A review of the effect of iron supplementation on the gut microbiota of children in developing countries and the impact of prebiotics.

Iron is essential for many physiological functions of the body, and it is required for normal growth and development. Iron deficiency (ID) is the most common form of micronutrient malnutrition and is particularly prevalent in infants and young children in developing countries. Iron supplementation is considered the most effective strategy to combat the risk of ID and ID anaemia (IDA) in infants, although iron supplements cause a range of deleterious gut-related problems in malnourished children. The purpose of this review is to assess the available evidence on the effect of iron supplementation on the gut microbiota during childhood ID and to further assess whether prebiotics offer any benefits for iron supplementation. Prebiotics are well known to improve gut-microbial health in children, and recent reports indicate that prebiotics can mitigate the adverse gut-related effects of iron supplementation in children with ID and IDA. Thus, provision of prebiotics alongside iron supplements has the potential for an enhanced strategy for combatting ID and IDA among children in the developing world. However, further understanding is required before the benefit of such combined treatments of ID in nutritionally deprived children across populations can be fully confirmed. Such enhanced understanding is of high relevance in resource-poor countries where ID, poor sanitation and hygiene, alongside inadequate access to good drinking water and poor health systems, are serious public health concerns.

Gamma aminobutyric acid production by commercially available probiotic strains.

AIMS: Certain bacteria can produce gamma aminobutyric acid (GABA) from glutamate in the human intestinal tract, leading to the possibility of altering GABA levels through diet. To this end, we assessed the ability of seven commercially available probiotic supplements to produce GABA. METHOD AND RESULTS: Probiotic strains were compared for GABA production in pure culture. The bacteria were inoculated at a concentration of 107 CFU ml-1 in 10 ml MRS supplemented with monosodium glutamate (1% w/v), both with and without oligofructose-enriched inulin (OFI) (1% w/v). Two strains with the highest production of GABA were further assessed for 48 h in pH-controlled anaerobic batch cultures inoculated with faecal bacteria. Liquid chromatography-mass spectrometry (LC-MS) was used for quantification of GABA and microbiota composition was determined through 16S rRNA gene sequencing. Levilactobacillus brevis LB01 (CGMCC 16921) and Lactiplantibacillus plantarum 299v (DSM 9843) were the most efficient producers of GABA. High GABA levels (28.32 mmol l-1 ± 0.29) were produced by the probiotic strain L. brevis LB01 at pH 5.4-5.6. This was significantly higher than the levels of GABA produced by L. plantarum (4.8 mmol l-1 ± 6.8) and a negative control (2.9 mM ± 3.1). The addition of OFI did not further stimulate GABA production under the conditions tested. The ability of these strains to produce GABA in-vitro was further evaluated in a faecal microbiota environment. Once again, L.brevis LB01 produced the highest levels of GABA (40.24 mmol l-1 ± 20.98). CONCLUSIONS: L. brevis LB01 was found to be the most efficient probiotic strain, of those tested, for GABA production.

Determination of the prebiotic activity of wheat arabinogalactan peptide (AGP) using batch culture fermentation.

PURPOSE: To test the prebiotic activity of wheat arabinogalactan-peptide (AGP), which is a soluble dietary fibre composed of arabinogalactan polysaccharide linked to a 15-residue peptide, which accounts for up to 0.4% of the dry weight of wheat flour. METHODS: The prebiotic activity of AGP prepared from white wheat flour was tested using in vitro fermentation by colonic bacteria in automated pH-controlled anaerobic stirred batch cultures and compared to fructooligosaccharide (FOS) and wheat flour arabinoxylan (AX). Bacterial populations were measured using fluorescence in situ hybridisation (flow-FISH) and short chain fatty acid (SCFA) concentrations were measured using HPLC. RESULTS: Fermentation of AGP resulted in a significant bifidogenic activity and increased concentrations of SCFAs, mainly acetate after 24 h of fermentation. CONCLUSIONS: These results were comparable to those obtained with AX and confirm the prebiotic potential of AGP. Furthermore, fermentation of a mixture of AGP and AX was faster compared to the single substrates and more similar to FOS, indicating that combinations of fermentable carbohydrates with different structures are potentially more effective as prebiotics than single substrates.

Development of chitosan-coated agar-gelatin particles for probiotic delivery and targeted release in the gastrointestinal tract.

This study reports the development of a novel and simple formulation for probiotic delivery using chitosan-coated agar-gelatin gel particles. This methodology involves the production of agar-gelatin particles by thermally treating a mixture of agar and gelatin solutions at high temperatures (121 °C) and subsequently coating with chitosan. The particles were able to protect the probiotic strain Lactobacillus plantarum NCIMB 8826 during incubation for 2 h in simulated gastric fluid (pH 2), as no statistically significant loss (P > 0.05) in cell concentration was observed, and also resist dissolution in simulated intestinal fluid (pH 7.2). Interestingly, this protection is related to the fact that the intense thermal treatment affected the physicochemical properties of agars and resulted in the formation of a strong and tight polymer network, as indicated by the X-ray diffraction (XRD) analysis. Using an in vitro faecal batch fermentation model simulating the conditions of the distal part of the large intestine (pH 6.7-6.9), it was demonstrated by quantitative real-time PCR that the majority of L. plantarum cells were released from the agar-gelatin particles within 30 to 48 h. Overall, this work led to the development of a novel methodology for the production of probiotic-containing particles, which is simpler compared with current encapsulation technologies and has a lot of potential to be used for the controlled release of probiotics and potentially other solid bioactives in the large intestine.Key Points• Chitosan gel particles is a simple and scalable method of probiotic encapsulation.• Autoclaving agar-gelatin particles increases their stability at low pH.• Chitosan gel particles protected L. plantarum during gastrointestinal conditions.• Probiotics could be controlled release in the colon using chitosan gel particles.

Impact of short-chain galactooligosaccharides on the gut microbiome of lactose-intolerant individuals.

Directed modulation of the colonic bacteria to metabolize lactose effectively is a potentially useful approach to improve lactose digestion and tolerance. A randomized, double-blind, multisite placebo-controlled trial conducted in human subjects demonstrated that administration of a highly purified (>95%) short-chain galactooligosaccharide (GOS), designated "RP-G28," significantly improved clinical outcomes for lactose digestion and tolerance. In these individuals, stool samples were collected pretreatment (day 0), after GOS treatment (day 36), and 30 d after GOS feeding stopped and consumption of dairy products was encouraged (day 66). In this study, changes in the fecal microbiome were investigated using 16S rRNA amplicon pyrosequencing and high-throughput quantitative PCR. At day 36, bifidobacterial populations were increased in 27 of 30 of GOS subjects (90%), demonstrating a bifidogenic response in vivo. Relative abundance of lactose-fermenting Bifidobacterium, Faecalibacterium, and Lactobacillus were significantly increased in response to GOS. When dairy was introduced into the diet, lactose-fermenting Roseburia species increased from day 36 to day 66. The results indicated a definitive change in the fecal microbiome of lactose-intolerant individuals, increasing the abundance of lactose-metabolizing bacteria that were responsive to dietary adaptation to GOS. This change correlated with clinical outcomes of improved lactose tolerance.

Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health.

Adhesion ability to the host is a classical selection criterion for potential probiotic bacteria that could result in a transient colonisation that would help to promote immunomodulatory effects, as well as stimulate gut barrier and metabolic functions. In addition, probiotic bacteria have a potential protective role against enteropathogens through different mechanisms including production of antimicrobial compounds, reduction of pathogenic bacterial adhesion and competition for host cell binding sites. The competitive exclusion by probiotic bacteria has a beneficial effect not only on the gut but also in the urogenital tract and oral cavity. On the other hand, prebiotics may also act as barriers to pathogens and toxins by preventing their adhesion to epithelial receptors. In vitro studies with different intestinal cell lines have been widely used along the last decades to assess the adherence ability of probiotic bacteria and pathogen antagonism. However, extrapolation of these results to in vivo conditions still remains unclear, leading to the need of optimisation of more complex in vitro approaches that include interaction with the resident microbiota to address the current limitations. The aim of this mini review is to provide a comprehensive overview on the potential effect of the adhesive properties of probiotics and prebiotics on the host by focusing on the most recent findings related with adhesion and immunomodulatory and antipathogenic effect on human health.

Evaluation of the prebiotic potential of arabinoxylans extracted from wheat distillers' dried grains with solubles (DDGS) and in-process samples.

Distillers' dried grains with solubles (DDGS) is a low-value agro-industrial by-product, rich in arabinoxylans (AX), which is produced by commercial distillery and bioethanol plants. In a first approach, we investigated the prebiotic potential of four fractions comprising arabinoxylan oligosaccharides (AXOS) and xylooligosaccharides (XOS) obtained by enzymatic hydrolysis of AX fractions derived from DDGS and wet solids (in-process sample of DDGS production process). Anaerobic batch cultures in controlled pH conditions were used to test the prebiotic activity of the samples. Results did not show significant differences between the enzymatic treatments used, and all AXOS/XOS were extensively fermented after 24 h. In addition, significant increases (P < 0.05) in Bifidobacterium and total short-chain fatty acids (SCFAs) were observed after 24 h of fermentation. Finally, DDGS-derived hydrolysates were separated on an anionic semi-preparative column to prepare AXOS/XOS fractions with degree of polymerisation (DP) greater than 3. Bifidogenic activity and an increase of SCFAs were again observed after 24 h of fermentation, although this time, the selectivity was higher and the fermentation slower, suggesting that the fermentation of this substrate could take place (at least partially) in the distal part of the colon with highly desirable beneficial effects.

Impact of inorganic iron and haem on the human gut microbiota; An in vitro batch-culture approach.

Although iron is an essential nutrient for humans, as well as for almost all other organisms, it is poorly absorbed (~15%) from the diet such that most passes through the upper gut into the large intestine. The colonic microbiota is thus exposed to, and potentially influenced by, such residual iron which could have an impact on human health. The aim of the research described here is to determine how the major forms of dietary iron (inorganic iron and haem) influence metabolic activity and composition of the human gut microbiota by utilizing an in vitro parallel, pH-controlled anaerobic batch culture approach. Controlled iron provision was enabled by the design of a 'modified' low-iron gut-model medium whereby background iron content was reduced from 28 to 5 µM. Thus, the impact of both low and high levels of inorganic and haem iron (18-180 µM and 7.7-77 µM, respectively) could be explored. Gut-microbiota composition was determined using next generation sequencing (NGS) based community profiling (16S rRNA gene sequencing) and flow-fluorescent in situ hybridization (FISH). Metabolic-end products (organic acids) were quantified using gas chromatography (GC) and iron incorporation was estimated by inductively coupled plasma optical emission spectroscopy (ICP-OES). Results showed that differences in iron regime induced significant changes in microbiota composition when low (0.1% w/v) fecal inoculation levels were employed. An increase in haem levels from 7.7 to 77 µM (standard levels employed in gut culture studies) resulted in reduced microbial diversity, a significant increase in Enterobacteriaceae and lower short chain fatty acid (SCFA) production. These effects were countered when 18 µM inorganic iron was also included into the growth medium. The results therefore suggest that high-dietary haem may have a detrimental effect on health since the resulting changes in microbiota composition and SCFA production are indicators of an unhealthy gut. The results also demonstrate that employing a low inoculum together with a low-iron gut-model medium facilitated in vitro investigation of the relationship between iron and the gut microbiota.

Impact of 2'-Fucosyllactose on Gut Microbiota Composition in Adults with Chronic Gastrointestinal Conditions: Batch Culture Fermentation Model and Pilot Clinical Trial Findings.

Intestinal dysbiosis has been described in patients with certain gastrointestinal conditions including irritable bowel syndrome (IBS) and ulcerative colitis. 2'-fucosyllactose (2'-FL), a prebiotic human milk oligosaccharide, is considered bifidogenic and butyrogenic. To assess prebiotic effects of 2'-FL, alone or in combination with probiotic strains (potential synbiotics), in vitro experiments were conducted on stool from healthy, IBS, and ulcerative colitis adult donors. In anaerobic batch culture fermenters, Bifidobacterium and Eubacterium rectale-Clostridium coccoides counts, and short-chain fatty acids (SCFAs) including butyrate increased during fermentation with 2'-FL and some of the 2'-FL/probiotic combinations. In a subsequent open-label pilot trial, the effect of a 2'-FL-containing nutritional formula was evaluated in twelve adults with IBS or ulcerative colitis. Gastrointestinal Quality of Life Index (GIQLI) total and gastrointestinal symptoms domain scores, stool counts of Bifidobacterium and Faecalibacterium prausnitzii, and stool SCFAs including butyrate, increased after six weeks of intervention. Consistent with documented effects of 2'-FL, the batch culture fermentation experiments demonstrated bifidogenic and butyrogenic effects of 2'-FL during fermentation with human stool samples. Consumption of the 2'-FL-containing nutritional formula by adults with IBS or ulcerative colitis was associated with improvements in intra- and extra-intestinal symptoms, and bifidogenic and butyrogenic effects.

Raw and Sous-Vide-Cooked Red Cardoon Stalks (Cynara cardunculus L. var. altilis DC): (Poly)phenol Bioaccessibility, Anti-inflammatory Activity in the Gastrointestinal Tract, and Prebiotic Activity.

The in vitro anti-inflammatory and prebiotic activity and the content and profile of bioaccessible (poly)phenols and catabolites of raw and sous-vide-cooked red cardoon (Cynara cardunculus L. var. altilis DC) were investigated during gastrointestinal (GI) digestion. Raw cardoon after in vitro GI digestion had 0.7% bioaccessible (poly)phenols, which protected against lipopolysaccharide-induced inflammation by counteracting IL-8, IL-6, TNF-α, and IL-10 secretions in differentiated Caco-2 cells. Contrarily, GI-digested sous vide cardoon showed higher (poly)phenol bioaccessibility (59.8%) and exerted proinflammatory effects in Caco-2 cells. (Poly)phenols were highly metabolized during the first 8 h of in vitro fermentation, and nine catabolites were produced during 48 h of fermentation. Colonic-fermented raw and sous-vide-cooked cardoon did not show anti-inflammatory activity in HT-29 cells but presented potential prebiotic activity, comparable to the commercial prebiotic FOS, by stimulating health-promoting bacteria such as Bifidobacterium spp. and Lactobacillus/Enterococcus spp. and by increasing the production of total SCFAs, especially acetate.

Malnutrition and Gut Microbiota in Children.

Malnutrition continues to threaten the lives of millions across the world, with children being hardest hit. Although inadequate access to food and infectious disease are the primary causes of childhood malnutrition, the gut microbiota may also contribute. This review considers the evidence on the role of diet in modifying the gut microbiota, and how the microbiota impacts childhood malnutrition. It is widely understood that the gut microbiota of children is influenced by diet, which, in turn, can impact child nutritional status. Additionally, diarrhoea, a major contributor to malnutrition, is induced by pathogenic elements of the gut microbiota. Diarrhoea leads to malabsorption of essential nutrients and reduced energy availability resulting in weight loss, which can lead to malnutrition. Alterations in gut microbiota of severe acute malnourished (SAM) children include increased Proteobacteria and decreased Bacteroides levels. Additionally, the gut microbiota of SAM children exhibits lower relative diversity compared with healthy children. Thus, the data indicate a link between gut microbiota and malnutrition in children, suggesting that treatment of childhood malnutrition should include measures that support a healthy gut microbiota. This could be of particular relevance in sub-Saharan Africa and Asia where prevalence of malnutrition remains a major threat to the lives of millions.

The tick saliva immunosuppressor, Salp15, contributes to Th17-induced pathology during Experimental Autoimmune Encephalomyelitis.

Salp15 is a tick saliva protein that inhibits CD4(+) T cell differentiation through its interaction with CD4. The protein inhibits early signaling events during T cell activation and IL-2 production. Because murine Experimental Autoimmune Encephalomyelitis development is mediated by central nervous system-infiltrating CD4(+) T cells that are specific for myelin-associated proteins, we sought to determine whether the treatment of mice with Salp15 during EAE induction would prevent the generation of proinflammatory T cell responses and the development of the disease. Surprisingly, Salp15-treated mice developed more severe EAE than control animals. The treatment of EAE-induced mice with the tick saliva protein did not result in increased infiltration of T cells to the central nervous system, indicating that Salp15 had not affected the permeability of the blood-brain barrier. Salp15 treatment did not affect the development of antibody responses against the eliciting peptide or the presence of IFNγ in the sera. The treatment with Salp15 resulted, however, in the increased differentiation of Th17 cells in vivo, as evidenced by higher IL-17 production from PLP(139-151)-specific CD4(+) T cells isolated from the central nervous system and the periphery. In vitro, Salp15 was able to induce the differentiation of Th17 cells in the presence of IL-6 and the absence of TGFß These results suggest that a conductive milieu for the differentiation of Th17 cells can be achieved by restriction of the production of IL-2 during T cell differentiation, a role that may be performed by TGFß and other immunosuppressive agents.

Comparative prebiotic activity of mixtures of cereal grain polysaccharides.

The main components of the non-starch polysaccharide (NSP) fraction of wheat flour are arabinoxylan (AX) and ß-glucan. These are also present in other cereal grains, but their proportions vary with AX being the major component in wheat and rye and ß-glucan in barley and oats. Therefore, it was hypothesised that these NSPs could act synergistically when fermented in vitro at the ratios present in the major foods consumed, resulting in increased prebiotic activity. AX and ß-glucan were therefore tested in in vitro fermentation studies to assess their prebiotic activity when used individually and/or in different ratios. Short-chain fatty-acids (SCFAs) produced from in vitro fermentation were measured using HPLC and bacterial populations were measured using flow cytometry with fluorescence in situ hybridisation (Flow-FISH). Fermentation of AX alone resulted in a significant bifidogenic activity and increased concentrations of SCFAs, mainly acetate, after 8-24 h of fermentation, however ß-glucan alone did not show prebiotic activity. The greatest prebiotic activity, based on concentration of total SCFAs and increases in total bacteria as well as beneficial Bifidobacterium and Clostridium coccoides/Eubacterium groups, was observed when AX and ß-glucan were combined at a 3:1 ratio, which corresponds to their ratios in wheat flour which is major source of cereal fibre in the diet. This indicates that the population of bacteria in the human GI tract may be modulated by the composition of the fibre in the diet, to maximise the prebiotic potential.

The role of RcsA in the adaptation and survival of Escherichia coli K92.

The Rcs phosphorelay is a two-component signal transduction system that senses stressful environmental signals such as desiccation or low temperatures, which serve as natural inducers in bacteria. RcsA is an important coregulator in this system involved in some functions regulated by the Rcs system, including biofilm formation and capsule synthesis. In this sense, we previously showed that RcsA is necessary for colanic acid synthesis in Escherichia coli K92. Here, using an E. coli K92ΔrcsA mutant lacking rcsA gene we further characterize the implications of RcsA on E. coli K92 survival under osmotic and oxidative stressful conditions, and bacterial attachment and biofilm formation on both biotic and abiotic surfaces. Our results show that RcsA protects E. coli K92 against osmotic and, especially, oxidative stress at low temperatures. In addition, RcsA did not interfere in biofilm formation in any surface tested, including polystyrene, stainless steel, silicone, Teflon, aluminum and glass. By contrast, deletion of rcsA increased bacterial attachment to the caco-2 cells monolayer used as biotic surface.

Genome Sequences of Potential Probiotic Lactobacillus rhamnosus Isolates from Human Infants.

Probiotics provide health benefits to their hosts, including modulation of host immune response, inhibition of colonization by pathogens, modulation of the gut microbiota, and epithelial barrier enhancement. Here, we present the draft genome sequences of two newly isolated Lactobacillus rhamnosus strains of probiotic potential from healthy human infants.

Milk- and solid-feeding practices and daycare attendance are associated with differences in bacterial diversity, predominant communities, and metabolic and immune function of the infant gut microbiome.

The development of the infant intestinal microbiome in response to dietary and other exposures may shape long-term metabolic and immune function. We examined differences in the community structure and function of the intestinal microbiome between four feeding groups, exclusively breastfed infants before introduction of solid foods (EBF), non-exclusively breastfed infants before introduction of solid foods (non-EBF), EBF infants after introduction of solid foods (EBF+S), and non-EBF infants after introduction of solid foods (non-EBF+S), and tested whether out-of-home daycare attendance was associated with differences in relative abundance of gut bacteria. Bacterial 16S rRNA amplicon sequencing was performed on 49 stool samples collected longitudinally from a cohort of 9 infants (5 male, 4 female). PICRUSt metabolic inference analysis was used to identify metabolic impacts of feeding practices on the infant gut microbiome. Sequencing data identified significant differences across groups defined by feeding and daycare attendance. Non-EBF and daycare-attending infants had higher diversity and species richness than EBF and non-daycare attending infants. The gut microbiome of EBF infants showed increased proportions of Bifidobacterium and lower abundance of Bacteroidetes and Clostridiales than non-EBF infants. PICRUSt analysis indicated that introduction of solid foods had a marginal impact on the microbiome of EBF infants (24 enzymes overrepresented in EBF+S infants). In contrast, over 200 bacterial gene categories were overrepresented in non-EBF+S compared to non-EBF infants including several bacterial methyl-accepting chemotaxis proteins (MCP) involved in signal transduction. The identified differences between EBF and non-EBF infants suggest that breast milk may provide the gut microbiome with a greater plasticity (despite having a lower phylogenetic diversity) that eases the transition into solid foods.

Transcriptional control of RfaH on polysialic and colanic acid synthesis by Escherichia coli K92.

The transcriptional antiterminator RfaH promotes transcription of long operons encoding surface cell components important for the virulence of Escherichiacoli pathogens. In this paper, we show that RfaH enhanced kps expression for the synthesis of group 2 polysialic acid capsule in E. coli K92. In addition, we demonstrate for the first time that RfaH promotes cps expression for the synthesis of colanic acid, a cell wall component with apparently no role on pathogenicity. Finally, we show a novel RfaH requirement for growth at low temperatures.

Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB.

We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.