculated with human feces. Major bacterial groups and production of short-chain fatty acids were compared with those for the prebiotic oligofructose and weakly fermented cellulose. Within treatments, a significant increase (P<.05) in bifidobacteria numbers was observed upon fermentation of all test carbohydrates, with the exception of cellulose. Toasting appeared to have an effect on growth of lactobacilli as only fermentation of raw wheat flakes resulted in a significant increase in levels of this group.

Determination of the in vivo prebiotic potential of a maize based wholegrain breakfast cereal: a human feeding study

Epidemiological studies have shown an inverse relationship between risk of CVD and intake of whole grain (WG)-rich food. Regular consumption of breakfast cereals can provide not only an increase in dietary WG but also improvements to cardiovascular health. Various mechanisms have been proposed, including prebiotic modulation of the colonic microbiota. In the present study, the prebiotic activity of a maize-derived WG cereal (WGM) was evaluated in a double-blind, placebo-controlled human feeding study (n 32). For a period of 21 d, healthy men and women, mean age 32 (sd 8) years and BMI 23·3 (sd 0·58) kg/m2, consumed either 48 g/d WG cereal (WGM) or 48 g placebo cereal (non-whole grain (NWG)) in a crossover fashion. Faecal samples were collected at five points during the study on days 0, 21, 42, 63 and 84 (representing at baseline, after both treatments and both wash-out periods). Faecal bacteriology was assessed using fluorescence in situ hybridisation with 16S rRNA oligonucleotide probes specific for Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum/perfringens subgroup, Lactobacillus–Enterococcus subgroup and total bacteria. After 21 d consumption of WGM, mean group levels of faecal bifidobacteria increased significantly compared with the control cereal (P = 0·001). After a 3-week wash-out period, bifidobacterial levels returned to pre-intervention levels. No statistically significant changes were observed in serum lipids, glucose or measures of faecal output. In conclusion, this WG maize-enriched breakfast cereal mediated a bifidogenic modulation of the gut microbiota, indicating a possible prebiotic mode of action

Colonization-induced host-gut microbial metabolic interaction

The gut microbiota enhances the host's metabolic capacity for processing nutrients and drugs and modulate the activities of multiple pathways in a variety of organ systems. We have probed the systemic metabolic adaptation to gut colonization for 20 days following exposure of axenic mice (n = 35) to a typical environmental microbial background using high-resolution (1)H nuclear magnetic resonance (NMR) spectroscopy to analyze urine, plasma, liver, kidney, and colon (5 time points) metabolic profiles. Acquisition of the gut microbiota was associated with rapid increase in body weight (4%) over the first 5 days of colonization with parallel changes in multiple pathways in all compartments analyzed. The colonization process stimulated glycogenesis in the liver prior to triggering increases in hepatic triglyceride synthesis. These changes were associated with modifications of hepatic Cyp8b1 expression and the subsequent alteration of bile acid metabolites, including taurocholate and tauromuricholate, which are essential regulators of lipid absorption. Expression and activity of major drug-metabolizing enzymes (Cyp3a11 and Cyp2c29) were also significantly stimulated. Remarkably, statistical modeling of the interactions between hepatic metabolic profiles and microbial composition analyzed by 16S rRNA gene pyrosequencing revealed strong associations of the Coriobacteriaceae family with both the hepatic triglyceride, glucose, and glycogen levels and the metabolism of xenobiotics. These data demonstrate the importance of microbial activity in metabolic phenotype development, indicating that microbiota manipulation is a useful tool for beneficially modulating xenobiotic metabolism and pharmacokinetics in personalized health care. IMPORTANCE: Gut bacteria have been associated with various essential biological functions in humans such as energy harvest and regulation of blood pressure. Furthermore, gut microbial colonization occurs after birth in parallel with other critical processes such as immune and cognitive development. Thus, it is essential to understand the bidirectional interaction between the host metabolism and its symbionts. Here, we describe the first evidence of an in vivo association between a family of bacteria and hepatic lipid metabolism. These results provide new insights into the fundamental mechanisms that regulate host-gut microbiota interactions and are thus of wide interest to microbiological, nutrition, metabolic, systems biology, and pharmaceutical research communities. This work will also contribute to developing novel strategies in the alteration of host-gut microbiota relationships which can in turn beneficially modulate the host metabolism.

Systemic multicompartmental effects of the gut microbiome on mouse metabolic phenotypes

To characterize the impact of gut microbiota on host metabolism, we investigated the multicompartmental metabolic profiles of a conventional mouse strain (C3H/HeJ) (n=5) and its germ-free (GF) equivalent (n=5). We confirm that the microbiome strongly impacts on the metabolism of bile acids through the enterohepatic cycle and gut metabolism (higher levels of phosphocholine and glycine in GF liver and marked higher levels of bile acids in three gut compartments). Furthermore we demonstrate that (1) well-defined metabolic differences exist in all examined compartments between the metabotypes of GF and conventional mice: bacterial co-metabolic products such as hippurate (urine) and 5-aminovalerate (colon epithelium) were found at reduced concentrations, whereas raffinose was only detected in GF colonic profiles. (2) The microbiome also influences kidney homeostasis with elevated levels of key cell volume regulators (betaine, choline, myo-inositol and so on) observed in GF kidneys. (3) Gut microbiota modulate metabotype expression at both local (gut) and global (biofluids, kidney, liver) system levels and hence influence the responses to a variety of dietary modulation and drug exposures relevant to personalized health-care investigations.

Analysis of time-related metabolic fluctuations induced by ethionine in the rat

The time-course of metabolic events following response to a model hepatotoxin ethionine (800 mg/kg) was investigated over a 7 day period in rats using high-resolution (1)H NMR spectroscopic analysis of urine and multivariate statistics. Complementary information was obtained by multivariate analysis of (1)H MAS NMR spectra of intact liver and by conventional histopathology and clinical chemistry of blood plasma. (1)H MAS NMR spectra of liver showed toxin-induced lipidosis 24 h postdose consistent with the steatosis observed by histopathology, while hypertaurinuria was suggestive of liver injury. Early biochemical changes in urine included elevation of guanidinoacetate, suggesting impaired methylation reactions. Urinary increases in 5-oxoproline and glycine suggested disruption of the gamma-glutamyl cycle. Signs of ATP depletion together with impairment of the energy metabolism were given from the decreased levels in tricarboxylic acid cycle intermediates, the appearance of ketone bodies in urine, the depletion of hepatic glucose and glycogen, and also hypoglycemia. The observed increase in nicotinuric acid in urine could be an indication of an increase in NAD catabolism, a possible consequence of ATP depletion. Effects on the gut microbiota were suggested by the observed urinary reductions in the microbial metabolites 3-/4-hydroxyphenyl propionic acid, dimethylamine, and tryptamine. At later stages of toxicity, there was evidence of kidney damage, as indicated by the tubular damage observed by histopathology, supported by increased urinary excretion of lactic acid, amino acids, and glucose. These studies have given new insights into mechanisms of ethionine-induced toxicity and show the value of multisystem level data integration in the understanding of experimental models of toxicity or disease.

In vitro fermentation and prebiotic potential of novel low molecular weight polysaccharides derived from agar and alginate seaweeds

Fermentation properties and prebiotic potential of novel low molecular weight polysaccharides (LMWPs) derived from agar and alginate bearing seaweeds was investigated. Ten LMWPs were supplemented to pH, temperature controlled anaerobic batch cultures inoculated with human feces from three donors, in triplicate. Microbiota changes were monitored using Fluorescent in-situ hybridization and short chain fatty acids, the fermentation end products were analysed using gas chromatography. Of the ten LMWPs tested, Gelidium seaweed CC2253 of molecular weight 64.64 KDa showed a significant increase in bifidobacterial populations from log(10) 8.06 at 0 h to log(10) 8.55 at 24 h (p = 0.018). For total bacterial populations, alginate powder CC2238 produced a significant increase from log(10) 9.01 at 0 h to log(10) 9.58 at 24 h (p = 0.032). No changes were observed in the other bacterial groups tested viz. Bacteroides, Lactobacilli/Enterococci, Eubacterium rectale/Clostridium coccoides and Clostridium histolyticum. The polysaccharides also showed significant increases in total SCFA production, particularly acetic and propionic acids, indicating that they were readily fermented. In conclusion, some LMWPs derived from agar and alginate bearing seaweeds were fermented by gut bacteria and exhibited potential to be used a novel source of prebiotics.

Microbial utilization and selectivity of pectin fractions with various structures

To evaluate the fermentation properties of oligosaccharides derived from pectins and their parent polysaccharides, a 5-ml-working-volume, pH- and temperature-controlled fermentor was tested. Six pectic oligosaccharides representing specific substructures found within pectins were prepared. These consisted of oligogalacturonides (average degrees of polymerization [DP] of 5 and 9), methylated oligogalacturonides (average DP of 5), oligorhamnogalacturonides (average DP of 10 as a disaccharide unit of galacturonic acid and rhamnose), oligogalactosides (average DP of 5), and oligoarabinosides (average DP of 6). The influence of these carbohydrates on the human fecal microbiota was evaluated. Use of neutral sugar fractions resulted in an increase in Bifidobacterium populations and gave higher organic acid yields. The Bacteroides-Prevotella group significantly increased on all oligosaccharides except oligogalacturonides with an average DP of 5. The most selective substrates for bifidobacteria were arabinan, galactan, oligoarabinosides, and oligogalactosides.

Assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1H NMR spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism. In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.